US007998744B2

(12) United States Patent (10) Patent N0.: US 7,998,744 B2 Stevenson et a]. (45) Date of Patent: Aug. 16,2011

(54) METHODS FOR DETERMINING Irizarry, et al., “Comprehensive high-throughput arrays for relative DYSREGULATION OF METHYLATION OF methylation (CHARM)”, Genome Research, 18:780-790, (2008). BRAIN EXPRESSED ON THE X James, et al., “Abnormal Trasmethylation/transsulfuration Metabo TO DIAGNOSE AUTISM lism and DNA Hypomethylation Among Parents of Children with SPECTRUM DISORDERS Autism”, JAutism Dev Disord, 38: 1966-1975, (2008). James, et al., “Metabolic biomarkers of increased oxidative stress and (75) Inventors: Roger E. Stevenson, Greenwood, SC impaired methylation capacity in children with autism”, Am J Clin (US); Julie R. Jones, Greenville, SC Nutr, 80:1611-1617, (2004). (US); Cindy D. Skinner, Greenwood, Jones, et al., “Hypothesis: Dysregulation of Methylation of Brain SC (US); Michael J. Friez, Cross Hill, Expressed Genes on the and Autism Spectrum Dis sc (Us) orders”, American Journal of Medical Genetics PartA, 146A:2213 2220, (2008). (73) Assignee: Greenwood Genetic Center, Inc., Mill, et al., “Epigenomic Pro?ling Reveals DNA-Methylation Greenwood, SC (US) Changes Associated with Maj or Psychosis”, The American Journal ofHuman Genetics, 82, 696-711, Mar. 2008. ( * ) Notice: Subject to any disclaimer, the term of this Morrow, et al., “Identifying Autism Loci and Genes by Tracing patent is extended or adjusted under 35 Recent Shared Ancestry”, Science, vol. 321, Jul. 11, 2008. Nagarajan, et al., “Reduced MeCP2 expression is frequent in autism USC 154(b) by 0 days. frontal cortex and correlates with aberrant MECP2 promoter methylation”, Epigenetics, 1(4):e1-11, (2006). (21) Appl.No.: 12/510,316 Nishimura, et al., “Genome-wide expression pro?ling of lymphoblastoid cell lines distinguishes different forms of autism and (22) Filed: Jul. 28, 2009 reveals shared pathways”, Human Molecular Genetics, vol. 16, No. 14, pp. 1682-1698, (2007). (65) Prior Publication Data Shimabukuro, et al., “Global hypomethylation of peripheral US 2010/0029009 A1 Feb. 4, 2010 leukocyte DNA in male patients with schizophrenia: A potential link between epigenetics and schizophrenia”, Journal of Psychiatric Related U.S. Application Data Research, 41, pp. 1042-1046, (2007). Qiao Y, et al., “Autism-associated familial microdeletion of Xpll. (60) Provisional application No. 61/084,063, ?led on Jul. 22”, Clin Genet, 74:134-144, (2008). 28, 2008. ArticleiAbrahams et al., “Advances in autism genetics: on the threshold of a new neurobiology,” Nature Reviews/Genetics, vol. 9, (51) Int. Cl. May 2008, pp. 341-355. G01N 33/50 (2006.01) Article-Allen et al., “Methylation of Hpail and Hhal Sites Near the (52) U.S. Cl...... 436/86; 436/94; 436/91; 436/501 Polymorphic CAG Repeat in the Human Androgen-Receptor Correlates with X Chromosome Inactivation,” Am. J Hum. Genet., (58) Field of Classi?cation Search ...... None vol. 51, 1992, pp. 1229-1239. See application ?le for complete search history. Article-Campbell et al., “A genetic variant that disrupts ME T tran scription is associated with autism,” PNAS, vol. 103, No. 45, Nov. 7, (56) References Cited 2006, pp. 16834-16839. Article-C. Sue Carter, “Sex differences in oxytocin and FOREIGN PATENT DOCUMENTS vasopressin: Implications for autism spectrum disorders?,” W0 WO 2005/024064 A2 3/2005 Behavioural Brain Research 176, 2007, pp. 170-186. ArticleiEngel et al., “Uniparental Disomy, Isodisomy, and Imprint OTHER PUBLICATIONS ing: Probable Effects in Man and Strategies for Their Detection,” Evans, Julie C. et al. “Early onset seizures and Rett-like features American Journal ofMedical Genetics, vol. 40, 1991, pp. 432-439. associated with mutations in CDKLS.” European Journal of Human ArticleiFolstein et al ., “Genetics of Autism: Complex Aetiology for a Heterogeneous Disorder,” Nature Reviews/Genetics, vol. 2, Dec. Genetics (2005) 13 1113-1120.* 2001, pp. 943-955. Kleefstra, T. et al. “Zinc ?nger 81 (ZNF81) mutations associated with X-linked mental retardation.” Journal of Medical Genetics (2004) 41 (Continued) 394-399.* McClean, Phillip. “DNA-Basics of Structure and Analysis.” accessed online at on Dec. 8, 2010, 1998* Assistant Examiner * Christopher A Hixson Purcell, A. E. et al. “Postmortem brain abnormalities of the glutamate neurotransmitter system in autism.” Neurology (2001) 57 1618 (74) Attorney, Agent, or Firm * Dority & Manning, P.A. 1628* Samuels, Benjamin Adam et al. “Cdl<5 Promotes Synaptogenesis by Regulating the Subcellular Distribution of the MAGUK Family (57) ABSTRACT Member CASK.” Neuron (2007) 56 823-837.* The discovery that alterations in methylation, which can Chiurazzi, et al., “XLMR genes: updated 2007”, European Journal of cause one or more genes on the single X chromosome in Human Genetics, 16, pp. 422-434, (2008). Craig, et al., “Application of microarrays to the analysis of the inac males to be partially silenced or overexpressed, constitute a tivation status of human X-linked genes expressed in lymphocytes”, predisposition to autism spectrum disorders is generally dis European Journal of Human Genetics, 12, pp. 639-646, (2004). closed herein. These alterations provide the rationale and Hu, et al., “Gene expression pro?ling of lymphoblastoid cell lines basis for methods to diagnose autism spectrum disorders. from monozygotic twins discordant in severity of autism reveals differential regulation of neurologically relevant genes”, BMC Genomics, 7: 118, (2006). 25 Claims, 7 Drawing Sheets US 7,998,744 B2 Page 2

