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Objective: To evaluate whether targeted sequencing and relative mutation dosage can be used to diagnose correctly inheritance of maternal β-thalassaemia mutations in cell-free DNA., Design: Feasibility study using samples collected in a prenatal clinic., Setting: South East Asia., Population: Couples where both partners were known to be carriers of one of four common β-thalassaemia mutations or an HbE mutation, and therefore at risk of carrying a fetus affected with β-thalassaemia., Methods: 49 samples previously identified as having inherited a paternal β-thalassaemia mutation were amplified using nested polymerase chain reaction (PCR), and then sequencing. Relative mutation dosage was used to classify the fetus as having inherited the wild-type or mutant maternal allele., Main Outcome Measures: Classification of the fetus as 'unaffected' (if the maternal wild-type allele was inherited) or 'affected' with β-thalassaemia (if the maternal mutant allele was inherited)., Results: A classification for inheritance of maternal allele was obtained in 48/49 samples (98.0%). A concordant call was made in 44/48 cases (91.7%): one false-positive and three false-negatives were obtained. Thus, we had an overall sensitivity of 87.5% [95% confidence interval (CI) 67.6-97.3%] and a specificity of 95.8% (95% CI 78.9-99.9%) for inheritance of maternal genotype., Conclusions: RMD for detection of inheritance of maternal β-thalassaemia mutations has potential for clinical use. Our sequential approach could be applied to other single-gene disorders., Tweetable Abstract: NIPT for β-thalassaemia achieved using nested-PCR followed by relative mutation dosage., (© 2017 Royal College of Obstetricians and Gynaecologists.)

Screening for α(0)-thalassemia is usually associated with a high false-positive rate, leading to an unnecessary PCR workload for accurate diagnosis. We have developed a modified Hb H inclusion test for use as a secondary screening. This test was performed on young red blood cell enriched fractions using dextran sedimentation. The study was performed in 100 subjects positive on initial screening. Confirmatory tests included Hb analysis and a multiplex PCR assay to identify α(0)-thalassemia deletions. A modified Hb H inclusion test was positive in 31 cases, 30 of whom were α(0)-thalassemia carriers (97%). The remaining case (3.0%) was homozygous for α(+)-thalassemia. The remaining 69 cases with a negative Hb H inclusion test included normal subjects, α(+)-thalassemia carriers and β-thalassemia carriers. Two of them (2/69, 3.0%) were found to be double heterozygotes for β(0)-thalassemia and α(0)-thalassemia. The overall sensitivity and specificity of the modified Hb H inclusion test for screening of α(0)-thalassemia were 94.0 and 99.0%, respectively. Therefore, we recommend the use of this test in combination with Hb analysis to exclude cases with αβ-thalassemia. This should lead to a significant reduction in the number of cases referred for PCR analysis of α(0)-thalassemia by about 50.0%., (© 2013 S. Karger AG, Basel.)

Objective: Current methods of prenatal diagnosis to detect beta-thalassemia are Sanger sequencing and reverse dot blot. These methods are time-consuming and can prolong assay turnaround time. We aim to develop a sensitive and rapid method to detect 27 beta-thalassemia mutations using pyrosequencing., Method: Pyrosequencing primer pairs and sequencing primers were designed to detect 27 most common beta-thalassemia mutations found in Singapore. Pyrosequencing was performed on 191 DNA samples with known beta-thalassemia mutations isolated from 143 peripheral blood and 48 prenatal samples (seven chorionic villus biopsies, 26 cultured amniocytes, 15 uncultured amniocytes). All mutations were validated with Sanger sequencing., Results: Pyrosequencing identified 210 alleles with beta-thalassemia mutations and 82 alleles without mutations with 100% sensitivity (lower 95% confidence interval [CI], 97.8%) and 100% specificity (lower 95% CI, 94.4%). All pyrosequences were concordant with Sanger-based sequences. Pyrosequencing was able to detect DNA concentrations as low as 2 ng, obviating the need for cell culture in volume-restricted samples. Sample receipt-to-report assay turnaround times were 16 to 18 h (Sanger sequencing) and 4 to 6 h (pyrosequencing)., Conclusion: Pyrosequencing is a rapid and sensitive method to detect common beta-thalassemia mutations without the need for cell culture, thus reducing the assay turnaround time., (© 2013 John Wiley & Sons, Ltd.)

