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• Obscurin is an important protein for muscle contraction, encoded by OBSCN. • Homozygous loss-of-function OBSCN variants predispose individuals to a risk of rhabdomyolysis. • Genetic testing of OBSCN is recommended for patients with unexplained rhabdomyolysis. Obscurin, encoded by the OBSCN gene, is a muscle protein consisting of three main splice isoforms, obscurin-A, obscurin-B, and obscurin kinase-only protein (also known as KIAA1639 or Obsc-kin). Obscurin is located at the M-band and Z-disks and interacts with titin and myomesin. It plays an important role in the stability and maintenance of the A- and M-bands and the subsarcolemmal organization of the microtubule network. Furthermore, obscurin is involved in Ca2+ regulation and sarcoplasmic reticulum function and is connected to several other muscle proteins. OBSCN gene variants have been reported to be relatively common in inherited cardiomyopathies. Here we reported two young patients with a history of cramps, myalgia, exercise intolerance, rhabdomyolysis, and myoglobinuria without any evidence of concomitant cardiomyopathy in association with novel OBSCN variants (c.24822C>A and c.2653+1G>C). Obscurin-deficient muscle fibers seem to have increased susceptibility to damage triggered by exercise that may lead to rhabdomyolysis. More studies are needed to clarify the diverse clinical phenotypes and the pathophysiology of OBSCN gene variants. [ABSTRACT FROM AUTHOR]

DNAJ/HSP40 co-chaperones are integral to the chaperone network, bind client proteins and recruit them to HSP70 for folding. We performed exome sequencing on patients with a presumed hereditary muscle disease and no genetic diagnosis. This identified four individuals from three unrelated families carrying an unreported homozygous stop gain (c.856A > T; p.Lys286Ter), or homozygous missense variants (c.74G > A; p.Arg25Gln and c.785 T > C; p.Leu262Ser) in DNAJB4. Affected patients presented with axial rigidity and early respiratory failure requiring ventilator support between the 1st and 4th decade of life. Selective involvement of the semitendinosus and biceps femoris muscles was seen on MRI scans of the thigh. On biopsy, muscle was myopathic with angular fibers, protein inclusions and occasional rimmed vacuoles. DNAJB4 normally localizes to the Z-disc and was absent from muscle and fibroblasts of affected patients supporting a loss of function. Functional studies confirmed that the p.Lys286Ter and p.Leu262Ser mutant proteins are rapidly degraded in cells. In contrast, the p.Arg25Gln mutant protein is stable but failed to complement for DNAJB function in yeast, disaggregate client proteins or protect from heat shock-induced cell death consistent with its loss of function. DNAJB4 knockout mice had muscle weakness and fiber atrophy with prominent diaphragm involvement and kyphosis. DNAJB4 knockout muscle and myotubes had myofibrillar disorganization and accumulated Z-disc proteins and protein chaperones. These data demonstrate a novel chaperonopathy associated with DNAJB4 causing a myopathy with early respiratory failure. DNAJB4 loss of function variants may lead to the accumulation of DNAJB4 client proteins resulting in muscle dysfunction and degeneration. [ABSTRACT FROM AUTHOR]

We have developed a simple diagnostic immunoassay to detect dysferlin expression on neutrophils in peripheral blood films (PBF) and tested its accuracy on samples obtained from healthy controls, patients with genetic diagnosis of dysferlinopathy and individuals with alternative genetic diagnoses. Further studies including participants in whom the disease status is unknown are required to predict how the test is likely to perform in clinical practice as a pre-screening for mutations in the DYSF gene and to validate the assay as a functional assessment for variants of uncertain significance (VUS). This study will have access to 200 samples from individuals recruited in the LATIN-SEQ project. LATIN-SEQ is an international research collaboration coordinated by the John Walton Muscular Dystrophy Research Centre in Newcastle upon Tyne, UK, that applies targeted whole exome sequencing to >2,000 patients with undiagnosed muscle weakness in Latin America. On completion of on-site training sessions for sample collection and quality check, PBF will be collected from adult participants who had onset of muscle weakness after age 15. PBFs will be assessed for quality and dysferlin expression in neutrophils will be blind-scored. Protein data will be compared to DYSF genetic status of participants when WES result interpretation takes place through LATIN-SEQ. The outcome of the study will demonstrate whether this assay is suitable to large population screening and as a test for VUS' pathogenicity. The study will also be useful to assess the requirements for a broader training program to ensure the reproducibility of the assay in diagnostic centers worldwide. [ABSTRACT FROM AUTHOR]

Autosomal dominant mutations in the RNA binding protein HNRPDL cause LGMDD3. LGMDD3 is a late onset dystrophy with vacuolar and aggregate myopathology. To date, two pathogenic variants p.Asp378Asn or p.Asp378His have been reported from 7 families of Spanish, Italian, South American and Chinese descent. Notably, these variants affect the same residue within the intrinsically disordered C-terminal domain of the protein. In this study, we report a USA family with a previously reported p.Asp378His variant. In addition, we report a patient with early adult onset LGMD and scapular winging carrying an unreported homozygous p.Tyr377Cys variant. Segregation confirmed that the patient's parents and sibling were unaffected yet heterozygous for the p.Tyr377Cys variant suggesting that this variant, unlike previous variants, requires homozygosity to be pathogenic. Muscle pathology from all affected patients had prominent rimmed vacuoles. Biophysical modeling of each variant demonstrates an increase in aggregation potential. Moreover, functional studies expressing each of the three variants in cells demonstrates an increased propensity to form stress granules at baseline and when treated with cellular stress as compared to HNRPDL-WT expression. Similar to another RNA binding protein associated with Welander distal myopathy, TIA1, that has been demonstrated to cause a similar disease in the heterozygous and homozygous state, we propose a gain of function mechanism. This study expands the genotype and phenotype of LGMDD3. In addition, it provides evidence that HNRPDL can cause an LGMD phenotype in the heterozygous and homozygous state. [ABSTRACT FROM AUTHOR]

Abstract: Congenital myasthenic syndromes (CMS) are a group of rare disorders that cause fatigable muscle weakness due to defective signal transmission at the neuromuscular junction, a specialized synapse between peripheral motor neurons and their target muscle fibers. There are now over 30 causative genes that have been reported for CMS. Of these, there are 10 that are associated with a limb‐girdle pattern of muscle weakness and are thus classed as LG‐CMS. Next‐generation sequencing and advanced methods of data sharing are likely to uncover further genes that are associated with similar clinical phenotypes, contributing to better diagnosis and effective treatment of LG‐CMS patients. This review highlights clinical and pathological hallmarks of LG‐CMS in relation to the underlying genetic defects and pathways. Tailored animal and cell models are essential to elucidate the exact function and pathomechanisms at the neuromuscular synapse that underlie LG‐CMS. The integration of genomics and proteomics data derived from these models and patients reveals new and often unexpected insights that are relevant beyond the rare genetic disorder of LG‐CMS and may extend to the functioning of mammalian synapses in health and disease more generally. [ABSTRACT FROM AUTHOR]

Congenital myasthenic syndromes (CMS) are a heterogeneous group of genetic disorders, all of which impair neuromuscular transmission. Epidemiological data and frequencies of gene mutations are scarce in the literature. Here we describe the molecular genetic and clinical findings of sixty-four genetically confirmed CMS patients from Spain. Thirty-six mutations in the CHRNE, RAPSN, COLQ, GFPT1, DOK7, CHRNG, GMPPB, CHAT, CHRNA1, and CHRNB1 genes were identified in our patients, with five of them not reported so far. These data provide an overview on the relative frequencies of the different CMS subtypes in a large Spanish population. CHRNE mutations are the most common cause of CMS in Spain, accounting for 27% of the total. The second most common are RAPSN mutations. We found a higher rate of GFPT1 mutations in comparison with other populations. Remarkably, several founder mutations made a large contribution to CMS in Spain: RAPSN c.264C > A (p.Asn88Lys), CHRNE c.130insG (Glu44Glyfs*3), CHRNE c.1353insG (p.Asn542Gluf*4), DOK7 c.1124_1127dup (p.Ala378Serfs*30), and particularly frequent in Spain in comparison with other populations, COLQ c.1289A > C (p.Tyr430Ser). Furthermore, we describe phenotypes and distinguishing clinical signs associated with the various CMS genes which might help to identify specific CMS subtypes to guide diagnosis and management. [ABSTRACT FROM AUTHOR]

