Positive Association of Engrailed 2 (EN2) Gene with Autism in the Indian Population

Background: Engrailed 2 (EN2) is located on chromosome 7q36.3, a genomic region linked to ASD and developmental language disorder, and has been implicated in the patterning of cerebellum during development. Studies on En2^-/- mice indicate the role of En2 in midbrain and cerebellum development as these mice display cerebellar hypoplasia, reduction in Purkinje cells with markedly reduced cerebellum and alterations in cerebellar serotonin levels. They also show impaired motor learning in the rotarod and complex socio-behavioral changes. Such deficits are reminiscent of symptoms observed in autistic individuals. Engrailed genes are necessary for serotonergic neuron development and defects in the serotoninergic system have been implicated in autism. Therefore, EN2 is considered a candidate gene for autism.

Objectives: The main focus is to investigate genetic association of EN2 with autism in the Indian population.

Methods: Autistic probands were recruited following DSM-IV criteria and CARS as the diagnostic instrument. The markers included are rs6150410 [9bp Ins/Del variant] and rs34808376 [GC/- Ins/Del polymorphism] in the EN2 promoter, rs3735653 (C/T) in exon 1, and two SNPs rs1861972 (A/G) and rs1861973 (C/T) in the intron. Genotyping was carried out by RFLP analysis following PCR. We used TDTPhase from the UNPHASED programme suite (version 2.403) for family-based association analysis. Pairwise linkage disequilibrium (LD) was computed with Haploview (version 4.1). “TFSearch” was used for identifying putative transcription factor binding sites.

Results: Since the two intronic SNPs are in absolute LD in this population, only one marker, rs1861973 was included for statistical analysis. We observed significant preferential transmission of the C allele of rs1861973 (hence A allele of rs1861972) from parents to the affected offspring (LRS=6.63, GS=0.006). Interestingly, the bias was highly significant for female offsprings (LRS=7.36, GS=0.0025). The study also revealed over-transmission of the Ins allele of rs6150410 (LRS=3.68, GS=0.03) and C allele of rs3735653 (LRS=3.4, GS=0.044) from mothers to affected sons. Significant transmission of the Ins allele from fathers to affected sons was noted for rs34808376 (LRS=3.4, GS=0.04). Comparatively strong LD was observed between different pairs of markers except for rs34808376 and rs3735653. The haplotype analysis demonstrated association of haplotypes formed between rs34808376-rs1861973 markers with autism. Based on these results, we investigated whether the SNPs impart any regulatory role during transcription. We identified a putative Sp1 transcription factor binding site in case of rs1861973 when the autism-associated C allele was present. In case of the Ins allele of rs6150410, we observed generation of three additional putative transcription factor binding sites.

Conclusions: The present study confirms in the Indian population previous results identifying the EN2 intronic SNP rs1861973 as an autism risk allele in other populations, and extends these results by identifying a sex-specific allelic transmission pattern that confers especial risk in females – a finding not reported earlier. Specific maternal/paternal transmission bias indicates possibility of genomic imprinting in the etiology, and imprinted regions have been identified in chromosome 7q. Bioinformatic analysis suggests that rs1861973 may exert its effect via transcriptional regulation of EN2. The current study suggests sex-specific involvement of Engrailed 2 gene in autism pathology in the Indian population.