Identification and functional characterization of novel GDAP1 variants in Chinese patients with Charcot-Marie-Tooth disease.

OBJECTIVE: To identify and characterize the pathogenicity of novel variants in Chinese patients with Charcot-Marie-Tooth disease. METHODS: Multiplex ligation-dependent probe amplification (MLPA) and whole-exome sequencing (WES) were performed in 30 unrelated CMT patients. Minigene assay was used to verify the effect of a novel splicing variant (c.694+1G>A) on pre-mRNA. Primary fibroblast cell lines were established from skin biopsies to characterize the biological effects of the novel variants p.L26R and p.S169fs. The mitochondrial structure was observed by an electron microscope. The expression level of protein was analyzed by Western Blotting. Mitochondrial dynamics and mitochondrial membrane potential (MMP, Δψm) were analyzed via immunofluorescence study. Mitochondrial ATP levels were analyzed via bioluminescence assay. The rate of oxygen consumption was measured with a Seahorse Bioscience XF-96 extracellular flux analyzer. RESULTS: We identified 10 pathogenic variants in three known CMT related genes, including three novel variants (p.L26R, p.S169fs, c.694+1G>A) and one known pathogenic variant (p.R120W) in GDAP1. Further, we described the clinical features of patients carrying pathogenic variants in GDAP1 and found that almost all Chinese CMT patients with GDAP1 variants present axonal type. The effect of c.694+1G>A on pre-mRNA was verified via minigene splice assay. Cellular biological effects showed ultrastructure damage of mitochondrial, reduced protein levels, different patterns of mitochondrial dynamics, decreased mitochondrial membrane potential (Δψm), ATP content, and defects in respiratory capacity in the patient carrying p.L26R and p.S169fs in GDAP1. INTERPRETATION: Our results broaden the genetic spectrum of GDAP1 and provided functional evidence for mitochondrial pathways in the pathogenesis of GDAP1 variants.

Genetic Spectrum and Variability in Chinese Patients with Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a progressive, fatal neurodegenerative disease characterized by selective impairment of upper and lower motor neurons. We aimed to investigate the genetic spectrum and variability in Chinese patients with ALS. A total of 24 familial ALS (FALS) and 21 early-onset sporadic ALS (SALS) of Chinese ancestry were enrolled. Targeted next-generation sequencing (NGS) was performed in the probands, followed by verification by Sanger sequencing and co-segregation analysis. Clinical features of patients with pathogenic or likely pathogenic variants were present. The mutation frequency of ALS-related genes was then analyzed in Chinese population. In this cohort, 17 known mutations (9 SOD1, 5 FUS, 2 TARDBP and one SETX) were identified in 14 FALS and 6 early-onset SALS. Moreover, 7 novel variants (SOD1 c.112G>C, OPTN c.811C>T, ERBB4 c.965T>A, DCTN1 c.1915C>T, NEFH c.2602G>A, NEK1 c.3622G>A, and TAF15 c.1535G>A) were identified. In southeastern Chinese FALS, the mutation frequency of SOD1, FUS, and TARDBP was 52.9%, 8.8%, 8.8% respectively. In early-onset SALS, FUS mutations were the most common (22.6%). In Chinese ALS cases, p.H47R is most frequent SOD1 mutations, while p.R521 is most common FUS mutation and p.M337V is most common TARDBP mutation. Our results revealed that mutations in SOD1, FUS and TARDBP are the most common cause of Chinese FALS, while FUS mutations are the most common cause of early-onset SALS. The genetic spectrum is different between Chinese ALS and Caucasian ALS.

Genetic spectrum and clinical profiles in a southeast Chinese cohort of Charcot-Marie-Tooth disease.

