Strautnieks, Sandra Sarmite; (1993) Mutation detection in X-linked disorders: Ornithine carbamoyl transferase deficiency and Pelizaeus-Merzbacher disease. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

This study was carried out to establish methods of mutation detection in the X-linked disorders: ornithine carbamoyl transferase (OCT) deficiency and Pelizaeus-Merzbacher disease (PMD) and to determine the molecular basis of these disorders. Two main approaches were used: single strand conformation polymorphism (SSCP) analysis and cytosine-guanine dinucleotide (CpG) site analysis. SSCP screening of the coding regions of the OCT and proteolipid protein genes in 5 families with late onset OCT deficiency and 14 families with PMD, identified sequence variations in 3 and 4 families respectively. On direct sequencing of the OCT gene 2 novel amino acid substitutions: Ala(176)Thr and Arg(97)Leu were identified and the fourth reported case of Arg(245)Trp. Sequencing of the proteolipid protein gene identified 3 novel changes: Leu(223)Pro, Thr(181)Pro and a G to T change at the -1 position of the 3’ acceptor splice site of exon 4. The fourth PMD family which gave a variant SSCP pattern is currently under analysis by a collaborating group. The second approach was the analysis of 5’CpG3’ dinucleotide sites located within the recognition sequences of the restriction enzymes: TaqI (TCGA), MspI (CCGG) and CfoI (GCGC), using the principal that mutation abolished digestion. Genomic DNA or PCR product of exons containing a restriction enzyme site, from patients with classical or late onset OCT deficiency, was digested and the products were analysed by Southern blotting or by separation on high percentage agarose gels respectively. Two point mutations: Arg(109)Gln and Pro(193)Leu, which removed the TaqI and MspI sites in exons 5 and 7 respectively, were identified.

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