Siriporn Keeratichamroen

Novel mutations in a Thai patient with methylmalonic acidemia

A Thai patient with methylmalonic acidemia (MMA) and no methylmalonyl-CoA mutase (MCM, EC 5.4.99.2) activity in leukocytes in the presence of deoxyadenosyl cobalamin (mut(0)) was found to be heterozygous for two novel mutations: 1048delT and 1706_1707delGGinsTA (G544X), inherited from her mother and father, respectively. The proband was also heterozygous for the polymorphism, A499T, which did not affect the activity of recombinant MCM.

Molecular analysis of the iduronate-2-sulfatase gene in Thai patients with Hunter syndrome

SummaryMolecular defects in the gene encoding the enzyme iduronate-2-sulfatase (IDS) result in Hunter disease (mucopolysaccharidosis type II, MPS II). To determine the molecular basis of MPS II in Thailand, the IDS gene was analysed in 20 Thai patients with Hunter syndrome from 18 unrelated families. A total of 19 different mutations, including 9 missense mutations, 3 nonsense mutations, 3 splice site alterations, 1 deletion, 2 indels, and 1 rearrangement were identified, 8 of which were novel (p.R101C, p.D148V, p.G224A, p.K227E, p.E254X, p.W337X, c.440_442delinsTT and c.720_731delinsTTTCAGATGTTCTCCCCAG). Evaluation of the IDS activity of two hemizygous variants identified in the same patient, p.R101C and p.R468Q, by expression of IDS with the individual mutations in COS 7 cells indicated that only the p.R468Q mutation affected IDS protein activity. Two exonic mutations, c.257C>T (p.P86L) and c.418G>A, were found to activate multiple cryptic splice sites, resulting in aberrantly spliced transcripts. Thus, MPS II in Thailand is caused by a diverse set of defects affecting both IDS protein production and activity.

Clinical and molecular findings in Thai patients with isolated methylmalonic acidemia

Isolated methylmalonic acidemia (MMA) is a genetically heterogeneous organic acid disorder caused by either deficiency of the enzyme methylmalonyl-CoA mutase (MCM), or a defect in the biosynthesis of its cofactor, adenosyl-cobalamin (AdoCbl). Herein, we report and review the genotypes and phenotypes of 14 Thai patients with isolated MMA. Between 1997 and 2011, we identified 6 mut patients, 2 cblA patients, and 6 cblB patients. The mut and cblB patients had relatively severe phenotypes compared to relatively mild phenotypes of the cblA patients. The MUT and MMAB genotypes were also correlated to the severity of the phenotypes. Three mutations in the MUT gene: c.788G>T (p.G263V), c.809_812dupGGGC (p.D272Gfs*2), and c.1426C>T (p.Q476*); one mutation in the MMAA gene: c.292A>G (p.R98G); and three mutations in the MMAB gene: c.682delG (p.A228Pfs*2), c.435delC (p.F145Lfs*69), and c.585-1G>A, have not been previously reported. RT-PCR analysis of a common intron 6 polymorphism (c.520-159C>T) of the MMAB gene revealed that it correlates to deep intronic exonization leading to premature termination of the open reading frame. This could decrease the ATP:cobalamin adenosyltransferase (ATR) activity resulting in abnormal phenotypes if found in a compound heterozygous state with a null mutation. We confirm the genotype-phenotype correlation of isolated MMA in the study population, and identified a new molecular basis of the cblB disorder.

Decreasing activity and altered protein processing of human iduronate-2-sulfatase mutations demonstrated by expression in COS7 cells.

Hunter syndrome, or mucopolysaccharidosis type II (MPS II; MIM:309900), is a rare X-linked recessive lysosomal storage disorder caused by defects of the gene coding for iduronate-2-sulfatase (IDS; EC 3.1.6.13) (Bach et al. 1973). IDS catabolizes dermatan sulfate and heparan sulfate in the lysosome. Lack of this enzyme leads to the systemic accumulation of these substances, eventually causing characteristic clinical

Apigenin inhibits growth and induces apoptosis in human cholangiocarcinoma cells

A promising nutraceutical, apigenin, was recently revealed to exhibit biological activity in inhibiting several types of cancer. The effects of apigenin on the growth inhibition and apoptosis of the cholangiocarcinoma HuCCA-1 cell line were investigated. Protein alterations subsequent to apigenin treatment were studied using a proteomic approach. The values of 20, 50 and 90% inhibition of cell growth (IC20, IC50 and IC90) were determined by MTT cell viability assay. Apoptotic cell death was detected using two different methods, a flow cytometric analysis (Muse Cell Analyzer) and DNA fragmentation assay. A number of conditions including attached and detached cells were selected to perform two-dimensional gel electrophoresis (2-DE) to study the alterations in the expression levels of treated and untreated proteins and identified by liquid chromatography (LC)/tandem mass spectrometry (MS/MS). The IC20, IC50 and IC90 values of apigenin after 48 h treatment in HuCCA-1 cells were 25, 75 and 200 µM, respectively, indicating the cytotoxicity of this compound. Apigenin induced cell death in HuCCA-1 cells via apoptosis as detected by flow cytometric analysis and exhibited, as confirmed with DNA fragmentation, characteristics of apoptotic cells. A total of 67 proteins with altered expression were identified from the 2-DE analysis and LC/MS/MS. The cleavage of proteins involved in cytoskeletal, cytokeratin 8, 18 and 19, and high expression of S100-A6 and S100-A11 suggested that apoptosis was induced by apigenin via the caspase-dependent pathway. Notably, two proteins, heterogeneous nuclear ribonucleoprotein H and A2/B1, disappeared completely subsequent to treatment, suggesting the role of apigenin in inducing cell death. The present study indicated that apigenin demonstrates an induction of growth inhibition and apoptosis in cholangiocarcinoma cells and the apoptosis pathway was confirmed by proteomic analysis.