# Patent

## Title
CC2D2A gene mutations associated with Joubert syndrome and diagnostic methods for identifying the same

## Abstract

The present invention provides a method of screening a subject for mutations in the CC2D2A gene that are associated with Joubert syndrome, an autosomal recessive form of mental retardation. The present invention also provides proteins that are associated with Joubert syndrome including proteins that includes an amino acid sequence that terminates in DHEGGSGMES (SEQ ID NO: 1). Also provided are nucleotide sequences encoding such proteins and methods of screening subjects to identify nucleotide sequences or proteins associated with Joubert syndrome.

## Background
This application is a continuation of U.S. application Ser. No. 13/346,069 filed on Jan. 9, 2012, now U.S. Pat. No. 8,598,330, which is a division of U.S. application Ser. No. 12/681,347 filed on Jul. 15, 2010, now U.S. Pat. No. 8,119,351, which is the 371 filing of International patent application no. PCT/CA2008/001760 filed on Oct. 3, 2008, which claims the benefit of U.S. application No. 60/977,803 filed on Oct. 5, 2007.


FIELD OF INVENTION
The present invention relates to gene mutations. More specifically, the present invention relates to gene mutations associated with mental retardation.
BACKGROUND OF THE INVENTION
Mental retardation (MR) is a condition that affects about 6 million American and over half a million Canadian children under the age of 14 years (Shea, 2006). MR is a general term for a heterogeneous group of disorders that are defined by deficits in cognitive and adaptive development. Frequently other terms used include “general learning disorder”, “mental handicap”, “learning disability”, “intellectual handicap”, and “intellectual disability” (Leonard & Wen, 2002), also “mentally challenged” and “developmental delay” (Shea, 2006). The prevalence of MR is commonly given as about 1% of the population (Szymanski & King, 1999), with a higher proportion of males to females affected (1.4:1; Murphy et al., 1998).
MR is commonly classified according to Intelligent Quotient (IQ). The Diagnostic & Statistical Manual of Mental Disorders (4th Edition; DSM-IV, 1994) identifies mild MR in the IQ range 50-55 to 70, moderate MR as 35-40 to 55-55, severe MR as 20-25 to 35-40, and profound MR as below 20-25. Attempts to understand the etiological basis of cases of MR are important because they may assist with the diagnosis of associated co-morbidities (eg. aortic stenosis in Williams syndrome), or may help with prenatal diagnostics and/or genetic counselling (eg. in fragile X syndrome), or may identify a treatable condition such as phenylketonuria (Shea, 2006). In addition, understanding the cause of MR may help families cope with a MR child and may help them access support infrastructure.
The contribution of genetics to MR has long been established. Conventionally, genetic forms of MR are subdivided into two major categories. Firstly, syndromic MR is characterized by cognitive deficits associated with other clinical and biological features. Secondly, non-syndromic form of MR in which cognitive impairment is the only manifestation of disease. Genetic factors are involved in the etiology of approximately two-thirds of mental retardation cases (Curry, 2002). In inherited forms of mental retardation, X-linkage or autosomal recessive inheritance patterns are the most plausible, since procreation from affected individuals is not common. To-date, more than 60 genes have been reported for X linked mental retardation (Chelly et al, 2006). But the molecular basis of autosomal recessive mental retardations are still poorly understood. Although autosomal recessive inheritance is estimated to be involved in nearly a quarter of all individuals with non-syndromic mental retardation (NSMR) (reviewed in Basel-Vanagaite et al,7), only four autosomal genes, the PRSS12 gene on chromosome 4q26 (neurotrypsin [MIM #606709]), the CC2D1A gene on chromosome 19p13.12 [MIM #610055], the CRBN gene on chromosome 3p26 (cereblon [MIM #609262]), and very recently GRIK2 on 6q16.1-q21 (ionotropic glutamate receptor 6 [MIM #138244]) have been reported so far to cause autosomal recessive NSMR.7-10 However, only a very few families or unrelated individuals with ARMR have been confirmed for each of these genes (PRSS12, N=1; CRBN, N=1; CC2D1A, N=9; GRIK2, N=1). The most recent of these genes, GRIK2, was discovered at one of 8 novel loci for autosomal recessive non-syndromic mental retardation (NSMR) recently mapped by homozygosity mapping in 78 consanguineous Iranian families. However, no disease gene or mutation has yet been reported for the other 7 loci (Najmabadi et al, 2007). Neurotrypsin (PRSS12) was the first gene reported in etiology of autosomal recessive non-syndromic mental retardation. The disease locus was mapped on chromosome 4q21-q25 by homozygosity mapping using a set 400 microsatellite markers across the genome. This interval encompasses about 29 genes of known function including the DKK2, PL34, CASP6, ANK2, CAMK2D TRPC3, and PRSS12 genes. A homozygous 4 by deletion in exon 7 of the PRSS12 gene was found cosegregating in all affected individuals, and resulted in a premature stop codon, 147 bp downstream of the deletion (Molinari et al, 2002). In another family with mild autosomal recessive non-syndromic mental retardation, a nonsense mutation causing a premature stop codon was identified in the CRBN gene that encodes for an ATP-dependent Lon protease. This C to T substitution changed an arginine residue to a stop codon in exon 11 (R419X) of this gene (Higgins et al, 2004). Mutations in the PRSS12 and CRBN genes have each been reported in only one family to-date. Recently a protein truncating mutation was identified in the CC2D1A gene in nine consanguineous families with severe autosomal recessive NSMR. The CC2D1A protein is involved in the calcium dependent phospholipid binding (Basel-Vanagaite et al, 2006).
A recent study has mapped 8 novel loci for autosomal recessive non-syndromic mental retardation (NSMR) by homozygosity mapping in 78 consanguineous Iranian families. However, no disease gene or mutation has yet been reported (Najmabadi et al, 2007). Another recently published study has mapped a new locus for autosomal recessive non-syndromic mental retardation to 1p21.1-p13.3 (Uyguner, 2007).
There is a need in the art to identify genetic markers associated with mental retardation. Further there is a need in the art to identify nucleotide sequences associated with mental retardation. There is also a need in the art for new diagnostic assays for mental retardation.
SUMMARY OF THE INVENTION
The present invention relates to gene mutations. More specifically, the present invention relates to gene mutations associated with mental retardation.
According to the present invention there is provided a protein comprising a fragment of SEQ ID NO:7. In a preferred embodiment the protein is truncated at amino acid 779 or earlier. In an alternate embodiment, the protein has all or part of the C2 domain abolished. Other proteins are also contemplated as described herein.
According to the present invention there is provided a protein comprising an amino acid sequence that terminates in DHEGGSGMES (SEQ ID NO:1).
According to the present invention, there is also provided the protein as defined above, wherein the amino acid sequence comprises SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4.
Also provided by the present invention is the protein as described above wherein the protein is between about 80% and 100% identical to SEQ ID NO:3.
The present invention also provides a nucleic acid encoding the protein as defined above.
Also provided by the present invention is the nucleic acid as defined above encoding the protein defined by SEQ ID NO:3 or 4.
The present invention also provides a nucleic acid comprising the complement of the nucleic acid defined above. In still a further non-limiting embodiment, the nucleic acid is capable of hybridizing to the nucleic acid defined above or its complement under stringent hybridization conditions.
The present invention also provides a nucleic acid as defined above comprising between 7 and 100 nucleotides. Further, the nucleic acid may be labeled at one or more sites.
Also provided by the present invention is a nucleotide sequence as defined above wherein the sequence is in a nucleotide construct comprising one or more regulatory elements.
The present invention also provides a method of screening a subject for a gene sequence associated with mental retardation comprising,

    
    
        a) obtaining a biological sample from the subject, the biological sample comprising DNA or RNA, and;
        b) assaying the sample for a nucleic acid encoding a protein comprising SEQ ID NO:1 at the C-terminus, wherein the presence of the nucleic acid indicates that the subject has a gene sequence associated with mental retardation.
    
