WO2009037481A1 - Methods for diagnosing and treating dementia - Google Patents

Abstract

The present invention relates to a method of screening for compounds of use in preventing or treating dementia wherein a cell having UBAPl is treated with a test compound and the effect of the test compound on the amount and/or function of UBAPl is assessed. Preferably the compounds of use in preventing or treating dementia increase the amount and/or function of UBAPl. Methods of assessing whether a subject has or is likely to develop a dementia comprising determining whether the subject has a mutation in the UBAPl gene are also provided. The invention also provides a non-human genetically modified animal having or predisposed to develop dementia, wherein the dementia results from an altered amount and/or function of UBAPl polypeptide.

Description

METHODS FOR DIAGNOSING AND TREATING DEMENTIA

The present invention related to methods of use in diagnosing neurological conditions and products that can be used to treat such conditions.

Dementia is the progressive decline in cognitive function due to damage or disease in the brain beyond what might be expected from normal aging. Particularly affected areas may be memory, attention, language, and problem solving. Especially in the later stages of the condition, affected persons may be disoriented in time (not knowing what day of the week, day of the month, month, or even what year it is), in place (not knowing where they are), and in person (not knowing who they are). The prevalence of dementia is rising as the global life expectancy is rising. Particularly in Western countries, there is increasing concern about the economic impact that dementia will have in future, older populaces. Dementia is a non-specific term encompassing many disease processes. At present there is no cure for many types of dementia.

Considerable progress in the understanding of the genetic and biological basis of dementia has been made over the past decade. However while Alzheimer's disease has been studied extensively over this period, our knowledge of the aberrant biology underlying frontotemporal lobar degeneration (FTLD) is still in its infancy.

FTLD is a pathologic process involving degeneration of gray matter in the frontal lobe and anterior portion of the temporal lobe of the cerebrum, with sparing of the parietal and occipital lobes. FTLD is the second most common form of dementia after Alzheimer's disease and is therefore a major cause of neurological problems in the elderly. The syndrome of FTLD encompasses the clinical subgroups of frontotemporal dementia (FTD), FTD with motor neuron disease, semantic dementia and primary progressive aphasia, and is characterized by changes in behaviour, personality and language with relative preservation of memory and perception.

Pathologically, there are two main histological profiles associated with FTLD. One of these is tauopathy, the accumulation of hyperphosphorylated tau in neurons and occasionally in glia. However, the most the most common neuropathology associated with FTLD, accounting for well over half of all cases, is that known as FTLD-U, in which there are neuronal cytoplasmic inclusions and neurites that are immunoreactive for ubiquitin (ub-ir) but not for tau. FTLD pathology of this type was first described in patients with motor neuron disease (MND) and dementia but has subsequently been recognized as a common neuropathological feature of FTLD in patients without motor symptoms. This ub-ir pathology is characteristically found in granule cells of dentate fascia of the hippocampus and in neurons of layer 2 of the frontal and temporal neocortex.

Significant progress in our understanding of one of the genetic causes of FTLD-U was made in 2006 when null-mutations of the pleiotropic growth factor progranulin (PGRN) was demonstrated to be significant cause of this disease subtype. A second major advancement was the identification of TDP-43 as the major ubiquitinated protein in the pathological lesions in FTLD-U. It was demonstrated that this protein is cleaved and phosphorylated in disease and importantly, is also the basis of the pathological features of MND suggesting a disease spectrum between these two conditions.

The genetics of FTLD is complex with 7 disease loci reported to date, these being on chromosomes 3, 9p (2 loci), 9q, 17q21 (2 loci) and 17q24. Only 4 of the genes within these loci are known. It has been reported that a mutation in the splice acceptor site of exon 6 of CHMP2B on chromosome 3 causes in a large Danish family with DLDH-type of histology. However, the inventors have shown that this is a rare genetic cause of FTLD. 15-20% of familial FTLD results from mutations in the MAPT gene on chromosome 17q21 , encoding the microtubule associated protein tau.

All cases with pathogenic MAPT mutations demonstrate prominent tau pathology. Interestingly, there are numerous families with autosomal dominant FTLD-U with linkage to chromosome 17q21 (MAPT region), in which no pathogenic MAPT mutations have been identified. The inventors have previously shown that this disease results from null-mutations of PGRN demonstrating there are 2 different genes for FTLD on chromosome 17q21.

It has also been established that Paget's disease with Inclusion Body Myopathy and FTD is caused by mutations in the VCP gene on chr9p; it is currently unclear to what extent, if any, this gene contributes to prototypical FTLD (i.e. without Paget's disease and Inclusion Body Myopathy). There have also been reports of linkage to chrl7q24 chr9p+q in pedigrees with FTD+MND. However, there have been no reports of other families linked to these regions, and the mutant genes have yet to be identified.

FTLD is the second most common form of dementia in individuals under the age of 65 where approximately half of all patients with FTLD present with a family history of a similar disorder indicating a significant genetic contribution to the etiology of this disease. Existing methods of diagnosing FTLD are based on a combination of neuropsychological test results, brain imaging studies, and physical findings. However there remains a clear need for developing further methods of diagnosing FTLD as well as assessing the likelihood that a subject will develop this disorder. Furthermore, at present there is no treatment for FTLD.

The inventors have undertaken further studies of the genetics of FTLD. Using a linkage disequilibrium approach the inventors genotyped one SNP per every haplotype block of all genes in the linkage region on chromosome 9q21 locus using a FTLD cohort

(N=214, MAPT and PGRN mutant -ve) and 286 controls using the Sequenom genotyping technology. Surprisingly the inventors found no evidence of association at the genotype or haplotype level, demonstrating no detectable disease risk gene(s) in that cohort. Moreover, the validity of the linkage to this region has now been questioned.

Nevertheless, linkage to chromosome 9p21 in families with FTD+MND was reported recently by 2 separate groups.

The inventors then repeated the same experimental design with the chr9p locus. 151 SNPs were analysed using Haploscore and plots of P values versus chromosomal position identified three gene regions (RECK, TEK and UBAPl) in that locus where P values consistently reached =0.01 for multiple window sizes. Using the tagger program additional SNPs were genotyped to cover all known haplotype blocks of these 3 genes. Analysis revealed that while both RECK and TEK failed to show an association for other SNPs, UBAPl (Ubiquitin Associated Protein 1) SNPs/haplotypes were consistently associated with a maximum sliding window P value of 0.00025. Of the 10 SNPs in the UBAPl haplotype, 7 were found to be associated with FTLD with a P value of =0.05, with the haplotype block demonstrating maximum OR of 1.71 (95% CI 1.20- 2.44, P=0.003). The inventors subsequently identified that three subjects diagnosed with FTLD have a mutation in the UBAPl gene, while no such mutation is present in any member of a control population of 280 subjects. This further strengthens the linkage between UBAPl and FTLD.

The inventors have therefore identified a linkage between the UBAPl and FTLD. Such a linkage was not known or suggested in the prior art, and is particularly surprising as UBAPl had previously been demonstrated to be implicated in a totally different type of disease (carcinomas).

The inventors have also examined the association of UBAPl polypeptide with the tau protein. As can be seen in the accompanying examples and figures, they have demonstrated that tau protein can be immunoprecipitated from neural cells using a UBAPl antibody. Moreover, tau and UBAPl polypeptide are colocalised in the parenchyma of neurons of subjects having FTLD. This suggests that UBAPl and tau polypeptides interact and are present in a cellular complex.

As discussed further below, it is likely that UBAPl is involved in the ubiquitin proteomsome system (UPS), facilitating degradation of proteins. Impairment of the functions of UBAPl is likely to be detrimental to a neurons health. Tau protein is the main component of neurofibrillary tangles, one of the pathological hallmarks of dementias such as FTLD and Alzheimer's disease. The inventors have demonstrated that tau and UBAPl interact using both immunoprecipitation and colocalisation with confocal microscopy (see accompanying examples). While not wishing to be bound to any particular theory, it is possible that a reduction in the function or amount of UBAPl would slow down the degradation of proteins, such as tau or other disease-related proteins (e.g. amyloid precursor protein), by the UPS. This would likely encourage the aggregation of these proteins and, in the case of tau, would probably lead to the formation of neurofibrillary tangles which could precipitate or accelerate disease.

Tau protein plays a key role in the development of Alzheimer's disease. In light of the above data demonstrating a link between UBAPl and FTLD, and that tau and UBAPl polypeptides interact, the inventors then investigated whether there is a linkage between UBAPl and Alzheimer's disease (AD).

Alzheimer's disease (AD) is a dementia that, in its most common form, is found in people over the age of 65. Approximately 24 million people worldwide have dementia of which the majority (~60%) is due to Alzheimer's.

Alzheimer's disease has been identified as a protein misfolding disease, or proteopathy, due to the accumulation of abnormally folded A-beta and tau proteins in the brains of AD patients. A-beta, also written AB, is a short peptide that is a proteolytic byproduct of the transmembrane protein amyloid precursor protein (APP), whose function is unclear but thought to be involved in neuronal development. The presenilins are components of a proteolytic complex involved in APP processing and degradation. Although amyloid beta monomers are soluble and harmless, they undergo a dramatic conformational change at sufficiently high concentration to form a beta sheet-rich tertiary structure that aggregates to form amyloid fibrils that deposit outside neurons in dense formations known as senile plaques or neuritic plaques, in less dense aggregates as diffuse plaques, and sometimes in the walls of small blood vessels in the brain in a process called amyloid angiopathy or congophilic angiopathy.

AD is also considered a tauopathy due to abnormal aggregation of the tau protein, a microtubule-associated protein expressed in neurons that normally acts to stabilize microtubules in the cell cytoskeleton. Like most microtubule-associated proteins, tau is normally regulated by phosphorylation; however, in AD patients, hyperphosphorylated tau accumulates as paired helical filaments that in turn aggregate into masses inside nerve cell bodies known as neurofibrillary tangles and as dystrophic neurites associated with amyloid plaques.

Clinical signs of Alzheimer's disease are characterized by progressive cognitive deterioration, together with declining activities of daily living and by neuropsychiatric symptoms or behavioural changes. It is the most common type of dementia. Plaques which contain misfolded peptides called amyloid beta (AB) are formed in the brain many years before the clinical signs of Alzheimer's are observed. Together, these plaques and neurofibrillary tangles form the pathological hallmarks of the disease. These features can only be discovered at autopsy and help to confirm the clinical diagnosis.

Existing medications can help reduce the symptoms of the disease, but importantly they cannot change the course of the underlying pathology.

While the biological cause of Alzheimer's is not precisely understood, genetic factors are clearly indicated as dominant mutations in three different genes account for the small number of cases of familial, early-onset AD have been identified. For the more common form of late onset AD, ApoE is the only clearly established susceptibility gene. All four genes can contain mutations or variants that confer increased risk for AD, but account for only 30% of the genetic picture of AD. These four genes have in common the fact that mutations in each lead to the excessive accumulation in the brain of AB, the main component of the senile plaques that litter the brains of AD patients.

Using a similar experimental protocol to that for the FTLD analysis, the inventors assessed whether there is a linkage between the UBAPl and AD. As set out in the accompanying example, they identified a positive association between UBAPl and AD in a cohort of 360 AD patients. This argues that UBAPl is a risk factor for Alzheimer's disease. Furthermore, as discussed above tau protein is the main component of neurofibrillary tangles, one of the pathological hallmarks of Alzheimer's disease, and the inventors have demonstrated that tau and UBAPl polypeptides interact using both immunoprecipitation and colocalisation with confocal microscopy.

The inventors have therefore identified UBAPl, a likely component of the ubiquitin proteomsome system, as having a key and previously unrecognised role in the development of dementias, including Alzheimer's disease and FTLD. This finding can be the basis for the development of new medicaments and prognostic markers of use in combating these debilitating diseases.

A first aspect of the invention provides a method of screening for compounds of use in preventing or treating dementia wherein a cell having UBAPl is treated with a test compound and the effect of the test compound on the amount and/or function of UBAPl is assessed. Ubiquitin Associated Protein 1 (UBAPl) encodes a protein of 502 residues, predicted to have a molecular weight of 55KDa. The gene is a member of the Ubiquitin-activated enzymes (UBA) family whose members include proteins having connections to ubiquitin and the ubiquitination pathway. The protein itself has two Ubiquitin- associated domains (UBA), between residues 389-430, and 451-498, and an Ubiquitin System Cue domain between residues 459-499, believed to be involved in the binding of ubiquitin-conjugating enzymes. The UBA domains are found in various proteins involved in the ubiquitin/proteosome pathway, growth control, receptor function, stress responses, DNA excision-repair and cell signalling via protein kinases suggesting a variety of possible roles for UBAPl .

UBAPl originates from a gene locus in a refined region on chromosome 9 undergoing loss of heterozygosity in nasopharyngeal carcinoma (NPC) and is presently considered to be an effective diagnosis candidate for NPC. Furthermore, decreased expression of UBAPl protein is a possible point of dysfunction along the pathogenesis pathway for NPC that may contribute to malignant transformation. Therefore UBAPl is thought in the prior art to be associated with NPC and until the present invention was not considered to be associated with the development of any form of dementia. However, it is important to point out that UBA domains are found in various proteins involved in the ubiquitin/proteosome pathway and therefore UBAPl can be considered an excellent candidate gene for FTLD pathology. In addition, as demonstrated herein UBAPl polypeptide interacts with tau protein, and the gene is genetically associated with both FTLD and Alzheimer's disease, suggesting a broader role for UBAPl in dementia.

Human UBAPl genomic DNA sequence can be located from a number of publicly available databases. For example, GenBank contains UBAPl DNA sequence as part of the sequence information from human Chromosome 9. Also, GenBank accession number NM O 16525 provides the UBAPl mRNA and polypeptide sequences set out below, as well as a link to the genomic DNA for the UBAPl gene. An example of the genomic DNA sequence for UBAPl is provided at the end of the examples section of the description below. The sequence of human mRNA encoding the UBAPl protein can be located from a number of different GenBank accessions, for example, NM016525, and is provided below.

UBAPl mRNA sequence

1 aaatgagtgg ggcggtgagg ggaaggagga gggaagtagg acttcaacat ggcggctgcg

61 gcactggcgg tggctacggt gacggcctgg cccggagcgg gcagagttgg aggtggtggc

121 gttcgctctc cctaggggct gtcgggagct cagcggggac cgagcctggg aggccggccg 181 gtgccagcac ctttcggctt ctgagacggc ggcagcagcg gcattcaggt tctaaatggc

241 ttctaagaag ttgggtgcag attttcatgg gactttcagt taccttgatg atgtcccatt

301 taagacagga gacaaattca aaacaccagc taaagttggt ctacctattg gcttctcctt

361 gcctgattgt ttgcaggttg tcagagaagt acagtatgac ttctctttgg aaaagaaaac

421 cattgagtgg gctgaagaga ttaagaaaat cgaagaagcc gagcgggaag cagagtgcaa 481 aattgcggaa gcagaagcta aagtgaattc taagagtggc ccagagggcg atagcaaaat

541 gagcttctcc aagactcaca gtacagccac aatgccacct cctattaacc ccatcctcgc

601 cagcttgcag cacaacagca tcctcacacc aactcgggtc agcagtagtg ccacgaaaca

661 gaaagttctc agcccacctc acataaaggc ggatttcaat cttgctgact ttgagtgtga

721 agaagaccca tttgataatc tggagttaaa aactattgat gagaaggaag agctgagaaa 781 tattctggta ggaaccactg gacccattat ggctcagtta ttggacaata acttgcccag

841 gggaggctct gggtctgtgt tacaggatga ggaggtcctg gcatccttgg aacgggcaac

901 cctagatttc aagcctcttc ataaacccaa tggctttata accttaccac agttgggcaa

961 ctgtgaaaag atgtcactgt cttccaaagt gtccctcccc cctatacctg cagtaagcaa

1021 tatcaaatcc ctgtctttcc ccaaacttga ctctgatgac agcaatcaga agacagccaa 1081 gctggcgagc actttccata gcacatcctg cctccgcaat ggcacgttcc agaattccct

1141 aaagccttcc acccaaagca gtgccagtga gctcaatggg catcacactc ttgggctttc

1201 agctttgaac ttggacagtg gcacagagat gccagccctg acatcctccc agatgccttc

1261 cctctctgtt ttgtctgtgt gcacagagga atcatcacct ccaaatactg gtcccacggt

1321 cacccctcct aatttctcag tgtcacaagt gcccaacatg cccagctgtc cccaggccta 1381 ttctgaactg cagatgctgt cccccagcga gcggcagtgt gtggagacgg tggtcaacat

1441 gggctactcg tacgagtgtg tcctcagagc catgaagaag aaaggagaga atattgagca

1501 gattctcgac tatctctttg cacatggaca gctttgtgag aagggcttcg accctctttt

1561 agtggaagag gctctggaaa tgcaccagtg ttcagaagaa aagatgatgg agtttcttca

1621 gttaatgagc aaatttaagg agatgggctt tgagctgaaa gacattaagg aagttttgct 1681 attacacaac aatgaccagg acaatgcttt ggaagacctc atggctcggg caggagccag

1741 ctgagaccag gccctgccta ggccctgccg cagaaccacc atccctggga ggccctgcag

1801 agcccacctg tggggaaaga gaaggggcag cttccggatt ttcttttggg ggttagaagg

1861 tcaggtgtgg agactgctcg ccagtctctg tgagcctagg ccctgagctg gggaggtggg

1921 gaagattcgg gcatgtgagt gcccccagaa ctgtcctggc tccttccgta ttaaacgcat 1981 ttgcattttg agaagtgtcc ttcccacttc agccctccgg agagactacc ctagtctttc 2041 tggggtgttt atgtcctcag ctgaagcctg gcctagttgc tgagaggggc tggggagatg

2101 gggcgggagg gccagactca gtgctgctgt ggagctaggt gcttccccct tcccctgaga

2161 ctggtggact gaactccagt caagttgagt tcaagtgaaa gattcttcca gggttttatt

2221 ttttcccctc ctaacaaagt ctcatagtgt taacactggt tctgcaatat ctctgaggtg 2281 caaagaatgc acttttccct atggggccca gagtttgcct tttctgccag gcagtcacca

2341 tgcttcccta ccccagcctg tttcttttgg cttggtttgg accacagtcc tctgctaccc

2401 agggttttag agcccctgct ctaggaaaca gtttaagaaa tcattggccc cttcccagca

2461 cattgaatgg gtaagcagac aggccatgat ttagttggcc agcactaact ccacctctgt

2521 tctccttgaa cagcttcccc tccagcccac tgctttagga tgacacaatg aataacacct 2581 agtcatagaa atcagtctct ctggtttgtt ttgtattatg ttgtacatca ttaaagatct

2641 aaatacaaag gatatacagt cttgaatcta aaataatttg ctaactaact attttgattc

2701 ttcagagaga actactaata aaaatctaaa aggtaaaaaa aaaaaaaaaa aaaaaaa

The sequence of the human UBAPl protein can be located from a number of different GenBank accessions, for example, NM O16525, and is provided below.

UPAB1 protein sequence

MASKKLGADFHGTFSYLDDVPFKTGDKFKTPAKVGLPIGFSLPDCLQWREVQYDFSLE KKTIEWAEEIKKIEEAEREAECKIAEAEAKVNSKSGPEGDSKMSFSKTHSTATMPPPIN

PILASLQHNSILTPTRVSSSATKQKVLSPPHIKADFNLADFECEEDPFDNLELKTIDEK

EELRNILVGTTGPIMAQLLDNNLPRGGSGSVLQDEEVLASLERATLDFKPLHKPNGFIT

LPQLGNCEKMSLSSKVSLPPIPAVSNIKSLSFPKLDSDDSNQKTAKLASTFHSTSCLRN

GTFQNSLKPSTQSSASELNGHHTLGLSALNLDSGTEMPALTSSQMPSLSVLSVCTEESS PPNTGPTVTPPNFSVSQVPNMPSCPQAYSELQMLSPSERQCVETVVNMGYSYECVLRAM

KKKGENIEQILDYLFAHGQLCEKGFDPLLVEEALEMHQCSEEKMMEFLQLMSKFKEMGF

ELKDIKEVLLLHNNDQDNALEDLMARAGAS

Also, further information on the UBAPl gene and associated publications can be identified from the HUGO Gene Nomenclature Committee website: http://www.genenames.org/ using the search term "UBAPl".

As demonstrated by the inventors, UBAPl polypeptide interacts with tau protein and the gene is genetically associated with dementia. They propose that a modulation, in particular reduction, in the function and/or amount of UBAPl would slow down the degradation of proteins, such as tau or other disease-related proteins (e.g. amyloid precursor protein), by the UPS, encouraging the aggregation of these proteins. In the case of tau, this would probably lead to the formation of neurofibrillary tangles, and this could precipitate or accelerate disease. In light of this, it is clear that compounds that modulate the function and/or amount of UBAPl would have particular utility in the prevention or treatment of dementia, particularly FTLD and Alzheimer's disease.

Against this background, the first aspect of the invention is a "screening method" to identify compounds of use in preventing or treating dementia. For the reasons outlined above, a compound that affects the amount and/or function of UBAPl is considered a compound that could be of use in preventing or treating dementia.

By "a cell having UBAPl" set out in the first aspect of the invention, we include cells including nucleic acid sequence encoding the UBAPl polypeptide. Such a nucleic acid sequence may be a "native" gene present in the genome of that cell, or it may be an extrachromosomal nucleic acid molecule. Examples of nucleic acid sequence encoding the UBAPl polypeptide are set out above.

Preferably the cell has a UBAPl polypeptide and the effect of a test compound on the amount and/or function of UBAPl polypeptide is assessed.

The cell could be any cell having UBAPl. However, it is preferred that the cell is a mammalian cell, most preferably a human cell. The cell could be any type. However, it is preferred that the cell is a neural cell, such as a neuroblastoma cell. Examples of such cells include SH-SY5Y and H4 neuroblastoma cells, which are further discussed below.

The step of assessing the "amount and/or function of UBAPl" may be performed using a number of different methods. For example, a method of assessing the effect of the test compound on the amount of UBAPl polypeptide is to quantify the amount of said polypeptide. Alternatively, the effect of the test compound in the first aspect of the invention can be determined by quantifying the amount of nucleic acid, preferably mRNA, encoding the UBAPl polypeptide.

Methods of assessing the amount of UBAPl polypeptide may be performed using a number of different methods, which are discussed below. In order to assess whether a cell exposed to the test compound has an altered amount of UBAPl polypeptide or nucleic acid encoding UBAPl polypeptide, the amount of UBAPl polypeptide and/or nucleic acid encoding UBAPl polypeptide in the cell can be compared to that of a "reference sample", i.e. a sample of protein or nucleic acid taken from a cell not exposed to the test compound. By comparing the amount of UBAPl polypeptide and/or nucleic acid encoding UBAPl polypeptide in a sample of protein or nucleic acid taken from an exposed cell, to the amount of UBAPl polypeptide and/or nucleic acid encoding UBAPl polypeptide in a sample of protein or nucleic acid in a non-exposed cell, it is possible to determine the effect of the test compound on the amount and/or function of UBAPl. This will show the test compound(s) to produce an elevation, reduction or no effect on expressed levels of the UBAPl polypeptide or nucleic acid, or a potentiation, inhibition or no effect on the function of UBAPl polypeptide.

Non-exhaustive examples of methods of determining the amount and/or function of polypeptide, or the amount of nucleic acid that encodes the polypeptide, are provided below. Further information regarding some of the experimental procedures set out below are described further in Sambrook et al. (2000) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

Assaying protein levels in a sample can be performed using any art-known method. Total protein levels within a sample can be measured using Bradford reagent, fluorescamine dye or by using the Lo wry method: these techniques are standard laboratory procedures.

It will be appreciated that the amount of a polypeptide may be measured by labelling a compound having affinity for that particular polypeptide. For example, antibodies, aptamers and the like may be labelled and used in an assay. Preferred for assaying protein levels in a biological sample are antibody-based techniques. Examples of immunoassays include immunofluorescence techniques known to the skilled technician, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay analyses. For example, the effect of a test compound on the amount of UBAPl expression can be measured using an antibody to this polypeptide, as part of techniques such as western blotting, immunohistochemistry and ELISA. Such an antibody is used in the accompanying examples.

Levels of mRNA encoding particular polypeptides may be performed using the RT- PCR method. Briefly, this method involves converting mRNA isolated from a sample to cDNA using a reverse transcriptase enzyme. The cDNA products are then subject to PCR according to conventional techniques. After a suitable number of rounds to achieve amplification, the PCR reaction product corresponding to the mRNA encoding the particular polypeptide is quantified. Variations on the RT-PCR method will be apparent to the skilled artisan. Any set of oligonucleotide primers which will amplify reverse transcribed target mRNA can be used and can be designed as will be well known to those skilled in the art.

Levels of mRNA encoding the particular polypeptide can also be assayed using northern blotting, a method well known to those skilled in the art.

Further methods which may be of use in measuring mRNA levels include in situ hybridisation, in situ amplification, nuclease protection, probe arrays and amplification based systems. In addition, microarray analysis, a technique well known to those skilled in the art, may also be used to assess the amount of mRNA encoding a particular polypeptide.

Using such techniques common in the art, it would be possible to determine the amount of expression of particular polypeptide.

Also, the expression of a certain gene can be measured using promoter-reporter constructs, a technique well known to the skilled person.

The screening methods of the invention may also include assessing the effect of the test compound on the function of UBAPl polypeptide. For example, an assay can be devised to measure the effect of a test compound on the ubiquitin and the ubiquitination pathway activities using UBAPl. Alternatively, the inventors have demonstrated that UBAPl polypeptide interacts with tau protein in vivo, and propose that this interaction is important for modulating cellular tau protein levels. Therefore, an assay for the function of UBAPl polypeptide could be based on measuring the effects of a test compound on UBAPl and tau polypeptide levels. That is, a test compound that modulates both UBAPl and tau polypeptide levels is considered to be a compound that affects UBAPl function.

As an example of such an assay, an appropriate cell line expressing tau and UBAPl (e.g. SH-SY5Y or H4 neuroblastoma cells, both of which are well known in the art and are available from, for example, the ATCC) would be grown in an appropriate medium.

Cells would be treated with an appropriate test compound or small molecule library for an appropriate period on time. Levels of tau and UBAPl would then be assayed using standard methods such as western blot or ELISA. Test compound that affect both UBAPl levels and tau levels would be selected as compounds that could be of use in treating dementia.

Tau protein is well known in the art. Reagents that can be used to measure tau protein levels are well known and are readily available; for example, the accompanying experimental data uses an antibody to tau which is readily commercially available.

Alternatively, a fluorescent in vivo cellular assay could be developed. For example, Dickey et al (2005) Curr Alzheimer Res. Apr ;2(2) :231 -8 describe a fluorescent in cell western assay that can be used to simultaneously measure tau protein levels in a sample along with another protein, for use in a high throughput drug screening assay for the detection of changes in tau levels. This assay could be readily adapted to simultaneously measure tau protein levels in a sample along with UBAPl polypeptide levels. As discussed above, a compound that modulates both UBAPl and tau polypeptide levels is considered to be a compound that affects UBAPl function.

As discussed above, a possible function UBAPl polypeptide is to promote the degradation of proteins such as tau via the UPS. A reduction in the amount and/or function of UBAPl polypeptide may lead to an accumulation of such protein which, in the case of tau, could precipitate or accelerate disease. Accordingly therefore, a test compound that increases the amount of UBAPl polypeptide and decreases the amount of tau protein would be an example of a compound that could be of use in preventing or treating dementia, in particular FTLD or Alzheimer's disease.

In addition, as shown herein UBAPl and tau polypeptide are known to interact in the cell. Therefore one measure of UBAPl function is the ability of the polypeptide to interact with tau. Again this function could be used as a basis for an assay.

For example, an assay can be devised to measure the effect of a test compound on UBAPl /tau interaction using an immunoprecipitation protocol. Here, an appropriate cell line expressing tau and UBAPl would be grown in an appropriate medium. Cells would be treated with an appropriate test compound or small molecule library for an appropriate period on time. UBAPl /tau interaction would then be assayed using standard methods such as the immunoprecipitation protocol outlined in the accompanying examples. Test compound that affect the amount of UBAPl /tau interaction would be selected as compounds that could be of use in treating dementia.

Furthermore, as shown in the accompanying examples, UBAPl and TDP-43 polypeptides have been shown to co-localise together in neuronal cytoplasmic inclusions in a case of familial FTLD. Therefore one function of UBAPl could be in the metabolism of TDP-43. Therefore one measure of UBAPl function is the ability of the polypeptide to co-localise with UBAPl, or to regulate the metabolism of TDP-43. Again this function could be used as a basis for an assay.

For example, an assay can be devised to measure the effect of a test compound on UBAPl /TDP-43 co-localistion using an immunoprecipitation protocol. Here, an appropriate cell line expressing TDP-43 and UBAPl would be grown in an appropriate medium. Cells would be treated with an appropriate test compound or small molecule library for an appropriate period on time. UBAPl /TDP-43 co-localistion would then be assayed using standard methods such as the immunoprecipitation protocol outlined in the accompanying examples. Test compound that affect the amount of UBAPl /TDP-43 co-localistion interaction would be selected as compounds that could be of use in treating dementia. The inventors have identified a genetic linkage between the UBAPl gene and frontotemporal lobar degeneration (FTLD) and Alzheimer's disease. As discussed above, FTLD and AD are types of dementia, and the inventors consider that the UBAPl gene may be used to identify therapeutic agents and as a diagnostic marker for the disorders within this broad category. Therefore the aspects of the invention provided herein are applicable to a wide range of dementias, including, FTLD, Alzheimer's disease, motor neuron disease, Parkinson's disease, dementia with Lewy bodies, prion diseases, progressive supranuclear palsy or multisystem atrophy.

An embodiment of the first aspect of the invention is wherein the dementia is characterised by tauopathy. Tauopathy is characterised by the accumulation of hyperphosphorylated tau in neurons and occasionally in glia.

A preferred embodiment of the first aspect of the invention is wherein the dementia is frontotemporal lobar degeneration (FTLD). The syndrome of FTLD encompasses the clinical subgroups of frontotemporal dementia (FTD), FTD with motor neuron disease, semantic dementia and primary progressive aphasia, and is characterized by changes in behaviour, personality and language with relative preservation of memory and perception. An embodiment of the first aspect of the invention is wherein the frontotemporal lobar degeneration is characterised by ub-ir.

The most the most common neuropathology associated with FTLD, accounting for well over half of all cases, is that known as FTLD-U, in which there are neuronal cytoplasmic inclusions and neurites that are immunoreactive for ubiquitin (ub-ir) but not for tau. FTLD pathology of this type was first described in patients with motor neuron disease (MND) and dementia but has subsequently been recognized as a common neuropathological feature of FTLD in patients without motor symptoms. This ub-ir pathology is characteristically found in granule cells of dentate fascia of the hippocampus and in neurons of layer 2 of the frontal and temporal neocortex. Significant progress in our understanding of one of the genetic causes of FTLD-U was made in 2006 when null-mutations of the pleiotropic growth factor progranulin (PGRN) was demonstrated by the inventors to be significant cause of this disease subtype. A second major advancement was the identification of TDP-43 as the major ubiquitinated protein in the pathological lesions in FTLD-U. It was demonstrated that this protein is cleaved and phosphorylated in disease and importantly, is also the basis of the pathological features of MND suggesting a disease spectrum between these two conditions.

A preferred embodiment of the first aspect of the invention is wherein the dementia is Alzheimer's disease.

The method of the first aspect of the invention relates to screening methods for drugs or lead compounds. The test compound may be a drug-like compound or lead compound for the development of a drug-like compound.

The term "drug-like compound" is well known to those skilled in the art, and may include the meaning of a compound that has characteristics that may make it suitable for use in medicine, for example as the active ingredient in a medicament. Thus, for example, a drug-like compound may be a molecule that may be synthesised by the techniques of organic chemistry, less preferably by techniques of molecular biology or biochemistry, and is preferably a small molecule, which may be of less than 5000 daltons and which may be water-soluble. A drug-like compound may additionally exhibit features of selective interaction with a particular protein or proteins and be bioavailable and/or able to penetrate target cellular membranes, but it will be appreciated that these features are not essential.

The term "lead compound" is similarly well known to those skilled in the art, and may include the meaning that the compound, whilst not itself suitable for use as a drug (for example because it is only weakly potent against its intended target, non-selective in its action, unstable, poorly soluble, difficult to synthesise or has poor bioavailability) may provide a starting-point for the design of other compounds that may have more desirable characteristics.

The screening methods of the invention can be used in "library screening" methods, a term well known to those skilled in the art. Thus, for example, the methods of the invention may be used to detect (and optionally identify) a test compound capable of affecting the amount and/or function of UBAPl polypeptide. Aliquots of a library may be tested for the ability to give the required result. Hence by "test compound" as used in the first aspect of the invention, we include where a cell is exposed to more than one compound at the same time, as is commonly performed in high throughput screening assays well known in the art.

An embodiment of the first aspect of the invention is wherein the method further comprises the step of selecting a compound that increases the amount and/or function of UBAPl; preferably UBAPl polypeptide.

By "increases" we include where the cell has, for example, 110%, 1250%, 130%, 140%, 150%, 200%, 250%, 500%, 1000%, or 10000% of the amount and/or function of the polypeptide or nucleic acid in the reference sample.

Of particular interest to this embodiment of the invention is the finding reported in the accompanying examples that analysis of UBAPl mRNA identifies disease associated haplotypes with significantly lower levels of expression compared to the reference haplotype. That this is observed in frontal lobe which is affected by pathology in FLTD and cerebellum, which is free from neuropathology, argues this not just a product of the neurodegenerative process. These data strongly suggest that the main variant that increases of FTLD risk operates by lowering UBAPl expression leading to loss/reduced function. Hence it is preferred that the selected compound that increases the amount of

UBAPl.

An embodiment of the first aspect of the invention is wherein the method further comprises the step of selecting a compound that decreases the amount and/or function of UBAPl; preferably UBAPl polypeptide.

By "decreases" we include where the cell has, for example, 90%, 80%, 70%, 60%, 50%, 25%, 10%, 5%, 1%, 0.1% or 0% of the amount and/or function of the polypeptide or nucleic acid in the reference sample.

A further embodiment of the first aspect of the invention is wherein the selected compound is formulated into a pharmaceutically acceptable composition. A discussion of pharmaceutical compositions is provided below. A second aspect of the invention provides a method of screening for compounds of use in preventing or treating dementia wherein a non-human animal is administered a test compound and the effect of the test compound on the amount and/or function of UBAPl is assessed; preferably UBAPl polypeptide.

The second aspect of the invention is also a "screening method". The embodiments of the first aspect of the invention discussed above, and various techniques for performing the screening method, also apply to the second aspect of the invention.

The non-human animal may be any non-human animal, including non-human primates such as baboons, chimpanzees and gorillas, new and old world monkeys as well as other mammals such as cats, dogs, rodents, pigs or sheep, or other animals such as poultry, for example chickens, fish such as zebrafish, or amphibians such as frogs. However, it is preferred that the animal is a rodent such as a mouse, rat, hamster, guinea pig or squirrel. Preferably the animal is mouse. Preferably the non-human animal has a nucleic acid sequence encoding UBAPl.

Techniques for isolating protein and nucleic acid samples from non-human animals to assess the amount and/or function of UBAPl are well known in the art.

According to a third aspect of the present invention, there is provided the use of an agent that modulates the amount or activation of UBAPl for the prevention or treatment of dementia

According to a fourth aspect of the present invention, there is provided the use of an agent that modulates the amount and/or activation of UBAPl in the manufacture of a medicament for the prevention or treatment of dementia.

According to a fifth aspect of the invention there is provided a method of preventing or treating dementia comprising administering to a subject a therapeutically effective quantity of an agent that modulates the amount and/or activation of UBAPl . The inventors, as explained above and in the Examples, have demonstrated that the UBAPl gene is genetically linked to dementia. This lead them to realise that agents of use in the third, fourth or fifth aspects of the invention, which modulate the amount or activation of UBAPl, are useful for preventing or treating dementia. The inventors were surprised to make these correlations because until the present application UBAPl has not been associated with any dementia disorders.

By "modulates" we mean that agent is effective for modulating the amount and/or activation of UBAPl such that the amount and/or activation of the UBAPl gene or gene product (mRNA and protein) is significantly increased or reduced in the subject when compared to that in a subject not administered the agent. Preferably the agent increases the amount and/or activation of UBAPl .

As reported herein, analysis of UBAPl mRNA identifies disease associated haplotypes with significantly lower levels of expression compared to the reference haplotype. This strongly suggests that the main variant that increases of FTLD risk operates by lowering UBAPl expression. Hence it is preferred that the agent increases the amount of UBAPl.

