EP0796342A2

EP0796342A2 - Genetic basis of factor xiii activity

Info

Publication number: EP0796342A2
Application number: EP95938560A
Authority: EP
Inventors: Alexander Fred Markham; Rashida Anwar; Krzystof Jozef Antoni Miloszewski
Original assignee: University of Leeds
Current assignee: University of Leeds
Priority date: 1994-12-08
Filing date: 1995-12-07
Publication date: 1997-09-24
Also published as: WO1996017953A2; GB9424823D0; JPH10510421A; WO1996017953A3

Abstract

The invention relates to a method for dianosing Factor XIII activity and in particular a genetic base method which identifies changes in the Factor XIII gene and correlates these changes with changes known to segretate with either an increase or decrease in Factor XIII activity.

Description

GENETIC BASIS OF FACTOR XIII ACTIVITY

The single inventive concept described in this application concerns a genetic understanding of Factor XIII activity which understanding can be applied so that in one aspect, that is where Factor XIII activity is low, or is genetically predicted as low, a method and product for safe guarding against haemorrhaging or miscarriage, where typically Factor XIII levels would normally fall, are provided; and in another aspect, where Factor XIII activity is high, or genetically predicted as high, a method and product for safeguarding against blood clot formation are provided.

It is well established that a number of agents are involved in the enzyme amplification cascade reaction associated with blood clotting. One agent of this cascade is Factor XIII which is found in plasma (as a pro-enzyme), in platelets and monocytes. It is essential for normal haemostasis.

Plasma Factor XIII circulates as a tetramer composed of 2 catalytic "a" sub units non covalently associated with 2 non-catalytic "b" sub units. In the presence of thrombin and calcium, the "a" and "b" sub units dissociate and Factor XHIa is converted to the active form by the removal of 37 N-terminal amino acids. The active form of Factor XHIa is a transglutaminase which cross-links fibrin monomers by introducing intermolecular covalent bonds between the alpha and gamma sub units of fibrin thus increasing the tensile strength of cross-linked fibrin. Activated Factor XIII also cross-links alpha 2-plasmin, fibronectin and collagen. It is thus able to produce clots of high mechanical strength with an increased resistance to degradation by plasmin.

The Factor XIII gene has been localised to chromosome 6 band p24-p25. The gene structure is found to extend to more than a 160Kb and contains 15 exons separated by 14 introns. Factor XIII cDNA, isolated from a human placental library, consists of 3,902 bases, of which, 2,196 bases are the coding sequence for 732 amino acids, 81 bases of 5' - and 1,625 bases of 3' - untranslated regions.

Polymorphisms in the genetic loci encoding the alpha and beta sub units of Factor XIII are surprisingly common. Despite this fact Factor XIII deficiency is a very rare disorder. Indeed, congenital deficiency of Factor XIII is a rare autosomal recessive disorder occurring with a frequency of 1 in 2 x 10⁶. This information would tend to imply that although the locus is polymorphic the majority of the polymorphisms are benign.

However, those polymorphisms that are linked with the disease are characterised by a lack of Factor XIII activity and a complete absence of the "a" sub unit, although deficiency of the "b" sub unit has also been described. The symptoms of the disease are prolonged bleeding, defective wound healing and a high risk of miscarriage.

The diagnosis of Factor XIII deficiency is difficult as routine coagulation tests, which depend on a demonstration of abnormal clotting time, are typically within normal limits. Indeed, clotting in Factor XIII deficiency is normal, it is the quality of the clot that is defective. It therefore follows that a diagnosis of the disease requires a means of measuring the quality of the clot.

In Factor XIII deficiency a plasma clot is simply a polymer of fibrin bound by ionic bonds only, which bonds can be dispersed by 1 % monochloroacetic acid or 5M urea. Clots cross-linked by Factor XIII are not soluble in these reagents. It therefore follows that if solubility in these reagents is found Factor XIII activity may be impaired or absent. Typically, this conventional diagnosis would be confirmed by a quantitative enzyme assay such as the dansylcadaverine-casein assay, which estimates the transglutaminase action of Factor XIII.

However, the quantitative analysis of transglutamanase activity is difficult to perform on a routine basis because of the small amounts of Factor XIII present in the plasma which small amounts form an initial part of the enzyme amplification cascade mechanism. Moreover, the level of Factor XIII within the plasma will vary according to the physiological status of the individual. For example, the total blood volume will significantly affect the quantitative assay and the total blood volume will be determined by the relative state of hydration/dehydration of the individual as a result of environmental conditions and rate of fluid intake/urine production.

A reliable determination of Factor XIII levels in plasma is therefore difficult to provide.

However, it is known that, within the population as a whole, levels of Factor Xπi activity are normally distributed. This would imply that some individuals in the population have a lower than average amount of Factor Xπi activity and conversely some individuals in the population have a higher than average amount of Factor XIII activity. It is with these two categories of the population that the present invention is concerned, and particularly, but not exclusively, it is with the extreme limits of these two categories. More specifically, in so far as the category with a lower than average amount of Factor XIII activity is concerned the invention relates to those individuals who have a lower than average amount of Factor XIII activity but who have not been conventionally diagnosed as Factor XIII deficient.

