HbA2 levels in β-thalassaemia carriers with the Filipino β0-deletion: are the levels higher than what is found with non-deletional forms of β0-thalassaemia?

AIMS: Classical carriers of β-thalassaemia are identified by a raised HbA2 level. Earlier studies indicated that the Filipino β-deletion has high raised HbA2 levels. The introduction of automated high performance liquid chromatography (HPLC) for thalassaemia screening is an important advance in technology for haematology laboratories. The BioRad Variant II Hb analyser is a common instrument used to quantify HbA2 levels in thalassaemia screening. This study aimed to determine HbA2 levels in carriers of Filipino β-mutation using the BioRad Variant II Hb analyser. METHODS: The Filipino β-deletion was identified using gap-polymerase chain reaction (PCR) in the parents of transfusion dependent β-thalassaemia patients who were homozygous for the Filipino β-deletion in the indigenous population of Sabah, Malaysia. Hb subtypes were quantified on the BioRad Variant II Hb analyser. Concurrent α-thalassaemia was identified by multiplex gap-PCR for deletions and amplification refractory mutation system (ARMS)-PCR for non-deletional mutations. RESULTS: The mean HbA2 level for Filipino β-thalassaemia trait was 5.9 ± 0.47 and with coinheritance of α-thalassaemia was 6.3 ± 0.44 (-α heterozygous) and 6.7 ± 0.36 (-α homozygous). The HbA2 levels were all >4% in keeping with the findings of classical β-thalassaemia trait and significantly higher than levels seen in non-deletional forms of β-thalassaemia. CONCLUSION: The HbA2 level measured on the BioRad Variant II Hb analyser was lower than the level in the first description of the Filipino β-thalassaemia. β-thalassaemia trait with coinheritance of α-thalassaemia (-α) is associated with significantly higher HbA2 level

Correlation of BACH1 and hemoglobin E/Beta-thalassemia globin expression

Objective: The diverse clinical phenotype of hemoglobin E (HbE)/β-thalassemia has not only confounded clinicians in matters of patient management but has also led scientists to investigate the complex mechanisms involved in maintaining the delicate red cell environment where, even with apparent similarities of α- and β-globin genotypes, the phenotype tells a different story. The BTB and CNC homology 1 (BACH1) protein is known to regulate α- and β-globin gene transcriptions during the terminal differentiation of erythroid cells. With the mutations involved in HbE/β-thalassemia disorder, we studied the role of BACH1 in compensating for the globin chain imbalance, albeit for fine-tuning purposes. Materials and Methods: A total of 47 HbE/β-thalassemia samples were analyzed using real-time quantitative polymerase chain reaction and correlated with age, sex, red blood cell parameters, globin gene expressions, and some clinical data. Results: The BACH1 expression among the β-thalassemia intermedia patients varied by up to 2-log differences and was positively correlated to age; α-, β-, and γ-globin gene expression level; and heme oxygenase 1 protein. BACH1 was also negatively correlated to reticulocyte level and had a significant correlation with splenectomy. Conclusion: This study indicates that the expression of BACH1 could be elevated as a compensatory mechanism to decrease the globin chain imbalance as well as to reduce the oxidative stress found in HbE/β-thalassemia

Concurrent inheritance of deletional a-thalassaemia in Malays with HbE trait

Introduction: HbE is the commonest beta haemoglobin (Hb) variant in Southeast Asia. It causes a reduction in synthesis of the beta-globin E chain. Studies indicate HbE coinherited with a-thalassaemia leads to milder clinical phenotype. This study investigates the commitant inheritance of a-thalassaemia in Malays with HbE. Methods: Four hundred and fourteen (414) blood samples were screened for haemoglobinopathy using primarily the first three steps of the BHES [ (B) blood counts, blood film; (H), HPLC; (E) electrophoresis; (S), stability ] protocol. Complete blood cell analyser, Hb typing with cation exchange high-performance liquid chromatography (HPLC) and Hb electrophoresis at an alkaline pH (pH 8.5) Forty-five (10.9%) were identified as HbE trait and DNA analysis was done for deletional a-thalassaemia using a single-tube multiplex-PCR assay. Results: Among the 45 subjects with HbE trait, 4 (8.9%) were found to have alpha-thalassaemia -2 (a) (a-37 kb deletion) and 1 (2.2%) the alpha-thalassaemia-1 (a0) (---SEA 20.5 kb deletion) defects respectively. Discussion: These findings show that 11.1% of Malays with HbE inherit alpha-thalassaemia concurrently. The most prevalent interaction found was a double heterozygote for HbE /a-thalassaemia 2, followed by HbE/a-thalassaemia 1. Conclusion: Molecular screening of deletional a-thalassaemia identified its concurrent inheritance in 11.1%o of Malays who were HbE carriers. This information will guide genetic counseling and the planning of treatment modalities in patients with HbE alpha-thalassaemia