Congenital myasthenic syndromes (CMS) are a rare group of inherited disorders caused by gene defects associated with the neuromuscular junction and potentially treatable with commonly available medications such as acetylcholinesterase inhibitors and β2 adrenergic receptor agonists. In this study, we identified and genetically characterized the largest cohort of CMS patients from India to date. Genetic testing of clinically suspected patients evaluated in a South Indian hospital during the period 2014–19 was carried out by standard diagnostic gene panel testing or using a two-step method that included hotspot screening followed by whole-exome sequencing. In total, 156 genetically diagnosed patients (141 families) were characterized and the mutational spectrum and genotype-phenotype correlation described. Overall, 87 males and 69 females were evaluated, with the age of onset ranging from congenital to fourth decade (mean 6.6 ± 9.8 years). The mean age at diagnosis was 19 ± 12.8 (1–56 years), with a mean diagnostic delay of 12.5 ± 9.9 (0–49 years). Disease-causing variants in 17 CMS-associated genes were identified in 132 families (93.6%), while in nine families (6.4%), variants in genes not associated with CMS were found. Overall, postsynaptic defects were most common (62.4%), followed by glycosylation defects (21.3%), synaptic basal lamina genes (4.3%) and presynaptic defects (2.8%). Other genes found to cause neuromuscular junction defects (DES , TEFM) in our cohort accounted for 2.8%. Among the individual CMS genes, the most commonly affected gene was CHRNE (39.4%), followed by DOK7 (14.4%), DPAGT1 (9.8%), GFPT1 (7.6%), MUSK (6.1%), GMPPB (5.3%) and COLQ (4.5%). We identified 22 recurrent variants in this study, out of which eight were found to be geographically specific to the Indian subcontinent. Apart from the known common CHRNE variants p.E443Kfs*64 (11.4%) and DOK7 p.A378Sfs*30 (9.3%), we identified seven novel recurrent variants specific to this cohort, including DPAGT1 p.T380I and DES c.1023+5G>A, for which founder haplotypes are suspected. This study highlights the geographic differences in the frequencies of various causative CMS genes and underlines the increasing significance of glycosylation genes (DPAGT1 , GFPT1 and GMPPB) as a cause of neuromuscular junction defects. Myopathy and muscular dystrophy genes such as GMPPB and DES , presenting as gradually progressive limb girdle CMS, expand the phenotypic spectrum. The novel genes MACF1 and TEFM identified in this cohort add to the expanding list of genes with new mechanisms causing neuromuscular junction defects. [ABSTRACT FROM AUTHOR]

Background: Late-onset Pompe disease is a rare genetic neuromuscular disorder caused by a primary deficiency of α-glucosidase and the associated accumulation of glycogen in lysosomal vacuoles. The deficiency of α-glucosidase can often be detected using an inexpensive and readily accessible dried blood spot test when Pompe disease is suspected. Like several neuromuscular disorders, Pompe disease typically presents with progressive weakness of limb-girdle muscles and respiratory insufficiency. Due to the phenotypic heterogeneity of these disorders, however, it is often difficult for clinicians to reach a diagnosis for patients with Pompe disease. Six hundred and six patients from a European population were recruited onto our study. Inclusion criteria stipulated that index cases must present with limb-girdle weakness or elevated serum creatine kinase activity. Whole exome sequencing with at least 250 ng DNA was completed using an Illumina exome capture and a 38 Mb baited target. A panel of 169 candidate genes for limb-girdle weakness was analysed for disease-causing variants. Results: A total of 35 variants within GAA were detected. Ten distinct variants in eight unrelated index cases (and four siblings not sequenced in our study) were considered disease-causing, with the patients presenting with heterogeneous phenotypes. The eight unrelated individuals were compound heterozygotes for two variants. Six patients carried the intronic splice site c.-13 T > G transversion and two of the six patients also carried the exonic p.Glu176ArgfsTer45 frameshift. Four of the ten variants were novel in their association with Pompe disease. Conclusions: Here, we highlight the advantage of using whole exome sequencing as a tool for detecting, diagnosing and treating patients with rare, clinically variable genetic disorders. [ABSTRACT FROM AUTHOR]

Mutations in the gene encoding the giant skeletal muscle protein titin are associated with a variety of muscle disorders, including recessive congenital myopathies ±cardiomyopathy, limb girdle muscular dystrophy (LGMD) and late onset dominant distal myopathy. Heterozygous truncating mutations have also been linked to dilated cardiomyopathy. The phenotypic spectrum of titinopathies is emerging and expanding, as next generation sequencing techniques make this large gene amenable to sequencing. We undertook whole exome sequencing in four individuals with LGMD. An essential splice site mutation, previously reported in dilated cardiomyopathy, was identified in all families in combination with a second truncating mutation. Affected individuals presented with childhood onset proximal weakness associated with joint contractures and elevated CK. Cardiac dysfunction was present in two individuals. Muscle biopsy showed increased internal nuclei and immunoblotting identified reduction or absence of calpain-3 and demonstrated a marked reduction of C-terminal titin fragments. We confirm the co-occurrence of cardiac and skeletal myopathies associated with recessive truncating titin mutations. Compound heterozygosity of a truncating mutation previously associated with dilated cardiomyopathy and a ‘second truncation’ in TTN was identified as causative in our skeletal myopathy patients. These findings add to the complexity of interpretation and genetic counselling for titin mutations. [ABSTRACT FROM AUTHOR]

Congenital myasthenic syndromes are a group of rare and genetically heterogenous disorders resulting from defects in the structure and function of the neuromuscular junction. Patients with congenital myasthenic syndrome exhibit fatigable muscle weakness with a variety of accompanying phenotypes depending on the protein affected. A cohort of patients with a clinical diagnosis of congenital myasthenic syndrome that lacked a genetic diagnosis underwent whole exome sequencing in order to identify genetic causation. Missense biallelic mutations in the MYO9A gene, encoding an unconventional myosin, were identified in two unrelated families. Depletion of MYO9A in NSC-34 cells revealed a direct effect of MYO9A on neuronal branching and axon guidance. Morpholino-mediated knockdown of the two MYO9A orthologues in zebrafish, myo9aa/ab, demonstrated a requirement for MYO9A in the formation of the neuromuscular junction during development. The morphants displayed shortened and abnormally branched motor axons, lack of movement within the chorion and abnormal swimming in response to tactile stimulation. We therefore conclude that MYO9A deficiency may affect the presynaptic motor axon, manifesting in congenital myasthenic syndrome. These results highlight the involvement of unconventional myosins in motor axon functionality, as well as the need to look outside traditional neuromuscular junction-specific proteins for further congenital myasthenic syndrome candidate genes. [ABSTRACT FROM AUTHOR]

Objective: Rare variants in certain transcription factors involved in cardiac development cause Mendelian forms of congenital heart disease. The purpose of this study was to systematically assess the frequency of rare transcription factor variants in sporadic patients with the cardiac outflow tract malformation tetralogy of Fallot (TOF). Methods and Results: We sequenced the coding, 5′UTR, and 3′UTR regions of twelve transcription factor genes implicated in cardiac outflow tract development (NKX2.5, GATA4, ISL1, TBX20, MEF2C, BOP/SMYD1, HAND2, FOXC1, FOXC2, FOXH, FOXA2 and TBX1) in 93 non-syndromic, non-Mendelian TOF cases. We also analysed Illumina Human 660W-Quad SNP Array data for copy number variants in these genes; none were detected. Four of the rare variants detected have previously been shown to affect transactivation in in vitro reporter assays: FOXC1 p.P297S, FOXC2 p.Q444R, FOXH1 p.S113T and TBX1 p.P43_G61del PPPPRYDPCAAAAPGAPGP. Two further rare variants, HAND2 p.A25_A26insAA and FOXC1 p.G378_G380delGGG, A488_491delAAAA, affected transactivation in in vitro reporter assays. Each of these six functionally significant variants was present in a single patient in the heterozygous state; each of the four for which parental samples were available were maternally inherited. Thus in the 93 TOF cases we identified six functionally significant mutations in the secondary heart field transcriptional network. Significance: This study indicates that rare genetic variants in the secondary heart field transcriptional network with functional effects on protein function occur in 3–13% of patients with TOF. This is the first report of a functionally significant HAND2 mutation in a patient with congenital heart disease. [ABSTRACT FROM AUTHOR]

