A. Cho

Utility of next generation sequencing in genetic diagnosis of early onset neuromuscular disorders

Background Neuromuscular disorders are a clinically, pathologically, and genetically heterogeneous group. Even for the experienced clinician, an accurate diagnosis is often challenging due to the complexity of these disorders. Here, we investigated the utility of next generation sequencing (NGS) in early diagnostic algorithms to improve the diagnosis for patients currently lacking precise molecular characterisation, particularly for hereditary myopathies. Methods 43 patients presenting with early onset neuromuscular disorders from unknown genetic origin were tested by NGS for 579 nuclear genes associated with myopathy. Results In 21 of the 43 patients, we identified the definite genetic causes (48.8%). Additionally, likely pathogenic variants were identified in seven cases and variants of uncertain significance (VUS) were suspected in four cases. In total, 19 novel and 15 known pathogenic variants in 17 genes were identified in 32 patients. Collagen VI related myopathy was the most prevalent type in our cohort. The utility of NGS was highlighted in three cases with congenital myasthenia syndrome, as early diagnosis is important for effective treatment. Conclusions A targeted NGS can offer cost effective, safe and fairly rapid turnaround time, which can improve quality of care for patients with early onset myopathies and muscular dystrophies; in particular, collagen VI related myopathy and congenital myasthenia syndromes. Nevertheless, a substantial number of patients remained without molecular diagnosis in our cohort. This may be due to the intrinsic limitation of detection for some types of mutations by NGS or to the fact that other causative genes for neuromuscular disorders are yet to be identified.

Clinical applications of next-generation sequencing-based gene panel in patients with muscular dystrophy: Korean experience

Muscular dystrophy (MD) is a genetically and clinically heterogeneous group of disorders. Here, we performed targeted sequencing of 18 limb‐girdle MD (LGMD)‐related genes in 35 patients who were highly suspected of having MD. We identified one or more pathogenic variants in 23 of 35 patients (65.7%), and a genetic diagnosis was performed in 20 patients (57.1%). LGMD2B was the most common LGMD type, followed by LGMD1B, LGMD2A, and LGMD2G. Among the three major LGMD types in this group, LGMD1B was correlated with the lowest creatine kinase (CK) levels and the earliest onset, whereas LGMD2B was correlated with the highest CK levels and the latest onset. Thus, next‐generation sequencing‐based gene panels can be a helpful tool for the diagnosis of MDs, particularly in young children and those displaying atypical symptoms.

Pitfalls of Multiple Ligation-Dependent Probe Amplifications in Detecting DMD Exon Deletions or Duplications.

Multiple ligation-dependent probe amplifications (MLPAs) are a key technology for the molecular diagnosis of Duchenne/Becker muscular dystrophy, which is mainly caused by large gene arrangements. However, little is known about the false-positive rates of MLPA for this disease. Here, we review MLPA analysis results from 398 patients suspected to have Duchenne/Becker muscular dystrophy. MLPA assay was used for screening the entire coding region. If these amplifications produced normal results, direct sequencing was performed to search for sequence variations and to determine single-exon deletions, duplications, or indeterminate results. Using MLPA, 290 cases (72.9%) showed exon deletion or duplication results. Among those, 75 cases (25.9%) resulted in a deletion or duplication of a single exon. Direct sequencing revealed that 11 single-exon deletion cases resulted in false-positives due to sequence variations within the patient population interfering with probe binding at the probe-hybridization sites. Abnormal MLPA results were closely related to the type of sequence change and the position within the probe-hybridization locus. The most common type was C-T transition (n = 19, 55.9%). Abnormal MLPA results correlated with CA mismatch and low melting temperature (≤75°C). False-positive events for large gene rearrangements involving a single exon in DMD accounted for approximately 15% (11/75). Therefore, careful design of MLPA probes is required to avoid false-positive results.

P.3.6 Antioxidant capacity is impaired in hyposialylated myotubes of GNE myopathy

GNE myopathy (also known as distal myopathy with rimmed vacuoles or hereditary inclusion body myopathy) is a rare autosomal recessive myopathy characterized by skeletal muscle atrophy and weakness that preferentially involve the distal muscles. The causative GNE gene encodes an essential enzyme in the biosynthesis of sialic acid and hyposialylation is thought to be a key factor in the underlying pathomechanism. We previously found that muscle atrophy appears in the young age of GNE myopathy model mice and is preventable by antioxidant administration, suggesting that oxidative stress would be associated with this primary myopathic phenotype. To link hyposialylation with the pathologic features, we investigated cellular response to oxidative stress in GNE myopathy myotubes. Skeletal muscle myotubes were prepared from Gne −/− hGNED176VTg mice and littermate controls. Intracellular reactive oxygen species (ROS) and cell viability were evaluated. First, we found that intracellular ROS generation was more profound in GNE myopathy myotubes compared to littermates. Then the susceptibility of GNE myopathy myotubes to increasing concentrations of menadione, a generator of ROS, with manipulating cellular sialylation was analyzed. We found that hyposialylated myotubes cultured in serum-free media are more vulnerable to menadione-induced oxidative stress. Dose-dependent increase in intracellular oxidants and cell mortality were weakened with the addition of NeuAc to culture media, which implies that normalization of sialylation levels improved intracellular antioxidant capacities. Our results provide a clue how hyposialylation makes the pathognomonic change in GNE myopathy and suggest that oxidative stress can be a therapeutic target of this progressive disease.

