The Association of Angiotensinogen (AGT M235T) Gene Polymorphism and Essential Hypertension in Thai Post-Menopausal Women

Objectives:This study aims to determine the relationship between angiotensinogen (AGT) M235T polymorphism and hypertension among post-menopausal Thai women.Materials and Methods: Case-control study was conducted. The study group was those who had hypertension or previously diagnosed and, the control were those who had no hypertension. Blood samples were taken for AGT M235T allelic characterization using allele specific oligonucleotides (ASO) PCR. results: Of 255 post-menopausal women, 128 had hypertension, regarded as “hypertension group”, the other 127 without hypertension, regarded as “control group”. The presence of AGT M235T polymorphism was 76.5% for homozygous mutation (73.4% for hypertension group and 79.5% for control group), 21.2% for heterozygous mutation (25.0% for hypertension group and 17.3% for control group, respectively) and 2.4% for homozygous wild-type (1.6% for hypertension group and 3.2% for control group, respectively). Distribution of MM, MT and TT genotypes was not significantly different between both group (p=0.251).Conclusions: Interestingly, overall TT genotype was much higher than that of TM and MM in post-menopausal Thai women. AGT M235T polymorphism was not significantly associated with hypertension, though TT genotype tended to give a small risk. They may not serve as a good genetic marker for essential hypertension among Thai population

THE ROLES OF CHANNELING AGENTS ON THE DRUG RELEASE FROM WAX MATRIX TABLETS PREPARED BY MELT GRANULATION

The study aimed to investigate the effect of the channeling agents and to explore the mechanisms by which they affect the drug release from wax matrix tablets.  The channeling agents were potato starch and icing sugar. The wax matrix tablet contained 4% w/w of chlorpheniramine maleate, a model drug. The matrix formers were carnauba wax, the mixtures of wax and potato starch or icing sugar with different weight ratio of wax: channeling agent, i.e., 1:1, 1:2, and 2:1. They were prepared by melt granulation and then compression into tablet. The in vitro drug release from the wax matrix tablets was studied using USP II (Paddle) method. Within 8 hours, only 22.26% of the drug released from the neat wax matrix tablet, whereas the drug release was significantly increased from tablets containing a channeling agent. The rate and the extent of the drug released increased linearly with the increasing amounts of potato starch. Unlike icing sugar, it had less effect on enhancing the drug release at concentrations of 33% and 50%, but at 67%, the dramatic increase of the drug release was attained. This discrepancy was attributed to the nature and different mechanisms of the two channeling agents on promoting the drug release. The SEM micrographs as well as the degree of water uptake and tablets erosion clearly demonstrated the roles of these two channeling agents on the drug release. The release profiles of the drug from all matrices followed Higuchi model showing the r2 of 0.97-0.99.Keywords: Carnauba wax; Chlorpheniramine maleate; Higuchi diffusion model;  Icing sugar; Melt granulation; Potato starch;Â

Identification of a Novel Single Nucleotide Polymorphism of HADHA Gene at a Referred Primer-binding Site During Pre-diagnostic Tests for Preimplantation Genetic Diagnosis

The pre-diagnostic test for preimplantation genetic diagnosis (PGD) of long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency was performed by polymerase chain reaction (PCR) and direct sequencing for hydroxyacyl-Coenzyme A dehydrogenase/3-ketoacyl-Coenzyme A thiolase/enoyl-Coenzyme A hydratase (HADHA) gene. We obtained unexpected genotyping results of HADHA gene by allele drop-out in the analysis of patients' genomic DNA samples with a referred PCR primer set. Upon further analysis with a re-designed primer set, we found a novel single nucleotide polymorphism (SNP) at the referred primer-binding site in the normal allele of HADHA gene (NT_022184, 5233296 a>t). We found that the frequency of this novel SNP was 0.064 in Korean population. Pre-diagnostic test using single lymphocytes and clinical PGD were successfully performed with the re-designed primer set. Nineteen embryos (95.0%) among 20 were successfully diagnosed to 5 homozygous mutated, 8 heterozygous carrier and 6 wild type. Among 6 normal embryos, well developed and selected 4 embryos were transferred into the mother's uterus, but a pregnancy was not achieved. We proposed that an unknown SNP at primer-binding sites would be a major cause of allele drop-out in the PGD for single gene disorder

