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Thesis (MScMedSc)--University of Stellenbosch, 2011. / ENGLISH SUMMARY: Background: Primary immunodeficiency disorders (PID) disrupt the proper functioning of the immune system. The prototypic PID is X-linked Agammaglobulinemia (XLA). This disorder is caused by mutations in the Bruton tyrosine kinase (Btk) gene and results in an arrest in B cell development which leads to a profound reduction of all classes of serum immunoglobulins (i.e antibodies). Patients with a lack of antibodies experience recurring bacterial infections during early childhood that can be fatal if not treated. Intravenous gammaglobulin replacement therapy (IVIg) is the standard treatment for XLA. It provides passive immunity thereby reducing the number and severity of infections as well as limiting many of the infectious complications. Early detection and treatment of XLA allows affected individuals to live a relatively normal life. Objective: The purpose of this study was to determine the molecular basis of XLA in South Africa using a direct sequence-based method to detect abnormalities in the Btk gene to aid clinical diagnosis of the disease. Methods : Male patients with a clinical diagnosis of XLA were included in this study. Genetic analysis was used to explore the exonic region of the Btk gene of 5 unrelated male patients and compared to 10 healthy controls. Family members were followed up to determine carrier status, where possible. Results: Mutational analysis revealed Btk abnormalities in 4 of the 5 patients leading to a definitive diagnosis of XLA. Two of the three mutations found in this study have been previously described while one mutation appears to be novel. The novel mutation is a one base pair deletion in exon 16 which leads to the truncation of the Btk protein. Despite the clinical findings suggestive of XLA, no mutation was identified in the exonic region of the Btk gene of the remaining patient, indicating that this patient might have a different form of PID. Maternal follow-up confirmed the maternal inheritance pattern as all mothers screened were carriers of the Btk mutation present in the affected individual. Discussion : Using a direct sequence-based method abnormalities were identified in the Btk gene of three patients. Molecular diagnosis coupled to clinical history of the patient provides a definitive XLA diagnosis. This study supports the use of molecular techniques in the diagnosis of PID and underlines the synergy that could be possible in a clinical setting. / AFRIKAANSE OPSOMMING: Agtergrond: Primêre immuungebrek siektes (PIGS) word gekenmerk aan ‘n gebrek aan teenliggame in die immuunsisteem wat lei tot herhaalde infeksies in jong kinders wat fataal kan wees indien dit nie vroegtydig behandel word nie. Die prototype van die bekende PIGS is X-gekoppelde Agammaglobulinemia (XGA). Die siekte word veroorsaak deur mutasies in die Bruton Tirosien kinase (Btk) geen en lei tot ʼn stilstand in B sel ontwikkeling en gevolglik ʼn vermindering van alle klasse van serum immuunoglobulins (teenliggaam). Intraveneuse gammaglobulien vervangingsterapie(IVIg) is die standaard behandeling vir XGA. Dit voorsien passiewe immunitiet en gevolglik verminder dit die getal en erns van infeksies en beperk baie van die aansteeklike komplikasies. Vroeë diagnose en behandeling van XGA laat toe dat geaffekteerde individue ʼn relatiewe normale lewe ly. Doel: Die doel van hierdie studie is om die molekulêre basis van XGA in Suid Afrika te ondersoek, deur gebruik te maak van direkte volgorde bepaling van die Btk geen in die hoop om die kliniese diagnose van die siekte aan te help. Metode : Manlike pasiente met ‘n kliniese diagnose wan XGA was by die studie ingesluit. Genetiese analise was gebruik om die “exonic” omgewing van die Btk geen te ondersoek van 5 onverwante manlike pasiente en vergelyk teenoor 10 gesonde kontrole. Waar moontlik was familie lede ogevolg om draers te bepaal. Resultaat: Mutasies in die Btk geen is geidentifiseer in 3 van die 4 pasiente, klinies gediagnoseer meet XGA. Die mutasies sluit 2 reeds beskryfde variante in en een nuwe mutasie, ‘n een basis paar delesie in ekson 16 van die Btk geen, Ten spyte van die kliniese profiel suggestief van XGA in die 5de pasient, was geen mutasies geidentifiseer in die “exconic” omgewing van die Btk geen nie, dit kan moontlik toegeskryf word aan die teenwoordigheid van ‘n ander vorm van PIGS in hierdie pasient. Opvolg analise op die DNA van die moeders van die pasiente het die moederlike oorerwings patroon van die siekte bevestig aangesien al die moeders draers van die geidentifiseerde mutasie in die Btk geen van die gaffekteerde individu was. Gevolgtrekking: Genetiese analise van die Btk geen blyk ʼn sensitiewe en spesefieke metode te wees om individue met XGA te diagnoseer. Hierdie studie ondersteun die gebruik van molekulêre metodes in die diagnose van PIGS en beklemtoon die moontlike sinergie wat kan bestaan tussen hierdie tipe benadering in die kliniese omgewing. / National Research Foundation / National Health Laboratory Services : Pathology Research Development Grant of NHLS Research Trust Grants

In comparison to men, the number of X-linked genes is doubled in women as they have two chromosomes X while men are hemizygotes for X-linked genes. This imbalance is compensated by X inactivation (XCI) process, also known as primary X-inactivation, occurring in the early stage of embryogenesis. X inactivation is a random process and females are mosaics of two cell populations. The ratio of expressed alleles in women can be random (50:50) or skewed (≥80:20). The skewed X inactivation may occur due to selection when one of the alleles is preferentially inactivated (secondary X inactivation). In this study XCI status in heterozygous females with various severity of phenotypic symptoms and traits in selected X linked inherited metabolic diseases is analysed, with the focus being Fabry disease - the deficiency of the enzyme alpha-galactosidase A encoded by GLA gene. Moreover, XCI in one family with X linked agammaglobulinemia is examined. Mutant alleles and XCI status based on various loci, different methodical approaches and different tissues is subjected to examination. For the first time, the direct analysis of GLA gene transcript to detect the allele ratio was used alongside with the single-nucleotide polymorphisms in the IDS and LAMP genes for allele-specific expression (ASE) and the AR, RP2 and...