Genetic variations and clinical spectrum of dystroglycanopathy in a large cohort of Chinese patients.

Dystroglycanopathy is a group of muscular dystrophies with deficient glycosylation of alpha-dystroglycan (α-DG). We recruited patients from 36 tertiary academic hospitals in China. In total, 143 patients with genetically diagnosed dystroglycanopathy were enrolled. Of these, limb girdle muscular dystrophy was the most common initial diagnosis (83 patients) and Walker-Warburg syndrome was the least common (1 patient). In 143 patients, mutations in FKRP gene were the most prevalent (62 patients), followed by POMT2, POMT1 (16), POMGNT1, ISPD (14), FKTN, GMPPB, B3GALNT2, DPM3, and DAG1. Several frequent mutations were identified in FKRP, POMT1, POMGNT1, ISPD, and FKTN genes. Many of these were founder mutations. Patients with FKRP mutations tended to have milder phenotypes, while those with mutations in POMGNT1 genes had more severe phenotypes. Mental retardation was a clinical feature associated with mutations of POMT1 gene. Detailed clinical data of 83 patients followed up in Peking University First Hospital were further analyzed. Our clinical and genetic analysis of a large cohort of Chinese patients with dystroglycanopathy expanded the genotype variation and clinical spectrum of congenital muscular dystrophies.

c.3G>A mutation in the CRYAB gene that causes fatal infantile hypertonic myofibrillar myopathy in the Chinese population.

Infantile hypertonic myofibrillar myopathy is characterized by the rapid development of rigid muscles and respiratory insufficiency soon after birth, with very high mortality. It is extremely rare, and only a few cases having been reported until now. Here we report four Chinese infants with fatal neuromuscular disorders characterized by abdominal and trunk skeletal muscle stiffness and rapid respiratory insufficiency progression. Electromyograms showed increased insertion activities and profuse fibrillation potentials with complex repetitive discharges. Immunohistochemistry staining of muscle biopsies showed accumulations of desmin in the myocytes. Powdery Z-bands with dense granules across sarcomeres were observed in muscle fibers using electron microscopy. All patients carry a homozygous c.3G>A mutation in the CRYAB gene, which resulted in the loss of the initiating methionine and the absence of protein. This study's findings help further understand the disease and highlight a founder mutation in the Chinese population.

Congenital muscular dystrophies in China.

Congenital muscular dystrophies (CMDs) are clinically and genetically heterogeneous conditions. We launched a nationwide study to determine the frequency of CMD in the Chinese population and assess the status of diagnosis and disease management for CMD in China. Cases were chosen from databases in 34 tertiary academic hospitals from 29 first-level administrative divisions (provinces, municipalities, autonomous regions, and special administrative regions), and medical records were reviewed to confirm the diagnoses. The study included 409 patients, of those patients who consented to genetic testing (n = 340), mutations were identified in 286 of them. The most common forms identified were LAMA2-related CMD (36.4%), followed by COL6-related CMD (23.2%) and α-dystroglycanopathy (21.0%). The forms of CMD related to mutations in LMNA and SEPN1 were less frequent (12.5% and 2.4%, respectively). We also recorded a significant difference in the diagnostic capabilities and disease management of CMD, with this being relatively backward in research centers from less developed regions. We provide, for the first time, comprehensive epidemiologic information of CMD in a large cohort of Chinese people. To our knowledge, this is the largest sample size of its kind so far highlighting the prevalence of CMD in China.

Effectiveness of High-Speed T2-Corrected Multiecho MR Spectroscopic Method for Quantifying Thigh Muscle Fat Content in Boys With Duchenne Muscular Dystrophy.