It is important at this stage to note that individuals who are conventionally diagnosed as Factor XIII deficient may be treated prophylactically and so receive, periodically, infusions of Factor XIII with a view to safeguarding against bleeding and generally improving the quality of life as such individuals. In addition, it is also important to note that conventionally diagnosed Factor XIII deficient females are unable to carry a pregnancy to term and therefore, whether receiving prophylactic treatment or not, such individuals are provided with more frequent, large dose Factor XIII infusions throughout pregnancy.

We have studied the Factor XIII gene of individuals suffering from the rare autosomal Factor XIII deficiency and we have identified a number of mutations which are causative in terms of Factor XIII deficiency. Since Factor XIII deficiency is autosomal recessive only those individuals with causative mutations or polymorphisms in each allele exhibit the disease. We have therefore begun assembling a data bank of causative mutations.

However, since it is known that Factor XIII activity within the population is normally distributed we have considered that, for example, those individuals with a lower than average amount of Factor XIII activity may have inherited a causative mutation from one parent only. Thus such individuals are heterozygotes for both the normal Factor XIII gene, or at least a polymorphism which is not causative, and a causative polymorphism. We therefore speculate that variations in the Factor XIII gene are responsible for the normal distribution of Factor XIII activity within the population. We also speculate that the inheritance of a causative polymorphism on one allele will reduce the level of Factor XIII activity below that of the average for the population. This means, that those individuals who have inherited one causative mutation for the disease will fall into a first category with a lower than average amount of Factor XIII activity but who are not conventionally diagnosed as Factor XIII deficient. This category of individuals will therefore be vulnerable to haemorrhaging and, in the case of females, miscarriage in circumstances where there naturally exists a predictable lowering of Factor XIII levels. Such a circumstance is found during the early stages of pregnancy where it is well known that Factor XHI levels decline.

Although not explained in these terms before, we consider that recurrent, spontaneous miscarriages in certain females are directly linked to Factor XIII deficiency and therefore although these females have not been conventionally diagnosed as Factor XIII deficient they carry a causative mutation on one allele. It therefore follows that the identification of this population of females and their treatment with periodic Factor XIII infusions would safeguard against recurrent, spontaneous miscarriage.

It is therefore an object of the invention to provide a means for diagnosing which members of the population, ideally female population, that have not been conventionally diagnosed as Factor XIII deficient, have a lower than average level of Factor XIII activity.

It is an object of the invention to provide a method for safeguarding against haemorrhaging and/or miscarriage in females during pregnancy where individuals and/or said females are not conventionally diagnosed as Factor XIII deficient.

It is a further object of the invention to provide the use of a product, the product being Factor XIII, to prevent haemorrhaging and/or miscarriage in females during pregnancy where individuals and/or said females have not been conventionally diagnosed as Factor XIII deficient.

Our investigations, and the subsequent identification of a number of causative mutations of Factor XIII deficiency have also led us to speculate that a second category, that is those individuals with a higher than average amount of Factor XIII activity may show no polymorphisms in the Factor XIII gene, or alternatively, may show no causative polymorphisms or may show at least one mutation or polymorphism which is typically associated with a high level of Factor XIII activity. Such individuals would have an increased propensity to produce clots and therefore an increased risk of coronary artery occlusion. It therefore follows that the identification of a lack of polymorphism in the

Factor XIII gene in either or both alleles, or alternatively, the presence of a benign polymorphism in either or both alleles, or the lack of a causative polymorphisms, or identification of a mutation or polymorphism in either or

_-- both alleles which is thought to segregate with increased Factor XIII activity, could be used as a diagnostic tool to predict the likelihood of an individual suffering from coronary/artery occlusion, or indeed, any other clot derived life threatening condition such as pulmonary embolus or deep vein thrombosis. Individuals suspected of being so threatened could then be treated prophylactically with anticoagulants such as heparin or warfarin to safe guard against such clot derived conditions. It is therefore a further object of the invention to provide a means for diagnosing which members of the population have a higher than average level of Factor XIII activity.

It is also an object of the invention to provide a method for safeguarding against the tendency to produce blood clots and so safeguard against clot derived conditions.

It is a further object of the invention to provide use of a product, the product being an anticoagulant, to safeguard against clot production in those individuals who are diagnosed as having a higher than average level of Factor XIII activity.

In its broadest aspect the invention therefore relates to a method of diagnosing the levels of Factor XIII activity of an individual within the population with a view to predicting the propensity of said individual towards bleeding, haemorrhage, miscarriage or clot formation. The genetic data described in this application facilitates this diagnosis and once the diagnosis is complete prophylactic treatment can then be determined with a view to safeguarding against the predictable affects of the determined level of Factor XIII activity.

The invention therefore lies in the realisation that Factor XIII activity is genetically determined and therefore the consequences of under or over activity can be predicted and prophylactically treated.

