Detection of pericentric inversion with breakpoint in DMD by whole genome sequencing.

BACKGROUND: Dystrophinopathies caused by variants in the DMD gene are a well-studied muscle disease. The most common type of variant in DMD are large deletions. Very rarely reported forms of variants are chromosomal translocations, inversions and deep intronic variants (DIVs) because they are not detectable by standard diagnostic techniques (sequencing of coding sequence, copy number variant detection). This might be the reason that some clinically and histologically proven dystrophinopathy cases remain unsolved. METHODS: We used whole genome sequencing (WGS) to screen the entire DMD gene for variants in one of two brothers suffering from typical muscular dystrophy with strongly elevated creatine kinase levels. RESULTS: Although a pathogenic DIV could not be detected, we were able to identify a pericentric inversion with breakpoints in DMD intron 44 and Xq13.3, which could be confirmed by Sanger sequencing in the index as well as in his brother and mother. As this variation affects a major part of DMD it is most likely disease causing. CONCLUSION: Our findings elucidate that WGS is capable of detecting large structural rearrangements and might be suitable for the genetic diagnostics of dystrophinopathies in the future. In particular, inversions might be a more frequent cause for dystrophinopathies as anticipated and should be considered in genetically unsolved dystrophinopathy cases.

Report of two siblings with spondylodysplastic Ehlers-Danlos syndrome and B4GALT7 deficiency.

BACKGROUND: The spondylodysplastic Ehlers-Danlos subtype (OMIM #130070) is a rare connective tissue disorder characterized by a combination of connective tissue symptoms, skeletal features and short stature. It is caused by variants in genes encoding for enzymes involved in the proteoglycan biosynthesis or for a zinc transporter. PRESENTATION OF CASES: We report two brothers with a similar phenotype of short stature, joint hypermobility, distinct craniofacial features, developmental delay and severe hypermetropia indicative for a spondylodysplastic Ehlers-Danlos subtype. One also suffered from a recurrent pneumothorax. Gene panel analysis identified two compound heterozygous variants in the B4GALT7 gene: c.641G > A and c.723 + 4A > G. B4GALT7 encodes for galactosyltransferase I, which is required for the initiation of glycosaminoglycan side chain synthesis of proteoglycans. CONCLUSIONS: This is a first full report on two cases with spondylodysplastic Ehlers-Danlos syndrome and the c.723 + 4A > G variant of B4GALT7. The recurrent pneumothoraces observed in one case expand the variable phenotype of the syndrome.

FLNC-Associated Myofibrillar Myopathy: New Clinical, Functional, and Proteomic Data.

OBJECTIVE: To determine whether a new indel mutation in the dimerization domain of filamin C (FLNc) causes a hereditary myopathy with protein aggregation in muscle fibers, we clinically and molecularly studied a German family with autosomal dominant myofibrillar myopathy (MFM). METHODS: We performed mutational analysis in 3 generations, muscle histopathology, and proteomic studies of IM protein aggregates. Functional consequences of the FLNC mutation were investigated with interaction and transfection studies and biophysics molecular analysis. RESULTS: Eight patients revealed clinical features of slowly progressive proximal weakness associated with a heterozygous c.8025_8030delCAAGACinsA (p.K2676Pfs*3) mutation in FLNC. Two patients exhibited a mild cardiomyopathy. MRI of skeletal muscle revealed lipomatous changes typical for MFM with FLNC mutations. Muscle biopsies showed characteristic MFM findings with protein aggregation and lesion formation. The proteomic profile of aggregates was specific for MFM-filaminopathy and indicated activation of the ubiquitin-proteasome system (UPS) and autophagic pathways. Functional studies revealed that mutant FLNc is misfolded, unstable, and incapable of forming homodimers and heterodimers with wild-type FLNc. CONCLUSIONS: This new MFM-filaminopathy family confirms that expression of mutant FLNC leads to an adult-onset muscle phenotype with intracellular protein accumulation. Mutant FLNc protein is biochemically compromised and leads to dysregulation of protein quality control mechanisms. Proteomic analysis of MFM protein aggregates is a potent method to identify disease-relevant proteins, differentiate MFM subtypes, evaluate the relevance of gene variants, and identify novel MFM candidate genes.

