Leukoencephalopathy with Brain stem and Spinal cord involvement and Lactate elevation (LBSL): Report of a new family and a novel DARS2 mutation.

Background: LBSL is a mitochondrial disorder caused by mutations in the mitochondrial aspartyl-tRNA synthetase gene DARS2, resulting in a distinctive pattern on brain magnetic resonance imaging (MRI) and spectroscopy. Clinical presentation varies from severe infantile to chronic, slowly progressive neuronal deterioration in adolescents or adults. Most individuals with LBSL are compound heterozygous for one splicing defect in an intron 2 mutational hotspot and a second defect that could be a missense, non-sense, or splice site mutation or deletion resulting in decreased expression of the full-length protein. Aim: To present a new family with two affected members with LBSL and report a novel DARS2 mutation. Results: An 8-year-old boy (Patient 1) was referred due to headaches and abnormal MRI, suggestive of LBSL. Genetic testing revealed a previously reported c.492 + 2 T > C mutation in the DARS2 gene. Sanger sequencing uncovered a novel variant c.228-17C > G in the intron 2 hotspot. Family studies found the same genetic changes in an asymptomatic 4-year-old younger brother (Patient 2), who was found on follow-up to have an abnormal MRI. mRNA extracted from patients' fibroblasts showed that the c.228-17C > G mutation caused skipping of exon 3 resulting in lower DARS2 mRNA level. Complete absence of DARS2 protein was also found in both patients. Summary: We present a new family with two children affected with LBSL and describe a novel mutation in the DARS2 intron 2 hotspot. Despite findings of extensive white matter disease in the brain and spine, the proband in this family presented only with headaches, while the younger sibling, who also had extensive white matter changes, was asymptomatic. Our in-vitro results confirmed skipping of exon 3 in patients and family members carrying the intron 2 variant, which is consistent with previous reported mutations in intron 2 hotspots. DARS2 mRNA and protein levels were also reduced in both patients, further supporting the pathogenicity of the novel variant.

Population-level deficit of homozygosity unveils CPSF3 as an intellectual disability syndrome gene.

Predicting the pathogenicity of biallelic missense variants can be challenging. Here, we use a deficit of observed homozygous carriers of missense variants, versus an expected number in a set of 153,054 chip-genotyped Icelanders, to identify potentially pathogenic genotypes. We follow three missense variants with a complete deficit of homozygosity and find that their pathogenic effect in homozygous state ranges from severe childhood disease to early embryonic lethality. One of these variants is in CPSF3, a gene not previously linked to disease. From a set of clinically sequenced Icelanders, and by sequencing archival samples targeted through the Icelandic genealogy, we find four homozygous carriers. Additionally, we find two homozygous carriers of Mexican descent of another missense variant in CPSF3. All six homozygous carriers of missense variants in CPSF3 show severe intellectual disability, seizures, microcephaly, and abnormal muscle tone. Here, we show how the absence of certain homozygous genotypes from a large population set can elucidate causes of previously unexplained recessive diseases and early miscarriage.

ECHS1 disease in two unrelated families of Samoan descent: Common variant - rare disorder.

Mutations in the short-chain enoyl-CoA hydratase (SCEH) gene, ECHS1, cause a rare autosomal recessive disorder of valine catabolism. Patients usually present with developmental delay, regression, dystonia, feeding difficulties, and abnormal MRI with bilateral basal ganglia involvement. We present clinical, biochemical, molecular, and functional data for four affected patients from two unrelated families of Samoan descent with identical novel compound heterozygous mutations. Family 1 has three affected boys while Family 2 has an affected daughter, all with clinical and MRI findings of Leigh syndrome and intermittent episodes of acidosis and ketosis. WES identified a single heterozygous variant in ECHS1 at position c.832G > A (p.Ala278Thr). However, western blot revealed significantly reduced ECHS1 protein for all affected family members. Decreased SCEH activity in fibroblasts and a mild increase in marker metabolites in urine further supported ECHS1 as the underlying gene defect. Additional investigations at the DNA (aCGH, WGS) and RNA (qPCR, RT-PCR, RNA-Seq, RNA-Array) level identified a silent, common variant at position c.489G > A (p.Pro163=) as the second mutation. This substitution, present at high frequency in the Samoan population, is associated with decreased levels of normally spliced mRNA. To our understanding, this is the first report of a novel, hypomorphic allele c.489G > A (p.Pro163=), associated with SCEH deficiency.