According to a first aspect of the invention there is therefore provided a method for diagnosing the level of Factor XIII activity of a given individual which method comprises:

a) taking a sample of cells from a given individual, b) determining the genetic sequence structure of both alleles of a Factor XIII gene from the individual, c) comparing said determined sequence structure with the known sequence structure of the Factor XIII gene, d) identifying any differences, e) comparing these differences with differences known to segregate with either a reduction in Factor XIII activity or an enhancement in Factor XIII activity, f) concluding as follows: i) in the instance where at least one difference is identified which difference typically segregates with a reduction in Factor XIII activity, diagnosing said individual as likely to suffer from the symptoms of Factor XIII deficiency in conditions where one would normally expect a reduction in the levels of Factor XIII, ii) in the instance where at least one difference is identified which difference typically segregates with an increase in Factor XIII activity, diagnosing said individual as likely to suffer from clot derived conditions, iii) in the instance where no differences are found in either allele, and where it has been shown that no difference in either allele results in an increased level of Factor XIII activity, diagnosing said individual as likely to suffer from clot derived conditions; vi) in the instance where no differences are found in either allele, and where it has been shown that no difference in either allele results in a decreased level of Factor XIII activity, diagnosing said individual as likely to suffer from the symptoms of Factor XIII deficiency in conditions where one would normally expect a reduction in the level of Factor XIII; and v) in the instance where a difference associated with increased Factor XIII activity is identified and a difference associated with decreased Factor XIII activity is identified diagnosing the individual according to the relative dominance of each of the said differences.

In a preferred embodiment of the invention the diagnosis concerns a comparison of those differences detailed in Table 3, which differences in Table 3 are known in the homozygote condition or in the compound heterozygote condition to be causative for Factor XIII deficiency, with the differences identified under d) above.

For the avoidance of doubt, the said known sequence structure of the Factor XIII gene referred to in c) above is the sequence structure in the absence of any polymorphisms.

According to a further aspect of the invention there is provided a method for preventing bleeding, and/or haemorrhage and/or miscarriage in females comprising; providing periodic Factor XIII infusions to individuals and/or said females who have been diagnosed as likely to suffer from the symptoms of Factor XIII deficiency in conditions where one would normally expect a reduction in the levels of Factor XIII.

According to a further aspect of the invention there is provided the use of Factor XIII in the manufacture of a medicament for the prevention of bleeding, and/or haemorrhage and/or miscarriage in females, for individuals and/or said females not conventionally diagnosed as Factor XIII deficient.

Ideally, said females will form part of a sub-population of females whose levels of Factor XIII activity are below the average for the population.

In view of the fact that Factor XIII is not known to give rise to complications or bring about allergic reactions or provide for the transmission of disease it may be commercially favourable to administer Factor XIII to all females who are pregnant and who are therefore exposed to a reduced level of Factor XIII and thus in its broadest concept the invention concerns the treatment of females who have not been conventionally diagnosed as Factor XIII deficient with Factor XIII at least during the early stages of pregnancy whilst the levels of Factor XIII are known to be low.

According to a further aspect of the invention there is provided a method for preventing clot-derived conditions in individuals who have been genetically diagnosed as having higher than average levels of Factor XIII activity comprising administering to said individuals anticoagulant.

According to a further aspect of the invention there is provided the use of anticoagulants in the manufacture of a medicament for the prevention of clot- derived conditions in individuals genetically diagnosed as having a higher than average amount of Factor XIII activity.

In the method of diagnosis of the invention preferably a peripheral blood sample is taken from an individual to be tested.

According to a yet further aspect of the invention there is provided primers which are complementary to those regions of the Factor XIII gene which undergo mutations related to an increase in Factor XIII activity and/or a decrease in Factor XIII activity.

Preferably at least one of the said mutations is located at the 3' end of a primer.

According to a yet further aspect of the invention there is provided a further method for diagnosing the level of Factor XIII activity of a given individual which method comprises:

a) taking a sample of cells from a given individual, b) determining whether at least one of the aforementioned primers of the invention binds to at least one or both alleles of the Factor XIII gene of said individual and in this way identifying whether there are any differences in the alleles of the Factor XIII gene of said individual when compared to the known sequence structure of the Factor XIII gene and c) concluding as follows: i) in the instance where at least one difference is identified which difference typically segregates with a reduction in Factor XIII activity, diagnosing said individual as likely to suffer from the symptoms of Factor XIII deficiency in conditions where one would normally expect a reduction in the levels of Factor XIII, ii) in the instance where at least one difference is identified which difference typically segregates with an increase in Factor XIII activity, diagnosing said individual as likely to suffer from clot derived conditions, iii) in the instance where no differences are found in either allele, and where it has been shown that no difference in either allele results in an increased level of Factor XIII activity, diagnosing said individual as likely to suffer from clot derived conditions; iv) in the instance where no differences are found in either allele, and where it has been shown that no difference in either allele results in a decreased level of Factor XIII activity, diagnosing said individual as likely to suffer from the symptoms of Factor XIII deficiency in conditions where one would normally expect a reduction in the levels of Factor XIII; and v) in the instance where a difference associated with increased

Factor XIII activity is identified and a difference associated with decreased Factor XIII activity is identified diagnosing the individual according to the relative dominance of each of the said differences.

Ideally the genetic sequence structure of the Factor XIII gene, from each parent, of the individual to be tested is determined using PCR techniques. For example, ideally, the genomic DNA of an individual to be tested is firstly amplified using PCR and more specifically Factor XIII "a" sub unit exons 1 to 15 are individually amplified using PCR primers.

