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The costs of whole-genome sequencing have rapidly decreased, and it is being increasingly deployed in large-scale clinical research projects and introduced into routine clinical care. This will lead to rapid diagnoses for patients with genetic disease but also introduces uncertainty because of the diversity of human genomes and the potential difficulties in annotating new genetic variants for individual patients and families. Here we outline the steps in organising whole-genome sequencing for patients in the neurology clinic and emphasise that close liaison between the clinician and the laboratory is essential.

Mutations in nuclear-encoded protein subunits of the mitochondrial ribosome are an increasingly recognised cause of oxidative phosphorylation system (OXPHOS) disorders. Among them, mutations in the MRPL44 gene, encoding a structural protein of the large subunit of the mitochondrial ribosome, have been identified in four patients with OXPHOS defects and early-onset hypertrophic cardiomyopathy with or without additional clinical features. A 23-year-old individual with cardiac and skeletal myopathy, neurological involvement, and combined deficiency of OXPHOS complexes in skeletal muscle was clinically and genetically investigated. Analysis of whole-exome sequencing data revealed a homozygous mutation in MRPL44 (c.467 T > G), which was not present in the biological father, and a region of homozygosity involving most of chromosome 2, raising the possibility of uniparental disomy. Short-tandem repeat and genome-wide SNP microarray analyses of the family trio confirmed complete maternal uniparental isodisomy of chromosome 2. Mitochondrial ribosome assembly and mitochondrial translation were assessed in patient derived-fibroblasts. These studies confirmed that c.467 T > G affects the stability or assembly of the large subunit of the mitochondrial ribosome, leading to impaired mitochondrial protein synthesis and decreased levels of multiple OXPHOS components. This study provides evidence of complete maternal uniparental isodisomy of chromosome 2 in a patient with MRPL44-related disease, and confirms that MRLP44 mutations cause a mitochondrial translation defect that may present as a multisystem disorder with neurological involvement.

Peripheral myelin protein 22 (PMP22) related neuropathies account for over 50% of inherited peripheral neuropathies. A gene copy variation results in CMT1A (duplication) and hereditary neuropathy with liability to pressure palsies (HNPP; single deletion). Point mutations comprise both phenotypes. The underlying pathological mechanisms are incompletely understood and biallelic mutations of PMP22 are very rare. We describe a 9-year-old girl who presented before the age of 1 year with severe locomotor delay. She now requires support for standing and walking in view of her severe sensory ataxia. Strikingly, her muscle power and bulk are close to normal in all segments. Nerve conduction studies showed sensory-motor velocities below 5 m/s. Genetic analysis revealed a homozygous sequence change in the PMP22 gene causing the loss of termination codon (c.483A > G; p.[*161Trpext*10]), extending the protein by 9 amino acids. Both heterozygous parents have neurophysiological abnormalities consistent with HNPP, consistent with this being a loss-of-function mutation. PMP22-deficient human models are rare but important to decipher the physiological function of the PMP22 protein in vivo. The predominance of large fiber sensory involvement in this and other rare similar cases suggests a pivotal role played by PMP22 in the embryogenesis of dorsal root ganglia in humans.

ObjectiveTo investigate the effectiveness of targeted next-generation sequencing (NGS) panels in achieving a molecular diagnosis in Charcot-Marie-Tooth disease (CMT) and related disorders in a clinical setting.MethodsWe prospectively enrolled 220 patients from 2 tertiary referral centers, one in London, United Kingdom (n = 120), and one in Iowa (n = 100), in whom a targeted CMT NGS panel had been requested as a diagnostic test.PMP22duplication/deletion was previously excluded in demyelinating cases. We reviewed the genetic and clinical data upon completion of the diagnostic process.ResultsAfter targeted NGS sequencing, a definite molecular diagnosis, defined as a pathogenic or likely pathogenic variant, was reached in 30% of cases (n = 67). The diagnostic rate was similar in London (32%) and Iowa (29%). Variants of unknown significance were found in an additional 33% of cases. Mutations inGJB1,MFN2, andMPZaccounted for 39% of cases that received genetic confirmation, while the remainder of positive cases had mutations in diverse genes, includingSH3TC2,GDAP1,IGHMBP2,LRSAM1,FDG4, andGARS, and another 12 less common genes. Copy number changes inPMP22,MPZ,MFN2,SH3TC2, andFDG4were also accurately detected. A definite genetic diagnosis was more likely in cases with an early onset, a positive family history of neuropathy or consanguinity, and a demyelinating neuropathy.ConclusionsNGS panels are effective tools in the diagnosis of CMT, leading to genetic confirmation in one-third of cases negative forPMP22duplication/deletion, thus highlighting how rarer and previously undiagnosed subtypes represent a relevant part of the genetic landscape of CMT.

The microtubule-associated protein tau gene (MAPT) 10+16 intronic mutation causes frontotemporal lobar degeneration (FTLD) by increasing expression of four-repeat (4R)-tau isoforms. We investigated the potential role for astrocytes in the pathogenesis of FTLD by studying the expression of 4R-tau. We derived astrocytes and neurons from induced pluripotent stem cells from two asymptomatic 10+16 carriers which, compared to controls, showed persistently increased 4R:3R-tau transcript and protein ratios in both cell types. However, beyond 300 days culture, 10+16 neurons showed less marked increase of this 4R:3R-tau transcript ratio compared to astrocytes. Interestingly, throughout maturation, both 10+16 carriers consistently displayed different 4R:3R-tau transcript and protein ratios. These elevated levels of 4R-tau in astrocytes implicate glial cells in the pathogenic process and also suggests a cell-type-specific regulation and may inform and help on treatment of pre-clinical tauopathies.

