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
Date
2000-03
Authors
Makubalo, Zola
Journal Title
Journal ISSN
Volume Title
Publisher
Stellenbosch : Stellenbosch University
Abstract
ENGLISH ABSTRACT: Progressive familial heart block type I (PFHBI) is a cardiac ventricular conduction disorder of
unknown cause associated with risk of sudden death, which has been described in several
South African families. Clinically, PFHBI is characterised by right bundle branch block on
ECG, which may progress to complete heart block, necessitating pacemaker implantation.
The disease shows an autosomal dominant pattern of inheritance with evidence of genetic
anticipation. Using genetic linkage analysis, the PFHBI-causative gene was mapped to a 10
eentimorgan (cM) gene-rich area of chromosome (C) 19q13.3, which has, subsequently, been
reduced 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 polymorphic
markers 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 STRs
by dot blot and Southern blot hybridisation using a radiolabelled (AAAT)lO oligonucleotide
probe. To characterise the harboured (AAAT)n STRs, the positively hybridising fragments
identified by Southern blot were sub-cloned, sequenced and primers designed from the unique
repeat-flanking sequences. These primers were used to genotype the (AAAT)n repeat locus to
assess its polymorphic nature in a panel of unrelated individuals. Alternatively, vectorette
PCR, a rapid method of identifying repeat sequences and obtaining the flanking sequences in
large inserts, was employed to develop polymorphic markers from the positively hybridising
clones. Selected exons of GSY1 and HRC were screened for the presence of potentially
disease-causing mutations by PCR-SSCP analysis and direct sequencing, respectively, in
PFHBI-affected and unaffected family members.
Of the available cosmid clones that gave strong signals on dot blot and Southern blot
hybridisation, three, 29395, 24493 and 20381, were located within the critical PFHBI area
and 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 tested
population. 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 motif
was 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 and
16 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-causing
mutation(s). However, no sequence variations were detected.
The interrupted (AAAT)n repeat identified in cosmid 29395 was not polymorphic, which
confirmed reports that complex repeats, especially those containing AAAT motifs of less than
6 repeats, are not polymorphic. One possible explanation for the absence of a repeated AAAT
motif in cosmids 24493 and 20381, which both gave positive hybridisation signals, is that the
low annealing temperature of the AfT -rich repeat-anchored primers used in vectorette peR
may have resulted in transient annealing to the diffuse single AAAT motifs detected on
sequencing. The screened regions of candidate genes GSYI and HRC were excluded from
carrying the disease-causing mutation(s).
The availability of new sequence data generated by the Human Genome Project will influence
future strategies to identify the PFHBI gene. Electronic searches will allow identification of
STR sequences for development of polymorphic markers and gene annotation will allow
selection of new candidate genes for mutation screening.
AFRIKAANSE OPSOMMING: Sien volteks vir opsomming
AFRIKAANSE OPSOMMING: Sien volteks vir opsomming
Description
Thesis (MSc)--Stellenbosch University, 2000.
Keywords
Heart -- Diseases -- Genetic aspects, Heart -- Diseases -- Molecular aspects, Coronary heart diseases, Dissertations -- Medicine