Search

Congenital iris abnormality is a feature of several genetic conditions, such as aniridia syndrome and anterior segment degeneration (ASD) disorders. Aniridia syndrome is caused by mutations in theGenomic DNA was extracted from peripheral blood of 24 patients belonging to 15 unrelated families and their available family members. Multiplex ligation-dependent probe amplification (MLPA) was used to detect the deletions or duplications in the 11p13-14 region, including theWe identified a total of 8 novel and 4 recurrent mutations in 20 of 24 affected individuals from 12 families. Among these mutations, one large deletion of the wholeThis study provides new data on causative mutations in individuals with abnormal development of iris tissue in Vietnam. These results contribute to clinical management and genetic counseling for affected people and their families.

Supplemental Digital Content is available in the text, CYP2D6 genetic variations could result in alteration of CYP2D6 enzyme activity, leading to dissimilarity among individuals in regard of drug metabolism. This study aims to detect all genetic variants, allele, and genotype frequencies of CYP2D6 gene in 136 unrelated healthy Kinh Vietnamese volunteers. All single nucleotide variants (SNVs) and structural variations (SVs) of CYP2D6 gene were identified by Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA) assay. Totally, 30 SNVs and 9 SVs including a whole gene deletion, 8 hybrid structures, and tandem arrangements were identified. Of the 7 novel SNVs detected, the 3157G>T (R329L) substitution was predicted to be deleterious by PROVEAN; the 3851G>A (W358X) variant resulted in a truncated protein; and the 2988G>A variant located in the intron 6 was predicted to be capable of modifying splicing motif by Human Splicing Finder. We determined 29 different genotypes of CYP2D6 from 136 individuals. The most common alleles were the CYP2D6∗10 (43.75%), ∗1 (18.75%), and tandem arrangement ∗36-∗10 (12.13%). This study provides best information on CYP2D6 polymorphism comprising the newly discovered SNVs, structural variations, and their frequencies in Kinh Vietnamese. These new data would be valuable in view of precise dosing of CYP2D6 metabolized drugs and giving better treatment outcome.

Tuberculosis (TB) is the most common mycobacterial infection in developing countries, requiring a rapid, accurate, and well-differentiated detection/diagnosis. For the rapid detection and discrimination of Mycobacterium tuberculosis complex (MTC) from nontuberculous mycobacteria (NTM), a novel, simple, and primer-combined single-step multiplex PCR using three primer pairs (6110F-6110R, 1081F-1081R, and 23SF-23SR; annealing on each of IS6110, IS1081, and 23S rDNA targets), hereafter referred to as a triplex PCR, has been developed and evaluated. The expected product for IS6110 is 416 bp, for IS1081 is 300 bp, and for 23S rDNA is 206 bp by single PCR, which was used to verify the specificity of primers and the identity of MTC using DNA extracted from the M. tuberculosis H37Rv reference strain (ATCC, USA) and other mycobacteria other than tuberculosis (MOTT) templates. The triplex PCR assay showed 100% specificity and 96% sensitivity; the limit of detection for mycobacteria was ~100 fg; and it failed to amplify any target from DNA of MOTT (50 samples tested). Of 307 blinded clinical samples, overall 205 positive M. tuberculosis samples were detected by single PCR, 142 by conventional culture, and 90 by AFB smear methods. Remarkably, the triplex PCR could subsequently detect 55 positive M. tuberculosis from 165 culture-negative and 115 from 217 AFB smear-negative samples. The triplex PCR, targeting three regions in the M. tuberculosis genome, has proved to be an efficient tool for increasing positive detection/ discrimination of this bacterium from clinical samples.

Purpose Retinoblastoma (Rb) is a rare and unique eye cancer that usually develops in the retinas of children less than 5 years old due to mutations in the RB1 gene. About 40% of affected individuals have the heritable form making genetics testing of the RB1 gene important for disease management. This study aims to identify germline mutations in RB1 in a cohort of patients with Rb from northern Vietnam. Methods Genomic DNA was extracted from peripheral blood of 34 patients with Rb (nine unilateral and 25 bilateral cases) and their available parents. Twenty-seven exons, flanking sequences, and the promoter region of RB1 gene were screened for mutations with direct PCR sequencing. Multiplex ligation-dependent probe amplification (MLPA) was applied for patients with negative sequencing results. In the mutation-positive patients, their available parental DNA was analyzed to determine the parental origin of the mutation. Results Germline mutations in RB1 were identified in 25 (73.53%) of 34 patients (four unilateral and 21 bilateral cases). Of these mutations, 19 were detected, including seven nonsense, six frameshift, four splice-site (one was identified in two siblings), and one missense, with Sanger sequencing. Three novel frameshift mutations were discovered in one unilateral and two bilateral patients. MLPA detected mutations in the RB1 gene in six bilateral cases, of whom five had a whole gene deletion (three familial cases) and one had a partial gene deletion (from exon 4 to exon 27) in one allele of the RB1 gene. Parental testing showed five mutations originated from the fathers and one was inherited from a mother who was mosaic for the mutation. Conclusions This study provides a data set of germline mutations in the RB1 gene in Vietnamese patients with retinoblastoma. Screening of mutations in the RB1 gene can help to identify heritable Rb and contribute to clinical management and genetic counseling for affected families.

