WO2010020695A1 - Monosomy 1p36 syndrome - Google Patents

Monosomy 1p36 syndrome Download PDF

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Publication number
WO2010020695A1
WO2010020695A1 PCT/EP2009/060869 EP2009060869W WO2010020695A1 WO 2010020695 A1 WO2010020695 A1 WO 2010020695A1 EP 2009060869 W EP2009060869 W EP 2009060869W WO 2010020695 A1 WO2010020695 A1 WO 2010020695A1
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rerl
expression
activity
syndrome
monosomy
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PCT/EP2009/060869
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French (fr)
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Wim Annaert
Guy Froyen
Dragana Spasic
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Vib Vzw
Katholieke Universiteit Leuven, K.U. Leuven R & D
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Application filed by Vib Vzw, Katholieke Universiteit Leuven, K.U. Leuven R & D filed Critical Vib Vzw
Publication of WO2010020695A1 publication Critical patent/WO2010020695A1/en

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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00Detection or diagnosis of diseases
    • G01N2800/38Pediatrics
    • G01N2800/385Congenital anomalies

Abstract

The present application relates to monosomy 1p36 syndrome, and more in particular to the role of the RER1 gene and protein therein. Methods are provided for diagnosing monosomy 1p36 syndrome on the basis of the presence/expression of (functional) RER1. Also disclosed are methods aimed to improve one or more symptoms of the monosomy 1p36 syndrome by restoring RER1 function or downstream signaling events.

Description

Monosomy Ip36 syndrome

Field of the invention

The present application relates to monosomy Ip36 syndrome, and more in particular to the role of the RERl gene and protein therein. Methods are provided for diagnosing monosomy Ip36 syndrome on the basis of the presence/expression of (functional) RERl. Also disclosed are methods aimed to improve one or more symptoms of the monosomy Ip36 syndrome by restoring RERl function or downstream signaling events wh ich are infl uenced by RE Rl fu nction, in particu lar the v- secretase/Notch pathway.

Background

Unbalanced chromosomal abnormalities account for about 20% of cases of mental retardation. A frequent class of cytogenetic abnormalities is deletion of the telomeric regions of chromosomes. These may cause substantial phenotypic abnormalities, because human telomeric regions are relatively gene rich as compared with other regions of the genome (Saccone et al., 1992). Monosomy Ip36, or Ip36 deletion syndrome (the deletion of the most distal (telomeric) band of the short arm of chromosome 1) is the most common terminal deletion syndrome. The prevalence of the Ip36 deletion is estimated to be 1 in 5000 births (Shaffer and Lupski, 2000), with a 2:1 female to male ratio (Slavotinek et al., 1999; Battaglia et al., 2008).

The constitutional deletion of Ip36 results in a syndrome with multiple congenital anomalies and mental retardation (Shapira et al. 1997). Apart from mental retardation or developmental delay, most patients display distinct facial characteristics (including deep-set eyes, flat nasal bridge, asymmetric ears, and pointed chin). Additional clinical characteristics include hearing loss, seizures, cardiomyopathy, growth delay, hypothyroidism, and orofacial clefting abnormalities (reviewed by Slavotinek et al., 1999; Shaffer and Heilstedt, 2001). Most of these problems can be treated, but when left untreated can lead to further difficulties. Thus, doctors need to recognize the clinical problems early in the patient's life, to provide maximum benefit of treatment. Disorders, such as hypothyroidism and hearing loss, have standard treatments. Recognition of developmental delay and other developmental issues, allows for early therapeutic intervention. However, treatment is not the same as cure.

Furthermore, chromosome Ip36 deletions have also been reported to occur in various neoplasms, including neuroblastoma, prostate cancer, lung cancer, malignant melanoma, hepatoma, cervical carcinoma, breast cancer, colorectal adenocarcinoma, ovarian cancer, and non Hodgkin lymphoma. The identification of deletions of Ip36 in a subset of diverse cancers led to the hypothesis that the Ip36 region contains a number of tumor-suppressor genes and that deletion of one or more of these genes is involved in the chain of events that results in malignancy (Blatt, 2001). Cancer is not typically listed as a symptom associated with the Ip36 syndrome, but this observation may be due to (i) the early age at which the diagnosis of monosomy Ip36 patients is made, (ii) the comparatively small number of su bjects in Ip36 studies compared with the incidence of various cancers such as neuroblastoma, or (iii) possible parent-of-origin effects among varying-sized deletions in the development of cancer, most notably neuroblastoma (Wu et al. 1999).

