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Homeobox transcription factor, ENGRAILED2 is an Autism Spectrum Disorder susceptibility gene

Jiyeon Choi
M.S., Korea University - 2004

Thesis Advisor: James H. Millonig, Ph.D.
Graduate Program in Cell & Developmental Biology

CABM, Room 010

Thursday, March 3, 2011
11:00 a.m.


Previously we demonstrated the common alleles of two intronic SNPs (underlined) rs1861972 (A/G) and rs1861973 (C/T) in homeobox transcription factor ENGRAILED2 (EN2) are associated with Autism Spectrum Disorder (ASD). Subsequent human genetic analyses identified the ASD associated A-C haplotype as the best candidate for functional study. My thesis characterizes the ASD associated A-C haplotype as a transcriptional regulator and provides further supports for EN2 as an ASD susceptibility gene.
To demonstrate the A-C haplotype is functional, luciferase assays were performed in mouse cerebellar granule neurons. The results determined the A-C haplotype increases expression and more active than the G-T haplotype. Mutation analyses demonstrated the A-C haplotype function requires both ASD-associated A and C alleles. Minimal element sufficient for the A-C haplotype function was then identified and used as a probe to pull out protein mediators. Mass spectrometry identified transcription factors specifically binding to the A-C haplotype, Cux1 and Nfib. Further validation in human cells demonstrated CUX1 and NFIB specifically bind to the A-C haplotype and they mediate the A-C haplotype function. These results identified the A-C haplotype as a transcriptional regulator, which supports EN2 as an ASD susceptibility gene.
Since the A-C haplotype is functional in vitro, I then asked if EN2 levels are altered in the cerebellum of ASD individuals. Human post-mortem analyses on 78 individuals (29 affected and 49 control) demonstrated EN2 mRNA levels are elevated in ASD individuals with AC/GT genotype (74%, p=.0005). These data demonstrated altered EN2 levels are associated with ASD in the context of rs1861972-rs1861973 genotype. The results further support EN2 as an ASD susceptibility gene.
Next I asked if other flanking genes are co-regulated with EN2 in the context of ASD or rs1861972-rs1861973 genotype. Human post-mortem analyses on five flanking genes demonstrated SONIC HEDGEHOG (SHH) and INSULIN INDUCED GENE1 display altered mRNA levels based on affection status. SHH and CANOPY1 HOMOLOG also display altered levels based on rs1861972-rs1861973 genotype. These data suggest adjacent genes of EN2 are co-regulated either through the A-C haplotype or in the context of ASD.
Finally I asked if other common variants of EN2 are associated with ASD. Association analysis on 12 common variants encompassing EN2 gene on AGREII and NIMH datasets indicated rs2361688 is also significantly associated with ASD (p=0.0008, three data sets combined).
Altogether, the present study provides further supports for EN2 as an ASD susceptibility gene.

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