Preferably exons 1 to 15 are amplified using those primers shown in Table 1 , or alternatively, using primers substantially similar to those shown in Table 1 having alterations, additions or deletions which do not affect the ability of said primers to amplify the exon to which each primer corresponds.

More specifically, the method of diagnosis for determining the genetic sequence structure of the Factor XIII gene of the individual to be tested involves the provision of cDNA relating to said gene. More preferably further still the said method involves amplification of said cDNA, ideally by the method of PCR.

In yet a further preferred method of diagnosis, PCR products produced from subjects carrying a deletion or insertion in one allele are sub-cloned into a vector, such as the pMOSBlue T-vector, prior to sequencing.

In yet a further preferred method of diagnosis the CDNA of the Factor XIII "a" gene is examined by dividing the entire coding region into 4 overlapping segments designated A, B, C and D and each segment is amplified, ideally by PCR, and therefore, preferably, using the oligonucleotides shown in Table 2, or alternatively, oligonucleotides substantially similar thereto having alterations, additions or deletions which do not affect the functional activity of the oligonucleotides.

Thus far, three mutations which may be the cause of "a" sub unit deficiency have been described. One sequence change is a two base deletion at a splice deletion acceptor site. This deletion does not grossly affect the splicing of the Factor XIII pre mRNA, but creates a translational frame shift resulting in early translation termination. The second mutation, is a G to A substitution at a splice donor site. The molecular mechanism of how this splice site mutation may be causing Factor XIII deficiency has not determined. The third is a nonsense mutation in which a C to T transition at nucleotide 598, in an arginine codon, results in stop codon TGA. We have identified a further eight mutations in patients from 5 unrelated families. The sequence changes found include MRNA abnormalities due to errors of splicing, a deletion/insertion event, a nonsense mutation, missense and silent mutations. These mutations are found in Table 3. Methods for describing how the data relating to the invention has been obtained will now be provided. However, it is important to note that the data obtained, thus far, are not exhaustive, rather the methods illustrate how further data relating to the invention can be compiled with a view to providing a data bank relating to Factor XIII activity. In the following methods section it will be explained how data was derived from individuals diagnosed as suffering from Factor XIII deficiency. Similar techniques can be used in order to obtain data from individuals suffering from coronary artery occlusion or clot derived disease conditions, or indeed any members of the population, with a view to providing data concerning Factor XIII activity and the occurrence of clinical conditions.

It is therefore apparent, that the collection of data in relation to the invention is ongoing, and indeed may continue to be so for many many years to come. It therefore follows that the inventive concept is not limited to the collection of data, but rather it concerns the realisation that genetic data relating to the Factor XIII gene can be used to predict Factor XIII activity and knowledge of this activity can be used to determine the clinical response of a given individual to either certain conditions, for example, a reduction in the levels of Factor XIII during the early stages of pregnancy, or alternatively, the propensity of the given individual to develop clot-derived disease conditions.

In the following methods Table 1 shows those primers suitable for amplifying exons 1 to 15 of the "a" sub unit of the Factor XIII gene.

Table 2 shows those primers used for amplifying designated regions of cDNA of the Factor XIII gene; and Table 3 is a summary of causative mutations involved in Factor XIII deficiency.

Table 4 shows those polymorphisms or mutations associated with an increase and a decrease in Factor XIII activity.

Patient N represents a normal genotype at the specified codon.

Patients A 1 (female) and A2 (male) have severe peripheral vascular disease.

Patients Bl and B2 are females who have had more than three recurrent miscarriages.

Figure 1 showing deficiency causing mutations for FXIIIa subunit.

Figure la shows PG family 001 Exon 3 sequenced with forward primer.

Figure lb shows SO family 005 Exon 14 sequenced with forward primer.

Figure lc shows DS family 003 Exon 11 sequenced with forward primer.

Figure Id shows DS family 003 Exon 3 sequenced with forward primer.

Figure le shows PC family 002 Exon 10 sequenced with forward primer.

Figure If shows DT family 004 Exon 3 sequenced with forward primer.

Figure 2 showing the Exon skipping events. Figure 2ai Lane 1 0X174 marker (Hae III cut) Lane 2 Normal Lane 3 Patient PG family 001 Lane 4 Patient DT family 004 RT-PCR Fragment A (amplified with oligo A 1 and A2 nested from Table 2).

Figure 2bi Lane 1 0X174 marker (Hae III cut)

Lane 2 Normal

Lane 3 Patient SO family 005 RT-PCR Fragment D (amplified with oligo Dl and D2 from Table 2).

Figure 2aii Sequence of RT-PCR Fragment A from normal vs mutant Sequenced with forward primer A l (from table 2).

Figure 2bii Sequence of RT-PCR Fragment D from normal vs mutant Sequenced with reverse primer (D2 from Table 2).

Figure 3 shows sequence data relating to codon 34.

Figure 4 shows sequence data relating to codon 564.

Figure 5 shows sequence data relating to codon 651.

MATERIALS AND METHODS Subjects:

Isolation of genomic DNA and total RNA: 30ml of peripheral blood was obtained from the subjects, using sodium heparin as an anticoagulant. Genomic DNA was extracted using standard procedures. The peripheral blood mononuclear cells (PBMC) were separated from whole blood using lymphoprep (Nycoma Pharma AS), according to the manufacturers instructions. Total RNA was isolated from the PBMC using the acid guanidinium thiocyanate-phenol method described by Chomczynski and Sacchi (1).

