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Silky Kamdar
MSc, University of Mumbai - 2002

Thesis Advisor: James H. Millonig, Ph.D.
Graduate Program in Neuroscience

CABM, Room 010

Tuesday, December 21, 2010
10:00 a.m.


Autism Spectrum Disorders (ASD) is a complex neuropsychiatric disorder with a strong genetic basis. Our previous research has focused on studying ENGRAILED2 (EN2) as an ASD candidate gene. EN2 is a homeobox transcription factor that regulates various aspects of CNS development including connectivity.
Previous work in the lab demonstrated the common alleles (underlined) of two intronic EN2 SNPs, rs1861972 (A/C) and rs1861973 (C/T), are associated with ASD (A-C haplotype in 518 families, P <.000001). In vitro molecular genetic experiments demonstrated the A-C haplotype increases gene expression via transcriptional regulation.
The above results lead us to ask if the A-C haplotype also functions in vivo. EN2 has a dynamic expression pattern throughout CNS development, which also leads us to ask if the A-C haplotype is functional at more than one developmental time points, in more than one cell type, and if the haplotype always increases gene expression during CNS development?
To investigate the in vivo developmental regulation of A-C haplotype, I first generated two types of transgenic mouse lines. I used ~20 kb of flanking EN2 cis-regulatory sequence, which drives expression of a fluorescent reporter: one has EN2 intron with ASD-associated A-C haplotype (A-C) and the other has the G-T haplotype (G-T) which is over-represented in unaffected siblings. In order to further characterize the in vivo function of EN2 intron I also generated a third transgene similar to the first two, except that it lacked the whole intron (intronless). 6 A-C lines and 8 G-T lines and 8 intronless lines with varying copy numbers were successfully established.
In order to first investigate when, where and how A-C functions in vivo, I performed expression analyses on the A-C and G-T lines. A total of over 200 animals from all 14 lines were analyzed for expression levels by performing RT-qPCR at five different developmental stages (embryonic days 9.5, 12.5, 17.5, postnatal day 6 and >30). Significant increase in levels of reporter gene expression was observed in A-C lines when compared to that in the G-T lines in postnatal (34% increase, P-value =0.01) and adult stages (100% increase, P-value <0.00001). No significant difference in reporter gene expression levels was observed in the embryonic stages E12.5, and E17.5. However, a spatial difference between the two haplotypes was observed in the mid-hindbrain region at E9.5 when analyzed by in situ hybridization, where A-C results in an expansion of the expression domain. After elimination of outliers identified by the spatial analyses a significant increase in expression levels (49% increase, P-value <0.005) was also observed with A-C.
The above observations demonstrate that the ASD associated A-C haplotype is functional in vivo, regulating the spatial expression pattern and levels at various stages of CNS development. These differences could lead to cell biological effects that alter CNS development and increase risk for ASD.

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