WO2010020695A1 - Monosomy 1p36 syndrome - Google Patents

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, 2^nd 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

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 not having a disorder characterized by RERl insufficiency, most in particular not having monosomy Ip36 syndrome. The definition of "sample" also applies to control sample, in particular regarding the wide variety of forms the sample can take. Typically, however, the control sample will be provided in a similar form, most particular in an identical form, as the test sample, as this allows a more accurate comparison of RERl expression and/or activity. This of course also applies to optional pre-treatment of the sample. Thus, according to particular embodiments, the test and control sample will be provided in an identical form, will optionally undergo identical pre-treatment steps, and expression and/or activity of RERl in the sample will be measured in an identical way. Nevertheless, this does not exclude the possibility that in specific cases, a different protocol will be used to evaluate RERl expression and/or activity in the test and control sample (e.g. when only limited samples are available). Such protocols are typically best suited for qualitative measurements as it can be difficult to accurately compare quantitative measurements obtained using different protocols, although this still may be a possibility.

According to particular embodiments, the evaluation of expression and/or activity is evaluated at the nucleic acid level, in particular at the mRNA level. Again, this can be done on an untreated sample, or the sample may be pre-treated first, e.g. to isolate the nucleic acid from fractions interfering with detection. A variety of methods are available for isolating nucleic acids from samples. Exemplary nucleic acid isolation techniques include (1) organic extraction followed by ethanol precipitation, e.g. using a phenol/chloroform organic reagent (e.g. Ausbel et al., eds., Current Protocols in Molecular Biology, John Wiley & Sons, New York (1995, including supplements through June 2003)), preferably using an automated DNA extractor, e.g., the Model 341 DNA Extractor available from Applied Biosystems (Foster City, Calif); (2) stationary phase adsorption methods (e.g. U.S. Pat. No. 5,234,809; Walsh et al., BioTechniques 10(4): 506-513 (1991); and (3) salt-induced DNA precipitation methods (e.g. Miller et al., Nucl. Acids Res., 16(3): 9-10 (1988)), such precipitation methods being typically referred to as "salting-out" methods. Commercially available kits can be used to expedite such methods, for example, Genomic DNA Purification Kit and the Total RNA Isolation System (both available from Promega, Madison, Wis.). Further, such methods have been automated or semi- automated using, for example, the ABI PRISM.TM. 6700 Automated Nucleic Acid Workstation (Applied Biosystems, Foster City, Calif.) or the ABI PRISM.TM. 6100 Nucleic Acid PrepStation and associated protocols, e.g., NucPrep.TM. Chemistry: Isolation of Genomic DNA from Animal and Plant Tissue, Applied Biosystems Protocol 4333959 Rev. A (2002), Isolation of Total RNA from Cultured Cells, Applied Biosystems Protocol 4330254 Rev. A (2002); and ABI PRISM. TM. Cell Lysis Control Kit, Applied Biosystems Protocol 4316607 Rev. C (2001).

According to particular embodiments, the expression and/or activity of RERl is evaluated via PCR.

Whereas the presence of the RERl gene in a sample may be assessed via a normal PCR protocol, expression will usually be evaluated at the mRNA level, to determine whether mRNA is transcribed from the gene. This can be done by a reverse transcription PCR (RT-PCR). Quantifying the amount of mRNA present in the sample can be done by performing a quantitative (or real-time) RT-PCR.

Nevertheless, either for PCR or non-PCR based methods, other quantification methods can also be used (e.g. Northern blot, Southern blot, quantification of products on a gel) and the manner of quantification, if quantification is desired, is not critical to the methods described herein.

According to alternative embodiments, RERl expression and/or activity is evaluated at the protein level. Protein isolation, purification and detection methods are extensively described in the art and include, but are not limited to, immunoprecipitation, protein sample fractionation, protein complex pull-downs, organelle isolation, cell lysis (typically fol lowed by protein precipitation a nd resolubilisation), Western blotting, dot blotting, mass spectrometry, ELISA, RIA and the like. Monoclonal or polyclonal antibodies against the Rerlp that may be used in several of these techniques can be generated using methods known in the art, optionally followed by methods of affinity maturation and purification. Alternatively, antibodies against Rerlp may be ordered from an antibody- generating company.

As mentioned before, Rerlp activity can be measured directly or indirectly. Indirect measuring of Rerlp activity may e.g. be achieved through the use of labeled or unlabeled substrates for γ-secretase (e.g. fluorogenic γ-secretase substrates (Calbiochem, San Diego, CA, USA)). The amount of proteolytic processing of the substrate is an indication of γ-secretase activity. Since Rerlp acts as a negative regulator of γ-secretase activity and expression levels of Rerlp were found to control γ-secretase assembly and activity (Spasic et al., 2007), a decrease in Rerlp expression and/or activity can be evaluated by the increased γ-secretase activity. Similarly, as γ-secretase is a positive regulator of Notch signaling (Fortini, 2002; Shih and Wang, 2007), a decrease in Rerlp expression and/or activity can be evaluated by the increased Notch signaling. As will be detailed in the examples, Rerlp activity is also important for normal ciliogenesis and is important for correct acetylation of tubulin. Thus, Rerlp activity may also be measured by evaluating acetylated tubulin levels (see e.g. example 6) or by determining presence and/or length of cilia in normally ciliated cells (see e.g. example 8). Without being bound to a particular mechanism, it is striking that Rerlp is involved in the assembly of multimeric complexes including, but not limited to γ-secretase, neurotransmitters, ion channels, multisubunit enzymes and receptors. Thus, disruption of the RERl gene or the loss of RERl gene product function is likely to result in pleiotropic effects, similar to what is observed in monosomy Ip36 syndrome (see also examples). Of note, RERl is situated in the region of Ip36 determined to be critical for several symptoms of monosomy Ip36 (Heilstedt et al., 2003). Knowing that γ-secretase plays an important role in development by cleaving various type I transmembrane proteins, and is linked to Alzheimer's disease (De Strooper, 2003), the fact that RERl negatively regulates γ-secretase links RERl with cognitive function, and impaired cognitive development is a hallmark symptom of monosomy Ip36 syndrome.

Moreover, proteolytic processing of Notch by γ-secretase is known to be important in the Notch signaling pathway. Notch signaling was found to govern ciliated cell differentiation (Liu et al., 2007; Stubbs et al., 2006; Hayes et al., 2007), and cochlear cilia are important for hearing. Hearing loss is also one of the major symptoms associated with monosomy Ip36 and can be explained by the loss of RERl function. This results in an increase in γ-secretase activity and thus an increase in Notch signaling. This, in turn, represses differentiation to ciliated cells (Liu et al., 2007) which would explain the hearing loss observed in monosomy Ip36 syndrome. All this is in line with (at least part of the) monosomy Ip36 syndrome being a ciliopathy.

Restoring RERl function, or at least some of its downstream effects, provides a novel therapeutic approach in monosomy Ip36 syndrome, or other diseases characterized by RERl malfunction.

