Clinical and genetic interpretation of uncertain DMD missense variants: evidence from mRNA and protein studies.

BACKGROUND: Pathogenic missense variants in the dystrophin (DMD) gene are rarely reported in dystrophinopathies. Most DMD missense variants are of uncertain significance and their pathogenicity interpretation remains complicated. We aimed to investigate whether DMD missense variants would cause aberrant splicing and re-interpret their pathogenicity based on mRNA and protein studies. METHODS: Nine unrelated patients who had an elevated serum creatine kinase level with or without muscle weakness were enrolled. They underwent a detailed clinical, imaging, and pathological assessment. Routine genetic testing and muscle-derived mRNA and protein studies of dystrophin and sarcoglycan genes were performed in them. RESULTS: Three of the 9 patients presented with a Duchenne muscular dystrophy (DMD) phenotype and the remaining 6 patients had a suspected diagnosis of Becker muscular dystrophy (BMD) or sarcoglycanopathy based on their clinical and pathological characteristics. Routine genetic testing detected only 9 predicted DMD missense variants in them, of which 6 were novel and interpreted as uncertain significance. Muscle-derived mRNA studies of sarcoglycan genes didn't reveal any aberrant transcripts in them. Dystrophin mRNA studies confirmed that 3 predicted DMD missense variants (c.2380G > C, c.4977C > G, and c.5444A > G) were in fact splicing and frameshift variants due to aberrant splicing. The 9 DMD variants were re-interpreted as pathogenic or likely pathogenic based on mRNA and protein studies. Therefore, 3 patients with DMD splicing variants and 6 patients with confirmed DMD missense variants were diagnosed with DMD and BMD, respectively. CONCLUSION: Our study highlights the importance of muscle biopsy and aberrant splicing for clinical and genetic interpretation of uncertain DMD missense variants.

SGCE promotes breast cancer stemness by promoting the transcription of FGF-BP1 by Sp1.

Breast cancer stem cells are mainly responsible for poor prognosis, especially in triple-negative breast cancer (TNBC). In a previous study, we demonstrated that ε-Sarcoglycan (SGCE), a type Ⅰ single-transmembrane protein, is a potential oncogene that promotes TNBC stemness by stabilizing EGFR. Here, we further found that SGCE depletion reduces breast cancer stem cells, partially through inhibiting the transcription of FGF-BP1, a secreted oncoprotein. Mechanistically, we demonstrate that SGCE could interact with the specific protein 1 transcription factor and translocate into the nucleus, which leads to an increase in the transcription of FGF-BP1, and the secreted FBF-BP1 activates FGF-FGFR signaling to promote cancer cell stemness. The novel SGCE-Sp1-FGF-BP1 axis provides novel potential candidate diagnostic markers and therapeutic targets for TNBC.

Identification of a shared, common haplotype segregating with an SGCB c.544 T > G mutation in Indian patients affected with sarcoglycanopathy.

Sarcoglycanopathy is the most frequent form of autosomal recessive limb-girdle muscular dystrophies caused by mutations in SGCB gene encoding beta-sarcoglycan proteins. In this study, we describe a shared, common haplotype co-segregating in 14 sarcoglycanopathy cases from 13 unrelated families from south Indian region with the likely pathogenic homozygous mutation c.544 T > G (p.Thr182Pro) in SGCB. Haplotype was reconstructed based on 10 polymorphic markers surrounding the c.544 T > G mutation in the cases and related family members as well as 150 unrelated controls from Indian populations using PLINK1.9. We identified haplotype H1 = G, A, G, T, G, G, A, C, T, G, T at a significantly higher frequency in cases compared to related controls and unrelated control Indian population. Upon segregation analysis within the family pedigrees, H1 is observed to co-segregate with c.544 T > G in a homozygous state in all the pedigrees of cases except one indicating a probable event of founder effect. Furthermore, Identical-by-descent and inbreeding coefficient analysis revealed relatedness among 33 new pairs of seemingly unrelated individuals from sarcoglycanopathy cohort and a higher proportion of homozygous markers, thereby indicating common ancestry. Since all these patients are from the south Indian region, we suggest this region to be a primary target of mutation screening in patients diagnosed with sarcoglycanopathy.

