Integration of Phenotype Term Prioritization and Gene Expression Analysis Reveals a Novel Variant in the PERP Gene Associated with Autosomal Recessive Erythrokeratoderma.

Hereditary palmoplantar keratodermas (PPKs) are a clinically and genetically heterogeneous group of disorders characterized by excessive epidermal thickening of palms and soles. Several genes have been associated with PPK including PERP, a gene encoding a crucial component of desmosomes that has been associated with dominant and recessive keratoderma. We report a patient with recessive erythrokeratoderma (EK) in which whole exome sequencing (WES) prioritized by human phenotype ontology (HPO) terms revealed the presence of the novel variant c.153C > A in the N-terminal region the PERP gene. This variant is predicted to have a nonsense effect, p.(Cys51Ter), resulting in a premature stop codon. We demonstrated a marked reduction in gene expression in cultured skin fibroblasts obtained from the patient. Despite the PERP gene is expressed at low levels in fibroblasts, our finding supports a loss-of-function (LoF) mechanism for the identified variant, as previously suggested in recessive EK. Our study underscores the importance of integrating HPO analysis when using WES for molecular genetic diagnosis in a clinical setting, as it facilitates continuous updates regarding gene-clinical feature associations.

A Novel Mutation Associated with Neonatal Lethal Cardiomyopathy Leads to an Alternative Transcript Expression in the X-Linked Complex I NDUFB11 Gene.

We report a neonatal patient with hypertrophic cardiomyopathy (HCM), lactic acidosis and isolated complex I deficiency. Using a customized next-generation sequencing panel, we identified a novel hemizygous variant c.338G>A in the X-linked NDUFB11 gene that encodes the NADH: ubiquinone oxidoreductase subunit B11 of the mitochondrial respiratory chain (MRC) complex I (CI). Molecular and functional assays performed in the proband's target tissues­skeletal and heart muscle­showed biochemical disturbances of the MRC, suggesting a pathogenic role for this variant. In silico analyses initially predicted an amino acid missense change p.(Arg113Lys) in the NDUFB11 CI subunit. However, we showed that the molecular effect of the c.338G>A variant, which is located at the last nucleotide of exon 2 of the NDUFB11 gene in the canonical 'short' transcript (sized 462 bp), instead causes a splicing defect triggering the up-regulation of the expression of an alternative 'long' transcript (sized 492 bp) that can also be detected in the control individuals. Our results support the hypothesis that the canonical 'short' transcript is required for the proper NDUFB11 protein synthesis, which is essential for optimal CI assembly and activity, whereas the longer alternative transcript seems to represent a non-functional, unprocessed splicing intermediate. Our results highlight the importance of characterizing the molecular effect of new variants in the affected patient's tissues to demonstrate their pathogenicity and association with the clinical phenotypes.

Biallelic mutations in NDUFA8 cause complex I deficiency in two siblings with favorable clinical evolution.

Isolated complex I (CI) deficiency is the most common cause of oxidative phosphorylation (OXPHOS) dysfunction. Whole-exome sequencing identified biallelic mutations in NDUFA8 (c.[293G > T]; [293G > T], encoding for an accessory subunit of CI, in two siblings with a favorable clinical evolution. The individuals reported here are practically asymptomatic, with the exception of slight failure to thrive and some language difficulties at the age of 6 and 9 years, respectively. These observations are remarkable since the vast majority of patients with CI deficiency, including the only NDUFA8 patient reported so far, showed an extremely poor clinical outcome. Western blot studies demonstrated that NDUFA8 protein was strongly reduced in the patients' fibroblasts and muscle extracts. In addition, there was a marked and specific decrease in the steady-state levels of CI subunits. BN-PAGE demonstrated an isolated defect in the assembly and the activity of CI with impaired supercomplexes formation and abnormal accumulation of CI subassemblies. Confocal microscopy analysis in fibroblasts showed rounder mitochondria and diminished branching degree of the mitochondrial network. Functional complementation studies demonstrated disease-causality for the identified mutation as lentiviral transduction with wild-type NDUFA8 cDNA restored the steady-state levels of CI subunits and completely recovered the deficient enzymatic activity in immortalized mutant fibroblasts. In summary, we provide additional evidence of the involvement of NDUFA8 as a mitochondrial disease-causing gene associated with altered mitochondrial morphology, CI deficiency, impaired supercomplexes formation, and very mild progression of the disease.

