The p.Ala2430Val mutation in filamin C causes a "hypertrophic myofibrillar cardiomyopathy".

Hypertrophic cardiomyopathy (HCM) often leads to heart failure. Mutations in sarcomeric proteins are most frequently the cause of HCM but in many patients the gene defect is not known. Here we report on a young man who was diagnosed with HCM shortly after birth. Whole exome sequencing revealed a mutation in the FLNC gene (c.7289C > T; p.Ala2430Val) that was previously shown to cause aggregation of the mutant protein in transfected cells. Myocardial tissue from patients with this mutation has not been analyzed before and thus, the underlying etiology is not well understood. Myocardial tissue of our patient obtained during myectomy at the age of 23 years was analyzed in detail by histochemistry, immunofluorescence staining, electron microscopy and western blot analysis. Cardiac histology showed a pathology typical for myofibrillar myopathy with myofibril disarray and abnormal protein aggregates containing BAG3, desmin, HSPB5 and filamin C. Analysis of sarcomeric and intercalated disc proteins showed focally reduced expression of the gap junction protein connexin43 and Xin-positive sarcomeric lesions in the cardiomyocytes of our patient. In addition, autophagy pathways were altered with upregulation of LC3-II, WIPI1 and HSPB5, 6, 7 and 8. We conclude that the p.Ala2430Val mutation in FLNC most probably is associated with HCM characterized by abnormal intercalated discs, disarray of myofibrils and aggregates containing Z-disc proteins similar to myofibrillar myopathy, which supports the pathological effect of the mutation.

De novo stop-loss variants in CLDN11 cause hypomyelinating leukodystrophy.

Claudin-11, a tight junction protein, is indispensable in the formation of the radial component of myelin. Here, we report de novo stop-loss variants in the gene encoding claudin-11, CLDN11, in three unrelated individuals presenting with an early-onset spastic movement disorder, expressive speech disorder and eye abnormalities including hypermetropia. Brain MRI showed a myelin deficit with a discrepancy between T1-weighted and T2-weighted images and some progress in myelination especially involving the central and peripheral white matter. Exome sequencing identified heterozygous stop-loss variants c.622T>C, p.(*208Glnext*39) in two individuals and c.622T>G, p.(*208Gluext*39) in one individual, all occurring de novo. At the RNA level, the variant c.622T>C did not lead to a loss of expression in fibroblasts, indicating this transcript is not subject to nonsense-mediated decay and most likely translated into an extended protein. Extended claudin-11 is predicted to form an alpha helix not incorporated into the cytoplasmic membrane, possibly perturbing its interaction with intracellular proteins. Our observations suggest that stop-loss variants in CLDN11 expand the genetically heterogeneous spectrum of hypomyelinating leukodystrophies.

Regulatory myeloid cells paralyze T cells through cell-cell transfer of the metabolite methylglyoxal.

Regulatory myeloid immune cells, such as myeloid-derived suppressor cells (MDSCs), populate inflamed or cancerous tissue and block immune cell effector functions. The lack of mechanistic insight into MDSC suppressive activity and a marker for their identification has hampered attempts to overcome T cell inhibition and unleash anti-cancer immunity. Here, we report that human MDSCs were characterized by strongly reduced metabolism and conferred this compromised metabolic state to CD8+ T cells, thereby paralyzing their effector functions. We identified accumulation of the dicarbonyl radical methylglyoxal, generated by semicarbazide-sensitive amine oxidase, to cause the metabolic phenotype of MDSCs and MDSC-mediated paralysis of CD8+ T cells. In a murine cancer model, neutralization of dicarbonyl activity overcame MDSC-mediated T cell suppression and, together with checkpoint inhibition, improved the efficacy of cancer immune therapy. Our results identify the dicarbonyl methylglyoxal as a marker metabolite for MDSCs that mediates T cell paralysis and can serve as a target to improve cancer immune therapy.

Homoplasmy of the Mitochondrial DNA Mutation m.616T>C Leads to Mitochondrial Tubulointerstitial Kidney Disease and Encephalopathia.