Charcot-Marie-Tooth (CMT) disease is a heterogeneous group of inherited sensorimotor neuropathies. To clarify the genetic spectrum and clinical profiles in Chinese CMT patients, we enrolled 150 unrelated CMT patients from southeast China. We performed multiplex ligation-dependent probe amplification (MLPA) testing in all patients and next-generation sequencing (NGS) among those patients without PMP22 rearrangements. We identified PMP22 duplications in 40 patients and deletions in 12 patients. In addition, we found 19 novel variants and 36 known mutations in 57 patients. Among these 55 variants, 45 pathogenic or likely pathogenic variants were identified in 48 cases, and 10 variants with uncertain significance were identified in 9 cases. Therefore, we obtained a genetic diagnosis in 63.8% (88/138) of CMT patients and 66.7% (100/150) of all included patients. PMP22, GJB1, and MFN2 are the most common causative genes in CMT1 (demyelinated form), intermediate CMT, and CMT2 (axonal form), respectively. In this study, we identified a higher proportion of intermediate CMT, a relatively high frequency of NDRG1 mutations and clinical features of later onset age in CMT1A patients. Our results broaden the genetic and clinical spectrum of CMT patients, which can help optimize the genetic and clinical diagnosis.

Clinical features and genetic spectrum in Chinese patients with recessive hereditary spastic paraplegia.

BACKGROUND: Although many causative genes of hereditary spastic paraplegia (HSP) have been uncovered in recent years, there are still approximately 50% of HSP patients without genetically diagnosis, especially in autosomal recessive (AR) HSP patients. Rare studies have been performed to determine the genetic spectrum and clinical profiles of recessive HSP patients in the Chinese population. METHODS: In this study, we investigated 24 Chinese index AR/sporadic patients by targeted next-generation sequencing (NGS), Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA). Further functional studies were performed to identify pathogenicity of those uncertain significance variants. RESULTS: We identified 11 mutations in HSP related genes including 7 novel mutations, including two (p.V1979_L1980delinsX, p.F2343 fs) in SPG11, two (p.T55 M, p.S308 T) in AP5Z1, one (p.S242 N) in ALDH18A1, one (p.D597fs) in GBA2, and one (p.Q486X) in ATP13A2 in 8 index patients and their family members. Mutations in ALDH18A1, AP5Z1, CAPN1 and ATP13A2 genes were firstly reported in the Chinese population. Furthermore, the clinical phenotypes of the patients carrying mutations were described in detail. The mutation (p.S242 N) in ALDH18A1 decreased enzyme activity of P5CS and mutations (p.T55 M, p.S308 T) in AP5Z1 induced lysosomal dysfunction. CONCLUSION: Our results expanded the genetic spectrum and clinical profiles of AR-HSP patients and further demonstrated the efficiency and reliability of targeted NGS diagnosing suspected HSP patients.

Identification and functional analysis of novel mutations in the SOD1 gene in Chinese patients with amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease characterized by selective involvement of motor neurons in the central nervous system (CNS). The most common causative gene of ALS in the Chinese population is the Cu/Zn superoxide dismutase 1 (SOD1) gene, which accounts for 20-42.9% of familial ALS (FALS) and 1-2% of sporadic ALS (SALS) cases. In this study, we identify three novel SOD1 mutations, Gly17Cys, Pro75Ser, and His121Gln, in four ALS pedigrees. A functional analysis was performed, and the results showed that all three mutations could lead to the formation of misfolded proteins. In addition, genotype-phenotype correlations in these patients are also described. Our study helps to characterize the genotype and phenotype of ALS with SOD1 mutations.

Subacute fulminant hepatic failure with intermittent fever.