Thus, according to a particular aspect, methods are provided for treatment of at least one symptom of a disorder characterized by insufficient RERl expression and/or activity in a subject in need thereof. More particularly, methods are provided for treatment of at least one symptom of monosomy Ip36 syndrome in a subject in need thereof. According to particular embodiments, the methods comprise a step of 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 specific embodiments, the methods comprise a step of upregulating RERl expression and/or activity; and/or downregulating γ-secretase expression and/or activity; and/or downregulating Notch signaling.

As γ-secretase is involved in the Notch signaling pathway, downregulating Notch signaling may be achieved by downregulating γ-secretase. Downregulating Notch signaling may also be achieved by targeting (downregulating/inhibiting) other components of the Notch signaling pathway, such as Notch ligands (e.g. Delta, in particular Deltal, and Jagged, in particular Jagged2), the Notch receptor (e.g. Notchl, Notch2, Notch4 or in particular Notch3), Notch-responsive transcription factors (e.g. CSL), or downstream effector genes (e.g. Hes or Her genes important in ciliogenesis). Rfx2 is a transcription factor repressed by Notch, and belongs to a gene family known as master regulators of ciliogenic gene expression (Liu et al., 2007). This transcription factor thus is an example of a gene that is positively regulated by RERl (through the decrease of Notch signaling via the control of γ-secretase availability). As will be detailed in the examples, at least some features of the monosomy Ip36 syndrome appear due to defective ciliogenesis, i.e. the syndrome is a ciliopathy.

According to yet more specific embodiments, the methods comprise a step of upregulating RERl expression and/or activity.

According to particular embodiments, the upregulating of RERl; of genes, proteins or protein complexes that are positively regulated by RERl; and/or the downregulating of genes, proteins or protein complexes that are negatively regulated by RERl results in improvement of at least one symptom of the disorder that is treated.

Accordingly, compounds are provided for use in the preparation of a medicament for a disorder characterized by insufficient RERl expression and/or activity, more in particular a medicament for at least one symptom of a disorder characterized by insufficient RERl expression and/or activity. According to particular embodiments, the disorder characterized by insufficient RERl expression and/or activity is monosomy Ip36 syndrome.

Compounds are also provided for use in the treatment of a disorder characterized by insufficient RERl expression and/or activity, more in particular for use in the treatment of at least one symptom of a disorder characterized by insufficient RERl expression and/or activity. According to particular embodiments, the disorder characterized by insufficient RERl expression and/or activity is monosomy Ip36 syndrome.

The compounds described above will upregulate RERl expression and/or activity; and/or upregulate expression or activity of a gene, protein or protein complex that is positively regulated by RERl; and/or downregulate expression or activity of a gene, protein or protein complex that is negatively regulated by RERl. According to specific embodiments, compounds are provided that upregulate RERl expression and/or activity, and/or downregulate γ-secretase expression and/or activity, and/or downregulate Notch signaling (in particular by downregulating expression and/or activity of a component of the Notch signaling pathway). According to further specific embodiments, compounds are provided that upregulate RERl expression and/or activity.

Upregulating of RERl expression and/or activity can be done via specific compounds. How to identify compounds that upregulate RERl expression and/or activity is described in WO2008/068302. Briefly, methods are provided therein to identify compounds that activate or enhance the RERl promoter. The read-out is done via the coupling of a reporter gene to the RERl promoter and evaluating the expression of the reporter gene.

Alternatively, RERl can be upregulated by gene therapy. Gene therapy protocols, intended to achieve therapeutic gene product expression in target cells, in vitro, but also particularly in vivo, have been extensively described in the art. These include, but are not limited to, intramuscular injection of plasmid DNA (naked or in liposomes), interstitial injection, instillation in airways, application to endothelium, intra-hepatic parenchyme, and intravenous or intra-arterial administration (e.g. intrahepatic artery, intra-hepatic vein). Various devices have been developed for enhancing the availability of DNA to the target cell. A simple approach is to contact the target cell physically with catheters or implantable materials containing DNA. Another approach is to utilize needle-free, jet injection devices which project a column of liquid directly into the target tissue under high pressure. These delivery paradigms can also be used to deliver viral vectors. Another approach to targeted gene delivery is the use of molecular conjugates, which consist of protein or synthetic ligands to which a nucleic acid-or DNA-binding agent has been attached for the specific targeting of nucleic acids to cells (Cristiano et al., 1993). Target cells will typically depend on which symptoms need to be treated and can be selected by the skilled person (e.g. cells in or near the ear to treat hearing problems).

Thus, according to some particular embodiments, RERl gene therapy vectors can be used to express a therapeutic amount of a RERl polypeptide (or other gene product, such as RNA) to ameliorate one or more symptoms of a disease characterized by insufficient RERl expression or activity, in particular monosomy Ip36 syndrome. Typically, the gene product is encoded by the coding sequence within the gene therapy vector (i.e. as a transgene), although in principle it is also possible to increase expression of an endogenous gene. A 'therapeutic amount' as used herein is an amount that ameliorates one or more symptoms of a disease. Such amount will typically depend on the gene product and the severity of the disease, but can be decided by the skilled person, possibly through routine experimentation. According to particular embodiments, the gene therapy vectors described in this application direct the expression of a therapeutic amount of the gene product for an extended period. Indeed, as long as therapeutic levels are achieved, no new treatment is necessary. Typically, therapeutic expression is envisaged to last at least 20 days, at least 50 days, at least 100 days, at least 200 days, and in some instances 300 days or more. Expression of the gene product (e.g. polypeptide) encoded by the coding sequence can be measured by any art-recognized means, such as by antibody-based assays, e.g. a Western Blot or an ELISA assay, for instance to evaluate whether therapeutic expression of the gene product is achieved. Expression of the gene product may also be measured in a bioassay that detects an enzymatic or biological activity of the gene product.

Gene therapy vectors can be episomal vectors (i.e., that do not integrate into the genome of a host cell), or can be vectors that integrate into the host cell genome. Examples of episomal vectors include (extrachromosomal) plasmids and so-called mini-circles, which are composed of the expression cassette only and are devoid of bacterial sequences, and examples of vectors that integrate into the host cell genome including viral vectors.

Representative plasmid vectors include pUC vectors, bluescript vectors (pBS) and pBR322 or derivatives thereof that are devoid of bacterial sequences (minicircles). Some of the plasmid vectors can be adapted to incorporate elements that enhance episomal plasmid persistence in the transfected cells. Such sequences include S/MARs that correspond to scaffold/matrix attached region modules linked to a transcription unit (Jenke et al., 2004; Manzini et al., 2006).

Representative viral vectors include vectors derived from adeno-associated virus, adenovirus, retroviruses and lentiviruses. Alternatively, gene delivery systems can be used to combine viral and non-viral components, such as nanoparticles or virosomes (Yamada et al., 2003).