First clinical and myopathological description of a myofibrillar myopathy with congenital onset and homozygous mutation in FLNC.

Filamin C (encoded by the FLNC gene) is a large actin-cross-linking protein involved in shaping the actin cytoskeleton in response to signaling events both at the sarcolemma and at myofibrillar Z-discs of cross-striated muscle cells. Multiple mutations in FLNC are associated with myofibrillar myopathies of autosomal-dominant inheritance. Here, we describe for the first time a boy with congenital onset of generalized muscular hypotonia and muscular weakness, delayed motor development but no cardiac involvement associated with a homozygous FLNC mutation c.1325C>G (p.Pro442Arg). We performed ultramorphological, proteomic, and functional investigations as well as immunological studies of known marker proteins for dominant filaminopathies. We show that the mutant protein is expressed in similar quantities as the wild-type variant in control skeletal muscle fibers. The proteomic signature of quadriceps muscle is altered and ultrastructural perturbations are evident. Moreover, filaminopathy marker proteins are comparable both in our homozygous and a dominant control case (c.5161delG). Biochemical investigations demonstrate that the recombinant mutant protein is less stable and more prone to degradation by proteolytic enzymes than the wild-type variant. The unusual congenital presentation of the disease clearly demonstrates that homozygosity for mutations in FLNC severely aggravates the phenotype.

Differential diagnosis of vacuolar myopathies in the NGS era.

Altered autophagy accompanied by abnormal autophagic (rimmed) vacuoles detectable by light and electron microscopy is a common denominator of many familial and sporadic non-inflammatory muscle diseases. Even in the era of next generation sequencing (NGS), late-onset vacuolar myopathies remain a diagnostic challenge. We identified 32 adult vacuolar myopathy patients from 30 unrelated families, studied their clinical, histopathological and ultrastructural characteristics and performed genetic testing in index patients and relatives using Sanger sequencing and NGS including whole exome sequencing (WES). We established a molecular genetic diagnosis in 17 patients. Pathogenic mutations were found in genes typically linked to vacuolar myopathy (GNE, LDB3/ZASP, MYOT, DES and GAA), but also in genes not regularly associated with severely altered autophagy (FKRP, DYSF, CAV3, COL6A2, GYG1 and TRIM32) and in the digenic facioscapulohumeral muscular dystrophy 2. Characteristic histopathological features including distinct patterns of myofibrillar disarray and evidence of exocytosis proved to be helpful to distinguish causes of vacuolar myopathies. Biopsy validated the pathogenicity of the novel mutations p.(Phe55*) and p.(Arg216*) in GYG1 and of the p.(Leu156Pro) TRIM32 mutation combined with compound heterozygous deletion of exon 2 of TRIM32 and expanded the phenotype of Ala93Thr-caveolinopathy and of limb-girdle muscular dystrophy 2i caused by FKRP mutation. In 15 patients no causal variants were detected by Sanger sequencing and NGS panel analysis. In 12 of these cases, WES was performed, but did not yield any definite mutation or likely candidate gene. In one of these patients with a family history of muscle weakness, the vacuolar myopathy was eventually linked to chloroquine therapy. Our study illustrates the wide phenotypic and genotypic heterogeneity of vacuolar myopathies and validates the role of histopathology in assessing the pathogenicity of novel mutations detected by NGS. In a sizable portion of vacuolar myopathy cases, it remains to be shown whether the cause is hereditary or degenerative.

Statin-induced myopathic changes in primary human muscle cells and reversal by a prostaglandin F2 alpha analogue.

