Detection of α-thalassaemia in neonates on cord blood and dried blood spot samples by capillary electrophoresis

Haemoglobin Bart’s (Hb Bart’s) level is associated with α-thalassaemia traits in neonates, enabling early diagnosis of α-thalassaemia. The study aimed to detect and quantify the Hb Bart’s using Cord Blood (CB) and CE Neonat Fast Hb (NF) progammes on fresh and dried blood spot (DBS) specimen respectively by capillary electrophoresis (CE). Methods: Capillarys Hemoglobin (E) Kit (for CB) and Capillarys Neonat Hb Kit (for NF) were used to detect and quantify Hb Bart’s by CE in fresh cord blood and dried blood spot (DBS) specimens respectively. High performance liquid chromatography (HPLC) using the β-Thal Short Programme was also performed concurrently with CE analysis. Confirmation was obtained by multiplex ARMS Gap PCR. Results: This study was performed on 600 neonates. 32/600 (5.3%) samples showed presence of Hb Bart’s peak using the NF programme while 33/600 (5.5%) were positive with CB programme and HPLC methods. The range of Hb Bart’s using NF programme and CB programme were (0.5–4.1%) and (0.5-7.1%), respectively. Molecular analysis confirmed all positive samples possessed α-thalassaemia genetic mutations, with 23/33 cases being αα/--SEA, four -α3.7/-α3.7, two αα/-α3.7 and three αα/ααCS. Fifty Hb Bart’s negative samples were randomly tested for α-genotypes, three were also found to be positive for α-globin gene mutations. Thus, resulting in sensitivity of 91.7% and 88.9% and specificity of 100% for the Capillarys Cord Blood programme and Capillarys Neonat Fast programme respectively. Conclusion: Both CE programmes using fresh or dried cord blood were useful as a screening tool for α-thalassaemia in newborns. All methods show the same specificity (100%) with variable, but acceptable sensitivities in the detection of Hb Bart.

Hb Lepore/β0-Thalassaemia With α+-Thalassaemia Interactions, A Potential Diagnostic Pitfall

Haemoglobin (Hb) Lepore is a variant Hb consisting of two α-globin and two δβ-globin chains. In a heterozygote, it is associated with clinical findings of thalassaemia minor, but interactions with other haemoglobinopathies can lead to various clinical phenotypes and pose diagnostic challenges. We reported a pair of siblings from a Malay family, who presented with pallor and hepatosplenomegaly at the ages of 21 months and 14 months old. The red cell indices and peripheral blood smears of both patients showed features of thalassaemia intermedia. Other laboratory investigations of the patients showed conflicting results. However, laboratory investigation results of the parents had led to a presumptive diagnosis of compound heterozygote Hb Lepore/β-thalassaemia and co-inheritance α+-thalassaemia (-α3.7). Hb Lepore has rarely been detected in Southeast Asian countries, particularly in Malaysia. These two cases highlight the importance of family studies for accurate diagnosis, hence appropriate clinical management and genetic counseling.

Molecular characteristic of alpha thalassaemia among patients diagnosed in UKM Medical Centre

Alpha (α) thalassaemia is the most common inherited disorder in Malaysia. The clinical severity is dependant on the number of α genes involved. Full blood count (FBC) and haemoglobin (Hb) analysis using either gel electrophoresis, high performance liquid chromatography (HPLC) or capillary zone electrophoresis (CE) are unable to detect definitively alpha thalassaemia carriers. Definitive diagnosis of α-thalassaemias requires molecular analysis and methods of detecting both common deletional and non-deletional molecular abnormailities are easily performed in any laboratory involved in molecular diagnostics. We carried out a retrospective analysis of 1623 cases referred to our laboratory in Universiti Kebangsaan Malaysia Medical Centre (UKMMC) for the diagnosis of α-thalassaemia during the period October 2001 to December 2012. We examined the frequency of different types of alpha gene abnormalities and their haematologic features. Molecular diagnosis was made using a combination of multiplex polymerase reaction (PCR) and real time PCR to detect deletional and non-deletional alpha genes relevant to southeast Asian population. Genetic analysis confirmed the diagnosis of α-thalassaemias in 736 cases. Majority of the cases were Chinese (53.1%) followed by Malays (44.2%), and Indians (2.7%). The most common gene abnormality was αα/--SEA (64.0%) followed by αα/-α3.7 (19.8%), -α3.7 /--SEA (6.9%), αα/ααCS (3.0%), --SEA/--SEA (1.2%), -α3.7/-α3.7 (1.1%), αα/-α4.2 (0.7%), -α4.2/--SEA (0.7%), -α3.7/-α4.2 (0.5%), ααCS/-- SEA (0.4%), ααCS/ααCd59 (0.4%), ααCS/ααCS (0.4%), -α3.7/ααCd59 (0.3%), αα/ααCd59 (0.1%), αα Cd59/ ααIVS I-1 (0.1%), -α3.7/ααCS (0.1%) and --SEA /ααCd59 (0.1%). This data indicates that the molecular abnormalities of α-thalassaemia in the Malaysian population is heterogenous. Although α-gene deletion is the most common cause, non-deletional α-gene abnormalities are not uncommon and at least 3 different mutations exist. Establishment of rapid and easy molecular techniques is important for definitive diagnosis of alpha thalassaemia, an important prerequisite for genetic counselling to prevent its deleterious complications.