Retroviruses and lentiviruses are RNA viruses that have the ability to insert their genes into host cell chromosomes after infection. Retroviral and lentiviral vectors have been developed that lack the genes encoding viral proteins, but retain the ability to infect cells and insert their genes into the chromosomes of the target cell (Miller, 1990; Naldini et al., 1996). The difference between a lentiviral and a classical Moloney-murine leukemia-virus (MLV) based retroviral vector is that lentiviral vectors can transduce both dividing and non-dividing cells whereas MLV-based retroviral vectors can only transduce dividing cells.

Adenoviral vectors are designed to be administered directly to a living subject. Unlike retroviral vectors, most of the adenoviral vector genomes do not integrate into the chromosome of the host cell. Instead, genes introduced into cells using adenoviral vectors are maintained in the nucleus as an extrachromosomal element (episome) that persists for an extended period of time. Adenoviral vectors will transduce dividing and nondividing cells in many different tissues in vivo including airway epithelial cells, endothelial cells, hepatocytes and various tumors (Trapnell, 1993).

Adeno-associated virus (AAV) is a small ssDNA virus which infects humans and some other primate species, not known to cause disease and consequently causing only a very mild immune response. AAV can infect both dividing and non-dividing cells and may incorporate its genome into that of the host cell. These features make AAV a very attractive candidate for creating viral vectors for gene therapy, although the cloning capacity of the vector is relatively limited.

Another viral vector is derived from the herpes simplex virus, a large, double-stranded DNA virus. Recombinant forms of the vaccinia virus, another dsDNA virus, can accommodate large inserts and are generated by homologous recombination.

How upregulating of genes, proteins or protein complexes that are positively regulated by RERl will typically depend on which genes, proteins or protein complexes need to be upregulated. Depending on the gene(s) or protein(s) of interest, compounds may be available for upregulating them. Compounds may also be identified using a similar strategy as described in WO2008/068302 for upregulating RERl, i.e. the coupling of a reporter gene to the promoter of a gene of interest to screen for compounds activating the promoter. Alternatively, gene therapy may also be used to upregulate the desired genes, using the same strategy as described above for RERl.

The same applies, mutatis mutandis, for the downregulating of genes, proteins or protein complexes that are negatively regulated by RERl. Thus, gene therapy may be used to lower expression of these genes, and compounds may be identified or available that downregulate the genes or proteins of interest. Moreover, 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.

As an example, compounds that downregulate or inhibit γ-secretase, a protein complex negatively regulated by RERl, are well known in the art and include, but are not limited to L-685,458 (e.g. Sigma- aldrich), S2188 (Sigma-Aldrich), pepstatin-A, MG132, a substrate-based difluoroketone (t- butoxycarbonyl-Val-lle-(S)-4-amino-3-oxo-2, 2-difluoropentanoyl-Val-lle-OMe), LY450139 (EIi Lilly, semagacestat), LY-411575, γ-secretase inhibitor I to XXI (Calbiochem), DAPT, IL-X (cbz-IL-CHO), tripeptide γ-secretase inhibitor (z-Leu-leu-Nle-CHO), arylsulfonamide (AS), dibenzazepine (DBZ), benzodiazepine (BZ), MK0752 (Merck), MRK-003, CHF5074 (Chiesi), NIC5-15 (Humanetics), GSI953 (Wyeth, begacestat), etc. As another example, compounds downregulating or inhibiting Notch signaling include, but are not limited to GSI, LY-411575, L-685458, MK0752 (Merck), MRK-003, arylsulfonamide (AS), dibenzazepine (DBZ), benzodiazepine (BZ), DAPT, anti-Notchl antibody (Viragen), Notch3 siRNA, etc.

Note that different compounds may act both on γ-secretase and other components of the Notch- signaling pathway. This has e.g. been demonstrated for MRK-003 (Konishi et al., 2007). Other compounds interfering with γ-secretase and or Notch function are also disclosed in e.g. Miele et al., 2006.

The symptoms that can be treated using the methods and compounds as described herein vary widely, due to the pleiotropic role of RERl in complex assembly. In particular the symptoms of monosomy Ip36 syndrome are well characterized. For instance, developmental delay/mental retardation are hallmarks of the syndrome. Approximately 90% of affected individuals have severe to profound mental retardation, whereas 10% have mild to moderate cognitive impairment. Expressive language is absent in 75% and limited to a few isolated words or at the level of first word associations in the remainder. Comprehension seems to be limited to a specific context. Intention to communicate, limited in early years, tends to improve over time, with extension of the gesture repertoire. Behavior disorders, present in 50%, include poor social interaction, temper tantrums, selfbiting of hands and wrists, a number of stereotypes, and, less frequently, hyperphagia.

Central nervous system defects, present in 88% of affected individuals, mainly include dilatation of the lateral ventricles and subarachnoid spaces; cortical atrophy; diffuse brain atrophy; and hypoplasia, thinning, and total or partial broadness of the corpus callosum. Other reported anomalies are delay in myelination, multifocal hyperintensity areas in the white matter (Battaglia et al., 2008), and periventricular nodular heterotopia (Neal et al., 2006).

Seizures occur in 44% to 58% of individuals with Ip36 deletion syndrome (Heilstedt et al., 2001 & 2003; Bahi-Buisson et al., 2008; Battaglia et al., 2008). Age at onset ranges from four days to two years, eight months. First seizures are either generalized (tonic, tonic-clonic, clonic, myoclonic) or partial (simple or complex). Almost 20% of all persons with the disorder have infantile spasms associated with hypsarrhythmia on EEG. Infantile spasms may either be the presenting seizure type or may follow other seizure types. Most seizure types are well controlled by standard pharmacotherapy. However, in one series (Bahi-Buisson et al., 2008) nearly one-third of persons developed drug-resistant epilepsy. A variety of EEG abnormalities are present in nearly all affected individuals. Feeding difficulties may be caused by hypotonia and/or oral facial clefts with related difficulty in sucking, poorly coordinated swallow with consequent aspiration, and/or gastroesophageal reflux and vomiting. Mild to severe oropharyngeal dysphagia has been observed on swallow studies in 72% of individuals (Heilstedt et al., 2003).

Congenital heart defects are noted in 43% to 71% of individuals. Structural heart defects reported are (in order of frequency) atrial and ventricular septal defects, valvular anomalies, patent ductus arteriousus, tetralogy of Fallot, coarctation of the aorta, infundibular stenosis of the right ventricle, and Ebstein anomaly (Heilstedt et al., 2003; Battaglia et al., 2008). Twenty-seven percent had a history of cardiomyopathy in infancy and childhood. Cardiomyopathy was of the non-compaction type in 23% and tended to improve over time (Battaglia et al., 2008).

Ophthalmologic abnormalities are also a characteristic observed in Ip36 deletion syndrome. Strabismus, nystagmus, refractive errors, and visual inattention are the most common ophthalmic manifestations (Heilstedt et al., 2003; Battaglia et al., 2008). Cataract, retinal albinism, and optic nerve coloboma have occasionally been observed.

