56 / 70
CARDIOVASCULAR JOURNAL OF AFRICA • Volume 30, No 5, September/October 2019
298
AFRICA
The workshop had a series of presentations in a progression
from laboratory findings to clinical experience, a discussion on
how single-nucleotide variants (SNV) could culminate in a similar
state, and treatment. The relevance of FH to a previously poorly
studied subgroup of the South African population was indicated
before a discussion and formulation of recommendations.
Laboratory diagnosis of FH (Prof MJ Kotze)
The central issue of a raised plasma LDL-C concentration
was indicated, along with the genes that are responsible for
this metabolic derangement. An autosomal dominant pattern
of inheritance relates to mutations in the LDL receptor,
apolipoprotein B100 (the ligand for the LDL receptor) and
PCSK9. This latter protein, in gain-of-function mutations, results
in greater degradation of LDL receptors. An autosomal recessive
form of LDL hypercholesterolaemia occurs with mutations in
the LDL receptor adaptor protein 1 (
LDLRAP1
) gene. Whereas
the preceding disorders are monogenic, it is also possible
that several mutations with relatively low impact on LDL-C
concentration can together result in LDL hypercholesterolaemia
in the range seen with the monogenic disorders.
In South Africa, three mutations were identified in the LDL
receptor in the Afrikaner population: in decreasing prevalence,
designated Afrikaner 1 (D206E), Afrikaner 2 (V408M) and
Afrikaner 3 (D154N). This meant that only a few mutations
needed to be sought specifically to confirm the disorder at
a genetic level after a clinical diagnosis. This was applied to
the diagnosis in children and even to prenatal diagnosis when
the diagnosis of homozygous FH could be considered for
termination of pregnancy. The polymerase chain reaction (PCR)
has made it possible for primers to amplify a selected series of
nucleotides of interest in a given gene, where after changes could
be identified.
The techniques used ranged from Sanger sequencing that
demonstrates a different nucleotide in the chain of nucleotides,
through restriction-length polymorphism where a change of
nucleotides either creates or abrogates the cutting site for
a sequence-specific nucleotide, or by amplification-resistance
mutations where an alteration in nucleotide sequence does not
hybridise with a primer that initiates amplification in the PCR.
Such investigations revealed a three-nucleotide deletion in a Pedi
patient, a six-nucleotide deletion in other patients of indigenous
African ancestry, a mutation in patients of Indian ancestry,
and several mutations in patients of mixed ancestry. A reverse
hybridisation strip assay was designed for founder mutations.
There are several reasons for making a precise genetic
diagnosis. Not only is the clinical diagnosis confirmed but cascade
screening is more accurate. New genes could be discovered when
known genes in LDL hypercholesterolaemia are excluded – this
led to the discovery of a locus on chromosome 1, which was
later identified as the gene for PCSK9. Genetic studies can also
determine genes that modulate FH, including interactions with
environmental factors,
8
as well as finding polygenic causes for
FH. A polygenic cause for FH was investigated in an Afrikaner
family suspected of having FH. After exclusion of the common
three LDL receptor (
LDLR
) mutations and a complete sequence
of the
LDLR
, whole-exome sequencing (WES) was performed.
None of the four above-mentioned genes was implicated as the
cause of FH by WES.
The Global Lipid Genetic Consortium (GLGC) six-SNV
panel for polygenic hypercholesterolaemia includes the following
genes:
APOE
(E2 and E4),
ABCG8
,
APOB
,
CELSR2
and
LDLR
. This investigation did not meet the criteria for making
the diagnosis of FH and indicates that not all causes of
hypercholesterolaemia can be ascertained by currently known
genes. As indicated in Fig. 1, the extension of screening to
the 12-nucleotide gene score in a pedigree suspected of FH
showed an incremental gene score that may be responsible for
hypercholesterolaemia.
Genetics of heterozygous FH in Cape Town
(Prof AD Marais)
The genetic investigation of FH over more than 20 years at a
referral hospital lipid clinic was reported. The heterozygous FH
phenotype was defined as definite if a tendon xanthoma was
present, and probable if there was LDL hypercholesterolaemia
>
5 mmol/l and a dominant pattern of inheritance of
premature heart disease and/or hypercholesterolaemia in the
family of the index case. The heterozygous phenotype of
LDL hypercholesterolaemia, tendon xanthomata and ischaemic
heart disease after the age of 25 years was contrasted with the
homozygous FHphenotype inwhichLDLhypercholesterolaemia
exceeds 12 mmol/l, tendon and cutaneous xanthomata sets in
during childhood and ischaemic heart disease mostly occurs
before the age of 25 years when no intervention is done.
The heterozygous FH phenotype includes the genes already
mentioned but in the experience of the clinic, also homozygotes
for sitosterolaemia (a rare autosomal recessive disorder).
Polygenic FH was not specifically investigated in Cape
Town but is a consideration in the light of recent experience.
The homozygous FH phenotype was attributable to two LDL
receptor defects while it was stressed that experience with
homozygous apolipoprotein B100 or PCSK9 was limited.
Although also manifesting dose-dependent effects, the
latter two causes of the homozygous FH phenotype may
be somewhat milder; this also appears to be the case for
autosomal recessive hypercholesterolaemia due to
LDLRAP1
mutations. Sitosterolaemia mostly presents as the homozygous
FH phenotype but may be milder if the diet is low in cholesterol
and plant sterols.
The Medical Research Council (MRC) of South Africa
contributed towards research in FH after full evaluation of
patients referred with severe hypercholesterolaemia. Not only
were the history, family tree and physical findings carefully
assessed, but secondary causes were specifically excluded and
electrophoresis confirmed that the hypercholesterolaemia was
due to elevations of LDL-C. Consent for research was obtained.
The known LDL receptor mutations were first sought before this
gene was explored exon by exon. Assessment for large-fragment
insertions and deletions was not available although some regions
were examined. Hereafter exons 26 and 29 of apolipoprotein B
were explored for mutations that disrupt ligand function.
PCSK9
was then explored exon by exon.
The systematic approach was done for the first 993 unrelated
patients with FH but hereafter the commonest mutations
were performed in new patients. The original methods were
PCR with restriction digests or single-strand conformational
polymorphism but in the past decade the introduction of high-