OTHER PUBLICATIONS ArticleiN. Carolyn Schanen, “Epigenetics of autism spectrum dis ArticleiCM. Freitag, “The genetics of autistic disorders and its orders,” Human Molecular Genetics, vol. 15, Review Issue No. 2, clinical relevance: a review of the literature,” Molecular Psychiatry, 2006, pp. R138-R150. ArticleiSchendel et al., “Birth Weight and Gestational Age Char vol. 12, 2007, pp. 2-22. acteristics of Children with Autism, Including a Comparison with ArticleiFriez et al., “Recurrent Infections, Hypotonia, and Mental Other Developmental Disabilities,” Pediatrics, vol. 121, No. 6, Jun. Retardation Caused by Duplication of MECPZ and Adjacent Region 2008, pp. 1155-1165. in Xq28,” Pediatrics, vol. 118, No. 6, Dec. 2006, pp. e1687-e1695. ArticleiAlbert Schinzel, “Microdeletion syndromes, balanced ArticleiM. E. Grant, “DNA methylation and mental retardation,” translocations, and gene mapping,”Journal ofMedical Genetics, vol. Clin. Genet., vol. 73, 2008, pp. 531-534. 25, 1988, pp. 454-462. Article (Letters to the Editor)iJohn P. Hussman, “Suppressed ArticleiSchroer et a1, “Autism and Maternally Derived Aberrations Gabaergic Inhibition as a Common Factor in Suspected Etiologies of of Chromosome 15q.” American Journal of Medical Genetics. vol. Autism,” Journal of Autism and Developmental Disorders, vol. 31, 76, 1998, pp. 327-336. No.2, 2001,pp. 247-248. ArticleiSebat et al., Strong Association of De Novo Copy Number ArticleiJames et al., “Metabolic Endophenotype and Related Geno Mutations with Autism, Science, vol. 316, Apr. 20, 2007, pp. 445 types are Associated with Oxidative Stress in Children with Autism,” 449. American Journal of Medical Genetics Part B (Neuropsychiatric ArticleiThomas et al., “Xp deletions associated with autism in three Genetics), vol. 141B, 2006, pp. 947-956. females,” Hum. Genet., vol. 104, 1999, pp. 43-48. ArticleiJiang et al., “A Mixed Epigenetic/ Genetic Model for ArticleiUllmann et al., “Array CGH Identi?es Reciprocal 16p13.1 Oligogenic Inheritance of Autism with a Limited Role for UBESA,” Duplications and Deletions That Predispo se to Autism and/ or Mental American Journal ofMedical Genetics, vol. 131A, 2004, pp. 1-10. Retardation,” Human Mutation, vol. 28, No. 7, 2007, pp. 674-682. ArticleiLivak et al., “Analysis of Relative Gene Expression Data ArticleiVan Esch et al., “Duplication of the MECPZ Region Is a Using Real-Time Quantitative PCR and the Z'AACT Frequent Cause of Severe Mental Retardation and Progressive Neu Method,”Methods, vol. 25, 2001, pp. 402-408. rological Symptoms in Males,”Am. J Hum. Genet. , vol. 77, 2005, pp. ArticleiLubs et al., “XLMR Syndrome Characterized by Multiple 442-453. Respiratory Infections, Hypertelorism, Severe CNS Deterioration ArticleiWeiss et al., “Association between Microdeletion and and Early Death Localizes to Distal Xq28,”American Journal of Microduplication at 16p11.2 andAutism,” TheNeW England Journal Medical Genetics, vol. 85, 1999, pp. 243-248. ofMedicine, vol. 358, No. 7, Feb. 14, 2008, pp. 667-675. ArticleiH. A. Lubs, “A Marker X Chromosome,” Am. J Hum. ArticleiWilson et al., “EpigenomicsiMapping the Methylome,” Genet., vol. 21, 1969, pp. 231-344. Cell Cycle, vol. 5, No. 2, Jan. 16, 2006, pp. 155-158. ArticleiMarshall at al., “Structural Variation of in ArticleiYu et al., “Fragile X Genotype Characterized by an Autism Spectrum Disorder,” TheAmerican Journal ofHuman Genet Unstable Region of DNA,” Science, vol. 252, May 24, 1991, pp. ics, vol. 82, Feb. 2008, pp. 477-488. 1 179- 1 181 . ArticleiMartin et al., “Analysis of Linkage Disequilibrium American Psychiatiric Association: Diagnostic and Statistical in-Aminobutyric Acid Receptor Subunit Genes in Autistic Manual of Mental Disorders, Fourth Edition, Text Revision. Ameri Disorder.,”American Journal ofMedical Genetics (Neuropsychiatric can Psychiatric Association, Washington, DC, 2000. Genetics), vol. 96, 2000, pp. 43-48. ArticleiBailey et al., “Autism as a strongly genetic disorder: evi ArticleiMeinkoth et al., “Hybridization of Nucleic Acids Immobi dence from a British twin study,” Psychological Medicine, vol. 25, lized on Solid Supports,”Analytical Biochemistry, vol. 138, 1984, pp. 1995, pp. 63-77. 267-284. ArticleiChen et al., “Establishment and Maintenance of DNA ArticleiMeins et al., “Submicroscopic duplication in Xq28 causes Methylation Patters in Mammals,” CTMI, vol. 301, 2006, pp. 179 increased expression of the ME CP2 gene in a boy with severe mental 201. retardation and features of Rett syndrome,” J Med. Genet., vol. 42, ArticleiForstein, et al., Infantile autism: a genetic study of 21 twin 2005, 6 pages. pairs. J. Child Psychol. Psychiatry 18:297, 1977. ArticleiMiles et al., “Development and Validation of a Measure of ArticleiMary F. Lyon, “Gene Action in the X-chromosome of the Dysmorphology: Useful for Autism Subgroup Classi?cation,” Mouse (Mus musculus L.),” Nature, vol. 190, Apr. 22, 1961, pp. American Journal ofMedical Genetics PartA, vol. 145A, 2008, pp. 372-373. 1 101-11 16. ArticleiRitvo et al., “Evidence for Autosomal Recessive Inherit ArticleiMorrow et al., “Identifying Autism Loci and Genes by ance in 46 Families with Multiple Incidences of Autism,” Am. J Tracing Recent SharedAncestry,” Science, vol. 321, Jul. 11, 2008, pp. Psychiatry, vol. 142, No. 2, Feb. 1985, pp. 187-192. 218-223. ArticleiDavid H. Skuse, “Imprinting, the X-chromosome, and the ArticleiNelson et al., “Neuropeptides and Neurotrophins in Neo Male Brain: Explaining Sex Differences in the Liability of Autism,” natal Blood of Children with Autism or Mental Retardation,” Ann. Pediatric Research, vol. 47, No. 1, Jan. 2000, 13 pages. Neurol., vol. 49, 2001, pp. 597-606. ArticleiSteffenburg et al., “A Twin Study of Autism in Denmark, ArticleiNicholls et al., “Genetic imprinting suggested by maternal Finland, Iceland, Norway and Sweden,” J Child Psychol., vol. 30, heterodisomy in non-deletion Prader-Willi syndrome,” Nature, vol. No. 3, 1989, pp. 405-416. 342, Nov. 16, 1989, pp. 281-285. Stevenson, et al., X-Linked Mental Retardation. Oxford Univ. Press, ArticleiOberlé et a1 ., “Instability of a 5 50- DNA Segments NewYork, 2000. and Abnormal Methylation in Fragile X Syndrome,” Research ArticleiGrant R. Sutherland, “Fragile Sites on Human Chromo Article, May 25, 1991, pp. 1097-1102. somes: Demonstration of Their Dependence on the Type of Tissue ArticleiPai et al., “A new X linked recessive syndrome of mental Culture Medium,” Science, vol. 197, 1977, pp. 265-266. retardation and mild dysmorphism maps to Xq28,” J Med. Genet., vol. 34, 1997, pp. 529-534. * cited by examiner

