Family‐based studies indicate association of Engrailed 2 gene with autism in an Indian population

Engrailed 2 (EN2) is a homeobox transcription factor involved in the patterning of cerebellum during brain development. Linkage analysis and studies on knockout mice support EN2, located on chromosome 7q36.3, as a potential risk locus for autism. Candidate gene approach also suggested association of EN2 with autism spectrum disorder (ASD) in various populations. Here, we have investigated the association of five markers [rs3735653 (C/T) in exon 1; rs34808376 (GC/‐) and rs6150410 (CGCATCCCC/‐) in promoter region; rs1861972 (A/G) and rs1861973 (C/T) in the intron] of the gene with autism and ASD in Indian population using family‐based approach. Probands have been recruited using Diagnostic and Statistical Manual of Mental Disorders Fourth Edition (DSM‐IV) diagnostic criteria. Genotypic distributions conform to Hardy–Weinberg equilibrium. Genotyping analysis showed that the intronic single nucleotide polymorphisms (SNPs) are in complete linkage disequilibrium showing A‐C and corresponding G‐T allelic association. We observed significant preferential transmission of C allele of rs1861973 from the parents to affected offspring [transmission disequilibrium test (TDT): narrow diagnosis likelihood ratio statistics (LRS) = 6.63, P = 0.006; broad diagnosis LRS = 4.47, P = 0.05]. Interestingly, gender‐based investigations showed significant transmission of C allele to the affected females [TDT: LRS = 7.36, P = 0.0025; haplotype‐based haplotype relative risk (HHRR): LRS = 7.16, P = 0.02]. A maternal overtransmission for these alleles was also noted (TDT: LRS = 3.65, P = 0.036; HHRR: LRS = 2.81, P = 0.036). Bioinformatic analysis using TFSearch showed generation of Sp1 binding site in the presence of C allele. While Del‐T haplotype formed from rs34808376‐rs1861973 markers showed increased non‐transmission, the Ins‐C showed significant transmission suggesting protective effect and risk, respectively, conferred by these haplotypes in autism etiology. These results suggest positive genetic correlation of EN2 with autism in the Indian population.

[1]  Saurabh Ghosh,et al.  Genetic analysis of reelin gene (RELN) SNPs: No association with autism spectrum disorder in the Indian population , 2008, Neuroscience Letters.

[2]  Karl Herrup,et al.  The mouse Engrailed genes: A window into autism , 2007, Behavioural Brain Research.

[3]  Emanuel Dicicco-Bloom,et al.  Support for the homeobox transcription factor gene ENGRAILED 2 as an autism spectrum disorder susceptibility locus. , 2005, American journal of human genetics.

[4]  E. Courchesne,et al.  Prediction and preparation, fundamental functions of the cerebellum. , 1997, Learning & memory.

[5]  A. Joyner,et al.  Examining pattern formation in mouse, chicken and frog embryos with an En-specific antiserum. , 1991, Development.

[6]  Patrick R Hof,et al.  Neuropathological findings in autism. , 2004, Brain : a journal of neurology.

[7]  J. Oberdick,et al.  Engrailed‐2 negatively regulates the onset of perinatal Purkinje cell differentiation , 2004, The Journal of comparative neurology.

[8]  Mark Daly,et al.  Haploview: analysis and visualization of LD and haplotype maps , 2005, Bioinform..

[9]  George M. Anderson,et al.  Platelet serotonin studies in hyperserotonemic relatives of children with autistic disorder. , 1993, Life sciences.

[10]  Geraldine Dawson,et al.  Heterogeneous association between engrailed‐2 and autism in the CPEA network , 2008, American journal of medical genetics. Part B, Neuropsychiatric genetics : the official publication of the International Society of Psychiatric Genetics.

[11]  J. Oberdick,et al.  Ectopic Overexpression of Engrailed-2 in Cerebellar Purkinje Cells Causes Restricted Cell Loss and Retarded External Germinal Layer Development at Lobule Junctions , 1998, The Journal of Neuroscience.

[12]  J. Terwilliger,et al.  A haplotype-based 'haplotype relative risk' approach to detecting allelic associations. , 1992, Human heredity.

[13]  Language Lateralization in a Bimanual Language , 2003 .

[14]  Allen R. Braun,et al.  Language Lateralization in a Bimanual Language , 2003, Journal of Cognitive Neuroscience.

[15]  W. Ewens,et al.  Transmission test for linkage disequilibrium: the insulin gene region and insulin-dependent diabetes mellitus (IDDM). , 1993, American journal of human genetics.

[16]  T. Heinemeyer,et al.  Databases on transcriptional regulation: TRANSFAC, TRRD and COMPEL , 1998, Nucleic Acids Res..