Distribution of alpha thalassaemia in 16 year old Malaysian Students in Penang, Melaka and Sabah

Objectives: Alpha thalassaemia is wide spread in Malaysia and is a public health problem. This study aimed to describe the carrier frequencies of α‒thalassaemia and its distribution among major ethnic groups in three states of Malaysia. Methods: Educational forums were organised and study was explained to students from three schools. Students were invited to take part in the screening with parent consent. A total of 8420 adolescent students aged 16 years volunteered to participate in the study. Peripheral blood samples were analysed for complete blood counts, haemoglobin quantification and typing, and serum ferritin levels. Genomic DNA was used for screening alpha thalassaemia alleles by PCR based molecular methods. Results: We identified seven α‒globin gene defects in 341 (4.08%) students: amongst them α+‒ and α0‒thalassaemias were detected in 232 (2.77%) and 107 (1.28%) students respectively. Genotype ‒α3.7/αα was the most prevalent among sub-populations of Malay, indigenous communities of Sahab and Indian, while ‒‒SEA/αα deletion is more prevalent in Malaysian Chinese. It is estimated that 63 pregnancies annually are at risk of Hb Bart’s hydrops fetalis. Conclusions: We have demonstrated the prevalence and mutation patterns of α‒thalassaemia in the 16 year olds in three states of Malaysia. High α0‒thalassaemia deletions amongst the study subjects place these carriers at an increased risk of conceiving fetuses with HbH disease and Hb Bart’s hydrops fetalis should they choose another heterozygous partner. It is therefore highly recommended to institute community screening programmes and provide prospective carriers with genetic counselling to help them make informed choices.

Co-inheritance of compound heterozygous Hb Constant Spring and a single –α3.7 gene deletion with heterozygous δβ thalassaemia: A diagnostic challenge

Haemoglobin Constant Spring (Hb CS) mutation and single gene deletions are common underlying genetic abnormalities for alpha thalassaemias. Co-inheritance of deletional and non-deletional alpha (α) thalassaemias may result in various thalassaemia syndromes. Concomitant co-inheritance with beta (β) and delta (δ) gene abnormalities would result in improved clinical phenotype. We report here a 33-year-old male patient who was admitted with dengue haemorrhagic fever, with a background history of Grave’s disease, incidentally noted to have mild hypochromic microcytic red cell indices. Physical examination revealed no thalassaemic features or hepatosplenomegaly. His full blood picture showed hypochromic microcytic red cells with normal haemoglobin (Hb) level. Quantitation of Hb using high performance liquid chromatography (HPLC) and capillary electrophoresis (CE) revealed raised Hb F, normal Hb A2 and Hb A levels. There was also small peak of Hb CS noted in CE. H inclusions was negative. Kleihauer test was positive with heterocellular distribution of Hb F among the red cells. DNA analysis for α globin gene mutations showed a single -α-3.7 deletion and Hb CS mutation. These fi ndings were suggestive of compound heterozygosity of Hb CS and a single -α-3.7 deletion with a concomitant heterozygous δβ thalassaemia. Co-inheritance of Hb CS and a single -α-3.7 deletion is expected to result at the very least in a clinical phenotype similar to that of two alpha genes deletion. However we demonstrate here a phenotypic modifi cation of α thalassemia presumptively as a result of co-inheritance with δβ chain abnormality as suggested by the high Hb F level.

Haemoglobinopathies in Southeast Asia.

In Southeast Asia α-thalassaemia, β-thalassaemia, haemoglobin (Hb) E and Hb Constant Spring (CS) are prevalent. The abnormal genes in different combinations lead to over 60 different thalassaemia syndromes, making Southeast Asia the locality with the most complex thalassaemia genotypes. The four major thalassaemic diseases are Hb Bart's hydrops fetalis (homozygous α-thalassaemia 1), homozygous β-thalassaemia, β-thalassaemia/Hb E and Hb H diseases. α-Thalassaemia, most often, occurs from gene deletions whereas point mutations and small deletions or insertions in the β-globin gene sequence are the major molecular defects responsible for most β-thalassaemias. Clinical manifestations of α-thalassaemia range from asymptomatic cases with normal findings to the totally lethal Hb Bart's hydrops fetalis syndrome. Homozygosity of β-thalassaemia results in a severe thalassaemic disease while the patients with compound heterozygosity, β-thalassaemia/Hb E, present variable severity of anaemia, and some can be as severe as homozygous β-thalassaemia. Concomitant inheritance of α-thalassaemia and increased production of Hb F are responsible for mild clinical phenotypes in some patients. However, there are still some unknown factors that can modulate disease severity in both α- and β-thalassaemias. Therefore, it is possible to set a strategy for prevention and control of thalassaemia, which includes population screening for heterozygotes, genetic counselling and foetal diagnosis with selective abortion of affected pregnancies.

Concurrent Inheritance of Deletional α-thalassaemia in Malays with HbE Trait

Introduction: HbE is the commonest beta haemoglovin (Hb) variant in Southeast Asia. It causes a reduction in synthesis of beta-globin E (βE) chain. Studies indicate HbE coinherited with α-thalassaemia leads to a milder clinical phenotype. This study investigates the concomitant inheritance of α-thalassaemia in Malays with HbE. Methods: Four hundred and fourteen (414) blood samples were screened for haemoglobinopathy using primarily the first 3 steps of the BHES [(B) blood counts, blood film: (H), HPLC; (E),elstrophoresis; (S),stability)] protocol. Complete blood counts were generated on an automated blood cell analyser, HB typing with cation exchange high-performance liquid chromatography (HPLC) and 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 α-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 (α⁺) (α-3.7 kb deletion) and 1 (2.2%) the alpha-thalassaemia-1 (α⁰) (—SEA 20.5kb 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/α-thalassaemia 2, followed by HbE/α-thalassaemia 1. Conclusion: Molecular screening of deletional α-thalassaemia identified its concurrent inheritance in 11.1% of Malays who were HbE carriers. This information will guide genetic counseling and the planning of treatment modalities in patients with HbE alpha-thalassaemia.