A common flanking variant is associated with enhanced stability of the FGF14-SCA27B repeat locus.

The factors driving or preventing pathological expansion of tandem repeats remain largely unknown. Here, we assessed the FGF14 (GAA)·(TTC) repeat locus in 2,530 individuals by long-read and Sanger sequencing and identified a common 5'-flanking variant in 70.34% of alleles analyzed (3,463/4,923) that represents the phylogenetically ancestral allele and is present on all major haplotypes. This common sequence variation is present nearly exclusively on nonpathogenic alleles with fewer than 30 GAA-pure triplets and is associated with enhanced stability of the repeat locus upon intergenerational transmission and increased Fiber-seq chromatin accessibility.

Two Novel Variants in PI4KA in a Family Presenting With Hereditary Spastic Paraparesis: A Case Report.

Objectives: To report novel biallelic PI4KA variants in a family presenting with pure hereditary spastic paraparesis. Methods: Two affected sisters presented with unsolved hereditary spastic paraparesis and underwent clinical and imaging assessments. This was followed by short-read next-generation sequencing. Results: Analysis of next-generation sequencing data uncovered compound heterozygous variants in PI4KA (NM_058004.4: c.[3883C>A];[5785A>C]; p.[(His1295Asn);(Thr1929Pro)]. Using ACMG guidelines, both variants were classified as likely pathogenic. Discussion: Here, next-generation sequencing revealed 2 novel compound heterozygous variants in the phosphatidylinositol 4-kinase alpha gene (PI4KA) in 2 sisters presenting with progressive pure hereditary spastic paraparesis. Pathogenic variants in PI4KA have previously been associated with a spectrum of disorders including autosomal recessive perisylvian polymicrogyria, with cerebellar hypoplasia, arthrogryposis, and pure spastic paraplegia. The cases presented in this study expand the phenotypic spectrum associated with PI4KA variants and contribute new likely pathogenic variants for testing in patients with otherwise unsolved hereditary spastic paraparesis.

Spinocerebellar ataxia 27B: episodic symptoms and acetazolamide response in 34 patients.

Ashton C et al report a retrospective multi-centre cohort of 34 patients from Canada, France, Austria and Australia with spinocerebellar ataxia 27B, describing the common feature of episodic ataxia and other episodic features, as well as the inefficacy of acetazolamide in these patients.

RFC1 in an Australasian neurological disease cohort: extending the genetic heterogeneity and implications for diagnostics.

Cerebellar ataxia, neuropathy and vestibular areflexia syndrome is a progressive, generally late-onset, neurological disorder associated with biallelic pentanucleotide expansions in Intron 2 of the RFC1 gene. The locus exhibits substantial genetic variability, with multiple pathogenic and benign pentanucleotide repeat alleles previously identified. To determine the contribution of pathogenic RFC1 expansions to neurological disease within an Australasian cohort and further investigate the heterogeneity exhibited at the locus, a combination of flanking and repeat-primed PCR was used to screen a cohort of 242 Australasian patients with neurological disease. Patients whose data indicated large gaps within expanded alleles following repeat-primed PCR, underwent targeted long-read sequencing to identify novel repeat motifs at the locus. To increase diagnostic yield, additional probes at the RFC1 repeat region were incorporated into the PathWest diagnostic laboratory targeted neurological disease gene panel to enable first-pass screening of the locus for all samples tested on the panel. Within the Australasian cohort, we detected known pathogenic biallelic expansions in 15.3% (n = 37) of patients. Thirty indicated biallelic AAGGG expansions, two had biallelic 'Maori alleles' [(AAAGG)exp(AAGGG)exp], two samples were compound heterozygous for the Maori allele and an AAGGG expansion, two samples had biallelic ACAGG expansions and one sample was compound heterozygous for the ACAGG and AAGGG expansions. Forty-five samples tested indicated the presence of biallelic expansions not known to be pathogenic. A large proportion (84%) showed complex interrupted patterns following repeat-primed PCR, suggesting that these expansions are likely to be comprised of more than one repeat motif, including previously unknown repeats. Using targeted long-read sequencing, we identified three novel repeat motifs in expanded alleles. Here, we also show that short-read sequencing can be used to reliably screen for the presence or absence of biallelic RFC1 expansions in all samples tested using the PathWest targeted neurological disease gene panel. Our results show that RFC1 pathogenic expansions make a substantial contribution to neurological disease in the Australasian population and further extend the heterogeneity of the locus. To accommodate the increased complexity, we outline a multi-step workflow utilizing both targeted short- and long-read sequencing to achieve a definitive genotype and provide accurate diagnoses for patients.

