Association of the ENGRAILED 2 (EN2) gene with autism in Chinese Han population.код для вставкиСкачать
American Journal of Medical Genetics Part B (Neuropsychiatric Genetics) 147B:434 –438 (2008) Association of the ENGRAILED 2 (EN2) Gene With Autism in Chinese Han Population Lifang Wang,1,2 Meixiang Jia,1,2 Weihua Yue,1,2 Fulei Tang,1,2 Mei Qu,1,2 Yan Ruan,1,2 Tianlan Lu,1,2 Handi Zhang,1,2 Hao Yan,1,2 Jing Liu,1,2 Yanqing Guo,1,2 Jishui Zhang,3 Xiaoling Yang,1,2* and Dai Zhang1,2* 1 Key laboratory for Mental Health, Ministry of Health, P.R. China Institute of Mental Health, Peking University, Beijing, P.R. China 3 Beijing Children’s Hospital Affiliated to Capital University of Medical Sciences, Beijing, P.R. China 2 Human ENGRAILED 2 (EN2) gene is localized to 7q36, an autism susceptibility locus. En2 knockout mice display hypoplasia of cerebellum and a decrease in the number of Purkinje cell, which are similar to those reported for individuals with autism. Furthermore, deficits in social behavior were detected in En2/ mice. Two recent studies have demonstrated that two intronic SNPs (rs1861972, rs1861973) in the EN2 gene are significantly associated with autism. To investigate whether this finding could be replicated in Chinese Han population, we performed the association study between eight single nucleotide polymorphisms (SNPs) of the EN2 gene and autism in 210 Chinese Han trios, using the familybased association test (FBAT). The present study demonstrated that a preferential transmission of the rs3824068 A-allele to affected offspring (A > G: Z ¼ 2.399, P ¼ 0.0165). After the Bonferroni correction, this statistical significance of preferential transmission did not remain. However, when haplotypes were constructed with multiple markers, a number of haplotypes including three two-marker haplotypes, nine three-marker haplotypes, one four-marker haplotype, and one six-marker haplotype, all of which contain the major allele A of rs3824068, displayed significantly associated with autism. These results were still significant after using the permutation method to obtain empirical P values. Thus, our data provide evidence that the EN2 gene may be implicated in the predisposition to autism in the Chinese Han population. ß 2007 Wiley-Liss, Inc. KEY WORDS: single nucleotide polymorphism (SNP); family-based association test (FBAT); haplotype Lifang Wang and Meixiang Jia contributed equally to this study. Grant sponsor: The National Natural Science Foundation of China; Grant numbers: 30270495; Grant sponsor: National Hightech R&D Program; Grant number: 2006AA02Z195. *Correspondence to: Prof. Xiaoling Yang and Prof. Dai Zhang, M.D., Ph.D., Institute of Mental Health, Peking University, 51, Hua Yuan Bei Road, Beijing 100083, China. E-mail: firstname.lastname@example.org; email@example.com Received 1 February 2007; Accepted 14 August 2007 DOI 10.1002/ajmg.b.30623 ß 2007 Wiley-Liss, Inc. Please cite this article as follows: Wang L, Jia M, Yue W, Tang F, Qu M, Ruan Y, Lu T, Zhang H, Yan H, Liu J, Guo Y, Zhang J, Yang X, Zhang D. 2008. Association of the ENGRAILED 2 (EN2) Gene With Autism in Chinese Han Population. Am J Med Genet Part B 147B:434–438. INTRODUCTION Autism is a severe neurodevelopmental disorder with childhood onset that is characterized by impairments in social interaction, communication, repetitive and stereotyped behaviors, and interests [American Psychiatric Association, 1994]. Twin and family studies indicate that genetic factors play an important role in the etiology of autism and the estimated heritability is over 90% [Folstein and Rutter, 1977; Bailey et al., 1995; Szatmari et al., 1998; Folstein and Rosen-Sheidley, 2001]. The cerebellum is one of the most consistent sites of neuroanatomic abnormality in autism. The cerebellar abnormalities including general hypoplasia and loss of Purkinje cells, are nearly universal in the published studies of human autopsy material [Williams et al., 1980; Ritvo et al., 