Search

The aetiology of dystonia disorders is complex, and next-generation sequencing has become a useful tool in elucidating the variable genetic background of these diseases. Here we report a deleterious heterozygous truncating variant in the inosine monophosphate dehydrogenase gene (IMPDH2) by whole-exome sequencing, co-segregating with a dominantly inherited dystonia-tremor disease in a large Finnish family. We show that the defect results in degradation of the gene product, causing IMPDH2 deficiency in patient cells. IMPDH2 is the first and rate-limiting enzyme in the de novo biosynthesis of guanine nucleotides, a dopamine synthetic pathway previously linked to childhood or adolescence-onset dystonia disorders. We report IMPDH2 as a new gene to the dystonia disease entity. The evidence underlines the important link between guanine metabolism, dopamine biosynthesis and dystonia.

Mutations in mitochondrial DNA encoded subunit of ATP synthase, MT-ATP6, are frequent causes of neurological mitochondrial diseases with a range of phenotypes from Leigh syndrome and NARP to ataxias and neuropathies. Here we investigated the functional consequences of an unusual heteroplasmic truncating mutation m.9154C>T in MT-ATP6, which caused peripheral neuropathy, ataxia and IgA nephropathy. ATP synthase not only generates cellular ATP, but its dimerization is required for mitochondrial cristae formation. Accordingly, the MT-ATP6 truncating mutation impaired the assembly of ATP synthase and disrupted cristae morphology, supporting our molecular dynamics simulations that predicted destabilized a/c subunit subcomplex. Next, we modeled the effects of the truncating mutation using patient-specific induced pluripotent stem cells. Unexpectedly, depending on mutation heteroplasmy level, the truncation showed multiple threshold effects in cellular reprogramming, neurogenesis and in metabolism of mature motor neurons (MN). Interestingly, MN differentiation beyond progenitor stage was impaired by Notch hyperactivation in the MT-ATP6 mutant, but not by rotenone-induced inhibition of mitochondrial respiration, suggesting that altered mitochondrial morphology contributed to Notch hyperactivation. Finally, we also identified a lower mutation threshold for a metabolic shift in mature MN, affecting lactate utilization, which may be relevant for understanding the mechanisms of mitochondrial involvement in peripheral motor neuropathies. These results establish a critical and disease-relevant role for ATP synthase in human cell fate decisions and neuronal metabolism.

OBJECTIVE: ITPR3, encoding inositol 1,4,5-trisphosphate receptor type 3, was previously reported as a potential candidate disease gene for Charcot-Marie-Tooth neuropathy. Here, we present genetic and functional evidence that ITPR3 is a Charcot-Marie-Tooth disease gene. METHODS: Whole-exome sequencing of four affected individuals in an autosomal dominant family and one individual who was the only affected individual in his family was used to identify disease-causing variants. Skin fibroblasts from two individuals of the autosomal dominant family were analyzed functionally by western blotting, quantitative reverse transcription PCR, and Ca2+ imaging. RESULTS: Affected individuals in the autosomal dominant family had onset of symmetrical neuropathy with demyelinating and secondary axonal features at around age 30, showing signs of gradual progression with severe distal leg weakness and hand involvement in the proband at age 64. Exome sequencing identified a heterozygous ITPR3 p.Val615Met variant segregating with the disease. The individual who was the only affected in his family had disease onset at age 4 with demyelinating neuropathy. His condition was progressive, leading to severe muscle atrophy below knees and atrophy of proximal leg and hand muscles by age 16. Trio exome sequencing identified a de novo ITPR3 variant p.Arg2524Cys. Altered Ca2+ -transients in p.Val615Met patient fibroblasts suggested that the variant has a dominant-negative effect on inositol 1,4,5-trisphosphate receptor type 3 function. INTERPRETATION: Together with two previously identified variants, our report adds further evidence that ITPR3 is a disease-causing gene for CMT and indicates altered Ca2+ homeostasis in disease pathogenesis. ispartof: ANNALS OF CLINICAL AND TRANSLATIONAL NEUROLOGY vol:7 issue:10 pages:1962-1972 ispartof: location:United States status: published

Background and ObjectivesTo determine the genetic cause of the disease in the previously reported family with adult-onset autosomal dominant distal myopathy (myopathy, distal, 3; MPD3).MethodsContinued clinical evaluation including muscle MRI and muscle pathology. A linkage analysis with single nucleotide polymorphism arrays and genome sequencing were used to identify the genetic defect, which was verified by Sanger sequencing. RNA sequencing was used to investigate the transcriptional effects of the identified genetic defect.ResultsSmall hand muscles (intrinsic, thenar, and hypothenar) were first involved with spread to the lower legs and later proximal muscles. Dystrophic changes with rimmed vacuoles and cytoplasmic inclusions were observed in muscle biopsies at advanced stage. A single nucleotide polymorphism array confirmed the previous microsatellite-based linkage to 8p22-q11 and 12q13-q22. Genome sequencing of three affected family members combined with structural variant calling revealed a small heterozygous deletion of 160 base pairs spanning the second last exon 10 of the heterogeneous nuclear ribonucleoprotein A1 (HNRNPA1) gene, which is in the linked region on chromosome 12. Segregation of the mutation with the disease was confirmed by Sanger sequencing. RNA sequencing showed that the mutant allele produces a shorter mutant mRNA transcript compared with the wild-type allele. Immunofluorescence studies on muscle biopsies revealed small p62 and larger TDP-43 inclusions.DiscussionA small exon 10 deletion in the gene HNRNPA1 was identified as the cause of MPD3 in this family. The new HNRNPA1-related phenotype, upper limb presenting distal myopathy, was thus confirmed, and the family displays the complexities of gene identification.