The natural history of infantile mitochondrial DNA depletion syndrome due to RRM2B deficiency.

PURPOSE: Mitochondrial DNA (mtDNA) depletion syndrome (MDDS) encompasses a group of genetic disorders of mtDNA maintenance. Mutation of RRM2B is an uncommon cause of infantile-onset encephalomyopathic MDDS. Here we describe the natural history of this disease. METHODS: Multinational series of new genetically confirmed cases from six pediatric centers. RESULTS: Nine new cases of infantile-onset RRM2B deficiency, and 22 previously published cases comprised a total cohort of 31 patients. Infants presented at a mean of 1.95 months with truncal hypotonia, generalized weakness, and faltering growth. Seizures evolved in 39% at a mean of 3.1 months. Non-neurological manifestations included respiratory distress/failure (58%), renal tubulopathy (55%), sensorineural hearing loss (36%), gastrointestinal disturbance (32%), eye abnormalities (13%), and anemia (13%). Laboratory features included elevated lactate (blood, cerebrospinal fluid (CSF), urine, magnetic resonance (MR), spectroscopy), ragged-red and cytochrome c oxidase-deficient fibers, lipid myopathy, and multiple oxidative phosphorylation enzyme deficiencies in skeletal muscle. Eight new RRM2B variants were identified. Patients with biallelic truncating variants had the worst survival. Overall survival was 29% at 6 months and 16% at 1 year. CONCLUSIONS: Infantile-onset MDDS due to RRM2B deficiency is a severe disorder with characteristic clinical features and extremely poor prognosis. Presently management is supportive as there is no effective treatment. Novel treatments are urgently needed.

Novel mutations in the mitochondrial complex I assembly gene NDUFAF5 reveal heterogeneous phenotypes.

Primary mitochondrial complex I deficiency is the most common defect of the mitochondrial respiratory chain. It is caused by defects in structural components and assembly factors of this large protein complex. Mutations in the assembly factor NDUFAF5 are rare, with only five families reported to date. This study provides clinical, biochemical, molecular and functional data for four unrelated additional families, and three novel pathogenic variants. Three cases presented in infancy with lactic acidosis and classic Leigh syndrome. One patient, however, has a milder phenotype, with symptoms starting at 27 months and a protracted clinical course with improvement and relapsing episodes. She is homozygous for a previously reported mutation, p.Met279Arg and alive at 19 years with mild neurological involvement, normal lactate but abnormal urine organic acids. We found the same mutation in one of our severely affected patients in compound heterozygosity with a novel p.Lys52Thr mutation. Both patients with p.Met279Arg are of Taiwanese descent and had severe hyponatremia. Our third and fourth patients, both Caucasian, shared a common, newly described, missense mutation p.Lys109Asn which we show induces skipping of exon 3. Both Caucasian patients were compound heterozygotes, one with a previously reported Ashkenazi founder mutation while the other was negative for additional exonic variants. Whole genome sequencing followed by RNA studies revealed a novel deep intronic variant at position c.223-907A>C inducing an exonic splice enhancer. Our report adds significant new information to the mutational spectrum of NDUFAF5, further delineating the phenotypic heterogeneity of this mitochondrial defect.

Activation of a cryptic splice site in the mitochondrial elongation factor GFM1 causes combined OXPHOS deficiency.

We report the clinical, biochemical, and molecular findings in two brothers with encephalopathy and multi-systemic disease. Abnormal transferrin glycoforms were suggestive of a type I congenital disorder of glycosylation (CDG). While exome sequencing was negative for CDG related candidate genes, the testing revealed compound heterozygous mutations in the mitochondrial elongation factor G gene (GFM1). One of the mutations had been reported previously while the second, novel variant was found deep in intron 6, activating a cryptic splice site. Functional studies demonstrated decreased GFM1 protein levels, suggested disrupted assembly of mitochondrial complexes III and V and decreased activities of mitochondrial complexes I and IV, all indicating combined OXPHOS deficiency.

