Mutation Spectrum of ß-Globin Gene in Patients with ß-Thalassemia at Tidar Hospital, Magelang, Central Java, Indonesia.

ß-Thalassemia is genetic disorder characterized by ß-globin chain deficiency resulting from mutations in the ß-globin coding gene. Both the quantity and quality of blood produced will be impacted by this condition. The distribution of mutation causing thalassemia is vary across ethnic and different regions in Indonesia. This study aims to identify the variant mutation in patients with ß-thalassemia at Tidar Hospital as representative samples of Javanese population, the largest ethnicity in Indonesia. Sixty-one blood samples were obtained from blood transfusion-dependent patients with ß-thalassemia. Mutation was identified using ARMS and RFLP PCR-based methods, and inconclusive samples were subjected to DNA sequencing. Results showed that the mutation variants were Cd 26/IVSI-5 (G > C) 47.54%, Cd 26/Cd 35 16.30%, Cd 26/IVSI-1 (G > T) 11.47%, Cd 26/IVSI-2 4.91%, IVSI-5 (G > C)/Cd 40 3.27%; 1.63%; IVSI-5 (G > C)/IVSI-1 (G > A) 1.63%; IVSI-5 (G > C)/Cap + 1 1.63%; Cd 26/Cd 15 1.63%; Cd 26/Cd 30 1.63%. We also found three homozygous of IVSI-1 (G > T), IVSI-5 (G > C) 6.55%, and Cd 35 1.63%. The most prevalent alleles would be recommended to be used as part of screening for ß-thalassemia in the Javanese ethnicity in Central Java, especially for families affected by thalassemia.

Phylogenetic and genetic variation analysis of lesser short-nosed fruit bat Cynopterus brachyotis (Müller 1838) on Java island, Indonesia, inferred from mitochondrial D-loop.

BACKGROUND: Cynopterus brachyotis (Müller 1838) is a generalist and widespread fruit bat species which inhabits different types of habitats in Southeast Asia. This species plays an essential role as a seed disperser and pollinator. Morphological study and phylogenetic analysis using mtDNA markers (cyt-b and D-loop) revealed that this species had two different forms in peninsular Malaysia and Borneo and six lineages in Southeast Asia that lead to new species formation. In addition, this species is also reported to have high genetic diversity in Malaysia and Thailand based on the D-loop sequence. However, a phylogenetic and genetic variation study of C. brachyotis in Indonesia has not been conducted yet. These two studies are important as additional information for taxonomic and population genetic studies of this species. Thus, we performed the phylogenetic and genetic diversity analysis of the C. brachyotis population collected from seven habitats on Java island, including open-fragmented habitats (urban, coffee and rubber plantations, pine forest, secondary forest, mangrove forest) and closed habitats (natural forest) using the mtDNA D-loop marker. RESULTS: The phylogenetic tree using the Bayesian inference (BI) and genetic distance using the Kimura-2 parameter (K-2P) demonstrated that 33 individuals of C. brachyotis from seven habitats on Java island overlapped between habitats and could not be distinguished according to their habitats and lineage. Intrapopulation and intraspecies analysis revealed high haplotype diversity of this species on Java island (Hd = 0.933-1.000). The haplotype network was split into two haplogroups, showing haplotype sharing between habitats. These phylogenetic and genetic variations analysis of C. brachyotis bats on Java island indicated that this species is widespread and adapt to different habitats. CONCLUSIONS: This study of C. brachyotis on Java island collected from seven different habitats has overlapped and genetically close and has high genetic variation. Our results provide the first reported study of C. brachyotis on Java island and provide data to understand the phylogenetic and genetic diversity of this species in Indonesia.

The Distribution of M2 Macrophage and Treg in Nasopharyngeal Carcinoma Tumor Tissue and the Correlation with TNM Status and Clinical Stage.

OBJECTIVE: This study aimed to identify the distribution of M2 macrophage and Treg in Nasopharyngeal Carcinoma (NPC) tumor tissue samples. The presence of these two groups of cells was further correlated to clinical stage, tumor size, the lymphatic node involvement, and metastasis. METHODS: The total of 50 formalin-fixed paraffin-embedded (FFPE) NPC tissue samples was collected retrospectively (27 samples) and prospectively (23 samples). Samples were FFPE tissue slices. Immunohistochemistry was done on the FFPE tissue slides using anti-CD-163 and anti-FoxP-3 antibodies for M2 macrophage and Treg detection, respectively. The M2 macrophage interpretation was performed by eye-balling method and the score was divided into 0 (negative), 1 (scant), 2 (focal), and 3 (abundant). The average number of Treg FOXP3+ cells in 5 high power fields (HPF) was calculated. The relationship of M2 macrophage and Treg was tested with Spearman's correlation. The relationship between M2 macrophage and Treg with clinical stage, tumor size, node involvement and metastasis was tested by chi square, with p<0.1. RESULTS: M2 macrophage and Treg were positive correlated (r=0.469, p<0.001). The presence of M2 macrophage and regulatory T cell (Treg) was significantly correlated to tumor size (p= 0.091 for M2 macrophage and p=0.022 for Treg) and clinical stage (p= 0.030 for M2 macrophage and p= 0.002 for Treg), but did not correlate with lymphatic node involvement and metastasis. CONCLUSIONS: In Epstein-Barr virus related NPC tumor microenvironment, the presence of M2 macrophage was correlated with Treg, and both types of the cells were correlated with tumor size and clinical stages.