ObjectiveTo characterize serum biomarkers in mitochondrial CHCHD10-linked spinal muscular atrophy Jokela type (SMAJ) for disease monitoring and for understanding of pathogenic mechanisms.MethodsWe collected serum samples from a cohort of 49 SMAJ patients, all carriers of the heterozygous c.197G>T p.G66V variant inCHCHD10. As controls, we used age- and sex-matched serum samples obtained from Helsinki Biobank. Neurofilament light (NfL) and glial fibrillary acidic protein (GFAP) were measured with Single molecule array (Simoa), and fibroblast growth factor 21 (FGF-21) and growth differentiation factor 15 (GDF-15) with enzyme-linked immunosorbent assay. For nontargeted serum metabolite profiling, samples were analyzed with liquid chromatography–high resolution mass spectrometry. Disease severity was evaluated retrospectively by calculating a symptom-based score.ResultsAxon degeneration marker NfL was unexpectedly not altered in the serum of SMAJ patients, whereas astrocytic activation marker GFAP was slightly decreased. Creatine kinase was elevated in most patients, in particular males. We identified six metabolites that were significantly altered in SMAJ patients’ serum compared to controls: increased creatine and pyruvate, and decreased creatinine, taurine, N-acetyl-carnosine and succinate. Creatine correlated with disease severity. Altered pyruvate and succinate indicated a metabolic response to mitochondrial dysfunction, however, lactate or mitochondrial myopathy markers FGF-21 or GDF-15 were not changed.ConclusionsBiomarkers of muscle mass and damage are altered in SMAJ serum, indicating a role for skeletal muscle in disease pathogenesis in addition to neurogenic damage. Despite the minimal mitochondrial pathology in skeletal muscle, signs of a metabolic shift can be detected in the serum of SMAJ patients.

The Polyneuropathy And Treatment with Hizentra (PATH) study required subjects with chronic inflammatory demyelinating polyneuropathy (CIDP) to show dependency on immunoglobulin G (IgG) and then be restabilized on IgG before being randomized to placebo or one of two doses of subcutaneous immunoglobulin (SCIG). Nineteen of the 51 subjects (37%) randomized to placebo did not relapse over the next 24 weeks. This article explores the reasons for this effect. A post‐hoc analysis of the PATH placebo group was undertaken. A literature search identified other placebo‐controlled CIDP trials for review and comparison. In PATH, subjects randomized to placebo who did not relapse were significantly older, had more severe disease, and took longer to deteriorate in the IgG dependency period compared with those who relapsed. Published trials in CIDP, whose primary endpoint was stability or deterioration, had a mean non‐deterioration (placebo effect) of 43%, while trials with a primary endpoint of improvement had a placebo response of only 11%. Placebo is an important variable in the design of CIDP trials. Trials designed to show clinical improvement will have a significantly lower effect of this phenomenon than those designed to show stability or deterioration.

Mitochondrial dysfunction elicits stress responses that safeguard cellular homeostasis against metabolic insults. Mitochondrial integrated stress response (ISRmt) is a major response to mitochondrial (mt)DNA expression stress (mtDNA maintenance, translation defects), but the knowledge of dynamics or interdependence of components is lacking. We report that in mitochondrial myopathy, ISRmt progresses in temporal stages and development from early to chronic and is regulated by autocrine and endocrine effects of FGF21, a metabolic hormone with pleiotropic effects. Initial disease signs induce transcriptional ISRmt (ATF5, mitochondrial one-carbon cycle, FGF21, and GDF15). The local progression to 2nd metabolic ISRmt stage (ATF3, ATF4, glucose uptake, serine biosynthesis, and transsulfuration) is FGF21 dependent. Mitochondrial unfolded protein response marks the 3rd ISRmt stage of failing tissue. Systemically, FGF21 drives weight loss and glucose preference, and modifies metabolism and respiratory chain deficiency in a specific hippocampal brain region. Our evidence indicates that FGF21 is a local and systemic messenger of mtDNA stress in mice and humans with mitochondrial disease.

INTRODUCTION: We aimed to explore the value of genetic screening for Fabry disease (FD) and hereditary ATTR amyloidosis in patients with idiopathic small fiber neuropathy (SFN) or mixed neuropathy in a clinical setting.METHODS: This was a Nordic multicenter study with 9 participating centers. Patients with idiopathic SFN or mixed neuropathy were included. Genetic sequencing of the TTR and GLA genes was performed.RESULTS: 172 patients were enrolled. Genetic screening was performed in 155 patients. No pathogenic mutations in the TTR gene were found. A single patient had a possible pathogenic variant, R118C, in the GLA gene, but the clinical investigation showed no firm signs of FD.DISCUSSION: Screening for hereditary ATTR amyloidosis and FD in patients with idiopathic SFN or mixed neuropathy without any additional disease-specific symptoms or clinical characteristics in a Nordic population appears to be of little value in a clinical setting. This article is protected by copyright. All rights reserved.

PATH study group., In patients with chronic inflammatory demyelinating polyneuropathy (CIDP), intravenous immunoglobulin (IVIG) is recommended to be periodically reduced to assess the need for ongoing therapy. However, little is known about the effectiveness of restabilization with IVIG in patients who worsen after IVIG withdrawal. In the Polyneuropathy And Treatment with Hizentra (PATH) study, the pre‐randomization period included sudden stopping of IVIG followed by 12 weeks of observation. Those deteriorating were then restabilized with IVIG. Of 245 subjects who stopped IVIG, 28 did not show signs of clinical deterioration within 12 weeks. Two hundred and seven received IVIG restabilization with an induction dose of 2 g/kg bodyweight (bw) IgPro10 (Privigen, CSL Behring, King of Prussia, Pennsylvania) and maintenance doses of 1 g/kg bw every 3 weeks for up to 13 weeks. Signs of clinical improvement were seen in almost all (n = 188; 91%) subjects. During IVIG restabilization, 35 subjects either did not show CIDP stability (n = 21, analyzed as n = 22 as an additional subject was randomized in error) or withdrew for other reasons (n = 14). Of the 22 subjects who did not achieve clinical stability, follow‐up information in 16 subjects after an additional 4 weeks was obtained. Nine subjects were reported to have improved, leaving a maximum of 27 subjects (13%) who either showed no signs of clinical improvement during the restabilization phase and 4 weeks post‐study or withdrew for other reasons. In conclusion, sudden IVIG withdrawal was effective in detecting ongoing immunoglobulin G dependency with a small risk for subjects not returning to their baseline 17 weeks after withdrawal., Editorial support was provided by Meridian HealthComms Ltd, funded by CSL Behring.