In vitro studies of autosomal dominant Alzheimer’s disease implicate longer amyloid-β peptides in disease pathogenesis; however, less is known about the behaviour of these mutations in vivo. In this cross-sectional cohort study, we used liquid chromatography-tandem mass spectrometry to analyse 66 plasma samples from individuals who were at risk of inheriting a mutation or were symptomatic. We tested for differences in amyloid-β (Aβ)42:38, Aβ42:40 and Aβ38:40 ratios between presenilin 1 (PSEN1) and amyloid precursor protein (APP) carriers. We examined the relationship between plasma and in vitro models of amyloid-β processing and tested for associations with parental age at onset. Thirty-nine participants were mutation carriers (28 PSEN1 and 11 APP). Age- and sex-adjusted models showed marked differences in plasma amyloid-β between genotypes: higher Aβ42:38 in PSEN1 versus APP (P, O’Connor et al. reveal significant differences in plasma Aβ ratios between PSEN1 and APP carriers, broadening understanding of the molecular drivers of Alzheimer’s disease. They also show associations between higher Aβ42:40 and Aβ42:38 ratios and earlier disease onset, supporting the pathogenicity of these peptide ratios.

ObjectiveNeurofilaments are the major scaffolding proteins for the neuronal cytoskeleton, and variants in NEFH have recently been described to cause axonal Charcot-Marie-Tooth disease type 2CC (CMT2CC).MethodsIn this large observational study, we present phenotype–genotype correlations on 30 affected and 3 asymptomatic mutation carriers from eight families.ResultsThe majority of patients presented in adulthood with motor-predominant and lower limb-predominant symptoms and the average age of onset was 31.0±15.1 years. A prominent feature was the development of proximal weakness early in the course of the disease. The disease progressed rapidly, unlike other Charcot-Marie-Tooth disease (CMT) subtypes, and half of the patients (53%) needed to use a wheelchair on average 24.1 years after symptom onset. Furthermore, 40% of patients had evidence of early ankle plantarflexion weakness, a feature which is observed in only a handful of CMT subtypes. Neurophysiological studies and MRI of the lower limbs confirmed the presence of a non-length-dependent neuropathy in the majority of patients.All families harboured heterozygous frameshift variants in the last exon of NEFH, resulting in a reading frameshift to an alternate open reading frame and the translation of approximately 42 additional amino acids from the 3' untranslated region (3′-UTR).ConclusionsThis phenotype–genotype study highlights the unusual phenotype of CMT2CC, which is more akin to spinal muscular atrophy rather than classic CMT. Furthermore, the study will enable more informative discussions on the natural history of the disease and will aid in NEFH variant interpretation in the context of the disease’s unique molecular genetics.

Neurofilaments are the major scaffolding proteins for the neuronal cytoskeleton, and variants inIn this large observational study, we present phenotype-genotype correlations on 30 affected and 3 asymptomatic mutation carriers from eight families.The majority of patients presented in adulthood with motor-predominant and lower limb-predominant symptoms and the average age of onset was 31.0±15.1 years. A prominent feature was the development of proximal weakness early in the course of the disease. The disease progressed rapidly, unlike other Charcot-Marie-Tooth disease (CMT) subtypes, and half of the patients (53%) needed to use a wheelchair on average 24.1 years after symptom onset. Furthermore, 40% of patients had evidence of early ankle plantarflexion weakness, a feature which is observed in only a handful of CMT subtypes. Neurophysiological studies and MRI of the lower limbs confirmed the presence of a non-length-dependent neuropathy in the majority of patients.All families harboured heterozygous frameshift variants in the last exon ofThis phenotype-genotype study highlights the unusual phenotype of CMT2CC, which is more akin to spinal muscular atrophy rather than classic CMT. Furthermore, the study will enable more informative discussions on the natural history of the disease and will aid in

Friedreich’s ataxia (FRDA) is a comparatively rare autosomal recessive neurological disorder primarily caused by the homozygous expansion of a GAA trinucleotide repeat in intron 1 of the FXN gene. The repeat expansion causes gene silencing that results in deficiency of the frataxin protein leading to mitochondrial dysfunction, oxidative stress and cell death. The GAA repeat tract in some cases may be impure with sequence variations called interruptions. It has previously been observed that large interruptions of the GAA repeat tract, determined by abnormal MboII digestion, are very rare. Here we have used triplet repeat primed PCR (TP PCR) assays to identify small interruptions at the 5′ and 3′ ends of the GAA repeat tract through alterations in the electropherogram trace signal. We found that contrary to large interruptions, small interruptions are more common, with 3′ interruptions being most frequent. Based on detection of interruptions by TP PCR assay, the patient cohort (n = 101) was stratified into four groups: 5′ interruption, 3′ interruption, both 5′ and 3′ interruptions or lacking interruption. Those patients with 3′ interruptions were associated with shorter GAA1 repeat tracts and later ages at disease onset. The age at disease onset was modelled by a group-specific exponential decay model. Based on this modelling, a 3′ interruption is predicted to delay disease onset by approximately 9 years relative to those lacking 5′ and 3′ interruptions. This highlights the key role of interruptions at the 3′ end of the GAA repeat tract in modulating the disease phenotype and its impact on prognosis for the patient.

In-vitrostudies of autosomal dominant Alzheimer’s disease (ADAD) implicate longer Aβ peptides in pathogenesis, however less is known about the behaviour of ADAD mutationsin-vivo. In this cross-sectional cohort study, we used liquid chromatography-tandem mass spectrometry to analyse 66 plasma samples from ADAD family members who were at-risk of inheriting a mutation or were already symptomatic. We tested for differences in plasma Aβ42:38, 38:40 and 42:40 ratios betweenPresenilin1 (PSEN1)andAmyloid Precursor Protein (APP)carriers. We examined the relationship between plasma andin-vitromodels of Aβ processing and, amongPSEN1carriers, tested for associations with parental age at onset (AAO). 39 participants were mutation carriers (28PSEN1and 11APP).Age- and sex-adjusted models showed marked differences in plasma Aβ betweenAPPandPSEN1: higher Aβ42:38 inPSEN1versusAPP(pAPPversusPSEN1(pAPPandPSEN1compared to non-carriers (both pPSEN1, sex-adjusted models demonstrated negative associations between (i)Aβ42:40 (ii)Aβ42:38 and parental AAO.In-vivodifferences in Aβ processing betweenAPPandPSEN1provide insights into ADAD pathophysiology which can inform therapy development.

