Detection of sequence diversity in the CYP2C19 gene of Xhosa South African individuals : an analytical and comparative study including in silico and functional analysis of the 5’ flanking region

Thesis (MSc (Genetics))--University of Stellenbosch, 2010.Thesis presented in partial fulfilment of the requirements for the degree of Master of Science (MSc) in Genetics at Stellenbosch University.ENGLISH ABSTRACT: The prevalence of adverse drug reactions (ADR) and treatment failure in South Africa requires urgent addressing and it is the aim of pharmacogenetics to aid in the alleviation of these ADRs and treatment failures. However, considering the high level of genetic diversity present in African populations, preliminary analysis of the genetic profiles of South African populations is required before pharmacogenetics can be successfully implemented in the South African context. Therefore this study aimed to characterise the gene encoding the drug metabolising enzyme, CYP2C19, in the South African Xhosa population. To identify the common CYP2C19 sequence variation present in the Xhosa population, semiautomated sequence analysis of CYP2C19 was performed on 15 healthy Xhosa individuals. The variation detected was then prioritised through various in silico analyses for further restriction fragment length polymorphism (RFLP) genotyping in an additional 85 healthy Xhosa individuals to confirm the frequencies of the prioritised variants in a larger cohort, while the copy number variation (CNV) present in the entire 100 Xhosa individuals was analysed with the use of duplex real-time PCR. To functionally validate the in silico data obtained for the 5’-upstream variants, dual luciferase reporter assays were utilised. In addition to these analyses, multi-species comparisons were used to highlight regions of high sequence similarity within the 5’-upstream regions, while CpG island analysis was utilised to identify possible CpG islands occurring within and around the CYP2C genes. Sequence analysis of the CYP2C19 gene revealed 30 variants, of which five were novel. Subsequent to RFLP analysis, the frequencies of the allele-defining variants detected in this population, namely CYP2C19*2, CYP2C19*9, CYP2C19*15 and CYP2C19*17 were found to be 0.21, 0.09, 0.09 and 0.10, respectively. Additionally, the novel non-synonymous V374I variant, which was designated CYP2C19*28, was found to occur at a frequency of 0.01. Dual luciferase reporter assays revealed that the construct containing the rs7902257 variant, demonstrated a significant decrease in the fold induction observed when compared to the “wild type” construct (P = 0.0077). This variant was designated CYP2C19*27 and was detected at a frequency of 0.33 in the Xhosa population. In addition to this, multi-species comparisons revealed four highly conserved regions, all of which were present within LINE L1 repetitive elements. Although putative CpG islands were identified in and around the CYP2C genes, no direct correlations could be made between the differences in expression observed between the genes and the presence of the CpG islands. The role of these islands with regards to the epigenetic regulation of these genes therefore remains to be elucidated. To our knowledge, this study provides the most comprehensive data for CYP2C19 in a South African population and shows that the Xhosa population displays a unique genetic profile, which differs from those of other populations, including the Cape Mixed Ancestry population of South Africa. Thus, novel genotyping platforms need to be developed in order to successfully apply pharmacogenetics to the diverse populations residing in South Africa.AFRIKAANSE OPSOMMING: Die doel van Farmakogenetika is om daadwerklike aandag aan die hoë voorkoms van nadelige geneesmiddel reaksies en mislukte behandelings te skenk en om hierdie voorkoms in Suid-Afrika te verlaag. Die bevolkingsgroepe van Afrika het hoë vlakke van genetiese diversiteit en dus hang die susksesvolle toepassing van Farmakogenetika in Suid-Afrika af van die voorlopige analise van die genetiese profiele van die Suid-Afrikaanse bevolkingsgroepe. Om hierdie rede was die doel van hierdie studie om die geneesmiddel metaboliseerings geen, CYP2C19, in ’n Suid-Afrikaanse Xhosa bevolkingsgroep te karakteriseer. Die CYP2C19 volgorde van 15 Xhosa individue is bepaal om die algemene variasie teenwoordig in die CYP2C19 geen te bevestig. Hierdie variasies is deur verskeie in silico analises geprioritiseer vir verder restriksie fragment lengte polimorfisme (RFLP) genotipering in 85 gesonde Xhosa individue om die frekwensie in ’n groter groep te bevestig, terwyl die kopie aantal variasie teenwoordig in hierdie 100 Xhosas geanaliseer is met Taqman® CNV toetse. Om die in silico data vir die 5’-stroomop variante funksioneel te bevestig, is daar gebruik gemaak van tweedeelige luciferase verklikker toetse. Verder is multi-spesie vergelykings gebruik om 5’-stroomop streke met hoë vlakke van ooreenstemming te identifiseer, terwyl CpG-eiland analise gebruik is om moontlike CpG-eilande in die omgewing van die CYP2C gene te identifiseer. Met behulp van volgorde bepaling van die CYP2C19 geen, is 30 variante geïdentifiseer. Uit hierdie variante was vyf vir die eerste keer met hierdie studie opgespoor. Met die gebruik van RFLP analise, is die alleel definerende variante naamlik CYP2C19*2, CYP2C19*9, CYP2C19*15 and CYP2C19*17, teen ’n frekwensie van 0.21, 0.09, 0.09 en 0.10 in die Xhosa bevolkingsgroep gevind. Verder was die nie-sinonieme variant, V374I, wat vir die eerst keer geïdentifiseer en CYP2C19*28 genoem is, teen ‘n frekwensie van 0.01 gevind. Tweedeelige luciferase verklikker toetse het bewys dat die konstruk met die rs7902257 variant ‘n beduidende afname in induksie in vergelyking met die “wilde tipe” konstruk gewys het (P = 0.0077). Hierdie variant was CYP2C19*27 genoem en was teen ’n frekwensie van 0.33 in die Xhosa bevolking gevind. Die multi-spesie vergelykings het vier gekonserveerde streke geïdentifiseer wat in LINE L1 herhalende elemente gevind is. Alhoewel CpG-eilande in die omgewing van die CYP2C gene gevind is, kon geen direkte korrelasies gemaak word tussen die veranderinge in uitdrukking van die gene en die teenwoordigheid van die CpG-eilande nie. Die rol van hierdie eilande met betrekking tot epigenetiese regulasie van hierdie gene moet dus nog ontrafel word. Tot ons kennis het hierdie projek die mees voledige inligting vir CYP2C19 in ’n Suid-Afrikaanse bevolkingsgroep gegee en het bewys dat die Xhosa bevolkingsgroep ‘n unieke genetiese profiel vertoon, wat van ander bevolkingsgroepe, insluitend die Kaapse Gemenge Herkoms populasie van Suid-Afrika, verskil het. Indien farmokogenetika suksesvol in die diverse bevolkingsgroepe van Suid-Afrika toegepas kan word, moet daar gebruik gemaak word van nuwe genotipering metodes