The agents may be used in the treatment of a number of different dementias, preferably frontotemporal lobar degeneration (FTLD) or Alzheimer's disease (AD); preferably the dementia is characterised by tauopathy.

Examples of agents which may be used according to the invention include where the agent may bind to the UBAPl polypeptide and increase or prevent UBAPl functional activity, e.g. antibodies and fragments and derivatives thereof (e.g. domain antibodies or

Fabs). Alternatively the agent may act as a competitive inhibitor to UBAPl by acting as an antagonist UBAPl . Alternatively the agent may be an activator of UBAPl by acting as an agonist of UBAPl . Alternatively the agent may inhibit or activate enzymes or other molecules in the UBAPl pathway. Alternatively the agent may bind to mRNA encoding UBAPl polypeptide in such a manner as to lead to an increase or reduction in that mRNA and hence a modulation in the amount of UBAPl polypeptide. Alternatively the agent may bind to a nucleic sequence encoding UBAPl in such a manner that it leads to an increase or reduction in the amount of transcribed mRNA encoding UBAPl polypeptide. For instance the agent may bind to coding or non-coding regions of the UBAPl gene or to DNA 5' or 3' of the UBAPl and thereby reduce or increase expression of the protein.

The agent may have been identified from the method of the first or second aspects of the invention as being of use in the prevention or treatment of dementia.

An embodiment of the third, fourth or fifth aspects of the invention is wherein the agent increases the amount and/or activation of UBAPl .

A further embodiment of the third, fourth or fifth aspects of the invention is wherein the agent is UBAPl polypeptide.

In such an embodiment, the UBAPl polypeptide may be administered directly to the subject. Alternatively, or additionally, in another embodiment of the invention, this may consists of administering a nucleic acid sequence encoding UBAPl to the subject, for example, by gene therapy. Gene therapy consists of the insertion or the introduction of a gene or genes into a subject in need of treatment. In accordance with the present invention, it is preferred that the gene UBAPl encoding the UBAPl polypeptide is used. Accordingly, it is preferred that at least one, and preferably, more than one, copy of the UBAPl gene will be introduced in to a subject to be treated.

It will be appreciated that there is some sequence variability between the sequence of the UBAPl gene and hence the UBAPl polypeptide between genuses and species. Hence, it is preferred that the sequence of the UBAPl gene used in the therapeutic aspects of the invention is from the same genus as that of the subject being treated. For example, if the subject to be treated is mammalian, then the methods according to the invention will use mammalian UBAPl gene, and hence mammalian UBAPl enzyme. It is especially preferred that the UBAPl gene used is from the same species as that of the subject being treated. For example, if the subject to be treated is human, then the method according to the invention will use the human UBAPl gene, and hence human UBAPl polypeptide, and so on. Preferably, the UBAPl gene used in the methods according to the invention is substantially homologous to the subject's native UBAPl gene, or a functional fragment thereof. Preferably, the degree of homology between the sequence of the UBAPl gene used in the method and the sequence of the subject's native UBAPl gene is at least 60% sequence identity, preferably, at least 75% sequence identity, preferably at least 85% identity; at least 90% identity; at least 95% identity; at least 97% identity; and most preferably, at least 99% identity.

Calculation of percentage identities between different amino acid/polypeptide/nucleic acid sequences may be carried out as follows. A multiple alignment is first generated by the ClustalX program (pairwise parameters: gap opening 10.0, gap extension 0.1, protein matrix Gonnet 250, DNA matrix IUB; multiple parameters: gap opening 10.0, gap extension 0.2, delay divergent sequences 30%, DNA transition weight 0.5, negative matrix off, protein matrix gonnet series, DNA weight IUB; Protein gap parameters, residue-specific penalties on, hydrophilic penalties on, hydrophilic residues GPSNDQERK, gap separation distance 4, end gap separation off). The percentage identity is then calculated from the multiple alignment as (N/T)*100, where N is the number of positions at which the two sequences share an identical residue, and T is the total number of positions compared. Alternatively, percentage identity can be calculated as (N/S)*100 where S is the length of the shorter sequence being compared. The amino acid/polypeptide/nucleic acid sequences may be synthesised de novo, or may be native amino acid/polypeptide/nucleic acid sequence, or a derivative thereof.

Suitably UBAPl polypeptide for provision as a therapeutic agent may be produced by known techniques. For instance, the protein may be purified from naturally occurring sources of UBAPl polypeptide. Indeed, such naturally occurring sources of UBAPl polypeptide may be induced to express increased levels of the protein, which may then be purified using well-known conventional techniques. Alternatively cells that do not naturally express UBAPl polypeptide may be induced to express such proteins. One suitable technique involves cellular expression of an UBAPl polypeptide/A/s construct. The expressed construct may subsequently be highly purified by virtue of the his "tag". Polynucleotide sequences encoding the UBAPl polypeptide are provided herein. It will be appreciated that UBAPl represents a favourable agent to be administered by techniques involving cellular expression of polynucleotide sequence encoding UBAPl. Such methods of cellular expression are particularly suitable for medical use in which the therapeutic effects of UBAPl are required over a prolonged period of time.

The UBAPl gene may further comprise elements capable of controlling and/or enhancing its expression in the cell being treated. For example, the UBAPl gene may be contained within a suitable vector to form a recombinant vector and preferably adapted to produce UBAPl polypeptide. The vector may for example be a plasmid, cosmid or phage. Such recombinant vectors are highly useful in the delivery systems of the invention for transforming cells with the nucleic acid molecule. Example of suitable vectors include pCMV6-XL5 (OriGene Technologies Inc), NTC retroviral vectors (Nature Technology Corporation), adeno-associated viral vectors (Avigen Technology).

For human gene therapy, vectors will be used to introduce genes coding for products with at least 50%, 60%, 70%, 80%, 90%, 95% or 99% identity with the UBAPl protein sequence provided herein.

State of the art vectors containing DNA coding for UBAPl protein may be introduced into the blood stream. Any state of the art advantages of gene therapy (for example, considerably improved viral vectors derived from adeno-associated viruses, retroviruses, particularly lentiviruses) may be used to introduce DNA sequences coding for UBAPl and homologies.

It is preferred that at least 2 administrations of 1-1000 million units/ml is given at certain intervals, depending on vectors used (the vectors will influence the stability of expression and persistence of UBAPl in organisms, from only several weeks to permanent expression) and individual requirements of the organism to be treated.

Recombinant vectors may comprise other functional elements to improve the gene therapy. For instance, recombinant vectors can be designed such that they will autonomously replicate in the cell in which they are introduced. In this case, elements that induce nucleic acid replication may be required in the recombinant vector. The recombinant vector may comprise a promoter or regulator to control expression of the UBAPl gene as required. Alternatively, the recombinant vector may be designed such that the vector and UPABl gene integrates into the genome of the cell, hi this case nucleic acid sequences, which favour targeted integration (e.g. by homologous recombination) may be desirable. Recombinant vectors may also have DNA coding for genes that may be used as selectable markers in the cloning process.

The UBAPl gene may (but not necessarily) be one, which becomes incorporated in the DNA of cells of the subject being treated.

The delivery system may provide the UBAPl gene the subject without it being incorporated in a vector. For instance, the nucleic acid molecule may be incorporated within a liposome or virus particle. Alternatively, a "naked" nucleic acid molecule may be inserted into a subject's cells by a suitable means e.g. direct endocytotic uptake.

The UBAPl nucleic acid molecule may be transferred to the cells of a subject to be treated by transfection, infection, microinjection, cell fusion, protoplast fusion or ballistic bombardment. For example, transfer may be by ballistic transfection with coated gold particles, liposomes containing the nucleic acid molecule, viral vectors (e.g. adenovirus) and means of providing direct nucleic acid uptake (e.g. endocytosis) by application of the gene directly.

UBAPl polypeptide or UBAPl gene may be combined in compositions having a number of different forms depending, in particular on the manner in which the composition is to be used. Thus, for example, the composition may be in the form of a powder, tablet, capsule, liquid, ointment, cream, gel, hydrogel, aerosol, spray, micelle, transdermal patch, liposome or any other suitable form that may be administered to a person or animal. It will be appreciated that the vehicle of the composition of the invention should be one which is well tolerated by the subject to whom it is given, and preferably enables delivery of the UBAPl polypeptide or UBAPl gene to the target cell, tissue, or organ. Hence, it is preferred that UBAPl polypeptide is delivered by means of a suitably protected carrier particle, for example, a micelle.

Compositions comprising UBAPl polypeptide or UBAPl gene according to the invention may be used in a number of ways. For instance, systemic administration may be required in which case the compound may be contained within a composition that may, for example, be ingested orally in the form of a tablet, capsule or liquid. Alternatively, the composition may be administered by injection into the blood stream. Injections may be intravenous (bolus or infusion) or subcutaneous (bolus or infusion). The compounds may be administered by inhalation (e.g. intranasally).

UBAPl polypeptide or UBAPl gene may also be incorporated within a slow or delayed release device. Such devices may, for example, be inserted on or under the skin, and the compound may be released over weeks or even months. Such devices may be particularly advantageous when long term treatment with a UBAPl polypeptide or UBAPl gene according to the invention is required and which would normally require frequent administration (e.g. at least daily injection).

It will be appreciated that the amount of a UBAPl polypeptide or UBAPl gene that is required is determined by its biological activity and bioavailability which in turn depends on the mode of administration, the physicochemical properties of the UBAPl polypeptide or UBAPl gene employed, and whether the UBAPl polypeptide or UBAPl gene is being used as a monotherapy or in a combined therapy. Also, the amount will be determined by the number and state of target cells to be treated. The frequency of administration will also be influenced by the above-mentioned factors and particularly the half-life of the UBAPl polypeptide or UBAPl gene within the subject being treated.

Optimal dosages to be administered may be determined by those skilled in the art, and will vary with the particular UBAPl polypeptide or UBAPl gene in use, the strength of the preparation, the mode of administration, and the advancement of the disease condition. Additional factors depending on the particular subject being treated will result in a need to adjust dosages, including subject age, weight, gender, diet, and time of administration.

Known procedures, such as those conventionally employed by the pharmaceutical industry (e.g. in vivo experimentation, clinical trials, etc.), may be used to establish specific formulations of UBAPl polypeptide or UBAPl gene according to the invention and precise therapeutic regimes (such as daily doses of the UBAPl polypeptide or UBAPl gene and the frequency of administration). Generally, a daily dose of between 0.01 μg/kg of body weight and 0.5 g/kg of body weight of UBAPl polypeptide or UBAPl gene according to the invention may be used for the prevention and/or treatment of dementia, depending upon which specific UBAPl polypeptide or UBAPl gene is used. More preferably, the daily dose is between 0.01 mg/kg of body weight and 200 mg/kg of body weight, and most preferably, between approximately 1 mg/kg and 100 mg/kg.

Daily doses may be given as a single administration (e.g. a single daily injection). Alternatively, the UBAPl polypeptide or UBAPl gene used may require administration twice or more times during a day. As an example, UBAPl polypeptide or UBAPl gene according to the invention may be administered as two (or more depending upon the severity of the condition) daily doses of between 25 mg and 7000 mg (i.e. assuming a body weight of 70kg). A patient receiving treatment may take a first dose upon waking and then a second dose in the evening (if on a two dose regime) or at 3 or 4 hourly intervals thereafter. Alternatively, a slow release device may be used to provide optimal doses to a patient without the need to administer repeated doses.

This invention provides a pharmaceutical composition comprising a therapeutically effective amount of a UBAPl polypeptide or UBAPl gene according to the invention and optionally a pharmaceutically acceptable vehicle. In one embodiment, the amount of the UBAPl polypeptide or UBAPl gene is an amount from about 0.01 mg to about

800 mg. In another embodiment, the amount of the UBAPl polypeptide or UBAPl gene is an amount from about 0.01 mg to about 500 mg. In another embodiment, the amount of the UBAPl polypeptide or UBAPl gene is an amount from about 0.01 mg to about

250 mg. In another embodiment, the amount of the UBAPl polypeptide or UBAPl gene is an amount from about 0.1 mg to about 60 mg. In another embodiment, the amount of the UBAPl polypeptide or UBAPl gene is an amount from about 0.1 mg to about 20 mg.

This invention provides a process for making a pharmaceutical composition comprising combining a therapeutically effective amount of an UBAPl polypeptide or UBAPl gene according to the invention and a pharmaceutically acceptable vehicle. A "therapeutically effective amount" is any amount of an UBAPl polypeptide or UBAPl gene according to the invention which, when administered to a subject provides prevention and/or treatment of dementia. A "subject" is a vertebrate, mammal, domestic animal or human being.

A "pharmaceutically acceptable vehicle" as referred to herein is any physiological vehicle known to those of ordinary skill in the art useful in formulating pharmaceutical compositions.

A further embodiment of the third, fourth or fifth aspects of the invention is where the agent decreases the amount and/or activation of UBAP 1.

Agent for use in the third, fourth or fifth aspects of the invention may bind to UBAPl polypeptide or to a nucleic acid encoding UBAPl polypeptide. Examples of nucleic acid and polypeptide sequences for UBAPl are shown above and at the end of the example section of the description.

When the agents binds to UBAPl polypeptide, it is preferred that the agent binds to an epitope defined by the protein that has been correctly folded into its native form. It will be appreciated, that there can be some sequence variability between species and also between genotypes. Accordingly other preferred epitopes will comprise equivalent regions from variants of the gene. Equivalent regions from further UBAPl polypeptides can be identified using sequence similarity and identity tools, and database searching methods, outlined herein. It is most preferred that the agent binds to a conserved region of the UBAPl polypeptide or a fragment thereof.

An embodiment of the third, fourth or fifth aspects of the invention is wherein the agent is an antibody or fragment thereof.

The use of antibodies as agents to modulate polypeptide activity is well known. Indeed, therapeutic agents based on antibodies are increasingly being used in medicine. It is therefore apparent that such agents have great utility as medicaments for the improving the prevention or treatment of dementia. Moreover, such antibodies can be used in the prognostic methods set out below in further aspects of the invention. Antibodies, for use in treating human subjects, may be raised against UBAPl polypeptide per se or a number of peptides derived from the UBAPl polypeptide, or peptides comprising amino acid sequences corresponding to those found in the UBAPl polypeptide.

It is preferred that the antibodies are raised against antigenic structures from human UBAPl polypeptide, and peptide derivatives and fragments thereof.

Antibodies may be produced as polyclonal sera by injecting antigen into animals. Preferred polyclonal antibodies may be raised by inoculating an animal (e.g. a rabbit) with antigen (e.g. all or a fragment of the UBAPl polypeptide) using techniques known to the art.

Alternatively the antibody may be monoclonal. Conventional hybridoma techniques may be used to raise such antibodies. The antigen used to generate monoclonal antibodies for use in the present invention may be the same as would be used to generate polyclonal sera.

In their simplest form, antibodies or immunoglobulin proteins are Y-shaped molecules usually exemplified by the γ-immunoglobulin (IgG) class of antibodies. The molecule consists of four polypeptide chains two identical heavy (H) chains and two identical (L) chains of approximately 5OkD and 25kD each respectively. Each light chain is bound to a heavy chain (H-L) by disulphide and non-covalent bonds. Two identical H-L chain combinations are linked to each other by similar non-covalent and disulphide bonds between the two H chains to form the basic four chain immunoglobulin structure (H-

L)₂.

Light chain immunoglobulins are made up of one V-domain (V_L) and one constant domain (C_L) whereas heavy chains consist of one V-domain and, depending on H chain isotype, three or four C-domains (C_HI , C_H2, C_H3 and C_H4).

At the N-terminal region of each light or heavy chain is a variable (V) domain that varies greatly in sequence, and is responsible for specific binding to antigen. Antibody specificity for antigen is actually determined by amino acid sequences within the V- regions known as hypervariable loops or Complementarity Determining Regions (CDRs). Each H and L chain V regions possess 3 such CDRs, and it is the combination of all 6 that forms the antibody's antigen binding site. The remaining V-region amino acids which exhibit less variation and which support the hypervariable loops are called frameworks regions (FRs).

The regions beyond the variable domains (C-domains) are relatively constant in sequence. It will be appreciated that the characterising feature of antibodies according to the invention is the V_H and V_L domains. It will be further appreciated that the precise nature of the C_H and C_L domains is not, on the whole, critical to the invention. In fact preferred antibodies for use in the invention may have very different C_H and C_L domains. Furthermore, as discussed more fully below, preferred antibody functional derivatives may comprise the Variable domains without a C-domain (e.g. scFV antibodies).

Preferred antibodies considered to be agents of use in the third, fourth or fifth aspects of the invention may have the V_L (first domain) and V_H (second domain) domains. A derivative thereof may have 75% sequence identity, more preferably 90% sequence identity and most preferably has at least 95% sequence identity. It will be appreciated that most sequence variation may occur in the framework regions (FRs) whereas the sequence of the CDRs of the antibodies, and functional derivatives thereof, should be most conserved.

A number of preferred embodiments of the agent of the third, fourth or fifth aspects of the invention relate to molecules with both Variable and Constant domains. However it will be appreciated that antibody fragments (e.g. scFV antibodies or FAbs) are also encompassed by the invention that comprise essentially the Variable region of an antibody without any Constant region.

A scFV antibody fragment considered to be an agent of the third, fourth or fifth aspects of the invention may comprise the whole of the V_H and V_L domains of an antibody raised against IFN polypeptide. The V_H and V_L domains may be separated by a suitable linker peptide. Antibodies, and particularly mAbs, generated in one species are known to have several serious drawbacks when used to treat a different species. For instance when murine antibodies are used in humans they tend to have a short circulating half-life in serum and may be recognised as foreign proteins by the immune system of a patient being treated. This may lead to the development of an unwanted human anti-mouse antibody (HAMA) response. This is particularly troublesome when frequent administration of an antibody is required as it can enhance its clearance, block its therapeutic effect, and induce hypersensitivity reactions. These factors limit the use of mouse monoclonal antibodies in human therapy and have prompted the development of antibody engineering technology to generate humanised antibodies.

Therefore, where the antibody capable of modulating the amount or activation of UBAPl is to be used as a therapeutic agent for preventing or treating dementia in a human subject, then it is preferred that antibodies and fragments thereof of non-human source are humanised.

Humanisation may be achieved by splicing V region sequences (e.g. from a monoclonal antibody generated in a non-human hybridoma) with C region (and ideally FRs from V region) sequences from human antibodies. The resulting 'engineered' antibodies are less immunogenic in humans than the non-human antibodies from which they were derived and so are better suited for clinical use.

Humanised antibodies may be chimaeric monoclonal antibodies, in which, using recombinant DNA technology, rodent immunoglobulin constant regions are replaced by the constant regions of human antibodies. The chimaeric H chain and L chain genes may then be cloned into expression vectors containing suitable regulatory elements and induced into mammalian cells in order to produce fully glycosylated antibodies. By choosing an appropriate human H chain C region gene for this process, the biological activity of the antibody may be pre-determined. Such chimaeric molecules may be used to treat or prevent dementia.

Further humanisation of antibodies may involve CDR-grafting or reshaping of antibodies. Such antibodies are produced by transplanting the heavy and light chain CDRs of a non-human antibody (which form the antibody's antigen binding site) into the corresponding framework regions of a human antibody.

Humanised antibody fragments represent preferred agents for use according to the invention. Human FAbs recognising an epitope on UBAPl polypeptide may be identified through screening a phage library of variable chain human antibodies. Techniques known to the art (e.g as developed by Morphosys or Cambridge Antibody Technology) may be employed to generate Fabs that may be used as agents according to the invention. In brief a human combinatorial Fab antibody library may be generated by transferring the heavy and light chain variable regions from a single-chain Fv library into a Fab display vector. This library may yield 2.1 x 10¹⁰ different antibody fragments. The peptide may then be used as "bait" to identify antibody fragments from then library that have the desired binding properties.

Domain antibodies (dAbs) represent another preferred agent that may be used according to this embodiment of the invention. dAbs are the smallest functional binding unit of antibodies and correspond to the variable regions of either the heavy or light chains of human antibodies. Such dAbs may have a molecule weight of around 13kDa (corresponding to about 1/10 (or less) the size of a full antibody).

According to another embodiment of the third, fourth or fifth aspects of the invention, peptides may be used to modulate the amount or activation of UBAPl. Such peptides represent other preferred agents for use according to the invention. These peptides may be isolated, for example, from libraries of peptides by identifying which members of the library are able to modulate the amount or activation of UBAPl polypeptide. Suitable libraries may be generated using phage display techniques.

Aptamers represent another preferred agent of the third, fourth or fifth aspects of the invention. Aptamers are nucleic acid molecules that assume a specific, sequence- dependent shape and bind to specific target ligands based on a lock-and-key fit between the aptamer and ligand. Typically, aptamers may comprise either single- or double- stranded DNA molecules (ssDNA or dsDNA) or single-stranded RNA molecules (ssRNA). Aptamers may be used to bind both nucleic acid and non-nucleic acid targets. Accordingly aptamers may be generated that recognise and so modulate the activity or amount of UBAPl. Suitable aptamers may be selected from random sequence pools, from which specific aptamers may be identified which bind to the selected target molecules with high affinity. Methods for the production and selection of aptamers having desired specificity are well known to those skilled in the art, and include the SELEX (systematic evolution of ligands by exponential enrichment) process. Briefly, large libraries of oligonucleotides are produced, allowing the isolation of large amounts of functional nucleic acids by an iterative process of in vitro selection and subsequent amplification through polymerase chain reaction.

Antisense molecules represent another preferred agent for use according to the third, fourth or fifth aspects of the invention. Antisense molecules are typically single- stranded nucleic acids, which can specifically bind to a complementary nucleic acid sequence produced by a gene and inactivate it, effectively turning that gene "off. The molecule is termed "antisense" as it is complementary to the gene's mRNA, which is called the "sense" sequence, as appreciated by the skilled person. Antisense molecules are typically are 15 to 35 bases in length of DNA, RNA or a chemical analogue. Antisense nucleic acids have been used experimentally to bind to mRNA and prevent the expression of specific genes. This has lead to the development of "antisense therapies" as drugs for the treatment of cancer, diabetes and inflammatory diseases. Antisense drugs have recently been approved by the US FDA for human therapeutic use. Accordingly, by designing an antisense molecule to polynucleotide sequence encoding UBAPl polypeptide it would be possible to reduce the expression of UBAPl polypeptide in a cell and thereby reduce in UBAPl activity.

Small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, represent further preferred agents for use according to the third, fourth or fifth aspects of the invention. It will be apparent that siRNA molecules that can reduce UBAPl expression may have utility in the preparation of medicaments for the prevention or treatment of dementia. siRNA are a class of 20-25 nucleotide-long RNA molecules are involved in the RNA interference pathway (RNAi), by which the siRNA can lead to a reduction in expression of a specific gene, or specifically interfere with the translation of such mRNA thereby inhibiting expression of protein encoded by the mRNA. siRNAs have a well defined structure: a short (usually 21-nt) double- strand of RNA (dsRNA) with 2-nt 3' overhangs on either end. Each strand has a 5' phosphate group and a 3' hydroxyl (-OH) group. In vivo this structure is the result of processing by Dicer, an enzyme that converts either long dsRNAs or hairpin RNAs into siRNAs. siRNAs can also be exogenously (artificially) introduced into cells by various transfection methods to bring about the specific knockdown of a gene of interest. Essentially any gene of which the sequence is known can thus be targeted based on sequence complementarity with an appropriately tailored siRNA. Given the ability to knockdown essentially any gene of interest, RNAi via siRNAs has generated a great deal of interest in both basic and applied biology. There are an increasing number of large-scale RNAi screens that are designed to identify the important genes in various biological pathways. As disease processes also depend on the activity of multiple genes, it is expected that in some situations turning off the activity of a gene with a siRNA could produce a therapeutic benefit. Hence their discovery has led to a surge in interest in harnessing RNAi for biomedical research and drug development. Recent phase I results of therapeutic RNAi trials demonstrate that siRNAs are well tolerated and have suitable pharmacokinetic properties. siRNAs and related RNAi induction methods therefore stand to become an important new class of drugs in the foreseeable future. siRNA molecules designed to nucleic acid encoding UBAPl polypeptide can be used to reduce the expression of UBAPl. Hence an embodiment of the third, fourth and fifth aspects of the invention is wherein the agent is a siRNA molecule having complementary sequence to UBAPl polynucleotide.

A polynucleotide sequence encoding an UBAPl polypeptide is provided above.

Using such information it is straightforward and well within the capability of the skilled person to design siRNA molecules having complementary sequence to UBAPl polynucleotide. For example, a simple internet search yields many websites that can be used to design siRNA molecules.

By "siRNA molecule" we include a double stranded 20 to 25 nucleotide-long RNA molecule, as well as each of the two single RNA strands that make up a siRNA molecule.

It is most preferred that the siRNA is used in the form of hair pin RNA (shRNA). Such shRNA may comprise two complementary siRNA molecules that are linked by a spacer sequence (e.g. of about 9 nucleotides). The complementary siRNA molecules may fold such that they bind together.

A ribozyme capable of cleaving RNA or DNA encoding UBAPl polypeptide represent another preferred agent of the third, fourth or fifth aspect of the invention.

An sixth aspect of the invention provides a method of making a pharmaceutical composition comprising mixing a compound identified from the screening methods of the invention with a pharmaceutically acceptable carrier.

It will be appreciated that the amount of an agent needed according to the invention is determined by biological activity and bioavailability which in turn depends on the mode of administration and the physicochemical properties of the agent. The frequency of administration will also be influenced by the abovementioned factors and particularly the half-life of the agent within the target tissue or subject being treated.

Known procedures, such as those conventionally employed by the pharmaceutical industry (e.g. in vivo experimentation, clinical trials etc), may be used to establish specific formulations of the agents and precise therapeutic regimes (such as daily doses and the frequency of administration).

Generally, a daily dose of between O.Olμg/kg of body weight and O.lg/kg of body weight of an agent may be used; more preferably the daily dose is between O.Olmg/kg of body weight and lOOmg/kg of body weight.

By way of example a suitable dose of an antibody according to the invention is lOμg/kg of body weight - 100mg/kg of body weight, more preferably about Olmg/kg of body weight - lOmg/kg of body weight and most preferably about 6mg/kg of body weight.

Daily doses may be given as a single administration (e.g. a single daily injection or a single dose from an inhaler). Alternatively the agent (e.g. an antibody or aptamer) may require administration twice or more times during a day. Medicaments according to the invention should comprise a therapeutically effective amount of the agent and a pharmaceutically acceptable vehicle.

A "therapeutically effective amount" is any amount of an agent according to the invention which, when administered to a subject inhibits or prevents cancer growth or metastasis.

A "subject" may be a vertebrate, mammal, domestic animal or human being. It is preferred that the subject to be treated is human. When this is the case the agents may be designed such that they are most suited for human therapy (e.g. humanisation of antibodies as discussed above). However it will also be appreciated that the agents may also be used to treat other animals of veterinary interest (e.g. horses, dogs or cats).

A "pharmaceutically acceptable vehicle" as referred to herein is any physiological vehicle known to those skilled in the art as useful in formulating pharmaceutical compositions.

In one embodiment, the medicament may comprise about 0.01 μg and 0.5 g of the agent. More preferably, the amount of the agent in the composition is between 0.01 mg and 200 mg, and more preferably, between approximately 0.1 mg and 100 mg, and even more preferably, between about lmg and lOmg. Most preferably, the composition comprises between approximately 2mg and 5mg of the agent.

Preferably, the medicament comprises approximately 0.1% (w/w) to 90% (w/w) of the agent, and more preferably, 1% (w/w) to 10% (w/w). The rest of the composition may comprise the vehicle.

Nucleic acid agents can be delivered to a subject by incorporation within liposomes. Alternatively the "naked" DNA molecules may be inserted into a subject's cells by a suitable means e.g. direct endocytotic uptake. Nucleic acid molecules may be transferred to the cells of a subject to be treated by transfection, infection, microinjection, cell fusion, protoplast fusion or ballistic bombardment. For example, transfer may be by ballistic transfection with coated gold particles, liposomes containing the DNA molecules, viral vectors (e.g. adenovirus) and means of providing direct DNA uptake (e.g. endocytosis) by application of the DNA molecules directly to the target tissue topically or by injection.

The antibodies, or functional derivatives thereof, may be used in a number of ways. For instance, systemic administration may be required in which case the antibodies or derivatives thereof may be contained within a composition which may, for example, be ingested orally in the form of a tablet, capsule or liquid. It is preferred that the antibodies, or derivatives thereof, are administered by injection into the blood stream.

Injections may be intravenous (bolus or infusion) or subcutaneous (bolus or infusion). Alternatively the antibodies may be injected directly to the liver.

Nucleic acid or polypeptide therapeutic entities may be combined in pharmaceutical compositions having a number of different forms depending, in particular on the manner in which the composition is to be used. Thus, for example, the composition may be in the form of a powder, tablet, capsule, liquid, ointment, cream, gel, hydrogel, aerosol, spray, micelle, transdermal patch, liposome or any other suitable form that may be administered to a person or animal. It will be appreciated that the vehicle of the composition of the invention should be one which is well tolerated by the subject to whom it is given, and preferably enables delivery of the therapeutic to the target cell, tissue, or organ.

In a preferred embodiment, the pharmaceutical vehicle is a liquid and the pharmaceutical composition is in the form of a solution. In another embodiment, the pharmaceutical vehicle is a gel and the composition is in the form of a cream or the like.

Compositions comprising such therapeutic entities may be used in a number of ways. For instance, systemic administration may be required in which case the entities may be contained within a composition that may, for example, be ingested orally in the form of a tablet, capsule or liquid. Alternatively, the composition may be administered by injection into the blood stream. Injections may be intravenous (bolus or infusion) or subcutaneous (bolus or infusion). The entities may be administered by inhalation (e.g. intranasally). Therapeutic entities may also be incorporated within a slow or delayed release device. Such devices may, for example, be inserted on or under the skin, and the compound may be released over weeks or even months. Such devices may be particularly advantageous when long term treatment with an entity is required and which would normally require frequent administration (e.g. at least daily injection).

The data provided herein shows that 7 UBAPl haplotypes were found to be associated with FTLD with a P value of =0.05, with the haplotype block demonstrating maximum OR of 1.71 (95% CI 1.20-2.44, P=0.003). The inventors also identified a positive association between UBAPl and AD in a cohort of 360 AD patients. This argues that UBAPl is a risk factor for Alzheimer's disease. Furthermore, as discussed above tau protein is the main component of neurofibrillary tangles, one of the pathological hallmarks of Alzheimer's disease, and the inventors have demonstrated that tau and UBAPl polypeptides interact using both immunoprecipitation and colocalisation with confocal microscopy.

Thus the presence of one or more mutations in UBAPl indicates that a subject has or is likely to develop a dementia, i.e. they have a higher than average likelihood of having or developing a dementia.

A seventh aspect of the invention provides a method of assessing whether a subject has or is likely to develop a dementia comprising determining whether the subject has a mutation in the UBAPl gene.

The method of this aspect of the invention includes determining whether a subject has a mutation in the UBAPl gene. If the subject has a mutation in the UBAPl gene, this indicates that subject has or is likely to develop dementia.

A mutant UBAPl nucleic acid is any UBAPl nucleic acid containing a mutation as compared to a wild type UBAPl nucleic acid. For example, a mutant human UBAPl nucleic acid can be a nucleic acid having the nucleotide sequence above having at least one mutation. By "mutation" as used herein with respect to nucleic acid, we include insertions of one or more nucleotides, deletions of one or more nucleotides, nucleotide substitutions, and combinations thereof, including mutations that occur in coding and non-coding regions (e.g., exons, introns, untranslated sequences, sequences upstream of the transcription start site of UBAPl mRNA, and sequences downstream of the transcription termination site of UBAPl mRNA).

Examples of mutations in UBAPl nucleic acid include those provided below in relation to this aspect of the invention.

A mutant UBAPl polypeptide is any UBAPl polypeptide containing an alteration to the amino acid sequence as compared to a wild type UBAPl polypeptide. For example, a mutant human UBAPl polypeptide can be a polypeptide having the amino acid sequence above having at least one alteration; for example this could be a substitution of one or more amino acid residues with other amino acid residues; this could be an insertion of one or more amino acid residues; this could be a deletion of one or more amino acid residues, and possibly a truncation of a large region of the UBAPl polypeptide.

By "UBAPl gene" we include the nucleic acid sequence set out above that encodes the UBAPl polypeptide or any fragment of that sequence. This can be genomic DNA sequence, mRNA sequence and cDNA sequence. UBAPl gene nucleic acid sequences include the untranslated regions extending both upstream of the transcription start site of UBAPl mRNA and downstream of the transcription termination site of UBAPImRNA by, for example, 5Kb. UBAPl gene nucleic acid sequences include all exon and intron sequences. We also include polymorphisms or variations in that nucleotide sequence that are naturally found between individuals of different ethnic backgrounds or from different geographical areas and which do not affect the function of the gene.

By "UBAPl gene" we also include "regulatory elements", including the 5' and 3' of the gene which is involved in regulating gene transcription. For instance, transcription factor binding sequences, the TATA box, the 5' promoter and 5' and 3' untranslated regions (UTRs). This definition also encompasses the DNA 5' of the first codon of the first exon of UBAPl. At least some of this sequence information is provided at the end of the example section of the description The method according to the present invention is an in vitro method and can be performed on a sample containing nucleic acid and/or polypeptide derived from the subject.

Various different approaches can be used to determine whether a subject has a mutation in the UBAPl gene. These include haplotype analysis of genomic DNA of the subject; determining the nucleic acid sequence of the UBAPl gene; determining the nucleic acid sequence of mRNA encoding the UBAPl polypeptide; determining whether the subject has a mutant UBAPl polypeptide.

A preferred method of determining whether a subject has a mutation in the UBAPl gene is to use haplotype analysis. Preferably, haplotype analysis is used when the method of the seventh aspect of the invention relates to FTLD.

Haplotype analysis is a powerful technique that can be used to determine whether a subject has, or is likely to have, a mutation in a specific gene. A haplotype is a set of genetic markers on a single chromatid that are statistically associated. It is thought that these associations, and the identification of a few alleles of a haplotype block, can unambiguously identify all other polymorphic sites in its region. Such information is very valuable for investigating the genetics behind common diseases and is collected by the International HapMap Project. Haplotypes of the UBAPl gene are individually associated with specific mutations in that gene.

In contrast to genotyping methods, a significant advantage of haplotype analysis is that it is not necessary to determine the sequence of the gene under investigation, e.g. UBAPl. Rather, certain haplotypes are statistically associated with a mutated version of the gene, i.e. a marker allele to disease gene association. Therefore, where a subject has a certain haplotype, then it can be concluded that the subject also has a mutated version of the gene; in the present case UBAPl.

A further significant advantage of haplotype analysis is that the presence of a mutated version of a gene can be detected in a subject even when the precise nucleotide mutation within that gene has not been established. Therefore it is possible to determine whether a subject has a mutation within the UBAPl gene without first having to determine the exact genetic mutation present.

Accordingly, in order to determine whether a subject has a mutation in the UBAPl gene, in this embodiment of the invention the haplotype of the subject is determined, and from that an assessment can be made as to the likelihood of that subject having a mutation in UBAP 1.

Preferably the haplotype analysis uses haplotype blocks. By "haplotype blocks" we mean a set of genetic markers within particular region of a chromosome that are statistically linked. Various different genetic markers can be located within the haplotype block, for example: nucleotide deletions, nucleotide insertions, nucleotide repeat sequences, nucleotide rearrangements, and single nucleotide polymorphisms (SNPs). Preferably the haplotype block has two or more SNPs markers.

In the present invention, the inventors have surprisingly found that the presence of a mutation in the UBAPl gene of a subject can be determined using a haplotype block, preferably comprising two or more SNPs. As way of an example, set out below are details of a number of SNPs that can included in a haplotype block which can form part of a haplotype analysis of UBAPl . Further information from each of the SNPs can be obtained from, for example, the webpage of the International HapMap Project: http://www.hapmap.org/. By searching the database on that website information regarding the nucleotide sequence of the SNP and the distribution of the haplotype can be obtained.

SNP Chromosomal position rs13283064 34142464 rslO971969 34154375 rs7018487 34157733 rs10971977 34170023 rs12375731 34194266 rs10814079 34216087 rs2380925 34239204 rs17258783 34243097 rs4574933 34243488 rsl0814083 34246347 Preferably the SNPs in the haplotype block are selected from the following group of SNPs: rsl3283064, rsl3283069, rs7018487, rslO971977, rsl2375731, rsl0814079, rs2380925, rsl7258783, rs4574933, rsl0814083.