Uniparental isodisomy as a cause of mitochondrial complex I respiratory chain disorder due to a novel splicing NDUFS4 mutation.

Uniparental disomy (UPD) is an underestimated cause of autosomal recessive disorders. In this study, we aim to raise awareness about the possibility of UPD in mitochondrial disorders - where it is a hardly described event -, by functionally characterizing a novel variant in a structural subunit of complex I (CI) of the mitochondrial oxidative phosphorylation system. Using next-generation sequencing, we identified a new intronic homozygous c.350 + 5G > A variant in the NDUFS4 gene in a one-year-old girl (being alive at the age of 7) belonging to a non-consanguineous family presenting with encephalopathy, psychomotor delay, lactic acidosis and a single CI deficiency, a less severe phenotype than those previously reported in most NDUFS4 patients. One parent lacked the variant, and microsatellite genotyping showed complete paternal uniparental isodisomy of the non-imprinted chromosome 5. We demonstrated in patient's skeletal muscle and fibroblasts splicing abnormalities, low expression of NDUFS4, undetectable NDUFS4 protein, defects in cellular respiration (decreased oxygen consumption and ATP production), and impaired assembly or stability of mitochondrial supercomplexes containing CI. Our findings support that c.350 + 5G > A variant is pathogenic, and reinforce that UPD, although rare, should be considered as a possible cause of mitochondrial diseases in order to provide accurate genetic counselling.

Novel NDUFA13 Mutations Associated with OXPHOS Deficiency and Leigh Syndrome: A Second Family Report.

Leigh syndrome (LS) usually presents as an early onset mitochondrial encephalopathy characterized by bilateral symmetric lesions in the basal ganglia and cerebral stem. More than 75 genes have been associated with this condition, including genes involved in the biogenesis of mitochondrial complex I (CI). In this study, we used a next-generation sequencing (NGS) panel to identify two novel biallelic variants in the NADH:ubiquinone oxidoreductase subunit A13 (NDUFA13) gene in a patient with isolated CI deficiency in skeletal muscle. Our patient, who represents the second family report with mutations in the CI NDUFA13 subunit, presented with LS lesions in brain magnetic resonance imaging, mild hypertrophic cardiomyopathy, and progressive spastic tetraparesis. This phenotype manifestation is different from that previously described in the first NDUFA13 family, which was predominantly characterized by neurosensorial symptoms. Both in silico pathogenicity predictions and oxidative phosphorylation (OXPHOS) functional findings in patient's skin fibroblasts (delayed cell growth, isolated CI enzyme defect, decreased basal and maximal oxygen consumption and as well as ATP production, together with markedly diminished levels of the NDUFA13 protein, CI, and respirasomes) suggest that these novel variants in the NDUFA13 gene are the underlying cause of the CI defect, expanding the genetic heterogeneity of LS.

Novel ATAD3A recessive mutation associated to fatal cerebellar hypoplasia with multiorgan involvement and mitochondrial structural abnormalities.

Lethal neonatal encephalopathies are heterogeneous congenital disorders that can be caused by mitochondrial dysfunction. Biallelic large deletions in the contiguous ATAD3B and ATAD3A genes, encoding mitochondrial inner membrane ATPases of unknown function, as well as compound heterozygous nonsense and missense mutations in the ATAD3A gene have been recently associated with fatal neonatal cerebellar hypoplasia. In this work, whole exome sequencing (WES) identified the novel homozygous variant c.1217 T > G in ATAD3A, predicting a p.(Leu406Arg) substitution, in four siblings from a consanguineous family presenting with fatal neonatal cerebellar hypoplasia, seizures, axial hypotonia, hypertrophic cardiomyopathy, hepatomegaly, congenital cataract, and dysmorphic facies. Biochemical phenotypes of the patients included hyperlactatemia and hypocholesterolemia. Healthy siblings and parents were heterozygous for this variant, which is predicted to introduce a polar chain within the catalytic domain of ATAD3A that shortens its beta-sheet structure, presumably affecting protein stability. Accordingly, patient's fibroblasts with the homozygous variant displayed a specific reduction in ATAD3A protein levels associated with profound ultrastructural alterations of mitochondrial cristae and morphology. Our findings exclude the causative role of ATAD3B on this severe phenotype, expand the phenotypical spectrum of ATAD3A pathogenic variants and emphasize the vital role of ATAD3A in mitochondrial biogenesis.