Highlights • We found a high asymptomatic rate in vulnerable people with epilepsy. • Enhanced surveillance allows to quickly contain outbreaks. • We report a low rate of COVID-19 morbidity and mortality in a long-term care facility. • Preventative measures allow reducing resident-to-resident and -to-caregiver transmission. • Children and young adults appear to have lower infection rates., In this cohort study, we aim to compare outcomes from coronavirus disease 2019 (COVID-19) in people with severe epilepsy and other co-morbidities living in long-term care facilities which all implemented early preventative measures, but different levels of surveillance. During 25-week observation period (16 March–6 September 2020), we included 404 residents (118 children), and 1643 caregivers. We compare strategies for infection prevention, control, and containment, and related outcomes, across four UK long-term care facilities. Strategies included early on-site enhancement of preventative and infection control measures, early identification and isolation of symptomatic cases, contact tracing, mass surveillance of asymptomatic cases and contacts. We measured infection rate among vulnerable people living in the facilities and their caregivers, with asymptomatic and symptomatic cases, including fatality rate. We report 38 individuals (17 residents) who tested severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-positive, with outbreaks amongst residents in two facilities. At Chalfont Centre for Epilepsy (CCE), 10/98 residents tested positive: two symptomatic (one died), eight asymptomatic on weekly enhanced surveillance; 2/275 caregivers tested positive: one symptomatic, one asymptomatic. At St Elizabeth’s (STE), 7/146 residents tested positive: four symptomatic (one died), one positive during hospital admission for symptoms unrelated to COVID-19, two asymptomatic on one-off testing of all 146 residents; 106/601 symptomatic caregivers were tested, 13 positive. In addition, during two cycles of systematically testing all asymptomatic carers, four tested positive. At The Meath (TM), 8/80 residents were symptomatic but none tested; 26/250 caregivers were tested, two positive. At Young Epilepsy (YE), 8/80 children were tested, all negative; 22/517 caregivers were tested, one positive. Infection outbreaks in long-term care facilities for vulnerable people with epilepsy can be quickly contained, but only if asymptomatic individuals are identified through enhanced surveillance at resident and caregiver level. We observed a low rate of morbidity and mortality, which confirmed that preventative measures with isolation of suspected and confirmed COVID-19 residents can reduce resident-to-resident and resident-to-caregiver transmission. Children and young adults appear to have lower infection rates. Even in people with epilepsy and multiple co-morbidities, we observed a high percentage of asymptomatic people suggesting that epilepsy-related factors (anti-seizure medications and seizures) do not necessarily lead to poor outcomes.

Background: Friedreich's ataxia is a rare autosomal recessive neurological disorder caused by the homozygous expansion of a GAA trinucleotide repeat in intron 1 of the FXN gene and frataxin protein deficiency. It has previously been observed that large interruptions of the GAA repeat tract, determined by abnormal MboII digestion, are very rare. Methods and Results: Here we have used triplet repeat primed PCR (TP PCR) assays to identify small interruptions at the 5' and 3' ends of the GAA repeat tract through alterations in the electropherogram trace signal. We found that contrary to large interruptions, small interruptions are more common, with 3' interruptions being most frequent. The patient cohort (n=101) was stratified into groups: 5' interruption, 3' interruption, both 5' and 3' interruptions or lacking interruption. Those patients with a 3' interruptions were associated with shorter GAA1 repeat tracts and later ages at disease onset. The age at disease onset was modelled by a group-specific exponential decay model. Conclusions and Significance: Based on this modelling, a 3' interruption is predicted to delay disease onset by approximately 9 years relative to those lacking 5' and 3' interruptions, highlightening the key role of interruptions at the 3' end of the GAA repeat tract in modulating the disease phenotype and its impact on prognosis for the patient.

Copyright: © 2021 by the authors. Friedreich’s ataxia (FRDA) is a comparatively rare autosomal recessive neurological disorder primarily caused by the homozygous expansion of a GAA trinucleotide repeat in intron 1 of the FXN gene. The repeat expansion causes gene silencing that results in deficiency of the frataxin protein leading to mitochondrial dysfunction, oxidative stress and cell death. The GAA repeat tract in some cases may be impure with sequence variations called interruptions. It has previously been observed that large interruptions of the GAA repeat tract, determined by abnormal MboII digestion, are very rare. Here we have used triplet repeat primed PCR (TP PCR) assays to identify small interruptions at the 5′ and 3′ ends of the GAA repeat tract through alterations in the electropherogram trace signal. We found that contrary to large interruptions, small interruptions are more common, with 3′ interruptions being most frequent. Based on detection of interruptions by TP PCR assay, the patient cohort (n = 101) was stratified into four groups: 5′ interruption, 3′ interruption, both 5′ and 3′ interruptions or lacking interruption. Those patients with 3′ interruptions were associated with shorter GAA1 repeat tracts and later ages at disease onset. The age at disease onset was modelled by a group-specific exponential decay model. Based on this modelling, a 3′ interruption is predicted to delay disease onset by approximately 9 years relative to those lacking 5′ and 3′ interruptions. This highlights the key role of interruptions at the 3′ end of the GAA repeat tract in modulating the disease phenotype and its impact on prognosis for the patient. European Union Seventh Framework Programme (FP7/2007–2013) under grant agreement number 242193/EFACTS; National Brain Appeal—Small Acorns Fund; The Wellcome Centre for Human Neuroimaging is supported by core funding from the Wellcome (203147/Z/16/Z); Department of Health’s National Institute for Health Research Biomedical Research Centres funding scheme; CRN; North Thames, National Institute for Health Research (NIHR). Medical Research Council (MR/N028767/1).

