Using a quantitative quadruple immunofluorescent assay to diagnose isolated mitochondrial Complex I deficiency

Isolated Complex I (CI) deficiency is the most commonly observed mitochondrial respiratory chain biochemical defect, affecting the largest OXPHOS component. CI is genetically heterogeneous; pathogenic variants affect one of 38 nuclear-encoded subunits, 7 mitochondrial DNA (mtDNA)-encoded subunits or 14 known CI assembly factors. The laboratory diagnosis relies on the spectrophotometric assay of enzyme activity in mitochondrially-enriched tissue homogenates, requiring at least 50 mg skeletal muscle, as there is no reliable histochemical method for assessing CI activity directly in tissue cryosections. We have assessed a validated quadruple immunofluorescent OXPHOS (IHC) assay to detect CI deficiency in the diagnostic setting, using 10 µm transverse muscle sections from 25 patients with genetically-proven pathogenic CI variants. We observed loss of NDUFB8 immunoreactivity in all patients with mutations affecting nuclear-encoding structural subunits and assembly factors, whilst only 3 of the 10 patients with mutations affecting mtDNA-encoded structural subunits showed loss of NDUFB8, confirmed by BN-PAGE analysis of CI assembly and IHC using an alternative, commercially-available CI (NDUFS3) antibody. The IHC assay has clear diagnostic potential to identify patients with a CI defect of Mendelian origins, whilst highlighting the necessity of complete mitochondrial genome sequencing in the diagnostic work-up of patients with suspected mitochondrial disease

A recessive homozygous p.Asp92Gly SDHD mutation causes prenatal cardiomyopathy and a severe mitochondrial complex II deficiency

Succinate dehydrogenase (SDH) is a crucial metabolic enzyme complex that is involved in ATP production, playing roles in both the tricarboxylic cycle and the mitochondrial respiratory chain (complex II). Isolated complex II deficiency is one of the rarest oxidative phosphorylation disorders with mutations described in three structural subunits and one of the assembly factors; just one case is attributed to recessively inherited SDHD mutations. We report the pathological, biochemical, histochemical and molecular genetic investigations of a male neonate who had left ventricular hypertrophy detected on antenatal scan and died on day one of life. Subsequent postmortem examination confirmed hypertrophic cardiomyopathy with left ventricular non-compaction. Biochemical analysis of his skeletal muscle biopsy revealed evidence of a severe isolated complex II deficiency and candidate gene sequencing revealed a novel homozygous c.275A>G, p.(Asp92Gly) SDHD mutation which was shown to be recessively inherited through segregation studies. The affected amino acid has been reported as a Dutch founder mutation p.(Asp92Tyr) in families with hereditary head and neck paraganglioma. By introducing both mutations into Saccharomyces cerevisiae, we were able to confirm that the p.(Asp92Gly) mutation causes a more severe oxidative growth phenotype than the p.(Asp92Tyr) mutant, and provides functional evidence to support the pathogenicity of the patient’s SDHD mutation. This is only the second case of mitochondrial complex II deficiency due to inherited SDHD mutations and highlights the importance of sequencing all SDH genes in patients with biochemical and histochemical evidence of isolated mitochondrial complex II deficiency

D-cycloserine-augmented one-session treatment of specific phobias in children and adolescents.

BACKGROUND: D-Cycloserine has potential to enhance exposure therapy outcomes. The current study presents a preliminary randomized, placebo-controlled double-blind pilot trial of DCS-augmented one-session treatment (OST) for youth (7-14 years) with specific phobia. A secondary aim of this pilot study was to explore the effects of youth age and within-session fear reduction as potential moderators of DCS outcomes in order to generate hypotheses for a larger trial. It was hypothesized that DCS would be associated with greater improvements than placebo, that children (7-10 years) would have greater benefits than adolescents (11-14 years), and that DCS effects would be stronger for participants with the greater within-session fear reduction during the OST. METHODS: Thirty-five children and adolescents were randomized to either OST combined with DCS (n = 17), or OST combined with placebo (PBO; n = 18) and assessed at 1 week, 1 month, and 3 month following treatment. RESULTS: There were no significant pre- to post-treatment or follow-up benefits of DCS relative to placebo. Secondary analyses of age indicated that relative to PBO, DCS was associated with greater improvements for children (but not adolescents) on measures of severity at 1-month follow-up. Children in the DCS condition also showed significantly greater improvement to 1 month on global functioning relative to other groups. Conversely, adolescents had significant post-treatment benefits in the PBO condition on symptom severity measures relative to DCS, and adolescents in the DCS condition had significantly poorer functioning at 3 months relative to all other groups. Finally, there was a trend for within-session fear reduction to be associated with moderating effects of DCS, whereby greater reduction in fear was associated with greater functioning at one-month follow-up for children who received DCS, relative to PBO. LIMITATIONS: The study sample was small and therefore conclusions are tentative and require replication. CONCLUSIONS: Age and within-session fear reduction may be important moderators of DCS-augmented one-session exposure therapy, which requires testing in a fully powered randomized controlled trial

Recessive germline SDHA and SDHB mutations causing leukodystrophy and isolated mitochondrial complex II deficiency

