Molecular analysis of the mitochondrial genome in South African patients with suspected mitochondrial disorders
Van Brummelen, Anna Catharina
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Human mitochondrial DNA (mtDNA) contains 37 genes, which encode 13 proteins (all subunits of the respiratory chain), 22 transfer ribonucleic acids (tRNA), and two ribosomal RNAs. The mtDNA mutation rate is approximately 10 times higher than that of nuclear DNA and mutations therefore accumulate more rapidly. mtDNA damage may result in mitochondrial dysfunction and consequently disease, especially in those tissues most reliant on energy. Therefore, these disorders are often associated with neuromuscular syndromes, are characterised by extensive clinical variation, and are difficult to diagnose. During this investigation 42 samples from 34 South African paediatric patients with suspected mitochondrial disorders were amplified, sequenced and screened for 24 pathogenic mtDNA mutations in the tRNA-Leu(UUR), tRNA-Lys and ATPase 6 mitochondrial genes. Whole mtDNA genome sequencing and screening were performed on four patients with mitochondrial disease criteria scores of eight. The nucleotide sequences were compared to the 2001 revised Cambridge reference sequence for any discrepancies. DNA isolated from whole blood was analysed, except for seven patients for whom DNA could be isolated from both blood and muscle. A total of 103 different reported polymorphisms, 44 different novel synonymous alterations and 17 different potentially pathogenic mutations were detected. None of the detected alterations were reported pathogenic mutations but the 956-965insCCCCC, T2416C, C3254T, G7979A and A13276G alterations should certainly be investigated further. Haplogroup analysis was performed for the four patients with whole mtDNA genome sequence data, as haplogroups can influence disease expression. Heteroplasmy was not detected for any of the alterations. However, it was demonstrated that low levels of heteroplasmy, detectable via restriction fragment length polymorphism analysis, remain undetected by cycle sequencing. Possible explanations for not detecting reported mutations could be that the pathogenic mutations are nuclear encoded, present in other tissues or that a novel aetiology accounts for mitochondrial disorders in the South African population.