Functional analysis of a novel POLγA mutation associated with a severe perinatal mitochondrial encephalomyopathy.

Mutations in the mitochondrial DNA polymerase gamma catalytic subunit (POLγA) compromise the stability of mitochondrial DNA (mtDNA) by leading to mutations, deletions and depletions in mtDNA. Patients with mutations in POLγA often differ remarkably in disease severity and age of onset. In this work we have studied the functional consequence of POLγA mutations in a patient with an uncommon and a very severe disease phenotype characterized by prenatal onset with intrauterine growth restriction, lactic acidosis from birth, encephalopathy, hepatopathy, myopathy, and early death. Muscle biopsy identified scattered COX-deficient muscle fibers, respiratory chain dysfunction and mtDNA depletion. We identified a novel POLγA mutation (p.His1134Tyr) in trans with the previously identified p.Thr251Ile/Pro587Leu double mutant. Biochemical characterization of the purified recombinant POLγA variants showed that the p.His1134Tyr mutation caused severe polymerase dysfunction. The p.Thr251Ile/Pro587Leu mutation caused reduced polymerase function in conditions of low dNTP concentration that mimic postmitotic tissues. Critically, when p.His1134Tyr and p.Thr251Ile/Pro587Leu were combined under these conditions, mtDNA replication was severely diminished and featured prominent stalling. Our data provide a molecular explanation for the patient´s mtDNA depletion and clinical features, particularly in tissues such as brain and muscle that have low dNTP concentration.

An ancestral 10-bp repeat expansion in VWA1 causes recessive hereditary motor neuropathy.

The extracellular matrix comprises a network of macromolecules such as collagens, proteoglycans and glycoproteins. VWA1 (von Willebrand factor A domain containing 1) encodes a component of the extracellular matrix that interacts with perlecan/collagen VI, appears to be involved in stabilizing extracellular matrix structures, and demonstrates high expression levels in tibial nerve. Vwa1-deficient mice manifest with abnormal peripheral nerve structure/function; however, VWA1 variants have not previously been associated with human disease. By interrogating the genome sequences of 74 180 individuals from the 100K Genomes Project in combination with international gene-matching efforts and targeted sequencing, we identified 17 individuals from 15 families with an autosomal-recessive, non-length dependent, hereditary motor neuropathy and rare biallelic variants in VWA1. A single disease-associated allele p.(G25Rfs*74), a 10-bp repeat expansion, was observed in 14/15 families and was homozygous in 10/15. Given an allele frequency in European populations approaching 1/1000, the seven unrelated homozygote individuals ascertained from the 100K Genomes Project represents a substantial enrichment above expected. Haplotype analysis identified a shared 220 kb region suggesting that this founder mutation arose >7000 years ago. A wide age-range of patients (6-83 years) helped delineate the clinical phenotype over time. The commonest disease presentation in the cohort was an early-onset (mean 2.0 ± 1.4 years) non-length-dependent axonal hereditary motor neuropathy, confirmed on electrophysiology, which will have to be differentiated from other predominantly or pure motor neuropathies and neuronopathies. Because of slow disease progression, ambulation was largely preserved. Neurophysiology, muscle histopathology, and muscle MRI findings typically revealed clear neurogenic changes with single isolated cases displaying additional myopathic process. We speculate that a few findings of myopathic changes might be secondary to chronic denervation rather than indicating an additional myopathic disease process. Duplex reverse transcription polymerase chain reaction and immunoblotting using patient fibroblasts revealed that the founder allele results in partial nonsense mediated decay and an absence of detectable protein. CRISPR and morpholino vwa1 modelling in zebrafish demonstrated reductions in motor neuron axonal growth, synaptic formation in the skeletal muscles and locomotive behaviour. In summary, we estimate that biallelic variants in VWA1 may be responsible for up to 1% of unexplained hereditary motor neuropathy cases in Europeans. The detailed clinical characterization provided here will facilitate targeted testing on suitable patient cohorts. This novel disease gene may have previously evaded detection because of high GC content, consequential low coverage and computational difficulties associated with robustly detecting repeat-expansions. Reviewing previously unsolved exomes using lower QC filters may generate further diagnoses.