In Southeast Asia α-thalassaemia, β-thalassaemia, haemoglobin (Hb) E and Hb Constant Spring (CS) are prevalent. The abnormal genes in different combinations lead to over 60 different thalassaemia syndromes, making Southeast Asia the locality with the most complex thalassaemia genotypes. The four major thalassaemic diseases are Hb Bart's hydrops fetalis (homozygous α-thalassaemia 1), homozygous β-thalassaemia, β-thalassaemia/Hb E and Hb H diseases. α-Thalassaemia, most often, occurs from gene deletions whereas point mutations and small deletions or insertions in the β-globin gene sequence are the major molecular defects responsible for most β-thalassaemias. Clinical manifestations of α-thalassaemia range from asymptomatic cases with normal findings to the totally lethal Hb Bart's hydrops fetalis syndrome. Homozygosity of β-thalassaemia results in a severe thalassaemic disease while the patients with compound heterozygosity, β-thalassaemia/Hb E, present variable severity of anaemia, and some can be as severe as homozygous β-thalassaemia. Concomitant inheritance of α-thalassaemia and increased production of Hb F are responsible for mild clinical phenotypes in some patients. However, there are still some unknown factors that can modulate disease severity in both α- and β-thalassaemias. Therefore, it is possible to set a strategy for prevention and control of thalassaemia, which includes population screening for heterozygotes, genetic counselling and foetal diagnosis with selective abortion of affected pregnancies.

Introduction: This study was conducted to examine ability of the Capillarys 2 haemoglobin (Hb) testing system to assist in presumptive diagnosis of common Hb variants found in Southeast Asia including five α-chain and nine β-chain variants., Methods: Blood samples with unknown Hb variants sent from other hospitals to our centre for identification were re-analysed using the Capillarys 2 Hb analyser (SEBIA). DNA analyses by allele specific PCR assays were used to establish the final diagnoses., Results: Five α-globin chain variants including Hbs Q-Thailand, Queens, Siam, Constant Spring and Paksé were detected. In heterozygous form, the machine demonstrated clearly two abnormal derivatives of Hbs A and A(2) for the former three variants. Small peaks of Hb Constant Spring and Hb Paksé were observed but could not be differentiated. In contrast, only one abnormal peak of Hb A was observed for β-globin chain variants including those with more negative charge (Hb J-Bangkok, Hb Hope and Hb Pyrgos) and less negative charge (Hb D-Punjab, Hb S, Hb Korle-Bu and Hb C). Hb Tak, an elongated β-chain variant was co-separated with Hb F whereas the Hb Malay co-migrated with Hb A in a subject with high Hb A(2) β- thalassaemia trait., Conclusion: The capillary electrophoresis system could clearly demonstrate the presence of abnormal Hbs and provide useful information for presumptive diagnoses in most cases. The Hb analysis results could help in selection of appropriate DNA testing for final diagnoses of these variants., (© 2011 Blackwell Publishing Ltd.)

Background: Prevention and control of thalassemia requires simple, rapid, and accurate screening tests for carrier couples who are at risk of conceiving fetuses with severe thalassemia., Methods: Single-tube multiplex real-time PCR with SYBR Green1 and high-resolution melting (HRM) analysis were used for the identification of α-thalassemia-1 Southeast Asian (SEA) and Thai type deletions and β-thalassemia 3.5-kb gene deletion. The results were compared with those obtained using conventional gap-PCR. DNA samples were derived from 28 normal individuals, 11 individuals with α-thalassemia-1 SEA type deletion, 2 with α-thalassemia-1 Thai type deletion, and 2 with heterozygous β-thalassemia 3.5-kb gene deletion., Results: HRM analysis indicated that the amplified fragments from α-thalassemia-1 SEA type deletion, α-thalassemia-1 Thai type deletion, β-thalassemia 3.5-kb gene deletion, and the wild-type β-globin gene had specific peak heights at mean melting temperature (T(m)) values of 86.89℃, 85.66℃, 77.24℃, and 74.92℃, respectively. The results obtained using single-tube multiplex real-time PCR with SYBR Green1 and HRM analysis showed 100% consistency with those obtained using conventional gap-PCR., Conclusions: Single-tube multiplex real-time PCR with SYBR Green1 and HRM analysis is a potential alternative for routine clinical screening of the common types of α- and β-thalassemia large gene deletions, since it is simple, cost-effective, and highly accurate.

Gel electrophoresis and ethidium bromide staining are routine methods in molecular laboratories. However, they are not ideally suited to large scale analyses in clinical laboratories. We used SYTO9 and high resolution melting (HRM) analyses for identification of the common beta(0)-thalassemia (beta(0)-thal) in Southeast Asia including the codons 17 (A>T), 41/42 (-TCTT) and 71/72 (+A) mutations. Multiplex amplification refractory mutation system-polymerase chain reaction (MARMS-PCR) was performed on 102 blood samples that had Hb A(2) levels between 3.1 and 9.9%. The SYTO9 HRM analysis showed specific characteristic peaks of each investigated type of beta(0)-thal mutation. Results of MARMS-PCR followed by SYTO9 HRM analyses were completely consistent with results of those analyzed by gel electrophoresis. Moreover, the beta(0)-thal 3.5 kb gene deletion could be identified in two samples with Hb A(2) levels of >4% by GREEN1 HRM analysis. The SYTO9 and SYBR GREEN1 HRM analysis is simple and rapid. This approach will facilitate laboratory diagnosis and genetic counseling for regions with a high prevalence of beta-thal.

