A Schematic Approach to Defining the Prevalence of COL VI Variants in Five Years of Next-Generation Sequencing.

OBJECTIVE: To define the prevalence of variants in collagen VI genes through a next-generation sequencing (NGS) approach in undiagnosed patients with suspected neuromuscular disease and to propose a diagnostic flowchart to assess the real pathogenicity of those variants. METHODS: In the past five years, we have collected clinical and molecular information on 512 patients with neuromuscular symptoms referred to our center. To pinpoint variants in COLVI genes and corroborate their real pathogenicity, we sketched a multistep flowchart, taking into consideration the bioinformatic weight of the gene variants, their correlation with clinical manifestations and possible effects on protein stability and expression. RESULTS: In Step I, we identified variants in COLVI-related genes in 48 patients, of which three were homozygous variants (Group 1). Then, we sorted variants according to their CADD score, clinical data and complementary studies (such as muscle and skin biopsy, study of expression of COLVI on fibroblast or muscle and muscle magnetic resonance). We finally assessed how potentially pathogenic variants (two biallelic and 12 monoallelic) destabilize COL6A1-A2-A3 subunits. Overall, 15 out of 512 patients were prioritized according to this pipeline. In seven of them, we confirmed reduced or absent immunocytochemical expression of collagen VI in cultured skin fibroblasts or in muscle tissue. CONCLUSIONS: In a real-world diagnostic scenario applied to heterogeneous neuromuscular conditions, a multistep integration of clinical and molecular data allowed the identification of about 3% of those patients harboring pathogenetic collagen VI variants.

Expanding the clinical and genetic spectrum of pathogenic variants in STIM1.

INTRODUCTION/AIMS: Stromal interaction molecule 1 (STIM1) is a reticular Ca2+ sensor composed of a luminal and a cytosolic domain. Autosomal dominant mutations in STIM1 cause tubular aggregate myopathy and Stormorken syndrome or its variant York platelet syndrome. In this study we aimed to expand the features related to new variants in STIM1. METHODS: We performed a cross-sectional study of individuals harboring monoallelic STIM1 variants recruited at five tertiary centers involved in a study of inherited myopathies analyzed with a multigene-targeted panel. RESULTS: We identified seven individuals (age range, 26-57 years) harboring variants in STIM1, including five novel changes: three located in the EF-hand domain, one in the sterile α motif (SAM) domain, and one in the cytoplasmatic region of the protein. Functional evaluation of the pathogenic variants using a heterologous expression system and measuring store-operated calcium entry demonstrated their causative role and suggested a link of new variants with the clinical phenotype. Muscle contractures, found in three individuals, showed variability in body distribution and in the number of joints involved. Three patients showed cardiac and respiratory involvement. Short stature, hyposplenism, sensorineural hearing loss, hypothyroidism, and Gilbert syndrome were variably observed among the patients. Laboratory tests revealed hyperCKemia in six patients, thrombocytopenia in two patients, and hypocalcemia in one patient. Muscle biopsy showed the presence of tubular aggregates in three patients, type I fiber atrophy in one patient, and nonspecific myopathic changes in two patients. DISCUSSION: Our clinical, histological, and molecular data expand the genetic and clinical spectrum of STIM1-related diseases.

VARS2-linked mitochondrial encephalopathy: two case reports enlarging the clinical phenotype.

