Rare coinheritance of hemoglobin vancleave with severe beta-thalassemia mutation in a patient with secondary erythrocytosis.

Hemoglobin (Hb) Vancleave (NM_000518.5:c.431 A > T; dbSNP: rs33918338) is an extremely rare structural hemoglobin variant worldwide, and studies are limited. This report describes the case of a 16-year-old male patient who presented with secondary erythrocytosis. The diagnosis of Hb Vancleave, in combination with codon 41/42 (-TTCT) (NM_000518.5:c.126_129del; dbSNP: rs80356821), was confirmed by direct sequencing. This report highlights the importance of sequencing in the differential diagnosis of beta-thalassemia syndrome in Malaysia.

Characterization of New Alpha Zero (α0) Thalassaemia Deletion (--GB) among Malays in Malaysian Population.

Malaysia is a multicultural and multiethnic country comprising numerous ethnic groups. From the total population of 32.7 million, Malays form the bulk of the Bumiputera in Malaysia comprise about 69.9%, followed by Chinese 22.8%, Indian 6.6%, and others 0.7%. The heterogeneous population and increasing numbers of non-citizens in this country affects the heterogeneity of genetic diseases, diversity, and heterogeneity of thalassaemia mutations. Alpha (α)-thalassaemia is an inherited haemoglobin disorder characterized by hypochromic microcytic anaemia caused by a quantitative reduction in the α-globin chain. A majority of the α-thalassaemia are caused by deletions in the α-globin gene cluster. Among Malays, the most common deletional alpha thalassaemia is -α3.7 deletion followed by --SEA deletion. We described the molecular characterization of a new --GB deletion in our population, involving both alpha genes in cis. Interestingly, we found that this mutation is unique among Malay ethnicities. It is important to diagnose this deletion because of the 25% risk of Hb Bart's with hydrops fetalis in the offspring when in combination with another α0- thalassaemia allele. MLPA is a suitable method to detect unknown and uncommon deletions and to characterize those cases which remain unresolved after a standard diagnostic approach.

Gene Mutation Spectrum among Alpha-Thalassaemia Patients in Northeast Peninsular Malaysia.

(1) Background: Alpha (α)-thalassaemia is a genetic disorder that affects 5% of the world population. Deletional or nondeletional mutations of one or both HBA1 and HBA2 on chromosome 16 will result in reduced production of α-globin chains, a component of haemoglobin (Hb) that is required for the formation of red blood cells (RBCs). This study aimed to determine the prevalence, haematological and molecular characterisations of α-thalassaemia. (2) Method: The parameters were based on full blood count, high-performance liquid chromatography and capillary electrophoresis. The molecular analysis involved gap-polymerase chain reaction (PCR), multiplex amplification refractory mutation system-PCR, multiplex ligation-dependent probe amplification and Sanger sequencing. (3) Results: With a total cohort of 131 patients, the prevalence of α-thalassaemia was 48.9%, leaving the remaining 51.1% with potentially undetected α gene mutations. The following genotypes were detected: -α3.7/αα (15.4%), -α4.2/αα (3.7%), --SEA/αα (7.4%), αCSα/αα (10.3%), αAdanaα/αα (0.7%), αQuong Szeα/αα (1.5%), -α3.7/-α3.7 (0.7%), αCSα/αCSα (0.7%), -α4.2/αCSα (0.7%), -SEA/αCSα (1.5%), -SEA/αQuong Szeα (0.7%), -α3.7/αAdanaα (0.7%), --SEA/-α3.7 (2.2%) and αCSα/αAdanaα (0.7%). Indicators such as Hb (p = 0.022), mean corpuscular volume (p = 0.009), mean corpuscular haemoglobin (p = 0.017), RBC (p = 0.038) and haematocrit (p = 0.058) showed significant changes among patients with deletional mutations, but not between patients with nondeletional mutations. (4) Conclusions: A wide range of haematological parameters was observed among patients, including those with the same genotype. Thus, a combination of molecular technologies and haematological parameters is necessary for the accurate detection of α-globin chain mutations.

Next-Generation Sequencing (NGS) and Third-Generation Sequencing (TGS) for the Diagnosis of Thalassemia.