    


In such a method, the step of assaying may comprise one or more hybridization assays, nucleotide sequencing, polymerase chain reactions (PCR) or any combination thereof.
The present invention also provides a method of screening as defined above wherein the sample that is assayed is a blood sample.
The present invention further provides a method of screening a subject for mutant protein associated with mental retardation, the method comprising,

    
    
        a) obtaining a biological sample from the subject,
        b) testing the sample for a protein that comprises SEQ ID NO:1 at the C-terminus thereof, wherein the presence of the protein indicates that the subject has a gene sequence that expresses a mutant protein associated with mental retardation.
    
    


The present invention also provides a method of screening a subject for mutant protein associated with mental retardation, the method comprising,

    
    
        a) obtaining a biological sample from the subject,
        b) testing the sample for a protein that comprises SEQ ID NO:1 at the C-terminus thereof, wherein the presence of the protein indicates that the subject has a gene sequence that expresses a mutant protein associated with mental retardation.
    
    


The present invention also provides a method of screening a subject for a gene sequence associated with mental retardation, the method comprising,

    
    
        a) obtaining a biological sample from the subject, the biological sample comprising DNA or RNA, and;
        b) assaying the sample for one or more mutations in a nucleotide sequence encoding a CC2D2A protein as defined by SEQ ID NO:7, an isoform or a naturally occurring allelic variant thereof, wherein the presence of said one or more mutations results in deletion in one or more amino acids of the protein or premature truncation of the protein and indicates that the subject has a gene sequence associated with mental retardation.
    
    


Also provided by the present invention is a method as defined above, wherein the one or more mutations are deletions, inversions, translocations, duplications, splice-donor site mutations, point-mutations or the like.
Also provided by the present invention is a method as defined above, wherein the one or more mutations occur in exon 19.
Also provided by the present invention is a method as defined above, wherein the one or more mutations abolish all or part of the C2 domain.
Also provided by the present invention is a method as defined above, wherein the one or more mutations result in truncation of the CC2D2A protein at amino acid 779 or earlier.
Also provided by the present invention is a method as defined above, wherein the one or more mutations add one or more nonsense amino acids to the protein.
The present invention also contemplates a kit comprising,

    
    
        a) a protein comprising an amino acid sequence that terminates in DHEGGSGMES (SEQ ID NO:1) and that is associated with mental retardation,
        b) a truncated version of the protein defined by SEQ ID NO:7 or a protein wherein all or part of the C2 domain is deleted;
        c) an antibody that selectively binds to the protein in a), b) or a) and b) but preferably not a similar CC2D2A wild-type protein that is not associated with mental retardation,
        d) one or more nucleic acid primers to amplify a nucleotide sequence encoding a protein or fragment thereof which comprises a mutation associated with mental retardation as provided herein,
        e) one or more nucleic acid probes of between about 9 and 100 nucleotides that hybridize to the nucleotide sequence encoding a protein or fragment thereof which comprises a mutation associated mental retardation as provided herein,
        f) one or more reagents comprising buffer(s), dATP, dTTP, dCTP, dGTP, DNA polymerase(s), or a combination thereof;
        g) instructions for assaying, diagnosing or determining the presence of a nucleotide sequence or protein that is associated with mental retardation in a subject,
        h) instructions for using any component or practicing any method as described herein, or;
        any combination or sub-combination thereof.
    
    


This summary of the invention does not necessarily describe all features of the invention.



BRIEF DESCRIPTION OF THE DRAWINGS
These and other features of the invention will become more apparent from the following description in which reference is made to the appended drawings wherein:
FIG. 1 shows representative protein and nucleotide sequences as defined herein including FIG. 1A) a first CC2D2A truncation protein associated with mental retardation plus retinitis pigmentosa, FIG. 1B) a second CC2D2A truncation protein associated with mental retardation plus retinitis pigmentosa, FIG. 1C) a representative nucleotide sequence encoding a CC2D2A truncation protein, FIG. 1D) a representative wild-type CC2D2A nucleotide sequence encoding a non-truncated protein, and FIG. 1E) a representative wild-type CC2D2 A protein.
FIG. 2 shows a graphic depiction of the pedigree of the family from the Mianwali district.
FIGS. 3A and 3B show a graphical depiction of the two-point and multi-point linkage analysis for the Mianwali family.
FIG. 4 shows haplotype analysis for the Mianwali family.
FIG. 5 shows an ideogrammatic representation of the mutation region in the CC2D2A gene.
FIG. 6 shows an ideogrammatic representation of the CC2D2A cDNA and the encoded protein.
FIG. 7 shows the express of the CC2D2A gene in several tissues.
FIG. 8 shows results that anti-serum from rabbit recognizes recombinant CC2D2A. Cos-7 cells were transfected with empty vector (1), CC2DAHisMyc (2) or PTCHD1HisMyc (3). A strong band is seen in lane 2 between 150 and 250 Kda but is not detected in control lanes 1 and 3. The theoretical size is 191 kDa (186 kDa CC2D2A plus 6 kDa HisMyc tag). Anti-serum was diluted 1:500 in 0.1% TBS-T in 5% milk. Secondary antibody was anti-rabbit IgG HRP (Jackson immunoresearch) diluted 1:20 000.