G.P.27 Muscle atrophy in the GNE myopathy mouse model is associated with oxidative stress

Abstract GNE myopathy (GM), which is also known as distal myopathy with rimmed vacuoles or hereditary inclusion body myopathy, is an autosomal recessive myopathy characterized by skeletal muscle atrophy and weakness that preferentially involve the distal muscles. The histopathologic features in muscle biopsy include the myofiber atrophy with the presence of rimmed vacuoles and intracellular amyloid deposits. Although it has been already demonstrated that sialic acid treatment can prevent the development of myopathic phenotype in GM model mouse, there remains an unexplained link between hyposialylation due to GNE mutations and the pathognomonic findings in the muscle. Previously we have reported that muscle atrophy is one of the early signs of myopathic phenotype in the mouse model. In this study, we evaluated several phenomena that contribute to atrophy in muscles and focused on reactive oxygen species (ROS) production. We measured the levels of hydroxyl radicals after induced muscle contraction in vivo. We found profound hydroxyl radical increments in GM mice as compared to littermates, indicating a decreased antioxidative capacity in the GM muscles. The implication of oxidative stress in disease mechanism was further supported by the finding that several genes responsive to oxidative stress were highly expressed in the GNE myopathy muscles. The evidence that muscle atrophy is associated with oxidative stress was further reinforced by the improvement in muscle strength in GM mice given α-tocopherol, and the increase in myofiber diameter in GM mice treated with N-acetylcysteine. These results not only provide important clues on understanding GM pathomechanism but more importantly demonstrate the potential of antioxidants in therapeutic regimen of GM patients.

Collagen VI-related myopathy: Expanding the clinical and genetic spectrum: Collagen VI-Related Myopathy

Introduction: We aimed to analyze the clinical and genetic characteristics of collagen VI‐related myopathy. Methods: We analyzed the clinical course and mutation spectrum in patients with collagen VI gene mutations among our congenital muscular dystrophy cohort. Results: Among 24 patients with mutations in collagen VI coding genes, 13 (54.2%) were categorized as Ullrich type, and 11 (45.8%) as non‐Ullrich type. Congenital orthopedic problems were similarly observed in both types, yet multiple joint contractures were found only in the Ullrich type. Clinical courses and pathology findings varied between patients. Mutations in COL6A1, COL6A2, and COL6A3 were found in 15 (65%), 3 (13%), and 5 (22%) patients, respectively, without genotype–phenotype association. Five novel variants were detected. Discussion: We verified clinical heterogeneity of collagen VI‐related myopathy, which emphasizes the importance of genetic testing. Genotype–phenotype association or early predictors for progression were not identified. Multiple joint contractures predict rapid deterioration. Muscle Nerve 58: 381–388, 2018

Severe hypotonia and postnatal growth impairment in a girl with a missense mutation in COL1A1: Implication of expanded phenotypic spectrum of type I collagenopathy

BACKGROUND It is known that type I collagenopathy has a broad-spectrum phenotypic variability. Here, we report a case of a Korean girl with a heterozygous COL1A1 mutation who had an atypical presentation. CASE PRESENTATION A 26-month-old girl presented with delayed motor development and failure to thrive. She had severe growth retardation. She exhibited right-sided plagiocephaly, blue sclerae, and facial dysmorphism, including a small pointed chin, frontal bossing, and a triangular face, but had microcephaly. Whole-exome sequencing revealed a novel de novo heterozygous sequence variant in COL1A1 (p.Gly1127Asp), which was validated by Sanger sequencing. Radiological finding showed generalized osteoporosis with progressive scoliosis of the spine without evidence of platyspondyly related to fractures and bowing of the long bones, and markedly delayed carpal bone age. Muscle pathology showed a marked size variation of myofibers and selective type 1 atrophy. CONCLUSIONS This study expanded the clinical and genetic spectrum of type I collagenopathy with a COL1A1 variant. Therefore, we suggest that type I collagenopathy should be considered in the patients who have some features of osteogenesis imperfecta simultaneously with atypical features such as facial dysmorphism.

G.P.23

Congenital myopathies and congenital muscular dystrophies are groups of clinically, pathologically, and genetically heterogeneous disorders. Even for the experienced clinicians, an accurate genetic diagnosis has often been challenging due to the heterogeneity and complexity of these groups of disorders. One gene can cause a wide variety of clinical and/or pathological features, while similar clinical features can be caused by mutations in different genes. Since next generation sequencing (NGS) is an effective diagnostic tool for the parallel investigation of a large number of genes, it has been increasingly used in recent clinical practices to diagnose these genetically and phenotypically heterogeneous diseases. Here, we present the result of a targeted NGS panel analysis in early onset myopathies. We selected 703 known pathogenic genes causing congenital myopathies, congenital muscular dystrophies, metabolic and mitochondrial myopathies, distal myopathies, channelopathies, neuromuscular junction disorders, and diseases of peripheral nerve. Total 42 infants or children with early onset ( COL6A1 (5), COL6A3 (1), LMNA (3), ACTA1 (2), MTM1 (1), DOK7 (1), and GARS (1). Our results suggest that targeted NGS has a significant potential to synergy with clinical and pathological analysis for an effective diagnosis of primary myopathies.