Bi-allelic GAD1 variants cause a neonatal onset syndromic developmental and epileptic encephalopathy.

Developmental and epileptic encephalopathies are a heterogeneous group of early-onset epilepsy syndromes dramatically impairing neurodevelopment. Modern genomic technologies have revealed a number of monogenic origins and opened the door to therapeutic hopes. Here we describe a new syndromic developmental and epileptic encephalopathy caused by bi-allelic loss-of-function variants in GAD1, as presented by 11 patients from six independent consanguineous families. Seizure onset occurred in the first 2 months of life in all patients. All 10 patients, from whom early disease history was available, presented with seizure onset in the first month of life, mainly consisting of epileptic spasms or myoclonic seizures. Early EEG showed suppression-burst or pattern of burst attenuation or hypsarrhythmia if only recorded in the post-neonatal period. Eight patients had joint contractures and/or pes equinovarus. Seven patients presented a cleft palate and two also had an omphalocele, reproducing the phenotype of the knockout Gad1-/- mouse model. Four patients died before 4 years of age. GAD1 encodes the glutamate decarboxylase enzyme GAD67, a critical actor of the γ-aminobutyric acid (GABA) metabolism as it catalyses the decarboxylation of glutamic acid to form GABA. Our findings evoke a novel syndrome related to GAD67 deficiency, characterized by the unique association of developmental and epileptic encephalopathies, cleft palate, joint contractures and/or omphalocele.

Homozygous Null TBX4 Mutations Lead to Posterior Amelia with Pelvic and Pulmonary Hypoplasia.

The development of hindlimbs in tetrapod species relies specifically on the transcription factor TBX4. In humans, heterozygous loss-of-function TBX4 mutations cause dominant small patella syndrome (SPS) due to haploinsufficiency. Here, we characterize a striking clinical entity in four fetuses with complete posterior amelia with pelvis and pulmonary hypoplasia (PAPPA). Through exome sequencing, we find that PAPPA syndrome is caused by homozygous TBX4 inactivating mutations during embryogenesis in humans. In two consanguineous couples, we uncover distinct germline TBX4 coding mutations, p.Tyr113∗ and p.Tyr127Asn, that segregated with SPS in heterozygous parents and with posterior amelia with pelvis and pulmonary hypoplasia syndrome (PAPPAS) in one available homozygous fetus. A complete absence of TBX4 transcripts in this proband with biallelic p.Tyr113∗ stop-gain mutations revealed nonsense-mediated decay of the endogenous mRNA. CRISPR/Cas9-mediated TBX4 deletion in Xenopus embryos confirmed its restricted role during leg development. We conclude that SPS and PAPPAS are allelic diseases of TBX4 deficiency and that TBX4 is an essential transcription factor for organogenesis of the lungs, pelvis, and hindlimbs in humans.

Motor neuron diseases caused by a novel VRK1 variant - A genotype/phenotype study.

BACKGROUND: Motor neuron disorders involving upper and lower neurons are a genetically and clinically heterogenous group of rare neuromuscular disorders with overlap among spinal muscular atrophies (SMAs) and amyotrophic lateral sclerosis (ALS). Classical SMA caused by recessive mutations in SMN1 is one of the most common genetic causes of mortality in infants. It is characterized by degeneration of anterior horn cells in the spinal cord, leading to progressive muscle weakness and atrophy. Non-SMN1-related spinal muscular atrophies are caused by variants in a number of genes, including VRK1, encoding the vaccinia-related kinase 1 (VRK1). VRK1 variants have been segregated with motor neuron diseases including SMA phenotypes or hereditary complex motor and sensory axonal neuropathy (HMSN), with or without pontocerebellar hypoplasia or microcephaly. RESULTS: Here, we report an association of a novel homozygous splice variant in VRK1 (c.1159 + 1G>A) with childhood-onset SMA or juvenile lower motor disease with brisk tendon reflexes without pontocerebellar hypoplasia and normal intellectual ability in a family with five affected individuals. We show that the VRK1 splice variant in patients causes decreased splicing efficiency and a mRNA frameshift that escapes the nonsense-mediated decay machinery and results in a premature termination codon. CONCLUSIONS: Our findings unveil the impact of the variant on the VRK1 transcript and further support the implication of VRK1 in the pathogenesis of lower motor neuron diseases.

