The Characteristics of Compound Heterozygosity for Hemoglobin G-Makassar with Hb E in Malaysia.

Background: Human hemoglobin of G-Makassar and hemoglobin E (Hb E) are hemoglobin variants that affect Beta (ß) globin. Hb G-Makassar is a very rare variant while Hb E is estimated to affect at least one million people worldwide. Both Hb G-Makassar and Hb E can be inherited in the heterozygous, homozygous or compound heterozygous state. This case series describes the characteristics of four individuals with compound heterozygosity for Hb G-Makassar/Hb E cases in Malaysia. To the best of our knowledge, these are the only four individuals with this genotype reported in the literature. Case Series: We present four cases of compound heterozygosity for Hb G-Makassar/Hb E identified from October 2014 to January 2021. All the cases were incidental findings whereby the screening Hb analysis showed the presence of peaks in both Hb S and Hb E zones on capillary electrophoresis (CE) and cation-exchange high-performance liquid chromatography (HPLC). Molecular analysis confirmed the findings of compound heterozygous Hb G-Makassar/Hb E. Two cases had a history of anemia secondary to unrelated conditions that resolved with treatment of the underlying cause. The other two cases were asymptomatic individuals who were detected through Malaysia's National Thalassemia Screening program. On the last follow-up, all the individuals were well, non-transfusion dependent, and had no reported history of chronic anemia, bleeding, hemolysis or thromboembolism complications. Conclusion: The cases reported here highlight the possibilities for rare compound heterozygous states in multi-ethnicity populations such as Malaysia. Compound heterozygous Hb G-Makassar/Hb E individuals are clinically silent with laboratory values suggesting microcytic and hypochromic red blood cells. Further local epidemiology or population studies with genotyping tests are required for a better understanding of the diversity of its clinical phenotype.

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.

Clinical and haematological characteristics of 38 individuals with Hb G-Makassar in Malaysia.

Haemoglobin (Hb) G-Makassar is a rare Hb variant. It presents a diagnostic challenge as it imitates sickle Hb (Hb S) in standard electrophoresis and high-performance liquid chromatography assays requiring DNA analysis to confirm diagnosis. Both have point mutations in codon 6, exon 1 in the ß-globin (HBB) gene with different pathogenicities. This study describes the clinical phenotype, haematology and genotype of Hb G-Makassar. Clinical and laboratory data of 38 cases of Hb G-Makassar over 8 years were analysed. Hb G-Makassar was confirmed by a direct sequencing of HBB gene and co-inheritance of α-thalassaemia determined through multiplex gap-PCR and multiplex Amplification Refractory Mutation System polymerase chain reaction. All cases were Malays, predominantly from Terengganu (n = 20, 52.6%). There were 14 (36.8%) males and 24 (63.2%) females with median age of 25 years. Majority (n = 33, 86.8%) had features of thalassaemia trait with mean ± SD for Hb, mean cell volume (MCV) and mean cell haemoglobin (MCH) as 13.21 g/dL ± 1.69, 73.06 ± 4.48 fL and 24.71 ± 1.82 pg, respectively. None had evidence of haemolysis or thromboembolic complications. Six genotypes were identified; ßG-Makassar/ß,αα/αα (n = 19, 50.0%), ßG-Makassar/ßE,αα/αα (n = 4, 10.5%), ßG-Makassar/ßNewYork,αα/αα (n = 1, 2.6%), ßG-Makassar/ß,αα/-α (n = 11, 28.9%), ßG-Makassar/ß,αα/αAdanaα (n = 2, 5.3%) and ßG-Makassar/ß,αα/-SEA (n = 1, 2.6%). The ßG-Makassar/ß,αα/αα showed that features of thalassaemia trait with mean ± SD for Hb, MCV and MCH were 13.74 g/dL ± 2.40, 76.18 ± 6.02 fL and 25.79 ± 2.41 pg, respectively. This is the largest study reporting a significant number of Hb G-Makassar in Malaysia. Although the mutation is similar to Hb S, the phenotype is benign.

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.

Evaluation of in silico predictors on short nucleotide variants in HBA1, HBA2, and HBB associated with haemoglobinopathies.

Haemoglobinopathies are the commonest monogenic diseases worldwide and are caused by variants in the globin gene clusters. With over 2400 variants detected to date, their interpretation using the American College of Medical Genetics and Genomics (ACMG)/Association for Molecular Pathology (AMP) guidelines is challenging and computational evidence can provide valuable input about their functional annotation. While many in silico predictors have already been developed, their performance varies for different genes and diseases. In this study, we evaluate 31 in silico predictors using a dataset of 1627 variants in HBA1, HBA2, and HBB. By varying the decision threshold for each tool, we analyse their performance (a) as binary classifiers of pathogenicity and (b) by using different non-overlapping pathogenic and benign thresholds for their optimal use in the ACMG/AMP framework. Our results show that CADD, Eigen-PC, and REVEL are the overall top performers, with the former reaching moderate strength level for pathogenic prediction. Eigen-PC and REVEL achieve the highest accuracies for missense variants, while CADD is also a reliable predictor of non-missense variants. Moreover, SpliceAI is the top performing splicing predictor, reaching strong level of evidence, while GERP++ and phyloP are the most accurate conservation tools. This study provides evidence about the optimal use of computational tools in globin gene clusters under the ACMG/AMP framework.

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.

Global Globin Network Consensus Paper: Classification and Stratified Roadmaps for Improved Thalassaemia Care and Prevention in 32 Countries.

The Global Globin Network (GGN) is a project-wide initiative of the Human Variome/Global Variome Project (HVP) focusing on haemoglobinopathies to build the capacity for genomic diagnosis, clinical services, and research in low- and middle-income countries. At present, there is no framework to evaluate the improvement of care, treatment, and prevention of thalassaemia and other haemoglobinopathies globally, despite thalassaemia being one of the most common monogenic diseases worldwide. Here, we propose a universally applicable system for evaluating and grouping countries based on qualitative indicators according to the quality of care, treatment, and prevention of haemoglobinopathies. We also apply this system to GGN countries as proof of principle. To this end, qualitative indicators were extracted from the IthaMaps database of the ITHANET portal, which allowed four groups of countries (A, B, C, and D) to be defined based on major qualitative indicators, supported by minor qualitative indicators for countries with limited resource settings and by the overall haemoglobinopathy carrier frequency for the target countries of immigration. The proposed rubrics and accumulative scores will help analyse the performance and improvement of care, treatment, and prevention of haemoglobinopathies in the GGN and beyond. Our proposed criteria complement future data collection from GGN countries to help monitor the quality of services for haemoglobinopathies, provide ongoing estimates for services and epidemiology in GGN countries, and note the contribution of the GGN to a local and global reduction of disease burden.