US. Patent Aug. 16, 2011 Sheet 3 017 US 7,998,744 B2

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-2502 (EIWJS 901) MISNEIlNI US 7,998,744 B2 1 2 METHODS FOR DETERMINING al. (2001) found several neuropeptides and neurotrophins DYSREGULATION OF METHYLATION OF (vasoactive intestinal peptide, calcitonin gene-related pep BRAIN EXPRESSED GENES ON THE X tide, brain-derived neurotrophic factor and neurotrophin 4/5) CHROMOSOME TO DIAGNOSE AUTISM to be elevated in neWborn blood spots from infants Who Were SPECTRUM DISORDERS later found to have autism. Con?rmation of these ?ndings has not been reported by other investigators. More recently, CROSS REFERENCE TO RELATED James et al. (2006, 2008) have proposed that metabolic vul APPLICATIONS nerability to oxidative stress may be an autism susceptibility factor, and Carter (2007) has suggested that the skeWed male: The present application claims ?ling bene?t of US. pro female ratio in autism may be explained by sex-speci?c visional patent application having the Ser. No. 61/084,063 responses to the neuropeptides, oxytocin and vasopressin. ?led on Jul. 28, 2008 entitled “Methods for Determining The genetic contribution to the causation/predisposition to Dysregulation of Methylation of Brain Expressed Genes on autism is considered to be substantial on the basis of high the X Chromosome to Diagnose Autism Spectrum Disor concordance in monoZygous tWins, a recurrence rate of about ders,” Roger E. Stevenson, Julie R. Jones, Cindy D. Skinner, 5% among siblings, the uniquely high malezfemale ratio and Michael J. FrieZ, inventors, Which is incorporated by (about 4:1 in most studies), the co-occurrence of autism With reference herein in its entirety. a number of single gene disorders and chromosome aberra tions, and the presence of behavioral disturbances among ?rst BACKGROUND degree relatives. These considerations aside, no speci?c 20 genetic cause has been found to explain more than 1-2% of Autism spectrum disorders include a group of serious and autism cases, and overall only in 10-20% of autism cases can enigmatic neurobehavioral disorders that usually become a cause be determined. apparent early in childhood and persist as lifelong disabilities. The strongest evidence for a heritable basis of autism Disturbances in three categories of behavior (reciprocal comes from tWin studies. Overall, these studies shoW high social interactions, verbal and nonverbal communications, 25 concordance of autism among monoZygous (MZ) tWins and and age appropriate activities and interests) are considered loW concordance among same sex diZygous (DZ) tWins, hallmarks of autism. resulting in greater than ninety percent heritability estimates. The number of children diagnosed With autism has greatly Four prominent studies dealing speci?cally With autistic dis increased in recent decades. At the midpoint of the 20th order (narroWly de?ned to exclude Asperger disorder and Century, autism Was narroWly de?ned and uncommonly diag 30 pervasive developmental disorder) report concordance of nosed (With a prevalence of about four per 10,000). Greater 36-96% in MZ tWins and 0-30% in same-sex DZ tWins (Fol awareness, availability of services, changes in diagnostic cri stein and Rutter 1977, Ritvo et al. 1985, Steffenburg et al. teria to include a broader spectrum of neurodevelopmental 1989, Bailey et al. 1995). abnormalities, and possibly other factors have contributed to Chromosomal abnormalities have been found in a number the ten-fold or greater increase in the frequency With Which 35 of individuals With autism. These include marker chromo autism spectrum disorder is diagnosed (current prevalence of somes, microdeletions and microduplications, rearrange 40-60 per 10,000). One extraordinary aspect of the epidemi ments, and autosomal fragile sites (Schroer et al. 1998, Ull ology is the three-fold to six-fold excess of males. mann et al. 2007, MorroW et al. 2008, Freitag 2007, Sebat et Autism appears causally heterogeneous. Although scien al. 2007, Weiss et al. 2008, Marshall et al. 2008). Taken tists have long abandoned the idea that autism is caused by 40 together, these observations do not suggest a single underly humorless and rigid parenting, they have been unable to iden ing chromosomal aberration, but rather that a variety of chro tify speci?c cause(s) in any substantial proportion of cases. mosomal changes may disturb brain development and func Standardized criteria for autism (DSM IV-TR) may be tion in a Way that leads to autism. Chromosome aberrations assessed based on parental, caregiver, and/ or examiner obser observed in more than one case of autism include: lq dele vations using the Autism Diagnostic IntervieW, Revised 45 tion, 15q deletion or duplication, 16p deletion or duplication, (ADI-R) and Autism Diagnostic Observation Schedule 17p deletion, 18q deletion, 22q deletion, and Xq28 deletion or (ADOS). A small percentage of patients Will have coexisting duplication. genetic disorders and an even smaller percentage a history of Several single gene entities have been found in association an environmental insult. With autism. Most notably are the fragile X syndrome, Rett Only meager evidence exists to suggest that environmental 50 syndrome, tuberous sclerosis, phenylketonuria, Angelman insults play a signi?cant role in the causation of autism. syndrome, and adenylosuccinate lyase de?ciency. Mutations Prenatal and postnatal infections (rubella, cytomegalovirus, in the neuroligins, neurexins, GABA receptors, reelin, herpes) have been documented in a feW cases. Little evidence ENGRAILED 2, SLC6A4 serotonin transporter, glutamate exists to suggest injury in the perinatal period as a causative receptor 6, DHCR7, DLXS, MET, RPL10, SHANK3, CNT factor, although loW birth Weight and premature birth has 55 NAP2, BDNF, and other genes have been associated With been noted as a risk factor (Schendel et al. 2008). Although autism and are properly considered candidate autism suscep autism has been reported among infants With prenatal expo tibility genes (Schanen 2006, Freitag 2007, Abrahams and sure to thalidomide, cocaine, alcohol, and valproate, most GeschWind 2008). infants prenatally exposed to these and other drugs or chemi A number of genome-Wide screens to identify chromo cal agents do not develop autism. Considerable attention has 60 somal regions linked to autism susceptibility have been been given to the concept that immunizations for measles, reported. The linkage evidence appears greatest for one or mumps, and rubella (MMR) might cause autism. HoWever, tWo loci on chromosome 7q, and loci on chromosomes lq, 2q, repeated study has not provided evidence to support this 3p, 3q, Sp, 6q, 9q, 11p, 15q, 16p, 17q, and 19p. The study of theory. candidate genes Within the linkage regions has failed to iden No laboratory ?nding is consistently abnormal, although 65 tify genes that clearly cause or strongly predispose to autism. plasma serotonin levels may be elevated in affected individu Although several X-linked genes (NLGN3, NLGN4, als and ?rst-degree relatives. In a promising study, Nelson et RPL10, FMR1, MECP2 and ten others noted With an asterisk US 7,998,744 B2 3 4 in the Table and FIG. 1) have been associated With autism or autism. Jiang et al. (2004) have proposed a mixed epigenetic autistic features in males, X-chromosomal loci have not been and genetic model for autism based on abnormal DNA implicated in autism by linkage analyses. This may be methylation at the 5' CpG island of UBE3A, on chromosome explained in part by existence of multiple X loci of impor 15, in one autism brain, decreased E6-AP (product of tance (heterogeneity) or by the uninformative nature of the UBE3A) in several autism brains, and sharing of paternal 15q sib-pairs used in the analysis. Of greater importance is that alleles in one cohort of autism sibpairs. MECP2, the X chro linkage analysis Would not detect epigenetic modi?cations of mosomal gene responsible for Rett syndrome, exerts its effect gene(s) on the X chromosome. by binding individual CpGs and recruiting other repression The recurrence rate in brothers and sisters of affected per related factors. A primary target of this effect is UBE3A, the sons is 3-8%. This recurrence rate is less than expected if all gene responsible for Angelman syndrome. This may explain cases Were caused by autosomal recessive gene mutations the clinical similarities betWeen Rett and Angelman syn (25%) or autosomal dominant gene mutations (50%). The drome. Hussman (2001) and Martin et al. (2000) have sug rate is not unlike that found in conditions considered to have gested that GABAergic inhibition predisposes to autism. multifactorial causation, such as neural tube defects and cleft Three GABA receptor subunits (GABA 0t, [3, y) are located lip/palate. Multifactorial causation implies a collaboration near the imprinted region of chromosome 15q. betWeen multiple genetic factors and environmental in?u Less Work has been performed on the putative autism loci ences. on 7q. Campbell et al. (2006) have found the C allele in the Soon after the discovery of the correct number of chromo promotor of MET receptor tyrosine kinase in the 7q3 1 autism somes in humans, the importance of gene dosage to human candidate region to be a risk factor in autism. The C allele development and health Was appreciated. Inactivation of an X 20 reduces MET expression and alters binding of