Ainoon Othman

In vitro expression of erythropoietin by transfected human mesenchymal stromal cells

BACKGROUND Mesenchymal stromal cells (MSC) are pluripotent progenitor cells that can be found in human bone marrow (BM). These cells have low immunogenicity and could suppress alloreactive T-cell responses. In the current study, MSC were tested for their capacity to carry and deliver the erythropoietin (EPO) gene in vitro. METHODS Expanded BM MSC was transfected with EPO-encoded plasmid pMCV1.2 and EPO-encoded MIDGE (minimalistic immunologically defined gene expression) vector by electroporation. The expressed EPO was used to induce hematopoietic stem cells (HSC) into erythroid colonies. RESULTS The results showed that the MIDGE vector was more effective and stable than the plasmid (pMCV1.2) in delivering EPO gene into MSC. The supernatants containing EPO obtained from the transfected cell culture were able to induce the differentiation of HSC into erythroid colonies. DISCUSSION MSC hold promise as a cell factory for the production of biologic molecules, and MIDGE vector is more effective and stable than the plasmid in nucleofection involving the EPO gene.

Homozygous variant of UGT1A1 gene mutation and severe neonatal hyperbilirubinemia

Background:  The aim of the present study was to compare, in a case–control study, the prevalence of nucleotide 211 guanine to adenine (G→A) mutation of uridine diphosphoglucuronosyl transferase (UGT1A1) gene in Malaysian Chinese newborns with and without severe hyperbilirubinemia (total serum bilirubin >250 µmol/L during first 48 h of life or ≥300 µmol/L thereafter), and to determine whether this mutation was a significant risk factor associated with severe hyperbilirubinemia.

A case series of α-thalassemia intermedia due to compound heterozygosity for Hb Adana [HBA2: c179G>A (or HBA1); p.Gly60Asp] with other α-thalassemias in Malay families.

Abstract Hb Adana [HBA2: c179G>A (or HBA1); p.Gly60Asp] is a rare hemoglobin (Hb) variant due to a mutation at codon 59 of the α2- or α1-globin gene resulting in a glycine to aspartic acid substitution. Two siblings with a unique coinheritance of Hb Adana and Hb Constant Spring (Hb CS, α142, Term→Gln, TAA>CAA; HBA2: c.427 T>C) (αcodon 59α/αCSα), were compared phenotypically with another two siblings carrying the Hb Adana mutation and a 3.7 kb deletion (αcodon 59α/−α3.7). Although they all had α-thalassemia intermedia (α-TI), the former were clinically more severe than the latter. The first pair of siblings presented at a much younger age than the second pair and showed lower Hb levels and significant extramedullay hemopoiesis. Another case of a hydropic fetus as a result of Hb H/Hb Adana is also described. Their clinical phenotypes and hematological parameters are all presented for comparison.

Risk Factors Associated with Unconjugated Neonatal Hyperbilirubinemia in Malaysian Neonates

OBJECTIVE To investigate the risk factors associated with neonatal hyperbilirubinemia in Malaysian neonates. METHODS A prospective study was conducted to investigate the effects of glucose-6-phosphate dehydrogenase (G6PD) mutation, variant uridine diphosphate glucuronosyltransferase UGT1A1 gene and hepatic organic anion transporter protein (OATP2) gene on a group of neonates. Hyperbilirubinemia was defined as a total serum bilirubin level of ≥250 µmol/l. RESULTS Of 318 neonates, 52 (16.4%) had hyperbilirubinemia. The incidence of G6PD mutation was 5.4% (15/280) among these infants. The incidence of G6PD mutation was significantly higher in the male neonates with hyperbilirubinemia (7.8%) when compared with the normal male neonates without hyperbilirubinemia (1.8%; p = 0.03). Logistic regression analysis showed that the significant risk factors for neonatal hyperbilirubinemia were Malay ethnicity [adjusted odds ratio (OR), 2.77; 95% confidence interval (CI): 1.31-5.86; p = 0.007] and G6PD mutation (adjusted OR, 3.29; 95% CI: 1.06-10.1820; p = 0.039). The gender, birth weight and gestation age of neonates, variant c.211G > A and variant of OATP2 gene were not significant. CONCLUSIONS Neonates with Malay ethnicity and G6PD mutation were at risk for hyperbilirubinemia.

Comparative global gene expression profile of human limbal stromal cells, bone marrow mesenchymal stromal cells, adipose-derived mesenchymal stromal cells and foreskin fibroblasts

Abstract Background: Limbal epithelial stem cells (LESC) have great potential in treating the blindness caused by corneal damage. LESC are maintained in stem cell niche called Palisade of Vogt. Limbal stromal (LS) cells

Genotyping of OATP2 Variants in a Group of Malaysian Neonates Using High-Resolution Melting Analysis

Organic anion transport polypeptide 2 (OATP2), also known as OATP-C, OATP1B1, and LST-1, is responsible for the transportation of organic anions into hepatocytes. It has been reported that OATP2 transports a broad range of endogenous and xenobiotic compounds such as bile acids, bilirubin, sulfate and glucuronide conjugates, thyroid hormones, peptides, and drugs like 3-hydroxyl-3 methylglutaryl-coenzymeA–reductase inhibitors (pravastatin, rosuvastatin, pitavastatinand, and methorexate).1–3 The gene for OATP2 is located at chromosome 12p12.14.1, and a number of single-nucleotide polymorphisms (SNPs) have been identified in both the encoding and regulation regions of the OATP2 gene in different populations of the world.3 Among the Asian populations, two OATP2 variants have been shown to be highly prevalent in the Chinese in mainland China, that is, c.388A>G (73.4%) and c.521T>C (14.0%).4 Additionally, two other silent mutations, c.571T>C and c.597C>T of the OATP2 gene, were also reported to be common in Asian populations, occurring at 26% and 50%, respectively, in the Chinese population,5 and 64.2% and 42.9% in a Japanese population.4–7 The importance of OATP2 genotyping may lie in the fact that variations in the OATP2 gene may have an impact on drug metabolism and perhaps on the pathogenesis of neonatal jaundice. One study has shown that the OATP2 c.388A>G variant may be a possible risk factor for severe neonatal hyperbilirubinemia.8 Malaysia is a multiethnic country comprising of major ethnic Malays, Chinese, and Indians. This study forms our initial effort to determine the existence of the OATP2 gene variation in the Malaysian population as a prequel to study its role in cases of unconjugated hyperbilirubinemia, a common condition in Malaysian newborns. We report here our study on the determination of allelic variations in Malaysian neonates using an HRM analysis and TaqMan Minor Groove Binder (MGB) assays. Until now, these OATP2 alleles have been genotyped with either a polymerase chain reaction (PCR)–restriction fragment length polymorphism (RFLP) assay (c.388A>G) or with an allele-specific PCR assay (c.521T>C).3,9 Recent study reported the use of real-time PCR with the fluorescence resonance energy transfer (FRET) assay for genotyping the c.388A>G and c.521T>C variant.10 To perform large epidemiological studies or for routine clinical use, a rapid genotype method is preferred. Therefore, the present study describes the application of HRM assays and TaqMan MGB assays. By using these methods, we were able to genotype these OATP2 polymorphisms in considerably less hands-on time and with a reduced contamination risk.