Genetic testing for mitochondrial disease: the United Kingdom best practice guidelines.

Primary mitochondrial disease describes a diverse group of neuro-metabolic disorders characterised by impaired oxidative phosphorylation. Diagnosis is challenging; >350 genes, both nuclear and mitochondrial DNA (mtDNA) encoded, are known to cause mitochondrial disease, leading to all possible inheritance patterns and further complicated by heteroplasmy of the multicopy mitochondrial genome. Technological advances, particularly next-generation sequencing, have driven a shift in diagnostic practice from 'biopsy first' to genome-wide analyses of blood and/or urine DNA. This has led to the need for a reference framework for laboratories involved in mitochondrial genetic testing to facilitate a consistent high-quality service. In the United Kingdom, consensus guidelines have been prepared by a working group of Clinical Scientists from the NHS Highly Specialised Service followed by national laboratory consultation. These guidelines summarise current recommended technologies and methodologies for the analysis of mtDNA and nuclear-encoded genes in patients with suspected mitochondrial disease. Genetic testing strategies for diagnosis, family testing and reproductive options including prenatal diagnosis are outlined. Importantly, recommendations for the minimum levels of mtDNA testing for the most common referral reasons are included, as well as guidance on appropriate referrals and information on the minimal appropriate gene content of panels when analysing nuclear mitochondrial genes. Finally, variant interpretation and recommendations for reporting of results are discussed, focussing particularly on the challenges of interpreting and reporting mtDNA variants.

Detection of novel mutations and review of published data suggests that hereditary spastic paraplegia caused by spastin (SPAST) mutations is found more often in males.

BACKGROUND: Hereditary spastic paraplegia (HSP) is characterised in its pure form by slowly progressive spastic paraparesis. Around 40% of autosomal dominant (AD) cases are caused by mutations in SPAST, encoding spastin. PATIENTS AND METHODS: The clinical and investigation details of all patients with a SPAST mutation identified through our centre were reviewed. All published reports of SPAST mutations where the sex of patients was given were subsequently analysed in order to determine whether there is evidence of one sex being preferentially affected. RESULTS: In total 22 probable pathogenic changes were detected, including 11 novel ones. One patient carried two adjacent missense mutations. The pathogenicity of a further novel missense mutation is uncertain. Most patients had a pure phenotype, although mild peripheral neuropathy was sometimes present. The total number of patients with SPAST mutations was 27, as three of the previously known mutations were present in more than one person. The excess of males over females in our population (17:10) prompted us to review all published studies where the sex of the patients was given (n=31). A significant excess of males was identified (ratio 1.29, p=0.0007). CONCLUSIONS: Our results are consistent with data suggesting that SPAST mutations mostly cause a pure HSP phenotype. The excess of males in our sample and in published reports suggests that penetrance or severity may be sex-dependent, and merits further investigation as it may have important implications for counselling.

Toll receptor polymorphisms and carotid artery intima-media thickness.

BACKGROUND AND PURPOSE: Inflammation is a key mechanism in atherosclerosis. Variation in genes encoding inflammatory responses may therefore influence atherosclerosis risk possibly through interaction with chronic infections and proinflammatory environmental risk factors such as smoking, diabetes, and obesity. The Toll-like receptor family (TLRs) genes TLR2 and TLR4, both involved in the inflammatory process, are potential candidates and TLR-4 has been previously associated with cardiovascular disease, although other studies have failed to confirm this. METHODS: A total of 3000 individuals from the prospective community-based Carotid Atherosclerosis Progression Study (CAPS) were genotyped for single nucleotide polymorphisms: TLR2 (Arg753Gln, -16934 A/T) and TLR4 (D299G, T399I). Associations were determined with common carotid artery intima-media thickness (IMT) at baseline and also progression of IMT over the 3-year follow-up period. Gene-environment interactions with high sensitive C-reactive protein, smoking, body mass index, and diabetes were determined. RESULTS: There was no association between single nucleotide polymorphisms or haplotypes in either TLR4 or TLR2 and either baseline IMT or progression of IMT over the 3-year follow up. There were no interactions among the three proinflammatory risk factors. No genotype or haplotype was associated with high sensitive C-reactive protein. CONCLUSIONS: In this large community population, we found no evidence for genetic variation in these two TLRs being risk factors for increased IMT either directly or through interaction with proinflammatory risk factors. We were unable to confirm associations with the TLR4 polymorphisms reported in previous smaller studies.

Genetic and acquired inflammatory conditions are synergistically associated with early carotid atherosclerosis.

BACKGROUND AND PURPOSE: If chronic inflammation plays a causal role in atherogenesis, individuals with proinflammatory gene variants would be expected to develop more atherosclerosis. We recently found a synergistic association between 3 functional proinflammatory gene polymorphisms/haplotypes and smoking on carotid intima-media thickness (IMT). We replicated this finding in a second large population and extended the analysis by inclusion of other inflammatory conditions (chronic infection and obesity/abnormal glucose tolerance). METHODS: Common carotid and femoral artery IMT was determined in the Bruneck Study population (n=810). Proinflammatory variants were determined in 3 genes (IL-6 [-174C, -572G, -597A haplotype], IL-1-receptor antagonist [VNTR *2], and endotoxin receptor CD-14 [-159C]). RESULTS: There was a significant relationship between gene-variant score and carotid IMT: age- and sex-adjusted mean IMT in subjects with 0, 1, and >or=2 gene variants was 936, 987 and 1047 microm, respectively (P=0.001), and synergistic effects of gene-variant score and smoking on IMT measurements (P=0.040). Analogous findings were obtained for obesity/abnormal glucose tolerance and chronic infection. Interactive effects of gene-variant score and a risk factor score composed of the acquired inflammatory conditions were highly significant (P<0.001 each). Results were similar for femoral artery IMT. CONCLUSIONS: These results provide support for a causal role of inflammation in carotid atherosclerosis, and emphasize the importance of gene-gene and gene-environment interactions in this pathogenic pathway. This may help to explain the substantial variability of disease expression in subjects with proinflammatory risk factors such as smoking, diabetes and chronic infection.