Haplotype analysis of β-Thalassaemia major and carriers with Filipino β°-deletion in Sabah, Malaysia

Objective: The Filipino β°-deletion has been reported as a unique mutation in East Malaysia with a severe phenotype due to the complete absence of β-globin chain synthesis. In this study, the haplotype patterns of the β-globin gene cluster were used to relate the human genetic variation to this specific β-thalassaemia mutation. Methods: The 376 study subjects included 219 β-thalassaemia major (β-TM) patients with homozygous Filipino β°-deletion and 157 carriers with heterozygous Filipino β°-deletion from 10 government hospitals in different regions of Sabah. Genomic DNA was isolated from whole blood using silica membrane based DNA purification protocol. Polymerase chain reaction restriction fragment length polymorphism analysis (PCR-RFLP) was conducted on five markers within the β-globin gene cluster to construct the haplotype patterns.Results: Four haplotypes (Haplotype I–IV) were identified with Haplotype I as the predominant haplotype with the highest frequency of 0.98, followed by Haplotype II, III and Haplotype IV with 0.02. Haplotype I was strongly linked with the Filipino β°-deletion among the indigenous population. Conclusion: Haplotype I as the predominant haplotype suggests the patients with the Filipino β°-deletion in Sabah have a similar origin

Effects of transfusion and splenectomy on globin chain expression in NTDT HbE/β-thalassaemia

Introduction: Majority of HbE/β-thalassaemia patients resembles the phenotype of non-transfusion dependent thalassaemia (NTDT). Current management strategies are highly diverse, and the objective of this study is to examine the effects of different treatments on multiple parameters in NTDT HbE/β-thalassaemia to further streamline the management of this disorder. Methods: In this cross-sectional study, we analysed the correlation between different treatment strategies with variable parameters including haematological parameter and globin gene expression. Statistical analyses were carried out using non-parametric tests such as Kruskal-Wallis and Mann-Whitney tests. Results: A total of 29 HbE/β-thalassaemia patients were included in the study. Data showed statistically significant differences were observed in the MCV, MCHC levels, reticulocyte count and log α/β fold change between the groups. Further analysis showed higher log α/β fold change in the transfusion only group compared to the non-treated group. Red blood cell count was found to be lower in transfused and splenectomised group compared to transfusion only. Significantly higher MCV level and reticulocyte count was seen in transfusion and splenectomised group compared to both non-treated and transfusion only groups and higher MCH level in the transfusion and splenectomised group compared to transfusion only group. Conclusion: In general, regardless of single or double combined therapies, HbE/β-thalassaemia showed variable changes in laboratory parameters to the therapies received particularly splenectomy

Screening for intermediate and severe forms of thalassaemia in discarded red blood cells: optimization and feasibility

Detection and quantification of Hb subtypes of human blood is integral to presumptive identification of thalassaemias. It has been used in neonatal screening of thalassaemia and Hb variants. The use of discarded red blood cells following processing of the cord blood for stem cells provides readily available diagnostic material for thalassaemia screening. In this study, we determined the range of Hb subtypes in 195 consecutive cord blood samples collected for cord blood banking. The 'cord blood samples' analysed were those of the remaining red blood cells after the cord blood was processed for stem cell storage. Quantification of Hb subtypes by high performance liquid chromatography (HPLC) was done on BioRad Variant II Hb testing system. Only 73 (36.5%) of the samples could be analyzed neat without dilution. With a 1:300 dilution with wash solution the acceptable area as recommended by the manufacturer for reading of a C-gram within the 1 to 3 million ranges were achieved in all. Eighteen (9%) 12 showed classical Hb Barts (γ4) prerun peaks were confirmed by Sebia Hydrasys automated Hb gel electrophoresis and quantified by Sebia Capillarys 2 capillary electrophoresis. Only 1 (0.5%) was presumptively identified with HbH disease. Due to the limited number of samples no beta-thalassaemia major, Hb E beta-thalassaemia and Hb Barts hydrops fetalis were found. The HPLC assay was possible at a cost US$ 5 per sample and a turnover time of 10 samples per hour without technical difficulties. This study reports an effective and valuable protocol for thalassaemia screening in red blood cells which would otherwise be discarded during cord blood processing. Cord blood with severe and intermediate forms of thalassaemia can be preselected and not stored

