Rattika Saetung

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

α‐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 α‐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 α‐thalassemia‐1 SEA allele can be easily distinguished from wild type α‐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.

Molecular and Clinical Features of Hb H Disease in Northern Thailand

Clinical assessment, hematological studies and molecular analyses were performed in 102 pediatric patients with Hb H disease in northern Thailand. A total of six mutations of the α-globin gene, which produced five genotypes, were detected. All patients had an α0-thalassemia (thal) deletion on one chromosome 16. All but one of these were of the South East Asian type (––SEA); one patient had the THAI deletion (––THAI). The deletional α+-thal mutations comprised 3.7 kb ( − α3.7) and 4.2 kb ( − α4.2) deletions which were found in 34 (33.3%) and 10 (9.8%) alleles, respectively. The nondeletional α+-thal mutations comprised 55 (53.9%) alleles of Hb Constant Spring (CS) (α142, TAA →CAA) and three (2.9%) alleles of Hb Pakse (α142, TAA→ TAT). Six patients with Hb H-CS disease also carried Hb E (AEBarts CS disease). The clinical features were diverse and the nondeletional genotypes were associated with more severe clinical and hematological features, including younger age at presentation, larger size of liver and spleen, lower hemoglobin (Hb) level, and higher transfusion requirements. The high proportion of nondeletional Hb H disease observed in this study was inconsistent with the previously reported gene frequencies of α-thal in the region, suggesting that many deletional Hb H patients with milder symptoms may have escaped recognition.

Detection and identification of β-thalassemia 3.5 kb deletion by SYBR Green1 and high resolution melting analysis

To the Editor: The b-globin gene cluster is located at the short arm of chromosome 11, with an arrangement of 5¢-e-c-c-d-b3¢. The whole cluster spans around 60 kb. A normal person has two copies of b-globin genes. A decrease or absence of b-globin leads to b-thalassemia (1). Most cases of b-thalassemia are because of the base substitutions, minor deletions and insertions in b-globin gene. Some cases are because of the larger deletions which vary from 0.1 kb to 10 kb (2, 3). The most frequent b-globin gene deletion found in Thai population is a 3.5 kb deletion. By means of restriction mapping technique, the length of the deletion was determined as approximately 3.5 kb. The 5¢ breakpoint was mapped to a 199 bp region flanked by the presence of HindII site and the loss of SphI site, and the 3¢ breakpoint was placed in a 267 bp region defined by the absence of AvaII site and an intact Fnu4HI site (4, 5). In Thailand, the prevalence of the b-thalassemia 3.5 kb deletion varies from 0.3 to 4.3% (6, 7). Even though the heterozygous of b-thalassemia 3.5 kb deletion results in minimal disease, the heterozygous combination between this deletion and other b-thalassemia mutations causes a severe disorder requiring regular blood transfusion for survival. Therefore, the presence of this deletion must be noted as it is undetectable by oligonucleotide primers reported to date for bthalassemia prenatal diagnosis. The conventional gapPCR analysis has been developed and used to diagnose b-thalassemia 3.5 kb deletion based on multiplex amplification at the breakpoint area of 3.5 kb deletion and the wild type b-globin gene allele (6). However, the technique requires post-PCR processing steps for gel electrophoresis and ethidium bromide staining. To resolve post-PCR processing steps, the real-time gap-PCR with SYBR Green1 and high resolution melting (HRM) analysis has been developed and used for detection of a-thalassemia-1 Southeast Asian (SEA) type (8). Therefore, the aim of this study is to develop this approach for diagnosis of bthalassemia 3.5 kb deletion. The analysis was performed on five DNA samples of normal individuals and six DNA samples of b-thalassemia 3.5 kb deletion (five heterozygouses and one homozygous). All samples were kindly provided by the Division of Hematology, Department of Pediatrics, Maharaj Nakorn Chiang-Mai Hospital, Chiang-Mai, Thailand. The genotype of b-thalassemia 3.5 kb deletion was firstly determined by conventional gap-PCR (6). This study was approved by the Faculty of Associated Medical Sciences Ethics Committee, Chiang-Mai University. DNA amplification was carried out in a 20 lL reaction volume containing: 10 lL of 2· SYBR Green1 PCR master mix (Bio-Rad Laboratories, Hercules, CA, USA), 0.38 lm of each primer as shown in Table 1 and 7 lL of DNA sample. The real-time PCR with SYBR Green1 was performed on Rotor-Gene 6000 (Corbett Research, Mortlake, New South Wales, Australia) as previously described (8). Briefly, the mixture was preheated at 95 C for 3 min and then the PCR was cycled 40 times at 95 C for 20 s, 62 C for 20 s, and 72 C for 20 s. Amplification cycles were followed by HRM cycle from 70 to 90 C at a rate of 0.1 C per 2 s. The temperature at which a peak occurs on the plot corresponds to the melting temperature (Tm) of DNA duplex. Amplified fragments with specific peak of Tm of normal individual, homozygous and heterozygous b-thalassemia 3.5 kb deletion were shown in Fig. 1. Only the amplified fragment from the wild type b-globin gene allele with a mean (standard deviation) specific peak at Tm of 76.52 (0.15) C was observed in five normal individuals (Fig. 1A). An amplified fragment from the b-thalassemia 3.5 kb deletion with the mean specific peak at Tm of 78.04 (0.05) C only was found in five replication tests of DNA sample from one patient with homozygous of bthalassemia 3.5 kb deletion (Fig. 1C). Both of those fragments with the mean specific peak at Tm of 76.24 (0.15) and 78.24 (0.09) C, respectively were observed in the five heterozygouses (Fig. 1B). Therefore, this study indicated the real-time gap-PCR with SYBR Green1 and HMR