transcription chromosome in normal females, as re?ected in formation of factors. Schanen (2006) revieWed the status of genes in the the sex chromatin body, Was recogniZed and considered to imprinted cluster on 7q21.3 ?nding several attractive candi equalize (at 1N) the dosage of X-linked genes betWeen dates, but none that had conclusive evidence for autism sus females and males (Lyon 1961). Trisomies, Which augmented ceptibility. Freitag (2007) suggested that RELN, LAMBl, the gene dosage (to 3N) for genes on individual chromo 25 and EN2 Were perhaps the three most promising autism can somes, Were found to be uniformly associated With mental didate genes on chromosome 7. defect and malformation, in most cases lethal before birth. Skuse (2000) has proposed that at least one loci on the X Deletions of small segments of the genome, Which reduced chromosome is imprinted, being expressed only from the gene dosage segmentally to 1N, Were likeWise found to be paternal chromosome. Patients With Turner syndrome (45,X) associated With malformations and mental retardation4e.g., 30 Who have a maternal X are more vulnerable to impairments in Cri du Chat, Miller-Dieker, Smith Magenis, velocardiofacial, language and social interactions, Whereas those With a pater Wolff-Hirschhorn, and other microdeletion syndromes nal X may be protected, having signi?cantly better social (SchinZel 1988, Pai et al. 2002). adjustment and superior language skills. Xp deletions of the These ?ndings and others reinforce the concept that dial boundary betWeen the Xp pseudoautosomal region and lelic expression of autosomal genes and monoallelic expres 35 marker DXS7103 have been found in three females With sion for genes on the sex chromosomes are the norm for autism (Thomas et al. 1999). humans. Silencing and overexpression of genes With normal DNA In the 1980s and 1990s, evidence Was found, initially in sequence on the single X chromosome in males and the active mice and then in humans, that diallelic expression Was not the X chromosome in females have been identi?ed as biological norm for all autosomal gene loci (N icholls et al. 1989, Engel 40 phenomena of signi?cance. The most prominent example of and DeLoZier-Blanchet 1991). Rather, expression of certain gene silencing occurs in the fragile X syndrome. The pathol genes, found in speci?c clusters on several autosomes, Was ogy is based on expansion and methylation of a CGG repeat noted to be monoallelic, While the second allele, although in the 5' untranslated region of the FMRl gene. Methylation present, Was silenced. Further, the expressed gene Was con and silencing of expression typically occur When the CGG sistently derived from the same parent and the silenced gene 45 repeat number exceeds 200 copies (Lubs 1969, Sutherland from the other. Such parent speci?c in?uence on gene activity 1977, Oberle et al. 1991, Yu et al. 1991). Overexpression of Was designated imprinting. MECP2 caused by duplication of the gene and adjacent Appreciation for the role of imprinted genes in the causa region in Xq28 has been documented in numerous cases of tion of human disease has groWn steadily since these initial males With mental retardation, hypotonia, and recurrent ?ndings. Currently, at least tWelve human chromosomes (1, 50 infections (Pai et al. 1997, Lubs et al. 1999, Van Esch et al. 4, 6, 7, 8, 10, 11, 14, 15, 18, 19, and 20) are knoWn to harbor 2005, FrieZ et al. 2006). Males With duplication of MECP2 gene loci that are imprinted. Genome-Wide, hoWever, less likeWise have an increased risk of autism spectrum disorders than 1% of autosomal genes appear to be imprinted, and (Meins et al. 2005, FrieZ et al. 2006). hence have monoallelic and parent speci?c expression. The various chromosomal alterations and gene mutations Several lines of evidence link imprinted regions and autism 55 currently reported in association With autism indicate the predisposition. Schanen (2006) has pointed out that the gene genetically heterogeneous nature of autism and taken loci With suggestive or possible linkage to autism overlap or together account for only a minority (less than 20%) of cases. are in close proximity to regions subject to genomic imprint ing. The evidence is strongest for loci on 7q and 15q. Dupli SUMMARY cation of proximal 15q, When derived from the mother, has 60 been associated With autism. When duplication from the same Objects and advantages of the invention Will be set forth in region is derived from the father, autism does not occur. part in the folloWing description, or may be obvious from the Deletions of the maternal copy of the same region on 15q, description, or may be learned through practice of the inven paternal disomy, and mutations of UBE3A cause Angelman tion. syndrome, a disorder that typically presents With autistic 65 According to one embodiment, a method for determining features and mental retardation. Hence, silencing or overex predisposition to or diagnosis of autism spectrum disorder in pression of genes from this region appears to predispose to an individual is generally disclosed. For example, the method US 7,998,744 B2 5 6 can include determining a cytosine methylation level of at Where each probe being speci?c for a biomarker (e.g., least three different test polynucleotide sequences. Each test mRNA, protein, etc.) of an X-chromosome gene. polynucleotide sequence comprises at least one gene on the X Other features and aspects of the present invention are chromosome, and each test polynucleotide sequence is discussed in greater detail beloW. obtained from the individual. The method also includes comparing the cytosine methy BRIEF DESCRIPTION OF THE FIGURES lation level of each test polynucleotide sequence to a control cytosine methylation range of a corresponding control poly A full and enabling disclosure of the present disclosure, nucleotide sequence. The control cytosine methylation range including the best mode thereof to one skilled in the art, is set forth more particularly in the remainder of the speci?cation, of the corresponding control polynucleotide sequence can be Which includes reference to the accompanying ?gures, in developed from data obtained from a control group compris Which: ing non-affected age matched individuals. A ?nding that the FIG. 1 represents data shoWing the overall methylation cytosine methylation level of at least about 20% of the test level of the entire X-chromosome for 5 individuals diagnosed polynucleotide sequences falls outside of the control cytosine With ASD and the overall methylation level of the entire methylation range of the corresponding control polynucle X-chromosome for 5 individuals not affected With ASD. otide sequences leads to a determination that the individual is FIG. 2 represents an ideogram of the X chromosome shoW predisposed to or affected With autism spectrum disorder. ing the location of the currently knoWn 85 brain expressed In one preferred embodiment, the cytosine methylation genes that can be a factor in development of autism When level comprises cytosines at CpG dinucleotide sites. hypermethylated or hypomethylated. In addition, the location The three or more different test polynucleotide sequences 20 of tWo housekeeping genes, [G6PD, GLA], tWo autism genes to be tested can include any genes of the X-chromosome and, [NLGN3, RPLlO], and one gene [AVPR2] of uncertain sig optionally, can also include ?anking sequences to the genes. ni?cance are shoWn in brackets. For instance, in one embodiment, all of the genes of the FIG. 3 represents scatterplots of the degree of methylation X-chromosome can be tested. In other embodiments, smaller of selected genes on the X chromosome in males With autism sets of genes can be tested. For example, the genes of the three 25 (solid squares) and unaffected males (open circles). The or more test polynucleotide sequences can include all XLMR squares encircled on the scatterplots for NLGN4, NLGN3, genes; the 85 XLMR genes of Table I (discussed further LlCAM, and AVPR2 identify males With autism Whose methylation values fall outside the range of values for unaf herein); a set of genes including NLGN4, NLGN3, LlCAM, fected males. and AVPR2; a set of genes including NLGN4, STK9, ARX, FIG. 4 represents an expression of LlCAM as determined NLGN3, AGTR2, FMRl, RPLlO, SLC6A8, MECP2, 30 by real-time PCR in a male With autism, his mother, his ARHGEF6, FACL4, MED12, JARIDlC, TM4SF21AP1S2; affected identical tWin brother, and tWo unaffected (normal) and so forth. males. The overexpression of LlCAM alone or in combina According to another embodiment, a method for determin tion With overexpression or underexpression of other brain ing predisposition to or diagnosis of autism spectrum disorder expressed genes on the X chromosome can predispose for in an individual that includes determining the presence or 35 autism. quantity of at least three different biomarkers in a test sample FIG. 5 represents an MA plot for X-chromosome sample obtained from the individual is generally disclosed. Each of non-redundant probes. Each dot represents the log [ratio of the at least three different biomarkers are speci?c for a dif probe intensity of the averaged autism (17 samples) vs. con ferent test X-chromosome gene. The method also includes trol (10 samples)] plotted against the log [probe intensity of determining an expression level of each of the test X-chro 40 the averaged autism (l7 samples)>

