Assays for Detecting WDR62 Mutations

Description

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. application Ser. No. 13/807,425, filed Jun. 17, 2013, which is the U.S. national phase application filed under 35 U.S.C. §371 claiming benefit to International Patent Application No. PCT/US2011/042647, filed on Jun. 30, 2011, which is entitled to priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 61/398,815, filed on Jul. 1, 2010, each of which application is hereby incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

The development of the human cerebral cortex is an orchestrated process involving the generation of neural progenitors in the periventricular germinal zones, cell proliferation characterized by symmetric and asymmetric mitoses, followed by migration of post-mitotic neurons to their final destinations in six highly ordered, functionally specialized layers (2008, Bystron et al., Nature Rev. Neurosci. 9:110-122; 2009, Rakic, Nature Rev. Neurosci. 10:724-735). An understanding of the molecular mechanisms guiding these intricate processes is in its infancy, substantially driven by the discovery of rare mutations that cause malformations of cortical development (2008, Guerrini et al., Trends Neurosci. 31:154-162; 2005, Guerrini, Epilepsia 46(suppl. 1):32-37; 2001, Guerrini and Carrozzo, Am. J. Med. Genet. 106:160-173; 2001, Mochida and Walsh, Curr. Opin. Neurol. 14:151-1563). Mapping of disease loci in putative Mendelian forms of malformations of cortical development has been hindered by marked locus heterogeneity, small kindred sizes and diagnostic classifications that may not reflect molecular pathogenesis.

Malformations of cortical development are a diverse group of often devastating structural brain disorders reflecting deranged neuronal proliferation, migration or organization. Application of traditional mapping approaches have proved to be particularly challenging for gene discovery in these syndromes, where kindreds with a single affected member are most common, linkage studies support high locus heterogeneity and recent genetic findings have fundamentally challenged previous diagnostic nosology (2008, Guerrini et al., Trends Neurosci. 31:154-162; 2001, Barkovich et al., Neurology 57:2168-2178; 2005, Barkovich et al., Neurology 65:1873-1887). Whole-exome sequencing using next generation platforms (2009, Choi et al., Proc. Natl Acad. Sci. USA 106:19096-19101; 2010, Ng et al., Nature Genet. 42:30-35; 2009, Ng et al., Nature 461:272-276) can markedly improve gene discovery efforts in these situations.

There is a need in the art for assays for detecting recessive mutations in genes involved in cortical development in both carrier subjects and affected subjects. The present invention addresses this need in the art.

SUMMARY OF THE INVENTION

The present invention relates to the discovery that recessive mutations in WD repeat domain 62 (WDR62) are involved in a wide spectrum of neurological diseases and disorders. In one embodiment, the invention is a method of determining whether a subject has a mutation in at least one allele of WDR62. In various embodiments, the method includes the steps of: obtaining a test sample from the subject, where the test sample comprises a WDR62 nucleic acid or a fragment thereof; comparing the WDR62 nucleic acid sequence in the test sample with a control WDR62 nucleic acid sequence, where when the WDR62 nucleic acid sequence in the test sample differs from the control WDR62 nucleic acid sequence, the subject is determined to have a WDR62 mutation in at least one allele of WDR62.

The mutation detected can be any mutation of WDR62 and includes the following mutations: W224S relative to SEQ ID NO:2; Q470X relative to SEQ ID NO:2; E526K relative to SEQ ID NO:2; E526X relative to SEQ ID NO:2; a 4-bp deletion (TGCC) in exon 31 beginning at codon 1402, leading to a premature stop codon at codon 1413 (V1402GfsX12); a nonsense mutation; a missense mutation; and and a 17-bp deletion in exon 30 leading to a frameshift at codon 1280 resulting in a premature termination codon following a novel peptide of 20 amino acids (G1280AfsX21).

In a preferred embodiment, the subject is a human. In various embodiments, the subject is a fetus, a child, an adolescent, an adult, a parent or a prospective parent. In some embodiments, the subject is a carrier subject having at least one mutation in only one allele of WDR62 and in other embodiments the subject is an affected subject having at least one mutation on each allele of WDR62.

In various embodiments, the affected subject has at least one neurological disease or disorder, including, but not limited to, intellectual disability, cerebral cortical malformation, microcephaly, agyria, pachygria, hypoplasia of the corpus callosum, lissencephaly, schizencephaly, polymicrogyria and cerebellar hypoplasia.

In some embodiments, assessment of the test sample involves the use of at least one of PCR, Northern analysis, Southern analysis, DNA array analysis, and direct sequence analysis. In one embodiment, the test sample from the subject comprises genomic DNA. In another embodiment, the test sample comprises chromosome 19 or a fragment thereof comprising 19q13.12.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of preferred embodiments of the invention will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

FIG. 1, comprising FIG. 1A-FIG. 1F, depicts the results of experiments identifying a 4-bp deletion in the WDR62 gene in a family with microcephaly and pachygria. FIG. 1A-FIG. 1D, Coronal (FIG. 1A) and axial (FIG. 1C) magnetic resonance images of a control subject compared with NG 26-1 (FIG. 1B, FIG. 1D) confirms the clinical diagnosis of microcephaly and shows a diffusely thickened cortex, an indistinct grey-white junction, pachgyria and underoperculization. All images are T2 weighted (photographically inverted). Scale bars, centimetres. FIG. 1E, a 4-bp deletion (red box) in the WDR62 is identified through exome sequencing (WT, wild type). FIG. 1F, Sanger sequencing confirms the deleted bases (in green). The altered amino-acid sequence (starting at position 1,402) leading to a premature stop-codon (X) is shown in red.

FIG. 2, comprising FIG. 2A-FIG. 2F, depicts additional WDR62 mutations. FIG. 2A-FIG. 2F, Pedigree structures with mutated bases (red) and the corresponding normal alleles (green) are marked on the chromatograms (left, mutant; right, wild type). FIG. 2A, Families NG 26 and NG 891 harbour the identical 4-bp deletion, whereas nonsense mutations leading to premature stop codons (X) are observed in NG 30 (FIG. 2B) and NG 294 (FIG. 2D). Missense mutations affecting conserved amino acids are seen in NG 190 (FIG. 2C) and NG 537 (FIG. 2F). In NG 339 (FIG. 2E), a 17-bp deletion leads to a premature stop codon. FIG. 2G, The locations of independent mutations are indicated on the genomic organization of WDR62.

FIG. 3, comprising FIG. 3A-FIG. 3L, depicts representative magnetic resonance images from patients demonstrating the wide spectrum of findings associated with mutations in WDR62. FIG. 3A, FIG. 3E, FIG. 3I, FIG. 3K, Axial (FIG. 3A), coronal (FIG. 3E), sagittal (FIG. 3K) MRI images and three-dimensional surface rendering (FIG. 3I) of a control subject are shown. FIG. 3B, Microlissencephalic features with microcephaly, diffusely thickened cortex, loss of grey-white junction and pachygyria. FIG. 3C, asymmetric microcephalic hemispheres with marked polymicrogyria (arrowheads). FIG. 3D, significant polymicrogyria (black arrowheads) and open-lip schizencephaly (red arrowhead). f, Unilateral cerebellar hypoplasia (arrowhead). FIG. 3G, Open-lip schizencephaly (red arrowhead) and the polymicrogyric cortex. FIG. 3H, unilateral brainstem atrophy (arrow). FIG. 3J, Three-dimensional surface rendering demonstrating craniofacial dysmorphology. FIG. 3L, microcephaly, pachygyria and abnormally shaped corpus callosum (arrowheads).

FIG. 4, comprising FIG. 4A-FIG. 4E, depicts the results of studies evaluating Wdr62 expression in the developing mouse brain. FIG. 4A, Wdr62 expression is enriched in the ventricular and subventricular zones (VZ and SVZ, respectively) as seen with in situ hybridization. FIG. 4B, WDR62 protein (red) distribution reveals a similar pattern. CP, cortical plate. FIG. 4C, FIG. 4D, WDR62 (red) localizes to the nuclei and is expressed by neural stem cells and intermediate progenitors, as marked by SOX2 and TBR2 expression (green), respectively. FIG. 4E, immunofluorescent staining for α-tubulin (cytoplasmic, blue), SOX2 (nuclear, green) and WDR62 (red) in E12.5 cortical neural progenitor cells reveals that the distribution of the WDR62 overlaps with that of SOX2 and is predominantly nuclear. (Nuclear staining by 4′,6-diamidino-2-phenylindole (DAPI) (blue) in FIG. 4B-FIG. 4D; rightmost panels are composite images in FIG. 4B-FIG. 4E).

FIG. 5 depicts two novel homozygous missense variants identified within the shared homozygosity regions of affected subjects of family NG 26. Left panel: On chromosome 12, position 130,807,115, an A to G transition results in a Thr to Ala substitution in the non-conserved 565th residue (marked with the red box) of Splicing Factor, Arginine/Serine-rich 8 (SFRS8). Right panel: Similarly, in IBTK (Inhibitor of Bruton Agammaglobulinemia Tyrosine Kinase), a G to C substitution on chromosome 6, position 82,980,721, results in a Pro to Ala substitution of the 716th residue of the protein (red box) which is not highly conserved across 44 vertebrate species (from www.genome.ucsc.edu: Kent W J, et al., Genome Res. 2002 June; 12(6): 996-1006.)

FIG. 6 depicts sequence traces of 7 families with WDR62 mutations. From left to right, the panels show the DNA sequences of the patients, control subjects, and the patients' parents, respectively. The predicted amino acids corresponding to each codon are represented above the nucleotide sequences, which are marked in bold letters above the chromatograms. For each sequence, the mutated base(s) are shown in red, as are resultant amino acid substitutions. For the wild type sequences, the altered bases are shown in green. Note that all patients are homozygous for the mutations while both parents are heterozygous. The following mutations are observed: (FIG. 6A) NG 26: V1402GfsX12, (FIG. 6B) NG 30: E526X, (FIG. 6C) NG 190: W224S, (FIG. 6D) NG 294: Q470X, (FIG. 6E) NG 339: G1280AfsX21, (FIG. 6F) NG 537: E526K, (FIG. 6G) NG 891: V1402GfsX12.

FIG. 7 depicts a sequence alignment of vertebrate WDR62 proteins. Missense mutations identified in NG 190 (W224S) and NG 537 (E526K) alter two highly conserved amino acid residues across 44 vertebrates (with the exception of Stickleback for position 526) (see www.genome.ucsc.edu; Kent, et al., 2002 June; 12(6):996-1006).

FIG. 8, comprising FIG. 8A-FIG. 8B depicts genome-wide linkage analysis of pedigree NG 190 with three affected and one unaffected family members. For linkage analysis, an autosomal recessive mode of inheritance with a phenocopy rate of 0.001, penetrance rates of 0.001 and 0.99 for heterozygous and homozygous conditions, respectively, were assumed. Disease causing allele frequency was set to 0.001. The vertical axis corresponds to LOD score and the horizontal axis shows genetic distance in centimorgans (cM). The linkage graphs for all autosomal chromosomes are shown. The maximum LOD score for the chromosome 19 locus spanning the WDR62 gene, located between 41,237,623 and 41,287,852 base pairs is 3.64. The LOD-3 interval is marked by markers rs3855681 and rs7359950, located at 16,259,314 (40.1 cM) and 44,455,605 (63.8 cM) base pairs, respectively.

FIG. 9, comprising FIG. 9A-FIG. 9I, depicts Wdr62 expression in the developing mouse brain. FIG. 9A-FIG. 9C, whole mount in situ hybridization at E9.5 to E11.5. Lateral (panels A and C) and frontal views (panel B) are shown. Wdr62 mRNA is detected in the telencephalon (tel), diencephalon (di), mesencephalon (mes), metencephalon (met), branchial arch (ba), nasal process (np), forelimb bud (flb), hindlimb bud (hlb), somites (s) and tail (t). FIG. 9D-FIG. 9F, coronal sections of the developing forebrain at three different rostrocaudal levels at embryonic day 14.5 (E14.5) are shown. Wdr62 expression is detected in proliferating neuronal progenitors in the neuroepithelium of the neocortex (nctx), striatum (str) and septum (se). FIG. 9G-FIG. 9H, sagittal (FIG. 9G) and axial (FIG. 9H) sections of the cerebellum at E17.5 and postnatal day 7 (P7) mouse. Wdr62 mRNA is detected in proliferating granule neuron precursors in the external granular layer (egl) of the developing cerebellum (cb). Expression is absent from the inferior colliculus (ic) and the medulla (med). FIG. 9I, coronal section of the forebrain of P21 mouse. Wdr62 mRNA expression is detected at low levels in the hippocampus (hip) and piriform cortex (pir). Scale bars: 0.2 mm (FIG. 9A, FIG. 9G to FIG. 9I), 0.5 mm (panels B to F).

FIG. 10, comprising FIG. 10A-FIG. 10B, depicts WDR62 expression in the developing human brain. FIG. 10A, immunohistochemical staining in 20 weeks of gestation human fetal neocortex: In the developing human brain, similar to the mouse brain, WDR62 is enriched in the ventricular and subventricular zones (VZ and SVZ, respectively) and weakly present in the cortical plate (CP) (scale bar: 200 μm). iVZ: inner ventricular zone; oSVZ: outer subventricular zone; IZ: intermediate zone; SP: subplate; MZ: marginal zone. FIG. 10B, in VZ cells near the ventricular surface, WDR62 is localized to nuclei (arrows) (scale bar: 5 μm).

FIG. 11, comprising FIG. 11A-FIG. 11B, depicts the results of experiments assessing localization. Immunofluorescent staining for α-tubulin (green), WDR62 (red, using mouse anti-WDR62 (Sigma-Aldrich) antibody), and counterstaining by DAPI (blue) in cultured E12.5 mouse cortical neural progenitors, HeLa, Neuro2a, and HEK-293FT cells. WDR62 localization is nuclear in cortical neural progenitors, HeLa, and Neuro2a cells. In HEK-293FT cells, WDR62 is localized to granules (arrows) as previously described by Wasserman et al, 2010 (Mol Bio Cell, 21:117-130, 2010). Scale bar: 10 μm. FIG. 11B, subcellular fractionation of mouse E14.5 neocortex and immunoblotting for WDR62 (using rabbit anti-WDR62 antibody, Novus) reveal that WDR62 is present in the nuclear fraction.

FIG. 12, comprising FIG. 12A-FIG. 12B, depicts the results of experiments using immunofluorescent staining of E15.5 mouse neocortex electroporated in utero with CAG-GFP at E13.5. FIG. 12A, in a GFP-filled VZ cell positioned away from the ventricular surface, WDR62 localization is nuclear and does not overlap with the single centrosome (arrow) marked by γ-tubulin (blue). Radial glial endfoot staining of γ-tubulin is indicated (asterisk). FIG. 12B, in a GFP-filled mitotic cell in metaphase at the ventricular surface, WDR62 is relocalized to the cytoplasm upon chromatin condensation and the breakdown of the nuclear membrane and does not apparently co-localize with the two centrosomes (arrows) marked by γ-tubulin. Scale bar: 5 μm in FIG. 12A, FIG. 12B. Composite images are shown as the right most images.

DETAILED DESCRIPTION

The present invention relates to the discovery that recessive mutations in WD repeat domain 62 (WDR62) are involved in a wide spectrum of neurological diseases and disorders, including, but not limited to, intellectual disability, cerebral cortical malformations, microcephaly, agyria, pachygria, hypoplasia of the corpus callosum, lissencephaly, schizencephaly, polymicrogyria and cerebellar hypoplasia. In various embodiments, the invention relates to a genetic screening assay of a subject to determine whether the subject has a mutation in at least one allele of WDR62. In some embodiments, the subject is a parent. In other embodiments, the subject is a prospective parent. In another embodiment, the subject is child. In a further embodiment, the subject is a fetus.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described.

As used herein, each of the following terms has the meaning associated with it in this section.

The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

“About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

The term “abnormal” when used in the context of organisms, tissues, cells or components thereof, refers to those organisms, tissues, cells or components thereof that differ in at least one observable or detectable characteristic (e.g., age, treatment, time of day, etc.) from those organisms, tissues, cells or components thereof that display the “normal” (expected) respective characteristic. Characteristics which are normal or expected for one cell or tissue type, might be abnormal for a different cell or tissue type.

