Pagnamenta, A.; (2006) Identification of nuclear genes responsible for respiratory chain disorders in childhood. Doctoral thesis , University of London.

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Abstract

The primary aim of this work was to identify novel mutations in genes that cause mitochondrial disorders. Due to the clinical and genetic heterogeneity among these patients, a variety of analytical approaches were used. In patients from pedigrees which exhibited maternal inheritance or in whom cybrid cell studies indicated a mitochondrial mutation, mitochondrial DNA (mtDNA) was sequenced. A novel mutation in a subunit of complex I (ND3) was identified in a patient with cardiomyopathy. The functional relationship between mutant load and complex I activity, lactate/pyruvate ratios and oxygen consumption was investigated. However, the pathogenic status of this mutation remains uncertain. Where a mtDNA mutation had been excluded or other factors such as consanguinity suggested involvement of a nuclear gene, results from respiratory chain (RC) enzyme assays were used to prioritise candidate genes for sequence analysis. A homozygous G555E mutation was discovered in the flavoprotein subunit of succinate dehydrogenase in a patient with late-onset Leigh syndrome and complex II deficiency. Another patient has previously been described with the same mutation but presented with a substantially more severe lethal infantile form of the disease. Fibroblast cultures derived from both patients were used to compare the activities and stability of RC enzymes in order to explain this phenotypic variability. In consanguineous pedigrees where analysis of candidate genes had proved unsuccessful, homozygosity mapping was used to search for novel nuclear genes involved with mitochondrial disorders. A novel gene for complex I deficiency was mapped to 5 genetic regions in an Israeli family using the new 10K SNP chip from Affymetrix. Analysis of eight candidate genes (including NDUFV2 and AFG3L2) was negative. In another highly consanguineous Israeli pedigree with mtDNA depletion, microsatellite analysis was used to narrow a candidate region on chromosome 13 down to 20Mb. A complex rearrangement was found in this region in a gene called SUCLA2. This gene codes for a citric acid cycle protein, which is thought to affect mitochondrial nucleotide pools via an interaction with nucleoside diphosphate kinase. Mutations in the gene for mitochondrial DNA polymerase gamma were found in 13/17 paediatric patients in the majority of whom Southern blots had indicated depletion of mtDNA, and also in a three-generation pedigree with premature external ophthalmoplegia associated with early menopause.

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