Autosomal-dominant tubulointerstitial kidney disease -(ADTKD) describes tubulointerstitial kidney disease with autosomal-dominant inheritance. In 2017, the term mitochondrial tubulointerstitial kidney disease (MITKD) was introduced for tubulointerstitial kidney disease caused by mitochondrial DNA (mtDNA) mutations. To date, there are few mutations described in literature causing MITKD, one of them is m.616T>C. A 5-year-old girl presented with chronic renal insufficiency and epilepsia. At the age of 3 years, status epileptic occurred and evolved into epilepsia partialis continua. At the age of 5 years, chronic renal failure (CKD II-III) was diagnosed due to tubulointerstitial kidney disease. Urine analysis showed elevated fractional excretions of sodium and chloride. Kidneys were enlarged and hyperechogenic. Blood pressure was elevated. The family history was unremarkable for renal and/or neurological disorders. Genetic testing was performed and revealed homoplasmy of the substitution m.616T>C in our patient's mtDNA. This mutation has been shown to cause chronic tubulointerstitial kidney disease leading to end-stage renal disease (ESRD) and epilepsia formerly. MITKD is a rare mitochondrial disease leading to ESRD and should be suggested in patients with epilepsia and renal insufficiency.

Mitochondrial DNA mutation analysis from exome sequencing-A more holistic approach in diagnostics of suspected mitochondrial disease.

Diagnostics for suspected mitochondrial disease (MD) can be challenging and necessitate invasive procedures like muscle biopsy. This is due to the extremely broad genetic and phenotypic spectrum, disease genes on both nuclear and mitochondrial DNA (mtDNA), and the tissue specificity of mtDNA variants. Exome sequencing (ES) has revolutionized the diagnostics for MD. However, the nuclear and mtDNA are investigated with separate tests, increasing costs and duration of diagnostics. The full potential of ES is often not exploited as the additional analysis of "off-target reads" deriving from the mtDNA can be used to analyze both genomes. We performed mtDNA analysis by ES of 2111 cases in a clinical setting. We further assessed the recall rate and precision as well as the estimation of heteroplasmy by ES data by comparison with targeted mtDNA next generation sequencing in 49 cases. ES identified known pathogenic mtDNA point mutations in 38 individuals, increasing the diagnostic yield by nearly 2%. Analysis of mtDNA variants by ES had a high recall rate (96.2 ± 5.6%) and an excellent precision (99.5 ± 2.2%) when compared to the gold standard of targeted mtDNA next generation sequencing. ES estimated heteroplasmy levels with an average difference of 6.6 ± 3.8%, sufficient for clinical decision making. Taken together, the mtDNA analysis from ES is of sufficient quality for clinical diagnostics. We therefore propose ES, investigating both nuclear and mtDNA, as first line test in individuals with suspected MD. One should be aware, that a negative result does not exclude MD and necessitates further test (in additional tissues).

Biallelic variants in the transcription factor PAX7 are a new genetic cause of myopathy.

PURPOSE: Skeletal muscle growth and regeneration rely on muscle stem cells, called satellite cells. Specific transcription factors, particularly PAX7, are key regulators of the function of these cells. Knockout of this factor in mice leads to poor postnatal survival; however, the consequences of a lack of PAX7 in humans have not been established. METHODS: Here, we study five individuals with myopathy of variable severity from four unrelated consanguineous couples. Exome sequencing identified pathogenic variants in the PAX7 gene. Clinical examination, laboratory tests, and muscle biopsies were performed to characterize the disease. RESULTS: The disease was characterized by hypotonia, ptosis, muscular atrophy, scoliosis, and mildly dysmorphic facial features. The disease spectrum ranged from mild to severe and appears to be progressive. Muscle biopsies showed the presence of atrophic fibers and fibroadipose tissue replacement, with the absence of myofiber necrosis. A lack of PAX7 expression was associated with satellite cell pool exhaustion; however, the presence of residual myoblasts together with regenerating myofibers suggest that a population of PAX7-independent myogenic cells partially contributes to muscle regeneration. CONCLUSION: These findings show that biallelic variants in the master transcription factor PAX7 cause a new type of myopathy that specifically affects satellite cell survival.

Diverse phenotype in patients with complex I deficiency due to mutations in NDUFB11.

