Pathogenic SLC25A26 variants impair SAH transport activity causing mitochondrial disease.

The SLC25A26 gene encodes a mitochondrial inner membrane carrier that transports S-adenosylmethionine (SAM) into the mitochondrial matrix in exchange for S-adenosylhomocysteine (SAH). SAM is the predominant methyl-group donor for most cellular methylation processes, of which SAH is produced as a by-product. Pathogenic, biallelic SLC25A26 variants are a recognized cause of mitochondrial disease in children, with a severe neonatal onset caused by decreased SAM transport activity. Here, we describe two, unrelated adult cases, one of whom presented with recurrent episodes of severe abdominal pain and metabolic decompensation with lactic acidosis. Both patients had exercise intolerance and mitochondrial myopathy associated with biallelic variants in SLC25A26, which led to marked respiratory chain deficiencies and mitochondrial histopathological abnormalities in skeletal muscle that are comparable to those previously described in early-onset cases. We demonstrate using both mouse and fruit fly models that impairment of SAH, rather than SAM, transport across the mitochondrial membrane is likely the cause of this milder, late-onset phenotype. Our findings associate a novel pathomechanism with a known disease-causing protein and highlight the quests of precision medicine in optimizing diagnosis, therapeutic intervention and prognosis.

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.

Pathological variants in TOP3A cause distinct disorders of mitochondrial and nuclear genome stability.

Topoisomerase 3α (TOP3A) is an enzyme that removes torsional strain and interlinks between DNA molecules. TOP3A localises to both the nucleus and mitochondria, with the two isoforms playing specialised roles in DNA recombination and replication respectively. Pathogenic variants in TOP3A can cause a disorder similar to Bloom syndrome, which results from bi-allelic pathogenic variants in BLM, encoding a nuclear-binding partner of TOP3A. In this work, we describe 11 individuals from 9 families with an adult-onset mitochondrial disease resulting from bi-allelic TOP3A gene variants. The majority of patients have a consistent clinical phenotype characterised by bilateral ptosis, ophthalmoplegia, myopathy and axonal sensory-motor neuropathy. We present a comprehensive characterisation of the effect of TOP3A variants, from individuals with mitochondrial disease and Bloom-like syndrome, upon mtDNA maintenance and different aspects of enzyme function. Based on these results, we suggest a model whereby the overall severity of the TOP3A catalytic defect determines the clinical outcome, with milder variants causing adult-onset mitochondrial disease and more severe variants causing a Bloom-like syndrome with mitochondrial dysfunction in childhood.

Biallelic Loss-of-Function NDUFA12 Variants Cause a Wide Phenotypic Spectrum from Leigh/Leigh-Like Syndrome to Isolated Optic Atrophy.

BACKGROUND: Biallelic loss-of-function NDUFA12 variants have hitherto been linked to mitochondrial complex I deficiency presenting with heterogeneous clinical and radiological features in nine cases only. OBJECTIVES: To fully characterize, both phenotypically and genotypically, NDUFA12-related mitochondrial disease. METHODS: We collected data from cases identified by screening genetic databases of several laboratories worldwide and systematically reviewed the literature. RESULTS: Nine unreported NDUFA12 cases from six pedigrees were identified, with presentation ranging from movement disorder phenotypes (dystonia and/or spasticity) to isolated optic atrophy. MRI showed basal ganglia abnormalities (n = 6), optic atrophy (n = 2), or was unremarkable (n = 1). All carried homozygous truncating NDUFA12 variants, three of which are novel. CONCLUSIONS: Our case series expands phenotype-genotype correlations in NDUFA12-associated mitochondrial disease, providing evidence of intra- and inter-familial clinical heterogeneity for the same variant. It confirms NDUFA12 variants should be included in the diagnostic workup of Leigh/Leigh-like syndromes - particularly with dystonia - as well as isolated optic atrophy.

Pitfalls of relying on genetic testing only to diagnose inherited metabolic disorders in non-western populations - 5 cases of pyruvate dehydrogenase deficiency from South Africa.

Pyruvate dehydrogenase complex (PDHC) deficiencies are a group of mainly infantile onset disorders stemming from defects in pyruvate catabolism. They are characterised by severe lactic acidosis and progressive neurodegeneration.Although the PDHA1 gene is implicated in most cases of PDHC deficiency worldwide, no pathogenic variants have been reported in South African patients to date, despite availability of PDHA1 sequencing in the state diagnostic setting. METHODS: DNA from five patients with low to absent PDHC activity in fibroblasts were subjected to PDHC deficiency gene panel analysis. Included in the panel were: PDHA1, PDHB, DLAT, DLD, PDHX, BOLA3, GLRX5, IBA57, LIAS, LIPT1, LIPT2, NFU1, PDP1, PDP2, SLC19A2, SLC19A3, SLC25A19, SLC25A26, TPK1 and FBXL4. RESULTS: No pathogenic variants were identified in 4 out of 5 cases investigated. A homozygous frame-shift mutation was detected in the BOLA3 gene in one patient, supporting a diagnosis of multiple mitochondrial dysfunction syndrome type 2. DISCUSSION: A single, novel, homozygous BOLA3 frame-shift mutation was detected in a black South African child with severe neurodegenerative disease and very low to absent PDHC enzyme activity. This finding of a homozygous mutation in a patient from a non-consanguineous background may indicate a need for further investigation in clinically similar cases as well as heterozygous carrier rates in unaffected individuals from the same ethnic background.The paucity of identifiable mutations in 4 out of 5 South African patients with confirmed PDHC deficiency highlights the dangers in relying on Western population based genetic panels for diagnosing rare metabolic disease in genetically understudied populations.