BACKGROUND: Mitochondrial respiratory chain consists of five complexes encoded by nuclear and mitochondrial genomes. Mitochondrial aminoacyl-tRNA synthetases are key enzymes in the synthesis of such complexes. Bi-allelic variants of VARS2, a nuclear gene encoding for valyl-tRNA (Val-tRNA) synthetase, are associated to several forms of mitochondrial encephalopathies or cardiomyoencephalopathies. Among these, the rare homozygous c.1100C > T (p.Thr367Ile) mutation variably presents with progressive developmental delay, axial hypotonia, limbs spasticity, drug-resistant epilepsy leading, in some cases, to premature death. Yet only six cases, of which three are siblings, harbouring this homozygous mutation have been described worldwide. CASE PRESENTATION: Hereby, we report two additional cases of two non-related young girls from Sardinia, born from non-consanguineous and healthy parents, carrying the aforesaid homozygous VARS2 variant. At onset both the patients presented with worsening psychomotor delay, muscle hypotonia and brisk tendon reflexes. Standard genetic tests were normal, as well as metabolic investigations. Brain MRI showed unspecific progressive abnormalities, such as corpus callosum hypoplasia (patient A) and cerebellar atrophy (patient A and B). Diagnosis was reached by adopting massive parallel next generation sequencing. Notably clinical phenotype of the first patient appears to be milder compared to previous known cases. The second patient eventually developed refractory epilepsy and currently presents with severe global impairment. Because no specific treatment is available as yet, both patients are treated with supporting antioxidant compounds along with symptomatic therapies. CONCLUSIONS: Given the paucity of clinical data about this very rare mitochondrial encephalopathy, our report might contribute to broaden the phenotypic spectrum of the disorder. Moreover, noteworthy, three out of five pedigrees so far described belong to the Northern Sardinia ethnicity.

Complex multisystem phenotype associated with the mitochondrial DNA m.5522G>A mutation.

Mitochondrial tRNAs are responsible for more than half of pathogenic point mutations in the mitochondrial genome (mtDNA). Different mutations give rise to widely differing phenotypes, ranging from isolated organ-specific diseases to multisystem conditions. Herein, we report a 40-year-old woman presenting with a complex multisystem phenotype including sensorineural hearing loss, retinopathy, severe dilated cardiomyopathy, non-insulin dependent diabetes mellitus, and renal failure. Sequence analysis of mtDNA identified the m.5522G>A mutation in MT-TW, the gene encoding mitochondrial tRNA for tryptophan. The heteroplasmic variant, thus far described once, was almost exclusively confined to skeletal muscle tissue, as shown by massive parallel sequencing and corroborated by an ad hoc designed PCR-based strategy. This patient, presenting a severe, multisystem involvement apparently sparing the brain, contributes to the genetic heterogeneity of mitochondrial diseases caused by mutations in mitochondrial tRNAs.

Dolichol-phosphate mannose synthase depletion in zebrafish leads to dystrophic muscle with hypoglycosylated α-dystroglycan.

Defective dolichol-phosphate mannose synthase (DPMS) complex is a rare cause of congenital muscular dystrophy associated with hypoglycosylation of alpha-dystroglycan (α-DG) in skeletal muscle. We used the zebrafish (Danio rerio) to model muscle abnormalities due to defects in the subunits of DPMS. The three zebrafish ortholog subunits (encoded by the dpm1, dpm2 and dpm3 genes, respectively) showed high similarity to the human proteins, and their expression displayed localization in the midbrain/hindbrain area and somites. Antisense morpholino oligonucleotides targeting each subunit were used to transiently deplete the dpm genes. The resulting morphant embryos showed early death, muscle disorganization, low DPMS complex activity, and increased levels of apoptotic nuclei, together with hypoglycosylated α-DG in muscle fibers, thus recapitulating most of the characteristics seen in patients with mutations in DPMS. Our results in zebrafish suggest that DPMS plays a role in stabilizing muscle structures and in apoptotic cell death.

Myoimaging in the NGS era: the discovery of a novel mutation in MYH7 in a family with distal myopathy and core-like features--a case report.

BACKGROUND: Myosin heavy chain 7 related myopathies are rare disorders characterized by a wide phenotypic spectrum and heterogeneous pathological features. In the present study, we performed clinical, morphological, genetic and imaging investigations in three relatives affected by autosomal dominant distal myopathy. Whilst earlier traditional Sanger investigations had pointed to the wrong gene as disease causative, next-generation sequencing allowed us to obtain the definitive molecular genetic diagnosis in the family. CASE PRESENTATION: The proposita, being found to harbor a novel heterozygous mutation in the RYR1 gene (p.Glu294Lys), was initially diagnosed with core myopathy. Subsequently, consideration of muscle magnetic resonance imaging (MRI) features and extension of family study led this diagnosis to be questioned. Use of next-generation sequencing analysis identified a novel mutation in the MYH7gene (p.Ser1435Pro) that segregated in the affected family members. CONCLUSIONS: This study identified a novel mutation in MYH7 in a family where the conclusive molecular diagnosis was reached through a complicated path. This case report might raise awareness, among clinicians, of the need to interpret NGS data in combination with muscle MRI patterns so as to facilitate the pinpointing of the main molecular etiology in inherited muscle disorders.