Gene Symbol Gene Name Gene Location XLMR Entity Function

ot-PIX* Rho guanine nucleotide Xq26.3 MRX46 Effector of the rho (ARHGEF6) exchange factor 6 GTPases AGTR2* -II receptor Xq23 XLMR-optic atrophy, Angiotensin II receptor type 2 MRX ALDP Adrenoleukodystrophy Xq28 Adrenoleuko- Peroxisomal transport (ABCDI) protein dystrophy protein APl S2* Sigma 2 subunit of Xp22.2 Turner, XLMR- Assembly of endocytic adaptor hydrocephaly-basal vesicles protein/complex ganglia calci?cation ARHGEF9 Rho guanine nucleotide Xql 1.2 XLMR-hypotonia Regulation of Rho exchange factor 9 seizures protein signal transduction ARX* Aristaless-related X Xp22. ll Hydranencephaly, Neuronal migration chromosome gene Partington, Proud, West, X-linked lissencephaly With abnormal genitalia, MRX29, 32, 33, 36, 43, 54, 76 ATP 6A8-9 receptor Xpl 1.4 XLMR-infantile Renin receptor (ATP6AP2) epilepsy ATP7A Copper transporting Xq2 l .l Menkes, occipital Copper transport ATPase 7A horn US 7,998,744 B2 11 12 TABLE l-continued

Gene Symbol Gene Name Gene Location XLMR Entity Function BCOR BCL6 corepressor Xp11.4 Lenz microphthalmia Histone/protein (1 type) deacetylation BRWD3 Bromodomain and WD Xq21.1 XLMR-macrocephaly- Transcription factor repeat domain- large ears containing protein 3 CUL4B Cullin 4B Xq24 XLMR- Cell cycle, ubiquitin hypogonadism- cycle, E3 ubiquitin tremor ligase DCX Doublecortin Xq23 X-linked Neuronal migration lissencephaly DDP Dystonia-deafness Xq22.1 Mohr-Tranebj aerg, Transcription factor (TIMM8A) peptide Jensen DKC1 Dyskerin Xq28 Dyskeratosis Cell cycle and nucleolar congenita functions DLG3 Neuroendocrine DLG Xq13.1 MRX NMDA-receptor, mediated signaling, synaptic plasticity DMD Dystrophin Xp21.2—p221.1 Duchenne Structure of skeletal muscle membrane FACL4* Fatty acid acyl CoA Xq23 MRX63, 68 Fatty acid CoA ligase 4 (ACSL4) synthetase type 4 FANCB Fanconi anemia Xp22.2 X-linked VACTERL- DNA repair complementation group hydrocephaly B protein FGD1 Faciogenital dysplasia Xp11.22 Aarskog-Scott Guanine nucleotide (FGDY) exchange factor FLNA (FLN1) Filamin 1 Xq28 Epilepsy With Actin-binding protein periventricular heterotopia FMR1* Fragile X mental Xq27.3 Fragile X RNA binding protein, retardation 1 gene regulation FMR2 Fragile X mental Xq28 Fragile XE Unknown retardation 2 FTSJ 1 Methyl transferase Xp11.23 MRX9 Methylase GDI1 Rab GDP-dissociation Xq28 MRX41, 48 Stabilizes GDP bound inhibitor 1 conformations GKD Glycerol kinase de?ciency Xp21.2 Glycerol kinase Metabolism, glycerol de?ciency uptake GPC3 Glypican 3 Xq26.2 Simpson-Golabi- Cell adhesion, motility Behmel GRIA3 Glutamate receptor Xq25 Chiyonobu XLMR Signal transduction, ion ionotropic AMPA 3 transport, glutamate signaling pathWay HADH2 Hydroxyacyl-coenzyme A Xp11.22 XLMR- Lipid metabolism dehydrogenase, type III choreoathetisis HCCS Holocytochrome C Xp22.2 MIDAS syndrome Energy production, synthase cytochrome homolyase HPRT Hypoxanthine guanine Xq26.2 Lesch-Nyhan Enzyme phosphoribosyl transferase HUWE1 E3 ubiquitin-protein ligase Xp11.22 MRX Ubiquitin-protein ligase, mRNA transport IDS Iduronate sulfatase Xq28 Hunter Lysosomal enzyme IGBP1 Immunoglobulin-binding Graham coloboma protein 1 IL1RAPL IL-1 receptor accessory Xp21.2 MRX34 UnknoWn protein-like JARID1C* Jumonj i, AT-rich Xp11.22 MRX Regulates transcription, interactive domain 1C chromatin remodeling KIAA1202 K1AA1202 protein Xp11.22 Stoccos dos Santos Roles in cellular (SHROOM4) architecture, neurulation, and ion channel function KIAA2022 KIAA2022 protein Xq13.2 Cantagrel spastic DNA synthesis, DNA paraplegia polymerase activity KLF8 Kruppel-like factor 8 Xp11.21 MRX (ZNF741) L1CAM Cell adhesion molecule, Xq28 XLHS, MASA, Neuronal migration, cell L1 XL-ACC, SPG2 adhesion LAMP2 Lysosomal associated Xq24 Danon Membrane, lysosome membrane protein 2 cardiomyopathy MAOA Monoamine oXidase A Xp11.3 Monoamine oXidase Enzyme A de?ciency MBTPS2 Intramembrane zinc Xp22.1 Ichthyosis follicularis, Protease activity, metalloprotease atrichia, activates signaling photophobia proteins US 7,998,744 B2 13 14 TABLE l-continued