Molecular and Haematological Characteristics of alpha-Thalassemia Deletions in Yogyakarta Special Region, Indonesia.

BACKGROUND: alpha-Thalassemia is caused primarily by deletions of one to two alpha-globin genes and is characterized by absent or deficient production of alpha-globin protein. The South-East Asia (SEA) deletion, 3.7-kb and 4.2-kb deletions are the most common causes. The present study aimed to observe the molecular characteristics of this common alpha-Thalassemia deletions and analyse its haematological parameter. METHODS: Blood samples from 173 healthy volunteers from thalassemia carrier screening in Yogyakarta Special Region were used. Haematological parameters were analysed and used to predict the carrier subjects. Genotype of suspected carriers was determined using multiplex gap-polymerase chain reaction and its haematological parameters were compared. The boundary site of each deletion was determined by analysing the DNA sequences. RESULTS: Seventeen (9.8%) of the volunteers were confirmed to have alpha-Thalassemia trait. Of these, four genotypes were identified namely -α3.7/αα (58.8%), -α4.2/αα (5.9%), -α3.7/-α4.2 (5.9%) and - -SEA/αα (29.4%). The 5' and 3' breakpoints of SEA deletion were located at nt165396 and nt184700 of chromosome 16, respectively. The breakpoint regions of 3.7-kb deletion were 176-bp long, whereas for 4.2-kb deletion were 321-bp long. The haematological comparison between normal and those with alpha-Thalassemia trait genotype indicated a significant difference in mean corpuscular volume (MCV) (p< 0.001) and mean corpuscular haemoglobin (MCH) (p< 0.001). As for identifying the number of defective genes, MCH parameter was more reliable (p= 0.003). CONCLUSION: The resultant molecular and haematological features provide insight and direction for future thalassemia screening program in the region.

Detection of HBB:c.92+5G>C and HBB:c.108delC mutations in ß-thalassemia carriers using high-resolution melting analysis.

The HBB:c.92+5G>C [known as IVSI-5 (GC)] and HBB:c.108delC [known as Cd 35 (del C)] are the ß-globin gene mutations that commonly found in Indonesia. Detection of the mutation in the ß-thalassemia carriers can be useful to prevent an increase in the number of ß-thalassemia patients. High-Resolution Melting Analysis (HRMA) is the method that can detect the mutation rapidly. The aim of this study was to detect HBB:c.92+5G>C and HBB:c.108delC mutations of the ß-thalassemia carriers using HRMA. DNA was isolated from blood archive of ten hematologically proved ß-thalassemia carriers. Detection of mutations was carried out by HRMA and then confirmed by sequencing. HRMA was performed by using two pairs of specific primers. One pair of primer targeted the region of HBB:c.92+5G>C and the other targeted the region of HBB:c.108delC. The results of detection of mutation using HRMA then were confirmed by sequencing. A specific primer pair covering the region of HBB:c.92+5G>C to HBB:c.108delC were used for sequencing. The results of HRMA showed that the HBB:c.92+5G>C and HBB:c.108delC mutations found in 50% and 30% samples, respectively. The HRMA results can be confirmed by sequencing in all samples. It can be concluded that HRMA can be used to detect HBB:c.92+5G>C heterozygote and HBB:c.108delC heterozygote mutations.

Comparison Between Three Molecular Diagnostics for the Identification of Heterozygous Hemoglobin E.