Mitochondrial complex I deficiency is the most frequent mitochondrial disorder presenting in childhood and the mutational spectrum is highly heterogeneous. The NDUFB11 gene is one of the recently identified genes, which is located in the short arm of the X-chromosome. Here we report clinical, biochemical, functional and genetic findings of two male patients with lactic acidosis, hypertrophic cardiomyopathy and isolated complex I deficiency due to de novo hemizygous mutations (c.286C > T and c.328C > T) in the NDUFB11 gene. Neither of them had any skin manifestations. The NDUFB11 gene encodes a relatively small integral membrane protein NDUFB11, which is essential for the assembly of an active complex I. The expression levels of this protein was decreased in both patient cells and a lentiviral complementation experiment also supported the notion that the complex I deficiency in those two patients is caused by NDUFB11 genetic defects. Our findings together with a review of the thirteen previously described patients demonstrate a wide spectrum of clinical features associated with NDUFB11-related complex I deficiency. However, histiocytoid cardiomyopathy and/or congenital sideroblastic anemia could be indicative for mutation in the NDUFB11 gene, while the clinical manifestation of the same mutation can be highly variable.

NAD(P)HX dehydratase (NAXD) deficiency: a novel neurodegenerative disorder exacerbated by febrile illnesses.

Physical stress, including high temperatures, may damage the central metabolic nicotinamide nucleotide cofactors [NAD(P)H], generating toxic derivatives [NAD(P)HX]. The highly conserved enzyme NAD(P)HX dehydratase (NAXD) is essential for intracellular repair of NAD(P)HX. Here we present a series of infants and children who suffered episodes of febrile illness-induced neurodegeneration or cardiac failure and early death. Whole-exome or whole-genome sequencing identified recessive NAXD variants in each case. Variants were predicted to be potentially deleterious through in silico analysis. Reverse-transcription PCR confirmed altered splicing in one case. Subject fibroblasts showed highly elevated concentrations of the damaged cofactors S-NADHX, R-NADHX and cyclic NADHX. NADHX accumulation was abrogated by lentiviral transduction of subject cells with wild-type NAXD. Subject fibroblasts and muscle biopsies showed impaired mitochondrial function, higher sensitivity to metabolic stress in media containing galactose and azide, but not glucose, and decreased mitochondrial reactive oxygen species production. Recombinant NAXD protein harbouring two missense variants leading to the amino acid changes p.(Gly63Ser) and p.(Arg608Cys) were thermolabile and showed a decrease in Vmax and increase in KM for the ATP-dependent NADHX dehydratase activity. This is the first study to identify pathogenic variants in NAXD and to link deficient NADHX repair with mitochondrial dysfunction. The results show that NAXD deficiency can be classified as a metabolite repair disorder in which accumulation of damaged metabolites likely triggers devastating effects in tissues such as the brain and the heart, eventually leading to early childhood death.

Clinical, biochemical and genetic spectrum of 70 patients with ACAD9 deficiency: is riboflavin supplementation effective?

BACKGROUND: Mitochondrial acyl-CoA dehydrogenase family member 9 (ACAD9) is essential for the assembly of mitochondrial respiratory chain complex I. Disease causing biallelic variants in ACAD9 have been reported in individuals presenting with lactic acidosis and cardiomyopathy. RESULTS: We describe the genetic, clinical and biochemical findings in a cohort of 70 patients, of whom 29 previously unpublished. We found 34 known and 18 previously unreported variants in ACAD9. No patients harbored biallelic loss of function mutations, indicating that this combination is unlikely to be compatible with life. Causal pathogenic variants were distributed throughout the entire gene, and there was no obvious genotype-phenotype correlation. Most of the patients presented in the first year of life. For this subgroup the survival was poor (50% not surviving the first 2 years) comparing to patients with a later presentation (more than 90% surviving 10 years). The most common clinical findings were cardiomyopathy (85%), muscular weakness (75%) and exercise intolerance (72%). Interestingly, severe intellectual deficits were only reported in one patient and severe developmental delays in four patients. More than 70% of the patients were able to perform the same activities of daily living when compared to peers. CONCLUSIONS: Our data show that riboflavin treatment improves complex I activity in the majority of patient-derived fibroblasts tested. This effect was also reported for most of the treated patients and is mirrored in the survival data. In the patient group with disease-onset below 1 year of age, we observed a statistically-significant better survival for patients treated with riboflavin.

PRUNE1 Deficiency: Expanding the Clinical and Genetic Spectrum.