Different mitochondrial genetic defects exhibit the same protein signature of metabolism in skeletal muscle of PEO and MELAS patients: A role for oxidative stress.

A major challenge in mitochondrial diseases (MDs) is the identification of biomarkers that could inform of the mechanisms involved in the phenotypic expression of genetic defects. Herein, we have investigated the protein signature of metabolism and of the antioxidant response in muscle biopsies of clinically and genetically diagnosed patients with Progressive External Ophthalmoplegia due to single large-scale (PEO-sD) or multiple (PEO-mD) deletions of mtDNA and Mitochondrial Encephalopathy Lactic Acidosis and Stroke-like episode (MELAS) syndrome, and healthy donors. A high-throughput immunoassay technique that quantitates the expression of relevant proteins of glycolysis, glycogenolysis, pentose phosphate pathway, oxidative phosphorylation, pyruvate and fatty acid oxidation, tricarboxylic acid cycle and the antioxidant response in two large independent and retrospectively collected cohorts of PEO-sD, PEO-mD and MELAS patients revealed that despite the heterogeneity of the genetic alterations, the three MDs showed the same metabolic signatures in both cohorts of patients, which were highly divergent from those of healthy individuals. Linear Discriminant Analysis and Support Vector Machine classifier provided a minimum of four biomarkers to discriminate healthy from pathological samples. Regardless of the induction of a large number of enzymes involved in ameliorating oxidative stress, the down-regulation of mitochondrial superoxide dismutase (SOD2) and catalase expression favored the accumulation of oxidative damage in patients' proteins. Down-regulation of SOD2 and catalase expression in MD patients is not due to relevant changes in the availability of their mRNAs, suggesting that oxidative stress regulates the expression of the two enzymes post-transcriptionally. We suggest that SOD2 and catalase could provide specific targets to improve the detoxification of reactive oxygen species that affects muscle proteins in these patients.

Missense mutations have unexpected consequences: The McArdle disease paradigm.

McArdle disease is a disorder of muscle glycogen metabolism caused by mutations in the PYGM gene, encoding for the muscle-specific isoform of glycogen phosphorylase (M-GP). The activity of this enzyme is completely lost in patients' muscle biopsies, when measured with a standard biochemical test which, does not allow to determine M-GP protein levels. We aimed to determine M-GP protein levels in the muscle of McArdle patients, by studying biopsies of 40 patients harboring a broad spectrum of PYGM mutations and 22 controls. Lack of M-GP protein was found in muscle in the vast majority (95%) of patients, irrespective of the PYGM genotype, including those carrying missense mutations, with few exceptions. M-GP protein biosynthesis is not being produced by PYGM mutations inducing premature termination codons (PTC), neither by most PYGM missense mutations. These findings explain the lack of PYGM genotype-phenotype correlation and have important implications for the design of molecular-based therapeutic approaches.

Genotypic and phenotypic features of all Spanish patients with McArdle disease: a 2016 update.