The contiguous gene deletion syndrome is presumably caused by haploinsufficiency of a number of genes. However, most clinical manifestations arising as a result of deletion of Ip36 are probably caused by the absence of one copy of a dose-sensitive gene (Shaffer and Heilstedt, 2001). Unlike other common deletion syndromes, patients with a chromosome Ip36 deletion have different sized pieces of chromosome missing. Most deletions are de novo. Apart from deletion size, the complexity of the chromosomal rearrangements also varies: not only terminal deletions are observed, but also interstitial deletions, more complex chromosomal rearrangements (including more than one deletion or deletions with duplications, triplications, insertions, and/or inversions), as well as a derivative chromosome 1 (i.e. a chromosome 1 in which the Ip telomeric region is replaced by another chromosome end). Individual patients, therefore, might be missing different genes, resulting in phenotype variability. Interestingly, the severity of associated disorders varies, whereas physical features are remarkably similar in patients.

Wu et al. (1999) and Heilstedt et al. (2003) suggested a complete genotype-phenotype correlation, identifying the critical regions for certain features and considering Ip36 deletion syndrome as a contiguous gene deletion syndrome. However, Gajecka et al. (2007) found no correlation between deletion size and number of observed clinical features in a large cohort; even individuals with small (<3 Mb) deletions of Ip36 presented with most of the features commonly associated with the syndrome. Redon et al. (2005) hypothesized that the features associated with Ip36 deletion syndrome may result from a position effect rather than a contiguous gene deletion syndrome.

Because of the large differences in deletion sizes and complexity, the syndrome is presumable caused by haploinsufficiency of a number of genes, complicated with variability of penetrance probably due to modifier genes. This is exemplified by studies on the delta subunit of GABA-A receptor (Windpassinger et al., 2002) or the beta subunit Kvβ2 of the voltage-gated K+-channel (Heilstedt et al., 2001) that can only partially explain the epilepsy phenotype. Still, the most common minimal deletion overlap suggests that a limited number of critical genes play a central role in the syndrome. Such potential critical regions (i.e. regions most commonly deleted) for certain clinical findings (e.g. clefting, hypothyroidism, cardiomyopathy, hearing loss, large fontanel, hypotonia) in monosomy Ip36 have been identified (Heilstedt et al., 2003). The terminal region of chromosome Ip36 is gene rich. However, only some of the genes will lead to a specific phenotype when deleted. Nonpenetrance, epigenetic, and stochastic factors are expected to influence certain clinical features (Heilstedt et al., 2003). Despite longtime ongoing efforts, no genes have been conclusively determined to be causative for any of the clinical features associated with Ip36 deletion syndrome.

At present, a testing strategy to confirm the diagnosis of monosomy Ip36 may involve cytogenetic studies, FISH (fluorescent in situ hybridization) and array-CGH (array-based comparative genomic hybridization). Although MLPA (multiplex ligation-dependent probe amplification) is clinically available, it is not a recommended method for detection of deletions of these sizes.

Conventional cytogenetic studies can be used to detect large deletions (i.e., >5 Mb) and more complex cytogenetic rearrangements (unbalanced chromosome translocations). However, because most human chromosomes end in light-staining GTG bands, the telomeric regions are difficult to visualize cytogenetically. Thus, telomere region-specific probes for FISH have been developed to identify small terminal deletions that otherwise might not be seen with conventional cytogenetic techniques (Knight et al. 1997). FISH using at least two subtelomeric region-specific probes (Vysis Ip subtel probe, Vysis p58 probe; D1Z2 Oncor probe or CEB108/T7) can identify parental rearrangements and may detect terminal and interstitial deletions and derivative chromosomes. However, FISH cannot detect an interstitial deletion proximal to the probes; cannot distinguish between a "true" terminal deletion and a more complex rearrangement; or cannot define the extent of the deletion. Array CGH can in principle be used to detect smaller deletions (i.e., <5 Mb) or interstitial deletions or complex rearrangements. Use of commercially available microarrays detects DNA copy-number changes in Ip36 deletion syndrome.

It would be advantageous to have an easier, less cumbersome and cheaper test for Ip36 deletion syndrome available (e.g. in the form of a PCR test). Further, it would be beneficial to be able to link a specific gene to at least some of the major symptoms observed in monosomy Ip36, as restoring gene function (or downstream effects of the missing gene) would provide a therapeutic approach to treat monosomy Ip36 or symptoms associated therewith.