DETAILED DESCRIPTION
The following description is of a preferred embodiment.
We have identified by homozygosity mapping followed by mutation screening a new gene involved in autosomal recessive mental retardation plus retinitis pigmentosa. The gene, CC2D2A (also termed KIAA1345; NCBI MIM entry 612013), contains a C2 domain near the C-terminal region of the protein, in addition to coiled-coil regions. The C2 motif is thought to be involved in calcium dependent phospholipid binding. CC2D1A, one of the three previously identified ARMR genes, also contains a single C2 domain towards the C-terminal end of the protein in addition to coiled-coil regions. Without wishing to be bound by theory or limiting in any manner, it is possible that these two proteins may have similar functions, and may be components of the same or parallel pathways that are important components for neuronal development, disruption of which leads to developmental delay.
Proteins and Amino Acids
According to the present invention, there is provided a protein comprising a fragment of SEQ ID NO:7. In a preferred embodiment the protein is truncated at amino acid 779 or earlier. In an alternate embodiment, the protein has all or part of the C2 domain abolished. Other proteins are also contemplated as described herein.
According to the present invention there is provided a protein that terminates in the amino acid sequence defined by DHEGGSGMES (SEQ ID NO:1), more preferably TLDHEGGSGMES (SEQ ID NO:2). The sequence as provided in SEQ ID NO:1 may comprise the C-terminal of a larger protein, for example, but not limited to the proteins defined in SEQ ID NO: 3 or SEQ ID NO:4.
The present invention also contemplates that the protein may comprise a fragment of SEQ ID NO:3, provided that the fragment terminates in the amino acid sequence defined by SEQ ID NO:1, for example, amino acids X-783 of SEQ ID NO:2, wherein X is for example, but not meant to be limiting 2, 5, 10, 20, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 710, 720, 730, 740, 750, 760, 770, 772 or any value therein between.
The present invention further contemplates proteins which are between 80% to 100% identical over a span of at least 11 continuous amino acids defined in SEQ ID NO:3 and which terminate in the amino acid sequence defined by DHEGGSGMES (SEQ ID NO:1). For example, the proteins may be 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO:3 and which terminates in SEQ ID NO: 1 at the C-terminus. Further, the proteins may comprise a percent identity or a range of identities defined by any two values provided above, or any value therein between.
Any method known in the art may be used for determining the degree of identity between polypeptide sequences. For example, but without wishing to be limiting, a sequence search method such as BLAST (Basic Local Alignment Search Tool; (Altschul S F, Gish W, Miller W, Myers E W, Lipman D J (1990) J Mol Biol 215, 403 410) can be used according to default parameters as described by Tatiana et al., FEMS Microbial Lett. 174:247 250 (1999), or on the National Center for Biotechnology Information web page at ncbi.nlm.gov/BLAST/, for searching closely related sequences. BLAST is widely used in routine sequence alignment; modified BLAST algorithms such as Gapped BLAST, which allows gaps (either insertions or deletions) to be introduced into alignments, or PSI-BLAST, a sensitive search for sequence homologs (Altschul et al., Nucleic Acids Res. 25:3389 3402 (1997); or FASTA, which is available on the world wide web at ExPASy (EMBL—European Bioinformatics Institute). Similar methods known in the art may be employed to compare DNA or RNA sequences to determine the degree of sequence identity.
Nucleic Acids
Also contemplated by the present invention is a nucleic acid comprising a sequence

    
    
        a) encoding a protein as defined above, or a fragment thereof;
        b) that is the complement of a sequence encoding the protein as defined above, or a fragment thereof,
        c) that is capable of hybridizing to a nucleic acid encoding the protein as defined above or fragment thereof under stringent hybridization conditions, or
        d) that is capable of hybridizing to a nucleic acid contained within 5′ or 3′ untranslated regions, intronic sequences, or upstream promoter or other regulatory sequences.
    
    


