Dystroglycanopathies are a clinically and genetically heterogeneous group of disorders that are typically characterised by limb-girdle muscle weakness. Mutations in 18 different genes have been ...associated with dystroglycanopathies, the encoded proteins of which typically modulate the binding of α-dystroglycan to extracellular matrix ligands by altering its glycosylation. This results in a disruption of the structural integrity of the myocyte, ultimately leading to muscle degeneration.
Deep phenotypic information was gathered using the PhenoTips online software for 1001 patients with unexplained limb-girdle muscle weakness from 43 different centres across 21 European and Middle Eastern countries. Whole-exome sequencing with at least 250 ng DNA was completed using an Illumina exome capture and a 38 Mb baited target. Genes known to be associated with dystroglycanopathies were analysed for disease-causing variants.
Suspected pathogenic variants were detected in DPM3, ISPD, POMT1 and FKTN in one patient each, in POMK in two patients, in GMPPB in three patients, in FKRP in eight patients and in POMT2 in ten patients. This indicated a frequency of 2.7% for the disease group within the cohort of 1001 patients with unexplained limb-girdle muscle weakness. The phenotypes of the 27 patients were highly variable, yet with a fundamental presentation of proximal muscle weakness and elevated serum creatine kinase.
Overall, we have identified 27 patients with suspected pathogenic variants in dystroglycanopathy-associated genes. We present evidence for the genetic and phenotypic diversity of the dystroglycanopathies as a disease group, while also highlighting the advantage of incorporating next-generation sequencing into the diagnostic pathway of rare diseases.
Ullrich congenital muscular dystrophy (UCMD), caused by collagen VI deficiency, is a common congenital muscular dystrophy. At present, the role of collagen VI in muscle and the mechanism of disease ...are not fully understood. To address this we have applied microarrays to analyse the transcriptome of UCMD muscle and compare it to healthy muscle and other muscular dystrophies. We identified 389 genes which are differentially regulated in UCMD relative to controls. In addition, there were 718 genes differentially expressed between UCMD and dystrophin deficient muscle. In contrast, only 29 genes were altered relative to other congenital muscular dystrophies. Changes in gene expression were confirmed by real-time PCR. The set of regulated genes was analysed by Gene Ontology, KEGG pathways and Ingenuity Pathway analysis to reveal the molecular functions and gene networks associated with collagen VI defects. The most significantly regulated pathways were those involved in muscle regeneration, extracellular matrix remodelling and inflammation. We characterised the immune response in UCMD biopsies as being mainly mediated via M2 macrophages and the complement pathway indicating that anti-inflammatory treatment may be beneficial to UCMD as for other dystrophies. We studied the immunolocalisation of ECM components and found that biglycan, a collagen VI interacting proteoglycan, was reduced in the basal lamina of UCMD patients. We propose that biglycan reduction is secondary to collagen VI loss and that it may be contributing towards UCMD pathophysiology. Consequently, strategies aimed at over-expressing biglycan and restore the link between the muscle cell surface and the extracellular matrix should be considered.
A high-sensitivity DNA microarray platform requiring nanograms of RNA input facilitates the application of transcriptome analysis to individual skeletal muscle (SM) tissue samples. Culturing myotubes ...from SM-biopsies enables investigating transcriptional defects and assaying therapeutic strategies. This study compares the transcriptome of aneurally cultured human SM cells versus that of tissue biopsies.
We used the Illumina expression BeadChips to determine the transcriptomic differences between tissue and cultured SM samples from five individuals. Changes in the expression of several genes were confirmed by QuantiGene Plex assay or reverse transcription real-time PCR. In cultured myotubes compared to the tissue, 1216 genes were regulated: 583 down and 633 up. Gene ontology analysis showed that downregulated genes were mainly associated with cytoplasm, particularly mitochondria, and involved in metabolism and the muscle-system/contraction process. Upregulated genes were predominantly related to cytoplasm, endoplasmic reticulum, and extracellular matrix. The most significantly regulated pathway was mitochondrial dysfunction. Apoptosis genes were also modulated. Among the most downregulated genes detected in this study were genes encoding metabolic proteins AMPD1, PYGM, CPT1B and UCP3, muscle-system proteins TMOD4, MYBPC1, MYOZ1 and XIRP2, the proteolytic CAPN3 and the myogenic regulator MYF6. Coordinated reduced expression of five members of the GIMAP gene family, which form a cluster on chromosome 7, was shown, and the GIMAP4-reduction was validated. Within the most upregulated group were genes encoding senescence/apoptosis-related proteins CDKN1A and KIAA1199 and potential regulatory factors HIF1A, TOP2A and CCDC80.