BACKGROUND: Primary microcephaly and profound global developmental delay have been considered the core clinical phenotype in patients with bi-allelic PRUNE1 mutations. METHODS: Linkage analysis and whole-exome sequencing (WES) in a multiplex family and extraction of further cases from a WES repository containing 571 children with severe developmental disabilities and neurologic symptoms. RESULTS: We identified bi-allelic PRUNE1 mutations in twelve children from six unrelated families. All patients who survived beyond the first 6 months of life had early-onset global developmental delay, bilateral spastic paresis, dysphagia and difficult-to-treat seizures, while congenital or later-evolving microcephaly was not a consistent finding. Brain MRI showed variable anomalies with progressive cerebral and cerebellar atrophies and T2-hyperintense brain stem lesions. Peripheral neuropathy was documented in five cases. Disease course was progressive in all patients and eight children died in the first or early second decade of life. In addition to the previously reported missense mutation p.(Asp106Asn), we observed a novel homozygous missense variant p.(Leu172Pro) and a homozygous contiguous gene deletion encompassing most of the PRUNE1 gene and part of the neighboring BNIPL gene. CONCLUSIONS: PRUNE1 deficiency causes severe early-onset disease affecting the central and peripheral nervous systems. Microcephaly is probably not a universal feature.

Characterization of a Leber's hereditary optic neuropathy (LHON) family harboring two primary LHON mutations m.11778G>A and m.14484T>C of the mitochondrial DNA.

Leber's hereditary optic neuropathy (LHON) is an inherited mitochondrial disease that usually leads to acute or subacute bilateral central vision loss. In 95% of cases, LHON is caused by one of three primary mutations of the mitochondrial DNA (mtDNA), m.11778G>A in the MT-ND4 gene, m.14484T>C in the MT-ND6 gene, or m.3460G>A in the MT-ND1 gene. Here we characterize clinically, genetically, and biochemically a LHON family with multiple patients harboring two of these primary LHON mutations, m.11778G>A homoplasmic and m.14484T>C heteroplasmic. The unusually low male-to-female ratio of affected family members is also seen among the other patients previously reported with two primary LHON mutations m.11778G>A and m.14484T>C. While the index patient had very late onset of symptoms at 75years and severe visual loss, her two daughters had both onset in childhood (6 and 9years), with moderate to mild visual loss. A higher degree of heteroplasmy of the m.14484T>C mutation was found to correlate with an earlier age at onset in this family. Ours is the first LHON family harboring two primary LHON mutations where functional studies were performed in several affected family members. A more pronounced bioenergetic defect was found to correlate with an earlier age at onset. The patient with the earliest age at onset had a more significant complex I dysfunction than all controls, including the LHON patient with only the m.11778G>A mutation, suggesting a synergistic effect of the two primary LHON mutations in this patient.

Adult, isolated respiratory chain complex IV deficiency with minimal manifestations.

OBJECTIVES: Isolated complex IV (cytochrome c oxidase) deficiency is one of the most frequent respiratory chain defects in mitochondrial disorders (MIDs) and usually occurs together with severe pediatric or rarely adult multisystem disease. Here we report an adult with isolated complex IV deficiency with unusually mild clinical manifestations. CASE REPORT: A 50-year-old man had developed generalized muscle aches and occasional twitching and stiffness of the musculature since age 48 years. He had a previous history of diabetes, acute hearing loss, hyperlipidemia, hyperuricemia, arterial hypertension, polyarthrosis, hypogonadism, and hypothyroidism. The family history was positive for diabetes (mother), CK elevation (brother), myalgias (brother), and proximal weakness of the upper limbs (mother). Work-up revealed hypoacusis, postural tremor and reduced tendon reflexes, recurrent mild hyper-CK-emia, neurogenic needle electromyography, and a muscle biopsy with mild non-specific changes. Biochemical investigations of the muscle homogenate revealed an isolated complex IV defect and reduced amounts of coenzyme Q (CoQ). He profited from CoQ supplementation, low-carbohydrate diet, and gluten-free diet. CONCLUSIONS: Isolated complex IV deficiency may present with only mild muscular, endocrine, or cardiac manifestations in adults. Coenzyme Q supplementation, low-carbohydrate diet, and gluten-free diet may have a beneficial effect at least on some of the manifestations.

Clinical, biochemical, and genetic spectrum of seven patients with NFU1 deficiency.