Mutations in TFAM, encoding mitochondrial transcription factor A, cause neonatal liver failure associated with mtDNA depletion.

In humans, mitochondrial DNA (mtDNA) depletion syndromes are a group of genetically and clinically heterogeneous autosomal recessive disorders that arise as a consequence of defects in mtDNA replication or nucleotide synthesis. Clinical manifestations are variable and include myopathic, encephalomyopathic, neurogastrointestinal or hepatocerebral phenotypes. Through clinical exome sequencing, we identified a homozygous missense variant (c.533C>T; p.Pro178Leu) in mitochondrial transcription factor A (TFAM) segregating in a consanguineous kindred of Colombian-Basque descent in which two siblings presented with IUGR, elevated transaminases, conjugated hyperbilirubinemia and hypoglycemia with progression to liver failure and death in early infancy. Results of the liver biopsy in the proband revealed cirrhosis, micro- and macrovesicular steatosis, cholestasis and mitochondrial pleomorphism. Electron microscopy of muscle revealed abnormal mitochondrial morphology and distribution while enzyme histochemistry was underwhelming. Electron transport chain testing in muscle showed increased citrate synthase activity suggesting mitochondrial proliferation, while respiratory chain activities were at the lower end of normal. mtDNA content was reduced in liver and muscle (11% and 21% of normal controls respectively). While Tfam mRNA expression was upregulated in primary fibroblasts, Tfam protein level was significantly reduced. Furthermore, functional investigations of the mitochondria revealed reduced basal respiration and spare respiratory capacity, decreased mtDNA copy number and markedly reduced nucleoids. TFAM is essential for transcription, replication and packaging of mtDNA into nucleoids. Tfam knockout mice display embryonic lethality secondary to severe mtDNA depletion. In this report, for the first time, we associate a homozygous variant in TFAM with a novel mtDNA depletion syndrome.

Mutations of human NARS2, encoding the mitochondrial asparaginyl-tRNA synthetase, cause nonsyndromic deafness and Leigh syndrome.

Here we demonstrate association of variants in the mitochondrial asparaginyl-tRNA synthetase NARS2 with human hearing loss and Leigh syndrome. A homozygous missense mutation ([c.637G>T; p.Val213Phe]) is the underlying cause of nonsyndromic hearing loss (DFNB94) and compound heterozygous mutations ([c.969T>A; p.Tyr323*] + [c.1142A>G; p.Asn381Ser]) result in mitochondrial respiratory chain deficiency and Leigh syndrome, which is a neurodegenerative disease characterized by symmetric, bilateral lesions in the basal ganglia, thalamus, and brain stem. The severity of the genetic lesions and their effects on NARS2 protein structure cosegregate with the phenotype. A hypothetical truncated NARS2 protein, secondary to the Leigh syndrome mutation p.Tyr323* is not detectable and p.Asn381Ser further decreases NARS2 protein levels in patient fibroblasts. p.Asn381Ser also disrupts dimerization of NARS2, while the hearing loss p.Val213Phe variant has no effect on NARS2 oligomerization. Additionally we demonstrate decreased steady-state levels of mt-tRNAAsn in fibroblasts from the Leigh syndrome patients. In these cells we show that a decrease in oxygen consumption rates (OCR) and electron transport chain (ETC) activity can be rescued by overexpression of wild type NARS2. However, overexpression of the hearing loss associated p.Val213Phe mutant protein in these fibroblasts cannot complement the OCR and ETC defects. Our findings establish lesions in NARS2 as a new cause for nonsyndromic hearing loss and Leigh syndrome.

Psychological Impact of Predictive Genetic Testing in VCP Inclusion Body Myopathy, Paget Disease of Bone and Frontotemporal Dementia.