Dermal fibroblasts from patients harboring mutations in the gap junction connexin43 gene have distinctly different mechanistic causes for the clinical presentations of oculodentodigital dysplasia., Oculodentodigital dysplasia (ODDD) is a rare genetic disease that affects the development of multiple organs in the human body. More than 70 mutations in the gap junction connexin43 (Cx43) gene, GJA1, are associated with ODDD, most of which are inherited in an autosomal dominant manner. Many patients exhibit similar clinical presentations. However, there is high intrafamilial and interfamilial phenotypic variability. To better understand this variability, we established primary human dermal fibroblast cultures from several ODDD patients and unaffected controls. In the present study, we characterized three fibroblast lines expressing heterozygous p.L7V, p.G138R, and p.G143S Cx43 variants. All ODDD fibroblasts exhibited slower growth, reduced migration, and defective cell polarization, traits common to all ODDD fibroblasts studied so far. However, we found striking differences in overall expression levels, with p.L7V down-regulated at the mRNA and protein level. Although all of the Cx43 variants could traffic to the cell surface, there were stark differences in gap junction plaque formation, gap junctional intercellular communication, Cx43 phosphorylation, and hemichannel activity among Cx43 variants, as well as subtle differences in myofibroblast differentiation. Together these findings enabled us to discover mutation-specific pathologies that may help to predict future clinical outcomes.

Following the involvement of CHCHD10 in FrontoTemporal-Dementia-Amyotrophic Lateral Sclerosis (FTD-ALS) clinical spectrum, a founder mutation (p.Gly66Val) in the same gene was identified in Finnish families with late onset spinal motor neuronopathy (SMAJ). SMAJ is a slowly progressive form of spinal muscular atrophy with a life expectancy within normal range. In order to understand why the p.Ser59Leu mutation, responsible for severe FTD-ALS, and the p.Gly66Val mutation could lead to different levels of severity, we compared their effects in patient cells. Unlike affected individuals bearing the p.Ser59Leu mutation, patients presenting with SMAJ phenotype have neither mitochondrial myopathy nor mtDNA instability. The expression of CHCHD10(S59L) mutant allele leads to disassembly of mitochondrial contact site and cristae organizing system (MICOS) with mitochondria] dysfunction and loss of cristae in patient fibroblasts. We also show that G66V fibroblasts do not display the loss of MICOS complex integrity and mitochondrial damage found in S59L cells. However, S59L and G66V fibroblasts show comparable accumulation of phosphorylated mitochondrial TDP-43 suggesting that the severity of phenotype and mitochondrial damage do not depend on mitochondrial TDP-43 localization. The expression of the CHCHD10(G66V) allele is responsible for mitochondrial network fragmentation and decreased sensitivity towards apoptotic stimuli, but with a less severe effect than that found in cells expressing the CHCHD10(S59L) allele. Taken together, our data show that cellular phenotypes associated with p.Ser59Leu and p.Gly66Val mutations in CHCHD10 are different; loss of MICOS complex integrity and mitochondrial dysfunction, but not TDP-43 mitochondrial localization, being likely essential to develop a severe motor neuron disease.

We thank Ravi Uniyal and colleagues for their comments on our results from the PATH trial1 on subcutaneous immunoglobulin (SCIg) for maintenance treatment in chronic inflammatory demyelinating polyneuropathy (CIDP). They express concern about the relapse rates in the treatment groups and hypothesise the cause being the pharmacokinetics of SCIg.

Variants in family 1 kinesin (KIF1A), which encodes a kinesin axonal motor protein, have been described to cause variable neurological manifestations. Recessive missense variants have led to spastic paraplegia, and recessive truncations to sensory and autonomic neuropathy. De novo missense variants cause developmental delay or intellectual disability, cerebellar atrophy and variable spasticity. We describe a family with father-to-son transmission of de novo variant in the KIF1A motor domain, in a phenotype of pure spastic paraplegia. Structural modeling of the predicted p.(Ser69Leu) amino acid change suggested that it impairs the stable binding of ATP to the KIF1A protein. Our study reports the first dominantly inherited KIF1A variant and expands the spectrum of phenotypes caused by heterozygous KIF1A motor domain variants to include pure spastic paraplegia. We conclude that KIF1A should be considered a candidate gene for hereditary paraplegias regardless of inheritance pattern.

Mutations in the mitochondrially located protein CHCHD10 cause motoneuron disease by an unknown mechanism. In this study, we investigate the mutations p. R15L and p. G66V in comparison to wild-type CHCHD10 and the non-pathogenic variant p. P34S in vitro, in patient cells as well as in the vertebrate in vivo model zebrafish. We demonstrate a reduction of CHCHD10 protein levels in p. R15L and p. G66V mutant patient cells to approximately 50%. Quantitative real-time PCR revealed that expression of CHCHD10 p. R15L, but not of CHCHD10 p. G66V, is already abrogated at the mRNA level. Altered secondary structure and rapid protein degradation are observed with regard to the CHCHD10 p. G66V mutant. In contrast, no significant differences in expression, degradation rate or secondary structure of non-pathogenic CHCHD10 p. P34S are detected when compared with wild-type protein. Knockdown of CHCHD10 expression in zebrafish to about 50% causes motoneuron pathology, abnormal myofibrillar structure and motility deficits in vivo. Thus, our data show that the CHCHD10 mutations p. R15L and p. G66V cause motoneuron disease primarily based on haploinsufficiency of CHCHD10.

