Hyung Jun Park

Targeted next-generation sequencing for the genetic diagnosis of dysferlinopathy

Dysferlinopathy comprises a group of autosomal recessive muscular dystrophies caused by mutations in the DYSF gene. Due to the large size of the gene and its lack of mutational hot spots, analysis of the DYSF gene is time-consuming and laborious using conventional sequencing methods. By next-generation sequencing (NGS), DYSF gene analysis has previously been validated through its incorporation in multi-gene panels or exome analyses. However, individual validation of NGS approaches for DYSF gene has not been performed. Here, we established and validated a hybridization capture-based target-enrichment followed by next-generation sequencing to detect mutations in patients with dysferlinopathy. With this approach, mean depth of coverage was approximately 450 fold and almost all (99.3%) of the targeted region had sequence coverage greater than 20 fold. When this approach was tested on samples from patients with known DYSF mutations, all known mutations were correctly retrieved. Using this method on 32 consecutive patient samples with dysferlinopathy, at least two pathogenic variants were detected in 28 (87.5%) samples and at least one pathogenic variant was identified in all samples. Our results suggested that the NGS-based screening method could facilitate efficient and accurate genetic diagnosis of dysferlinopathy.

The Role of Insulin Resistance in Diabetic Neuropathy in Koreans with Type 2 Diabetes Mellitus: A 6-Year Follow-Up Study

Purpose We previously reported that insulin resistance, low high-density lipoprotein (HDL) cholesterol, and glycaemic exposure Index are independently associated with peripheral neuropathy in Korean patients with type 2 diabetes mellitus. We followed the patients who participated in that study in 2006 for another 6 years to determine the relationship between insulin resistance and neuropathy. Materials and Methods This study involved 48 of the original 86 Korean patients with type 2 diabetes mellitus who were referred to the Neurology clinic for the assessment of diabetic neuropathy from January 2006 to December 2006. These 48 patients received management for glycaemic control and prevention of diabetic complications in the outpatient clinic up to 2012. We reviewed blood test results and the nerve conduction study findings of these patients, taken over a 6-year period. Results Low HDL cholesterol and high triglycerides significantly influenced the development of diabetic neuropathy. Kitt value (1/insulin resistance) in the previous study affected the occurrence of neuropathy, despite adequate glycaemic control with HbA1c <7%. Insulin resistance affected the development of diabetic neuropathy after 6 years: insulin resistance in 2006 showed a positive correlation with a change in sural sensory nerve action potential in 2012. Conclusion Diabetic neuropathy can be affected by previous insulin resistance despite regular glycaemic control. Dyslipidaemia should be controlled in patients who show high insulin resistance because HDL cholesterol and triglycerides are strongly correlated with later development of diabetic neuropathy.

The Usefulness of Proximal Radial Motor Conduction in Acute Compressive Radial Neuropathy

Background and Purpose The objective of this study was to determine diagnostic and prognostic values of proximal radial motor conduction in acute compressive radial neuropathy. Methods Thirty-nine consecutive cases of acute compressive radial neuropathy with radial conduction studies-including stimulation at Erbs point-performed within 14 days from clinical onset were reviewed. The radial conduction data of 39 control subjects were used as reference data. Results Thirty-one men and eight women (age, 45.2±12.7 years, mean±SD) were enrolled. All 33 patients in whom clinical follow-up data were available experienced complete recovery, with a recovery time of 46.8±34.3 days. Partial conduction block was found frequently (17 patients) on radial conduction studies. The decrease in the compound muscle action potential area between the arm and Erbs point was an independent predictor for recovery time. Conclusions Proximal radial motor conduction appears to be a useful method for the early detection and prediction of prognosis of acute compressive radial neuropathy.

