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Richard Sun,

Jim Millonig's Lab

Autism spectrum disorder (ASD) is comprised of three separate diagnoses: Autistic Disorder (AD), Asperger’s syndrome (AS), and Pervasive Developmental Delay-Not Otherwise Specified (PDD-NOS). Evidence from recent studies indicates a strong but rather complex genetic basis for ASD involving about 3-15 interacting genes. The most commonly affected CNS structure in autistic individuals is the cerebellum. A reduction in the number of Purkinje cells as well as cerebellar hypoplasia have been observed in autopsy studies. In addition, standard MRI studies reveal altered cerebellar developmental patterns. Functional MRI studies display abnormal cerebellar activation patterns in autistic individuals. It is likely these developmental defects contribute to the behavioral manifestations of ASD.

Recently, Dr. Millonig’s lab has identified ENGRAILED2 (EN2) as an ASD susceptibility gene (OMIM #131310). In the mouse, Engrailed-2 (En-2), is expressed in parasagittal stripe-like domains in the cerebellum. Disruptions of cerebellar circuitry have been observed in En2 mutant lines. It is my hypothesis that other genes with functional roles in cerebellar circuitry are also associated with ASD. Mouse ALDOLASE C (ALDOC) and Purkinje cell protein-2 (Pcp-2) are also expressed in stripe-like domains and are likely to have similar functions in establishing and/or maintaining this cerebellar circuitry. In linkage studies, human ALDOLASE C (ALDOC) and PURKINJE CELL PROTEIN-2 (PCP2) map near markers that display significant linkage with ASD. Consequently I am analyzing PCP2 and ALDOC for association with ASD.

The genotyping and analysis of single nucleotide polymorphisms (SNPs) allows for the identification of risk alleles that have localized near SNPs during the course of evolution. When a SNP have a specific allele that is in linkage disequilibrium (LD) with the risk allele, then both the SNP allele and the risk allele will be co-inherited more often in affected than in unaffected individuals. Hence, by knowing the genotypes of polymorphic SNPs on candidate genes, it is then possible to identify putative risk alleles.

Family DNA samples are supplied by the Autism Genetic Resource Exchange (AGRE) and the association of ALDOC and PCP2 with ASD were be analyzed within the AGRE I dataset. SNPs on both ALDOC and PCP2 were identified with dbSNP. The ALDOC gene resides on chromosome 17 and is 3.7Kb in length. Two of the 5 SNPs reside in ALDOC exons. The PCP2 gene resides on chromosome 19 and is 2.0Kb in length. 1 of the 7 SNPs is exonic in PCP2. Initial sequencing of PCP2 on 20 chromosomes reveals 3 polymorphic (rs2279040, rs2279042, and rs1862514) and four non-polymorphic SNPs. The sequencing of ALDOC on 20 chromosomes reveals no polymorphic SNPs but polymorphisms are observed in the upstream rs618791 SNP and the downstream rs652677 SNP. As a result, only SNPs showing polymorphisms were genotyped and used for subsequent analysis. Regions encompassing the identified SNPs were amplified via multiplex PCR and all DNA of individuals in AGRE I was genotyped via Luminex cytometry, a high-throughput, accurate, and rapid method for multiplex genotyping of SNPs.

Association between SNPs and ASD were evaluated under either the narrow diagnostic spectrum (AD) or the broad spectrum (AD, AS, and PDD-NOS) where the threshold of significant association is P<0.05. SNPs (rs2279040, rs2279042, and rs1862514) in PCP2 were not found to be significantly associated with ASD under both spectrums as alleles (PÎ[0.25,0.57]). Significant association was also not observed when these genes were analyzed as haplotypes (PÎ[0.29,0.61]). Association for ALDOC was not observed in both spectrums individually and as haplotypes (allelic: PÎ[0.35,0.59; haplotype: PÎ[0.24,1.00]). These results indicate that the risk allele(s) for ASD is not localized on or in the vicinity of these genes.

These results do not exclude the possibility that regulatory regions for either PCP2 or ALDOC may be associated with ASD but are not in LD with the genes themselves. Further study of these two genes as susceptibility loci can be aided through the haplotype map of the human genome constructed by the Internaional HapMap Consortium. A unique feature of the HapMap Project is its identification of tagSNPs, which are SNPs that would best represent all other SNPs within specific LD blocks. Since it is likely that a regulatory region and the regulated gene are located within the same LD block, further association analysis of a tagSNP with ASD would provide evidence for the location of non-coding regulatory regions.

References

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. Support for the Homeobox Transcription Factor Gene ENGRAILED 2 as an Autism Spectrum Disorder Susceptibility Locus. Am J Hum Genet. 2005 Nov;77(5):851-68. Epub 2005 Oct 5.

Gharani N, Benayed R, Mancuso V, Brzustowicz LM, Millonig JH. Association of the homeobox transcription factor, ENGRAILED 2, 3, with autism spectrum disorder. Mol Psychiatry. 2004 May;9(5):474-84.

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