Skeletal anomalies found in 40% of individuals with Ip36 deletion syndrome (Battaglia et al., 2008) include delayed bone age, scoliosis, rib anomalies, and lower-limb asymmetry.

Hearing loss, mostly of the sensorineural type, can be detected in 47% to 82% of individuals with Ip36 deletion syndrome (Heilstedt et al., 2003; Battaglia et al., 2008).

Genitourinary malformations can be seen in 22% of affected individuals and include unilateral renal pelvis with hydronephrosis of the upper pole, kidney ectopia with right kidney cyst, and unilateral pelvic ectasia (Battaglia et al., 2008). Cryptorchidism, hypospadias, scrotal hypoplasia, and micropenis are seen in a minority of males (Battaglia et al., 2008). Small labia minora and small clitoris, labia majora hypertrophy, and uterine hypoplasia have been reported in females (Battaglia et al., 2008).

Hypothyroidism has been reported in 15% to 20% of persons of varied ages with deletion Ip36 syndrome in whom TSH and T4 levels were studied (Heilstedt et al., 2003; Battaglia et al., 2008).

Other abnormalities reported in a few individuals with Ip36 deletion syndrome include the following: Telangiectatic skin lesions and hyperpigmented macules (Keppler-Noreuil et al., 1995) Polydactyly (Keppler-Noreuil et al., 1995) Congenital spinal stenosis (Reish et al., 1995)

Congenital fiber type disproportion myopathy (Okamoto et al., 2002) Redundant skin on the nape of the neck (Wang and Chen, 2004) - Intestinal malrotation and annular pancreas (Minami et al., 2005) Hypertrophic pyloric stenosis

Anteriorly placed or imperforate anus, hooked or bilobed gallbladder, and small spleen (Battaglia et al., 2008)

Neuroblastoma (Laureys et al., 1990, Biegel et al., 1993, Anderson et al., 2001) - Pemphigus vulgaris (Halpern et al., 2006)

Thus, according to particular embodiments, the methods or compounds described herein can be used to treat at least one symptom selected from 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.

According to further particular embodiments, the symptom is selected from the group consisting of neurological defects, developmental delay, mental retardation, hypotonia, skeletal anomalies, hearing loss, and neuroblastoma.

Recent findings in genetic research have suggested that a large number of genetic disorders, both genetic syndromes and genetic diseases, that were not previously related in the medical literature, may be, in fact, highly related in the root cause of the widely-varying set of medical symptoms that are clinically visible in the disorder. These have been grouped as an emerging class of diseases called ciliopathies. The underlying cause may be a dysfunctional molecular mechanism in the primary cilia structures, organelles which are present in many diverse cellular types throughout the human body. Cilia defects adversely affect "numerous critical developmental signaling pathways" essential to cellular development and thus offer a plausible hypothesis for the often multi-symptom nature of a large set of syndromes and diseases. Strikingly, RERl is herein identified as a protein involved in ciliogenesis, and the broad scala of symptoms associated with monosomy Ip36 syndrome is indeed typical of a ciliopathy. Most notably, cilial dysfunction has been associated with male infertility (flagellum of human sperm is a modified cilium), left-right anatomic abnormalities and congenital heart disease (proper cilial function is responsible for the normal left-right asymmetry in mammals), as well as with kidney or renal disorders (e.g. polycystic kidney disease), retinal and ophtalmological disorders, mental retardation, Polydactyly, obesity, deafness etc. (Badano et al., 2006), all of which have been observed in monosomy Ip36 syndrome. The novel insight that the syndrome is a ciliopathy opens up new possibilities for diagnosis and treatment of the disorder.

It is understood by the skilled person that some of these symptoms will most effectively be treated when the subject is very young. This is especially true for symptoms that may be difficult to reverse (e.g. skeletal anomalies). According to particular embodiments, the diagnostic and therapeutic methods are performed on very young subjects, such as prenatal subjects, newborns, subjects not older than one, two, three, four, five, six, seven, eight, nine, ten, eleven or twelve months.

It is to be understood that although particular embodiments, specific configurations as well as materials and/or molecules, have been discussed herein for cells and methods according to the present invention, various changes or modifications in form and detail may be made without departing from the scope and spirit of this invention. The following examples are provided to better illustrate particular embodiments, and they should not be considered limiting the application. The application is limited only by the claims.

Examples

Introduction

Given the crucial role of Rerlp in the quality control of the early biosynthetic route, and the evidence that it mediates assembly into functional multisubunit enzymes, receptors and transporters, including those controlling neuronal excitability, RERl presents itself as a critical candidate gene in the Ip36 deletion syndrome.

Cellular observations The presence of RERl within the deletions of almost all Ip36 deletion syndrome patients raises the question whether down-regulation of its protein product contributes to one or more of the clinical characteristics. At the cellular level, it was first demonstrated that Rerlp levels were reduced in fibroblasts derived from patients to 50% the levels observed in fibroblasts from controls (Figure 3A). A similar down-regulation was obtained in HeIa cells using RNAi (Figure 2). Increased γ-secretase activity in fibroblasts obtained from patients (Figure 3B), which is an indirect proof of significantly reduced Rerlp levels (Spasic et al., 2007), was also noticed. These findings are further elaborated using more Ip36 patient samples. Zebrafish model system

In order to dissect the potential contribution of Rerlp to the complex phenotype of monosomy Ip36, the down-regulation of Rerlp was mimicked in a Danio rerio (zebrafish) model system.

Example 1. Expression pattern of RERl in zebrafish (Fig. 4)

In order to check the expression pattern of Rerl gene in zebrafish whole mount in situ hybridization was performed with two antisense RNA probes, one corresponding to the first 360 nucleotides of mRNA and the second probe matching the last 345 nucleotides. Zebrafish embryos were fixed at different developmental stages (1-cell stage, 1000-cell stage, 15-, 24-, 48-, 72-h.p.f. (hours post fertilization) and 5-d.p.f. (days post fertilization) fish), stained with DIG-UTP labeled RNA probes and incubated with AP anti-DIG antibody. Rerl showed ubiquitous expression at the very early developmental stages (1000-cell stage and 15-h.p.f.). At 24-h.p.f. Rerl gene expression is confined to somite boundaries, pectoral fin buds and the whole brain region. Furthermore, at 48-h.p.f. and two other later stages (72-h.p.f. and 5-d.p.f.) Rerl is expressed in the ear, lateral line organ and in a particular brain region called the optic tectum (Fig. 4). This expression pattern was independently underscored by staining with antibody against zebrafish Rerlp. An antibody against last C-terminal 15 amino acids (RTYRGKDDTGKTFAS (SEQ ID NO: I)) of zebrafish Rerlp was generated in the GenScript Corporation (Piscataway, NJ, USA). Subsequently the serum was affinity purified using immobilized peptide antigen.

