Mutation screening of candidate genes and the development of polymorphic markers residing on chromosome 19q13.3, the progressive familial heart block I gene search area

[摘要] ENGLISH ABSTRACT:Progressive familial heart block type I (PFHBI) is a cardiac ventricular conduction disorder ofunknown cause associated with risk of sudden death, which has been described in severalSouth African families. Clinically, PFHBI is characterised by right bundle branch block onECG, which may progress to complete heart block, necessitating pacemaker implantation.The disease shows an autosomal dominant pattern of inheritance with evidence of geneticanticipation. Using genetic linkage analysis, the PFHBI-causative gene was mapped to a 10eentimorgan (cM) gene-rich area of chromosome (C) 19q13.3, which has, subsequently, beenreduced to 7cM by fine mapping with polymorphic dinucleotide (CA)n short tandem repeat(STR) markers. Several attractive candidate genes, including muscle glycogen synthase(GSY 1) and histidine-rich calcium binding protein (HRC), lie within this region.The aim of the present study was two-fold: 1) to identify and characterise tetranucleotide(AAAT)n STRs within the PFHBI critical region that could be developed as polymorphicmarkers for use in genetic fine mapping and 2) to screen selected regions of GSY 1and HRC,positional candidate genes, for the presence ofPFHBI-causing mutation(s).Cosmids harbouring CI9q13.3 insert DNA were screened for the presence of (AAAT)n STRsby dot blot and Southern blot hybridisation using a radiolabelled (AAAT)lO oligonucleotideprobe. To characterise the harboured (AAAT)n STRs, the positively hybridising fragmentsidentified by Southern blot were sub-cloned, sequenced and primers designed from the uniquerepeat-flanking sequences. These primers were used to genotype the (AAAT)n repeat locus toassess its polymorphic nature in a panel of unrelated individuals. Alternatively, vectorettePCR, a rapid method of identifying repeat sequences and obtaining the flanking sequences inlarge inserts, was employed to develop polymorphic markers from the positively hybridisingclones. Selected exons of GSY1 and HRC were screened for the presence of potentiallydisease-causing mutations by PCR-SSCP analysis and direct sequencing, respectively, inPFHBI-affected and unaffected family members.Of the available cosmid clones that gave strong signals on dot blot and Southern blothybridisation, three, 29395, 24493 and 20381, were located within the critical PFHBI areaand were used for marker development. An interrupted (AAAT)n repeat motif (n less than 5)was identified in cosmid 29395, however, the repeat locus was not polymorphic in the testedpopulation. No (AAAT)n motif, single or repeated was observed in the partial sequence of the sub-cloned fragment of cosmid 24493. Using vectorette peR, no repeated (AAAT)n motifwas identified on sequencing the generated products in either cosmid 24493 or 2038l.However, diffuse single AAAT motifs were detected in both cosmids. Exons 4, 5, 11, 12 and16 of GSY 1, containing domains that are conserved across species, and the conserved eterminus-encoding exons 2-6 of HRC were selected for screening for potential PFHBI-causingmutation(s). However, no sequence variations were detected.The interrupted (AAAT)n repeat identified in cosmid 29395 was not polymorphic, whichconfirmed reports that complex repeats, especially those containing AAAT motifs of less than6 repeats, are not polymorphic. One possible explanation for the absence of a repeated AAATmotif in cosmids 24493 and 20381, which both gave positive hybridisation signals, is that thelow annealing temperature of the AfT -rich repeat-anchored primers used in vectorette peRmay have resulted in transient annealing to the diffuse single AAAT motifs detected onsequencing. The screened regions of candidate genes GSYI and HRC were excluded fromcarrying the disease-causing mutation(s).The availability of new sequence data generated by the Human Genome Project will influencefuture strategies to identify the PFHBI gene. Electronic searches will allow identification ofSTR sequences for development of polymorphic markers and gene annotation will allowselection of new candidate genes for mutation screening.

[发布机构] Stellenbosch University