The molecular characterisation of the annexin II gene in pre-eclampsia

De Jager, Jacoba Martina (2009-12)

Thesis (MSc(Genetics))--University of Stellenbosch, 2009.

Thesis

ENGLISH ABSTRACT: The hypertensive conditions of pregnancy (including pre-eclampsia (PE)) is the leading cause of primary obstetric death in South Africa and affects at least five percent of pregnancies in the Western Cape province. Reduced levels of placental protein 13 (PP13) early in pregnancy are associated with a higher incidence of PE in later gestation. PP13 and annexin II have been co-localised to the brush border membrane of syncytiotrophoblasts, and form a complex that is transported to the maternal circulation. It is speculated that genetic variation in the gene encoding annexin II (ANXA2) could underlie the reduced PP13 levels. The aim of this study was to screen the ANXA2 gene, including the proximal promoter region, in two South African population groups, (Mixed Ancestry and Black) from the Western Cape, to identify whether variants in the ANXA2 gene confer susceptibility to PE. The study cohort comprised of 120 pre-eclamptic maternal, 94 pre-eclamptic fetal and 54 healthy control individuals. Genomic DNA of patient and control individuals was extracted for PCR amplification of ANXA2 and Multiphor SSCP/HD analysis was performed for mutation detection. The conformational variants identified were subjected to automated DNA sequencing and subsequently to RFLP analysis, to confirm the genotypes in the remainder of the cohort. Nine previously identified variants (c.-31 T>C, c.292 G>T; p.Val98Leu, c.975 C>T;p.Gly325Gly, c.-12+75 C>A, c.-11-43 G>A, c.-11-13 A>T, c.48+67 C>T, c.449-17 G>A, c.683-56 G>A) and 16 novel variants (c.-442 C>G, c.-191 G>C, c.-189_-188insGCCGG, c.-135 C>G, c.-92 A>T, c.222 C>T; p.Ala74Ala, c.600 C>T; p.Asp110Asp, c.934 G>A; p.Gly312Ser, c.244-42 G>C, c.244-76 C>G, c.528+38 C>T, c.589-5 C>T, c.682+49 C>T, c.961-30 A>G, c.961-24 C>G, c.*1057 A>G) were identified upon screening the ANXA2 gene. Statistical analysis identified significant association at five loci: SNP c.-92 A>T located within the ANXA2 5‟UTR, exonic SNP c.222 C>T; p.Ala74Ala and three intronic SNPs c.244-76 C>G, c.449-17 G>A and c.589-5 C>T. Three of the five variants (c.-92 A>T, c.244-76 C>G, c.589-5 C>T) were significantly associated with PE (P<0.05) and could contribute to PE susceptibility in these two SA iv populations, whereas the other two variants (c.222 C>T; p.Ala74Ala, c.449-17 G>A) revealed a possible protective effect, suggesting a reduced risk of developing PE. In silico analysis predicted the disruption and creation of several putative transcription factor binding sites by three SNPs in the ANXA2 gene, which could subsequently affect ANXA2 functioning. This study provides evidence for genetic variation in the ANXA2 gene, which warrants functional experimental validation in an attempt to investigate the function of these SNPs in molecular, cellular and physiological processes underlying PE. Identifying an association between variants in the ANXA2 gene and PE could contribute to the development of an additional early biomarker. The early identification of PE would promote the South African health system by providing the appropriate health care support and monitoring of high risk pregnancies, which could ultimately result in improved pregnancy outcome.