SGCD Missense Variant in a Lagotto Romagnolo Dog with Autosomal Recessively Inherited Limb-Girdle Muscular Dystrophy.

An 8-month-old female Lagotto Romagnolo dog was presented for a 1-month history of an initial severe reluctance to move, rapidly progressing to a marked stiff gait and progressive muscular weakness and evolving to tetraparesis, which persuaded the owner to request euthanasia. A primary muscle pathology was supported by necropsy and histopathological findings. Macroscopically, the muscles were moderately atrophic, except for the diaphragm and the neck muscles, which were markedly thickened. Histologically, all the skeletal muscles examined showed atrophy, hypertrophy, necrosis with calcification of the fibers, and mild fibrosis and inflammation. On immunohistochemistry, all three dystrophin domains and sarcoglycan proteins were absent. On Western blot analysis, no band was present for delta sarcoglycan. We sequenced the genome of the affected dog and compared the data to more than 900 control genomes of different dog breeds. Genetic analysis revealed a homozygous private protein-changing variant in the SGCD gene encoding delta- sarcoglycan in the affected dog. The variant was predicted to induce a SGCD:p.(Leu242Pro) change in the protein. In silico tools predicted the change to be deleterious. Other 770 Lagotto Romagnolo dogs were genotyped for the variant and all found to be homozygous wild type. Based on current knowledge of gene function in other mammalian species, including humans, hamsters, and dogs, we propose the SGCD missense variant as the causative variant of the observed form of muscular dystrophy in the index case. The absence of the variant allele in the Lagotto Romagnolo breeding population indicates a rare allele that has appeared recently.

A Genetics Pearl for Counseling Patients with Epsilon-Sarcoglycan Myoclonus-Dystonia.

Background: Epsilon-sarcoglycan (SGCE) myoclonus-dystonia is autosomal dominant (AD) with reduced penetrance due to maternal imprinting 95% of the time. Patients may lack family history delaying diagnosis and treatment. Additionally, counseling patients on their risk of passing on the variant differs for females versus males. Case Report: A woman in her thirties with typical phenotype of myoclonus-dystonia but lacking an AD pedigree was found to have a pathogenic variant in the SGCE gene. She was counseled that her daughters each have a 2.5% chance of expressing the phenotype. Discussion: Understanding the genetics of SGCE-myoclonus-dystonia enables effective genetic counseling and arrival at a timely diagnosis and treatment. Summary: In an era of advancing genetic analysis and precision medicine-based treatments, neurologists will be faced with increasing responsibility to properly counsel patients on the results of genetic testing. This case highlights a genetics pearl for counseling patients with epsilon-sarcoglycan myoclonus-dystonia, an autosomal dominant condition with penetrance differing by sex.

Differential histological features and myogenic protein levels in distinct muscles of d-sarcoglycan null muscular dystrophy mouse model.

Skeletal muscle (SkM) comprises slow and fast-twitch fibers, which differ in molecular composition, function, and systemic energy consumption. In addition, muscular dystrophies (DM), a group of diverse hereditary diseases, present different patterns of muscle involvement, progression, and severity, suggesting that the regeneration-degeneration process may differ depending on the muscle type. Therefore, the study aimed to explore the expression of proteins involved in the repair process in different muscles at an early stage of muscular dystrophy in the δ-sarcoglycan null mice (Sgcd-null), a limb-girdle muscular dystrophy 2 F model. Hematoxylin & Eosin (H&E) Staining showed a high number of central nuclei in soleus (Sol), tibialis (Ta), gastrocnemius (Gas), and extensor digitorum longus (Edl) from four months Sgcd-null mice. However, fibrosis, determined by trichrome of Gomori modified staining, was only observed in Sgcd-null Sol. In addition, the number of Type I and II fibers variated differentially in the Sgcd-null muscles vs. wild-type muscles. Besides, the protein expression level of ß-catenin, myomaker, MyoD, and myogenin also presented different expression levels in all the Sgcd-null muscles studied. In summary, our study reveals that muscles with different metabolic characteristics showed distinct expression patterns of proteins involved in the muscle regeneration process. These results could be relevant in designing therapies for genetic and acquired myopathy.