A common flanking variant is associated with enhanced meiotic stability of the FGF14 -SCA27B locus.

The factors driving initiation of pathological expansion of tandem repeats remain largely unknown. Here, we assessed the FGF14 -SCA27B (GAA)•(TTC) repeat locus in 2,530 individuals by long-read and Sanger sequencing and identified a 5'-flanking 17-bp deletion-insertion in 70.34% of alleles (3,463/4,923). This common sequence variation was present nearly exclusively on alleles with fewer than 30 GAA-pure repeats and was associated with enhanced meiotic stability of the repeat locus.

Uncovering the significance of expanded CD8+ large granular lymphocytes in inclusion body myositis: Insights into T cell phenotype and functional alterations, and disease severity.

Introduction: Inclusion body myositis (IBM) is a progressive inflammatory myopathy characterised by skeletal muscle infiltration and myofibre invasion by CD8+ T lymphocytes. In some cases, IBM has been reported to be associated with a systemic lymphoproliferative disorder of CD8+ T cells exhibiting a highly differentiated effector phenotype known as T cell Large Granular Lymphocytic Leukemia (T-LGLL). Methods: We investigated the incidence of a CD8+ T-LGL lymphoproliferative disorder in 85 IBM patients and an aged-matched group of 56 Healthy Controls (HC). Further, we analysed the phenotypical characteristics of the expanded T-LGLs and investigated whether their occurrence was associated with any particular HLA alleles or clinical characteristics. Results: Blood cell analysis by flow cytometry revealed expansion of T-LGLs in 34 of the 85 (40%) IBM patients. The T cell immunophenotype of T-LGLHIGH patients was characterised by increased expression of surface molecules including CD57 and KLRG1, and to a lesser extent of CD94 and CD56 predominantly in CD8+ T cells, although we also observed modest changes in CD4+ T cells and γδ T cells. Analysis of Ki67 in CD57+ KLRG1+ T cells revealed that only a small proportion of these cells was proliferating. Comparative analysis of CD8+ and CD4+ T cells isolated from matched blood and muscle samples donated by three patients indicated a consistent pattern of more pronounced alterations in muscles, although not significant due to small sample size. In the T-LGLHIGH patient group, we found increased frequencies of perforin-producing CD8+ and CD4+ T cells that were moderately correlated to combined CD57 and KLRG1 expression. Investigation of the HLA haplotypes of 75 IBM patients identified that carriage of the HLA-C*14:02:01 allele was significantly higher in T-LGLHIGH compared to T-LGLLOW individuals. Expansion of T-LGL was not significantly associated with seropositivity patient status for anti-cytosolic 5'-nucleotidase 1A autoantibodies. Clinically, the age at disease onset and disease duration were similar in the T-LGLHIGH and T-LGLLOW patient groups. However, metadata analysis of functional alterations indicated that patients with expanded T-LGL more frequently relied on mobility aids than T-LGLLOW patients indicating greater disease severity. Conclusion: Altogether, these results suggest that T-LGL expansion occurring in IBM patients is correlated with exacerbated immune dysregulation and increased disease burden.

A novel phenotype of AChR-deficiency syndrome with predominant facial and distal weakness resulting from the inclusion of an evolutionary alternatively-spliced exon in CHRNA1.