Purpose: This study reviews Asian immigration in California and the effect it has had on public health in the state in terms of genetic disease detection. This is documented in terms of the numbers of cases of thalassemia detected, including Hemoglobin (Hb) E/beta-thalassemia, beta-thalassemia major, and Hb H disease., Patients and Methods: California has been screening all newborns for hemoglobinopathies since 1990 and tests approximately 530,000 newborns per year. Samples are collected on filter paper during the first I to 2 days of life and sent to one of eight contract laboratories. The screening methodology is cation exchange high-performance liquid chromatography. Confirmatory testing is performed at Children's Hospital Oakland hemoglobin laboratory using a variety of methods., Results: Approximately five to seven cases each of Hb E/beta-thalassemia and beta-thalassemia major are detected annually. Most cases are of Southeast Asian origin. Prevalence rate of Hb E/beta-thalassemia among Southeast Asians is approximately 1 in 2,200 births. A pilot program for Hb H disease screening was successful and this disorder has now been incorporated in newborn screening, detecting approximately 40 cases per year., Conclusions: Increases in Asian immigration and births in the U.S., particularly California, have been dramatic during the past 10 years and have led to detection of previously rare diseases like Hb E/beta-thalassemia. It has also changed the way other thalassemic disorders are viewed, such as Cooley anemia, which previously affected mainly individuals of Mediterranean origin. Now, most affected patients are of Asian origin.

Hemoglobin E beta-thalassemia is an important cause of childhood chronic disease in Southeast Asia. It is characterized by the presence of hemoglobin E and F, and the amount of hemoglobin E ranges from 35% to 75%. The patients are generally classified as having thalassemia intermedia because they have inherited a beta-thalassemia allele and hemoglobin E, which acts as a mild beta+-thalassemia. However, a remarkable variability in the clinical expression, ranging from a mild form of thalassemia intermedia to transfusion-dependent conditions, is observed. Severe hemoglobin E beta-thalassemia may have clinical features of thalassemia major. Phenotypes of thalassemia major can be predicted from the early onset of clinical symptoms and the requirement of regular blood transfusion from infancy for survival. Coinheritance of alpha-thalassemia alleviated the severity of beta-thalassemia disease in patients with at least one allele of mild beta-thalassemia genotype.

The provision of a prenatal diagnosis service for thalassaemia is becoming more demanding. In an ethnically-diverse community, the number of mutations has increased. Requests for prenatal testing continue to come at advanced stage in pregnancy, often without the underlying mutation having been identified. Although controls are included in PCR assays, errors can still occur. The alternative to DNA testing, i.e., an alpha/beta globin chain synthesis ratio on a fetal blood sample, is now less readily available. In the circumstances described, the laboratory must adopt a more efficient and reliable approach to DNA mutation analysis. With currently available technology, this improvement is more likely to come through increased automation. To achieve this aim, we have moved to capillary electrophoresis. With capillary electrophoresis we are able to use a PCR-based screening strategy which can detect up to 11 beta thalassaemia mutations. The actual prenatal test is undertaken using two independent PCRs thereby reducing the potential for error. Despite the advantages of PCR, approximately 12 per cent of beta thalassaemia and about nine per cent of alpha thalassaemia cases require further study in our experience. In this situation, capillary electrophoresis has again proven helpful since a DNA scanning approach, such as single strand conformation polymorphism, can be automated to identify the region of DNA to be sequenced.

The overall laboratory features of the common RBC disorders occurring in Southeast Asians is summarized in Table 4. These erythrocyte disorders will continue to be important public health issues, and it has been predicted that most new cases of thalassemia in the United States will occur in this population group. The fertility rate in Southeast Asian families is very high, with an average of more than five children delivered by each married woman. This number of children is consistent with perceptions of ideal family size, and, to date, no evidence suggests any change in the size of Southeast Asian families who now reside in the United States. Moreover, attitudes about health care, reasons why one seeks medical attention, and a variety of other cultural issues may impair the effectiveness of genetic counseling and other preventive measures designed to reduce the incidence of serious blood diseases. Genetic screening and prenatal diagnosis clearly have led to a markedly decreased incidence of homozygous thalassemia disorders in high-risk Mediterranean populations throughout the world. With further assimilation into Western culture, a similar disease may occur in the Southeast Asian population also.

During recents the number of immigrants to Norway from Africa and South East Asia has risen considerably. These persons come from countries with a high prevalence of haemoglobinopathies. Most of the immigrants with haemoglobinopathies are silent carriers, but some have a serious disease or have offspring with serious disease. This situation calls for increasing awareness and knowledge of haemoglobinopathies, especially the thalassemias and sickle cell trait. Genetic counselling is particularly important, since the majority of these immigrants marry within small ethnic groups. This paper gives a survey of the most frequent haemoglobinopathies likely to be encountered in Norway.