Example 2. Characterization RERl-morpholino's using qPCR

Downregulation of Rerl in zebrafish is achieved through microinjections of morpholino antisense oligonucleotides (MO) at the one-to-four-cell-stage embryos. Two different morpholinos, splice-modifying, which causes splicing defects of the mRNA, and translation-blocking, are used at various concentrations (Ing, 2ng, 3ng and 6ng). Splice-m o d i f y i n g M O ( 5 '- CCACCCCTAATACAAACAAACAAAC-3' (SEQ I D NO: 2)) targets the splice acceptor site between the second intron and third exon, while translation-blocking MO (5'-CCGGCACTGTCTCCTTCTGGCATTC-S' (SEQ ID NO: 3)) targets the translation start. Binding of splice-modifying MO to the endogenous pre- mRNA should lead to a deletion of exon 3 thus introducing a frameshift resulting in an instable truncated Rerl protein. 5'-mismatch morpholino controls of both spl ice mod ifyi ng MO (5'- CCAgCCgTAATACAAAgAAtCAtAC-3' ( S E Q I D N O : 4 )) and translation-b l o c k i n g M O ( 5 '- CCcGCACTcTCTgCTTgTGGgATTC-3' (SEQ ID NO: 5)) were used at the same concentrations. Total RNA was extracted from injected embryos 1, 4 a nd 5 days after MO injection and used for reverse- transcriptase PCR. One day after injection a new RT-PCR product (485bp) is present in Rerl MO- injected embryos, which corresponds to exon 3-deleted transcript. The original full-length transcript (585bp) appears in Rerl MO-injected embryos starting from day 4.

Example 3. Gross developmental changes in zebrafish morphants: Detail of curly tail (and U-shape somite)

Rerl knockdown resulted in embryos with characteristic curled shape of the body which is apparent already at 24 hours of development. This particular phenotype, known in the literature as "curly tail down" is caused by defects in different genes known as ϋ-type genes since the major features are U-shaped instead of normal V-shaped somites.

Delay in ear development and hearing loss

Larvae with downregulated Rerl levels were scored for the acoustic startle reflex starting at 72-h.p.f. Uninjected larvae, as well as larvae injected with control and Rerl morpholino were stimulated with a series of taps or vibrational stimuli. While larvae from both control groups showed an escape response, Rerl morphants did not respond to acoustic stimuli, indicating they were deaf. All deaf morphants recovered by day 5 of their development which corresponded to the appearance of the original Rerl RNA transcript.

Decrease in ciliated cells of lateral lines as monitored by FM1-43 staining Immuno- and F M 1-43 dye stainings in zebrafish

Whole mount immunostainings were performed with embryos fixed at different developmental stages using antibodies such as anti-acetylated tubulin, widely used as cilia marker, anti-Rerl and phalloidin (to mark actin), followed by light-microscopical imaging. Rerl colocalized with acetylated tubulin staining in ciliated tissues, such as hair cells of the inner ear, lateral line organ or pronephros. In order to establish whether the lateral line(s) is (are) affected in Rerl morphants, free swimming larvae were immersed in FM1-43 dye for 30 seconds, followed by quick rinses. Using this procedure, FM1-43 was found to be restricted to hair cells in neuromasts of the lateral line. All stainings were done with the uninjected, control- and morpholino-injected embryos. Neuromasts of both anterior and posterior lateral line were counted at day 3, 4 and 5 of development. 3-d. p. f. larvae showed 50% less neuromasts on the head (anterior lateral line) and no changes in the number on the tail (posterior lateral line) after Rerl downregulation. As already described for the deafness, larvae recovered neuromasts staining by day 5 of their development. Example 4. Defect left-right asymmetry during heart development

Specific localization of Rerl in ciliated tissues points to a possible role of Rerl in determining left-right (LR) asymmetry of the inner organs, such as heart, gut, liver and pancreas. This is due to a crucial role of ciliated cells within a transient structure in the development known as Kuppfer's vesicle. Movement of these cilia in the same direction results in asymmetrical gene expression between left and right side of the embryo. In order to check whether LR asymmetry is affected in Rerl morphants a cmlc2-transgenic zebrafish line was used (where cmlc2 stands for cardiac myosin light chain 2 and represents a line with heart-specific fluorescence). Only in the Rerl morphants reverse (ranging from 25-42%) or no heart looping (from 14-30%) was scored at 48-h.p.f. suggesting involvement of the Rerl gene in left-right asymmetric development of the heart. 3-5% of Rerl MO-injected embryos showed presence of rudiments (this term is used for hearts that are recognized as hearts because of their contractile behavior). 40% of Rerl morphants had normal heart looping. LR asymmetry of other organs such as liver, pancreas, gut will be checked after Rerl downregulation by using specific in situ probes.

Conclusion

RERl downregulation recapitulates features of the monosomy Ip36 syndrome in zebrafish

Morpholino-oligonucleotide (MO)-mediated down-regulation of Dr-Rerl resulted in a striking 'curly-tail' phenotype, which made the fish to swim unidirectionally in circles, indicating a body axis asymmetry. The curly tail is caused by a subtle defect in somite formation, i.e. they are U-shaped instead of V-shaped and this pattern is mediated through the somite boundaries (Brand et al., 1996). Interestingly, Rerlp is highly expressed in this outer cell layer. Downregulation of integrin5α, highly expressed in somite boundaries too, also results in U-shaped somites (Koshida et al., 2005). In HeIa cells in which RERl is knocked down by RNAi, levels of integrin heterodimers involving integrin5α are less abundant. These data thus link changes at the cellular level with a disturbed somite development in zebrafish. A second observation in Rerl MO-treated fish was a clear problem with balance, which prompted us to look into more detail to sensory organs in fish. RERl mRNA and protein levels, were highly abundant in the ear and lateral organs. This could explain the apparent deafness, and loss of lateral line clusters and swim bladder explaining the improper balance. Of note, hearing loss is one of the most common clinical phenotypes in Ip36 deletion syndrome patients. Strikingly, RERl expression (both mRNA as well as at the protein level) coincides with cells or cell clusters that intensively stain with FM1-43 dye. This dye identifies ciliated cells in lateral lines, the ear and the pronephros, among other tissues. The cilia of ciliated cells can equally be immunostained using anti-acetylated tubulin. Downregulation of RERl in zebrafish, using morpholinos, not only results in a decrease of FM1-43 staining but also of anti-acetylated tubulin, demonstrating that these cells or cell clusters in the different organs did not normally develop cilia. These cilia are needed in mechanosensing, e.g. in the ear and lateral line (balance) as well as for fluid propulsion in the pronephros. In the latter, downregulation of RERl leads to cyst formation in the zebrafish pronephros. Furthermore, the ciliated Kuppfer's vesicle is needed for proper left-right symmetry in the development of different organs such as heart (example 4). Also here, downregulation of RERl leads to severe problems in heart development resulting in reverse or even no heart looping.

