Analysis of genetic variants in the 5’ regulatory region of the ALAS1 gene in South African patients with Variegate Porphyria (VP)
Year: 2007
Type: dissertation
Study design: Observational Study
Abstract: The porphyrias are a group of genetic disorders arising from mutations in either one of the final seven genes encoding the haeme synthesis enzymes.These disease-causing mutations lead to an enzyme deficiency that disrupts normal haeme production, resulting in clinical features due to the subsequent accumulation of porphyrin precursors.Like most of the porphyrias, variegate porphyria (VP) is characterized by high inter-and intra-familial clinical variability, with no apparent genotype-phenotype correlation.The delta-aminolevulinate synthase-1 gene (ALAS1) is an apparent candidate gene to explain the variable clinical expression observed in VP, since it encodes the first and rate-determining enzyme of haeme synthesis.Several studies have defined important regulatory elements for the human-, rat-and chicken ALAS1 gene that regulate expression patterns of this gene.It was hypothesized that in VP individuals, variants within/near critical regulatory sites might alter the transcription rate of this gene, and consequently increase/decrease the amount of haeme precursors accumulating as a result of the defective haeme synthesis enzyme.The aim of this study was to identify genetic variants that could influence gene expression in the proximal promoter area of the ALAS1 gene, as well as the two ALAS1-drug responsive enhancer sequences (ADRES) located further upstream.DNA (2133 bp per patient) of 19 clinically defined VP patients was analysed by polymerase chain reaction (PCR) and semiautomated DNA sequencing.Subsequently, in silico analyses using appropriate software programs, and in vitro studies using the luciferase reporter system, were performed to investigate the functionality of the identified variants on ALAS1 gene transcription.Two novel single nucleotide polymorphisms (SNPs), a C>T transition at nucleotide -853 and a T>A transversion at nucleotide position -1253, were identified in three VP patients from a single family.No correlation between the sequence variants and clinical symptoms could be observed, suggesting that these two variants are not definitive factors in determining the variable expression of VP.However, due to the small sample size of the VP cohort used, the possibility of variation in ALAS1 gene expression contributing to the complex process of phenotypic presentation in VP and other porphyrias could not be excluded.In silico analysis revealed that the -853C/T variant is located within three bp from a potential half-palindromic estrogen response element (ERE), the estrogen receptor (ERα) binding site, while the -1253T/A variant is located 3' to a putative non-consensus stimulatory protein-1 (Sp1) site.For in vitro analyses, ALAS1 promoter mutation constructs were generated and cotransfection experiments performed with an ERα-expression vector in HepG2 cells.Results revealed that the ALAS1 wild-type promoter is transcriptionally up-regulated in response to estrogen stimulation and that ERα is required for the observed enhanced activity.Cotransfections with the promoter mutation constructs indicated that the -853T allele amplifies the effect of estrogen, whereas the -1253A allele reduced transcriptional activity irrespective of the presence of estrogen.This study provides evidence for a novel mechanism of haeme regulation via ALAS1 in response to estrogen.These in vivo and in silico findings may also contribute to a better understanding of the variable sympomatic expression of the porphyrias, such as VP, with possible alleviating and aggravating effects mediated by the -1253A and -853T variants respectively.OPSOMMING Die porfirieë is `n groep genetiese siektes wat ontstaan het as gevolg van mutasies in enige een van die finale sewe gene wat kodeer vir die heemsintese ensieme.Hierdie siekteveroorsakende mutasies gee aanleiding tot `n ensiem tekort wat normale heemsintese ontwrig en kliniese simptome veroorsaak as gevolg van `n opeenhoping van heemvoorlopers.Soos meeste ander porfirieë, word variegate porfirie (VP) gekenmerk deur aansienlike inter-en intrafamiliële kliniese verskille, met geen ooglopende genotipe-fenotipe korrelasie nie.Die deltaaminolevuliniese suur sintase-I geen (ALAS1) is `n potensiële kandidaat geen wat die variërende kliniese ekspressie van VP kan verduidelik, aangesien dit vir die eerste en tempobepalende ensiem van die heempadweg kodeer.`n Aantal studies het belangrike regulatoriese elemente, wat die uitdrukkings-patrone van ALAS1 reguleer, in die mens-, rot-en hoender ALAS1 geen beskryf.Daar word dus veronderstel dat in VP individue, variante in/naby kritiese regulatoriese areas die transkripsie vlakke van hierdie geen sal verander, en vervolgens die aantal heemvoorlopers, wat ophoop as gevolg van die defektiewe heemsintese ensiem, sal verhoog/verlaag.Die doelwit van die huidige studie was om genetiese variante wat geen uitdrukking beïnvloed, in die proksimale promotor area van die ALAS1 geen, asook in die twee ALAS1-middel reagerendverhogende volgordes (ADRES) verder stroomop, te identifiseer.Die DNS (2133 bp per pasiënt) van 19 klinies gedefinieerde VP pasiënte is geanaliseer deur polimerase ketting reaksie (PKR) en semi-outomatiese DNS volgorde bepaling.Gevolglik is in silico analises, deur gebruik te maak van toepaslike sagteware programme, en in vitro analises met die lusiferase verklikker sisteem uitgevoer, om sodoende die funksionaliteit van hierdie variante op ALAS1 transkripsie te ondersoek.Twee nuwe puntmutasies is geïdentifiseer, naamlik, `n C>T transisie by nukleotied posisie -853, en `n T>A transversie by nukleotied posisie -1253, in drie VP pasiënte wat afkomstig is van dieselfde familie.Geen verband kon gevind word tussen die betrokke variante en simptome nie, wat `n aanduiding is dat hierdie variante nie `n bepalende faktor is in die variërende uitdrukking van VP nie.As gevolg van die beperkte aantal VP pasiënt-monsters wat beskikbaar was, kan die moontlikheid dat variasie in ALAS1 geenuitdrukking bydra tot die komplekse proses van die fenotipiese uitdrukking in VP en ander porfirieë, egter nie uitgeskakel word nie.In silico analise het aangedui dat die -853C/T variant binne drie bp van `n half-palindromiese estrogeen reaktiewe element (ERE), die bindings setel vir die estrogeen reseptor (ERα), geposisioneer is, terwyl die -1253T/A variant aan die 3' kant van `n moontlike nie-konsensus stimulerende proteïen-1 (Sp1) setel geleë is.Vir in vitro analises is ALAS1 promotor-mutasie konstrukte geskep, en ko-transfeksie eksperimente uitgevoer met `n ERα-ekspressie vektor in HepG2 selle.Resultate het aangedui dat die ALAS1 wilde-tipe promoter, in reaksie tot estrogeen, transkripsioneel verhoog word, en dat ERα noodsaaklik is vir die waargenome verhoging in aktiwiteit.Ko-transfeksies met die promoter-mutasie konstrukte het getoon dat die -853T alleel die effek van estrogeen amplifiseer, terwyl die -1253A alleel transkripsie verlaag, ongeag van die teenwoordigheid van estrogeen.Hierdie studie verskaf bewyse vir `n nuwe meganisme van heem regulering, via ALAS1 in reaksie op estrogeen.Die in vivo en in silico bevindinge mag ook moontlik bydra tot `n beter begrip van die variërende simptomatiese uitdrukking van die porfirieë, soos VP, met moontlike verbeterde en verswarende effekte veroorsaak deur onderskeidelik die -1253A en -853T variante. My supervisor, Prof
Author: Nelita du Plessis
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
Funder: Medical Research Council
OpenAlex ID: W854175306