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Detection of Haemoglobin S using Multiplex Ligation-Dependent Probe Amplification and Flow-through Hybridization Techniques: Experience in a Tertiary Hospital

Original article

Abstrak

Hemoglobin S (HbS, α2β26GluVal) merupakan variasi hemoglobin yang terbentuk hasil daripada mutasi GAG GTG pada kodon 6 gen β-globin. Hemoglobinopati haemoglobin S (HbS) jarang ditemui di kalangan penduduk Malaysia tetapi selalunya dijumpai di kalangan pendatang asing dari Afrika. Walau bagaimanapun beberapa kes didapati dalam kaum India dan Melayu. Kajian ini meninjau keputusan makmal pesakit HbS dan penggunaan “multiplex ligation-dependent probe amplification” (MLPA) dan “flow-through hybridization” (FTH) dalam mengesan mutasi HbS. HbS dikenalpasti melalui kromatografi cecair prestasi tinggi (HPLC) dan/atau elektroforesis kapilari serta elektroforesis hemoglobin. Analisis molekul dijalankan menggunakan kaedah MLPA, FTH dan penjujukan Sanger.  Dua warga Afrika, tiga Melayu dan dua India berusia antara 2-31 tahun telah dikenalpasti. Lima pesakit adalah HbS homozigot, seorang kompaun heterozigot HbS/β-talasemia dan seorang lagi pembawa HbS. Tahap hemoglobin (Hb) kes HbS homozigot adalah antara 7.4-10.2 g/dL dengan aras HbS dan HbF diantara 58.3-94.7% dan 1.5-35.5%. Hb untuk kes kompaun heterozigot HbS/β-talasemia adalah 5.8 g/dL dan normal pada pembawa HbS. Aras HbS, HbF dan HbA2 untuk HbS/β-talasemia dan pembawa HbS adalah 67%, 27.2% dan 4.2%, dan 38.6%, 0.1% and 2.8% setiap satu. Kedua-dua kaedah MLPA dan FTH berjaya mengesan mutasi HbS dalam semua kes, manakala cuma FTH dapat menentukan zygositi mutasi HbS dan β-talasemia dalam satu ujian yang sama.

Abstract

Haemoglobin S (HbS, α2β26GluVal) is a variant haemoglobin resulted from GAGGTG mutation on codon 6 of HBB gene. HbS haemoglobinopathy is uncommon in Malaysia and mainly seen in immigrants. However, Malaysian Indians and Malays are rarely affected. This study reviewed the laboratory findings of patients with HbS and the utilization of multiplex ligation-dependent probe amplification (MLPA) and flow-through hybridization (FTH) in the detection of HbS mutation. HbS was identified and quantified by high performance liquid chromatography (HPLC), capillary electrophoresis (CE) and cellulose acetate gel electrophoresis. Molecular analysis was performed using MLPA, FTH and Sanger Sequencing. Two Africans, three Malays and two Indian individuals aged between 2-31 years were identified from our laboratory. Five patients were homozygous HbS, one was compound heterozygous HbS/β-thalassemia and one was a carrier of HbS. The patients with homozygous HbS had their haemoglobin (Hb) ranging from 7.4-10.2 g/dL with HbS and HbF levels of 58.3-94.7% and 1.5-35.5%, respectively. The Hb of compound heterozygous HbS/β-thalassaemia patients was 5.8 g/dL and was normal in heterozygous HbS. HbS, HbF and HbA2 levels for the HbS/β-thalassaemia and the carrier were 67%, 27.2% and 4.2%, and 38.6%, 0.1% and 2.8%, respectively. Both MLPA and FTH successfully detected HbS mutation in all cases but only FTH could identify the zygosity of the HbS mutation together with underlying concomitant β-thalassaemia in a single test.