Primary acetylcholine receptor deficiency is the most common subtype of congenital myasthenic syndrome, resulting in reduced amount of acetylcholine receptors expressed at the muscle endplate and impaired neuromuscular transmission. AChR deficiency is caused mainly by pathogenic variants in the ε-subunit of the acetylcholine receptor encoded by CHRNE, although pathogenic variants in other subunits are also seen. We report the clinical and molecular features of 13 patients from nine unrelated kinships with acetylcholine receptor deficiency harbouring the CHRNA1 variant NM_001039523.3:c.257G>A (p.Arg86His) in homozygosity or compound heterozygosity. This variant results in the inclusion of an alternatively-spliced evolutionary exon (P3A) that causes expression of a non-functional acetylcholine receptor α-subunit. We compare the clinical findings of this group to the other cases of acetylcholine receptor deficiency within our cohort. We report differences in phenotype, highlighting a predominant pattern of facial and distal weakness in adulthood, predominantly in the upper limbs, which is unusual for acetylcholine receptor deficiency syndromes, and more in keeping with slow-channel syndrome or distal myopathy. Finally, we stress the importance of including alternative exons in variant analysis to increase the probability of achieving a molecular diagnosis.

Deep Intronic FGF14 GAA Repeat Expansion in Late-Onset Cerebellar Ataxia.

BACKGROUND: The late-onset cerebellar ataxias (LOCAs) have largely resisted molecular diagnosis. METHODS: We sequenced the genomes of six persons with autosomal dominant LOCA who were members of three French Canadian families and identified a candidate pathogenic repeat expansion. We then tested for association between the repeat expansion and disease in two independent case-control series - one French Canadian (66 patients and 209 controls) and the other German (228 patients and 199 controls). We also genotyped the repeat in 20 Australian and 31 Indian index patients. We assayed gene and protein expression in two postmortem cerebellum specimens and two induced pluripotent stem-cell (iPSC)-derived motor-neuron cell lines. RESULTS: In the six French Canadian patients, we identified a GAA repeat expansion deep in the first intron of FGF14, which encodes fibroblast growth factor 14. Cosegregation of the repeat expansion with disease in the families supported a pathogenic threshold of at least 250 GAA repeats ([GAA]≥250). There was significant association between FGF14 (GAA)≥250 expansions and LOCA in the French Canadian series (odds ratio, 105.60; 95% confidence interval [CI], 31.09 to 334.20; P<0.001) and in the German series (odds ratio, 8.76; 95% CI, 3.45 to 20.84; P<0.001). The repeat expansion was present in 61%, 18%, 15%, and 10% of French Canadian, German, Australian, and Indian index patients, respectively. In total, we identified 128 patients with LOCA who carried an FGF14 (GAA)≥250 expansion. Postmortem cerebellum specimens and iPSC-derived motor neurons from patients showed reduced expression of FGF14 RNA and protein. CONCLUSIONS: A dominantly inherited deep intronic GAA repeat expansion in FGF14 was found to be associated with LOCA. (Funded by Fondation Groupe Monaco and others.).

Partial loss-of-function variant in neuregulin 1 identified in family with heritable peripheral neuropathy.

Neuregulin 1 signals are essential for the development and function of Schwann cells, which form the myelin sheath on peripheral axons. Disruption of myelin in the peripheral nervous system can lead to peripheral neuropathy, which is characterized by reduced axonal conduction velocity and sensorimotor deficits. Charcot-Marie-Tooth disease is a group of heritable peripheral neuropathies that may be caused by variants in nearly 100 genes. Despite the evidence that Neuregulin 1 is essential for many aspects of Schwann cell development, previous studies have not reported variants in the neuregulin 1 gene (NRG1) in patients with peripheral neuropathy. We have identified a rare missense variant in NRG1 that is homozygous in a patient with sensory and motor deficits consistent with mixed axonal and de-myelinating peripheral neuropathy. Our in vivo functional studies in zebrafish indicate that the patient variant partially reduces NRG1 function. This study tentatively suggests that variants at the NRG1 locus may cause peripheral neuropathy and that NRG1 should be investigated in families with peripheral neuropathy of unknown cause.

Isolation of Live Leukocytes from Human Inflammatory Muscles.

In inflammatory myopathies, the self-reactive immune cells involved in muscle aggression have been studied mostly using histological assessment of muscle biopsy sections; this methodology provides the advantage of visualizing and identifying cells within the tissue, but it does not allow further investigation. To gain access to live and isolated cells, many studies utilized blood samples; however, in the absence of biological tools to discriminate the leukocytes associated with the autoimmune process from those that emerged from responses against pathogens, the information observed on circulating immune cells often lacks in specificity, and thus result interpretation may prove difficult. In order to selectively retrieve self-reactive immune cells, we developed a protocol to isolate live leukocytes from human muscle biopsies, which allows for further analysis using a large range of methodologies. The protocol uses enzymatic digestion to release live leukocytes from freshly collected skeletal muscle samples, followed by filtration and separation of the leukocytes from the myocytes by density gradient centrifugation. The isolated cells can be submitted immediately to various analysis strategies to characterize ex vivo the specific cellular and molecular mechanisms responsible for self-directed immune muscle aggression or may be placed in culture for expansion.