Statin-related muscle side effects are a constant healthcare problem since patient compliance is dependent on side effects. Statins reduce plasma cholesterol levels and can prevent secondary cardiovascular diseases. Although statin-induced muscle damage has been studied, preventive or curative therapies are yet to be reported. We exposed primary human muscle cell populations (n = 22) to a lipophilic (simvastatin) and a hydrophilic (rosuvastatin) statin and analyzed their expressome. Data and pathway analyses included GOrilla, Reactome and DAVID. We measured mevalonate intracellularly and analyzed eicosanoid profiles secreted by human muscle cells. Functional assays included proliferation and differentiation quantification. More than 1800 transcripts and 900 proteins were differentially expressed after exposure to statins. Simvastatin had a stronger effect on the expressome than rosuvastatin, but both statins influenced cholesterol biosynthesis, fatty acid metabolism, eicosanoid synthesis, proliferation, and differentiation of human muscle cells. Cultured human muscle cells secreted ω-3 and ω-6 derived eicosanoids and prostaglandins. The ω-6 derived metabolites were found at higher levels secreted from simvastatin-treated primary human muscle cells. Eicosanoids rescued muscle cell differentiation. Our data suggest a new aspect on the role of skeletal muscle in cholesterol metabolism. For clinical practice, the addition of omega-n fatty acids might be suitable to prevent or treat statin-myopathy.

Expanding the importance of HMERF titinopathy: new mutations and clinical aspects.

OBJECTIVE: Hereditary myopathy with early respiratory failure (HMERF) is caused by titin A-band mutations in exon 344 and considered quite rare. Respiratory insufficiency is an early symptom. A collection of families and patients with muscle disease suggestive of HMERF was clinically and genetically studied. METHODS: Altogether 12 new families with 19 affected patients and diverse nationalities were studied. Most of the patients were investigated using targeted next-generation sequencing; Sanger sequencing was applied in some of the patients and available family members. Histological data and muscle MRI findings were evaluated. RESULTS: Three families had several family members studied while the rest were single patients. Most patients had distal and proximal muscle weakness together with respiratory insufficiency. Five heterozygous TTN A-band mutations were identified of which two were novel. Also with the novel mutations the muscle pathology and imaging findings were compatible with the previous reports of HMERF. CONCLUSIONS: Our collection of 12 new families expands mutational spectrum with two new mutations identified. HMERF is not that rare and can be found worldwide, but maybe underdiagnosed. Diagnostic process seems to be complex as this study shows with mostly single patients without clear dominant family history.

A new case expanding the mutation and phenotype spectrum of TMEM5-related alpha-dystroglycanopathy.

Dystroglycanopathies are a diverse group of neuromuscular disorders caused by aberrant glycosylation of alpha-dystroglycan. TMEM5 is one of many glycosyltransferases recently described to be associated with alpha-dystroglycanopathies. We report the case of a 15-year-old boy suffering from a congenital muscular dystrophy with elevated serum creatine kinase levels and an almost complete absence of alpha-dystroglycan in muscle biopsy. The clinical course was milder than any previously reported case and did not include brain or eye defects. Standard next-generation sequencing analysis revealed a homozygous mutation in the donor splice site region of exon 5 in TMEM5 (c.914+6 T>G). Available in-silico prediction tools anticipated a reduced efficiency of the splice site. Subsequent cDNA sequencing confirmed the expression of a truncated transcript of TMEM5 lacking exon 5, hence leading to an in-frame deletion in the exostosin domain of the protein. This report expands the clinical and mutation spectrum of alpha-dystroglycanopathies.

Distribution and Structure of DM2 Repeat Tract Alleles in the German Population.