Methods of performing haplotype analysis are well known in the art. Further information can be found from, for example, the HapMap webpage given above. A discussion of the methods used by the present inventors is provided in the accompanying example.

An embodiment of the method of the invention is wherein the step of determining whether the subject has a mutation in the UBAPl gene comprises genotyping the UBAPl gene.

The step of genotyping UBAPl includes examining the nucleotide sequence of that gene to identify whether one or more genetic variations in the sequence are present. Such genetic variations can include mutations which, as discussed above, insertions of one or more nucleotides, deletions of one or more nucleotides, nucleotide substitutions, and combinations thereof, including mutations that occur in coding and non-coding regions. SNPs are also genetic variations, and hence this method of the invention can be used to identify whether any SNPs are present in UBAPl.

As shown in the accompanying examples, the inventors have identified a number of SNPs in the UBAPl genomic DNA sequence are statistically associated with the development of dementia. Therefore, while any SNPs or genetic variations can be studied in this aspect of the invention, in particular, SNPs rsl3283064, rslO971969, rs7018487, rslO971977, rsl2375731, rsl0814079, rs2380925, rsl7258783, rs4574933 and rsl0814083 are associated with FTLD; rslO971977, rsl2375731, rs2380925 and rs 10972030 are associated with Alzheimer's disease.

Therefore in an embodiment of this aspect of the invention, where the subject is assessed for the likelihood of FTLD, the method can comprise genotyping UBAPl to detect the presence of SNPs rsl3283064, rslO971969, rs7018487, rslO971977, rsl2375731, rsl0814079, rs2380925, rsl7258783, rs4574933 and rsl0814083. In a further embodiment of this aspect of the invention, where the subject is assessed for the likelihood of Alzheimer's disease, the method can comprise genotyping UBAPl to detect the presence of SNPs rslO971977, rsl2375731, rs2380925 and rsl0972030.

Methods of genotypic analysis are well known to those skilled in the art. The genotype may preferably be determined by testing a sample from the subject. Preferably the sample contains genomic DNA. Methods of providing samples of genomic DNA from a subject are discussed above and can be routinely performed by the skilled person.

The nucleic acid sequence for UBAPl is provided herein and as part of the GenBank accession entries given above. This information can be used to design materials, such as oligonucleotide primers or probes specific for each allele that can be used when determining the genotype of the UBAPl gene of a subject. The design of such oligonucleotide primers is routine in the art and can be performed by the skilled person with reference to the information provided herein without any inventive contribution. If required, the primer(s) or probe(s) may be labelled to facilitate detection.

Techniques that may be used to detect mutations include:- (1) Direct sequencing of the polymorphic region of interest (e. g. using commercially available kits such as the Cysts Thermo Sequence dye terminator kit-Amersham Pharmacia Biotech); (2) Sequence Specific Oligonucleotide Hybridization (SSO) (involving dot or slot blotting of amplified DNA molecules comprising the polymorphic region; hybridisation with labelled probes which are designed to be specific for each polymorphic variant; and detection of said labels); (3) Heteroduplex and single-stranded conformation polymorphism (SSCP) Analysis (involving analysis of electrophoresis band patterns of denatured amplified DNA molecules comprising the polymorphic region); (4) Sequence Specific Priming (SSP) [also described as Amplification Refractory Mutation System (ARMS)]; (5) Mutation Scanning [e. g. using the PASSPORT Mutation Scanning Kit (Amersham Pharmacia Biotech)]; (6) Chemical Cleavage of Mismatch Analysis; (7) Non-isotopic RNase Cleavage Assay (Ambion Ltd.); (8) Enzyme Mismatch Cleavage Assay; and (9) Single Nucleotide Extension Assay. It will be apparent to the person skilled in the art that there are a large number of analytical procedures, which may be used to detect whether there is a mutation in the UBAPl gene. A further method of genotypic analysis is the PCR-SSOP (sequence-specific oligonucleotide probe) typing system using, for example, the Dynal AutoRELI 48™ system.

Reference Strand mediated Conformational Analysis (RSCA) can also be used for UBAPl genotyping. A PCR reaction is performed on a sample of DNA isolated from a subject using primers that flank a region of the UBAPl gene. The amplified product is then hybridized with fluorescent-labeled reference DNA molecules at a temperature that permits annealing to occur, even when mismatches are present. Mismatches between the reference strand and the sample DNA result in the formation of bulges or "bubbles" in the heteroduplex that is formed. The number and location of the bulges give the heteroduplex a unique mobility on a polyacrylamide gel, and can be used to determine whether there is a mutation in the UBAPl gene.

A further method is sequence based typing (SBT). SBT combines a low-resolution SSP-PCR reaction followed by high resolution allele typing using automated DNA sequencing. In summary, DNA isolated from a subject is used as a template for a PCR reaction that amplifies a region of the UBAPl gene to create a primary amplification product. That product is then purified to remove excess reaction reagents, though there are single-tube reactions available in which this purification step is not required. The primary amplification product is then used as a template for sequencing reactions. Once complete, the sequence reactions are analysed by a sequencer, and the products analysed to determine whether there is a mutation in the UBAPl gene.

Where PCR amplification is required as part of the method of UBAPl genotyping, PCR primers may be designed such that they are suitable for amplifying a region of the UBAPl gene. The design of suitable PCR primers is a routine laboratory technique.

Furthermore, it is possible that genomic rearrangements can lead to mutations in the UBAPl gene. Methods of determining genomic rearrangements include Southern blotting (essentially as performed as set out in Sambrook et al (1989). Molecular cloning, a laboratory manual, 2^nd edition, Cold Spring Harbor Press, Cold Spring Harbor, New York) or quantitative PCR. A further embodiment of this aspect of the invention is wherein the method comprises determining the nucleic acid sequence of mRNA encoding the UBAPl polypeptide.

Methods of isolating mRNA molecules from a sample are routine in the art and well known to the skilled person. Once isolated, the nucleotide sequence of the mRNA molecule can be determined, preferably from a cDNA sample prepared from mRNA isolated from the subject. The sequence of cDNA molecules can be determined according to the genotyping methods set out above.

A further embodiment of this aspect of the invention is wherein the method comprises determining whether the subject has a mutant UBAPl polypeptide. That is, if a sample from a subject has a mutant UBAPl polypeptide, then that subject has a mutation in the UBAPl gene.

The polypeptide sequence for UBAPl is provided herein. This information can be used to design materials, such as antibodies or further specific binding molecules, that may be required for the methods set out below.

Determining whether a subject has a mutant UBAPl polypeptide may be conducted by isolating then sequencing UBAPl protein from a sample derived from that subject. Methods of purifying proteins are well known in the art and can be readily applied to the method of the invention. For example, a molecule that selectively binds to the UBAPl protein, e.g. an antibody or a fragment of an antibody, can be used to purify the UBAPl protein from the sample from the subject. Then, using well-known peptide sequencing methods, such as N-terminal sequencing, the amino acid sequence of the isolated UBAPl protein can be determined and compared to that of the UBAPl protein provided herein.

Alternatively, the presence mutant UBAPl polypeptide in the sample can be detected using immunological methods. In a preferred embodiment, the presence of a mutant UBAPl polypeptide in the sample can be detected using an antibody that selectively binds to a mutant UBAPl polypeptide.

Antibodies which can selectively bind to mutant UBAPl polypeptides can be made, for example, using peptides that include amino acid sequences particular to that mutation.

Various procedures known within the art may be used for the production of polyclonal or monoclonal antibodies directed against a polypeptide, or against derivatives, fragments, analogs homologs or orthologs thereof (see, for example, Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, incorporated herein by reference), and are well known to those skilled in the art.

Screening assays to determine binding specificity of such an antibody are well known and routinely practiced in the art. For a comprehensive discussion of such assays, see Harlow et al. (Eds), Antibodies A Laboratory Manual; Cold Spring Harbor Laboratory; Cold Spring Harbor, N.Y. (1988), Chapter 6.

Suitable monoclonal antibodies to selected antigens may be prepared by known techniques, for example those disclosed in "Monoclonal Antibodies: A manual of techniques ", H Zola (CRC Press, 1988) and in "Monoclonal Hybridoma Antibodies: Techniques and Applications", J G R Hurrell (CRC Press, 1982). Such methods include the use of hybridomas, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In a hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes can be immunized in vitro.

It will be appreciated that other antibody-like molecules may be used in the method of the inventions including, for example, antibody fragments or derivatives which retain their antigen-binding sites, synthetic antibody-like molecules such as single-chain Fv fragments (ScFv) and domain antibodies (dAbs), and other molecules with antibody- like antigen binding motifs. Sequence analysis of the coding region of UBAPl has identified three mutations in that sequence. Two of these lead to amino acid substitutions: a P to L change at position 96 (P96L); and a E to K change at position 87 (E87K). A further mutation leads to a stop codon being introduced after the M at position 413 (M413X), and therefore results in a truncated UBAPl polypeptide; this mutation is also known as S391Afs21X .

A further mutation in UBAPl identified from sequence analysis is a P to L change at position 256 (P256L).

The P96L mutation is caused by a C to T change at position 522 of the UBAPl nucleotide sequence as set out above in GenBank Accession NM 016525. The E87K mutation is caused by a G to A change at position 494 of the UBAPl nucleotide sequence as set out above in GenBank Accession NM 016525. The M413X (S391Afs21X) mutation is caused by a stop codon being introduced at amino acid position 413 (M413X), and therefore results in a truncated UBAPl polypeptide. The stop codon is introduced as a result of a single nucleotide deletion in the codon encoding the proline at position 390. Preferably the deletion is of a C nucleotide at position 1404 or 1405 according to the numbering of the UBAPl nucleic acid set out above; preferably nucleotide 1404. This deletion causes a "frame shift" in the open reading frame, such that the methionine residue at position 413 is altered to a stop codon. The amino acid sequence of the M413X mutation is provided below.

Therefore a preferred embodiment of the invention is wherein the method determines whether the subject has a P96L, E87K or M413X (S391Afs21X) mutation in UBAPl. Preferably, this embodiment is used when the method of the seventh aspect of the invention relates to FTLD.

A further preferred embodiment of the invention is wherein the method determines whether the subject has C to T change at position 522, a G to A change at position 494 or a deletion of a C nucleotide at position 1404. Preferably, this embodiment is used when the method of the seventh aspect of the invention relates to FTLD. As described herein, the presence of UBAPl nucleic acid containing one or more mutations (e.g., one or more mutations listed above) in a subject can indicate that that subject has dementia or is likely to develop dementia. In some cases, the presence of UBAPl nucleic acid containing one or more mutations in a human can indicate that that human has dementia, especially when that human is between the ages of 35 and 75, has a family history of dementia, and/or presents symptoms of dementia. Symptoms of dementia can include changes in behaviour such as changes that result in impulsive, repetitive, compulsive, or even criminal behaviour. For example, changes in dietary habits and personal hygiene can be symptoms of dementia. Symptoms of dementia also can include language dysfunction, which can present as problems in expression of language, such as problems using the correct words, naming objects, or expressing oneself. Difficulties reading and writing can also develop, hi some cases, the presence of UBAPl nucleic acid containing one or more mutations in a subject, together with positive results of other diagnostic tests, can indicate that the subject has dementia. For example, the presence of a mutation in UBAPl nucleic acid together with results from a neurological exam, neurophysical testing, cognitive testing, and/or brain imaging can indicate that a mammal has dementia. Other diagnostic tests can include, without limitation, tests for mutations in MAPT and/or apolipoprotein E (APOE) nucleic acid.

In some cases, any subject containing a mutation in UBAPl nucleic acid can be classified as having an elevated risk of developing dementia. For example, a subject having one or more than one mutation in UBAPl nucleic acid (e.g., one or more than one mutation set out above) can be classified as having an elevated risk of developing dementia when the human is any age (e.g., less than 65, 60, 55, 50, 45, 40, or 35 years old), does or does not appear to have symptoms of dementia, or has or has not had a positive or negative diagnostic test for dementia. In some cases, a human having one or more mutations in UBAPl nucleic acid can be classified as having an elevated risk of developing dementia when the human also has one or more mutations in MAPT or APOE nucleic acid and is less than, for example, 35 years old or does not appear to have symptoms of dementia.

The inventors have identified a genetic linkage between the UBAPl gene and frontotemporal lobar degeneration (FTLD) and Alzheimer's disease. As discussed above, FTLD and AD are types of dementia, and the inventors consider that the UBAPl gene may be used as a diagnostic marker and a therapeutic agent for the disorders within this broad category. Therefore the aspects of the invention provided herein are applicable to a wide range of dementias, including Alzheimer's disease, motor neuron disease, Parkinson's disease, dementia with Lewy bodies, prion diseases, progressive supranuclear palsy or multisystem atrophy.

However a preferred embodiment of the seventh aspect of the invention is wherein the dementia is frontotemporal lobar degeneration (FTLD). The syndrome of FTLD encompasses the clinical subgroups of frontotemporal dementia (FTD), FTD with motor neuron disease, semantic dementia and primary progressive aphasia, and is characterized by changes in behaviour, personality and language with relative preservation of memory and perception

An embodiment of the seventh aspect of the invention is wherein the dementia, including frontotemporal lobar degeneration and Alzheimer's disease, is characterised by tauopathy. Tauopathy is characterised by the accumulation of hyperphosphorylated tau in neurons and occasionally in glia.

An embodiment of the seventh aspect of the invention is wherein the frontotemporal lobar degeneration is characterised by ub-ir.

An embodiment of the seventh aspect of the invention is wherein the dementia is Alzheimer's disease.

While it can be appreciated that the method of the invention can be applied to animal subjects of veterinary interest, it is preferred that the subject to be tested is a human subject.

The method according to the present invention is an in vitro method and can be performed on a sample containing nucleic acid and/or polypeptide derived from the subject.

The method of the invention is particularly suitable for being carried out on genomic DNA, particularly on isolated genomic DNA. Such genomic DNA may be isolated from blood or tissue samples (e. g. hair, oral buccal swabs, nail or skin, blood, plasma, bronchoalveolar lavage fluid, saliva, sputum, cheek-swab or other body fluid or tissue), or from other suitable sources, using conventional methods. The nucleic acid containing sample that is to be analysed can either be a treated or untreated biological sample isolated from the individual. A treated sample, may be for example, one in which the nucleic acid contained in the original biological sample has been isolated or purified from other components in the sample (tissues, cells, proteins etc), or one where the nucleic acid in the original sample has first been amplified, for example by polymerase chain reaction. Thus, it will be appreciated that the sample may equally be a nucleic acid sequence corresponding to the sequence in the sample, that is to say that all or a part of the region in the sample nucleic acid may firstly be amplified using any convenient technique e.g. PCR, before analysis of allelic variation.

The method of the invention can also be carried out on protein samples obtained from a subject. Such protein may be isolated from blood or tissue samples as specified above, or from other suitable sources using conventional methods.

Therefore an embodiment of this aspect of the invention is wherein the UBAPl gene or polypeptide is derived from a sample of genomic DNA or polypeptide from the subject. Preferably the sample is derived from blood or tissue samples.

The identification of one or more UBAPl mutations (e.g., one or more mutations listed in below) in an allele can be used to determine whether a subject has or is likely to develop a dementia. Such a method may be performed when a subject has already exhibited clinical symptoms of dementia, i.e. as an adjuvant to existing techniques for diagnosing such neurological disorders. Alternatively, the method may be performed as a means of assessing whether the subject has a predisposition towards developing dementia. This enables a medical practitioner to take appropriate action to prevent or lessen the likelihood of onset of the disease or disorder or to allow appropriate treatment of the disease or disorder.

The inventors have determined that the UBAPl gene is associated with dementia, specifically FTLD, and Alzheimer's disease. As set out above, this finding is the basis for the method of the seventh aspect of the invention in which the presence of a mutation is the UBAPl gene is indicative of a subject having, or being predisposed to developing, a dementia.

The linkage of the UBAPl gene to dementia suggests that, as well as mutations in that gene being linked to dementia, the amount of UBAPl polypeptide present in a subject can be used to determine if a subject has is predisposed to developing, a dementia.

According to an eighth aspect of the invention there is provided a method of assessing whether a subject has or is likely to develop a dementia comprising determining whether the subject has an altered amount of UBAPl polypeptide and/or nucleic acid encoding UBAPl polypeptide.

The method of this aspect of the invention includes determining whether a subject has a modified amount of UBAPl polypeptide and/or nucleic acid encoding UBAPl polypeptide. If the subject has an altered amount of said polypeptide and/or nucleic acid, this indicates that the subject has or is likely to develop dementia.

The method of this aspect of the invention can be performed on sample taken from the subject. Suitable samples that may be used in this method of the invention include those which contain representative samples of the patient's polypeptide and/or nucleic acid as set out above.

In order to determine whether the subject has an altered amount of UBAPl polypeptide or nucleic acid encoding UBAPl polypeptide, the amount of UBAPl polypeptide and/or nucleic acid encoding UBAPl polypeptide in the subject is compared to that of a "reference sample", i.e. a sample of protein or nucleic acid taken from a subject that does not have dementia. By comparing the amount of UBAPl polypeptide and/or nucleic acid encoding UBAPl polypeptide in a sample of protein or nucleic acid taken from a control subject, to the amount of UBAPl polypeptide and/or nucleic acid encoding UBAPl polypeptide in a sample of protein or nucleic acid taken from the subject, it is possible to determine whether the subject has an altered amount of UBAPl polypeptide and/or nucleic acid encoding UBAPl polypeptide. In one embodiment of this aspect of the invention, the amount of UBAPl polypeptide or nucleic acid encoding UBAPl polypeptide is elevated.

By "elevated" we include where the subject has, for example, 150%, 200%, 250%, 500%, 1000%, or 10000% of the amount of the polypeptide or nucleic acid in the control subject.

In an alternative embodiment of this aspect of the invention, the amount of UBAPl polypeptide or nucleic acid encoding UBAPl polypeptide is reduced.

By "reduced" we include where the subject has, for example, 50%, 25%, 10%, 5%, 1%, 0.1% or 0% of the amount of the polypeptide or nucleic acid in the control subject.

Of particular interest to this embodiment of the invention is the finding reported in the accompanying examples that analysis of UBAPl mRNA identifies disease associated haplotypes with significantly lower levels of expression compared to the reference haplotype. These data strongly suggest that the main variant that increases of FTLD risk operates by lowering UBAPl expression leading to loss/reduced function.

Methods of determining the amount and/or function of polypeptide or the amount of nucleic acid are provided above in relation to the first aspect of the invention.

This method may be useful in the diagnosis of dementia or as a basis of genetic counselling.

While it can be appreciated that the method of the invention can be applied to animal subjects of veterinary interest, it is preferred that the subject to be tested is a human subject.

The method of the invention can also be carried out on protein samples obtained from a subject. Such protein may be isolated from blood or tissue samples as specified above, or from other suitable sources using conventional methods. Therefore an embodiment of this aspect of the invention is wherein the amount of UBAPl polypeptide or nucleic acid encoding said polypeptide is derived from a sample of genomic DNA or polypeptide from the subject. Preferably the sample is derived from blood or tissue samples.

A preferred embodiment of the eighth aspect of the invention is wherein the dementia is characterised by tauopathy. A preferred embodiment of the eighth aspect of the invention is wherein the dementia is frontotemporal lobar degeneration (FTLD) or

Alzheimer's disease. As discussed above, FTLD and Alzheimer's disease are types of dementia, and the inventors consider that the UBAPl gene may be used as a diagnostic marker and a therapeutic agent for the disorders within this broad category. A preferred embodiment of the eighth aspect of the invention is wherein the subject is a human subject.

The inventors have determined that the UBAPl gene is associated with dementia, specifically FTLD and Alzheimer's disease. As set out above, this finding is the basis for the method of the eighth aspect of the invention in which the presence of a mutation is the UBAPl gene is indicative of a subject having, or being predisposed to developing, a dementia.

A ninth aspect of the invention provides a non-human genetically modified animal having or predisposed to develop dementia, wherein the dementia results from an altered amount and/or function of UBAPl polypeptide.

As set out above, UBAPl is associated with dementia. Animals with an altered amount and/or function of UBAPl polypeptide can be expected to display dementia and may therefore be useful in screening for potential therapeutic agents for preventing or treating dementia.

As set out above, sequence analysis of the coding region of UBAPl has identified three mutations in that sequence. Two of these lead to amino acid substitutions: a P to L change at position 96 (P96L); and a E to K change at position 87 (E87K). A further mutation leads to a stop codon being introduced after the M at position 413 (M413X), and therefore results in a truncated UBAPl polypeptide; this mutation is also known as S391Afs21X . A further mutation in UBAPl identified from sequence analysis is a P to L change at position 256 (P256L).

The P96L mutation is caused by a C to T change at position 522 of the UBAPl nucleotide sequence as set out above in GenBank Accession NM Ol 6525. The E87K mutation is caused by a G to A change at position 494 of the UBAPl nucleotide sequence as set out above in GenBank Accession NM 016525. The M413X

(S391Afs21X) mutation is caused by a stop codon being introduced at amino acid position 413 (M413X), and therefore results in a truncated UBAPl polypeptide. The stop codon is introduced as a result of a single nucleotide deletion at position 1404 or

1405 according to the numbering of the UBAPl nucleic acid set out above; preferably nucleotide 1404.

Therefore a preferred embodiment of this aspect of the invention is wherein the non- human genetically modified animal has a mutation(s) in UBAPl equivalent to the P96L, E87K and/or M413X (S391Afs21X) mutation in human UBAPl described above.

However, alternatively the non-human genetically modified animal may have been specifically genetically engineered to have a copy of the human UBAPl gene, but having one or more mutations in that gene. Therefore a further preferred embodiment of this aspect of the invention is wherein the non-human genetically modified animal has a human UBAPl nucleic acid sequence having a P96L, E87K and/or M413X mutation as described above. In order to allow the effect of the mutations in the human UBAPl gene to be fully manifested in the animal, it is preferred that the native homologue of human UBAPl has been removed or otherwise mutated such that no native UBAPl polypeptide is produced.

The non-human animal may be any non-human animal, including non-human primates such as baboons, chimpanzees and gorillas, new and old world monkeys as well as other mammals such as cats, dogs, rodents, pigs or sheep, or other animals such as poultry, for example chickens, fish such as zebrafish, or amphibians such as frogs. However, it is preferred that the animal is a rodent such as a mouse, rat, hamster, guinea pig or squirrel. Preferably the animal is mouse. By "dementia" we include those disorders discussed above in relation to the first aspect of the invention. A preferred embodiment is wherein the dementia is characterised by tauopathy. Preferably the dementia is FTLD or Alzheimer's disease.

By "altered amount and/or function" we include that, in comparison to a normal animal of the same species or strain, the animal of the ninth aspect of the invention has a reduced or elevated amount of UBAPl polypeptide and/or a reduced or elevated amount of the UBAPl polypeptide can function in the way that the same polypeptide operates in the comparative animal.

For example, the animal of this aspect of the invention may have the same amount of polypeptide per se, but the polypeptide is in a non-functional state. Preferably the animal of the ninth aspect of the invention has a reduced amount and/or function of UBAPl polypeptide.

Alternatively, the altered amount of UBAPl polypeptide may be due to an altered amount of nucleic acid encoding the UBAPl polypeptide.

Methods of determining the amount and/or function of polypeptide or the amount of nucleic acid are provided herein in relation to other aspects of the invention.

Preferably, "altered amount and/or function of includes where the animal has a reduced amount, i.e. 50%, 25%, 10%, 5%, 1%, 0.1% or 0% of the amount and/or function of the polypeptide, or nucleic acid, in the normal animal. Preferably the non-human animal has no functional UBAPl polypeptide.

Of particular interest to this embodiment of the invention is the finding reported in the accompanying examples that analysis of UBAPl mRNA identifies disease associated haplotypes with significantly lower levels of expression compared to the reference haplotype. This strongly suggest that the main variant that increases of FTLD risk operates by lowering UBAPl expression leading to loss/reduced function. Alternatively, the animal may have an elevated amount, i.e. 150%, 200%, 250%, 500%, 1000%, or 10000% of the amount and/or function of the polypeptide, or nucleic acid, in the normal animal.

The non-human animal of this aspect of the invention may have an altered amount and/or function of UBAPl polypeptide due to the animal being genetically modified so as to have an agent which can modify said polypeptide function. For example the animal could be genetically modified to express a peptide or antibody which can bind to the UBAPl and prevent function or sub-cellular localisation. The non-human animal of this aspect of the invention may have an altered amount of nucleic acid encoding UBAPl polypeptide due to the animal being genetically modified so as to have an agent which can cause or induce degradation of said nucleic acid, for example a ribozyme which can target the nucleic acid, or an antisense molecule which can bind to the UBAPl nucleic acid. By "antisense" we include RNA interference (RNAi) technologies.

Alternatively, the animal may be genetically modified in such a manner as to alter the native UBAPl gene.

The term "genetically modified" is well known to those skilled in the art. The term includes animals having introduced native or foreign nucleic acid. The animal may have had a modification made to its genome.

There are a number of different methods that can be employed to generate a non-human genetically modified animal according to this aspect of the invention. These will be discussed in turn below. Preferred methods include those in which the gene encoding the said polypeptide is altered or removed so as to produce little or none of said polypeptide. Other methods include inhibiting the transcription of the said gene or preventing any mRNA encoded by said gene from being translated due to the animal being genetically modified so as to have an agent which can modify said polypeptide transcription, translation and/or function. Preferably, the methods set out below are employed to generate a non-human genetically modified animal according to this aspect of the invention in which the function of the UBAPl polypeptide altered.

"Homologous recombination" is a technique well known to those skilled in the art. Animals in which an endogenous gene has been inactivated by homologous recombination are referred to as "knockout" animals. Hence this aspect of the invention includes wherein the amount and/or function of UBAPl polypeptide is altered by mutated one or more gene(s) encoding UBAPl by homologous recombination.

"Insertional mutagenesis" is also a term well known to those skilled in the art. Examples of such mutagenesis include transposon-tagging, homing endonuclease genes (HEGs). In such methods a region of DNA is introduced into a gene such that the controlling or coding region of the gene is disrupted. Such methods can be used to disrupt one or more genes encoding UBAPl polypeptide. As a result the animal will no longer be able to synthesise UBAPl polypeptide, i.e. there will be a reduction in the amount of this polypeptide.

Chemical or physical mutagenesis can also be used in the method of this aspect of the invention. Here, a gene is mutated by exposing the genome to a chemical mutagen, for example ethyl methylsulphate (EMS) or ethyl Nitrosurea (ENU), or a physical mutagen, for example X-rays. Such agents can act to alter the nucleotide sequence of a gene or, in the case of some physical mutagens, can rearrange the order of sequences in a gene.

Practical methods of using chemical or physical mutagenesis in animals are well known to those skilled in the art. Such methods can be used to disrupt one or more genes encoding UBAPl polypeptide. As a result the animal may no longer be able to synthesise UBAPl polypeptide, i.e. there will be a reduction in the amount and/or function of this polypeptide; alternatively the mutation may cause overactivity of the mutated polypeptide, i.e. there will be a increase in the amount and/or function of UBAPl polypeptide; alternatively, the mutation may cause an altered function of the mutated polypeptide.

Homologous recombination, insertional mutagenesis and chemical or physical mutagenesis can be used to generate a non-human animal which is heterozygous for the target gene, e.g. UBAPl gene (^{+ ~}). Such animals may be of particular use if the homozygous non-human animal has too severe a phenotype.

The non-human animal of this aspect of the invention could be genetically modified to include an antisense molecule or siRNA molecule that can affect the expression of UBAPl.

Antisense oligonucleotides are single-stranded nucleic acids, which can specifically bind to a complementary nucleic acid sequence. By binding to the appropriate target sequence, an RNA-RNA, a DNA-DNA, or RNA-DNA duplex is formed. These nucleic acids are often termed "antisense" because they are complementary to the sense or coding strand of the gene. Recently, formation of a triple helix has proven possible where the oligonucleotide is bound to a DNA duplex. It was found that oligonucleotides could recognise sequences in the major groove of the DNA double helix. A triple helix was formed thereby. This suggests that it is possible to synthesise sequence-specific molecules which specifically bind double-stranded DNA via appropriate formation of major groove hydrogen bonds.

By binding to the target nucleic acid, the above oligonucleotides can inhibit the function of the target nucleic acid. This could, for example, be a result of blocking the transcription, processing, poly(A)addition, replication, translation, or promoting inhibitory mechanisms of the cells, such as promoting RNA degradations.

By "antisense" we also include all methods of RNA interference, which are regarded for the purposes of this invention as a type of antisense technology.

A further method of generating a non-human animal of this aspect of the invention is wherein the animal is genetically modified so as to have a ribozyme capable of cleaving RNA or DNA encoding UBAPl polypeptide.

A further method of generating a non-human animal of this aspect of the invention is wherein the animal is genetically modified so as to have an agent that acts as antagonist to UBAPl amount and/or function. The term "antagonist" is well known to those skilled in the art. By "antagonist" we include in this definition any agent that acts to alter the level and/or functional ability of UBAPl polypeptide. An example of an antagonist would include a chemical ligand that binds to and affects UBAPl function, and in broader terms this could also include an antibody, or antibody fragment, that binds to one of the said polypeptides such that the polypeptide cannot effect its normal function. The antagonist may also alter the subcellular localisation of UBAPl polypeptide. In this way, the amount of functional polypeptide is reduced.

A further method of generating a non-human animal of this aspect of the invention is wherein the animal is genetically modified so as to have a dominant inactive form of a UBAPl polypeptide.

To date human UBAPl polypeptides and nucleotide sequences have been characterized and are set out above. By "UBAPl polypeptides" we include the human and mouse UBAPl polypeptides as well as further homologues, orthologues or paralogies of UBAPl polypeptides.

Further UBAPl polypeptides are disclosed in GenBank. Methods by which homologues, orthologues or paralogues of polypeptides can be identified are well known to those skilled in the art: for example, in silico screening or database mining.

Preferably, such polypeptides have at least 40% sequence identity, preferably at least

60%, at least 70%, at least 80%, at least 90% or at least 95% sequence identity to the polypeptide sequence of UBAPl polypeptide. Methods of determining the percent sequence identity between two polypeptides are well known in the art.

The term "nucleic acid encoding UBAPl polypeptide" includes both DNA and RNA molecules, including mRNA. By encode we mean that the sequence of bases in the nucleic acid molecule is such that, on transcription and/or translation, it encodes a polypeptide having the sequence of a UBAPl polypeptide.

The various elements required for a technician to perform the method of the seventh aspect of the invention may be incorporated in to a kit. Thus, according to an tenth aspect of the invention there is provided a kit for assessing whether a subject has or is likely to develop a dementia comprising means for determining whether the subject has a mutation in the UBAPl gene.

By "means for determining whether the subject has a mutation in the UBAPl gene" we include the molecules given in the seventh aspect of the invention that can be used to determine whether the subject has a mutation in the UBAPl gene. Preferably the molecule is an oligonucleotide probe or antibody.

The kit of the tenth aspect of the invention may also comprise relevant buffers and regents for conducting such methods.

The buffers and regents provided with the kit may be in liquid form and preferably provided as pre-measured aliquots. Alternatively, the buffers and regents may be in concentrated (or even powder form) for dilution.

The various elements required for a technician to perform the method of the eighth aspect of the invention may be incorporated in to a kit.

Thus, according to an eleventh aspect of the invention there is provided a kit for assessing whether a subject has or is likely to develop a dementia comprising means for determining whether the subject has an altered amount of UBAPl polypeptide and/or nucleic acid encoding UBAPl polypeptide.

By "means for determining whether the subject has an altered amount of UBAPl polypeptide and/or nucleic acid encoding UBAPl polypeptide" we include the molecules given in the eighth aspect of the invention that can be used to determine the amount of UBAPl polypeptide and/or nucleic acid encoding UBAPl polypeptide. Preferably the molecule is an oligonucleotide probe or antibody.

The kit of the eleventh aspect of the invention may also comprise relevant buffers and regents for conducting such methods. The buffers and regents provided with the kit may be in liquid form and preferably provided as pre-measured aliquots. Alternatively, the buffers and regents may be in concentrated (or even powder form) for dilution.

A further aspect of the invention provides an isolated polypeptide having the amino acid sequence:

MASKKLGADFHGTFSYLDDVPFKTGDKFKTPAKVGLPIGFSLPDCLQWREVQYDFSLE KKTIEWAEEIKKIEEAEREAECKIAEAEAKVNSKSGLEGDSKMSFSKTHSTATMPPPIN PILASLQHNSILTPTRVSSSATKQKVLSPPHIKADFNLADFECEEDPFDNLELKTIDEK EELRNILVGTTGPIMAQLLDNNLPRGGSGSVLQDEEVLASLERATLDFKPLHKPNGFIT LPQLGNCEKMSLSSKVSLPPIPAVSNIKSLSFPKLDSDDSNQKTAKLASTFHSTSCLRN GTFQNSLKPSTQSSASELNGHHTLGLSALNLDSGTEMPALTSSQMPSLSVLSVCTEESS PPNTGPTVTPPNFSVSQVPNMPSCPQAYSELQMLSPSERQCVETWNMGYSYECVLRAM KKKGENIEQILDYLFAHGQLCEKGFDPLLVEEALEMHQCSEEKMMEFLQLMSKFKEMGF ELKDIKEVLLLHNNDQDNALEDLMARAGAS

The polypeptide of this aspect of the invention corresponds to the UBAPl polypeptide having a P96L mutation. The invention also includes a nucleic acid molecule encoding the polypeptide of this aspect of the invention. Such a polypeptide or nucleic acid molecule would have utility, for example, in preparing agents of use in the diagnostic methods of the invention.

A further aspect of the invention provides an isolated polypeptide having the amino acid sequence:

MASKKLGADFHGTFSYLDDVPFKTGDKFKTPAKVGLPIGFSLPDCLQWREVQYDFSLE KKTIEWAEEIKKIEEAEREAECKIAEAKAKVNSKSGPEGDSKMSFSKTHSTATMPPPIN PILASLQHNSILTPTRVSSSATKQKVLSPPHIKADFNLADFECEEDPFDNLELKTIDEK EELRNILVGTTGPIMAQLLDNNLPRGGSGSVLQDEEVLASLERATLDFKPLHKPNGFIT LPQLGNCEKMSLSSKVSLPPIPAVSNIKSLSFPKLDSDDSNQKTAKLASTFHSTSCLRN GTFQNSLKPSTQSSASELNGHHTLGLSALNLDSGTEMPALTSSQMPSLSVLSVCTEESS PPNTGPTVTPPNFSVSQVPNMPSCPQAYSELQMLSPSERQCΓVETVVNMGYSYECVLRAM KKKGENIEQILDYLFAHGQLCEKGFDPLLVEEALEMHQCSEEKMMEFLQLMSKFKEMGF ELKDIKEVLLLHNNDQDNALEDLMARAGAS

The polypeptide of this aspect of the invention corresponds to the UBAPl polypeptide having a E87K mutation. The invention also includes a nucleic acid molecule encoding the polypeptide of this aspect of the invention. Such a polypeptide or nucleic acid molecule would have utility, for example, in preparing agents of use in the diagnostic methods of the invention.

A further aspect of the invention provides an isolated polypeptide having the amino acid sequence:

MASKKLGADFHGTFSYLDDVPFKTGDKFKTPAKVGLPIGFSLPDCLQWREVQYDFSLE KKTIEWAEEIKKIEEAEREAECKIAEAEAKVNSKSGPEGDSKMSFSKTHSTATMPPPIN PILASLQHNSILTPTRVSSSATKQKVLSPPHIKADFNLADFECEEDPFDNLELKTIDEK EELRNILVGTTGPIMAQLLDNNLPRGGSGSVLQDEEVLASLERATLDFKPLHKPNGFIT LPQLGNCEKMSLSSKVSLPPIPAVSNIKSLSFPKLDSDDSNQKTAKLASTFHSTSCLRN GTFQNSLKPSTQSSASELNGHHTLGLSALNLDSGTEMPALTSSQMPSLSVLSVCTEESS PPNTGPTVTPPNFSVSQVPNMPSCPQAYSELQMLSPASGSVWRRWSTWATRTSVSSEP

The polypeptide of this aspect of the invention corresponds to the UBAPl polypeptide having a M413X (S391Afs2 IX) mutation. The invention also includes a nucleic acid molecule encoding the polypeptide of this aspect of the invention. Such a polypeptide or nucleic acid molecule would have utility, for example, in preparing agents of use in the diagnostic methods of the invention.

Methods of preparing polypeptide and nucleic acid sequence of the above aspects of the invention are routine in the art.

All of the features described herein (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined with any of the above aspects in any combination, except combinations where at least some of such features and/or steps are mutually exclusive. The invention will now be further described with reference to the following Example and figures in which:

Figure 1: Sliding Window Haplotype analysis for the combined Dutch and Manchester UBAPl data. Green line corresponds to a p value of 0.05.

