The search for the PFHBI gene : refining the target area and identification and analysis of candidate gene transcripts

[摘要] ENGLISH ABSTRACT:Progressive familial heart block I (PFHBI) is an inherited autosomal dominant cardiacconduction disorder which segregates in a large South African (SA) pedigree, twosmaller SA families and a Lebanese family. It specifically affects conduction in theventricles and is of unknown cause. Clinically, PFHBI is detected on electrocardiogram(ECG) by evidence of bundle-branch disease, i.e., as right bundle branch block, leftanterior or posterior hemiblock, or complete heart block with broad QRS complexes. ThePFHBI-causative gene was mapped to a lOcM region on chromosome 19ql3.3 usinglinkage analysis, and the locus was subsequently reduced to 7cM by genetic finemapping.The present study involved a multi-strategy approach to search for the PFHBI gene. Theobjectives were the further reduction of the PFHBI locus by genetic fine mapping usingpublished and novel markers, searching for short gene transcripts from publicly availabledatabases and the generation of an integrated map of the locus to which genes weremapped. Prioritised genes were screened for PFHBI-causing mutations and, in addition,the PFHBI locus was searched for the presence of a G protein-encoding gene (PI 15-RhoGEF), a connexin (Cx) gene and any genes containing a CTG repeat expansion motif,since these genes are plausible PFHBI candidate genes.Genotyping and fine genetic mapping using known and novel polymorphic dinucleotide(CA)n and novel tetranucleotide (A3G)n repeat markers across the PFHBI locus wereperformed. Publicly available databases, such as LLNL (Livermore, USA), andGENEMAP (NCBI) were searched for ESTs which, in turn, were extended usingclustering programmes, such as UNIGENE (NCBI) and STACK (SANBI), and theresulting consensus sequences were subsequently BLAST-searched against the proteindatabases. Using the available data, an integrated physical and genetic map of the PFHBIlocus was generated and, as the HGP progressed, a number of novel genes were placedthereon. Subsequently, genes were prioritised on the basis of position, function and expression profile.Genetic fine mapping reduced the PFHBI locus from 7cM to 4cM. The EST approachyielded 38 ESTs, of which 24 ESTs matched proteins, such as activating transcriptionfactor 5 (ATF5), actin-binding protein (KPTN) and zinc finger protein 473 (ZFP473)(May 2003). All the map data generated experimentally and computationally were placedon the PFHBI map. The PI 15-RhoGEF was excluded as a PFHBI candidate gene andalthough homologous sequences to connexin 37 (Cx37) was located on both chromosome19 radiation hybrid clones (RHG12 and ORIM-7), it was not identified on the DNAclones spanning the PFHBI locus. No evidence of an expansion of a CTG repeat motifsequence in PFHBI-affected individuals was found. Five highly prioritised candidategenes, namely, 5CZ2-associated X protein (BAX), potassium voltage-gated channelShaker-related subfamily member 7 (KCNA7'), potassium inwardly-rectifying channel,subfamily J, member 14 (KIR2.4), lin-7 homolog B {LIN-7B) and glycogen synthase 1(GSYI) were selected for mutation screening. No disease associated mutations wereidentified in the exonic and flanking intronic regions of these genes.In summary, this study reduced the PFHBI locus substantially and generated a detailedmap of the region. A number of attractive candidate genes were excluded from causingPFHBI; however, several plausible candidate genes are still present at this gene-richlocus and remain to be screened. Identifying the PFHBI-causative gene and associatedmutation will provide a platform for further studies to understand the pathophysiology,not only of PFHBI, but also of other more commonly occurring conduction disturbances.

[发布机构] Stellenbosch University