Further pitfalls in the diagnosis of mtDNA mutations: homoplasmic mt-tRNA mutations.

BACKGROUND: Mitochondrial DNA (mtDNA) mutations are important causes of human genetic disease, with mutations in tRNA genes particularly prevalent. In many patients, mutations are heteroplasmic, affecting a population of mtDNA molecules. Establishing the pathogenicity of homoplasmic mitochondrial tRNA (mt-tRNA) mutations, in which the mutation is present in every mtDNA molecule, is extremely difficult. These mutations must conform to specific pathogenic criteria, documenting unequivocally a functional defect of the mutant mt-tRNA. AIMS: To investigate the pathogenic nature of two homoplasmic mt-tRNA(Thr) deletions, m.15940delT (previously reported as pathogenic) and m.15937delA, by assessing the steady state levels of the mutant mt-tRNA in tissue and cell-line samples from six unrelated families, in which affected individuals were thoroughly investigated for mitochondrial DNA disease on the basis of clinical presentations. Rates of de novo mitochondrial protein synthesis were also examined in control and m.15937delA mutant fibroblasts. RESULTS: Our data strongly suggest that both single nucleotide deletions are neutral polymorphisms; no obvious defects were apparent in either steady state mt-tRNA(Thr) levels or rates of mitochondrial protein synthesis. CONCLUSIONS: These findings have important implications for the investigation of other families with suspected mtDNA disease, in particular the requirement to fulfil strict and established pathogenic criteria in order to avoid misattribution of pathogenicity to mt-tRNA variants.

The significance of reduced respiratory chain enzyme activities: clinical, biochemical and radiological associations.

BACKGROUND: Mitochondrial diseases are an important group of neurometabolic disorders in children with varied clinical presentations and diagnosis that can be difficult to confirm. AIM: To report the significance of reduced respiratory chain enzyme (RCE) activity in muscle biopsy samples from children. METHODS: Retrospective odds ratio was used to compare clinical and biochemical features, DNA studies, neuroimaging, and muscle biopsies in 18 children with and 48 without reduced RCE activity. RESULTS: Children with reduced RCE activity were significantly more likely to have consanguineous parents, to present with acute encephalopathy and lactic acidaemia and/or within the first year of life; to have an axonal neuropathy, CSF lactate >4 mmol/l; and/or to have signal change in the basal ganglia. There were positive associations with a maternal family history of possible mitochondrial cytopathy; a presentation with failure to thrive and lactic acidaemia, ragged red fibres, reduced fibroblast fatty acid oxidation and with an abnormal allopurinol loading test. There was no association with ophthalmic abnormalities, deafness, epilepsy or myopathy. CONCLUSION: The association of these clinical, biochemical and radiological features with reduced RCE activity suggests a possible causative link.

Fumarate hydratase mutations and predisposition to cutaneous leiomyomas, uterine leiomyomas and renal cancer.

Germline heterozygous loss-of-function mutations of fumarate hydratase (FH) predispose to the autosomal dominant syndrome of multiple cutaneous and uterine leiomyomatosis (MCUL). Forty-five distinct FH mutations have been identified in 76 of 89 (85%) reported probands with skin leiomyomas. This suggests that MCUL is a genetically homogeneous condition and that most patients presenting with skin leiomyomas will have underlying FH mutations. FH mutations identified include 26/45 (58%) missense; 12/45 (27%) frameshift, 4/45 (9%) nonsense changes and 3/45 (7%) different whole gene deletions. In MCUL kindreds, the majority of females with FH mutations have both skin and uterine leiomyomas. A proportion of individuals with FH mutations have associated renal cancer, a variant known as hereditary leiomyomatosis and renal cell cancer (HLRCC). If selection bias is removed, the prevalence of renal cancer in MCUL lies between one of 46 (2%) families who were not radiologically screened, and two of 32 (6%) families who were radiologically screened. Truncating, particularly frameshift, mutations appear to be significantly associated with renal cancer (P = 0.003), suggesting a possible basis for selective screening. There may also be a significantly increased rate of renal cancer in females (P = 0.004), suggesting a possible role for hormonal factors. Review of the literature suggests that, unlike most individuals presenting with skin leiomyomas, the majority of patients presenting with uterine leiomyomas or renal cancer will not have underlying FH mutations.

Accumulation of Krebs cycle intermediates and over-expression of HIF1alpha in tumours which result from germline FH and SDH mutations.

The nuclear-encoded Krebs cycle enzymes, fumarate hydratase (FH) and succinate dehydrogenase (SDHB, -C and -D), act as tumour suppressors. Germline mutations in FH predispose individuals to leiomyomas and renal cell cancer (HLRCC), whereas mutations in SDH cause paragangliomas and phaeochromocytomas (HPGL). In this study, we have shown that FH-deficient cells and tumours accumulate fumarate and, to a lesser extent, succinate. SDH-deficient tumours principally accumulate succinate. In situ analyses showed that these tumours also have over-expression of hypoxia-inducible factor 1alpha (HIF1alpha), activation of HIF1alphatargets (such as vascular endothelial growth factor) and high microvessel density. We found no evidence of increased reactive oxygen species in our cells. Our data provide in vivo evidence to support the hypothesis that increased succinate and/or fumarate causes stabilization of HIF1alpha a plausible mechanism, inhibition of HIF prolyl hydroxylases, has previously been suggested by in vitro studies. The basic mechanism of tumorigenesis in HPGL and HLRCC is likely to be pseudo-hypoxic drive, just as it is in von Hippel-Lindau syndrome.