Neurogenetic fetal akinesia and arthrogryposis: genetics, expanding genotype-phenotypes and functional genomics.

BACKGROUND: Fetal akinesia and arthrogryposis are clinically and genetically heterogeneous and have traditionally been refractive to genetic diagnosis. The widespread availability of affordable genome-wide sequencing has facilitated accurate genetic diagnosis and gene discovery in these conditions. METHODS: We performed next generation sequencing (NGS) in 190 probands with a diagnosis of arthrogryposis multiplex congenita, distal arthrogryposis, fetal akinesia deformation sequence or multiple pterygium syndrome. This sequencing was a combination of bespoke neurogenetic disease gene panels and whole exome sequencing. Only class 4 and 5 variants were reported, except for two cases where the identified variants of unknown significance (VUS) are most likely to be causative for the observed phenotype. Co-segregation studies and confirmation of variants identified by NGS were performed where possible. Functional genomics was performed as required. RESULTS: Of the 190 probands, 81 received an accurate genetic diagnosis. All except two of these cases harboured class 4 and/or 5 variants based on the American College of Medical Genetics and Genomics guidelines. We identified phenotypic expansions associated with CACNA1S, CHRNB1, GMPPB and STAC3. We describe a total of 50 novel variants, including a novel missense variant in the recently identified gene for arthrogryposis with brain malformations-SMPD4. CONCLUSIONS: Comprehensive gene panels give a diagnosis for a substantial proportion (42%) of fetal akinesia and arthrogryposis cases, even in an unselected cohort. Recently identified genes account for a relatively large proportion, 32%, of the diagnoses. Diagnostic-research collaboration was critical to the diagnosis and variant interpretation in many cases, facilitated genotype-phenotype expansions and reclassified VUS through functional genomics.

Rehabilitation for ataxia study: protocol for a randomised controlled trial of an outpatient and supported home-based physiotherapy programme for people with hereditary cerebellar ataxia.

INTRODUCTION: Emerging evidence indicates that rehabilitation can improve ataxia, mobility and independence in everyday activities in individuals with hereditary cerebellar ataxia. However, with the rarity of the genetic ataxias and known recruitment challenges in rehabilitation trials, most studies have been underpowered, non-randomised or non-controlled. This study will be the first, appropriately powered randomised controlled trial to examine the efficacy of an outpatient and home-based rehabilitation programme on improving motor function for individuals with hereditary cerebellar ataxia. METHODS AND ANALYSIS: This randomised, single-blind, parallel group trial will compare a 30-week rehabilitation programme to standard care in individuals with hereditary cerebellar ataxia. Eighty individuals with a hereditary cerebellar ataxia, aged 15 years and above, will be recruited. The rehabilitation programme will include 6 weeks of outpatient land and aquatic physiotherapy followed immediately by a 24- week home exercise programme supported with fortnightly physiotherapy sessions. Participants in the standard care group will be asked to continue their usual physical activity. The primary outcome will be the motor domain of the Functional Independence Measure. Secondary outcomes will measure the motor impairment related to ataxia, balance, quality of life and cost-effectiveness. Outcomes will be administered at baseline, 7 weeks, 18 weeks and 30 weeks by a physiotherapist blinded to group allocation. A repeated measures mixed-effects linear regression model will be used to analyse the effect of the treatment group for each of the dependent continuous variables. The primary efficacy analysis will follow the intention-to-treat principle. ETHICS AND DISSEMINATION: The study has been approved by the Monash Health Human Research Ethics Committee (HREC/18/MonH/418) and the Human Research Ethics Committee of the Northern Territory Department of Health and Menzies School of Health Research (2019/3503). Results will be published in peer-reviewed journals, presented at national and/or international conferences and disseminated to Australian ataxia support groups. TRIAL REGISTRATION NUMBER: ACTRN12618000908235.