AFRIKAANSE OPSOMMING: Die hipertensiewe siektes van swangerskap (insluitende pre-eklampsie (PE)) is die belangrikste direkte oorsaak van moedersterftes in Suid-Afrika en dit kom voor by ongeveer 5% van swangerskape in die Wes-Kaap provinsie. Verlaagde plasentale proteïen 13 (PP13) vlakke tydens vroeë swangerskap word verbind met „n hoër voorkoms van PE in latere swangerskap. PP13 en anneksin II kom albei op die borselgrens membraan van synsitiotrofoblaste voor waar hulle „n kompleks vorm wat na die moederlike sirkulasie vervoer word. Daar word gespekuleer dat die onderliggende oorsaak vir laer PP13 vlakke as gevolg van genetiese variasie in die geen wat anneksin II kodeer (ANXA2) kan wees. Die doel van hierdie studie was om die ANXA2 geen, insluitende die proksimale promoter area, in twee Suid-Afrikaanse populasie groepe, (Kleurling en Swart) van die Wes-Kaap, te skandeer met die doel om variante in die ANXA2 geen te identifiseer en ‟n moontlike assosiasie met die vatbaarheid vir PE te bepaal. Hierdie studie populasie het bestaan uit 120 pre-eklamptiese vroue, 94 neonate van pre-eklamptiese ma‟s en 54 gesonde kontrole individue. Genomiese DNS van die pasiënte en kontrole individue is geëkstraeer vir polimerase kettingreaksie amplifikasie van die ANXA2 geen, waarna Multiphor enkelstring konformasie polimorfisme heterodupleks analise uitgevoer is met die doel om DNS variante te identifiseer. Die verskillende konformasies waargeneem is onderwerp aan semi-geoutomatiseerde DNS volgorde bepalingsanalise en gevolglik restriksie fragment lengte polimorfisme analise om genotipes in die res van die studiegroep te bevestig. Vyf-en-twintig variante is geïdentifiseer met die skandering van die ANXA2 geen, waarvan nege voorheen geïdentifiseer is (c.-31 T>C, c.292 G>T; p.Val98Leu, c.975 C>T;p.Gly325Gly, c.-12+75 C>A, c.-11-43 G>A, c.-11-13 A>T, c.48+67 C>T, c.449-17 G>A, c.683-56 G>A) en 16 nuwe variante is (c.-442 C>G, c.-191 G>C, c.-189_-188insGCCGG, c.-135 C>G, c.-92 A>T, c.222 C>T; p.Ala74Ala, c.600 C>T; p.Asp110Asp, c.934 G>A; p.Gly312Ser, c.244-42 G>C, c.244-76 C>G, c.528+38 C>T, c.589-5 C>T, c.682+49 C>T, c.961-30 A>G, c.961-24 C>G, c.*1057 A>G). Statistiese analise het „n statisties beduidende assosiasie met vyf SNPs geïdentifiseer: SNP c.-92 A>T geleë in die ANXA2 5‟UTR, die koderende SNP c.222 C>T; p.Ala74Ala en drie SNPs c.244-76 C>G, c.449-17 G>A and c.589-5 C>T geleë in die nie-koderende areas. Drie van hierdie vyf SNPs (c.-92 A>T, c.244-76 C>G, c.589-5 C>T) het statisties beduidende assosiasie met PE (P<0.05) getoon en kan bedra tot die vatbaarheid vir PE in hierdie twee Suid-Afrikaanse populasies, terwyl die ander twee SNPs (c.222 C>T; p.Ala74Ala, c.449-17 G>A) „n moontlike beskermende effek gedui het, wat „n verlaagde risiko vir die ontwikkeling van PE voorstel. In silico analise het voorspel dat verskeie voorgestelde transkripsiefaktor bindingsetels onderbreek of geskep sal word in die teenwoordigheid van drie SNPs in die ANXA2 geen, wat gevolglik die funksionering van ANXA2 kan affekteer. Hierdie studie verskaf bewyse vir genetiese variasie in die ANXA2 geen, wat verdere funksionele eksperimentele ondersoeke vereis om die funksie van hierdie SNPs in molekulêre, sellulêre en fisiologiese prosesse onderliggend aan PE te bepaal. Die identifisering van ‟n assosiasie tussen variante in die ANXA2 geen en PE kan bydra tot die ontwikkeling van ‟n addisionele vroeë genetiese merker. Die vroeë identifisering van PE kan die Suid-Afrikaanse gesondheidsisteem geweldig baat deurdat die geskikte gesondheidsorg en ondersteuning asook deurgaanse monitering van hoë risiko swangerskappe verskaf sal kan word. Dit kan uiteindelik lei tot ‟n verbeterde uitkoms vir swangerskappe in Suid-Afrika.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/1940
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