Summary of the invention

It is an object of the invention to provide methods for diagnosing disorders characterized by insufficient RERl function (e.g. through deletion, mutation or instability of the RERl gene), in particular monosomy Ip36 syndrome. To this end, methods of diagnosis of disorders characterized by insufficient RERl function, in particular monosomy Ip36 syndrome are provided, comprising the steps of: providing a sample of a subject suspected of having a disorder characterized by insufficient RERl expression and/or activity, in particular monosomy Ip36 syndrome; - evaluating the expression and/or activity of RERl in the sample; wherein an absence of or a decrease in RERl expression and/or activity is indicative of the presence of the disorder characterized by insufficient RERl expression and/or activity, in particular monosomy Ip36 syndrome.

According to particular embodiments, the methods can be extended with a step of comparing the expression and/or activity of RERl in the sample with the expression and/or activity of RERl in a control sample, wherein an absence of or a decrease in RERl expression and/or activity as compared to the control sample is indicative of the presence of the disorder characterized by insufficient RERl expression and/or activity, in particular monosomy Ip36 syndrome.

Expression and/or activity of RERl can be evaluated at the mRNA level or the protein level. According to specific embodiments, the expression and/or activity of RERl is evaluated via PCR, in particular via

RT-PCR.

According to alternative specific embodiments, the expression of RERl is evaluated via Western blotting, in particular using Rerl specific polyclonal or monoclonal antibodies.

According to particular embodiments, the expression and/or activity of RERl is evaluated indirectly, by evaluating expression and/or activity of molecules downstream of RERl, such as γ-secretase, Notch or other components of the Notch signaling pathway such as for example Notch ligands (including, but not limited to Deltal, Jagged2), the Notch receptors themselves (e.g. Notchl, Notch2, Notch3, Notch4), or downstream Notch effector genes like Hes or Her genes. Evaluating RERl indirectly may be particularly advantageous in cases where easy activity tests are available, such as e.g. for evaluating v- secretase activity. Of note, RERl is a negative regulator of γ-secretase (and thus of Notch signaling), thus an increase in γ-secretase activity (or Notch signaling) is indicative of a decrease in RERl expression and/or activity. The expression and/or activity of RERl may also be evaluated through specific phenotypic manifestations, such as by monitoring acetylated tubulin levels or determining cilia number or length. Typically, cilia number or length will be determined from cells like fibroblasts, in each case cells that are normally ciliated. In a further aspect of the invention, restoring of RERl function, or of at least some of its downstream effects, can be used to treat at least one symptom of the disorder characterized by insufficient RERl expression and/or activity, in particular monosomy Ip36 syndrome. 'At least one symptom' implies that the methods presented herein may also be applied to treat more than one symptom, or to treat one or more symptoms.

Thus, methods are provided of treating at least one symptom of a disorder characterized by insufficient RERl expression and/or activity, in particular monosomy Ip36 syndrome, in a subject in need thereof, comprising - upregulating RERl expression and/or activity; and/or upregulating expression and/or activity of a gene, protein or protein complex that is positively regulated by RERl; and/or downregulating expression and/or activity of a gene, protein or protein complex that is negatively regulated by RERl.

According to further specific embodiments, the downregulating expression and/or activity of a gene, protein or protein complex that is negatively regulated by RERl can be done by downregulating v- secretase expression and/or activity; and/or by downregulating Notch signaling. According to yet further specific embodiments, the downregulating Notch signaling is done by downregulating Notch expression and/or activity, more in particular by downregulating Notch3 expression and/or activity.

In line with this, compounds are also provided for use in treatment of at least one symptom of a disorder characterized by insufficient RERl expression and/or activity, in particular monosomy Ip36 syndrome, which compounds upregulate RERl expression and/or activity; and/or upregulate expression and/or activity of a gene, protein or protein complex that is positively regulated by RERl; and/or downregulate expression and/or activity of a gene, protein or protein complex that is negatively regulated by RERl. Again, specific compounds may downregulate γ-secretase expression and/or activity, and/or downregulate expression and/or activity of components in the Notch signaling pathway.

