Abstract.A study on thalassemia intermedia and major patients in Jakarta was initiated to obtain a comprehensive picture of metabolic dysregulation, iron overload, oxidative stress, and cell damage. Data are presented from a group of 14 transfusiondependent patients in an age range of 11-25 years (T) and another group of 9 frequently transfused (for at least 15 years) patients aged 17-30 years (L). A third group comprised 6 patients (aged 7 to 14 years) who had not yet obtained transfusions (N). The 21 controls (C) were voluntary students without diagnosis or clinical signs of thalassemia up to 30 years of age. The study was approved by the Ethical Clearance Board of the Medical Faculty and all blood samples from controls and patients were obtained on fully informed consent. Levels of antioxidants (vitamins A, C, E and β-carotene) and reactive thiols are considerably decreased in transfused patients, whereas signs of iron overload and cell damage are increased (serum iron, ferritin, transferrin saturation, SGOT, SGPT, γ-GT, bilirubin). Results can be summarized that non-transfused thalassemia intermedia patients exert slight signs of oxidative stress, and increased hemoglobin degradation but no significant indication of tissue or cell damage. This picture differs considerably from transfusion-dependent thalassemia major patients: highly significant decrease in antioxidants and thiols and tremendous iron overload and cell damage. The picture is even worsened in long-term transfused patients. Iron chelation after transfusion is not sufficient in Indonesia, because it is normally (with few exceptions) applied only once together with transfusion. Hence, one major reason of the bad condition of transfusion-dependent thalassemia patients in Indonesia appears to be frequent transfusions (on the average one per month) and insufficient chelation of one treatment per month together with transfusion.

Multiple common variants in transmembrane protease serine 6 (TMPRSS6) were associated with the plasma iron concentration in genome-wide association studies, but their effect in young children where anemia and iron deficiency (ID) were prevalent has not been reported, particularly taking account of iron intake. This study aims to investigate whether TMPRSS6 SNPs (rs855791 and rs4820268) and iron intake are associated with a low iron and hemoglobin concentration in under-two-year-old children. The study analyzed the baseline of a randomized trial (NUPICO, ClinicalTrials.gov NCT01504633) in East Lombok, Indonesia. Children aged 6–17 months (n = 121) were included in this study. The multiple linear regressions showed that TMPRSS6 decreased serum ferritin (SF) by 4.50 g/L per copy minor allele (A) of rs855791 (p = 0.08) and by 5.00 μg/L per copy minor allele (G) of rs4820268 (p = 0.044). There were no associations between rs855791 and rs4820268 with soluble transferrin receptor (sTfR) and hemoglobin (Hb) concentration (rs855791; p = 0.38 and p = 0.13, rs4820268; p = 0.17 and p = 0.33). The finding suggests the need for further studies to explore whether the nutrient recommendation for iron should be based on genetic characteristics, particularly for children who have mutation in TMPRSS6.

Erythrocyte membrane structural parameters were studied in transfusion-dependent beta-thalassemia patients, in long-term transfused patients (regularly transfused < 15 years), and in those who had not yet obtained transfusions. Controls were voluntary students up to 30 years of age without diagnosis or clinical signs of thalassemia. Membranes were isolated and investigated by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electron paramagnetic resonance (EPR) spectroscopy. Data obtained from the thiol-reactive spin label N-ethyl-maleimidoproxyl reveal immobilization of protein environment in erythrocyte membranes from thalassemic patients. SDS-PAGE shows both degradation and aggregation of membrane proteins. Thalassemic erythrocyte membranes exert higher order parameters in the hydrophobic region as determined by 16-doxyl-stearic acid. Rotational correlation times of this spin label increase only in transfused patients. Polarity is higher in membranes of all patients than in controls. In the polar interface, order parameters obtained from 5-doxyl-stearic acid increase in non-transfused and decrease in transfusion-dependent patients as compared with controls. Transfused patients exert increasing membrane order in the hydrophobic region and counter-currently decreasing order in the polar interface indicating loss of membrane integrity along with the loss of fluidity and polarity gradients and the loss the energetic barrier function of the membrane.

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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 β(T)mRNA, was detected in the sequencing analysis of the whole mRNA (cDNA). However, the β(T)mRNA 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 β(T)mRNA. Thus, their more severe phenotype was not caused by the "dominant type" β-thal in which a considerable amount of the β(T)mRNA 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.

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.

Mitochondrial gene mutation plays a role in the development of type two diabetes mellitus (T2DM). A point mutation in the mitochondrial gene Nicotinamide adenine dinucleotide dehydrogenase 1 (mtDNA ND1) gene mainly reported as the most common mutation related to T2DM. However, several studies have identified another SNP (singlenucleotide polymorphisms) in the RNA region of mtDNA from patients from specific ethnic populations in Indonesia. Building on those findings, this study aimed to use PCR and DNA sequencing technology to identify nucleotides in RNA and ND1 fragment from 20 Gorontalonese and 20 Javanese T2DM patients, that may trigger T2DM expression. The results showed successful amplification of RNA along 294 bp for all samples. From these samples, we found two types of point mutation in Javanese patients in the G3316A and T3200C points of the rRNA and ND1 gene. In samples taken from Gorontalonese patients, no mutation were found in the RNA or ND1 region. We conclude that T2DM was triggered differently in our two populations. While genetic mutation is implicated for the 20 Javanese patients, T2DM pathogenesis in the Gorontalonese patients must be traced to other genetic, environmental, or behavioral factors.

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.