Figure 2: Confocal images of neuronal staining of tau-2 (a) and UBAPl (b) and colocalisation (d) in hippocampus in FTLD.

Figure 3: Immunoprecipitation of UBAPl pulling down. Progranulin (arrow). A - total protein input; B - no antibody IP control; C - IP of progranulin.

Figure 4: Immunoprecipitation oftau using UBAPl antibody..

Figure 5: Colocalisation oftau and UBAPl in the parenchyma of neurons in FTLD

Figure 6: Sliding window analysis with a maximal global P value of 0.0002 using a sliding window size of 7.

Figure 7. Immunohistochemistry and western blotting

Figure 8: UBAPl expression levels

Example 1: Genetic variation of the gene Ubiquitin Associated protein 1 increases the risk of developing frontotemporal lobar degeneration or other neurodegenerative diseases.

Using a linkage disequilibium approach we genotyped one SNP per every haplotype block of all genes in the linkage region on chromosome 9p21. Genotyping was performed with the Sequenom MassArray system

(http://www.sequenom.com/seq_svstems.html#analvzer). 151 SNPs were analysed using Haploscore (Schaid, D.J., et al.. Am J Hum Genet, 2002. 70(2): p. 425-34) and plots of P values versus chromosomal position identified three gene regions (RECK, TEK and UBAPl) where P values consistently reached =0.01 for multiple window sizes. Using the tagger program (de Bakker et al, supra) additional SNPs were genotyped to cover all known haplotype blocks of these 3 genes. Analysis revealed that while both RECK and TEK failed to show an association for other SNPs, UBAPl (Ubiquitin Associated Protein 1) SNPs/haplotypes were consistently associated with a maximum sliding window P value of 0.00025. Of the 10 SNPs in the UBAPl haplotype, 7 were found to be associated with FTLD with a P value of =0.05, with the haplotype block demonstrating maximum OR of 1.71 (95% CI 1.20-2.44, P=0.003).

We successfully replicated this finding with a Dutch FTLD cohort cases (N=230, controls N=484) with a maximum haplotype association of OR 1.39 (95% CI 1.02-1.89, P= 0.033). The combined analysis resulted in a haplotype association of P=0.014, with a maximum OR of 1.42 (95% CI 1.12-1.80, P=0.003).

UBAPl, originally cloned from a tumour suppressor locus (Qian et al, supra), has two Ubiquitin-associated domains (UBA), between residues 389-430, and 451-498, and an Ubiquitin System Cue domain between residues 459-499 and is likely involved in the binding of ubiquitin-conjugating enzymes. The UBA domains are found in various proteins involved in the ubiquitin/proteosome pathway and is therefore an excellent candidate gene for FTLD. Sequence analysis of the open reading frames has identified 3 mutations (P96L, E87K, M413X, all in fully conserved residues in other species), however, we do not have access to other family members to demonstrate segregation but these are absent in 280 controls.

Without definitively linked chr9p families to analyse we cannot be absolutely sure UBAPl is the gene responsible for the linkage to this region (Morita et al, supra; Vance et al, supra), however, our data confirms UBAPl is a risk factor for FTLD in two different populations. It is even conceivable there are 2 genes for FTLD on chr9p similarly to MAPT and PGRN on chrlq21. Furthermore, polyclonal antibodies we have made demonstrates that UBAPl staining is present in neurons and astrocytes in grey and white matter, and appear as discrete granules or larger globules usually clustered into larger conglomerates within the perikaryon (Fig 2). Intriguingly, these same structures are also immunoreactive to the phospho-specific anti-tau antibody Tau-2 (Fig 2) and Cyclin Tl a known PGRN binding protein (Hoque, M., et al.,. MoI Cell Biol, 2003. 23(5): p. 1688-702). Furthermore, we have demonstrated we can immunoprecipitate progranulin with UBAPl suggesting these 2 proteins interact in a functional complex (Fig 3).

Example 2: Genetic variation of the gene Ubiquitin Associated protein 1 increases the risk of developing Alzheimer's disease.

Using the same experimental techniques as set out in Example 1 above, we investigated whether there was a linkage between the UBAPl gene and Alzheimer's disease. Analysis revealed that 4 UBAPl SNPs were associated with Alzheimer's disease is a cohort of 360 patients: rslO971977, rsl2375731, rs2380925 and rsl0972030 are associated with Alzheimer's disease. The statistical data demonstrating this linkage is shown in the tables presented at the end of the example section of the description. From this information the inventors have concluded that UBAPl is also associated with the likelihood of developing Alzheimer' s disease.

Example 3: UBAPl association with tau protein.

Immunoprecipitation of Tau using UBAPl antibody

Cells were lysed by incubating on ice with 200-500μl ice cold RIPA buffer (I x PBS, 1% NP-40, 0.5% Deoxycholate, 0.1% SDS) with added PMSF (1:200 dilution of stock) for 30 minutes to lhr on ice followed by a spin at 15000 rpm for 5 mins at 4 ⁰C to pellet debris. The supernatant (Total cell lysate) was removed and stored on ice or in the freezer. Lysate was incubated at 4 ⁰C for 1 hr or overnight on rotary shaker with lOμl polyclonal UBAPl antibody. 50μl protein G beads slurry was added to the cell lysate/antibody mixture and incubated at 4 ⁰C for 1-2 hours on rotary shaker. Beads were washed 5 times with ImI ice-cold RIPA buffer and centrifuged at 200Og for 1 min at 4ύC each wash. Beads were then boiled in SDS sample buffer to elute the protein which were then analysed by Western blot. The results of this experiment are shown in Figure 4.

It can be seen from Figure 4 that the UBAPl antibody can immunoprecipitate cellular tau protein. Colocalisation oftau and UBAPl

4μM brain sections from subjects with FTLD were dewaxed with xylene then rehydrated through ethanols and rinsed in distilled water. Antigen retrieval was performed by microwaving sections for 20 minutes in 1OmM Sodium citrate buffer and allowing to cool. Sections were rinsed twice with PBS then permeablized by incubating for 20 mins in 0.2% Triton-X100. After 3 washes with PBS, non-specific epitopes were blocked by incubating in normal serum for 20 mins. Sections were then incubated in primary antibodies (rabbit anti-UBAPl-359 (diluted 1:200 in PBS); mouse anti-Tau2 (diluted 1:2000 in PBS)) overnight at 4 ⁰C then rinsed 3 times with PBS containing 0.01% Tween-20 (PBS-TT). Sections were incubated in secondary antibodies (Alexa- 488 conjugated goat anti-mouse antibody; Alexa-546 conjugated goat anti-rabbit antibody (diluted 1:200 in PBS)) for lhr at RT then rinsed 3 times with PBS-TT and mounted in Vectashield^a fluorescent mounting medium (Vectorlabs, UK) and coveslips sealed. Fluorescence was imaged and processed using excitation with argon (488nm) and green helium neon (543nm) lasers and sequential line-scanned to prevent fluorophore bleedthrough with a Leica DM IRE2 inverted laser scanning confocal microscope and LCS software (Leica Microsystems, UK). The results of this experiment are shown in Figure 5.

It can be seen from Figure 5 that the UBAPl and tau proteins are colocalised in the parenchyma of neurons from FTLD-affected patients.

Example 4: Ubiquitin Associated Protein 1 is a risk factor for frontotemporal lobar

degeneration

INTRODUCTION

Frontotemporal lobar degeneration (FTLD) is the term used for the clinical syndrome characterized by changes in behaviour, personality and language with relative preservation of memory and perception¹. FTLD is a common form of dementia in individuals under the age of 65 and around half of all patients present with a family history of a similar disease . The genetic aetiology of FTLD is complex with 7 loci identified to date on chromosomes 3, 9p (two loci), 9q, 17q,21 (two locii) and 17q24 ³. Four of the genes that account for these linkages have been identified being CHMP2B on chromosome 3, VCP on chromosome 9p and MAPT and progranulin on chromosome 17q21 and collectively these account for 10-20% of FTLD ^4"8. There are two common neuropathological subgroups observed in FTLD being tauopathy, the accumulation of abnormally phosphorylated and insoluble aggregates of tau protein in neurons and sometimes glia, the other is referred to as FTLD-U where neuronal inclusions containing TAR DNA binding protein (TDP-43) are present in varying quantities ⁹. FTLD-U has been further divided into three main subgroups determined by the distribution of the TDP-43 pathology ⁹'¹⁰. Familial tauopathy is often associated with mutations in MAPT whereas null-mutations of progranulin have been shown to lead to FTLD-U ⁶'⁷. Cases with mutations in progranulin have been designated type 3 FTLD-U whereas those cases from families linked to chromosome 9p have been identified as having type 2 pathology ⁹. One characteristic that unites the two differing histological subtypes in FTLD is that both tau and TDP-43 inclusions are ubiquitinated to varying degrees. The observation that many proteins that accumulate within neurons and/or glia in neurodegenerative diseases and are ubiquitinated has lead to the suggestion that dysfunction of the ubiquitin proteosome system could be an aetiological factor in this group of conditions ^π.

The identification of the remaining unknown genes will help elucidate the important aberrant biological pathways underlying FTLD. Therefore, in order to investigate whether we could identify any evidence of genetic association between genes in the chromosome 9p linkage region in our FTLD cohort from the North West of Great Britain we undertook a large scale linkage disequilibrium mapping approach of the minimal region defined from published families ¹²'¹³.

Subjects

Manchester cohort: Genomic DNA was available from 259 FTLD patients (median age at onset 60 years, range 23 to 83) fulfilling diagnostic criteria for FTLD (ref). The series comprised of 139 men (median age 60 years, range 35 to 79), and 120 women (median age years, range 23 to 83). All patients were recruited between 1987 and 2007 through longitudinal neuropsychological and clinical assessment within the Cerebral Function Unit of the University of Manchester. All patients known to harbour a mutation in MAPT or PGRN genes were excluded, as were those shown to display tau pathology (i.e. neurofibrillary tangles or Pick bodies) at postmortem. The final study group comprised of 214 patients (mean age at onset 59.6 years, range 23 to 83). The series comprised of 116 men (mean age at onset 59.1 years, range 35 to 79), and 98 women (mean age at onset 60.1 years, range 23 to 83). By the time of this study 44 patients had come to postmortem. In all, 119 patients (64 men and 55 women, mean age at onset 58.0 years (standard deviation 9.7 years), range 23 to 82) were diagnosed clinically with frontotemporal dementia (FTD). Of the remainder, 33 patients (20 men, 13 women; mean age at onset 60.1 years (7.7 years), range 43 to 74) were diagnosed with FTD and motor neurone disease (MND). 35 patients were diagnosed with semantic dementia (SD) (16 men, 19 women; mean age at onset 59.6 years (6.6), range 47 to 72), 22 with progressive non-fluent aphasia (PNFA) (12 men, 10 women; mean age at onset 70.0 (7.3) years, range 51 to 77), 3 with progressive apraxia (PAX) (2 men, 1 women; mean age at onset 69.6 (14.0) years, range 55 to 83 years) and two male patients with PNFA and MND (mean age at onset 78.5 years (range 78 and 79 years). Control data were drawn from a cohort of 286 mentally normal people (at the time of sample acquisition). All samples were of UK origin, and were recruited with Ethical Committee approval, and provided informed consent. Dutch cohort: 214 patients with FTLD comprising of 118 females and 96 males with a mean age at onset 57.9 +/- 9.0 (30-76) and a disease duration of illness 8.3 +/- 3.9 with age at death being 65.0+/- 9.6 where known. 138 of these had a clinical diagnosis of FTD, 38 had semantic dementia, 23 progressive non-fluent aphasia and 15 had FTD + MND. Dutch controls consisted of 149 males and 149 females with an average age of 61.06 +/-2.89 years at the time of collection.

SNP selection

First Round Screening Using the linkage data of Morita et al, 2006 ¹⁴ and Vance et al, 2006 ¹⁵ a 10.26Mb region on chromosome 9p between markers D9S2154 and D9S1874 was identified as being a possible locus containing a gene risk factor for FTLD and MND. To map the region using case-control linkage disequilibrium analysis a total of 190 frequency validated SNPs were selected from public databases including the NCBI SNP databases (www.ncbi.nlm.nih.gov/SNP), Ensembl (www.ensembl.org) and HapMap (phase 2.0 data) (www.hapmap.org - CEPH population), using a gene centric approach. A total of 133 genes (as of genome build 35) were found in the region, Linkage disequilibrium (LD) maps were obtained from HapMap, where multiple genes fell within a single haplotype block, tag SNP 's were selected to reduce the total number of SNPs required for genotyping using the tagger application ¹⁶. A minimum of one SNP per gene or two SNPs per haplotype block were prioritized to give as complete coverage as possible of the region. Any SNPs also in coding regions or with a potential biological function were prioritized for genotyping selection, as were SNPs with a minor allele frequency of 0.2 or greater.

Second Round Screening

By analysis of individual markers and sliding window haplotype analysis three regions were chosen for further linkage study. As before SNPs were chosen using the tagger application to identify tag SNPs that capture the majority of variation across the candidate region. Where a gene fell across multiple haplotype blocks snps were chosen that spanned both blocks, additionally 5kb up- and downstream of the gene was also included in the screening area so as to increase the likelihood of genotyping any putative regulatory regions.

Genotyping Analysis

DNA samples were randomly assigned across 2 genotyping plates with 5 cell line samples duplicated across the plates, and an additional 7 duplicate samples on each plate to test for genotyping consistency. 15ng of each DNA was genotyped using the Sequenom MassArray genotyping technology according to manufactures instructions.

Genotyping quality control for each SNP assay included concordance of genotypes between replicate samples both internally per genotyping plate and externally between plates. An equal call of genotypes had to be observed in both the case and control groups of samples (the average ration of genotyping calls between the cases and controls being 1.002), with a minimum of 75% samples with an assigned genotype. All SNPs were required to be in Hardy- Weinberg in the control population with a minimum P value of 0.05, while cases could be out of equilibrium for inclusion into the study, no SNP was out of equilibrium in the cases and not in the controls.

Statistical analysis Simple χ² and Hardy- Weinberg Equilibrium P values for each SNP variant were calculated using Haploview ¹⁷. Haplotype block structure was examined using haploview ^1?, a block defined using the confidence intervals option, where 95% confidence bounds on D' are generated, a block defined if 95% of informative comparisons are in strong linkage disequilibrium . For comparison with the generated data, data corresponding to genomic positions 26856230 to 37152282 on chromosome 9 were downloaded from the international haplotype mapping project (http://www.hapmap.org/). In order to correspond with the samples genotyped in the present study only CEPH samples were analyzed. The CEPH samples comprise of Utah (USA) residents with ancestry from northern and western Europe. For multiple testing correction the false discovery rate as implemented using the Qvalue algorithm run through R was implemented ¹⁹. To reconstruct haplotypes from genotype data Phase version 2.1 was used ²⁰^₅ using 10,000 iterations and a burn in of 10,000. For single variant and haplotype odds ratios (OR) and 95% confidence intervals (95% CI) were estimated using un-conditional logistic regression adjusting for age and sex using Stata (version 9), using the most common variant/haplotype as the baseline. CLUMP ²² was used to assess the χ2 significance of the haplotypes between groups. Haplo.Stats (version 1.2.2) was used to carry out sliding-window haplotype analysis, used to estimate haplotype effects under the generalized linear model (http://mayoresearch.mayo.edu/mayo/research/biostat/schaid.cfm).

Sequencing

Sequencing was performed using standard PCR methods using BigDye 3.1 and a

AB 13730 sequencer.

Antibodies and Western Blotting.

An affinity purified polyclonal rabbit anti-UBAPl peptide antibody (UBAP 1-359) was generated by Eurogentec raised against peptides matching the C-terminal coding sequence of UBAPl (CQDNALEDLMARAGAS). Also, rabbit polyclonal UBAPl antibody from Proteintech was used. For TDP-43 immunohistochemistry mouse anti- TARDBP monoclonal antibody (Clone MOl; Abnova) was employed.. Alexa-488 conjugated goat anti-mouse and Alexa-546 conjugated goat anti-rabbit secondary antibodies were obtained from Invitrogen Molecular Probes, UK. Insoluble protein fractions from human brain were generated using the TDP-43 protocol² .

Immunohistochemistry

4μM sections were dewaxed with xylene then rehydrated through ethanols and rinsed in distilled water. Antigen retrieval was performed by microwaving sections for 20 minutes in 1OmM Sodium citrate buffer and allowing to cool. Sections were rinsed twice with PBS then permeablized by incubating for 20 mins in 0.2% Triton-X100. After 3 washes with PBS, non-specific epitopes were blocked by incubating in normal serum for 20 mins. Sections were then incubated in primary antibodies (rabbit anti- UBAP1-359 (diluted 1 :200 in PBS); mouse anti-TDP-43 (diluted 1 :2000 in PBS)) overnight at 4°C then rinsed 3 times with PBS containing 0.01% Tween-20 (PBS-TT). Sections were incubated in secondary antibodies (Alexa-488 conjugated goat anti- mouse antibody; Alexa-546 conjugated goat anti-rabbit antibody (diluted 1 :200 in PBS)) for lhr at RT then rinsed 3 times with PBS-TT and mounted in Vectashield™ fluorescent mounting medium (Vectorlabs, UK) and coveslips sealed. Fluorescence was imaged and processed using excitation with argon (488nm) and green helium neon (543nm) lasers and sequential line-scanned to prevent fluorophore bleedthrough with a Leica DM IRE2 inverted laser scanning confocal microscope and LCS software (Leica Microsystems, UK).

RESULTS

First Round screening

A total of 190 SNP 's were genotyped across the 10.296 Mbp region of 9p determined by linkage analysis to be associated with disease. After dropping those assays that did not meet the stringent quality control measures 151 SNPs were left for analysis, possessing an average genotype call of 92.43% (76.3-98.6% call). On examination of P values calculated using a case-control analysis, 10 P values were found to be significant (P = <0.05). None of these P values remained significant after correction for multiple testing (using the false discovery rate) (data not shown). This is likely in part due to the relatively small number of cases available for analysis, combined with the large number of tests carried out, and as such will not all be false positive results. To assess the effect of these SNPs, haplotype associations were calculated using haploview. 7 regions displayed strong linkage with a haplotype combination associated with FTLD (P = <0.05), associated with the genes TEK, LRRN6C, DDX58, UBAPl, OR2S2, RECK and PAX5. To assess the haplotype association further we preformed a sliding window analysis of the genomic regions, including 2-7 snps in each block. We observed the strongest effect when we included markers rs4574933 (UBAPl) (global P value of = 0.0005), rs664513 (TEK) P = 0.02, and rsl 359885 (RECK) P = 0.0052, whereas the other markers (and associated regions of linkage) failed to produce significance upon sliding window analysis.

Second Round Screening

Using the hapmap and tagger programs an additional 50 SNPs were chosen to examine the haplotype structure of the genomic regions spanning RECK (N = 5, 36026906 - 36114448bp), TEK (N = 31, 27099441 -27220165bp) and UBAPl (N = 14, 34169011 - 34242521 bp). Haplotype analysis both excluding and including the first round SNP assays was carried out using both haploview and haploscore. Upon analysis the haplotype block structure of both TEK and RECK showed concordance with that seen in the CEPH HAPMAP data, however neither gene displayed a haplotype that was associated with a significant effect for FTLD (data not shown), hi contrast, in addition to the first round rs4574933 SNP, and additional 6 SNPs were found to have an allele association with FTLD (table 1). Sliding window analysis confirmed this with a maximal global P value of 0.0002 using a sliding window size of 7 (figure 6). This 7 SNP haplotype includes 6 of the 7 significant SNP assays (rslO971977 to rsl0814083), corresponding to the genomic region that contains and surrounds the UBAPl coding sequence. Haploview analysis confirmed that the haplotype structure around UBAPl agreed with that from by the CEPH hapmap data, with a 10 SNP haplotype block that spans all of the UBAPl genomic sequence (Fig 6). Indeed inheritance of the TATTAGATGC block was associated with an increased risk of FTLD of OR 1.71 (95% CI 1.20 - 2.44) in the Manchester cohort.

In order to evaluate whether UBAPl also conferred disease risk for FTLD in separate independent cohorts and to evaluate whether the effect we observed was a type 1 error we genotyped the second round SNPs in two additional independent FTLD cohorts from The Netherlands and from the USA. Significant association was observed for multiple SNPs in both of these cohorts. Importantly, the same 2 SNPs (rs7018487 and rs 10814079) were consistently significantly associated with FTLD across all three populations (Table 2). In addition, haplotype analysis also identified significantly associated haplotypes (OR 1.39 95%CI 1.02-1.89, P=0.033 to 1.71 95%CI 1.2-2.4, P=0.003) in all three popultations (Table 3). This provides strong evidence that common genetic variation at the UBAPl locus confers risk for FTLD.

UBAPl sequence analysis The entire open reading frame of UBAPl was sequenced on both the Manchester and Dutch cohorts. Five variants were identified in separate familial cases (E87K, P96L, S391Afs21X and H149Q in the Manchester samples and a P256L in the Dutch cohort). Four of these were absent from 450 controls and are possibly mutations, however, the H149Q was found in controls (frequency X) and is therefore likely to be a polymorphism. Unfortunately, samples from other family members were unavailable to test for segregation. Nevertheless, the E87K, P96L, P256L and S391Afs21X are all in fully conserved regions of UBAPl (data not shown) suggesting functional significance.

Immunohistochemistry and western blotting We stained 40 cases of FTLD with the 2 different UBAPl antibodies and both produced identical results. UBAPl immunoreactivity was present in pyramidal and non-pyramidal neurons throughout the cerebral cortex in the form of small granules or larger globules or a mixture of both. These were evenly scattered throughout the perikaryon and showed no focal accumulation at any particular region of their neurons. Occasional glial cells, probably astrocytes, also showed similar but smaller accumulations of UBAP-I immunoreactivity. In all but one case the UBAPl failed to stain any pathological inclusions i.e. TDP-43 cytoplasmic or intranulcear inclusions, tangles or Pick bodies. However, we did observed robust staining of TDP-43 positive cytoplasmic inclusions dystrophinc neurites with both UBAPl antibodies in a single familial case (Fig 7). We were able to demonstrate colocalisation of TDP-43 and UBAPl in these pathological structures using double labelling immunoflorescence microscopy (Fig 7).

Expression analysis Analysis of the levels of UBAPl mRNA in frontal cortex and cerebellum in FTLD-U from the Manchester Brain Bank revealed that the two disease associated haplotypes produced significantly (p value) less mRNA than the reference haplotype (Fig 8).

DISCUSSION

We have undertaken a large scale linkage disequilibrium mapping study of the published minimal linkage region for FTLD linked to chromosome 9p using our large FTLD cohort from the North West of Great Britain. This analysis has identified UBAPl as a significant risk factor for FTLD. Genetic association studies of human disease are often complicated by type 1 errors, i.e. false positive association, and are fraught with poor replication. However, we have successfully replicated this finding in an additional two independent unrelated cohorts from the Netherlands and the USA and this argues against this association being a type 1 error. Sequence analysis of the open reading frame of UBAPl has identified several putative mutations at fully conserved residues, however, samples from other family members were not available to demonstrate segregation. As these patients are all currently living we are unable to determine the precise histological changes, i.e. TDP-43 or tauopathy, are associated with these mutations. Nevertheless, due to the small number of individuals with these mutations it is clear that the main pathogenic variant(s) driving the observed association has yet to be identified.

Analysis of UBAPl mRNA identifies disease associated haplotypes with significantly lower levels of expression compared to the reference haplotype. That this is observed in frontal lobe which is affected by pathology in FLTD and cerebellum, which is free from neuropathology, argues this not just a product of the neurodegenerative process. These data strongly suggest that the main variant that increases of FTLD risk operates by lowering UBAPl expression leading to loss/reduced function. This hypothesis is consistent with one of the putative mutations we have identified (S391Afs21X) which removes both of the ubiquitin associated domains of UBAPl and this presumably has a negative effect on the function of this protein.

UBAPl encodes a protein of 502 residues, predicted to have a molecular weight of 55KDa and was originally cloned from a tumour suppressor locus ²⁴. While little is known of the actual function of the protein, the gene is likely a member of the Ubiquitin-activated enzymes family whose members include proteins having connections to ubiquitin and the ubiquitination pathway. The protein itself has two Ubiquitin-associated domains (UBA), between residues 389-430, and 451-498, and an Ubiquitin System Cue domain between residues 459-499, believed to be involved in the binding of ubiquitin-conjugating enzymes. The UBA domains are found in various proteins, including p62 which is found in certain TDP-43 positive inclusions in FTLD ²⁵, and are involved numerous processes including the ubiquitin/proteosome pathway, growth control, receptor function, stress responses, DNA excision-repair and cell signalling via protein kinases ²⁶. It is believed that the ubiquitin proteomsome system (UPS) plays a vital role in protecting the CNS from the accumulation of toxic proteins ^u . Furthermore, it has been demonstrated that mutations of certain genes involved in the UPS can lead to Parkinson's disease and clearly link a dysfunctional UPS to neurodegeneration ²⁷. Therefore UBAPl is an excellent candidate gene for FTLD.

The identification that UBAPl and TDP-43 proteins co-localise in together in neuronal cytoplasmic inclusions in a case of familial FTLD is important as it directly implicates UBAPl in the metabolism of TDP-43. It also suggests that UBAPl related FTLD will likely be of the FTLD-U neuropathological subtype, it is a very common feature that the protein products of genetic risk factors for neurodegenerative disease are found in the cytoplasmic pathological inclusions of these diseases and our current data are supportive of this hypothesis in at least some cases of FTLD . The family with UBAPl positive neuropathology is the same family recently reported to have a mutation in the IFT74 gene²⁹. Given the lack of replication of mutations in this gene it is now thought unlikely that IFT74 is the gene responsible for linkage to this region and that the original variant is not pathogenic²⁹'³⁰. Furthermore, the presence of UBAPl in the neuropathological lesions in the case argues for a functional role of UBAPl in the pathogenesis of disease in this individual. Nevertheless, sequence analysis of the open reading frame of UBAPl in a disease haplotype carrying affected member from this family has failed to identify any variation so far. However, this analysis does not exclude pathological copy number or regulatory sequence variants in this pedigree. Our expression data suggests a reduction in expression being aetiologically relevant and therefore we are investigating such regions for variation. The identification of UBAPl as a risk factor for FTLD is an important discovery because for the first time it provides evidence for a link between the UPS and this group of conditions. This observation, therefore, suggests the UPS is a potential future therapeutic target for FTLD. It will be interesting to establish whether UBAPl mutations lead to a TDP-43 or tauopathy based histology and to establish whether this protein has a wider role in neurodegenerative disease. Moreover, it will be important to investigate UBAPl in further populations and in families with FTLD definitively linked to chr9p. Finally, if pathogenic variations are absent these latter families this does not refute our finding of UBAPl being an independent risk factor for FTLD in this region. If this scenario was true it would be analogous to situation on chromosome 17 where the two genes, MAPT and PGRN, causing FTLD are only 1.7Mb apart '⁷.

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Tables associated with this example Table 1

Table 2

MANCHESTER DUTCH MAYO

Major Allele Major Allele Major Allele major frequency major frequency major frequency allele (Case. allele (Case, allele (Case,

(Controls) Control) ChI 2 (Controls) Control) Chi 2 (Controls) Control) Chi 2

IS2275003 0 SOS, 0526 0345 0557 0504, 0528 059 0442 0481, 0519 0618 0431 rs4879753 0670, 0648 0394 0530 0638, 0654 0245 062 0647, 0644 0997 nil 3283064 0674, 0756 7049 0.008 0728, 0744 0392 0531 0767, 0698 291 0088 rs10971969 0819.0844 0753 0386 0910. 0831 0768 0838, 0835 0996 re 701S4B7 0 726, 0658 4268 0.039 0701, 0637 0.023 0624, 0 704 4656 0031 rs10971977 0475.0597 13297 0.000 0535, 0566 0989 0319 0611, 0526 3493 0061

IS12375731 0508, 0596 6225 0.013 0558, 0578 0414 0519 0592, 0547 1 267 026 rs10814079 0734, 0654 6291 0.012 0697, 0637 4613 0.031 0636, 0733 5304 0.021 rs2380925 0487, 0600 10926 0.001 0536, 0567 0315 0590, 0547 1 124 0289 nil 7258783 0746, 0800 3497 0062 0797, 0798 0001 0977 0828, 0771 2379 0 123

IS4574933 0700, 0792 10358 0.001 0790, 0793 0001 09751 0813, 0775 1 325 0249 rs10814083 0728, 0674 2637 0104 0705, 0639 5479 0019 0626, 0703 4294 0 038 rs10972030 0654, 0582 4646 0.031 0627, 0621 0017 0895 0565, 0621 1 469 0225 rs17350373 0547, 0579 0808 0369 0561, 0563 0003 0957 0595.0560 0536 0464 rs11788425 0527, 0560 0778 0377 0556, 0564 0059 0808 0572, 0550 0262 0609

1S12377 0976, 0965 0725 0394 0993.0970 5762 0.016 0954, 0981 3164 0075

Three populations Two population One population Trend Table 3

Manchester Cohort

Controls Cases OR 95% Cl P haptotype N % N %

CAGAGCGCAT 190 34.55 112 27.52 1

CATAGGGCA C 135 24.55 84 20.64 1.05 0.73-1.51 0.768

TATTAGATGC 107 19.45 108 26.54 1.71 1.20-2.44 0.003

CGTTAGACA C 88 16 80 19.66 1.54 1.05-2.26 0.026

TATTAGACAC 30 5.45 23 5.65 1.3 0.72-2.34 0.384

OTHER 22 21

Mayo

Cohort

Controls Cases OR 95% Cl P

N % N %

CAGAGCGCAT 107 29.4 130 36.93 1.41 1.04-1.94 0.028

Dutch

Cohort

Controls Cases OR 95% Cl P

N % N %

CATAGGGCA C 196 20.48 108 23.79 1.39 1.02-1.89 0.033

Genomic DNA sequence of UBAPl i aaatgagtgg ggcggtgagg ggaaggagga gggaagtagg acttcaacat ggcggctgcg

