Quantitative analysis of fourteen bufadienolides in Venenum Bufonis crude drug and its Chinese patent medicines by ultra-high performance liquid chromatography coupled with tandem mass spectrometry.

ETHNOPHARMACOLOGICAL RELEVANCE: Venenum Bufonis, a product of the secretions of Bufo gargarizans Cantor or B. melanostictus Schneider, possessed an array of pharmacological activities, such as cardiotonic, anti-tumor, antinociceptive, anti-inflammatory, anesthetic and antimicrobial activities. However, there were few efficient methods for quality evaluation of Venenum Bufonis medicinal materials and its related Chinese patent medicines. AIM OF THE STUDY: To establish an effective method for quality assessment of crude drugs and Chinese proprietary medicines of Venenum Bufonis, and explore the relationship of primary compounds - target - pathway - disease through a series of network databases. MATERIALS AND METHODS: An ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-QqQ-MS/MS) method was developed and validated to simultaneously determine 14 bufadienolides for quantitative analysis of 71 batches of crude drugs and 20 kinds of Chinese patent medicines of Venenum Bufonis. Multiple reaction monitoring with good specificity and accuracy was applied to monitor the 14 bufadienolides in positive mode. RESULTS: The methodology was validated with good specificity, precision, stability, repeatability and recovery. The low limits of quantification were in the range of 0.1-2.7 ng/mL. The relative standard deviation values for intra- and inter-day precisions ranged from 0.98% to 6.3% and from 2.39% to 6.76%, respectively. The recovery was varied from 87.78% to 110.57% for crude drugs and 88.32%-100.96% for Chinese proprietary medicine (Shexiang Baoxin Pill). The contents of 14 analytes in 71 batches of crude drugs and 20 sorts of Chinese proprietary medicines were procured, the results showed that the contents of crude drugs collected from the market exhibited great variations. Furthermore, 13 batches of crude drugs were identified as counterfeit with no bufadienolides detected. In addition, the total contents of bufadienolides in the same drug showed great difference among products from various manufacturers or brands. Subsequently, 9 bufadienolides with the higher contents were applied to screen the anti-tumor effect by network pharmacology, and 8 pathways which had prior correlation with bufadienolides were disclosed. CONCLUSION: This method could be used for quality assessment of crude drugs and Chinese patent medicines of Venenum Bufonis, and the data could be served as the fundamental basis for drug research and development of Venenum Bufonis.

Novel collagen VI mutations identified in Chinese patients with Ullrich congenital muscular dystrophy.

BACKGROUND: We determined the clinical and molecular genetic characteristics of 8 Chinese patients with Ullrich congenital muscular dystrophy (UCMD). METHODS: Clinical data of probands were collected and muscle biopsies of patients were analyzed. Exons of COL6A1, COL6A2 and COL6A3 were analyzed by direct sequencing. Mutations in COL6A1, COL6A2 and COL6A3 were identified in 8 patients. RESULTS: Among these mutations, 5 were novel [three in the triple helical domain (THD) and 2 in the second C-terminal (C2) domain]. We also identified five known missense or in-frame deletion mutations in THD and C domains. Immunohistochemical studies on muscle biopsies from patients showed reduced level of collagen VI at the muscle basement membrane and mis-localization of the protein in interstitial and perivascular regions. CONCLUSIONS: The novel mutations we identified underscore the importance of THD and C2 domains in the assembly and function of collagen VI, thereby providing useful information for the genetic counseling of UCMD patients.

[Clinical and mutation analyses of a Chinese family with Bethlem myopathy].