Autosomal dominant inherited Myotonic dystrophy type 1 and 2 (DM1 and DM2) are the most frequent muscle dystrophies in the European population and are caused by repeat expansion mutations. For Germany cumulative empiric evidence suggests an estimated prevalence of DM2 of roughly 9 in 100,000, therefore being as prevalent as DM1. In DM2, a (CCTG)n repeat tract located in the first intron of the CNBP gene is expanded. The CCTG repeat tract is part of a complex repeat structure comprising not only CCTG tetraplets but also repeated TG dinucleotides and TCTG tetraplet elements as well as NCTG interruptions. Here, we provide the distribution of normal sized alleles in the German population, which was found to be highly similar to the Slovak population. Sequencing of 34 unexpanded healthy range alleles in DM2 positive patients (heterozygous for a full expansion) revealed that the CCTG repeat tract is usually interrupted by at least three tetraplets which according to current opinion is supposed to render it stable against expansion. Interestingly, only the largest analyzed normal allele had 23 uninterrupted CCTGs and consequently could represent an instable early premutation allele. In our diagnostic history of DM2 cases, a total of 18 premutations were detected in 16 independent cases. Here, we describe two premutation families, one with an expansion from a premutation allele and the other with a contraction of a full expansion down to a premutation allele. Our diagnostic results support the general assumption that the premutation range of unstable CCTG stretches lies obviously between 25 and 75 CCTGs. However, the clinical significance of premutation alleles is still unclear. In the light of the two described families we suggest incomplete penetrance. Thus, as it was proposed for other repeat expansion diseases (e.g., Huntington's disease), a fluid transition of penetrance is more likely rather than a clear cut CCTG number threshold.

The metabolic and endocrine characteristics in spinal and bulbar muscular atrophy.

OBJECTIVE: Spinal and bulbar muscular atrophy (SBMA) is caused by an abnormal expansion of the CAG repeat in the androgen receptor gene. This study aimed to systematically phenotype a German SBMA cohort (n = 80) based on laboratory markers for neuromuscular, metabolic, and endocrine status, and thus provide a basis for the selection of biomarkers for future therapeutic trials. METHODS: We assessed a panel of 28 laboratory parameters. The clinical course and blood biomarkers were correlated with disease duration and CAG repeat length. A subset of 11 patients was evaluated with body fat MRI. RESULTS: Almost all patients reported muscle weakness (99%), followed by dysphagia (77%), tremor (76%), and gynecomastia (75%) as major complaints. Creatine kinase was the most consistently elevated (94%) serum marker, which, however, did not relate with either the disease duration or the CAG repeat length. Paresis duration and CAG repeat length correlated with dehydroepiandrosterone sulfate after correction for body mass index and age. The androgen insensitivity index was elevated in nearly half of the participants (48%). CONCLUSIONS: Metabolic alterations in glucose homeostasis (diabetes) and fat metabolism (combined hyperlipidemia), and sex hormone abnormalities (androgen insensitivity) could be observed among SBMA patients without association with the neuromuscular phenotype. Dehydroepiandrosterone sulfate was the only biomarker that correlated strongly with both weakness duration and the CAG repeat length after adjusting for age and BMI, indicating its potential as a biomarker for both disease severity and duration and, therefore, its possible use as a reliable outcome measure in future therapeutic studies.

Assessing the influence of age and gender on the phenotype of myotonic dystrophy type 2.

This study aims to provide a detailed clinical characterization of a large cohort of myotonic dystrophy type 2 (DM2) patients investigating the influence of age and gender as modifying factors of DM2 phenotype. A retrospective study was conducted on 307 patients with genetically confirmed DM2. The following data were analyzed: (1) demographics, (2) clinical features (first symptom, muscular complaints, and multisystemic involvement), (3) diagnostics (serological tests, electromyography, and muscle biopsy). In this cohort (186 females, 121 males), a proximal weakness was the leading symptom at onset (55.4%), followed by myalgia (35.5%) and myotonia (25.4%). Proximal weakness was more common in women than men (64.9 vs. 43.8%, p = 0.0006), whereas being male was associated with higher odds for developing myalgia [OR 2.94 (95% CI 1.53-5.67)]. Patients with muscle weakness at onset were older than those with myalgia and myotonia (p < 0.0001), while each additional disease year was associated with 10% decrease in the odds of developing myotonia [OR 0.9 (95% CI 0.87-0.93)] and 6% decrease of myalgia [OR 0.94 (95% CI 0.91-0.97)]. Cataract and thyroid diseases occurred more frequently in women (p = 0.002 and p = 0.002, respectively). Early onset of DM2 is an independent risk factor for the occurrence of multisystemic involvement [OR 0.94 (95% CI 0.90-0.98)]. In this updated clinical description of DM2 emerges a profound gender and age influence on the phenotype, emphasizing that female gender and ageing may be associated with a higher disease burden. These age- and gender-specific differences should be considered in diagnostics, management, and future clinical studies of DM2.

