Interaction of hemoglobin Grey Lynn (Vientiane) with a non-deletional α+-thalassemia in an adult Thai proband

Hemoglobin (Hb) Grey Lynn is a Hb variant caused by a substitution of Phe for Leu at position 91 of α1-globin chain, originally described in individual of unknown ethnic background. This article addresses the interaction of Hb Grey Lynn with a non-deletional α+-thalassemia found in Thailand, a hitherto un-described condition. The proband was adult Thai woman referred for investigation of mild anemia with Hb 90 g/L. Hb analyses using low pressure liquid chromatography raised a suspicion of abnormal Hb presence, which was failed to demonstrate by cellulose acetate electrophoresis and capillary electrophoresis. DNA sequencing identified a CTT (Leu) to TTT (Phe) mutation at codon 91 corresponding to the Hb Grey Lynn (Vientiane) [α91(FG3)Leu>Phe (α1) on α1-globin gene and a C deletion between codons 36 and 37 on α2-globin gene causing α+-thalassemia. As compared to those observed in a compound heterozygote for Hb Grey Lynn / α0-thalassemia reported previously, higher MCV (81.7 fL) and MCH (26.3 pg) values with a lower level of Hb Grey Lynn (19.7%) were observed in the proband. The normochromic normocytic anemia observed could be due to the interaction of Hb Grey Lynn with α+-thalassemia. The two mutations could be identified using PCR-RFLP and allele-specific PCR assays developed

Evaluation of staff performance and interpretation of the screening program for prevention of thalassemia

IntroductionThalassemia screening program has been implemented for years in Southeast Asia, but no external quality assessment program has been established. We have developed and initiated the proficiency testing (PT) program for the first time in Thailand with the aim to assess the screening performance of laboratory staff and their competency in interpretation of the screening results. Materials and methodsThree PT cycles per year were organized. From the first to the third cycle of the PT scheme, a total number of participant laboratories increased from 59 to 67. In each cycle, 2 PT items (assigned as blood samples of the couple) were provided. Performance evaluation was based on the accuracy of screening results, i.e. mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and the dichlorophenolindophenol (DCIP) test for haemoglobin E, including the competency in interpretation of screening results and assessment of foetal risk. Performance was assessed by comparing the participants’ result against the assigned value. ResultsOf all 3 cycles, most laboratories reported acceptable MCV and MCH values. From the first to the third cycle, incorrect DCIP test and misinterpretation rates were decreased while incorrect risk assessment varied by cycle to cycle. Combining the accuracy of thalassemia screening and the competency in interpretation and risk assessment, approximately half of participants showed excellent performance. ConclusionImproved performance observed in many laboratories reflects the achievement and benefit of the PT program which should be regularly provided

Imputation of missing genotypes: an empirical evaluation of IMPUTE

<p>Abstract</p> <p>Background</p> <p>Imputation of missing genotypes is becoming a very popular solution for synchronizing genotype data collected with different microarray platforms but the effect of ethnic background, subject ascertainment, and amount of missing data on the accuracy of imputation are not well understood.</p> <p>Results</p> <p>We evaluated the accuracy of the program IMPUTE to generate the genotype data of partially or fully untyped single nucleotide polymorphisms (SNPs). The program uses a model-based approach to imputation that reconstructs the genotype distribution given a set of referent haplotypes and the observed data, and uses this distribution to compute the marginal probability of each missing genotype for each individual subject that is used to impute the missing data. We assembled genome-wide data from five different studies and three different ethnic groups comprising Caucasians, African Americans and Asians. We randomly removed genotype data and then compared the observed genotypes with those generated by IMPUTE. Our analysis shows 97% median accuracy in Caucasian subjects when less than 10% of the SNPs are untyped and missing genotypes are accepted regardless of their posterior probability. The median accuracy increases to 99% when we require 0.95 minimum posterior probability for an imputed genotype to be acceptable. The accuracy decreases to 86% or 94% when subjects are African Americans or Asians. We propose a strategy to improve the accuracy by leveraging the level of admixture in African Americans.</p> <p>Conclusion</p> <p>Our analysis suggests that IMPUTE is very accurate in samples of Caucasians origin, it is slightly less accurate in samples of Asians background, but substantially less accurate in samples of admixed background such as African Americans. Sample size and ascertainment do not seem to affect the accuracy of imputation.</p