Multiple acyl-CoA dehydrogenation deficiency is genetically heterogenous metabolic disease with mutations in genes involved in electron transfer to the mitochondrial respiratory chain. Disease symptoms vary from severe neonatal form to late-onset presentation with metabolic acidosis, lethargy, vomiting, muscle pain and weakness. Riboflavin therapy has been shown to ameliorate diseases symptoms in some of these patients. Recently, mutations in FAD synthase have been described to cause multiple acyl-CoA dehydrogenation deficiency. We describe here the effect of riboflavin supplementation therapy in a previously reported adult patient with multiple acyl-CoA dehydrogenation deficiency having compound heterozygous gene variations in FLAD1 (MIM: 610595) encoding FAD synthase. We present thorough clinical history including laboratory investigations, muscle MRI, muscle biopsy and spiroergometric analyses comprising of a follow-up of 20 years. Our data suggest that patients with adult-onset multiple acyl-CoA dehydrogenation deficiency with FLAD1 gene mutations also benefit from long-term riboflavin therapy. (C) 2017 Elsevier B.V. All rights reserved.

© The Author 2017. Published by Oxford University Press. All rights reserved.De novo mutations in ATAD3A (ATPase family AAA-domain containing protein 3A) were recently found to cause a neurological syndrome with developmental delay, hypotonia, spasticity, optic atrophy, axonal neuropathy, and hypertrophic cardiomyopathy. Using whole-exome sequencing, we identified a dominantly inherited heterozygous variant c.1064G>A (p. G355D) in ATAD3A in a mother presenting with hereditary spastic paraplegia (HSP) and axonal neuropathy and her son with dyskinetic cerebral palsy, both with disease onset in childhood. HSP is a clinically and genetically heterogeneous disorder of the upper motor neurons. Symptoms beginning in early childhood may resemble spastic cerebral palsy. The function of ATAD3A, a mitochondrial inner membrane AAA ATPase, is yet undefined. AAA ATPases form hexameric rings, which are catalytically dependent on the co-operation of the subunits. The dominant-negative patient mutation affects the Walker A motif, which is responsible for ATP binding in the AAA module of ATAD3A, and we show that the recombinant mutant ATAD3A protein has a markedly reduced ATPase activity. We further show that overexpression of the mutant ATAD3A fragments the mitochondrial network and induces lysosome mass. Similarly, we observed altered dynamics of the mitochondrial network and increased lysosomes in patient fibroblasts and neurons derived through differentiation of patient-specific induced pluripotent stem cells. These alterations were verified in patient fibroblasts to associate with upregulated basal autophagy through mTOR inactivation, resembling starvation. Mutations in ATAD3A can thus be dominantly inherited and underlie variable neurological phenotypes, including HSP, with intrafamiliar variability. This finding extends the group of mitochondrial inner membrane AAA proteins associated with spasticity.

Charcot–Marie–Tooth disease (CMT) is a group of hereditary peripheral neuropathies. The dominantly inherited axonal CMT2 displays striking genetic heterogeneity, with 17 presently known disease genes. The large number of candidate genes, combined with lack of genotype–phenotype correlations, has made genetic diagnosis in CMT2 time-consuming and costly. In Finland, 25% of dominant CMT2 is explained by either a GDAP1 founder mutation or private MFN2 mutations but the rest of the families have remained without molecular diagnosis. Whole-exome and genome sequencing are powerful techniques to find disease mutations for CMT patients but they require large amounts of sequencing to confidently exclude heterozygous variants in all candidate genes, and they generate a vast amount of irrelevant data for diagnostic needs. Here we tested a targeted next-generation sequencing approach to screen the CMT2 genes. In total, 15 unrelated patients from dominant CMT2 families from Finland, in whom MFN2 and GDAP1 mutations had been excluded, participated in the study. The targeted approach produced sufficient sequence coverage for 95% of the 309 targeted exons, the rest we excluded by Sanger sequencing. Unexpectedly, the screen revealed a disease mutation only in one family, in the HSPB1 gene. Thus, new disease genes underlie CMT2 in the remaining families, indicating further genetic heterogeneity. We conclude that targeted next-generation sequencing is an efficient tool for genetic screening in CMT2 that also aids in the selection of patients for genome-wide approaches.

We describe a founder mutation in the gene encoding ganglioside-induced differentiation associated-protein 1 (GDAP1), leading to amino acid change p.H123R, as a common cause of autosomal dominant axonal Charcot-Marie-Tooth (CMT2) neuropathy in Finland. The mutation explains up to 14 % of CMT2 in Finland, where most patients with axonal neuropathy have remained without molecular diagnosis. Only three families out of 28 were found to carry putative disease mutations in the MFN2 gene encoding mitofusin 2. In addition, the MFN2 variant p.V705I was commonly found in our patients, but we provide evidence that this previously described mutation is a common polymorphism and not pathogenic. GDAP1-associated polyneuropathy caused predominantly a mild and slowly progressive phenotype. Besides distal leg muscle weakness, most patients showed mild proximal weakness, often with asymmetry and pes cavus. Our findings broaden the understanding of GDAP1 mutations in CMT2 phenotypes and provide support for the use of whole-exome sequencing in CMT gene diagnostics.