In the case of the pronephros, the classical interdigitating 'salt-and-pepper' pattern of ciliated (fluid propulsion) and non-ciliated (for ion transport) cells is caused by a lateral inhibition mechanism mediated through Notch3-signaling (Liu et al., 2007). Notch signaling (through γ-secretase mediated intramem brane proteolysis) inhibits the expression of its ligand Jagged2 as well as rfx2 and downstream genes regulating the ciliogenesis program. Directly or indirectly, it also regulates the expression of ion channel genes allowing the Notch3-expressing cells to acquire the transporting cell- phenotype. As demonstrated, downregulation of RERl results in increased γ-secretase activity in HeLa cells (using siRNA, Figure 2) as wel l as i n monosomy Ip36 patient cells (Figure 3). Hence, downregulation of RERl in ciliated cells in different organs of zebrafish, leading to enhanced v- secretase activity may increase Notch3 signaling leading to a stronger inhibition of the ciliogenesis program in these organs - again consistent with the monosomy Ip36 syndrome being a ciliopathy.

Initial experiments also point to a delay in cartilage formation, which together with the body axis asymmetry points to another major clinical phenotype of the syndrome. Moreover, morpholino knockdown is only temporary as they become diluted during development of the embryo. In the case of RERl targeted morpholinos, RERl expression levels are restored from day 5-6 onwards. Interestingly, the symptoms improve as RERl function is restored, indicating that - at least some - defects are reversible through restoring RERl function.

Of note, very recently, the zebrafish was identified as an interesting model to study ciliopathies (Tobin and Beales, 2008).

Example 5. Rerlp affects ciliogenesis in CL4 cells

Porcine kidney epithelial LLC-CL4 cells were used as models for ciliated cells. They were grown on Transwell membranes in order to generate differentiated epithelial monolayers including the formation of apical cilia. In order to immunolocalize cilia we used an antibody directed against acetylated tubulin as this is a commonly used cilia marker. As acetylated tubulin is present throughout the cell and to distinguish the cilia-associated pool, we acquired short confocal z-stacks from an area just above the apical membrane of the epithelial cell layer. Downregulation of Rerlp in CL4 cells dramatically decreased the length of cilia which is represented in Figure 5A (left). Although the number of ciliated cells did not change when Rerlp was downregulated, the average cilia length decreased 50% (Fig. 5B (left)). When quantifying the frequencies in cilia length distributions, it was found that long cilia are almost exclusively present in control cells, while the percentage of cells with short cilia increases significantly with Rerlp knockdown (Fig 5B, right graph). That Rerlp downregulation indeed affects the cilia length in CL4 cells was further confirmed by quantifying the total area within each field occupied by acetylated tubulin staining, which resulted in similar reduction of up to 50% (Fig 5C). The downregulation efficiency was validated by western blot which showed 70% reduction in Rerlp protein levels (Fig 5A right).

When CL4 cells were analyzed by scanning electron microscopy (SEM), the absence of long cilia was striking in the Rerlp downregulated cells (Fig 5D). Moreover, a dramatically decreased number of microvilli was noticed suggesting an additional or more genuine role for Rerlp in apical morphology.

Example 6. Tubulin acetylation is altered by the knockdown of Rerlp in RPE cells

Acetylation is one of the posttranslational modifications of tubulin that is known to stabilize the microtubules. It is a dynamic process with a regulated balance between two independent processes of acetylation and deacetylation. This prompted us to examine in more detail in our cell models how Rerlp influences these processes. While by immunostaining of acetylated tubulin we observed shorter cilia in CL4 cells (Fig 5A), western blot analysis of the total RPE (human retinal pigment epithelium) cell extracts showed higher total levels of acetylated tubulin when Rerlp is downregulated (Fig 6A). In order to study the dynamics of tubulin polymerization and acetylation, we treated cells with nocodazole, an antimitotic agent that disrupts the microtubules by binding to β tubulin. Furthermore, we combined this with the addition of taxol, a compound that stabilizes polymerized microtubules, in the lysis buffer (2OmM Tris- HCI, pH 6.8, 0.14M NaCI, 0.5% Nonidet P-40, ImM MgCI₂, 2mM EGTA) which allowed us to separate the soluble fraction of tubulin from the polymerized fraction by a simple centrifugation step (with polymerized tubulin ending up in the pellet and soluble tubulin in the supernatant).

We next downregulated Rerlp (achieving 83% efficiency of downregulation) followed by treatment with 0.5μM of nocodazole for 30 min. After nocodazole washout, we followed the dynamics of tubulin repolymerization and acetylation at different time points (5, 10 and 20 minutes of recovery). Cells were then lysed with taxol-containing buffer and further processed for western blot analysis (Fig 6A). Quantifications of both α-tubulin and acetylated tubulin in soluble and polymerized fractions are shown in figure 6B and C. The levels of polymerized α-tubulin, which represents the total amount of tubulin that is built in microtubules, are almost the same between control and cells with Rerlp knockdown at the initial time point (Fig 6C, with polymerized fractions, compare the unfilled triangles and squares). However, at the same time point, the levels of acetylated tubulin are higher when Rerlp is downregulated (Fig 6C, black triangles and squares). This points to the fact that acetylation on tubulin occurs more frequently in the absence of Rerlp. The dynamics of depolymerization stimulated by nocodazole- treatment follows the same kinetics. During repolymerization, cells with Rerlp knockdown are showing again a more sharp increase in acetylation. Although, the differences seem to be much more pronounced than the ones observed at the initial time point, 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. This effect on acetylation is also confirmed by our immunofluoroscence data (not shown). Our results suggest that Rerlp downregulation probably regulates the levels and/or localization of the acetyltransferase in the cell. Moreover, Rerlp also seems to be a negative regulator of this yet-to-be-identified enzyme, reminiscent of its role in γ-secretase complex assembly (Spasic et al., 2007).

Example 7. Effect of Rerl knockdown on zebrafish cilia

Downregulation of Rerlp in differentiated cells, which form cilia, results in two prominent features: shorter cilia and increased levels of acetylated tubulin within the cytoplasm. The knockdown of the same gene in zebrafish causes shortening of cilia as well. Indeed all ciliated organs such as pronephros, inner ear, olfactory pits and neuromasts of the lateral line, develop with severe reduction in cilia length. In order to check whether this phenotype is not just a delay in embryonic development often caused by injecting morpholino oligonucleotides, uninjected control and morphants were fixed at different developmental stages (starting from 22hpf (hours post fertilization) until 30hpf every two-to- three hours and in addition at 48hpf). The cilia length within two ciliated organs, the pronephros and the inner ear, was used as readout after double immunostaining for acetylated tubulin, which marks cilia, and Rerlp. The cilia of both organs were consistently shorter when Rerlp was downregulated regardless of developmental stage, indicating that the phenotype is a true manifestation of the Rerlp absence during development (data not shown). In addition, levels of acetylated tubulin within the developing ear were markedly increased throughout all stages, confirming the findings from the cells. Moreover, the same effect of Rerlp downregulation on acetylation was earlier spotted within the developing neuromasts of the lateral line, pointing to the common mechanism. In all examined cases, the higher levels of acetylated tubulin accumulate within the cytoplasm of ciliated cells. Example 8. Reduced ciliogenesis in monosomy Ip36 patient fibroblasts