Molecular basis of transfusion dependent beta-thalassemia major patients in Sabah

Beta-thalassemia is one of the most prevalent inherited diseases and a public health problem in Malaysia. Malaysia is geographically divided into West and East Malaysia. In Sabah, a state in East Malaysia, there are over 1000 estimated cases of β-thalassemia major patients. Accurate population frequency data of the molecular basis of β-thalassemia major are needed for planning its control in the high-risk population of Sabah. Characterization of β-globin gene defects was done in 252 transfusion dependent β-thalassemia patients incorporating few PCR techniques. The study demonstrates that β-thalassemia mutations inherited are ethnically dependent. It is important to note that 86.9% of transfusion-dependent β-thalassemia major patients in Sabah were of the indigenous population and homozygous for a single mutation. The Filipino β0-deletion was a unique mutation found in the indigenous population of Sabah. Mutations common in West Malaysia were found in 11 (4.3%) patients. Four rare mutations (Hb Monroe, CD 8/9, CD 123/124/125 and IVS I-2) were also found. This study is informative on the population genetics of β-thalassemia major in Saba

Retrospective evaluation of whole exome and genome mutation calls in 746 cancer samples

Funder: NCI U24CA211006Abstract: The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) curated consensus somatic mutation calls using whole exome sequencing (WES) and whole genome sequencing (WGS), respectively. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2,658 cancers across 38 tumour types, we compare WES and WGS side-by-side from 746 TCGA samples, finding that ~80% of mutations overlap in covered exonic regions. We estimate that low variant allele fraction (VAF < 15%) and clonal heterogeneity contribute up to 68% of private WGS mutations and 71% of private WES mutations. We observe that ~30% of private WGS mutations trace to mutations identified by a single variant caller in WES consensus efforts. WGS captures both ~50% more variation in exonic regions and un-observed mutations in loci with variable GC-content. Together, our analysis highlights technological divergences between two reproducible somatic variant detection efforts

Molecular characterisation of beta thalassaemia in patients from Sabah, Malaysia

Sabah has the largest number of β-thalassaemia major (β-TM) patients in Malaysia with estimated over 1000 cases of transfusion dependent β-TM patients. However,complete molecular characterisation of thalassaemia major patients has not been done. The objective for this study is to characterise the molecular spectrum in Sabah population through β- and α-globin gene genotyping, identifying XmnI Gγ-polymorphism, haplotyping for β-globin gene cluster and to develop an ideal diagnostic algorithm and tools which is suitable for this population. In this study, 252 β-TM patients (Group I) and 165 carriers (Group II) were recruited from ten different hospitals in Sabah. Filipino β0-deletion was the predominant mutation identified in the Kadazandusun, Rungus, Murut, Sungai and Bajau. A total of 219 (86.9%) β-TM patients were identified as homozygous Filipino β0-deletion. HbE and Hb Malay were found as the most common Hb variants to co-inherit with Filipino β0-deletion. Some common mutations in West Malaysia were found to coexist with Filipino β0-deletion. This can be due to intermarriage between different ethnic groups. Carriers showed the frequency of Filipino β0-deletion at 95.2% (n=157). Only seven (4.2%) carriers were found with point mutations commonly seen in West Malaysia. High frequency of co-inheritance of -α3.7 deletion was found in the Sabah β-thalassaemia population. Co-inheritance of heterozygous -α3.7 deletion was found in 67 (26.6%) β-TM patients and 42 (25.3%) carriers. Co-inheritance of homozygous -α3.7 deletion was found in seven (2.8%) β-TM patients and six (3.6%) carriers. This may be related to the natural selection and protection for survival from severe malaria (Plasmodium Falciparum). Only type I of -α3.7 deletion was observed in this study population, indicating that the population has a single origin. XmnI Gγ polymorphism was reported with higher Gγ-globin gene expression. Clinical presentation will be ameliorated in homozygous states. In this study, XmnI (-/-) genotype was found in 237 (94%) β-TM patients and 156 (94.4%) carriers, indicating low existence of this polymorphism as an ameliorating factor. In haplotyping analysis, seven haplotype patterns were inferred in 417 samples consisting of 252 β-TM patients and 165 carriers. Hp I (+ - - - -) was the predominant pattern demonstrated in 98.14% of the population. This suggested a unicentric origin and an apparent single origin with low genetic diversity. This is the first report to demonstrate Hp I in the Sabah population with Filipino β0-deletion. Two new diagnostic tools, Taqman and HRM analysis were developed using realtime detection for Filipino β0-deletion. Taqman analysis was found more ideal as a diagnostic tool by having high specificity and sensitivity although it is more expensive. An added advantage is that there is no requirement for post-PCR processing. Multiplex ligation-dependent probe amplification (MLPA) analysis is an efficient technique for the screening of large deletions which can be included in the diagnosis algorithm provided technical expertise and necessary funding are available. This study reveals a notable regional specificity of the β- and α-thalassaemia mutations, which are Filipino β0-deletion and -α3.7 deletion. XmnI polymorphism is uncommon in this study population. From the haplotype analysis and type of -α3.7 deletion, the findings suggested that the Sabah population with β-thalassaemia may belong to the same stock with similar origin. Taqman analysis is more ideal as a diagnostic tool. The findings from this study are informative for molecular diagnosis in the Sabah population with β-thalassaemia