Recessive Charcot-Marie-Tooth and multiple sclerosis associated with a variant in MCM3AP.

Variants in MCM3AP, encoding the germinal-centre associated nuclear protein, have been associated with progressive polyneuropathy with or without intellectual disability and ptosis in some cases, and with a complex phenotype with immunodeficiency, skin changes and myelodysplasia. MCM3AP encoded protein functions as an acetyltransferase that acetylates the replication protein, MCM3, and plays a key role in the regulation of DNA replication. In this study, we report a novel variant in MCM3AP (p.Ile954Thr), in a family including three affected individuals with characteristic features of Charcot-Marie-Tooth neuropathy and multiple sclerosis, an inflammatory condition of the central nervous system without known genetic cause. The affected individuals were homozygous for a missense MCM3AP variant, located at the Sac3 domain, which was predicted to affect conserved amino acid likely important for the function of the germinal-centre associated nuclear protein. Our data support further expansion of the clinical spectrum linked to MCM3AP variant and highlight that MCM3AP should be considered in patients with accompaniment of recessive motor axonal Charcot-Marie-Tooth neuropathy and multiple sclerosis.

Expression of the GBGT1 Gene and the Forssman Antigen in Red Blood Cells in a Palestinian Population.

BACKGROUND: The Forssman antigen (FORS1 Ag) is expressed on human red blood cells (RBCs). We investigated its presence on RBCs from Palestinian subjects and Swedish subjects by serological testing and by sequencing part of exon 7 of the GBGT1 gene, which encodes Forssman synthase. MATERIALS AND METHODS: Blood samples from Palestinian subjects (n = 211 adults and n = 73 newborns) and from Swedish subjects (n = 47 adults) were analyzed in the study. RBCs from the Palestinian samples were typed for the FORS1 Ag using a monoclonal anti-Forssman antibody. The GBGT1 gene was genotyped by DNA sequencing (all adult samples) or by using amplification refractory mutation system PCR (newborn samples). RESULTS: All of the studied samples were negative for the FORS1 Ag by serologic typing. DNA sequencing of the 3' end of exon 7 of the GBGT1 gene, which includes Arg296, showed that all samples had the wild-type Arg296 sequence, which is associated with an inactive form of Forssman synthase. We detected four single nucleotide polymorphisms in the adult samples; two were silent (p.Tyr232=, p. Gly290=), and two were missense (p. Arg243Cys, p. Arg243His). The allele frequencies ranged from 0.2 to 3.6%. The p. Arg243Cys SNP was a novel SNP that was detected in one Palestinian sample. CONCLUSION: Our results confirmed the allelic diversity of GBGT1 and identified a novel nucleotide polymorphism in this gene, p. Arg243Cys. Our results also confirmed that the FORS blood group system is a low-frequency system.

Ataxia-telangiectasia-like disorder in a family deficient for MRE11A, caused by a MRE11 variant.

OBJECTIVE: We report 3 siblings with the characteristic features of ataxia-telangiectasia-like disorder associated with a homozygous MRE11 synonymous variant causing nonsense-mediated mRNA decay (NMD) and MRE11A deficiency. METHODS: Clinical assessments, next-generation sequencing, transcript and immunohistochemistry analyses were performed. RESULTS: The patients presented with poor balance, developmental delay during the first year of age, and suffered from intellectual disability from early childhood. They showed oculomotor apraxia, slurred and explosive speech, limb and gait ataxia, exaggerated deep tendon reflex, dystonic posture, and mirror movement in their hands. They developed mild cognitive abilities. Brain MRI in the index case revealed cerebellar atrophy. Next-generation sequencing revealed a homozygous synonymous variant in MRE11 (c.657C>T, p.Asn219=) that we show affects splicing. A complete absence of MRE11 transcripts in the index case suggested NMD and immunohistochemistry confirmed the absence of a stable protein. CONCLUSIONS: Despite the critical role of MRE11A in double-strand break repair and its contribution to the Mre11/Rad50/Nbs1 complex, the absence of MRE11A is compatible with life.