The at least one symptom of the disorder characterized by insufficient RERl expression and/or activity, in particular monosomy Ip36 syndrome, that can be treated using the compounds described herein or by performing the methods described herein is particularly selected from the list of: neurological defects, developmental delay, mental retardation, hypotonia, seizures, epilepsy, feeding difficulties, oropharyngeal dysphagia, congenital heart defects, cardiovascular abnormalities, ophthalmological abnormalities, skeletal anomalies, hearing loss, genitourinary malformations, hypothyroidism, and neuroblastoma..

Brief description of the Figures

Figure 1 presents RERl as an integral membrane protein with four transmembrane (TM) domains.

Figure 2 shows that RERl expression negatively regulates γ-secretase activity. (A): Aβ secretion from APP-C99 transfected HeLa cells. After 24 h of overexpression or 48 h of down-regulation (RNAi, specific duplex; NS, nonspecific control) of hRerlp in combination with overexpression of APP-C99, HeLa cells were metabolically labeled for 4 h as described previously (Annaert et al., 1999). Total secreted Aβ and APP-C99 were, respectively, immunoprecipitated from media and extracts and quantified by phosphorimaging. The ratio of Aβ to APP-C99 is significantly decreased or increased when hRerlp levels are up- or down-regulated (mean ± SEM; n = 5; t test *, P < 0.03; **, P < 0.05). (B) Cell-free γ-secretase assay showing AICD production in vitro. Extracts from control and hRerlp knockdown HeLa cells were mixed with affinity-purified recombinant APP-C99-FLAG (from transfected γ-secretase-deficient MEFs) and incubated at 37°C. Newly produced AICD-FLAG is clearly increased after hRerlp knockdown, indicating enhanced levels of γ-secretase activity. From Spasic et al., 2007. Aβ, amyloid β; APP-C99, C-terminal 99 amino acids of amyloid precursor protein, a direct γ-secretase substrate; MEF, mouse embryonic fibroblast; AICD, APP intracellular domain.

Figure 3 demonstrates that RERl expression is down to 50% (A) and γ-secretase activity (as shown by AICD production) increased (B) in (fibroblasts derived from) monosomy Ip36 patients as compared to control fibroblasts. Levels of actin are shown as control. AICD, APP intracellular domain.

Figure 4 shows the expression pattern of RERl in zebrafish (Danio rerio) using in situ hybridization. From left to right, top to bottom are shown: 1000 cell stage, 15 hours post fertilization

(h.p.f.) embryo, 24 h.p.f. embryo with indication of somite boundaries, 48 h.p.f. embryo, 72 h.p.f. embryo with indication of neuromasts and pectoral fin, 72 h.p.f. embryo with indication of ear, 72 h.p.f. embryo with indication of neuromasts and optic tectum, 5 days post fertilization (d.p.f.) embryo with indication of optic tectum. Places of high RERl expression during development are indicated through arrows (somite boundaries, pectoral fin, neuromasts (lateral line), ear, and optic tectum)

Figure 5. Rerlp is required for ciliogenesis in LLC-CL4 cells. (A) lmmunostaining with acetylated tubulin, which is a marker for cilium, in control and Rerlp downregulated cells is showing that cilia are much shorter when Rerlp is depleted (upper panel). Scale bar, lOμm. Lower graph depicts the efficiency of downregulation checked by western blot (70% reduction in Rerlp levels normalized to GAPDH). (B) Quantification of cilia number and length from four fields (upper panel). Lower panel shows the frequency of cells with certain length distribution. While long cilia are almost exclusively absent from cells with Rerlp knockdown, the percentage of cells with short cilia is significantly higher. (C) Graph with the quantification of the area occupied by acetylated tubulin, which actually represents the area covered by cilia, confirms 50% decrease observed in panel B when Rerlp levels are reduced. Measurements of cilia length and area covered by acetylated tubulin staining were done with ImageJ program. (D) Scanning EM figures of control and Rerlp downregulated cells at different magnifications. Note the striking differences in the cell morphology as well as the appearance of cilia and microvilli.