In an embodiment the nucleic acid comprises a nucleotide sequence that encodes the amino acid sequence defined by SEQ ID NO: 1, more preferably SEQ ID NO:2. In still a further embodiment, there is provided a nucleic acid comprising a nucleotide sequence that encodes the amino acid sequence defined by SEQ ID NO:3, SEQ ID NO:4 or a fragment thereof that comprises SEQ ID NO:1. For example, but not wishing to be limiting in any manner, the nucleic acid may comprise SEQ ID NO: 3. These representative sequences are exemplary and are not meant to be exhaustive or limiting in any manner.
In a further embodiment of the present invention, there is provided a nucleic acid of at least about 7 nucleotides which binds to a mutant form of the CC2D2A gene, the mutant form encoding a protein comprising the amino acid sequence defined above, for example SEQ ID NO:1, but that does not bind to the wild-type form of a CC2D2A gene that encodes a protein that terminates in a sequence other than SEQ ID NO:1. For example, but not to be considered limiting in any manner, a mutant form of the CC2D2A gene may comprise SEQ ID NO: 5, and a wild-type form of a CC2D2A gene may comprise SEQ ID NO:6. Such nucleic acids may be used as probes in screening methods to identify subjects that are carriers or that have a genetic mutation associated with mental retardation.
While the present invention contemplates probes comprising nucleotide sequences of at least about 7 nucleotides, preferably the probe comprises greater than 7 nucleotides, for example, but not limited to 9, 11, 15, 17, 21, 25, 27, 30, 40, 50, 100 or more nucleotides. Further the size of the probes may be defined by a range of any two values as provided above or any two values in between. Also, the probe may be labeled by an appropriate moiety as would be known in the art, for example, but not limited to one or more fluorophores, radioactive groups, chemical substituents, enzymes, antibodies or the like to facilitate identification in hybridization assays and other assays or tests.
The nucleic acids as provided herein may be employed to produce proteins which are associated with mental retardation, as probes to identify or diagnose subjects with mental retardation, identify or diagnose subjects carrying a mutation which causes or predisposes the subject or its offspring to mental retardation, antisense or short inhibitory RNA that may be used to modulate production of protein from genes associated with mental retardation or a combination thereof.
The present invention contemplates nucleic acids that hybridize to nucleotide sequences that encode proteins as provided herein under stringent hybridization conditions. Stringent hybridization conditions as described above may be, for example but not limited to hybridization overnight (from about 16-20 hours) hybridization in 4×SSC at 65° C., followed by washing in 0.1×SSC at 65° C. for an hour, or 2 washes in 0.1×SSC at 65° C. each for 20 or 30 minutes. Alternatively, an exemplary stringent hybridization condition could be overnight (16-20 hours) in 50% formamide, 4×SSC at 42° C., followed by washing in 0.1×SSC at 65° C. for an hour, or 2 washes in 0.1×SSC at 65° C. each for 20 or 30 minutes, or overnight (16-20 hours); or hybridization in Church aqueous phosphate buffer (7% SDS; 0.5M NaPO4 buffer pH 7.2; 10 mM EDTA) at 65° C., with 2 washes either at 50° C. in 0.1×SSC, 0.1% SDS for 20 or 30 minutes each, or 2 washes at 65° C. in 2×SSC, 0.1% SDS for 20 or 30 minutes each for unique sequence regions.
The present invention is further directed to a nucleotide construct comprising the nucleic acid as described above operatively linked to one or more regulatory elements or regulatory regions. By “regulatory element” or “regulatory region”, it is meant a portion of nucleic acid typically, but not always, upstream of a gene, and may be comprised of either DNA or RNA, or both DNA and RNA. Regulatory elements may include those which are capable of mediating organ specificity, or controlling developmental or temporal gene activation. Furthermore, “regulatory element” includes promoter elements, core promoter elements, elements that are inducible in response to an external stimulus, elements that are activated constitutively, or elements that decrease or increase promoter activity such as negative regulatory elements or transcriptional enhancers, respectively. By a nucleotide sequence exhibiting regulatory element activity it is meant that the nucleotide sequence when operatively linked with a coding sequence of interest functions as a promoter, a core promoter, a constitutive regulatory element, a negative element or silencer (i.e. elements that decrease promoter activity), or a transcriptional or translational enhancer.
By “operatively linked” it is meant that the particular sequences, for example a regulatory element and a coding region of interest, interact either directly or indirectly to carry out an intended function, such as mediation or modulation of gene expression. The interaction of operatively linked sequences may, for example, be mediated by proteins that interact with the operatively linked sequences.
Regulatory elements as used herein, also includes elements that are active following transcription initiation or transcription, for example, regulatory elements that modulate gene expression such as translational and transcriptional enhancers, translational and transcriptional repressors, and mRNA stability or instability determinants. In the context of this disclosure, the term “regulatory element” also refers to a sequence of DNA, usually, but not always, upstream (5′) to the coding sequence of a structural gene, which includes sequences which control the expression of the coding region by providing the recognition for RNA polymerase and/or other factors required for transcription to start at a particular site. An example of a regulatory element that provides for the recognition for RNA polymerase or other transcriptional factors to ensure initiation at a particular site is a promoter element. A promoter element comprises a core promoter element, responsible for the initiation of transcription, as well as other regulatory elements that modify gene expression. It is to be understood that nucleotide sequences, located within introns, or 3′ of the coding region sequence may also contribute to the regulation of expression of a coding region of interest. A regulatory element may also include those elements located downstream (3′) to the site of transcription initiation, or within transcribed regions, or both. In the context of the present invention a post-transcriptional regulatory element may include elements that are active following transcription initiation, for example translational and transcriptional enhancers, translational and transcriptional repressors, and mRNA stability determinants.
The regulatory elements, or fragments thereof, may be operatively associated (operatively linked) with heterologous regulatory elements or promoters in order to modulate the activity of the heterologous regulatory element. Such modulation includes enhancing or repressing transcriptional activity of the heterologous regulatory element, modulating post-transcriptional events, or both enhancing/repressing transcriptional activity of the heterologous regulatory element and modulating post-transcriptional events. For example, one or more regulatory elements, or fragments thereof, may be operatively associated with constitutive, inducible, tissue specific promoters or fragment thereof, or fragments of regulatory elements, for example, but not limited to TATA or GC sequences may be operatively associated with the regulatory elements of the present invention, to modulate the activity of such promoters within plant, insect, fungi, bacterial, yeast, or animal cells.
There are several types of regulatory elements, including those that are developmentally regulated, inducible and constitutive. A regulatory element that is developmentally regulated, or controls the differential expression of a gene under its control, is activated within certain organs or tissues of an organ at specific times during the development of that organ or tissue. However, some regulatory elements that are developmentally regulated may preferentially be active within certain organs or tissues at specific developmental stages, they may also be active in a developmentally regulated manner, or at a basal level in other organs or tissues within a plant as well.
By “promoter” it is meant the nucleotide sequences at the 5′ end of a coding region, or fragment thereof that contain all the signals essential for the initiation of transcription and for the regulation of the rate of transcription. There are generally two types of promoters, inducible and constitutive promoters.
An inducible promoter is a promoter that is capable of directly or indirectly activating transcription of one or more DNA sequences or genes in response to an inducer. In the absence of an inducer the DNA sequences or genes will not be transcribed. Typically the protein factor that binds specifically to an inducible promoter to activate transcription is present in an inactive form which is then directly or indirectly converted to the active form by the inducer. The inducer can be a chemical agent such as a protein, metabolite, growth regulator, or a physiological stress imposed directly by heat, cold, or toxic elements or indirectly through the action of a pathogen or disease agent such as a virus.
A constitutive promoter directs the expression of a gene throughout the various parts of an organism and/or continuously throughout development of an organism. Any suitable constitutive promoter may be used to drive the expression of the proteins or fragments thereof as described herein. Examples of known constitutive promoters include but are not limited to those associated with the CaMV 35S transcript. (Odell et al., 1985, Nature, 313: 810-812).
The term “constitutive” as used herein does not necessarily indicate that a gene is expressed at the same level in all cell types, but that the gene is expressed in a wide range of cell types, although some variation in abundance is often observed.
The gene construct of the present invention can further comprise a 3′ untranslated region. A 3′ untranslated region refers to that portion of a gene comprising a DNA segment that contains a polyadenylation signal and any other regulatory signals capable of effecting mRNA processing or gene expression. The polyadenylation signal is usually characterized by effecting the addition of polyadenylic acid tracks to the 3 prime end of the mRNA precursor.
The gene construct of the present invention can also include further enhancers, either translation or transcription enhancers, as may be required. These enhancer regions are well known to persons skilled in the art, and can include the ATG initiation codon and adjacent sequences. The initiation codon must be in phase with the reading frame of the coding sequence to ensure translation of the entire sequence. The translation control signals and initiation codons can be from a variety of origins, both natural and synthetic. Translational initiation regions may be provided from the source of the transcriptional initiation region, or from the structural gene. The sequence can also be derived from the regulatory element selected to express the gene, and can be specifically modified so as to increase translation of the mRNA.
The present invention further includes vectors comprising the nucleic acids as described above. Suitable expression vectors for use with the nucleic acid sequences of the present invention include, but are not limited to, plasmids, phagemids, viral particles and vectors, phage and the like. For insect cells, baculovirus expression vectors are suitable. For plant cells, viral expression vectors (such as cauliflower mosaic virus and tobacco mosaic virus) and plasmid expression vectors (such as the Ti plasmid) are suitable. The entire expression vector, or a part thereof, can be integrated into the host cell genome.
Those skilled in the art will understand that a wide variety of expression systems can be used to produce the proteins or fragments thereof as defined herein. With respect to in vitro production, the precise host cell used is not critical to the invention. The proteins or fragments thereof can be produced in a prokaryotic host (e.g., E. coli or B. subtilis) or in a eukaryotic host (e.g., Saccharomyces or Pichia; mammalian cells, such as COS, NIH 3T3, CHO, BHK, 293, or HeLa cells; insect cells; or plant cells). The methods of transformation or transfection and the choice of expression vector will depend on the host system selected and can be readily determined by one skilled in the art. Transformation and transfection methods are described, for example, in Ausubel et al. (1994) Current Protocols in Molecular Biology, John Wiley & Sons, New York; and various expression vectors may be chosen from those provided, e.g., in Cloning Vectors: A Laboratory Manual (Pouwels et al., 1985, Supp. 1987) and by various commercial suppliers.
In addition, a host cell may be chosen which modulates the expression of the inserted sequences, or modifies/processes the gene product in a specific, desired fashion. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the activity of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen by one skilled in the art to ensure the correct modification and processing of the expressed protein.
Methods of Screening
The present invention also provides a method of screening a subject for a gene sequence associated with mental retardation, the method comprising,

    
    
        a) obtaining a biological sample from the subject, the biological sample comprising DNA or RNA, and;
        b) assaying the sample for one or more mutations in a nucleotide sequence encoding a CC2D2A protein as defined by SEQ ID NO:7, an isofonn or a naturally occurring allelic variant thereof, wherein the presence of said one or more mutations results in deletion in one or more amino acids of the protein or premature truncation of the protein and indicates that the subject has a gene sequence associated with mental retardation.
    
    


In the method as defined above, the one or more mutations include without limitation, deletions, inversions, translocations, duplications, splice-donor site mutations, point-mutations or the like. The one or more mutations may occur in any region of the protein, for example, exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or higher. In a preferred embodiment, the one or more mutations occur in exon 19. In an alternate embodiment, the one or more mutations abolish all or part of the C2 domain, for example, between 5% and 100% of the C2 domain including 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% thereof. In still a further embodiment, the one or more mutations result in truncation of the CC2D2A protein as defined by SEQ ID NO:7 in the C2 domain, for example, but not limited to at about amino acid 779. However, without wishing to be limiting in any manner, the present invention contemplates premature truncation at any earlier or later amino acid in the sequence.
The present invention also provides a method of screening a subject for a gene sequence associated with mental retardation, the method comprising,

    
    
        a) obtaining a biological sample from the subject, the biological sample comprising DNA or RNA, and;
        b) assaying the sample for one or more mutations the gene sequence encoding CC2D2A protein as defined by SEQ ID NO:6, an isoform or a naturally occurring allelic variant thereof, wherein the presence of said one or more mutations results in deletion in one or more amino acids of the protein or premature truncation of the protein and indicates that the subject has a gene sequence associated with mental retardation.
    