Prevalence of antibodies to a new histo-blood system: the FORS system.

BACKGROUND: In 1987, three unrelated English families were reported with a putative blood subgroup called Apae. Swedish researchers later found evidence leading to abolishment of the Apae subgroup and establishment instead of the FORS blood group system (System 31 - ISBT, 2012). It is important to know the prevalence of antibodies in order to make the best decisions in transfusion medicine. Cells expressing the Forssman saccharide, such as sheep erythrocytes, are needed to detect the anti-Forssman antibody. The aim of this study was to define the prevalence of human anti-Forssman antibody. MATERIALS AND METHODS: Plasma samples from 800 individuals were studied. Sheep erythrocytes or Forssman "kodecytes" were mixed with the plasma samples using the tube technique. Plasma from an Apae individual was used as a negative control and monoclonal anti-Forssman antibody (M1/22.25.8HL cell line supernatant) was used as the positive control. RESULTS: Of the 800 individuals tested, one was negative for the presence of anti-Forssman antibody. We compared the anti-Forssman antibody reaction pattern between genders and found that males have weaker reactions than females, both at room temperature (p=0.026) and at 37 °C (p=0.043). We also investigated the reaction pattern of anti-Forssman antibody in relation to ABO and Rh blood group types without finding any significant differences. DISCUSSION: Sheep erythrocytes are suitable for searching for human anti-Forssman antibody. The quantity of anti-Forssman antibodies in plasma is higher in females than in males. In the population (n=800) studied here, we found one individual lacking the anti-Forssman antibody. These results contribute to the data already published, confirming that FORS is a rare blood group.

Histopathological changes in skeletal muscle associated with chronic ischaemia.

Muscle biopsy is an essential part in the diagnostic workup in patients with suspected neuromuscular disorders. It is therefore important to be aware of morphological alterations that can be caused by systemic factors or natural ageing. Chronic limb ischaemia is frequent in elderly individuals. This study was performed to examine histopathological and mitochondrial changes in muscle in patients with chronic critical limb ischaemia. Muscle biopsy of skeletal muscle of the lower limb of patients with chronic ischaemia leading to amputation was performed and compared with muscle biopsies of healthy, age-matched controls. The histopathological abnormalities included fibrosis, necrosis, atrophy, glycogen depletion, internal nuclei, rimmed vacuoles, fibre type grouping, cytochrome c oxidase deficient fibres, MHC-I upregulation, and signs of microangiopathy. The only alteration found in age-matched controls was a few cytochrome c oxidase deficient fibres. There were also increased levels of multiple mitochondrial DNA deletions in ischaemic muscles compared with controls. Critical limb ischaemia is associated with significant histopathological changes in muscle tissue and also increased levels of mitochondrial DNA deletions. Since the alterations mimic different primary myopathic changes, chronic ischaemia is important to consider as a differential diagnosis in elderly individuals, investigated with muscle biopsy for muscle disease.

Truncating CHRNG mutations associated with interfamilial variability of the severity of the Escobar variant of multiple pterygium syndrome.

BACKGROUND: In humans, muscle-specific nicotinergic acetylcholine receptor (AChR) is a transmembrane protein with five different subunits, coded by CHRNA1, CHRNB, CHRND and CHRNG/CHRNE. The gamma subunit of AChR encoded by CHRNG is expressed during early foetal development, whereas in the adult, the γ subunit is replaced by a ε subunit. Mutations in the CHRNG encoding the embryonal acetylcholine receptor may cause the non-lethal Escobar variant (EVMPS) and lethal form (LMPS) of multiple pterygium syndrome. The MPS is a condition characterised by prenatal growth failure with pterygium and akinesia leading to muscle weakness and severe congenital contractures, as well as scoliosis. RESULTS: Our whole exome sequencing studies have identified one novel and two previously reported homozygous mutations in CHRNG in three families affected by non-lethal EVMPS. The mutations consist of deletion of two nucleotides, cause a frameshift predicted to result in premature termination of the foetally expressed gamma subunit of the AChR. CONCLUSIONS: Our data suggest that severity of the phenotype varies significantly both within and between families with MPS and that there is no apparent correlation between mutation position and clinical phenotype. Although individuals with CHRNG mutations can survive, there is an increased frequency of abortions and stillbirth in their families. Furthermore, genetic background and environmental modifiers might be of significance for decisiveness of the lethal spectrum, rather than the state of the mutation per se. Detailed clinical examination of our patients further indicates the changing phenotype from infancy to childhood.