Figure 6. Increase in tubulin acetylation is consistent throughout microtubule repolymerization. (A) Western blot showing the dynamics of microtubule depolymerization and repolymerization in RPE cells after knockdown of Rerlp. To study this microtubule dynamics, the cells were treated with 0.5μM nocodazole for 30 min and then left for 5, 10 and 20 minutes to recover and repolymerize microtubules. Cells were then lysed in a taxol (5OnM) containing buffer, centrifuged to separate the soluble (S) and polymerized (P) fraction and further processed for western blot analysis. The graphs show the quantifications of both α-tubulin and acetylated tubulin in the soluble (B) and polymerized fractions (C). The levels of acetylated tubulin at the initial time point are higher when Rerlp is downregulated (graph with polymerized fractions, compare black triangles with black squares). The dynamics of depolymerization follows the same kinetics compared to control. During repolymerization, cells with Rerlp knockdown show again increase in acetylation. Although the differences seem to be much more pronounced, the levels of α-tubulin in polymerized fraction are also higher during recovery in the cells with Rerlp knockdown compared to control cells. This reflects that there is a consistent increase in the process of acetylation when Rerlp is downregulated.

Figure 7. Reduced ciliogenesis in monosomy Ip36 patient fibroblasts (A)

Doubleimmunostaining of control and patient fibroblast cells with Rerlp and acetylated tubulin is showing decreased ciliogenesis in the patient cells. Scale bar, lOμm. (B) Quantification of the average cilia number (upper panel) and cilia length (lower panel) obtained from 100 cells shows a significant reduction in the number and size of the cilium in the patient cells. (C) Histogram is representing the frequency of cells with certain length distribution. The percentage of patient fibroblasts with short cilia is higher compared to control while the long cilia are almost completely absent in the patient fibroblasts. Cilia length measurements were done with ImageJ program. Detailed description

Definitions

The present invention will be described with respect to particular embodiments and with reference to certain drawings but the invention is not limited thereto but only by the claims. Any reference signs in the claims shall not be construed as limiting the scope. The drawings described are only schematic and are non-limiting. In the drawings, the size of some of the elements may be exaggerated and not drawn on scale for illustrative purposes. Where the term "comprising" is used in the present description and claims, it does not exclude other elements or steps. Where an indefinite or definite article is used when referring to a singular noun e.g. "a" or "an", "the", this includes a plural of that noun unless something else is specifically stated.

Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.

The following terms or definitions are provided solely to aid in the understanding of the invention. Unless specifically defined herein, all terms used herein have the same meaning as they would to one skilled in the art of the present invention. Practitioners are particularly directed to Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, Plainsview, New York (1989); and Ausubel et al., Current Protocols in Molecular Biology (Supplement 47), John Wiley & Sons, New York (1999), for definitions and terms of the art. The definitions provided herein should not be construed to have a scope less than understood by a person of ordinary skill in the art.

'Monosomy Ip36 syndrome' or '(chromosome) Ip36 deletion syndrome' as used in the application refers to the constitutional deletion of (part of) the Ip36 chromosomal region in one of the chromosomes, resulting in a syndrome with multiple congenital anomalies and mental retardation, first delineated by Shapira et al. (1997). The condition is designated as #607872 in the OMIM database

Figure imgf000009_0001
Deletions can be terminal, interstitial, more complex or the result of a derivative chromosome (i.e. part of the Ip36 chromosomal region is replaced by another chromosomal segment).

A 'subject' as used herein refers to an individual mammal, more in particular an individual human. Particularly envisaged are subjects that are young, i.e. 12 years or less, 10 years or less, 8 years or less, 6 years or less, 4 years or less, 2 years or less, 18 months or less, 12 months or less, 11 months or less, 10 months or less, 9 months or less, 8 months or less, 7 months or less, 6 months or less, 5 months or less, 4 months or less, 3 months or less, 2 months or less, or 1 month or less. Specific subjects also include newborns and pre-natal subjects (note that in these cases, a sample of the subject may also be acquired by a sample of the mother, e.g. an amniotic fluid sample).

'Expression' or 'gene expression' as used in the application refers to the process by which inheritable information from a gene, such as the DNA sequence, is made into a functional gene product, such as protein or RNA. This definition thus encompasses, but is not limited to, transcription and/or translation of a gene. Evaluating expression may encompass processes such as detecting or measuring the presence of gene products, or determining the expression levels, i.e. the (relative or absolute) amount of gene product present. Evaluating expression may be done qualitatively (i.e. whether or not there is expression in a sample) and/or quantitatively (determining the amount of expression, or expression levels). Evaluating expression may involve comparison with a positive control (e.g. to assess whether gene products can be detected in the sample, in particular whether the detection method works), a negative control or a blank (typically to assess whether no false positive signal is being generated), one or more standards (either internal or external standards, typically to allow more accurate quantification), or a combination thereof. The positive control may additionally or alternatively be an internal positive control, typically a gene product known to be present in the sample (e.g. to assess whether gene products can be detected in the sample, in particular whether the detection method works or whether gene products are indeed present in the sample). Detection of expression and/or activity is well known in the art, and a skilled person is capable of choosing appropriate controls and/or standards.