Cord blood screening for Α-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 (36.6%) had thalassemia genes or Hb variants. Seventeen different genotypes were found. Nine cases (1.6%) of Hb H disease (five deletional Hb H diseases, two Hb H/Constant Spring diseases, one deletional Hb H disease/Hb E, carrier and one Hb H/Constant Spring disease/Hb E carrier) and one Hb E-beta-thalassemia were identified. IEF could clearly distinguish Hb H diseases and carriers of two alpha-globin gene defects from normal individuals according to the presence of Hb Barts and its percentage. For carriers of a single alpha-globin gene defect, Hb Barts was either absent or present in a small amount and was therefore not reliable for screening. The presence of an additional band at the Hb A(2) position in the newborns signified an Hb E carrier. One case of an absent Hb A and a presence of Hb E was identified as Hb E-beta-thalassemia. Two Hb Q-Thailand carriers were seen with two additional Hb fractions, presumably combinations of gamma-globin and beta-globin with the alpha-globin variant. Newborns with Hb H disease had lower Hb, MCV, and MCH levels than normal. MCV and MCH were also useful for differentiation of carriers of two alpha-globin gene defects, but not for carriers of Hb E or single alpha-globin gene defect. IEF was a reliable method for neonatal cord blood screening for alpha-thalassemia and Hb variants.

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

A total of 218 β‐thalassemia (thal) genes from 109 β‐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 β‐globin promoter region which have not been reported in Thailand before [−31 (A→G) and −87 (C→A)]. Although these mutations are described as β+‐thal, the compound heterozygote with one of the common β0‐thal mutations exhibits the phenotype of β‐thal major. The frequency of β‐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 β‐globin gene mutations from this study will be useful for planning a prenatal diagnosis program especially for this region of Thailand.

Analysis of Real-Time SYBR-Polymerase Chain Reaction Cycle Threshold for Diagnosis of the α-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 α-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 (ΔCT) and CT ratio, showed that lower CT values generated by primer sets P1/P2 and P1/P3 were observed in normal and Hb Barts 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 ΔCT and CT ratio between normal, heterozygous and Hb Barts 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 α-thalassemia-1 (α-thal-1) with the SEA type (– –SEA) deletion.

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

To report the use of real‐time gap‐PCR using SYTO9 with high‐resolution melting analysis (HRMA) in prenatal diagnosis of α‐thalassemia 1.