Background Anoctaminopathies are muscle diseases caused by recessive mutations in the ANO5 gene. The effects of anoctaminopathy on oxidative capacity have not previously been studied in a controlled setting. Objective To characterize oxidative capacity in a clinically and genetically well-defined series of patients with anoctaminopathy. Methods We sequenced the ANO5 gene in 111 Finnish patients with suspected LGMD2. Patients with positive findings underwent close clinical examination, including electromyography, muscle MRI, and, in selected cases, muscle biopsy. Oxidative capacity was analyzed using spiroergometry and compared to age-matched healthy controls. Results We characterized 12 newly identified and 2 previously identified patients with ANO5 mutations from 11 families. Our material was genetically homogeneous with most patients homozygous for the Finnish founder variant c.2272C>T (p.Arg758Cys). In one family, we found a novel p.Met470Arg variant compound heterozygous with p.Arg758Cys. Lower limb muscle MRI revealed progressive fatty degeneration of specific posterior compartment muscles. Patients' spiroergometric profiles showed that anoctaminopathy significantly impaired oxidative capacity with increasing ventilation. Conclusions Our findings support earlier reports that anoctaminopathy progresses slowly and demonstrate that the disease impairs the capacity for aerobic exercise.

Mitochondrial myopathy (MM) with progressive external ophthalmoplegia (PEO) is a common manifestation of mitochondrial disease in adulthood, for which there is no curative therapy. In mice with MM, ketogenic diet significantly delayed progression of the disease. We asked in this pilot study what effects high‐fat, low‐carbohydrate “modified Atkins” diet (mAD) had for PEO/MM patients and control subjects and followed up the effects by clinical, morphological, transcriptomic, and metabolomic analyses. All of our five patients, irrespective of genotype, showed a subacute response after 1.5–2 weeks of diet, with progressive muscle pain and leakage of muscle enzymes, leading to premature discontinuation of the diet. Analysis of muscle ultrastructure revealed selective fiber damage, especially in the ragged‐red‐fibers (RRFs), a MM hallmark. Two years of follow‐up showed improvement of muscle strength, suggesting activation of muscle regeneration. Our results indicate that (i) nutrition can modify mitochondrial disease progression, (ii) dietary counseling should be part of MM care, (iii) short mAD is a tool to induce targeted RRF lysis, and (iv) mAD, a common weight‐loss method, may induce muscle damage in a population subgroup. ![][1] High‐fat, low‐carbohydrate modified Atkins diet (mAD) is a common weight‐loss method, found to ameliorate mitochondrial myopathy in mice. In human patients, mAD induces muscle damage, especially of ragged‐red fibers, the most affected by the disease. [1]: /embed/graphic-1.gif

Item does not contain fulltext Facioscapulohumeral dystrophy (FSHD) is associated with somatic chromatin relaxation of the D4Z4 repeat array and derepression of the D4Z4-encoded DUX4 retrogene coding for a germline transcription factor. Somatic DUX4 derepression is caused either by a 1-10 unit repeat-array contraction (FSHD1) or by mutations in SMCHD1, which encodes a chromatin repressor that binds to D4Z4 (FSHD2). Here, we show that heterozygous mutations in DNA methyltransferase 3B (DNMT3B) are a likely cause of D4Z4 derepression associated with low levels of DUX4 expression from the D4Z4 repeat and increased penetrance of FSHD. Recessive mutations in DNMT3B were previously shown to cause immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome. This study suggests that transcription of DUX4 in somatic cells is modified by variations in its epigenetic state and provides a basis for understanding the reduced penetrance of FSHD within families.

ObjectiveWe used patient-specific neuronal cultures to characterize the molecular genetic mechanism of recessive nonsense mutations in neurofilament light (NEFL) underlying early-onset Charcot-Marie-Tooth (CMT) disease.MethodsMotor neurons were differentiated from induced pluripotent stem cells of a patient with early-onset CMT carrying a novel homozygous nonsense mutation in NEFL. Quantitative PCR, protein analytics, immunocytochemistry, electron microscopy, and single-cell transcriptomics were used to investigate patient and control neurons.ResultsWe show that the recessive nonsense mutation causes a nearly total loss of NEFL messenger RNA (mRNA), leading to the complete absence of NEFL protein in patient's cultured neurons. Yet the cultured neurons were able to differentiate and form neuronal networks and neurofilaments. Single-neuron gene expression fingerprinting pinpointed NEFL as the most downregulated gene in the patient neurons and provided data of intermediate filament transcript abundancy and dynamics in cultured neurons. Blocking of nonsense-mediated decay partially rescued the loss of NEFL mRNA.ConclusionsThe strict neuronal specificity of neurofilament has hindered the mechanistic studies of recessive NEFL nonsense mutations. Here, we show that such mutation leads to the absence of NEFL, causing childhood-onset neuropathy through a loss-of-function mechanism. We propose that the neurofilament accumulation, a common feature of many neurodegenerative diseases, mimics the absence of NEFL seen in recessive CMT if aggregation prevents the proper localization of wild-type NEFL in neurons. Our results suggest that the removal of NEFL as a proposed treatment option is harmful in humans.

Objectives Mutations in the CHCHD10 gene, which encodes a mitochondrially targeted protein, have emerged as an important cause of motor neuron disease and frontotemporal lobar degeneration. The aim of this study was to assess the clinical variability in a large family carrying the p.Gly66Val mutation of the CHCHD10 gene. This mutation has recently been reported to cause late-onset spinal muscular atrophy (SMAJ) or sensorimotor axonal Charcot–Marie–Tooth neuropathy (CMT2) in the Finnish population. Materials and methods Nine affected members of an extended Finnish pedigree were included in the study. Detailed clinical and neurophysiological examinations were performed. The CHCHD10 p.Gly66Val mutation was examined by Sanger sequencing. Results The heterozygous p.Gly66Val mutation was present in all affected individuals from whom a DNA sample was available. The clinical phenotype varied from proximal sensorimotor neuropathy to spinal muscular atrophy and in one case resembled motor neuron disease ALS at its early stages. The age of onset varied from 30 to 73 years. Conclusions Our data demonstrate that even within the same family, the p.Gly66Val variant can cause variable phenotypes ranging from CMT2-type axonal neuropathy to spinal muscular atrophy, which may also present as an ALS-like disease. The spectrum of CHCHD10-related neuromuscular disease has widened rapidly, and we recommend keeping the threshold for genetic testing low particularly when dominant inheritance or mitochondrial pathology is present.

