Abstract 107 Poster Session III, Monday, 5/3 (poster 134)

Myocarditis and Dilated Cardiomyopathy (DCM) are common causes of morbidity and mortality in children. Many studies have implicated the enteroviruses, and particularly the Coxsackievirus B family, as etiologic agents of the acquired forms of these diseases. However, over the past few years we have shown the group C adenoviruses to be as commonly detected in the myocardium of children and adults with these diseases. It has remained something of a conundrum why two such divergent virus families cause these diseases. The recent description of the common human Coxsackievirus B-adenovirus receptor (HCAR) offers at least a partial explanation. We have speculated that polymorphisms in this gene could be associated with altered susceptibility to the development of cardiac disease. Although the cDNA sequence for this gene has been reported, the genomic structure has not.

We screened a bacterial artificial chromosomal (BAC) library using a radiolabeled polymerase chain reaction (PCR) product derived the 3′ end of the HCAR cDNA sequence. This identified 16 BACs (numbered 1 - 16) which were further characterized by PCR amplification of seven contiguous regions of the entire cDNA sequence. Based upon this two patterns of amplification were observed. Fourteen of the BACs yielded PCR products of the size or close to the size predicted from the cDNA sequence with each of the sets of PCR primers. The other 2 BACs (# 9 and 13) yielded PCR products of the predicted size with 4 of the sets of primers (the 3′ non-translated region) while the remaining sets failed to produce a PCR product under the conditions used. Further, DNA sequence analysis of the amplification products using the primer set closest to the 3′ end revealed that BACs 9 and 13 had sequences that matched the published cDNA sequences while the other 14 had sequences that were identical to each other but different to the published sequence. These data suggested that BACs 9 and 13 encoded the functional gene while the remainder encoded pseudogenes. These BACs are currently being sequenced to identify the intron-exon boundary and promoter sequences: to date we have identified 3 introns.

Fluorescent in situ hybridization (FISH) analysis with BACs 9 and 13 localized the HCAR gene to chromosome 21q11.2. FISH experiments with two of the other BACs produced signals on an additional 4 chromosomes (suggesting the presence of at least 4 pseudogenes in the human genome). We are currently screening monochromosomal hybrid panels to identify the number and chromosomal localization.

Once the DNA sequences of HCAR promoter and intron/exon boundaries have been determined we propose to use single stranded DNA conformation polymorphism analysis to screen acquired, idiopathic and familial myocarditis/DCM patients to determine whether mutation in this gene have a role in conferring susceptibility to the development of these diseases.