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 A2 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 A2 was quantified, and proportions ≥3.5% were further assessed for beta thalassemia mutation using HRM analysis. Twelve types of beta thalassemia mutations, including hemoglobin E, were identified. There were 23 couples who were detected as at-risk. All at-risk couples were identified within 7 working days after sample receipt. Prenatal diagnosis revealed 6 affected fetuses. One fetus was homozygous CD17 (AT), and five fetuses exhibited beta0 – thalassemia/ hemoglobin E disease. These results were consistent with the outcomes calculated using the Hardy-Weinberg equation. Antenatal blood tests for mutation detection using high-resolution DNA melting analysis after hemoglobin A2 estimation is a feasible laboratory method for the recruitment of couples with a fetus that is at risk for beta thalassemia. This modified strategy is cost-effective and may be beneficial for use in a beta thalassemia prevention program

DNA methylation profiles of plasma DNA between pregnant and non-pregnant women were compared at three levels.

<p>There are (A) overall, (B) individual CpG sites, and (C) DNA methylation patterns. Grey and white boxes in the boxplot represent the pregnant and non-pregnant groups, respectively. P and N represents the group of pregnant and non-pregnant women, respectively. 0 and 1 represent unmethylated and methylated status for CpG sites, respectively.</p

Methylation-sensitive high resolution melting profiles of the <i>HBAP1</i> gene between the placenta and the plasma of non-pregnant women are different.

<p>Green and red lines represent the placenta and non-pregnant plasma, respectively.</p

Decreased DNA methylation of a CpG site in the <i>HBAP1</i> gene in plasma DNA from pregnant women

<div><p>Objective</p><p>The objective of this study is to identify potential CpG site(s) or DNA methylation pattern(s) in the pseudo α-globin 1 gene (<i>HBAP1</i> gene), the gene which locates in α-thalassemia-1 deletion mutation, to differentiate plasma DNA between pregnant and non-pregnant women.</p><p>Method</p><p>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. The differential region was used to compare DNA methylation profile of plasma DNA between pregnant and non-pregnant women by bisulfite amplicon sequencing in three levels: overall, individual CpG sites and individual molecules (DNA methylation patterns).</p><p>Result</p><p>Using MethBase data, four consecutive CpG sites in the <i>HBAP1</i> gene were identified as regions of differential DNA methylation between placenta and peripheral blood. The confirmation by MS-HRM showed the differential DNA methylation profile between the placenta and plasma from non-pregnant women. The comparison of DNA methylation profiles between the plasma of pregnant and non-pregnant women showed that, in the overall levels of the four CpG sites, DNA methylation of pregnant women was detected at lower levels than non-pregnant women. In the individual CpG site level, only the second CpG site showed differential DNA methylation between the groups. In the DNA methylation pattern level, there was no strongly significant differences in DNA methylation patterns between the pregnant and non-pregnant groups.</p><p>Conclusion</p><p>Our result demonstrated that, in the plasma from pregnant women, only one of the four CpG sites displays a decrease in DNA methylation compared with non-pregnant women. It indicates that this CpG site might be useful for determining the presence or absence of fetal wild-type α-globin gene cluster allele in maternal plasma.</p></div

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. The method could be successfully employed for NIPST starting with the 7th week of gestation. The results showed that 19 pregnant women were negative for PIBTM (53%). After an on-track and on-time of one year, including postnatal thalassemia blood tests, none of the babies showed symptoms or signs of beta thalassemia disease. We concluded that the modified ARMS RT-PCR method was an accurate, cost-effective and feasible method for use as a NIPST for at-risk couples with the potential of having a baby with CHBT

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. Methods: Pregnant women and their spouses who are at risk of bearing an offspring with β-thalassaemia disease from common MIB mutations (CD 41/42-TCTT, CD17A>T, IVS1-1G>T and CD26G>A) were enrolled. The ddPCR assay sets were constructed for each of the four mutations. All cell-free DNA samples were first screened for the paternally inherited β-thalassaemia (PIB) mutation. The PIB-negative samples were considered as non-disease and were not further analyzed. For PIB-positive samples, DNA fragments of 50-300 base pairs in size were isolated and purified, and further analyzed for MIB mutation. The allelic ratio between the mutant and the wild-type was used to determine the presence of MIB in cffDNA. All cases underwent a prenatal diagnosis by amniocentesis for a definite diagnosis. Results: Forty two couples at risk were enrolled. Twenty two samples were positive for PIBs. Among these 22 samples, there were 10 cases with allelic ratio >1.0 (MIB positive). All foetuses with over-represented mutant alleles were further diagnosed with β-thalassaemia disease; eight with compound heterozygous and two with homozygous mutations. The 20 PIB-negative and 12 MIB-negative foetuses were non-affected. Interpretation & Conclusions: The results of this study suggest that NIPT utilizing the ddPCR assay can be effectively used for the screening and diagnosis of foetal β-thalassaemia in at risk pregnancies

