Chris Adhiyanto

Oxidation Status of β-Thalassemia Minor and Hb H Disease, and Its Association with Glycerol Lysis Time (GLT50)

Abstract β-Thalassemia (β-thal), especially β-thalassemia major (β-TM), is reported to be related to reactive oxygen species (ROS) and enhanced oxidation status. It is reflected by increased malondialdehyde (MDA), by membrane lipid peroxidation and decreased by the newly developed total antioxidant capacity (TAC). However, there is less evidence for β-thal minor and Hb H (β4) disease on its association with oxidation status. On the other hand, hemolysis by glycerol lysis time (GLT50) is invariably prolonged in thalassemia. The reason for the prolongation of GLT50 is not well understood. The aim of this study was to investigate the oxidation state in β-thal minor and Hb H disease and to find out the association of the oxidation with the prolongation of GLT50. Blood samples from 39 subjects (33 with β-thal minor, six with Hb H disease) were collected from individuals living in Japan. The clinical screening tests and molecular identification of the thalassemias were performed. Malondialdehyde and TAC were measured using spectrophotometric analyses. In β-thal minor and Hb H disease, the plasma MDA level was significantly elevated and the TAC reduced. A highly reversed correlation between MDA and TAC was noted. Their GLT50 levels were evidently prolonged, and the GLT50 has significant correlations with MDA and TAC. β-Thalassemia minor and mild Hb H disease are evidently in a milieu of reduced redox state, and GLT50 prolongation in β-thal minor and Hb H disease has a close correlation with the oxidation state, possibly by oxidative impairment of the membrane protein of the red cell.

A New Krüppel-Like Factor 1 Mutation (c.947G > A or p.C316Y) in Humans Causes β-Thalassemia Minor

Abstract Here we describe a Japanese patient with mild β-thalassemia (β-thal) with an intact β-globin gene but a new missense mutation of c.947G > A or p.C316Y in the erythroid Krüppel-Like Factor (KLF1) gene which is strongly associated with the expression of the β-globin gene. The association of the KLF1 mutation with β-thal, is here described. The p.C316Y mutation occurred at one of the cysteines that constitute the second zinc finger motif of KLF1, and would have changed the zinc finger conformation to impair the DNA binding properties or the promoter function of the β-globin gene. Our expression study found that the mutant KLF1 gene had a markedly negative effect on the β-globin gene expression, or 7.0% of that of its normal counterpart. A presumed heterozygous state, or equimolar presence of the mutant and normal KLF1s reduced the expression rate to 70.0% of the normal alone. This degree of the decrease may explain the very mild phenotype of the patient’s β-thal. Furthermore, the patient’s whole-exome analysis using next-generation sequencing revealed that the β-thal defect is caused by only this KLF1 gene mutation. The Hb A2 and Hb F levels that are frequently elevated in KLF1 mutations were elevated by 4.1 and 1.3%, respectively, in this case. The contribution to their elevation by KLF1: p.C316Y is uncertain.

A New β0-Thalassemia Mutation (codon 102, AAC>ATCAC) in Coexistence with a Heterozygous P4.2 Nippon Gene

A new β-thalassemia (β-thal) frameshift mutation was found at codon 102 (AAC>ATCAC) in a 17-year-old Japanese male and his 14-year-old sister. Both demonstrated a more severe phenotype than the usual β-thal minor with mild hemolytic involvement. No mRNA derived from the thalassemic allele, or βTmRNA, was detected in the sequencing analysis of the whole mRNA (cDNA). However, the βTmRNA from the whole βmRNA was specifically amplified by amplification refractory mutation system (ARMS), and was actually found to be present. Furthermore, quantitative polymerase chain reaction (q-PCR) demonstrated a negligible amount of βTmRNA. Thus, their more severe phenotype was not caused by the “dominant type” β-thal in which a considerable amount of the βTmRNA would be expected. In fact, our proband had a total βmRNA level that was mostly normal. Thus, the cause of a β-thal phenotype by the frameshift mutation was ascribed to the reduced amount of mRNA. We further searched for the cause of their severe phenotype. However, factors that exacerbated the phenotype of β-thal, such as α-globin gene triplication, coexisting iron deficiency and infection were not found. Finally, we noticed that the red cell morphology revealed ovalocytosis and small numbers of stomatocytes that were seen in the hereditary spherocytosis (HS), especially by P4.2 mutations. The sequence of the P4.2 gene disclosed heterozygous P4.2 Nippon, or missense mutation at codon 142 (GCT>ACT) on exon 3, the most common mutation of Japanese HS. Frequent mutations of other membrane proteins, Band 3 and ankyrin that are common cause of HS in the Japanese population, other than P4.2, were not detected. When HS by P4.2 Nippon develops it is homozygous, and no P4.2 protein is observed in sodium dodecilsulphate-polyacrylamide gel electrophoresis (SDS-PAGE), while in our case the amount of the P4.2 was almost normal in the SDS-PAGE. However, there are several reports that revealed more severe phenotypes of β-thal by the coexisting abnormality of membrane protein. It is uncertain, but the presence of heterozygous P4.2 Nippon may be associated with the exacerbation of the phenotype of β-thal minor.