Lethal multiple pterygium syndrome, the extreme end of the RYR1 spectrum.

BACKGROUND: Lethal multiple pterygium syndrome (LMPS, OMIM 253290), is a fatal disorder associated with anomalies of the skin, muscles and skeleton. It is characterised by prenatal growth failure with pterygium present in multiple areas and akinesia, leading to muscle weakness and severe arthrogryposis. Foetal hydrops with cystic hygroma develops in affected foetuses with LMPS. This study aimed to uncover the aetiology of LMPS in a family with two affected foetuses. METHODS AND RESULTS: Whole exome sequencing studies have identified novel compound heterozygous mutations in RYR1 in two affected foetuses with pterygium, severe arthrogryposis and foetal hydrops with cystic hygroma, characteristic features compatible with LMPS. The result was confirmed by Sanger sequencing and restriction fragment length polymorphism analysis. CONCLUSIONS: RYR1 encodes the skeletal muscle isoform ryanodine receptor 1, an intracellular calcium channel with a central role in muscle contraction. Mutations in RYR1 have been associated with congenital myopathies, which form a continuous spectrum of pathological features including a severe variant with onset in utero with fetal akinesia and arthrogryposis. Here, the results indicate that LMPS can be considered as the extreme end of the RYR1-related neonatal myopathy spectrum. This further supports the concept that LMPS is a severe disorder associated with defects in the process known as excitation-contraction coupling.

Developmental MYH3 Myopathy Associated with Expression of Mutant Protein and Reduced Expression Levels of Embryonic MyHC.

OBJECTIVE: An essential role for embryonic MyHC in foetal development has been found from its association with distal arthrogryposis syndromes, a heterogeneous group of disorders characterised by congenital contractions. The latter probably result from severe myopathy during foetal development. Lack of embryonic muscle biopsy material and suitable animal models has hindered study of the pathomechanisms linking mutations in MYH3 to prenatal myopathy. METHODS AND RESULTS: We determined the pathomechanisms of developmental myopathy caused by recurrent p.Thr178Ile MYH3 heterozygosity, using patient-derived skeletal muscle cells in culture as an experimental disease model to emulate early embryonic development. These cultured cells were processed for discrimination and quantitative analysis of mutant and wild-type MYH3 alleles and MyHC transcripts, real-time RT-qPCR, sequence analysis, immunofluorescence microscopy, immunoblot, and proteomic assessments. Involvement of the ubiquitin proteasome system was investigated in patients with p.Thr178Ile mutations in MYH3 and MYH2. We found equal overall expression of mutant and wild-type MyHC mRNAs and proteins. Compared to the controls, however, expression of embryonic MyHC transcripts and proteins was reduced whereas expression of myosin-specific E3 ubiquitin ligase (MuRF1) was increased. We also found delayed myofibrillogenesis and atrophic myotubes but structured sarcomeres. CONCLUSION: In conclusion, this study suggests that developmental p.Thr178Ile MYH3 myopathy is associated with a combined pathomechanism of insufficient dosage of functional embryonic MyHC and production of mutant protein.

Mitochondrial pathology in inclusion body myositis.