Nearly all interphase and non-dividing cells in vertebrates contain or develop a so-called primary cilium. For instance, fibroblasts grown at high density followed by serum starvation generate primary cilia. Therefore we cultured human fibroblasts derived from monosomy Ip36 patients (which express less than 50% of the Rerl protein (quantitative western blot analysis) and mRNA level (measured by qPCR) as well as control human fibroblasts under these conditions in order to check whether heterozygous deletion of rerl would affect ciliogenesis. Using acetylated tubulin as a marker for immunolocalizing cilia, we quantified their number and length for 100 control cells (9 fields) and 100 patient fibroblast cells (13 fields). Figure 7A shows a representative field of the control and the patient cells immunostained for Rerlp and acetylated tubulin. While all the control cells had a primary cilium, only 75% of patient cells were ciliated (Fig 7B left). The cilia were also shorter compared to control (Fig 7B right). In addition, and consistent with the results obtained in CL4 cells, there is a higher percentage of patient cells with shorter cilia while longer cilia are completely absent (Figure 7C, see also figure 5B). Since the phenotype of cells wherein Rerlp is downregulated closely resembles that of cells derived from monosomy Ip36 patients, and given the phenotype of zebrafish in which RERl is downregulated, it is highly probable that the RERl gene is the dose-sensitive gene which may give rise to most clinical manifestations arising as a result of deletion of Ip36 (cf. Shaffer and Heilstedt, 2001).

References

Annaert WG, Levesque L, Craessaerts K, Dierinck I, Snellings G, Westaway D, George-Hyslop PS, Cordell B, Fraser P, De Strooper B. Presenilin 1 controls gamma-secretase processing of amyloid precursor protein in pre-golgi compartments of hippocampal neurons. J Cell Biol. 1999; 147(2):277-94.

Anderson J, Kempski H, Hill L, Rampling D, Gordon T, Michalski A. Neuroblastoma in monozygotic twins-a case of probable twin-to-twin metastasis. Br J Cancer. 2001; 85(4):493-6.

Badano JL, Mitsuma N, Beales PL, Katsanis N. The ciliopathies: an emerging class of human genetic disorders. Annu Rev Genomics Hum Genet. 2006; 7:125-48.

Bahi-Buisson N, Guttierrez-Delicado E, Soufflet C, Rio M, Daire VC, Lacombe D, Heron D, Verloes A, Zuberi S, Burglen L, Afenjar A, Moutard ML, Edery P, Novelli A, Bernardini L, Dulac O, Nabbout R, Plouin

P, Battaglia A. Spectrum of epilepsy in terminal Ip36 deletion syndrome. Epilepsia. 2008; 49(3):509-15.

Battaglia A, Hoyme HE, Dallapiccola B, Zackai E, Hudgins L, McDonald-McGinn D, Bahi-Buisson N, Romano C, Williams CA, Brailey LL, Zuberi SM, Carey JC. Further delineation of deletion Ip36 syndrome in 60 patients: a recognizable phenotype and common cause of developmental delay and mental retardation. Pediatrics. 2008; 121(2):404-10.

Biegel JA, White PS, Marshall HN, Fujimori M, Zackai EH, Scher CD, Brodeur GM, Emanuel BS. Constitutional Ip36 deletion in a child with neuroblastoma. Am J Hum Genet. 1993;52(l):176-82.

Blatt J. Tumor suppressor genes on the short arm of chromosome 1 in neuroblastoma. Pediatr Hematol Oncol. 2001; 18(l):3-5.

Cristiano RJ, Smith LC, Kay MA, Brinkley BR, Woo SL. Hepatic gene therapy: efficient gene delivery and expression in primary hepatocytes utilizing a conjugated adenovirus-DNA complex. Proc Natl Acad Sci U S A. 1993; 90(24):11548-52.

De Strooper B. Aph-1, Pen-2, and Nicastrin with Presenilin generate an active gamma-Secretase complex. Neuron. 2003; 38(1):9-12.

Fortini ME. Gamma-secretase-mediated proteolysis in cell-surface-receptor signalling. Nat Rev MoI Cell Biol. 2002; 3(9):673-84.

Gajecka M, Mackay KL, Shaffer LG. Monosomy Ip36 deletion syndrome. Am J Med Genet C Semin Med Genet. 2007; 145C(4):346-56. Halpern AV, Bansal A, Heymann WR. Pemphigus vulgaris in a patient with Ip36 deletion syndrome. J Am Acad Dermatol. 2006; 55(5 Suppl):S98-9.

Hayes JM, Kim SK, Abitua PB, Park TJ, Herrington ER, Kitayama A, Grow MW, Ueno N, Wallingford JB. Identification of novel ciliogenesis factors using a new in vivo model for mucociliary epithelial development. Dev Biol. 2007; 312(l):115-30.

Heilstedt HA, Burgess DL, Anderson AE, Chedrawi A, Tharp B, Lee O, Kashork CD, Starkey DE, Wu YQ, Noebels JL, Shaffer LG, Shapira SK. Loss of the potassium channel beta-subunit gene, KCNAB2, is associated with epilepsy in patients with Ip36 deletion syndrome. Epilepsia. 2001; 42(9):1103-ll.

Heilstedt HA, Ballif BC, Howard LA, Lewis RA, Stal S, Kashork CD, Bacino CA, Shapira SK, Shaffer LG. Physical map of Ip36, placement of breakpoints in monosomy Ip36, and clinical characterization of the syndrome. Am J Hum Genet. 2003; 72(5):1200-12.

Jenke AC, Stehle IM, Herrmann F, Eisenberger T, Baiker A, Bode J, Fackelmayer FO, Lipps HJ. Nuclear scaffold/matrix attached region modules linked to a transcription unit are sufficient for replication and maintenance of a mammalian episome. Proc Natl Acad Sci U S A. 2004; 101(31):11322-7.

Keppler-Noreuil KM, Carroll AJ, Finley WH, Rutledge SL. Chromosome Ip terminal deletion: report of new findings and confirmation of two characteristic phenotypes. J Med Genet. 1995; 32(8):619-22.

Knight SJ, Horsley SW, Regan R, Lawrie NM, Maher EJ, Cardy DL, Flint J, Kearney L. Development and clinical application of an innovative fluorescence in situ hybridization technique which detects submicroscopic rearrangements involving telomeres. Eur J Hum Genet. 1997 Jan-Feb;5(l):l-8.

Konishi J, Kawaguchi KS, Vo H, Haruki N, Gonzalez A, Carbone DP, Dang TP. Gamma-secretase inhibitor prevents Notch3 activation and reduces proliferation in human lung cancers. Cancer Res. 2007; 67(17):8051-7.

