Sakorn Pornprasert

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.

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

Detection of α-thalassemia-1 Southeast Asian and Thai type deletions and β-thalassemia 3.5-kb deletion by single-tube multiplex real-time PCR with SYBR Green1 and high-resolution melting analysis.

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 (Tm) 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.

DIAGNOSIS OF THALASSEMIA ON DRIED BLOOD SPOT SAMPLES BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

High performance liquid chromatography (HPLC) on fresh lysates is the standard test for identification of thalassemia. Samples in the form of dried blood spot(s) (DBS) mailed to reference laboratories where HPLC is available could be an alternative. Hemoglobin (Hb) on DBS at day 1, 7, 15 and 30 were analyzed by HPLC and compared to those analyzed from fresh liquid whole blood at day 0. A 100% consistent interpretation of β-thalassemia (β-thal) trait and β-thal/Hb E disease between liquid whole blood and DBS was observed when analyzing Hb A2 on DBS at a level of 2.7–9.9% in conjunction with a lower MCV (<80 fL) and MCH (<27 pg) and analyzing β-thal/Hb E by using Hb E and Hb F at a level of 30–48% and >10%, respectively. Therefore, our results show that detection of thalassemia carriers using DBS is possible and is the alternative of choice in a low resource setting.

Comparison Between Capillary Electrophoresis and High Performance Liquid Chromatography for Detection and Quantification of Hb Constant Spring [Hb CS; α142, Term→Gln (TAA>CAA IN α2)]

Hb Constant Spring [Hb CS; α142, Term→Gln (TAA>CAA in α2)] is often missed by routine laboratory testing since its mRNA as well as gene product are unstable and presented at a low level in peripheral blood. This study aimed to analyze the efficacy of capillary electrophoresis (CE) and high performance liquid chromatography (HPLC) for detecting and quantifying Hb CS in 19 heterozygotes and 14 homozygotes with Hb CS as well as 10 Hb H-CS disease subjects who were detected by molecular analysis. In the CE electrophoregram, Hb CS was seen at zone 2 and was observed in all samples, while the chromatogram of Hb CS peaks was found in 26.32% heterozygotes, 42.86% homozygotes and 90% Hb H-CS disease subjects, respectively. In addition, the Hb CS levels in each group of subjects quantified by CE were significantly higher than those quantified by HPLC. Based on the CE method, the lowest Hb CS level was found in the heterozygotes, whereas the highest level was found in the Hb H-CS disease patients. Therefore, the CE method was superior to the HPLC method for detecting Hb CS. Furthermore, the level of Hb CS quantified by CE proved useful in screening heterozygotes and homozygotes with Hb CS as well as Hb H-CS disease.

Development of TaqMan real-time polymerase chain reaction for the detection and identification of Penicillium marneffei.

Penicillium marneffei is a dimorphic fungus, which is endemic in Southeast Asia and responsible for emerging opportunistic infections. Diagnosis of penicilliosis may be difficult when few yeast cells are present, while a gold standard diagnosis technique requires long‐term culture. In order to provide a more rapid and accurate diagnosis, we developed a TaqMan real‐time PCR to detect and identify P. marneffei DNA coding for 5.8S rRNA in purified yeast DNA and clinical samples. All P. marneffei DNA preparations could be detected using specific primers and TaqMan probe. The assay has a sensitivity to detect at least 10 yeast cells in seeded blood. Moreover, it can detect P. marneffei DNA in peripheral blood samples and blood‐culture bottles. Therefore, the real‐time PCR assay may represent a potential tool for early diagnosis of penicilliosis marneffei.

SYTO9 and SYBR Green1 with High Resolution Melting Analysis for Molecular Confirmatory Testing of the Common Southeast Asian β0-thalassemia Mutations

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 β0-thalassemia (β0-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 A2 levels between 3.1 and 9.9%. The SYTO9 HRM analysis showed specific characteristic peaks of each investigated type of β0-thal mutation. Results of MARMS-PCR followed by SYTO9 HRM analyses were completely consistent with results of those analyzed by gel electrophoresis. Moreover, the β0-thal 3.5 kb gene deletion could be identified in two samples with Hb A2 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 β-thal.

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.

Red Cell Indices and Formulas Used in Differentiation of β-Thalassemia Trait from Iron Deficiency in Thai School Children

Abstract Red cell indices and formulas have been established as simple, fast, and inexpensive means for discrimination between the β-thalassemia (β-thal) trait and iron deficiency. However, there were no reports of the diagnostic reliability of different red cell indices and formulas in discrimination of β-thal trait from iron deficiency in the Thai population. The aim of this study was to examine the diagnostic accuracy of five red cell indices [red blood cell (RBC) count, mean corpuscular volume (MCV), mean corpuscular hemoglobin (Hb) (MCH), mean corpuscular Hb concentration (MCHC), and red cell distribution width (RDW)] and eight formulas (Sirdah, Green & King, RDW Index, Menzler, England & Fraser, Ehsani, Srivastava, and Shine & Lal). Their sensitivity, specificity, positive and negative prognostic value and efficiency, were analyzed in 77 Thai school children, 21 with the β-thal trait and 56 with iron deficiency. The Sirdah and Srivastava formulas proved to be the most reliable indexes as they had 100.0% sensitivity and negative predictive value, the highest efficiency (97.4%), and the highest Youden’s Index value (96.4%). Therefore, these formulas could be used in initial discrimination of the β-thal trait from iron deficiency in Thai school children.

Detection of alpha(0)‐thalassemia South‐East Asian‐type deletion by droplet digital PCR

The α(0)‐thalassemia South‐East Asian (SEA)‐type deletion is the most common genetic disorder in the Asian population. Couples who are both carriers have a 25% chance of conceiving Barts hydrops fetalis. Therefore, results from carrier screening and prenatal diagnosis frequently need to be available rapidly. The aim of this study was to implement a droplet digital polymerase chain reaction (ddPCR) for diagnosis of α(0)‐thalassemia SEA‐type deletion.