Gene Symbol Gene Name Gene Location XLMR Entity Function MECP2* Methyl-CpG binding Xq28 Rett, MRX16, 79 Binds methylated CpGs protein 2 MED12* Mediator of RNA Xq13.1 Opitz FG syndrome, Transcription (HOPA) polymerase II Lujan syndrome regulation, RNA transcription, subunit 12 polymerase II transcription mediator activity, ligand dependent nuclear receptor transcription coactivator activity, vitamin D receptor and thyroid hormone receptor binding MID1 Midline 1 Xp22.2 Opitz G/BBB Zinc ?nger gene MTM1 Myotubularin Xq28 Myotubular myopathy Tyrosine phosphatase NDP Norrie Xp11.3 Norrie Neuroectodermal cell interaction NDUFA1 NADH dehydrogenase Xq24 Mitochondrial Energy production, (ubiquinone) 1 alpha complex 1 oxidoreductase subcomplex de?ciency activity NEMO NF—KB essential Xq28 Incontinentia pigmenti Activates the (IKB 6KG) modulator transcription factor NF-KB NHS Nance-Horan syndrome Xp22.2—22. 13 Nance-Horan i gene NLGN3* Neuroligin 3 Xq13.1 Autism Cell adhesion NLGN4* Neuroligin 4 Xp22.32—22.31 Autism Cell adhesion NXFS Nuclear RNA export Xq22.1 XLMR-short stature- mRNA processing, factor 5 muscle Wasting mRNA export from nucleus OCRL1 Oculorenal Xq25 LoWe Enzyme OFD1 Oral-facial-digital Xp22.2 Oral-facial-digital 1 Unknown syndrome 1 OPHN1 Oligophrenin 1 Xq12 MRX6O GTPase activating protein OTC Ornithine Xp 1 1 .4 Ornithine transcarbamylase Enzyme transcarbamylase de?ciency PAK3 P21-activated kinase Xq23 MRX30, 47 Rac/Cdc 42 effector PCDH19 Protocadherin 19 Xq22 Epilepsy and mental retardation limited to females PDHA1 Pyruvate dehydrogenase Xp22.12 Pyruvate dehydro- Enzyme genase de?ciency PGK1 Phosphoglycerokinase 1 Xq13.3 Phosphoglycero- Enzyme kinase de?ciency PHF6 PHD-like zinc ?nger gene 6 Xq26.2 Borjeson-Forssman- Unknown Lehmann PHFS PFD ?nger protein 8 Xp11.22 XLMR-clefting Regulates transcription, binds DNA PLP Proteolipid protein Xq22.2 PMP, SPG1 Myelination PORCN Drosophila porcupine Xp11.23 Goltz syndrome Wnt receptor signaling homolog pathWay, acryl transferase activity, integral to membrane of endoplasmic reticulum PQBP1 Polyglutamine tract Xp11.23 Renpenning, Polyglutamine binding, binding protein 1 Sutherland-Haan, regulates Hamel cerebro- transcription palatocardiac, Golabi-Ito-Hall, Porteous, MRXS 5 PRPS1 Phosphoribosyl Xq22.3 Arts syndrome, Ribonucleotide pyrophosphate PRPS1 monophosphate synthetase 1 superactivity biosynthesis RPL10* Ribosomal protein L10 Xq28 Autism Protein synthesis, ribosomal protein RSK2 Threonine-serine kinase 2 Xp22.12 Cof?n—LoWry, MRX19 Kinase signaling pathWay SLC16A2 T3 transporter Xq13.2 Allan-Herndon- T3 receptor (MCT8) Dudley SLC6A8* Creatine transporter Xq28 XLMR With seizures Creatine transporter SLC9A6 Sodium-hydrogen Xq26.3 Christianson, X-linked Sodium-hydrogen exchanger NHE6 Angelman-like antiporter activity, syndrome lysosome organization and biogenesis, regulation ofendosome volume US 7,998,744 B2 15 16 TABLE l-continued

Gene Symbol Gene Name Gene Location XLMR Entity Function

SMClN SMC1 structural Xp11.22 X-linked Cornelia de Cell cycle, mitotic SMC1L1 maintenance of Lange syndrome spindle organization chromosomes 1-like and biogenesis, chromosome segregation SMS Spermine synthase Xp22.11 Snyder-Robinson Synthesis of spermine SOX3 SRY-box 3 Xq27.1 XLMR-growth Pituitary ?anction, hormone de?ciency transcription factor SRPX2 Sushi repeat containing Xq22.1 XLMR-Rolandic Signal transduction, protein, X-linked epilepsy-speech growth factor 2 dyspraxia STK9* Serine-threonine kinase 9 Xp22.12 XLMR with seizures Unknown (CDKLS) Rett-like SYN1 Synapsin 1 Xp11.23 Epilepsy- Synaptic vesicle protein macrocephaly TM4SF2* Transmembrane 4 Xp 1 1 .4 MRX5 8 Interacts with integrins superfamily member 2 UBE2A Ubiquitin-conjugating Xq24 XLMR-nail dystrophyseizures Ubiquitin cycle, enzyme E2A ubiquitin-protein ligase UPF3B UPF3 regulator of Xq24 MRX, Lujan/FG mRNA catabolism, nonsense transcript phenotype nonsense mediated homolog B decay XNP (XH2, X-linked nuclear protein, Xq21.1 ot-thalassemia mental Transcription factor, ATRX) X-linked helicase 2 retardation, helicase activities, Carpenter-Waziri, Chudley-Lowry, Holmes-Gang, Juberg-Marsidi, XLMR-hypotonic facies, XLMR spastic paraplegia ZDHHC9 Zinc ?nger, DHHC- Xq25-Xq26.1 XLMR-macrocephaly- 7 domain containing Marfanoid habitus protein 9 ZDHHC15 Zinc ?nger DHHC Xq13 .3 MRX91 domain-containing protein 15 ZNF41 Zinc ?nger 41 Xp11.3 MRX Zinger ?nger ZNF674 Zinc ?nger protein 674 Xp11.3 XLMR-retinal Transcription regulation dystrophy-short stature and MRX ZNF81 Zinc ?nger 81 Xp11.23 MRX45 Zinc ?nger