Background Tetralogy of Fallot (TOF) is common in individuals with hemizygous deletions of chromosome 22q11.2 that remove the cardiac transcription factor TBX1. Objective To assess the contribution of common and rare TBX1 genetic variants to TOF. Design Rare TBX1 variants were sought by resequencing coding exons and splice-site boundaries. Common TBX1 variants were investigated by genotyping 20 haplotype-tagging SNPs capturing all the common variations present at the locus. Association analysis was performed using the program UNPHASED. Patients TBX1 exons were sequenced in 93 patients with non-syndromic TOF. Single nucleotide polymorphism analysis was performed in 356 patients with TOF, their parents and healthy controls. Results Three novel variants not present in 1000 chromosomes from healthy ethnically matched controls were identified. One of these variants, an in-frame 57 base-pair deletion in the third exon which removed 19 evolutionarily conserved residues, decreased transcriptional activity by 40% in a dual luciferase assay (p=0.008). Protein expression studies demonstrated that this mutation affected TBX1 protein stability. After correction for multiple comparisons, no significant associations between common genetic variants and TOF susceptibility were found. Conclusion This study demonstrates that rare TBX1 variants with functional consequences are present in a small proportion of non-syndromic TOF. [ABSTRACT FROM AUTHOR]

Consanguineous marriages have a prevalence rate of 24% in Turkey. These carry an increased risk of autosomal recessive genetic conditions, leading to severe disability or premature death, with a significant health and economic burden. A definitive molecular diagnosis could not be achieved in these children previously, as infrastructures and access to sophisticated diagnostic options were limited. We studied the cause of neurogenetic disease in 246 children from 190 consanguineous families recruited in three Turkish hospitals between 2016 and 2020. All patients underwent deep phenotyping and trio whole exome sequencing, and data were integrated in advanced international bioinformatics platforms. We detected causative variants in 119 known disease genes in 72% of families. Due to overlapping phenotypes 52% of the confirmed genetic diagnoses would have been missed on targeted diagnostic gene panels. Likely pathogenic variants in 27 novel genes in 14% of the families increased the diagnostic yield to 86%. Eighty-two per cent of causative variants (141/172) were homozygous, 11 of which were detected in genes previously only associated with autosomal dominant inheritance. Eight families carried two pathogenic variants in different disease genes. De novo (9.3%), X-linked recessive (5.2%) and compound heterozygous (3.5%) variants were less frequent compared to non-consanguineous populations. This cohort provided a unique opportunity to better understand the genetic characteristics of neurogenetic diseases in a consanguineous population. Contrary to what may be expected, causative variants were often not on the longest run of homozygosity and the diagnostic yield was lower in families with the highest degree of consanguinity, due to the high number of homozygous variants in these patients. Pathway analysis highlighted that protein synthesis/degradation defects and metabolic diseases are the most common pathways underlying paediatric neurogenetic disease. In our cohort 164 families (86%) received a diagnosis, enabling prevention of transmission and targeted treatments in 24 patients (10%). We generated an important body of genomic data with lasting impacts on the health and wellbeing of consanguineous families and economic benefit for the healthcare system in Turkey and elsewhere. We demonstrate that an untargeted next generation sequencing approach is far superior to a more targeted gene panel approach, and can be performed without specialized bioinformatics knowledge by clinicians using established pipelines in populations with high rates of consanguinity. [ABSTRACT FROM AUTHOR]

Alterations in RNA-splicing are a molecular hallmark of several neurological diseases, including muscular dystrophies, where mutations in genes involved in RNA metabolism or characterized by alterations in RNA splicing have been described. Here, we present five patients from two unrelated families with a limb-girdle muscular dystrophy (LGMD) phenotype carrying a biallelic variant in SNUPN gene. Snurportin-1, the protein encoded by SNUPN , plays an important role in the nuclear transport of small nuclear ribonucleoproteins (snRNPs), essential components of the spliceosome. We combine deep phenotyping, including clinical features, histopathology and muscle MRI, with functional studies in patient-derived cells and muscle biopsies to demonstrate that variants in SNUPN are the cause of a new type of LGMD according to current definition. Moreover, an in vivo model in Drosophila melanogaster further supports the relevance of Snurportin-1 in muscle. SNUPN patients show a similar phenotype characterized by proximal weakness starting in childhood, restrictive respiratory dysfunction and prominent contractures, although inter-individual variability in terms of severity even in individuals from the same family was found. Muscle biopsy showed myofibrillar-like features consisting of myotilin deposits and Z-disc disorganization. MRI showed predominant impairment of paravertebral, vasti, sartorius, gracilis, peroneal and medial gastrocnemius muscles. Conservation and structural analyses of Snurportin-1 p.Ile309Ser variant suggest an effect in nuclear-cytosol snRNP trafficking. In patient-derived fibroblasts and muscle, cytoplasmic accumulation of snRNP components is observed, while total expression of Snurportin-1 and snRNPs remains unchanged, which demonstrates a functional impact of SNUPN variant in snRNP metabolism. Furthermore, RNA-splicing analysis in patients' muscle showed widespread splicing deregulation, in particular in genes relevant for muscle development and splicing factors that participate in the early steps of spliceosome assembly. In conclusion, we report that SNUPN variants are a new cause of limb girdle muscular dystrophy with specific clinical, histopathological and imaging features, supporting SNUPN as a new gene to be included in genetic testing of myopathies. These results further support the relevance of splicing-related proteins in muscle disorders. [ABSTRACT FROM AUTHOR]

Duchenne and Becker muscular dystrophies, caused by pathogenic variants in DMD, are the most common inherited neuromuscular conditions in childhood. These diseases follow an X-linked recessive inheritance pattern, and mainly males are affected. The most prevalent pathogenic variants in the DMD gene are copy number variants (CNVs), and most patients achieve their genetic diagnosis through Multiplex Ligation-dependent Probe Amplification (MLPA) or exome sequencing. Here, we investigated a female patient presenting with muscular dystrophy who remained genetically undiagnosed after MLPA and exome sequencing. RNA sequencing (RNAseq) from the patient's muscle biopsy identified an 85% reduction in DMD expression compared to 116 muscle samples included in the cohort. A de novo balanced translocation between chromosome 17 and the X chromosome (t(X;17)(p21.1;q23.2)) disrupting the DMD and BCAS3 genes was identified through trio whole genome sequencing (WGS). The combined analysis of RNAseq and WGS played a crucial role in the detection and characterisation of the disease-causing variant in this patient, who had been undiagnosed for over two decades. This case illustrates the diagnostic odyssey of female DMD patients with complex structural variants that are not detected by current panel or exome sequencing analysis. [ABSTRACT FROM AUTHOR]

Cytoplasmic and nuclear iron-sulfur (Fe-S) enzymes that are essential for genome maintenance and replication depend on the cytoplasmic Fe-S assembly (CIA) machinery for cluster acquisition. The core of the CIA machinery consists of a complex of CIAO1, MMS19 and FAM96B. The physiological consequences of loss of function in the components of the CIA pathway have thus far remained uncharacterized. Our study revealed that patients with biallelic loss of function in CIAO1 developed proximal and axial muscle weakness, fluctuating creatine kinase elevation, and respiratory insufficiency. In addition, they presented with CNS symptoms including learning difficulties and neurobehavioral comorbidities, along with iron deposition in deep brain nuclei, mild normocytic to macrocytic anemia, and gastrointestinal symptoms. Mutational analysis revealed reduced stability of the variants compared with WT CIAO1. Functional assays demonstrated failure of the variants identified in patients to recruit Fe-S recipient proteins, resulting in compromised activities of DNA helicases, polymerases, and repair enzymes that rely on the CIA complex to acquire their Fe-S cofactors. Lentivirus-mediated restoration of CIAO1 expression reversed all patient-derived cellular abnormalities. Our study identifies CIAO1 as a human disease gene and provides insights into the broader implications of the cytosolic Fe-S assembly pathway in human health and disease. [ABSTRACT FROM AUTHOR]