1986; Courchesne et al., 1988; Bauman, 1991; Courchesne et al., 1994a,b; Hashimoto et al., 1995; Bailey et al., 1998; Kemper and Bauman, 1998]. Structural MRI studies have demonstrated hypoplasia of one or more regions in the cerebellar vermis and hemispheres [Murakami et al., 1989; Hashimoto et al., 1995; Courchesne et al., 2001]. Furthermore, functional MRI studies have indicated the abnormal patterning of cerebellar activation during a motor task and attention tasks in autism patients [Allen and Courchesne, 2003; Allen et al., 2004]. Together, these findings from histological and imaging studies suggest that genes involved in cerebellar development are candidate genes in autism. ENGRAILED 2 (EN2) gene is a homeobox transcription factor homologous to the Drosophila melanogaster engrailed gene with an essential role in the development of the midbrain and cerebellum. En2 knockout and transgenic mice displayed hypoplasia of cerebellum and decreased number of Purkinje cell that are similar to those postmortem reported for individuals with autism [Millen et al., 1994; Kuemerle et al., 1997; Baader et al., 1998]. In addition, when En2 gene was ectopically expressed in Purkinje cells, during late embryonic and postnatal cerebellar development, the cerebellum was greatly reduced in size and Purkinje cell numbers throughout the cerebellum was reduced by more than one-third relative to normal animals [Baader et al., 1998]. Misexpression of mouse En2 in primary cortical cultures elicited a reduction in neuronal differentiation, as reflected by the number of process-bearing neurons that express bIII-tubulin [Benayed et al., 2005]. Furthermore, a recent study investigated the behavior of En2 knockout mice. Deficits in social behavior were detected in En2/ mice across maturation that included decreased play, reduced social sniffing and allogrooming, and EN2 Gene and Autism less aggressive behavior. Deficits in two spatial learning and memory tasks were also observed [Cheh et al., 2006]. Additionally, in human beings cerebellar hypoplasia could result from a mutation or deletion in the EN2 gene [Zec et al., 1997; Sarnat et al., 2002]. All these results supported the possibility that EN2 gene might be involved in the pathogenesis of autism. Human EN2 gene is localized to chromosome 7q36.3, an autism susceptibility locus. One Finnish study reported linkage to a combined phenotype of autism spectrum disorder and dysphasia obtained at marker D7S550, located approximately 170 kb distal of EN2 [Auranen et al., 2002]. The other two studies also reported the linkage for D7S483, located about 5.5 Mb proximal of EN2 locus [Liu et al., 2001; Alarcon et al., 2002]. Additionally multiple chromosomal abnormalities interrupting chromosome 7q have been identified in cases with autism or autistic features. To date, studies have both supported and refuted the validity of EN2 as a susceptibility gene for autism [Petit et al., 1995; Zhong et al., 2003]. Two recent family-based studies have shown that two intronic SNPs (rs1861972, rs1861973) and the A-C (rs1861972-rs1861973) haplotype demonstrated significant association with autism, suggesting a role for EN2 as a susceptibility gene in the etiology of autism [Gharani et al., 2004; Benayed et al., 2005]. In this study, we attempted to investigate the association between the EN2 gene and autism in Chinese Han Population using the family-based association test (FBAT). MATERIALS AND METHODS Subjects The sample for this study consisted of 210 Chinese Han family trios (singleton autistic disorder patients and their unaffected biological parents). These families were recruited at the Institute of Mental Health, Peking University, China. Of the 210 autistic child probands, 196 were male and 14 were female. The mean age of the children at the time of testing was 6.5 years (range 2–17 years). Diagnoses of autism were established by senior psychiatrists. All patients fulfilled the DSM-IV criteria for autistic