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Contains fulltext : 204158.pdf (Publisher’s version ) (Closed access) Mitochondrial disease (MD) is a group of rare inherited disorders with clinical heterogeneous phenotypes. Recent advances in next-generation sequencing (NGS) allow for rapid genetic diagnostics in patients who experience MD, resulting in significant strides in determining its etiology. This, however, has not been the case in many patient populations. We report on a molecular diagnostic study using mitochondrial DNA and targeted nuclear DNA (nDNA) NGS of an extensive cohort of predominantly sub-Saharan African pediatric patients with clinical and biochemically defined MD. Patients in this novel cohort presented mostly with muscle involvement (73%). Of the original 212 patients, a muscle respiratory chain deficiency was identified in 127 cases. Genetic analyses were conducted for these 127 cases based on biochemical deficiencies, for both mitochondrial (n = 123) and nDNA using panel-based NGS (n = 86). As a pilot investigation, whole-exome sequencing was performed in a subset of African patients (n = 8). These analyses resulted in the identification of a previously reported pathogenic mitochondrial DNA variant and seven pathogenic or likely pathogenic nDNA variants (ETFDH, SURF1, COQ6, RYR1, STAC3, ALAS2, and TRIOBP), most of which were identified via whole-exome sequencing. This study contributes to knowledge of MD etiology in an understudied, ethnically diverse population; highlights inconsistencies in genotype-phenotype correlations; and proposes future directions for diagnostic approaches in such patient populations.

Mitochondrial DNA (mtDNA) variation has been implicated in many common complex diseases, but inconsistent and contradicting results are common. Here we introduce a novel mutational load hypothesis, which also considers the collective effect of mainly rare variants, utilising the MutPred Program. We apply this new methodology to investigate the possible role of mtDNA in two cardiovascular disease (CVD) phenotypes (hypertension and hyperglycaemia), within a two-population cohort (n = 363; mean age 45 ± 9 yrs). Very few studies have looked at African mtDNA variation in the context of complex disease, and none using complete sequence data in a well-phenotyped cohort. As such, our study will also extend our knowledge of African mtDNA variation, with complete sequences of Southern Africans being especially under-represented. The cohort showed prevalence rates for hypertension (58.6%) and prediabetes (44.8%). We could not identify a statistically significant role for mtDNA variation in association with hypertension or hyperglycaemia in our cohort. However, we are of the opinion that the method described will find wide application in the field, being especially useful for cohorts from multiple locations or with a variety of mtDNA lineages, where the traditional haplogroup association method has been particularly likely to generate spurious results in the context of association with common complex disease.

Thorough investigation of the glycine conjugation pathway has been neglected. No defect of the glycine conjugation pathway has been reported and this could reflect the essential role of glycine conjugation in hepatic metabolism. Therefore, we hypothesised that genetic variation in the open reading frame (ORF) of the GLYAT gene should be low and that deleterious alleles would be found at low frequencies. This hypothesis was investigated by analysing the genetic variation of the human GLYAT ORF using data available in public databases. We also sequenced the GLYAT ORF of a small cohort of South African Afrikaner Caucasian individuals. In total, data from 1537 individuals was analysed. The two most prominent GLYAT haplotypes in all populations analysed, were S156 (70%) and T17S156 (20%). The S156C199 and S156H131 haplotypes, which have a negative effect on the enzyme activity of a recombinant human GLYAT, were detected at very low frequencies. In the Afrikaner Caucasian cohort a novel Q61L SNP occurring at a high frequency (12%) was detected. The results of this study indicated that the GLYAT ORF is highly conserved and supported the hypothesis that the glycine conjugation pathway is an essential detoxification pathway. These findings emphasise the importance of future investigations to determine the in vivo capacity of the glycine conjugation pathway for the detoxification of benzoate and other xenobiotics.

Hereditary tyrosinemia type 1 (HT1) is an autosomal recessive disorder affecting fumarylacetoacetate hydrolase (FAH), the last enzyme in the tyrosine catabolism pathway. The liver mosaicism observed in HT1 patients is due to the reversion to the wild type of one allele of the original point mutation in fah. It is generally accepted that these reversions are true back mutations; however, the mechanism is still unresolved. Previous reports excluded intragenic recombination, mitotic recombination, or homologous recombination with a pseudogene as possible mechanisms of mutation reversion in HT1. Sequence analysis did not reveal DNA motifs, tandem repeats or other sequence peculiarities that may be involved in mutation reversion. We propose the hypothesis that a point mutation instability mutator (PIN) phenotype brought about by the sustained stress environment created by the accumulating metabolites in the cell is the driver of the true back mutations in HT1. The metabolites accumulating in HT1 create a sustained stress environment by activating the extracellular signal-regulated kinase (ERK) and AKT survival pathways, inducing aberrant mitosis and development of death resistant cells, depleting glutathione, and impairing DNA ligase IV and possibly DNA polymerases δ and ε. This continual production of proliferative and stress-related survival signals in the cellular environment coupled with the mutagenicity of FAA, may instigate a mutator phenotype and could end in tumorigenesis and/or mutation reversion. The establishment of a PIN-mutator phenotype therefore not only seems to be a possible mechanism underlying the true back mutations, but also contributes to explaining the clinical heterogeneity seen in hereditary tyrosinemia type 1. http://dx.doi.org/10.1007/s10545-011-9401-x

Background Chronic Fatigue Syndrome (CFS) is a prevalent debilitating condition that affects approximately 250,000 people in the UK. There is growing interest in the role of mitochondrial function and mitochondrial DNA (mtDNA) variation in CFS. It is now known that fatigue is common and often severe in patients with mitochondrial disease irrespective of their age, gender or mtDNA genotype. More recently, it has been suggested that some CFS patients harbour clinically proven mtDNA mutations. Methods MtDNA sequencing of 93 CFS patients from the United Kingdom (UK) and South Africa (RSA) was performed using an Ion Torrent Personal Genome Machine. The sequence data was examined for any evidence of clinically proven mutations, currently; more than 200 clinically proven mtDNA mutations point mutations have been identified. Results We report the complete mtDNA sequence of 93 CFS patients from the UK and RSA, without finding evidence of clinically proven mtDNA mutations. This finding demonstrates that clinically proven mtDNA mutations are not a common element in the aetiology of disease in CFS patients. That is patients having a clinically proven mtDNA mutation and subsequently being misdiagnosed with CFS are likely to be rare. Conclusion The work supports the assertion that CFS should not be considered to fall within the spectrum of mtDNA disease. However, the current study cannot exclude a role for nuclear genes with a mitochondrial function, nor a role of mtDNA population variants in susceptibility to disease. This study highlights the need for more to be done to understand the pathophysiology of CFS. Electronic supplementary material The online version of this article (doi:10.1186/s12881-017-0387-6) contains supplementary material, which is available to authorized users.