BACKGROUND: Genetic variants that cause rare disorders may remain elusive even after expansive testing, such as exome sequencing. The diagnostic yield of genome sequencing, particularly after a negative evaluation, remains poorly defined. METHODS: We sequenced and analyzed the genomes of families with diverse phenotypes who were suspected to have a rare monogenic disease and for whom genetic testing had not revealed a diagnosis, as well as the genomes of a replication cohort at an independent clinical center. RESULTS: We sequenced the genomes of 822 families (744 in the initial cohort and 78 in the replication cohort) and made a molecular diagnosis in 218 of 744 families (29.3%). Of the 218 families, 61 (28.0%) -- 8.2% of families in the initial cohort -- had variants that required genome sequencing for identification, including coding variants, intronic variants, small structural variants, copy-neutral inversions, complex rearrangements, and tandem repeat expansions. Most families in which a molecular diagnosis was made after previous nondiagnostic exome sequencing (63.5%) had variants that could be detected by reanalysis of the exome-sequence data (53.4%) or by additional analytic methods, such as copy-number variant calling, to exome-sequence data (10.8%). We obtained similar results in the replication cohort: in 33% of the families in which a molecular diagnosis was made, or 8% of the cohort, genome sequencing was required, which showed the applicability of these findings to both research and clinical environments. CONCLUSIONS: The diagnostic yield of genome sequencing in a large, diverse research cohort and in a small clinical cohort of persons who had previously undergone genetic testing was approximately 8% and included several types of pathogenic variation that had not previously been detected by means of exome sequencing or other techniques. (Funded by the National Human Genome Research Institute and others.). [ABSTRACT FROM AUTHOR]

• We report a recurrent intronic homozygous c.1023+5G> A desmin mutation in indian patients. • Second report of recessive desminopathy associated with mixed limb girdle CMS – myofibrillar myopathy. • Partial loss of desmin leading to NMJ dysfunction and CMS phenotype. • Leaky splice site with intron retention leads to partial desmin loss. Recessive desminopathies are rare and often present as severe early-onset myopathy. Here we report a milder phenotype in three unrelated patients from southern India (2 M, 1F) aged 16, 21, and 22 years, who presented with childhood-onset, gradually progressive, fatigable limb-girdle weakness, ptosis, speech and swallowing difficulties, without cardiac involvement. Serum creatine kinase was elevated, and repetitive nerve stimulation showed decrement in all. Clinical improvement was noted with pyridostigmine and salbutamol in two patients. All three patients had a homozygous substitution in intron 5: DES(NM_001927.4):c.1023+5G> A , predicted to cause a donor splice site defect. Muscle biopsy with ultrastructural analysis suggested myopathy with myofibrillar disarray, and immunohistochemistry showed partial loss of desmin with some residual staining, while western blot analysis showed reduced desmin. RT-PCR of patient muscle RNA revealed two transcripts: a reduced normal desmin transcript and a larger abnormal transcript suggesting leaky splicing at the intron 5 donor site. Sequencing of the PCR products confirmed the inclusion of intron 5 in the longer transcript, predicted to cause a premature stop codon. Thus, we provide evidence for a leaky splice site causing partial loss of desmin associated with a unique phenotypic presentation of a milder form of desmin-related recessive myopathy overlapping with congenital myasthenic syndrome. [ABSTRACT FROM AUTHOR]

The RASopathies are a group of genetic rare diseases caused by mutations affecting genes involved in the RAS/MAPK (RAS–mitogen activated protein kinase) pathway. Among them, PTPN11 pathogenic variants are responsible for approximately 50% of Noonan syndrome (NS) cases and, albeit to a lesser extent, of Leopard syndrome (LPRD1), which present a few overlapping clinical features, such as facial dysmorphism, developmental delay, cardiac defects, and skeletal deformities. Motor impairment and decreased muscle strength have been recently reported. The etiology of the muscle involvement in these disorders is still not clear but probably multifactorial, considering the role of the RAS/MAPK pathway in skeletal muscle development and Acetylcholine Receptors (AChR) clustering at the neuromuscular junction (NMJ). We report, herein, four unrelated children carrying three different heterozygous mutations in the PTPN11 gene. Intriguingly, their phenotypic features first led to a clinical suspicion of congenital myasthenic syndrome (CMS), due to exercise-induced fatigability with a variable degree of muscle weakness, and serum proteomic profiling compatible with a NMJ defect. Moreover, muscle fatigue improved after treatment with CMS-specific medication. Although the link between PTPN11 gene and neuromuscular transmission is unconfirmed, an increasing number of patients with RASopathies are affected by muscle weakness and fatigability. Hence, NS or LPDR1 should be considered in children with suspected CMS but negative genetic workup for known CMS genes or additional symptoms indicative of NS, such as facial dysmorphism or intellectual disability. [ABSTRACT FROM AUTHOR]

• Neuromuscular junction proteins are essential for transmission effectiveness. • Cholinergic receptor nicotinic δ subunit mutations are rare but possibly fatal. • Congenital myasthenic syndromes neonatal onset can be severe and life-threatening. • Myasthenic syndromes diagnosis is tricky, extensive genetic approach is required. Congenital myasthenic syndromes (CMS) are a group of inherited disorders caused by mutations in genes encoding proteins essential for neuromuscular transmission. CMS is characterized by fatigable muscle weakness with onset at birth or in early childhood; rarely, symptoms may present later. The most frequently involved proteins are choline acetyltransferase, the endplate species of acetylcholinesterase and the acetylcholine receptor subunits. Defects in the cholinergic receptor nicotinic delta subunit (CHRND) are a rare cause for CMS but they should be considered in patients with a severe, early onset disease, with respiratory distress. We describe two sisters, clinically and genetically diagnosed with CMS, carrying two heteroallelic variants in the CHRND gene: c.730C>T; p.(Arg244Cys) and c.1304T>C; p.(Leu435Pro). The first variant has already been described yet no clinical relevance has been proved; the second one, is a novel variant documented here for the first time. These two cases expand the clinical spectrum of CMS linked to CHRND mutations. [ABSTRACT FROM AUTHOR]

Objective: The Popeye domain containing 3 (POPDC3) gene encodes a membrane protein involved in cyclic adenosine monophosphate (cAMP) signaling. Besides gastric cancer, no disease association has been described. We describe a new muscular dystrophy associated with this gene.Methods: We screened 1,500 patients with unclassified limb girdle weakness or hyperCKemia for pathogenic POPDC3 variants. Five patients carrying POPDC3 variants were examined by muscle magnetic resonance imaging (MRI), muscle biopsy, and cardiac examination. We performed functional analyses in a zebrafish popdc3 knockdown model and heterologous expression of the mutant proteins in Xenopus laevis oocytes to measure TREK-1 current.Results: We identified homozygous POPDC3 missense variants (p.Leu155His, p.Leu217Phe, and p.Arg261Gln) in 5 patients from 3 ethnically distinct families. Variants affected highly conserved residues in the Popeye (p.Leu155 and p.Leu217) and carboxy-terminal (p.Arg261) domains. The variants were almost absent from control populations. Probands' muscle biopsies were dystrophic, and serum creatine kinase levels were 1,050 to 9,200U/l. Muscle weakness was proximal with adulthood onset in most patients and affected lower earlier than upper limbs. Muscle MRI revealed fat replacement of paraspinal and proximal leg muscles; cardiac investigations were unremarkable. Knockdown of popdc3 in zebrafish, using 2 different splice-site blocking morpholinos, resulted in larvae with tail curling and dystrophic muscle features. All 3 mutants cloned in Xenopus oocytes caused an aberrant modulation of the mechano-gated potassium channel, TREK-1.Interpretation: Our findings point to an important role of POPDC3 for skeletal muscle function and suggest that pathogenic variants in POPDC3 are responsible for a novel type of autosomal recessive limb girdle muscular dystrophy. ANN NEUROL 2019;86:832-843. [ABSTRACT FROM AUTHOR]

Background: Late-onset Pompe disease is a rare genetic neuromuscular disorder caused by a primary deficiency of α-glucosidase and the associated accumulation of glycogen in lysosomal vacuoles. The deficiency of α-glucosidase can often be detected using an inexpensive and readily accessible dried blood spot test when Pompe disease is suspected. Like several neuromuscular disorders, Pompe disease typically presents with progressive weakness of limb-girdle muscles and respiratory insufficiency. Due to the phenotypic heterogeneity of these disorders, however, it is often difficult for clinicians to reach a diagnosis for patients with Pompe disease. Six hundred and six patients from a European population were recruited onto our study. Inclusion criteria stipulated that index cases must present with limb-girdle weakness or elevated serum creatine kinase activity. Whole exome sequencing with at least 250 ng DNA was completed using an Illumina exome capture and a 38 Mb baited target. A panel of 169 candidate genes for limb-girdle weakness was analysed for disease-causing variants.Results: A total of 35 variants within GAA were detected. Ten distinct variants in eight unrelated index cases (and four siblings not sequenced in our study) were considered disease-causing, with the patients presenting with heterogeneous phenotypes. The eight unrelated individuals were compound heterozygotes for two variants. Six patients carried the intronic splice site c.-13 T > G transversion and two of the six patients also carried the exonic p.Glu176ArgfsTer45 frameshift. Four of the ten variants were novel in their association with Pompe disease.Conclusions: Here, we highlight the advantage of using whole exome sequencing as a tool for detecting, diagnosing and treating patients with rare, clinically variable genetic disorders. [ABSTRACT FROM AUTHOR]