As used herein the terms “defect,” “alteration,” “variation,” or “mutation,” refers to a mutation in WDR62 that affects the function, activity, expression (transcription or translation) or conformation of the polypeptide that it encodes. Mutations encompassed by the present invention can be any mutation of WDR62 gene that results in the disruption of the function, activity, expression or conformation of the encoded polypeptide, including the complete absence of expression of the encoded protein and can include, for example, missense and nonsense mutations, insertions, deletions, frameshifts and premature terminations. Without being so limited, mutations encompassed by the present invention may alter splicing the mRNA (splice site mutation) or cause a shift in the reading frame (frameshift).

As used herein, the term “control nucleic acid” is meant to refer to a nucleic acid sample (e.g., RNA, DNA) that does not come from a subject known to have a mutation in WDR62 (control subject). For example, the control can be a wild type WDR62 nucleic acid sequence which does not contain a variation in its nucleic acid sequence. Also, as used herein, a control can be a fragment or portion of WDR62 that does not include the defect/variation that is the mutation of interest (that is, the mutation to be detected in an assay).

The term, “fragment,” as used herein, indicates that the portion of the gene, DNA, mRNA or cDNA is a polynucleotide of a length that is sufficient to identify it as a fragment of WDR62. In one representative embodiment, a fragment comprises one or more exons of the WDR62 gene. In another representative embodiment, a fragment comprises part of an exon of the WDR62 gene. In some embodiments, the fragment can also include an intron/exon junction of the WDR62 gene.

As used herein, “homologous” refers to the subunit sequence similarity between two polymeric molecules, e.g., between two nucleic acid molecules, e.g., two DNA molecules or two RNA molecules, or between two polypeptide molecules. When a subunit position in both of the two molecules is occupied by the same monomeric subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then they are homologous at that position. The homology between two sequences is a direct function of the number of matching or homologous positions, e.g., if half (e.g., five positions in a polymer ten subunits in length) of the positions in two compound sequences are homologous then the two sequences are 50% homologous, if 90% of the positions, e.g., 9 of 10, are matched or homologous, the two sequences share 90% homology. By way of example, the DNA sequences 3′ATTGCC5′ and 3′TATGGC share 50% homology.

As used herein, “homology” is used synonymously with “identity.” In addition, when the term “homology” is used herein to refer to the nucleic acids and proteins, it should be construed to be applied to homology at both the nucleic acid and the amino acid levels. The determination of percent identity between two nucleotide or amino acid sequences can be accomplished using a mathematical algorithm. For example, a mathematical algorithm useful for comparing two sequences is the algorithm of Karlin and Altschul (1990, Proc. Natl. Acad. Sci. USA 87:2264-2268), modified as in Karlin and Altschul (1993, Proc. Natl. Acad. Sci. USA 90:5873-5877). This algorithm is incorporated into the NBLAST and XBLAST programs of Altschul, et al. (1990, J. Mol. Biol. 215:403-410), and can be accessed, for example, at the National Center for Biotechnology Information (NCBI) world wide web site having the universal resource locator www.ncbi.nlm.nih.gov/BLAST/. BLAST nucleotide searches can be performed with the NBLAST program (designated “blastn” at the NCBI web site), using the following parameters: gap penalty=5; gap extension penalty=2; mismatch penalty=3; match reward=1; expectation value 10.0; and word size=11 to obtain nucleotide sequences homologous to a nucleic acid described herein. BLAST protein searches can be performed with the XBLAST program (designated “blastn” at the NCBI web site) or the NCBI “blastp” program, using the following parameters: expectation value 10.0, BLOSUM62 scoring matrix to obtain amino acid sequences homologous to a protein molecule described herein.

To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (1997, Nucleic Acids Res. 25:3389-3402). Alternatively, PSI-Blast or PHI-Blast can be used to perform an iterated search which detects distant relationships between molecules (id.) and relationships between molecules which share a common pattern. When utilizing BLAST, Gapped BLAST, PSI-Blast, and PHI-Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See www.ncbi.nlm.nih.gov. The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically exact matches are counted.

As used herein a “probe” is defined as a nucleic acid capable of binding to a target nucleic acid of complementary sequence through one or more types of chemical bonds, usually through complementary base pairing, usually through hydrogen bond formation. As used herein, a probe may include natural (i.e. A, G, U, C, or T) or modified bases (7-deazaguanosine, inosine, etc.). In addition, a linkage other than a phosphodiester bond may join the bases in probes, so long as it does not interfere with hybridization. Thus, probes may be peptide nucleic acids in which the constituent bases are joined by peptide bonds rather than phosphodiester linkages.

The term “match,” “perfect match,” “perfect match probe” or “perfect match control” refers to a nucleic acid that has a sequence that is perfectly complementary to a particular target sequence. The nucleic acid is typically perfectly complementary to a portion (subsequence) of the target sequence. A perfect match (PM) probe can be a “test probe”, a “normalization control” probe, an expression level control probe and the like. A perfect match control or perfect match is, however, distinguished from a “mismatch” or “mismatch probe.”

The term “mismatch,” “mismatch control” or “mismatch probe” refers to a nucleic acid whose sequence is not perfectly complementary to a particular target sequence. As a non-limiting example, for each mismatch (MM) control in a high-density probe array there typically exists a corresponding perfect match (PM) probe that is perfectly complementary to the same particular target sequence. The mismatch may comprise one or more bases. While the mismatch(es) may be located anywhere in the mismatch probe, terminal mismatches are less desirable because a terminal mismatch is less likely to prevent hybridization of the target sequence. In a particularly preferred embodiment, the mismatch is located at or near the center of the probe such that the mismatch is most likely to destabilize the duplex with the target sequence under the test hybridization conditions.

A homo-mismatch substitutes an adenine (A) for a thymine (T) and vice versa and a guanine (G) for a cytosine (C) and vice versa. For example, if the target sequence was: AGGTCCA, a probe designed with a single homo-mismatch at the central, or fourth position, would result in the following sequence: TCCTGGT.

In one embodiment, pairs are present in perfect match and mismatch pairs, one probe in each pair being a perfect match to the target sequence and the other probe being identical to the perfect match probe except that the central base is a homo-mismatch. Mismatch probes provide a control for non-specific binding or cross-hybridization to a nucleic acid in the sample other than the target to which the probe is directed. Thus, mismatch probes indicate whether hybridization is or is not specific. For example, if the target is present, the perfect match probes should be consistently brighter than the mismatch probes because fluorescence intensity, or brightness, corresponds to binding affinity. (See e.g., U.S. Pat. No. 5,324,633, which is incorporated herein for all purposes.) Finally, the difference in intensity between the perfect match and the mismatch probe (I(PM)−I(MM)) provides a good measure of the concentration of the hybridized material. See PCT No WO 98/11223, which is incorporated herein by reference for all purposes.

Nucleic acids according to the present invention may include any polymer or oligomer of pyrimidine and purine bases, preferably cytosine, thymine, and uracil, and adenine and guanine, respectively. (See Albert L. Lehninger, Principles of Biochemistry, at 793-800 (Worth Pub. 1982) which is herein incorporated in its entirety for all purposes). Indeed, the present invention contemplates any deoxyribonucleotide, ribonucleotide or peptide nucleic acid component, and any chemical variants thereof, such as methylated, hydroxymethylated or glucosylated forms of these bases, and the like. The polymers or oligomers may be heterogeneous or homogeneous in composition, and may be isolated from naturally occurring sources or may be artificially or synthetically produced. In addition, the nucleic acids may be DNA or RNA, or a mixture thereof, and may exist permanently or transitionally in single-stranded or double-stranded form, including homoduplex, heteroduplex, and hybrid states.

An “oligonucleotide” or “polynucleotide” is a nucleic acid ranging from at least 2, preferably at least 8, 15 or 25 nucleotides in length, but may be up to 50, 100, 1000, or 5000 nucleotides long or a compound that specifically hybridizes to a polynucleotide. Polynucleotides include sequences of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) or mimetics thereof which may be isolated from natural sources, recombinantly produced or artificially synthesized. A further example of a polynucleotide of the present invention may be a peptide nucleic acid (PNA). (See U.S. Pat. No. 6,156,501 which is hereby incorporated by reference in its entirety.) The invention also encompasses situations in which there is a nontraditional base pairing such as Hoogsteen base pairing which has been identified in certain tRNA molecules and postulated to exist in a triple helix. “Polynucleotide” and “oligonucleotide” are used interchangeably in this disclosure.

A “genome” is all the genetic material of an organism. In some instances, the term genome may refer to the chromosomal DNA. Genome may be multichromosomal such that the DNA is cellularly distributed among a plurality of individual chromosomes. For example, in human there are 22 pairs of chromosomes plus a gender associated XX or XY pair. DNA derived from the genetic material in the chromosomes of a particular organism is genomic DNA. The term genome may also refer to genetic materials from organisms that do not have chromosomal structure. In addition, the term genome may refer to mitochondria DNA. A genomic library is a collection of DNA fragments representing the whole or a portion of a genome. Frequently, a genomic library is a collection of clones made from a set of randomly generated, sometimes overlapping DNA fragments representing the entire genome or a portion of the genome of an organism.

The term “chromosome” refers to the heredity-bearing gene carrier of a cell which is derived from chromatin and which comprises DNA and protein components (especially histones). The conventional internationally recognized individual human genome chromosome numbering system is employed herein. The size of an individual chromosome can vary from one type to another within a given multi-chromosomal genome and from one genome to another. In the case of the human genome, the entire DNA mass of a given chromosome is usually greater than about 100,000,000 bp. For example, the size of the entire human genome is about 3×10⁹ bp. The largest chromosome, chromosome no. 1, contains about 2×10⁸ by while the smallest chromosome, chromosome no. 22, contains about 5.3×10⁷ bp.

A “chromosomal region” is a portion of a chromosome. The actual physical size or extent of any individual chromosomal region can vary greatly. The term “region” is not necessarily definitive of a particular one or more genes because a region need not take into specific account the particular coding segments (exons) of an individual gene.

An “allele” refers to one specific form of a genetic sequence (such as a gene) within a cell, an individual or within a population, the specific form differing from other forms of the same gene in the sequence of at least one, and frequently more than one, variant sites within the sequence of the gene. The sequences at these variant sites that differ between different alleles are termed “variants”, “polymorphisms”, or “mutations.”

A “disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal's health continues to deteriorate.

In contrast, a “disorder” in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal's state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal's state of health.

As used herein the term “isolated,” such as in the expression “isolated nucleic acid” or “isolated polypeptide” means altered “by the hand of man” from its natural state (i.e. if it occurs in nature, it has been changed or removed from its ordinary context) or it has been synthesized in a non-natural environment (e.g., artificially synthesized). These terms do not require absolute purity (such as a homogeneous preparation). For example, a protein/peptide naturally present in a living organism is not “isolated”, but the same protein separated from the coexisting materials of its natural state is “isolated” as this term is employed herein.

As used herein, an “instructional material” includes a publication, a recording, a diagram, or any other medium of expression which can be used to communicate the usefulness of a compound, composition, vector, or delivery system of the invention in the kit for effecting alleviation of the various diseases or disorders recited herein. Optionally, or alternately, the instructional material can describe one or more methods of alleviating the diseases or disorders in a cell or a tissue of a mammal. The instructional material of the kit of the invention can, for example, be affixed to a container which contains the identified compound, composition, vector, or delivery system of the invention or be shipped together with a container which contains the identified compound, composition, vector, or delivery system. Alternatively, the instructional material can be shipped separately from the container with the intention that the instructional material and the compound be used cooperatively by the recipient.

The terms “array” and “microarray” refers broadly to both “DNA microarrays” and “DNA chip(s),” and encompasses all art-recognized solid supports, and all art-recognized methods for affixing nucleic acid molecules thereto or for synthesis of nucleic acids thereon. Preferred arrays typically comprise a plurality of different nucleic acid probes that are coupled to a surface of a substrate in different, known locations. These arrays, also described as “microarrays” or colloquially “chips” have been generally described in the art, for example, U.S. Pat. Nos. 5,143,854, 5,445,934, 5,744,305, 5,677,195, 5,800,992, 6,040,193, 5,424,186 and Fodor et al., 1991, Science, 251:767-777, each of which is incorporated by reference in its entirety for all purposes. Arrays may generally be produced using a variety of techniques, such as mechanical synthesis methods or light directed synthesis methods that incorporate a combination of photolithographic methods and solid phase synthesis methods. Techniques for the synthesis of these arrays using mechanical synthesis methods are described in, e.g., U.S. Pat. Nos. 5,384,261, and 6,040,193, which are incorporated herein by reference in their entirety for all purposes. Although a planar array surface is preferred, the array may be fabricated on a surface of virtually any shape or even a multiplicity of surfaces. Arrays may be nucleic acids on beads, gels, polymeric surfaces, fibers such as fiber optics, glass or any other appropriate substrate. (See U.S. Pat. Nos. 5,770,358, 5,789,162, 5,708,153, 6,040,193 and 5,800,992, which are hereby incorporated by reference in their entirety for all purposes.)

Assays for amplification of the known sequence are also disclosed. For example primers for PCR may be designed to amplify regions of the sequence. For RNA, a first reverse transcriptase step may be used to generate double stranded DNA from the single stranded RNA. The array may be designed to detect sequences from an entire genome; or one or more regions of a genome, for example, selected regions of a genome such as those coding for a protein or RNA of interest; or a conserved region from multiple genomes; or multiple genomes, Arrays and methods of genetic analysis using arrays is described in Cutler, et al., 2001, Genome Res. 11(11): 1913-1925 and Warrington, et al., 2002, Hum Mutat 19:402-409 and in US Patent Pub No 20030124539, each of which is incorporated herein by reference in its entirety.

Arrays may be packaged in such a manner as to allow for diagnostic use or can be an all-inclusive device; e.g., U.S. Pat. Nos. 5,856,174 and 5,922,591 incorporated in their entirety by reference for all purposes. Arrays are commercially available from, for example, Affymetrix (Santa Clara, Calif.) and Applied Biosystems (Foster City, Calif.), and are directed to a variety of purposes, including genotyping, diagnostics, mutation analysis, marker expression, and gene expression monitoring for a variety of eukaryotic and prokaryotic organisms. The number of probes on a solid support may be varied by changing the size of the individual features. In one embodiment the feature size is 20 by 25 microns square, in other embodiments features may be, for example, 8 by 8, 5 by 5 or 3 by 3 microns square, resulting in about 2,600,000, 6,600,000 or 18,000,000 individual probe features.

Hybridization “probes” are oligonucleotides capable of binding in a base-specific manner to a complementary strand of nucleic acid. Such probes include peptide nucleic acids, as described in Nielsen et al., 1991, Science 254, 1497-1500, and other nucleic acid analogs and nucleic acid mimetics. See U.S. Pat. No. 6,156,501.

The term “hybridization” refers to the process in which two single-stranded nucleic acids bind non-covalently to form a double-stranded nucleic acid; triple-stranded hybridization is also theoretically possible. Complementary sequences in the nucleic acids pair with each other to form a double helix. The resulting double-stranded nucleic acid is a “hybrid.” Hybridization may be between, for example tow complementary or partially complementary sequences. The hybrid may have double-stranded regions and single stranded regions. The hybrid may be, for example, DNA:DNA, RNA:DNA or DNA:RNA. Hybrids may also be formed between modified nucleic acids. One or both of the nucleic acids may be immobilized on a solid support. Hybridization techniques may be used to detect and isolate specific sequences, measure homology, or define other characteristics of one or both strands.

The stability of a hybrid depends on a variety of factors including the length of complementarity, the presence of mismatches within the complementary region, the temperature and the concentration of salt in the reaction. Hybridizations are usually performed under stringent conditions, for example, at a salt concentration of no more than 1 M and a temperature of at least 25° C. For example, conditions of 5×SSPE (750 mM NaCl, 50 mM NaPhosphate, 5 mM EDTA, pH 7.4) or 100 mM MES, 1 M Na, 20 mM EDTA, 0.01% Tween-20 and a temperature of 25-50° C. are suitable for allele-specific probe hybridizations. In a particularly preferred embodiment, hybridizations are performed at 40-50° C. Acetylated BSA and herring sperm DNA may be added to hybridization reactions.

The term “label” as used herein refers to a luminescent label, a light scattering label or a radioactive label. Fluorescent labels include, but are not limited to, the commercially available fluorescein phosphoramidites such as Fluoreprime (Pharmacia), Fluoredite (Millipore) and FAM (ABI). See U.S. Pat. No. 6,287,778.