Neuroligins are cell-adhesion molecules located at the postsynaptic side of the synapse. Neuroligins interact with beta-neurexins and this interaction is involved in the formation of functional synapses. Mutations in two X-linked neuroligin genes, NLGN3 and NLGN4, have recently been implicated in pathogenesis of autism. The neuroligin gene family consists of five members (NLGN1 at 3q26, NLGN2 at 17p13, NLGN3 at Xq13, NLGN4 at Xp22, and NLGN4Y at Yq11), of which NLGN1 and NLGN3 are located within the best loci observed in our previous genome-wide scan for autism in the Finnish sample. Here, we report a detailed molecular genetic analysis of NLGN1, NLGN3, NLGN4, and NLNG4Y in the Finnish autism sample. Mutation analysis of 30 probands selected from families producing linkage evidence for Xq13 and/or 3q26 loci revealed several polymorphisms, but none of these seemed to be functional. Family-based association analysis in 100 families with autism spectrum disorders yielded only modest associations at NLGN1 (rs1488545, P=0.002), NLGN3 (DXS7132, P=0.014), and NLGN4 (DXS996, P=0.031). We conclude that neuroligin mutations most probably represent rare causes of autism and that it is unlikely that the allelic variants in these genes would be major risk factors for autism.

Limb-girdle muscular dystrophy 2D (LGMD2D) is caused by mutations in the alpha-sarcoglycan gene (SGCA). The most frequently reported mutation, 229CGC>TGC (R77C) in exon 3 of SGCA, results in the substitution of arginine by cysteine. We present here the clinical, immunohistochemical, and genetic data of 11 Finnish patients with LGMD2D caused by mutations in SGCA. Mutational analysis showed 10 patients homozygous and 1 compound heterozygous for R77C. A wide spectrum of SGCA mutations has been reported previously. Our results show an enrichment of R77C in Finland, further underlined by the observed carrier frequency of 1 per 150. According to the annual birth rate of approximately 60,000 in Finland, one LGMD2D patient with a homozygous mutation is expected to be born every 1 or 2 years on average. The presence of an ancient founder mutation is indicated by the fact that all patients shared a short common haplotype extending > or = 790 kilobases. Our results emphasize the need to include the SGCA gene R77C mutation test in routine DNA analyses of severe dystrophinopathy-like muscular dystrophies in Finland, and suggest that the applicability of this test in other populations should be studied as well.

Nutrient availability is the major regulator of life and reproduction, and a complex cellular signaling network has evolved to adapt organisms to fasting. These sensor pathways monitor cellular energy metabolism, especially mitochondrial ATP production and NAD(+)/NADH ratio, as major signals for nutritional state. We hypothesized that these signals would be modified by mitochondrial respiratory chain disease, because of inefficient NADH utilization and ATP production. Oral administration of nicotinamide riboside (NR), a vitamin B3 and NAD(+) precursor, was previously shown to boost NAD(+) levels in mice and to induce mitochondrial biogenesis. Here, we treated mitochondrial myopathy mice with NR. This vitamin effectively delayed early- and late-stage disease progression, by robustly inducing mitochondrial biogenesis in skeletal muscle and brown adipose tissue, preventing mitochondrial ultrastructure abnormalities and mtDNA deletion formation. NR further stimulated mitochondrial unfolded protein response, suggesting its protective role in mitochondrial disease. These results indicate that NR and strategies boosting NAD(+) levels are a promising treatment strategy for mitochondrial myopathy.

Further evidence for linkage of autosomal-dominant medullary cystic kidney disease on chromosome 1q21.BackgroundAutosomal-dominant medullary cystic kidney disease (ADMCKD) is characterized by the development of cysts at the corticomedullary border of the kidneys. It resembles nephronophthisis (NPH) with an autosomal-recessive mode of inheritance. Genetic linkage has been shown either on chromosome 1q21 (ADMCKD1) or 16p12 (ADMCKD2), and families exist who are not linked to the aforementioned loci. No disease-causing gene underlying this disorder has been reported.MethodsThe Finnish Transplantation Register and hospital records were searched to identify all of the ADMCKD families in the Finnish population. Detailed clinical information of the patients was collected. Linkage analysis was used to study whether the Finnish families originating from a homogenous population showed genetic linkage to the ADMCKD1 or ADMCKD2 loci. Also, the coding region of a strong candidate gene, natriuretic peptide receptor A (NPRA), located on the chromosome 1q21 critical region, was sequenced using polymerase chain reaction sequencing with an ABI 377XL Automated DNA sequencer (Applera Corp., Norwalk, CT, USA).ResultsFive of the six families showed linkage to the previously identified region of chromosome 1q21. Family 6 with hyperuricemia as a prominent clinical feature was linked to neither of the ADMCKD loci. Wide interfamiliar and intrafamiliar variability in the clinical picture of the patients was detected. The NPRA gene mutation was excluded as a causative gene by sequencing.ConclusionThis study locates the gene for ADMCKD1 close to a marker D1S1595 in a region