The MAPT 10+16 intronic mutation causes frontotemporal lobar degeneration (FTLD) by increasing expression of four-repeat (4R)-tau isoforms. We investigated the potential role for astrocytes in the pathogenesis of FTLD by studying the expression of 4R-tau. We derived astrocytes and neurons from induced pluripotent stem cells from two asymptomatic 10+16 carriers and, compared to controls, showed persistently increased 4R:3R-tau transcript and protein ratios in both cell types. However, beyond 300 days culture, 10+16 neurons showed less marked increase of this 4R:3R-tau transcript ratio compared to astrocytes. Interestingly, throughout maturation, both 10+16 carriers consistently displayed different 4R:3R-tau ratios at transcript and protein levels. Our study shows elevated levels of 4R-tau in astrocytes implicating glial cells in the pathogenic process and also suggests a cell-type-specific regulation and may inform and help on treatment of preclinical tauopathies.

BackgroundRepeat expansion (RE) disorders affect ~1 in 3000 individuals and are clinically heterogeneous diseases caused by expansions of short tandem DNA repeats. Genetic testing is often locus-specific, resulting in under diagnosis of atypical clinical presentations, especially in paediatric patients without a prior positive family history. Whole genome sequencing (WGS) is emerging as a first-line test for rare genetic disorders, but until recently REs were thought to be undetectable by this approach.MethodsWGS pipelines for RE disorder detection were deployed by the 100,000 Genomes Project and Illumina Clinical Services Laboratory. Performance was retrospectively assessed across the 13 most common neurological RE loci using 793 samples with prior orthogonal testing (182 with expanded alleles and 611 with alleles within normal size) and prospectively interrogated in 13,331 patients with suspected genetic neurological disorders.FindingsWGS RE detection showed minimum 97·3% sensitivity and 99·6% specificity across all 13 disease-associated loci. Applying the pipeline to patients from the 100,000 Genomes Project identified pathogenic repeat expansions which were confirmed in 69 patients, including seven paediatric patients with no reported family history of RE disorders, with a 0.09% false positive rate.InterpretationWe show here for the first time that WGS enables the detection of causative repeat expansions with high sensitivity and specificity, and that it can be used to resolve previously undiagnosed neurological disorders. This includes children with no prior suspicion of a RE disorder. These findings are leading to diagnostic implementation of this analytical pipeline in the NHS Genomic Medicine Centres in England.FundingMedical Research Council, Department of Health and Social Care, National Health Service England, National Institute for Health Research, Illumina Inc

Spinocerebellar ataxia type 3 (SCA3), caused by a CAG repeat expansion in the ataxin-3 gene (ATXN3), is characterized by neuronal polyglutamine (polyQ) ATXN3 protein aggregates. Although there is no cure for SCA3, gene-silencing approaches to reduce toxic polyQ ATXN3 showed promise in preclinical models. However, a major limitation in translating putative treatments for this rare disease to the clinic is the lack of pharmacodynamic markers for use in clinical trials. Here, we developed an immunoassay that readily detects polyQ ATXN3 proteins in human biological fluids and discriminates patients with SCA3 from healthy controls and individuals with other ataxias. We show that polyQ ATXN3 serves as a marker of target engagement in human fibroblasts, which may bode well for its use in clinical trials. Last, we identified a single-nucleotide polymorphism that strongly associates with the expanded allele, thus providing an exciting drug target to abrogate detrimental events initiated by mutant ATXN3. Gene-silencing strategies for several repeat diseases are well under way, and our results are expected to improve clinical trial preparedness for SCA3 therapies.

Mutations in nuclear-encoded protein subunits of the mitochondrial ribosome are an increasingly recognised cause of oxidative phosphorylation system (OXPHOS) disorders. Among them, mutations in the MRPL44 gene, encoding a structural protein of the large subunit of the mitochondrial ribosome, have been identified in four patients with OXPHOS defects and early-onset hypertrophic cardiomyopathy with or without additional clinical features. A 23-year-old individual with cardiac and skeletal myopathy, neurological involvement, and combined deficiency of OXPHOS complexes in skeletal muscle was clinically and genetically investigated. Analysis of whole-exome sequencing data revealed a homozygous mutation in MRPL44 (c.467 T G), which was not present in the biological father, and a region of homozygosity involving most of chromosome 2, raising the possibility of uniparental disomy. Short-tandem repeat and genome-wide SNP microarray analyses of the family trio confirmed complete maternal uniparental isodisomy of chromosome 2. Mitochondrial ribosome assembly and mitochondrial translation were assessed in patient derived-fibroblasts. These studies confirmed that c.467 T G affects the stability or assembly of the large subunit of the mitochondrial ribosome, leading to impaired mitochondrial protein synthesis and decreased levels of multiple OXPHOS components. This study provides evidence of complete maternal uniparental isodisomy of chromosome 2 in a patient with MRPL44-related disease, and confirms that MRLP44 mutations cause a mitochondrial translation defect that may present as a multisystem disorder with neurological involvement.