Cultured muscle cells display reductive metabolic and muscle-system transcriptome adaptations as observed in muscle atrophy and they activate tissue-remodeling and senescence/apoptosis processes.
Between 8% and 22% of female carriers of DMD mutations exhibit clinical symptoms of variable severity. Development of symptoms in DMD mutation carriers without chromosomal rearrangements has been ...attributed to skewed X-chromosome inactivation (XCI) favouring predominant expression of the DMD mutant allele. However the prognostic use of XCI analysis is controversial. We aimed to evaluate the correlation between X-chromosome inactivation and development of clinical symptoms in a series of symptomatic female carriers of dystrophinopathy.
We reviewed the clinical, pathological and genetic features of twenty-four symptomatic carriers covering a wide spectrum of clinical phenotypes. DMD gene analysis was performed using MLPA and whole gene sequencing in blood DNA and muscle cDNA. Blood and muscle DNA was used for X-chromosome inactivation (XCI) analysis thought the AR methylation assay in symptomatic carriers and their female relatives, asymptomatic carriers as well as non-carrier females.
Symptomatic carriers exhibited 49.2% more skewed XCI profiles than asymptomatic carriers. The extent of XCI skewing in blood tended to increase in line with the severity of muscle symptoms. Skewed XCI patterns were found in at least one first-degree female relative in 78.6% of symptomatic carrier families. No mutations altering XCI in the XIST gene promoter were found.
Skewed XCI is in many cases familial inherited. The extent of XCI skewing is related to phenotype severity. However, the assessment of XCI by means of the AR methylation assay has a poor prognostic value, probably because the methylation status of the AR gene in muscle may not reflect in all cases the methylation status of the DMD gene.
Thyroid hormone is crucial in the development of different organs, particularly the brain. MCT8 is a specific transporter of triiodothyronine (T3) hormone and MCT8 gene mutations cause a rare ...X-linked disorder named MCT8 deficiency, also known as Allan-Herndon-Dudley syndrome, characterized by psychomotor retardation and hypotonia. Typically, elevation of T3 and delayed myelination in cerebral magnetic resonance imaging are found.
We present a 24-month-old boy, born from non-consanguineous healthy parents, with severe motor and cognitive delay and global hypotonia, being unable to hold head upright or sit without support. Deep tendon reflexes were absent bilaterally at the ankles. T3 was elevated and thyroxine slightly decreased, consistent with MCT8 deficiency. Genetic studies confirmed the diagnosis.
Although a rare disease (MCT8 mutations have been reported in about 50 families all around the world), we illustrate the importance of excluding Allan-Herndon-Dudley syndrome in the evaluation of floppy male infants with development delay, without history of perinatal asphyxia. The simple evaluation of thyroid status, including T3, T4 and TSH can guide the diagnosis, avoiding a number of useless, expensive and invasive investigations and allowing appropriate genetic counseling to the affected families.
We investigated the manifestations of CMT4C disease in a genetically homogeneous group of patients homozygous for the recently identified Gypsy founder mutation p.Arg1109X in SH3TC2. We observed a ...surprising degree of variation in age at onset, rate of progression, extent and severity of motor and sensory involvement, scoliosis, and cranial nerve involvement, suggesting that the phenotypic spectrum of CMT4C disease is much broader than the classical diagnostic criteria. Phenotype similarity in first degree relatives and increasing heterogeneity in more distantly related subjects point to the involvement of genetic modifiers, possibly variants in the genes encoding protein partners interacting with SH3TC2.
Spinal muscular atrophy (SMA) is a frequent autosomal recessive disease characterized by degeneration of the motor neurons of the spinal cord causing proximal paralysis with muscle atrophy. The ...region on chromosome 5q13 encompassing the disease gene is particularly unstable and prone to large-scale deletions whose characterization recently led to the identification of the survival motor neuron (SMN) gene. We now present a genetic analysis of 54 unrelated Spanish SMA families that has revealed a 4-basepair (bp) deletion (AGAG) in exon 3 of SMN in four unrelated patients. This deletion, which results in a frameshift and a premature stop codon, occurs on the same haplotype background, suggesting that a single mutational event is involved in the four families. The other patients showed either deletions of the SMN gene (49/54) or a gene conversion event changing SMN exon 7 into its highly homologous copy (cBCD541, 1/54). This observation gives strong support to the view that mutations of the SMN gene are responsible for the SMA phenotype as it is the first frameshift mutation reported in SMA.
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.
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