The term “solid support,” “support,” and “substrate” as used herein are used interchangeably and refer to a material or group of materials having a rigid or semi-rigid surface or surfaces. In one embodiment, at least one surface of the solid support will be substantially flat, although in some embodiments it may be desirable to physically separate synthesis regions for different compounds with, for example, wells, raised regions, pins, etched trenches, or the like. According to other embodiments, the solid support(s) will take the form of beads, resins, gels, microspheres, or other geometric configurations. See U.S. Pat. No. 5,744,305 for exemplary substrates.

The term “target” as used herein refers to a molecule that has an affinity for a given probe. Targets may be naturally-occurring or man-made molecules. Also, they can be employed in their unaltered state or as aggregates with other species. Targets may be attached, covalently or noncovalently, to a binding member, either directly or via a specific binding substance. Targets are sometimes referred to in the art as anti-probes. As the term targets is used herein, no difference in meaning is intended.

A “probe target pair” is formed when two macromolecules have combined through molecular recognition to form a complex.

U.S. Pat. Nos. 5,800,992 and 6,040,138 describe methods for making arrays of nucleic acid probes that can be used to detect the presence of a nucleic acid containing a specific nucleotide sequence. Methods of forming high-density arrays of nucleic acids, peptides and other polymer sequences with a minimal number of synthetic steps are known. The nucleic acid array can be synthesized on a solid substrate by a variety of methods, including, but not limited to, light-directed chemical coupling, and mechanically directed coupling. For additional descriptions and methods relating to arrays see U.S. patent application Ser. No. 10/658,879, 60/417,190, 09/381,480, 60/409,396, 5,861,242, 6,027,880, 5,837,832, 6,723,503 and PCT Pub No 03/060526 each of which is incorporated herein by reference in its entirety.

The terms “patient,” “subject,” “individual,” and the like are used interchangeably herein, and refer to any animal, or cells thereof whether in vitro or in situ, amenable to the methods described herein. In certain non-limiting embodiments, the patient, subject or individual is a human.

“Sample” or “biological sample” as used herein means a biological material isolated from a subject. The biological sample may contain any biological material suitable for detecting a WDR62 sequence mutation, and may comprise cellular and/or non-cellular material obtained from the individual.

Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

Standard codon/amino acid designators:

		3-Letter	1-Letter
Codon	Amino Acid	Abbreviation	Abbreviation
TTT	Phenylalanine	Phe	F
TTC	Phenylalanine	Phe	F
TTA	Leucine	Leu	L
TTG	Leucine	Leu	L
TCT	Serine	Ser	S
TCC	Serine	Ser	S
TCA	Serine	Ser	S
TCG	Serine	Ser	S
TAT	Tyrosine	Tyr	Y
TAC	Tyrosine	Tyr	Y
TAA	Termination	Ter	X
TAG	Termination	Ter	X
TGT	Cysteine	Cys	C
TGC	Cysteine	Cys	C
TGA	Termination	Ter	X
TGG	Tryptophan	Trp	W
CTT	Leucine	Leu	L
CTC	Leucine	Leu	L
CTA	Leucine	Leu	L
CTG	Leucine	Leu	L
CCT	Proline	Pro	P
CCC	Proline	Pro	P
CCA	Proline	Pro	P
CCG	Proline	Pro	P
CAT	Histidine	His	H
CAC	Histidine	His	H
CAA	Glutamine	Gln	Q
CAG	Glutamine	Gln	Q
CGT	Arginine	Arg	R
CGC	Arginine	Arg	R
CGA	Arginine	Arg	R
CGG	Arginine	Arg	R
ATT	Isoleucine	Ile	I
ATC	Isoleucine	Ile	I
ATA	Isoleucine	Ile	I
ATG	Methionine	Met	M
ACT	Threonine	Thr	T
ACC	Threonine	Thr	T
ACA	Threonine	Thr	T
ACG	Threonine	Thr	T
AAT	Asparagine	Asn	N
AAC	Asparagine	Asn	N
AAA	Lysine	Lys	K
AAG	Lysine	Lys	K
AGT	Serine	Ser	S
AGC	Serine	Ser	S
AGA	Arginine	Arg	R
AGG	Arginine	Arg	R
GTT	Valine	Val	V
GTC	Valine	Val	V
GTA	Valine	Val	V
GTG	Valine	Val	V
GCT	Alanine	Ala	A
GCC	Alanine	Ala	A
GCA	Alanine	Ala	A
GCG	Alanine	Ala	A
GAT	Aspartate	Asp	D
GAC	Aspartate	Asp	D
GAA	Glutamate	Glu	E
GAG	Glutamate	Glu	E
GGT	Glycine	Gly	G
GGC	Glycine	Gly	G
GGA	Glycine	Gly	G
GGG	Glycine	Gly	G

DESCRIPTION

The present invention relates to the discovery that recessive mutations in WD repeat domain 62 (WDR62) are involved in a wide spectrum of neurological diseases and disorders, including, but not limited to, intellectual disability, cerebral cortical malformations, microcephaly, agyria, pachygria, hypoplasia of the corpus callosum, lissencephaly, schizencephaly, polymicrogyria, seizures and cerebellar hypoplasia.

In various embodiments, the invention relates to a genetic screening assay of a subject to determine whether the subject has a mutation in WDR62. In some embodiments, the subject is a parent. In other embodiments, the subject is a prospective parent. In another embodiment, the subject is child. In a further embodiment, the subject is a fetus.

The present invention provides methods of assessing for the presence or absence of a genetic mutation in WDR62, as well as methods of diagnosing a subject having a mutation in WDR62, and methods of assessing a subject for carrier status for a mutation in WDR62. As described herein, certain mutations of WDR62 are associated with a wide spectrum of intellectual disabilities and cerebral cortical malformations, including, but not limited to, microcephaly, pachygria with cortical thickening, hypoplasia of the corpus callosum, lissencephaly, schizencephaly, polymicrogyria and cerebellar hypoplasia. The mutations in WDR62 described herein are alterations (e.g., deletions, insertions, or transitions) in the nucleic acid sequence of WDR62. The position of the mutations in the sequence of WDR62 are numbered in relation to the nucleic acid or amino acid sequence. That is, the numbered position of an altered nucleotide, or amino acid, is the position number of that nucleotide, or amino acid, in the nucleic acid or amino acid sequence. WDR62 maps to chromosome 19q13.12 and encodes 1,523 amino acids. The nucleic acid and amino acid sequence of WDR62 is set forth in GenBank accession number NM_001083961 (herein SEQ ID NOS:1 and 2). WDR62 maps to chromosome 19q13.12 and encodes a polypeptide having 1,523 amino acids. The WDR62 mutations useful in the methods of the invention include, but are not limited to, the following: W224S; Q470X; E526K; E526X; a 4-bp deletion (TGCC) in exon 31 beginning at codon 1402, leading to a premature stop codon at codon 1413 (V1402GfsX12); a nonsense mutation leading to a premature stop codon; a missense mutation affecting a conserved amino acid; and a 17-bp deletion in exon 30 leading to a frameshift at codon 1280 resulting in a premature termination codon following a novel peptide of 20 amino acids (G1280AfsX21).

In the methods of the invention, a test sample from a subject is assessed for the presence of one or more mutations in WDR62. In some embodiments, the subject is a human subject, and may be of any race and any age, including fetus, infant, juvenile, adolescent, and adult. Representative subjects include those who have not previously been diagnosed as being affected by a mutation in WDR62 or as being a carrier of a mutation in WDR62, as well as those who have been determined to be at risk for having a mutation in WDR62 or for being a carrier of a mutation in WDR62, and those who have been initially diagnosed as being affected by mutation in WDR62 where confirming information is desired.

In one embodiment, the test sample is a sample containing at least a fragment of a nucleic acid of WDR62, including WDR62 DNA or a fragment of WDR62 DNA, WDR62 mRNA or a fragment of WDR62 mRNA, and WDR62 cDNA or a fragment of WDR62 cDNA, from the subject. The term, “fragment,” as used herein, indicates that the portion of the gene, DNA, mRNA or cDNA is a polynucleotide of a length that is sufficient to identify it as a fragment of WDR62. In one representative embodiment, a fragment comprises one or more exons of the WDR62 gene. In another representative embodiment, a fragment comprises part of an exon of the WDR62 gene. In some embodiments, the fragment can also include an intron/exon junction of the WDR62 gene.

The test sample is prepared from a biological sample obtained from the subject. The biological sample can be a sample from any source which contains nucleic acid (e.g., DNA (e.g., chromosomal nucleic acid) or RNA), such as a blood, amniotic fluid, cerebrospinal fluid, or tissue such as, by way of example, skin, muscle, buccal mucosa, conjunctival mucosa, placenta, gastrointestinal tract or other organs. A biological sample of nucleic acid from fetal cells or tissue can be obtained by appropriate methods, such as by amniocentesis or chorionic villus sampling (direct or cultured). In certain embodiments, a biological sample containing genomic DNA is used. A biological sample can be used as the test sample; alternatively, a biological sample can be processed to enhance access to nucleic acids, or copies of nucleic acids (e.g., copies of nucleic acids comprising WDR62), and the processed biological sample can then be used as the test sample. For example, in one embodiment, cDNA is prepared from a biological sample comprising mRNA, for use in the methods. Alternatively or in addition, if desired, an amplification method can be used to amplify nucleic acids comprising all or a fragment of WDR62 in a biological sample, for use as the test sample in the assessment for the presence or absence of a mutation in WDR62.

The test sample is assessed to determine whether one or more mutations are present in the WDR62 sequence of the subject. In general, detecting a mutation may be carried out by determining the presence or absence of nucleic acids containing a mutation of interest in the test sample.

In some embodiments, hybridization methods, such as Southern analysis, Northern analysis, or in situ hybridizations, can be used (see Current Protocols in Molecular Biology, Ausubel, F. et al., eds., John Wiley & Sons, including all supplements). For example, the presence of a mutation can be indicated by hybridization of nucleic acid in the genomic DNA, RNA, or cDNA to a nucleic acid probe. A “nucleic acid probe”, as used herein, can be a DNA probe or an RNA probe; the nucleic acid probe can contain at least one polymorphism of interest, as described herein. The probe can be, for example, the gene, a gene fragment (e.g., one or more exons), a vector comprising the gene, a probe or primer, etc. For representative examples of use of nucleic acid probes, see, for example, U.S. Pat. Nos. 5,288,611 and 4,851,330.

To detect one or more mutations of interest, a hybridization sample is formed by contacting the test sample with at least one nucleic acid probe. A preferred probe for detecting mRNA or genomic DNA is a labeled nucleic acid probe capable of hybridizing to mRNA or genomic DNA of WDR62. The nucleic acid probe can be, for example, a full-length nucleic acid molecule, or a portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to appropriate target mRNA, cDNA or genomic DNA. The hybridization sample is maintained under conditions which are sufficient to allow specific hybridization of the nucleic acid probe to mRNA, cDNA or genomic DNA of WDR62. “Specific hybridization,” as used herein, indicates exact hybridization (e.g., with no mismatches). Specific hybridization can be performed under high stringency conditions or moderate stringency conditions, as appropriate. In a preferred embodiment, the hybridization conditions for specific hybridization are high stringency. Specific hybridization, if present, is then detected using standard methods. If specific hybridization occurs between the nucleic acid probe and WDR62 gene, mRNA or cDNA in the test sample, the mutation that is present in the nucleic acid probe is also present in the WDR62 of the subject. More than one nucleic acid probe can also be used concurrently in this method. Specific hybridization of any one of the nucleic acid probes is indicative of the presence of the mutation of interest, as described herein.

In Northern analysis (see Current Protocols in Molecular Biology, Ausubel, F. et al., eds., John Wiley & Sons, supra), the hybridization methods described above are used to identify the presence of a mutation of interest. For Northern analysis, a test sample comprising RNA is prepared from a biological sample from the subject by appropriate means. Specific hybridization of a nucleic acid probe, as described above, to RNA from the subject is indicative of the presence of a mutation of interest, as described herein.

Alternatively, a peptide nucleic acid (PNA) probe can be used instead of a nucleic acid probe in the hybridization methods described herein. PNA is a DNA mimic having a peptide-like, inorganic backbone, such as N-(2-aminoethyl)glycine units, with an organic base (A, G, C, T or U) attached to the glycine nitrogen via a methylene carbonyl linker (see, for example, 1994, Nielsen et al., Bioconjugate Chemistry 5:1). The PNA probe can be designed to specifically hybridize to a WDR62 sequence comprising one or more mutations of interest. Hybridization of the PNA probe to a WDR62 sequence is indicative of the presence of the polymorphism of interest.

In another embodiment of the methods of the invention, mutation analysis by restriction digestion can be used to detect a WDR62 mutation, if the mutation results in the creation or elimination of a restriction site. A sample containing nucleic acid from the subject is used. Polymerase chain reaction (PCR) can be used to amplify all or a fragment of WDR62 (and, if necessary, the flanking sequences) in the sample. RFLP analysis is conducted as described (see Current Protocols in Molecular Biology, supra). The digestion pattern of the relevant fragments indicates the presence or absence of mutation in WDR62.

Direct sequence analysis can also be used to detect specific mutations in WDR62. A sample comprising DNA or RNA is used, and PCR or other appropriate methods can be used to amplify all or a fragment of WDR62, and/or its flanking sequences, if desired. The sequence WDR62, or a fragment thereof (e.g., one or more exons), or cDNA, or fragment of the cDNA, or mRNA, or fragment of the mRNA, is determined, using standard methods. The sequence of the gene, gene fragment, cDNA, cDNA fragment, mRNA, or mRNA fragment is compared with the known nucleic acid sequence of WDR62, as appropriate. The presence of a mutation can then be identified.

Allele-specific oligonucleotides can also be used to detect the presence of a mutation of WDR62, through, for example, the use of dot-blot hybridization of amplified oligonucleotides with allele-specific oligonucleotide (ASO) probes (see, for example, 1986, Saiki et al., Nature 324:163-166). An “allele-specific oligonucleotide” (also referred to herein as an “allele-specific oligonucleotide probe”) is an oligonucleotide of approximately 10-50 base pairs, preferably approximately 15-30 base pairs, that specifically hybridizes to the WDR62 sequence, and that contains a mutation. An allele-specific oligonucleotide probe that is specific for a particular mutation can be prepared, using standard methods (see Current Protocols in Molecular Biology, supra). To identify a mutation, a sample comprising nucleic acid is used. PCR can be used to amplify all or a fragment of WDR62. The nucleic acid containing the amplified WDR62 sequence (or fragment of WDR62) is dot-blotted, using standard methods (see Current Protocols in Molecular Biology, supra), and the blot is contacted with the oligonucleotide probe. The presence of specific hybridization of the probe to the amplified WDR62 nucleic acid is then detected. Specific hybridization of an allele-specific oligonucleotide probe to nucleic acid from the subject is indicative of the presence of a mutation of interest.

In another embodiment of the invention, fluorescence resonance energy transfer (FRET) can be used to detect the presence of a mutation. FRET is the process of a distance-dependent excited state interaction in which the emission of one fluorescent molecule is coupled to the excitation of another. A typical acceptor and donor pair for resonance energy transfer consists of 4-[[4-(dimethylamino) phenyl]azo]benzoic acid (DABCYL) and 5-[(2-aminoethylamino]naphthalene sulfonic acid (EDANS). EDANS is excited by illumination with 336 nm light, and emits a photon with wavelength 490 n.times.n. If a DABCYL moiety is located within 20 angstroms of the EDANS, this photon will be efficiently absorbed. DABCYL and MANS will be attached to two different oligonucleotide probes designed to hybridize head-to-tail to nucleic acid adjacent to and/or overlapping the site of one of the mutations of interest. Melting curve analysis is then applied: cycles of denaturation, cooling, and re-heating are applied to a test sample mixed with the oligonucleotide probes, and the fluorescence is continuously monitored to detect a decrease in DABCYL fluorescence or an increase in EDANS fluorescence (loss of quenching). While the two probes remain hybridized adjacent to one another, FRET will be very efficient. Physical separation of the oligonucleotide probes results in inefficient FRET, as the two dyes are no longer in close proximity. The presence or absence of a mutation of interest can be assessed by comparing the fluorescence intensity profile obtained from the test sample, to fluorescence intensity profiles of control samples comprising known mutations of interest in WDR62.

