Non-invasive prenatal screening & diagnosis of β-thalassaemia in an affected foetus.

Background & Objectives: Non-invasive prenatal testing (NIPT) of maternally inherited alleles of β-thalassaemia (MIB) remains to be a challenge. Furthermore, current techniques are not available for use as routine tests. NIPT for β-thalassaemia disease was developed by using a specific droplet digital polymerase chain reaction (ddPCR) assay to analyze the cell-free foetal DNA (cffDNA) derived from maternal plasma.

Noninvasive Prenatal Diagnosis of Beta-Thalassemia Disease by Using Digital PCR Analysis of Cell-Free Fetal DNA in Maternal Plasma.

Introduction: Prenatal diagnosis of thalassemia disease was usually based on invasive technique. Noninvasive diagnosis using cell-free fetal DNA (cff-DNA) was described with various laboratory techniques. The aim of this study was to identify the performance of dPCR for analyzing cff-DNA in maternal plasma to diagnose fetal beta-thalassemia diseases.

An in-depth analysis of the mitochondrial phylogenetic landscape of Cambodia.

Cambodia harbours a variety of human aboriginal populations that have scarcely been studied in terms of genetic diversity of entire mitochondrial genomes. Here we present the matrilineal gene pool of 299 Cambodian refugees from three different ethnic groups (Cham, Khmer, and Khmer Loeu) deriving from 16 Cambodian districts. After establishing a DNA-saving high-throughput strategy for mitochondrial whole-genome Sanger sequencing, a HaploGrep based workflow was used for quality control, haplogroup classification and phylogenetic reconstruction.

Noninvasive prenatal screening test for compound heterozygous beta thalassemia using an amplification refractory mutation system real-time polymerase chain reaction technique.

We propose using a modified amplification refractory mutation system real-time polymerase chain reaction (ARMS RTPCR) technique to exclude the invasive prenatal diagnosis for a non-paternally inherited beta thalassemia mutation in couples atrisk for having a baby with CHBT. The ARMS RT-PCR method was performed for 36 at-risk couples by using isolated fetal cell-free DNA from maternal plasma. The modified ARMS RT-PCR primers targeted one of the following paternally inherited beta thalassemia mutation: -28 A→G, CD17 A→T, CD 26 G→A, IVS1-1 G→T and CD 41-42 -CTTT.

Molecular characteristics of thalassemia and hemoglobin variants in prenatal diagnosis program in northern Thailand.

Molecular analysis of globin genes is an essential process for prenatal diagnosis (PND) of severe thalassemia. This study aimed to describe the molecular characteristics of thalassemia and hemoglobin (Hb) variants in PND program in northern Thailand. The type and frequency of globin gene mutations from 1290 couples at risk of fetal severe thalassemia diseases that were tested at Thalassemia Laboratory at Chiang Mai University from 2012 to 2017 were retrospectively reviewed.

The shortcut strategy for beta thalassemia prevention.

We propose antenatal blood tests using high-resolution DNA melting (HRM) analysis for beta thalassemia mutation detection after hemoglobin A estimation as a modified strategy for the identification of beta thalassemia at-risk couples. Antenatal blood samples of 1,115 couples were transferred from the antenatal care clinic. Hemoglobin A was quantified, and proportions ≥3.

Decreased DNA methylation of a CpG site in the HBAP1 gene in plasma DNA from pregnant women.

Objective: The objective of this study is to identify potential CpG site(s) or DNA methylation pattern(s) in the pseudo α-globin 1 gene (HBAP1 gene), the gene which locates in α-thalassemia-1 deletion mutation, to differentiate plasma DNA between pregnant and non-pregnant women.

Method: DNA methylation profiles of placenta and peripheral blood from the MethBase database were compared to screen differentially methylated regions. This region was confirmed the differential by methylation-sensitive high resolution melt (MS-HRM) analysis.

Association of Tissue-Specific DNA Methylation Alterations with α-Thalassemia Southeast Asian Deletion.

In the wild-type allele, DNA methylation levels of 10 consecutive CpG sites adjacent to the upstream 5'-breakpoint of α-thalassemia Southeast Asian (SEA) deletion are not different between placenta and leukocytes. However, no previous study has reported the map of DNA methylation in the SEA allele. This report aims to show that the SEA mutation is associated with DNA methylation changes, resulting in differential methylation between placenta and leukocytes.

High-resolution melting analysis for prenatal diagnosis of beta-thalassemia in northern Thailand.

