Thesis (MSc)--University of Stellenbosch, 2008. / ENGLISH ABSTRACT: Iron metabolism disorders comprise the most common disorders in humans. Hereditary
haemochromatosis (HH) is a common condition resulting from inappropriate iron absorption.
The most common form of the disease (Type 1) is associated with mutations in the HFE gene.
The C282Y homozygous genotype accounts for approximately 80% of all reported cases of
HH within the Caucasian population. A second HFE mutation, H63D, is associated with less
severe disease expression. The C282Y mutation is extremely rare in Asian and African
populations. The H63D mutation is more prevalent and has been observed in almost all
populations.
Iron overload resulting from haemochromatosis is predicted to be rare in Asian Indian
populations and is not associated with common HFE mutations that are responsible for HH in
the Caucasian population. The aberrant genes associated with HH in India have not yet been
identified.
The present study attempted to identify variants in six iron regulatory genes that were
resulting in the Type 1 HH phenotype observed in two Asian Indian probands from a highly
consanguineous family.
The promoter and coding regions of the HMOX1, HFE, HAMP, SLC40A1, CYBRD1 and HJV
genes were subjected to mutation analysis. Gene fragments were amplified employing the
polymerase chain reaction (PCR) and subsequently subjected to heteroduplex single-strand
conformational polymorphism (HEX-SSCP) analysis. Samples displaying aberrations were
then analysed using bi-directional semi-automated DNA sequencing analysis to identify any
known or novel variants within the six genes. Variants disrupting restriction enzyme
recognition sites were genotyped employing restriction fragment length polymorphism
(RFLP) analysis.
Mutation analysis of the six genes revealed 24 previously identified variants, five novel
variants (HFE: 5’UTR-840T→G, CYBRD1: 5’UTR-1813C→T, 5’UTR-1452T→C, 5’UTR-
1272T→C; HJV: 5’UTR-534G→T, 5’UTR-530G→T), one previously described microsatellite and two novel repeats. Variants identified within the SLC40A1, CYBRD1 and
HJV genes do not seem to be associated with the iron overload phenotype.
A previously described HAMP variant (5’UTR-335G→T) was observed in the homozygous
state in both probands. This variant seems to be the genetic aberration responsible for iron
overload in this Indian family. The severe juvenile haemochromatosis phenotype usually
associated with HAMP mutations, was not exhibited by the two Indian probands. Their
symptoms resembled those observed in classic Type 1 HH. It is suggested that variants
identified in the HMOX1 and HFE genes are modifying the effect of the HAMP variant and
resulting in the less severe disease phenotype. Although this variant has only been identified
in one Indian family, it could shed some light in the hunt for the iron-loading gene in India. / AFRIKAANSE OPSOMMING: Oorerflike hemochromatose (OH) is ‘n algemene siektetoestand wat ontstaan as gevolg van
oneffektiewe opname van yster in die liggaam. Die mees algemene vorm van die siekte (Tipe
1) word geassosieer met mutasies in die HFE-geen. Die C282Y homosigotiese genotipe is
verantwoordelik vir ongeveer 80% van alle gerapporteerde gevalle van OH binne die
Kaukasiese bevolking. ‘n Tweede HFE mutasie, H63D, word geassosieer met minder ernstige
siekte simptome. Die C282Y mutasie is besonder skaars in Asiese en Afrika bevolkings.
Daar word bespiegel dat oorerflike ysteroorlading as gevolg van hemochromatose skaars is in
Asiese Indiër bevolkings en word nie geassosieer met algemene HFE mutasies wat
verantwoordelik is vir OH in Kaukasiese bevolkings nie. Die abnormale gene wat wél
geassosieer word met OH in Indië is tot dusver nog nie identifiseer nie.
Die doel van hierdie studie was om die variante in ses yster-regulerende gene te identifiseer
wat die Tipe 1 OH fenotipe in hierdie familie veroorsaak. Hierdie fenotipe is waargeneem in
twee Asies Indiese familielede afkomstig van ‘n bloedverwante familie.
Die promotor en koderingsareas van die HMOX1, HFE, HAMP, SLC40A1, CYBRD1 en HJV
gene is gesif vir mutasies. Geen fragmente is geamplifiseer met behulp van die polimerase
kettingsreaksie (PKR) en daarna aan heterodupleks enkelstring konformasie polimorfisme
(HEX-SSCP) analise blootgestel. PKR produkte wat variasies getoon het, is daarna
geanaliseer deur tweerigting semi-geoutomatiseerde DNS volgorde-bepalingsanalise om
enige bekende of nuwe variante binne die ses gene te identifiseer. Variante waar restriksie
ensiem herkenningsetels teenwoordig is, is verder analiseer met behulp van die restriksie
fragment lengte polimorfisme (RFLP) analise sisteem.
Mutasie analise van die ses gene het 24 bekende variante, vyf nuwe variante (HFE: 5’UTR-
840T→G, CYBRD1: 5’UTR-1813C→T, 5’UTR-1452T→C, 5’UTR-1272T→C, HJV:
5’UTR-534G→T, 5’UTR-530G→T), een bekende herhaling en twee nuwe herhalings gewys.
Variante wat binne die SLC4041, CYBRD1 en HJV gene geïdentifiseer is, blyk nie om by te
dra tot die ysteroorladings-fenotipe nie. Die bekende HAMP variant (5’UTR-335G→T) is waargeneem in die homosigotiese toestand
in beide van die aangetaste individue. Hierdie variant blyk om die genetiese fout te wees wat
verantwoordelik is vir die ysteroorlading in die betrokke Indiese familie. Die erge juvenielehemochromatose
fenotipe wat meestal geassosieer word met HAMP-mutasies, is nie
waargeneem in hierdie familie nie. Hul simptome kom ooreen met die simptome van die
klassieke Tipe 1 OH. Dit blyk moontlik te wees dat die variante identifiseer in die HMOX1 en
HFE gene die impak van die HAMP variant modifiseer en die matiger siekte-fenotipe tot
gevolg het. Alhoewel hierdie variant slegs in een Indiese familie geïdentifiseer is, kan dit lig
werp op die soektog na die veroorsakende ysterladingsgeen in Indië.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/21458 |
| Date | 03 1900 |
| Creators | Hallendorff, Michelle-Angelique |
| Contributors | Zaahl, M. G., Hift, R. J., Stellenbosch University. Faculty of Agrisciences. Dept. of Genetics. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | Unknown |
| Type | Thesis |
| Format | xv, 168 leaves : ill. |
| Rights | Stellenbosch University |
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