Acitretin-responsive ichthyosis in Chanarin-Dorfman syndrome with a novel mutation in the ABHD5/CGI-58 gene.

Chanarin-Dorfman syndrome (CDS) is a rare nonlysosomal neutral lipid storage disorder characterized by congenital ichthyosis, lipid vacuoles in leukocytes (Jordan's anomaly), and hepatomegaly. The authors herein report an 18-month-old boy with ichthyosis and hepatomegaly diagnosed with CDS and confirmed to have a novel c.506-3C>G mutation in the ABHD5/CGI-58 gene. Our case also illustrates that retinoids such as acitretin could be useful in the treatment of skin manifestations in CDS even in the presence of liver derangement.

CCDC78: Unveiling the Function of a Novel Gene Associated with Hereditary Myopathy.

CCDC78 was identified as a novel candidate gene for autosomal dominant centronuclear myopathy-4 (CNM4) approximately ten years ago. However, to date, only one family has been described, and the function of CCDC78 remains unclear. Here, we analyze for the first time a family harboring a CCDC78 nonsense mutation to better understand the role of CCDC78 in muscle. METHODS: We conducted a comprehensive histopathological analysis on muscle biopsies, including immunofluorescent assays to detect multiple sarcoplasmic proteins. We examined CCDC78 transcripts and protein using WB in CCDC78-mutated muscle tissue; these analyses were also performed on muscle, lymphocytes, and fibroblasts from healthy subjects. Subsequently, we conducted RT-qPCR and transcriptome profiling through RNA-seq to evaluate changes in gene expression associated with CCDC78 dysfunction in muscle. Lastly, coimmunoprecipitation (Co-Ip) assays and mass spectrometry (LC-MS/MS) studies were carried out on extracted muscle proteins from both healthy and mutated subjects. RESULTS: The histopathological features in muscle showed novel histological hallmarks, which included areas of dilated and swollen sarcoplasmic reticulum (SR). We provided evidence of nonsense-mediated mRNA decay (NMD), identified the presence of novel CCDC78 transcripts in muscle and lymphocytes, and identified 1035 muscular differentially expressed genes, including several involved in the SR. Through the Co-Ip assays and LC-MS/MS studies, we demonstrated that CCDC78 interacts with two key SR proteins: SERCA1 and CASQ1. We also observed interactions with MYH1, ACTN2, and ACTA1. CONCLUSIONS: Our findings provide insight, for the first time, into the interactors and possible role of CCDC78 in skeletal muscle, locating the protein in the SR. Furthermore, our data expand on the phenotype previously associated with CCDC78 mutations, indicating potential histopathological hallmarks of the disease in human muscle. Based on our data, we can consider CCDC78 as the causative gene for CNM4.

Subclinical myopathy in a child with neutral lipid storage disease and mutations in the PNPLA2 gene.

We report a 14-year-old-boy with markedly elevated serum creatine kinase (CK) levels, in whom massive triglyceride storage was found in peripheral blood leukocytes and in muscle biopsy. Sequencing PNPLA2, the gene encoding the adipose triglyceride lipase (ATGL) and responsible for the neutral lipid storage disease with myopathy (NLSDM), we identified two heterozygous mutations, including a previously reported nonsense and a novel missense mutation in the patatin domain of the gene. Lipid storage myopathy can be clinically silent in childhood and presenting only with hyperCKemia.

Pontocerebellar hypoplasia type 6 caused by mutations in RARS2: definition of the clinical spectrum and molecular findings in five patients.