Targeted population screening of late onset Pompe disease in unspecified myopathy patients for Korean population

We performed targeted population screening of late onset Pompe disease (LOPD) in unspecified myopathy patients, because early diagnosis is difficult due to its heterogeneous clinical features. We prospectively enrolled 90 unrelated myopathic patients who had one or more signs out of five LOPD-like clinical findings (proximal weakness, axial weakness, lingual weakness, respiratory difficulty, idiopathic hyperCKemia). Acid alpha glucosidase activity was evaluated with dried blood spot and mixed leukocyte simultaneously. For a final diagnosis of LOPD, 16 patients with decreased enzyme activity were genotyped by GAA molecular analysis. We found two patients with LOPD (2.2%), and the remaining 14 patients had at least one G576S or E689K mutation, known as the pseudodeficiency allele. Acid alpha glucosidase activity of LOPD patients was significantly lower than that of patients with at least one pseudodeficiency allele (p = 0.017). This study is the first LOPD screening study for targeted Korean population, and more generally, an Asian population. Our findings suggest that for diagnosis of LOPD in Asian population, modified cutoff value of acid alpha glucosidase activity with dry blood spot considering that of patients having heterozygote pathogenic variants or pseudodeficiency alleles may reduce time and cost requirements and increase the comfort of patients.

Clinical characterization and genetic analysis of Korean patients with X‐linked Charcot‐Marie‐Tooth disease type 1

Mutations in the gap junction protein beta 1 gene (GJB1) cause X‐linked Charcot‐Marie‐Tooth disease type 1 (CMTX1). CMTX1 is representative of the intermediate type of CMT, having both demyelinating and axonal neuropathic features. We analyzed the clinical and genetic characterization of 128 patients with CMTX1 from 63 unrelated families. Genetic analysis revealed a total of 43 mutations including 6 novel mutations. Ten mutations were found from two or more unrelated families. p.V95M was most frequently observed. The frequency of CMTX1 was 9.6% of total Korean CMT family and was 14.8% when calculated within genetically identified cases. Among 67 male and 61 female patients, 22 females were asymptomatic. A high‐arched foot, ataxia, and tremor were observed in 87%, 41%, and 35% of the patients, respectively. In the male patients, functional disability scale, CMT neuropathy score, and compound muscle action potential of the median/ulnar nerves were more severely affected than in the female patients. This study provides a comprehensive summary of the clinical features and spectrum of GJB1 gene mutations in Korean CMTX1 patients.

Clinical Usefulness of Ultrasonography-Guided Laryngeal Electromyography

OBJECTIVES/HYPOTHESIS To investigate the proper approach and technical method of ultrasonography-guided laryngeal electromyography (US-guided LEMG). STUDY DESIGN This is a prospective study. METHODS Twenty patients who underwent US-guided LEMG were enrolled. US-guided LEMG was cooperatively performed by one otolaryngologist, one neurologist, and one radiologist. The location of the needle electrode was confirmed with US after electrode insertion into the laryngeal intrinsic muscle. The US transducer was applied on the neck by a transverse/midline and transverse/oblique approach to identify the cricothyroid (CT), thyroarytenoid (TA) muscles, and the location of the needle electrode. RESULTS CT muscles were easily identified on US in all 20 patients. TA muscles were identified in 17 patients (85%). The transverse/oblique approach was helpful to detect TA muscle in case of calcified thyroid cartilage or anatomic variation. CONCLUSIONS US-guided LEMG, which enables the exact insertion of the needle electrode, improves the reliability of examination and is helpful in early detection and to prevent complications.

Molecular Genetic Diagnosis of a Bethlem Myopathy Family with an Autosomal-Dominant COL6A1 Mutation, as Evidenced by Exome Sequencing

Background We describe herein the application of whole exome sequencing (WES) for the molecular genetic diagnosis of a large Korean family with dominantly inherited myopathy. Case Report The affected individuals presented with slowly progressive proximal weakness and ankle contracture. They were initially diagnosed with limb-girdle muscular dystrophy (LGMD) based on clinical and pathologic features. However, WES and subsequent capillary sequencing identified a pathogenic splicing-site mutation (c.1056+1G>A) in COL6A1, which was previously reported to be an underlying cause of Bethlem myopathy. After identification of the genetic cause of the disease, careful neurologic examination revealed subtle contracture of the interphalangeal joint in the affected members, which is a characteristic sign of Bethlem myopathy. Therefore, we revised the original diagnosis from LGMD to Bethlem myopathy. Conclusions This is the first report of identification of COL6A1-mediated Bethlem myopathy in Korea, and indicates the utility of WES for the diagnosis of muscular dystrophy.