How a paternal uniparental isodisomy of chromosome 17 leads to autosomal recessive limb-girdle muscular dystrophy R3.

Uniparental isodisomy is a condition where both chromosomes of a pair are inherited from one parental homologue. If a deleterious variant is present on the duplicated chromosome, its homozygosity can reveal an autosomal recessive disorder in the offspring of a heterozygous carrier. Limb-girdle muscular dystrophy (LGMD) R3 is an autosomal recessive inherited disease that is associated with alpha-sarcoglycan gene (SGCA) variants. We report the first published case of LGMDR3 due to a homozygous variant in SGCA unmasked by uniparental isodisomy. The patient is an 8-year-old who experienced delayed motor milestones but normal cognitive development. He presented with muscle pain and elevated plasma creatine kinase. Sequencing of the SGCA gene showed a homozygous pathogenic variant. Parents were not related and only the father was heterozygous for the pathogenic variant. A chromosomal microarray revealed a complete chromosome 17 copy number neutral loss of heterozygosity encompassing SGCA, indicating paternal uniparental isodisomy.

The P2X7 purinoceptor in pathogenesis and treatment of dystrophino- and sarcoglycanopathies.

Dystrophinopathy and sarcoglycanopathies are incurable diseases caused by mutations in the genes encoding dystrophin or members of the dystrophin associated protein complex (DAPC). Restoration of the missing dystrophin or sarcoglycans via genetic approaches is complicated by the downsides of personalised medicines and immune responses against re-expressed proteins. Thus, the targeting of disease mechanisms downstream from the mutant protein has a strong translational potential. Acute muscle damage causes release of large quantities of ATP, which activates P2X7 purinoceptors, resulting in inflammation that clears dead tissues and triggers regeneration. However, in dystrophic muscles, loss of α-sarcoglycan ecto-ATPase activity further elevates extracellular ATP (eATP) levels, exacerbating the pathology. Moreover, seemingly compensatory P2X7 upregulation in dystrophic muscle cells, combined with high eATP leads to further damage. Accordingly, P2X7 blockade alleviated dystrophic damage in mouse models of both dystrophinopathy and sarcoglycanopathy. Existing P2X7 blockers could be re-purposed for the treatment of these highly debilitating diseases.

Early recognition of SGCE-myoclonus-dystonia in children.

AIM: To evaluate early dystonic features in children and adolescents with SGCE-myoclonus-dystonia. METHOD: In this cross-sectional study, 49 patients (26 females and 23 males) with SGCE-myoclonus-dystonia (aged 15y 2mo, SD 12y) with childhood-onset (2y 10mo, SD 1y 10mo) dystonia were examined using a standardized video recorded protocol. Dystonia was rated using the Writer's Cramp and Gait Dystonia Rating Scales. Disability and impairment for handwriting and walking were also rated. RESULTS: Dystonia was present at rest (n=1), posture (n=12), and during specific motor tasks (n=45) such as writing (n=35), walking (n=23), and running (n=20). Most children reported disability while performing these tasks. Early dystonic patterns were identified for writer's cramp and gait dystonia, the latter named the 'circular shaking leg', 'dragging leg', and 'hobby-horse gait' patterns. Sensory tricks were used by five and eight children to improve dystonia and myoclonus during writing and walking respectively. The rating scales accurately measured the severity of action dystonia and correlated with self-reported disability. INTERPRETATION: Children with SGCE-myoclonus-dystonia show recognizable dystonic patterns and sensory tricks that may lead to an early diagnosis and timely therapeutic approach. Isolated writer's cramp is a key feature in childhood and should prompt SCGE analysis. The proposed action dystonia scales could be used to monitor disease course and response to treatment. WHAT THIS PAPER ADDS: Most children with SGCE-myoclonus-dystonia got writer's cramp and had walking and running dystonia. Writer's cramp was a key feature and should prompt SGCE genetic investigation. 'Circular shaking leg', 'dragging leg', and 'hobby-horse gait' were recognized as early gait patterns. Children used sensory tricks to improve myoclonus and dystonia, suggesting common pathophysiological mechanisms. Action dystonia rating scales are valid tools to assess severity in children.