In another embodiment, arrays of oligonucleotide probes that are complementary to target nucleic acid sequence segments from a subject can be used to identify mutations in WDR62. For example, in one embodiment, an oligonucleotide array can be used. Oligonucleotide arrays typically comprise a plurality of different oligonucleotide probes that are coupled to a surface of a substrate in different known locations. These oligonucleotide arrays, also known as “Genechips,” have been generally described in the art, for example, U.S. Pat. No. 5,143,854 and PCT patent publication Nos. WO 90/15070 and 92/10092. These arrays can generally be produced using mechanical synthesis methods or light directed synthesis methods which incorporate a combination of photolithographic methods and solid phase oligonucleotide synthesis methods. See Fodor et al., Science, 251:767-777 (1991), Pirrung et al., U.S. Pat. No. 5,143,854 (see also PCT Application No. WO 90/15070) and Fodor et al., PCT Publication No. WO 92/10092 and U.S. Pat. No. 5,424,186. Techniques for the synthesis of these arrays using mechanical synthesis methods are described in, e.g., U.S. Pat. No. 5,384,261.

After an oligonucleotide array is prepared, a nucleic acid of interest is hybridized with the array and scanned for mutations. Hybridization and scanning are generally carried out by methods described herein and also in, e.g., Published PCT Application Nos. WO 92/10092 and WO 95/11995, and U.S. Pat. No. 5,424,186, the entire teachings of which are incorporated by reference herein. In brief, a target nucleic acid sequence which includes one or more previously identified mutations or markers is amplified by well-known amplification techniques, e.g., PCR. Typically, this involves the use of primer sequences that are complementary to the two strands of the target sequence both upstream and downstream of the mutation. Asymmetric PCR techniques may also be used. Amplified target, generally incorporating a label, is then hybridized with the array under appropriate conditions. Upon completion of hybridization and washing of the array, the array is scanned to determine the position on the array to which the target sequence hybridizes. The hybridization data obtained from the scan is typically in the form of fluorescence intensities as a function of location on the array.

Although often described in terms of a single detection block, e.g., for detection of a single mutation, arrays can include multiple detection blocks, and thus be capable of analyzing multiple, specific mutations. In alternate arrangements, it will generally be understood that detection blocks may be grouped within a single array or in multiple, separate arrays so that varying, optimal conditions may be used during the hybridization of the target to the array. This allows for the separate optimization of hybridization conditions for each situation. Additional description of use of oligonucleotide arrays for detection of polymorphisms can be found, for example, in U.S. Pat. Nos. 5,858,659 and 5,837,832, the entire teachings of which are incorporated by reference herein.

Other methods of nucleic acid analysis can be used to detect mutations of interest. Representative methods include direct manual sequencing (1988, Church and Gilbert, Proc. Natl. Acad. Sci. USA 81:1991-1995; 1977, Sanger et al., Proc. Natl. Acad. Sci. 74:5463-5467; Beavis et al. U.S. Pat. No. 5,288,644); automated fluorescent sequencing; single-stranded conformation polymorphism assays (SSCP); clamped denaturing gel electrophoresis (CDGE); denaturing gradient gel electrophoresis (DGGE) (1981, Sheffield et al., Proc. Natl. Acad. Sci. USA 86:232-236), mobility shift analysis (1989, Orita et al., Proc. Natl. Acad. Sci. USA 86:2766-2770; 1987, Rosenbaum and Reissner, Biophys. Chem. 265:1275; 1991, Keen et al., Trends Genet. 7:5); restriction enzyme analysis (1978, Flavell et al., Cell 15:25; 1981, Geever, et al., Proc. Natl. Acad. Sci. USA 78:5081); heteroduplex analysis; chemical mismatch cleavage (CMC) (1985, Cotton et al., Proc. Natl. Acad. Sci. USA 85:4397-4401); RNase protection assays (1985, Myers, et al., Science 230:1242); use of polypeptides which recognize nucleotide mismatches, such as E. coli mutS protein (see, for example, U.S. Pat. No. 5,459,039); Luminex xMAP™ technology; and/or allele-specific PCR, for example.

These and other methods can be used to identify the presence of one or more mutations of interest in WDR62. For example, in certain embodiments, the methods can be used to assess both the first and the second alleles of WDR62 of a subject for the presence of one or more mutations. The terms, “first” and “second” alleles are arbitrarily applied to the two alleles; that is, either allele may be designated as the “first” allele, and the other allele is then designated as the “second” allele.

In another embodiment of the invention, the methods of assessing a test sample for the presence or absence of a mutation in WDR62, as described herein, are used to diagnose in a subject affected by a disorder associated with a mutation in WDR62. The two alleles of the affected subject may have the same mutation present, or may have different mutations. Furthermore, more than one mutation may be found in one or both alleles. In these methods, at least one mutation is found in at least one of the two alleles of WDR62 (the “first” allele). In addition, in affected subjects, at least one mutation in WDR62 is present on the other allele of WDR62 (the “second” allele).

In a further embodiment of the invention, the methods of assessing a test sample for the presence or absence of a mutation in WDR62, as described herein, are used to diagnose carrier status of a subject for a mutation in WDR62. The term, “carrier status,” indicates that the subject carries mutation of interest in only one allele of WDR62, and thus is considered a carrier for this recessive disorder. In these methods, at least one mutation is found in only one of the two alleles of WDR62 (in the “first” allele). In addition, no mutations in WDR62 are found in the second allele, although it should be noted that benign sequence changes may also be present in either or both alleles of WDR62.

The present invention also pertains to kits useful in the methods of the invention. Such kits comprise components useful in any of the methods described herein, including for example, hybridization probes or primers (e.g., labeled probes or primers), reagents for detection of labeled molecules, restriction enzymes (e.g., for RFLP analysis), allele-specific oligonucleotides, means for amplification of WDR62 nucleic acids, or means for analyzing the nucleic acid sequence of WDR62 and instructional materials. For example, in one embodiment, the kit comprises components useful for analysis of WDR62 mutations. In a preferred embodiment of the invention, the kit comprises components for detecting one or more of the mutations of WDR62.

EXPERIMENTAL EXAMPLES

The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compounds of the present invention and practice the claimed methods. The following working examples therefore, specifically point out the preferred embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.

Example 1

Whole Exome Sequencing Identifies Recessive WDR62 Mutations in Severe Brain Malformations

It is demonstrated herein using whole-exome sequencing that recessive mutations in WD repeat domain 62 (WDR62) are the cause of a wide spectrum of severe cerebral cortical malformations including microcephaly, pachygria with cortical thickening as well as hypoplasia of the corpus callosum (see 2010, Bilguvar et al., Nature 467:207-210). Some patients with mutations in WDR62 had evidence of additional abnormalities including lissencephaly, schizencephaly, polymicrogyria and, in one instance, cerebellar hypoplasia, all traits traditionally regarded as distinct entities.

In mice and humans, WDR62 transcripts and protein are enriched in neural progenitors within the ventricular and subventricular zones. Expression of WDR62 in the neocortex is transient, spanning the period of embryonic neurogenesis. Unlike other known microcephaly genes, WDR62 does not apparently associate with centrosomes and is predominantly nuclear in localization, as demonstrated herein. These findings unify previously disparate aspects of cerebral cortical development and highlight the use of whole-exome sequencing to identify disease loci in settings in which traditional methods have proved challenging.

The materials and methods employed in these experiments are now described.

Human Subjects

The study protocol was approved by the Yale Human Investigation Committee. Approvals from institutional review boards for genetic studies, and written consent from all study subjects, were obtained at the participating institutions.

MRI Sequences

MRI examinations presented were performed with a 3-T scanner (Trio, Siemens).

Illumina Genotyping

Whole-genome genotyping of the samples was performed on the Illumina Platform with Illumina Human 370K Duo or 610K Quad Beadchips using the manufacturer's protocol. The image data were normalized and the genotypes were called using data analysis software (Bead Studio, Illumina). Linkage analysis was performed using Allegro version 2.0 software (DeCode Genetics).

Sanger Sequencing

The exons and exon-intron boundaries of WDR62 were determined using the University of California, Santa Cruz (UCSC) Genome Browser (genome.ucsc.edu); unique primers were designed using Sequencher 4.8 (Gene Codes) and synthesized by Invitrogen. The fragments were amplified, purified and direct re-sequencing was performed using ABI's 9800 Fast Thermocyclers. The amplicons were analysed on an 3730xL DNA Analyser (Applied Biosystems).

Targeted Sequence Capture

Genomic DNA of sample NG 26-1 was captured on a NimbleGen 2.1M Human Exome Array (based on the build of 30 Apr. 2008 of the consensus coding sequence (CCDS) database) with modifications to the manufacturer's protocol (2009, Choi et al., Proc. Natl Acad. Sci. USA 106:19096-19101). The pre- and post-capture libraries were compared by quantitative PCR for the determination of the relative fold enrichment of the targeted sequences.

Exome Sequencing

Single-read cluster generation was performed on the Cluster Station (Illumina). The captured, purified and clonally amplified library targeting the exome from patient NG 26-1 was sequenced on Genome Analyser IIx. Two lanes of single-read sequencing at a read length of 74 by was performed following the manufacturer's protocol. Image analysis and base calling was performed by Illumina Pipeline version 1.5 with default parameters, installed on Yale University's High Performance Computing Cluster.

Targeted Exome Sequencing

Genomic DNA of sample NG 26-1 was captured on a NimbleGen 2.1M Human Exome Array with modifications to the manufacturer's protocol (2009, Choi et al., Proc. Natl Acad. Sci. USA 106:19096-19101), followed by single-read cluster generation on the Cluster Station (Illumina). The captured, purified and clonally amplified library targeting the exome from patient NG 26-1 was then sequenced on Genome Analyser IIx. Two lanes of single-read sequencing at a read length of 74 by was performed following the manufacturer's protocol.

Exome Sequence Analysis

The sequence reads obtained were aligned to the human genome (hg18) using Maq (2008, Li et al., Genome Res. 18:1851-1858) and BWA (2009, Li et al., Bioinformatics 25:1754-1760) software. The percentage alignment of the reads to both the reference genome as well as the targeted region, exome, was calculated using perl scripts (2009, Choi et al., Proc. Natl Acad. Sci. USA 106:19096-19101). Similarly, perl scripts were used for the detection of mismatch frequencies and error positions. SAMtools (2009, Li et al., Bioinformatics 25:2078-2079) was used for the detection of single-nucleotide variations on the reads aligned with Maq. The indels were detected on the reads aligned with BWA for its ability to allow for gaps during the alignment. Shared homozygous segments of the affected subjects were detected using Plink software version 1.06 (2007, Purcell et al., Am. J. Hum. Genet. 81:559-575), and the variants were filtered for shared homozygosity. The variants were annotated for novelty compared with both dbSNP (build 130) and nine personal genome databases and previous exome sequencing experiments performed by the human genomics group. Novel variants were further evaluated for their impact on the encoded protein, conservation across 44 vertebrate species, Caenorhabditis elegans and Drosophila melanogaster, expression patterns and potential overlap with known microRNAs.

Functional Annotation

Published microarray data sets of E9.5, E11.5 and E13.5 mouse brain tissue (GSE8091) were downloaded from the GEO database (www.ncbi.nlm.nih.gov/projects/geo/query/acc.cgi) (2008, Hard et al., 8:1257-1265) and processed using R statistical program (Affy package) (2003, Irizarry et al., Nucleic Acids Res. 31:e15). Genes that correlated highly with Wdr62 (Bonferroni corrected P<0.01) were functionally annotated using DAVID tools (david.abcc.ncifcrf.gov) (2009, Huang et al., Nature Protocols 4:44-57).

Animals

Experiments were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee at Yale University School of Medicine.

In Situ Hybridization

Sections and wholemount embryos were processed for non-radioactive in situ hybridization as described previously with minor modifications (2009, Stillman et al., J. Comp. Neurol. 513:21-37). An RNA probe complementary to mouse Wdr62 (bases 3,525-4,480, relative to SEQ ID NO: 3, of the mouse Wdr62 complementary DNA, NCBI Reference Sequence: NM_146186) was prepared and labelled with digoxigenin-1 l-uridine-5′-triphosphate. Embryos and tissue sections were analysed using a Zeiss Stemi dissecting microscope or a Zeiss AxioImager fitted with a Zeiss AxioCam MRc5 digital camera. Images were captured using AxioVision AC software (Zeiss) and assembled using Adobe Photoshop.

Immunostaining and Confocal Imaging

E15.5 embryos were obtained from timed-pregnant CD-1 mice (Charles River). For timed pregnancies, midday of the day of vaginal plug discovery was considered E0.5. Dissected brains were fixed by immersion in 4% paraformaldehyde for 16 h at 4° C. and sectioned at 70 μm using a vibratome (Leica VT1000S). Human fetal brains at 19 and 20 weeks' gestation were obtained under the guidelines approved by the Yale Institutional Review Board (protocol number 0605001466) from the Human Fetal Tissue Repository at the Albert Einstein College of Medicine (CCI number 1993-042), fixed by immersion in 4% paraformaldehyde for 36 h, cryoprotected and frozen, and cryosectioned at 60 μm. For mouse sections, an unconjugated donkey anti-mouse IgG Fab fragment (Jackson Immuno Research Laboratories, 1:200) was added to block endogenous mouse IgG. Primary antibodies were diluted in blocking solution at the following concentrations: mouse anti-WDR62 (Sigma-Aldrich), 1:400; rabbit anti-SOX2 (Millipore), 1:500; rabbit anti-TBR2 (Abcam), 1:500; chicken anti-GFP (Abcam), 1:1,500; rat anti-α-tubulin (Abcam), 1:500; rabbit anti-γ-tubulin (Sigma), 1:250; standard methods were followed. Confocal images were collected using laser-scanning microscope (Zeiss LSM 510). For diaminobenzidine staining, brain sections were incubated with biotinylated secondary antibodies and processed using the ABC and diaminobenzidine kits (Vector Laboratories). Images were acquired using a digital scanner (Aperio).

Cell Culture

For neural progenitor cultures, dorsal telencephalon was dissected from E12.5 mouse embryos and enzymatically dissociated and re-suspended as previously described (2005, Abelson et al., Science 310:317-320). For cell lines, Neuro2a, HeLa and HEK-293FT cells were plated on glass coverslips coated with poly-L-ornithine (15 μg ml⁻¹) at 5×10⁵ cells per square centimetre in 24-well plates. Sixteen hours after plating, the cells were fixed by immersion in 4% paraformaldehyde for 15 min at room temperature and processed for immunostaining.

Subcellular Fractionation and Western Blotting

Dorsal telencephalon was dissected from E14.5 mouse embryos and fractionated using the CelLytic nuclear extraction kit (Sigma). The manufacturer's protocol was followed with the exception that cell lysis was achieved by addition of 0.5% Triton X-100. Immunoblotting was done with primary antibodies diluted at the following concentrations: rabbit anti-WDR62 (Novus), 1:1,000; rat anti-α-tubulin (Abcam), 1:5,000.

In Utero Electroporation

CAG-GFP plasmid DNA was transfected into ventricular zone progenitors of E13.5 embryos by in utero electroporation as previously described (2008, Kwan et al., Proc. Natl Acad. Sci. USA 105:16021-16026). At E15.5, the embryos were collected and fixed for immunostaining.

Clinical Histories of Patients with WDR62 Mutations

This study was approved by the Yale Human Investigation Committee (9406007680 (OCt. 24, 2009) and 0908005592 (Aug. 17, 2009)). Consents were obtained from all study participants by the referring physicians. IRB protocol numbers and approval dates are as follows: Istanbul: NO:C-033 (Dec. 22, 2009); Hacettepe: 2008ABH67540017 (Sep. 27, 2007); Kayseri: 2009/55 (Sep. 3, 2009); Ege: B.30.2.EGE.0.20.05.00/OM/1093-1432, #09-5.1/16, (Jun. 23, 2009).

NG 26-1 (Mutation: V1402GfsX12)

The patient is a 4 year 6 month old female who was the product of a consanguineous union. She was brought to medical attention at 4 months of age due to small head size. At that time, her head circumference was 33 cm and she was given a diagnosis of microcephaly. Metabolic and TORCH workups were negative. She was last seen in clinic at 2 years and 3 months of age. Her head circumference was 38 cm. She showed micrognathia and a bulbous nose, and suffered from severe mental retardation. She was able to say a few words including “cat”, “dad”, “come”, and “new”, and responded to basic verbal commands. She was not toilet trained nor able to feed herself. She was able to walk and run, but could not ascend or descend stairs. Her vision and hearing were noted to be unremarkable. She has no spasticity in any of her extremities and has never experienced any seizures.