Thalassemia is one of the most heterogeneous diseases, with more than a thousand mutation types recorded worldwide. Molecular diagnosis of thalassemia by conventional PCR-based DNA analysis is time- and resource-consuming owing to the phenotype variability, disease complexity, and molecular diagnostic test limitations. Moreover, genetic counseling must be backed-up by an extensive diagnosis of the thalassemia-causing phenotype and the possible genetic modifiers. Data coming from advanced molecular techniques such as targeted sequencing by next-generation sequencing (NGS) and third-generation sequencing (TGS) are more appropriate and valuable for DNA analysis of thalassemia. While NGS is superior at variant calling to TGS thanks to its lower error rates, the longer reads nature of the TGS permits haplotype-phasing that is superior for variant discovery on the homologous genes and CNV calling. The emergence of many cutting-edge machine learning-based bioinformatics tools has improved the accuracy of variant and CNV calling. Constant improvement of these sequencing and bioinformatics will enable precise thalassemia detections, especially for the CNV and the homologous HBA and HBG genes. In conclusion, laboratory transiting from conventional DNA analysis to NGS or TGS and following the guidelines towards a single assay will contribute to a better diagnostics approach of thalassemia.

Molecular and hematological studies in a cohort of beta zero South East Asia deletion (ß°-thal SEA) from Malaysian perspective.

Abstract: We report the haematological parameters and molecular characterization of beta zero (ß°) South East Asia (SEA) deletion in the HBB gene cluster with unusually high levels of Hb F compared to a classical heterozygous beta zero (ß°)-thalassaemia. Methods: Retrospective study on 17 cases of (ß°) South East Asia (SEA) deletion from 2016 to 2019 referred to Institute for Medical Research were conducted. The clinical information and haematological profiles were evaluated. The mutation was analyzed, and the results were compared with other ß°-thalassaemia groups. For HBB gene genotyping, all the cases were subjected for multiplex gap-PCR, 5 cases were subjected for HBB gene sequencing for exclusion of compound heterozygous with other beta variants. Co-inheritance of α-thalassaemia were determined using multiplex gap-PCR and multiplex ARMS-PCR. Results: Seventeen cases were positive for ß°-thal SEA deletion. Fifteen cases were heterozygous and two were compound heterozygous for ß°-thal SEA deletion. The results were compared with 182 cases of various heterozygous ß° deletions and mutations. The mean Hb for heterozygous ß°-thal SEA deletion (13.44 ± 1.45 g/dl) was normal and significantly higher than heterozygous IVS 1-1 and Codon 41/42 (post hoc test, p < 0.05). The medians for the MCV and MCH of ß°-thal SEA deletion were significantly higher than for all heterozygote ß°-thalassaemia traits (Mann Whitney test, p < 0.05). Patients with ß°-thal SEA deletion had elevated levels of Hb A2 consistent with ß-thalassaemia traits, with Hb F levels consistent with HPFH or δß-thalassaemia carriers. The median for Hb A2 was 4.00 + 1.00%, similar to that observed in other ß°-thalassaemia groups except for IVS 1-1 mutation (median 5.30 + 0.45%) and ß°-Filipino (∼45 kb deletion) deletion (median 6.00 + 0.58). Interestingly, we found that Hb F levels for ß°-thal SEA deletion were statistically higher than other ß°-thalassaemia mutations (median 19.00 + 5.50%, p < 0.05), except for the ß°-thal 3.5 kb deletion group. Conclusion: We conclude that ß°-thal SEA deletion has a unique haematological parameters of beta zero thalassaemia trait. We affirm to classifying this deletion as SEA-HPFH based on previous studies considering the phenotype features rather than the molecular defect of ß°-thal SEA deletion, as this will make it easier to offer genetic counselling to affected individuals.

Multiplex amplification refractory mutation system (MARMS) for the detection of ß-globin gene mutations among the transfusion-dependent ß-thalassemia Malay patients in Kelantan, Northeast of Peninsular Malaysia.