Loss-of-function mutations in SGCE found in Japanese patients with myoclonus-dystonia.

SGCE myoclonus-dystonia is a monogenic form of dystonia with an autosomal dominant mode of inheritance that co-occurs with a myoclonic jerk. In this study, we present 12 Japanese patients from nine families with this disease. Targeted next-generation sequencing covering major causative genes for monogenic dystonias identified nine distinct SGCE mutations from each of the families: three nonsense, two frameshift, two missense, one in-frame 15 bp deletion, and one splice donor site mutations, of which four were previously unreported. One missense mutation (c.662G>T, p.Gly221Val) was located at the 3' end of exon 5 (NM_001099400), which was predicted to cause aberrant splicing according to in silico predictions. Minigene assays performed together with the c.825+1G>C mutation demonstrated complete skipping of exon 5 and 6, respectively, in their transcripts. The other missense (c.1345A>G, p.Met449Val) and 15 bp deletion (c.168_182del, p.Phe58_Leu62del) mutations showed a significant reduction in cell membrane expression via HiBiT bioluminescence assay. Therefore, we concluded that all the detected mutations were disease-causing. Unlike the other detected mutations, p.Met449Val affects only isoform 3 (NP_001092870 encoded by NM_001099400) among the variously known isoforms of SGCE. This isoform is brain-specific and is mostly expressed in the cerebellum, which supports recent studies showing that cerebellar dysfunction is a key element in the pathophysiology of SGCE myoclonus-dystonia.

Cortical neuronal hyperexcitability and synaptic changes in SGCE mutation-positive myoclonus dystonia.

Myoclonus dystonia is a childhood-onset hyperkinetic movement disorder with a combined motor and psychiatric phenotype. It represents one of the few autosomal dominant inherited dystonic disorders and is caused by mutations in the ε-sarcoglycan (SGCE) gene. Work to date suggests that dystonia is caused by disruption of neuronal networks, principally basal ganglia-cerebello-thalamo-cortical circuits. Investigation of cortical involvement has primarily focused on disruption to interneuron inhibitory activity, rather than the excitatory activity of cortical pyramidal neurons. Here, we have sought to examine excitatory cortical glutamatergic activity using two approaches: the CRISPR/Cas9 editing of a human embryonic cell line, generating an SGCE compound heterozygous mutation, and three patient-derived induced pluripotent stem cell lines, each gene edited to generate matched wild-type SGCE control lines. Differentiation towards a cortical neuronal phenotype demonstrated no significant differences in either early- (PAX6, FOXG1) or late-stage (CTIP2, TBR1) neurodevelopmental markers. However, functional characterization using Ca2+ imaging and microelectrode array approaches identified an increase in network activity, while single-cell patch clamp studies found a greater propensity towards action potential generation with larger amplitudes and shorter half-widths associated with SGCE mutations. Bulk RNA sequencing analysis identified gene ontological enrichment for 'neuron projection development', 'synaptic signalling' and 'synaptic transmission'. Examination of dendritic morphology found SGCE mutations to be associated with a significantly higher number of branches and longer branch lengths, together with longer ion-channel dense axon initial segments, particularly towards the latter stages of differentiation (Days 80 and 100). Gene expression and protein quantification of key synaptic proteins (synaptophysin, synapsin and PSD95), AMPA and NMDA receptor subunits found no significant differences between the SGCE mutation and matched wild-type lines. By contrast, significant changes to synaptic adhesion molecule expression were identified, namely higher presynaptic neurexin-1 and lower postsynaptic neuroligin-4 levels in the SGCE mutation carrying lines. Our study demonstrates an increased intrinsic excitability of cortical glutamatergic neuronal cells in the context of SGCE mutations, coupled with a more complex neurite morphology and disruption to synaptic adhesion molecules. These changes potentially represent key components to the development of the hyperkinetic clinical phenotype observed in myoclonus dystonia, as well a central feature to the wider spectrum of dystonic disorders, potentially providing targets for future therapeutic development.