NG 30-1 (Mutation: E526X)

The patient is a 7-year-old female who is the product of a consanguineous union. The pregnancy was uneventful and the neonatal period was unremarkable. The patient presented to medical attention at 9 months of age due to small head size. On examination, she was found to have motor retardation. Her head circumference at that time was noted to be 38.5 cm and she was diagnosed with microcephaly. She had an unrevealing metabolic workup. At the age of 4, she began experiencing generalized seizures which were controlled with levetiracetam. Her last clinic visit was at the age of 6. At that visit, she was able to ambulate independently, was able to understand only basic verbal commands, had limited vocabulary, and was noted to have moderate mental retardation based on clinical examination.

NG 190-1 (Mutation: W224S)

The patient is a 6 year 5 month old boy who is the product of a consanguineous union. His periand neonatal periods were unremarkable. He presented to medical attention at the age of 2 due to hyperactivity, seizures, and inability to sleep. The seizures were generalized, tonic/clonic, lasting approximately 1-2 minutes each and occurring on average twice a day. At that time on neurologic exam, he was able to speak 1-2 word sentences. Motor tone and bulk were grossly normal. Reflexes were within normal limits and cranial nerves were intact. At the most recent clinic visit in 2009, he continued to experience 4-8 seizures per day and was being treated with valproic acid. Physical exam revealed microcephaly and micrognathia. His head circumference was noted to be 42 cm and he had severe mental retardation based on clinical observation. He was not toilet trained, could only speak a single word, “dad”, could not feed himself, and was only able to ambulate with the support of others.

NG 190-5 (Mutation: W224S)

This patient is an 8 year 7 month old female who is the product of a consanguineous marriage and the cousin of NG 190-1. The patient presented to medical attention at the age of 3 due to seizures. She is microcephalic, hyperactive, and has dysconjugate gaze. On the most recent exam her head circumference was 44 cm and she was noted to have moderate mental retardation based on clinical observation. She demonstrated poor verbal skills, but was able to carry out simple activities of daily living. She had normal tone, reflexes, and no dysmetria on exam. She was able to walk independently, and had no obvious dysmorphic features. She was grossly less affected than her brother (NG 190-6) and cousin (NG 190-1).

NG 190-6 (Mutation: W224S)

This patient is a 12 year, 11 month old boy who is the product of a consanguineous marriage and is the brother of patient NG 190-5. He has a history of seizures and mental and motor retardation. He is noted to have microcephaly (current head circumference is 45 cm) and self-mutilating behaviors. On last exam, his gaze was described as dysconjugate, muscle tone was increased, and reflexes were hyperactive. He was assessed as having severe mental retardation based on clinical exam, but could ambulate independently. His symptoms are notably more severe than his sister's.

NG 294-1 (Mutation: Q470X)

The patient is a 14 year, 6 month old male who is the product of a consanguineous marriage. He has two normal siblings. His perinatal history is significant for preterm birth at 32 weeks of gestation. He was hospitalized at 27 days for bilirubinemia at which time he was found to have genu varum (bow leggedness) and microcephaly. He has had two deformity correction surgeries since that time, a hernia repair at 2 months, and cryptorchidism repair at 8 years of age. He has celiac disease, arachnodactly, microcephaly and severe mental retardation diagnosed by clinical observation. He has never suffered a seizure.

NG 339-1 (Mutation: G1280AfsX21)

The patient is a 10 year, 10 month old female who is the product of a consanguineous union. She presented to medical attention at 3 months of age due to failure to thrive and small head size. At the time of presentation her head circumference was 34.5 cm with obvious microcephaly. On neurologic examination, she had good head control. She recognized her mother and was noted to have a social smile. Her deep tendon reflexes (DTR's) were 3+ in all four extremities and she had increased muscle tone throughout. She has one healthy sibling. No current clinical information is available.

NG 537-1 (Mutation: E526K)

The patient is a 15 year, 5 month old female who is the product of consanguineous marriage. Peri- and neonatal periods were unremarkable except for meconium aspiration. She was delayed to acquire motor skills in the first three years of life but ultimately presented to medical attention at the age of 3.5 years due to poor verbal skills. Head circumference at this time was 43 cm, consistent with microcephaly. She was noted to have severe mental retardation, but the remainder of the neurologic exam at the time was normal. She was placed on anti-epileptic medication for a brief period of time during her childhood due to abnormal electroencephalograms (EEG's), however, she never suffered an overt seizure. The medication was discontinued. At her last clinic visit in 2009, her head circumference was 51 cm. On physical exam, she was noted to have microcephaly, prognathism, dysconjugate gaze, and dysarthria. She was able to ambulate independently, demonstrated full strength in all muscle groups, and had normal reflexes.

NG 891-1 (Mutation: V1402GfsX12)

The patient is a 2 year 4 month old male who was born to consanguineous parents. He had a normal prenatal and neonatal period and was the product of an uneventful vaginal delivery. He presented to medical attention at 20 months of age due to relatively small head size compared to his healthy sibling. At the time of presentation, he was 9,500 gr (50-75th percentile) and 83 cm (50^th percentile). His head circumference, however, was 41 cm (<3 percentile). He was noted on clinical exam to have developmental delay and severe psychomotor retardation but has not suffered from seizures.

The results of the experiments are now described.

It is demonstrated herein using whole-exome sequencing that recessive mutations in WD repeat domain 62 (WDR62) are the cause of a wide spectrum of severe cerebral cortical malformations including microcephaly, pachygria with cortical thickening as well as hypoplasia of the corpus callosum. Some patients with mutations in WDR62 had evidence of additional abnormalities including lissencephaly, schizencephaly, polymicrogyria and, in one instance, cerebellar hypoplasia, all traits traditionally regarded as distinct entities.

In mice and humans, WDR62 transcripts and protein are enriched in neural progenitors within the ventricular and subventricular zones. Expression of WDR62 in the neocortex is transient, spanning the period of embryonic neurogenesis. Unlike other known microcephaly genes, WDR62 does not apparently associate with centrosomes and is predominantly nuclear in localization, as demonstrated herein. These findings unify previously disparate aspects of cerebral cortical development and highlight the use of whole-exome sequencing to identify disease loci in settings in which traditional methods have proved challenging.

Whole-exome sequencing using next generation technology was applied to the index case of a small consanguineous kindred (NG 26) from eastern Turkey that presented for medical attention owing to failure to reach developmental milestones and was found on clinical examination to have microcephaly. Neuroimaging studies identified a complex array of developmental abnormalities including pachygria and thickened cortex (FIG. 1A-FIG. 1D and FIG. 3C).

Initially, whole-genome genotyping of the two affected members was performed to identify shared homozygous segments (each >2.5 centimorgans (cM)) that together composed 80.11 cM (Table 1). Given the substantial length of these shared segments, whole-exome sequencing of the index case was next performed using Nimblegen solid-phase arrays and the Illumina Genome Analyser IIx instrument (2009, Choi et al., Proc. Natl Acad. Sci. USA 106:19096-19101). A mean coverage of 44× was achieved, and 94% of all targeted bases were read more than four times, sufficient to identify novel homozygous variants with high specificity (Table 2). Two novel homozygous missense variants and one novel homozygous frameshift mutation were identified within the shared homozygosity intervals (FIG. 5 and Table 3). The frameshift mutation occurred in WDR62, deleting four base pairs (bp) in exon 31 (FIG. 1E). The full-length WDR62 (NM_001083961) maps to chromosome 19q13.12 and encodes 1,523 amino acids. The identified mutation causes a frameshift in codon 1,402, resulting in a premature stop codon at position 1,413 (FIG. 1F). The mutation was confirmed to be homozygous in both affected subjects and to be heterozygous in both parents using Sanger sequencing (FIG. 2A and FIG. 2). It was not observed in 1,290 Turkish control chromosomes.

TABLE 1
					Number	Length	Length
Chromosome	Start	End	SNP Start	SNP End	of SNPs	(cM)	(Mb)

1	240,862,637	247,177,330	rs10926796	rs6704311	623	10.90	6.31
6	82,149,868	87,794,412	rs2120536	rs7740936	510	2.96	5.64
12	125,564,137	132,288,869	rs16920745	rs7975069	907	20.79	6.72
19	8,931,837	14,582,019	rs2547067	rs6511944	510	9.16	5.65
19	36,896,151	47,035,366	rs10417470	rs3922888	894	13.17	10.14
X	147,386,161	154,582,606	rs5980537	rs557132	478	23.13	7.20
Total					3,922	80.11	41.66

TABLE 2
Read	Number		Number	Reads Mapped	Reads Mapped	Average Coverage	Bases Covered ≧4X
Length	of lanes	Read Type	of Reads	to the Genome	to the Exome	Across The Exome	within the Exome
74	2	Single end	37.1M	97.3%	57.40%	44.3X	94.34%
* In two lanes of single-end 74 base pair reads, we obtained 37 million reads, 21 million of which mapped to the exome, and 466,000 of which mapped to the 969,174 exomic base pairs (out of 41.66 million genomic base pairs) in shared regions of homozygosity between the two affected siblings (Supplementary Table 1). The sequence error rate (per base per read) was 0.34%. Sensitivity and specificity for detection of homozygous variation from the reference sequence was high (both >97%) as determined by comparison of the sequencing data to the results of SNP genotyping as a reference.

TABLE 3
		Base	Quality		Major allele	Minor allele			Amino acid	Amino acid
Chr	Position	change	Score	Coverage	(no PCR duplicates)	(no PCR duplicates)	Gene	Status	change	position

6	82,980,721	G to C	228	43	11	5	IBTK	Missense	P716A	716/1353
12	130,807,115	A to G	96	20	7	0	SFRS8	Missense	T565A	565/951
19	41,287,310	-TGCC	163/391	6	4	0	WDR62	Frame-shift	V1402GfsX12	1402/1523
		deletion

Because this homozygous mutation in WDR62 was particularly compelling, it was investigated whether mutations in this gene might account for additional cases of malformations of cortical development. As the index case was ascertained with an initial diagnosis of pachygyria, a group of 30 probands who carried diagnoses of agyria or pachygyria and were products of consanguineous unions (inbreeding coefficient >1.5% (2007, Purcell et al., Am. J. Hum. Genet. 81:559-575)), were focused on. Among these patients, whole-genome genotyping identified eight with homozygosity of at least 2 cM spanning the WDR62 locus. One of these affected subjects, NG 891-1, was found to have the identical homozygous haplotype spanning the WDR62 locus and had the same 4-bp deletion (FIG. 2A and FIG. 6). Although there was no known relatedness between the two pedigrees, the kinship coefficient of NG 891-1 with NG 26-1 and NG 26-4 was 2.47% and 3.72%, consistent with fourth-degree relatedness (for example, first cousins once removed).

Further Sanger sequencing of the complete coding region of WDR62 in the seven remaining kindreds revealed five additional novel homozygous mutations (FIG. 2B-FIG. 2F). The affected member of kindreds NG 30 and NG 294 had homozygous nonsense mutations at codons 526 (E526X) and 470 (Q470X), respectively (FIG. 2B, FIG. 2D); subject NG 339-1 had a homozygous 17-bp deletion leading to a frameshift at codon 1,280 that resulted in a premature termination codon following a novel peptide of 20 amino acids (FIG. 2E); subjects NG 190-1 and NG 537-1 respectively had novel homozygous missense variants W224S and E526K (FIG. 2C, FIG. 2F), which occurred at positions highly conserved among vertebrates and were predicted to be deleterious by the Polyphen algorithm (FIG. 7). Moreover, after identification of the W224S mutation in NG 190, two additional relatives affected with microcephaly and mental retardation (kinship coefficients of 7.47% and 5.81%), both of whom also proved to be homozygous for the same mutation, were ascertained. The resulting lod score for linkage to the trait within the expanded kindred was 3.64; the chromosome segment containing WDR62 was the sole homozygous region shared among all three affected subjects (FIG. 12).

All of the newly identified mutations, except E526K, were absent from 1,290 Turkish and 1,500 caucasian control chromosomes. The heterozygous E526K variant was detected in three apparently unrelated Turkish subjects who were neurologically normal (allele frequency 0.2%). As an additional control measure in the evaluation of these homozygous mutations, the coding region of the gene in 12 consanguineous patients with non-neurological conditions who were found to have segments of homozygosity of at least one million base pairs spanning WDR62 was sequenced. None of these 12 subjects were found to have protein coding changes in WDR62. Similarly, only four heterozygous novel missense variants in WDR62 in the sequence of 100 whole exomes of subjects with non-neurological diseases were identified (Table 4). Public databases (dbSNP) showed no validated nonsense or frameshift alleles at this locus. Finally, no copy number variants overlapping the coding regions of WDR62 in the own set of 11,320 whole-genome genotypes were observed and only one deletion identified by bacterial artificial chromosome (BAC) array is reported in the Database of Genomic Variants (projects.tcag.ca/variation/).

TABLE 4
			Base	Amino Acid	Amino Acid
Sample ID	Chr	Position	Change	Change	Location
RKH005	chr19	41,284,406	A > T	D991V	991/1523
PTH120	chr19	41,285,904	G > A	A1152T	1157/1523
PTH108	chr19	41,284,409	C > T	S992L	992/1523
LMB06	chr19	41,285,506	T > C	V1083A	1083/1523

All of the index cases with WDR62 mutations presented for medical attention with mental retardation and were found to have prominent microcephaly on physical examination; some also suffered from seizures. Re-examination of the high field strength (3 T) magnetic resonance imaging (MRI) scans of the affected subjects by independent neuroradiologists who were blind to previous diagnoses identified hallmarks of a wide range of severe cortical malformations (summarized in Table 5. All nine patients had extreme microcephaly, pachygyria and hypoplasia of the corpus callosum (FIG. 3). In addition, they demonstrated radiographic features consistent with lissencephaly, including varying degrees of cortical thickening and loss of grey-white junction (FIG. 3). Under-opercularization (shallow Sylvian fissures) (FIG. 1B) was observed in six affected subjects. Two of the subjects had striking polymicrogyria that predominantly affected one hemisphere (FIG. 3C, FIG. 3D, FIG. 3G); in one this was associated with a unilateral open-lip schizencephaly characterized by a cleft surrounded by grey matter that extended into the ventricle (FIG. 3D, FIG. 3G). Other malformations observed included hippocampal dysmorphology with vertical orientation in six cases and a single case of unilateral dysgenesis of the cerebellum (FIG. 3F). There were no abnormalities of the brainstem, with the exception of unilateral atrophy observed in one patient, most likely secondary to Wallerian degeneration from the severe cerebral abnormalities observed (FIG. 3H).

TABLE 5
	CORTICAL
PATIENT ID	THICKENING	MICROENCEPHALY	PACHYGYRIA	POLYMICROGRYRIA	SCHIZENCEPHALY
NG 26-1	+	+	+	R temperal and	g
				RL occipito-parfetal
NG 30-1	+	+	+	g	g
NG 190-1	+	+	+	L fronto-	L parfetal
				temporal-parfetal
NG 190-5	+	+	+	g	g
NG 190-6	+	+	+	g	g
NG 294-1	+	+	+	g	g
NG 339-1	+	+	+	g	g
NG 537-1	+	+	+	g	g
NG 891-1	+	+	+	g	g

UNDER-

CORPUS CALLOSUM

	PATIENT ID	OPERCULARIZATION	CEREBELLUM	HIPPOCAMPUS	ROSTRUM	SPLENIUM
	NG 26-1	+	Normal	Normal	Mild	Mild
					hypoplasia	hypoplasia
	NG 30-1	g	Normal	Dysmorpic	Absent	Moderete
						hypoplasia
	NG 190-1	+	Normal	Dysmorpic	Absent	Marked
						hypoplasia
	NG 190-5	g	R hypoplasia	Dysmorpic	Absent	Marked
						hypoplasia
	NG 190-6	g	Normal	Dysmorpic	Mild	Normal
					hypoplasia
	NG 294-1	+	Normal	Dysmorpic	Mild	Marked
					hypoplasia	hypoplasia
	NG 339-1	+	Normal	Dysmorpic	Absent	Marked
						hypoplasia
	NG 537-1	+	Normal	Normal	Mild	Normal
					hypoplasia
	NG 891-1	+	Normal	Normal	Mild	Moderate
					hypoplasia	hypoplasia

Given the wide range of cortical malformations associated with WDR62 mutations, its expression in the developing mouse brain was investigated. Notably, during early development, in wholemount embryos from embryonic day (E)9.5 to E11.5, Wdr62 expression is prominent in neural crest lineages (FIG. 9A-FIG. 9C). Wdr62 also shows striking expression in the ventricular and subventricular zones during the period of cerebral cortical neurogenesis (E11.5-16.5), with expression decreasing in intensity by E17.5 (FIG. 4A, FIG. 9D-FIG. 9F). In the cerebellum, Wdr62 is strongly expressed in precursors of granule neurons at late embryonic and early postnatal stages; by postnatal day 9 (P9) Wdr62 expression is dramatically reduced (FIG. 9G, FIG. 9H). By postnatal day 21 (P21), low levels of Wdr62 expression are detected only in the hippocampus and the piriform cortex, and transcription is absent among differentiated cortical neurons (FIG. 9I).