61 gcactggcgg tggctacggt gacggcctgg cccggagcgg gcagagttgg aggtggtggc

121 gttcgctctc cctaggggct gtcgggagct cagcggggac cgagcctggg aggccggccg

181 gtgccagcac ctttcggctt ctgagacggc ggcagcagcg gcattcaggt gaggggggcc

241 tccctctggg ggagggggaa gaggggcgga gttgggggag gggggctggt actgggagac

301 tgggaaacgg gatggggggc cgagcgaggt gaagggcgcc ggagctagga ggtgggaggg

361 aaaagtggcc ggagatccgc gattcgggga ggattctggg agaagggtcg gagtgagggg

421 accggagttg aaggggcgga ggggaagtgg gatgcggaaa aatgagttcg gattaaggag

481 actaataccc tactggaaga caggcatagt agacatctat cgggctggac tgagaactcg

541 gtgggaacta gaggatgtca gggaggggga cccagatttt agtaaagaag gtgagtggtg

601 gggagttgag ggtgagctgg ggacgtgcgg tgtagattta gttctccctg aggcaatgag

661 attggggaga aaaggtggag actagaccag aggtgagtga tctttgggga tatctttggc

721 cttgtctggt cttctccagg tatgtttatc cgcctttttt gtttcttggg tggaggctta

781 cctatacagt ccacctgggc ctcaggaacc gccccagtct ttcttgtgga acgtcaggtt

841 catttatttg agctatatca gtgatctttg acctttgttg ccaaatgtaa ttgtttccag

901 actagcgtct taaaagtatg gttgggcata aaaatacgtt ttatgttaaa ctgaaaaaaa

961 aattgttact tttagtcacc atgcagtgtt gcgaaaccag atttggtaat ttagttaagt

1021 attacgtata ttctgttcta actttactcg ttagtgcttt gtctcttcag ttattagcat

1081 ttgagaattt gaatagatat gatggatgtt ttccttttta attttgtcag atttgatggt

1141 gggaaaaaca agatacttag tctttagtga tatatagttc tttctagtta atgtcccaac

1201 ctaaactaaa agtttccgta ttggccgggc gctgtggctc acgtctgaaa tcccagcact

1261 ttgggaggcc gaggcgggcg gatcacgagg tcaagagttc aagaccagcc tgaccaacat

1321 gttgaaaccc tttctctact aaaaagccag ccgggcgtgg tggcaggcgc ctgtaatccc

1381 agctactcgg gtggccgagg caggagaatg gcttgaacct gggaggcgga ggtagcagtg

1441 agcctagatc gctgcattgc actccagcct gggcaacaga gcgagactcc gactcaaaaa

1501 aaaaaaaaaa gtttacgtat tatggcaaag aatgtgaaat gtgtcaagtc ttaaaagaat

1561 tacattgtcc gcattaactt actataagtg aaattttatt aaatttttgt ttctagttct

1621 attaattcat ggtgtttatt aaatagctac tgtgtgttgc aataataaga gtcattataa

1681 ttacagaaat gctaaactta atgtttcttt agtgagtgac catcgctgtc attgcacagc

1741 agataacttt actaacctgc tagaacctca ttggaaaaaa aacttttttt tttttttgtt

1801 tctttttatt tttttccaga gggagtctcg gtctgttgcc caggctggag tgcagtggcg

1861 cgatcttggc ttgctgcaac ctccgcctcc tgggttcaag tgactgtcct gcctcagcct

1921 cccgagtagc tgagattata gacacccacc actacggctg gctaattttt gtattttagt

1981 agagacgggg tttcaccatg ttggtcaggc tggtctcgaa cttctgacct caaataatcc

2041 tccggccttg gtctccgaaa gtgctgggat tacaggcgtg agccaccgca cccggcctgt

2101 tttctttttt tttttttttt tgagacagag tttcgctctg tgcccaggct ggagtgcagt

2161 ggcgcaatct cagctcactg caagctccgc ctcccgggct tgccattctc ctgcctcagc

2221 ctcctcagta gctgcgatta caggcgcctg ccacacacct ggctaatttt ttaaaatatt

2281 tttttagtag agacggggtt tcaccgtgtt agccaggatg gtgtccatct gctgacctcg

2341 tgatccaccc gtcttggcct cccagagtgc tgggattaca ggcgtgagcc accgcgcccg

2401 gcctttttta tttttatttg tattttattt tattttttga gacggagtct ccctctgtcg

2461 cccaggctgg agtgcagtgg cgcgatctcg gctctctgca agctctgcct cccgcgttca

2521 cgccattctc ctgcctcagc ctcctgagta gctgggacta cgggctcccg ccaccacgcc

2581 cggctaattt tttgtatttt tagtagagac ggggtttcac tgggttagcc aggatggtct

2641 cgatctcctg acctcgtgat ccgcccgcct tggcctccca aagtgctggg attacaggcg

2701 tgagccaccg tgccaggcct tttttttttt ttttttaatg aaggcaaagg aaaaaaatta

2761 aactctgtaa atatgaaaac ttcttcagtt tctttcgttt agtatttaag actgatttcc

2821 taatcatggc atttcttctg tgtattcttc tgaaatattt tttcattttg attttgaaga

2881 cagagtattc tgaaaagata ataatagatc tgtagacaag tctcatcaat gattgttagt

2941 gcatttttta attaaaattt ttttttttgt agagaagggg tctcttatgt tgcccagtct

3001 gattttgaac tcctgagctc aagtgatcct cccaccacag cctcccaaac tgctgggagg

3061 catgagcagc tagtgcacgt atttttgact ggtgaatgtc acattatttg gtgtatcttt

3121 taactatggt atttaaggat ccctgacgtt tatttattta tttatttatt tatttattta

3181 tttatttatt gagactgagt ctcgcactgt tgcccaggct ggagtgctgt ggcgtgatct

3241 cagctcactg caacctccat ttcccaggtt caagtgattc tcctgcctca gcctcccaag

3301 tagctgggat tacaggcacc caccaccaca cccggctaat ttttatagtt ttagtagcga

3361 cggggtttca caacgttggc caggctggtc ttgaactcct gacctcaaat gacctacccg 3421 cctcggcctc ccaaagtgca gggattacag acgtgagcca tggcgcccag atggatccct 3481 gacgtttaat tcagtgacta aaattcttat tattttcagc attatagttg aaatatcatt 3541 agccttgagt gtaaatgcca atattacaag cttttcgtgt tcttttttct ttttctttct 3601 ttccttcttt ctttctttct ttctttcttt ctttctttct ttctttcttt ctttctttct 3661 ttctttcttt ctctctctct ttcttttctt ttctttctct ctctttctct ctctcctttc 3721 tttcttttca ctcagtcgcc caggctgggg tacagtggct cgatctctgc tcactgcaag 3781 ctccgcctcc tgggttcacg ccattctcct gcctcagcct cctgaatagc tgggactaca 3841 ggtgcccacc accacgcctg gctaattttt tgtagtttta gtagagatgg ggtttcaccg 3901 tgttatccag gatggtctcg atctcctggc taattttttg tagttttagt agagatgagg 3961 tttcaccgtg ttagccagga tggtctcgat ttcctgacct cttgatccgc ccatctcagc 4021 cttccaaagt gctgggatta taggcgtgag ccactgtgcc tggcctctgt tctttagtta 4081 tgttattgaa tgtattgaaa tttttcaaat gtagatcatt ttaagagtaa tcttttttcc 4141 tacagattca ttaaaaatat ttattcatca tttatcaggt ggttttcttt agacagtctt 4201 tctcaagaaa tgcaaaaagg taatacagag tgccttaaaa cattaaatag gcctggcgcc 4261 atggctcatg cctgtaatcc cagcactttg gggggccgag gcgggcggat catgaggtca 4321 ggagattgag accatcctgg ctaacacggt gaaatcccgt ctgtactaaa aaatacaaaa 4381 aaattagccg ggcgtggtgg tgtgtgcctg tagtcccagc tactcgggag gctgaggcag 4441 gagaatggcg tgaacccggc cactccagag gcggagcttg cagtgagctg agatagtgcc 4501 actacactcc agcctgggca acagagcgag actccgtctc aaaaaaaaaa aaaaaacatt 4561 aaataaacct tttttctaac aattaaaaaa aaagaaaaaa actcaagctg aaaagaagct 4621 tagaaatcac ctgggtgtag tttcccacac agaatgggaa tccctgcttc tataccaaaa 4681 caaatgaaaa tccatcctct tttccagatt tctaaggaag taataggatt tttcagaatt 4741 tctttttttt tgtttgtttt ttgttttttt tttttgaaac ggagctttgc tcttgttgcc 4801 caggctggag tgcaatggcg ccatctcagc tcaccgcaac tgccgcctcc cgggttcaag 4861 tgattctcct accccaggct cccgagtagc tggaattaca ggcatgtgcc accatgcccg 4921 gctaattttg tgtttttagt agagacgggg tttctccatg ttggtcaggc tggtctcaaa 4981 ctcttcaact caggtgatcc atccgcttcg gcctcccaaa gtgctgggat tacaggtgtg 5041 agatagcgct cccggcccag aatttcttat aggaaaagta tgttcaatta gaaaaaagaa 5101 gcaagcagag tagtgctctt ccaacctgta acatacagtg tcctgtaaca ttagaaaata 5161 acttgaaggt aacagtaata ttttcctatg aatcgagagt tacgatttca ggttttagaa 5221 attttaggtg gaagtagcaa gattcacaaa ggaaagggtg ctgacactgg gtatgtacta 5281 atgagccagg attgttaaac actttctttc cttttttttt tagatagtct cgctctgggc 5341 cgggcgcggt ggctcaggtc tgtaatccca gcactttggg aggccgaggc gggcggatca 5401 caaggtcagg agatcgagac catcctggct aacacggtga aaccctgtct ctactaaaaa 5461 tagaaaaaat tagccgggcc tggtggcggg cacctgtagt cccagctact cgggaggctg 5521 aggcaggaga atggcgtgaa cccgggaggt ggagcttgca gtgagccaag atcacgccac 5581 tgcactccag cctgggcaat agagcgagac tccgtctgaa aaaaaaaaaa aaagagagtc 5641 tcgctctgtt gccaggctgg agtgcagtgg cacaatctcg gctcactgta acctccacct 5701 cccaggttca agagattctc ctgcctcagc ctcctgagta gctgggacta cagatggtgt 5761 gccaccatgt ccggctaatt tttgtatttt tagtagagac aaggtttcac catgttggcc 5821 aggatggtct tgatctcttg acctcgtgat cctcccacct cggcctccca aagtgctggg 5881 attacaggcg tgcgccaccg cgcccagcca atcctttttt tttttttttt tttttttttg 5941 agatgaagtc tcgctcttat tccccaggct ggagtgcaat ggtgtgatct cggctcactg 6001 caacctctac ctcccgagtt caagcgattc tcctgtgtca gcctccctag tagctgggat 6061 tacaggcgcc tgccaccacg cctgctaatt tttgtatttt tagtagagat ggtgtttcac 6121 cttattcacc aggctggtct cgaacttcgg acctcaggtg atctgcctgc ctcggcctcc 6181 caaagtgctg ggattacagg catgagccac tgcactcggc cctgttaaaa cactttacat 6241 aatctcagtt cttggccaga cacagtggct catgcctgta gtcccagcag tttgggaatc 6301 ccaggcgggc ggattgcttg agcccagaag ttcaagacca gcctggacaa cacagtgaga 6361 cctgtctcta cagaagtcac agaaaaatta gctgggattg gtggttcatg cctgtagtcc 6421 caggtacacg agaggctgag gtaggaggat cacctgagct caggaagtcg aggctgcagt 6481 gagctgagat catgcccctg cactccagcc tgggcaacag agcaagatcc tacctcaaaa 6541 gaaataatct cgggcgggtg ctgtgggtca tgcctgtaat cctggcactt ggggaggccc 6601 aggcaggtgg atcacctgag gtcaggattt caagaccagt ctggccaaca cggtgaaacc 6661 ccgtctctac taaaaagaca aaaattagct gggcgtggtg gcaggtgcct gtaatcccag 6721 ctactcagga ggctgaggca ggagaattgc ttgaacctgg gaggcggtgg tggcagtaag 6781 cctagatcat gccactgcac tccagcctgg ctgacagaac gcatctctgt ctcaaaaaaa 6841 gaaaaacaaa caaacaaaca cattcagttc tcacacttga attaggtagg taggttgtgt 6901 tatgcgtgct ttttatgtat tgaggaaaca ggatcacaga ggttgtgtaa cttaactaag 6961 gttacaacta ggtagactga atttgaactg aaatctgtcc aacctcaatg cctatgctgc 7021 attcttttat gccattttgc ccctcttagg tggtcacagg accattccaa ttcctataag 7081 gtataagcac tagttttaaa gtaataccat tgtgaactgc ttgttctcat tgaaataata 7141 gtgtaactgg aatgataaat agttcattaa aattttagtc acattatgtc atcagaattc 7201 taggaggaaa tgaatacctg attattttgt acttcacaaa ataaactgga taaactggat 7261 aaatgactga aagtttcttt tttatattgc tgagtcagat tggatccggt tcattaaggg 7321 ccaaaatttg tatttggcag taatcctcat gtgttctcag aatgcttgat tggtcattat 7381 cagttgctta atgcttacca aacacttcca gtgttttgag tcactgaatt ggctcatggt 7441 aattgttaat cttgtggtta ctgagtcaag agccagcagt gtgtgttttg gctgcttcac 7501 ttcctcctca acacttgata ttgtcagatt ttaaattaat tttttttttc taatctggtg 7561 ggtttgaaat tgtatctata gtgtttttta tcagcatttc ctgaagctgg gcatcttttc 7621 atgtattttc gttggtcatt tgtgcttctg cgaagtgtat attcaatttc gttgcctatt 7681 tttttggtct tttctctttt tctgatttat ttttaagagt tctttatata ttctggatac 7741 taattatttg ttaatttata ggttgcaaat atcctttccc aatttgtgac ttgtcttttc 7801 atctcttcat agtcttctgg tgagaaaagc tcctaattgt aatgaactga atttttcagt 7861 ctttcatggt ttgtgttttg tgtttttttt tattttttat tttttatttt ttgagatgga 7921 gtttcgctct tgttgcccat gcgggagtgc aatggcgtga tcttggctca ctgcagcctc 7981 cgcctcctgg gttcaagcaa ttctcctgtc tcagcctccc gagtagctgg gattacaggt 8041 gcctgccacc acgcccagct aatttctgta tttttagtag agacagggtt tcatcatatt 8101 ggtcaggctg gtcttgaact cctgacctca ggtgatccac ccgcctgggc ctcccaaagt 8161 gctgggatta caggcgtgag ccactgcgcc cggctggttt gtgcttgttg tatcaaattt 8221 gggaaatcct tctttgctgc caggcatgtt gttttaataa atacagtagt atacctatct 8281 ataatgtggt atatctcatc tgacatttat ttccaatatt tgcttgatta caaaggcaga 8341 cttcttagaa gttgctttct tattatcttt ggagttatag ggccacaggg ttttaaaagg 8401 aggttggatt gtgccttaca tgtgggtttc atacattgtc caaactgaga caccaacaga 8461 tttgtgaatg agatgtgcat cttattaaat gttaatgtga tttgacaatg agacctttct 8521 ttgtgtggac agtattatat acaattgata gtgtgtcatt cagtctaagc tgttagcact 8581 ttcactaaaa tatattcaaa ttttttttga tgtctagttt ttacagtgtt gggctttatc 8641 aagaatagtt gtggattgtg gaagagctga tatggaaaca gaggaatgta ttttgctttt 8701 ttttttaaac tctgtccggt tccttggact tgtgtgtctc ctatcttatc aagaataatt 8761 gtagattgtg gaagagttga tatggaaaca tcttagaaga atgttt'tttg ctttttttaa 8821 aaaaaaaaac aaaactgtcc agttccttgg acttgtgtgt ctcctatcta ccattaaacc 8881 cttatgactt aaggtatatg tataaataca tatatacatc ttggtggcat tgatttttct 8941 atgttgtgta ttaaagctgc taaaaaacat tttttctttt tttcaatgag aagactagtg 9001 attactttga ctttaggaat tattcttcta aggagtcaag tatttgatct gggcgaggat 9061 cacactgaaa tataaaacat ctgagtaatt tgggttcact aaaaaaaaaa aaaaagaaat 9121 gtaaaacaac caccagatta aaaatacact acaaatactg tttacaacca ccagattaaa 9181 aatacaacac aaatactgtt tatttataca aatagtatac ctttgacatt tatgaaaggc 9241 atactcttag atcattggga atccgtactt atgtaactac catagtttat agtgtccttc 9301 atgaaataca gaaaagtgta acaggaaatt tttagttacc atcagatttg atgggtaaat 9361 agaagagagc aacaaagtaa agcaatttga atgctaaact tagtcttcag cttttttttt 9421 tttttttttt tttttaaaga gatgggatct tgccctggta cccaggttgg tcttaaatgc 9481 ctggcctcaa gtaattcacc tgcttcggcc tccccaagtg ctgggattac agacgtgagc 9541 caccatgcct ggtgctttca gctaccttta acttcctgcc tttttctgtt gtttgttact 9601 accactggcc tattccagcc tcctttagta acctattgga aactcaagat tagtctgtct 9661 tgggccgggt gcgttggctc acgcctgtaa tcccagcact ttgggaggct gaggcgtgtg 9721 gatcacctga ggtcaggagt tcgagaccag cctggccaac atggtgaaac cctgtctctc 9781 ctaaaaatag aaaaattagc caggcatggt ggtggattcc tgtaatccca gctactcagg 9841 aggctgaggc aggagaattg cttgaagccg ggaggcagag gtttcggtga gccgagatcg 9901 taccattgca ctccagcctg gccgacaaga gtgaaactcc atctccaaaa aataaaaaat 9961 aaaaaagatt aggctggctt tgcctgtctt ttgcttcatc tcatcatcat ggtcttggtc 10021 ccaggtgggg tttgcatcct tactggtaag aatggaatct tcctaatcct aaaatctttc 10081 tatagtttgg gagtgttaac atttccttca gtagtttcat ggggaaggat atgggcagta 10141 acttccccat ttctttcttc tagtagatca ttatatcttt tttttttttt tttaaagaca 10201 gagtcttgct gtgtcgccta gcttggagtg cagtggtgcg atcttgactc attgcaacct 10261 cctcctccag ggttcaagtg attctttcac ctcagcctcc cgagtagctg ggattacagg 10321 cacgtgccac catgcctggc taatttttat atttttagta gaaatggggt ttcaccatgt 10381 tggctaggct ggtcgcaaac tcctgaactc aaatgatcca cctgcctcat cctcccaaag 10441 tgctgggatt acaggcatga gccaccgcac ccggccgatc attatgtctt ataggatacc 10501 tcatgagaga agtgatctgg ctgcatgggt ggaattcaag gtcaaatggt cagccaaatt 10561 aaaaatcact gctggagcag actttagggg tagtttgtga actttcaaat ttggaaatta 10621 ttattattat tattattttt tgagacggag ttttgctctt gttgcccagg ctggagtgca 10681 atggtacaat cttggcttac tgcagcctct gcctcttggg ttcaagcagt tctcctgcct 10741 cagtctcccg agtagctggg attacgggcg tgctccacca tgctcgatta attttgtatt 10801 tttagtagag acggtgtttc tccgtgttgg tctggctgga cttgaactcc tgacctcagg 10861 tgatctaccc gcctcagcct cccaaagtgc tgggattaca ggtgtgagcc actgctcctg 10921 gccagacata tttaattctt tcatcaaagg tttattatat ttccgagata ttctagccaa 10981 aggaaacaaa cttaacaact tttgttttct gaaaatactt tagcaaaaat agaagggaga 11041 ggatcaggaa aaataactaa tggataccag gcttaatacc tgggtgatga aataatctgt 11101 acaacaaccc ccatgacaca agtttaccta tgtaacaaac cttcacatgt acccctgaac 11161 ttaaaagtta aaaaaattta atgttaataa aatcttaaac aaaaatcacg gaaggtcttt 11221 aattgtttat gtcgtgtcct atccgttttc ttgaagaaga aatattagct agttcttgta 11281 atttatttat tttttggggt ggagtcccac tctgtcaccc agactggagt gcagtggtgt 11341 ggtcttggct cactgcaacc tctgcctcct gggtccaagc gattctcctg cctcagcctc 11401 ctgagtagct gggattacag gcccttgcca ccatgcctgg ctaaattttt tatttttagt 11461 ggagatgggg tttcaccatg ttggccaggc tggtcttgaa ctcctgatct caggggatcc 11521 accttcctca gcttcccaaa gtgctgggat tacagacttg agccaccatg cctggcagtt 11581 cttgtaattt aatagggaat cttttttcct ttctttcttt cttttttttt ttttttttgg 11641 agacagggtc tcgctctgtt gcccaggctg gagtgcagtg atgcagtcac agctcactgc 11701 tgcagccttg acttcctggg ttcaggtgat tctcctacct cagcctcccg agtagctggg 11761 actgcaggct catgccacca tgcctggctc tttttttttt tttttttgta ttttttgtag 11821 agatggggtt tcatcatgtt gcacaggctg gtctcaaact cctaagttca ggtgatccac 11881 ctgcctcggc ctcccaaagt gttggaatta caggtgtgag tcacagtgcc cagcctgctc 11941 tggatatatt actggcatta tatttagtgg aagagcatat atatattaga gtaatggaga 12001 tttatttcat ttagtagtat aagaaattat gtttaggtgc agttctaatt tgagacattc 12061 tgtatctgtc ctgtctgata tgtctccaac attgacagtg gagatataaa acattttcat 12121 cagtgtagaa agttctgctg gacagcactg ttgtaggtca gtgattctca aagttttagt 12181 gtgtgtatgg ataacttgga tgtattatta aaatgtagat tctgagtcag tagctctggt 12241 gtgggcttga gagtttgtat ttatttattt atagagatgg ggtcttgcta tgttccctgg 12301 tctgaaactt ctggcctcaa gcagtcttcc caccttggcc tcctaaagca ccaggattgt 12361 gggtgtgatc cactgtgcct ggcctgtgta aactttaaaa tagtgtcttt agccatgagg 12421 gatgatattg taatggcaga actctggaca gtgagttgaa agatcgaaag ttgtagttca 12481 ctttttccac catcttgaag cttttagagg cctgctggga ataggacttc taaaaggcaa 12541 atatgtctgg aaggctgtgg tccaaggcca ttttttgttg gctgtaagtg tggtctccag 12601 aatcaaagag agcacacaac tgtttttaaa tcgaaggtgt ttatgcctga gatgaagctg 12661 aattctgttt gggcaagaga tttgtgctta tgtatacaaa gcaaagaaca acacagtgac 12721 tcctggtggc aaaccaaaca aaaccagagt aatctgtgga aagataattt gcgcccatgg 12781 aaacagacat gccaaattcc aaaggaatct tcctgctgag gccgtttgga cacagaattc 12841 acgtgatgtt gtacccctca aggatttaaa ctactaaaaa gtaaaagtgg atttgtgaac 12901 aaaaaaagtc ctagttcagc tttcctgttt aaaaaccaac ctctctcagc cttggatttt 12961 tttttttaat tttttaaatt taaaaaaaat ttttttaatt aaattttttt ttttagtgat 13021 ggggttttgc tgtgttgccc aggttagtct ctaattcctg acctcaagtg atcgtccctc 13081 catggcctcc taaagtactg ggattacagg catgagccac ggtccccggc ctgtataaac 13141 tttttaaata gtgtcttttg ctatgagagc ttgtattgta atggcagaac tgtggacagt 13201 gagttgaaag atctaaagtt caggccaggc gcggtggctc atgcctgtaa tcccagcact 13261 ttgggaggct gaggtgggtg gatcatgagg tcaggaatgt gagaccagcc tgaccaacat 13321 ggtgaaaccc cgtctctact gaaaatacaa aaattagccg ggtatggtgg cgcacgcctg 13381 tgatcccagc tactcaggag gctgagacag gagaatcact tgaacccggg aggtggaggt 13441 tgcagtgagc cgaggtcacg ccactgcact ctagcctggg tggatagaat gagactccat 13501 ctcaaaaaaa aaaaaaaaaa aaaaagatct acacttctag ttcatctttc cagttttaaa 13561 accaatctct ctcagccttg aactcttttt ttatttttca attttcttta gggatggggg 13621 tcttgctatg ttgcccagtt tggtctcaaa ctcctggcct cgagcatacc tcccaccttg 13681 gcttcccaaa gtgctgggat tataggcatg agccaccaag cctggctaat gtattttttt 13741 aaaatcacaa gttcataatg atatttatta tttgtttaac ttttagcccc tgcaaagaaa 13801 tataatttag attttagaat tataggactt taaatatttt gcctttcctc ttttatataa 13861 aaatattagt tctttacaac tataattact tatttttcta tattggactc atataaaaag 13921 ggttttaaaa tagtaatact gctattatag ctgataagta caatctaaaa tctttgctat 13981 tctttatgtt cttaaaatac tgtgtaaggg agggccaggc gcagtggccc acgtctgtaa 14041 ttcccagaac tttgggaggc gaggcaggca gatcacttga ggtcaggagt ttgagaacag 14101 cttggccaat gtggcaaaac cccatctctc tactaaaaat acaaaaaatt agccaggcgt 14161 ggtggtatgc acctagaatc ccagttactc aggggggctg aggcaggagg atcgcttgaa 14221 cccaggaggt ggaggttgca gtgagctgag atcacgccac tgcgctcccg cctgggtgac 14281 agagtgagac tctgtctcaa aaaaataaaa ataataaaaa tattgtgtag gggaagctct 14341 caaaccacgt tcttcctcta ctctcacacc accacaacaa tcatcaacac agaacacatc 14401 tgtgaccaaa agtgtggggg tttttcacct ctgcaccaag cagtggacac cagctcggtg 14461 ttctccaatt caggttgggg gctcagtctt caagactgcc tgcacacccc tggacaccag 14521 ttgcatgtct ggggccctgg atcttatgac ccactgtctt catgttggtg ttcccacgat 14581 cccctctttg ggtttggtta atttgctgga gcatctcaca gaactcaggg aaacactgaa 14641 atttattggt ttattctgaa gaatcttaca aaggatatag atgaagggat gcatagagca 14701 aggtatgggg gaagaggtgt ggagcttcta tacccttctt ggggcaccac catccaggaa 14761 cctccaggtg ttcagctatc cagaagttct ctgaacccag tcctcttggg cttttgtgga 14821 agcttcatga tgtcaggatg ccttctctca gggtataggg cagtcccctc tctggggagg 14881 gtgttaagac ccacaatcag aaaggtgggg aaagattata gtcctttctt ggggcaggtg 14941 aaagtagggc agaggaagag gttttgtttc ctgagacctg cccctgagac ctaacacacc 15001 caatattgta tcagaagact gtaactaggg ctgtgggaat tatgaaccag gaacagtgga 15061 tgaaaaccaa tatatgtcat aacactacaa atatatttta ctgatgtata caattagaat 15121 accgagttct aaagtcactt caaataatta tattctctgg tcattttact aattttctgt 15181 aaagttaggt ttatttgttt cattttgtat tcattttgag ggctaaaaaa ttatattgct 15241 aagaatgtgt aatagctatt ttgaaaagtt agaatacaga gatgtataaa aagaaaaatt 15301 ctctctcttt tttttttttt gagatggagt ctcgctctgt tgccctggct ggagtgcagt 15361 ggcacaatct cggctcgctg taaactccgc ctcccaggtt cacgccattc tcctgcctca 15421 gcctcctcag cagctgggac cacaggcacc tgccaccatg cccggctaat tttttgtatt 15481 tttagtagag atggggtttc actgttttag ccaggatggt ctcgatctcc cgacctcgtg 15541 atcctcctgc ctcggcctcc caaagtgctg ggattatagg cgtgagccac cgcacctgac 15601 caataaaaat tttctttaag ccaccattgg aaatatccac tattagaata tttatataaa 15661 atatttgtgg agctcacatt tattgaaaac attatgccat gtgttcagat tgttctaagc 15721 actttgtatg cattatctca tttgatcctt acaataactt tatgagatag ttactattat 15781 ctctatttta tttttctttt ttcagccagt caaatttagc agtggggggt tgtataccaa 15841 ctttagtaac actgctttga ataagttctg atgagccact accattggac cagcctgtta 15901 ttcccatttg attgattaaa actcaaatgt taacttgtac aagggatacg taacctagta 15961 agttgcagaa ctgagacttg acccctgaca attcttgctc aaactcactt tattcatccc 16021 tgtgtattgt ttttatcttt ttttctgtgc ttacacatac acggattttt ttcttttgtc 16081 atttgagaac agagattttt taaatgtatt tcatacattc aggtaagttt tttttttttt 16141 ctctaacagt gtcttataag aagtttttga tttcattgaa gtccccttta gtttttcctt 16201 tatggtttgt gcttttggtg ttatatctaa gaatttttgc ttaatctaag gtcagaaaga 16261 tgttctcttc ttgaagtttt atactttcag gtttgacttt tttgtctatg attcattttg 16321 aattaattat atatgacgtc atatggatgg aagatttttt ttgtttgcac atagatatct 16381 attttagcac tttttgttga aaagattacc ctttctaaaa tattttgtcc atgtctatat 16441 ttggacctgt tctattctat tgatctgttt gtctatcttt atgccagtat cacactatct 16501 tgataattgt agctttataa taaattttga aaccagaaaa gaaagtttcc caaagttgtt 16561 acgactgtag agcctttgga tttttctttt tttttttttt ctgagacgga gtttcactct 16621 tgttgcccag gctggagtgc aatggcgcga tctcggctca ccgcaacctc cgcctcccag 16681 gttcaagcga ttctcctgcc tcagcctccc gagtagctgg gattacagac atgtgccact 16741 atgcccggct aattttgtat ttttagtaga gacggggttt ctccatgttg gtcaggctgg 16801 tctcgaactc ctgacctcag gtgatccacc tgcctcggct tcccaaagtg ctgggattac 16861 aggcatgagc caccacgtcc ggccagtcct ttggatttct atacaaattt tttggttttt 16921 tttttttttt tttttttttt tttttttttt ttgagataga gtctcactat gttgcctagg 16981 ctggcctcca acttttgggc tcaagtgatc ctcctacctc agtctcctga gtagctgcaa 17041 ctacacaaac acgctaccat gtctggctcc atatgatttt taaaattagc ttgccaagtt 17101 tctataaaaa gctgctggct ttttatttat ttatttattt atttgagacc aggtcttgct 17161 ctgactccta ggctggagtg cagtggtgtg gtcatggctc actgcagcct cgacctttcg 17221 ggctgaagtt tatcctccta cctcagcctc ttgagtagct gggaccacag gtgtgtgcca 17281 ctaagcttgg ctaattaaaa acaatttttt tttttttttg gagacggaat ttcactctta 17341 ttgcccaggc tggagtgcaa tggcgtgatc tcggctcact gcaacctctg cctcctgggt 17401 tcaagcgatt ctcctgcctc agcctcccga gtagctggga ttacaggtat gcatcaccac 17461 gcccggctaa ttttttgtat ttttagtaga gatggggttt cgctgtgttg accaggctgg 17521 tctcgaactc ctgacctcag gtgatctgcc ctactcggcc tcccaaagtg ctgggattac 17581 ggccacggcg cccagcctaa ataatttttt ttaatataga cacggtctca ctatgttgcc 17641 ctggctgatc ctgaactttt ggcctcaagt gatcctcctg tctcagcctc ccaaagcacc 17701 gggattacaa gcgtgagcca ccatgcctag cttcactgat tcttcagctt gattgagtct 17761 gctgttgaaa ttctctattg aattatttgg ttcagtcatt atattcttcc gctctaagat 17821 ttgcttttta ttatttgttt ctttgttttc attttgttca tgtattgtct ttctaatatt 17881 gttatttgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgttttta attgagatgg 17941 agtctcactc tgtaacccag gctggagtgc agtgacacaa tcttggctca ctgcaacctc 18001 cacctcctgg gttcaagtga ttctcccgcc tcagcctccc gaatagctga gactacaggc 18061 gtgtgccacc acacccggct aatttttgta tttttagtag agacggggtt tcactatgtt 18121 ggccaggctg gcgttgaact cttgaccttg tgatccgctt gccttggcct cccaaagtgc 18181 tggaattaca gatgtgagcc actgtgcccg gccagttatt tgtgttttat tatagttcac 18241 ggaccttctt taataggatt atttatttat ttattatttt gtaaaaattt ttattaaaat 18301 tttttttttc ttttatagag acgggtttca ctatgttgtg ctagtctcaa acagccgggg 18361 ctcaagcaat ctgcccgcct tggcctccca aagtgctggg attacaagtg agagccactg 18421 cacctggcca agaggattat tctgaattct ttgtcagtca gttctataga tctccatttc 18481 tttagggttg ttttttggca ttttattagt tttcttttgt ggtatcatgt ttccttggtt 18541 cttttttttt cttttgagac agatttttgc tctgtcatct aggctggagt gcagtggcac 18601 gatctcagct cactgtagcc tctgcctcct gggttcaagt gattgtcctg cctcagcctc 18661 cccagtagct ggggttacaa gcatgtgcta ccatgcctgg ctaatttttg tagttttagt 18721 agagatgggg tttcaccatg ttggccaggc ttgtcttgaa ctcctgacct caggtgatcc 18781 acctgcctca gcctcctgaa gtgctaggat tacaggcgtg agccactaca cttggcctcc 18841 ttgattctta atgatctttt tgtccttgtg ttggtttctg tgcatttgag gaagtagtca 18901 cttcttttag tctttaccaa ttcatttctg tgaggaaaga ccttcaccag tcagcctaac 18961 ctggggttct ggatgggtca gctggtagta tctgtgggca agcagggcgt gctgctggaa 19021 tctcttgttg aacttggtag ctgcccatac tctgaggtta ggtggggctt gttctctggg 19081 gtctgaggcc agtgcaggtc tgttggctgg gctctgaggt taggaggggc agctggctgg 19141 gctccatgtt taggccaggc tgctctgtga cttgggaggg ctgcaggctg tactctggaa 19201 tttccccggt caggtgaggc tacagggtat gcaccataga tgggtaggac tataggctgc 19261 attctgcaat caggtggggt tgtatgttaa gtttagctgt tgaatggggg cattggctga 19321 tttttttgcc tggatggggc tgctggccct gctccaaggt tagacgggtt ctgtggccgg 19381 gctgcctggt tgggcaggca ggttgcctgg ttggtcctgt ggagttggag tttcagctat 19441 actattaggt gaggccatag gctatgctca gctgaggtca gactggatca ctggcacagg 19501 acccaagcca ggcagaatgt tgactgtgct tgttggatag acatgccgca gatgggcaga 19561 gctggtggtt gggctctctg gttgggtgag gccactggca gggatgtggt ctcattgtca 19621 tgatttgcat actgtttgct gtgagcccca cccccttctt tgttcctagc tgactccagt 19681 ggtctagccc tcccatttct tccctatgag gcaagacaga catggatctc ctggaaagca 19741 ccctggaaag ctgggaaagt tggggaagct gaatgtctgc cttgggcttt ctttttccca 19801 ctgcagaaac tataggccca ggggatcact ctctggcccc atgtgggcct ggtgaggggc 19861 gatgcagtca aaaagtcaag ccactcttac ccgtctaatg gggcttttgg tatttgtgct 19921 ccaagggagt gcttaagccc taggctctgg aattttcaca aaggtgttct tgtctgtgga 19981 taattactaa ttgacatttc tgtgatagtg actagaacct ggacctccta ttccaccatc 20041 atgttcttgg actctttttt attatttatt tatttttttg agacggagcc tcgctctgtc 20101 gcgcaggctg gagtgcagtg atgcgatttc ggctcactgc aacctctgcc tcccgggttc 20161 aagcagttct cctgcctcag cctcccgagt agctgggact acaggggcac accaccacgc 20221 ccagctaata ttttgtattt ttagtagaga tggggtttca ccgtattagc caggatggtc 20281 tcgaacccct gaccttgtga tccccccgcc ttggcctccc aaagtgctgg gattacaggc 20341 atgagccacc gcgcccagcc tattatttat ttttaatttt aatttttttg agacactatt 20401 tctattttgg aataatttta gatttacaga acatttgtag agatagtaca gagagtacag 20461 agagttcctg gctatcccta actcagtttt ccctaatgtt aaccatctta cattaccatg 20521 atacatttgt caaaactgaa aaacctacct tggtacgtta gtatcaacta aactctggac 20581 tgtatttaga tttcccctgt ttttctttct tcttcttttt tttttctctc tctttttttg 20641 agacagggtc tcattgtgtc tccccaggct ggagtacagt ggcatgatca tgtcctactg 20701 caaccttgac tacccctggc tcaggtgatc ctcctgcctc agcctcccta gtagctggga 20761 ctacaggcac gtgccaccat gcctggctga tttttttttt tttttttttg tagagactgg 20821 gtttcaccat gttgcccagg ctggtctgga actcctgggc ttaagagatc cgatccttct 20881 gcctcggcct tgcaaagtac tgagattaca ggtgtgaacc actgcaccca acccaggttt 20941 tctacaattt ttttttttct atttcaggat ccaatctagg ataactaaat tgtgtttagt 21001 tgttatactt tcttagtttc ttctggtctg tgatagtttc ttagtccttc cccccgctcc 21061 cagtaatttg acagctttgg ggaaaactgg tcaggtcctc aatttggttt tgtctgctgt 21121 tttcctcgta attagactgg gcttgtggat ttttggaaaa atacctgagg taaagtacct 21181 gtctcagtta tatgaaatca gtatggactt gagtatttag tttatatttt ggattaaagt 21241 cccacattga tttattttgt tgctcaagtt gttttatctt tggccattga gagctctttc 21301 agattggcct tggccttaga atctgtcatt tctccagaga atagggcttg tttctctgtc 21361 agtctgatct tcattccttt gtaaagactt catttgtggt atcttgataa cttttaggag 21421 ccaccctttt aggagggggt ttgttagtgc tattacggtt ttatttttta agagattatg 21481 attaggatgg atagatgttt agtttgccaa cctgatctaa acttttaatt tttgctaaac 21541 agaatttgta gcatttagag ctatatgctt tagaattttt cattgtatga tgtttaacat 21601 acagaaaaaa tatgaatgta aaacatctaa gttatgaggc ataataataa acatttgcaa 21661 gcttattagt tgtcttgaaa actagagcat taccaataca ttactattta tttatttgtc 21721 tgtttctgtc ctatcctatc cttttactac tccacagagt tagctgttct cccgaatttt 21781 gggtttatca tttccttcct tttataaacc acatgatata tattcctaaa taatatattg 21841 tttatttttg cttaattgtg agctttataa aaatgttaaa ctgtatatag gcttaagtga 21901 ttttattttt ttttgagatg gagtttcgct cttgttgccc aggctggagt gtgatggcgt 21961 gatcttggct caccgcaatc tccgcctccc gggttcaagt gattctcctg cctcagcctc 22021 ccgagtgagt agctgggatt acaggcatgc gccaccatgc ctgcctaatt ttttgtagtt 22081 ttagtggaga cgtggtttct ccatgttggt caggctggtc tggaactcct gacctcaggt 22141 gatccaccag ccttggcctc ccaaagtgct gggattacag gtgtgagcca tggcacccag 22201 ccttgtttta gttctttatt ttagaaagtt ccaaactcat aaaagtagat ttggctgggc 22261 atgactcatg cctgtaatcc tagcactttg ggagggtgag gtggccagat cacctgaagt 22321 caggaagttc gagaccagtc tggccaacat ggtgaaaccc caactctact aaaaatacaa 22381 aaattaacga gtgtggtggt gtgtgcctat aatcccagct acttgggata ctgaggcagg 22441 agaatcactt gaaccccaga ggcagaggtt gcagtgagct gagatcgtgc cactgcagtc 22501 cagcctgggt ggcagactga gactgtcaca acagcaacaa caacaacagc aacaacaaaa 22561 atgtagatta atcctgcttt gtaaaaactc ttattttttt ctactcacac accaccacag 22621 caattatcaa cacagaaaaa gaattctgtg gccaaatgtg tgggcagttt tccccacaca 22681 ccaagcagct gccaccagtt gggtgttctc cagttcaatg ccgacactgt ctacctggag 22741 atagtgtcag atcccactgg ttgaatgcgc agtctccaag attgccccct agcactcatc 22801 agtcacaagt ctgggcctca agtgcttctg accaccagct tcactcatgt cagggttccc 22861 tcggtcctct ctttaggttt aatttgccag agtgtttcac agaactcaga gaaactcttt 22921 tatgtttact ggtttattct aaaggcgatt gcaaagtata cagatgaaga gatttgtctg 22981 gcaaggtagg ggggaaggtg cactgacttt ccatatcctc cctggggtgt caccatccac 23041 gaacctccgt ccaggtgttc agctagccag aagctctcca aatccagtcc tcttgagttt 23101 ttatggaagc ttcatgatgt tagcattcct tctcccagag tacagagtgg gagtctccgt 23161 ctttttcttt tgagacacgc tttcactctg ttgcccagct ggagtgcagt ggcaccatct 23221 tgggtcactg caacctccac ctccgagtag ctgggattac aggcatgcac caccacgccc 23281 ggctaatttt tgtattttta gtagagatgg ggttttacca tgttggtaag gctggtctca 23341 aactcctgac ctcaagtgat cctcccgcct cagcctccca aaatgctggg attgtaggag 23401 tgagccaccc tgccctgcct gtgagagggg cttaagactc acaatgagaa aggggtattg 23461 ggagagatag agttctacct taggacagtt gaagggaggg tgggaaaaga tttgagagat 23521 tctgttttct gaggcctgac atacctagca ttataacaga agactccagc aagggctatg 23581 ggagttatgg gccagaagct gtagacagaa acgtgtgtgt gtgtgtgtgt gtgtgtgtgt 23641 gtgtgtgtgt gtgtgtgtgt gtgtgtcccc gtccccgtat atgtatgtat cataacacca 23701 tacagcccca ataactgtca atcttgtttt atatacatac cctctgccca gttattttag 23761 agcaaatccc agtcatatca tttaacctgt caagttattt ttgtatatct ctgaaagatc 23821 acataaatta ctataatagc attatcctac ctatagaaaa tgaacttatt ccttaatatt 23881 atcaaatatt catgttttct caattttttt attgtgtttt cctcatgtgt ttggattggg 23941 actcaaataa ggtctgtaag ttagtgattg cttgagatgt attccttaag atctttttca 24001 gtctgtaggt tctcttttca tcttgctttt tttgtgttga aggaaactga atcacttttt 24061 ctaaaagttt gctacagtca ggattttgca gtttaattca ctactatttt aattaggatt 24121 tgggtatgta tggataaaac ataaataatc agctaggtgt ggtggtacat tcctatagtc 24181 ccagatactt gggaggctga ggcaggaaga tcttttgggc tcaggagttt gaggctgcag 24241 tgagcaatgt tcatgccact tcactccaac ctgggctgtt cagagcaaga ccctgtctct 24301 aaaaacaagg atcaacaaaa attaaatatg ctttcatttc tggttctttt ttctcttttc 24361 tctgtcagtt tttggtatca gtgttatgct gatctcatac gagaacttgg aattcagcaa 24421 cattcattca gcaggagtta ctgagcacta ctgtggccgg gcactatgct tggtgtaatg 24481 gtgaatgaga cagaagtggt atttgtcctc gtagtgcata aaggcaagtg gggaagacag 24541 acaataaaca aatcctgaat aaattgataa gctacaaata tatatttgtt tccattttat 24601 gtgttggaat agattaaata aaataggaat aatctgtttc ttaaatattt aatagaactt 24661 taaagacatc tgttctgaca tttattttag catacacata tttcataagc atgtgaaatt 24721 acttaagaac cccttttctc tccccataaa tctttgacac cgtgtgaagt tgttccatcc 24781 ttatattgtt tttcctttag gcctacagtg ttcccctggg gatttgagtt ttcaagtttt 24841 gtgaacaagg ctgtattatt tctgtgggaa aaatcattca ggtttcacat gatggaaaca 24901 gaatgataca atccactgag tttgaagact ccaggaagaa caaaaaatta tatttgcaat 24961 tacttatttc aggtcaaatt aatagcttct agtatgataa cttttttttg ttttaaaatt 25021 aaccagttaa gagactgggc gtggtggttc atgcctgcaa tcccagagct ttaggaggcc 25081 acagtgggtg gatcagttga ggtcaggagt ttgagaccag cctaggcaac gtggtgaaat 25141 cctgtctcta ctaaaaatac aaaaattagc tgggtgtgat ggcccacgcc tgtaatccca 25201 gctactcggg aggctaaggc acaagaatct tgctgctgca ctccagagtg cagacgacag 25261 agcaagactc tgtctcaaga caacaacaac aaaaaactga ccagttagta ttgtattagt 25321 tacttgtaga ggaaaatagt atcaaatagt aaggattttc ttataaaaaa caaaaaaagg 25381 gtgtggattg tatatacttg ctgagtcaag tatacttgtc tttctgagtc aagtaagtat 25441 acacagtcca cacccatttt tttttttgaa gaaaacaccc gtttgttttt atgtgccctc 25501 agaattaaga tgaagtcttg tgctatcttt ttaatatgta taagactaga aaaaccagtg 25561 aaattcatag ttagaaaata atctatctct agtcctttaa ttctggaaag aattccagag 25621 atcatttact ccagtagtag tcttgtttta tggaagaagc cctaaaagtt cttttttttt 25681 aaatttcaga ggtttttacc cattctacaa gtcttttaat tgcctcaggt gctcttgtct 25741 ttcttgcctt ctggtatgaa ttgtgatatg atttttggag ttgggaagga aattatattt 25801 ttggttcaat aacttgttga cattattttt ttgccaacca gtctttattc agttgcaaga 25861 attaaattga taggttcgtg gtcagatttc ctcttgagga cactgcactg aactctctga 25921 agaaaatact ttaaataaat tgtaatgaat tggttaagat gctgacttga aatttctgat 25981 tggctaatgc tttaattttt attttttttg agacaagttc tcactctgtt acccagagtg 26041 agagtgcagg ctggagtgca gtggcgccat cactgttcac tgcagcttcg acctccccag 26101 gctcaggtgg tcccctgcct caacctcctg agtagctggg actacaggtg tgtgccacca 26161 cacctggcca gtttttgtat