OBJECTIVE. The purpose of this study was to investigate the potential of high-speed T2-corrected multiecho (HISTO) MR spectroscopy (MRS) for rapidly quantifying the fat content of thigh muscles in children with Duchenne muscular dystrophy (DMD). SUBJECTS AND METHODS. This study prospectively enrolled 58 boys with DMD (mean age, 7.5 years; range, 4-11 years) and 30 age-matched healthy boys (mean age, 7.2 years; range, 4-11 years) at one institution over a 1-year period. T1- and T2-weighted, multiecho Dixon, and HISTO sequences were performed on the right adductor magnus and vastus lateralis muscles. The fat fractions of these muscles were acquired from HISTO and multiecho Dixon images. An experienced radiologist graded the degree of fat infiltration of the adductor magnus and vastus lateralis muscles on axial T1-weighted images. The Bland-Altman method was used to assess the consistency and repeatability of the HISTO sequence. Pearson linear correlation analysis was used to determine the correlation coefficient relating HISTO fat fraction to multiecho Dixon fat fraction values. Spearman rank correlation analysis was used to assess the relation between the HISTO fat fraction values and T1-weighted image fat infiltration grades. The independent t test was used to compare the HISTO fat fraction values of the boys with DMD with those of the healthy control subjects. RESULTS. Bland-Altman analysis showed that 95.5% of the HISTO fat fraction values of the adductor magnus were within the 95% CI. HISTO fat fraction and multiecho Dixon fat fraction values of the adductor magnus and vastus lateralis muscles were highly positively correlated (adductor magnus, r = 0.983; vastus lateralis, r = 0.967; p < 0.0001). HISTO fat fraction values were also highly positively correlated with the grades of fat infiltration on T1-weighted images (adductor magnus, r = 0.911; vastus lateralis, r = 0.937; p < 0.0001). The HISTO fat fraction of the adductor magnus muscle was 33.3% ± 22.6% and of the vastus lateralis muscle was 25.6% ± 20.3% in patients with DMD. The corresponding values were 2.9% ± 2.1% and 2.3% ± 1.9% in the control group. The differences were statistically significant (p < 0.0001). CONCLUSION. The HISTO sequence is a rapid and feasible noninvasive MRS technique for quantifying the fat infiltration of thigh muscles in children with known or suspected DMD. It is useful for diagnosis and for assessment of disease activity and prognosis.

A mutation in the filamin c gene causes myofibrillar myopathy with lower motor neuron syndrome: a case report.

BACKGROUND: Myofibrillar myopathies (MFMs) are a genetically heterogeneous group of muscle disorders. Mutations in the filamin C gene (FLNC) have previously been identified in patients with MFM. The phenotypes of FLNC-related MFM are heterogeneous. CASE PRESENTATION: The patient was a 37-year-old male who first experienced weakness in the distal muscles of his hand, which eventually spread to the lower limbs and proximal muscles. Serum creatine kinase levels were moderately elevated. Obvious neuropathic changes in the electromyographic exam and edema changes in lower distal limb magnetic resonance imaging were observed. Histopathological examination revealed the presence of abnormal protein aggregates and angular atrophy in some muscle fibers. Ultrastructural analysis showed inordinate myofibrillar structures and dissolved myofilaments. DNA sequencing analysis detected a heterozygous missense mutation (c.7123G > A, p.V2375I) in the immunoglobulin (Ig)-like domain 21 of FLNC. CONCLUSIONS: FLNC mutation c.7123G > A, p.V2375I in the immunoglobulin (Ig)-like domain 21 can be associated with distal myopathy with typical MFM features and lower motor neuron syndrome. Although electromyographic examination of our patient showed obvious neuropathic changes, MFM could not be excluded. Therefore, genetic testing is necessary to make an accurate diagnosis.

FKRP mutations, including a founder mutation, cause phenotype variability in Chinese patients with dystroglycanopathies.