Recessive mutations in the mitochondrial arginyl-transfer RNA synthetase (RARS2) gene have been associated with early onset encephalopathy with signs of oxidative phosphorylation defects classified as pontocerebellar hypoplasia 6. We describe clinical, neuroimaging and molecular features on five patients from three unrelated families who displayed mutations in RARS2. All patients rapidly developed a neonatal or early-infantile epileptic encephalopathy with intractable seizures. The long-term follow-up revealed a virtual absence of psychomotor development, progressive microcephaly, and feeding difficulties. Mitochondrial respiratory chain enzymes in muscle and fibroblasts were normal in two. Blood and CSF lactate was abnormally elevated in all five patients at early stages while appearing only occasionally abnormal with the progression of the disease. Cerebellar vermis hypoplasia with normal aspect of the cerebral and cerebellar hemispheres appeared within the first months of life at brain MRI. In three patients follow-up neuroimaging revealed a progressive pontocerebellar and cerebral cortical atrophy. Molecular investigations of RARS2 disclosed the c.25A>G/p.I9V and the c.1586+3A>T in family A, the c.734G>A/p.R245Q and the c.1406G>A/p.R469H in family B, and the c.721T>A/p.W241R and c.35A>G/p.Q12R in family C. Functional complementation studies in Saccharomyces cerevisiae showed that mutation MSR1-R531H (equivalent to human p.R469H) abolished respiration whereas the MSR1-R306Q strain (corresponding to p.R245Q) displayed a reduced growth on non-fermentable YPG medium. Although mutations functionally disrupted yeast we found a relatively well preserved arginine aminoacylation of mitochondrial tRNA. Clinical and neuroimaging findings are important clues to raise suspicion and to reach diagnostic accuracy for RARS2 mutations considering that biochemical abnormalities may be absent in muscle biopsy.

Sex influences clinical phenotype in valosin-containing protein mutations: A case family report and systematic literature review.

OBJECTIVE: Mutations in the valosin-containing protein (VCP) gene cause autosomal dominant multisystem proteinopathy 1 (MSP1), characterized by a variable combination of inclusion body myopathy (IBM), Paget's disease of bone (PDB), and frontotemporal dementia (FTD). Here we report a novel VCP missense mutations in an Italian family with FTD as the prevalent manifestation and compare our results with those described in the literature. METHODS: We described the clinical, molecular, and imaging data of the studied family. We also conducted a systematic literature search with the aim of comparing our findings with previously reported VCP-related phenotypes. RESULTS: A novel heterozygous VCP missense mutation (c 0.473 T > C/p.Met158Thr) was found in all the affected family members. The proband is a 69-year-old man affected by progressive muscle weakness since the age of 49. Muscle MRI showed patchy fatty infiltration in most muscles, and STIR sequences revealed an unusual signal increase in distal leg muscles. At age 65, he presented a cognitive disorder suggestive of behavioral variant FTD. A bone scintigraphy also revealed PDB. The patient's mother, his maternal aunt and her daughter had died following a history of cognitive deterioration consistent with FTD; the mother also had PDB. No relatives had any muscular impairments. Reviewing the literature data, we observed a different sex distribution of VCP-related phenotypes, being FTD prevalence higher among women as compared to men (51.2 % vs 31.2 %) and IBM prevalence higher among men as compared to women (92.1 % vs 72.8 %). DISCUSSION: This study broadened our clinical, genetic, and imaging knowledge of VCP-related disorders.

Using Cluster Analysis to Overcome the Limits of Traditional Phenotype-Genotype Correlations: The Example of RYR1-Related Myopathies.

Thanks to advances in gene sequencing, RYR1-related myopathy (RYR1-RM) is now known to manifest itself in vastly heterogeneous forms, whose clinical interpretation is, therefore, highly challenging. We set out to develop a novel unsupervised cluster analysis method in a large patient population. The objective was to analyze the main RYR1-related characteristics to identify distinctive features of RYR1-RM and, thus, offer more precise genotype-phenotype correlations in a group of potentially life-threatening disorders. We studied 600 patients presenting with a suspicion of inherited myopathy, who were investigated using next-generation sequencing. Among them, 73 index cases harbored variants in RYR1. In an attempt to group genetic variants and fully exploit information derived from genetic, morphological, and clinical datasets, we performed unsupervised cluster analysis in 64 probands carrying monoallelic variants. Most of the 73 patients with positive molecular diagnoses were clinically asymptomatic or pauci-symptomatic. Multimodal integration of clinical and histological data, performed using a non-metric multi-dimensional scaling analysis with k-means clustering, grouped the 64 patients into 4 clusters with distinctive patterns of clinical and morphological findings. In addressing the need for more specific genotype-phenotype correlations, we found clustering to overcome the limits of the "single-dimension" paradigm traditionally used to describe genotype-phenotype relationships.