The genetic landscape of axonal neuropathies in the middle-aged and elderly: Focus on MME.

OBJECTIVE: To test the hypothesis that monogenic neuropathies such as Charcot-Marie-Tooth disease (CMT) contribute to frequent but often unexplained neuropathies in the elderly, we performed genetic analysis of 230 patients with unexplained axonal neuropathies and disease onset ≥35 years. METHODS: We recruited patients, collected clinical data, and conducted whole-exome sequencing (WES; n = 126) and MME single-gene sequencing (n = 104). We further queried WES repositories for MME variants and measured blood levels of the MME-encoded protein neprilysin. RESULTS: In the WES cohort, the overall detection rate for assumed disease-causing variants in genes for CMT or other conditions associated with neuropathies was 18.3% (familial cases 26.4%, apparently sporadic cases 12.3%). MME was most frequently involved and accounted for 34.8% of genetically solved cases. The relevance of MME for late-onset neuropathies was further supported by detection of a comparable proportion of cases in an independent patient sample, preponderance of MME variants among patients compared to population frequencies, retrieval of additional late-onset neuropathy patients with MME variants from WES repositories, and low neprilysin levels in patients' blood samples. Transmission of MME variants was often consistent with an incompletely penetrant autosomal-dominant trait and less frequently with autosomal-recessive inheritance. CONCLUSIONS: A detectable fraction of unexplained late-onset axonal neuropathies is genetically determined, by variants in either CMT genes or genes involved in other conditions that affect the peripheral nerves and can mimic a CMT phenotype. MME variants can act as completely penetrant recessive alleles but also confer dominantly inherited susceptibility to axonal neuropathies in an aging population.

A novel RFC1 repeat motif (ACAGG) in two Asia-Pacific CANVAS families.

Cerebellar ataxia, neuropathy and vestibular areflexia syndrome (CANVAS) is a progressive late-onset, neurological disease. Recently, a pentanucleotide expansion in intron 2 of RFC1 was identified as the genetic cause of CANVAS. We screened an Asian-Pacific cohort for CANVAS and identified a novel RFC1 repeat expansion motif, (ACAGG)exp, in three affected individuals. This motif was associated with additional clinical features including fasciculations and elevated serum creatine kinase. These features have not previously been described in individuals with genetically-confirmed CANVAS. Haplotype analysis showed our patients shared the same core haplotype as previously published, supporting the possibility of a single origin of the RFC1 disease allele. We analysed data from >26 000 genetically diverse individuals in gnomAD to show enrichment of (ACAGG) in non-European populations.

The Impact of Next-Generation Sequencing on the Diagnosis, Treatment, and Prevention of Hereditary Neuromuscular Disorders.

The impact of high-throughput sequencing in genetic neuromuscular disorders cannot be overstated. The ability to rapidly and affordably sequence multiple genes simultaneously has enabled a second golden age of Mendelian disease gene discovery, with flow-on impacts for rapid genetic diagnosis, evidence-based treatment, tailored therapy development, carrier-screening, and prevention of disease recurrence in families. However, there are likely many more neuromuscular disease genes and mechanisms to be discovered. Many patients and families remain without a molecular diagnosis following targeted panel sequencing, clinical exome sequencing, or even genome sequencing. Here we review how massively parallel, or next-generation, sequencing has changed the field of genetic neuromuscular disorders, and anticipate future benefits of recent technological innovations such as RNA-seq implementation and detection of tandem repeat expansions from short-read sequencing.

Genomic sequencing highlights the diverse molecular causes of Perrault syndrome: a peroxisomal disorder (PEX6), metabolic disorders (CLPP, GGPS1), and mtDNA maintenance/translation disorders (LARS2, TFAM).