Next, WDR62 protein expression it was examined using a previously characterized antibody (2010, Wasserman et al., Mol. Biol. Cell 21:117-130) (FIG. 4B-FIG. 4D). Both in the mouse and human fetal brain, WDR62 was enriched within the ventricular and subventricular zones, consistent with the in situ hybridization findings (FIG. 4 and FIG. 10). These stainings suggested that WDR62 localizes predominantly to the nucleus in neuronal cells, which was confirmed by immunofluorescence microscopy using cell cultures and western blotting with subcellular fractionation of cortical embryonic mouse cells with a second antibody (FIG. 4E and FIG. 11). Genes previously implicated in microcephaly encode centrosomal proteins (2005, Bond et al., Nature Genet. 37:353-355; 2009, Kumar et al., Am. J. Hum. Genet. 84:286-290; 2009, Thornton et al., Trends Genet. 25:501-510); thus it is noteworthy that WDR62 is apparently not associated with the centrosome during mitosis (FIG. 12).

The findings described herein implicate WDR62 in the pathogenesis of a spectrum of cortical abnormalities that until now have largely been conceptualized to be distinct (2008, Guerrini et al., Trends Neurosci. 31:154-162; 2001, Barkovich et al., Neurology 57:2168-2178; 2005, Barkovich et al., Neurology 65:1873-1887), suggesting that these diverse features can have unified underlying causation. It is noteworthy that WDR62 lies in a 10-million-bp interval that had previously been identified as a microcephaly locus, MCPH2 (1999, Roberts et al., Eur. J. Hum. Genet. 7:815-820). Although there were no imaging studies presented in the previous mapping of this locus, the findings described herein suggest that WDR62 is the MCPH2 gene and extend the phenotype beyond microcephaly.

To seek further insight into the biological function of WDR62, the expression data of early embryonic development of mouse brain (GSE8091) (2008, Hartl et al., 8:1257-1265) for genes with expression profiles significantly correlated with that of WDR62 was examined (Bonferroni corrected P<0.01, n=1,104). Functional annotation suggested that positively correlated genes were enriched for those encoding nuclear proteins (Benjamini adjusted P=6.23×10⁻³⁰), RNA processing proteins (Benjamini adjusted P=1.90×10⁻³¹) and cell-cycle proteins (Benjamini adjusted P=3.25×10⁻¹⁸). Negatively correlated genes encoded neuronal differentiation proteins (Benjamini adjusted P=1.40×10⁻⁷). Several genes linked to developmental brain malformations, such as DCX, DCC and BURB1B, were found in these enrichment sets (Table 6).

TABLE 6
	Wdr62
Symbol	correlation	P value	Malformation	References

Dc	−0.95	3.00E−08	X-linked lissencephaly-1; Double	Cell 92: 51-61,1998, Cell 92: 63-72, 1998
			Cortex Syndrome
Tubo1	−0.94	5.80E−08	Lissencephaly type 3	Cell 128: 45-57, 2007; Hum.
				Mutat. 28: 1055-1064, 2007
Dcc	−0.94	9.71E−08	Congenital Mirror Movements	Science 328: 592-only, 2010
			(Bimanual Synergia)
Bub1b	0.93	1.18E−07	Mosaic Variegated Aneuploidy	Am. J. Med. Genet. 140A: 358-367, 2006
			Syndrome (MVA), microcephaly
Cc2d2	0.94	5.47E−08	Joubert Syndrome 9;	Am. J. Hum. Genet. 82: 1011-1018, 2008;
			Type 6 Meckel Syndrome	Am. J. Hum. Genet. 82: 1361-1367, 2008
Col18o1	0.95	2.36E−08	Type 1 Knobloch Syndrome	Hum. Molec. Genet. 9: 2051-2058, 2000;
				Hum. Mutat. 23: 77-84, 2004
indicates data missing or illegible when filed

The results disclosed herein demonstrate that Whole-exome sequencing is particularly valuable for gene discovery in those conditions in which mapping has been confounded by locus heterogeneity and uncertainty about the boundaries of diagnostic classification, pointing to a bright future for its broad application to medicine.

Sequences

Homo sapiens WD repeat domain 62 (WDR62) nucleotide sequence, NCBI

Reference

Sequence: NM_001083961

(SEQ ID NO: 1)

tttcccgcgg ctgttcgctg ttccagtggg tcgtggcggt ggcggcagcg gcggttaggg   60

gatgtaacgg tcgcccgcct ccggcgtgac gatggcggcc gtagggtccg gaggctatgc  120

gcggaacgat gcaggggaga agctgccctc tgtcatggcg ggagttccgg cgcggagggg  180

ccagtcctcc ccgccccccg ccccaccaat ctgcctacgg cggcggacgc gactctcgac  240

ggcctccgag gagacggtgc agaaccgggt gtcactcgag aaggtgcttg gcatcacagc  300

ccagaacagc agtggcctaa cctgtgaccc cggcacaggc catgtggcct acctggcagg  360

ctgtgtggtg gtgattttgg accccaagga gaacaagcag cagcacatct ttaacaccgc  420

caggaagtct ctcagtgctc tggccttctc ccctgatggg aagtacatag tgacagggga  480

gaatgggcat aggcctgctg tgcgcatctg ggatgtggag gagaagaatc aggtggcgga  540

gatgctaggc cacaagtatg gtgtggcgtg tgtggccttc tcacccaata tgaagcacat  600

cgtgtccatg ggctaccaac atgacatggt gctcaacgtc tgggactgga agaaagacat  660

cgtagtggcc tccaacaagg tatcttgtag agtcattgcc ctctccttct cagaggacag  720

cagctatttt gtcactgttg ggaaccgcca tgtgaggttc tggttcttgg aagtctccac  780

tgagacaaag gtgacgagca cagtgcccct tgtagggcgc tcgggcatcc tgggcgagct  840

gcacaacaac atcttctgtg gtgtggcctg cggtcggggc cggatggcgg gcagtacctt  900

ctgtgtgtcc tactcgggcc tcctctgcca gttcaatgag aagagggtgc tggagaagtg  960

gatcaacctg aaggtctccc tgtcttcctg cctctgtgtc agccaggagc tcatcttctg 1020

tggctgcaca gatgggatag tccgcatctt ccaggcccat agcctgcact acctcgccaa 1080

cctgcccaag ccacactacc ttggggtaga cgtggcacag ggcctggagc ccagcttcct 1140

cttccacagg aaggcggaag cagtctaccc agatacagtg gcactgacct tcgaccccat 1200

ccaccagtgg ctgtcctgcg tgtataagga ccacagcatc tacatctggg atgtcaagga 1260

catcaacaga gtgggcaagg tgtggtcaga gctcttccac agctcctacg tttggaacgt 1320

ggaggtgtat cctgagtttg aagaccagag agcttgtttg ccatcaggat cctttctgac 1380

ttgttcttca gacaacacca ttcgcttctg gaacttggac agcagccctg attctcactg 1440

gcagaaaaac atcttcagca ataccctgct gaaggtcgtg tacgtggaga atgacatcca 1500

gcacctgcag gacatgtcac acttcccaga ccgggggagc gagaatggga cacccatgga 1560

cgtgaaagcc ggggtgcggg tcatgcaggt cagtcctgac ggccagcatt tggcttcagg 1620

cgaccgaagt ggaaatctga ggatccacga gctgcacttc atggacgagc tggtcaaggt 1680

ggaggcccat gatgctgagg tgctgtgcct ggagtactcc aagccagaga cggggctgac 1740

cttgctggcc tcagccagtc gggaccggct gatccatgtg ctgaacgtgg agaagaacta 1800

caacctggag cagacgctgg atgaccactc ctcctccatc accgccatca agttcgctgg 1860

caacagagac atccagatga tcagctgtgg ggctgacaag agcatctact ttcgcagtgc 1920

ccagcagggt tcggatggac tacactttgt ccgtacccac cacgtagcag agaaaaccac 1980

cttgtatgac atggacattg acatcaccca gaagtacgtg gccgtggcct gccaggaccg 2040

caatgtgaga gtctacaaca ctgtgaacgg gaagcagaag aagtgctaca agggctccca 2100

gggtgacgaa gggtccttgc tgaaggtcca tgtggacccc tcaggcacct tcctggccac 2160

cagctgctct gacaaaagca tctcagtgat tgacttttac tcgggcgagt gcattgccaa 2220

gatgtttggc cattcagaaa ttattaccag catgaagttc acctatgact gtcatcactt 2280

gatcacagta tctggagaca gctgcgtgtt catctggcac ctgggcccgg agatcaccaa 2340

ctgcatgaag cagcacttgc tggagattga ccaccggcag cagcagcagc acacaaatga 2400

caagaagcgg agtggccacc ccaggcagga tacgtatgtg tccacaccta gtgagattca 2460

ctccctgagc cctggagagc aaacagagga tgatctggag gaagagtgtg agccagaaga 2520

gatgctgaag acaccatcca aagatagctt ggatccagat cctcgttgcc tgctaaccaa 2580

cggcaagctg ccactgtggg caaagcggct gctaggggac gatgatgtgg cagatggctt 2640

ggccttccac gccaagcgca gctaccagcc ccacggccgc tgggcagagc gggccggcca 2700

agagcccctc aagaccatcc tggatgccca ggacctggat tgctacttta cccccatgaa 2760

gcccgagagt ctggagaact ccattctgga ttcactggag ccacagagcc tggccagcct 2820

gctgagtgag tcagagagtc cccaggaagc tggccgcggg cacccctcct tcctgcccca 2880

gcagaaggaa tcatctgagg ccagtgagct catcctctac tctctggagg cagaagtgac 2940

agtcacaggg acagacagcc agtattgcag gaaggaggtg gaggccgggc ctggagacca 3000

gcagggcgac tcctacctca gggtgtcctc cgacagccca aaggaccaga gcccgcctga 3060

ggactcgggg gagtcagagg ccgacctgga gtgcagcttc gcagccatcc actccccagc 3120

tccgcctcct gaccctgccc ctcggtttgc cacgtcgctg ccccatttcc caggatgcgc 3180

aggtcccaca gaagatgagc tgtccctgcc cgagggaccc agcgtcccca gcagctccct 3240

accccagact ccggagcagg agaagttcct ccgccaccac tttgagacac tgactgagtc 3300

cccctgcaga gagctcttcc ccgcagctct gggagacgtg gaggcctctg aagctgaaga 3360

ccacttcttc aacccacgcc tgagtatctc cacgcagttc ctctcaagcc tccagaaggc 3420

atccaggttc acccatacct tccctccccg ggcaacccag tgccttgtga agtctccaga 3480

ggtcaagctc atggaccgag gcggaagcca gcccagagca ggtactggct acgcctcccc 3540

agacaggacc cacgtcctcg ctgcagggaa ggctgaagag accctggagg cctggcgccc 3600

accacctccc tgccttacga gcctggcgtc ctgtgtccct gcttcctccg tgctgcccac 3660

agacaggaat ctcccaacgc ccacatctgc acccacccca ggcctggctc agggtgtcca 3720

tgccccctcc acctgttcct acatggaggc cactgccagc tcccgtgcca ggatatcacg 3780

cagcatctcc ctcggtgaca gtgagggccc tatcgtggcc acactggccc agcccctccg 3840

taggccatcg tccgttgggg agctggcctc cttgggccag gagcttcagg ccatcaccac 3900

cgcgacaaca cccagtttgg acagtgaggg ccaagagcct gccctgcgtt cctggggcaa 3960

ccacgaggcc cgggccaacc tgagactgac cctgtcaagt gcctgtgatg ggctcctgca 4020

gccccccgtg gatacccagc ctggcgtcac cgtccctgca gtgagcttcc cagcccctag 4080

ccctgtggaa gagagcgccc tgaggctcca cggctctgcc tttcgcccaa gtctcccagc 4140

tcctgagtcc cctggccttc ctgcccaccc cagtaacccc cagcttccag aggcccggcc 4200

tggcatccct ggcggcactg cctccctcct ggagcccacc tccggtgcac ttggtctgtt 4260

acagggcagc cctgcccgct ggagtgagcc ctgggtgccg gttgaagccc tgcccccatc 4320

tccccttgag ctgagcaggg tggggaacat cttgcacagg ctgcagacca ccttccaaga 4380

agccctcgac ctttaccgtg tgttggtctc cagtggccag gtggacaccg ggcagcagca 4440

ggcacggact gagctggtct ccaccttcct gtggatccac agccagctgg aggctgaatg 4500

cctggtgggg actagtgtgg ccccagccca ggctctgccc agcccaggac ccccgtcccc 4560

accgacgctg taccccctgg ccagcccaga cctgcaggcc ctgctggaac actactcgga 4620

gctgctggtg caggccgtgc ggaggaaggc acgggggcac tgagggcgca gcccctccac 4680

cgcagccctg ctgcttctga ggacttaggt attttaagcg aataaactga cagctttgag 4740

gaatga                                                            4746

Homo sapiens WD repeat domain 62 (WDR62) nucleotide sequence, NCBI

Reference

Sequence: NM_001083961

(SEQ ID NO: 2)