disorder. The cases were assessed using childhood autism rating scale (CARS) [Schopler et al., 1980] and autism behavior checklist (ABC) [Krug et al., 1980]. Children with fragile X syndrome, tuberous sclerosis, a previously identified chromosomal abnormality, dysmorphic features, or any other neurological condition suspected to be associated with autism were excluded. All subjects provided written informed consent for participation in this study. The study was approved by the Ethics Committee of the Health Science Center, Peking University. Genotyping Genomic DNA was extracted using the phenol–chloroform method. We selected eight single nucleotide polymorphisms 435 (SNPs) from the dbSNP (http://www.ncbi.nlm.nih.gov/SNP/) and the international HapMap project (http://www.hapmap.org/), located at the region from exon 1 to exon 2 of EN2 gene. These SNPs included rs3735653 (nonsynonymous change), rs3824068, rs2361688, rs3824067, rs1861972, rs1861973, rs4717034 (intron), and rs2361689 (synonymous change) that span the 3.9kb. All those with frequencies of minor alleles greater than 5% were used as genetic markers in this study. Four SNPs (rs3735653, rs1861973, rs4717034, rs2361689) were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis and direct DNA sequencing was used to genotype the other SNPs (rs3824068, rs2361688, rs3824067, rs1861972). The information of primers and restriction enzymes is given in Table I. The PCR amplification was performed in a 25 ml volume containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 200 mM of each dNTP, 0.3 mM of each primer, 1 U of Taq DNA polymerase, and 40 ng of the genomic DNA. The conditions used for PCR amplification were an initial denaturation phase at 948C for 5 min, followed by 36 cycles at 948C for 30 sec, annealing at 62.5–688C for 30 sec, and extension at 728C for 30 sec, followed by a final extension phase at 728C for 7 min. Each 15 ml sample of PCR products was completely digested with 3 U of restriction enzyme overnight. Digestion products were separated by electrophoresis in 2% agarose gels and then stained with ethidium bromide. Gels were read blindly by two independent raters with discrepancies resolved by re-genotyping. For rs3824068, rs2361688, rs3824067, and rs1861972, the primer sequences were 50 -GTGGTTGGAAACCCAGACAGA-30 for the forward primer and 50 -TTTGGACAGGGTCGCTGTAAG-30 for the reverse primer. The PCR products were sequenced by DNA sequencing after cleaning the PCR product using a BigDye Terminator Cycle Sequencing Ready Reaction Kit with Ampli Taq DNA polymerase (PE Biosystem). The inner primers were used for the cycle-sequencing reaction, and the fragments were separated by electrophoresis on an ABI PRISM 377-96 DNA Sequencer (Applied Biosystem, Foster city, CA). Mendelian inconsistencies identified by error checking were resolved with repeat genotyping. A number of sample genotypes could not be assigned due to repeated PCR failure or unclear genotype results. These consist of 4 genotypes for SNP rs3735653; 10 for SNP rs3824068, rs2361688 and rs4717034; 11 for SNP rs3824067 and rs1861972; and 4 for SNP rs1861973. Statistical Analysis Deviation from the Hardy–Weinberg equilibrium (HWE) for genotype frequency distributions was analyzed using the Chisquare goodness-of-fit test. Prior to analyses, Mendelian inconsistencies were checked using the PEDCHECK program, version 1.1 [O’Connell and Weeks, 1998]. Haplotype inconsistencies were identified by the SIMWALK2 program, version TABLE I. Detailed Information of the PCR-RFLP Analysis for the Four SNPs Primer sequence (50 ! 