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. The A primers were for detection of beta thalassemia mutations in the HBB promoter region, the B primers for mutations in exon I, the C primers for exon II, the D primers for exon III and the E primers for the 3.4 kb deletion mutation. The mutations were diagnosed by comparing the complete melting curve profiles of a wild type control with those for each mutant sample. With the PCR-HRM technique, fourteen types of beta thalassemia mutations were detected. Each mutation had a unique and specific melting profile. The mutations included 36.4% (52 cases) codon 41/42-CTTT, 26.6% (38 cases) codon 17 A-T, 11.2% (16 cases) IVS1-1 G-T, 8.4% (12 cases) codon 71/72 +A, 8.4% (12 cases) of the 3.4 kb deletion and 3.5% (5 cases) -28 A-G. The remainder included one instance each of -87 C-A, -31 A-C, codon 27/28 +C, codon 30 G-A, IVS1-5 G-C, codon 35 C-A, codon 41-C and IVSII -654 C-T. Of the total cases, 85.8% of the mutations could be detected by primers B and C. The PCR-HRM method provides a rapid, simple and highly feasible strategy for mutation screening of beta thalassemia traits.

High resolution DNA melting analysis: an application for prenatal control of alpha-thalassemia.

OBJECTIVE: To report the use of real-time gap-PCR using SYTO9 with high-resolution melting analysis (HRMA) in prenatal diagnosis of alpha-thalassemia 1. MATERIALS AND METHODS: Real-time gap-PCR using SYTO9 with HRMA was performed in 33 DNA samples from chorionic villi sampling (8 normal, 16 heterozygous, and 9 homozygous) to determine the alpha-thalassemia 1 gene [normal and Southeast Asia (-SEA) allele]. RESULT: The dissociation curve analysis in normal and - SEA allele gave a peak of T(m) at 91.80 +/- 0.14 degrees C and 88.67 +/- 0.08 degrees C, respectively. Normal genotype and homozygous alpha-thalassemia 1 showed a single peak of T(m) that corresponded to their alleles. The heterozygotes gave both peaks with higher normal peak and smaller - SEA peak. Thirty one samples showed consistent results with the conventional gap-PCR. Two samples with ambiguous results were confirmed to be maternal DNA contamination on real-time quantitative PCR and microsatellite assay. HRMA from both samples showed similar pattern to that of heterozygotes. However, they showed much smaller normal peak compared with the - SEA peak, which is in contrast to those of heterozygotes and can readily be distinguished. CONCLUSION: HRMA with SYTO9 is feasible for prenatal diagnosis of alpha-thalassemia. It had potential advantage of prompt detection maternal DNA contamination.

Prenatal diagnosis of beta-thalassemia/Hb E by hemoglobin typing compared to DNA analysis.

To determine the accuracy of prenatal diagnosis of beta-thalassemia (beta-thal)/Hb E disease using fetal hemoglobin (Hb) typing compared to DNA analysis, automated DNA sequencing was performed on 98 blood samples from fetuses diagnosed as beta-thal/Hb E by Hb typing. Thirteen samples from homozygous Hb E fetuses were also collected. The Hb patterns obtained by high performance liquid chromatography (HPLC) from both groups were analyzed. The codon 26 (G>A) mutation was identified in all 98 samples. The beta-globin gene mutation was identified in 97 cases by DNA sequencing and the 3.4 kb deletion by polymerase chain reaction (PCR) in one case. The result from DNA analysis was in agreement with the HPLC result in all samples. In beta-thal/Hb E fetuses, the Hb A level was 0-0.3% and mean Hb A(2)(E) level was 1.3 +/- 0.3%. In homozygous Hb E fetuses, the Hb A level was 0% and mean Hb A(2)(E) level was 2.48 +/- 0.6%. The Hb pattern obtained by HPLC on fetal blood is a reliable and accurate method for prenatal diagnosis of this disease.

Analysis of real-time SYBR-polymerase chain reaction cycle threshold for diagnosis of the alpha-thalassemia-1 Southeast Asian type deletion: application to carrier screening and prenatal diagnosis of Hb Bart's hydrops fetalis.

Without gel electrophoresis and specific probes, the two tubes real-time SYBR-polymerase chain reaction (SYBR-PCR) was setup by using different primer sets: P1/P2 for the detection of wild type alpha-globin gene alleles and P1/P3 for detection of the allele bearing the Southeast Asian (SEA) type (--SEA) deletion. Analyses of the cycle threshold (CT) values obtained by each primer set together with a delta-cycle threshold (DeltaCT) and CT ratio, showed that lower CT values generated by primer sets P1/P2 and P1/P3 were observed in normal and Hb Bart's hydrops fetalis subjects, respectively. In heterozygous subjects the CT values generated by both sets of primers were similar to each other. There was no overlapping of DeltaCT and CT ratio between normal, heterozygous and Hb Bart's hydrops fetalis subjects. Therefore, the two tubes real-time SYBR-PCR could represent a rapid, cost effective, high-throughput assay for screening of carriers and prenatal diagnosis of alpha-thalassemia-1 (alpha-thal-1) with the SEA type (--SEA) deletion.