Molecular basis of non-deletional HPFH in Thailand and identification of two novel mutations at the binding sites of CCAAT and GATA-1 transcription factors

Abstract High Hb F determinants are genetic defects associated with increased expression of hemoglobin F in adult life, classified as deletional and non-deletional forms. We report the first description of non-deletional hereditary persistence of fetal hemoglobin (HFPH) in Thailand. Study was done on 388 subjects suspected of non-deletional HPFH with elevated Hb F expression. Mutations in the Gγ- and Aγ-globin genes were examined by DNA analysis and rapid diagnosis of HPFH mutations were developed by PCR-based methods. Twenty subjects with five different mutations were identified including three known mutations, − 202 Aγ (C>T) (n = 3), − 196 Aγ (C>T) (n = 3), and − 158 Aγ (C>T) (n = 12), and two novel mutations, − 117 Aγ (G>C) (n = 1) and − 530 Gγ (A>G) (n = 1). Interaction of the − 117 Aγ (G>C) and Hb E (HBB:c.79G>A) resulted in elevation of Hb F to the level of 13.5%. Two plain heterozygous subjects with − 530 Gγ (A>G) had marginally elevated Hb F with 1.9% and 3.0%, whereas the proband with homozygous − 530 Gγ (A>G) had elevated Hb F of 11.5%. Functional prediction indicated that the − 117 Aγ (G>C) and − 530 Gγ (A>G) mutations dramatically alter the binding of transcription factors to respective γ-globin gene promotors, especially the CCAAT and GATA-1 transcription factors. Diverse heterogeneity of non-deletional HFPH with both known and new mutations, and complex interactions of them with other forms of thalassemia are encountered in Thai population

A large cohort of hemoglobin variants in Thailand: molecular epidemiological study and diagnostic consideration.

Hemoglobin (Hb) variants are structurally inherited changes of globin chains. Accurate diagnoses of these variants are important for planning of appropriate management and genetic counseling. Since no epidemiological study has been conducted before, we have investigated frequencies, molecular and hematological features of Hb variants found in a large cohort of Thai subjects.Study was conducted on 26,013 unrelated subjects, inhabiting in all geographical parts of Thailand over a period of 11 years from January 2002-December 2012. Hb analysis was done on high performance liquid chromatography (HPLC) or capillary electrophoresis (CE). Mutations causing Hb variants were identified using PCR and related techniques.Among 26,013 subjects investigated, 636 (2.4%) were found to carry Hb variants. Of these 636 subjects, 142 (22.4%) carried α-chain variants with 13 different mutations. The remaining included 451 (70.9%) cases with 16 β-chain variants, 37 (5.8%) cases with Hb Lepore (δβ-hybrid Hb) and 6 (0.9%) cases with a single δ-chain variant. The most common α-globin chain variant was the Hb Q-Thailand (α⁷⁴GAC-CAC, Asp-His) which was found in 101 cases (15.8%). For β-globin chain variants, Hb Hope (β¹³⁶GGT-GAT, Gly-Asp) and Hb Tak (β¹⁴⁶+AC, Ter-Thr) are the two most common ones, found in 121 (19.0%) and 90 (14.2%) cases, respectively. Seven Hb variants have never been found in Thai population. Hb analysis profiles on HPLC or CE of these variants were illustrated to guide presumptive diagnostics.Hb variants are common and heterogeneous in Thai population. With varieties of thalassemias and hemoglobinopathies in the population, interactions between them leading to complex syndromes are common and render their diagnoses difficult in routine practices. Knowledge of the spectrum, molecular basis, genotype-phenotype correlation and diagnostic features should prove useful for prevention and control of the diseases in the region

Hemoglobin Constant Spring among Southeast Asian Populations: Haplotypic Heterogeneities and Phylogenetic Analysis.

