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Identification of gene associated with reading disability and uses therefor

Identification of gene associated with reading disability and uses therefor description/claims

The present application claims the benefit of U.S. Provisional Application No. 60/610,023, filed Sep. 14, 2004, by Jeffrey R. Gruen and Haiying Meng, entitled “DCDC2 Mutations Cause Dyslexia” and U.S. Provisional Application No. 60/685,101, filed May 26, 2005, by Jeffrey R. Gruen and Haiying Meng, entitled “DCDC2 Mutations Cause Dyslexia.” The referenced applications are incorporated herein in their entirety by reference.

This invention was made with United States government support under grant R01 NS43530, awarded by the National Institutes of Health. The United States government has certain rights in the invention.

Reading disability (RD), also known as developmental dyslexia and also known as dyslexia, is one of the most common of the complex neurobehavioral disorders, with prevalence rates ranging from 5 to 17 percent (1). It is characterized by an impairment of reading ability in subjects with normal intelligence and adequate educational opportunities. A range of neuroimaging studies, including diffusion tensor and functional magnetic resonance imaging, show that dyslexics have altered brain activation patterns compared to fluent readers when challenged with reading tasks (2). Partial remediation in language processing deficits results in improved reading, ameliorates disrupted function in brain regions associated with phonologic processing, and produces additional compensatory activation in other brain areas (3). These studies also implicate specific brain locations where genes integral to reading and language are expressed, and which likely are altered in RD.

Over the past 30 years clinical studies have shown that up to 50% of children of dyslexic parents, 50% of siblings of dyslexics, and 50% of parents of dyslexic children are affected (4). Estimates of heritability range from 44 to 75% (5). The first RD susceptibility region, DYX1, was reported on chromosome 15 in 1983 (6). Subsequently, loci were described on chromosomes 1, 2p15-16, 3p13, 6p (7-21), 6q, 7q32, 11, 15q21, and 18p11.2. It is still unclear which and/or how many genes contribute to RD and additional information would be useful for developing diagnostic, preventive and therapeutic approaches to this disorder.

The present invention relates to identification of a human gene, DCDC2 (MIM: 605755), associated with susceptibility for developing reading disability (RD), which is useful in identifying or aiding in identifying individuals at risk for developing RD, as well as for diagnosing or aiding in the diagnosis of RD. Forms of the DCDC2 gene that harbor variations that are associated with susceptibility for developing RD or lead to differences in RD are referred to, interchangeably, herein as DCDC2 variants, variant DCDC2 DNA or variant DCDC2 genes. As described in detail herein, Applicants identified an intronic polymorphic deletion of DCDC2 and alleles of dbSTS ID 808238 within the region that the deletion spans that are in significant disequilibrium with multiple RD traits. DCDC2 in which there is a deletion, such as the intronic polymorphic deletion described herein, and DCDC2 alleles that are associated with RD are examples of DCDC variants. The polymorphic deletion encodes tandem repeats of putative brain-related transcription factor binding sites in intron 2 of DCDC2. RT-PCR data show that DCDC2 localizes to the region of the brain where fluent reading occurs and RNAi studies show that down regulating DCDC2 leads to alteration in neuronal migration, again within the brain regions of interest. Results demonstrate that DCDC2 is a gene correlated with RD.

In summary, Applicants saturated the region of the genome around JA04, which led to the identification of an intronic polymorphic deletion of DCDC2. Alleles of dbSTS ID 808238 within the region that the deletion spans are in significant disequilibrium with multiple RD traits. RT-PCR data suggest that DCDC2 localizes to the region of the brain where fluent reading occurs and RNAi studies show that down regulating DCDC2 leads to alteration in neuronal migration, again within the brain regions of interest. Applicants' findings support the role of DCDC2 as a gene for harboring variations that lead to differences in RD.

Thus, the present invention relates to a human gene associated with susceptibility for developing RD, which is useful in identifying or aiding in identifying individuals at risk for developing RD, as well as for diagnosing or aiding in the diagnosis of RD. It also relates to methods for identifying or aiding in identifying individuals at risk for developing RD; methods for diagnosing or aiding in the diagnosis of RD; polynucleotides (e.g., probes, primers) useful in the methods; diagnostic kits containing such probes or primers; antibodies that bind wild type DCDC2 or altered DCDC2 gene product (e.g., protein); methods of treating or aiding in treating an individual at risk for or suffering from RD and compositions, such as pharmaceutical compositions, useful for treating an individual at risk for or suffering from RD; methods for determining appropriate and, preferably, optimal treatment for individuals, including response to educational interventions, curricula, written materials, tutoring, specialized classes and pharmaceuticals related to pharmacogenetics. The methods and compositions of the present invention can be used alone or in combination with other methods and compositions used for such purposes. For example, a method of diagnosing or aiding in the diagnosis of RD of the present invention can be used in conjunction with testing and behavioral assessments presently used for determining if an individual has RD. The methods of the present invention provide DNA (genetic) diagnostic tests useful in assessing RD in individuals, as well as in populations, such as the general population.

