Jones, Alison Mary; (1992) Molecular studies in x-linked immune deficiency diseases. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

A large number of inherited immunodeficiencies are known to exist, including seven X-linked immunodeficiency syndromes. For some of these disorders the underlying mechanisms are well understood, but for the majority the molecular defects remain to be elucidated. Even where the underlying abnormality has been characterised, treatments often remain far from adequate, and many disorders are still fatal. Reliable methods of prenatal diagnosis and carrier detection are therefore essential for families at risk. In order to improve these methods and to develop improved forms of treatment, it is important to identify and characterise the genes responsible for these disorders. This study involves two of the X-linked immunodeficiencies, one which is well understood, and one for which there is no knowledge about the underlying defect. X-linked chronic granulomatous disease (X-CGD) is caused by absence of the large subunit of phagocyte cytochrome b-245, which results in defective intracellular killing of ingested micro-organisms. The defective gene was isolated in 1986. In this study a sample of normal females and members of families known to carry X-CGD were analysed for the existence of restriction fragment length polymorphisms (RFLPs) detected by the X-CGD cDNA, which might allow first trimester prenatal diagnosis, previously not available. A new RFLP was identified, and, in combination with one previously described, will allow prenatal diagnosis by chorionic villus sample analysis in approximately 45% of families. Two patients affected by X-CGD were found to carry deletions. One of these was detected only by altered band patterns on Southern blots hybridised with X-CGD cDNA. The other patient also exhibited McLeod red blood cell phenotype and possessed no X-CGD hybridising species at all. For both of these families prenatal diagnosis can be offered on the basis of these alterations. X-linked severe combined immunodeficiency (X-SCID) is characterised by severe defects in both cellular and humoral immunity, resulting in inevitable death within the first year of life in the absence of bone marrow transplantation. Nothing is known about the gene responsible for X-SCID or its protein product, but the approximate chromosomal localisation (Xq13-21) has been determined by family linkage analysis. The identification and characterisation of the X-SCID gene should lead to elucidation of the underlying molecular mechanism, and development of more rational forms of therapy, including somatic gene therapy. Data presented in this thesis represent the early stages of a long-term project aimed at isolating the X-SCID gene. A total of twelve single copy DNA probes, which map to Xq12-21, were used for long-range mapping by pulsed field gel electrophoresis. A short-range 1Mb map, including five probes in Xq12, has been constructed, which will not be relevant to X-SCID, but may be of value in isolation of other genes in the region. Preliminary data are presented for probes mapping to Xq13-21. A total of approximately 15Mb of DNA has been covered by non-overlapping Mlu I fragments, which emphasises the very large size of the region containing the gene.

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