Blood biomarkers have great potential to advance clinical care and accelerate trials in Alzheimer’s disease (AD). Plasma phospho-tau181 (p-tau181) is a promising blood biomarker however, it is unknown if levels increase in presymptomatic AD. Therefore, we investigated the timing of p-tau181 changes using 153 blood samples from 70 individuals in a longitudinal study of familial AD (FAD). Plasma p-tau181 was measured, using an in-house single molecule array assay. We compared p-tau181 between symptomatic carriers, presymptomatic carriers, and non-carriers, adjusting for age and sex. We examined the relationship between p-tau181 and neurofilament light and estimated years to/from symptom onset (EYO), as well as years to/from actual onset in a symptomatic subgroup. In addition, we studied associations between p-tau181 and clinical severity, as well testing for differences between genetic subgroups. Twenty-four were presymptomatic carriers (mean baseline EYO −9.6 years) while 27 were non-carriers. Compared with non-carriers, plasma p-tau181 concentration was higher in both symptomatic (p

The rare autosomal dominant Charcot-Marie-Tooth type 2B (CMT2B) is associated with mutations in the RAB7A gene, involved in the late endocytic pathway. CMT2B is characterized by predominant sensory loss, ulceromutilating features, with lesser-to-absent motor deficits. We characterized clinically and genetically a family harboring a novel pathogenic RAB7A variant and performed structural and functional analysis of the mutant protein. A 39-year-old woman presented with early-onset walking difficulties, progressive distal muscle wasting and weakness in lower limbs and only mild sensory signs. Electrophysiology demonstrated an axonal sensorimotor neuropathy. Nerve biopsy showed a chronic axonal neuropathy with moderate loss of all caliber myelinated fibers. Next-generation sequencing (NGS) technology revealed in the proband and in her similarly affected father the novel c.377A&gt, G (p.K126R) heterozygous variant predicted to be deleterious. The mutation affects the biochemical properties of RAB7 GTPase, causes altered interaction with peripherin, and inhibition of neurite outgrowth, as for previously reported CMT2B mutants. However, it also shows differences, particularly in the epidermal growth factor receptor degradation process. Altogether, our findings indicate that this RAB7A variant is pathogenic and widens the phenotypic spectrum of CMT2B to include predominantly motor CMT2. Alteration of the receptor degradation process might explain the different clinical presentations in this family.

We report the first clinical-radiological-genetic-molecular-pathological study of a kindred with c.823-10G>T MAPT intronic variant (rs63749974) associated with frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). We describe the clinical spectrum within this family and emphasize the association between MAPT gene variants and motor neuron disease. This report of a second family with FTDP-17 associated with c.823-10G>T MAPT variant strongly supports pathogenicity of the variant and confirms it is a 4-repeat (4R) tauopathy. This intronic point mutation, probably strengthens the polypyrimidine tract and alters the splicing of exon 10 (10 nucleotides into intron 9) close to the 3' splice site.

Mutations in the Presenilin 1 (PSEN1) gene are the most common cause of autosomal dominant familial Alzheimer's disease. We report the clinical, imaging and postmortem findings of kindred carrying a novel duplication mutation (Ile168dup) in the PSEN1 gene. We interpret the pathogenicity of this novel variant and discuss the additional neurological features (pyramidal dysfunction, myoclonus and seizures) that accompanied cognitive decline. This report broadens the clinical phenotype of PSEN1 insertion mutations while also highlighting the importance of considering duplication, insertion and deletion mutations in cases of young onset dementia.

Objectives To analyse and describe the clinical and genetic spectrum of Charcot-Marie-Tooth disease (CMT) caused by mutations in the neurofilament light polypeptide gene ( NEFL ). Methods Combined analysis of newly identified patients with NEFL -related CMT and all previously reported cases from the literature. Results Five new unrelated patients with CMT carrying the NEFL mutations P8R and N98S and the novel variant L311P were identified. Combined data from these cases and 62 kindreds from the literature revealed four common mutations (P8R, P22S, N98S and E396K) and three mutational hotspots accounting for 37 (55%) and 50 (75%) kindreds, respectively. Eight patients had de novo mutations. Loss of large-myelinated fibres was a uniform feature in a total of 21 sural nerve biopsies and ‘onion bulb’ formations and/or thin myelin sheaths were observed in 14 (67%) of them. The neurophysiological phenotype was broad but most patients with E90K and N98S had upper limb motor conduction velocities Conclusions NEFL -related CMT is clinically and genetically heterogeneous. Based on this study, however, we propose mutational hotspots and relevant clinical–genetic associations that may be helpful in the evaluation of NEFL sequence variants and the differential diagnosis with other forms of CMT.

Pyruvate dehydrogenase complex (PDC) deficiency caused by mutations in the X-linked PDHA1 gene has a broad clinical presentation, and the pattern of X-chromosome inactivation has been proposed as a major factor contributing to its variable expressivity in heterozygous females. Here, we report the first set of monozygotic twin females with PDC deficiency, caused by a novel, de novo heterozygous missense mutation in exon 11 of PDHA1 (NM_000284.3: c.1100A>T). Both twins presented in infancy with a similar clinical phenotype including developmental delay, episodes of hypotonia or encephalopathy, epilepsy, and slowly progressive motor impairment due to pyramidal, extrapyramidal, and cerebellar involvement. However, they exhibited clear differences in disease severity that correlated well with residual PDC activities (approximately 60% and 20% of mean control values, respectively) and levels of immunoreactive E1α subunit in cultured skin fibroblasts. To address whether the observed clinical and biochemical differences could be explained by the pattern of X-chromosome inactivation, we undertook an androgen receptor assay in peripheral blood. In the less severely affected twin, a significant bias in the relative activity of the two X chromosomes with a ratio of approximately 75:25 was detected, while the ratio was close to 50:50 in the other twin. Although it may be difficult to extrapolate these results to other tissues, our observation provides further support to the hypothesis that the pattern of X-chromosome inactivation may influence the phenotypic expression of the same mutation in heterozygous females and broadens the clinical and genetic spectrum of PDC deficiency. Electronic supplementary material The online version of this article (10.1007/s00439-019-02075-9) contains supplementary material, which is available to authorized users.