High-resolution melting (HRM) analysis is a rapid mutation analysis which assesses the pattern of reduction of fluorescence signal after subjecting the amplified PCR product with saturated fluorescence dye to an increasing temperature. We used HRM analysis for prenatal diagnosis of beta-thalassemia disease in northern Thailand. Five PCR-HRM protocols were used to detect point mutations in five different segments of the beta-globin gene, and one protocol to detect the 3.

Q Sepharose micro-column chromatography: A simple screening method for identifying beta thalassemia traits and hemoglobin E carriers.

Objectives: For beta thalassemia control program in pregnancy, mass screening of the carrier state by determination of the hemoglobin (Hb) A and Hb E proportions and mutation analysis is a preferred method for making prenatal diagnoses. Q Sepharose micro-column chromatography, developed for the determination of Hb A and Hb E for screening purposes, was compared with high performance liquid chromatography (HPLC) to ascertain its relative accuracy and reliability.

Design And Methods: Results using Q Sepharose micro-column chromatography in 350 blood specimens, including 50 samples genetically proven to be beta thalassemia heterozygotes, were compared to HPLC for validation.

Iron overload in non-transfusion-dependent thalassemia: association with genotype and clinical risk factors.

In the present study, we sought to determine the prevalence of iron overload in patients with non-transfusion-dependent thalassemia (NTDT) and its association with genotype and other clinical risk factors, and to evaluate the correlation between serum ferritin (SF) and liver iron concentration (LIC). Myocardial and liver iron concentration was measured by MRI using a T2* gradient multi-echo sequence in NTDT patients, aged 10-50 years. Of 91 patients, 54 (59 %) had hepatic iron overload.

Hypercoagulable state as demonstrated by thromboelastometry in hemoglobin E/beta-thalassemia patients: Association with clinical severity and splenectomy status.

Introduction: Patients with hemoglobin E/beta-thalassemia disease (E/β) are at risk of thromboembolism. Rotational thromboelastometry (ROTEM®) can be used to determine a hypercoagulable state. The objective was to describe the hemostatic and thromboelastometric changes in pediatric patients with E/β with different clinical severity, in comparison with healthy children as controls.

DETECTION OF DELETION α(+)-THALASSEMIA MUTATION [-α (3.7), -α (4.2)] BY QUANTITATIVE PCR ASSAY.

In Thailand, Hb H (α0(-thal/α(+)-thal) disease is highly prevalent. We designed 3 primer sets (A, B and C) to detect -α (3.7) and -α (4.

Molecular Diagnosis of α⁰-Thalassemia Through Urine DNA: A Novel DNA Source to Facilitate Prevention Programs in Remote Geographical Areas.

We assessed whether urinary DNA sediment was a feasible sample type for the molecular diagnosis of α-thalassemia (α-thal) mutations. Urine samples (5-10 mL) were collected from 218 male and female volunteers. The cells were centrifuged, and DNA was isolated according to the protocol of a commercial DNA isolation kit.

Pulmonary hypertension in non-transfusion-dependent thalassemia: Correlation with clinical parameters, liver iron concentration, and non-transferrin-bound iron.

Background: Pulmonary hypertension is a major cardiac complication in non-transfusion-dependent thalassemia (NTDT). Several clinical and laboratory parameters, including iron overload, have been shown to have a positive correlation with the incidence of pulmonary hypertension. Non-transferrin-bound iron (NTBI) is a form of free-plasma iron that is a good indicator of iron overload.

The correlation of α-globin gene mutations and the XmnI polymorphism with clinical severity of Hb E/β-thalassemia.

Clinical severity assessment and molecular analysis of β-, α-globin genes and the -158 (C > T) XmnI polymorphism of the (G)γ-globin gene were performed in 80 pediatric patients with Hb E (HBB: c.79G > A)/β-thalassemia (β-thal) to investigate the effects of coinheritance of α-thalassemia (α-thal) and other molecular determinants on their clinical severity. The mean age was 9.

The associations of SEA-alpha thalassemia 1, XmnI-Ggamma polymorphism and beta-globin gene mutations with the clinical severity of beta-thalassemia syndrome in northern Thailand.

Background: At least three genetic factors including beta-thalassemia mutations, alpha-thalassemia, and XmnI-Ggamma polymorphism were shown to modify clinical symptoms in beta-thalassemia disease.

Objective: To determine associations of beta-thalassemia mutations, SEA-alpha thalassemia 1, and XmnI-Ggamma polymorphism, and clinical severity of beta-thalassemia in northern Thailand.