Reevaluation of the South Asian MYBPC3Δ25bp Intronic Deletion in Hypertrophic Cardiomyopathy.

BACKGROUND: The common intronic deletion, MYBPC3Δ25, detected in 4% to 8% of South Asian populations, is reported to be associated with cardiomyopathy, with ≈7-fold increased risk of disease in variant carriers. Here, we examine the contribution of MYBPC3Δ25 to hypertrophic cardiomyopathy (HCM) in a large patient cohort. METHODS: Sequence data from 2 HCM cohorts (n=5393) was analyzed to determine MYBPC3Δ25 frequency and co-occurrence of pathogenic variants in HCM genes. Case-control and haplotype analyses were performed to compare variant frequencies and assess disease association. Analyses were also undertaken to investigate the pathogenicity of a candidate variant MYBPC3 c.1224-52G>A. RESULTS: Our data suggest that the risk of HCM, previously attributed to MYBPC3Δ25, can be explained by enrichment of a derived haplotype, MYBPC3Δ25/-52, whereby a small subset of individuals bear both MYBPC3Δ25 and a rare pathogenic variant, MYBPC3 c.1224-52G>A. The intronic MYBPC3 c.1224-52G>A variant, which is not routinely evaluated by gene panel or exome sequencing, was detected in ≈1% of our HCM cohort. CONCLUSIONS: The MYBPC3 c.1224-52G>A variant explains the disease risk previously associated with MYBPC3Δ25 in the South Asian population and is one of the most frequent pathogenic variants in HCM in all populations; genotyping services should ensure coverage of this deep intronic mutation. Individuals carrying MYBPC3Δ25 alone are not at increased risk of HCM, and this variant should not be tested in isolation; this is important for the large majority of the 100 million individuals of South Asian ancestry who carry MYBPC3Δ25 and would previously have been declared at increased risk of HCM.

Use of FGF-21 as a Biomarker of Mitochondrial Disease in Clinical Practice.

Recent work has suggested that fibroblast growth factor-21 (FGF-21) is a useful biomarker of mitochondrial disease (MD). We routinely measured FGF-21 levels on patients who were investigated at our centre for MD and evaluated its diagnostic performance based on detailed genetic and other laboratory findings. Patients' FGF-21 results were assessed by the use of age-adjusted z-scores based on normalised FGF-21 values from a healthy population. One hundred and fifty five patients were investigated. One hundred and four of these patients had molecular evidence for MD, 27 were deemed to have disorders other than MD (non-MD), and 24 had possible MD. Patients with defects in mitochondrial DNA (mtDNA) maintenance (n = 32) and mtDNA rearrangements (n = 17) had the highest median FGF-21 among the MD group. Other MD patients harbouring mtDNA point mutations (n = 40) or mutations in other autosomal genes (n = 7) and those with partially characterised MD had lower FGF-21 levels. The area under the receiver operating characteristic curve for distinguishing MD from non-MD patients was 0.69. No correlation between FGF-21 and creatinine, creatine kinase, or cardio-skeletal myopathy score was found. FGF-21 was significantly associated with plasma lactate and ocular myopathy. Although FGF-21 was found to have a low sensitivity for detecting MD, at a z-score of 2.8, its specificity was above 90%. We suggest that a high serum concentration of FGF-21 would be clinically useful in MD, especially in adult patients with chronic progressive external ophthalmoplegia, and may enable bypassing muscle biopsy and directly opting for genetic analysis. Availability of its assay has thus modified our diagnostic pathway.

Alternative RNA splicing complexes containing the scaffold attachment factor SAFB2.

The scaffold attachment factor SAFB1 and its recently discovered homologue SAFB2 might provide an important link between pre-mRNA splicing, intracellular signalling and transcription. Using novel mono-specific antisera, we found endogenous SAFB2 protein has a different spatial distribution from SAFB1 within the nucleus where it is found in much larger nuclear complexes (up to 670 kDa in size), and a distinct pattern of expression in adult human testis. By contrast, SAFB1 protein predominantly exists either as smaller complexes or as a monomeric protein. Our results suggest stable core complexes containing components comprised of SAFB1, SAFB2 and the RNA binding proteins Sam68 and hnRNPG exist in parallel with free SAFB1 protein. We found that SAFB2 protein, like SAFB1, acts as a negative regulator of a tra2beta variable exon. Despite showing an involvement in splicing, we detected no stable interaction between SAFB proteins and SR or SR-related splicing regulators, although these were also found in stable higher molecular mass complexes. Each of the detected alternative splicing regulator complexes exists independently of intact nucleic acids, suggesting they might be pre-assembled and recruited to nascent transcripts as modules to facilitate alternative splicing, and/or they represent nuclear storage compartments from which active proteins are recruited.