Introduction of the AmpliChip CYP450 Test to a South African cohort : a platform comparative prospective cohort study

The original publication is available at http://www.biomedcentral.com/1471-2350/14/20Abstract Background Adverse drug reactions and lack of therapeutic efficacy associated with currently prescribed pharmacotherapeutics may be attributed, in part, to inter-individual variability in drug metabolism. Studies on the pharmacogenetics of Cytochrome P450 (CYP) enzymes offer insight into this variability. The objective of this study was to compare the AmpliChip CYP450 Test® (AmpliChip) to alternative genotyping platforms for phenotype prediction of CYP2C19 and CYP2D6 in a representative cohort of the South African population. Methods AmpliChip was used to screen for thirty-three CYP2D6 and three CYP2C19 alleles in two different cohorts. As a comparison cohort 2 was then genotyped using a CYP2D6 specific long range PCR with sequencing (CYP2D6 XL-PCR + Sequencing) platform and a PCR-RFLP platform for seven CYP2C19 alleles. Results Even though there was a low success rate for the AmpliChip, allele frequencies for both CYP2D6 and CYP2C19 were very similar between the two different cohorts. The CYP2D6 XL-PCR + Sequencing platform detected CYP2D6*5 more reliably and could correctly distinguish between CYP2D6*2 and *41 in the Black African individuals. Alleles not covered by the AmpliChip were identified and four novel CYP2D6 alleles were also detected. CYP2C19 PCR-RFLP identified CYP2C19*9,*15, *17 and *27 in the Black African individuals, with *2, *17 and *27 being relatively frequent in the cohort. Eliminating mismatches and identifying additional alleles will contribute to improving phenotype prediction for both enzymes. Phenotype prediction differed between platforms for both genes. Conclusion Comprehensive genotyping of CYP2D6 and CYP2C19 with the platforms used in this study, would be more appropriate than AmpliChip for phenotypic prediction in the South African population. Pharmacogenetically important novel alleles may remain undiscovered when using assays that are designed according to Caucasian specific variation, unless alternate strategies are utilised.Publishers' Versio

Genome-wide Scan Identifies Association Between an Interferon Regulatory Factor Variant and Interferon-beta Induced Liver Injury in Multiple Sclerosis Patients

10.1016/j.vascn.2017.09.01988173-17

Common variation near IRF6 is associated with IFN-beta-induced liver injury in multiple sclerosis

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Association Between SLC16A5 Genetic Variation and Cisplatin-Induced Ototoxic Effects in Adult Patients With Testicular Cancer

10.1001/jamaoncol.2017.0502JAMA ONCOLOGY3111558-156

CYP2D6 as a treatment decision aid for ER-positive non-metastatic breast cancer patients: a systematic review with accompanying clinical practice guidelines

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CIAO1 and MMS19 de fi ciency : A lethal neurodegenerative phenotype caused by cytosolic Fe-S cluster protein assembly disorders

Purpose: The functionality of many cellular proteins depends on cofactors; yet, they have only been implicated in a minority of Mendelian diseases. Here, we describe the first 2 inherited disorders of the cytosolic iron-sulfur protein assembly system. Methods: Genetic testing via genome sequencing was applied to identify the underlying disease cause in 3 patients with microcephaly, congenital brain malformations, progressive developmental and neurologic impairments, recurrent infections, and a fatal outcome. Studies in patient-derived skin fibroblasts and zebrafish models were performed to investigate the biochemical and cellular consequences. Results: Metabolic analysis showed elevated uracil and thymine levels in body fluids but no pathogenic variants in DPYD, encoding dihydropyrimidine dehydrogenase. Genome sequencing identified compound heterozygosity in 2 patients for missense variants in CIAO1, encoding cytosolic iron-sulfur assembly component 1, and homozygosity for an in-frame 3-nucleotide deletion in MMS19, encoding the MMS19 homolog, cytosolic iron-sulfur assembly component, in the third patient. Profound alterations in the proteome, metabolome, and lipidome were observed in patient-derived fibroblasts. We confirmed the detrimental effect of deficiencies in CIAO1 and MMS19 in zebrafish models. Conclusion: A general failure of cytosolic and nuclear iron-sulfur protein maturation caused pleiotropic effects. The critical function of the cytosolic iron-sulfur protein assembly machinery for antiviral host defense may well explain the recurrent severe infections occurring in our patients. (c) 2024 The Authors. Published by Elsevier Inc. on behalf of American College of Medical Genetics and Genomics. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)