Familial hypercholesterolaemia workshop for leveraging point-of-care testing and personalised medicine in association with the Lipid and Atherosclerosis Society of Southern Africa.

Familial hypercholesterolaemia (FH) is a common autosomal dominantly inherited disorder in which impaired clearance of plasma low-density lipoprotein cholesterol causes premature atherosclerotic vascular disease and tendon xanthomata. This workshop aimed to consolidate information on the diagnosis and management of FH in South Africa. The genetic causes include mutations in the LDL receptor, apolipoprotein B100 and proprotein convertase subtilisin/kexin type 9 (PCSK9). Additionally, the concatenation of multiple gene variants can result in polygenic FH. Therapeutic measures include a healthy lifestyle, statins and cholesterol-absorption inhibitors that will achieve control of the dyslipidaemia in the majority of cases. The recently introduced monoclonal antibodies to PCSK9 can improve achievement of target concentration in severe cases. FH is present in all sectors of the South African population but there is sparse documentation in the indigenous African populations. FH should be actively sought, diagnosed and treated with judicious pharmacotherapy and screening of relatives.

Pharmacogenetics of Aromatase Inhibitors in Endocrine Responsive Breast Cancer: Lessons Learnt from Tamoxifen and CYP2D6 Genotyping.

BACKGROUND: Genetics play a significant role in drug metabolism of endocrine therapy of breast cancer. These aspects have been studied extensively in patients on tamoxifen, but the pharmacogenetics of aromatase inhibitors are less established. In contrast to the protective effect of tamoxifen, aromatase inhibitors are linked with an increased risk for bone loss and fractures. OBJECTIVE: This review outlines key issues in the implementation of pharmacogenetics of cytochrome P450 and tamoxifen as a model for optimal use of aromatase inhibitors in postmenopausal women with estrogen receptor positive breast cancer. METHODS: Lessons learnt from the association between tamoxifen and CYP2D6 genotyping were applied to identify polymorphisms with the potential to change clinical decision-making in patients on aromatase inhibitors. The ability of next generation sequencing to supersede single-gene analysis was furthermore evaluated in a subset of breast cancer patients on aromatase inhibitors selected from a central genomics database. RESULTS: Methodological flaws in major randomised controlled trials and continued referral to incorrect results in expert consensus statements are important factors delaying the implementation of CYP2D6 pharmacogenetics in tamoxifen treatment. This highlighted the importance of a clinical pipeline including comprehensive genotyping, to define the target population most likely to benefit from aromatase inhibitor pharmacogenetics. CONCLUSION: The clinical utility of CYP2D6 genotyping is well-established in patients at increased risk of tamoxifen resistance due to cumulative risk. The pharmacogenetics of CYP19A1 requires further clarification in terms of bone risk assessment for appropriate use in the treatment algorithm of high-risk patients at the onset of aromatase inhibitors.

Postmenopausal Breast Cancer, Aromatase Inhibitors, and Bone Health: What the Surgeon Should Know.

Breast cancer, as the most common malignancy in women, remains a major public health issue despite countless advances across decades. Endocrine therapy is the cornerstone of treatment of the hormone-sensitive subtype of breast cancer. The use of aromatase inhibitors (AIs) in the postmenopausal women has extended the survival beyond that of Tamoxifen, but harbors a subset of side effects, most notably accelerated bone loss. This, however, does not occur in all women undergoing treatment. It is vital to identify susceptible patients early, to limit such events, employ early treatment thereof, or alter drug therapy. International trials on AIs, predominantly performed in North American and European females, provide little information on what to expect in women in developing countries. Here, surgeons often prescribe and manage endocrine therapy. The prescribing surgeon should be aware of the adverse effect of the endocrine therapy and be able to attend to side effects. This review highlights clinical and biochemical factors associated with decrease in bone mineral density in an, as yet, unidentified subgroup of postmenopausal women. In the era of personalized medical care, appropriate management of bone health by surgeons based on these factors becomes increasingly important.

Apolipoprotein E genotyping and questionnaire-based assessment of lifestyle risk factors in dyslipidemic patients with a family history of Alzheimer's disease: test development for clinical application.