30 ) SNP rs3735653 rs1861973 rs4717034 rs2361689 0 0 5 -GGGCGGCTCGTGGTGTTTCTA-3 50 -ACCGTGCAGCGAGAGCGTCTT-30 50 -AGCCGATTCATACACCGCAC-30 50 -ACCACCCTTTCCCCAGACAT-30 50 -TACCGCCATCCCTGTTCCTGA-30 50 -AGGCACCGGGTAAGGATTCTG-30 50 -CCGAGTTCCAGACCAACAG-30 50 -TCCTCACCAAGCCAACAC-30 Product (bp) RFLP 722 Alu I 376 PfIf I 315 BstU I 693 Eco0109 I Allele (bp) T (722) C (62/314) T (315) T (693) PCR-RFLP, polymerase chain reaction-restriction fragment length polymorphism; SNP, single nucleotide polymorphism. C (135/587) T (62/107/207) C (112/203) C (167/526) 436 Wang et al. 2.91 [Sobel and Lange, 1996]. The power of sample size for association tests was evaluated using the Genetic Power Calculator program (http://statgen.iop.kcl.ac.uk/gpc/) [Purcell et al., 2003]. For the disease locus, a prevalence of 0.001, a genotype relative risk Aa ¼ 2 and AA ¼ 2, and a D-prime ¼ 1 were used to perform the analyses. For each marker in the analysis, the allele frequency used was that reported in Table II. We used the FBAT program (v. 1.5.1) (www.biostat. harvard.edu/fbat/default.html) to perform single and multilocus tests of association [Rabinowitz and Laird, 2000]. The FBAT program uses a generalized score statistic to perform transmission disequilibrium tests, including haplotype analyses. Moreover, the FBAT program provides pairwise linkage disequilibrium (LD) analysis to detect an inter-marker relationship, using D’ value. SNP pairs were considered to be in strong LD if D’ values were >0.70. The individual haplotype tests were conducted under ‘‘biallelic’’ mode in haplotype FBAT, meanwhile the global haplotype tests of association were performed under ‘‘multiallelic’’ mode in haplotype FBAT. FBATs were performed under an additive model. Bonferroni correction for multiple testing was carried out to reduce type I errors. For multilocus comparison, the permutation test has been applied. RESULTS Eight SNPs in EN2 gene were genotyped in 210 Chinese Han autism trios. The genotype distributions of the eight SNPs of parents did not deviate from HWE (data not shown). The power of different SNPs in our study was between 0.7501 and 0.8820. Allele frequencies and the results of FBAT for SNPs are shown in Table II. As for rs3824068, univariate FBAT demonstrated that variant allele showed a preferential transmission (A > G: Z ¼ 2.399, P ¼ 0.0165). But after the Bonferroni correction, this statistical significance of preferential transmission did not remain. To further analyze the pattern of linkage disequilibrium (LD) in our sample, we computed pairwise LD for all possible combination of the eight SNPs using D’ value. Seven SNPs (rs3735653, rs3824068, rs2361688, rs3824067, rs1861972, rs1861973, rs4717034) were found to be in strong LD between each other (D’ values ranging from 0.85 to 1.0) except for rs2361689 (Table III). In addition, the specific and global-haplotype FBAT tests of association were performed. Positive results were showed in Table IV. When two-marker haplotype analyses were per- formed, three haplotypes displayed significant association. Haplotype constructed from the A allele of rs3824068 and the C allele of rs4717034 revealed significant excess transmission both in the specific and global haplotype FBAT (P ¼ 0.0074 and 0.0096, respectively). The haplotype A-G (rs3824068rs2361688) and haplotype constructed from the A allele of rs3824068 and the T allele of rs3824067 demonstrated an excess transmission (P ¼ 0.0136 and 0.0136, respectively). In our study three-marker haplotype analyses involving A-C (rs3824068-rs4717034) demonstrated excess transmission from parents to affected offspring for rs3735653-rs3824068rs4717034 (T-A-C), rs3824068-rs2361688-rs4717034 (A-G-C), rs3824068-rs3824067-rs4717034 (A-T-C), rs3824068-rs1861972rs4717034 (A-A-C), and rs3824068-rs1861973-rs4717034 (A-C-C). Four other haloptypes consisting of rs3824068-rs2361688 (A-G) and rs3824068-rs3824067 (A-T), respectively, and a four-marker haplotype A-G-T-C (rs3824068-rs2361688rs3824067-rs4717034) displayed statistically significant association (Table IV). Furthermore, when haplotype was constructed with six markers, an A-G-T-A-C-C haplotype (rs3824068-rs2361688-rs3824067-rs1861972-rs1861973rs4717034) demonstrated significant association with autism (Z ¼ 2.633, P ¼ 0.0085), the results were still significant when the global haplotype FBAT was performed (w2 ¼ 10.877, P ¼ 0.0280). These results were still significantly associated with autism, after using the permutation method to obtain empirical P values. DISCUSSION In the present study, we investigated the association of EN2 gene and autism in Chinese Han population. Eight SNPs that span 3.9 kb of EN2 were selected. The rare allele frequencies for the eight tested SNP were similar to those of previous study [Benayed et al., 2005] except the SNP rs1861972 and rs1861973. When haplotypes were constructed with two, three, and four markers, we demonstrated that excess transmission of a number of haplotypes from parents to affected offspring was significant in this family-based association study. Furthermore, an A-G-T-A-C-C haplotype (rs3824068-rs2361688rs3824067-rs1861972-rs1861973-rs4717034) demonstrated significant association with autism, the results remained significant when the global haplotype FBAT was performed (w2 ¼ 10.877, P ¼ 0.0280). TABLE II. Results of FBAT for Eight SNPs in 210 Chinese Han Family Trios Marker rs3735653 rs3824068 rs2361688 rs3824067 rs1861972 rs1861973 rs4717034 rs2361689 Allele Afreqa Afreqb Families S E(S) Z P C T G A A G A T A G C T C T C T 0.354 0.646 0.342 0.658 0.068 0.932 0.177 0.823 0.932 0.068 0.933 0.067 0.826 0.174 0.188 0.812 0.381 0.619 0.381 0.619 0.074 0.926 0.182 0.818 0.927 0.073 0.926 0.074 0.811 0.189 0.204 0.796 135 135 136 136 52 52 107 107 51 51 52 52 107 107 108 108 107.000 163.000 102.000 170.000 28.000 76.000 59.000 155.000 75.000 27.000 76.000 28.000 157.000 57.000 62.000 154.000 118.000 152.000 118.000 154.000 30.000 74.000 62.000 152.000 73.000 29.000 74.000 30.000 149.500 64.500 70.000 146.000 1.649 1.649 2.399 2.399 0.516 0.516 0.539 0.539 0.525 0.525 0.516 0.516 1.321 1.321 1.393 1.393 0.0992 0.0992 0.0165 0.0165 0.6056 0.6056 0.5900 0.5900 0.5994 0.5994 0.6056 0.6056 0.1866 0.1866 0.1637 0.1637 Values shown in bold are statistically significant. FBAT, family-based association test; Afreq, allele frequency. a Allele frequency of patients. b Allele frequency of parents; Families, number of informative families; S, test statistics for the observed number of transmitted alleles; E(S), expected value of S under the null hypothesis (i.e., no linkage or association). EN2 Gene and Autism 437 TABLE III. Measure of Pairwise Linkage Disequilibrium D (D’) Between Eight SNPs in EN2 gene rs3824068 rs2361688 rs3824067 rs1861972 rs1861973 rs4717034 rs2361689 rs3735653 rs3824068 rs2361688 rs3824067 rs1861972 rs1861973 rs4717034 0.212 (0.90) 0.040 (0.85) 0.106 (0.95) 0.039 (0.85) 0.040 (0.85) 0.015 (0.90) 0.114 (0.91) 0.044 (0.96) 0.110 (0.98) 0.044 (0.96) 0.044 (0.96) 0.108 (0.93) 0.117 (0.93) 0.012 (0.91) 0.068 (0.99) 0.069 (1.00) 0.013 (0.91) 0.014 (0.24) 0.013 (1.00) 0.012 (0.91) 0.142 (0.97) 0.122 (0.85) 0.068 (0.99) 0.013 (0.96) 0.014 (0.23) 0.013 (0.91) 0.014 (0.24) 0.122 (0.81) The result of our study is not quite consistent with the four previous studies that have investigated EN2 as an autism susceptibility locus. Petit et al.  reported the significant association (P < 0.01) between a Pvu II polymorphism located 50 of the EN2 promoter and infantile autism in a case-control study on a Northern French population. Then the second study was performed by Zhong et al. in 196 multiplex families. Transmission disequilibrium test did not show any association between the nonsynonymous SNP rs3735653 from exon 1 which is only 2.9 kb from the PvuII polymorphism and autistic disorder. There was also no linkage or association between language and stereotypic behavior quantitative traits and the exon 1 variant [Zhong et al., 2003]. Two recent studies have demonstrated association between two allelic variants in the human EN2 gene and autism spectrum disorder, suggesting a role for EN2 gene as a susceptibility locus in autism [Gharani et al., 2004]. Their researches demonstrated that the two intronic SNPs, rs1861972 and rs1861973, are consistently inherited more frequently in individuals