Inclusion Body Myopathy associated with Paget's disease of bone and Fronto-temporal Dementia, also known as multisystem proteinopathy is an autosomal dominant, late onset neurodegenerative disorder caused by mutations in Valosin containing protein (VCP) gene. This study aimed to assess uptake and decision making for predictive genetic testing and the impact on psychological well-being. Individuals who had participated in the gene discovery study with a 50 % a priori risk of inheriting VCP disease were sent a letter of invitation offering genetic counseling and testing and were also invited to participate in this psychosocial study. A total of 102 individuals received an invitation and 33 individuals participated in genetic counseling and testing (32.3 %) with 29 completing baseline questionnaires. Twenty completed the follow-up post-test Hospital Anxiety and Depression Scale questionnaire including 13 of the 18 who had tested positive. Mean risk perception at baseline was 50.1 %. Reasons for testing included planning for the future, relieving uncertainty, informing children and satisfying curiosity. At baseline, one quarter of the participants had high levels of anxiety. However, scores were normal one year following testing. In this small cohort, one third of individuals at 50 % risk chose pre-symptomatic testing. Although one quarter of those choosing testing had high anxiety at baseline, this was not evident at follow-up.

Mutation in the nuclear-encoded mitochondrial isoleucyl-tRNA synthetase IARS2 in patients with cataracts, growth hormone deficiency with short stature, partial sensorineural deafness, and peripheral neuropathy or with Leigh syndrome.

Mutations in the nuclear-encoded mitochondrial aminoacyl-tRNA synthetases are associated with a range of clinical phenotypes. Here, we report a novel disorder in three adult patients with a phenotype including cataracts, short-stature secondary to growth hormone deficiency, sensorineural hearing deficit, peripheral sensory neuropathy, and skeletal dysplasia. Using SNP genotyping and whole-exome sequencing, we identified a single likely causal variant, a missense mutation in a conserved residue of the nuclear gene IARS2, encoding mitochondrial isoleucyl-tRNA synthetase. The mutation is homozygous in the affected patients, heterozygous in carriers, and absent in control chromosomes. IARS2 protein level was reduced in skin cells cultured from one of the patients, consistent with a pathogenic effect of the mutation. Compound heterozygous mutations in IARS2 were independently identified in a previously unreported patient with a more severe mitochondrial phenotype diagnosed as Leigh syndrome. This is the first report of clinical findings associated with IARS2 mutations.

Abnormalities in glycogen metabolism in a patient with alpers' syndrome presenting with hypoglycemia.

Intermittent hypoglycemia has been described in association with Alpers' syndrome, a disorder caused by mutations in the mitochondrial DNA polymerase gamma gene. In some patients hypoglycemia may define the initial disease presentation well before the onset of the classical Alpers' triad of psychomotor retardation, intractable seizures, and liver failure. Correlating with the genotype, POLG pathogenicity is a result of increased mitochondrial DNA mutability, and mitochondrial DNA depletion resulting in energy deficient states. Hypoglycemia therefore could be secondary to any metabolic pathway affected by ATP deficiency. Although it has been speculated that hypoglycemia is due to secondary fatty acid oxidation defects or abnormal gluconeogenesis, the exact underlying etiology is still unclear. Here we present detailed studies on carbohydrate metabolism in an Alpers' patient who presented initially exclusively with intermittent episodes of hypoglycemia and ketosis. Our results do not support a defect in gluconeogenesis or fatty acid oxidation as the cause of hypoglycemia. In contrast, studies performed on liver biopsy suggested abnormal glycogenolysis. This is shown via decreased activities of glycogen brancher and debrancher enzymes with normal glycogen structure and increased glycogen on histology of the liver specimen. To our knowledge, this is the first report documenting abnormalities in glycogen metabolism in a patient with Alpers' syndrome.

Severity of cardiomyopathy associated with adenine nucleotide translocator-1 deficiency correlates with mtDNA haplogroup.