The cholesterol-raising properties of the apolipoprotein E (APOE) epsilon-4 (ε-4) allele has been validated in the South African population. Mounting evidence supports the added value of APOE genotyping for the evaluation of cardiovascular risk in dyslipidemic patients beyond its established role in the diagnosis of late-onset Alzheimer's disease (AD). The aim of this study was to determine the potential benefits of combining AD family history with questionnaire-based lifestyle assessment to facilitate the clinical interpretation of APOE genotyping results. A total of 580 unrelated South African individuals prospectively enrolled in a chronic disease screening program incorporating a genetic component (2010-2015) was selected for inclusion in this study based on the presence (75) or absence (505) of AD family history. Biochemical assessment of their lipid profiles was performed according to standard laboratory protocols. All study participants were genotyped for the APOE ε-2/ε-3/ε-4 alleles using allele-specific TaqMan real-time polymerase chain reaction technology. In patients without a family history of AD, APOE genotype modified the relationship between alcohol intake and body mass index (p = 0.026), with a significant positive correlation noted between these parameters being limited to ε-4 allele carriers. APOE genotype also modified the association between alcohol intake and total serum cholesterol in patients with a positive family history of AD (p = 0.026). We demonstrated the benefits of a questionnaire-based approach for assessment of lifestyle risk factors to facilitate clinical interpretation of APOE genotyping results for targeted intervention in a genetic subgroup of dyslipidemic patients at increased risk for AD.

Apolipoprotein E ε-4 as a genetic determinant of Alzheimer's disease heterogeneity.

Alzheimer's disease (AD) displays a high degree of heterogeneity in terms of its etiology, presentation, prognosis, and treatment response. This can partly be explained by high-penetrance mutations in the amyloid precursor protein, presenilin 1 and presenilin 2 genes causing amyloid beta aggregation, which is a major pathogenic mechanism in the development of early-onset AD in a small subgroup of patients. Late-onset AD is considered a polygenic disorder in which cumulative risk resulting from interaction with modifiable environmental risk factors may be responsible for the majority of cases. The ε-4 allele of the apolipoprotein E (APOE) gene has emerged as the most significant genetic risk factor for late-onset AD, influencing nearly every pathogenic domain affected in AD. It is a major risk factor for cerebral amyloid angiopathy, recognized as a common pathological finding in an AD subtype associated with white matter dysfunction. The APOE ε-4 allele is also a known risk factor for ischemic stroke, which can result in vascular dementia or contribute to subcortical vascular dysfunction. In this review, we evaluate the clinical relevance of APOE genotyping in relation to cholesterol metabolism and available evidence on risk reduction strategies applicable to AD.

A radiological method to evaluate alveolar bone regeneration in the Chacma baboon (Papio ursinus).

INTRODUCTION: The evaluation of alveolar bone healing may have a role in dental implantology, in prosthodontics in the post-extraction phase and in monitoring fracture repair. There are several radiological techniques described to evaluate alveolar bone regeneration. However, most are expensive and time consuming. OBJECTIVES: To develop and evaluate a radiological method utilising readily available equipment to measure alveolar bone regeneration. MATERIALS AND METHODS: An apparatus was designed to enable the acquisition of standardized x-ray images, consisting of a disposable impression tray, digital positioning system, aluminum step wedge, digital x-ray sensor, Rinn apparatus and laboratory putty. Bone biopsies were collected from each oral quadrant in each of five Chacma baboons (Papio ursinus). Accurately standardised x-ray images of the biopsy sites were taken pre-operatively, directly post-operatively and again after three and six week intervals. These images were analysed using a graded histogram provided in a computer software program. RESULTS: The average gray-scale value on the histogram of the selected biopsy area was determined on the series of standardised images. The average values for the three biopsied sites per quadrant were expressed as percentages of pre-operative density. The results ndicated a mean ncrease of 6.3% (+/- 1.4%) (mean +/- 1 SEM) in bone density after three weeks and 12.6% (+/- 1.7%) six weeks post-operatively. CONCLUSION: A standardised radiologica examination method was developed which, together with a computer ised evaluation technique, could be applied to accurately determine relative bone density. This method was shown to provide comparative bone density values during the regeneration process of alveolar bone over a six week period.