Background Isolated complex II deficiency is a rare form of mitochondrial disease, accounting for approximately 2% of all respiratory chain deficiency diagnoses. The succinate dehydrogenase (SDH) genes (SDHA, SDHB, SDHC and SDHD) are autosomally-encoded and transcribe the conjugated heterotetramers of complex II via the action of two known assembly factors (SDHAF1 and SDHAF2). Only a handful of reports describe inherited SDH gene defects as a cause of paediatric mitochondrial disease, involving either SDHA (Leigh syndrome, cardiomyopathy) or SDHAF1 (infantile leukoencephalopathy). However, all four SDH genes, together with SDHAF2, have known tumour suppressor functions, with numerous germline and somatic mutations reported in association with hereditary cancer syndromes, including paraganglioma and pheochromocytoma. Methods and results Here, we report the clinical and molecular investigations of two patients with histochemical and biochemical evidence of a severe, isolated complex II deficiency due to novel SDH gene mutations; the first patient presented with cardiomyopathy and leukodystrophy due to compound heterozygous p.Thr508Ile and p.Ser509Leu SDHA mutations, while the second patient presented with hypotonia and leukodystrophy with elevated brain succinate demonstrated by MR spectroscopy due to a novel, homozygous p.Asp48Val SDHB mutation. Western blotting and BN-PAGE studies confirmed decreased steady-state levels of the relevant SDH subunits and impairment of complex II assembly. Evidence from yeast complementation studies provided additional support for pathogenicity of the SDHB mutation. Conclusions Our report represents the first example of SDHB mutation as a cause of inherited mitochondrial respiratory chain disease and extends the SDHA mutation spectrum in patients with isolated complex II deficiency

Nevirapine- Versus Lopinavir/Ritonavir-Based Initial Therapy for HIV-1 Infection among Women in Africa: A Randomized Trial

In a randomized control trial, Shahin Lockman and colleagues compare nevirapine-based therapy with lopinavir/ritonavir-based therapy for HIV-infected women without previous exposure to antiretroviral treatment

Early‐onset coenzyme Q10 deficiency associated with ataxia and respiratory chain dysfunction due to novel pathogenic COQ8A variants, including a large intragenic deletion

Coenzyme Q10 (CoQ10) deficiency is a clinically and genetically heterogeneous subtype of mitochondrial disease. We report two girls with ataxia and mitochondrial respiratory chain deficiency who were shown to have primary CoQ10 deficiency. Muscle histochemistry displayed signs of mitochondrial dysfunction—ragged red fibers, mitochondrial paracrystalline inclusions, and lipid deposits while biochemical analyses revealed complex II+III respiratory chain deficiencies. MRI brain demonstrated cerebral and cerebellar atrophy. Targeted molecular analysis identified a homozygous c.1015G>A, p.(Ala339Thr) COQ8A variant in subject 1, while subject 2 was found to harbor a single heterozygous c.1029_1030delinsCA variant predicting a p.Gln343_Val344delinsHisMet amino acid substitution. Subsequent investigations identified a large‐scale COQ8A deletion in trans to the c.1029_1030delinsCA allele. A skin biopsy facilitated cDNA studies that confirmed exon skipping in the fibroblast derived COQ8A mRNA transcript. This report expands the molecular genetic spectrum associated with COQ8A ‐related mitochondrial disease and highlights the importance of thorough investigation of candidate pathogenic variants to establish phase. Rapid diagnosis is of the utmost importance as patients may benefit from therapeutic CoQ10 supplementation

Pathogenic variants in MT-ATP6: A UK-based Mitochondrial Disease Cohort Study

Distinct clinical syndromes have been associated with pathogenic MT-ATP6 variants. In this cohort study, we identified 125 individuals (60 families) including 88 clinically affected individuals and 37 asymptomatic carriers. Thirty-one individuals presented with Leigh syndrome and seven with Neuropathy Ataxia Retinitis Pigmentosa. The remaining 50 patients presented with variable non-syndromic features including ataxia, neuropathy and learning disability. We confirmed maternal inheritance in 39 families, and demonstrated tissue segregation patterns and phenotypic threshold are variant-dependent. Our findings suggest that MT-ATP6-related mitochondrial disease is best conceptualised as a spectrum disorder and should be routinely included in genetic ataxia and neuropathy gene panels. This article is protected by copyright. All rights reserved

A systematic policy approach to changing the food system and physical activity environments to prevent obesity

As obesity prevention becomes an increasing health priority in many countries, including Australia and New Zealand, the challenge that governments are now facing is how to adopt a systematic policy approach to increase healthy eating and regular physical activity. This article sets out a structure for systematically identifying areas for obesity prevention policy action across the food system and full range of physical activity environments. Areas amenable to policy intervention can be systematically identified by considering policy opportunities for each level of governance (local, state, national, international and organisational) in each sector of the food system (primary production, food processing, distribution, marketing, retail, catering and food service) and each sector that influences physical activity environments (infrastructure and planning, education, employment, transport, sport and recreation). Analysis grids are used to illustrate, in a structured fashion, the broad array of areas amenable to legal and regulatory intervention across all levels of governance and all relevant sectors. In the Australian context, potential regulatory policy intervention areas are widespread throughout the food system, e.g., land-use zoning (primary production within local government), food safety (food processing within state government), food labelling (retail within national government). Policy areas for influencing physical activity are predominantly local and state government responsibilities including, for example, walking and cycling environments (infrastructure and planning sector) and physical activity education in schools (education sector). The analysis structure presented in this article provides a tool to systematically identify policy gaps, barriers and opportunities for obesity prevention, as part of the process of developing and implementing a comprehensive obesity prevention strategy. It also serves to highlight the need for a coordinated approach to policy development and implementation across all levels of government in order to ensure complementary policy action