Detection of alpha-thalassemia-1 Southeast Asian type using real-time gap-PCR with SYBR Green1 and high resolution melting analysis.

Alpha-thalassemia-1 Southeast Asian (SEA) type is the most common genetic disorder in the Asian population. Couples who are both carriers have a 25% chance of conceiving Bart's hydrops fetalis. Therefore, results from carrier screening and prenatal diagnosis frequently need to be available rapidly. A rapid technique for diagnosis of alpha-thalassemia-1 SEA type was implemented. The technique used is based on real-time gap-PCR and high resolution melting (HRM) analysis of the amplified fragment using the Rotor-Gene 6000. The DNA samples used for amplification were obtained from whole blood, cord blood, and chorionic villus sampling (CVS). With this method, the alpha-thalassemia-1 SEA allele can be easily distinguished from wild type alpha-globin gene allele. The real-time gap-PCR and HRM analysis offers additional benefits including minimal labor, rapid turnaround time, and a decreased risk of PCR carryover contamination. It is cost-effective and safe, does not require fluorescently labeled probe and hazardous chemicals. Moreover, it is accurate showing 100% concordance with conventional gap-PCR analysis.

A successful strategy for Preimplantation Genetic Diagnosis of beta-thalassemia and simultaneous detection of Down's syndrome using multiplex fluorescent PCR.

OBJECTIVES: Preimplantation Genetic Diagnosis (PGD) is an alternative to prenatal diagnosis providing couples the chance to start a pregnancy with an unaffected fetus. The objective of the present study was to develop and apply quick, sensitive and accurate single cell PCR protocols for PGD of beta-thalassemia and Down's syndrome detection. MATERIAL AND METHOD: Two couples carrying beta-thalassemia codon41-42 mutation underwent routine IVF procedures. Embryo biopsy was performed on Day-3 post-fertilisation and single cell multiplex fluorescent PCR was employed for mutation analysis, contamination detection and diagnosis of trisomy 21 cases. RESULTS: Seventeen embryos were tested in two clinical PGD cycles. This resulted in the first birth following PGD for a single gene disorder in Thailand and South East Asia, confirmed by prenatal testing. Two embryos were shown to be affected by Down's syndrome. CONCLUSION: Successful strategy for PGD of beta-thalassemia and Down's syndrome detection using multiplex fluorescent PCR was introduced.

Homogeneity of beta(0)-thalassemia codon 17 (A-->T) alleles in Northern Thailand using a direct DNA sequencing method.

The aim of this study was to characterize beta-globin gene micro-haplotype polymorphisms (frameworks) associated with a beta-thalassemia mutations common in Northern Thailand using a direct DNA sequencing method. A total of 11 beta-thalassemia major patients homozygous for the codon 17 (A-->T) mutation admitted to Chiang Mai University Hospital were examined. All 22 alleles were found to contain the Asian framework 3A. The homogeneity of the framework associated with the codon 17 (A-->T) mutation indicates a relatively recent origin of the codon 17 (A-->T) mutation. Similar studies in other East Asian populations may provide information concerning the origin and the migrational spread of this beta-thalassemia mutation.

Analysis of beta-thalassemia mutations in northern Thailand using an automated fluorescence DNA sequencing technique.

A total of 218 beta-thalassemia (thal) genes from 109 beta-thal major patients were characterized using an automated fluorescence DNA sequencing technique. Eight different mutations were identified in all 218 alleles (100%). Four common mutations accounted for 96.8% [49.5% were codons 41/42 (-TTCT), 34.4% were codon 17 (A --> T), 6.9% were IVS-I-1 (G --> T) and, 6.0% were codons 71/72 (+A)]. There were three cases of -28 (A --> G) and one of IVS-II-654 (C --> T), mutations that have been previously described in Thai subjects. We also identified two mutations in the beta-globin promoter region which have not been reported in Thailand before [-31 (A --> G) and -87 (C --> A)]. Although these mutations are described as beta+-thal, the compound heterozygote with one of the common beta(o)-thal mutations exhibits the phenotype of beta-thal major. The frequency of beta-thal genes in northern Thailand were similar to the northeastern region, but different from those reported in southern and central Thailand, where IVS-I-5 (G --> C) and IVS-II-654 (C --> T) were the second most common anomalies, respectively. The spectrum of beta-globin gene mutations from this study will be useful for planning a prenatal diagnosis program especially for this region of Thailand.