Disorders of the mitochondrial energy metabolism are clinically and genetically heterogeneous. An increasingly recognized subgroup is caused by defective mitochondrial iron-sulfur (Fe-S) cluster biosynthesis, with defects in 13 genes being linked to human disease to date. Mutations in three of them, NFU1, BOLA3, and IBA57, affect the assembly of mitochondrial [4Fe-4S] proteins leading to an impairment of diverse mitochondrial metabolic pathways and ATP production. Patients with defects in these three genes present with lactic acidosis, hyperglycinemia, and reduced activities of respiratory chain complexes I and II, the four lipoic acid-dependent 2-oxoacid dehydrogenases and the glycine cleavage system (GCS). To date, five different NFU1 pathogenic variants have been reported in 15 patients from 12 families. We report on seven new patients from five families carrying compound heterozygous or homozygous pathogenic NFU1 mutations identified by candidate gene screening and exome sequencing. Six out of eight different disease alleles were novel and functional studies were performed to support the pathogenicity of five of them. Characteristic clinical features included fatal infantile encephalopathy and pulmonary hypertension leading to death within the first 6 months of life in six out of seven patients. Laboratory investigations revealed combined defects of pyruvate dehydrogenase complex (five out of five) and respiratory chain complexes I and II+III (four out of five) in skeletal muscle and/or cultured skin fibroblasts as well as increased lactate (five out of six) and glycine concentration (seven out of seven). Our study contributes to a better definition of the phenotypic spectrum associated with NFU1 mutations and to the diagnostic workup of future patients.

Mutations in TTC19: expanding the molecular, clinical and biochemical phenotype.

BACKGROUND: TTC19 deficiency is a progressive neurodegenerative disease associated with isolated mitochondrial respiratory chain (MRC) complex III deficiency and loss-of-function mutations in the TT19 gene in the few patients reported so far. METHODS: We performed exome sequencing and selective mutational analysis of TTC19, respectively, in patients from three unrelated families presenting with initially unspecific clinical signs of muscular hypotonia and global developmental delay followed by regression, ataxia, loss of speech, and rapid neurological deterioration. One patient showed severe lactic acidosis at the neonatal age and during intercurrent illness. RESULTS: We identified homozygous mutations in all three index cases, in two families novel missense mutations (c.544 T > C/p.Leu185Pro; c.917 T > C/p.Leu324Pro). The younger sister of the severely affected patient 3 showed only mild delay of motor skills and muscular hypotonia so far but is also homozygous for the same mutation. Notably, one patient revealed normal activities of MRC complex III in two independent muscle biopsies. Neuroimaging of the severely affected patients demonstrated lesions in putamen and caudate nuclei, cerebellar atrophy, and the unusual finding of hypertrophic olivary nuclei degeneration. Reviewing the literature revealed striking similarities regarding neuroimaging and clinical course in pediatric patients with TTC19 deficiency: patterns consistent with Leigh or Leigh-like syndrome were found in almost all, hypertrophic olivary nucleus degeneration in all patients reported so far. The clinical course in pediatric patients is characterized by an initially unspecific developmental delay, followed by regression, progressive signs and symptoms of cerebellar, basal ganglia and brainstem affection, especially loss of speech and ataxia. Subsequently, neurological deterioration leading to a vegetative state occurs. CONCLUSIONS: Our findings add to the phenotypic, genetic, and biochemical spectrum of TTC19 deficiency. However, TTC19 deficient patients do show characteristic clinical and neuroimaging features, which may facilitate diagnosis of this yet rare disorder. Normal MRC complex III activity does not exclude the diagnosis.

Spectrum of combined respiratory chain defects.

Inherited disorders of mitochondrial energy metabolism form a large and heterogeneous group of metabolic diseases. More than 250 gene defects have been reported to date and this number continues to grow. Mitochondrial diseases can be grouped into (1) disorders of oxidative phosphorylation (OXPHOS) subunits and their assembly factors, (2) defects of mitochondrial DNA, RNA and protein synthesis, (3) defects in the substrate-generating upstream reactions of OXPHOS, (4) defects in relevant cofactors and (5) defects in mitochondrial homeostasis. Deficiency of more than one respiratory chain enzyme is a common finding. Combined defects are found in 49 % of the known disease-causing genes of mitochondrial energy metabolism and in 57 % of patients with OXPHOS defects identified in our diagnostic centre. Combined defects of complexes I, III, IV and V are typically due to deficiency of mitochondrial DNA replication, RNA metabolism or translation. Defects in cofactors can result in combined defects of various combinations, and defects of mitochondrial homeostasis can result in a generalised decrease of all OXPHOS enzymes. Noteworthy, identification of combined defects can be complicated by different degrees of severity of each affected enzyme. Furthermore, even defects of single respiratory chain enzymes can result in combined defects due to aberrant formation of respiratory chain supercomplexes. Combined OXPHOS defects have a great variety of clinical manifestations in terms of onset, course severity and tissue involvement. They can present as classical encephalomyopathy but also with hepatopathy, nephropathy, haematologic findings and Perrault syndrome in a subset of disorders.