PCR amplification of genomic DNA: FXIIIa subunit exons 1 to 15 were individually amplified by 35 cycles of PCR in a mixture of lOOμl containing 50ng genomic DNA, 20μM of dNTPs, l μM of each primer, l.Ounit of Taq DNA polymerase (Promega) and reaction buffer (Promega). Each cycle consisted of denaturation of 92° C for lmin, annealing at 58° C for lmin (except for exon 5 and exon 13 amplimers which were annealed at 42° C for lmin) and extension at 72° C for 2min. Following a final step of extension at 72° C for 5min, 5μl of the PCR product was analysed by agarose gel electrophoresis and ethidium bromide staining.

Synthesis of cDNA and PCR amplification of cDNA (RT-PCR): First strand cDNA was generated from 1 μg of total RNA using random hexamers (Boehringer Mannheim) and Molony murine leukaemia virus reverse transcriptase (Promega) under standard conditions. lOng of the reverse transcribed RNA was amplified by PCR as described above.

Sequence analysis of PCR products: PCR products were desalted and purified from unincorporated nucleotides using Wizard DNA clean up columns (Promega) according to the manufacturers instructions. 25-50 fmoles of DNA were then used as template for direct cycle sequencing using the Promega fmol cycle sequencing kit and gamma ³³P-end labelled amplimers. PCR products produced from subjects carrying a deletion or insertion in one allele were subcloned using the pMOSBlue T-vector kit (Amersham) prior to sequencing as before.

RESULTS

For each of the probands from five unrelated families, all 15 exons of the Factor Xllla gene were amplified with the primers described in Table 1. The PCR products obtained from each exon in each individual were found to be of the expected sizes (data not shown) indicating no large insertions or deletions within exonic regions of the Factor Xllla gene in these patients. FXIIIa CDNA was examined from all patients. The entire coding region was divided into four overlapping segments called A, B, C and D, and each fragment was amplified by RT-PCR using the oligonucleotides shown in Table 2. The RT-PCR products relating to fragments A, B, C and D from all patients were of the expected sizes with the exception of fragment A from proband PG of family 001 and fragment D from proband SO of family 005. These are detailed in the section on mRNA abnormalities below. The PCR products obtained from genomic DNA and cDNA were sequenced directly with each amplimer and the sequence changes detected in all probands are summarised in Table 3.

Gross mRNA abnormalities

Three families proved to carry mutations affecting splice sites, with the proband from each family being homozygous for the sequence change. Sequence analysis of the amplified exon 3 from genomic DNA of proband PG from family 001 revealed a mutation in the donor splice site, at the last position of the exon, the normal sequence ATTG/gtga being changed to ATTT/gtga (Fig la). The RT-PCR fragment A, which spans exon 2 to exon 5 (Table 2), amplified from cDNA of this patient was found to be about 200bp shorter than the expected molecular size (Fig 2ai). Direct sequence analysis of this RT-PCR product showed 189bp missing between positions 215 and 403 of the mature FXIIIa mRNA (Fig 2aϋ).

This corresponds to an exon skipping event involving exon 3, with a perfect splicing together of exons 2 and 4. In addition, the single Sau3A restriction site, GATC, located within the exon 3 region of fragment A, was shown to have been lost upon digestion of fragment A with Sau3A (data not shown). Even though further RT-PCRs performed using primers complementary to exon 3 enabled the faint detection of transcripts containing sequences from exon 3 (results not shown), it can be assumed from the RT-PCR result that >95% of FXIIIa mRNA produced in this individual is devoid of exon 3 sequence. This also confirms the homozygosity of patient PG for this mutation. This identical mutation was also identified in proband DT of family 004. Patient DT was heterozygous for this mutation, inheriting it from the maternal line. This heterozygosity is also apparent in the RT-PCR fragment A where the truncated cDNA as well as the normal length are present (lane 4 in Fig 2ai). This exon 3 skipping event has left the translational frame intact and the resultant in-frame 'deletion' of 189 bases in the mature mRNA can be predicted to cause a shortening of the mature Xllla polypeptide by 63 amino acid residues.

The second splice site mutation was identified in proband SO of family 005.

Sequence analysis of the amplified genomic DNA from this patient displayed a mutation in the donor splice site of exon 14 at the last base of the exon, the normal sequence TCCG/gtaa being changed to TCCA/gtaa (Fig lb). This sequence change is in agreement with that found by Board et al (2) for this specific patient. This base change is expected to result in a loss of the Mspl restriction site CCGG, and this was confirmed by digesting the amplified exon 14 DNA with Mspl (data not shown). The RT-PCR product from this region of the cDNA (fragment D covering exon 12 to exon 15) was discovered to be about 140bp shorter than the expected size of 718bp (Fig 2bi). Direct sequence analysis of this RT-PCR product showed 137bp missing between positions 1993 and 2129 of the mature FXIIIa mRNA (Fig 1). Direct sequence analysis of this RT-PCR product showed 137bp missing between positions 1993 and 2129 of the mature FXIIIa mRNA (Fig 2bii). As before, an exon skipping event is observed, involving exon 14 this time, with exons 13 and 15 splicing together perfectly (Fig 2iib). This exon 14 skipping results in a frame shift and a translation termination codon, TGA, is produced following the amino acid lysine at codon 635. Thus a truncated form of FXIIIa polypeptide would be produced, lacking the last 103 amino acid residues.