Fast-track strategy for the prevention of Hb Bart’s hydrops fetalis syndrome

We propose a fast-track strategy [direct blood DNA analysis using a quantitative real-time polymerase chain reaction (PCR) technique] for the early risk detection and prenatal diagnosis of α(0)-thalassemia (SEA and Thai deletion). Blood DNA samples were obtained from a volunteer group of 1235 ANC couples. They were assessed using quantitative real-time PCR to detect carriers of α(0)-thalassemia (SEA and Thai deletion). At-risk couples were identified, and further prenatal diagnosis by amniocentesis was implemented. Fetal DNA was isolated from the amniotic cells and characterized by quantitative real-time PCR to detect the α(0)-thalassemia mutation, which was reconfirmed using the droplet digital PCR method. Fifteen at-risk couples were identified. The timing of prenatal diagnosis was appropriate for all couples and four of the fetuses were diagnosed with Bart’s hydrops fetalis. The results were compatible with those calculated using the Hardy-Weinberg equation for a recessively inherited single gene disorder. The conclusion was that the fast-track strategy could shorten screening policy timelines, promoting early risk detection for couples and early prenatal diagnosis. The fast-track strategy might be beneficial for the prevention of hemoglobin Bart’s hydrops fetalis syndrome.   针对 α(0) 地中海贫血（东南亚和泰国类型的贫血）的早期风险检测和产前诊断，我们提出了一种快速跟踪方法：使用定量实时聚合酶链反应 (PCR)，进行直接血液 DNA 分析。血液 DNA 样本取自 1,235 对 ANC 夫妇的志愿者组。使用定量实时 PCR 对其进行评估，以检测 α(0) 地中海贫血（东南亚和泰国类型的贫血）的载体。确定危险群夫妇之后，实施羊膜穿刺术，进行进一步的产前诊断。从羊膜细胞中分离出胎儿 DNA，使用定量实时 PCR 进行特征标度，以检测 α(0) 地中海贫血突变，然后使用液滴数字 PCR 法再次确认。共确定出十五对危险群夫妇。产前诊断这一时机适用于所有夫妇，其中四例胎儿确诊为巴特水肿胎儿。我们使用 Hardy-Weinberg 方程式来计算隐性遗传单基因疾病，结果一致。结论是，此快速跟踪方法可以缩短筛选策略时间表、方便夫妇进行早期风险检测、便于早期产前诊断。此快速跟踪方法或有利于预防血红蛋白巴特水肿胎儿综合征

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. Methylation-sensitive high-resolution analysis was used to compare DNA methylation among placenta, leukocytes, and unmethylated control DNA. The result indicates that the DNA methylation between placenta and leukocyte DNA is different and shows that the CpG status of both is not fully unmethylated. Mapping of individual CpG sites was performed by targeted bisulfite sequencing. The DNA methylation level of the 10 consecutive CpG sites was different between placenta and leukocyte DNA. When the 10th CpG of the mutation allele was considered as a hallmark for comparing DNA methylation level, it was totally different from the unmethylated 10th CpG of the wild-type allele. Finally, the distinct DNA methylation patterns between both DNA were extracted. In total, 24 patterns were found in leukocyte samples and 9 patterns were found in placenta samples. This report shows that the large deletion is associated with DNA methylation change. In further studies for clinical application, the distinct DNA methylation pattern might be a potential marker for detecting cell-free fetal DNA

Human Oncogenic Epstein–Barr Virus in Water and Human Blood Infection of Communities in Phayao Province, Thailand

Water can contain pathogenic viruses. Many studies on RNA virus sources have shown that water can transmit them. However, there are few reports on pathogenic DNA virus transmission through water, such as adenovirus, which pose a widespread public health risk. Therefore, this study aimed to show waterborne viral transmission by detecting viruses in pooled human whole blood samples, tap water, and natural water from Mueang District, Phayao Province, Thailand, using a metagenomic approach. Viral prevalence in whole blood samples was measured by polymerase chain reaction (PCR) and quantitative PCR (qPCR), and environmental factors that affect viral infection were assessed. Metagenomics results showed that Epstein–Barr virus (EBV) members were among the prominent cancer-associated oncogenic DNA viruses detected in human blood and all water types similar to the EBV reference sequence (NC_007605). There were 59 out of 813 (7.26%) human whole blood samples that were positive for EBV DNA based on PCR and qPCR for the EBNA-1 and EBNA-2 genes. Water- and blood-borne human oncogenic EBV should be a concern in tap water treatment and blood transfusion in patients, respectively. Therefore, the detection of EBV in water suggests that transmission via water is possible and should be investigated further