Cardiovascular Journal of Africa: Vol 26 No 2 (March/April 2015)

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CARDIOVASCULAR JOURNAL OF AFRICA • Volume 26, No 2, March/April 2015 64 AFRICA
‘restrictive features’.1-3 Mutations in this gene have been described
to cause RCM, HCM and dilated cardiomyopathy and specific
mutations have on occasion been associated with more than
one of these phenotypes.4 We focused on screening TNNI3 in a
South African panel of HCM-affected probands for mutations,
which included these two probands, using a high-resolution melt
(HRM) approach.
Methods
Institutional approval was granted for this study by the Ethical
Review Committee of the Faculty of Medicine and Health
Science at Stellenbosch University (N04/C3/062). Informed,
written consent was obtained from all participants or, on the
behalf of minors/children enrolled in the study, from their
next of kin, caretakers or guardians. Clinical investigations
were conducted according to the principles expressed in the
Declaration of Helsinki.
The study cohort included the two index individuals with
RCM with focal ventricular hypertrophy and restrictive features
as well as 113 South African HCM-unrelated probands, all
previously diagnosed with HCM using standard criteria, with
or without known HCM-causing mutations. The two index
individuals and first-degree family members underwent a physical
examination, 12-lead electrocardiography and transthoracic
two-dimensional echocardiography (Doppler, tissue Doppler).
Past medical records were attained for deceased family
members and, if relevant, in living individuals. Rhythm and
conduction abnormalities were defined using established criteria.
Echocardiograms were analysed using measurement conventions
as outlined in the American Society of Echocardiography5 and
the British Society of Echocardiography
guidelines for the assessment of
patients with HCM.
DNA extraction and mutation analysis: DNA was extracted
from peripheral blood obtained from the participants as
previously described.6 A control group consisted of anonymous
blood samples from 100 mixed-ancestry individuals of varying
age and gender obtained from the Western Province Blood
Transfusion Services.
PCR amplification: the TNNI3 gene reference sequence
(accession number: NM_000363.4) was obtained from NCBI
Entrez Nucleotides Database
. All primers were designed using Integrated DNA
Technologies Software, Primer Quest .
Primer sequences are given in Table 1. The NCBI basic local
alignment search tool (BLAST)
was used to examine primer specificity.
Polymerase chain reaction (PCR) was performed in a reaction
mixture consisting of 30 ng of genomic DNA, kapa readymix
(Kapa Biosystems Inc, Massachusetts, USA) (1XKapa buffer, 1.5
mMMgCl2, 200 μM dNTPs, 1.0 U of kapa polymerase enzyme),
0.2 μM of each forward and reverse primer, 5% formamide, 2 μM
SYTO9 fluorescent dye (Invitrogen, California, USA), and H2O
to a final volume of 50 μl. PCR amplifications were performed
using the GeneAmp® PCR System 2700 (Applied Biosystems
Inc, California, USA). The standard PCR cycle consisted of
initial denaturation at 95°C for three minutes, followed by 35
cycles of denaturation at 95°C for 30 seconds, annealing at the
Ta for the specific PCR primer sets for 30 seconds, elongation at
72°C for 30 seconds, and a final elongation step at 72°C for five
minutes.
HRM analysis: PCR products were subsequently subjected
to HRM analysis on a Rotor-Gene 6000 analyser (Corbet Life
Sciences, Brisbane, Australia). The samples were held at 50°C for
one minute to ensure that the DNA was double stranded before
being melted by increasing the temperature from 75 to 95°C in
0.1°C increments. As the DNA separated into single strands, the
shift in fluorescence was measured. A characteristic denaturing
profile, which is based on the length and GC content of the
amplicon, was visualised for each DNA sample.
Nucleotide sequencing: representative DNA samples for
each distinct melting curve, identified by HRM analysis, were
bi-directionally sequenced using the BigDye® Terminator v3.1
cycle sequencing kit (Applied Biosystems Inc), followed by
electrophoresis on an ABI 3130XL genetic analyser (Applied
Biosystems Inc). Sequences were analysed using BioEdit
sequence alignment editor software v7.0.9.0.7 The sequences
were aligned to the TNNI3 reference sequence (accession
number: NM_000363.4) in NCBI
), using the ClustalW v1.4 programme and analysed
for mismatches to the reference sequence.
The effect of sequence variants on restriction enzyme
recognition sites was determined using
, and NEBcutter,
. Restriction enzyme analysis was then used to
confirm genotypes, where possible. Digestion reactions consisted
of 10 μl of the PCR product, 1 × appropriate restriction enzyme
buffer, five units of the restriction enzyme and H2O to a final
volume of 20 μl. Samples were then incubated at the temperature
and for the duration recommended by the manufacturer (New
England Biolabs Inc, Massachusetts).
Haplotype construction: haplotypes were typed on different
loci with microsatellite markers provided by the Ensemble
database. Linkage to a specific region on a certain chromosome
was determined with four microsatellite markers, namely
(D19S926, D19S418, D19S880, D19S605) for TNNI3. The
forward primer for each marker was fluorescently labelled with
either FAM, HEX or Cy5 for easy genotyping. Each marker
Table 1. Oligonucleotide primers used for the
amplification of relevant exons in the TNNI3 gene
Position
(exon) Primers (5′-3′) Tm
(C°)
Ta
(C°)
Size
(bp)
1F CCGTTATCTGGCATAGTGG 56.6
54 338
1R AGAGTCCCTACGCCTACCT 55.5
2_3F GACACAGCCCACCACTAA 55.3
54 366
2_3R ACTCCCAGGGTCTTGGAT 56.8
4F ACTCAGGGCTCAAGTTGG 56.2 54 239
4R CACCCATTCTCAAGCTCC 56.6
5F CACGCCTGGTCTTTATCC 56.6 54 222
5R AGAAACCTCGCATCCTTG 56.3
6F CCCAACAACACACACCAC 56.4
54 177
6R AAGTCCCAGCCATCTCAC 55.9
7F GGAAATGGAAGGAGAAGTACC 56.7
52 257
7R CCTCAGCATCCTCTTTCC 55.7
8F GGAGACCAAGAAGAGACCC 56.1
54 230
8R GCCTAAGCCCTGGGTAAT 56.7
bp: base pairs; C°: degrees Celsius; F: forward, R: reverse; Ta: anneal-
ing temperature; T m: melting temperature.