Development of reverse dot blot hybridisation strip assays to identify common beta-thalassaemia alleles in Malays and Chinese in Malaysia

Beta-thalassemia is the most common autosomal genetic disorder in Malaysia particularly among Malays and Chinese-Malaysians. The heterozygous carrier frequency of β-thalassaemia in Malaysia is estimated to be 4.5% from micro-mapping studies. The spectrum of β-thalassaemia mutations differs in each ethnic group in Malaysia. There are four to five common mutations responsible for more than 95% of the mutations seen in each ethnic group respectively. The current diagnostic method in Malaysia, amplification refractory mutation system (ARMS-PCR), is only able to identify one mutation in each reaction. It is found labour intensive and time consuming when few mutations need to be identified. Therefore, there is a need to have an effective and accurate laboratory method that can identify common mutations simultaneously in each ethnic group.In this study, the reverse dot blot hybridization (RDBH) technique was used in development of strip assays for characterisation of the β-thalassaemia mutations. Two strip assays were designed specifically for Malays and Chinese-Malaysians respectively with each strip to identify six common mutations simultaneously. The mutations identified with the strip assays were validated with the gold standard method, ARMS-PCR. A total of 177 patients (354 alleles) from University Malaya Medical Centre (UMMC) and the Institute of Medical Research (IMR) in Malaysia were studied. One hundred and thirty seven were Malays (274 alleles) and 40 were Chinese-Malaysians (80 alleles) respectively. One hundred and nineteen (86.9%) Malay patients consisting of 238 alleles were identified by the RDBH-Strip M(6). In the Malays, the most common β-thalassaemia mutations identified was CD 26, followed by IVS I-5, IVS I-1, CD 19 and the least with CD 8/9. In view of possible inter-marriage with Chinese, the RDBH-Strip C(6) was used to identify the 18 unidentified alleles in the Malays. The mutations identified were common Chinese mutations, CD 41/42 (5 heterozygous), CD 17 (2 heterozygous), -29 (2 heterozygous) and CD 71/72 (1 homozygous). Thus, a total of 129 (94.6%) Malay patients consisting of 258 alleles were identified using the RDBH-Strip Assays [RDBH-Strip M(6) and RDBH-Strip C(6)] . In the Chinese-Malaysians by the RDBH-Strip C(6), mutations were identified in 32 (80%) patients consisting of 64 alleles. IVS II-654 and CD 41/42 were the two most common β-thalassaemia mutations amongst Chinese-Malaysians, followed by CD17 and -28. In the Chinese-Malaysians, RDBH-Strip M(6) identified CD 26 (3 heterozygous) and IVS I-5(1 heterozygous). Thus, a total of 36 (90.0%) Chinese-Malaysians patients consisting of 72 alleles were identified using the RDBH-Strip Assays [RDBH-Strip C(6) and RDBH-Strip M(6)] . The RDBH-Strip Assays developed in this project study identified 93.2% of the mutations seen in the Malays and Chinese-Malaysians. There were remaining 11 heterozygous beta-thalassaemia carriers (eight Malays and four Chinese) whose mutations could not be identified. These unknown mutations require DNA sequencing for ultimate diagnosis. ARMS-PCR was used to confirm and validate the presence of the six mutations used in the RDBH-Strip Assays. It amplified each mutation as a separate and distinct PCR product. The Strip Assays showed 100% sensitivity and specificity through validation by ARMS-PCR. Therefore, the Strip Assay can be defined as a reliable diagnostic tool for accurate beta-thalassaemia mutation identification in Malays and Chinese Malaysians. In conclusion, the developed RDBH- Strip Assays [M(6) and C(6)] are accurate and rapid diagnostic tools for the identification of beta-thalassaemia mutations in the Malays and Chinese-Malaysians