Mutations of both nuclear and mitochondrial DNA (mtDNA)-encoded mitochondrial proteins can cause cardiomyopathy associated with mitochondrial dysfunction. Hence, the cardiac phenotype of nuclear DNA mitochondrial mutations might be modulated by mtDNA variation. We studied a 13-generation Mennonite pedigree with autosomal recessive myopathy and cardiomyopathy due to an SLC25A4 frameshift null mutation (c.523delC, p.Q175RfsX38), which codes for the heart-muscle isoform of the adenine nucleotide translocator-1. Ten homozygous null (adenine nucleotide translocator-1(-/-)) patients monitored over a median of 6 years had a phenotype of progressive myocardial thickening, hyperalaninemia, lactic acidosis, exercise intolerance, and persistent adrenergic activation. Electrocardiography and echocardiography with velocity vector imaging revealed abnormal contractile mechanics, myocardial repolarization abnormalities, and impaired left ventricular relaxation. End-stage heart disease was characterized by massive, symmetric, concentric cardiac hypertrophy; widespread cardiomyocyte degeneration; overabundant and structurally abnormal mitochondria; extensive subendocardial interstitial fibrosis; and marked hypertrophy of arteriolar smooth muscle. Substantial variability in the progression and severity of heart disease segregated with maternal lineage, and sequencing of mtDNA from five maternal lineages revealed two major European haplogroups, U and H. Patients with the haplogroup U mtDNAs had more rapid and severe cardiomyopathy than those with haplogroup H.

Heteroplasmic mutations of the mitochondrial genome cause paradoxical effects on mitochondrial functions.

Mitochondrial genome (mtDNA) mutation causes highly variable clinical features, and its pathogenesis is not fully understood. In this study, we analyzed the heteroplasmic mtDNA mutation C4936T (p.T156I) in ND2 of complex I and the homoplasmic mtDNA mutation A9181G (p.S219G) in ATPase 6 of complex V. Using cybrid technology, we found that in a high-glucose medium in which cultured cells mainly depend on anaerobic glycolysis for energy, the C4936T mutation inhibited cell growth by 50%. Oxygen consumption and reactive oxygen species production were also reduced by 60 and 75%, respectively. Because the subject also had conjunctiva carcinoma, we further tested whether the C4936T mutation was associated with tumor formation. In an anchorage-dependant growth test, we found that only cells with a high level of C4936T mutation formed colonies. In contrast, when the cells grew in a galactose medium in which cells were forced to generate ATP through oxidative phosphorylation, the C4936T mutation protected cells from apoptosis probably caused by the A9181G mutation. Our results suggest that the phenotype caused by mtDNA mutations may depend on the availability of the nutrients. This gene-environment interaction may contribute to the complexity of pathogenesis and clinical phenotypes caused by mtDNA mutation.

Mitochondrial and ion channel gene alterations in autism.

To evaluate the potential importance in autistic subjects of copy number variants (CNVs) that alter genes of relevance to bioenergetics, ionic metabolism, and synaptic function, we conducted a detailed microarray analysis of 69 autism probands and 35 parents, compared to 89 CEU HapMap controls. This revealed that the frequency CNVs of≥100kb and CNVs of≥10 Kb were markedly increased in probands over parents and in probands and parents over controls. Evaluation of CNVs≥1Mb by chromosomal FISH confirmed the molecular identity of a subset of the CNVs, some of which were associated with chromosomal rearrangements. In a number of the cases, CNVs were found to alter the copy number of genes that are important in mitochondrial oxidative phosphorylation (OXPHOS), ion and especially calcium transport, and synaptic structure. Hence, autism might result from alterations in multiple bioenergetic and metabolic genes required for mental function. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).

Mitochondrial DNA variant associated with Leber hereditary optic neuropathy and high-altitude Tibetans.