tttttgtaga gacaggttgt cactatgttg ctcaggctag 26221 tcttgaactc ctgggctcaa gtgatcttcc agtctcccaa agtgctggga ttacaggcct 26281 gagcagtcac acccagccct ggctaatgct ttgaagtcat gactcccatt ctgattttta 26341 actagtcatt actttcctgt tcttccatta tggtagtctc acaagactta taatttcaga 26401 ctttttattg gtaactacaa gagattattg ttttagcttt ttcattttaa agatattgtt 26461 ttgtctagtt gtattctgaa attacatgtt aggtataatt attagttgta caattagcta 26521 ttaaattttc agaaagtttg aagggagaga gtaagtgaaa agccactcct gctaaagatt 26581 agacactgta ctagatgctt ggaggattaa gaggagacca gagctaaggg aaatctttgt 26641 ctggatttta ttgacccatt ttggaattag ctgcagatct taagaaatca gattgggttg 26701 gcaggacgtg gtggctcatg tctgtaatct cagcactttg ggaggctgag gcgggaggat 26761 cacgaggtca agagattgag accagcctgg ccaacatggt gaaaccccgt ctctactaaa 26821 aatacaaaaa ttagccaggt gtggtggcgg gcacctgtag tcccagctac tcaggaggct 26881 gagacaggag aatggcatga accccggagg tggaggtttc agtgagccaa gatcgtgcca 26941 ctgcactcca gcctggtgac agagcgagac tccgtctcaa aaaacaaaca aacaaaaaac 27001 agtttgggtc aataatttgc gtttacagta ctagtttatg tacttatttg tcactttgta 27061 atttttaggg agttgctttt caactgtaat ctgtttttat taaactattt gaaatgagct 27121 ttgtattcag tcactagttg tgtttctccc ttccaagtac taaccaggct caaccctgct 27181 tctgagatca gacaagatca ggagcgttca gggtggtttg gttgtagaca ctaattgctt 27241 tgcttataag ccttaaggat accttacaat taataacttt ggtaaaacct catttgaaaa 27301 ataagactaa gcctgagcca aacggcaaaa tcccatctct acaaaaaatt agccgagcat 27361 ggtgttgggt gcctgtggtc ccagctactc aggaggctga ggtgggagga tcacttgagc 27421 tcgggaggtt gaggctgcag tgagtcaaga ttgtgccact gcattccagc atgggtgaca 27481 gagtgagacc ctgtctcaaa aaaaagaaaa gaaaaaagta agactaaaat cagttagtga 27541 caaaacttat gtattacagc atcttgactt aaaaaaaaaa caaaccaaaa cccttcattt 27601 tccgagtttt tcataagcca ggtattttct taaatttgaa gtcttagaaa cttaggttat 27661 tttggctcgc tagctgtatg tgtagagtga aaatagtatt tctacatttg gtcagttgca 27721 taatgttact atacctttga tccatctgga atttatcttg gtatgcagtg tgaagcaagc 27781 atcggattct gcttttttct aggtgactac ccagttgtcc catgacttga ttttaaaaag 27841 cagctttatt atataccaaa tgtttatatt ttgggtattt ttagggattt ctactttgtt 27901 ccactaatct gtatttgtac cagtacgaca cagctttcag tattgtatct tctaatattt 27961 taatatctga taggctagtc tccattttct tcggagattt tttctttttt ttctgcgttt 28021 gcttaattgt tatttctttt tttttttttt ttttttgaga tggaattttg ctcttgtcgc 28081 ccaggctgga gtgcaatggt gtgagctcag ctcactgcaa cctctgcctc ccaggtttaa 28141 gtgattctcc tgcctcagcc tctggagtgg gttaggatta cgggctcccg ccacagctaa 28201 tttttgtatt tttagtagag atggggtttc accatgttgg ccaggctggt ctcaaactcc 28261 tgaccttagg tgatctaccc acctcagcct cccaaagtgc tgggattaca ggtgtgggtc 28321 accacgcctg gccgcttaat tgttatttct acatgaactt tagaatcagc ttgtctaatt 28381 aaaaaaaaaa aaaaagaaag aaaaacatgg tacttcagtt ttcatttgca tcatggcaaa 28441 tttgtatagc aacttagaat tgatcttaat aatgttgaat atcccgctca agagtattgt 28501 atgtttttct aattgtctaa gttttctttt gtgtctttca gtacaggttt agttttctcc 28561 ctttatgtct tcagtttatt cctagttctt aactgctgtg gtatattggt atgttgtgtg 28621 tttctcttat atttcttttt atttatttct tttttttttt taaagagatg ggatcttgca 28681 atgttgccca ggctggactc gaactcctgg gcttaagcag ttctcttgtt tcagcacatt 28741 atatttatta tctggccatg tatatatgaa ggctgtttca gagtattaat ttgtttcaga 28801 atattaattt tgtatccaga aatagtttat cgtattctct tgtcagtttt tgggttttcc 28861 tgggaaagtt tcttatctga gtttttatgc tttttatttc tcccatctaa tatgaaataa 28921 caatgctaag caattgtgat aataatggat atctttgtct tgttgctgtc cttcgtggga 28981 atacttctgt agtctcccca ctagtcagca taatctggta tttggtatta gttttttgtt 29041 ttttaaaact catgtaaagg agatagccat atgatttttc ttctttgagc attaatgctg 29101 aattatatca gcagaattgt taatgttggt gttggactgt atttatgcaa aaaatctcac 29161 ttggacatgg tttattccta aaatgtactg gtaattttgg ttgttacttt tttttttaat 29221 caaggatttt catatctgta tttatatatt caatttttcc gaagtttttt ggcaccaata 29281 ttatactcat tttgtaaata caaattagac acttttctat attttggatc cattaaaatc 29341 ccactggagg ccgggcacag tggcttatgc ctgtaatccc agcactttgg gaggccgagg 29401 caggcagatc acctgaggtc aggagttcga gaccatccta gccaccatgg tgaaaccctg 29461 tctctactaa aaataaaaaa attagccggg cgcggtggct cacgcctgta atcccagcac 29521 tttgggaggc cgaggcgggc agatcatgag gtcaggagat tgagaccatc ctggttaaca 29581 cagtgaaatc ccgtctctac taaaaataca aaaaaattag ccggacgtgg tggcgggcgc 29641 ctgtagtccc agctacttgg gaggctgagg caggtcaatg gcgtgaacct gggaggcgga 29701 gtttgcagtg agccgagatg gcaccactgc actccagcct gggcgacaga gtgagactct 29761 gtctcaaaaa aaaaaaaaaa aattagctgg gtgtggtggt gggtacctgt aatcccagct 29821 actcgggagg ctgaggcggg agaattgctt gagcctggat ggtggaggct caagcctgga 29881 tggagtgagc cgagatcaag ctggttatta actcagcttc gtattttagt tctatttttt 29941 gaggtggagt ctcactctgt tgctcaggct ggagtgcagt ggcgtgatct cggctcaccg 30001 caacctccgc ttcctgggtt caagccattc tcctgcctca gcctcccgtg tagctgggat 30061 tacaggcata tgccatgaga cctggctaat attttttgta tttttagtag agatggagtt 30121 tcaccatgtt ggccaggctg gtctcaaact cctgacctca agtgatctgc ctgccttggc 30181 cgcctcccaa agtgctggga ttacaggcgt gagccaccac gcctggcctg actcagctac 30241 ttctgatttt ggaaatttat catggttttc tttgtgtcct aatatttaca caagttttgt 30301 aacatgttct ttgggtattt taaaaagtgt gtcctgcttt caagatgctt ttgatctaac 30361 aatataaatt aaatctagat tagagctaaa ttagatttgt tatctatttc tgcctagttt 30421 ttgtttccta tcatttttgc cttttgagtg gtaatgctat gttatttggg gcaattcagt 30481 attgatattc agtggtttgc actctttatt gttaataaag taaaacacaa tggtttgcac 30541 tctttattgt taataaagta aaacacctgt tacgtactcc atgaagtcta taacctagag 30601 tcatttgtat gcttactggt ctagaaacca ctgatttatt ctttgttctt ttttctctct 30661 ctgaagtcgt tatctttcat tagttaccta tttttttttt tgtttgtgtt ttttccagcg 30721 ctctgttgaa ttacatatta ttgtatttcc ttttaatcct cttataggtg cttgttgcat 30781 accagttttt ttcccccaat tacaaagtat gtgtgtgttg atcatagata aatgtatgca 30841 tctatgctca gtacaggaaa cctggaaaac agcacaaaag gaaaaaccaa aaggctaaat 30901 ttgtgatccc accattataa aaatatctta tccttatttg tttccatatc cacataaaca 30961 tacttctctc ttttccacaa tgtcagggcc tattaaatct tgtttctata cgctagtgac 31021 tccaattttc ttttttaatt tgcgataaaa cgggtgctta ggaaaatctg aaccagtatt 31081 ttattccagc tgaattaaaa gaaattgatg cataaagaaa agcttactgg agctattgaa 31141 atgttcctca gtatgaatca gggtactgtg acctcttgtt ggccttccat tttcagccaa 31201 gtacgccgat gtgcagcgga cagtaataag ggggctaagt tttgcaaaca cagagcttgt 31261 ggacagtgat agatcctgaa agtagtgtct cttgctaatt ctcagactta acctgactga 31321 aagcaaggcc tcacaaattg aattcagtct tcacagttac gttcttaact aacacaaaaa 31381 aatattctaa tactattctg ttcaaataaa aatttacgtt ttctggccag gtgcggtggc 31441 tcacacctgt aatctcagca ctttgggagg ccaagatggg tggatcacct gaggtcagga 31501 gtttgagacc agcctggcca acatggcaaa aacccatctc tactaaatac aaaaaaatta 31561 gctgggcatg gtggtgtgtg cctgtagtac cagctacctg ggaggctgag gcaggaggat 31621 tgcttgaacc tgggaggcag agggttgcag tgagccgaga ttgtgtcacc acactccagc 31681 ctgagtgaca gagggagact ccatctttaa aaaaaaaaaa aatttttttt ttttacatgt 31741 tttctatagg tatactagtt tactggtaga gtggttggaa atgttttgcc tttttgaaag 31801 ttatatttaa ttcaccaacc tttttttttc tttaaacaca tcgttgctgg tttgaataat 31861 ttgtcaacct ttgtggagct ctttctgcat gtcaggcagt atataaaagt atcttttgtt 31921 aatttttata ataagaatca tttgagatta ctttatatgt ggtatagggg aatggtttcc 31981 aggcttgggt tttatggatc agtaagaaaa aaaatgggat gatgaacata tgattgctag 32041 cttttaattt tgtcaatata gacattggta tcatttatct tgaatactca atatttaatt 32101 tggaaacatg aatatttttc cttcatttat tctctcatta tagatttatg gtctatctag 32161 agttggctgg ttattcagtc tactaccttt attttactga tgagggagct gaggctccaa 32221 aatgcctttg agaccccaga gagaagacag aaaacttctt tctcatgaat atattatagc 32281 ctaatattga catgttacta gcagttacaa acataaatgt tgctatgact tgtatgggag 32341 ttaatttttc ttcaggcttg aaatgtgatg attcccaaag ccatcttttt gtgtaaatta 32401 gataatttcc tggattaatt tgtatcttgg gaaattactg attgcagcta ttattttaga 32461 agtgtgactg atagcttact gagattctga gttgctattt tgttttggga agcattttta 32521 gagagtatta taataatttg acagatatgc aaaaggaaag gaagtgaaaa tgagagaaca 32581 ttaatacagt tgcccttggg ctttctcctc cccttctcag agggcctggt ccttcgcaag 32641 gtgtttttta ttattacttt ttaaaaattt cctggtctcc tatccctgcc tccttcaact 32701 tttccttttc ccatttcttc ccattcctta acccatctct tcttctttca ttctggctcc 32761 tccttctctc cctgcatttc tcttgttacc tgtttcccca tctcttttta ttcttagatt 32821 tattctttgt tcttttttct gatgtcttta accagtaatt cagattttaa gttaccgggt 32881 tttcttcctg tagcatttga actgaagtga tactgagctg gaaaaataga atatcactga 32941 taagttgttg gttttgggat ttctaagttt tattagtgta aaagttcttg aattatgaat 33001 gtgtgttcca atccatacca ccagctaaaa aagaaaaaga atatgtttta ttgttatttt 33061 tgattgatac tttcccttta atagaagcat atacttattt tagtggaaat ttatgattga 33121 ttataaattt ttttccctgt gattaattat agctttacat tagtggcaaa catttctttc 33181 agtcttgttt tgtgcacatt ttttacataa ggcaatttaa tttaatttat taaaacattt 33241 taaaatttat aattattggc caggaatggt ggcttatacc tgtaatccta gcactttggg 33301 aggccaaggc aggaggatag cttgagttca gaagttcaaa accagcctgg ttaacatagt 33361 gagacctcat ttctattaaa acacacacac acacacacac acacacacac acacttatag 33421 ttattttcta tttattatat gctttttaaa aataaaataa tttttttagt tccatgtcta 33481 ctcctatgtt atgtgccttt agtagacact cttagtgcca tcacaacgtt atcttctgga 33541 ttttccatta tttaatcatt tttccataat tgagtattta gataatagga gagtattgta 33601 aaataaatgg gtttagggaa aatacaaccc tcctagctta aatcaggagg aattggatac 33661 cctgaacaga ccaataacaa gcagcaagat tgaaatggta atttaaaaat taccaacaaa 33721 aaagtccagg accagatggc ttcacagcag aattctacca gacattcaaa gaagagttgg 33781 taccaatcct tctgacacta ttccacaaga taaagaagga accatgccta attcattgta 33841 taaagccagc atcaccctac caaaaccagg aaaggacata accaaaaaag aaaactacag 33901 actaatatcc ttgatgaaca tagatgctaa aatccttaac aaaatactag ctaaccaaat 33961 ctagcaacat atcaaaaaga taatccacca tgatcaagtg ggtttcatac cagggatgca 34021 gggatggttt aacatattta agtcaataaa tgtgatacac cacataaaca gaattaaaaa 34081 caaaagtcac atgatcatct caatagatgc agaaaaagca tttgacaaaa tccagcatcc 34141 ctttatgatt agaactctca gcaaaatcag catacgaggg acatacctca atgtaatgaa 34201 agccatctct gggccaggtg cagtggctca catcacacct gtaatcccag cactttggga 34261 ggccgaggca ggtggattac ctgaggtcag gagtttgaga ccagcctggc caacatggtg 34321 aaaccccgtc tcatctaaaa atacaaaaat tagctcggca tggtggcagg cgcttgcaat 34381 cccagctact caggagactg agacaggaga atcgcttgaa cccaggaggt ggaagttgca 34441 gtgagccgag atcgcaccat tgcactccag catgggcgac agagcgagac tctgtctcaa 34501 aaacaaaaac aaaaaaaccc aaccatctat gacacaccta cagccaacct aatactgaat 34561 ggggaaaagt ggaaagcatt acctgtgaga actagaacaa gacaaggatg cctgttctca 34621 ccactcctct tcaacatagt actggaagtc ctagccagaa cattcagaca agggaaagaa 34681 agaaagggca tccaaactgg taaagaggaa gtcaaactgt cactgtttgc tgacaatatg 34741 atcgtttacc ttgaaaaccc taaagcctcc tccagaaagc tcctagaact gataaaagaa 34801 ttcagcaaag tttctggata caagattaat gtacacaaat cagaagctct tctatacacc 34861 aacagtgacc aagcagagaa tcaaataaaa aactcaaccc cttttacaac agctgcaaaa 34921 aaaaaaaata cttaggaata tacctaacta aggagtcaaa agacctctac aaggaaaact 34981 acaaaacact gctgaaagaa atcatagatg gcacaaacaa atggaagcac atcccatgct 35041 catggatggg tagaatcaat attgtgaaaa tgaccatact gccaaaagca atctacaaat 35101 tcaatgtaat ccccatcaaa ataccaccat cattcttcac agaattagaa acaacatttc 35161 taaaattcat atggaacaaa aaaagaacct gcatagccaa agcaagacta agcaaagaga 35221 acaaatctgg aggcatcaca ctacctgatt tcaaactata ctataaggcc atagtcacca 35281 aaacagcacg gtactggtat aaaaataggc acacagacca atggaacaga atagagaacc 35341 cagaaataaa cccaaatact tacagccaat tgatcttcga caaagcaaac aaaaacataa 35401 agtggggaaa ggataccctt tttaacaata gcactgggat aattggctgg ccatatgtag 35461 gacaatgaaa ctggatccta tacaagatag attaaggact taacgacctg aagctataaa 35521 aaattctaga aggtatcatt ggaaaaaccc ttctagacat tggcttaggc gaggatttca 35581 tgaccaagaa cccaaaagca aatgcaataa aaacaaagat aaatagctgg gacccaatta 35641 agctaaagag cttttgcaca gtaaaaggaa tagtcagcag agtaaacaga caacccacag 35701 agtgggagaa aatcttcata atgtatacat ctgacaaagg actgatatcc agaatctaca 35761 accaactcag taagaaaaaa acaatcccat caaaaagtgg gctaaggaca tgaatagaca 35821 gttctcaaaa gaggatatac agatggccaa caaacatgaa aaatgcccaa catcactaat 35881 gatcagggaa gtgcacatca aaaccacaat gtgataccac cttactcctg caagaatggc 35941 cataataaaa aaataaaaaa acagtagatg ttggcgtgga tgcagtgatc aggcaacaca 36001 tttacactgc tgtgggaatg taaactagta cagccactct ggaaaacagt gtggagattc 36061 cttaaagaac taagagtaga actaccattt gatccagcag tcccactact ggatatctac 36121 ccagaggaaa agaagtcatt tgaaaaagat gcttgcacat gtatgtttat agcagcacaa 36181 ttcacaagtg taaaatcatg gaagcaaccc aaatgcctat caacgaatgg ataaactgct 36241 gtgtgtatat atgtatatat gatagtgtgt gtgtgtgtat atatatgtat ataagtatgt 36301 atatatgtgt catatatata tatatatgat ggaatactgc tcagctgtaa aaaggaatga 36361 attagcattt gcagtgactt ggatgagatt ggagactgtt ctaagtaaag taactcagga 36421 atggaaaacc aaacatctaa tgttctcact gataggtgaa agctaagcta tgaggacgca 36481 aaggcataag aatgatacag tggactttgg tgacctgggg ggaagggttg gaggggggtg 36541 agggataaaa gaccacaaat agggtgcagc gtatactgct cgggtgatgg gtgcaccgaa 36601 atctcacaaa tcaccactaa ggaacttatg taactgaata ccacctgtac cccaataact 36661 tatggaaaaa taaataataa aaaaagggtt taaaaaagta taggaagtta ttggtgaaac 36721 gtgtgtacat atatatatgt acacacacac acacacatac atgtatgtac atatatataa 36781 aagatgttct gggggaagaa aactgaccct aaataggtat taaaatgcct ttgagctttg 36841 agagactgaa gttttgctgc atagtggacc ttagaaaaat ctttatgtgc ccaaatatag 36901 atttctcata ttcttaagga ctgtggaaaa taatatataa tatgggaaaa aaatatttta 36961 tcagctctat tgaggcatga tttaaaaaat tttacattat ttactaataa agagaactgt 37021 attttatttt tcatttacta ttccttgtgg tcttctatct agttgttcac tgcctcattg 37081 cctctctgat tttcaaactt ggaagttatt gacacgcgtc tttctggccc tctcctctgg 37141 agcacctctc tgctgccaat ttttttctgc catttttttt tttgtaattt aatttaaaaa 37201 aaaacttact gaggtgaaat tcacatagca tgaaattaac catgttaaaa tgatcaattt 37261 agtggcattt agtactctca tagtgttgtg taatcaccat atctggttct gaaacctttc 37321 cttcactcca aaggagaccc ttaccaacta cacagtttct ctttcccaga cccaggcaac 37381 cgccagtctg ctttcagtct ctttggattt tccctttctg gacacctcat atgaatggca 37441 tcacacaata tttgtgaact tttatgtctt ctttttttta ttttaaattt tttttagagc 37501 tggggtctca ctctgttgcc caggctgcag tgcagtggtg cgatcatagc tcactgcagc 37561 cttttaccac ctggactcaa gtgaacctga ttagctggga ctacaggcat gcaccaccac 37621 accatgctaa tttttttttt tttttttttt ttttttttga gactggctct cttgcccaag 37681 ctggagtgca gtggcttaat catggctcac ggcaaccttt gcctcctggg ctcaagccat 37741 cctcccacct cagcctccca agtagctggg actacaggtg cgtaccacca tgctcggcta 37801 atgtttgtat gtttagtaga gatggggttt ggccatatta cccagggtgg tctcaaactc 37861 ctgagctcta gttaattttg aaatatagaa tatattattg ttaactgtag tcaccttact 37921 gtacaataga ataccagaac ttattcttcc tatgtgatgg taactttaat cattcatcaa 37981 cttctcccca tcccactccc tgcccttccc tttactaccc tccccacctc tccggcctct 38041 ggtaattact attatactct ctactctttt gggaagggtg tttgctattt atttatgata 38101 aatattccac atctgtgatt ctccacagtc agaagttctt tgagaggatg ccatcagcct 38161 tggtcagtca gagaatggag tcatctgatt taccatcctg tgaatggccc taggcagata 38221 gcataggtga ttgttgttac tgttttgaga cagaatctct gttgcccaag ctggagtgca 38281 gtggcatgat ctcagctcac tgcagcctct gcctcctggg ttcaagtgat tcttctgcct 38341 cagcctccca agtagttgag actacaggca tgtgccacca tgcccggcta atttttgtat 38401 ttttgtagat actgggtttc accatgttgg ccaggctggt ctcgaattcc tgacctcagg 38461 tgatctgcct tcttcggctt ctcaaagttc taagattaca gtcgtgagcc accgtgcctg 38521 gctggtggcg gtggtggttt ttttaatttt tttaattttt gttttttcaa cttttatttt 38581 gattcggggg cacatgtgcg gtttgttacc tgagtaacaa aaaaaaaagc ctctgccttt 38641 atgagataaa cttttttata ttctgtatga gtaagatcat acaaaatttg tctttctgtg 38701 tctggcttgt ttcagttaac acaaagtcct gcagattcat tcacattgtc ataaatgaca 38761 ggatttcgtt cttttttttt tttttttttt tttttttttt tgagacagac agtcttgctg 38821 agttgcccag gctggagtgc agtggcacca tctcggctca ctgcaacctc cgtctcccag 38881 cctcaagtga ttctcatgct ggatttcatt cattttttta tggtgaacag ttgagccttt 38941 taaaaaagcc aagcatctgg ctgggtgcag tagctcgcgt ctgtgatccc agcactttgg 39001 gaggccgagg cgggtggatc acaaggtcaa gagatccaga ttatcctggc caacatggtg 39061 aaaccccgtc tctactaaaa atacaaaaaa ttagccaggc gtggtggcgg gggcctgtag 39121 tcccagctac tccggaggct gaggcaggag aatggcttga acccgggagg cggtggttgc 39181 agtgagccga gatcatgcca ctgcactcca gcctggtgac agagcgagac tccatctcaa 39241 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aagccaagca catccattta 39301 aattttggtg gaacttcttt ataacattcc tggtgtcagc atattctagg tgttgtttgg 39361 aaaattattt attaaatcaa agactcagcc tctctcaagt tctttacctc aacaacagtt 39421 gcttcttcaa acagctgata tcacacaatt tttattgtgg ttatagaaat gaaacactaa 39481 ttttctgcat atgtctgtga gcatattcac aactcattgc tggaagttta gataccgcta 39541 ctttcccaat ttaggaaaac agatttctgg tatgtgccta cgtgggaagg ttgaggctgc 39601 taagagtctt gcttctgggt tttagaagtc tcactgtgag accgggtgca gtggctcatg 39661 cctgtaattg cagcactttg ggaggctggg gtgagtggat cacttgagcc caggagttta 39721 agaccagcct atccaacatg gcaaaacccc gtttctacta aaaatacaaa ataattagcc 39781 gggtgtgatg gcacactcct gtaatcccag ctactcaggg aagctgaggc atgagaattg 39841 tttgaacctg ggaggcagag gttgcaataa agattgtgcc actgcactcc agcctgggtg 39901 acagcaagat gcctgtgaat gttttctgca catataaaaa ggttaaatca ttttcagtat 39961 ggtttgctgg ttctttgaaa attgataata catacacttt ataaatttaa ttctccatga 40021 actacaatat caaacagaat acactctatc tctacaccat gctgaaaaat ggaaactttt 40081 tttttttctt tttggtaaca ggatcttgct gtgtcgccta ggctggagtg cagtggcaca 40141 atcactggtc actgcagcct tgacttcctg ggctcaggtg attctcctac ctcagcctcc 40201 cgagtagcag ggactacagg catgcaacac cacaccggca aattttttgt atctttgtag 40261 agatgaggtt tcaccatgtt gcccaggctg gtcttcgaac tcctgggctc aagtaatcca 40321 cctgcctcag ccttccaaaa tgtgggatta catgtgtaag tcaccacgcc tggctgaaaa 40381 cagaaacttt tcaagaaaag ttttaaaaga aactaccaaa ttaatagatg ttaccatata 40441 acgttcagaa atacaaatat tttatgaatt atttccagtc ctattacttg gaggcaacca 40501 ttgtaaatat tttttatgta caagttgagt atccctcatc taaatccttg gcaccagaag 40561 tgtttcagat ttcagatttt ctaacttttt gcctcatact tattggttca gcatccctaa 40621 tctgaaaaaa atgcccctgt gaacattgag ttttgtcttg tctccctccc ctcccctcct 40681 ttcctctcct ctcctctccc ctcccttccc ctcccctccc cctccccctt cccctcccct 40741 cccctcccct ctcccctccc ttctcctctc ccctttccca tctccacttt cccctctccc 40801 ctcccccttc cctctccctt ccccttcccc tcccccttcc ctctcccttc ccctccccct 40861 tccctctccc ttcccctccc ctctcccttc ccacctcccc cctctcccct cccctcccct 40921 cccttctccc CtCtCCCCtC tcccctcccc tcccccttcc ctctcccttc ccctctgctc 40981 ccctcccacc tccccctacc cctccccctc ccctcctctc ccctcccctt cccctcccct 41041 ccccctcccc tctcccctct cccctcccct tccctccccc tcccctcccc tcttccctcc 41101 ccctcccctc ccctcccctc ccctcccctt tcctctcctg ccctctcctc tcttttctta 41161 gacagggtct tgtgctgttg cacaggctag agttcagtgg caccttctta actcactgca 41221 gcctcaaatc ctgagcttaa gtgatcatct cacatcagcc tcccaagtag ctaggacaat 41281 aggcatgcgt caccacgccc agcgaatttt tgattttcat taattaatta tttttgtgtg 41341 tgagacaggg tctcgctctg ttgcccaggc tgtagtgcag tggctcactg caacctcaat 41401 ctcccgggca caagcgatcc tcccatctca gcctcccaag tagctgggac tacaggtgta 41461 tgccaacaca cccagctaat tttttcaatt ttttataggg acagggtctc cctatgttgc 41521 ccaggctggt ctcgaactcc tgtgctcaag tgatcctccc agctcggcct cacaaagtac 41581 tgggatatag gagtgagcca ccaagcctgg cctcggctaa tttttaattt ttttaatata 41641 gagatggggt ctcactatgt tgctggtctc aaattcttga gctcaagtga tcctcccacc 41701 tctgccgccc aaagcactgg gattacaggc atgagccaca gtgcctggat cttttttttg 41761 gactttggga gtgagacgtg aggattctgt cagccctaaa accttaggtc ttctggtttc 41821 ttttttgtac tctcaatttt cttcgtacta ccttcaaggt ataatgtgcc taagaaatat 41881 ttccttgttt ttcaggttct aaatggcttc taagaagttg ggtgcagatt ttcatggtaa 41941 gtggactatt agaatcatgg tttaagttat gatttgatca gaggatttgg aatcacttag 42001 catagtttta agtacaagtg cccctttttg tctcttttaa tgaacagctt gacaaaaata 42061 aagttgtaca acaatgtatc aagtatatct gaaaggtctt cagagaactt gaagtatttc 42121 ttcaaatggg tagttaaact ttagagtgtc tctggttatt gtacttgaaa ctactagtga 42181 tagattagga gtctgcctgt tctggttctt cagcagcatt gaaggtatag ttactggctg 42241 ggcgcggtgg ttcaagcctg taatcccaac actttgggag gctgaggcgg gtggatcacg 42301 aggtcaggag atcgagacca tcctgtctaa cacggtgaaa ccccgtctct actaaaaata 42361 caaaaaatta gccgggcgtg ttggcgggcg cctgtagtcc tagctactcg ggaggctgag 42421 ggcagaagaa tggcgtgaac ccgggaggcg gagcttgcag tgagccgaga tcgcaccact 42481 gcactccagc ctgggggaca gagcgagact ccattaaaaa aaaaaaaaaa aaggtatagt 42541 tacctttccc tttcctgttc cttggtcata gctcttctcc atatgtgtgt gtgtgaaaaa 42601 ttctaaccct ttggttaatg ctttataatg ttttagttta gttccctgtg ccccatcaaa 42661 ggaagtaatt tactacaaat caatcactat attacagagg tatcaatact gatatttata 42721 attttgaatt tgaaattaag agtttctctg tcatcttttc ctaagataca accttccaaa 42781 atggaataca ataataaact actatctgcc tttggagggg aatggaggga ggtggaggta 42841 gctaagaagt taaaattata cccttttttc atacagtaga aggtaagaat ttaaaaccag 42901 aagtagccaa gtaacacaat tattttaaga gaggttttaa agccaatctt ccatgttaaa 42961 aattatatca gccaggcaca gtggcatgtg cctgcagtcc cagctactca gggggctaag 43021 gtgggaggat tgcttgatcc cagaagtttg aagttgcagt gatcatgcca ctgcactcca 43081 gcctgggtga cagagtgaga ccctgactca aacaaaaaaa atacatatat atatcacatc 43141 attttaagaa tgatttacta gactgcctgg ggacgtgttg gtatgctctt ctgagctgcc 43201 agtggttgct tgataagatc actcagctga gtcagagtgg tgtagcggtg attgtgattt 43261 tacttgctta tatattttac acaacagcct ttgaattttg atttgtcagt gtgcatatac 43321 ttacatagat aacctgcctg agtttttaaa aatccttttg aagttattca aactctggaa 43381 ttagcattcc ttaaagaggt caggcatctt atttttcagg tgagctattt cctattattt 43441 atggattatt acaggtcttc ttaaaagtat tcaaatgata gtagaaaggc agatctgggc 43501 agggcacggt ggctcatgcc tgtaatccca acagtagatt aggaggttga ggtgagagga 43561 tcgcttgagg ccaggagttc aagaccagcc tcagcaacat agtgagacgc tgtctctaca 43621 aaaaaatttt aaaaattagc tgggtatgct ggtatgtgcc tgtagtctca gctactcggg 43681 aggctgagag gtgggagtag tgctgagccc gggaggtcaa ggctgcagtg agccatgatc 43741 acgccactgc actccagcca gaatcacatg agagcctgtc tcaaacaaac aaacaaaaaa 43801 tgattcttgc cactgagctt aagaaaagaa aaagggaaaa aaaggcagat ctgaattccc 43861 tctagatcct accttttcaa gggagaaaag agaggacaga gccaagggca gaggaaaagc 43921 ttagggagag aaaatagcaa aaatgaaaaa tttacactta tttcaaaaga tagactttct 43981 gttttgaatc tttggaacat ctgttttgat cagactgaaa atagttggac cacatgtttt 44041 gtgtttcaac tgaacattcc agagagaaga ttataattct gaaggtgtct gttcataaag 44101 actggtattt cccatatctc cttaaattcc taaccaggct gggcatggtg gctcacgcct 44161 gtaatcccag cactttggga ggccgaggcg ggcggatcac ctgaggtcag gagttcaaga 44221 ccagcctgac caacatggag aaaccctgtc tctattaaaa atacaaaatt agccgggtgt 44281 agtgacgcat gcctgtaatc ccagctactc gggaggctga ggaaggagaa tcgcttgaac 44341 ctgggaggca gaggttgcag tgaactgaga tggtgccaat gcactccatt ccatctcaaa 44401 aaaaaaaaaa aaagaattct taaccaaagt gttattttgt ttgtttgttt gtttgttttt 44461 gagacagagt ctcgctctgt cgcccaggct ggagtgcagt agcacgatct tggctcactg 44521 caagctctgc ctcctgggct cacgccattc tctgccttag cctcccgagt agctgggact 44581 acaggtgcct gccaccatgc ccagctaatt tttttgtatt ttttagtaga ggcggggttt 44641 taccgtatta gccaggatgg tctcgatctc ctgacctcgt gatccgcctg ccttggcctc 44701 ccaaagtgtt ggggttacag gcgtgagcca ccgtgcccgg cccaaagtgt tattgtttaa 44761 atgaaacttt ctacctgagt aaaatttagg agaaaaattt tctttctccc ttaggctgct 44821 tctcctttaa cagggctctt tttgctgagt tcctggttga atttcactgg ctttacttgt 44881 tttttttgtt tttgtttttt tgttttttgg gttttttttg ggaacaaaac aataaacacc 44941 ttcattacat gggtgaagac aaaacgagga tttatttgcc tttccggacc ttgattttcc 45001 taagatagaa ctccaactcc ttcccctcta gcacatagcc gtctgtttgg ccacactgtc 45061 ccggccttga agtgatgcac gcaagaagct tgccctgctg gaactgctcc tccaggagac 45121 tgctgatttt ggcctttttt tttcctttca tcgatttctt ctgaaatttt ttagatcatt 45181 ttttgtttaa aatctcttct tcctcaggag tcagcttgtc tcctttcttg tggcccaggg 45241 gtagcgcata gtgggactcg taccactgtc catacggtgt gctgtcgatg agtatggtgt 45301 gctgtcaatg agcatgaagc aattcttcac cagggtcttg ttatggacca gctcgttatt 45361 aaacgcattg tagacaacat cgatgatcct tgttttgcaa atacaacgct gagccccagg 45421 agaaattccc tacatgcagc cttagggcac ggtatttctt gttacctcct caaacatgga 45481 ctgtgtggac gtggcagggg ccaatgttgg tgttggcagc tgggcacccc tgctcatact 45541 tccgcttctt gtggtagggc tttctcttgc ccccggtctt gcggtgcttg tgtcagttct 45601 cccgagagat gcccattgct cggcgctggc tggaaagagc tagttgggtt tttagagtaa 45661 agtatcctat ttttagatat caggtagttt aaataataga aatgaaaggc agtagatgga 45721 gaatatttaa taggcaattg tgcaggcatg tgagtacaga aaataaagtt tctgctctct 45781 caaggctaaa attagaaagc caggaactaa ggctactcct tccacttctc aggaagctgc 45841 ttagcctttc ttcactatat gtctctgtgt ctgtgatcca ttattattca gttcatagac 45901 ttgctttctt ggcctaatag tgtagagtac acagggctat aaatggccac cccagttaag 45961 gtgctgcttg ctacttgatc tttcagtttc agtggctagc atcatagaga tagagatttt 46021 atatcttttg gtcaaggcaa actgatggca tagccttctt agcttatgag cctgtggatt 46081 ggtcctccaa agttagatat ctgactgatc cattgagagg ttaatggatg tgggtaactt 46141 ttctgtggca cttggggtgt ggacaaggag gtaataactg ctgtcatgac ggatattaaa 46201 atgtattttt ttttcttttt aatatttggg actgactagg tctggtgctg tgctgtcttt 46261 gactagcact tgatttacct ctgagttcat tattttcaac taaagttgaa caacttcact 46321 gttactagtg accatatatc cctaaataac agaataaggg aaacaaacag ttatttctca 46381 aacttctaga aatattatcc atttcttata aaaactccat acccttcgct tagtttggat 46441 aaggactttc ttggagcctg gtgctttaaa atctcaatcc tctggctggg cgcggtggct 46501 catgcctgta atcacagcac tttgggaggt tgaggcaggc ggatcatgag gtcaggagat 46561 ccagaccatc ctggctaaca tggtgaaacc ctgtctctac taaaaataca aaaaaaaatt 46621 agccgagcat ggtggctggt gcctgtagtc ccagctactt gggaggctga ggcaggagaa 46681 tggcgtgaac ccaggaggaa gagcttgcag tgagccgaga tcacaccact gcactccagc 46741 ctgggtgaca gagcaagacg ccatcttaaa aaaaaaaaaa aaagaaaaga aaaaagaaat 46801 ctcaatcctc tttcactgat tcttattttc atctaggaga aaggttattt atctggaaaa 46861 tgtgttgaga ctctaattta tgaaaaaata gaccccgtga taagatttga taaaactagc 46921 ccccctcctt aaatcacttt atcgaggtat aattgatata taaaaagctg tacatattta 46981 catgtttaat gtatacatct tgatgagttt gaggataagt atacactcat gaaaccatca 47041 ccatcattaa ggcataaaca ttcattacct tctaaagttt cctcctactc tattgtgtgt 47101 gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtggtggg ctggagggat 47161 tgcttgaggc caggagtttg agaccagcct gggcaacata gaaaaacccc gtttcttgtt 47221 tttttttttt tttttgagat ggagtcccgc tctgtcgccc atgctggagt gcagtggtgc 47281 catctctgct cactgcaacc tctgcctcct caagcaattg ctcaagcaat tctcctgtct 47341 cagccccctg agtagctggg attataggtg cgcaccacca tgcctggcta atttttgtat 47401 ttttagtaga gacagggttt cgccatgttg gccaggctgg ttttgaactc ttgacctcag 47461 gtgatccacc tgcgtctgcg tcccaaagtt ctgggattac aggcatgagc caccgcgcct 47521 ggccttgttt ctacaaaaaa ttaaaaatta gccaggtgtg gtggagcttc tctatagtcc 47581 tagctgctca ggagcctggg acaggaggat tgcttaagcc tgggagttca aggttatagt 47641 gagctatgat cacatatgca ttaggttgtt tggtattgtt ccagatatct cttaacactg 47701 ttcaattttt tttttccctt tttctctctt tgtatttcag tttggctaat ttcttttgac 47761 ctaacttcaa gtcccaagtc tttcttatgc tgattattgt cttcggatta gcatagcctg 47821 gtttatgaac catcaaaatt tttcttttct gataatgcat ttttcacttg tggcatttcc 47881 atttgtctct ttatatttcc atctctctgc tgaagttccc atctgctcac acatgtttag 47941 tttttccatt agacccttta acatatttat tattgttatt ttaaagctcc ctgtctgatg 48001 tttacaatat cttggctctc cctgaatttg ggtctgatga ctggttcctc tcttaattct 48061 ggatcacatt ttcttgcttc tttctgtgtc ttataatttt taattgaatg ccagacaatg 48121 tatattaaaa agctgatttt gtagattata tggcttgttt tgttgttagg atatgagtaa 48181 cattttcttg tgatttcaca tcttacaaca gatatctatg ttattttgta tatttatgat 48241 tttgccagtg ttacctggct tgtggcttta agatagtttt ttttgtttta tttatttatt 48301 tatttatttt taagtccttt gctgcaggag gcgctgatgg ctttaagatg gtgataacca 48361 gtcatggaat tacagatttt cacttaattt taagtttgat cttcttttta aaaaaacctc 48421 aatttgaatc tcctcacaat aatacctgaa ttaaatatta tgcatttgct atcctctatt 48481 ggggatttct ccccccacct ggaattgtac tttttttaga aaaaacgggc agattgcaga 48541 atcataggca tatcagcaat ttagaaagaa acaaagcaaa tgaagtcttc ccagagttta 48601 tcctgagtca ttacatattc agcccactca ctatcttcca ccaaagtggt atgcagaacc 48661 taacttcaag gaactgccac aaccgcagcc atgaggcatg cacttagtgg ctatttaaaa 48721 aattgaactt aggaaaatgg gtgcaaatat tttaggaaga ttttcagtaa taaggactga 48781 aactaaaagg tatttgaggt tgaggcagaa aattgttagc tataatattt taaattctag 48841 atacattgga taaaaatgga caattccact ttgggaggct gaggtgggag gattgcttga 48901 gtctgggaat ttgagaccag cctgggcaac aaagtgagcc ctcagctcta caaaaaaatt 48961 taaaaaatta gctgggcatg gtggtgcatg cctgtagtct cagctactca ggaggctgag 49021 gtgggaggat cacttgagcc caggagaatg aggctgcagt gagtggtgat gacgtcactg 49081 cactccagcc tgggtgagag agcaagaccc tgtcttaaaa aagaagctgg gtgcgatggc 49141 tcacgccttt aatcccagca ctttgggagg ccgaggtggg cggatcatga ggtaaggaga 49201 tcgagaccat cctggctaac acggtgaaac cccatctcta ctaaaaatac aaaacattag 49261 ccaggcgtgg tggcaggcac ctgtagtccc agctactcag gaggctgagg caggagaata 49321 gcgtgaaccc aggaggcgga gcttgcagtg agccgagatt gtgccactgc actccagcct 49381 gggcgacagt gcgagacttc atctcaaaaa aaaaaaaaaa aagaaaaaga aaagctaatt 49441 cctgaggcat aattggttat atttggaaga catatatttt gtaattctca cagcaccatc 49501 attaatagta cttacggtgg tctctatcca ttattacaaa tactgctttc tttgtttccc 49561 cccccccctt ttttttttgg agatggagtc tcagtcaccc aggctggagt tcagtggcgt 49621 gattttcgct cactgccgcc tccgcctcct gggttcaagt gatcctcctg cctcagtttc 49681 ccgagtagct gggattacag gcgtgcatca ccatgcctag ctaatttttt tttttttttg 49741 tatttttagt agagacgggg gtttcaccat gttggccagg ctggtcttga aatcctgacc 49801 ttgagtgatc tatccgcctt ggcctcccaa agtgctggga ttacagctgt gagccactgt 49861 gcctggccac gttcccatct ctttaaatgc ctttttgccc tttatcccta cctcctcctt 49921 ccagtaatca ctgaactatt tgttcattct ctcttaataa agcttttaaa gtacgaagac 49981 cctggagggt tgggggatgt actaaataaa tcaccttggc attggcttta ttagtgaaag 50041 gaaactgatt tttaaaccag atacctgcaa ttggtgctag tgaagggaaa cggcagccct 50101 ttaaaggtat tctacctcat cttttctgtc cctaagccag gacctagttt catttataga 50161 atataagttt gtcagtgtat atcacactgt agtagtttgc tacgtgaaag gtaggcaagg 50221 agatcacttc tcttatagga tttttatctt tttttttttt tttgagatgg agtcttgttc 50281 tgttgcccag gctgcagtgc aatgacgtga tcttggctca ctgcaacctc tgcatcccag 50341 gttcaagcga ttctcctgcc tcagcctccc gagtagctgg gattacaggc acacaccacc 50401 atgctcggct aatttttgta tttttggtag agatggggtt tcaccatgtt ggccaggatg 50461 gtcttgatct cctgacctcg tgatcctccc acctcagcct cccaaagtgc tgggattaca 50521 ggcatgagcc actgtactcg gcctgacatg gagtcttgct ctgtcactag gctggagtgc 50581 agtggagcca tctcggctca ctgcagcctc tgactccctg gttcaagaga ttctcctgcc 50641 tcagcctcct gagtagctgg gattacagga acgtgctacc acgcccagct aattttttgt 50701 atttttagta gagatggggt ttcaccatgt tggccaggat ggtctcaatc tcctgacctc 50761 atgatctgct tgcctcggcc tcccaaaatg ctgggattac aggcataagt caccatgctt 50821 ggcccctaat ttttgtattt ttagtagata cggggtttta ccatgttggc caggctggtc 50881 tcgaactcct gacctcaggt gatccacccg cctctgcctc ccaaagtgtt gggattacag 50941 gcgtgagcca ccacgctggc ctcatatctt tagcacagta cttttgtagt ttttctccaa 51001 ctccttgtta ccaatctaca atagaacttg gttatttaag aaaatttagt tgttattagt 51061 attattattg aaatggagtc tcgccctgtc gcccaggctg gagtgcagtg gtgtgatctc 51121 agctcactgc aacttctgcc tcccgggtta aagcaattct cctgcttcag cttcccgagt 51181 agctgggatt acatgggcac gccaccatgc ccagctaatt tttttgtatt tttagtagag 51241 acggggtttc accatgctgg ccaggctggt ctcgaactcc tgacctcatg atccgcctgc 51301 cttggcctct caaattgctg ggattacagg cgtgagctgc cgcacctggc caaatatagt 51361 tattattaat gtggatagat tttatcattt ttgtgtatca tctgtgctta ttagtgttaa 51421 acaaaagact taattgtgtg cttgttaact tgactgtaaa tctttccaat gattcttagt 51481 gccaaagttt cagctttgtt tccagttaat caggatacta gccaaagtta gaatctttca 51541 ataacatttg agtccctatt ttcaaagaaa acagacaaaa attctttaaa attagaaaga 51601 gcttttgttc taatctttta aaatataagc attttataag tgccatagag tttaagtgtt 51661 gtaatcaaca cttagttttg ttggtaagct gtgatggcac ccggcacttt tttggtggtg 51721 ctggggcggg ggatgacatc tgtctggtac atttttgtaa ctttgttcag atctcagaca 51781 tatgcttgta tcatgtatga gtaatctgtt aaaaacattt tcatgactag ccttgtcatt 51841 tttgggcaaa taattttcaa ttttcatatt aaatataagt tggaactggg tgtgatagct 51901 catgcttgta atcccagcaa tttgagaggc cgaggcggga agaatgcttg agcccaggag 51961 ttggagacca gcctggacga cataatgaga ccctgtctta gctactcagg aggctgaggt 52021 gggaggatta tttgggaccc agagggcaag gatgcagtga gccatgatag caccactgca 52081 ctccagcctg ggcaaatgtc tctttaaaaa aattatgtgt gtgtgtgtgt gtgtgtgtgt 52141 gtgtgtgtgt gtgtgtgtgt gtgttggggt cggggttaga ttgaatccag aagaagtata