BACKGROUND: Viral hepatitis B accounts for over 80% of acute hepatic failures in China and the patients die mainly of its complications. A patient with hepatic failure and fever is not uncommon, whereas repeated fever is rare. METHODS: A 32-year-old female was diagnosed with subacute hepatic failure and hepatitis B viral infection because of hyperbilirubinemia, coagulopathy, hepatic encephalopathy, serum anti-HBs-positive without hepatitis B vaccination, and typical intrahepatic pathological features of chronic hepatitis B. Plasma exchange was administered twice and she awoke with hyperbilirubinemia and discontinuous fever. RESULTS: Urethritis was confirmed and medication-induced fever and/or spontaneous bacterial peritonitis (Gram-negative bacillus infection) was suspected. The patient was treated with antibiotics, steroids and a Chinese herbal medicine, matrine, for three months and she recovered. CONCLUSION: The survival rate of patients with hepatic failure might be improved with comprehensive supporting measures and appropriate, timely management of complications.

[Lamivudine, interferon-alpha and oxymatrine treatment for the surviving hepatic failure patients with hepatitis B].

OBJECTIVE: To investigate the effect of lamivudine, interferon alpha and oxymatrine treatment for surviving hepatic failure patients with hepatitis B. METHODS: 200 hepatitis B patients, including 100 subacute or acute-on-chronic hepatic failure survivals (group A), and 100 chronic (group B, n=100) hepatic failure survivals, were enrolled in this study. Patients in group A received interferon alpha (n=35), lamivudine (n=33) , or combinational lamivudine and oxymatrine (n=32) therapy for six months; Patients in group B received lamivudine (n=49), or combinational lamivudine and oxymatrine (n=51) therapy for six months, respectively. After the treatment, all patients were followed-up for six months. RESULTS: At the end of follow-up, all patients in group A survived, while in group B three patients (6.1%) receiving lamivudine, and four (7.8%, P>0.05) receiving combinational therapy died; more than 90% of all survivals had their HBV DNA loss. The HBeAg/anti-HBe seroconversion rate in patients of group A treated with interferon alpha (9/17, 52.9%) was higher than that in patients treated with combinational lamivudine and matrine (5/16, 31.3%, P<0.05), which was higher than that in the patients treated with lamivudine alone (1/17, 5.9%, P<0.01), and the Knodell histological activity index score in patients treated with lamivudine (7.2+/-0.8, P<0.05) was lower than that in patients treated with interferon alpha (8.2+/-1.3, P<0.05), and the best efficacy was found in receiving combinational therapy (6.9+/-0.7, P<0.01); Lamivudine or lamivudine in combination with matrine significantly inhibited the intrahepatic inflammatory activities, but had no effect on the existing fibrosis in group B patients. CONCLUSION: Long term nucleotide analogues treatment may delay the progress of fibrosis in hepatitis B-induced hepatic failure survivals, and the administration of matrine in time may further enhance the anti-fibrotic effect of nucleotide analogues.

[Glycocorticosteroid administration prevents fulminant hepatic failure occurrence in patients with chronic hepatitis B of severe degree].

OBJECTIVE: To prevent chronic severe hepatitis, even more fulminant hepatic failure (FHF) occurrence in patients with chronic hepatitis B of severe degree using steroid. METHODS: 120 patients were randomized into conventional supporting treatment and steroid treatment groups. The latter, 62 patients were given intravenously hydrocortisone sodium succinate at the dose of 150 mg to approximately 200 mg everyday plus support care. RESULTS: The rate of deteriorating to chronic severe hepatitis in steroid treatment group was significantly lower than that of conventional group (22% vs 48%, x(2) =7.60, P<0.01). 53.6% (15/28) patients with chronic severe hepatitis in conventional group died, while only 28.6% (4/14) in steroid treatment group succumbed to terminal liver disease (x(2)=0.02, P>0.05). There was no difference between the two groups regarding to complications incidence: gastrointestinal bleeding and infections except for some controllable serious reverse events, such as candidiasis, diabetes, herpes zoster and pulmonary tuberculosis found in some patients in steroid-treated group. CONCLUSION: These results suggest that steroid administration with improved support care not only is likely to prevent chronic severe hepatitis occurrence in patients with chronic viral hepatitis of severe degree, but also shows some efficacy for FHF, which warrant further investigation.