Non-alcoholic fatty liver disease (NAFLD) in the Western Cape: a descriptive analysis.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is the most prevalent chronic liver disease in Western countries, but the disease profile has not yet been described in South Africa. NAFLD affects all spheres of society, especially the poorest and least educated. Aim. To investigate the demographics and clinical and biochemical features of South African patients diagnosed with non-alcoholic fatty liver and non-alcoholic steatohepatitis (NASH) in the Western Cape, South Africa. DESIGN/METHOD: Overweight/obese subjects were screened by ultrasound and those with fatty liver/hepatomegaly were included. Liver biochemistry, insulin resistance (using the insulin resistance homeostasis model assessment method for insulin resistance, HOMA-IR) and body mass index were assessed and liver biopsies were performed on patients older than 45 years with persistently abnormal liver function and/or hepatomegaly. RESULTS: We screened 233 patients: 69% coloured, 25% Caucasian, 5% black and 1% Asian. The majority (73%) were female. NAFLD was confirmed histologically in 111 patients, of whom 36% had NASH and 17% advanced liver fibrosis. No black patient had advanced fibrosis. Subjects with NASH had higher mean triglyceride (p=0.03) and cholesterol (p=0.01) levels than subjects with NAFL. All patients were insulin resistant/diabetic. HOMA-IR and not the degree of obesity was strongly associated with advanced fibrosis (p=0.09). CONCLUSION: This study is the first to describe the clinical characteristics of NAFLD in South Africa, albeit only in the Western Cape population. Insulin resistance was the universal factor present. The degree of obesity was not associated with severity of disease. The role of genetic risk factors in disease development and severity remains to be defined.

Pathogenic Mechanisms Underlying Iron Deficiency and Iron Overload: New Insights for Clinical Application.

Iron uptake, utilisation, release and storage occur at the gene level. Individuals with variant forms of genes involved in iron metabolism may have different requirements for iron and are likely to respond differently to the same amount of iron in the diet, a concept termed nutrigenetics. Iron deficiency, iron overload and the anemia of inflammation are the commonest iron-related disorders. While at least four types of hereditary iron overload have been identified to date, our knowledge of the genetic basis and consequences of inherited iron deficiency remain limited. The importance of genetic risk factors in relation to iron overload was highlighted with the identification of the HFE gene in 1996. Deleterious mutations in this gene account for 80-90% of inherited iron overload and are associated with loss of iron homeostasis, alterations in inflammatory responses, oxidative stress and in its most severe form, the disorder hereditary haemochromatosis (HH). Elucidation of the genetic basis of HH has led to rapid clinical benefit through drastic reduction in liver biopsies performed as part of the diagnostic work-up of affected patients. Today, detection of a genetic predisposition in the presence of high serum ferritin and transferrin saturation levels is usually sufficient to diagnose HH, thereby addressing the potential danger of inherited iron overload which starts with the same symptoms as iron deficiency, namely chronic fatigue. This review provides the scientific back-up for application of pathology supported genetic testing, a new test concept that is well placed for optimizing clinical benefit to patients with regard to iron status.

Iron and the folate-vitamin B12-methylation pathway in multiple sclerosis.

UNLABELLED: Some subjects with multiple sclerosis (MS) present with low blood iron parameters. Anecdotal reports and a single patient study suggest that iron supplementation may be beneficial in these subjects. Myelin is regenerated continually, but prerequisites for this process are iron and a functional folate-vitamin B12-methylation pathway. The aim of this study was to determine iron status, folate and homocysteine in MS subjects, and to evaluate the effect on MS symptoms if deficiencies were addressed. RESULTS: In relapsing-remitting MS subjects, serum iron concentration correlated significantly with age at diagnosis (r=0.49; p=0.008). In Caucasian female MS subjects, serum iron and ferritin concentrations were significantly lower than in matched controls. In a 6-month pilot study, 12 subjects taking a regimen of nutritional supplements designed to promote myelin regeneration, improved significantly neurologically as measured by the Kurzke EDSS (Total Score means 3.50 to 2.45, 29.9%; p=0.021). These were significantly improved (p=0.002) compared to 6 control group patients taking multivitamins (Kurzke Score increased by 13.9% from 4.83 to 5.50). Both groups had significantly reduced homocysteine concentrations at 6 months, suggesting that methylation is necessary but not sufficient for myelin regeneration.

Analysis of the three common mutations in the CARD15 gene (R702W, G908R and 1007fs) in South African colored patients with inflammatory bowel disease.

