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CARDIOVASCULAR JOURNAL OF AFRICA • Volume 27, No 3, May/June 2016
AFRICA
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associated with a benign outcome.
12
The clinical pattern and
outcome of HCM in Africans is not known. The aim of this
study was to delineate the clinical features, spectrum of disease-
causing mutations and outcome of HCM in African patients.
Methods
Consecutive patients diagnosed with HCM at the Cardiac Clinic,
Groote Schuur Hospital (GSH), Cape Town, South Africa
were prospectively enrolled into a longitudinal cohort study of
familial cardiomyopathy, from 1 February 1996 to 31 August
2012. The diagnosis of HCM was based on the presence of a
hypertrophied, non-dilated left ventricle in the absence of other
diseases capable of producing the degree of observed LVH (i.e.
left ventricular wall thickness
>
14 mm on echocardiography).
13
Clinical data were collected at six-monthly visits during the study
period.
The study was designed in keeping with the principles of the
Helsinki Declaration, and was approved by the University of
Cape Town Human Research Ethics Committee. All participants
gave informed, written consent to participate in the study.
All patients had comprehensive clinical assessment,
complementedbychest radiography, electrocardiography, detailed
two-dimensional and Doppler colour-flow echocardiography,
and cardiac catheterisation, when appropriate. The primary
imaging modality used for diagnosis in all patients was
transthoracic two-dimensional and Doppler echocardiography.
Patients found to have outflow tract gradients below 40 mmHg
underwent Valsalva manoeuvre. Patients with cardiovascular
risk factors, angina or subjects over 40 years old frequently
underwent coronary angiography, at the discretion of the
attending clinician.
A comprehensive database that incorporated patient
demographic details, medical history, co-morbidity, medical
therapy, clinical, electrographic and echocardiographic details
was utilised. Normal values for echocardiographic measurement
were based on age and body-surface area, as described by Lauer
et al
.
14
Genotyping
Peripheral blood was collected from HCM probands for DNA
extraction using standard methods. Mutation screening was
undertaken by pyrosequencing of the coding regions and exon/
intron boundaries of the following 15 genes that are associated
with HCM: cardiac myosin-binding protein C (
MYBPC3
),
cardiac
β
-myosin heavy chain (
MYH7
), cardiac troponin T
type 2 (
TNNT2
), cardiac troponin I type 3 (
TNNI3
), regulatory
light chain of myosin (
MYL2
), essential light chain of myosin
(
MYL3
), tropomyosin 1 (
TPM1
), phospholamban (
PLN
),
α
-actin (
ACTC1
), cysteine and glycine-rich protein 3 (
CSRP3
),
AMP-activated protein kinase (
PRKAG2
),
α
-galactosidase
(
GLA
), four-and-a-half LIM domains 1 (
FHL1
), lamin A/C
(
LMNA
) and lysosome-associatedmembrane protein 2 (
LAMP2
)
(Table 1).
15
Exons and intron/exon boundaries (
±
10 base pairs) of the 15
cardiomyopathy-related genes were amplified by microdroplet
polymerase chain reaction (PCR) using RDT 1000 technology
(Rain Dance Technologies, Billerica, MA 01821, USA). Libraries
were prepared using the Rapid Library 454 FLX protocol, which
included adding molecular identifiers to each sample. Samples
were pooled and then sequenced using the Roche 454 FLX next-
generation sequencing platform. Samples were processed and
analysed using NextGENe version 2.2.0 (SoftGenetics).
Prior to analysis, reads were trimmed and low-quality
reads were removed. Reads were aligned to .gbk files and
variants seen in
<
20% were annotated. Variants were filtered,
taking into account coverage, read balance, allele balance
and homopolymers. Samples with coverage below 10 were
considered failures. Unclassified variants were Sanger sequenced
to confirm their presence; known polymorphisms were not
Sanger sequenced.
The Cape Town population controls were used to determine
the population frequencies of all novel variants identified in the
15 genes. One hundred and ninety-five anonymous blood donors
from the Western Province Blood Transfusion Service provided
consent for blood samples to be taken for DNA extraction. The
control DNA consisted of samples from 95 persons of mixed
ancestry, 50 black Africans and 50 white South Africans.
Statistical analysis
Simple descriptive statistics were used for data interpretation
and to draw inferences about the population of patients studied.
Results of continuous variables are given as means
±
SD.
Categorical variables are represented as number and percentage.
Pearson’s chi-squared or Fisher’s exact test were used to compare
the relative frequency of characteristics between individuals. All
p-
values were two-sided, and
p
≥
0.05 was considered not to
indicate statistical significance.
Survival analysis testing between groups was compared using
log-rank testing, and the Kaplan–Meier survival curves were
constructed using the product-limit method. Age-, gender- and
race-adjusted survival curves for the general South African
population were derived and compared with the Kaplan–Meier
survival rates for the patients with HCM. Analysis included
Table 1. List of genes that were subjected to
mutation screening in this study
Genes
Ensemble gene number
Chromosome: base range
MYBPC3
ENSG00000134571
chr11:47352958–47374253
MYH7
ENSG00000092054
chr14:23881948–23904870
TNNT2
ENSG00000118194
chr1:201328143–201346805
TNNI3
ENSG00000129991
chr19:55663137–55669100
TPM1
ENSG00000140416
chr15:63334838–63364111
MYL2
ENSG00000111245
chr12:111348626–111358404
MYL3
ENSG00000160808
chr3:46899357–46904973
ACTC1
ENSG00000159251
chr15:35080297–35087927
PLN
ENSG00000198523
chr6:118869442–118881586
CSRP3
ENSG00000129170
chr11:19203578–19223589
FHL1
ENSG00000022267
chr X:135229559-135293518:1
PRKAG2
ENSG00000106617
chr 7:151253197-151574210
GLA
ENSG00000102393
X:100652791-100662913
LMNA
ENSG00000160789
chr1:156084461–156109878
LAMP2
ENSG00000005893
chr X:119561682-119603220
MYBPC3
, myosin-binding protein 3;
MYH7
, beta-myosin heavy chain;
TNNT2
, troponin T;
TNNI3
, troponin I;
MYL2
, essential myosin light chain;
MYL3
, regulatory myosin light chain;
TPM1
, tropomyosin 1 alpha;
PLN
,
phospholamban;
ACTC1
, actin;
CSRP3
, cysteine- and glycine-rich protein 3;
PRKAG2
, 5
′
-AMP-actvated protein kinase;
GLA
, alpha-galactosidase;
FHL1
,
four-and-a-half LIM domains 1;
LMNA
, lamin A/C;
LAMP2
, lysosome-associ-
ated membrane protein.