Inclusion body myositis (IBM) is usually associated with a large number of cytochrome c oxidase (COX)-deficient muscle fibers and acquired mitochondrial DNA (mtDNA) deletions. We studied the number of COX-deficient fibers and the amount of mtDNA deletions, and if variants in nuclear genes involved in mtDNA maintenance may contribute to the occurrence of mtDNA deletions in IBM muscle. Twenty-six IBM patients were included. COX-deficient fibers were assayed by morphometry and mtDNA deletions by qPCR. POLG was analyzed in all patients by Sanger sequencing and C10orf2 (Twinkle), DNA2, MGME1, OPA1, POLG2, RRM2B, SLC25A4 and TYMP in six patients by next generation sequencing. Patients with many COX-deficient muscle fibers had a significantly higher proportion of mtDNA deletions than patients with few COX-deficient fibers. We found previously unreported variants in POLG and C10orf2 and IBM patients had a significantly higher frequency of an RRM2B variant than controls. POLG variants appeared more common in IBM patients with many COX-deficient fibers, but the difference was not statistically significant. We conclude that COX-deficient fibers in inclusion body myositis are associated with multiple mtDNA deletions. In IBM patients we found novel and also previously reported variants in genes of importance for mtDNA maintenance that warrants further studies.

Broad phenotypic variability in patients with complex I deficiency due to mutations in NDUFS1 and NDUFV1.

We report clinical, metabolic, genetic and neuroradiological findings in five patients from three different families with isolated complex I deficiency. Genetic analysis revealed mutations in NDUFS1 in three patients and in NDUFV1 in two patients. Four of the mutations are novel and affect amino acid residues that either are invariant among species or conserved in their properties. The presented clinical courses are characterized by leukoencephalopathy or early death and expand the already heterogeneous phenotypic spectrum. A literature review was performed, showing that patients with mutations in NDUFS1 in general have a worse prognosis than patients with mutations in NDUFV1.

LC-MS/MS characterization of combined glycogenin-1 and glycogenin-2 enzymatic activities reveals their self-glucosylation preferences.

Glycogen synthesis is initiated by self-glucosylation of the glycosyltransferases glycogenin-1 and -2 that, in the presence of UDP-glucose, form both the first glucose-O-tyrosine linkage, and then stepwise add a series of α1,4-linked glucoses to a growing chain of variable length. Glycogen-1 and -2 coexist in liver glycogen preparations where the proteins are known to form homodimers, and they also have been shown to interact with each other. In order to study how glycogenin-1 and -2 interactions may influence each other's glucosylations we setup a cell-free expression system for in vitro production and glucosylation of glycogenin-1 and -2 in various combinations, and used a mass spectrometry based workflow for the characterization and quantitation of tryptic glycopeptides originating from glycogenin-1 and -2. The analysis revealed that the self-glucosylation endpoint was the incorporation of 4-8 glucose units on Tyr 195 of glycogenin-1, but only 0-4 glucose units on Tyr-228 of glycogenin-2. The glucosylation of glycogenin-2 was enhanced to 2-4 glucose units by the co-presence of enzymatically active glycogenin-1. Glycogenin-2 was, however, unable to glucosylate inactive glycogenin-1, at least not an enzymatically inactivated Thr83Met glycogenin-1 mutant, recently identified in a patient with severe glycogen depletion.

Phenotypes of myopathy-related beta-tropomyosin mutants in human and mouse tissue cultures.

Mutations in TPM2 result in a variety of myopathies characterised by variable clinical and morphological features. We used human and mouse cultured cells to study the effects of ß-TM mutants. The mutants induced a range of phenotypes in human myoblasts, which generally changed upon differentiation to myotubes. Human myotubes transfected with the E41K-ß-TM(EGFP) mutant showed perinuclear aggregates. The G53ins-ß-TM(EGFP) mutant tended to accumulate in myoblasts but was incorporated into filamentous structures of myotubes. The K49del-ß-TM(EGFP) and E122K-ß-TM(EGFP) mutants induced the formation of rod-like structures in human cells. The N202K-ß-TM(EGFP) mutant failed to integrate into thin filaments and formed accumulations in myotubes. The accumulation of mutant ß-TM(EGFP) in the perinuclear and peripheral areas of the cells was the striking feature in C2C12. We demonstrated that human tissue culture is a suitable system for studying the early stages of altered myofibrilogenesis and morphological changes linked to myopathy-related ß-TM mutants. In addition, the histopathological phenotype associated with expression of the various mutant proteins depends on the cell type and varies with the maturation of the muscle cell. Further, the phenotype is a combinatorial effect of the specific amino acid change and the temporal expression of the mutant protein.