BACKGROUND: We recently described the genotype/phenotype features of all Spanish patients diagnosed with McArdle disease as of January 2011 (n = 239, prevalence of ~1/167,000) (J Neurol Neurosurg Psychiatry 2012;83:322-8). Several caveats were however identified suggesting that the prevalence of the disease is actually higher. METHODS: We have now updated main genotype/phenotype data, as well as potential associations within/between them, of all Spanish individuals currently diagnosed with McArdle disease (December 2016). RESULTS: Ninety-four new patients (all Caucasian) have been diagnosed, yielding a prevalence of ~1/139,543 individuals. Around 55% of the mutated alleles have the commonest PYGM pathogenic mutation p.R50X, whereas p.W798R and p.G205S account for 10 and 9% of the allelic variants, respectively. Seven new mutations were identified: p.H35R, p.R70C, p.R94Q, p.L132WfsX163, p.Q176P, p.R576Q, and c.244-3_244-2CA. Almost all patients show exercise intolerance, the second wind phenomenon and high serum creatine kinase activity. There is, however, heterogeneity in clinical severity, with 8% of patients being asymptomatic during normal daily life, and 21% showing limitations during daily activities and fixed muscle weakness. A major remaining challenge is one of diagnosis, which is often delayed until the third decade of life in 72% of new patients despite the vast majority (86%) reporting symptoms before 20 years. An important development is the growing proportion of those reporting a 4-year improvement in disease severity (now 34%) and following an active lifestyle (50%). Physically active patients are more likely to report an improvement after a 4-year period in the clinical course of the disease than their inactive peers (odds ratio: 13.98; 95% confidence interval: 5.6, 34.9; p < 0.001). Peak oxygen uptake is also higher in the former (20.7 ± 6.0 vs. 16.8 ± 5.3 mL/kg/min, p = 0.0013). Finally, there is no association between PYGM genotype and phenotype manifestation of the disease. CONCLUSIONS: The reported prevalence of McArdle disease grows exponentially despite frequent, long delays in genetic diagnosis, suggesting that many patients remain undiagnosed. Until a genetic cure is available (which is not predicted in the near future), current epidemiologic data support that adoption of an active lifestyle is the best medicine for these patients.

Secondary coenzyme Q10 deficiencies in oxidative phosphorylation (OXPHOS) and non-OXPHOS disorders.

We evaluated the coenzyme Q10 (CoQ) levels in patients who were diagnosed with mitochondrial oxidative phosphorylation (OXPHOS) and non-OXPHOS disorders (n=72). Data from the 72 cases in this study revealed that 44.4% of patients showed low CoQ concentrations in either their skeletal muscle or skin fibroblasts. Our findings suggest that secondary CoQ deficiency is a common finding in OXPHOS and non-OXPHOS disorders. We hypothesize that cases of CoQ deficiency associated with OXPHOS defects could be an adaptive mechanism to maintain a balanced OXPHOS, although the mechanisms explaining these deficiencies and the pathophysiological role of secondary CoQ deficiency deserves further investigation.

Taking advantage of an old concept, "illegitimate transcription", for a proposed novel method of genetic diagnosis of McArdle disease.

PURPOSE: McArdle disease is a metabolic disorder caused by pathogenic mutations in the PYGM gene. Timely diagnosis can sometimes be difficult with direct genomic analysis, which requires additional studies of cDNA from muscle transcripts. Although the "nonsense-mediated mRNA decay" (NMD) eliminates tissue-specific aberrant transcripts, there is some residual transcription of tissue-specific genes in virtually all cells, such as peripheral blood mononuclear cells (PBMCs). METHODS: We studied a subset of the main types of PYGM mutations (deletions, missense, nonsense, silent, or splicing mutations) in cDNA from easily accessible cells (PBMCs) in 12 McArdle patients. RESULTS: Analysis of cDNA from PBMCs allowed detection of all mutations. Importantly, the effects of mutations with unknown pathogenicity (silent and splicing mutations) were characterized in PBMCs. Because the NMD mechanism does not seem to operate in nonspecific cells, PBMCs were more suitable than muscle biopsies for detecting the pathogenicity of some PYGM mutations, notably the silent mutation c.645G>A (p.K215=), whose effect in the splicing of intron 6 was unnoticed in previous muscle transcriptomic studies. CONCLUSION: We propose considering the use of PBMCs for detecting mutations that are thought to cause McArdle disease, particularly for studying their actual pathogenicity.Genet Med 18 11, 1128-1135.