• Muscle MRI reveals consistent features shared by patients with distal hereditary motor neuropathies that can be helpful for the diagnosis process. • A reticular pattern of fat replacement progressing towards fat islands is the most common finding observed across all patients. • Distal muscles of the lower legs are the most affected, especially the posterior compartment of the legs including soleus and gastrocnemius muscles. • A distal to proximal gradient of fat replacement is frequent in this cohort of patients, suggesting there is a dying-back phenomenon of axon degeneration. • Hyperintensities in STIR signal are common, following a distal to proximal gradient in many cases suggesting that there is an active process of denervation in several patients. Distal motor neuropathies (dHMN) are an heterogenous group of diseases characterized by progressive muscle weakness affecting predominantly the distal muscles of the lower and upper limbs. Our aim was to study the imaging features and pattern of muscle involvement in muscle magnetic resonance imaging (MRI) in dHMN patients of suspected genetic origin (dHMN). We conducted a retrospective study collecting clinical, genetic and muscle imaging data. Muscle MRI included T1-weighted and T2 weighted Short Tau Inversion Recovery images (STIR-T2w) sequences. Muscle replacement by fat was quantified using the Mercuri score. Identification of selective patterns of involvement was performed using hierarchical clustering. Eighty-four patients with diagnosis of dHMN were studied. Fat replacement was predominant in the distal lower leg muscles (82/84 cases), although also affected thigh and pelvis muscles. Asymmetric involvement was present in 29% of patients. The superficial posterior compartment of the leg, including the soleus and gastrocnemius muscles, was the most affected area (77/84). We observed a reticular pattern of fatty replacement progressing towards what is commonly known as "muscle islands" in 79.8%. Hyperintensities in STIR-T2w were observed in 78.6% patients mainly in distal leg muscles. Besides features common to all individuals, we identified and describe a pattern of muscle fat replacement characteristic of BICD2, HSPB1 and DYNC1H1 patients. We conclude that muscle MRI of patients with suspected dHMN reveals common features helpful in diagnosis process. [ABSTRACT FROM AUTHOR]

Filamin-A-interacting protein 1 (FILIP1) is a structural protein that is involved in neuronal and muscle function and integrity and interacts with FLNa and FLNc. Pathogenic variants in filamin-encoding genes have been linked to neurological disorders (FLNA) and muscle diseases characterized by myofibrillar perturbations (FLNC), but human diseases associated with FILIP1 variants have not yet been described. Here, we report on five patients from four unrelated consanguineous families with homozygous FILIP1 variants (two nonsense and two missense). Functional studies indicated altered stability of the FILIP1 protein carrying the p.[Pro1133Leu] variant. Patients exhibit a broad spectrum of neurological symptoms including brain malformations, neurodevelopmental delay, muscle weakness and pathology and dysmorphic features. Electron and immunofluorescence microscopy on the muscle biopsy derived from the patient harbouring the homozygous p.[Pro1133Leu] missense variant revealed core-like zones of myofibrillar disintegration, autophagic vacuoles and accumulation of FLNc. Proteomic studies on the fibroblasts derived from the same patient showed dysregulation of a variety of proteins including FLNc and alpha-B-crystallin, a finding (confirmed by immunofluorescence) which is in line with the manifestation of symptoms associated with the syndromic phenotype of FILIP1opathy. The combined findings of this study show that the loss of functional FILIP1 leads to a recessive disorder characterized by neurological and muscular manifestations as well as dysmorphic features accompanied by perturbed proteostasis and myopathology. [ABSTRACT FROM AUTHOR]

Highlights • Our study expands the knowledge on clinical presentation of patients with mutations in the SGCA gene. • This report widens the diagnostic algorithm available for patients with primary axial myopathy. • Clinical data notably enriches the interpretation of exome analysis. • Partial expression of alpha-sarcoglycan could be enough to modify the phenotype. Abstract Mutations in the SGCA gene cause limb girdle muscular dystrophy type 2D (LGMD2D). We report a family with three affected siblings with a mild phenotype consisting of late onset glutei and axial muscle weakness produced by a new mutation in the SGCA gene leading to a partial expression of the alpha-sarcoglycan protein. The MRI showed muscle atrophy involving paraspinal, pelvic and thigh muscles and a dystrophic pattern was observed in the muscle biopsy. Exome sequencing revealed a homozygous intronic deletion of SGCA and mRNA analysis showed the presence of three different transcripts. The presence, though in a lower proportion, of wild type transcript leads to a milder presentation of the disease. Although clinical symptoms did not entirely correspond with a sarcoglycanopathy, a compatible muscle MRI drove us to look for changes in the sarcoglycan genes. These cases are an example of how clinical, radiological and pathological data enriches the interpretation of exome analysis. [ABSTRACT FROM AUTHOR]

Highlights • We report two novel compound heterozygous mutations in the POMK gene. • The mutations cause limb-girdle muscular dystrophy-dystroglycanopathy (type C12). • The probands have a phenotype consistent with mild limb-girdle muscular dystrophy. • At ages 10 and 13 years, their disease courses seem slow or even non-progressive. Abstract We describe two Finnish siblings in whom an incidentally detected elevated creatine kinase activity eventually led to a diagnosis of limb-girdle muscular dystrophy-dystroglycanopathy (Type C12; MDDGC12). When diagnosed at age 10 and 13 years, they were mildly affected with a slow or non-progressive disease course. The main symptoms comprised infrequent hip cramps triggered by flexion, neck cramps triggered by yawning, transient growing pains, calf hypertrophy and mild proximal muscle weakness. Their cognitive and motor developments were unremarkable and they were physically active. Whole-exome sequencing revealed compound heterozygous mutations, both of which were novel, in the protein O-mannosyl kinase (POMK) gene in both siblings; a missense mutation, p.Pro322Leu (c.965C > T), and a nonsense mutation, p.Arg46Ter (c.136C > T). The results were confirmed by Sanger sequencing, showing that the parents were heterozygous carriers of one mutation each. This report adds to the literature by providing phenotype and genotype data on this ultra-rare POMK -related dystroglycanopathy. [ABSTRACT FROM AUTHOR]

Recessive mutations in MEGF10 (multiple epidermal growth factor 10) have been reported in a severe early onset disorder named Early Myopathy, Areflexia, Respiratory Distress and Dysphagia, and a milder form with cores in the muscle biopsy; and a possible genotype–phenotype correlation determining the clinical presentation has been suggested. We undertook exome sequencing in a 66 year old male with a 20 year history of progressive proximal and distal weakness of upper and lower limbs, facial weakness and dysphagia, who developed respiratory failure requiring ventilation while still ambulant in his 50s. Muscle biopsy demonstrated myopathic changes with aggregation of myofibrillar proteins. Mutations in MEGF10 were identified: a novel essential splice site (c.1426+1G>T) and a novel missense variant (c.352T>C, p.(Cys118Arg)). We performed a detailed review of all reported MEGF10 cases (n = 20), and confirmed the presence of a genotype–phenotype correlation, namely that with ≥1 null mutation onset of respiratory dysfunction occurs in the first year of life, whereas with 2 missense mutations, respiratory dysfunction occurs at 10 years old or much later, as in the patient reported here. Our findings expand the phenotype of MEGF10 mutations to include onset in the 5th decade, and discuss the spectrum of MEGF10 related disease. [ABSTRACT FROM AUTHOR]