Subnormal levels of POLγA cause inefficient initiation of light-strand DNA synthesis and lead to mitochondrial DNA deletions and progressive external ophthalmoplegia [corrected].

The POLG1 gene encodes the catalytic subunit of mitochondrial DNA (mtDNA) polymerase γ (POLγ). We here describe a sibling pair with adult-onset progressive external ophthalmoplegia, cognitive impairment and mitochondrial myopathy characterized by DNA depletion and multiple mtDNA deletions. The phenotype is due to compound heterozygous POLG1 mutations, T914P and the intron mutation c.3104 + 3A > T. The mutant genes produce POLγ isoforms with heterozygous phenotypes that fail to synthesize longer DNA products in vitro. However, exon skipping in the c.3104 + 3A > T mutant is not complete, and the presence of low levels of wild-type POLγ explains patient survival. To better understand the underlying pathogenic mechanisms, we characterized the effects of POLγ depletion in vitro and found that leading-strand DNA synthesis is relatively undisturbed. In contrast, initiation of lagging-strand DNA synthesis is ineffective at lower POLγ concentrations that uncouples leading strand from lagging-strand DNA synthesis. In vivo, this effect leads to prolonged exposure of the heavy strand in its single-stranded conformation that in turn can cause the mtDNA deletions observed in our patients. Our findings, thus, suggest a molecular mechanism explaining how POLγ mutations can cause mtDNA deletions in vivo.

A novel mitochondrial tRNA Arg mutation resulting in an anticodon swap in a patient with mitochondrial encephalomyopathy.

We report a mutation in the anticodon of the tRNA(Arg) gene (m.10437 G>A), resulting in an anticodon swap from GCU to ACU, which is the anticodon of tRNA(Trp), in a boy with mitochondrial encephalomyopathy. Enzyme histochemical analysis of muscle tissue and biochemical analysis of isolated muscle mitochondria demonstrated cytochrome c oxidase (COX) deficiency. Restriction fragment length polymorphism analysis showed that 90% of muscle and 82% of urinary epithelium mtDNA harbored the mutation. The mutation was not identified in blood, fibroblasts, hair roots, or buccal epithelial cells and it was absent in the asymptomatic mother, suggesting that it was a de novo mutation. Single-fiber PCR analysis showed that the proportion of mutated mtDNA correlated with enzyme histochemical COX deficiency. This mutation adds to the three previously described disease-causing mutations in tRNA(Arg), but it is the first mutation occurring in the anticodon of tRNA(Arg).

Phenotypic and genotypic variability in Alpers syndrome.

BACKGROUND: Alpers syndrome is one of the most common phenotypes of mitochondrial disorders in early childhood and has been associated with pathogenic mutations in POLG1. AIMS: To investigate the phenotypic-genotypic correlations in Alpers syndrome and to identify potential differences among patients with Alpers syndrome with or without pathogenic POLG1 mutations. METHODS: Patients with the phenotype of Alpers syndrome who were referred to our pediatric hospital during 1984-2007 and were diagnosed with mitochondrial encephalomyopathy underwent further biochemical, morphological and genetic investigations. RESULTS: A total of 19 patients were included in the study, of whom six had pathogenic POLG1 mutations including a novel mutation (c.907 G>A, p.Gly303Arg). Complete mtDNA sequencing in the subgroup without POLG1 mutations showed 5 novel and 5 very rare mtDNA variants considered as rare polymorphisms. Compared to POLG1(-) patients, the POLG1(+) patients more frequently had seizures at onset, which often became refractory. Ataxia and stroke-like episodes were much more common, while microcephaly and spasticity were encountered almost solely in the POLG1(-) group. Hepatic and ophthalmological involvement developed in 79% and 88% of patients, respectively. Most of the patients in both groups had predominant deficiency of complex I. In addition to the major degenerative changes in the cerebral cortex, the basal ganglia, thalamus and white matter were also involved to variable extent. CONCLUSION: Alpers syndrome is a heterogeneous syndrome that should be considered in patients with early-onset progressive cortical encephalopathy regardless of liver involvement. The phenotype is different depending on the presence or absence of POLG1 mutations.