The synergistic effect of numerous environmental endocrine disrupting chemicals (EDCs) has raised research concern among researchers. To extend previous studies, the measured additional potential interactions among bisphenol A (BPA), 4-nonylphenol (NP), 4-tert octylphenol (OP) and isobutylparaben (IBP) in mouse model were observed. Pregnant Swiss-albino mice were treated with binary combined chemicals (5, 50 or 500 mg kg(-1) b.wt. day(-1)) from gestation day (GD) 1 to 21. Interestingly, maternal exposure to these EDCs caused fluctuation in GD time, live ratio, female/male ratio, body and organ weights of mouse offspring at postnatal day (PND) 1, 21, and 41 days. At most doses early reduced 0.85 to 1.87 GD compared to controls. Besides females/males ratio showed a significant difference in BPA+ OP, BPA+IBP groups. Female body weight at PND 21 and 41, showed a significant reduction at all combined levels, whereas male offspring showed reduction in weight at dose 50 mg kg(-1) b.wt. day(-1). The potential effects of synergic estrogenicity detected histopathological abnormities, such as ovary analysis revealed increase of corpora lutea, cystic follicles and an endometrial hypertrophy and morphometric changes in uteri measurement. Taken together, these results provided an additional insight into synergistic effects of EDCs toxicology on reproductive tracts.

Proteinase A is a papain-like cysteine endopeptidase of vetch (Vicia sativa L.) which was assumed to initiate storage-globulin breakdown just after the onset of seed germination. This enzyme was purified from cotyledons of vetch seedlings. On gelatin-containg SDS gels, active proteinase A migrated with an apparent molecular mass of 21 kDa, whereas after heat denaturation its molecular size on SDS/PAGE was 29 kDa. Although proteinase A is capable of hydrolyzing storage globulins in vitro it could not be localized in the protein-body fraction of cotyledons from germinating seeds. cDNA clones encoding proteinase A precursor have been obtained by PCR. The precursor is composed of an N-terminal signal sequence followed by a propeptide, the region encoding mature proteinase A, and a C-terminal KDEL sequence. Mature proteinase A with a derived molecular mass of 25,244 Da does not have the KDEL sequence. The derived amino acid sequence of the proteinase A precursor is 78.2% identical to sulfhydryl-endopeptidase (SH-EP), a cysteine endopeptidase from germinating Vigna mungo seedlings. Northern blot analysis indicated that proteinase A mRNA appears de novo in cotyledons of 1-day-germinated vetch seeds, where its amount increases up to day 6. No proteinase A mRNA was detected in other vetch organs, not even in the embryo axis, which contains stored globulins. By means of antibodies raised against the purified and against recombinantly produced proteinase A, the 29-kDa bands of mature proteinase A were detected in cotyledon extracts of 6-day-germinated seeds when globulin degradation has already far proceeded. The reported data do not agree with the proposed triggering role of proteinase A in storage-globulin breakdown during germination.

To determine which amino acid residues are essential for the catalytic activity of soybean P-amylase, deoxyoligonucleotide site-directed mutagenesis was employed against aspartyl, glutamyl, and cysteinyl residues located in highly conserved regions found in P-amylase family to date. Both substitution of aspartic acid at position 101 and that of glutamic acid at position 186 of the enzyme by neutral and acidic amino acids, respectively, led to the complete elimination of activity, but did not induce any significant changes in circular dichroic spectra or the binding affinity for cyclomaltohexaose, a substrate analogue. Taking account of the results obtained here, the above two amino acid residues are involved in the catalytic site of soybean P-amylase. The replacement of glutamic acid at position 345 decreased activity to below 6% of the non-mutant level, implying that this residue may also play a crucial role in /I-amylase activity, although it may not be involved at the catalytic site itself. In contrast, substitution of cysteinyl residue at position 95 by a serinyl residue led to a drastic reducing of the optimal temperature (from 50°C to 30”C), suggesting that this cysteinyl residue is responsible for the thermal stability of the enzyme. 8- Amylase (1 ,4-a-D-glucan maltohydrolase) hydrolyzes a-l,4-glucosidic linkage of starch and glycogen with liberation of P-anomeric maltose from the non-reducing ends. This enzyme is found in many plant and bacteria species. Although the physicochemical properties of the enzymes from sweet potato, barley, and soybean have been well investigated [l], the catalytic mechanism of the enzyme has not yet been elucidated. Comparison of deduced sequences of P-amylases originating from plant and bacteria revealed three highly conserved sequences which very likely are involved in the segment assembly of the catalytic site of /I-amylase [2-41. In the case of a-amylase (1,4-a-~-glucan glucanohydrolase) that catalyzes the hydrolysis of 1,4-a-D-glucans, the aspartyl and glutamyl residues contained in highly conserved regions have been identified as catalytic residues by X-ray crystallography [5 -71 and by site-directed mutagenesis [8, 91. Although the action pattern of P-amylase differs from that of a-amylase and there is no significant similarity between the primary sequences, all of the aspartyl and glutamyl residues located in the conserved regions of P-amylase family can be considered possible catalytic residues. Recently, the results of affinity labeling of soybean P-amylase with 2,3epoxy propyl a-D-glucopyranoside suggested that Glu186 was involved in the catalytic site [lo]. In addition, since the enzyme was inactivated by sulfhydryl reagents such as iodoacetamide, N-ethylmaleimide and mercuribenzoate [l , 111, the cysteinyl residues located in the regions may also participate in the catalytic reaction.