Material And Method: Thirty-two beta-thalassemia major and 28 beta-thalassemia intermedia attending the Thalassemia Clinic at Maharaj Nakorn Chiang Mai Hospital, Chiang Mai, Thailand were recruited The beta-globin gene mutations and SEA-alpha thalassemia 1 were determined by MS-PCR and Gap-PCR, respectively.

Prevalence and molecular characterization of glucose-6-phosphate dehydrogenase deficiency in northern Thailand.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is one of the most common inherited enzymopathies in endemic areas of malaria including Southeast Asia. The molecular features of G6PD deficiency are similar among Southeast Asian population, with differences in the type of the prominent variants in each region. This study determined the prevalence and molecular characteristics of G6PD deficiency in northern Thailand.

Large-scale mitochondrial DNA analysis in Southeast Asia reveals evolutionary effects of cultural isolation in the multi-ethnic population of Myanmar.

Background: Myanmar is the largest country in mainland Southeast Asia with a population of 55 million people subdivided into more than 100 ethnic groups. Ruled by changing kingdoms and dynasties and lying on the trade route between India and China, Myanmar was influenced by numerous cultures. Since its independence from British occupation, tensions between the ruling Bamar and ethnic minorities increased.

Genotyping of beta thalassemia trait by high-resolution DNA melting analysis.

Beta thalassemia is a common hereditary hemalogogical disease in Thailand, with a prevalence of 5-8%. In this study, we evaluated the high resolution DNA melting (HRM) assay to identify beta thalassemia mutation in samples from 143 carriers of the beta thalassemia traits in at risk couples. The DNA was isolated from venous blood samples and tested for mutation under a series of 5 PCR-HRM (A, B, C, D and E primers) protocols.

Detection of Hb H disease genotypes common in northern Thailand by quantitative real-time polymerase chain reaction and high resolution melting analyses.

We used quantitative real-time polymerase chain reaction (qPCR) and high resolution melting (HRM) analyses for the detection of the common α-thalassemia (α-thal) genotypes in 40 northern Thai Hb H (β4) patients. The α(0)-thal [- -(SEA) (Southeast Asian) deletion] was detected by a multiplex gap real-time PCR. To determine the α(+)-thal, three primer pairs were designed.

Preimplantation genetic diagnosis of alpha-thalassemia-SEA using novel multiplex fluorescent PCR.

Purpose: Preimplantation genetic diagnosis (PGD) is an alternative to prenatal diagnosis (PND) giving couples at risk a chance to start a pregnancy with a disease-free baby. This study aimed to develop a new PGD protocol for alpha-thalassemia(-SEA) mutation, the commonest Mendelian disorder.

Patients And Methods: Multiplex fluorescent PCR was employed for mutation, contamination and linkage analysis.

Southeast Asian diversity: first insights into the complex mtDNA structure of Laos.

Background: Vast migrations and subsequent assimilation processes have shaped the genetic composition of Southeast Asia, an area of close contact between several major ethnic groups. To better characterize the genetic variation of this region, we analyzed the entire mtDNA control region of 214 unrelated donors from Laos according to highest forensic quality standards. To detail the phylogeny, we inspected selected SNPs from the mtDNA coding region.

Analysis of real-time PCR cycle threshold of alpha-thalassemia-1 Southeast Asian type deletion using fetal cell-free DNA in maternal plasma for noninvasive prenatal diagnosis of Bart's hydrops fetalis.

Background: Noninvasive prenatal diagnosis based on detection of fetal cell-free DNA is hampered when mother and father are both carriers for the same autosomal recessive mutation.

Objective: To compare the diagnosis of Bart's hydrops fetalis using conventional Gap-PCR analysis of fetal cells/tissues with the measurement of quantitative difference (deltaCp) between alpha-thalassemia-1 SEA type deletion gene (C(T-mutant)) and wild type alpha-globin gene (C(T-wild type)) in plasma of pregnancies by using the Taqman real-time quantitative PCR.

Material And Method: Plasma DNA samples were collected from three groups of pregnancies whose fetuses have known thalasemia status (7 normal, 11 heterozygote alpha-thalassemia-1 SEA type deletion, and 7 Bart's hydrops fetalis).

Cord blood screening for alpha-thalassemia and hemoglobin variants by isoelectric focusing in northern Thai neonates: correlation with genotypes and hematologic parameters.

We describe the screening of newborns for thalassemia and Hb variants by using isoelectric focusing (IEF) in a population from northern Thailand where hemoglobinopathies are highly prevalent. The report focuses on findings of alpha-thalassemia, Hb E, and other hemoglobin variants, and their correlation with genotypes and hematologic parameters. Two-hundred and seven out of 566 newborns (36.