COQ4 mutations cause a broad spectrum of mitochondrial disorders associated with CoQ10 deficiency.

Primary coenzyme Q10 (CoQ10) deficiencies are rare, clinically heterogeneous disorders caused by mutations in several genes encoding proteins involved in CoQ10 biosynthesis. CoQ10 is an essential component of the electron transport chain (ETC), where it shuttles electrons from complex I or II to complex III. By whole-exome sequencing, we identified five individuals carrying biallelic mutations in COQ4. The precise function of human COQ4 is not known, but it seems to play a structural role in stabilizing a multiheteromeric complex that contains most of the CoQ10 biosynthetic enzymes. The clinical phenotypes of the five subjects varied widely, but four had a prenatal or perinatal onset with early fatal outcome. Two unrelated individuals presented with severe hypotonia, bradycardia, respiratory insufficiency, and heart failure; two sisters showed antenatal cerebellar hypoplasia, neonatal respiratory-distress syndrome, and epileptic encephalopathy. The fifth subject had an early-onset but slowly progressive clinical course dominated by neurological deterioration with hardly any involvement of other organs. All available specimens from affected subjects showed reduced amounts of CoQ10 and often displayed a decrease in CoQ10-dependent ETC complex activities. The pathogenic role of all identified mutations was experimentally validated in a recombinant yeast model; oxidative growth, strongly impaired in strains lacking COQ4, was corrected by expression of human wild-type COQ4 cDNA but failed to be corrected by expression of COQ4 cDNAs with any of the mutations identified in affected subjects. COQ4 mutations are responsible for early-onset mitochondrial diseases with heterogeneous clinical presentations and associated with CoQ10 deficiency.

Sengers syndrome: six novel AGK mutations in seven new families and review of the phenotypic and mutational spectrum of 29 patients.

BACKGROUND: Sengers syndrome is an autosomal recessive condition characterized by congenital cataract, hypertrophic cardiomyopathy, skeletal myopathy and lactic acidosis. Mutations in the acylglycerol kinase (AGK) gene have been recently described as the cause of Sengers syndrome in nine families. METHODS: We investigated the clinical and molecular features of Sengers syndrome in seven new families; five families with the severe and two with the milder form. RESULTS: Sequence analysis of AGK revealed compound heterozygous or homozygous predicted loss-of-function mutations in all affected individuals. A total of eight different disease alleles were identified, of which six were novel, homozygous c.523_524delAT (p.Ile175Tyrfs*2), c.424-1G > A (splice site), c.409C > T (p.Arg137*) and c.877 + 3G > T (splice site), and compound heterozygous c.871C > T (p.Gln291*) and c.1035dup (p.Ile346Tyrfs*39). All patients displayed perinatal or early-onset cardiomyopathy and cataract, clinical features pathognomonic for Sengers syndrome. Other common findings included blood lactic acidosis and tachydyspnoea while nystagmus, eosinophilia and cervical meningocele were documented in only either one or two cases. Deficiency of the adenine nucleotide translocator was found in heart and skeletal muscle biopsies from two patients associated with respiratory chain complex I deficiency. In contrast to previous findings, mitochondrial DNA content was normal in both tissues. CONCLUSION: We compare our findings to those in 21 previously reported AGK mutation-positive Sengers patients, confirming that Sengers syndrome is a clinically recognisable disorder of mitochondrial energy metabolism.

Phenotypic spectrum of eleven patients and five novel MTFMT mutations identified by exome sequencing and candidate gene screening.

