CARDIOVASCULAR JOURNAL OF AFRICA • Vol 24, No 6, July 2013
232
AFRICA
South African cLQTS probands, including the 23 probands of
the families that are carriers of the founder p.A341V mutation,
for mutations in the five most frequently implicated cLQTS
genes,
KCNQ1
,
KCNH2
,
SCN5A
,
KCNE1
and
KCNE2,
by
multiplex capillary electrophoresis single-strand conformation
polymorphism (multi-CE-SSCP). A sub-group was further
analysed by the direct sequencing of these genes.
Methods
Forty-four LQTS probands (23 known founder mutation carriers
and 21 unrelated patients shown not to carry the founder
mutation) [77% female; mean (SD) age at first event 10 (6)
years, were ascertained to have cLQTS, mean (SD) QTc 501 (59)
ms]. See Table 1 for clinical and demographic information. The
diagnosis was based on the 1993 standard diagnostic criteria
22
and included a physical examination, a standard 12-lead ECG
and a personal interview. All probands were of the RWS type.
All patients provided written informed consent (parental in the
case of minors). The study was approved by the Health Research
Ethics Committee of the University of Stellenbosch.
Molecular genetic procedures
DNA was isolated from peripheral blood using a commercially
available procedure (Qiagen GmBH, Germany). Primers were
designed to amplify all the exons of the five LQTS-causing
genes under investigation, including the intron/exon boundaries.
The primers were synthesised by the Synthetic DNA Laboratory
(University of Cape Town, Cape Town, South Africa) and
Applied Biosystems (Copenhagen, Denmark).
PCR amplification was performed by published protocol for
SCN5A.
23
Primer sequences and PCR conditions for
KCNQ1,
KCNH2, KCNE1
and
KCNE2
are available upon request
). Primers used for generating PCR products for
sequencing were tagged with M13 sequence. PCR products were
qualitatively assessed on 2% agarose gels by standard procedures
prior to multi-CE-SSCP and direct sequencing.
Multi-CE-SSCP
PCR primers were labelled at their 5
′
end with one of the
following fluorophors: 6FAM
TM
, VIC
®
, NED
TM
or PET
TM
(Applied
Biosystems, Foster City, California, USA). The labelled PCR
amplicons were mixed and diluted according to published
protocol
23
for
SCN5A
. Amplicons of
KCNQ1, KCNH2, KCNE1
and
KCNE2
were mixed and diluted in a similar manner; the
specifics of these mixtures are available upon request.
Multi-CE-SSCP was performed on an ABI Prism
TM
3100
Genetic Analyser (PE Applied Biosystems, Foster City,
California, USA) with GeneScan polymer (Applied Biosystems,
Foster City, California, USA) at 18°C and 30°C. PCR amplicons
were sequenced to identify the variants responsible for alterations
in the electrophoretic mobility detected by multi-CE-SSCP
analysis.
Direct sequencing
A sub-group of 32 probands was additionally analysed by direct
DNA sequencing. The probands of the founder families were
included to assess the frequency of compound heterozygozity in
our cohort. PCR products were purified by exonuclease 1 and
shrimp alkaline phosphatase reaction.
Sequencing of both strands was performed using BigDye
®
Terminator v1.1 (Applied Biosystems, UK) and M13 primers
(sense
5
′
-CAGTTCTCACAGGAGCCACA-3
′
) and (antisense
5
′
-AGGTGAACTGGAACCACAGG-3
′
) (Taq Copenhagen,
Denmark). Sequences were analysed with the ABI 3730 DNA
analyser (Applied Biosystem, UK).
Bio-informatics
Nucleotide sequences were aligned to GenBank (http://
) reference sequences
KCNQ1
(NM_000218.2/NP_000209.2),
KCNH2
(NM_000238.2/
NP_000229.1),
SCN5A
(NM_000335.2/NP_000326.2),
KCNE1
(NM_000219.2/NP_000210.2) and
KCNE2
(NM_172201.1/
NP_75195.1). Multiple sequence alignments of multiple species
were performed with Clustal W (version 1.82) (
ac.uk/clustalw/#). SNPs were compared to the NCBI dbSNP
database (
) and the
NHLBI Exome Sequencing Project Exome Variant Server (EVS)
(
). These data were used as
in
silico
control.
Criteria for disease association
A genetic variant was considered a disease-causing mutation if
it resulted in an amino acid substitution, interfered with a splice
site or had previously been shown to be associated with cLQTS
and was not found in
in silico
controls and had not previously
been described as a polymorphism. Sequence variants also found
in controls were considered polymorphisms and are not reported
here.
Results
We identified 14 different disease-causing mutations in 34 of the
44 probands tested (Table 2). Three mutations were identified
among the 23 founder probands; all harboured the South African
founder mutation KCNQ1:p.A341V, while two probands (8.5%)
harboured an additional mutation in a second cLQTS-causing
gene, which may contribute to disease. These are cases of double
heterozygosity. Furthermore, 11 mutations were identified in 13
non-founder probands.
The multi-CE-SSCP results corroborated standard gel-based
PCR-SSCP results (not shown), however, in 32 cases where
the DNA concentration was sufficient, direct DNA sequencing
led to the identification of four disease-causing mutations
(KCNQ1:p.Y315C; KCNQ1:p.A344V; KCNH2:c.917-3T
>
C
and KCNH2:p.R328C), which were missed by multi-CE-SSCP.
In the case of KCNQ1:pA344V, two cases had been detected by
SSCP, but an additional case was identified by direct sequencing.
TABLE 1. DEMOGRAPHICAND CLINICAL
CHARACTERISTICS OF 44 INDEX PATIENTS
Cohort
% female
QTc
mean (SD)
Age at first event
mean (SD)
Total
77
501 (59)
10 (6)
A341V carriers
81
513 (48)
8 (4)
Other mutation carriers
73
507 (69)
11 (5)
No mutation detected
75
452 (35)
17 (7)