with autistic spectrum disorders (ASD) than unaffected siblings. The A-C haplotype (rs1861972-rs1861973) had consistently demonstrated similar of more significant association than either SNP individually. That was initially reported in 167 pedigrees. And the significant association was observed in two additional data sets and in the entire sample of 518 families [Benayed et al., 2005]. However, our study did not replicate these positive results. No significant association was obtained for SNPs rs1861972 and rs1861973. The A-C haplotype of rs1861972-rs1861973 did not demonstrated significant association (P ¼ 0.5716). Variant allele for rs3824068 showed a preferential transmission (A > G: Z ¼ 2.399, P ¼ 0.0165). It is consistent with the study by Benayed et al.  that minimal association is detected for rs3824068 individually (w2 ¼ 4.372, P ¼ 0.036). But after the Bonferroni correction, the statistical significance of preferential transmission did not remain. In contrast, our study demonstrated the significant excess transmission of a number of haplotypes from parents to affected offspring. There might be several reasons for these discordant findings. First, our study suggests that there may be more than one functional allele contributing to disease susceptibility in the EN2 region and may help explain the inconsistency between studies in the associated marker alleles. Second, a large ethnic difference in the frequencies of the polymorphisms may exist. Benayed et al.  reported that for rs1861972 the minor allele frequency (MAF) was 0.269 and 0.317 in the AGRE II and NIMH data sets, respectively. And for rs1861973, the MAF was 0.279 and 0.295, respectively. Whereas in our study, the MAF was 0.073 for rs1861972; and for rs1861973 the MAF was 0.074. So the number of informative families (at least one parent was heterozygous) for SNP rs1861972 and rs1861973 was only 51, which decreased the ability to detect association. This may explain the reason why we did not replicate the positive results of previous research. Third, different ethnic distributions could contribute to different genetic associations. Studies from different geographical locations, across ethnic groups are difficult to generate consistent results. Fourth, difference in statistical methods used between the studies should be considered [Nicodemus et al., 2007]. Moreover, autism is a complex disease and characterized by genetic heterogeneity. In summary, we found no association between individual SNPs of EN2 and autism in our family-based association study with Bonferroni tests. However, we observed a number of haplotypes including three two-marker haplotypes, nine threemarker haplotypes, one four-marker haplotype and one sixmarker haplotype, all of which contain the major allele A of rs3824068, revealed significantly associated with autism. Because haplotype has more accuracy and statistical power than individual SNPs in LD-based association studies, it is suggested that EN2 might be involved in the etiology of autism in Chinese Han population. Further researches for association TABLE IV. Haplotype Analysis for the Genetic Association Between EN2 and Autism Specific Haplotype FBAT Marker Allele rs3824068-rs4717034 A-C rs3735653-rs3824068-rs4717034 T-A-C rs3824068-rs2361688-rs4717034 A-G-C rs3824068-rs3824067-rs4717034 A-T-C rs3824068-rs1861972-rs4717034 A-A-C rs3824068-rs1861973-rs4717034 A-C-C rs3824068-rs2361688 A-G rs3824068-rs2361688-rs1861972 A-G-A rs3824068-rs2361688-rs4717034 A-G-C rs3824068-rs3824067 A-T rs3735653-rs3824068-rs3824067 T-A-T rs3824068-rs3824067-rs4717034 A-T-C rs3824068-rs2361688-rs3824067-rs4717034 A-G-T-C rs3824068-rs2361688-rs3824067-rs1861972-rs1861973-rs4717034 A-G-T-A-C-C Global Haplotypea FBAT S E(S) Z P w2(df) P 181.500 168.364 183.500 181.956 182.441 183.500 185.000 182.703 183.500 186.000 173.860 181.956 184.956 182.897 164.000 154.381 166.000 163.995 165.456 166.500 169.000 167.814 166.000 170.000 160.359 163.995 166.995 