Expanding the clinical-pathological and genetic spectrum of RYR1-related congenital myopathies with cores and minicores: an Italian population study.

Mutations in the RYR1 gene, encoding ryanodine receptor 1 (RyR1), are a well-known cause of Central Core Disease (CCD) and Multi-minicore Disease (MmD). We screened a cohort of 153 patients carrying an histopathological diagnosis of core myopathy (cores and minicores) for RYR1 mutation. At least one RYR1 mutation was identified in 69 of them and these patients were further studied. Clinical and histopathological features were collected. Clinical phenotype was highly heterogeneous ranging from asymptomatic or paucisymptomatic hyperCKemia to severe muscle weakness and skeletal deformity with loss of ambulation. Sixty-eight RYR1 mutations, generally missense, were identified, of which 16 were novel. The combined analysis of the clinical presentation, disease progression and the structural bioinformatic analyses of RYR1 allowed to associate some phenotypes to mutations in specific domains. In addition, this study highlighted the structural bioinformatics potential in the prediction of the pathogenicity of RYR1 mutations. Further improvement in the comprehension of genotype-phenotype relationship of core myopathies can be expected in the next future: the actual lack of the human RyR1 crystal structure paired with the presence of large intrinsically disordered regions in RyR1, and the frequent presence of more than one RYR1 mutation in core myopathy patients, require designing novel investigation strategies to completely address RyR1 mutation effect.

Mitochondrial myopathy in a child with a muscle-restricted mutation in the mitochondrial transfer RNAAsn gene.

We report an 11-year-old boy with exercise-related myopathy, and a novel mutation m.5669G>A in the mitochondrial tRNA Asparagine gene (mt-tRNA(Asn), MTTN). Muscle biopsy studies showed COX-negative, SDH-positive fibers at histochemistry and biochemical defects of oxidative metabolism. The m.5669G>A mutation was present only in patient's muscle resulting in the first muscle-specific MTTN mutation. Mt-tRNA(Asn) steady-state levels and in silico predictions supported the pathogenicity of this mutation. A mitochondrial myopathy should be considered in the differential diagnosis of exercise intolerance in children.

Cerebellar hypoplasia and brainstem thinning associated with severe white matter and basal ganglia abnormalities in a child with an mtDNA deletion.

Cerebellar and brainstem hypoplasia may occur in different conditions, including those disorders designated as pontocerebellar hypoplasia (PCH). In particular, when PCH is combined with severe supratentorial white matter involvement and cerebral atrophy, mutations in the mitochondrial arginyl-tRNA synthethase (RARS2) gene causing PCH6 are possible. We describe a patient with a lethal mitochondrial encephalomyopathy due to a mtDNA deletion and no alterations in RARS2, whose magnetic resonance (MR) findings mimicked PCH6. A thorough diagnostic work-up for mitochondrial disorders should be carried out when facing with a PCH-like and severe white matter and basal ganglia involvement on brain MR imaging in children, even if clinical and laboratory mitochondrial "stigmata" are scant or nonspecific.

Protein aggregates and autophagy involvement in a family with a mutation in Z-band alternatively spliced PDZ-motif protein.

Z-band alternatively spliced PDZ-motif protein (ZASP) is a sarcomeric component expressed both in cardiac and skeletal muscles. Mutations in the LDB3/ZASP gene cause cardiomyopathy and myofibrillar myopathy. We describe a c.76C>T / p.[Pro26Ser] mutation in the PDZ motif of LDB3/ZASP in two siblings exhibiting late-onset myopathy with axial, proximal and distal muscles involvement and marked variability in clinical severity in the absence of a significant family history for neuromuscular disorders. Notably, we identified involvement of the psoas muscle on MRI and muscle CT, a feature not previously documented. Proband's muscle biopsy showed an increase of ZASP expression by western blotting. Muscle fibres morphological features included peculiar sarcolemmal invaginations, pathological aggregates positive to ZASP, ubiquitin, p62 and LC3 antibodies, and the accumulation of autophagic vacuoles, suggesting that protein aggregate formation and autophagy are involved in this additional case of zaspopathy.

Early Findings in Neonatal Cases of RYR1-Related Congenital Myopathies.

