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

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. Results: Our aim was to search for genetic footprints of Myanmar’s geographic, historic and sociocultural characteristics and to contribute to the picture of human colonization by describing and dating of new mitochondrial DNA (mtDNA) haplogroups. Therefore, we sequenced the mtDNA control region of 327 unrelated donors and the complete mitochondrial genome of 44 selected individuals according to highest quality standards. Conclusion: Phylogenetic analyses of the entire mtDNA genomes uncovered eight new haplogroups and three unclassified basal M-lineages. The multi-ethnic population and the complex history of Myanmar were reflected in its mtDNA heterogeneity. Population genetic analyses of Burmese control region sequences combined with population data from neighboring countries revealed that the Myanmar haplogroup distribution showed a typical Southeast Asian pattern, but also Northeast Asian and Indian influences. The population structure of the extraordinarily diverse Bamar differed from that of the Karen people who displayed signs of genetic isolation. Migration analyses indicated a considerable genetic exchange with an overall positive migration balance from Myanmar to neighboring countries. Age estimates of the newly described haplogroups point to the existence of evolutionary windows where climatic and cultural changes gave rise to mitochondrial haplogroup diversification in Asia

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

<p>Abstract</p> <p>Background</p> <p>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. For <it>a posteriori </it>data quality control, quasi-median network constructions and autosomal STR typing were performed. In order to describe the mtDNA setup of Laos more thoroughly, the data were subjected to population genetic comparisons with 16 East Asian groups.</p> <p>Results</p> <p>The Laos sample exhibited ample mtDNA diversity, reflecting the huge number of ethnic groups listed. We found several new, so far undescribed mtDNA lineages in this dataset and surrounding populations. The Laos population was characteristic in terms of haplotype composition and genetic structure, however, genetic comparisons with other Southeast Asian populations revealed limited, but significant genetic differentiation. Notable differences in the maternal relationship to the major indigenous Southeast Asian ethnolinguistic groups were detected.</p> <p>Conclusions</p> <p>In this study, we portray the great mtDNA variety of Laos for the first time. Our findings will contribute to clarify the migration history of the region. They encourage setting up regional and subpopulation databases, especially for forensic applications. The Laotian sequences will be incorporated into the collaborative EMPOP mtDNA database <url>http://www.empop.org</url> upon publication and will be available as the first mtDNA reference data for this country.</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

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

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

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