[17]  Jing Liu,et al.  Association of the ENGRAILED 2 (EN2) gene with autism in Chinese Han population , 2008, American journal of medical genetics. Part B, Neuropsychiatric genetics : the official publication of the International Society of Psychiatric Genetics.

[18]  Pinchen Yang,et al.  Association of the Homeobox Transcription Factor Gene ENGRAILED 2 with Autistic Disorder in Chinese Children , 2008, Neuropsychobiology.

[19]  J. Bower,et al.  Cerebellum Implicated in Sensory Acquisition and Discrimination Rather Than Motor Control , 1996, Science.

[20]  P. Thuras,et al.  Purkinje Cell Size Is Reduced in Cerebellum of Patients with Autism , 2002, Cellular and Molecular Neurobiology.

[21]  A. Joyner,et al.  Abnormal embryonic cerebellar development and patterning of postnatal foliation in two mouse Engrailed-2 mutants. , 1994, Development.

[22]  A. Joyner,et al.  Pattern Deformities and Cell Loss in Engrailed-2Mutant Mice Suggest Two Separate Patterning Events during Cerebellar Development , 1997, The Journal of Neuroscience.

[23]  D. Curtis,et al.  An extended transmission/disequilibrium test (TDT) for multi‐allele marker loci , 1995, Annals of human genetics.

[24]  A. Joyner,et al.  Two enhancer regions in the mouse En-2 locus direct expression to the mid/hindbrain region and mandibular myoblasts. , 1993, Development.

[25]  K. Degenhardt,et al.  A role for Engrailed-2 in determination of skeletal muscle physiologic properties. , 2001, Developmental biology.

[26]  P. Strick,et al.  Activation of a cerebellar output nucleus during cognitive processing. , 1994, Science.

[27]  A. Joyner,et al.  Expression patterns of the homeo box-containing genes En-1 and En-2 and the proto-oncogene int-1 diverge during mouse development. , 1988, Genes & development.

[28]  J. Townsend,et al.  Attention coordination and anticipatory control. , 1997, International review of neurobiology.

[29]  S. Folstein,et al.  Chromosome 7q: where autism meets language disorder? , 2000, American journal of human genetics.

[30]  C. Barthélémy,et al.  Association study with two markers of a human homeogene in infantile autism. , 1995, Journal of medical genetics.

[31]  R. Benayed,et al.  Association of the homeobox transcription factor, ENGRAILED 2, 3, with autism spectrum disorder , 2004, Molecular Psychiatry.

[32]  Linda M. Brzustowicz,et al.  Autism-Associated Haplotype Affects the Regulation of the Homeobox Gene, ENGRAILED 2 , 2009, Biological Psychiatry.

[33]  L. Peltonen,et al.  A genomewide screen for autism-spectrum disorders: evidence for a major susceptibility locus on chromosome 3q25-27. , 2002, American journal of human genetics.

[34]  Eric Courchesne,et al.  Brainstem, cerebellar and limbic neuroanatomical abnormalities in autism , 1997, Current Opinion in Neurobiology.

[35]  D. Geschwind,et al.  Evidence for a language quantitative trait locus on chromosome 7q in multiplex autism families. , 2002, American journal of human genetics.

[36]  C. Gillberg,et al.  Genome-wide scan for autism susceptibility genes. Paris Autism Research International Sibpair Study. , 1999, Human molecular genetics.

[37]  S. Folstein,et al.  Genetics of austim: complex aetiology for a heterogeneous disorder , 2001, Nature Reviews Genetics.

[38]  Pak Chung Sham,et al.  Genetic Power Calculator: design of linkage and association genetic mapping studies of complex traits , 2003, Bioinform..

[39]  Christopher Gillberg,et al.  Genome-Wide Scan for Autism Susceptibility Genes , 1999 .

[40]  D. O'Leary,et al.  Engrailed genes control developmental fate of serotonergic and noradrenergic neurons in mid- and hindbrain in a gene dose-dependent manner , 2005, Molecular and Cellular Neuroscience.

[41]  F. Serajee,et al.  No association between the EN2 gene and autistic disorder , 2003, Journal of medical genetics.

[42]  F. Dudbridge.  Pedigree disequilibrium tests for multilocus haplotypes , 2003, Genetic epidemiology.

[43]  J. Millonig,et al.  En2 knockout mice display neurobehavioral and neurochemical alterations relevant to autism spectrum disorder , 2006, Brain Research.

[44]  L. Peltonen,et al.  A heterogeneity-based genome search meta-analysis for autism-spectrum disorders , 2006, Molecular Psychiatry.