'Activity' or 'functional activity' as used in the application refers to the exertion of a biological function by a gene product, and evaluating activity involves the studying of such function. For instance, a protein may be tested in an assay specifically designed to measure activity. Evaluating activity of a gene product may however also be done by inhibiting the gene product in the sample and evaluating whether there is a difference with the sample before it was inhibited or with another sample wherein the gene product is not inhibited. Methods and products for inhibiting gene products are well known in the art, and include, but are not limited to, antisense RNA, RNAi, siRNA, morfolinos, antibodies, nanobodies, peptide inhibitors, small molecule inhibitors and the like. Another alternative approach is indirectly evaluating gene product activity, e.g. by evaluating the activity of another gene product influenced by altered activity of the gene product of interest. For instance, Rerlp acts as a negative regulator of γ-secretase activity (Spasic et al., 2007), thus a decrease in Rerlp activity can be evaluated by the increased γ-secretase activity. The latter can be detected (and even quantified) e.g. in an assay using labeled or unlabeled substrates for γ-secretase.

Just like the evaluation of expression, the evaluation of activity can be absolute or relative, qualitative and/or quantitative, and may encompass comparison with one or more blanks, internal and/or external controls (positive and/or negative controls), internal and/or external standards, or a combination thereof.

'RERl' as used herein refers to the "retention in endoplasmic reticulum 1" gene and protein, more particular the human RERl (GenelD 11079; RefSeqs NM_007033 (mRNA) and NP_008964 (protein)). Unless particularly specified otherwise, the term 'RERl' is intended to encompass the RERl gene as well as its products, such as the RERl RNA (most particularly, the RERl mRNA) and the RERl protein (also referred to as 'Rerlp'). The human RERl gene is situated in the Ip36 chromosomal region. It encodes a transmembrane protein (Fig. 1). The cargo retrieval receptor Rerlp acts as a quality control mechanism by recognizing critically spaced polar residues within the transmembrane domain of its interacting proteins. Assembly of multimeric complexes is tightly controlled by quality control mechanisms in the ER and extended up to the Golgi. Unassembled subunits can be retained or retrieved to the ER through interaction with ER-to-Golgi or Golgi-to-ER cargo receptors (such as Rerlp). Only upon proper combination of individual subunits into a functional complex, specific retention/retrieval motifs in cytosolic or transmembrane domains are masked allowing assembled complexes to pass through the Golgi (Michelsen et al., 2005).

The term 'γ-secretase' as used herein refers to a multisubunit complex consisting of presenilinl or 2 (PSl or 2), nicastrin (NCT), PEN-2 (presenilin enhancer-2) and APH-I (anterior pharynx defective-1) (De Strooper, 2003). These proteins are minimally required to assemble a functional complex. The y- secretase complex cleaves type I integral membrane proteins like amyloid precursor protein and Notch in a process of regulated intramembrane proteolysis. It was shown recently that Rerlp expression levels control the formation of gamma-secretase subcomplexes and, concomitantly, total cellular gamma-secretase activity by competing with APH-I for binding to nicastrin (Spasic et al., 2007). Thus, Rerlp acts as a negative regulator of γ-secretase assembly and activity.

However, Rerlp function is likely not restricted to γ-secretase in mammals and, as in yeast, is implicated too in the assembly of other multimeric complexes including neurotransmitter receptors and ion channels. 'Notch signaling' as used in the application refers to a highly conserved cell signaling system present in most multicellular organisms. Vertebrates possess four different notch receptors, referred to as Notchl to Notch4. The Notch receptor is a single-pass transmembrane receptor protein. Ligand proteins binding to the extracellular domain induce proteolytic cleavage and release of the intracellular domain, which enters the cell nucleus to alter gene expression. Ligands of Notch include Jagged and Delta proteins. The Notch intracellular domain activates the transcription factor CSL, which induces transcription of target genes such as Hes genes and Her genes. As γ-secretase cleavage is required to activate Notch signaling, Rerlp acts as a negative regulator of Notch signaling by controlling the availability of γ-secretase complexes.