Clinical features and genetic analysis of SGCE myoclonus-dystonia: A case report.

Myoclonus-dystonia caused by mutations in the SGCE gene is clinically characterized by early onset, myoclonus, and dystonia. Here we describe a family in which several members exhibit varying degrees of myoclonus and dystonia, caused by a novel heterozygous mutation in the SGCE gene.

Homozygous Inversion on Chromosome 13 Involving SGCG Detected by Short Read Whole Genome Sequencing in a Patient Suffering from Limb-Girdle Muscular Dystrophy.

New techniques in molecular genetic diagnostics now allow for accurate diagnosis in a large proportion of patients with muscular diseases. Nevertheless, many patients remain unsolved, although the clinical history and/or the muscle biopsy give a clear indication of the involved genes. In many cases, there is a strong suspicion that the cause must lie in unexplored gene areas, such as deep-intronic or other non-coding regions. In order to find these changes, next-generation sequencing (NGS) methods are constantly evolving, making it possible to sequence entire genomes to reveal these previously uninvestigated regions. Here, we present a young woman who was strongly suspected of having a so far genetically unsolved sarcoglycanopathy based on her clinical history and muscle biopsy. Using short read whole genome sequencing (WGS), a homozygous inversion on chromosome 13 involving SGCG and LINC00621 was detected. The breakpoint in intron 2 of SGCG led to the absence of γ-sarcoglycan, resulting in the manifestation of autosomal recessive limb-girdle muscular dystrophy 5 (LGMDR5) in the young woman.

Immunofluorescence signal intensity measurements as a semi-quantitative tool to assess sarcoglycan complex expression in muscle biopsy.

Sarcoglycanopathies are highly heterogeneous in terms of disease progression, muscular weakness, loss of ambulation and cardiac/respiratory involvement. Their clinical severity usually correlates with the residual protein amount, which makes protein quantification extremely relevant. Sarcoglycanopathy diagnosis is genetic, but skeletal muscle analysis - by both immunohistochemistry and Western blot (WB) - is still mandatory to establish the correct diagnostic process. Unfortunately, however, WB analysis cannot be performed if the bioptic specimen is scarce. This study provides a sensitive tool for semi-quantification of residual amount of sarcoglycans in patients affected by sarcoglycanopathies, based on immunofluorescence staining on skeletal muscle sections, image acquisition and software elaboration. We applied this method to eleven sarcoglycanopathies, seven Becker muscular dystrophies and four age-matched controls. Fluorescence data analysed in patients and compared to age-matched controls showed a significant reduction of the mutated sarcoglycan expression and a variable reduction of the other sarcoglycans. Fluorescence normalized data analysed in relation to the age of onset of the disease, showed a negative correlation of α-sarcoglycan fluorescent signal versus fibrosis in patients with an early age of onset and a negative correlation between δ-sarcoglycan signal and fibrosis in both intermediate and late age of onset groups. The availability of a method that allows objective quantification of the sarcolemmal proteins, faster and less consuming than WB analysis and able to detect low residual sarcoglycan expression with great sensitivity, proves useful to better define both patient prognosis and expected disease evolution. The proposed method could be employed also to monitor the efficacy of therapeutic interventions and during clinical trials.

Comparative genomic analyses of multiple backcross mouse populations suggest SGCG as a novel potential obesity-modifier gene.