52201 aaagcatgtc tttatttatt tatttatttt gagatggagt ttcactcttg ttgcccaggc

52261 tggagtgcac tggtgcgttc ttggctcact gcaacttctg cctcccaggt tcctgcctca

52321 gcctcccgag tagctgggat tacaggcatg tgccaccatg tctggctaat ttcttgtatt 52381 tttagtagag atggggtttc accattgttg gccaggctgg tctcgaactc atgacctcca 52441 cctgccttgg cctcccaaag ttctgggatt acaggcgtga gccaccggtc ccggccaaaa 52501 gcatgtcttt attattggtt cttcattcat tgaacaaatt ccatgagcca tacattatac 52561 tgagttttgg tgatatagat atgaaacatt tctcccctgt taagggacac tcaaaatttt 52621 tttgtttttt ttttttgttt gtttgttttt gagatagagt ctcgctctgt cacccagcct 52681 ggagtgcagt ggtgcgatct tgactcactg caagctccgc ctcccgggtt cacgacattc 52741 ttctgcctca gcctccccag tagctgggac tacaggcgcc cccactgcgc ccggctcatt 52801 ttttgtattt ttagtagaga cggggtttca ccatggtctt gatctcctga cctcatgatc 52861 cgcccacctc agcctcccaa agtgctggga ttacaggcgt gagccactgt gcccggccgg 52921 gacactcaaa ttcttgctca gactctctga agggaggtta aggaaatgaa ctattaacaa 52981 tacaaagagg taagtcctaa ggtggtagta ttaactggct attctgaggt tgagaaagga 53041 gtcgttcttg aggctcactg caccttccgc ctcctaggtt caagcagttc tcctgcttca 53101 gcctcccaag tagctgggac tacaggcgtg tgccactacg cccggtgcag ttttgtattt 53161 tcagtagaga gggggtttca ccatgttggc caggttagtc tagaactcct gacctcagat 53221 gatccacctg ccttggcctc ccaaaggggt gagactacag gcatgggcca ctgcacccgg 53281 cctgagtttc ttcttaaagg gctaaataat aattactcaa gtaaataagg gtaggaatgt 53341 gataggcatt gccagccatg aaattagcac atctaaaagg atgaccacac ttggaaaatt 53401 cctagggtgt taataaatat ggtgatttcc tagagtgtta aattactaaa ttgatcacag 53461 ttgtaaattt aatttgctag ctaagtgagc gatagttcaa atgttttagt tctggccaga 53521 agttagatgg gaaagagatg gaaaagacac ccattccccc aagggttggg agtagggggc 53581 attcttccta atggagagtt tcaggtgtat ctgcagaata cctatcccag gaaatgggag 53641 tgatgacaag attacaaata ttttctgacc ttgataattc ttgagatcta cacagttttc 53701 gtaaggaaag aagtcatctt catggaagag gtataattgt cttgttctgt gtctgtgagc 53761 acccaagaaa atagtgtgag gctgagaggt aaaccttcct ttaccagctg ccaatgttga 53821 aaaacaaaat ttattttatc aactgtattt cattgtccac aatggttctg cctgtaaatt 53881 agcctcccag agctgctcca gaaaagaaaa catggccctg tttggagtat atagctatcg 53941 agagtctcat ataagaggcg ttttcctttt atctgacccc gtgaatagat ggtcatgttc 54001 tggtctttcc atgttgttca gttatgatgg gcaaagaacc tgtaggagat taaagcagct 54061 ccattgtgta tggcttgtgc tcctgatgat cttactcaga atcccatgat tttgtcgggt 54121 tttccccttt tcatgtaagt gttgtgctgc ttaaggattt ccatgttgtt ctagaatgta 54181 gtgtttttta agctagagta attctttctc ttcttttttt tttttttgag acagagtctt 54241 gctctgttgc ccaggctgga gtgcagtggc gcgatctcgg ctgactgcaa cctccgtctc 54301 ctgggttcaa gcgattctcc tgccttagcc tcctgagtag ctgggtttac aggcatgcac 54361 caccatgcct ggccagtttt tgtgttttta gtagagacgg ggtttcacca tgttggtcag 54421 gctgggagta attctttata ttaattgaaa atgatttttt tctaaagtag agaatcctaa 54481 tcaaggagat tcttttctgg ggagtctcat ccattcagta tttattaaat gctgatgtgt 54541 gccaggcaat actgaaggtt ttgaaaatat gtcattgaat aagatagact taaatacttg 54601 ccctatattt ctttgggaaa gagaaacaat aaatataatt aaaaagtcaa tttataatgt 54661 tagaaggcaa taagtgttat ggggaaaagg taaatggtaa tatgagagta ttgtagtgag 54721 ggtggggtag tacttttaag tagggcgggt cagggtaagc ctcactgaga gggtaacatt 54781 tgagcaaaaa cttgaaggag tgagtcatgt gacagaagag tgttccagtt agaaggaaca 54841 gttggtacag agtatttgag gtggattaag cctgtcatct ttaaaagcag cagtcaccat 54901 tacttagtac agtgagccag aggatgactg taaggagatg agggcagaga ggctgtgggt 54961 gatgtgagta ctttggcttt tacactaagt gccattgaaa ataaactcct aataagactg 55021 aatcagtaga atttccttct cagagattta gggtcaagga aaaggtaagc agtcagagaa 55081 ggaactctgt aacttctaga cttaggatag caaaaattaa tgcatatccg ttagacataa 55141 gattatggca aaacaaataa tgaagttctt tgctgatatt ttctacttta tttttgatta 55201 tagggacttt cagttacctt gatgatgtcc catttaagac aggagacaaa ttcaaaacac 55261 cagctaaagt tggtctacct attggcttct ccttgcctga ttgtttgcag gttgtcagag 55321 aagtacaggt aagtggtaat ttttagttaa agtttagtgc atttaaaagt ttaaagagta 55381 agaatttgat gtatagtgaa atagaaaaaa ttacagttgt gagctgaatc atgttttggt 55441 caacgatgga ccacatatac aatggtggtc ccataaaatt ataaggggct gggcgtggtg 55501 gctcacgcct gtaatcctaa cactgtgggt ggccaaggtg ggaggatcac ttgagctcag 55561 gagtttgaga ccaggctggg caacttaatg agacctcatc tctactaggg gaaaaaaaaa 55621 tgatagatat atatatatat attcctatca catagtgacg tagtagccat tgtaacatgg 55681 tagcgcaatg cattactcaa gtgtttgtgg tgatgctggt gtaaacacat ctgctgcatt 55741 gacagtctta caaaagtata gcacatacaa ttcagtatag tatgtaatac aaataaatga 55801 ctatgttact catttatgta ttactatgct atacttttta gcattatctt tctacttatt 55861 aaaaaagtta actgtagaac agcctcaggt aggtccttca ggagatattc cacaggaagg 55921 cattgtgatc ataggagatg acagctccat gcatgtattg ctcctgaaga ccatacagtg 55981 aaacaagatg ggaaggtgga agacagtgat actgatgatc ctataggcct aggctgatgt 56041 gtgtgtggct tcatttttaa gaaagaagtt taaaaagtaa aaaaataaaa aatttaaaga 56101 tcaaaaagct tatagaataa gagtataaag aaaaatgttt tggaagataa aaaacatttt 56161 aggctaagta ttagttcgga agagtaaaaa agttaaaaaa atttaatgtt tataaagtta 56221 aaaaggcaca gtaagttaga gttattattt agagaaaaat tgttttttac atttgtgtat 56281 atgtgtatat atatatgtgt gtgtgtgtgt gtgtgtgtgt atatatatat gtatgtattt 56341 ttttgagacg gagtctcact ctgtcgccca ggctggagtg cagcggtgca atcttggctc 56401 actgcaagct ctgcctcctg ggttcacacc attctcctgc ctcagcctcc cgagtagctg 56461 ggactacagg cgcccgccac catgcctggc taattttttg tattttttag tagagacggg 56521 gtttcactgt attagccagg atggtctcaa tctcctgacc tcgtgatctg cctgccttgg 56581 cctcccaaag tgctggaatt acaggcgtga gccactgcgc ccggtcaaaa aattgttttt 56641 tacaaattta gtatagccta agtgtacagt gtttataaag tcaagggtac tactgtacag 56701 taatgtccta ggccttcaca tttaccattc gcttactcac tcacccagag caacttccag 56761 tcctacaagc tccatttatg gtaagtgccc tatacaggtg gaccattttt aatctttttt 56821 tttttttttc tttttttgag acggagtttc attcttgttg cccaggctgg agtgcaatgg 56881 cgcgatctcg tcaagaagag agactctttc tgtctctatg taacctccgc ctccacctcc 56941 tgggttcaag caattctcct gcctcagcct tctaagtagc tgggattaca ggtgtgcact 57001 accatgcccg gctaattttg tatttttagt ggagtcaggg tttcaccatg ttagtcaggc 57061 tggtctcgag ctcctgactt cagttgatcc gcccacctcg acctcccaaa atgctgggat 57121 tacaagcgtg agccactgca cccagcctat ttttaatctt ttataccata tttttactgt 57181 accttttcta tgtttagata ggtttagata cataaatact tgccattatg ctacaattgc 57241 ctacagtatt cagcatagta acatgctata caggtttata gcctaggagc aatactgtac 57301 catagcaata ccgtatagcc taggtgtata gcaggctgta ccatctaggt ttgtgtaaat 57361 acactctgcc ctcaccgatg aaatcaccta acatgcattt tttagagcgt atccttgtca 57421 ttatgactgt attatcttat ttttgtttct tatagctcct attgttgcct ggctcacatt 57481 gttatacaca catacagatt ttgtgtcctc tgttagattg tatattcctg gggccctgta 57541 cctcacacag accaaattcc cattgcccat ttaactagct ttttcacctc tttccctttc 57601 tctgctaatg tcctcattct cttttcagtc actcaagtta gatttgctgt gtctcacatt 57661 gcagttcagc ctgacctcat ttttcacctg ggctgttgca gtagcctctt taattggccc 57721 ttctgcttgc ggtgttttcc ttctattttt tacatagttt ctagaatgat cttaaagttt 57781 taaattgtca catccctttt cataactgtt caaaagggtt caatgactaa ccagtcctta 57841 ttgagtatag tctagacata aggttatgac aaagcaattg agctcatcac ctcctccatg 57901 ttccctccaa accacgttac tagtagtctg ttagaccttg tacgttatct ctctatatgt 57961 tgcttctgct catgagccgt tatctccacc tggaatattc tttttctcac tatactgaaa 58021 actccgttca aatctattct agattgcgtg taaatgctat ctcttcgagg gcttctttct 58081 cagtcctccc atctttaagg ctcagttata tagaaatgta ctgagttact ccttgaaagc 58141 aacaaatagc cctgtgttac ttacttactg cagtacacat ttactaaacg catgtttatg 58201 agaggtggtc tactgggcac tgcaggtaga gataaatgcc ttaatcactg ccctcaaggt 58261 acttacagtt gagagaagtt acctagagca acagaaaaat atggtacttt aactcttctt 58321 cctgtgactt aagaagattt ttaaaaatgc agctttcagg aaaagaagtt ttgtttttct 58381 ggaattcact aaggcatcac cattttattc taaaaggtat tacttttttt atcttccaaa 58441 gtctatatgc agtaagtaat cataatgagg gaacttcagt tacttttggg agaaaaaaaa 58501 tatttattga gatataattc acataccatg aaatttacct ttttttaaaa aaaaactttt 58561 tttggtttat ttgtagacac agtctcactc tgtcactcag ggtggagtct agtggcacag 58621 tcatggctca ctgcagcctc gacctccctg agcttggtga tcctccctcc tcagcctcct 58681 gagtagctgg gactattggc atgcaccacc atgcctggct aatttttgta ttttttgtag 58741 agatggggtt tcatcatgtt gcccaggctg gtcttgagct cctggcctca agtgatctgc 58801 ccacctcagc ctcccaaagt gctgggatta caggtgtcag ccaccatgcc cggccaaaat 58861 tcaccttttt aaggtataca tacagttcag tgatttttaa tatattcaca tagttgtgta 58921 ataatcatta ctatctaatt cccgaacatt ttcatcaccc caaaaggaaa cccagtgtcc 58981 actagaagtc actccccaca gcctctggca gcctcctcgc agcctctggc aaccagtatc 59041 tactttctgt ctctatgggt ttgcctattc tggccatttc atataaatgg aattctgcag 59101 tagcagcctt ggtgactggc ttattcactt agcataatgt tttcaaggtt tatccacgtt 59161 gtatgaatga ggtattagta cttcatttat ttttgtaggt gaataatatt tcattgtttg 59221 gatataacac actttgttta tctgttcatc agttgatggg acattgtggt agtttctaat 59281 tttcggctat tatgaataat gatgctctga acatctgtaa acagattttt gtgtggacac 59341 atgttttcag ttttcttggg tataatgtat aggaatggca ttgctgggtc atagtaactc 59401 tatgtttaac attttaaaga actgctattt ttccaaagtg gctgcacaat tttacaatcc 59461 cagcagcact acaagagggt accaatttct tcacttcctt gcccactctt attaactcgt 59521 tttcattata tacattcttg tgggtgagaa gttgtctctc attgtggttt ttatttgcat 59581 ttctttcatg tctaatggtg tcaagcatct taagtatctt taatactgaa ttaatgttcc 59641 ttttgtgtat ggagacttta ttgggccaga agagagatag acctactttg taacgttttt 59701 ccatgtttta tttgtgtaag aattttttta ttggcaataa caaatagtca ttacttacct 59761 ttgtacatga tactattagc aatatgattt aaaaacacct gtcttgtttc tcattattca 59821 ctgctttact gatgccttca gaaaacttgt tttgattccc aaattgtcga tttaggcttt 59881 gctgggaatt tcccatttgg tcagtctgaa gcaccaataa aagcatggta tgtgctcact 59941 gtgctcatag cacccactga taacaaaagg agatcaaagc taagagctcc attccaatgt 60001 gggccatgtg aaatattttt gttctctaat gcacaaagca atcttttttt gagatggagt 60061 cttgctcttt tgcccaggct ggagtgcagt ggcgccatct tggctcactg caacctccac 60121 ctcccaggtt caagagattc tcctgcctta gcctcctgag tagctgggac tacggcatgt 60181 gccaccacac ctggctaatt tttgtatttt tagtagaaga cagggttttg ccatgttgac 60241 caggctggtc ccgaactcct gacctcaagt gatccgccca ccttggcctc tcaaaggagg 60301 ccaagcctgt ctggcctaca aagcagcctt aaactttgac tgtctggagt ctccatgggg 60361 ttgtagtgtt gtatctggtc ccctccgtac ccagagaaca gtcagaccta gagctctaga 60421 cagatggctt gaaagttcct ataactgtca tatttgcctt tgcatgacaa tacaaactca 60481 acttttcagc taaaatttct ttagagtgag ccagtaaaac atacacagcc cttgcttcta 60541 gggtccagaa agcttctgtt gaatgtggct cacgtgaggt agcccttagt gtcatggttt 60601 agaacatcct ctggtgtcac tcatacatgt gctggagaat tgacctgtaa tagctatata 60661 atcttggaca gattttttaa cttctgcaag cctcaatttc ttcatctgta aagggaggat 60721 gaggatgatt agcttttaag gttgttagaa gagttaaata atgcatttat aatgcttact 60781 ctggttcctg gaactccata aatacaagat gacattattt attattatac tccaaaacca 60841 cattcttttc tactttaata tttctgccat cctcagcccc ctccctccct gcaccgcacc 60901 ctgctttttc tgcgttccaa gtattctaca ttactttggt aactaaaaaa caaaagttca 60961 ataaatgttt gctagaaaag gaaagaaatt tccaattttt ttaaaataaa acttcaaaga 61021 acaatgttta tagggcaatg ggaagttagt tatagcctcc atttttatta ggggaaccaa 61081 ttgcaaaatt gggttttctc cacagttaga agagcagagt cattgccact gctcttttct 61141 cattgttcca ttagtggtag aaaggaagcc cagtgatccc cccatatgtc tatctgaggc 61201 acagcagctg gctcaggatg taaagtaact tcttgtcatg tgcctttcta ggcttctctt 61261 ttgttgtttg aatttgaatg gcttgagtat gaccaggggc tctttcctat gtttcttctc 61321 taaaattctg gtgttagcac gttagacctc tcttttcccc cttttttact tttagtgtgt 61381 ctctcacctt ggacattaac acagatactg tttagctcta gtgctttggg gtgaaagcca 61441 aagattttgt tttttcctgt cctatgtgaa tgttgcttta tataataggt ctgtttctct 61501 taagtgtgca aaataaattt ttgtaaattg agctatactt aagatattgt taaagaacca 61561 tgaagtggct ctttgtgtaa tttgctgtgt aaaaatctta atctgatttc ttataagttt 61621 ttctgcattt taactgcaac ctgttcatca taatctaata gatagtggtt taagagcata 61681 gaatctgtag tcagactgcc tgggttcaaa tccaggctct tccatttagc taccagcttt 61741 atgcttggtt gtagatcctg ttaagtagga ataatagtag ttactttaaa aggttattga 61801 gaaaatctag agagttaatg cataaaaagt gtttagaatc atgcatggca ccttacaggt 61861 atttcttaca tgtttggggc tatcattacc tagcatggga tttgtttagg acattgtgct 61921 agataataga gatccaagga gggtctgtag ccaggttgct gcccacaaga ggcttgtggg 61981 aacatagttt ggttaaatct tacaaggaca ttggaagcct tactcttttt aagtggggat 62041 catcccacac gccttaaatg ctgccttgtc cggttatcct cttgcaccag gagtgaggaa 62101 ggagtgaatt gggtggtggg gtaaagatgg ccacctgggt tcacaccccc ttcctctgct 62161 atatctttgc agtatgactt ctctttggaa aagaaaacca ttgagtgggc tgaagagatt 62221 aagaaaatcg aagaagccga gcgggaagca gagtgcaaaa ttgcggaagc agaagctaaa 62281 gtgaattcta agagtggccc agagggcgat agcaaaatga gcttctccaa gactcacagt 62341 acagccacaa tgccacctcc tattaacccc atcctcgcca gcttgcagca caacagcatc 62401 ctcacaccaa ctcgggtcag cagtagtgcc acgaaacaga aagttctcag cccacctcac 62461 ataaaggcgg atttcaatct tgctgacttt gagtgtgaag aagacccatt tgataatctg 62521 gagttaaaaa ctattgatga gaaggaagag ctgagaaata ttctggtagg aaccactgga 62581 cccattatgg ctcagttatt ggacaataac ttgcccaggg gaggctctgg gtctgtgtta 62641 caggatgagg aggtcctggc atccttggaa cgggcaaccc tagatttcaa gcctcttcat 62701 aaacccaatg gctttataac cttaccacag ttgggcaact gtgaaaagat gtcactgtct 62761 tccaaagtgt ccctcccccc tatacctgca gtaagcaata tcaaatccct gtctttcccc 62821 aaacttgact ctgatgacag caatcagaag acagccaagc tggcgagcac tttccatagc 62881 acatcctgcc tccgcaatgg cacgttccag aattccctaa agccttccac ccaaagcagt 62941 gccagtgagc tcaatgggca tcacactctt gggctttcag ctttgaactt ggacagtggc 63001 acagagatgc cagccctgac atcctcccag atgccttccc tctctgtttt gtctgtgtgc 63061 acagaggaat catcacctcc aaatactggt cccacggtaa gtcttttaaa tcccccgccg 63121 actcccatat tttcctgatg acagggatta tgttttttct tgttgtttgg ttgatttttt 63181 tcgagacagg gtctcattct gttgcccagg ctggaatgca gtggtgtgat cgtaccttat 63241 tgtagcctcg acctcccggg ctcaagcctg ttgggacaac aggcacatgc catcacacct 63301 aatttttgtg ttttttgtag agatgcggtt ttgacacgtt gcccaggctg gtctcaaact 63361 cctgacttca agttatctgc ctgccttggc cttccaaagt gctgggatta caggtgtgag 63421 ccaccaagac tggcagtaca gaacaacaca ttattgcatt ctagaataga aatcaataga 63481 agtcaagaac cttttgctac accagcttac agatttcttt tcctttcttt tttttgagac 63541 agagtcttgc tctgtcgccc aggctggagt gcagtggcac catctcggct cactgccaac 63601 ctccacctct cacgcaattc tcctgcctca gcttcccgaa tagctgggac tacaggcata 63661 tgccaccaca ctcagctaat ttttgtattt ttagtagaga cggggtttta ccacgtttcc 63721 gaggctggtc tcaaactcct gaccttagtt gatccacctg cctccacctc ccaaattcct 63781 gggattacag gggtgagccg ctgtgtccgg ccagcttaca gattttttaa tgtaaaagca 63841 catcaagaga ctatttagag ccaaaaagga gcctgagtat attggtggga tatctgtcag 63901 agaccataac aagcctgcta ttagctggta aaaagataca aaatcttggc ctgctttaca 63961 agtcccagta atagcattct tggtcaatga tagtctgtgt gaatgcttct attgtcagag 64021 agctcatagc tgcttgtaaa cttccataaa ccttggcccg ggtaaacttg atagagtgcc 64081 gaacaccatt attatcttct tggagtgggc ttcattctaa ttcctcttgg tcagttgttg 64141 ggctctggtc agcactgtac ttcttccttt tcatggtact ttgaatgctt tagagaaatt 64201 aaaatctccc tggaaaagat cagggatcct tggccaacac tcccttcctc tcatttaaag 64261 ttagattaac catttagaga tcaaagccaa taaactaaca gcagagatct ttctcaaact 64321 agaacgcttg tcattctttg accagcctga tgatgggagt ttctttctga cctgttcatc 64381 tcaggatggg aagaggcctg cttcttgttg gcttgagaat ttgctgcctt ccacctgcag 64441 gtataaacag agctggagtt ctggtgtttc ttttttcttt tttttttttt tgagacagat 64501 tctaattctg tcacccaggc tggaatgtag tggtgtgatc tctgctcact gcaacctcca 64561 cctcccaggg tcgggcgatt gtcctgcctc tcgcagcctg ccaagtagct gggattacag 64621 gcatgtgcta tcgtgctcgg ctcatttttg tatttttagc agagataggg tttcgccatg 64681 ttggccaggc tggtctggaa ctcctgtgct caagtgatcc acccgcctca gcctcccaaa 64741 gtgctgggat tacaggtgtg agccacagtg gctggccaat taaatattga ctataatcac 64801 cctgttgtct tatcaaatgg tagatcttat tctatttttt tctttgcttt tttcttcaac 64861 agagtctcgc tctgtcgccc aggctggagt gcagtggcgt gattttggct cactgcaacc 64921 tctgcctcct gggttcaagg gattcttctg cctcagcctc cagagtagca gggattacag 64981 gcgcctgcca ccatgcgcag ctaatttttg tatttttagt agagacgggg tttcaccatc 65041 ttggccaggc tggtcttgaa ctcctgacct cgtgatccgc ccgcctcggc ctcccaaatg 65101 ctgggattac aggtgtgagt caccgcacac agccattctt tctatttttt ttgtacctat 65161 taaccaccag cccccgacta ctacccttct cagcccctgt ggtaaccatc cttctagtgt 65221 ctatgtccat gggttcaatt gctttgattt ttagatcaca cacataagtg agaacatgtg 65281 atgtttgtct ttctatgcct ggcttatttc acttaacata atgatctcca gttccatcca 65341 tgttgttgca aatgacagga tctcattctt tttcatggct gaacggtact ccattgtgta 65401 cgagcaccac atttttttaa tccatccttg tttaagttct ttcactttac tctttttttt 65461 tttctttgga gacagatttt gctcttgttg ttcaagctgg cgtgcaatgt tgcgatctcg 65521 gctcactgca acctccgcct cccgggttca agtgattctc ctgcctcagc ctcccatgta 65581 gctgggatta caggcatgtg ccaccacgcc tggctaattg ttctattttt agtacagacg 65641 gggtttcatc atattggtca ggctggtctc aaactcttga cctcaggtga tgcacctgcc 65701 tcggcctccc aaagtgctgg gattataggt gtgagccact gcacctggcc tatccattca 65761 tctcttgatg gacacttagg ttgcttccaa atcttggcta ttgtgaacag ggctgcaaca 65821 aacatgagag tgcagatctt gtttatgttt tactttctgg gacatatcct taacttacaa 65881 cttcaaaaaa aaactactaa atcttacaac cttttatttt atttaattta tttttccccc 65941 tgaacttgat agtaggtgat tattttttaa gagatgggat ttttgtcccc taggctgggg 66001 tgcagcttac tgcctcagcc tcctgtctgg gactacaggt gtgcaccact atgcccagct 66061 gatttttgta gagttggggg tctcactgtt tgcccaggct ggtcttgaac tcctgggctc 66121 aagtgatcct cctgcctcag cctcccaaag tgttgggatt acaagcatga gccaccatgc 66181 ccagcctaaa tcttactacc ttttaaatac aatttttact ttatcatatt ttaaatttcc 66241 aagtgctttg tctattccct cttttggttt ggtgttttca tgctggtggc tttggctttc 66301 tattcacgtt taagtatgag gcactaaaaa aataacagtc acaaagtttg tgtgtcagag 66361 gtgtgcttgc tgatcattaa gcttcactat aggttgaaag ttaattggct ttgccgggga 66421 agtccccaaa atgtaaacat gtgaagggct tgggtgaggg tttctccaga gaattctggc 66481 tgaggactga tgttggggaa tgctggtgtg ttgcttttac ttaatccttg tttaagttct 66541 ttcacttcac tctgccccac actgtgcctg gtgcttagtc ctctttggtc tctggttccc 66601 attttctgga gactatacct tctgcctcct gcacagggtg gggatgaatg gggactgttt 66661 caccaagggc tttactgctc tgatcactga ctgtgtgtat ctttgcctta tcccaacagt 66721 acctggtgct tccagagtct gagcccctct tagggatcct aatgggctga tagactgact 66781 cccccctttc ctacttactt acttagttta taacttcatt gctggcaatt accatccctt 66841 cattatcttt ccacattcta aaatttcatt gaaatatatc acaccagtaa tcccagcatt 66901 ttgggaggcc gaggtgggag gattgcttga gcccaggact tcgagatcag cctgggcagc 66961 atactgagat cttgtctttg tttctttatt taaaaagaat aagaaatata ttgtccactg 67021 ctaccccctt tccttttatt gctttaaaga tgaatacagt tttaattctt tatttttaaa 67081 tttttattat tttttttgag acagggtctt gctctgtaat cacggctcac tgcagcctcg 67141 acctcctggg ctcaagtgat cctcctgcct cagccttctg ggtagctggg actacaggca 67201 cgtgccacca cacctggcta actttttgtg ttttgtagag atgagtctca actatgttgt 67261 ccaggctggt ctcaaaattc tgagcttaag cagtccttct gcctcagcct cccaaagtgc 67321 tgggattata ggcatgagcc attgctcctg gccttagttt gtttatgatc attttagtgg 67381 gcttttggga agaagagatg gtaaagacat tttagtgtgt taaatgaaat tctccctttg 67441 aaaattctaa tggacttcaa tttattccac aactgttgat gaagaccagt aagtgtaaag 67501 ccaagagtca ggccctgggg atttagaaat gggcatgaca tggtaactga cctctctgca 67561 agctcacact aatgtaatgt gataagtact ttgccagcag tgtgactaaa gctctgtggg 67621 accacaggtg gcagtgtgtc cgctgagttg tgcacacaca ttagcagtgg gttaaaagga 67681 ggtttatgga aagacttggt agaagctgtc ttgaggttag gcttgagggc cacataggta 67741 cagaaaggac tgccaccttc tgaggcctga gagcatgctg agactcttca gaagcttgag 67801 cactaatatt tgtctccttt cacaaacgta ctttccctca aacactcata tttaatttgg 67861 aaagatttca taataattct catttttgtc tagtttctac ttggttacaa atatggagca 67921 cttcagtcat cgagtatgaa aatccctttt tatgccagag tcaacctttg cccctgcagg 67981 aagtaaattt tactattctt ttattgaaaa aataatgaca aatggctacc atgaatgcag 68041 ttaatggtgt ttaaaaagtc atatctagat tactcctaag agccttatat atatttgaaa 68101 atggtaccct ggagtaaata aatttggtga gtaactggag aaactaaaca ctgttataat 68161 tttgatgagg gatcctttca aaccttgtct gtccgtttgg ggtctgctgg attagcctgt 68221 tgcctgtttg attcatgaaa catttatatg aaggtgacat cttattttgt ttgtccctgt 68281 tcaagaaaag tatcatctgt taggatagat gatactttta aatcatccat ttaaaatcag 68341 tcatttaata tcccgtgcat ctttaaaaaa tattgtagag catgtacaat taataaatat 68401 atcttttttt cttatttatt tatttattta gagacggtgt ctcgctgtgt caaccaggct 68461 ggagtgcagt ggcgcaatct tggcccactg agacctccgc cttctgggtt caagtgatcc 68521 tcctacctca gcctcccaag tagttgggat tacaggaatg caccaccaca cttggcccat 68581 tttgtatttt tagtagagat ggggtttctc cgtgttggtc aggctggtct tgaactcccg 68641 acctcaggtg gtctgcctgc cttgcccccc aaagtgctgg gattacaggt gtgagacacc 68701 tcacccgctg ttttttcact taattcagat gctttaaata attaacagtc gtatgttcat 68761 tatttgctaa cctaacaccc tgtgctggat atttggtagg ttgtccctaa aatttattac 68821 aaatgatgtt gagatggata tattcctata cataaatatt ggtttgcatt attattattt 68881 cttcagacta gtttcctaga aataggtaac tggatcaaag aatctgagca gttttctgga 68941 tttatgccat gtttacctaa agcataccaa ataacatttc caccagtagg gtatgtgaga 69001 gtactgcatg tatgtttaaa atagatacga tttttttttt tttcagacag tctcactctg 69061 tcacccaggc tggagtgcaa tggcatggtc tccactcact gcaacctcca cctcccgggt 69121 tcaagcgatt ttcccacctc agcctcccga gtagctggga ctacaggcat gtgccaccac 69181 acccggctga tttttgtatt tttagtagag atggggtttc actatgttgg ccaggctggt 69241 ctcaaactcc tgacctcatg atccacctgc ctcggcctcc caaagtgctg ggattacagg 69301 cgtgagccac cgcacctggc tagatatgat tttattaaca tttgtaatgt gcagtattct 69361 gggtgtatgc ctgcatccca ccaggcattt aggaggcatt atgagaaggg caggcagttg 69421 aagaatagtc ctgactcctt cctggctggg gaatgtcatg tgagagctgg cattgctctg 69481 tggctgttgg tttctttagc ccaggttaaa gcccgtctct aaagagggag acacaatctt 69541 agacctgcca agtggctaga ctggtgtgat ccaatccctg tgccttattg tgactcttgt 69601 accataatga cagccctgtg cttgggctgc tgagtgtgtg ctgtgggtga gatggttaag 69661 caccaacaga atgggctctg aggcctatag ccatcctggc tttccccagc agactttgag 69721 gagagcatct ggttacatct gtataatttc gaacttgctt ctaggacaga gggcttccta 69781 ccacctaacc atgctccctc tccccaccaa taaaacaagg aaataaaaaa atttttgtgt 69841 gaacatcttc cctggttctt gggacctgcg ccaagctgta aaagggcttg tgacctcgtt 69901 cctgtggtca gtgggtcctt tggcctggga gtgtgctgtc ttcctgcctt gggcttgatt 69961 tgtgatcaga agtggtgggg gagttaatca cggcactctg tacgcctatc cgactggggt 70021 tagtaagttc cagtggcagg gagcaggctg cctgtagctg gtgggcaggt ccctagagtc 70081 cagattccag tctgtgggat ccagcctggt gtggctagta tgccttctgt cctggcactg 70141 taggatggca gctttggcag gaaacgaagc acagtacatg atgtagggca aaggtgtgga 70201 gtaccctcaa aaccctgggt tcctgagctg tccaggttcc taagctgtcc atgcatggga 70261 accacactca cagcctctgc cgaagagtag agactttggg atagagttag ctccaaagtg 70321 ggagtatttg aagtgaaaaa tgatgcctca gaactgatac ttcagcctgg gaaagggata 70381 atgctggaga gaaatcgcag ccagcttgga catcagtccc gggggcagcc acagcttctt 70441 tcttcaactc aaatcctgga gatttgggca tgcaagggcc tgtcgtgttg tggcgctttc 70501 tcaatcacca aagtgggact agggcagagc aggacagcca caattgcagg tgacctccca 70561 cctttttccc tctcagctgg cactccccta cccctctttg ttccttttca gggtgtctga 70621 ccagcaacct ggcgatttct tccaacctga ccgcttttct tggggcttga atagtgtcag 70681 aaatgccaac ccctggacta ggctgcctca gtgacttctt tgggggccca ggtcttcttg 70741 ccttaatctt cttgtttttc cactaggtca cccctcctaa tttctcagtg tcacaagtgc 70801 ccaacatgcc cagctgtccc caggcctatt ctgaactgca gatgctgtcc cccagcgagc 70861 ggcagtgtgt ggagacggtg gtcaacatgg gctactcgta cgagtgtgtc ctcagagcca 70921 tgaagaagaa aggagagaat attgagcagg tgagcggttg gtcagccagg agggcaggct 70981 cagacctgtg gagctggagt agtgtcctcc cctgtcctag agcccctcag acttgtagca 71041 ccaacctcct ttcctgtggg tttgtgagga cacggggcat gttttgggga aagccagcag 71101 ccttgggagc agtacttctt ttggaagcta ttccgggacc caggctgaga gacagctttg 71161 ggagaattgg agctgttctc caactacccc ctcctttctc tgcctctcca aaaggggcag 71221 agctaggctt ggggtatggt caaatgtctc tttccaggaa ttttcatcct gtcatctatg 71281 cccagagtgt tgaattaggg tactggagtg ttagaccttg gtgacatttt ctgacttggg 71341 ctgttttctt agagtagccg cctgcccagg ctgtggagtt gaccattctg gtgggcgtgt 71401 cagctgtggc tgcaactcct ttggaaaatg cctgctcctg tttagaaggc tgtgcctgtg 71461 ttcattaaag agtcactggg gtcctgttgt gttggacaca gtcaaggcac aaaatcaaat 71521 cctatccagt atctgacggc ctgggtgggg cggagaacgg acttcacaca ctagtttgtt 71581 gaggtctctt ggaaagcaca gcaaagagca gcagtaggtg ctaaacccct tgtttctttt 71641 cttagattct cgactatctc tttgcacatg gacagctttg tgagaagggc ttcgaccctc 71701 ttttagtgga agaggctctg gaaatgcacc agtgttcaga agaaaaggtg ggaatcttca 71761 tgtttcttgg gaaccctctg gaaaaggagg gaccaagtat ccatcctggt tttctccctt 71821 ggcccacaca caaccttttt gctttgccag tgactacagg tacaagtatt tgaagggcag 71881 aatctcccaa gtattcagac aggccgaggc ctgcgtttgg cttggtggtt gaagcattgg 71941 gttgaaaact tcagaatccc agtgaaggga agggaatatt ggcctgggct aaaagccaaa 72001 cttcttagat gattagggag ggagattgct ggactttttg cctagactgc tctttgaaga 72061 gcagccattt ctgggttcag tatccttcct tggtgggaag atggatgtgg gtggtgggac 72121 ccagacagag ctcagcaaca gtgagctggg actgggctga ctcctgaagg ttgatggccg 72181 tcaccaaacc cgtttgcctt ggcttccacc acagcccacc tctgcttgct aggggacctt 72241 atggagactc acggggctct gggggaagga tgtagacatt ctgctctccc ccagtccccg 72301 tcccacacac acactccttc actcagctgt gctgtgaccc cacccttttc tttaatctgt 72361 gttctcttct ctcccttaga tgatggagtt tcttcagtta atgagcaaat ttaaggagat 72421 gggctttgag ctgaaagaca ttaaggaagt tttgctatta cacaacaatg accaggacaa 72481 tgctttggaa gacctcatgg ctcgggcagg agccagctga gaccaggccc tgcctaggcc 72541 ctgccgcaga accaccatcc ctgggaggcc ctgcagagcc cacctgtggg gaaagagaag 72601 gggcagcttc cggattttct tttgggggtt agaaggtcag gtgtggagac tgctcgccag 72661 tctctgtgag cctaggccct gagctgggga ggtggggaag attcgggcat gtgagtgccc 72721 ccagaactgt cctggctcct tccgtattaa acgcatttgc attttgagaa gtgtccttcc 72781 cacttcagcc ctccggagag actaccctag tctttctggg gtgtttatgt cctcagctga 72841 agcctggcct agttgctgag aggggctggg gagatggggc gggagggcca gactcagtgc 72901 tgctgtggag ctaggtgctt cccccttccc ctgagactgg tggactgaac tccagtcaag 72961 ttgagttcaa gtgaaagatt cttccagggt tttatttttt cccctcctaa caaagtctca