    


The present invention also provides a method of screening a subject for a gene sequence associated with mental retardation, the method comprising

    
    
        a) obtaining a biological sample from the subject, the biological sample comprising DNA or RNA, and;
        b) assaying the sample for a nucleic acid encoding a protein comprising SEQ ID NO:1 at the C-terminus, wherein the presence of the nucleic acid indicates that the subject has a gene sequence associated with mental retardation.
    
    


It is to be understood in the method as described above, that step b) may comprise assaying for a nucleic acid encoding a protein comprising SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or a fragment thereof.
By the terms “assaying the sample” it is meant characterizing the sample provided by the subject for a nucleic acid that encodes a protein as defined above and is meant to include without limitation hybridization assays, nucleotide sequencing, nucleotide PCR including, but not limited to RT-PCR, etc or any combination thereof.
The sample obtained from the subject may comprise any tissue or biological fluid sample from which DNA or RNA may be obtained. For example, but not wishing to be limiting, DNA may be obtained from blood, hair follicle cells, skin cells, cheek cells, saliva cells, tissue biopsy, or the like. In a preferred embodiment, the sample is blood.
The present invention also contemplates screening methods which identify and/or characterize the proteins as defined above within biological samples from subjects. Such samples may or may not comprise DNA or RNA. For example, such screening or testing methods may employ immunological methods, for example, but not limited to antibody binding assays such as ELISAs or the like, protein sequencing, electrophoretic separations to identify the proteins as described above in a sample. As will be evident to a person of skill in the art, the screening methods allow for the differentiation of the proteins as defined herein from wild type proteins known in the art.
Accordingly, in a further embodiment, the present invention also provides a method of screening a subject for mutant protein associated with mental retardation, the method comprising

    
    
        a) obtaining a biological sample from the subject,
        b) testing the sample for a protein that comprises SEQ ID NO:1 at the C-terminus thereof, wherein the presence of the protein indicates that the subject has a gene sequence that expresses a mutant protein associated with mental retardation.
    
    