40 Hypomethylation or hypermethylation of these 87 brain plary brain-expressed genes on the X chromosome which expressed genes or ?anking regions of these 87 genes (85 may predispose to autism when hypermethylated or hypom XLMR genes plus NLGN3 and RPL10) singly or in combi ethylated. Two housekeeping genes, G6PD and GLA, which nation may predispose to autism, whereas usual mutations in are used as controls, are also shown in brackets. Two autism the 85 XLMR genes (e.g., changes in the coding sequence of 45 genes, NLGN3 and RPL10, are also shown in brackets. The the genes) cause mental retardation. For instance, when location of AVPR2, an X-linked gene of uncertain importance examining any group of three or more X-chromosome genes in autism and mental retardation, is also shown in brackets. and ?anking regions thereof, either hypomethylation or According to the present disclosure, any suitable method hypermethylation of at least about 20% of the group can be can be used for detecting the presence of hypomethylation or 50 evidence of a predisposition to or diagnosis of ASD. Of hypermethylation of brain expressed gene(s) in an individual. course, larger groups of genes are encompassed in the disclo Suitable methods for detecting altered methylation of brain sure as well. By way of example, any group of seven, ten, or expressed genes on the X chromosome, include, but are not more of genes on the X chromosome and corresponding limited to, DNA bisul?te sequencing, pyrosequencing, poly ?anking regions can be examined, and either hypomethyla 55 merase chain reaction (PCR)/ digestion with restriction endo tion or hypermethylation of at least about 20% of the group nucleases, methylation-speci?c PCR, realtime PCR, South can be evidence of a predisposition to or diagnosis of ASD. ern blot analysis, mass spectrometry, multiplex ligation Moreover, it should be noted that hypomethylation or hyper dependent probe ampli?cation (MLPA), chromatin methylation of a larger percentage of any examined group of immunoprecipitation (ChIP), methylation microarray, high genes can also signal the predisposition to or diagnosis of 60 performance liquid chromatography (HPLC), high perfor ASD. For instance, evidence of either hypomethylation or mance capillary electrophoresis (HPCE), methylation-sensi hypermethylation on at least about 50%, 60%, 70% or 80% of tive single-nucleotide primer extension, methylation-sensi an examined group of genes can be evidence of a predispo tive single-stranded conformational polymorphism, sition to or actually affected with ASD. methylation-sensitive restriction endonucleases, ligation The distribution of the XLMR genes of Table 1 on the X 65 mediated PCR, methylation-speci?c in situ hybridization, chromosome is shown in FIG. 2. Speci?cally, FIG. 2 shows an incomplete primer extension mixture, competitive primer ideogram that identi?es and shows the location of the exem binding site analysis, solid-phase primer extension, denatur US 7,998,744 B2 17 18 ing gradient gel electrophoresis, enzymatic regional methy such as about 10, about 25, about 40 or about 100 controls can lation assay, combined bisul?te restriction analysis (CO be utiliZed to create a suitable range of methylation values for BRA), methylLight, and the like. a particular CpG site. For example, one particular embodiment involves compar In general, ASD affected persons have more than one ing the degree of methylation (methylation value) of a spe- 5 hypermethylated or hypomethylated X-linked gene. As such, the more hypermethylated or hypomethylated genes that a ci?c CpG dinucleotide associated With a particular gene of an person has, the more likely the chances of that person devel individual to be tested to a pre-determined range of methyla oping autism. Thus, a comparison of methylation values of tion values of that particular CpG site from a control group of CpG sites associated With several genes from the same per non-affected persons. The pre-determined range can be, for 0 son, as discussed above, can be effective in determining the example Within one standard deviation of the average value of 1 predisposition of that person to autism. that particular CpG site found in the control group. The It is Well established that the level of expression of X methylation value of the particular CpG site can readily be chromosome genes is correlated With and perhaps even con determined according to methods Well knoWn to one of ordi trolled by methylation of CpG sites in the promoter regions nary skill in the art. If the methylation value falls outside of (see, e.g., Chen and Li, 2006, Curr T0. Microbiol Immunol, this pre-determined range, then the person from Which the 5 301:179-201; Wilson, et al., Biochim Biophys Acla, 1775: tested CpG site originated may be predisposed to or even 138-162; both of Which are incorporated herein by reference). suffering from undiagnosed autism. This type of “positive” Moreover, it is believed that in males even subtle alterations in result can alert the tester, doctors, parents, and other caregiv normal methylation patterns may signi?cantly affect tran ers that the person has an increased likelihood of developing scriptional ef?ciency, and this may be particularly important autism or its symptoms. At a minimum, a “positive” result can in brain tissue, Where tighter gene regulation can be expected. indicate that the individual is a candidate for additional test Furthermore, it is apparent that the methylation status of key ing for autism. CpG’s may actually act as a sWitch for expression ef?ciency One method of comparing the methylation value of a par regardless of the status of other possible sites of methylation. ticular CpG site of an individual to a pre-determined range of Accordingly, in conjunction With or alternative to deter methylation values of that particular CpG site from a control mining the methylation level of genes in determination of group of non-affected persons involves plotting the methyla predisposition to or diagnosis of ASD, the present disclosure tion value of the sample CpG site against a scatterplot of the is also directed to detecting overexpression or underexpres methylation values of that particular CpG site taken from a sion of X-chromosome genes in an individual. For instance, plurality of non-affected persons. For example, a plot can be disclosed herein is a method including determining the used to create a chart having on the X-axis the age of the expression level of a group of three or more X-chromosome person from Which the DNA originated and on the Y-axis the genes, Wherein a signi?cantly different expression level methylation value of the CpG site being analyZed. When betWeen tested samples and control samples indicates a pre analyZing the resulting scatterplot chart, if the methylation disposition to or diagnosis of ASD. value of the patient falls above or beloW the methylation In one embodiment, the present disclosure is directed to values of non-affected persons, then the tested CpG site may microarrays that can be used to determine the expression level be from an individual that is predisposed to or even suffering of genes to indicate a predisposition to or diagnosis of ASD. from undiagnosed autism. By Way of example, if the methy Bene?cially, disclosed arrays can be particularly designed to lation value of the tested individual is statistically different examine RNA from cell culture or from native lymphocytes (e. g., P value of 20.05) from the average methylation value of for overexpression or underexpres sion of predetermined gene the comparison data, then the individual may be predisposed 0 sets associated With ASD. For example, according to dis to or affected With ASD. closed methods, an array, e.g., an oligo microarray or a PCR When conducting a comparison of the methylation value of array, can be used to determine expression levels With empha a particular CpG site from an individual against those values sis on a speci?ed set of genes as disclosed herein. of that CpG site taken from a control group of non-affected Through comparison of the gene expression level of human persons, the number of controls (non-affected persons) may lymphoblast cells With controls, exemplary protocols of vary, as is generally knoWn in the art. HoWever, in order to be Which are described in the Example section, beloW, 44 X of increased value, a statistically signi?cant number of con chromosome genes have been determined to exhibit signi? trols are generally utiliZed. For instance, at least about 2 cant differences in expression in affected individuals (Table 2, controls can be utiliZed. In other embodiments, more controls beloW). TABLE 2

Autism/ Gene Name Functional Description Location Control

ELKl ELKl , member of ETS nuclear target for the ras-raf-MAPK Xp11.2 1.2 oncogene family signaling cascade PLP2 proteolipid protein 2 unknown Xp11.23 0.8 (colonic epithelium enriched) PQBPI polyglutamine binding PQBPI is a nuclear polyglutamine-binding Xp11.23 1.3 protein 1 protein that contains a WW domain ZNF81 Zinc ?nger protein 81 unknown Xp11.23 1.4 CFP complement factor active in the alternative complement Xpl 1.3—p11.23 1.4 properdin pathWay of the innate immune system DDX3X DEAD (Asp-Glu-Ala-Asp) involving alteration of RNA secondary Xp11.3—p11.23 1.2 box polypeptide 3, X structure such as translation initiation, linked nuclear and mitochondrial splicing, and ribosome and spliceosome assembly US 7,998,744 B2 19 20 TABLE 2-c0ntinued