Background: Tibial muscular dystrophy (TMD), a late-onset dominant distal myopathy, is caused by yet unknown mutations on chromosome 2q, whereas MD with myositis (MDM) is a muscular dystrophy of the mouse, also progressing with age and linked to mouse chromosome 2. For both disorders, linkage studies have implicated titin as a potential candidate gene.Methods: The authors analyzed major candidate regions in the titin gene by sequencing and Southern blot hybridization, and performed titin immunohistochemistry on TMD patient material to identify the underlying mutation. Western blot studies were performed on the known titin ligands in muscle samples of both disorders and controls, and analysis of apoptosis was also performed.Results: The authors identified almost complete loss of calpain3, a ligand of titin, in the patient with limb-girdle MD (LGMD) with a homozygous state of TMD haplotype when primary calpain3 gene defect was excluded. Apoptotic myonuclei with altered distribution of transcription factor NF-kB and its inhibitor IkBα were encountered in muscle samples of patients with either heterozygous or homozygous TMD haplotype. Similar findings were confirmed in the MDM mouse.Conclusions: These results imply that titin mutations may be responsible for TMD, and that the pathophysiologic pathway following calpain3 deficiency may overlap with LGMD2A. The loss of calpain3 could be a downstream effect of the deficient TMD gene product. The significance of the secondary calpain3 defect for the pathogenesis of TMD was emphasized by similar calpain3 deficiency in the MDM mouse, which is suggested to be a mouse model for TMD. Homozygous mutation at the 2q locus may thus be capable of producing yet another LGMD.

X-linked vacuolar myopathies can be divided into two forms: one that is associated with cardiomyopathy and mental retardation (XVCM-MR) and a second form, termed X-linked myopathy with excessive autophagy (XMEA), that spares cardiac muscle and has no central nervous system involvement. In this article, we demonstrate linkage between XMEA and markers on chromosome Xq28 and assign the XMEA gene locus to the most telomeric 10.5 cM of chromosome X. We also show that XVCM-MR is not allelic to XMEA.

Muscle-eye-brain disease belongs to congenital muscular dystrophies with central nervous system abnormalities. The etiology of MEB is still unknown, but abnormal immunoreactivity for laminin-2 has been reported. To evaluate disease progression in muscle tissue, 32 biopsy specimens from 17 muscle-eye-brain patients were analysed. The samples of four patients were studied by immunohistochemical techniques and by quantitative Western blotting. The samples showed a great variation in the muscle pathology. Regenerative fibers and mild fiber size variation were present in over 60%. At infancy, necrotic and regenerative fibers were common, while fat infiltration was the most prominent finding in the age group over five years. In quantitative studies, the amount of laminin alpha 2 chain was clearly reduced to 10-20% of normal. In contrast, laminin beta 2 chain was overexpressed in the Western blotting studies. These findings may reflect a yet unidentified primary disturbance in the basement membrane composition and function.

Objective: To elaborate the diagnostic methods used as “gold standard” in one of the most common glycogen storage diseases (GSDs), Tarui disease (GSDVII). Methods: Two siblings with disease suggestive of GSD underwent thorough clinical analysis, including muscle biopsy, muscle MRI, exercise tests, laboratory examinations, and wholeexome sequencing (WES). Results: Both siblings had juvenile-onset exercise intolerance with cramping and infrequent myoglobinuria. Muscle biopsy showed extralysosomal glycogen accumulation, but because of normal phosphofructokinase histochemistry, GSDVII was thought to be excluded. However, WES revealed a causative homozygous PFKM gene defect, R39Q, in both siblings, establishing the diagnosis of GSDVII, which was confirmed by very low residual phosphofructo-1-kinase (PFK) enzyme activity in biochemical studies. Conclusions: We suggest that in patients with suspicion of GSD and extralysosomal glycogen accumulation, biochemical activity assay of PFK followed by molecular genetics should be performed even when enzyme histochemistry is normal. Neurol Genet 2015;1:e7; doi: 10.1212/ NXG.0000000000000007

Objective: We describe the phenotype consistent with axonal Charcot-Marie-Tooth disease type 2 (CMT2) in 4 families with a c.197G>T (p.(Gly66Val)) variant in CHCHD10 . Methods: We sequenced the CHCHD10 gene in a cohort of 107 families with CMT2 of unknown etiology. The patients were characterized by clinical examination and electroneuromyography. Muscle MRI and biopsy of the muscle or nerve were performed in selected cases. Neuropathologic autopsy was performed in 1 case. Results: The c.197G>T variant in CHCHD10 was found in 6 families, 4 of which included multiple individuals available for detailed clinical study. Variants in this gene have recently been associated with amyotrophic lateral sclerosis-frontotemporal dementia, mitochondrial myopathy, or spinal muscular atrophy Jokela type (SMAJ), but not with CMT2. Our patients had a late-onset distal axonal neuropathy with motor predominance, progressing to involve sensory nerves. Neurophysiologic and neuropathologic studies confirmed the diagnosis of sensorimotor axonal neuropathy with no loss of anterior horn neurons. Muscle biopsies showed occasional cytochrome c oxidase–negative fibers, combined with small amounts of mitochondrial DNA deletions. Conclusions: CHCHD10 c.197G>T (p.(Gly66Val)) is a cause of sensorimotor axonal neuropathy. This gene should be considered in patients presenting with a pure CMT2 phenotype, particularly when motor symptoms predominate.