Congenital myasthenic syndromes (CMSs) are heterogeneous genetic disorders that result from impaired signal transmission at the neuromuscular junction (NMJ). Congenital disorders of glycosylation (CDG) are a challenging group of diseases affecting different organs in particular the central nervous system and the skeletal muscle. Only recently CDG have been implicated in some forms of post-synaptic autosomal recessive CMS. The protein encoded by the DPAGT1 gene is an enzyme that catalyzes the first step in the dolichol-linked oligosaccharide pathway for glycoprotein biosynthesis. Mutations in DPAGT1 were described in patients with CMS and tubular aggregates on muscle biopsy. We report a 16 year old young man, first child of a consanguineous family, who presented with walking difficultywith onset by the age of 3 year. The motor milestones were slightly delayed. There was speech difficulty which fluctuated during the day. On examination, he had generalized muscle weakness with proximal groups being more affected, excessive lordosis, waddling gait, and contractures of the Achilles tendons. There was no bulbar or extra-ocular muscle involvement. He had a mild intellectual disability with an IQ score of 50–55. Tensilon test was positive. Electromyography revealed decrementing response to repetitive nerve stimulation. He responded well to pyridostigmine, 3,4-diaminopyridine and salbutamol treatment. We carried out whole exome sequencing and identified a novel homozygous DPAGT1 mutation (c.509A>T). The non-synonymous change (p.Tyr170Phe) is predicted to be deleterious by in silico analysis and affects an aminoacid residue that is highly conserved across species. In conclusion we report a novel mutation on the DPAGT1 gene responsible for a form of “limb-girdle CMS”. Comparing with the previously reported cases this family has mental retardation as an unusual feature and a good response to a combined treatment with pyridostigmine and 3,4-diaminopyridine. [ABSTRACT FROM AUTHOR]

The International Centre for Genomic Medicine in Neuromuscular Diseases (ICGNMD) is a consortium constituted by four UK research centres and five low/middle income countries collaborating in diagnosis and research in NMDs. As an ICGNMD member and UK reference centre for limb-girdle muscular dystrophies, we aimed to provide genetic diagnosis and compare the genetic architecture of muscular dystrophies across regions. However, here, we have focused on the analysis of an Indian cohort. A total of 177 MD patients from India underwent genetic analysis. Mutational screening was pursued by whole exome sequencing and analysed following in-house bioinformatic pipeline. Variants were filtered applying standard criteria and classified following ACMG guidelines. Cases diagnostic status were divided into 4 categories according to the pathogenicity classification of the identified variants and their correlation with the patient's clinical presentation: 53.1% Solved; 15.8% Maybe-VUS (variant/s of unknown significance); 7.9% Maybe-Phenotype doesn't perfectly match (disagreements between observed/expected clinical presentation); and 23.2% Unsolved. Most solved cases (66/94) were caused by variants in DYSF and CAPN3. The "Maybe" categories might get solved with intrafamilial segregations, functional studies and/or further clinical discussion with local partners. Interestingly, "Maybe-Phenotype doesn't perfectly match" might represent new clinical presentations for known genes, and such seems to be the case for 5 patients carrying disease-causing variants in GNE and exhibiting proximal weakness, rather than the classical distal involvement. The genome-wide re-analysis of unsolved cases might lead to the discovery of new disease-causing genes. Finally, ICGNMD provided much-needed genetic diagnosis to 94 patients from India and has proven successful in expanding our understanding of pathogenic variants for muscular dystrophies. [ABSTRACT FROM AUTHOR]

Next-generation sequencing has enhanced the rate of genetic diagnosis in neuromuscular diseases. Nevertheless, half of the patients remain without a molecular diagnosis. One factor that can impede the acquisition of a genetic diagnosis is the presence of mosaicism: the coexistence of at least two different genetic cell populations within an organism. We present two cases of neuromuscular pediatric patients in which a phenotype-driven analysis guided the careful interrogation of candidate genes. Patient A showed a phenotype of congenital myopathy (CM). Trio genome sequencing detected a heterozygous likely pathogenic variant in RYR1 (c.14566G>A; p.Ala4856Thr), inherited from his unaffected mother. It was considered the potential existence of a missing second variant in trans in the proband, but also hypothesized that the variant might be mosaic in the mother. The comparison between alternative allele frequencies (AAF) showed that, while the affected child had an AAF of 0.60, the mother had an AAF of only 0.23. A quantification through ddPCR from blood-derived DNA revealed a 17% of the alternative allele in the mother's sample, whereas in the individual's sample, 49% was observed. Patient B was born with congenital hip dislocation, hypotonia and distal hyperlaxity. Upon clinical examination of the parents, it was detected a left hemicorporal hypotrophy and hand retractions in the father. A neuromuscular gene panel in patient B and his father was inconclusive. RNAseq performed on the muscle sample of the father revealed an exon 9 skipping of COL6A1 in 15% of the transcripts. In genome sequencing it was detected an exon 9 deletion of COL6A1 in 10% of the reads in the father and 50% in patient B (p.Gly269_Pro286del), which was validated through a custom MLPA. Our study highlights the importance of phenotype-driven analysis and careful interrogation of the likely relevant genes and variants and illustrates the genetic mosaicism as a possible explanation behind genetically unsolved affected individuals. [ABSTRACT FROM AUTHOR]

Ullrich congenital muscular dystrophy and Bethlem myopathy (BM) are a spectrum of diseases caused by molecular alterations in COL6A1, COL6A2, COL6A3 and COL12A1. BM is the milder end of the spectrum as patients present mild symptoms and remain ambulant till adulthood. Here, we present a familial case of BM, where the 5 affected members over two generations, presented onset of symptoms in childhood, slow progressive proximal muscle weakness of upper and lower limbs, joint contractures, and difficulty walking. The index case showed muscle MRI findings suggestive of collagenopathy. A DNA sample from the index case was initially analysed by whole exome sequencing (WES). No single nucleotide nor indel variants in the COL6 and COL12 genes were identified. Exome data was then analysed applying a pipeline for copy number and structural variants as part of the Solve-RD project. Candidate variants were segregated and further studied at the mRNA level. Re-analysis of WES data detected a large heterozygous insertion in exon 10 of COL6A2 , and its exact size and complete sequence was determined by PCR-sanger sequencing. The variant consists of an insertion of ∼321bp between positions c.989_990 (hg19:chr21:47536717), 10bp before the 3' end of exon 10. Two key elements of the inserted sequence indicate that it stems from an Alu element: a target site duplication and the conserved A5TACA6 motif. Segregation analysis demonstrated that the insertion co-segregates with the presence of clinical symptoms, while the non-affected members of the family included in this study did not carry the variant. RNA analysis of the index patient's fibroblasts showed the existence of an alternative transcript lacking exon 10, leading to an in-frame deletion. Here, we have confirmed the clinical diagnosis of BM by the insertion of an Alu element in COL6A2. To our knowledge, this constitutes the second case so far of collagenopathies caused by Alu elements, but the first mapping in the exonic region of the gene. [ABSTRACT FROM AUTHOR]

Dystrophinopathies, including Duchenne and Becker muscular dystrophies, are the most common inherited neuromuscular conditions in childhood. These diseases are caused by pathogenic variants in the DMD gene and present an X-linked recessive inheritance pattern, thus, mainly males are affected. The most prevalent pathogenic variants are Copy Number Variants (CNVs) and genetic diagnosis is mostly achieved through MLPA and exome sequencing of the DMD gene. Here, we aimed to identify the genetic diagnosis of a female patient presenting with muscular dystrophy, resembling a dystrophinopathy. We performed MLPA, exome sequencing, aCGH, X-chromosome inactivation assay, and genome sequencing on DNA extracted from patient's peripheral blood. Routine histological immunostaining and total RNA sequencing were performed from the patient's muscle biopsy. Histological analysis revealed dystrophic features and a reduction in α, β, γ, δ-sarcoglycan, β-dystroglycan and dystrophin staining. MLPA, exome sequencing and aCGH were negative. RNA sequencing outlier expression analysis identified the DMD gene as a statistically significant downregulated gene with an 85% reduction in expression compared to all muscle samples analysed in the cohort. Trio whole genome sequencing revealed a de novo balanced translocation between chromosome 17 and the X chromosome (t(X;17)(p21.1;q23.2), disrupting both the DMD and BCAS3 genes. X-inactivation from patient's peripheral blood showed a non-random X-chromosome inactivation pattern (73%-27%). A combined analysis of RNA and whole genome sequencing played a crucial role in the detection, characterisation, and interpretation of the disease-causing variant in this patient, who had been followed up for several years. This case illustrates the diagnostic odyssey in female DMD patients with complex structural variants that are not detected by panel or even exome sequencing. [ABSTRACT FROM AUTHOR]