The caspase recruitment domain-containing protein 15 gene (CARD15) was recently identified as an important susceptibility gene for Crohn's disease (CD). The purpose of this study was to assess the likelihood that the three most common CARD15 mutations, R702W, G908R and 1007fs, contribute to inflammatory bowel disease (IBD) susceptibility in the South African colored population. The study cohort included 76 IBD patients, 41 with CD and 35 with ulcerative colitis (UC), and 100 population-matched controls. Mutations R702W, G908R and 1007fs were present at relatively low frequencies (<20%) in our study population. No statistically significant differences were furthermore, observed for these mutations between UC and CD patients or when compared with normal control individuals. Two additional mutations were identified, one novel (A661P) and one previously described (A725G), with the latter being identified in 4 of 35 (11%) UC patients. Statistically significant differences were obtained between UC and control individuals when comparing both allele (p<0.004, chi2 with Yates' correction=8.01) and genotype frequencies (p<0.004, chi2 with Yates' correction=8.14) for the A725G mutation, suggesting a possible role for this variant in disease expression.

Molecular diagnosis of hereditary hemochromatosis: application of a newly-developed reverse-hybridization assay in the South African population.

A recently developed strip-assay for hemochromatosis provides a rapid method for simultaneous detection of multiple mutations, which among others includes the HFE gene mutations V53M, V59M, H63D, H63H, S65C, Q127H, E168Q, and C282Y, previously detected in the general South African population using gel-based mutation-screening methods. The objective of the study was to determine the frequency of the relatively rare mutations in samples selected for altered iron parameters or a family history of hereditary hemochromatosis (HH) as part of the validation process of the assay for routine diagnostic purposes. The study population consisted of 451 individuals previously screened for mutations C282Y and H63D by restriction enzyme analysis in order to confirm or possibly exclude a diagnosis of HH. These individuals were subjected to mutation screening using the commercially available hemochromatosis strip-assay. Previous positive results for mutations C282Y and H63D in 233 individuals confirmed the accuracy of the reverse-hybridization assay. Mutation S65C was detected in 13 Caucasians, including three compound heterozygotes. These constituted 2% (13/600) of the chromosomes without mutations C282Y or H63D. The African-specific HFE mutation V53M was detected in one out of 11 (9%) African subjects screened. Mutation E168Q was detected in a single Caucasian individual together with mutation H63D. Our data demonstrate the value of the strip-based technology in providing a rapid and reliable comprehensive test for simultaneous analysis of multiple mutations.

Allelic variation in the promoter region of the LDL receptor gene: analysis of an African-specific variant in the FP2 cis-acting regulatory element.

DNA samples of 2303 individuals from nine different population groups were screened for variant -175g-->t in the promoter region of the low-density lipoprotein receptor (LDLR) gene. The -175g-->t variant detected at carrier frequencies of 3-10% in different African population groups was absent in the Caucasian and Asian (Chinese) individuals studied. In contrast to previous findings in Black South Africans where this polymorphism predominated in patients with familial hypercholesterolaemia (FH), it occurred at a significantly lower frequency in hypercholesterolaemics from the recently admixed Coloured population of South Africa compared with population-matched controls (P<0.0001). Haplotype and mutation analysis excluded the likelihood that this finding is due to association with a specific disease-related mutation in FH patients, although reversal of the positive association with FH observed in the Black population may, at least in part, be due to admixture linkage disequilibrium. Transient transfection studies in HepG2 cells demonstrated that the -175t allele is associated with a non-significant decrease ( approximately 7%) of LDLR transcription in the absence of sterols. The data presented in this study raise the possibility that the -175g-->t polymorphism may have subtle effects that become clinically important within certain genetic and/or environmental contexts.

Significance of novel endothelin-B receptor gene polymorphisms in Hirschsprung's disease: predominance of a novel variant (561C/T) in patients with co-existing Down's syndrome.