Objective Several genome-wide screens have been performed in autism spectrum disorders resulting in the identification of numerous putative susceptibility loci. Analyses of pooled primary data should result in an increased sample size and the different study samples have a potential to strengthen the evidence for some earlier identified loci, reveal novel loci, and even to provide information of the general significance of the locus. The objective of this study was to search for potential susceptibility loci for autism, which are supported by two independent samples. Methods We performed a combined analysis of the primary genome scan data of the Autism Genetic Resource Exchange (AGRE) and Finnish autism samples to reveal susceptibility loci potentially shared by these study samples. Results In the initial combined data analysis, the best loci (p < 0.05) were observed at 1p12-q25, 3p24-26, 4q21-31, 5p15-q12, 6q14-21, 7q33-36, 8q22-24, 17p12-q21, and 19p13-q13. The combined analysis of Finnish and AGRE families showed the most promising shared locus on 3p24-26 with nonparametric logarithm of odds (NPL) score of 2.20 (p = 0.011). The combined data analysis did not provide increased linkage evidence for the earlier identified loci on 3q25-27 or 17p12-q21. However, the 17p12-q21 locus remained promising also in the combined sample (NPLall =2.38, p = 0.0076). Interpretation Our study of 314 autism families highlights the importance of further analyses on 3p24-26 locus involving comprehensive molecular genetic analyses of oxytocin receptor gene (OXTR), a positional and functional candidate gene for autism. Ann Neurol 2005

Recent molecular studies on autism and related disorders have supported a multilocus etiology for the disease spectrum. To maximize genetic and cultural homogeneity, we have focused our molecular studies to families originating from a subisolate of Central Finland. Genealogical studies enabled the identification of a megapedigree comprising of 12 core families with autism and Asperger syndrome (AS). We analyzed two chromosomal regions on Iq and 3q showing highest lod scores in our genome-wide scan, as well as the AUTS1 locus on chromosome 7q. For markers on 3q25-27, more significant association was observed in families from subisolate compared to families from the rest of Finland. In contrast, no clear evidence for association on AUTS1 locus was obtained. The wide interval showing association, in particular, on chromosome 3q suggests a locus for autism spectrum of disorders on this chromosomal region.

The role of genetic factors in the etiology of the autistic spectrum of disorders has clearly been demonstrated. Ten chromosomal regions, on chromosomes 1p, 4p, 6q, 7q, 13q, 15q, 16p, 17q, 19q and 22q have potentially been linked to autism.1–8 We have analyzed these chromosomal regions in a total of 17 multiplex families with autism originating from the isolated Finnish population by pairwise linkage analysis and sib-pair analysis. Mild evidence for putative contribution was found only with the 1p chromosomal region in the susceptibility to autism. Our data suggest that additional gene loci exist for autism which will be detectable in and even restricted to the isolated Finnish population.

SummaryCongenital chloride diarrhea (CLD) is an inherited intestinal disorder caused by mutations in the down-regulated in adenoma gene. In Finland, the disease is prevalent because of a founder effect, and all but one of the CLD-associated chromosomes carry the same mutation, V317del. In Poland, another area with a high incidence of CLD, as many as seven different mutations have been detected so far. A third known cluster of CLD, around the Persian Gulf, has not been genetically studied. We studied the allelic diversity of CLD in Poland, in Saudi Arabia and Kuwait, and in three isolated families from North America and Hong Kong. Altogether, eight novel mutations were identified, making a total of 19 known CLD gene mutations. The Polish major mutation I675–676ins was found in 47% of the Polish CLD-associated chromosomes. Haplotype analysis and clustering of the I675–676ins mutation supported a founder effect and common ancestral origin. As in Finland, a major founder effect was observed in Arab patients: 94% of the CLD-associated chromosomes carried a nonsense mutation, G187X, which occurred in either a conserved ancestral haplotype or its derivative. Our data confirm that the same locus is mutated in all cases of CLD studied so far. In Poland, a relatively common founder mutation is likely to highlight a set of rare mutations that would very rarely produce homozygosity alone. This suggests that mutations in the CLD locus are not rare events. Although the disease is thought to be rare, undiagnosed patients may not be uncommon.

To identify genetic loci for autism-spectrum disorders, we have performed a two-stage genomewide scan in 38 Finnish families. The detailed clinical examination of all family members revealed infantile autism, but also Asperger syndrome (AS) and developmental dysphasia, in the same set of families. The most significant evidence for linkage was found on chromosome 3q25-27, with a maximum two-point LOD score of 4.31 ( Z max dom ) for D3S3037, using infantile autism and AS as an affection status. Six markers flanking over a 5-cM region on 3q gave Z max dom >3, and a maximum parametric multipoint LOD score (MLS) of 4.81 was obtained in the vicinity of D3S3715 and D3S3037. Association, linkage disequilibrium, and haplotype analyses provided some evidence for shared ancestor alleles on this chromosomal region among affected individuals, especially in the regional subisolate. Additional potential susceptibility loci with two-point LOD scores >2 were observed on chromosomes 1q21-22 and 7q. The region on 1q21-22 overlaps with the previously reported candidate region for infantile autism and schizophrenia, whereas the region on chromosome 7q provided evidence for linkage 58 cM distally from the previously described autism susceptibility locus (AUTS1).

Objective: To discuss the diagnostic criteria for Rett syndrome based on mutational screening of the methyl-CpG-binding protein 2 gene ( MECP2 ) in patients with classic Rett syndrome and patients with Rett-like features. Methods: Thirty-nine patients with classical Rett syndrome, one with preserved speech variant (PSV), and 12 patients with developmental delay and some features of Rett syndrome were recruited for sequence analysis of the MECP2 gene coding region. The phenotype of the patients was correlated with the presence and type of the mutation as well as the X-chromosome inactivation (XCI) pattern. Results: found in 100% of the patients with classical Rett syndrome originating from Finland. One novel mutation, P127L, was detected in a patient with PSV. No mutations were found in other cases. The XCI status was found to be random in 72% of the patients with classical Rett syndrome, including the patient with PSV and all patients with developmental delay informative for the analysis. Conclusions: An MECP2 mutation can be found in almost every patient with classical Rett syndrome. More patients need to be analyzed in order to clarify the mutation prevalence in patients with atypical Rett syndrome and in patients with mental retardation.