165.950 2.677 2.240 2.704 2.740 2.634 2.635 2.469 2.313 2.704 2.469 2.170 2.740 2.773 2.633 0.0074 0.0251 0.0068 0.0061 0.0084 0.0084 0.0136 0.0207 0.0068 0.0136 0.0300 0.0061 0.0056 0.0085 11.440 (3) 12.150 (5) 11.553 (4) 10.825 (3) 9.494 (4) 11.210 (4) 6.114 (2) 8.903 (3) 11.553 (4) 8.251 (3) 10.178 (4) 10.825 (3) 11.656 (4) 10.877 (4) 0.0096 0.0328 0.0210 0.0127 0.0499 0.0243 0.0470 0.0306 0.0210 0.0411 0.0375 0.0127 0.0201 0.0280 S, test statistics for the observed number of transmitted alleles; E(S), expected value of S under the null hypothesis (i.e., no linkage or association). a Global haplotype represents the haplotype using all possible variants. 438 Wang et al. analysis in other samples and for the function of EN2 are needed. Gharani N, Benayed R, Mancuso V, Brzustowicz LM, Millonig JH. 2004. Association of the homeobox transcription factor, ENGRAILED 2, 3,with autism spectrum disorder. Mol Psychiatry 9:474–484. ACKNOWLEDGMENTS Hashimoto T, Tayama M, Murakawa K, Yoshimoto T, Miyazaki M, Harada M, Kuroda Y. 1995. Development of the brainstem and cerebellum in autistic patients. J Autism Dev Disord 25:1–18. We thank all the patients and their families for their support and participation. Kemper TL, Bauman M. 1998. Neuropathology of infantile autism. J Neuropathol Exp Neurol 57:645–652. REFERENCES Alarcon M, Cantor RM, Liu J, Gilliam TC, Geschwind DH. 2002. Evidence for a language quantitative trait locus on chromosome 7q in multiplex autism families. Am J Hum Genet 70:60–71. Allen G, Courchesne E. 2003. Differential effects of developmental cerebellar abnormality on cognitive and motor functions in the cerebellum: An fMRI study of autism. Am J Psychiatry 160:262–273. Allen G, Muller RA, Courchesne E. 2004. Cerebellar function in autism: Functional magnetic resonance image activation during a simple motor task. Biol Psychiatry 56:269–278. American Psychiatric Association. 1994. Diagnostic and Statistical Manual of Mental Disorders. 4th edition. Washington, DC: American Psychiatric Association. Auranen M, Vanhala R, Varilo T, Ayers K, Kempas E, Ylisaukko-Oja T, Sinsheimer JS, Peltonen L, Jarvela I. 2002. A genomewide screen for autism-spectrum disorders: Evidence for a major susceptibility locus on chromosome 3q 25-27. Am J Hum Genet 71:777–790. Baader SL, Sanlioglu S, Berrebi AS, Parker-Thornburg J, Oberdick J. 1998. Ectopic overexpression of engrailed-2 in cerebellar Purkinje cells causes restricted cell loss and retarded external germinal layer development at lobule junctions. J Neurosci 18:1763–1773. Bailey A, Le Couteur A, Gottesman I, Bolton P, Simonoff E, Yuzda E, Rutter M. 1995. Autism as a strongly genetic disorder: Evidence from a British twin study. Psychol Med 25:63–77. Bailey A, Luthert P, Dean A, Harding B, Janota I, Montgomery M, Rutter M, Lantos P. 1998. A clinicopathological study of autism. Brain 121(Pt 5):889–905. Bauman ML. 1991. Microscopic neuroanatomic abnormalities in autism. Pediatrics 87:791–796. Benayed R, Gharani N, Rossman I, Mancuso V, Lazar G, Kamdar S, Bruse SE, Tischfield S, Smith BJ, Zimmerman RA, Dicicco-Bloom E, Brzustowicz LM, Millonig JH. 2005. Support for the homeobox transcription factor gene ENGRAILED 2 as an autism spectrum disorder susceptibility locus. Am J Hum Genet 77:851–868. Krug DA, Arick J, Almond P. 1980. Behavior checklist for identifying severely handicapped individuals with high levels of autistic behavior. J Child Psychol Psychiatry 21:221–229. Kuemerle B, Zanjani H, Joyner A, Herrup K. 1997. Pattern deformities and cell loss in Engrailed-2 mutant mice suggest two separate patterning events during cerebellar development. J Neurosci 17:7881–7889. Liu J, Nyholt DR, Magnussen P, Parano E, Pavone P, Geschwind D, Lord C, Iversen P, Hoh J, Ott J, Gilliam TC. 2001. A genomewide screen for autism susceptibility loci. Am J Hum Genet 69:327–340. Millen KJ, Wurst W, Herrup K, Joyner AL. 1994. Abnormal embryonic cerebellar development and patterning of postnatal foliation