Collagen XII-related disease (COL12 -RD), also classified as myopathic Ehlers-Danlos syndrome (EDS), is a congenital onset disorder with overlapping connective tissue and muscle involvement. Biallelic loss-of-function (LoF), as well as dominantly acting monoallelic variants in COL12A1 , have been recognized to cause COL12 -RD. While there have been additional reports of patients with dominant COL12A1 variants, fewer cases of the presumed more severe recessive COL12 -RD have been documented. We present clinical, immunocytochemical, and imaging data on eight additional patients from seven families, with biallelic variants in COL12A1. All patients presented with a consistent constellation of symptoms including congenital onset disease characterized by hypotonia, dysmorphic features including most notably gingival hypertrophy, prominent distal joint hyperlaxity with co-occurring contractures of large joints, and muscle involvement of variable degrees, evident both clinically and on muscle imaging. Five patients presented with a severe congenital phenotype manifesting with profound weakness, significantly delayed or minimal attainment of motor milestones, respiratory insufficiency, and feeding difficulties. Three patients presented with milder disease manifesting with mild to moderate muscle weakness and delayed milestones but were able to achieve independent ambulation. Patients were found to have biallelic LoF COL12A1 variants, except for one family with compound heterozygosity for a missense variant (p.I1393Ffs*11/ p.A1110D). Consistent with the variable clinical spectrum, in vitro collagen XII immunocytochemistry analysis in fibroblasts ranged from complete absence of collagen XII expression in a patient with severe disease, to a mild reduction in a patient with milder disease. This series aids to further establish COL12A1 as a recessive myopathic EDS gene and to expand the clinical spectrum to include a milder EDS phenotype. [ABSTRACT FROM AUTHOR]

Dystroglycan is an essential component of the dystrophin-glycoprotein complex that links the actin-associated cytoskeleton to the extracellular matrix (ECM). Dystroglycan, encoded by the DAG1 gene, is proteolytically cleaved into α-Dystroglycan, a highly glycosylated protein that binds to ECM ligands, and β-Dystroglycan, a transmembrane protein non-covalently linked to α-Dystroglycan. Biallelic variants in DAG1 cause primary dystroglycanopathies with a wide phenotypic variability. Through exome sequencing we identified heterozygous truncating DAG1 variants in 18 individuals from 10 unrelated families. Five variants were stop gains and five were frameshift; with the majority localized in α-Dystroglycan (90%). Segregation analysis was available for seven families, with one variant being de novo, three dominantly inherited and three inherited from an asymptomatic parent. WGS performed in six patients excluded the presence of additional deep intronic DAG1 variants. The most common clinical features were the presence of high CK levels (18/18), myalgia (9/18) and muscle hypertrophy (3/18). Mild levels of learning or social difficulties were also observed in some patients (4/18). Histological analysis was performed in eight patients and showed the presence of mild dystrophic or myopathic changes. Dystroglycan immunostaining, either β-Dystroglycan or α-Dystroglycan, was reduced in four out of five patients. RNAseq data from muscle biopsies from two unrelated patients showed the expression of both wild-type and truncated alleles, indicating that nonsense-mediated decay is not triggered by these alleles. To date, few studies have reported the presence of heterozygous truncating DAG1 variants in asymptomatic or pauci-symptomatic individuals. Here, we describe 18 additional individuals with AD DAG1 hyperCKemia with mild muscular manifestations. Expression of both the truncated and wild-type alleles argues against haploinsufficiency as the underlying pathomechanisms in DAG1 -AD individuals. [ABSTRACT FROM AUTHOR]

Parent-of-origin (or imprinting) effects relate to the situation in which traits are influenced by the allele inherited from only one parent and the allele from the other parent has little or no effect. Given SNP genotype data from case-parent trios, the parent of origin of each allele in the offspring can often be deduced unambiguously; however, this is not true when all three individuals are heterozygous. Most existing methods for investigating parent-of-origin effects operate on a SNP-by-SNP basis and either perform some sort of averaging over the possible parental transmissions or else discard ambiguous trios. If the correct parent of origin at a SNP could be determined, this would provide extra information and increase the power for detecting the effects of imprinting. We propose making use of the surrounding SNP information, via haplotype estimation, to improve estimation of parent of origin at a test SNP for case-parent trios, case-mother duos, and case-father duos. This extra information is then used in a multinomial modeling approach for estimating parent-of-origin effects at the test SNP. We show through computer simulations that our approach has increased power over previous approaches, particularly when the data consist only of duos. We apply our method to two real datasets and find a decrease in significance of p values in genomic regions previously thought to possibly harbor imprinting effects, thus weakening the evidence that such effects actually exist in these regions, although some regions retain evidence of significant effects. [ABSTRACT FROM AUTHOR]

Objectives: To present phenotype features of a large cohort of congenital myasthenic syndromes (CMS) and correlate them with their molecular diagnosis. Methods: Suspected CMS patients were divided into three groups: group A (limb, bulbar or axial weakness, with or without ocular impairment, and all the following: clinical fatigability, electrophysiology compatible with neuromuscular junction involvement and anticholinesterase agents response), group B (limb, bulbar or axial weakness, with or without ocular impairment, and at least one of additional characteristics noted in group A) and group C (pure ocular syndrome). Individual clinical findings and the clinical groups were compared between the group with a confirmed molecular diagnosis of CMS and the group without molecular diagnosis or with a non‐CMS molecular diagnosis. Results: Seventy‐nine patients (68 families) were included in the cohort: 48 in group A, 23 in group B and 8 in group C. Fifty‐one were considered confirmed CMS (30 CHRNE, 5 RAPSN, 4 COL13A1, 3 DOK7, 3 COLQ, 2 GFPT1, 1 CHAT, 1 SCN4A, 1 GMPPB, 1 CHRNA1), 7 probable CMS, 5 non‐CMS and 16 unsolved. The chance of a confirmed molecular diagnosis of CMS was significantly higher for group A and lower for group C. Some individual clinical features, alterations on biopsy and electrophysiology enhanced specificity for CMS. Muscle imaging showed at least mild alterations in the majority of confirmed cases, with preferential involvement of soleus, especially in CHRNE CMS. Conclusions: Stricter clinical criteria increase the chance of confirming a CMS diagnosis, but may lose sensitivity, especially for some specific genes. [ABSTRACT FROM AUTHOR]

Background: Goltz syndrome (GS) is a X-linked disorder defined by defects of mesodermal- and ectodermal-derived structures and caused by PORCN mutations. Features include striated skin-pigmentation, ocular and skeletal malformations and supernumerary or hypoplastic nipples. Generally, GS is associated with in utero lethality in males and most of the reported male patients show mosaicism (only three non-mosaic surviving males have been described so far). Also, precise descriptions of neurological deficits in GS are rare and less severe phenotypes might not only be caused by mosaicism but also by less pathogenic mutations suggesting the need of a molecular genetics and functional work-up of these rare variants.Results: We report two cases: one girl suffering from typical skin and skeletal abnormalities, developmental delay, microcephaly, thin corpus callosum, periventricular gliosis and drug-resistant epilepsy caused by a PORCN nonsense-mutation (c.283C > T, p.Arg95Ter). Presence of these combined neurological features indicates that CNS-vulnerability might be a guiding symptom in the diagnosis of GS patients. The other patient is a boy with a supernumerary nipple and skeletal anomalies but also, developmental delay, microcephaly, cerebral atrophy with delayed myelination and drug-resistant epilepsy as predominant features. Skin abnormalities were not observed. Genotyping revealed a novel PORCN missense-mutation (c.847G > C, p.Asp283His) absent in the Genome Aggregation Database (gnomAD) but also identified in his asymptomatic mother. Given that non-random X-chromosome inactivation was excluded in the mother, fibroblasts of the index had been analyzed for PORCN protein-abundance and -distribution, vulnerability against additional ER-stress burden as well as for protein secretion revealing changes.Conclusions: Our combined findings may suggest incomplete penetrance for the p.Asp283His variant and provide novel insights into the molecular etiology of GS by adding impaired ER-function and altered protein secretion to the list of pathophysiological processes resulting in the clinical manifestation of GS. [ABSTRACT FROM AUTHOR]