Met Ala Ala Val Gly Ser Gly Gly Tyr Ala Arg Asn Asp Ala Gly Glu

1               5                   10                  15

Lys Leu Pro Ser Val Met Ala Gly Val Pro Ala Arg Arg Gly Gln Ser

20                  25                  30

Ser Pro Pro Pro Ala Pro Pro Ile Cys Leu Arg Arg Arg Thr Arg Leu

35                  40                  45

Ser Thr Ala Ser Glu Glu Thr Val Gln Asn Arg Val Ser Leu Glu Lys

50                  55                  60

Val Leu Gly Ile Thr Ala Gln Asn Ser Ser Gly Leu Thr Cys Asp Pro

65                  70                  75                  80

Gly Thr Gly His Val Ala Tyr Leu Ala Gly Cys Val Val Val Ile Leu

85                  90                  95

Asp Pro Lys Glu Asn Lys Gln Gln His Ile Phe Asn Thr Ala Arg Lys

100                 105                 110

Ser Leu Ser Ala Leu Ala Phe Ser Pro Asp Gly Lys Tyr Ile Val Thr

115                 120                 125

Gly Glu Asn Gly His Arg Pro Ala Val Arg Ile Trp Asp Val Glu Glu

130                 135                 140

Lys Asn Gln Val Ala Glu Met Leu Gly His Lys Tyr Gly Val Ala Cys

145                 150                 155                 160

Val Ala Phe Ser Pro Asn Met Lys His Ile Val Ser Met Gly Tyr Gln

165                 170                 175

His Asp Met Val Leu Asn Val Trp Asp Trp Lys Lys Asp Ile Val Val

180                 185                 190

Ala Ser Asn Lys Val Ser Cys Arg Val Ile Ala Leu Ser Phe Ser Glu

195                 200                 205

Asp Ser Ser Tyr Phe Val Thr Val Gly Asn Arg His Val Arg Phe Trp

210                 215                 220

Phe Leu Glu Val Ser Thr Glu Thr Lys Val Thr Ser Thr Val Pro Leu

225                 230                 235                 240

Val Gly Arg Ser Gly Ile Leu Gly Glu Leu His Asn Asn Ile Phe Cys

245                 250                 255

Gly Val Ala Cys Gly Arg Gly Arg Met Ala Gly Ser Thr Phe Cys Val

260                 265                 270

Ser Tyr Ser Gly Leu Leu Cys Gln Phe Asn Glu Lys Arg Val Leu Glu

275                 280                 285

Lys Trp Ile Asn Leu Lys Val Ser Leu Ser Ser Cys Leu Cys Val Ser

290                 295                 300

Gln Glu Leu Ile Phe Cys Gly Cys Thr Asp Gly Ile Val Arg Ile Phe

305                 310                 315                 320

Gln Ala His Ser Leu His Tyr Leu Ala Asn Leu Pro Lys Pro His Tyr

325                 330                 335

Leu Gly Val Asp Val Ala Gln Gly Leu Glu Pro Ser Phe Leu Phe His

340                 345                 350

Arg Lys Ala Glu Ala Val Tyr Pro Asp Thr Val Ala Leu Thr Phe Asp

355                 360                 365

Pro Ile His Gln Trp Leu Ser Cys Val Tyr Lys Asp His Ser Ile Tyr

370                 375                 380

Ile Trp Asp Val Lys Asp Ile Asn Arg Val Gly Lys Val Trp Ser Glu

385                 390                 395                 400

Leu Phe His Ser Ser Tyr Val Trp Asn Val Glu Val Tyr Pro Glu Phe

405                 410                 415

Glu Asp Gln Arg Ala Cys Leu Pro Ser Gly Ser Phe Leu Thr Cys Ser

420                 425                 430

Ser Asp Asn Thr Ile Arg Phe Trp Asn Leu Asp Ser Ser Pro Asp Ser

435                 440                 445

His Trp Gln Lys Asn Ile Phe Ser Asn Thr Leu Leu Lys Val Val Tyr

450                 455                 460

Val Glu Asn Asp Ile Gln His Leu Gln Asp Met Ser His Phe Pro Asp

465                 470                 475                 480

Arg Gly Ser Glu Asn Gly Thr Pro Met Asp Val Lys Ala Gly Val Arg

485                 490                 495

Val Met Gln Val Ser Pro Asp Gly Gln His Leu Ala Ser Gly Asp Arg

500                 505                 510

Ser Gly Asn Leu Arg Ile His Glu Leu His Phe Met Asp Glu Leu Val

515                 520                 525

Lys Val Glu Ala His Asp Ala Glu Val Leu Cys Leu Glu Tyr Ser Lys

530                 535                 540

Pro Glu Thr Gly Leu Thr Leu Leu Ala Ser Ala Ser Arg Asp Arg Leu

545                 550                 555                 560

Ile His Val Leu Asn Val Glu Lys Asn Tyr Asn Leu Glu Gln Thr Leu

565                 570                 575

Asp Asp His Ser Ser Ser Ile Thr Ala Ile Lys Phe Ala Gly Asn Arg

580                 585                 590

Asp Ile Gln Met Ile Ser Cys Gly Ala Asp Lys Ser Ile Tyr Phe Arg

595                 600                 605

Ser Ala Gln Gln Gly Ser Asp Gly Leu His Phe Val Arg Thr His His

610                 615                 620

Val Ala Glu Lys Thr Thr Leu Tyr Asp Met Asp Ile Asp Ile Thr Gln

625                 630                 635                 640

Lys Tyr Val Ala Val Ala Cys Gln Asp Arg Asn Val Arg Val Tyr Asn

645                 650                 655

Thr Val Asn Gly Lys Gln Lys Lys Cys Tyr Lys Gly Ser Gln Gly Asp

660                 665                 670

Glu Gly Ser Leu Leu Lys Val His Val Asp Pro Ser Gly Thr Phe Leu

675                 680                 685

Ala Thr Ser Cys Ser Asp Lys Ser Ile Ser Val Ile Asp Phe Tyr Ser

690                 695                 700

Gly Glu Cys Ile Ala Lys Met Phe Gly His Ser Glu Ile Ile Thr Ser

705                 710                 715                 720

Met Lys Phe Thr Tyr Asp Cys His His Leu Ile Thr Val Ser Gly Asp

725                 730                 735

Ser Cys Val Phe Ile Trp His Leu Gly Pro Glu Ile Thr Asn Cys Met

740                 745                 750

Lys Gln His Leu Leu Glu Ile Asp His Arg Gln Gln Gln Gln His Thr

755                 760                 765

Asn Asp Lys Lys Arg Ser Gly His Pro Arg Gln Asp Thr Tyr Val Ser

770                 775                 780

Thr Pro Ser Glu Ile His Ser Leu Ser Pro Gly Glu Gln Thr Glu Asp

785                 790                 795                 800

Asp Leu Glu Glu Glu Cys Glu Pro Glu Glu Met Leu Lys Thr Pro Ser

805                 810                 815

Lys Asp Ser Leu Asp Pro Asp Pro Arg Cys Leu Leu Thr Asn Gly Lys

820                 825                 830

Leu Pro Leu Trp Ala Lys Arg Leu Leu Gly Asp Asp Asp Val Ala Asp

835                 840                 845

Gly Leu Ala Phe His Ala Lys Arg Ser Tyr Gln Pro His Gly Arg Trp

850                 855                 860

Ala Glu Arg Ala Gly Gln Glu Pro Leu Lys Thr Ile Leu Asp Ala Gln

865                 870                 875                 880

Asp Leu Asp Cys Tyr Phe Thr Pro Met Lys Pro Glu Ser Leu Glu Asn

885                 890                 895

Ser Ile Leu Asp Ser Leu Glu Pro Gln Ser Leu Ala Ser Leu Leu Ser

900                 905                 910

Glu Ser Glu Ser Pro Gln Glu Ala Gly Arg Gly His Pro Ser Phe Leu

915                 920                 925

Pro Gln Gln Lys Glu Ser Ser Glu Ala Ser Glu Leu Ile Leu Tyr Ser

930                 935                 940

Leu Glu Ala Glu Val Thr Val Thr Gly Thr Asp Ser Gln Tyr Cys Arg

945                 950                 955                 960

Lys Glu Val Glu Ala Gly Pro Gly Asp Gln Gln Gly Asp Ser Tyr Leu

965                 970                 975

Arg Val Ser Ser Asp Ser Pro Lys Asp Gln Ser Pro Pro Glu Asp Ser

980                 985                 990

Gly Glu Ser Glu Ala Asp Leu Glu Cys Ser Phe Ala Ala Ile His Ser

995                 1000             1005

Pro Ala  Pro Pro Pro Asp Pro  Ala Pro Arg Phe Ala  Thr Ser Leu

1010                 1015                 1020

Pro His  Phe Pro Gly Cys Ala  Gly Pro Thr Glu Asp  Glu Leu Ser

1025                 1030                 1035

Leu Pro  Glu Gly Pro Ser Val  Pro Ser Ser Ser Leu  Pro Gln Thr

1040                 1045                 1050

Pro Glu  Gln Glu Lys Phe Leu  Arg His His Phe Glu  Thr Leu Thr

1055                 1060                 1065

Glu Ser  Pro Cys Arg Glu Leu  Phe Pro Ala Ala Leu  Gly Asp Val

1070                 1075                 1080

Glu Ala  Ser Glu Ala Glu Asp  His Phe Phe Asn Pro  Arg Leu Ser

1085                 1090                 1095

Ile Ser  Thr Gln Phe Leu Ser  Ser Leu Gln Lys Ala  Ser Arg Phe

1100                 1105                 1110

Thr His  Thr Phe Pro Pro Arg  Ala Thr Gln Cys Leu  Val Lys Ser

1115                 1120                 1125

Pro Glu  Val Lys Leu Met Asp  Arg Gly Gly Ser Gln  Pro Arg Ala

1130                 1135                 1140

Gly Thr  Gly Tyr Ala Ser Pro  Asp Arg Thr His Val  Leu Ala Ala

1145                 1150                 1155

Gly Lys  Ala Glu Glu Thr Leu  Glu Ala Trp Arg Pro  Pro Pro Pro

1160                 1165                 1170

Cys Leu  Thr Ser Leu Ala Ser  Cys Val Pro Ala Ser  Ser Val Leu

1175                 1180                 1185

Pro Thr  Asp Arg Asn Leu Pro  Thr Pro Thr Ser Ala  Pro Thr Pro

1190                 1195                 1200

Gly Leu  Ala Gln Gly Val His  Ala Pro Ser Thr Cys  Ser Tyr Met

1205                 1210                 1215

Glu Ala  Thr Ala Ser Ser Arg  Ala Arg Ile Ser Arg  Ser Ile Ser

1220                 1225                 1230

Leu Gly  Asp Ser Glu Gly Pro  Ile Val Ala Thr Leu  Ala Gln Pro

1235                 1240                 1245

Leu Arg  Arg Pro Ser Ser Val  Gly Glu Leu Ala Ser  Leu Gly Gln

1250                 1255                 1260

Glu Leu  Gln Ala Ile Thr Thr  Ala Thr Thr Pro Ser  Leu Asp Ser

1265                 1270                 1275

Glu Gly  Gln Glu Pro Ala Leu  Arg Ser Trp Gly Asn  His Glu Ala

1280                 1285                 1290

Arg Ala  Asn Leu Arg Leu Thr  Leu Ser Ser Ala Cys  Asp Gly Leu

1295                 1300                 1305

Leu Gln  Pro Pro Val Asp Thr  Gln Pro Gly Val Thr  Val Pro Ala

1310                 1315                 1320

Val Ser  Phe Pro Ala Pro Ser  Pro Val Glu Glu Ser  Ala Leu Arg

1325                 1330                 1335

Leu His  Gly Ser Ala Phe Arg  Pro Ser Leu Pro Ala  Pro Glu Ser

1340                 1345                 1350

Pro Gly  Leu Pro Ala His Pro  Ser Asn Pro Gln Leu  Pro Glu Ala

1355                 1360                 1365

Arg Pro  Gly Ile Pro Gly Gly  Thr Ala Ser Leu Leu  Glu Pro Thr

1370                 1375                 1380

Ser Gly  Ala Leu Gly Leu Leu  Gln Gly Ser Pro Ala  Arg Trp Ser

1385                 1390                 1395

Glu Pro  Trp Val Pro Val Glu  Ala Leu Pro Pro Ser  Pro Leu Glu

1400                 1405                 1410

Leu Ser  Arg Val Gly Asn Ile  Leu His Arg Leu Gln  Thr Thr Phe

1415                 1420                 1425

Gln Glu  Ala Leu Asp Leu Tyr  Arg Val Leu Val Ser  Ser Gly Gln

1430                 1435                 1440

Val Asp  Thr Gly Gln Gln Gln  Ala Arg Thr Glu Leu  Val Ser Thr

1445                 1450                 1455

Phe Leu  Trp Ile His Ser Gln  Leu Glu Ala Glu Cys  Leu Val Gly

1460                 1465                 1470

Thr Ser  Val Ala Pro Ala Gln  Ala Leu Pro Ser Pro  Gly Pro Pro

1475                 1480                 1485

Ser Pro  Pro Thr Leu Tyr Pro  Leu Ala Ser Pro Asp  Leu Gln Ala

1490                 1495                 1500

Leu Leu  Glu His Tyr Ser Glu  Leu Leu Val Gln Ala  Val Arg Arg

1505                 1510                 1515

Lys Ala  Arg Gly His

1520

Mus musculus WD repeat domain 62 (WDR62) nucleotide sequence, NCBI

Reference

Sequence: NM_146186

(SEQ ID NO: 3)