Mutations in the fukutin-related protein (FKRP) gene have been associated with dystroglycanopathies, which are common in Europe but rare in Asia. Our study aimed to retrospectively analyze and characterize the clinical, myopathological and genetic features of 12 Chinese patients with FKRP mutations. Three patients were diagnosed with congenital muscular dystrophy type 1C (MDC1C) and nine patients were diagnosed with limb girdle muscular dystrophy type 2I (LGMD2I). Three muscle biopsy specimens had dystrophic changes and reduced glycosylated α-dystroglycan staining, and two showed reduced expression of laminin α2. Two known and 13 novel mutations were identified in our single center cohort. Interestingly, the c.545A>G mutation was found in eight of the nine LGMD2I patients as a founder mutation and this founder mutation in Chinese patients differs from the one seen in European patients. Moreover, patients homozygous for the c.545A>G mutation were clinically asymptomatic, a less severe phenotype than in compound heterozygous patients with the c.545A>G mutation. The 13 novel mutations of FKRP significantly expanded the mutation spectrum of MDC1C and LGMD2I, and the different founder mutations indicate the ethnic difference in FKRP mutations.

Mutation in the caveolin-3 gene causes asymmetrical distal myopathy.

Mutations in the gene encoding caveolin-3 (CAV3) can cause a broad spectrum of clinical phenotypes, including limb girdle muscular dystrophy, rippling muscle disease, distal myopathy (MD), idiopathic persistent elevation of serum creatine kinase and cardiomyopathy. MD is a relatively rare subtype of caveolinopathy. Here, we report a sporadic case of a middle-aged female Chinese patient with MD in which a CAV3 mutation was identical to that previously reported in cases of rippling muscle disease. T1-weighted enhanced skeletal muscle MRI of the lower limbs showed an abnormal signal in the distal and proximal muscles. A muscle biopsy revealed moderate dystrophic changes, and immunohistochemical staining showed reduced CAV-3 expression in the plasmalemma. Genetic analysis revealed a heterozygous c.136G > A (p.Ala46Thr) CAV3 mutation that appeared to be de novo because it was absent from the patient's parents. This study suggested that the CAV3 c.136G > A (p.Ala46Thr) mutation can cause MD as well as different phenotypes in different individuals, suggesting that additional unknown loci must affect the disease phenotypes.

[A novel mutation in GCH1 gene causes dopa-responsive dystonia].

OBJECTIVE: To identify potential mutation of the GCH1 gene in a Chinese family affected with dopa-responsive dystonia. METHODS: Genomic DNA of patients was extracted from peripheral blood samples. The 6 exons of the GCH1 gene and at least 100 bp of flanking intronic sequences were amplified with PCR. Potential mutations were screened by direct sequencing. Identified mutation was verified with denaturing high performance liquid chromatography (DHPLC) in 100 healthy controls. RESULTS: All patients were found to be heterozygous for a novel c.597delT (p.Ala200LeufsX5) deletion in the exon 5 of the GCH1 gene. The deletion of T has resulted in formation of a shorter (203 amino acids) truncated non-functional guanosine triphosphate cyclohydrolase I. The same mutation was not found in the 100 controls. CONCLUSION: A novel GCH1 gene frameshifing mutation probably underlies the dopa-responsive dystonia in this Chinese family.

Clinical and MRI features of neurological complications after influenza A (H1N1) infection in critically ill children.

BACKGROUND: Influenza A (H1N1) can cause severe neurological complications. OBJECTIVE: The purpose of this study was to analyze clinical and MRI features of neurological complications after H1N1 infection in critically ill children. MATERIALS AND METHODS: We retrospectively analyzed clinical and neuroimaging findings in 17 children who were hospitalized in an intensive care unit with severe neurological complications after H1N1 infection in South China between September 2009 and December 2011. All children underwent pre- and post-contrast-enhanced brain MRI. Postmortem studies were performed in two children. RESULTS: Six children died, five because of acute necrotizing encephalopathy (ANE) and one because of intracranial fungal infection. Eleven recovered; their manifestations of H1N1 were meningitis (3), encephalitis (1) and influenza encephalopathy (7). MRI features of ANE included multiple symmetrical brain lesions demonstrating prolonged T1 and T2 signal in the thalami, internal capsule, lenticular nucleus and pontine tegmentum. Postmortem MRI in two children with acute necrotizing encephalopathy showed diffuse prolonged T1 and T2 signal in the bilateral thalami, brainstem deformation and tonsillar herniation. CONCLUSION: Fatal neurological complications in children after H1N1 infection include ANE and opportunistic fungal infection. MRI is essential for identification of neurological complications and for clinical evaluation.