It is to be understood in the method as described above, that step b) may comprise testing for a protein comprising SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or a fragment thereof.
Kits
Also provided by the present invention is a kit comprising a protein as provided herein, for example, but not limited to one that comprises SEQ ID NO:1 at the C-terminus thereof, and that is associated with mental retardation, an antibody that selectively binds to a protein as provided herein, for example, but not limited to one that comprises SEQ ID NO:1 at the C-terminus thereof, and that is associated with mental retardation, rather than a similar wild-type protein that is not associated with mental retardation, one or more nucleic acid primers to amplify a nucleotide sequence encoding a protein or fragment thereof which comprises a mutation associated with mental retardation as provided herein, one or more nucleic acid probes of between about 9 and 100 nucleotides that hybridize to the nucleotide sequence encoding a protein or fragment thereof which comprises a mutation associated mental retardation as provided herein, one or more reagents including, but not limited to buffer(s), dATP, dTTP, dCTP, dGTP, DNA polymerase(s), instructions for assaying, diagnosing or determining the presence of a nucleotide sequence or protein in a subject that is associated with mental retardation, instructions for using any component or practicing any method as described herein, or any combination thereof.
The present invention will be further illustrated in the following examples.
EXAMPLES
Example 1
Materials and Methods
Patients
The family ascertained in this study is from the province of Punjab in Pakistan. Appropriate informed consent was obtained from all participants in the study. Clinical examination of affected individuals revealed that the early motor development was delayed, occipito-frontal circumference (OFC) was within normal range and face appears normal. There was no dysmorphic feature, no hepatosplenomegaly, no murmur, and no skin abnormalities. Structural MRI of the brain was performed for two affected individuals, which was generally normal, with some indication of mild cerebellar atrophy in one of the affected individuals. Molar-toot sign (MTS) was also present—a hallmark sign of Joubert syndrome.
Sample Collection and DNA extraction
Blood samples were collected from five affected and 12 unaffected members of the family. Genomic DNA was extracted from peripheral blood leukocytes by standard methods. Lymphoblast cell line was successfully established for only one family member.
SNP Homozygosity or Autozgosity Mapping
DNA samples of five affected and one unaffected were analyzed using the Affymetrix GeneChip Mapping 500K array. These arrays allow analysis of  ̃500,000 SNPs with a median physical distance of 2.5 kb and an average physical distance of 5.8 Kb between SNPs. The average heterozygosity of these SNPs is 0.30. However, in our experiments we just used NspI chip from GeneChip Mapping 500K set which allowed us to genotyping  ̃260,000 SNPs in our samples. Sample processing, labelling and hybridization were performed in accordance with the manufacturer's instructions (Affymetrix Mapping 500K Assay Manual). The arrays were scanned with a GeneChip Scanner and the data was processed using GeneChip® Operating Software (GCOS) and GeneChip® Genotyping Analysis Software (GTYPE) Software (ver. 3.0.2) to generate SNP allele calls.
Copy Number Analysis
Copy Number variations (CNVs) that include deletions, and duplication events were inferred by comparative analysis of hybridization intensities using dChip analyzer (Li and Wong, 2003; Zhao, 2004, Zhao, 2005). After normalization, we used Hidden Markov Model (HMM) to infer the DNA copy number from the raw signal data.
DNA Analysis with Microsatellite Markers and Linkage Analysis
12 fluorescent labelled microsatellite markers across the 4p region were PCR amplified using standard protocol and were electrophoresed on an ABI 3730x1 DNA analyzer. The genotypes were called using Genemapper software (Applied Biosystems) and linkage analysis was performed using MLINK software.
Cytogenetic Analysis
Karyotyping was performed by harvesting the lymphoblast cell line using standard cytogenetic procedure. Slides were made from fixed cells in the Thermotron, aged and then GTG banded.
DNA Sequencing and Mutation Screening
PCR primers were designed using Primer 3 (v. 0.3.0) to amplify all exons and intron-exon boundaries of known genes within the critical region. PCRs were performed using standard conditions, and products were purified and sequenced directly using the BigDye Terminator v3.1 Cycle Sequencing Ready Reaction Kit (Applied Biosystems)
Expression Analysis
Expression analysis for the CC2D2A gene was performed by RT-PCR, using the forward primer from exon 18 (5′-ACAGTCAGTCGGCCACTAGG) (SEQ ID NO:8) and reverse primer from exon 25 (5′-GTTCTGCCAGCTTGAAAAGG) (SEQ ID NO:9), thus spanning exon 19 containing the splice mutation in the Mianwali family.
Bioinformatic Analysis of CC2D2A
Promoter analysis for CC2D2A was performed using PromoterIspector from Genomatix. Homology searches was performed using the BLAST algorithm at the National Center for Biotechnology Information (Altschul et al. 1997) and the UCSC Human Genome Project Working Draft. (Protein domain predictions were performed using the SMART program (Simple Modular Architecture Research Tool) from the European Molecular Biology Laboratory (EMBL), the PSORT II suite of programs at University of Tokyo, the SOSUI algorithm and the COILS Program from the Swiss EMBNet. In order to identify regions of the proteins that have been highly conserved across evolution so that additional potentially functional relevant motifs may be identified in CC2D2A, comparative sequence analysis was performed for CC2D2A orthologues (known and predicted from genomic or cDNA sequences) across a variety of species using CLUSTAL-W.
Results
The locus for mental retardation and retinitis pigmentosa, as described herein and throughout has been accorded MIM entry 612285 at NCBI.
Homozygosity Mapping/Autozygosity Mapping
Homozygosity mapping (Autozygosity mapping) revealed a common  ̃11.2 Mb homozygous and haploidentical region on short arm of chromosome 4 (4p15.2-p15.33) in four affected individuals but not in the normal individual, nor in the fifth affected individual. This was the only large (>0.5 Mb) homozygous and haploidentical region present in four out of five affecteds, and no such regions were present in all five. The 11.2 Mb critical region containing  ̃39 genes (UCSC Genome Browser) was fine mapped by sequencing the flanking SNPs, rs2191685 and rs7664104 (at 14.001 and 25.203 Mb from the p telomere respectively (UCSC March 2006).
Copy Number Variants (CNVs) Analysis
As homozygosity mapping (autozygosity mapping) did not reveal any common region in all the affected individuals, we further hypothesized that there might be a large chromosomal deletion or duplication segregating with phenotype. Interestingly, copy number analysis indicated duplication of entire X chromosome in the fifth affected individual, who did not share the disease haplotype with other four affected members of the family. Subsequent cytogenetic analysis has indicated the karyotype 48,XXXX. This rare tetrasomy is almost always associated with mental retardation, and thus suggests that this female is a phenocopy.
Linkage Analysis
Linkage to the locus on chromosome 4p15.33-p15.2 was confirmed by genotyping 17 members of the family using the 12 microsatellite markers across the 4p region. Linkage was calculated using MLINK software, and a maximum two-point logarithm of odds (LOD) score of 3.59 at theta=0.0 was obtained at marker D4S419.
Mutation Screening
The 11.2 Mb critical region contains about 39 annotated genes (UCSC 2004). Because the gene for phenylketonuria type 2 (PKU2; MIM +262630), quinoid dihydropteridine reductase (QDPR), lies within the critical region, we initially, considered this as a potential candidate gene, and hence we screened all the coding sequence and splice sites of QDPR. An upstream homozygous base substitution was identified, but this was also homozygous in one of the unaffected parents, so did not segregate with disease. Furthermore, biochemical analysis of patient's blood samples also excluded this gene. By sequence analysis of all other known genes in the region, a splice donor site mutation (IVS19+1:G to C) in exon 19 of the CC2D2A genes was identified. The mutation segregates with phenotype and is predicted to result in addition of 3 nonsense aminoacids (M, E, S) and then premature truncation of protein. The CC2D2A (NM—001080522) gene gives a mRNA  ̃5 Kb in length, and consists of at least 13 different isoforms with up to 37 exons spanning  ̃131.5 Kb of genomic DNA (from 15,080,760-15,212,278 bp; UCSC March 2006) on 4p15.33. The CC2D2A protein contains up to 1620 amino acids (depending on exon usage) and a C2 domain is predicted in this gene from 1042-1202. From RT-PCR followed by sequence analysis using lymphoblast-derived cDNA from the affected individuals, we determined that the splice mutation results in the skipping of exon 19. The splicing of exon 18 to exon 20 results in a frame shift, with 13 nonsense amino acids added (ECPSHLKLMAVTS (SEQ ID NO: 14)*) beyond exon 18 before a premature stop codon truncates the protein at amino acid 740, thus abolishing the C2 domain. We screened 460 chromosomes of healthy controls, also from Pakistan, and were unable to identify find this mutation.
Bioinformatic Analysis of the CC2D2A Gene and Encoded Protein
Genomic organization of CC2D2A on 4p15.33 is shown in FIG. 5. Analysis of the sequence at and around the CC2D2A gene using the ElDorado and PromoterInspector programs from Genomatix indicated the presence of a putative promoter sequence of 773 bp from 15,080,088-15,080,860 bp (from the 4p telomere; UCSC March 2006). Analysis of the protein sequence using Simple Modular Architecture Research Tool (SMART) indicated the presence of a C2 domain from residue 1042 to 1202 (in the 1620 amino acid isoform). The C2 domain, also known as protein kinase C conserved region 2 (CalB), is a Ca2+-dependent membrane-targeting module present in many proteins involved in membrane trafficking or signal transduction. Transmembrane (TM) prediction using TMpred suggested two possibilities, either 1 strong TM helix (from residues 1278 to 1297), or no clear TM domains. Coiled-coil analysis using COILS v.21 predicted three stretches of the protein with >90% probability of coiled-coil structure: amino acid residues 442-463, 472-492 and 533-580 (using a 21-residue window). SOSUI predicts the protein to be soluble, with average hydrophobicity of −0.655. No signal peptide was identified, and k-NN prediction suggests a nuclear localization within the cell (91.3% nuclear, 4.3% cytoskeletal, 4.3% plasma membrane). Secondary structure is predicted to be mainly alpha helix and random coil, with some extended strand (Network Protein Sequence analysis). No signal peptides were detected. In addition, initial analysis indicates that CC2D2A appears to have a number of potential CaMKII recognition sites (9 RXXS/T and 3 S/TXD sites), as well as numerous putative PKC phosphorylation sites. Two putative nuclear localization signals were also detected using the PredictProtein suite (QRAKKKKRK (SEQ ID NO: 12) at residue 587 and RPRRK (SEQ ID NO: 13) at 1021). Alignment analysis indicates no homology with CC2D1 A and IB. There is 34% homology with CC2D2B, but only towards the C-terminal end (residues 1250-1620) of CC2D2A. CC2D2B has neither coiled coil nor C2 domains.
Comparative Sequence Analysis of CC2D2A
We performed cross-species sequence analysis using either full length coding sequences, or orthologues predicted from genomic DNA sequence and expressed sequenced tags (EST) from 18 species. Comparative analysis of the protein sequence using CLUSTALW shows a high degree of conservation across vertebrate evolution (FIG. 5). The human sequence is 99.4% identical to that of chimp, 96.9% to rhesus monkey, 88.8% to horse, 84.8% to mouse and rat, 75.7% to opossum, 70.7% to chicken, 60% to xenopus, 59.6% to zebrafish, 44.7% to sea urchin. The human protein is 21.6% identical to C. elegans protein K07G5.3 (NP—492026), and 29.9% with the Drosophila protein CG18631 (NP—611230), with the strongest overlap and homology occurring towards the C-terminal end (from amino acid 1301 in human, 894 in C. elegans, and 200 in Drosophila, to the carboxyl terminus), suggesting conserved functionality to this region in addition to the C2 domain. The WormBase information on this protein indicates that it is expressed in the nervous system, including head and tail neurons (as well as other unidentified cells in the head and tail) during both adult and larval stages. Knock-down through RNAi of the K07G5.3 gene was non-lethal.
The Ka/Ks ratio calculated from pairwise comparisons of the full-length cDNA sequence across species revealed that overall, the CC2D2A gene sequence is conserved. Interspecies comparisons of closely related species also revealed a high level of overall conservation. Ka and Ks values calculated from pairwise comparisons of the C2 domain in primates were higher than the Ka and Ks for the full-length primate sequence comparisons. In contrast, the mouse-rat C2 domain comparison gave a Ka value that was lower than the Ka for the full length mouse-rat comparison. However, the Ks value was slightly higher for the rodent C2 domain than for the rodent full length sequence. To further examine whether the C2 domain had a different evolutionary profile than the flanking sequences, 500 basepairs upstream and downstream of this region (Flanking Regions 1 and 2, hereafter FR1 and FR2) were analyzed in pairwise comparisons. In the primate lineage of human, chimp and rhesus, both the Ka and Ks values were higher for the C2 domain than for FR1 or FR2. In the mouse-rat comparison, the Ka for the C2 domain was lower than the Ka for both FR1 and FR2, while the Ks for the C2 domain was higher than the Ks for FR1, but similar to that for FR2.
The C terminal domain (encompassing the last 1113 nucleotides of the coding sequence in the human) was then compared with the first 1115 nucleotides at the N terminal domain, in order to examine the rates of evolution at the conserved C terminal. In all pairwise comparisons, the Ka/Ks ratios for the C terminal were significantly lower than for the N terminal, providing additional evidence that the C terminal is well conserved across species.
Expression Analysis
RT-PCR analysis indicates that CC2D2A is expressed in many tissues (FIG. 7). In a panel of cDNAs from 12 tissues expression was detected in each tissue, albeit at varying levels, with maximum expression seen in prostate, pancreas, kidney, lung and liver, with lower expression in spleen, small intestine, colon, skeletal muscle, ovary, thymus and heart. Brain expression was also strong. Further, expression of the CC2D2A-GFP fusion protein in Cos-7 cells appeared to be almost exclusively cytoplasmic, despite the predicted presence of potential nuclear localization signals.
Antibody to CC2D2A
The anti-CC2D2A antibodies were raised against 2 epitopes injected into rabbits carried out as a service by OPEN Biosystems (Huntsville, Ala.). Two epitopes were utilized due to the sheer size of the predicted CC2D2A protein  ̃186 kDa. The sequence utilized was from human sequence of CC2D2A (NP—001073991). The first epitope is in the centre but N-terminal to the Coiled-Coil and C2 Domain, RSKRFRLLHLRSQEVPEFRNYK (SEQ ID NO:10), as well as the mutation in the Mianwali family, whereas the second epitope is on the C-terminal tail, EDDHRAELLKQLGDYRFSGFPL (SEQ ID NO:11). These epitopes are also 100% conserved in the mouse orthologue of CCD2A (NP—758478) making the anti-bodies potentially cross reactive for human and mouse CC2D2A. The rabbit anti-serum was purified using affinity purification.
Example 2
CC2D2A in Relation to Joubert Syndrome
We have reported the identification through homozygosity/autozygosity mapping followed by gene sequencing in a Pakistani family with mental retardation and retinitis pigmentosa of a truncating mutation within the gene CC2D2A. Without wishing to be limiting or bound by theory, it is also believed that the symptoms in this family overlap with Joubert syndrome (MIM 213300).
Joubert syndrome, also known as Joubert-Boltshauser syndrome, JS, is a rare autsomal recessive disorder first described in a French-Canadian family (Joubert et al, 1969). JS is a clinically and genetically heterogeneous group of disorders characterized by hypoplasia of the cerebellar vermis with the hallmark neuroradiologic “molar tooth sign” (MTS), and accompanying neurologic symptoms, including abnormal breathing pattern and developmental delay. Other variable features include nystagmus, retinal dystrophy, dysmorphic facial features, hypotonia, ataxia, occipital encephalocele, renal disease, oculo colobomas, hepatic abnormalities and polydactyly. Characteristic dysmorphic facial features often noted are described as: large head, prominent forehead, high rounded eyebrows, epicanthal folds, ptosis (occasionally), upturned nose with evident nostrils, open mouth (the mouth tends to have an oval shape early on, a ‘rhomboid’ appearance later, and finally can appear triangular with downturned mouth angles), tongue protrusion and rhythmic tongue motions, and occasionally low-set and tilted ears (Maria et al, 1999). Neuroophthalmologic examination shows oculomotor apraxia.
MTS has been observed in a group of syndromes now termed Joubert syndrome and related disorder (JSRD). JSRD includes the following:

    
    
        1. classical JS (MIM213300)
        2. JS plus Leber congenital amaurosis (LCA)
        3. JS plus nephronophthisis (NPHP [MIM 256700]
        4. JS plus LCA plus NPHP (also termed cerebello-oculo-renal syndrome, CORS, MIM 608091)
        5. cerebellar vermis hypo/aplasia, oligophrenia, congenital ataxia, ocular coloboma, hepatic fibrosis (COACH [MIM 216360]) syndrome
        6. oral-facial-digital syndrome type VI (MIM 277170)

and other features such as polydactyly and encephalocele also present in each of the six subgroups.

    
    


Many genes identified for JSRD localize to cilia, and thus JSRD is thought to be a disorder of cilia function, or ciliopathy. JSRD mutations have been found in AHI1, CEP290, NPHP1, RPGRIPIL, TMEM67 and ARL13B.
In addition, another syndrome, Meckel syndrome (also known as Meckel-Gruber syndrome; MKS; MIM 249000) shares some of the clinical features of JSRD, Mutations causing MKS have been found in TMEM67, RPGRIPL1, and now in CC2D2A (Tallila et al, 2008). MKS is generally lethal.
In terms of prevalence of JS, in the United States this has been estimated as approximately 1:100 000 (Parisi et al, 1999-2006), although this is likely to be an underestimate, as the clinical signs or MRI findings are poorly recognised and diagnosed, particularly in the more mildly affected individuals (Parisi et al, 2007).
Two of the four affected members of the Pakistani family (the oldest (male, 27 yrs), and the youngest (female, 18 months) underwent full neurological examination and MRI by an independent physician. Whilst many of the JSRD features were not noted, the physician did report the presence of MTS in the MRI of the young girl, and suggested Joubert syndrome as a diagnosis, but excluded because of the lack of other features. Subsequently, all 4 affected members of the family were seen by an ophthalmologist, as the eldest male clearly had visual impairment including night blindness. All 4 showed nystagmus. The elder 3 had night blindness, and progressive retinitis pigmentosa. The youngest (just 3 years at examination) had astigmatism.
The disorder in this family appears related to Joubert syndrome, having the hallmark MTS, mental retardation, nystagmus and retinopathy (and ataxia and cerebellar atrophy, at least in the older patient examined). Thus, in an embodiment of the present invention, there is provided a method of screening a subject for Joubert Syndrome or a nucleotide sequence associated with Joubert Syndrome by assaying for a nucleic acid or protein as described herein. Early diagnosis or identifying subjects at risk for Joubert Syndrome is desirable as many of the symptoms associated with Joubert Syndrome can be treated and/or inhibited by therapeutic intervention.
All citations are hereby incorporated by reference.
The present invention has been described with regard to one or more embodiments. However, it will be apparent to persons skilled in the art that a number of variations and modifications can be made without departing from the scope of the invention as defined in the claims.
REFERENCES