in two mouse Engrailed-2 mutants. Development 120:695–706. Murakami JW, Courchesne E, Press GA, Yeung-Courchesne R, Hesselink JR. 1989. Reduced cerebellar hemisphere size and its relationship to vermal hypoplasia in autism. Arch Neurol 46:689–694. Nicodemus KK, Luna A, Shugart YY. 2007. An evaluation of power and type I error of single-nucleotide polymorphism transmission/disequilibriumbased statistical methods under different family structures, missing parental data, and population stratification. Am J Hum Genet 80:178– 185. O’Connell JR, Weeks DE. 1998. PedCheck: A program for identification of genotype incompatibilities in linkage analysis. Am J Hum Genet 63:259–266. Petit E, Herault J, Martineau J, Perrot A, Barthelemy C, Hameury L, Sauvage D, Lelord G, Muh JP. 1995. Association study with two markers of a human homeogene in infantile autism. J Med Genet 32:269–274. Purcell S, Cherny SS, Sham PC. 2003. Genetic power calculator: Design of linkage and association genetic mapping studies of complex traits. Bioinformatics 19:149–150. Rabinowitz D, Laird N. 2000. A unified approach to adjusting association tests for population admixture with arbitrary pedigree structure and arbitrary missing marker information. Hum Hered 50:211–223. Ritvo ER, Freeman BJ, Scheibel AB, Duong T, Robinson H, Guthrie D, Ritvo A. 1986. Lower Purkinje cell counts in the cerebella of four autistic subjects: Initial findings of the UCLA-NSAC Autopsy Research Report. Am J Psychiatry 143:862–866. Cheh MA, Millonig JH, Roselli LM, Ming X, Jacobsen E, Kamdar S, Wagner GC. 2006. En2 knockout mice display neurobehavioral and neurochemical alterations relevant to autism spectrum disorder. Brain Res 1116:166–176. Sarnat HB, Benjamin DR, Siebert JR, Kletter GB, Cheyette SR. 2002. Agenesis of the mesencephalon and metencephalon with cerebellar hypoplasia: Putative mutation in the EN2 gene--report of 2 cases in early infancy. Pediatr Dev Pathol 5:54–68. Courchesne E, Yeung-Courchesne R, Press GA, Hesselink JR, Jernigan TL. 1988. Hypoplasia of cerebellar vermal lobules VI and VII in autism. N Engl J Med 318:1349–1354. Schopler E, Reichler RJ, DeVellis RF, Daly K. 1980. Toward objective classification of childhood autism: Childhood autism rating scale (CARS). J Autism Dev Disord 10:91–103. Courchesne E, Saitoh O, Townsend JP, Yeung-Courchesne R, Press GA, Lincoln AJ, Haas RH, Schriebman L. 1994a. Cerebellar hypoplasia and hyperplasia in infantile autism. Lancet 343:63–64. Sobel E, Lange K. 1996. Descent graphs in pedigree analysis: Applications to haplotyping, location scores, and marker sharing statistics. Am J Hum Genet 58:1323–1337. Courchesne E, Townsend J, Saitoh O. 1994b. The brain in infantile autism: Posterior fossa structures are abnormal. Neurology 44:214–223. Szatmari P, Jones MB, Zwaigenbaum L, MacLean JE. 1998. Genetics of autism: Overview and new directions. J Autism Dev Disord 28:351–368. Courchesne E, Karns CM, Davis HR, Ziccardi R, Carper RA, Tigue ZD, Chisum HJ, Moses P, Pierce K, Lord C, Lincoln AJ, Pizzo S, Schreibman L, Haas RH, Akshoomoff NA, Courchesne RY. 2001. Unusual brain growth patterns in early life in patients with autistic disorder: An MRI study. Neurology 57:245–254. Williams RS, Hauser SL, Purpura DP, DeLong GR, Swisher CN. 1980. Autism and mental retardation: Neuropathologic studies performed in four retarded persons with autistic behavior. Arch Neurol 37:749–753. Folstein S, Rutter M. 1977. Infantile autism: A genetic study of 21 twin pairs. J Child Psychol Psychiatry 18:297–321. Zec N, Rowitch DH, Bitgood MJ, Kinney HC. 1997. Expression of the homeobox-containing genes EN1 and EN2 in human fetal midgestational medulla and cerebellum. J Neuropathol Exp Neurol 56:236– 242. Folstein SE, Rosen-Sheidley B. 2001. Genetics of autism: Complex aetiology for a heterogeneous disorder. Nat Rev Genet 2:943–955. Zhong H, Serajee FJ, Nabi R, Huq AH. 2003. No association between the EN2 gene and autistic disorder. J Med Genet 40:e4.