Identification of a Novel Mutation in the β-Globin Gene 3′ Untranslated Region [+1,506 (A>C)] in a Japanese Male with a Heterozygous β-Thalassemia Phenotype

β-Thalassemia (β-thal) is characterized by the absent or reduced production of β-globin chains. The precise molecular lesion that causes decreased β-globin synthesis in β+-thal is difficult to predict when mutations occur in the locus control region (LCR), the promoter, the introns or 3′ untranslated regions (3′UTRs). Among them, the role of the 3′UTR of β-globin gene in mRNA stability is poorly understood, mainly due to very few cases that have mutations in this region. So far, only three mutations have been reported in the 3′UTR of β-globin gene. Although, it is speculated that some of these reported mutations could be associated with mRNA stability, the precise molecular basis still remains unclear. We report here a novel mutation in the β-globin gene 3′UTR [+1,506 (A>C)] in a 31-year-old Japanese male with hematological parameters suggestive of heterozygous β-thal. Further functional studies on this novel mutation reported here, may help in understanding of the regulation and expression of the β-globin gene and its products.

The +1,506 (A>C) Mutation in the 3′ Untranslated Region Affects β-Globin Expression

The 3 ′ untranslated region (3 ′UTR) is known to be important to mRNA stability but the stabilization mechanism on the β-globin gene is not fully elucidated. We speculated in our previous report that +1,506 (A>C) mutation (HGVS nomenclature: *32A>C) on the β-globin 3 ′UTR causes β-thalassemia (β-thal) in order to destabilize the mRNA. To investigate further, we studied the expression efficiency for the mutation with a luciferase assay. We made recombinant pGL4.74 vectors in which the luciferase 3 ′UTR was replaced with the wild-type and mutant 3 ′UTR of the β-globin gene. For a comparison experiment, recombinant vectors were made not only for this mutation but also six other mutations in the β-globin 3 ′UTR which bring about β-thal or affect mRNA stability. The +1,506 mutation led to a 30.0% lower protein expression than normal in this assay. We concluded that this mutation destabilizes mRNA and consequently decreases the β-globin amount to finally cause β-thal. Our study highlights the crucial area of β-globin 3 ′UTR for protein expression.

Internet-based approach to population screening for common hemoglobinopathies in United Arab Emirates

This article reports on efforts to overcome common hurdles that were faced during population-based screening for common hemoglobinopathies in the United Arab Emirates. An Internet-based approach was designed and implemented to increase the acceptance of the screening program. The process involved: an awareness campaign, a simple bilingual (Arabic/English) online consent form and registration process, the use of a barcode for sample labeling, an equipment upgrade, electronic communication of a successful registration process, test results, and a counseling process. Before the implementation of the Internet-based system, great concern was noted among the clients in terms of the availability of accurate and timely test results, the need for pretest and post-test counseling, and the way that their personal health information was handled. Lapses in information exchange between the clients who participated in the screening program for the carrier state of inherited disorders and the screening laboratory posed significant challenges. The emphasis on confidentiality and the ease of access to services was instrumental in increasing the level of acceptance of these services in our community. Based on an analysis of > 10,000 samples, we conclude that Internet-based reporting holds much promise for improving the quality of care that clients receive.

Identification of Delta-Aminolevulinic Acid Dehydratase (ALAD) Gene Polymorphism in Students of Elementary School in Kalideres, Jakarta

Polymorphism in ALAD genes is form of adaptation mechanism environment changes due to pollutants Pb (Lead) and have an impact on the health risks. Accumulation of Pb in the lowest air pollutants may be a risk to children’s health. Children with height range between 100 – 140 cm (6 to 9 years old) may have risks being exposed to Pb. Particulates Pb are found in ±1 meter above ground. The study was done to determine frequencies of genetic polymorphisms ALAD in population students of elementary school in Kalideres, Jakarta. The method used in this study was a descriptive method with laboratory test techniques using PCR-ARMS (Amplification Refractory Mutation System) technique and the completion of questionnaires by the parents for respondent characteristics. The samples were 60 students who were 51.7% were ALAD wildtypes, 48.3% heterozygotic genotypes, and no mutants were found in the population of elementary school children in Kalideres, Jakarta. This study is necessary to identify the individual’s genetic susceptibility mainly in polymorphism