Autism/ Gene Name Functional Description Location Control CASK calcium/calmodulin- calcium/calmodulin-dependent serine Xp11.4 0.7 dependent serine protein protein kinase; member of the membrane kinase (MAGUK family) associated guanylate kinase (MAGUK) protein family; scaffolding proteins associated With intercellular junctions RP2 retinitis pigmentosa 2 (X- cause of X-linked retinitis pigmentosa; may Xp11.4—p11.21 1.2 linked recessive) be involved in beta-tubulin folding. DMD dystrophin responsible for Duchenne (DMD) and Xp21.2 0.6 Becker (BMD) Muscular Dystrophies; dystrophin (as encoded by the Dp427 transcripts) is a large, rod-like cytoskeletal protein Which is found at the inner surface ofmuscle ?bers; dystrophin is part ofthe dystrophin-glycoprotein complex (DGC), Which bridges the inner cytoskeleton (F actin) and the extra-cellular matrix CDKLS cyclin-dependent kinase- Ser/Thr protein kinase; mutations Xp22 1.5 like 5 associated With X-linked infantile spasm syndrome (ISSX); also knoWn as X-linked West syndrome and Rett syndrome (RTT) MID1 midline 1 (OpitZ/BBB may act as anchor points to microtubules; Xp22 0.7 syndrome) mutations associated With the X-linked form of OpitZ syndrome, Which is characterized by midline abnormalities such as cleft lip, laryngeal cleft, heart defects, hypospadias, and agenesis ofthe corpus callosum MAP7D2 MAP7 domain containing 2 unknown Xp22.12 0.3 GPR64 G protein-coupled receptor unknoWn Xp22.13 0. 6 64 SCML1 sex comb on midleg-like 1 unknoWn Xp22.2—p22.1 0.6 (Drosophila) CLCN4 chloride channel 4 unknoWn but may contribute to the Xp22.3 2.7 pathogenesis of neuronal disorders AMELX* amelogenin amelogenin family ofextracellular matrix Xp22.31—p22.1 0.8 (amelogenesis imperfecta proteins; involved in biomineraliZation 1, X-linked) during tooth enamel development; mutations cause X-linked amelogenesis imperfecta WWC3 WWC family member 3 unknoWn Xp22.32 2.4 ARHGEF9 Cdc42 guanine nucleotide R_ho—like GTPases that act as molecular Xq11.1 1.4 exchange factor (GEF) 9 sWitches by cycling from the active GTP bound state to the inactive GDP-bound state; regulators of the actin cytoskeleton and cell signaling FLJ44635 TPT1-like protein unknoWn Xq13.1 1.3 NLGN3 neuroligin 3 neuronal cell surface protein; may act as Xq13.1 1.3 splice site-speci?c ligand for beta-neurexins and may be involved in the formation and remodeling of central nervous system synapses; mutations in this gene may be associated With autism and Asperger syndrome KIF4A kinesin family member 4A microtubule-based motor proteins that Xq13.1 0.8 generate directional movement along microtubules KIAA2022 KIAA2022 unknoWn Xq13.3 1.6 RNF12 ring ?nger protein 12 ubiquitin protein ligase that targets LIM Xq13-q21 1.2 domain binding 1 (LDB1/CLIM) and causes proteasome-dependent degradation of LDB1 ZMATl Zinc ?nger, matrin type 1 unknoWn Xq21 0.6 DIAPH2 diaphanous homolog 2 defects in this gene have been linked to Xq21.33 0.8 (Drosophila) premature ovarian failure 2 GPRASP1 G protein-coupled unknoWn Xq22.1 1.2 receptor associated sorting protein 1 CXorf39 chromosome X open unknoWn Xq22.2 1.2 reading frame 39 NUP62CL nucleoporin 62 kDa C- unknoWn Xq22.3 0.7 terminal like TBC1D8B TBC1 domain family, unknoWn Xq22.3 0.7 member 8B (With GRAM domain) AGTR2* angiotensin II receptor, plays a role in the central nervous system Xq22-q23 0.8 type2 and cardiovascular functions that are US 7,998,744 B2 21 22 TABLE 2-continued

Autism/ Gene Name Functional Description Location Control mediated by the renin-angiotensin system; this receptor mediates programmed cell death (apoptosis). AMOT angiomotin may mediate the inhibitory effect of angiostatin Xq23 1.9 on tube formation and the migration of endothelial cells toward growth factors during the formation ofnew blood vessels KLHL13 kelch-like 13 (Drosophila) unknown Xq23-q24 1.4 LONRF3 LON peptidase N-terminal may be involved in protein-protein and Xq24 0.7 domain and ring ?nger 3 protein-DNA interaction WDR44 WD repeat domain 44 unknown Xq24 1.3 UTP14A UTP14, U3 small unknown Xq25 1.3 nucleolar ribonucleoprotein, homolo g A (yeast) SH2D1A* SH2 domain protein 1A plays a major role in the bidirectional Xq25-q26 0.8 stimulation ofT and B cells; acting as an inhibitor of the signaling lymphocyte activation molecule; mutations in this gene cause lymphoproliferative syndrome X linked type 1 or Duncan disease, a rare immunode?ciency characterized by extreme susceptibility to infection with Epstein-Barr virus, with symptoms including severe mononucleosis and malignant lymphoma BCORL1 BCL6 co-repressor-like 1 unknown Xq25—q26.1 1.3 FHL1 four and a half LIM LIM proteins, named for ‘L1N11, ISL1, and Xq26 0.5 domains 1 MEC3,’ are de?ned by the possession ofa highly conserved double zinc ?nger motif called the LIM domain CT45—4* cancer/testis antigen unknown Xq26.3 0.8 CT45-4 LOC644538* hypothetical protein unknown Xq26.3 0.8 LOC64453 8 RP13- cancer/testis antigen unknown Xq26.3 0.7 3 6C9.1* CT45 RP13- cancer/testis antigen CT45-5 unknown Xq26.3 0.8 3 6C9.6* LDOC1 leucine zipper, down- may regulate the transcriptional response Xq27 0.6 regulated in cancer 1 mediated by the nuclear factor kappa B (NF-kappaB) SLC6A8 solute carrier family 6 transports creatine into and out of cells; Xq28 0.6 (neurotransmitter transporter, defects in this gene can result in X-linked creatine), member 8 creatine de?ciency syndrome

Of these 44 genes, 21 have been found to be up-regulated in the 44 genes of FIG. 6 were further examined. Upon exami affected individuals, with expression increases ranging from 45 nation, it was determined that the differently expressed genes about 1.2 to about 2.7 fold, and 23 were found to be down tend to cluster in several chromosomal locations including regulated in affected individuals, with expression decreases Xp11.2 to Xp11.4,Xp21 to Xp22, Xq23 to Xq 24 and Xq 25. ranging from about 20% to about 70%. FIG. 6 is an ideogram Accordingly, in one embodiment, disclosed methods and representation of the X chromosome location of these 44 products are directed to determination of expression levels of genes and illustrates either increased or decreased expression 50 genes found in one or more of these locations, and markers for each gene in individuals diagnosed with ASD. Accord can encompass all or a portion of the genes within that area. ingly, in one embodiment, probes for markers of these 44 The 5 genes of Table 2 having the highest expression level genes can be utilized in forming a device, e.g., a microarray, and the 5 genes having the lowest expression level are DMD, for determining the expression level of these 44 genes in a test CDKLS, MAP7D2, GPR64, SCMLl, CLCN4, WWC3, subject. Comparison of the expression levels of these genes 55 KIAA2022, AMOT, FHLl. According to one embodiment, with normal expression levels can provide information with this set of 10 genes can be utilized in forming a device for regard to the predisposition of the tested individual for ASD. detection of ASD or ASD predisposition. For example, statistically signi?cant differences in expres As described further in the Examples section, below, eight sion levels for three or more of these 44 genes can signify an of the above-listed 44 genes exhibited signi?cantly altered increased likelihood of the test subject having a predisposi 60 expression in the greatest number of autism cases tested. tion for or being affected with ASD. Accordingly, in one embodiment, a set of markers for use as A set of markers for use as described herein is not limited described herein can include markers for these eight genes, to materials for expression detection of all of the 87 genes of i.e., CLCN4, WDR44, NLGN3, CDKLS, K1AA2022, Table 1 or all of the 44 genes of Table 2, however, and larger AMOT, MAP7D2, and TBC1D8B. or smaller gene sets of the X-chromosome can be examined. 65 It has also been noted that the 44 genes of Table 2 are For example, in order to obtain a smaller set of genes for use enriched with 11 genes known to play a role in metabolism in determining predisposition toward or diagnosis of ASD, and transcription. The 11 genes known to play a role in