Defects of mitochondrial oxidative phosphorylation (OXPHOS) are associated with a wide range of clinical phenotypes and time courses. Combined OXPHOS deficiencies are mainly caused by mutations of nuclear genes that are involved in mitochondrial protein translation. Due to their genetic heterogeneity it is almost impossible to diagnose OXPHOS patients on clinical grounds alone. Hence next generation sequencing (NGS) provides a distinct advantage over candidate gene sequencing to discover the underlying genetic defect in a timely manner. One recent example is the identification of mutations in MTFMT that impair mitochondrial protein translation through decreased formylation of Met-tRNA(Met). Here we report the results of a combined exome sequencing and candidate gene screening study. We identified nine additional MTFMT patients from eight families who were affected with Leigh encephalopathy or white matter disease, microcephaly, mental retardation, ataxia, and muscular hypotonia. In four patients, the causal mutations were identified by exome sequencing followed by stringent bioinformatic filtering. In one index case, exome sequencing identified a single heterozygous mutation leading to Sanger sequencing which identified a second mutation in the non-covered first exon. High-resolution melting curve-based MTFMT screening in 350 OXPHPOS patients identified pathogenic mutations in another three index cases. Mutations in one of them were not covered by previous exome sequencing. All novel mutations predict a loss-of-function or result in a severe decrease in MTFMT protein in patients' fibroblasts accompanied by reduced steady-state levels of complex I and IV subunits. Being present in 11 out of 13 index cases the c.626C>T mutation is one of the most frequent disease alleles underlying OXPHOS disorders. We provide detailed clinical descriptions on eleven MTFMT patients and review five previously reported cases.

Alterations of red cell membrane properties in neuroacanthocytosis.

Neuroacanthocytosis (NA) refers to a group of heterogenous, rare genetic disorders, namely chorea acanthocytosis (ChAc), McLeod syndrome (MLS), Huntington's disease-like 2 (HDL2) and pantothenate kinase associated neurodegeneration (PKAN), that mainly affect the basal ganglia and are associated with similar neurological symptoms. PKAN is also assigned to a group of rare neurodegenerative diseases, known as NBIA (neurodegeneration with brain iron accumulation), associated with iron accumulation in the basal ganglia and progressive movement disorder. Acanthocytosis, the occurrence of misshaped erythrocytes with thorny protrusions, is frequently observed in ChAc and MLS patients but less prevalent in PKAN (about 10%) and HDL2 patients. The pathological factors that lead to the formation of the acanthocytic red blood cell shape are currently unknown. The aim of this study was to determine whether NA/NBIA acanthocytes differ in their functionality from normal erythrocytes. Several flow-cytometry-based assays were applied to test the physiological responses of the plasma membrane, namely drug-induced endocytosis, phosphatidylserine exposure and calcium uptake upon treatment with lysophosphatidic acid. ChAc red cell samples clearly showed a reduced response in drug-induced endovesiculation, lysophosphatidic acid-induced phosphatidylserine exposure, and calcium uptake. Impaired responses were also observed in acanthocyte-positive NBIA (PKAN) red cells but not in patient cells without shape abnormalities. These data suggest an "acanthocytic state" of the red cell where alterations in functional and interdependent membrane properties arise together with an acanthocytic cell shape. Further elucidation of the aberrant molecular mechanisms that cause this acanthocytic state may possibly help to evaluate the pathological pathways leading to neurodegeneration.

Mitochondrial depletion syndromes in children and adults.

To highlight differences between early-onset and adult mitochondrial depletion syndromes (MDS) concerning etiology and genetic background, pathogenesis, phenotype, clinical presentation and their outcome. MDSs most frequently occur in neonates, infants, or juveniles and more rarely in adolescents or adults. Mutated genes phenotypically presenting with adult-onset MDS include POLG1, TK2, TyMP, RRM2B, or PEO1/twinkle. Adult MDS manifest similarly to early-onset MDS, as myopathy, encephalo-myopathy, hepato-cerebral syndrome, or with chronic progressive external ophthalmoplegia (CPEO), fatigue, or only minimal muscular manifestations. Diagnostic work-up or treatment is not at variance from early-onset cases. Histological examination of muscle may be normal but biochemical investigations may reveal multiple respiratory chain defects. The outcome appears to be more favorable in adult than in early-onset forms. Mitochondrial depletion syndromes is not only a condition of neonates, infants, or juveniles but rarely also occurs in adults, presenting with minimal manifestations or manifestations like in the early-onset forms. Outcome of adult-onset MDS appears more favorable than early-onset MDS.