Laureys G, Speleman F, Opdenakker G, Benoit Y, Leroy J. Constitutional translocation t(l;17)(p36;ql2- 21) in a patient with neuroblastoma. Genes Chromosomes Cancer. 1990; 2(3):252-4.

Liu Y, Pathak N, Kramer-Zucker A, Drummond IA. Notch signaling controls the differentiation of transporting epithelia and multiciliated cells in the zebrafish pronephros. Development. 2007; 134(6):llll-22. Manzini S, Vargiolu A, Stehle IM, Bacci ML, Cerrito MG, Giovannoni R, Zannoni A, Bianco MR, Forni M, Donini P, Papa M, Lipps HJ, Lavitrano M. Genetically modified pigs produced with a nonviral episomal vector. Proc Natl Acad Sci U S A. 2006; 103(47):17672-7.

Michelsen K, Yuan H, Schwappach B. Hide and run. Arginine-based endoplasmic-reticulum-sorting motifs in the assembly of heteromultimeric membrane proteins. EMBO Rep. 2005; 6(8):717-22.

Miele L, Miao H, Nickoloff BJ. NOTCH signaling as a novel cancer therapeutic target. Curr Cancer Drug Targets. 2006; 6(4):313-23.

Miller AD. Retrovirus packaging cells. Hum Gene Ther. 1990; 1(1):5-14.

Minami K, Boshi H, Minami T, Tamura A, Yanagawa T, Uemura S, Takifuji K, Kurosawa K, Tsukino R, Izumi G, Yoshikawa N. Ip36 deletion syndrome with intestinal malrotation and annular pancreas. Eur J Pediatr. 2005; 164(3):193-4.

Naldini L, Blomer U, Gallay P, Ory D, Mulligan R, Gage FH, Verma IM, Trono D. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science. 1996; 272(5259):263-7.

Neal J, Apse K, Sahin M, Walsh CA, Sheen VL Deletion of chromosome Ip36 is associated with periventricular nodular heterotopia. Am J Med Genet A. 2006; 140(15):1692-5.

Okamoto N, Toribe Y, Nakajima T, Okinaga T, Kurosawa K, Nonaka I, Shimokawa O, Matsumoto N. A girl with Ip36 deletion syndrome and congenital fiber type disproportion myopathy. J Hum Genet. 2002;47(10):556-9.

Redon R, Rio M, Gregory SG, Cooper RA, Fiegler H, Sanlaville D, Banerjee R, Scott C, Carr P, Langford C, Cormier-Daire V, Munnich A, Carter NP, Colleaux L. Tiling path resolution mapping of constitutional Ip36 deletions by array-CGH: contiguous gene deletion or "deletion with positional effect" syndrome? J Med Genet. 2005; 42(2):166-71.

Reish O, Berry SA, Hirsch B. Partial monosomy of chromosome Ip36.3: characterization of the critical region and delineation of a syndrome. Am J Med Genet. 1995; 59(4):467-75.

Saccone S, De Sario A, Delia VaIIe G, Bernardi G. The highest gene concentrations in the human genome are in telomeric bands of metaphase chromosomes. Proc Natl Acad Sci U S A. 1992; 89(ll):4913-7.

Shaffer LG, Heilstedt HA. Terminal deletion of Ip36. Lancet. 2001; 358 Suppl:S9. Shaffer LG, Lupski JR. Molecular mechanisms for constitutional chromosomal rearrangements in humans. Annu Rev Genet. 2000; 34:297-329.

Shapira SK, McCaskill C, Northrup H, Spikes AS, Elder FF, Sutton VR, Korenberg JR, Greenberg F, Shaffer LG. Chromosome Ip36 deletions: the clinical phenotype and molecular characterization of a common newly delineated syndrome. Am J Hum Genet. 1997; 61(3):642-50.

Shih IeM, Wang TL. Notch signaling, gamma-secretase inhibitors, and cancer therapy. Cancer Res. 2007; 67(5):1879-82.

Slavotinek A, Shaffer LG, Shapira SK. Monosomy Ip36. J Med Genet. 1999; 36(9):657-63.

Spasic D, Raemaekers T, Dillen K, Declerck I, Baert V, Serneels L, Fϋllekrug J, Annaert W. Rerlp competes with APH-I for binding to nicastrin and regulates gamma-secretase complex assembly in the early secretory pathway. J Cell Biol. 2007; 176(5):629-40.

Stubbs JL, Davidson L, Keller R, Kintner C. Radial intercalation of ciliated cells during Xenopus skin development. Development. 2006; 133(13):2507-15.

Tobin JL, Beales PL. Restoration of renal function in zebrafish models of ciliopathies. Pediatr Nephrol. 2008; 23(ll):2095-9.

Trapnell BC. Adenoviral vectors for gene transfer. Adv. Drug Del. Rev. 1993; 12: 185-199.

Wang BT, Chen M. Redundant skin over the nape in a girl with monosomy Ip36 caused by a de-novo satellited derivative chromosome: a possible new feature? Clin Dysmorphol. 2004; 13(2):107-9.

Windpassinger C, Kroisel PM, Wagner K, Petek E. The human gamma-aminobutyric acid A receptor delta (GABRD) gene: molecular characterisation and tissue-specific expression. Gene. 2002; 292(1- 2):25-31.

Wu Y-Q, Heilstedt H, Bedell JA, May KM, Starkey DE, McPherson JD, Shapira SK, Shaffer LG. Molecular refinement of the Ip36 deletion syndrome reveals size diversity and a preponderance of maternally derived deletions. Hum MoI Genet 1999; 8:313-321.

Yamada T, Iwasaki Y, Tada H, Iwabuki H, Chuah MK, VandenDriessche T, Fukuda H, Kondo A, Ueda M, Seno M, Tanizawa K, Kuroda S. Nanoparticles for the delivery of genes and drugs to human hepatocytes. Nat Biotechnol. 2003; 21(8):885-90.

Claims

Claims

1. A method of diagnosis of monosomy Ip36 syndrome, comprising 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.

2. The method according to claim 1, further comprising 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 monosomy Ip36 syndrome.

3. The method according to claim 1 or 2, wherein the expression and/or activity of RERl is evaluated at the mRNA level.

4. The method according to any one of claims 1 to 3, wherein the expression and/or activity of RERl is evaluated via PCR, in particular via RT-PCR.

5. The method according to claim 1 or 2, wherein the expression and/or activity of RERl is evaluated at the protein level.

6. The method according to claim 1 or 2, wherein the expression and/or activity of RERl is evaluated by monitoring acetylated tubulin levels or determining cilia number or length.

7. A method of treating at least one symptom of monosomy Ip36 syndrome in a subject in need thereof, comprising upregulating RERl expression and/or activity; and/or downregulating γ-secretase expression and/or activity; and/or downregulating Notch signaling.

8. A compound that upregulates RERl expression and/or activity, and/or downregulates γ-secretase expression and/or activity for use in treatment of at least one symptom of monosomy Ip36 syndrome.

9. The method according to claim 7, or compound according to claim 8, wherein the at least one symptom is 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.