Perrault syndrome is a rare heterogeneous condition characterised by sensorineural hearing loss and premature ovarian insufficiency. Additional neuromuscular pathology is observed in some patients. There are six genes in which variants are known to cause Perrault syndrome; however, these explain only a minority of cases. We investigated the genetic cause of Perrault syndrome in seven affected individuals from five different families, successfully identifying the cause in four patients. This included previously reported and novel causative variants in known Perrault syndrome genes, CLPP and LARS2, involved in mitochondrial proteolysis and mitochondrial translation, respectively. For the first time, we show that pathogenic variants in PEX6 can present clinically as Perrault syndrome. PEX6 encodes a peroxisomal biogenesis factor, and we demonstrate evidence of peroxisomal dysfunction in patient serum. This study consolidates the clinical overlap between Perrault syndrome and peroxisomal disorders, and highlights the need to consider ovarian function in individuals with atypical/mild peroxisomal disorders. The remaining patients had variants in candidate genes such as TFAM, involved in mtDNA transcription, replication, and packaging, and GGPS1 involved in mevalonate/coenzyme Q10 biosynthesis and whose enzymatic product is required for mouse folliculogenesis. This genomic study highlights the diverse molecular landscape of this poorly understood syndrome.

Targeted gene panel use in 2249 neuromuscular patients: the Australasian referral center experience.

OBJECTIVE: To develop, test, and iterate a comprehensive neuromuscular targeted gene panel in a national referral center. METHODS: We designed two iterations of a comprehensive targeted gene panel for neuromuscular disorders. Version 1 included 336 genes, which was increased to 464 genes in Version 2. Both panels used TargetSeqTM probe-based hybridization for target enrichment followed by Ion Torrent sequencing. Targeted high-coverage sequencing and analysis was performed on 2249 neurology patients from Australia and New Zealand (1054 Version 1, 1195 Version 2) from 2012 to 2015. No selection criteria were used other than referral from a suitable medical specialist (e.g., neurologist or clinical geneticist). Patients were classified into 15 clinical categories based on the clinical diagnosis from the referring clinician. RESULTS: Six hundred and sixty-five patients received a genetic diagnosis (30%). Diagnosed patients were significantly younger that undiagnosed patients (26.4 and 32.5 years, respectively; P = 4.6326E-9). The diagnostic success varied markedly between disease categories. Pathogenic variants in 10 genes explained 38% of the disease burden. Unexpected phenotypic expansions were discovered in multiple cases. Triage of unsolved cases for research exome testing led to the discovery of six new disease genes. INTERPRETATION: A comprehensive targeted diagnostic panel was an effective method for neuromuscular disease diagnosis within the context of an Australasian referral center. Use of smaller disease-specific panels would have precluded diagnosis in many patients and increased cost. Analysis through a centralized laboratory facilitated detection of recurrent, but under-recognized pathogenic variants.

Cerebellar ataxia, neuropathy, vestibular areflexia syndrome due to RFC1 repeat expansion.

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.

Congenital Titinopathy: Comprehensive characterization and pathogenic insights.

OBJECTIVE: Comprehensive clinical characterization of congenital titinopathy to facilitate diagnosis and management of this important emerging disorder. METHODS: Using massively parallel sequencing we identified 30 patients from 27 families with 2 pathogenic nonsense, frameshift and/or splice site TTN mutations in trans. We then undertook a detailed analysis of the clinical, histopathological and imaging features of these patients. RESULTS: All patients had prenatal or early onset hypotonia and/or congenital contractures. None had ophthalmoplegia. Scoliosis and respiratory insufficiency typically developed early and progressed rapidly, whereas limb weakness was often slowly progressive, and usually did not prevent independent walking. Cardiac involvement was present in 46% of patients. Relatives of 2 patients had dilated cardiomyopathy. Creatine kinase levels were normal to moderately elevated. Increased fiber size variation, internalized nuclei and cores were common histopathological abnormalities. Cap-like regions, whorled or ring fibers, and mitochondrial accumulations were also observed. Muscle magnetic resonance imaging showed gluteal, hamstring and calf muscle involvement. Western blot analysis showed a near-normal sized titin protein in all samples. The presence of 2 mutations predicted to impact both N2BA and N2B cardiac isoforms appeared to be associated with greatest risk of cardiac involvement. One-third of patients had 1 mutation predicted to impact exons present in fetal skeletal muscle, but not included within the mature skeletal muscle isoform transcript. This strongly suggests developmental isoforms are involved in the pathogenesis of this congenital/early onset disorder. INTERPRETATION: This detailed clinical reference dataset will greatly facilitate diagnostic confirmation and management of patients, and has provided important insights into disease pathogenesis. Ann Neurol 2018;83:1105-1124.