Clinical and Pathologic Findings of Korean Patients with RYR1-Related Congenital Myopathy

Background and Purpose This study was designed to investigate clinical and pathologic characteristics of five Korean patients with RYR1-related congenital myopathy (CM). Methods Five patients from unrelated families were diagnosed with RYR1-related CM via direct or targeted sequencing of RYR1. Their clinical, mutational, and pathologic findings were then analyzed. Results Seven different mutations were identified, including two novel mutations: c.5915A>T and c.12250C>T. All of the patients presented at infancy with proximal dominant weakness and delayed motor milestones. Other clinical findings were scoliosis in three patients, winged scapula in two, hip dislocation in one, and pectus excavatum in one. Ophthalmoplegia was observed in one patient with a novel recessive mutation. Two of three muscle specimens revealed a myopathic pattern with core. Conclusions We have identified a novel compound heterozygous RYR1 mutation and demonstrated clinical and pathologic findings in five Korean patients with RYR1-related CM.

Electron Microscopy Pathology of ADSSL1 Myopathy

Dear Editor, Inherited muscular disorder is a heterogeneous group of genetic disorders that primarily affect skeletal muscle fibers. We have recently reported compound heterozygous mutations in ADSSL1 in two Korean families with adolescent-onset distal myopathy.1 However, ultrastructural changes associated with adenylosuccinate synthetase-like 1 (ADSSL1) myopathy have not been demonstrated previously. Here we report a third Korean family with ADSSL1 mutations and describe the ultrastructural features of ADSSL1 myopathy. A 15-year-old girl (Supplementary Fig. 1A and B in the online-only Data Supplement, II2) presented with gait disturbance. She was the second child of healthy, nonconsanguineous parents. She had first noticed difficulty running when aged 8 years, and the muscle weakness had progressed very slowly. When we examined her at an age of 15 years, she could still ambulate independently. A neurological examination revealed diffuse muscle weakness, predominantly in the leg muscles. She did not exhibit facial weakness, sensory deficits, joint contractures, or a high-arch palate. Her serum creatine kinase (CK) level was 281 IU/l (reference value <185 IU/l). The findings of needle electromyography were compatible with generalized myopathy. Her electrocardiography findings were normal. Lower-limb MRI showed subtle fatty replacement of the tibialis anterior and gastrocnemius muscles (Supplementary Fig. 1C-G in the online-only Data Supplement). The vastus lateralis muscle was biopsied when she was 15 years old. The myofibers showed marked size and shape variations along with frequent internalization of the sarcolemmal nuclei and splitting; vacuolization of the sarcoplasm, degenerating myofibers, and abnormal (whorled) arrangement of the myofibrils were also noted (Fig. 1A and B). In addition, focal endomysial fibrosis and fatty infiltration were present. Periodic acid-Schiff and Oil Red O staining did not produce abnormal positive reactions. Modified Gomori trichrome and nicotinamide adenine dinucleotide-tetrazolium reductase staining revealed whorled fibers and occasional rimmed vacuoles without ragged red fibers, cytoplasmic bodies, or nemaline rods (Fig. 1C and D). The ATPase reaction with preincubations at different pH values and immunostaining with myosin heavy chain (fast), myosin heavy chain (slow), and myosin IIa revealed a predominance of type I fibers. An electron microscopy examination showed myofibers with varying degrees of disorganization, and disorientation of the myofibrils with loss of their normal striation pattern (Fig. 1E). The myofibers showing diffuse or localized myofibrillar destruction contained remnants of the myofibrils, irregular electron-dense material of Z-line origin, and rounded and swollen mitochondria (Fig. 1F). In addition, various abnormalities of the Z-disc (including thickening) were noted, as well as displaced and replicated triads. To identify the genetic cause, exome sequencing was performed on the proband (Supplementary Table 1 in the online-only Data Supplement). Screening of myopathy-related genes revealed 27 functionally significant variants (Supplementary Table 2 in the online-only Data Supplement). We subsequently identified compound heterozygous c.910G>A (p.D340N) and Hyung Jun Park Jee Eun Lee Gyeong Seon Choi Heasoo Koo Soo Jeong Han Jeong Hyun Yoo Young-Chul Choi Kee Duk Park