BACKGROUND AND OBJECTIVES: Hemoglobin E is a variant hemoglobin caused due to the base substitution G→A at codon 26 in the ß-globin-coding gene that is followed by the alteration of glutamic acid (GAG) to lysine (AAG). Various types of molecular analysis methods such as tetra-primer amplification refractory mutation system (T-ARMS-PCR), Tm-shift real-time polymerase chain reaction (Tm-shift qPCR) and high-resolution melting analysis (HRMA) are commonly used to detect several mutations in the ß-globin-coding gene. This study was conducted to compare the detection result of Cd 26 (G→A) mutation in the ß-globin-coding gene of heterozygous HbE between the above-mentioned methods. MATERIALS AND METHODS: DNA samples were isolated from blood archive of heterozygous HbE and analyzed for the detection of the mutation using HRMA and Tm-shift on a real-time PCR instrument, whereas T-ARMS analysis was performed on a conventional PCR equipment. High resolution melt v3.1 software and Bio-Rad CFX Manager software were used to analyze the result of HRMA and Tm-shift qPCR, whereas the T-ARMS-PCR result was analyzed by observing the number and size of DNA bands on gel electrophoresis. RESULTS: Among 21 samples, the Cd 26 mutation was detected in numbers 18, 19 and 21 by HRMA, Tm-shift qPCR and T-ARMS-PCR. DNA Sequencing confirmed Cd 26 mutation on 5 ambiguous samples and revealed two homozygous mutation. CONCLUSION: The Cd 26 (G→A) mutation was detected in proportions 100, 91 and 86% by T-ARMS-PCR, Tm-shift qPCR and HRMA, respectively.

The snapping shrimp dactyl plunger: a thermomechanical damage-tolerant sandwich composite.

The dactyl plunger of Alpheus sp. was found to be a layered composite, with mineral-rich outer and inner layers and a chitin-rich middle layer of high porosity. The chitin-rich middle layer is itself composed of several porous chitin laminae. Modelling heat conduction through the plunger cross-section revealed that the chitin-rich layer is able to insulate heat and retard its progress through the material. Heat accumulates in the plunger after a series of successive snaps and as such, its thermally resistant design can be considered most useful under the conditions of successive snapping. The plunger has a concurrent mechanical damage-tolerant design with biogenic mineral layers, viscous (chitin-mineral) interfaces, energy-dissipating porous chitin, and sidewalls composed of ordered, layered aragonite. The snapping shrimp plunger has a design that may protect it and internal soft tissues from thermomechanical damage during plunger-socket compression prior to cavitation bubble release.

α-globin Alteration in α-thalassemia Disorder: Prediction and Interaction Defect.

BACKGROUND AND OBJECTIVE: The α-thalassemia is an inherited blood disorder affecting quality and quantity of hemoglobin. It caused mostly by deletion of one or two α-globin genes and characterized by deficient production of α-globin chain in hemoglobin leading from mild anemia to lethal. The α-globin gene with partial deletion could reduce chain production or produce abnormal chain. Its effect depends on mechanism of chain production affected. This study aimed to analyze the effect of partial deletion in α-globin gene influencing the mechanisms to produce functional α-globin chain in α-thalassemia cases. MATERIALS AND METHOD: The three mutant genes from genebank were selected and processed. The analysis performed in deleted sequences determination, mRNA sequences, protein structures and protein chains interaction to form hemoglobin by SWISS MODEL, CHIMERA and SABLE Polyview 2D. RESULTS: The result showed 76 amino acids deleted in one mutant α-globin gene (V00516.1). The mutation gave effect in every mechanism of the α-globin chain conformation and production. It affected protein conformation by losing over half the helical chains. It reduced the function completely, in which, disturb hemoglobin A (HbA) production with emergence of ß-sheets conformation. CONCLUSION: The analysis concluded that the protein produced by the α-globin gene with partial deletion lost its function and unable to form hemoglobin.

Study of Genetic Marker of Cuscuses (Marsupialia: Phalangeridae) from Maluku and Papua Based on Cytochrome b Gene Sequences.

Cuscuses is marsupials animal (Phalangeridae) which has limited spread in eastern Indonesia (Sulawesi, Maluku, Papua and Timor islands), Australia and Papua New Guinea. The ex-situ and in-situ conservation of cuscuses under captivating condition is an alternative solution to protect from extinction. This study aimed to determine nucleotide sequences and genetic marker on cyt b gene with sequencing method of each species on two provinces. Whole genome DNA was extracted from 22 samples of cuscuses obtained from different habitats, Maluku (13 individuals) and Papua (8 individuals) according to the protocol of Qiamp DNA Blood Mini Kit (Qiagen) and then it was used as template for amplification of cyt b gene by using PCR method. The PCR product were then purified using column chromatography and were used as template for sequencing reaction. Results sequencing of cyt b gene were analyzed using MEGA program versions 6.0. The PCR product gives results nucleotides of 982 bp according to database GeneBank and sequencing product gives results nucleotides of 771 bp. Nucleotides alignment of Phalanger members was found 24 nucleotides distinguishing and Spilocuscus members was found 11 nucleotides distinguishing, which can be used as genetic marker between Phalanger and Spilocuscus members from Papua and Maluku.