Several genes have been implicated in the pathogenesis of Hirschsprung's disease (HSCR). In a previous study performed, five novel (V202M, E480K, IVS10-2A/G, D771N, IVS19-9C/T) mutations and one previously described mutation (P937L) have been identified in the RET proto-oncogene in 20% of the study population. To further investigate the involvement of other genes, mutation analysis of the endothelin-B receptor (EDNRB) gene was performed in 52 unrelated sporadic HSCR patients, including 38 non-syndromic and 14 patients with HSCR and Down's syndrome. Six novel (178G/A, 552C/T, 561C/T, 702C/T, IVS3-6C/T and IVS4 + 3A/G) sequence variants and one previously described (831G/A) polymorphism were identified. Statistically significant differences were achieved for six (178G/A, 552C/T, 561C/T, 702C/T, IVS3-6C/T and 831G/A) of these variants. The T-allele of the 561C/T polymorphism was over represented in the HSCR/Down's syndrome patient group (36% representing 5 of 14) compared to normal controls (6% representing 5 of 84) (p < 0.002, chi(2) with Yates correction = 12.14), suggesting that the 561C/T variant is associated with a low penetrance effect in patients with this complex phenotype. Detection of the 178G/A polymorphism in only non-syndromic HSCR patients, provide further support for an important role of specific sequence variants in the EDNRB gene in the HSCR/Down's syndrome phenotype.

Familial adenomatous polyposis in two Black South African families.

The apparent low incidence of colon cancer in the Black population of South Africa has been ascribed to a non-Western diet. The present authors report the identification of two common 5-bp deletions at codons 1309 and 1061 of the adenomatous polyposis coli (APC) gene in a Xhosa and Zulu patient, respectively. The in vitro transcription/translation test (PTT) and a non-radioactive heteroduplex method, which facilitates resolution of enzymatically amplified DNA by agarose gel electrophoresis, were used for mutation detection. This study represents the first report of APC mutations in indigenous Black individuals clinically diagnosed with familial adenomatous polyposis coli (FAP). The two deletion mutations are responsible for FAP in 35% of affected South Africans, a frequency similar to that described in several other non-African populations. The apparently low incidence of colon cancer in the African population may be ascribed either to the rare occurrence of the 'second hit' needed for polyp formation or to a lower incidence of mutations in the APC gene.

Single nucleotide polymorphisms of the protoporphyrinogen oxidase gene: inter-population heterogeneity of allelic variation.

Five single nucleotide polymorphisms (SNPs) in the protoporphyrinogen oxidase gene (PPOX) were used for inter-population comparisons of six South African populations and two non-South African Caucasian populations. Novel polymorphisms identified in the promoter region and exon 11 of the PPOX gene, as well as three known variants in exon 1 and intron 2, were analysed using single-strand conformation polymorphism (SSCP) and restriction enzyme analyses. Significant population differences were found for four of the five polymorphisms analysed. A G-to-A transition was found at nucleotide position -1081 and is the first polymorphism to be identified in the 5' promoter region of the gene. A novel A-to-C substitution at nucleotide position 3880 in exon 11 was not detected in subjects of European descent. This study represents the first inter-population comparison of allelic variation at the PPOX locus. The significant differences observed between populations demonstrate the importance of population considerations when marker association studies are performed at this locus.

Prenatal diagnosis of familial hypercholesterolemia: importance of DNA analysis in the high-risk South African population.

In this report on the outcome of the first prenatal diagnosis performed for familial hypercholesterolemia (FH) in a South African family, we aim to demonstrate the value of a population-directed screening strategy to identify FH patients in populations with an enrichment for certain low-density lipoprotein receptor (LDLR) gene mutations. Prenatal diagnosis was offered to an Afrikaner couple, both partners heterozygous for the FH mutation D206E, whose first child was diagnosed with heterozygous FH and the second with homozygous FH. Genomic DNA isolated from parental peripheral blood and subsequently amniotic fluid was amplified by the polymerase chain reaction (PCR) and subjected to mutation analysis. Heterozygosity for mutation D206E was confirmed in both parents, whilst this mutation was not detected in DNA directly amplified from amniotic fluid. To exclude the possibility of a false-negative result due to the limited number of cells in the uncultured amniotic fluid sample, cells were also cultured in vitro, and the DNA extracted and subjected to a second round of analysis. This confirmed the absence of mutation D206E in the fetus. This case illustrates the application of a DNA-based mutation detection technique as a simple and rapid diagnostic aid that can be carried out at a relatively early gestational stage. Prenatal diagnosis of FH, aimed at the detection of homozygous cases, is particularly feasible in populations and families with molecularly defined LDLR gene mutations.