OBJECTIVE: To explore the clinical features and gene mutation of a Chinese family with Bethlem myopathy in three generations. METHODS: The clinical data of proband and his family members was collected. Genomic DNA from the patient and his family members was extracted routinely from peripheral blood leukocytes. Polymerase chain reaction and DNA direct sequencing were employed to analyze COL6A1, A2 and A3 genes to determine the mutation. And the relationship between genotype and phenotype was analyzed. Furthermore, the patient's skin fibroblast was cultured and immunofluorescent staining was performed with anti-collagen VI antibody. And the expression pattern of type VI collagen in extracellular matrix between the control and the patient's fibroblast was compared. RESULTS: In this family, 9 patients conformed to the clinical diagnosis of Bethlem myopathy. The features included motor development delay after late infantile period, generalized muscle weakness, walking unstability, distal hyper laxity, proximal joint contractures, skin changes (including hypertrophic scars) and normal intellectual development. Serum creatine kinase (CK) level became mildly elevated and electromyography showed myogenic injury. Disease progressed slowly but the lifespan was not affected. Mutation in exon 2 of COL6A1 gene with c.111-129 deletion was detected in 7 patients in this family. Immunofluorescent staining of type VI collagen in cultured skin fibroblast showed reduced expression of collagen VI in extracellular matrix in the patient compared with the control. CONCLUSIONS: Our study has defined the clinical features of Bethlem myopathy. According to molecular genetic analysis, 7 patients in this family have in-frame deletion mutations of COL6A1 and they conform to autosomal dominant inheritance. And genetic counseling and prenatal diagnosis are available. This is the first Chinese report of Bethlem myopathy family.

[Clinical and mutation analysis of a Chinese family with muscle eye brain disease].

OBJECTIVE: To study the clinical feature of a Chinese family with muscle-eye-brain disease (MEB) and the mutation of protein O-linked-mannose beta-1, 2-N-acetylglucosaminyltransferase 1 gene (POMGNT1). METHODS: Clinical data of the proband and his family members were collected. Genomic DNA from the patient and his parents was extracted using standard procedures from the peripheral blood leukocytes. Polymerase chain reaction and DNA direct sequencing were employed to analyze all of the exons to determine the mutation, and the relationship between genotype and phenotype was analyzed. RESULTS: The proband was diagnosed as floppy baby, presented with delayed psychomotor development and myopathic face. His serum creatine kinase (CK) level elevated moderately and brain MRI showed cerebral and cerebellar gyrus abnormalities with white matter signal intensity changes, cerebellar cysts and cerebellar and brain stem hypoplasia, consistent with congenital muscular dystrophy with eye brain disorder. Further test with DNA detected a compound heterozygous mutation of c.1896 1 G to C before exon 22 which may induce splicing error, and missense mutation c.1319T to G, p.L440R in exon 16. Both parents had a heterozygous mutation at the mutation sites. CONCLUSION: According to our study, the family is diagnosed as MEB. The proband carried compound heterozygous mutations in the POMGNT1 gene, and his parents are heterozygous carriers, which is consistent with autosomal recessive inheritance. The child is definitely diagnosed as having muscle eye brain disease.

[Genotype, phenotype analysis and follow-up study on patients with Duchenne/Becker muscular dystrophy].

OBJECTIVE: To improve the diagnosis and management of Duchenne/Becker muscular dystrophy(DMD/BMD). METHODS: Clinical features of 90 cases of DMD/BMD were collected. Genomic DNA was extracted using standard procedures from the peripheral blood leukocytes, and multiplex ligation-dependent probe amplification (MLPA) was applied to detect DMD gene to identify genetic mutation. For those patients whose deletion/duplication mutation was not identified, FKRP gene mutation analysis was performed using PCR-DNA direct sequence. All the cases were followed up. RESULTS: Among the 90 cases of clinically diagnosed DMD/BMD, exons deletion of DMD was detected in 58 cases (64.44%), and exons duplication in 9 (10.00%). Among the 34 mothers with an affected boy but without previous genetic conformation, 17 were confirmed to be carriers with gene deletion/duplication. None of the 23 cases, without detected DMD gene deletion/duplication, carried FKRP gene mutation. Fourteen children were given short-term intermittent prednisone therapy (0.75 mg/kg daily during the first 10 days of each month). The course was not long enough and the sample size was too small to conclude any benefits or side effects. Prenatal diagnosis was provided for one mother in her next pregnancy detecting a female carrier fetus. CONCLUSION: DMD gene deletions mainly occurs between exons 45 and 54, while duplications mostly at 5'-terminus. Identification of the characteristics and types of gene mutation may facilitate the recognition and prognosis prediction of DMD/BMD. MLPA is a non-complex and quick diagnostic tool for DMD/BMD and its carriers, and also helpful in genetic counseling.