In one embodiment, the present invention provides polynucleotides useful for detecting or aiding in detecting, in a sample, a DCDC2 variant(s). A DCDC2 variant (also referred to as variant DCDC2 DNA or a variant DCDC2 gene) comprises at least one alteration in or difference from wild type DCDC2. The alteration or difference can be any nucleotide polymorphism of a coding region, exon, exon-intron boundary, signal peptide, 5-prime untranslated region, promoter region, enhancer sequence, 3-prime untranslated region or intron that is associated with RD. These polymorphisms include, but are not limited to, changes in the amino acid sequence of the proteins encoded by the DCDC2 gene, produce alternative splice products, create truncated products, introduce a premature stop codon, introduce a cryptic exon, alter the degree or expression to a greater or lesser extent, alter tissue specificity of DCDC2 expression, introduce changes in the tertiary structure of the proteins encoded by DCDC2, introduce changes in the binding affinity or specificity of the proteins expressed by DCDC2 or alter the function of the proteins encoded by DCDC2.

In another embodiment, the present invention provides methods and compositions useful for identifying or aiding in identifying individuals at risk for developing RD. In a further embodiment, the methods and compositions of the invention may be used for the treatment of an individual who has (is suffering from) RD or is at risk for developing RD. The invention also encompasses diagnostic kits for detecting, in a sample from an individual, variant DCDC2 DNA, such as a DCDC2 allele that is correlated with RD in humans. Such kits are useful in identifying or aiding in identifying individuals at risk for developing RD, as well as for diagnosing or aiding in the diagnosis of RD in an individual.

In one embodiment, the invention provides an isolated polynucleotide for the detection of a DCDC2 allele that is correlated with RD in humans, the polynucleotide comprising a nucleic acid molecule that specifically detects variant DCDC2 DNA that is correlated with the occurrence of RD in humans. Isolated polynucleotides are useful for detecting, in a sample from an individual, DCDC2 gene variants that are correlated with RD in humans. In certain embodiments, the isolated polynucleotide is a probe that hybridizes, under highly stringent conditions, to all or a portion of a DCDC2 gene that is correlated with the occurrence of RD in humans (all or a portion of a variant DCDC2 gene). In certain embodiments, the isolated probe hybridizes, under highly stringent conditions, to all or a portion of a DCDC2 gene that is associated with susceptibility for developing RD in humans but does not hybridize to a DCDC2 gene that is not associated with susceptibility for developing RD in humans. In further embodiments, the isolated polynucleotide is a primer that hybridizes, under highly stringent conditions, adjacent, upstream, or downstream to an alteration in a DCDC2 gene that is associated with susceptibility for developing RD in humans. Alternatively, polynucleotides of the present invention can be primers or probes that are useful to identify wild type DCDC2, wild type DCDC2 gene or wild type DCDC2 DNA, as defined herein. Such polynucleotides, for example, recognize or hybridize to all or a portion of wild type DCDC2, wild type DCDC2 gene or wild type DCDC2 DNA.

The polynucleotides described herein (e.g., a polynucleotide probe or a polynucleotide primer) may be a DNA or RNA molecule. The subject polynucleotide may be single-stranded or double-stranded. Polynucleotide probes and primers of the invention may be from about 5 nucleotides to about 3000 nucleotides. In certain embodiments, the polynucleotide probes and primers of the invention are from about 8 nucleotides to about 500 nucleotides. In further embodiments, the polynucleotide probes and primers of the invention are from about 10 to about 250 nucleotides, from about 10 to about 100 nucleotides, from about 10 to about 80 nucleotides, from about 10 to about 50 nucleotides, from about 10 to about 40 nucleotides, from about 10 to about 30 nucleotides, from about 10, 11, 12, 13 or 15 nucleotides to about 20, 21, 22, 23, 24 or 25 nucleotides. The subject polynucleotides may comprise one or more non-natural or modified nucleotides. Non-natural or modified nucleotides include, without limitation, radioactively, fluorescently, or chemically labeled nucleotides, and protein nucleic acids. Included within the scope of the present invention is any polynucleotide useful to identify or detect wild type or variant DCDC2 sequences. Based on the information provided herein, one of ordinary skill in the art can design and produce polynucleotide probes and primers using methods known in the art.