It is an object of the invention to provide methods to diagnose the presence of disorders involving aberrant or insufficient RERl function, in particular monosomy Ip36 syndrome. Disorders involving insufficient RERl function can be due to mutations in the RERl gene, partial or complete deletion of the RERl gene, instability of the RERl gene (e.g. by deletion of surrounding regions, resulting in decreased transcription or unstable transcripts), expression of aberrant Rerlp, decreased or absent expression of Rerlp, or due to a combination of these. According to particular embodiments, the methods comprise the steps of:

- providing a sample of a subject suspected of having insufficient RERl function;

- evaluating the expression and/or activity of RERl in the sample; wherein an absence of or a decrease in RERl expression and/or activity is indicative of the insufficient RERl function.

According to further particular embodiments, the methods comprise the steps of:

- providing a sample of a subject suspected of having monosomy Ip36 syndrome;

- evaluating the expression and/or activity of RERl in the sample; wherein an absence of or a decrease in RERl expression and/or activity is indicative of the presence of monosomy Ip36 syndrome.

The term "sample" or "biological sample" is used in a broad sense herein and is intended to include a wide range of biological materials as well as compositions derived or extracted from such biological materials. The sample may be any suitable preparation in which RERl is to be detected, either as a nucleic acid (DNA, RNA) or as a protein. The sample may comprise, for instance, a body tissue or fluid such as but not limited to blood (including plasma and platelet fractions), spinal fluid, mucus, sputum, saliva, semen, stool or urine or any fraction thereof. Exemplary samples include whole blood, red blood cells, white blood cells, buffy coat, hair, nails and cuticle material, swabs, including but not limited to buccal swabs, throat swabs, vaginal swabs, urethral swabs, cervical swabs, throat swabs, rectal swabs, lesion swabs, abscess swabs, nasopharyngeal swabs, and the like, lymphatic fluid, amniotic fluid, cerebrospinal fluid, peritoneal effusions, pleural effusions, fluid from cysts, synovial fluid, vitreous humor, aqueous humor, bursa fluid, eye washes, eye aspirates, plasma, serum, pulmonary lavage, lung aspirates, biopsy material of any tissue in the body. The skilled artisan will appreciate that lysates, extracts, or any material(s) obtained from any of the exemplary biological samples listed above are also considered as samples. Tissue culture cells, including explanted material, primary cells, secondary cell lines, and the like, as well as lysates, extracts, supernatants or materials obtained from any cells, tissues or organs, are also within the meaning of the term biological sample as used herein. These lists are not intended to be exhaustive. According to particular embodiments, the sample is provided in vitro, i.e. the method does not require contact with the subject suspected of having insufficient RERl function, in particular monosomy Ip36 syndrome.

In a particular embodiment of the invention, the sample is pre-treated to facilitate the detection of RERl in the sample with the detection method. For instance, typically a pre-treatment of the sample resulting in a semi-isolation or isolation of RERl (e.g. RNA or protein) or ensuring the amplification of RERl is envisaged. Many methods and kits are available for pre-treating samples of various types.

The pre-treatment or isolation methods can further comprise a detergent extraction step, an enzyme digestion step, e.g. digestion with a proteolytic enzyme and/or an enzymatic amplification step, e.g. by PCR, and/or a shearing/sonication step for fragmentation.

Typically, the preparation or pre-treatment of the sample will be determined by the detection method. The sample may be in any appropriate form such as a solid, a solution or suspension or a gas, suitably prepared to enable evaluation of expression and/or activity of RERl. The detection sample can be at any suitable pH. As a non-limiting example, when detection of expression using PCR is envisaged, a typical sample will be provided in liquid form, at a pH at which the polymerase used is active.

According to particular embodiments, the methods provided herein also encompass a step of comparing the expression and/or activity of RERl in the sample with the expression and/or activity of RERl in a control sample. An absence of or decrease in RERl expression and/or activity as compared to the control sample is indicative of a disorder related to insufficient RERl function, in particular monosomy Ip36. Evaluation of the expression and/or activity of RERl in the control sample can be done beforehand (e.g. comparison is with the stored results of a control sample, or detecting expression and/or activity is done prior to the analysis of the test sample), concomitantly (e.g. evaluation of RERl expression and/or activity is done in parallel in the two samples), or after RERl expression and/or activity has been measured in the test sample (e.g. comparison is with the stored results of a test sample, e.g. to provide an additional control). A "control sample" as used herein refers to a sample of a subject not having a disorder characterized by aberrant RERl expression and/or activity, in particular