ggctttcccg cggccattcg ctattcgagt gggtgctggc tacaggcggc tgttaagaag   60

cgtaacggac actggtctcc gacagcatga tggctgcctt agcggccgga ggttatacgc  120

ggagtgacac gatagaaaag ctgtcctctg tcatggcggg agttccggcg cggagaaacc  180

agtcctcccc gcctcctgcc ccaccgctct gcctccggcg gcggacgcga ctcgcggcgg  240

ctcccgagga cactgtgcag aaccgggtga cacttgagaa ggtgcttggc atcacagccc  300

agaacagcag cgggctaacc tgtgaccctg gcacaggcca tgtggcctac ttagcagggt  360

gcgtggtggt ggtcttgaac cccaaggaga acaagcagca gcatatattt aacacaacca  420

ggaagtccct gagtgctctg gccttctccc cagatgggaa gtacatagtg acaggagaga  480

atggacaccg gccagctgtg cgcatctggg atgtggagga gaagactcaa gtggcagaga  540

tgctgggcca caagtatggt gtggcctgtg tggctttctc acccaatatg aagcacatcg  600

tgtccatggg ctaccaacat gacatggtcc tcaatgtttg ggattggaag aaagacattg  660

tggtggcttc caacaaggtg tcatgtagag tcatcgctct ctccttctct gaggacagca  720

gctattttgt caccgttggg aatcggcatg tgaggttttg gttcttagaa gcctctactg  780

aggccaaggt aaccagcaca gtgcccctgg taggacgctc aggcatcctg ggtgagctgc  840

acaacaatat cttttgtggc gtggcctgcg gccggggccg gatggcaggc aataccttct  900

gtgtgtccta ctctggcctc ctctgccagt tcaatgagaa aagggtgctg gacaagtgga  960

tcaacctgaa ggtctccttg tcttcctgcc tgtgtgtcag tgacgagttg atcttctgtg 1020

gatgcacaga cgggatagtc cgcatcttcc aggcccacag cctactctac ctcaccaacc 1080

tgcccaaacc acactacctg ggagtggacg tggcccacgg actggactcc agcttcctct 1140

tccacagaaa agcagaagca gtctacccag atacagtggc cctgaccttt gaccccgtcc 1200

accagtggct gtcctgtgtt tacaaagacc acagcatcta catctgggat gtcaaggaca 1260

ttgatgaagt cagcaagata tggtcagagc tcttccacag ctcctttgtc tggaatgtag 1320

aggtgtaccc tgaatttgaa gaccagagag cttgcctgcc gtccgggact tttctgactt 1380

gttcctcaga caataccatc cgcttctgga atttggatag cgcctctgac actcgatggc 1440

aaaagaacat cttcagcgat tctctgctga aggtggtcta tgtagagaat gacatccagc 1500

acctgcagga cctgtctcac ttcccagacc ggggcagtga gaacggcact cccatggata 1560

tgaaagctgg ggttcgagtc atgcaggtca gtcctgacgg ccagcacttg gcttcaggcg 1620

accgcagtgg aaatctgagg atccacgagc tgcacttcat ggatgagctg atcaaggtgg 1680

aggcccacga tgctgaggtg ctgtgcctgg agtactccaa gcctgagaca ggagtgactt 1740

tgctggcttc agccagtcgg gacagactca tccatgtgtt aaacgtggag aagaactaca 1800

acctggagca gaccctggac gaccactcct cctccatcac agccattaag tttgctggca 1860

ccagagatgt ccagatgatc agttgtggag ctgacaagag catctacttt cgcagtgccc 1920

agcaggcctc ggatggactg cactttgtcc gtacccacca cgtagcagag aagaccacct 1980

tgtatgacat ggatatcgac atcacacaga agtatgtggc agtggcctgc caggaccgca 2040

acgtaagggt ctacaacacc gtgagtggga aacagaagaa gtgctacaag ggttcccagg 2100

gcgacgaagg gtccctgctg aaggtccacg tggacccctc aggcaccttc ctggccacaa 2160

gctgctctga caaaagcatc tccttgattg acttttactc gggcgagtgt gttgccaaga 2220

tgtttggcca ttcggaaatt gtcactggca tgaagttcac ctacgactgc cgccacttga 2280

tcacagtatc tggagacagc tgtgtgttca tctggcacct gggcccggag atcaccacct 2340

gcatgaagca gcacttgctg gagatcaacc accaggagca gcagcagcag cccaaggacc 2400

agaagtggag tggccctccc agccaggaga catatgcatc cacaccaagt gagattcgtt 2460

ccctgagccc tggagagcag acagaggatg agatggagga ggaatgtgag ccagaagagt 2520

tgctgaaaac accatccaaa gacagcttgg acccagatcc tcgatgcctg ctgacaaatg 2580

gcaagctgcc actctgggca aagcggctgc taggggatga tgatgtggca gacagctcag 2640

ctttccacgc caagcgcagc taccagccac atggccgttg ggcagagcgg gctgagcagg 2700

aacccctcaa gaccatcctg gatgcctgga gcctggattc ctactttaca cccatgaagc 2760

ctgaaaatct ccaagactct gttctggact cagtagagcc acagaacctg gcaggcctgc 2820

taagtgagtg ttcactcggt aatggacaca cgtctccggg agaaggcttg gtgagctacc 2880

tacttcaccc agagttaggg agtcccaaag aggacaaccg aggccacccc tcctacctgc 2940

ctctacagag ggaagccacc gaggcaagcg aactcatcct ctgctcccca gaggcggaag 3000

tgtcacttac agggatgcac agggagtatt atgaggagga gacagaggca ggacctgaag 3060

accagcaagg cgacacctat ctcagggtct cttccgtcag ttcaaaggat cagagccccc 3120

ctgaggactc aggggagtca gaggctgaac tggagtgcag ctttgccgcc gcccacagct 3180

cagctcctca aacggaccct ggccctcacc tcaccatgac agcaggtaag ccagagtacc 3240

caagtacaga agagctttcc cagcctgagc tgccaggctt gggcaatggc tccttacccc 3300

agacacctga gcaggagaag ttcctccgcc accattttga gacacttact gatgccccta 3360

ctgaagagct ctttcatgga tccctgggag acataaagat ctcagagact gaggactatt 3420

tcttcaatcc ccggctgagc atatccaccc agttcctctc ccgcctccag aagacctcca 3480

ggtgccctcc ccgactgccc ctgcacctta tgaagtctcc agaggctcag cctgtgggcc 3540

aagggggcaa ccagcccaaa gcagggcccc tgagagcagg tactggctac atgtcctcag 3600

atgggaccaa cgtcctctct gggcagaagg ctgaagaaac tcaagaggcc ttgagtctac 3660

tggacaggaa gcctccaaca cccacatctg tactgaccac aggccgggag caaagtatct 3720

ccgccccatc ttcgtgttct tacctggagt ccacaacgag ctcacatgcc aagacgacac 3780

gcagcatctc tcttggggac agtgagggcc ctgtgacagc tgagctaccc cagtcacttc 3840

acaagccctt atcccctggc caggaactcc aagccatccc cactacagtg gcactgacct 3900

ccagcatcaa agaccacgag cctgcaccgc tttcctgggg caaccatgag gcccgagcca 3960

gcctgaaact gaccttatcc agtgtctgtg agcagctgct ctctccacct ccacaggagc 4020

cacccatcac ccatgtgtgg tctcaggaac ctgtggatgt cccacctagt atggcagtca 4080

cagtagccag cttctgtgca cccagccctg tagacatgag caccctggga ctccacagtt 4140

ctatgtttct cccaaagacc tcagcctctg ggcccctaac ccctcctgcc cacctccaac 4200

ttctagagac caggtctagg gtgcctggca gcactgctgc tctcctggag cccacccccg 4260

atgcatcagg tgtgatcgca gacagccctg gacactggga cacagaagtc cctaccccag 4320

aactcctagg cagtgtggaa tcagtcttgc acaggctgca gactgccttc caagaagctc 4380

ttgaccttta ccgcatgctg gtctccagca gccagctggg tcctgagcag cagcaggcac 4440

agaccgagct ggcctccacc ttccactgga tcctcaacca gctagaagcc agcaactgta 4500

tggctgcagc taacttggcc ccaccccaga cactgcctag cccagaccct ctgtccctac 4560

ctacactttg tcccctggcc agcccaaacc tgcaggccct gctggaacac tactcagagt 4620

tgctggtgca agcagtgagg aggaaggcaa ggggtgactg agaacctgaa gcccctctac 4680

caaagccccg ctgccctgga gaaatagatt tgtaaaccaa taaactgaca gttgtttcct 4740

ggaa                                                              4744

Mus musculus WD repeat domain 62 (WDR62) amino acid sequence, NCBI

Reference

Sequence: NM_146186

(SEQ ID NO: 4)

Met Met Ala Ala Leu Ala Ala Gly Gly Tyr Thr Arg Ser Asp Thr Ile

1               5                   10                  15

Glu Lys Leu Ser Ser Val Met Ala Gly Val Pro Ala Arg Arg Asn Gln

20                  25                  30

Ser Ser Pro Pro Pro Ala Pro Pro Leu Cys Leu Arg Arg Arg Thr Arg

35                  40                  45

Leu Ala Ala Ala Pro Glu Asp Thr Val Gln Asn Arg Val Thr Leu Glu

50                  55                  60

Lys Val Leu Gly Ile Thr Ala Gln Asn Ser Ser Gly Leu Thr Cys Asp

65                  70                  75                  80

Pro Gly Thr Gly His Val Ala Tyr Leu Ala Gly Cys Val Val Val Val

85                  90                  95

Leu Asn Pro Lys Glu Asn Lys Gln Gln His Ile Phe Asn Thr Thr Arg

100                 105                 110

Lys Ser Leu Ser Ala Leu Ala Phe Ser Pro Asp Gly Lys Tyr Ile Val

115                 120                 125

Thr Gly Glu Asn Gly His Arg Pro Ala Val Arg Ile Trp Asp Val Glu

130                 135                 140

Glu Lys Thr Gln Val Ala Glu Met Leu Gly His Lys Tyr Gly Val Ala

145                 150                 155                 160

Cys Val Ala Phe Ser Pro Asn Met Lys His Ile Val Ser Met Gly Tyr

165                 170                 175

Gln His Asp Met Val Leu Asn Val Trp Asp Trp Lys Lys Asp Ile Val

180                 185                 190

Val Ala Ser Asn Lys Val Ser Cys Arg Val Ile Ala Leu Ser Phe Ser

195                 200                 205

Glu Asp Ser Ser Tyr Phe Val Thr Val Gly Asn Arg His Val Arg Phe

210                 215                 220

Trp Phe Leu Glu Ala Ser Thr Glu Ala Lys Val Thr Ser Thr Val Pro

225                 230                 235                 240

Leu Val Gly Arg Ser Gly Ile Leu Gly Glu Leu His Asn Asn Ile Phe

245                 250                 255

Cys Gly Val Ala Cys Gly Arg Gly Arg Met Ala Gly Asn Thr Phe Cys

260                 265                 270

Val Ser Tyr Ser Gly Leu Leu Cys Gln Phe Asn Glu Lys Arg Val Leu

275                 280                 285

Asp Lys Trp Ile Asn Leu Lys Val Ser Leu Ser Ser Cys Leu Cys Val

290                 295                 300

Ser Asp Glu Leu Ile Phe Cys Gly Cys Thr Asp Gly Ile Val Arg Ile

305                 310                 315                 320

Phe Gln Ala His Ser Leu Leu Tyr Leu Thr Asn Leu Pro Lys Pro His

325                 330                 335

Tyr Leu Gly Val Asp Val Ala His Gly Leu Asp Ser Ser Phe Leu Phe

340                 345                 350

His Arg Lys Ala Glu Ala Val Tyr Pro Asp Thr Val Ala Leu Thr Phe

355                 360                 365

Asp Pro Val His Gln Trp Leu Ser Cys Val Tyr Lys Asp His Ser Ile

370                 375                 380

Tyr Ile Trp Asp Val Lys Asp Ile Asp Glu Val Ser Lys Ile Trp Ser

385                 390                 395                 400

Glu Leu Phe His Ser Ser Phe Val Trp Asn Val Glu Val Tyr Pro Glu

405                 410                 415

Phe Glu Asp Gln Arg Ala Cys Leu Pro Ser Gly Thr Phe Leu Thr Cys

420                 425                 430

Ser Ser Asp Asn Thr Ile Arg Phe Trp Asn Leu Asp Ser Ala Ser Asp

435                 440                 445

Thr Arg Trp Gln Lys Asn Ile Phe Ser Asp Ser Leu Leu Lys Val Val

450                 455                 460

Tyr Val Glu Asn Asp Ile Gln His Leu Gln Asp Leu Ser His Phe Pro

465                 470                 475                 480

Asp Arg Gly Ser Glu Asn Gly Thr Pro Met Asp Met Lys Ala Gly Val

485                 490                 495

Arg Val Met Gln Val Ser Pro Asp Gly Gln His Leu Ala Ser Gly Asp

500                 505                 510

Arg Ser Gly Asn Leu Arg Ile His Glu Leu His Phe Met Asp Glu Leu

515                 520                 525

Ile Lys Val Glu Ala His Asp Ala Glu Val Leu Cys Leu Glu Tyr Ser

530                 535                 540

Lys Pro Glu Thr Gly Val Thr Leu Leu Ala Ser Ala Ser Arg Asp Arg

545                 550                 555                 560

Leu Ile His Val Leu Asn Val Glu Lys Asn Tyr Asn Leu Glu Gln Thr

565                 570                 575

Leu Asp Asp His Ser Ser Ser Ile Thr Ala Ile Lys Phe Ala Gly Thr

580                 585                 590

Arg Asp Val Gln Met Ile Ser Cys Gly Ala Asp Lys Ser Ile Tyr Phe

595                 600                 605

Arg Ser Ala Gln Gln Ala Ser Asp Gly Leu His Phe Val Arg Thr His

610                 615                 620

His Val Ala Glu Lys Thr Thr Leu Tyr Asp Met Asp Ile Asp Ile Thr

625                 630                 635                 640

Gln Lys Tyr Val Ala Val Ala Cys Gln Asp Arg Asn Val Arg Val Tyr

645                 650                 655

Asn Thr Val Ser Gly Lys Gln Lys Lys Cys Tyr Lys Gly Ser Gln Gly

660                 665                 670

Asp Glu Gly Ser Leu Leu Lys Val His Val Asp Pro Ser Gly Thr Phe

675                 680                 685

Leu Ala Thr Ser Cys Ser Asp Lys Ser Ile Ser Leu Ile Asp Phe Tyr

690                 695                 700

Ser Gly Glu Cys Val Ala Lys Met Phe Gly His Ser Glu Ile Val Thr

705                 710                 715                 720

Gly Met Lys Phe Thr Tyr Asp Cys Arg His Leu Ile Thr Val Ser Gly

725                 730                 735

Asp Ser Cys Val Phe Ile Trp His Leu Gly Pro Glu Ile Thr Thr Cys

740                 745                 750

Met Lys Gln His Leu Leu Glu Ile Asn His Gln Glu Gln Gln Gln Gln

755                 760                 765

Pro Lys Asp Gln Lys Trp Ser Gly Pro Pro Ser Gln Glu Thr Tyr Ala

770                 775                 780

Ser Thr Pro Ser Glu Ile Arg Ser Leu Ser Pro Gly Glu Gln Thr Glu

785                 790                 795                 800

Asp Glu Met Glu Glu Glu Cys Glu Pro Glu Glu Leu Leu Lys Thr Pro

805                 810                 815

Ser Lys Asp Ser Leu Asp Pro Asp Pro Arg Cys Leu Leu Thr Asn Gly

820                 825                 830

Lys Leu Pro Leu Trp Ala Lys Arg Leu Leu Gly Asp Asp Asp Val Ala

835                 840                 845

Asp Ser Ser Ala Phe His Ala Lys Arg Ser Tyr Gln Pro His Gly Arg

850                 855                 860

Trp Ala Glu Arg Ala Glu Gln Glu Pro Leu Lys Thr Ile Leu Asp Ala

865                 870                 875                 880

Trp Ser Leu Asp Ser Tyr Phe Thr Pro Met Lys Pro Glu Asn Leu Gln

885                 890                 895

Asp Ser Val Leu Asp Ser Val Glu Pro Gln Asn Leu Ala Gly Leu Leu

900                 905                 910

Ser Glu Cys Ser Leu Gly Asn Gly His Thr Ser Pro Gly Glu Gly Leu

915                 920                 925

Val Ser Tyr Leu Leu His Pro Glu Leu Gly Ser Pro Lys Glu Asp Asn

930                 935                 940

Arg Gly His Pro Ser Tyr Leu Pro Leu Gln Arg Glu Ala Thr Glu Ala

945                 950                 955                 960

Ser Glu Leu Ile Leu Cys Ser Pro Glu Ala Glu Val Ser Leu Thr Gly

965                 970                 975

Met His Arg Glu Tyr Tyr Glu Glu Glu Thr Glu Ala Gly Pro Glu Asp

980                 985                 990

Gln Gln Gly Asp Thr Tyr Leu Arg Val Ser Ser Val Ser Ser Lys Asp

995                 1000                 1005

Gln Ser  Pro Pro Glu Asp Ser  Gly Glu Ser Glu Ala  Glu Leu Glu

1010                 1015                 1020

Cys Ser  Phe Ala Ala Ala His  Ser Ser Ala Pro Gln  Thr Asp Pro

1025                 1030                 1035

Gly Pro  His Leu Thr Met Thr  Ala Gly Lys Pro Glu  Tyr Pro Ser

1040                 1045                 1050

Thr Glu  Glu Leu Ser Gln Pro  Glu Leu Pro Gly Leu  Gly Asn Gly

1055                 1060                 1065

Ser Leu  Pro Gln Thr Pro Glu  Gln Glu Lys Phe Leu  Arg His His

1070                 1075                 1080

Phe Glu  Thr Leu Thr Asp Ala  Pro Thr Glu Glu Leu  Phe His Gly

1085                 1090                 1095

Ser Leu  Gly Asp Ile Lys Ile  Ser Glu Thr Glu Asp  Tyr Phe Phe

1100                 1105                 1110

Asn Pro  Arg Leu Ser Ile Ser  Thr Gln Phe Leu Ser  Arg Leu Gln

1115                 1120                 1125

Lys Thr  Ser Arg Cys Pro Pro  Arg Leu Pro Leu His  Leu Met Lys

1130                 1135                 1140

Ser Pro  Glu Ala Gln Pro Val  Gly Gln Gly Gly Asn  Gln Pro Lys

1145                 1150                 1155

Ala Gly  Pro Leu Arg Ala Gly  Thr Gly Tyr Met Ser  Ser Asp Gly

1160                 1165                 1170

Thr Asn  Val Leu Ser Gly Gln  Lys Ala Glu Glu Thr  Gln Glu Ala

1175                 1180                 1185

Leu Ser  Leu Leu Asp Arg Lys  Pro Pro Thr Pro Thr  Ser Val Leu

1190                 1195                 1200

Thr Thr  Gly Arg Glu Gln Ser  Ile Ser Ala Pro Ser  Ser Cys Ser

1205                 1210                 1215

Tyr Leu  Glu Ser Thr Thr Ser  Ser His Ala Lys Thr  Thr Arg Ser

1220                 1225                 1230

Ile Ser  Leu Gly Asp Ser Glu  Gly Pro Val Thr Ala  Glu Leu Pro

1235                 1240                 1245

Gln Ser  Leu His Lys Pro Leu  Ser Pro Gly Gln Glu  Leu Gln Ala

1250                 1255                 1260

Ile Pro  Thr Thr Val Ala Leu  Thr Ser Ser Ile Lys  Asp His Glu

1265                 1270                 1275

Pro Ala  Pro Leu Ser Trp Gly  Asn His Glu Ala Arg  Ala Ser Leu

1280                 1285                 1290

Lys Leu  Thr Leu Ser Ser Val  Cys Glu Gln Leu Leu  Ser Pro Pro

1295                 1300                 1305

Pro Gln  Glu Pro Pro Ile Thr  His Val Trp Ser Gln  Glu Pro Val

1310                 1315                 1320

Asp Val  Pro Pro Ser Met Ala  Val Thr Val Ala Ser  Phe Cys Ala

1325                 1330                 1335

Pro Ser  Pro Val Asp Met Ser  Thr Leu Gly Leu His  Ser Ser Met

1340                 1345                 1350

Phe Leu  Pro Lys Thr Ser Ala  Ser Gly Pro Leu Thr  Pro Pro Ala

1355                 1360                 1365

His Leu  Gln Leu Leu Glu Thr  Arg Ser Arg Val Pro  Gly Ser Thr

1370                 1375                 1380

Ala Ala  Leu Leu Glu Pro Thr  Pro Asp Ala Ser Gly  Val Ile Ala

1385                 1390                 1395

Asp Ser  Pro Gly His Trp Asp  Thr Glu Val Pro Thr  Pro Glu Leu

1400                 1405                 1410

Leu Gly  Ser Val Glu Ser Val  Leu His Arg Leu Gln  Thr Ala Phe

1415                 1420                 1425

Gln Glu  Ala Leu Asp Leu Tyr  Arg Met Leu Val Ser  Ser Ser Gln

1430                 1435                 1440

Leu Gly  Pro Glu Gln Gln Gln  Ala Gln Thr Glu Leu  Ala Ser Thr

1445                 1450                 1455

Phe His  Trp Ile Leu Asn Gln  Leu Glu Ala Ser Asn  Cys Met Ala

1460                 1465                 1470

Ala Ala  Asn Leu Ala Pro Pro  Gln Thr Leu Pro Ser  Pro Asp Pro

1475                 1480                 1485

Leu Ser  Leu Pro Thr Leu Cys  Pro Leu Ala Ser Pro  Asn Leu Gln

1490                 1495                 1500

Ala Leu  Leu Glu His Tyr Ser  Glu Leu Leu Val Gln  Ala Val Arg

1505                 1510                 1515

Arg Lys  Ala Arg Gly Asp

1520

The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.