Ryanodine receptor type 1-related congenital myopathies are the most represented subgroup among congenital myopathies (CMs), typically presenting a central core or multiminicore muscle histopathology and high clinical heterogeneity. We evaluated a cohort of patients affected with Ryanodine receptor type 1-related congenital myopathy (RYR1-RCM), focusing on four patients who showed a severe congenital phenotype and underwent a comprehensive characterization at few months of life. To date there are few reports on precocious instrumental assessment. In two out of the four patients, a muscle biopsy was performed in the first days of life (day 5 and 37, respectively) and electron microscopy was carried out in two patients detecting typical features of congenital myopathy. Two patients underwent brain MRI in the first months of life (15 days and 2 months, respectively), one also a fetal brain MRI. In three children electromyography was performed in the first week of life and neurogenic signs were excluded. Muscle MRI obtained within the first years of life showed a typical pattern of RYR1-CM. The diagnosis was confirmed through genetic analysis in three out of four cases using Next Generation Sequencing (NGS) panels. The development of a correct and rapid diagnosis is a priority and may lead to prompt medical management and helps optimize inclusion in future clinical trials.

The Diagnostic Approach to Mitochondrial Disorders in Children in the Era of Next-Generation Sequencing: A 4-Year Cohort Study.

Mitochondrial diseases (MDs) are a large group of genetically determined multisystem disorders, characterized by extreme phenotypic heterogeneity, attributable in part to the dual genomic control (nuclear and mitochondrial DNA) of the mitochondrial proteome. Advances in next-generation sequencing technologies over the past two decades have presented clinicians with a challenge: to select the candidate disease-causing variants among the huge number of data provided. Unfortunately, the clinical tools available to support genetic interpretations still lack specificity and sensitivity. For this reason, the diagnosis of MDs continues to be difficult, with the new "genotype first" approach still failing to diagnose a large group of patients. With the aim of investigating possible relationships between clinical and/or biochemical phenotypes and definitive molecular diagnoses, we performed a retrospective multicenter study of 111 pediatric patients with clinical suspicion of MD. In this cohort, the strongest predictor of a molecular (in particular an mtDNA-related) diagnosis of MD was neuroimaging evidence of basal ganglia (BG) involvement. Regression analysis confirmed that normal BG imaging predicted negative genetic studies for MD. Psychomotor regression was confirmed as an independent predictor of a definitive diagnosis of MD. The findings of this study corroborate previous data supporting a role for neuroimaging in the diagnostic approach to MDs and reinforce the idea that mtDNA sequencing should be considered for first-line testing, at least in specific groups of children.

The spectrum of GNE mutations: allelic heterogeneity for a common phenotype.

Hereditary inclusion body myopathy (IBM2) was mainly reported in Middle Eastern Jewish patients. Distal myopathy with rimmed vacuoles has been described as a worldwide distributed distal myopathy. Both diseases are caused by mutations of the UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) gene. Herein we report two patients: an Egyptian Muslim patient with the "common" Middle Eastern mutation (M712T), rarely described in non-Jewish patients; and an Italian patient carrying a novel GNE mutation (L179F) in the epimerase domain. Our patients share common clinical and histopathological features, with some interesting aspects. The first patient presented a clinical deterioration during her first pregnancy confirming that an increased requirement of sialic acid during pregnancy may trigger a clinical worsening. The second patient showed a slowly progressive deterioration, different from other patients carrying mutations in the epimerase domain, who had a severe and rapid progression.

Respiratory muscle involvement in HNRNPDL LGMD D3 muscular dystrophy: an extensive clinical description of the first Italian patient.

Limb girdle muscular dystrophy is a genetically inherited condition that primarily affects skeletal muscle leading to progressive, predominantly proximal muscle weakness at presentation. Autosomal dominant LGMD represent 10% of all LGMDs. HNRNPDL-related muscular dystrophy, LGMD1G/LGMD D3 (MIM#609115), is an extremely rare autosomal dominant adult onset myopathy described in a handful of families. Here we fully characterized the muscular and respiratory involvement of a 58 years old Italian woman presenting the previously reported pathogenic variant c.1132G > C p.(Asp378Asn) in the HNRNPDL gene.