In one embodiment, the polynucleotide primer of the invention hybridizes vicinal to an alteration or difference (nucleotide polymorphism) in a DCDC2 gene that is associated with susceptibility for developing RD in humans. For example, hybridization may occur in such a manner that fewer than 10 nucleotides separate the alteration and the end of the hybridized primer proximal to the alteration. In specific embodiments, hybridization occurs in such a manner that 1-3 nucleotides separate the alteration and the end of the hybridized primer proximal to the alteration. In certain embodiments, the polynucleotide primer hybridizes immediately adjacent to the alteration. In another embodiment, the polynucleotide primer of the invention hybridizes upstream or downstream from an alteration in the DCDC2 gene that is correlated with the occurrence of RD in humans. For example, hybridization may occur in such a manner that the end of the hybridized primer proximal to the alteration is 10, 25, 50, 100, 250, 1000, 5000, or up to 10,000 nucleotides upstream or downstream from an alteration in the DCDC2 gene. The invention described herein also provides a pair of polynucleotide primers that specifically detect a mutation in the DCDC2 gene that is correlated with the occurrence of RD in humans, wherein the first polynucleotide primer hybridizes to one side of an alteration (e.g., one side of the deletion described herein, such as the 5-prime side) and the second polynucleotide primer hybridizes to the other side of the alteration (e.g., the other side of the deletion described herein, such as the 3 prime side). A pair of polynucleotide primers that hybridize to a region of DNA that comprises an alteration in the DCDC2 gene that is associated with susceptibility for developing RD in humans may hybridize to the region in such a manner that the ends of the hybridized primers proximal to the alteration are from about 20 to about 10,000 nucleotides apart.

Variants of the DCDC2 gene that predispose an individual to RD may be detected by the methods and compositions described herein. In particular embodiments, variant alleles, such as those depicted in Supplementary Table 3 may be detected. As used herein, the terms “wild type DCDC2”, wild type DCDC2 gene” and “wild type DCDC2 DNA” refer to DNA that is not associated with susceptibility for developing RD in humans.

In certain aspects, the invention provides a method of detecting, in a sample obtained from an individual, a DCDC2 allele that is associated with susceptibility for developing RD in humans. Such a method may comprise: (a) combining the sample with a polynucleotide probe that hybridizes, under highly stringent conditions, to a DCDC2 allele that is correlated with RD in humans, but does not hybridize to a DCDC2 gene that is not associated with susceptibility for developing RD in humans and (b) determining whether hybridization occurs. The occurrence of hybridization indicates that a DCDC2 gene that is associated with susceptibility for developing RD in humans is present in the sample. Alternatively, the method may comprise: (a) combining the sample with a polynucleotide probe that uses the polymerase chain reaction to amplify, under stringent conditions, a DCDC2 allele that is associated with susceptibility for developing RD in humans, and (b) sequencing the allele, such as by conventional fluorescent tagged dideoxy terminator sequencing, wherein if the allele comprises the sequence of variant DCDC2 DNA, a DCDC2 allele that is associated with susceptibility for developing RD in humans is present in the sample.

Samples used in the methods described herein may comprise cells from the eye, epidermis, epithelium, blood, tears, saliva, mucus, urine, stool, sperm, ova, or any other tissues or bodily fluids from which sufficient DNA or RNA can be obtained. In a specific embodiment, cells obtained from a buccal swab are used. The sample should be sufficiently processed to render DNA or RNA present available for assaying in the methods described herein. For example, samples may be processed such that DNA from the sample is available for amplification by DNA polymerases or other enzymes that increase the total DNA content or for hybridization to another polynucleotide. The processed samples may be crude lysates where available DNA or RNA is not purified from other cellular material, or may be purified to specifically isolate DNA or RNA. Samples may be processed by any means known in the art that renders DNA or RNA available for assaying in the methods described herein. Methods for processing samples may include, without limitation, mechanical, chemical, enzymatic, or molecular means of lysing and/or purifying cells and cell lysates. Processing methods may include chromatographic methods such as ion exchange (e.g., cation and anion), size exclusion, affinity, and hydrophobic interaction chromatography.

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