Small alterations within the coding region

Missense mutations: Two missense mutations were identified. Proband PC family 002 was found to be homozygous for a sequence change in codon 408. Arg (408) was changed from CGG to CAG, resulting in an amino acid change to Gin (Fig lc). The second missense mutation was discovered in codon 60 of probands DS and LS of family 003. who were heterozygous for this base change. In this case the sequence AAC was found to be changed to ABA leading to an amino acid change from An to Lays (Fig Id).

Nonsense mutation: One nonsense mutation was identified in exon 11 of probands DS and LS of family 003, with both probands being heterozygous for this sequence change (Fig le). The C - A transition at nucleotide 1410 converted Tyr (441) to a stop codon (TAA). This mutation was transmitted through the paternal line in these patients.

Deletion/Insertion sequence change: Sequence analysis of amplified exon 3 from genomic DNA of proband DT from family 004 indicated the presence of two separate sequences, within the same PCR product. This suggested a deletion or an insertion even in one of the alleles. This was determined by subcloning the PCR product into the pMOSBlue T-vector prior to sequence analysis. The GG dinucleotide at positions 375 and 376 in the mature FXIIIa mRNA was found to be replaced by the sequence TCGTCC (Fig If). This deletion/insertion event results in a loss of the Sau3 A site, GATC, at position 376, and this was confirmed by Sau3A restriction analysis of the amplified exon 3 DNA (results not presented). Proband DT was heterozygous for this sequence change, inheriting it from the paternal line.

Silent mutations: Two silent mutations were found. Proband PG of family 001 showed a change in codon 331 from CCC to CCA and in codon 567 from GAG to GAA. This patient was homozygous for the change in codon 331, but heterozygous for the change in codon 567. In addition to the sequence changes described in the coding region of the FXIIIa gene, all patients were found to have a T to C substitution 23bp 5' to die 5'-end of exon 10, in intron 9, and a T deletion from the six T residues at 24bp to 29bp downstream of exon 7, in intron 7 (16;data not presented). Both these sequence changes do not appear to affect the splicing of the FXIIIa preRNA in these patients, as the RT-PCR fragments B and C, covering exon 7 and 10, respectively, were the expected size in each case. Association of Specific Polymorphisms in the Factor Xllla gene with (a) increased propensity to induce clots and (b) recurrent miscarriage

Using the above methods ie amplification of the Factor Xllla gene exonic regions by the polymerase chain reaction and nucleotide sequence analysis of the PCR products as described above. Polymorphisms or mutations which segregate with either an increased propensity to produce clots or recurrent miscarriage were identified.

These results are shown in Table 4 and Figures 3, 4 and 5.

From the results shown in Table 4 and Figures 3, 4 and 5 it can be seen that Pro564 and Gln651 are associated with increased Factor XIII activity and therefore increased risk of clot formation. In contrast, Leu34 is associated with reduced Factor XIII activity and hence recurrent miscarriage.

DISCUSSION

In the present study, 10 mutations are reported in the FXIIIa gene from 5 unrelated families. These sequence changes are discussed in relation to their effect on the product of translation. The G - T mutation found at the donor splice site of exon 3 in proband PG of family 001 and 004 leads to aberrant splicing of the FXIIIa mRNA, and hence truncated FXIIIa subunit polypeptide in these individuals. The amino acid residues missing from this polypeptide would be Glu-43 through He- 105 from the ^β-sandwich domain. Hence the β-sandwich domain structure in the truncated polypeptides may greatly differ from the expected two four-stranded anti-parallel β-sandwich (3). The altered structure is likely to influence the ability of the subunit to dimerise, which may in turn affect the in vivo half-life of the protein (4). In addition, the β-sandwich structure assumed in this truncated polypeptide may prevent the activation peptide from associating with the core domain in the other subunit of the dimer and this may have consequences for the ability of the protein to become activated (5). The Asn60Lys change found in probands DS and LS of family 003 is also likely to have similar affects on the β-sandwich structure, since the change is from a polar amino acid to a highly charged residue with a longer side chain.

The second splice donor site mutation, identified in proband SO of family 005, also leads to aberrant splicing resulting in truncated FXIIIa mRNA. The polypeptide synthesise would like the whole of barrel 2 with the exception of the first 6 amino acid. Although this domain is not implicated in the activation of the protein, it is thought to be an important structural element (6).

The nonsense mutation observed in probands DS and LS of family 003 would result in early translation termination producing a truncated polypeptide, lacking 291 amino acid residues from the carboxyl terminus. This correlates to all of barrels 1 and 2 as well as part of the carboxyl terminus of the core domain being missing. In view of FXIIIa X-ray crystallography data, the consequence of this would obviously be lack of correct structure formation in the active site cavity as well as loss of dimerisation.