Mutation and biochemical analysis in carnitine palmitoyltransferase type II (CPT II) deficiency.

Carnitine palmitoyltransferase type II (CPT II) deficiency has three basic phenotypes, late-onset muscular (mild), infantile/juvenile hepatic (intermediate) and severe neonatal. We have measured fatty acid oxidation and CPT II activity and performed mutation studies in 24 symptomatic patients representing the full clinical spectrum of disease. Severe and intermediate phenotypes show a clear correlation with biochemical indices and genetic analysis revealed causative mutations in most patients. Studies of mild phenotypes suggest a more complex interaction, with higher residual fatty acid oxidation, a wider range of CPT II activity (10-60%) but little evidence of genotype-phenotype correlation. Residual CPT II mutant protein from myopathic patients shows thermal instability at 41 degrees C. The common 'polymorphisms' V3681 and M647V are strikingly overrepresented in the myopathic patients, the implication being that they may significantly influence the manifestation of clinical disease and could therefore potentially be considered as a susceptibility variants. Among myopathic individuals, males comprised 88% of patients, suggesting increased susceptibility to clinical disease. A small number of symptomatic patients appear to have significant residual CPT II activity (42-60%) The synergistic interaction of partial deficiencies of CPT II, muscle adenosine monophosphate deaminase and possibly other enzymes of muscle energy metabolism in the aetiology of episodic myopathy deserves wider consideration.

Genetic and functional analyses of FH mutations in multiple cutaneous and uterine leiomyomatosis, hereditary leiomyomatosis and renal cancer, and fumarate hydratase deficiency.

Germline mutations of the fumarate hydratase (FH, fumarase) gene are found in the recessive FH deficiency syndrome and in dominantly inherited susceptibility to multiple cutaneous and uterine leiomyomatosis (MCUL). We have previously reported a number of germline FH mutations from MCUL patients. In this study, we report additional FH mutations in MCUL and FH deficiency patients. Mutations can readily be found in about 75% of MCUL cases and most cases of FH deficiency. Some of the more common FH mutations are probably derived from founding individuals. Protein-truncating FH mutations are functionally null alleles. Disease-associated missense FH changes map to highly conserved residues, mostly in or around the enzyme's active site or activation site; we predict that these mutations severely compromise enzyme function. The mutation spectra in FH deficiency and MCUL are similar, although in the latter mutations tend to occur earlier in the gene and, perhaps, are more likely to result in a truncated or absent protein. We have found that not all mutation-carrier parents of FH deficiency children have a strong predisposition to leiomyomata. We have confirmed that renal carcinoma is sometimes part of MCUL, as part of the variant hereditary leiomyomatosis and renal cancer (HLRCC) syndrome, and have shown that these cancers may have either type II papillary or collecting duct morphology. We have found no association between the type or site of FH mutation and any aspect of the MCUL phenotype. Biochemical assay for reduced FH functional activity in the germline of MCUL patients can indicate carriers of FH mutations with high sensitivity and specificity, and can detect reduced FH activity in some patients without detectable FH mutations. We conclude that MCUL is probably a genetically homogeneous tumour predisposition syndrome, primarily resulting from absent or severely reduced fumarase activity, with currently unknown functional consequences for the smooth muscle or kidney cell.

Familial hypercholesterolaemia: pilot study to identify children at risk.

AIMS: To evaluate a more effective method of identifying children with familial hypercholesterolaemia by screening a population at high risk. METHODS: Domiciliary measurement of random cholesterol concentration was made in 200 children who were first or second degree relatives of subjects with premature onset coronary artery disease. Measurements were taken by a health visitor using a portable analyser. RESULTS: Twelve new cases of familial hypercholesterolaemia were identified during the first nine months of the study. Random cholesterol concentrations were within the normal range (< 5.2 mmol/l) in 70.5% of samples tested. Forty two (21%) of patients tested had a borderline cholesterol (5.2-5.9 mmol/l) but 50% of these fell within the normal range when fasting capillary samples were analysed. Children with significant hypercholesterolaemia on random testing (concentrations of > 5.9 mmol/l) (8.5%) also had fasting venous blood assayed for high density lipoprotein (HDL) cholesterol and tri-glyceride in the laboratory. Results indicated that 6.5% of patients screened were at high risk of cardiovascular disease (ratio of total: HDL cholesterol of > 4.5), and 1% had a moderately increased risk (ratio 3.5-4.5). CONCLUSIONS: Children with familial hypercholesterolaemia can be identified from a selected "high risk" population by measuring random capillary cholesterol concentration.