See Paisán-Ruiz and Jen (doi:10.1093/brain/awaa015) for a scientific commentary on this article. Cortese et al. describe the full disease phenotype, including progression of ataxia, in 100 confirmed carriers of RFC1 repeat expansions. RFC1 repeat expansion should be considered in all cases of sensory ataxic neuropathy, particularly if cerebellar dysfunction, vestibular involvement and cough coexist., Ataxia, causing imbalance, dizziness and falls, is a leading cause of neurological disability. We have recently identified a biallelic intronic AAGGG repeat expansion in replication factor complex subunit 1 (RFC1) as the cause of cerebellar ataxia, neuropathy, vestibular areflexia syndrome (CANVAS) and a major cause of late onset ataxia. Here we describe the full spectrum of the disease phenotype in our first 100 genetically confirmed carriers of biallelic repeat expansions in RFC1 and identify the sensory neuropathy as a common feature in all cases to date. All patients were Caucasian and half were sporadic. Patients typically reported progressive unsteadiness starting in the sixth decade. A dry spasmodic cough was also frequently associated and often preceded by decades the onset of walking difficulty. Sensory symptoms, oscillopsia, dysautonomia and dysarthria were also variably associated. The disease seems to follow a pattern of spatial progression from the early involvement of sensory neurons, to the later appearance of vestibular and cerebellar dysfunction. Half of the patients needed walking aids after 10 years of disease duration and a quarter were wheelchair dependent after 15 years. Overall, two-thirds of cases had full CANVAS. Sensory neuropathy was the only manifestation in 15 patients. Sixteen patients additionally showed cerebellar involvement, and six showed vestibular involvement. The disease is very likely to be underdiagnosed. Repeat expansion in RFC1 should be considered in all cases of sensory ataxic neuropathy, particularly, but not only, if cerebellar dysfunction, vestibular involvement and cough coexist.

Late-onset ataxia is common, often idiopathic, and can result from cerebellar, proprioceptive, or vestibular impairment; when in combination, it is also termed cerebellar ataxia, neuropathy, vestibular areflexia syndrome (CANVAS). We used non-parametric linkage analysis and genome sequencing to identify a biallelic intronic AAGGG repeat expansion in the replication facsstor C subunit 1 (RFC1) gene as the cause of familial CANVAS and a frequent cause of late-onset ataxia, particularly if sensory neuronopathy and bilateral vestibular areflexia coexist. The expansion, which occurs in the poly(A) tail of an AluSx3 element and differs in both size and nucleotide sequence from the reference (AAAAG)(11) allele, does not affect RFC1 expression in patient peripheral and brain tissue, suggesting no overt loss of function. These data, along with an expansion carrier frequency of 0.7% in Europeans, implies that biallelic AAGGG expansion in RFC1 is a frequent cause of late-onset ataxia.

ObjectivesHereditary sensory neuropathy type 1 (HSN1) is a rare, slowly progressive neuropathy causing profound sensory deficits and often severe motor loss. L-serine supplementation is a possible candidate therapy but the lack of responsive outcome measures is a barrier for undertaking clinical trials in HSN1. We performed a 12-month natural history study to characterise the phenotype of HSN1 and to identify responsive outcome measures.MethodsAssessments included Charcot-Marie-Tooth Neuropathy Score version 2 (CMTNSv2), CMTNSv2-Rasch modified, nerve conduction studies, quantitative sensory testing, intraepidermal nerve fibre density (thigh), computerised myometry (lower limbs), plasma 1-deoxysphingolipid levels, calf-level intramuscular fat accumulation by MRI and patient-based questionnaires (Neuropathic Pain Symptom Inventory and 36-Short Form Health Survey version 2 [SF-36v2]).Results35 patients with HSN1 were recruited. There was marked heterogeneity in the phenotype mainly due to differences between the sexes: males generally more severely affected. The outcome measures that significantly changed over 1 year and correlated with CMTNSv2, SF-36v2-physical component and disease duration were MRI determined calf intramuscular fat accumulation (mean change in overall calf fat fraction 2.36%, 95% CI 1.16 to 3.55, p=0.0004), pressure pain threshold on the hand (mean change 40 kPa, 95% CI 0.7 to 80, p=0.046) and myometric measurements of ankle plantar flexion (median change −0.5 Nm, IQR −9.5 to 0, p=0.0007), ankle inversion (mean change −0.89 Nm, 95% CI −1.66 to −0.12, p=0.03) and eversion (mean change −1.61 Nm, 95% CI −2.72 to −0.51, p=0.006). Intramuscular calf fat fraction was the most responsive outcome measure.ConclusionMRI determined calf muscle fat fraction shows validity and high responsiveness over 12 months and will be useful in HSN1 clinical trials.

Objective To characterize the phenotype in individuals with OPA3-related autosomal dominant optic atrophy and cataract (ADOAC) and peripheral neuropathy (PN). Methods Two probands with multiple affected relatives and one sporadic case were referred for evaluation of a PN. Their phenotype was determined by clinical ± neurophysiological assessment. Neuropathologic examination of sural nerve and skeletal muscle, and ultrastructural analysis of mitochondria in fibroblasts were performed in one case. Exome sequencing was performed in the probands. Results The main clinical features in one family (n = 7 affected individuals) and one sporadic case were early-onset cataracts (n = 7), symptoms of gastrointestinal dysmotility (n = 8), and possible/confirmed PN (n = 7). Impaired vision was an early-onset feature in another family (n = 4 affected individuals), in which 3 members had symptoms of gastrointestinal dysmotility and 2 developed PN and cataracts. The less common features among all individuals included symptoms/signs of autonomic dysfunction (n = 3), hearing loss (n = 3), and recurrent pancreatitis (n = 1). In 5 individuals, the neuropathy was axonal and clinically asymptomatic (n = 1), sensory-predominant (n = 2), or motor and sensory (n = 2). In one patient, nerve biopsy revealed a loss of large and small myelinated fibers. In fibroblasts, mitochondria were frequently enlarged with slightly fragmented cristae. The exome sequencing identified OPA3 variants in all probands: a novel variant (c.23T>C) and the known mutation (c.313C>G) in OPA3. Conclusions A syndromic form of ADOAC (ADOAC+), in which axonal neuropathy may be a major feature, is described. OPA3 mutations should be included in the differential diagnosis of complex inherited PN, even in the absence of clinically apparent optic atrophy.