The aim of this study was to adapt MARMS with some modifications to detect beta mutation in our cohort of thalassemia patients. We focused only on transfusion-dependent thalassemia Malay patients, the predominant ethnic group (95%) in the Kelantanese population. Eight mutations were identified in 46 out of 48 (95.83%) beta thalassemia alleles. Most of the patients (54.2%) were compound heterozygous with co-inheritance Cd 26 (G>A). The frequencies of spectrum beta chain mutation among these patients are presented in Table 2. Among the transfusion dependent beta thalassemia Malay patients studied, 26 patients were found to be compound heterozygous and the main alleles were Cd 26 (G>A). Compound heterozygous mutation of Cd 26 (G>A) and IVS 1-5 (G>C) were 12 (46.2%), Cd 26 (G>A) and Cd 41/42 (TTCT) were 9 (34.6%), Cd 26 (G>A) and IVS 1-1 (G>C) were 2 (7.7%) respectively. Meanwhile the minority were made of a single compound heterozygous of Cd 26 (G>A) and Cd 71/72, Cd 26 (>A) and Cd 17 (A>T), Cd 26 (G>A) and -28 (G>A) respectively. Twenty out of forty six patients were shown to have homozygous of IVS 1-5 (G>C) were 2 (10.0%), Cd 26 (G>A) were 15 (75.0%), Cd 19 (A>G) were 1 (5.0%), and IVS 1-1 (G>T) were 2 (10.0%). The beta chain mutations among the Kelantanese Malays followed closely the distribution of beta chain mutations among the Thais and the Malays of the Southern Thailand. The G-C transition at position 5 of the IVS 1-5 mutation was predominant among the Malay patients. In conclusion, this method has successfully identified the mutation spectrum in our cohort of transfusion-dependent beta thalassemia patients, and this method is equally effective in screening for mutation among thalassemia patients.

Distribution of alpha thalassaemia gene variants in diverse ethnic populations in malaysia: data from the institute for medical research.

Alpha thalassaemia is highly prevalent in the plural society of Malaysia and is a public health problem. Haematological and molecular data from 5016 unrelated patients referred from various hospitals to the Institute for Medical Research for α thalassaemia screening from 2007 to 2010 were retrieved. The aims of this retrospective analysis were to describe the distribution of various alpha thalassaemia alleles in different ethnic groups, along with their genotypic interactions, and to illustrate the haematological changes associated with each phenotype. Amongst the patients, 51.2% (n = 2567) were diagnosed with α thalassaemia. Of the 13 α thalassaemia determinants screened, eight different deletions and mutations were demonstrated: three double gene deletions, --(SEA), --(THAI), --(FIL); two single-gene deletions, α-³·7 and -α4·²; and three non-deletion mutations, Cd59G > A (haemoglobin [Hb] Adana), Cd125T > C (Hb Quong Sze) and Cd142 (Hb Constant Spring). A high incidence of α-³·7 deletion was observed in Malays, Indians, Sabahans, Sarawakians and Orang Asli people. However, the --SEA deletion was the most common cause of alpha thalassaemia in Chinese, followed by the α-³·7 deletion. As many as 27 genotypic interactions showed 1023 α thalassaemia silent carriers, 196 homozygous α⁺ thalassaemia traits, 973 heterozygous α° thalassaemia carriers and 375 patients with Hb H disease. Statistical analysis showed a significant difference in the distribution of α thalassaemia determinants amongst the various ethnic groups. Hence, the heterogeneous distribution of common determinants indicated that the introduction of an ethnicity-targeted hierarchical α thalassaemia screening approach in this multi-ethnic Malaysian population would be effective.

Detection of ß-globin Gene Mutations Among ß-thalassaemia Carriers and Patients in Malaysia: Application of Multiplex Amplification Refractory Mutation System-Polymerase Chain Reaction.

BACKGROUND: ß-thalassaemia is one of the most common single-gene disorders worldwide. Each ethnic population has its own common mutations, accounting for the majority of cases, with a small number of mutations for the rarer alleles. Due to the heterogeneity of ß-thalassaemia and the multi-ethnicity of Malaysians, molecular diagnostics may be expensive and time consuming. METHODS: A simple polymerase chain reaction (PCR) approach involving a multiplex amplification refractory mutation system (MARMS) and one amplification refractory mutation system (ARMS), consisting of 20 ß-globin gene mutations, were designed and employed to investigate ß-thalassaemia patients and carriers. RESULTS: Out of 169 carriers tested with the MARMS, Cd 41/42 (-TTCT), Cd 26 (A-G) HbE, IVS 1-1 (G-T), and IVS 1-5 (G-C) were the most common mutations, accounting for 78.1%. Among the Malays, Cd 26 (A-G) HbE, Cd 41/42 (-TTCT), IVS 1-1 (G-T), and IVS 1-5 (G-C) were the most common mutations, accounting for 81.4%, whereas Cd 41/42 (-TTCT) and IVS 2-654 (C-T) were most common among the Chinese (79.1%). CONCLUSION: We propose the use of this cheap, easy to interpret, and simple system for the molecular diagnostics of ß-thalassaemia among Malaysians at the Institute for Medical Research (IMR).