Evaluation of genome coverage and fidelity of multiple displacement amplification from single cells by SNP array

The scarce amount of DNA contained in a singe cell is a limiting factor for clinical application of preimplantation genetic diagnosis mainly due to the risk of misdiagnosis caused by allele dropout and the difficulty in obtaining copy number variations in all 23 pairs of chromosomes. Multiple displacement amplification (MDA) has been reported to generate large quantity of products from small amount of templates. Here, we evaluated the fidelity of whole-genome amplification MDA from single or a few cells and determined the accuracy of chromosome copy number assessment on these MDA products using an Affymetrix 10K 2.0 SNP Mapping Array. An average coverage rate (86.2%) from single cells was obtained and the rates increased significantly when five or more cells were used as templates. Higher concordance for chromosome copy number from single cells could be achieved when the MDA amplified product was used as reference (93.1%) than when gDNA used as reference (82.8%). The present study indicates that satisfactory genome coverage can be obtained from single-cell MDA which may be used for studies where only a minute amount of genetic materials is available. Clinically, MDA coupled with SNP mapping array may provide a reliable and accurate method for chromosome copy number analysis and most likely for the detection of single-gene disorders as well

Preimplantation Genetic Diagnosis for Ornithine Transcarbamylase Deficiency by Simultaneous Analysis of Duplex-nested PCR and Fluorescence In Situ Hybridization : A Case Report

Ornithine transcarbamylase (OTC) deficiency is an X-linked co-dominant disorder. A couple, with a previous history of a neonatal death and a therapeutical termination due to OTC deficiency, was referred to our center for preimplantation genetic diagnosis (PGD). The female partner has a nonsense mutation in the exon 9 of the OTC gene (R320X). We carried out nested polymerase chain reaction (PCR) for R320X mutation and fluorescence in situ hybridization (FISH) for aneuploidy screening. Among a total of 11 embryos, two blastomeres per embryo from 9 embryos were biopsied and analyzed by duplex-nested PCR and FISH, and one blastomere per embryo from 2 embryos by only duplex-nested PCR. As a result of PCR and restriction fragment length polymorphism analysis, four embryos were diagnosed as unaffected embryos having the normal OTC gene. Among these embryos, only one embryo was confirmed as euploidy for chromosome X, Y and 18 by FISH analysis. A single normal embryo was transferred to the mother, yielding an unaffected pregnancy and birth of a healthy boy. Based on our results, PCR for mutation loci and FISH for aneuploidy screening with two blastomeres from an embryo could provide higher accuracy for the selection of genetically and chromosomally normal embryos in the PGD for single gene defects

An update of preimplantation genetic diagnosis in gene diseases, chromosomal translocation, and aneuploidy screening

Preimplantation genetic diagnosis (PGD) is gradually widely used in prevention of gene diseases and chromosomal abnormalities. Much improvement has been achieved in biopsy technique and molecular diagnosis. Blastocyst biopsy can increase diagnostic accuracy and reduce allele dropout. It is cost-effective and currently plays an important role. Whole genome amplification permits subsequent individual detection of multiple gene loci and screening all 23 pairs of chromosomes. For PGD of chromosomal translocation, fluorescence in-situ hybridization (FISH) is traditionally used, but with technical difficulty. Array comparative genomic hybridization (CGH) can detect translocation and 23 pairs of chromosomes that may replace FISH. Single nucleotide polymorphisms array with haplotyping can further distinguish between normal chromosomes and balanced translocation. PGD may shorten time to conceive and reduce miscarriage for patients with chromosomal translocation. PGD has a potential value for mitochondrial diseases. Preimplantation genetic haplotyping has been applied for unknown mutation sites of single gene disease. Preimplantation genetic screening (PGS) using limited FISH probes in the cleavage-stage embryo did not increase live birth rates for patients with advanced maternal age, unexplained recurrent abortions, and repeated implantation failure. Polar body and blastocyst biopsy may circumvent the problem of mosaicism. PGS using blastocyst biopsy and array CGH is encouraging and merit further studies. Cryopreservation of biopsied blastocysts instead of fresh transfer permits sufficient time for transportation and genetic analysis. Cryopreservation of embryos may avoid ovarian hyperstimulation syndrome and possible suboptimal endometrium