Late-onset ataxia is common, often idiopathic, and can result from cerebellar, proprioceptive, or vestibular impairment; when in combination, it is also termed cerebellar ataxia, neuropathy, vestibular areflexia syndrome (CANVAS). We used non-parametric linkage analysis and genome sequencing to identify a biallelic intronic AAGGG repeat expansion in the replication factor C subunit 1 (RFC1) gene as the cause of familial CANVAS and a frequent cause of late-onset ataxia, particularly if sensory neuronopathy and bilateral vestibular areflexia coexist. The expansion, which occurs in the poly(A) tail of an AluSx3 element and differs in both size and nucleotide sequence from the reference (AAAAG)11 allele, does not affect RFC1 expression in patient peripheral and brain tissue, suggesting no overt loss of function. These data, along with an expansion carrier frequency of 0.7% in Europeans, implies that biallelic AAGGG expansion in RFC1 is a frequent cause of late-onset ataxia.

Objective To describe 5 cases of Parkinson9s disease lacking any detectable histopathology. Background The diagnosis of Parkinson9s disease is supported histologically by the findings of α-synuclein immunopositive Lewy bodies and neurites and severe substantia nigra cell loss. Bradykinesia as defined by slowness of initiation of movement and a progressive reduction in speed and amplitude on finger tapping is a clinical correlate of pars compacta nigral degeneration. There are very few published cases of Parkinson9s disease in which no pathological abnormality was found, and some of these cases were in hindsight thought to have probably been cases of indeterminate senile tremor or dystonic tremor. Methods Retrospective case notes review of the Queen Square Brain Bank archival collection and detailed neuropathological analysis of the selected cases. Results 5 cases considered to have Parkinson9s disease by neurologists throughout the entirety of their illness that lacked any histopathological findings known to be associated with Parkinson9s syndromes were identified out of a total number of 773 brains with a final clinical diagnosis of Parkinson9s disease in the Queen Square Brain Bank. Retrospective case note analysis did not suggest dystonic tremor or indeterminate tremor in any of them. There was a reduction in tyrosine hydroxylase (TH) density in the striatum in these cases when compared with healthy controls, but not in the substantia nigra. Conclusions Striatal dopamine deficiency without nigral cell loss is the most likely explanation for the clinical findings; other possible explanations include slowness due to comorbidities misinterpreted as bradykinesia, a tardive syndrome related to undisclosed previous neuroleptic exposure, or ‘soft age-related’ parkinsonian signs. These cases emphasise the need to regularly review the diagnosis in cases of suspected Parkinson9s disease and highlight the need for precision in the neurological examination particularly of elderly patients. These cases may represent a distinct entity of diagnostic exclusion and may be considered one explanation for the radiological phenomenon of SWEDD (scans without evidence of dopaminergic deficit).

Mitofusin 2 (MFN2) mutations are the most common cause of axonal Charcot-Marie-Tooth disease (CMT2). The majority are inherited in an autosomal dominant manner but recessive and semi-dominant kindreds have also been described. We previously reported a deletion of exons 7 and 8 resulting in nonsense-mediated decay, segregating with disease when present in trans with another pathogenic MFN2 mutation. Detailed clinical and electrophysiological data on a series of five affected patients from four kindreds and, when available, their parents and relatives were collected. MFN2 Sanger sequencing, multiplex ligation probe amplification, and haplotype analysis were performed. A severe early-onset CMT phenotype was seen in all cases: progressive distal weakness, wasting, and sensory loss from infancy or early childhood. Optic atrophy (four of five) and wheelchair dependency in childhood were common (four of five). All were compound heterozygous for a deletion of exons 7 and 8 in MFN2 with another previously reported pathogenic mutation (Phe216Ser, Thr362Met, and Arg707Trp). Carrier parents and relatives were unaffected (age range: 24-82 years). Haplotype analysis confirmed that the deletion had a common founder in all families.

Objective: To establish the phenotypic spectrum of KIF5A mutations and to investigate whether KIF5A mutations cause axonal neuropathy associated with hereditary spastic paraplegia (HSP) or typical Charcot-Marie-Tooth disease type 2 (CMT2). Methods: KIF5A sequencing of the motor-domain coding exons was performed in 186 patients with the clinical diagnosis of HSP and in 215 patients with typical CMT2. Another 66 patients with HSP or CMT2 with pyramidal signs were sequenced for all exons of KIF5A by targeted resequencing. One additional patient was genetically diagnosed by whole-exome sequencing. Results: Five KIF5A mutations were identified in 6 unrelated patients: R204W and D232N were novel mutations; R204Q, R280C, and R280H have been previously reported. Three patients had CMT2 as the predominant and presenting phenotype; 2 of them also had pyramidal signs. The other 3 patients presented with HSP but also had significant axonal neuropathy or other additional features. Conclusion: This is currently the largest study investigating KIF5A mutations. By combining next-generation sequencing and conventional sequencing, we confirm that KIF5A mutations can cause variable phenotypes ranging from HSP to CMT2. The identification of mutations in CMT2 broadens the phenotypic spectrum and underlines the importance of KIF5A mutations, which involve degeneration of both the central and peripheral nervous systems and should be tested in HSP and CMT2.