Aims: MICU1 encodes the gatekeeper of the mitochondrial Ca2+ uniporter, MICU1 and biallelic loss‐of‐function mutations cause a complex, neuromuscular disorder in children. Although the role of the protein is well understood, the precise molecular pathophysiology leading to this neuropaediatric phenotype has not been fully elucidated. Here we aimed to obtain novel insights into MICU1 pathophysiology. Methods: Molecular genetic studies along with proteomic profiling, electron‐, light‐ and Coherent anti‐Stokes Raman scattering microscopy and immuno‐based studies of protein abundances and Ca2+ transport studies were employed to examine the pathophysiology of MICU1 deficiency in humans. Results: We describe two patients carrying MICU1 mutations, two nonsense (c.52C>T; p.(Arg18*) and c.553C>T; p.(Arg185*)) and an intragenic exon 2‐deletion presenting with ataxia, developmental delay and early onset myopathy, clinodactyly, attention deficits, insomnia and impaired cognitive pain perception. Muscle biopsies revealed signs of dystrophy and neurogenic atrophy, severe mitochondrial perturbations, altered Golgi structure, vacuoles and altered lipid homeostasis. Comparative mitochondrial Ca2+ transport and proteomic studies on lymphoblastoid cells revealed that the [Ca2+] threshold and the cooperative activation of mitochondrial Ca2+ uptake were lost in MICU1‐deficient cells and that 39 proteins were altered in abundance. Several of those proteins are linked to mitochondrial dysfunction and/or perturbed Ca2+ homeostasis, also impacting on regular cytoskeleton (affecting Spectrin) and Golgi architecture, as well as cellular survival mechanisms. Conclusions: Our findings (i) link dysregulation of mitochondrial Ca2+ uptake with muscle pathology (including perturbed lipid homeostasis and ER–Golgi morphology), (ii) support the concept of a functional interplay of ER–Golgi and mitochondria in lipid homeostasis and (iii) reveal the vulnerability of the cellular proteome as part of the MICU1‐related pathophysiology. [ABSTRACT FROM AUTHOR]

Pur-α protein (PURA) syndrome manifests in early childhood with core features such as neurodevelopmental and speech delay, feeding difficulties, epilepsy, and hypotonia at birth. We identified three cases with PURA syndrome in a cohort of patients with unexplained muscular weakness, presenting with a predominantly neuromuscular and ataxic phenotype. We further characterize the clinical presentation of PURA syndrome including myopathic facies and muscular weakness as the main clinical symptoms in combination with elevated serum creatine kinase levels. Furthermore, we report two novel variants located in the conservative domains PUR-I and PUR-II. For the first time, we present the muscle biopsies of PURA syndrome patients, showing myopathic changes, fiber size variability, and fast fiber atrophy as the key features. PURA syndrome should be taken into consideration as a differential diagnosis in pediatric patients with unexplained muscle weakness. [ABSTRACT FROM AUTHOR]

• Despite symptom onset in childhood, late onset presentations are frequent. • The pathognomic second wind phenomenon may not be volunteered by the patient. • Rhabdomyolysis and myoglobinuria are not universally present in all McArdle patients. • Fixed muscle weakness is common, particularly in patients older than 40 years. • The condition may be associated with comorbid cardiac and psychological disease. This retrospective study evaluated the phenotypic and genotypic features of 14 patients with McArdle disease attending the West of Scotland adult muscle clinic. Although all patients experienced exercise-induced cramps, exercise intolerance and hyperCKaemia, only 71% (n = 10) experienced the second wind phenomenon, rhabdomyolysis and/or myoglobinuria. We observed a high rate of fixed muscle weakness (50%; n = 7), coronary artery disease (36%; n = 5), and psychological comorbidity (50%; n = 7). Although 79% had symptom onset in the first decade of life, the mean age at presentation and at genetic diagnosis was 43.8 years and 47.7 years, respectively. 93% had at least one copy of the common PYGM pathogenic variant, c.148C > T , p.(Arg50*), with 50% (n = 7) of the cohort being homozygous. Our cohort highlights the phenotypic variability seen in McArdle disease and underscores the potential for late-onset presentations. It emphasises the need for improved awareness and recognition of this condition amongst neurologists, rheumatologists and general physicians. A history of exercise intolerance and second wind phenomenon may not always be volunteered by the patient, underscoring the need to ask specific questions in clinic to extrapolate the relevant symptoms in this patient cohort. [ABSTRACT FROM AUTHOR]

• SD should be considered in the diagnosis of lower motor neuron diseases. • These clinical cases illustrate the mildest and heterogenic presentation of SD. • An early diagnosis is essential for genetic counseling and for upcoming treatments. Defects in the HEXB gene which encodes the β-subunit of β-hexosaminidase A and B enzymes, cause a GM2 gangliosidosis, also known as Sandhoff disease, which is a rare lysosomal storage disorder. The most common form of the disease lead to quickly progressing mental and motor decline in infancy; however there are other less severe forms with later onset that can also involve lower motor neurons. The diagnosis of this disease is based on low serum β-hexosaminidases A and B levels and confirmed using genetic test. We report two siblings with compound heterozygous HEXB mutations whose phenotype was extremely mild consisting in stuttering in both cases associated to mild proximal weakness in one of the cases, broadening the clinical spectrum of late onset Sandhoff disease. [ABSTRACT FROM AUTHOR]

• An intronic insertion in the LAMA2 gene was found to contribute to a patient with partial laminin α2 chain deficiency. • An elevated CK activity, brain white matter lesions and muscle biopsy findings pointed towards partial laminin α2 chain deficiency long before genetic confirmation. • This patient with partial laminin α2 chain deficiency presented with mild weakness in early childhood and continuous slow improvement. Recessive mutations in the LAMA2 gene lead to congenital muscular dystrophy type 1A and limb girdle muscular dystrophy R23 with complete or partial laminin α2 chain deficiency. Complete laminin α2 chain deficiency presents with early onset of severe hypotonia and generalized weakness, whereas partial deficiency shows a milder and more variable course with limb girdle weakness. Here, we report a child with mildly delayed motor development, elevated serum creatine kinase levels (>1000 U/l) and brain white matter hypointensity, indicative of laminin α2 chain deficiency. In addition to a stop gain variant in exon 39, the patient was found to carry an intronic insertion of 72 bp in intron 38 of the LAMA2 gene in trans. RNA analysis revealed that this insertion results in abnormally spliced as well as wild type transcript, which explains the partial laminin α2 chain deficiency observed in the muscle biopsy. [ABSTRACT FROM AUTHOR]

The myosin-directed chaperone UNC-45B is essential for sarcomeric organization and muscle function from Caenorhabditis elegans to humans. The pathological impact of UNC-45B in muscle disease remained elusive. We report ten individuals with bi-allelic variants in UNC45B who exhibit childhood-onset progressive muscle weakness. We identified a common UNC45B variant that acts as a complex hypomorph splice variant. Purified UNC-45B mutants showed changes in folding and solubility. In situ localization studies further demonstrated reduced expression of mutant UNC-45B in muscle combined with abnormal localization away from the A-band towards the Z-disk of the sarcomere. The physiological relevance of these observations was investigated in C. elegans by transgenic expression of conserved UNC-45 missense variants, which showed impaired myosin binding for one and defective muscle function for three. Together, our results demonstrate that UNC-45B impairment manifests as a chaperonopathy with progressive muscle pathology, which discovers the previously unknown conserved role of UNC-45B in myofibrillar organization. [ABSTRACT FROM AUTHOR]

We identified the known c.1_9del mutation in the PLEC gene in four unrelated females from consanguineous families of Turkish origin. All individuals presented with slowly progressive limb-girdle weakness without any dermatological findings, and dystrophic changes observed in their muscle biopsies. Additionally, the neurological examination revealed ptosis, facial weakness, fatigability, and muscle cramps in all four cases. In two patients, repetitive nerve stimulation showed a borderline decrement and a high jitter was detected in all patients by single-fiber electromyography. Clinical improvement was observed after treatment with pyridostigmine and salbutamol was started. We further characterize the phenotype of patients with limb-girdle muscular dystrophy R17 clinically, by muscle magnetic resonance imaging (MRI) features and by describing a common 3.8 Mb haplotype in three individuals from the same geographical region. In addition, we review the neuromuscular symptoms associated with PLEC mutations and the role of plectin in the neuromuscular junction. [ABSTRACT FROM AUTHOR]