To nominate novel disease genes for obesity and type 2 diabetes (T2D), we recently generated two mouse backcross populations of the T2D-susceptible New Zealand Obese (NZO/HI) mouse strain and two genetically different, lean and T2D-resistant strains, 129P2/OlaHsd and C3HeB/FeJ. Comparative linkage analysis of our two female backcross populations identified seven novel body fat-associated quantitative trait loci (QTL). Only the locus Nbw14 (NZO body weight on chromosome 14) showed linkage to obesity-related traits in both backcross populations, indicating that the causal gene variant is likely specific for the NZO strain as NZO allele carriers in both crosses displayed elevated body weight and fat mass. To identify candidate genes for Nbw14, we used a combined approach of gene expression and haplotype analysis to filter for NZO-specific gene variants in gonadal white adipose tissue, defined as the main QTL-target tissue. Only two genes, Arl11 and Sgcg, fulfilled our candidate criteria. In addition, expression QTL analysis revealed cis-signals for both genes within the Nbw14 locus. Moreover, retroviral overexpression of Sgcg in 3T3-L1 adipocytes resulted in increased insulin-stimulated glucose uptake. In humans, mRNA levels of SGCG correlated with body mass index and body fat mass exclusively in diabetic subjects, suggesting that SGCG may present a novel marker for metabolically unhealthy obesity. In conclusion, our comparative-cross analysis could substantially improve the mapping resolution of the obesity locus Nbw14. Future studies will throw light on the mechanism by which Sgcg may protect from the development of obesity.

The therapeutic potential of soluble activin type IIB receptor treatment in a limb girdle muscular dystrophy type 2D mouse model.

Limb girdle muscular dystrophy type 2D (LGMD2D) is characterized by progressive weakening of muscles in the hip and shoulder girdles. It is caused by a mutation in the α-sarcoglycan gene and results in absence of α-sarcoglycan in the dystrophin-glycoprotein complex. The activin type IIB receptor is involved in the activin/myostatin pathway, with myostatin being a negative regulator of muscle growth. In this study, we investigated the effects of sequestering myostatin by a soluble activin type IIB receptor (sActRIIB) on muscle growth in Sgca-null mice, modelling LGMD2D. Treatment was initiated at 3 weeks of age, prior to the disease onset, or at 9 weeks of age when already in an advanced stage of the disease. We found that early sActRIIB treatment resulted in increased muscle size. However, this led to more rapid decline of muscle function than in saline-treated Sgca-null mice. Furthermore, no histopathological improvements were seen after sActRIIB treatment. When initiated at 9 weeks of age, sActRIIB treatment resulted in increased muscle mass too, but to a lesser extent. No effect of the treatment was observed on muscle function or histopathology. These data show that sActRIIB treatment as a stand-alone therapy does not improve muscle function or histopathology in Sgca-null mice.

Clinical, genetic profile and disease progression of sarcoglycanopathies in a large cohort from India: high prevalence of SGCB c.544A > C.

The clinico-genetic architecture of sarcoglycanopathies in Indian patients is reported only as short series. In the present study, we aimed to investigate the clinical picture, genetic basis, and disease progression of patients genetically confirmed to have sarcoglycanopathy. Next-generation sequencing was performed in 68 probands with suspected sarcoglycanopathy. A total of 35 different variants were detected in the sarcoglycan genes in 68 probands (M = 37; age range, 5-50 years). Consanguinity was present in 44 families. Thirty-two variants are predicted to be pathogenic/likely pathogenic, among which 25 (78.13%) are reported, and 7 (21.87%) are novel. The clinical diagnosis was confirmed in a total of 64 (94.12%) probands with biallelic variations [SGCA(n=18); SGCB(n=34); SGCG(n=7); SGCD(n=5)]. The most common mutation was c.544A > C (p.Thr182Pro) in SGCB, and detected in 20 patients (29.42%). The majority of pathogenic mutations are homozygous (n = 30; 93.75%). Variants in 4 cases are of uncertain significance. Thirty-three patients lost ambulation at a mean age of 15.12 ± 9.47 years, after 7.76 ± 5.95 years into the illness. Only 2 patients had cardiac symptoms, and one had respiratory muscle involvement. The results from this study suggest that mutations in SGCB are most common, followed by SGCA, SGCG, and SGCD. The novel variations identified in this study expand the mutational spectrum of sarcoglycanopathies. To the best of our knowledge, this is the first study from India to describe a large cohort of genetically confirmed patients with sarcoglycanopathy and report its disease progression.