CaseA 20-year-old female had symptom onset age 7 of bilateral foot drop and distal lower limb atrophy. Nerve conduction studies demonstrated a length dependent axonal motor and sensory neuropathy. At 16 her vision deteriorated to 3/36 right, 6/60 left. Visual evoked potentials were borderline compatible with bilateral optic atrophy. Metabolic and structural causes were excluded.ExaminationAt age 18 examination demonstrated bilateral foot drop and a positive knee bob sign. There was asymmetrical distal limb atrophy. Fundi were pale. Reflexes were brisk in upper limbs and knees with absent ankles and mute plantars.ResultsThe 56-gene Bristol axonal neuropathy panel revealed a non-pathogenic TRPV4 mutation. Mutations in OPA1, OPA3, POLG, ATPase 6 and 8 were not identified. Exome sequencing in the research setting demonstrated compound heterozygous SLC25A46 mutations. One mutation was confirmed in each of her parents.DiscussionMutations in SLC25A46 are associated with spectrum of disorders encompassing pontocerebellar hypoplasia (including lethal congenital cases), Leigh Syndrome, Progressive myoclonic ataxia with optic atrophy and neuropathy, and hereditary motor and sensory neuropathy type 6B, sometimes with associated features such as cerebellar ataxia and spasticity. We present a case of CMT2 with optic atrophy due to heterozygous mutations and discuss the literature.

Priya Gami, Christina Murray, Lucia Schottlaender, Conceicao Bettencourt, Eduardo De Pablo Fernandez ... Tamas Revesz ... et al.

Charcot-Marie-Tooth disease (CMT) refers to a group of inherited neuropathies with a broad range of phenotypes, inheritance patterns and causative genes. The number of disease genes identified in CMT has expanded rapidly over the past few decades, such that more than 60 CMT-associated genes have now been discovered. This rise in genetic discovery can be attributed to the development of next-generation sequencing (NGS) technology, which allows the entire exome or genome to be sequenced in a matter of days. In this Review, we discuss how NGS is being employed in the diagnostic evaluation of patients with CMT and how the genetic advances in CMT are influencing clinical practice. In particular, we explore how genetic advances have broadened the phenotype of CMT and related disorders and how NGS allows a large number of CMT genes to be screened simultaneously early in the evaluation of an unexplained neuropathy. Finally, we discuss the different methods of NGS that can be used in CMT and related disorders, and propose a simple diagnostic algorithm in which clinical assessment and neurophysiology are used to guide the application of phenotype specific 'panels'.

An expanded hexanucleotide repeat in the C9orf72 gene is the most common genetic cause of frontotemporal dementia and amyotrophic lateral sclerosis (c9FTD/ALS). We now report the first description of a homozygous patient and compare it to a series of heterozygous cases. The patient developed early-onset frontotemporal dementia without additional features. Neuropathological analysis showed c9FTD/ALS characteristics, with abundant p62-positive inclusions in the frontal and temporal cortices, hippocampus and cerebellum, as well as less abundant TDP-43-positive inclusions. Overall, the clinical and pathological features were severe, but did not fall outside the usual disease spectrum. Quantification of C9orf72 transcript levels in post-mortem brain demonstrated expression of all known C9orf72 transcript variants, but at a reduced level. The pathogenic mechanisms by which the hexanucleotide repeat expansion causes disease are unclear and both gain- and loss-of-function mechanisms may play a role. Our data support a gain-of-function mechanism as pure homozygous loss of function would be expected to lead to a more severe, or completely different clinical phenotype to the one described here, which falls within the usual range. Our findings have implications for genetic counselling, highlighting the need to use genetic tests that distinguish C9orf72 homozygosity. Electronic supplementary material The online version of this article (doi:10.1007/s00401-013-1147-0) contains supplementary material, which is available to authorized users.

Summary The molecular basis of cytochrome c oxidase (COX, complex IV) deficiency remains genetically undetermined in many cases. Homozygosity mapping and whole-exome sequencing were performed in a consanguineous pedigree with isolated COX deficiency linked to a Leigh syndrome neurological phenotype. Unexpectedly, affected individuals harbored homozygous splice donor site mutations in NDUFA4, a gene previously assigned to encode a mitochondrial respiratory chain complex I (NADH:ubiquinone oxidoreductase) subunit. Western blot analysis of denaturing gels and immunocytochemistry revealed undetectable steady-state NDUFA4 protein levels, indicating that the mutation causes a loss-of-function effect in the homozygous state. Analysis of one- and two-dimensional blue-native polyacrylamide gels confirmed an interaction between NDUFA4 and the COX enzyme complex in control muscle, whereas the COX enzyme complex without NDUFA4 was detectable with no abnormal subassemblies in patient muscle. These observations support recent work in cell lines suggesting that NDUFA4 is an additional COX subunit and demonstrate that NDUFA4 mutations cause human disease. Our findings support reassignment of the NDUFA4 protein to complex IV and suggest that patients with unexplained COX deficiency should be screened for NDUFA4 mutations., Graphical Abstract, Highlights • Mutations in NDUFA4, assigned to encode a complex I subunit, cause human COX deficiency • Confirmed interaction between NDUFA4 and the COX holoenzyme in control muscle • The COX holoenzyme without NDUFA4 is detectable with no abnormal subassemblies in patient muscle • NDUFA4 is essential for complex IV activity, but is not required for assembly of the COX holoenzyme, Isolated cytochrome c oxidase (COX) deficiency is a frequent finding in human mitochondrial disease. Mutations in nuclear-encoded structural subunits are extremely rare, and, in many cases, the molecular basis remains undetermined. Recent evidence in cell lines has suggested that NDUFA4, previously assigned to encode a complex I subunit, actually encodes a structural component of COX. Hanna and colleagues now demonstrate that NDUFA4 mutations cause human COX deficiency, thus confirming NDUFA4 as a COX subunit that is essential for the enzyme’s activity.