The distinction between mild pathogenic mtDNA mutations and population polymorphisms can be ambiguous because both are homoplasmic, alter conserved functions, and correlate with disease. One possible explanation for this ambiguity is that the same variant may have different consequences in different contexts. The NADH dehydrogenase subunit 1 (ND1) nucleotide 3394 T > C (Y30H) variant is such a case. This variant has been associated with Leber hereditary optic neuropathy and it reduces complex I activity and cellular respiration between 7% and 28% on the Asian B4c and F1 haplogroup backgrounds. However, complex I activity between B4c and F1 mtDNAs, which harbor the common 3394T allele, can also differ by 30%. In Asia, the 3394C variant is most commonly associated with the M9 haplogroup, which is rare at low elevations but increases in frequency with elevation to an average of 25% of the Tibetan mtDNAs (odds ratio = 23.7). In high-altitude Tibetan and Indian populations, the 3394C variant occurs on five different macrohaplogroup M haplogroup backgrounds and is enriched on the M9 background in Tibet and the C4a4 background on the Indian Deccan Plateau (odds ratio = 21.9). When present on the M9 background, the 3394C variant is associated with a complex I activity that is equal to or higher than that of the 3394T variant on the B4c and F1 backgrounds. Hence, the 3394C variant can either be deleterious or beneficial depending on its haplogroup and environmental context. Thus, this mtDNA variant fulfills the criteria for a common variant that predisposes to a "complex" disease.

Mutations in DNMT1 cause hereditary sensory neuropathy with dementia and hearing loss.

DNA methyltransferase 1 (DNMT1) is crucial for maintenance of methylation, gene regulation and chromatin stability. DNA mismatch repair, cell cycle regulation in post-mitotic neurons and neurogenesis are influenced by DNA methylation. Here we show that mutations in DNMT1 cause both central and peripheral neurodegeneration in one form of hereditary sensory and autonomic neuropathy with dementia and hearing loss. Exome sequencing led to the identification of DNMT1 mutation c.1484A>G (p.Tyr495Cys) in two American kindreds and one Japanese kindred and a triple nucleotide change, c.1470-1472TCC>ATA (p.Asp490Glu-Pro491Tyr), in one European kindred. All mutations are within the targeting-sequence domain of DNMT1. These mutations cause premature degradation of mutant proteins, reduced methyltransferase activity and impaired heterochromatin binding during the G2 cell cycle phase leading to global hypomethylation and site-specific hypermethylation. Our study shows that DNMT1 mutations cause the aberrant methylation implicated in complex pathogenesis. The discovered DNMT1 mutations provide a new framework for the study of neurodegenerative diseases.

An Indian boy with nephropathic cystinosis: a case report and molecular analysis of CTNS mutation.

Cystinosis is a rare autosomal recessive lysosomal storage disorder characterized by excessive accumulation of cystine within the lysosome. Cystinosis is caused by mutations in the lysosomal cystine transporter, cystinosin (CTNS). The CTNS gene consists of 12 exons and encodes for an integral lysosomal membrane protein with seven transmembrane domains. A majority of cystinotic patients are of European descents, and only a few cases have been reported from other ethnic groups. Here we report a case of nephropathic cystinosis in an Indian boy born to consanguineous parents. Major symptoms of the patient include weight loss, vomiting, dehydration, and cystine crystals in the cornea. Ichthyosis on the arms and legs is also observed. Sequencing analysis of all the CTNS exons revealed that the proband is homozygous for a 3-bp in-frame deletion in exon 10 (c.809_811delCCT), resulting in the loss of a conserved p.Ser270del within the fifth transmembrane domain of CTNS. His parents are both heterozygous for the same mutation. This work represents the first molecular characterization of cystinotic patients from India. Interestingly, a p.Ser270del resulting from c.809_811delCCT in CTNS had been identified in a European patient. Therefore, it appears that this mutation arose independently in the two different continents.

A novel NDUFA1 mutation leads to a progressive mitochondrial complex I-specific neurodegenerative disease.