Effect of haematological alterations on thalassaemia investigation in HIV‐1‐infected Thai patients receiving antiretroviral therapy

To evaluate the effect of haematological alterations resulting from antiretroviral therapy (ART) on the diagnosis of thalassaemia carriers in HIV‐1‐infected Thai patients.

Prenatal Diagnosis of β-Thalassemia/Hb E by Hemoglobin Typing Compared to DNA Analysis

To determine the accuracy of prenatal diagnosis of β-thalassemia (β-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 β-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 β-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 β-thal/Hb E fetuses, the Hb A level was 0–0.3% and mean Hb A2(E) level was 1.3 ± 0.3%. In homozygous Hb E fetuses, the Hb A level was 0% and mean Hb A2(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.

Comparison of real‐time polymerase chain reaction SYBR Green1 with high resolution melting analysis and TaqMan MGB probes for detection of α‐thalassemia‐1 South‐East Asian type on dried blood spots

To the Editor: a-Thalassemia-1 South-East Asian (SEA) type is the main a-thalassemia abnormality in South-East Asia (1, 2). The gap-polymerase chain reaction (PCR) analysis currently used to diagnose a-thalassemia-1 SEA type requires post-PCR processing steps and hazardous chemicals (3–5). In an effort to develop a more straightforward diagnostic test, quantitative real-time PCR with the specific probe has been used for detection of a-thalassemia-1 SEA type (6, 7). However, using the specific probe is relatively expensive. As a cost-effective method, realtime PCR with SYBR Green1 followed by melting curve analysis (8, 9) and real-time gap-PCR with SYBR Green1 followed by high resolution melting (HRM) analysis (10) have been developed and used to enhance the speed of detection of a-thalassemia-1 SEA type. In lowresource settings, real-time PCR diagnostic protocols are considered too costly and complex. Dried blood spot (DBS) has been applied for many molecular investigations as it carries less of a biohazard risk than liquid samples and is easier to ship (11–15). Therefore, reliable detection of a-thalassemia-1 SEA type on DBS by realtime PCR has a potential to control a serious genetically transmitted disease in low-resource settings. The gap-PCR with SYBR Green1 followed by HRM analysis and TaqMan MGB probe-based assay were set up and analyzed for rapid detection of a-thalassemia-1 SEA type on DBS. The analysis was performed on 64 peripheral blood and six cord blood samples obtained from the Division of Hematology, Department of Pediatrics, Maharaj Nakorn Chiang-Mai Hospital. According to the thalassemia screening protocol, a-thalassemia-1 SEA type was first determined by conventional gap-PCR analysis and samples were sent to the Faculty of Associated Medical Sciences, Chiang-Mai University for DBS preparation on filter paper (Schleicher and Schuell; BioScience, Keene, NH, USA). This study was approved by the Faculty of Associated Medical Sciences Ethics Committee, Chiang-Mai University. DNA from liquid blood and cord blood samples was extracted using the Chelex method (Chelex 100 Resin; Bio-Rad Laboratories, Hercules, CA, USA) (16). DNA from DBS was extracted following the procedure described by Fischer et al. (17). The a-thalassemia-1 SEA type was analysed using two detection systems of realtime PCR. The gap-PCR with SYBR Green1 and HRM analysis was performed on Rotor-Gene 6000 (Corbett Research, Mortlake, NSW, Australia) as previously described (10). Whereas, the TaqMan real-time PCR based on specific primers and MGB-probes as shown in Table 1 was performed on ABI PRISM 7300 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). The amplified fragments with specific peak of Tm and interpretative criteria for detection of a-thalassemia-1 SEA type by real-time gap-PCR with SYBR Green1 and HRM analysis were previously described (10). Briefly, amplified fragments from a-thalassemia-1 SEA allele had the specific peak at Tm of 88 ± 1 C while amplified fragments from wild type a-globin gene allele had the specific peak at Tm of 91 ± 1 C. Whereas, the specific fluorescent signals from TaqMan MGB-based real-time PCR and interpretative criteria for detection of a-thalassemia-1 SEA type were shown in Fig. 1. In