73021 tagtgttaac actggttctg caatatctct gaggtgcaaa gaatgcactt ttccctatgg

73081 ggcccagagt ttgccttttc tgccaggcag tcaccatgct tccctacccc agcctgtttc

73141 ttttggcttg gtttggacca cagtcctctg ctacccaggg ttttagagcc cctgctctag

73201 gaaacagttt aagaaatcat tggccccttc ccagcacatt gaatgggtaa gcagacaggc

73261 catgatttag ttggccagca ctaactccac ctctgttctc cttgaacagc ttcccctcca

73321 gcccactgct ttaggatgac acaatgaata acacctagtc atagaaatca gtctctctgg

73381 tttgttttgt attatgttgt acatcattaa agatctaaat acaaaggata tacagtcttg

73441 aatctaaaat aatttgctaa ctaactattt tgattcttca gagagaacta ctaataaaaa

73501 tctaaaaggt a

Table of genetic association data for UBAPl

1. DUTCH RESULTS

ALL CASES rs2275003 Controls Cases OR 95% CI

N % N %

CC 120 27.15 59 26.11 1

CT 227 51.36 110 48.67 0.98 0.67-1.44 0.941

TT 95 21.49 57 25.22 1.22 0.77-1.91 0.389

CT+TT 322 72.85 167 73.89 1.05 0.73-1.51 0.773

N/A 42 4

rs4879753

AA 192 41.56 88 40.55 1

AG 220 47.62 101 46.54 1 0.70-1.41 0.993

GG 50 10.82 28 12.9 1.22 0.72-2.06 0.456

AG+GG 270 58.44 129 59.45 1.04 0.75-1.44 0.804

N/A 22 13

rsl3283064

CC 250 55.07 121 53.54 1

CT 176 38.77 87 38.5 1.02 0.72-1.42 0.902

TT 28 6.17 18 7.96 1.32 0.70-2.49 0.378

CT+TT 204 44.93 105 46.46 1.06 0.77-1.46 0.707

N/A 30 4

rs 10971969

AA 313 68.79 143 65.6 1

AG 130 28.57 67 39.73 1.12 0.79-1.60 0.506

GG 12 2.64 8 3.67 1.45 0.58-3.64 0.419

AG+GG 142 31.21 75 34.4 1.15 0.82-1.62 0.407

N/A 29 12

rs7018487

TT 192 41.74 105 47.95 1

GT 202 43.91 97 44.29 0.87 0.62-1.23 0.453

GG 66 14.35 17 7.76 0.47 0.26-0.84 0.011

GT+GG 268 58.26 114 52.05 0.77 0.56-1.07 0.128

N/A 24 11

rs 10971977

GG 145 32.22 62 27.43 1

AG 219 48.67 118 52.21 1.26 0.86-1.82 0.223 AA 86 19.11 46 20.3₅ 1.2₅ 0.78-1.99 0.346

AG+ AA 3O₅ 67.78 164 72.₅7 1.2₅ 0.88-1.78 0.203

N/A 34 4

rsl237₅731

GG 149 32.82 63 29.17 1

AG 227 50 115 53.24 1.19 0.82-1.73 0.339

AA 78 17.18 38 17.59 1.15 0.70-1.87 0.568

AG+ AA 3O₅ 67.18 1₅3 70.83 1.18 0.83-1.68 0.342

N/A 30 14

rsl0814079

GG 192 42.48 107 46.93 1

CG 192 42.48 104 45.61 0.97 0.69-1.36 0.868

CC 68 15.04 17 7.46 0.44 0.25-0.80 0.007

260 57.52 121 53.07 0.83 0.60-1.15 0.27

N/A 32 2

rs2380925

GG 150 32.26 61 27.6 1

GA 227 48.82 115 52.04 1.24 0.85-1.80 0.248

AA 88 18.92 45 20.36 1.25 0.78-2.00 0.336

GA+AA 315 67.74 160 72.4 1.23 0.87-1.77 0.217

N/A 19 9

rs 17258783

CC 284 62.69 145 63.88 1

CT 155 34.22 72 31.72 0.9 0.64-1.28 0.59

TT 14 3.09 10 4.41 1.39 0.60-3.22 0.431

CT+TT 169 37.31 82 36.12 0.95 0.68-1.32 0.763

N/A 31 3

rs4574933

AA 281 61.62 136 62.67 1

AG 161 35.31 71 32.72 0.91 0.64-1.28 0.598

GG 14 3.07 10 4.61 1.47 0.63-3.40 0.362

AG+GG 175 38.38 81 37.33 0.95 0.68-1.33 0.793

N/A 28 13

rs 10814083

CC 197 42.73 106 48.85 1

TC 195 42.3 94 43.32 0.89 0.63-1.25 0.527

TT 69 14.97 17 7.83 0.45 0.25-8.81 0.008

TC+TT 264 57.27 111 51.15 0.78 0.56-1.08 0.136

N/A 23 13 rs 10972030

CC 163 37.3 85 37.78 1

TC 217 49.66 112 49.78 0.98 0.69-1.40 0.954

TT ₅7 13.04 28 12.44 0.94 0.55-1.58 0.823

TC+TT 274 62.7 140 62.22 0.97 0.70-1.36 0.904

N/A 47 5

rsl 7350373

GG 137 30.24 74 32.46 1

GA 236 52.1 108 47.37 0.84 0.58-1.21 0.371

AA 80 17.66 46 20.18 1.06 0.67-1.68 0.79

GA+AA 316 69.76 154 67.54 0.9 0.64-1.27 0.556

N/A 31 2

rsl 1788425

AA 139 30.28 69 31.94 1

AG 240 52.29 102 47.22 0.8₅ 0.59-1.23 0.411

GG 80 17.43 45 20.83 1.13 0.71-1.80 0.599

AG+GG 320 69.72 147 68.06 0.92 0.65-1.31 0.663

N/A 25 14

rsl2377

CC 435 93.95 205 98.56 1

CA 28 6.05 3 1.44 0.22 0.06-0.75 0.016

AA 0 0 0 0

CA+AA 0 0 0 0

N/A 21 22

Haplotype analysis

Phase was used to impute haplotypes for all the samples, using 10000 iterations.

The 10 snps that correspond to the haplotype are marked on the markers table at the top of this document in bold

Dutch results

ALL CASES

Controls Cases OR 95% CI P

N % N %

CAGAGCGCAT 347 36.26 137 30.18 1

CATAGGGCAC 196 20.48 1408 23.79 1.39 1.02-1.89 0.033

TATTAGATGC 194 20.27 91 20.04 1.18 0.86-1.63 0.288

CGTTAGACAC 170 17.76 87 19.16 1.29 0.93-1.79 0.118

TATTAGACAC 50 5.22 31 6.83 1.57 0.96-2.56 0.071

OTHER 11 6 2. MANCHESTER CASES

ALLCASES rs2275003 Controls ^• Cases OR 95% CI P

N % N %

CC 70 24.73 62 29.67 1

CT 146 51.59 96 45.93 0.74 0.48-1.13 0.172

TT 67 23.67 51 24.4 0.85 0.52-1.41 0.552

CT+TT 213 75.27 147 70.33 0.779 0.52-1.16 0.223

N/A 5 3

rs4879753

AA 110 43.31 86 45.74 1

AG 109 42.91 80 42.55 0.93 0.62-1.40 0.759

GG 35 13.78 22 11.7 0.8 0.43-1.46 0.478

AG+GG 144 56.69 102 54.26 0.9 0.61-1.32 0.61

N/A 32 26

rsl3283064

CC 158 58.09 91 46.67 1

CT 95 34.93 81 41.54 1.48 0.99-2.19 0.05

TT 19 6.99 23 11.79 2.1 1.08-4.06 0.027

CT+TT 114 41.91 104 53.33 1.58 1.09-2.29 0.015

N/A 14 19

rslO971969

AA 175 70.85 118 66.67 1

AG 67 27.13 54 30.51 1.19 0.77-1.83 0.414

GG 5 2.02 5 2.82 1.48 0.42-5.23 0.54

AG+GG 72 29.15 59 33.33 1.21 0.80-1.84 0.358

N/A 39 37

rs7018487

TT 1 11 43.36 101 54.3 1

GT 115 44.92 68 36.56 0.64 0.43-0.97 0.01

GG 30 1 1.72 17 9.14 0.62 0.32-1.19 0.036

GT+GG 145 56.64 85 45.7 0.64 0.44-0.94 0.023

N/A 30 28

rslO971977

GG 98 35.77 48 24.37 1

AG 131 47.81 91 46.19 1.41 0.91-2.19 0.117

AA 45 16.42 58 29.44 2.63 1.56-4.42 0

AG+AA 176 64.23 149 75.63 1.72 1.14-2.60 0.009

N/A 12 17 rsl2375731

GG 94 36.72 53 29.28 1

AG 1 17 45.7 78 43.09 1.18 0.75-1.83 0.458

AA 45 17.58 50 27.62 1.97 1.16-3.33 0.011

AG+AA 162 63.28 128 70.72 1.4 0.93-2.10 0.106

N/A 30 33

rslO814O79

GG 113 41.54 109 55.33 1

CG 130 47.79 71 36.04 0.56 0.38-0.83 0.004

CC 29 10.66 17 8.63 0.6 031-1.16 0.136

159 58.46 88 44.67 0.57 0.39-0.83 0.003

N/A 14 17

«2380925

GG 100 37.74 50 26.88 1

GA 1 18 44.53 81 43.55 1.37 0.88-2.13 0.16

AA 47 17.74 55 29.57 2.34 139-3.92 0.001

GA+AA 165 62.26 136 73.12 1.64 1.09-2.47 0.016

N/A 21 28

rsl7258783

CC 176 65.67 108 56.54 1

CT 77 28.73 69 36.13 1.46 0.97-2.18 0.06

TT 15 5.6 14 7.33 1.52 0.70-3.27 0.284

CT+TT 92 34.33 83 43.46 1.47 1.00-2.15 0.048

N/A 18 23

rs4574933

AA 184 65.02 108 51.43 1

AG 80 28.27 78 37.14 1.66 1.12-2.45 0.011

GG 19 6.71 24 11.43 2.15 1.12-4.11 0.02

AG+GG 99 34.98 102 48.57 1.75 1.21-2.52 0.002

N/A 3 4

rsl0814083

CC 109 44.13 101 55.49 1

TC 115 46.56 63 34.62 0.59 0.39-0.89 0.012

TT 23 9.31 18 9.89 0.84 0.43-1.65 0.623

TC+TT 138 55.87 81 44.51 0.63 0.43-0.93 0.02

N/A 39 32

rsl 0972030 CC 96 3₅.69 84 43.08 1

TC 121 44.98 87 44.62 0.82 0.54-1.22 0.338

TT ₅2 19.33 24 12.31 0.52 0.29-0.92 0.027

TC+TT 173 64.31 111 56.92 0.73 0.50-1.06 0.107

N/A 17 19

rsl7350373

GG 91 34.21 55 28.65 1

GA 126 47.37 100 52.08 1.31 0.85-2.00 0.209

AA 49 18.42 37 19.27 1.24 0.75-2.14 0.421

GA+AA 175 65.79 137 71.35 1.29 0.86-1.93 0.208

N/A 20 22

rsl 1788425

AA 85 32.82 50 27.47 1

AG 120 46.33 92 50.55 1.3 0.83-2.02 0.241

GG 54 20.85 40 21.98 1.25 0.73-2.15 0.401

AG+GG 174 67.18 132 72.53 1.28 0.85-1.95 0.231

N/A 27 32

rsl2377

CC 267 93.36 202 95.28

CA 18 6.29 10 4.72 0.73 0.33-1.62 0.446

AA 1 0.35 0 0

CA+AA 19 6.64 0 0 0.69 0.31-1.52 0.366

N/A 0 2

MANCHESTER CASES

ALL CASES

Controls Cases OR 95% CI P haplotype N % N %

CAGAGCGCAT 188 34.18 1 16 28.16 1

CATAGGGCAC 135 24.55 84 20.39 1 0.70-1.44 0.963

TATTAGATGC 108 19.64 109 26.46 1.63 1.14-2.32 0.006

CGTTAGACAC 89 16.18 80 19.42 1.45 0.99-2.13 0.053

TATTAGACAC 30 5.45 23 5.58 1.24 0.68-2.24 0.471

OTHER 22 21

3. AD CASES

rs2275003 Controls Cases OR 95% CI P

N % N %

CC 70 24.73 97 26.65 1

CT 146 51.59 180 49.45 0.9 0.61-1.31 0.592

TT 67 23.67 87 23.9 0.95 0.61-1.47 0.822 CT+TT 213 75.27 2₆7 73.35 0.91 0.64-1.30 0.635

N/A 5

rs4879753

AA 110 43.31 147 40.38 1

AG 109 42.91 170 46.7 1.17 0.83-1.66 0.352

GG 35 13.78 47 12.91 1.01 0.61-1.67 0.927

AG+GG 144 56.69 217 59.62 1.13 0.82-1.57 0.435

N/A 32

rsl3283064

CC 158 58.09 191 52.47 1

CT 95 34.93 142 39.01 1.22 0.87-1.70 0.236

TT 19 6.99 31 8.52 1.34 0.73-2.48 0.334

CT+TT 114 41.91 173 47.53 1.24 0.90-1.70 0.175

N/A 14

rs 10971969

AA 175 70.85 245 66.58 1

AG 67 27.13 112 30.43 1.17 0.82-1.68 0.374

GG 5 2.02 11 2.99 1.57 0.53^.60 0.41

AG+GG 72 29.15 123 33.42 1.2 0.84-1.70 0.298

N/A 39

rs7018487

TT 111 43.36 158 43.17 1

GT 115 44.92 174 47.54 1.07 0.76-1.50 0.68

GG 30 11.72 34 9.29 0.8 0.46-1.38 0.43

GT+GG 145 56.64 208 56.83 1.01 0.73-1.40 0.919

N/A 30

rsl0971977

GG 98 35.77 104 28.97 1

AG 131 47.81 176 49.03 1.26 0.88-1.80 0.195

AA 45 16.42 79 22.01 1.61 1.02-2.55 0.039

AG+ AA 176 64.23 255 71.03 1.35 0.97-1.89 0.075

N/A 12

rsl2375731

GG 94 36.72 105 28.85 1

AG 117 45.7 178 48.9 1.36 0.94-1.95 0.095

AA 45 17.58 81 22.25 1.57 0.99-2.48 0.051

AG+ AA 162 63.28 259 71.15 1.42 1.01-1.99 0.042

N/A 30 rs 10814079

GG 1 13 41.54 157 43.49 1

CG 130 47.79 170 47.09 0.94 0.67-1.31 0.721

CC 29 10.66 34 9.42 0.84 0.48-1.46 0.546

159 58.46 204 56.51 0.92 0.67-1.26 0.624

N/A 14

rs2380925

GG 100 37.74 105 29.01 1

GA 118 44.53 175 48.34 1.41 0.98-2.02 0.06

AA 47 17.74 82 22.65 1.62 1.03-2.54 0.034

GA+AA 165 62.26 257 70.99 1.47 1.05-2.06 0.024

N/A 21

rs 17258783

CC 176 65.67 222 61.16 1

CT 77 28.73 122 33.61 1.24 0.87-1.75 0.223

TT 15 5.6 19 5.23 1 0.49-2.03 0.991

CT+TT 92 34.33 141 38.84 1.2 0.86-1.66 0.272

N/A 18

rs4574933

AA 184 65.02 217 59.62 1

AG 80 28.27 128 35.16 1.35 0.96-1.90 0.08

GG 19 6.71 19 5.22 0.84 0.43-1.64 0.627

AG+GG 99 34.98 147 40.38 1.25 0.91-1.73 0.161

N/A 3

rs 10814083

CC 109 44.13 160 43.36 1

TC 1 15 46.56 173 46.88 1.03 0.73-1.45 0.845

TT 23 9.31 36 9.76 1.07 0.60-1.91 0.803

TC+TT 138 55.87 209 56.64 1.04 0.75-1.44 0.807

N/A 39

rs 10972030

CC 96 35.69 153 42.74 1

TC 121 44.98 162 45.25 0.84 0.60-1.20 0.354

TT 52 19.33 43 12.01 0.52 032-0.84 0.008

TC+TT 173 64.31 205 57.26 0.75 0.54-1.03 0.085

N/A 17

rsl 7350373 GG 91 34.21 100 27.32 GA 12₆ 47.37 185 50.55 1.33 0.92-1.92 0.118

AA 49 18.42 81 22.13 1.5 0.95-2.36 0.078

GA+AA 17₅ 6₅.79 266 72.68 1.38 0.98-1.94 0.063

N/A 20

rsl 1788425

AA 85 32.82 102 27.49 1

AG 120 46.33 184 49.6 1.26 0.87-1.83 0.207

GG 54 20.85 85 22.91 1.31 0.83-2.04 0.233

AG+GG 174 67.18 269 72.51 1.28 0.90-1.80 0.16

N/A 27

rsl2377

CC 267 93.36 336 90.81 1

CA 18 6.29 34 9.19 1.5 0.83-2.72 0.176

AA 1 0.35 0 0

CA+AA 19 6.64

N/A 0

AD

Controls Cases OR 95% CI P haplotype N % N %

CAGAGCGCAT 188 34.18 247 33.42 1

CATAGGGCAC 135 24.55 149 20.16 0.84 0.62-1.13 0.255

TATTAGATGC 108 19.64 166 22.46 1.16 0.86-1.59 0.318

CGTTAGACAC 89 16.18 134 18.13 1.14 0.52-1.59 0.416

TATTAGACAC 30 5.45 43 5.82 1.09 0.65-1.80 0.735

OTHER

Claims

1. A method of screening for compounds of use in preventing or treating dementia wherein a cell having UBAPl is treated with a test compound and the effect of the test compound on the amount and/or function of UBAPl is assessed.

2. The method of claim 1 wherein the dementia is characterised by tauopathy.

3. The method of claim 1 or 2 wherein the dementia is FTLD.

4. The method of claim 1 or 2 wherein the dementia is Alzheimer's disease.

5. The method of any of the previous claims wherein the method further comprises the step of selecting a compound that increases the amount and/or function of UBAPl.

6. The method of any of claims 1 to 4 wherein the method further comprises the step of selecting a compound that decreases the amount and/or function of UBAPl.

7. The method of claim 5 or 6 wherein the selected compound is formulated into a pharmaceutically acceptable composition.

8. A method of screening for compounds of use in preventing or treating dementia wherein a non-human animal is administered a test compound and the effect of the test compound on the amount and/or function of UBAPl is assessed.

9. The use of an agent that modulates the amount and/or activation of UBAPl for the prevention or treatment of dementia.

10. The use of claim 9 wherein the agent increases the amount and/or activation of UBAPl.

11. The use of claim 9 or 10 wherein the agent is UBAPl polypeptide or a nucleic acid molecule encoding UBAPl polypeptide.

12. The use of claim 9 wherein the agent decreases the amount and/or activation of UBAPl.

13. The use of claim 9 to 12 wherein the agent binds to UBAPl polypeptide or nucleic acid encoding UBAPl polypeptide.

14. The use of claim 9 to 12 wherein the agent is an antibody or fragment thereof.

15. The use of claim 9 to 12 wherein the agent is a peptide.

16. The use of claim 9 to 12 wherein the agent is an aptamer.

17. The use of claim 9 to 12 wherein the agent is an antisense molecule or a siRNA.

18. The use of any of claims 9 to 17 wherein the subject is human.

19. The use of any of claims 9 to 18 wherein the dementia is characterised by tauopathy.

20. The use of any of claims 9 to 19 wherein the dementia is frontotemporal lobar degeneration (FTLD).

21. The use of any of claims 9 to 19 wherein the dementia is Alzheimer's disease.

22. A method of preventing or treating dementia comprising administering to a subject a therapeutically effective quantity of an agent that modulates the amount and/or activation of UB API.

23. A method of assessing whether a subject has or is likely to develop a dementia comprising determining whether the subject has a mutation in the UBAPl gene.

24. The method of claim 23 wherein the step of determining whether a subject has a mutation in the UBAPl gene uses haplotype analysis.

25. The method of claim 24 wherein the haplotype analysis is based on a haplotype block.

26. The method of claim 25 wherein the haplotype block comprises two or more SNPs.

27. The method of claim 26 wherein the SNPs are selected from the following group of SNPs: rsl3283064, rsl3283069, rs7018487, rslO971977, rsl2375731, rsl0814079, rs2380925, rsl7258783, rs4574933, rsl0814083.

28. The method of claim 23 wherein the step of determining whether a subject has a mutation in the UBAPl gene comprising genotyping the UBAPl gene of that subject.

29. The method of claim 28 comprising determining whether the subject has one or more of the following UBAPl mutations: P96L, E87K or M413X (S391Afs21X).

30. The method of claim 23 comprising determining whether the subject has a mutant UBAPl polypeptide.

31. The method of any of claims 23 to 30 wherein the UBAPl gene or polypeptide is derived from a sample of genomic DNA or polypeptide from the subject.

32. The method of claim 31 wherein the sample is derived from blood or tissue samples.

33. The method of any of claims 23 to 32 wherein the dementia is characterised by tauopathy.

34. The method of any of claims 23 to 33 wherein the dementia is frontotemporal lobar degeneration (FTLD).

35. The method of claim 34 wherein the FTLD is characterised by ub-ir

36. The method of claims 23 to 33 wherein the dementia is Alzheimer's disease.

37. The method of claims 23 to 36 wherein the subject is a human subject.

38. A method of assessing whether a subject has or is likely to develop a dementia comprising determining whether the subject has an altered amount of UBAPl polypeptide and/or nucleic acid encoding UBAPl polypeptide.

39. The method of claim 38 wherein if the subject has an altered amount of said polypeptide and/or nucleic acid, then this indicates that the subject has or is likely to develop dementia.

40. The method of claim 38 or 39 wherein the amount of UBAPl polypeptide or nucleic acid encoding UBAPl polypeptide is elevated.

41. The method of claim 38 or 39 wherein the amount of UBAPl polypeptide or nucleic acid encoding UBAPl polypeptide is reduced.

42. The method of any of claims 38 to 41 wherein the dementia is characterised by tauopathy.

43. The method of any of claims 38 to 42 wherein the dementia is frontotemporal lobar degeneration (FTLD) or Alzheimer's disease.

44. The method of any of claims 38 to 43 wherein the subject is a human subject.

45. A non-human genetically modified animal having or predisposed to develop dementia, wherein the dementia results from an altered amount and/or function of UBAPl polypeptide.

46. The non-human animal of claim 45 wherein the animal is a rodent.

47. The non-human animal of claim 45 or 46 wherein the dementia is characterised by tauopathy.

Ill

48. The non-human animal of any of claims 45 to 47 wherein the dementia is frontotemporal lobar degeneration (FTLD) or Alzheimer's disease.

49. The non-human animal of any of claims 45 to 48 wherein the amount and/or function of UBAPl polypeptide is elevated.

50. The non-human animal of any of claims 45 to 48 wherein the amount and/or function of UBAPl polypeptide is reduced.

51. The non-human animal of any of claims 45 to 50 wherein the genetically modified animal has a mutation(s) in UBAPl equivalent to the P96L, E87K and/or M413X (S391Afs21X) mutation in human UBAPl.

52. A kit for assessing whether a subject has or is likely to develop a dementia comprising means for determining whether the subject has a mutation in the UBAPl gene.

53. A kit for assessing whether a subject has or is likely to develop a dementia comprising means for determining whether the subject has an altered amount of UBAPl polypeptide and/or nucleic acid encoding UBAPl polypeptide.