Mitochondrial diseases have been shown to result from mutations in mitochondrial genes located in either the nuclear DNA (nDNA) or mitochondrial DNA (mtDNA). Mitochondrial OXPHOS complex I has 45 subunits encoded by 38 nuclear and 7 mitochondrial genes. Two male patients in a putative X-linked pedigree exhibiting a progressive neurodegenerative disorder and a severe muscle complex I enzyme defect were analyzed for mutations in the 38 nDNA and seven mtDNA encoded complex I subunits. The nDNA X-linked NDUFA1 gene (MWFE polypeptide) was discovered to harbor a novel missense mutation which changed a highly conserved glycine at position 32 to an arginine, shown to segregate with the disease. When this mutation was introduced into a NDUFA1 null hamster cell line, a substantial decrease in the complex I assembly and activity was observed. When the mtDNA of the patient was analyzed, potentially relevant missense mutations were observed in the complex I genes. Transmitochondrial cybrids containing the patient's mtDNA resulted in a mild complex I deficiency. Interestingly enough, the nDNA encoded MWFE polypeptide has been shown to interact with various mtDNA encoded complex I subunits. Therefore, we hypothesize that the novel G32R mutation in NDUFA1 is causing complex I deficiency either by itself or in synergy with additional mtDNA variants.

Molecular analysis of ANT1, TWINKLE and POLG in patients with multiple deletions or depletion of mitochondrial DNA by a dHPLC-based assay.

ANT1, TWINKLE and POLG genes affect mtDNA stability and are involved in autosomal dominant PEO, while mutations in POLG are responsible for numerous clinical presentations, including autosomal recessive PEO, sensory ataxic neuropathy, dysarthria and ophthalmoparesis (SANDO), spino-cerebellar ataxia and epilepsy (SCAE) or Alpers syndrome. In this study, we report on the mutational analysis of ANT1, TWINKLE and POLG genes in 15 unrelated patients, using a dHPLC-based protocol. This series of patients illustrates the large array of clinical presentations associated with mtDNA stability defects, ranging from isolated benign PEO to fatal Alpers syndrome. A total of seven different mutations were identified in six of 15 patients (40%). Six different recessive mutations were found in POLG, one in TWINKLE while no mutation was identified in ANT1. Among the POLG mutations, three are novel and include two missense and one frameshift changes. Seventeen neutral changes and polymorphisms were also identified, including four novel neutral polymorphisms. Overall, this study illustrates the variability of phenotypes associated with mtDNA stability defects, increases the mutational spectrum of POLG variants and provides an efficient and reliable detection protocol for ANT1, TWINKLE and POLG mutational screening.

Facioscapulohumeral muscular dystrophy (FSHD) myoblasts demonstrate increased susceptibility to oxidative stress.

Facioscapulohumeral muscular dystrophy is an autosomal dominant disorder resulting from an unusual genetic mechanism. The mutation, a deletion of 3.3 kb subtelomeric repeats, appears to disrupt the regional regulation of 4q35 g ene expression. The specific gene(s)responsible for facioscapulohumeral muscular dystrophy have not been identified. However, the 'vacuolar/necrotic' phenotype exhibited by facioscapulohumeral muscular dystrophy myoblasts suggests that aberrant gene expression occurs early in facioscapulohumeral muscular dystrophy muscle development. In order to test this hypothesis, global gene expression profiling and in vitro characterization of facioscapulohumeral muscular dystrophy and control myoblasts were carried out. Genes involved in several cellular processes such as oxidative stress were found to be dysregulated. In vitro studies confirmed this susceptibility to oxidative stress, as proliferative stage facioscapulohumeral muscular dystrophy myoblasts exhibit greatly reduced viability when exposed to the oxidative stressor paraquat. This effect was not seen in either normal or disease control myoblasts, or in any of the cell lines upon differentiation to multinucleated myotubes. Immunocytochemical studies of the cyclin dependent kinase inhibitor p21 demonstrated increased expression in facioscapulohumeral muscular dystrophy myoblasts, suggesting an early cell cycle arrest. Another process distinguishing facioscapulohumeral muscular dystrophy from controls involves the transcription of extracellular matrix components. Expression of elastin, decorin, lumican and the extracellular matrix remodeling factor TIMP3 were reduced in facioscapulohumeral muscular dystrophy myoblasts. These studies suggest that facioscapulohumeral muscular dystrophy muscular dystrophy results from a defect in early myogenesis, manifested as increased susceptibility to oxidative stress, morphological aberrations and early cell cycle arrest.