The highly charged Arg-408 within the core domain, being in the immediate vicinity of the catalytic triad, specifically Asp- 396 (5), may be involved in hydrogen bonding or salt bridge formation with other amino acid residues around the active site. Since glutamine is a polar non-basic amino acid, it is probable that the Arg408Gln change discovered in proband PC of family 002 results in a conformational change around the catalytic triad, which in turn would grossly affect the activity of the protein. The true affect of this, and the Asn60Lys, point mutation can best be examined by the expression of these mutants alleles in mammalian cells and analysis of the activity of the 3-D X-ray crystallography structure of the altered proteins.

The deletion/insertion seen in proband DT of family 004 results in a translational frameshift such that a polypeptide of 190 amino acid residues is synthesised. Only the first 96 residues of this polypeptide correlate with the FXIIIa subunit protein sequence. It is interesting to note that the nucleotide sequence TCGTCC also exists at positions 354 to 369 in the mature FXIIIa mRNA, and the conversion from GG to TCGTCC may have arisen due to a DNA replication error in this particular family, particularly as it is transmitted through the paternal line.

Of all the FXIIIa subunit deficiency causing sequence changes identified so far, 4 out of the 7 families studied (20,22 and this study) show mutations at splice sites, corresponding to 3/8 mutations. The G - T mutation at the donor splice site of exon 3 was identified in two families (001 and 004) and may prove to be a common sequence change. Also probands from 3 of the families (001, 002 and 005) were found to be homozygous for the deficiency causing mutation, suggesting these sequence changes may be more common than those found in the compound heterozygotes in patients of families 003 and 004. Overall the FXIIIa subunit deficiency appears to be genetically heterogeneous.

Six normal polymorphisms have been described at the level of amino acid (17-19), giving rise to four different alleles. In this study, probands PG, PC and DT of families 001, 022 and 004, respectively, were found to have the *IB allele, proband SO of family 005 the *1A allele and probands DS and LS of family 003 the *1A/*1B alleles, as defined by Suzuki (7). No further polymorphisms, at the amino acid level, were identified in this study.

In summary, 8 mutations were identified in 6 patients from 5 unrelated families. 4 of the sequence changes are predicted to produce truncated polypeptides. The translational capacity of the FXIIIa mRNA containing the 2 silent mutations is not expected to be influenced by codon usage factors. The affects on the structure-function relationships of the AspόOLys and Arg408Gln changes can best be analysed by expression of these mutant alleles in mammalian cells, and examination of the proteins produced.

In addition, we have also identified specific polymorphisms or mutations in the Factor Xllla gene which correlate with either an increased propensity to produce clots or recurrent miscarriage.

Using the methods described in this application it has been possible to identify mutations in the Factor XIII gene. This has enabled us to compile tables of mutations, which in the homozygous state or compound heterozygote state, are causative for Factor XIII deficiency. One would expect to find causative mutations for Factor XIII deficiency in individuals suffering from Factor XIII deficiency. Similarly, one would expect to find causative mutations for clot derived disease conditions in individuals suffering from such conditions, and/or, the lack of any causative mutations for Factor XIII deficiency in individuals suffering from such conditions. As mentioned above, since Factor XIII is not known to give rise to complications or have any deleterious effect, it may be advisable to administer Factor XIII to all individuals of the population likely to experience, under certain predictable circumstances, a reduction in Factor XIII levels.

In contrast, anticoagulants can have deleterious effects and therefore their administration has to be carefully considered and we therefore propose that the information contained in this patent application is another step in that process of carefully consideration since it is possible to genetically predict the level of Factor XIII activity of a given individual and thus predict the likely consequences, deleterious of otherwise, of administering anticoagulants to an individual.

The inventive concept described in this patent application therefore has far reaching consequences.

REFERENCES

1. Chomczynski P., and Sacchi N., (1987) Single step method of RNA extraction by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162, 156-159.

2. Board P. G., Coggan M., and Miloszewski K. J. A., (1992) Identification of a point mutation in factor Xllla subunit deficiency. Blood 80, 937-941. 3. Yee V. C, Pederson L. C, Trong I. L., Bishop P. D., Stenkamp R. E. and Teller D. C, (1994) Three-dimensional structure of a transglutaminase Human blood coagulation factor XIII. Proc. Natl. Acad. Sci USA 91 7296- 7300.

4. Fear J. D., Miloszewski K. J. A., and Losowsky M. S., (1983) The half-life of factor XIII in the management of inherited deficiency. Thromb. Haemost 49 102-105.

5. Pederson L. C, Yee V. C, Bishop P. D., Trong I. L., Teller D. C, and Stenkamp R.E., (1994) Transglutaminase factor XIII uses proteinase-like catalytic triad to crosslink macromolecules. Protein Science 3 1131-1135.

6. Song Y., Taubenfeld S. M., Sheng DeQ and Matseuda G. R, (1994) Characterisation of a monoclonal antibody directed against the carboxyl terminus of human factor XIII. An epitope exposed upon denaturation and conserved across species lines. Thromb. Haemost 71 62-67.

7. Suzuki K., Iwata M., Ito S., Matsui K., Uchida A., and Mizoi Y., (1994) Molecular basis for subtypic differences of the 'a' subunit of coagulation factor XIII with description of the genesis of the subtypes. Hum. Genet. 94, 129-135.