Mutation c.943G>T (p.Ala315Ser) in FGFR2 Causing a Mild Phenotype of Crouzon Craniofacial Dysostosis in a Three-Generation Family.

Crouzon syndrome craniofacial dysostosis type I [OMIM 123500] is caused by mutations in the gene encoding fibroblast growth factor receptor-2 (FGFR2). An overlapping phenotype with Muenke and Crouzon syndrome with acanthosis nigricans (FGFR3 mutations) is known. The clinical diagnosis can be corroborated by molecular studies in about 80-90% of the cases. No clear genotype/phenotype correlation has been identified yet. Here, we describe a second family with a mild phenotype in which the FGFR2 mutation c.943G>T leading to the amino acid substitution p.Ala315Ser was detected. Five affected family members showed craniofacial dysostosis without overt craniosynostosis. They all had midface hypoplasia. Crouzonoid appearance with mild protrusion of bulbi was only apparent in our index patient as well as obstructive sleep apnea episodes leading to reduced oxygen saturation; therefore, surgical intervention was suggested. One other affected family member additionally had iris coloboma.

Deep intronic variants introduce DMD pseudoexon in patient with muscular dystrophy.

Dystrophinopathies are X-linked muscle diseases caused by mutations in the large DMD gene. The most common mutations are detected by standard diagnostic techniques. However, some patients remain without detectable mutation, most likely due to changes in the non-coding sequence. We report on a boy with complete absence of dystrophin in muscle biopsy but no causative mutation according to standard diagnostics. To search for deep intronic variations (DIV) in the DMD gene we isolated mRNA from muscle tissue and amplified overlapping cDNA fragments using RT-PCR. One cDNA product revealed an augmented fragment size showing an insertion of 77 bp between the exons 7 and 8 by sequencing. We sequenced the flanking sequences of gDNA and found two hemizygous single nucleotide variants (c.650-39575 A>C and c.650-39498 A>G) surrounding the inserted fragment. Both variants create cryptic splice sites which initiate the formation of a pseudoexon that produces a frameshift in the DMD gene.

Leg Muscle Involvement in Facioscapulohumeral Muscular Dystrophy: Comparison between Facioscapulohumeral Muscular Dystrophy Types 1 and 2.

BACKGROUND: Facioscapulohumeral muscular dystrophy (FSHD) presents with 2 genetically distinct types. We describe for the first time the MRI patterns of leg muscle involvement in type 2 and compare it with type 1. METHODS: The intramuscular fat content was assessed on lower extremity axial T1-weighted MRI scans in 6 FSHD1 and 5 FSHD2 patients. RESULTS: Overall, the muscle involvement profile did not differ substantially between FSHD1 and FSHD2. In the thigh, the dorsomedial compartment including the semimembranosus, semitendinosus and adductor magnus was the most affected. The quadriceps was mostly spared, but isolated involvement of the rectus femoris was common. Fat infiltration in the distal soleus and the medial gastrocnemius with sparing of the lateral gastrocnemius was a common finding; involvement of the tibialis anterior was less frequent. A proximal-to-distal increase in fat content was frequently present in some muscles. CONCLUSION: Muscle involvement appears to be independent of type, confirming a similar pathophysiological pathway in FSHD1 and FSHD2.

The Caveolin-3 G56S sequence variant of unknown significance: Muscle biopsy findings and functional cell biological analysis.