[Genetic analysis and prenatal diagnosis for a family with megalencephalic leukoencephalopathy and subcortical cysts].

OBJECTIVE: To identify potential mutation in the MLC1 gene in a Chinese family affected with megalencephalic leukoencephalopathy and subcortical cysts (MLC), and to provide prenatal diagnosis. METHODS: Genomic DNA of the patients, their parents and younger sister were extracted from peripheral blood. That of the fetus was extracted from an amniotic fluid sample. A total of 12 exons and at least 100 bp flanking the intronic sequence of the MLC1 gene were amplified with PCR. MLC1 mutations were screened by sequencing. Linkage analysis was performed for the family to assure accuracy of prenatal diagnosis. RESULTS: The two patients were both heterozygote for c.177_178delG (p.Ser60AlafsX5) mutation in exon 2 and c.598-2A>C change in intron 7. The c.177_178delG mutation was inherited from the father, and the c.598-2A>C mutation was inherited from the mother. The younger sister and the fetus have both inherited c.177_178delG from the father but did not inherit c.598-2A>C from the mother. Prenatal diagnosis suggested the fetus to be a carrier for a MLC1 mutation. Linkage analysis was consistent with the result of mutation detection. The fetus was born normal as predicted. CONCLUSION: The c.598-2A>C is a novel splicing mutation. Prenatal diagnosis through DNA sequencing and linkage analysis were performed for the first time on Chinese patients with MLC.

Novel Intronic Mutations Introduce Pseudoexons in DMD That Cause Muscular Dystrophy in Patients.

Background: Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are two subtypes of muscular dystrophy diseases caused by pathogenic mutations in the DMD gene. Until now, more than 4,600 disease-causing mutations in DMD have been reported. However, only 33 mutations were deep intronic, cases with this type of mutations were limited. Methods: In this study, we used a combination of complementary DNA (cDNA) and target DNA sequencing analysis in addition to conventional whole-exome sequencing (WES). Results: Three novel hemizygous mutations IVS11 + 17811C > G (c.1331 + 17811C > G), IVS21 + 3252A > G (c.2803 + 3252A > G) and IVS40 + 362A > G (c.5739 + 362A > G) were identified in DMD patients, while a reported hemizygous mutation IVS62-285A > G (c.9225-285A > G) was found in the BMD patient. These DMD mutations lead to pseudoexon insertions, causing the generation of truncated and dysfunctional dystrophin. Conclusion: This study defines three novel and one reported intronic mutations, which can result in DMD/BMD. We also emphasize the need to combine WES and cDNA-based methods to detect the variant in the very large DMD gene in which the mutational spectrum is complex.

Initial application of three-dimensional speckle-tracking echocardiography to detect subclinical left ventricular dysfunction and stratify cardiomyopathy associated with Duchenne muscular dystrophy in children.