First Identification of Compound Heterozygous FKRP Mutations in a Korean Patient with Limb-Girdle Muscular Dystrophy

Dystroglycanopathies are a genetically and clinically heterogeneous group of muscular dystrophies that are characterized by reduced glycosylation of α-dystroglycan. Among 18 causative genes, the fukutin-related protein (FKRP; MIM 606596) gene is the most common cause in Caucasians.1 However, FKRP mutations have rarely been found in the Asian population,2 and have not been reported previously in Korea. Herein we report novel compound FKRP mutations in a patient with myopathy, which represents the first reported case in Korea. A 43-year-old female (II-4) (Fig. 1A) was the fourth child of nonconsanguineous healthy Korean parents who achieved normal motor milestones after birth. She first noticed difficulty in climbing stairs at age 7 years, but muscle weakness did not progress. At the age of 23 years the patient perceived the progression of muscle weakness and was admitted to our institute. She displayed proximal weakness, especially in the pelvic girdle muscles. Mild winged scapulae and lordosis were also observed. No sensory deficits, joint contracture, tongue hypertrophy, or mental retardation were apparent. Her serum creatine kinase level was markedly elevated, at 2,439 IU/L (reference value: <185 IU/L). An electrophysiological study revealed a generalized myogenic process. No cardiac abnormality was observed. At the last examination, when she was 43 years old, she could walk with assistance. Fig. 1 Pedigree, sequencing chromatograms, and conservation profile of the patient. A: Pedigree of a Korean patient with compound heterozygous FKRP mutations. The filled symbol indicates the affected member; open symbols indicate unaffected members. B: Sequencing ... The genetic cause of this patients myopathy was evaluated by performing targeted sequencing of 69 myopathy-causative genes including FKRP (Supplementary Information, Supplementary Table 1, 2, and 3 in the online-only Data Supplement). DNA fragments in the target regions were enriched by solution-based hybridization capture, followed by sequencing with an Illumina Hiseq2000 platform. The sequencing data were analyzed using a standard pipeline. Variants were then filtered further based on the patients phenotype. Compound heterozygous FKRP mutations of c.857G>C and c.1170_1171delGC were identified (Fig. 1B). The c.1170_1171delGC mutation has been reported previously in a Japanese patient with congenital muscular dystrophy.2 However, the c.857G>C mutation was novel, and was not detected in 352 healthy controls, dbSNP138, or the 1000 Genomes Database (September 2014 release). In silico analysis using SIFT and PolyPhen2 predicted this mutation to be disease-causing. Genomic evolutionary rate profiling (GERP) indicated that the affected nucleotide is highly conserved (score=4.16), as became apparent upon inspection of the amino acid sequence in the altered region of the protein (Fig. 1C). Thus, the compound heterozygous FKRP mutations of c.1170_1171delGC and c.857G>C were determined to be the underlying cause of the myopathy in this patient. FKRP mutations result in various clinical presentations from severe brain abnormality to mild limb-girdle muscular dystrophy.1 This variability makes it difficult to diagnose FKRP-related muscular dystrophy based on clinical findings alone. Therefore, a step-by-step, classical approach with assessment of muscle pathology, muscle immunoanalysis, and mutational analysis is needed for the diagnosis of FKRP-related muscular dystrophy. The findings of a recent study in Taiwan that employed this approach suggested that FKRP-related muscular dystrophy is underdiagnosed, and not rare in the Asian population.3 However, this classical approach often fails to identify the causative gene due to pathological variability and the limited power of muscle immunoanalysis. Advances in next-generation sequencing, including targeted sequencing, have enabled the rapid and cost-effective analysis of many causative genes in uncategorized muscular dystrophy.4 In the present case, compound heterozygous FKRP mutations were identified using such next-generation sequencing. In conclusion, novel compound heterozygous FKRP mutations were identified in a patient with limb-girdle muscular dystrophy; this is the first reported case in Korea.