Claims

1. A method for diagnosing the level of Factor XIII activity of a given individual which method comprises:

a) taking at least one cell from a given individual, b) determining the genetic sequence structure of both alleles of a Factor XIII gene from the individual, c) comparing said determined sequence structure with the known sequence structure of the Factor XIII gene, d) identifying any differences, e) comparing these differences with differences known to segregate with either a reduction in Factor XIII activity or an enhancement in Factor XIII activity, f) concluding as follows: i) in the instance where at least one difference is identified which difference typically segregates with a reduction in Factor XIII activity, diagnosing said individual as likely to suffer from the symptoms of

Factor XIII deficiency in conditions where one would normally expect a reduction in the levels of Factor XIII, ii) in the instance where at least one difference is identified which difference typically segregates with an increase in Factor XIII activity, diagnosing said individual as likely to suffer from clot derived conditions, iii) in the instance where no differences are found in either allele, and where it has been shown that no difference in either allele results in an increased level of Factor XIII activity, diagnosing said individual as likely to suffer from clot derived conditions; vi) in the instance where no differences are found in either allele, and where it has been shown that no difference in either allele results in a decreased level of Factor XIII activity, diagnosing said individuals as likely to suffer from the symptoms of Factor XIII deficiency in conditions where one would normally expect a reduction in the level of Factor XIII; and v) in the instance where a difference associated with increased Factor XIII activity is identified and a difference associated with decreased Factor XIII activity is identified diagnosing the individual according to the relative dominance of each of the said differences.

2. A method according to claim 1 wherein the comparison under e) above comprises comparing the said differences with the information provided in Table 4.

3. A method according to claim 1 wherein the comparison under e) above comprises comparing the said differences with the information provided in

Table 3.

4. A method according to claim 1 wherein the comparison under e) above comprises comparing said differences with the information in Tables 3 and

4.

5. A method for preventing bleeding; and/or haemorrhage; and/or miscarriage in females comprising: providing periodic Factor XIII infusions to individuals who have been diagnosed as likely to suffer from the symptoms of Factor XIII deficiency in conditions where one would normally expect a reduction in the level of Factor XIII.

6. A method according to claim 5 wherein said individuals include females which form part of a sub-population of females whose levels of Factor XIII activity are below the average for the population.

7. The use of Factor XIII in the manufacture of a medicament for the prevention of bleeding; and/or haemorrhage; and/or miscarriage; in females not conventionally diagnosed as Factor XIII deficient.

8. The use of Factor XIII in the manufacture of a medicament for the prevention of bleeding and/or haemorrhage in males not conventionally diagnosed as Factor XIII deficient.

9. Use according to claims 7 or 8 wherein said medicament is for preventing said ailments in circumstances where Factor XIII levels fall.

10. A prenatal regime comprising administering Factor XIII to all females who are pregnant.

11. A method for preventing clot-derived conditions in individuals who have been genetically diagnosed as having higher than average levels of

Factor XIII activity comprising administering to said individuals anti¬ coagulant.

12. The use of an anti-coagulant in the manufacture of a medicament for the prevention of clot-derived conditions in individuals genetically diagnosed as having a higher than average level of Factor XIII activity.

13. Primers which are complementary to those regions of the Factor XIII gene which exhibit changes related to an increase in Factor XIII activity and/or a decrease in Factor XIII activity.

14. A method for diagnosing the level of Factor XIII activity of a given individual which method comprises:

a) taking at least one cell from a given individual, b) determining whether at least one of the primers according to claim 12 binds to at least one or both alleles of the Factor XIII gene of said individual and in this way identifying whether there are any differences in the alleles of the Factor XIII gene of said individual when compared to the known sequence structure of the Factor XIII gene and c) concluding as follows: i) in the instance where at least one difference is identified which difference typically segregates with a reduction in Factor XIII activity, diagnosing said individual as likely to suffer from the symptoms of Factor XIII deficiency in conditions where one would normally expect a reduction in the levels of Factor XIII, ii) in the instance where at least one difference is identified which difference typically segregates with an increase in Factor XIII activity, diagnosing said individual as likely to suffer from clot derived conditions, iii) in the instance where no differences are found in either allele, and where it has been shown that no difference in either allele results in an increased level of Factor XIII activity, diagnosing said individual as likely to suffer from clot derived conditions; iv) in the instance where no differences are found in either allele, and where it has been shown that no difference in either allele results in a decreased level of Factor XIII activity, diagnosing said individual as likely to suffer from the symptoms of Factor XIII deficiency in conditions where one would normally expect a reduction in the levels of Factor XIII; and v) in the instance where a difference associated with increased

15. A method according to claims 1 or 14, wherein the method under b) above involves the provision of cDNA relating to said gene.

16. A method according to claim 15, wherein the method under b) above further involves amplification of said cDNA.

17. A method according to claim 16, wherein said amplification is undertaken using PCR methods.

18. A method according to claims 1 or 14, wherein the cDNA of the

Factor XIII "a" gene is examined by dividing the entire coding region into four overlapping segments designated A, B, C and D and each segment is amplified using the ogUoneucleotides shown in Table 2 or alternatively, ogUoneucleotides substantially similar thereto having alterations, additions or deletions which do not affect the functional activity of the ogUoneucleotides.