Three-dimensional (3D) speckle-tracking echocardiography (STE) is a new imaging modality used for quantitative analysis of left ventricular (LV) function. The aim of this study is to assess the value of 3D STE in early detection of subclinical myocardial involvement in children with Duchenne muscular dystrophy (DMD). Fifty-six children with DMD (mean age, 8.8 ± 1.9 years) and 31 age-matched control subjects were studied. Patients were subdivided into two groups by age: ≤ 8 or > 8 years. Standard echocardiography examinations were performed to measure LV size and ejection fraction (EF). 3D STE was performed to assess LV 3D global strain and LV end-diastolic volume (EDV), end-systolic volume (ESV), and EF. Standard and 3D echocardiography measures were compared between children with DMD and those in the control group as well as between different patient groups. The areas under the receiver-operating characteristic (ROC) curve were calculated to determine the capability of 3D global strain indices to discriminate between patients and control subjects. No significant difference was detected in either LVEF derived from M-mode or 3D echocardiography between the two groups, and they were both within the normal range. Compared with control subjects, children with DMD had significantly reduced LV 3D global longitudinal strain (GLS; - 16.6 ± 4.7 vs. - 19.5 ± 3.7, p = 0.003), global circumferential strain (GCS; - 13.7 ± 2.9 vs. - 15.8 ± 2.6, p = 0.001), global radial strain (GRS; 42.5 ± 9.7 vs. 50.3 ± 10.4, p = 0.001), and global area strain (GAS; - 25.3 ± 4.9 vs. - 30.7 ± 4.1, p = 0.000). The older DMD children (age > 8 years) had lower GLS (- 15.1 ± 4.43 vs. - 18.6 ± 4.35, p < 0.05), GCS (- 12.8 ± 3.48 vs. - 14.8 ± 2.83, p < 0.001), GAS (- 23.8 ± 4.7 vs. - 29.0 ± 5.4, p < 0.001), and GRS (40.7 ± 8.8 vs. 47.3 ± 11.5, p < 0.05) than younger patients (age ≤ 8 years). The AUC of GAS was 0.80, and the cutoff value of - 29.5 had a sensitivity of 85.7% and a specificity of 71.0% for differentiating DMD patients from control. 3D speckle-tracking echocardiography is useful for detecting subclinical myocardial dysfunction and stratifying cardiomyopathy in children with DMD.

CA2: the most vulnerable sector to bicuculline exposure in rat hippocampal slice cultures.

The vulnerability of the CA2 sector to chronic exposure to bicuculline was investigated in rat hippocampal slice cultures. Selective neuronal cell death was observed only in the CA2 sector after exposure to 6 microM bicuculline for 12 h, but the effect of the cell toxicity extended to the CA3 sector after 24 h. The effect was increased by adding 20 microM roscovitine but was reduced by adding 200 nM omega-agatoxin IVA. Bicuculline also induced a calcium influx into neuronal cells mainly in the CA2 sector. These results suggest that CA2 is the most vulnerable sector to bicuculline exposure in hippocampal slice cultures, and that neuronal cell death in the CA2 sector involves the P/Q-type voltage-dependent calcium channel.

Patient with spinal muscular atrophy with respiratory distress type 1 presenting initially with hypertonia.

Spinal muscular atrophy with respiratory distress type 1 (SMARD1) is a rare autosomal recessive neuromuscular disorder caused by mutations in the IGHMBP2 gene and characterized by life-threatening respiratory distress due to irreversible diaphragmatic paralysis between 6weeks and 6months of age. In this study, we describe a two-month-old boy who presented with hypertonia at first and developed to hypotonia progressively, which was in contrast to the manifestations reported previously. Bone tissue compromise was also observed as one of the unique symptoms. Muscle biopsy indicated mild myogenic changes. He was misdiagnosed until genetic screening to be confirmed as SMARD1. SMARD1 is a clinical heterogeneous disease and this case broadens our perception of its phenotypes.

Detection of glutamate in optically trapped single nerve terminals by Raman spectroscopy.

Glutamate is the major excitatory neurotransmitter and is of particular interest in light of current models of memory and learning. The paper describes the first in situ detection of glutamate in single nerve terminals (synaptosomes), which is achieved by using laser trapping Raman spectroscopy. The near-infrared laser light captures a single synaptosome obtained from a Wister rat brain. The release of glutamate in a single laser-trapped synaptosome was detected by subtracting the Raman spectrum before depolarization from that after depolarization with the addition of the K(+)-channel blocker, 4-aminopyridine. The result indicated that the single synaptosome released approximately 3 amol of glutamate and that the release rate depended on the 4-aminopyridine concentration.