PURPOSE: In the era of next-generation sequencing, we are increasingly confronted with sequence variants of unknown significance. This phenomenon is also known for variations in Caveolin-3 and can complicate the molecular diagnosis of the disease. Here, we aimed to study the ambiguous character of the G56S Caveolin-3 variant. EXPERIMENTAL DESIGN: A comprehensive approach combining genetic and morphological studies of muscle derived from carriers of the G56S Caveolin-3 variant were carried out and linked to biochemical assays (including phosphoblot studies and proteome profiling) and morphological investigations of cultured myoblasts. RESULTS: Muscles showed moderate chronic myopathic changes in all carriers of the variant. Myogenic RCMH cells expressing the G56S Caveolin-3 protein presented irregular Caveolin-3 deposits within the Golgi in addition to a regular localization of the protein to the plasma membrane. This result was associated with abnormal findings on the ultra-structural level. Phosphoblot studies revealed that G56S affects EGFR-signaling. Proteomic profiling demonstrated alterations in levels of physiologically relevant proteins which are indicative for antagonization of G56S Caveolin-3 expression. Remarkably, some proteomic alterations were enhanced by osmotic/mechanical stress. CONCLUSIONS AND CLINICAL RELEVANCE: Our studies suggest that G56S might influence the manifestation of myopathic changes upon the presence of additional cellular stress burden. Results of our studies moreover improve the current understanding of (genetic) causes of myopathic disorders classified as caveolinopathies.

Identification of variants in MBNL1 in patients with a myotonic dystrophy-like phenotype.

The myotonic dystrophies (DMs) are the most common inherited muscular disorders in adults. In most of the cases, the disease is caused by (CTG)n/(CCTG)n repeat expansions (EXPs) in non-coding regions of the genes DMPK (dystrophia myotonica-protein kinase) and CNBP (CCHC-type zinc-finger nucleic acid-binding protein). The EXP is transcribed into mutant RNAs, which provoke a common pathomechanism that is characterized by misexpression and mis-splicing. In this study, we screened 138 patients with typical clinical features of DM being negative for EXP in the known genes. We sequenced DMPK and CNBP - associated with DM, as well as CELF1 (CUGBP, Elav-like family member 1) and MBNL1 (muscleblind-like splicing regulator 1) - associated with the pathomechanism of DM, for pathogenic variants, addressing the question whether defects in other genes could cause a DM-like phenotype. We identified variants in three unrelated patients in the MBNL1 gene, two of them were heterozygous missense mutations and one an in-frame deletion of three amino acids. The variants were located in different conserved regions of the protein. The missense mutations were classified as potentially pathogenic by prediction tools. Analysis of MBNL1 splice target genes was carried out for one of the patients using RNA from peripheral blood leukocytes (PBL). Analysis of six genes known to show mis-splicing in the skeletal muscle gave no informative results on the effect of this variant when tested in PBL. The association of these variants with the DM phenotype therefore remains unconfirmed, but we hope that in view of the key role of MBNL1 in DM pathogenesis our observations may foster further studies in this direction.

Dysferlinopathy in Switzerland: clinical phenotypes and potential founder effects.

BACKGROUND: Dysferlin is reduced in patients with limb girdle muscular dystrophy type 2B, Miyoshi myopathy, distal anterior compartment myopathy, and in certain Ethnic clusters. METHODS: We evaluated clinical and genetic patient data from three different Swiss Neuromuscular Centers. RESULTS: Thirteen patients from 6 non-related families were included. Age of onset was 18.8 ± 4.3 years. In all patients, diallelic disease-causing mutations were identified in the DYSF gene. Nine patients from 3 non-related families from Central Switzerland carried the identical homozygous mutation, c.3031 + 2 T>C. A possible founder effect was confirmed by haplotype analysis. Three patients from two different families carried the heterozygous mutation, c.1064_1065delAA. Two novel mutations were identified (c.2869 C>T (p.Gln957Stop), c.5928 G>A (p.Trp1976Stop)). CONCLUSIONS: Our study confirms the phenotypic heterogeneity associated with DYSF mutations. Two mutations (c.3031 + 2 T>C, c.1064_1065delAA) appear common in Switzerland. Haplotype analysis performed on one case (c. 3031 + 2 T>C) suggested a possible founder effect.