    Basel-Vanagaite, L.; Attia, R.; Yahav, M.; Ferland, R. J.; Anteki, L.; Walsh, C. A.; Olender, T.; Straussberg, R.; Magal, N.; Taub, E.; Drasinover, V.; Alkelai, A.; Bercovich, D.; Rechavi, G.; Simon, A. J.; Shohat, M. (2006). The CC2D1A, a member of a new gene family with C2 domains, is involved in autosomal recessive non-syndromic mental retardation. J. Med. Genet. 43:203-210.
    Cantagrel V, Silhavy J L, Bielas S L, Swistun D, Marsh S E, Bertrand J Y, Audollent S, Attié-Bitach T, Holden K R, Dobyns W B, Traver D, Al-Gazali L, Ali B R, Lindner T H, Caspary T, Otto E A, Hildebrandt F, Glass I A, Logan C V, Johnson C A, Bennett C, Brancati F; International Joubert Syndrome Related Disorders Study Group, Valente E M, Woods C G, Gleeson J G. Mutations in the cilia gene ARL13B lead to the classical form of Joubert syndrome. Am J Hum Genet. 2008 August; 83(2):170-179.
    Chelly J, Khelfaoui M, Francis F, Cherif B, Bienvenu T (2006). Genetics and pathophysiology of mental retardation. Eur J Hum Genet 14:701-713.
    Curry C J. (2002). Rational evaluation of the adolescent with mental retardation. Adolesc Med 13:331-343, vii. Review.
    Higgins, J. J.; Pucilowska, J.; Lombardi, R. Q.; Rooney, J. P. (2004). A mutation in a novel ATP-dependent Lon protease gene in a kindred with mild mental retardation. Neurology 63:1927-1931.
    Joubert M, Eisenring J J, Robb J P, Andermann F. Familial agenesis of the cerebellar vermis. A syndrome of episodic hyperpnea, abnormal eye movements, ataxia, and retardation. Neurology. 1969 September; 19(9):813-25.
    Maria, B. L.; Boltshauser, E.; Palmer, S. C.; Tran, T. X.: Clinical features and revised diagnostic criteria in Joubert syndrome. J. Child Neurol. 14: 583-591, 1999
    Molinari, F.; Rio, M.; Meskenaite, V.; Encha-Razavi, F.; Auge, J.; Bacq, D.; Briault, S.; Vekemans, M.; Munnich, A.; Attie-Bitach, T.; Sonderegger, P.; Colleaux, L. (2002). Truncating neurotrypsin mutation in autosomal recessive nonsyndromic mental retardation. Science 298:1779-1781.
    Murphy C C Boyle C, Schendel D, Decoufié P, Yeargin-Allsopp M (1998). Epidemiology of mental retardation in children. Ment Retard Dev Diabil Res Rev 4:6-13.
    Najmabadi H, Motazacker M M, Garshasbi M, Kahrizi K, Tzschach A, Chen W, Behjati F, Hadavi V, Nieh S E, Abedini S S, Vazifehmand R, Firouzabadi S G, Jamali P, Falah M, Seifati S M, Gruters A, Lenzner S, Jensen L R, Ruschendorf F, Kuss A W, Ropers H H: Homozygosity mapping in consanguineous families reveals extreme heterogeneity of non-syndromic autosomal recessive mental retardation and identifies 8 novel gene loci. Hum Genet 2007; 121:43-48
    Noor, A.; Windpassinger, C.; Patel, M.; Stachowiak, B.; Mikhailov, A.; Azam, M.; Irfan, M.; Siddiqui, Z. K.; Naeem, F.; Paterson, A. D.; Lutfullah, M.; Vincent, J. B.; Ayub, M. “CC2D2A, encoding a coiled-coil and C2 domain protein, causes autosomal-recessive mental retardation with retinitis pigmentosa.” Am. J. Hum. Genet. 82: 1011-1018, 2008. PubMed ID: 18387594.
    Parisi M A, Glass I A: Joubert syndrome; In: GeneReviews at GeneTests-GeneClinics: Medical Genetics Information Resource [database online]. Copyright, University of Washington, Seattle. 1997-2006.
    Parisi M A, Doherty D, Chance P F, Glass I A. Joubert syndrome (and related disorders) (OMIM 213300). Eur J Hum Genet. 2007 May; 15(5):511-21.
    Shea, S. E. (2006). Mental retardation in children ages 6-16. Sem Ped Neurol 13:262-270.
    Szymanski, L., and King, B. H. (1999). Practice parameters for the assessment and treatment of children, adolescents and adults with mental retardation and comorbid mental disorders. J Am Acad Child Adolesc Psychiatry 38:5S-31S.
    Tallila, J.; Jakkula, E.; Peltonen, L.; Salonen, R.; Kestila, M.: Identification of CC2D2A as a Meckel syndrome gene adds an important piece to the ciliopathy puzzle. Am. J. Hum. Genet. 82: 1361-1367, 2008.
    Uyguner O, Kayserili H, Li Y, Karaman B, Nurnberg G, Hennies H, Becker C, Nurnberg P, Basaran S, Apak M Y, Wollnik B. 2007 A new locus for autosomal recessive non-syndromic mental retardation maps to 1p21.1-p13.3. Clin Genet 71:212-219


URLS


    Promoterinspector: http://www.genomatix.de
    BLAST: http://www.ncbi.nlm.nih.gov/BLAST
    UCSC: http://genome.ucsc.edu
    SMART: http://smart.embl-heidelberg.de
    PSORT: http://psort.ims.u-tokyo.ac.jp/cgi-bin/runpsort.pl
    SOSUI: http://sosui.proteome.bio.tuat.ac.jp/sosuiframe0.html
    COILS: http://www.ch.embnet.org/software/COILS_form.html









TABLE 1





Linkage analysis of microsatellite markers across the 4p region.


Markers shaded grey are within the homozygous critical


region shared by the four affected individuals.

## Claims

1. A method of detecting a mutation associated with Joubert Syndrome in a human subject, comprising:
assaying a biological sample obtained from a human subject using at least one primer to detect a deletion of exon 19 in an RNA transcript or cDNA derived from a CC2D2A gene in the biological sample,
wherein the assaying is performed by nucleotide sequencing,
wherein the deletion of exon 19 in the RNA transcript or cDNA derived from the CC2D2A gene indicates the presence of a mutation associated with Joubert Syndrome.

2. The method of claim 1, wherein the deletion of exon 19 in the RNA transcript or cDNA derived from the CC2D2A gene is due to a splice site mutation in exon 19.

3. The method of claim 1, wherein the encoded product of said CC2D2A gene terminates in SEQ ID NO: 1 or SEQ ID NO:2.

4. The method of claim 1, wherein the biological sample is a blood sample.

5. The method of claim 1, wherein the nucleotide sequencing comprises sequencing a DNA fragment obtained by reverse transcriptase polymeric chain reaction of the transcript or by polymerase chain reaction on the cDNA.

6. The method of claim 1, wherein the at least one primer binds to a mutant form of the CC2D2A gene.

7. The method of claim 1, wherein the at least one primer comprises SEQ ID NO:8.

8. The method of claim 1, wherein the at least one primer comprises SEQ ID NO:9.

9. A method of detecting a mutation in the CC2D2A gene comprising:
obtaining a biological sample in a human subject,
assaying the biological sample using at least one primer to detect a deletion of exon 19 in an RNA transcript or cDNA derived from a CC2D2A gene in the biological sample,
wherein the assaying is performed by nucleotide sequencing,
wherein detecting a deletion of exon 19 in the CC2D2A gene indicates the presence of a mutation in the CC2D2A gene.

10. The method of claim 9, wherein the at least one primer binds to a mutant form of the CC2D2A gene.

11. The method of claim 9, wherein the at least one primer comprises SEQ ID NO:8.

12. The method of claim 9, wherein the at least one primer comprises SEQ ID NO:9.