BACKGROUND:Hemoglobin Constant Spring (Hb CS) is an abnormal Hb caused by a mutation at the termination codon of α2-globin gene found commonly among Southeast Asian and Chinese people. Association of Hb CS with α°-thalassemia leads to a thalassemia intermedia syndrome commonly encountered in the region. We report chromosome background and addressed genetic origins of Hb CS observed in a large cohort of Hb CS among Southeast Asian populations. MATERIALS AND METHODS:A study was done on 102 Vietnamese (aged 15-49 year-old) and 40 Laotian (aged 18-39 year-old) subjects with Hb CS and results compared with 120 Hb CS genes in Thailand. Hematological parameters were recorded and Hb analysis was performed using capillary electrophoresis. Hb CS mutation and thalassemia genotypes were defined by DNA analysis. Six DNA polymorphisms within α-globin gene cluster including 5'Xba I, Bgl I, Inter-zeta HVR, AccI, RsaI and αPstI 3', were determined using PCR-RFLP assay. RESULTS:Nine different genotypes of Hb CS were observed. In contrast to the Thai Hb CS alleles which are mostly linked to haplotype (+-S + + -), most of the Vietnamese and the Laotian Hb CS genes were associated with haplotype (+-M + + -), both of which are different from that of the European Hb CS. CONCLUSIONS:Hb CS is commonly found in combination with other thalassemias among Southeast Asian populations. Accurate genotyping of the cases requires both hematologic and DNA analyses. At least two independent origins are associated with the Hb CS gene which could indirectly explain the high prevalence of this Hb variant in the region

Effect of health education on severe thalassemia prevention and control in communities in Cambodia

Abstract Background Severe thalassemia diseases are a major health problem in Southeast Asia. In Cambodia, there has never been a significant program for prevention or control of severe thalassemia. We, therefore, studied the effect of a health education program on severe thalassemia prevention and control in Phnom Penh, Cambodia. Methods A quasi-experimental study in several communities around Phnom Penh was done. The respective intervention and control group comprised 124 and 117 people, between 18 and 40 years of age, male and female. Pre- and post-tests using a validated and reliable questionnaire were performed in the intervention group and one test was done in the control group. A health education program was organized to give important information to the intervention group and, at the end of the process, to the control group. The outcomes were evaluations of their knowledge and attitude vis-à-vis severe thalassemia prevention and control, and participating in thalassemia screening. Results Among participants in the intervention group, 105 (84.7%) considered undergoing blood screening vs. 65 (55.6%) in the control group (p-value < 0.001). In the intervention group, the respective mean scores for knowledge and attitude to a prevention and control program for severe thalassemia before and after health education were 2.6 VS 6.5 (p-value < 0.001) and 4.6 VS 6.5 (p-value < 0.001). Conclusions The intention to undergo screening was significantly higher in the intervention group than the control group. Knowledge and attitude towards prevention and control of severe thalassemia was significantly improved in the intervention group. Health education clearly heightens awareness and improves consideration of screening for prevention and control of severe thalassemia

Molecular basis of a high Hb A2/Hb Fβ-thalassemia trait: a retrospective analysis, genotype-phenotype interaction, diagnostic implication, and identification of a novel interaction with α-globin gene triplication

Background β0-thalassemia deletion removing 5´β-globin promoter usually presents phenotype with high hemoglobin (Hb) A2 and Hb F levels. We report the molecular characteristics and phenotype-genotype correlation in a large cohort of the β0-thalassemia with 3.4 kb deletion. Methods A total of 148 subjects, including 127 heterozygotes, 20 Hb E-β-thalassemia patients, and a double heterozygote with α-globin gene triplication, were recruited. Hb and DNA analysis were performed to identify thalassemia mutations and four high Hb F single nucleotide polymorphisms (SNPs) including four base pair deletion (-AGCA) at Aγ-globin promoter, rs5006884 on OR51B6 gene, −158 Gγ-XmnI, BCL11A binding motifs (TGGTCA) between 3´Aγ-globin gene and 5´δ-globin gene. Results It was found that heterozygous β0-thalassemia and Hb E-β0-thalassemia with 3.4 kb deletion had significantly higher Hb, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin and Hb F values as compared with those with other mutations. Co-inheritance of heterozygous β0-thalassemia with 3.4 kb deletion and α-thalassemia was associated with even higher MCV and MCH values. The Hb E-β0-thalassemia patients carried a non-transfusion-dependent thalassemia phenotype with an average Hb of around 10 g/dL without blood transfusion. A hitherto undescribed double heterozygous β0-thalassemia with 3.4 kb deletion and α-globin gene triplication presented as a plain β-thalassemia trait. Most of the subjects had wild-type sequences for the four high Hb F SNPs examined. No significant difference in Hb F was observed between those of subjects with and without these SNPs. Removal of the 5´β-globin promoter may likely be responsible for this unusual phenotype. Conclusions The results indicate that β0-thalassemia with 3.4 kb deletion is a mild β-thalassemia allele. This information should be provided at genetic counseling and prenatal thalassemia diagnosis