Balkan Journal of Medical Genetics

MOLECULAR DIAGNOSTICS OF β-THALASSEMIA
Atanasovska B1, Bozhinovski G1, Chakalova L1, Kocheva S2, Karanfilski O3, Plaseska-Karanfiska D1,*
*Corresponding Author: Professor Dr. Dijana Plaseska-Karanfilska, Research Centre for Genetic Engineering and Biotechnology “Georgi D. Efremov”, Macedonian Academy of Sciences and Arts, Krste Misirkov 2, Skopje 1000, Republic of Macedonia; Tel: +389(0)2 3235410; Fax: +389 (0)2 3115434; E-mail: dijana@manu.edu.mk
page: 61

INTRODUCTION

Hemoglobinopathies are caused by genetic defects affecting the globin genes encoding for the a and b chains of the hemoglobin (Hb) molecule. In the Mediterranean region in particular, there is a high incidence of mutations disturbing the function of the HBB gene [1]. Some of the mutations reduce or eliminate the expression of the HBB gene leading to net Hb deficiency and b-thalassemia (b-thal) [2]. Other mutations give rise to abnormal Hb variants such as Hb S [b6(A3)Glu®Val, GAG>GTG] and Hb Lepore-Boston-Washington (Hb LBW; d87- bIVS-II-8) [2-4]. The severity of the clinical symptoms depends on the molecular consequences of the genetic abnormality or combination thereof and is modulated by other genetic and environmental factors [2,5,6]. The best practice in hemoglobinopathy diagnostics involves molecular identification of the causative mutations. Molecular diagnostics of b-thal has been a major focal point of the activities at the International Reference Laboratory for Haemoglobinopathies, Research Centre for Genetic Engineering and Biotechnology (RCGEB) “Georgi D. Efremov”, Skopje, Republic of Macedonia at Skopje, Republic of Macedonia. Thousands of cases have been tested for the presence of mutations throughout the years. This study has helped determine the frequencies of the most common mutations in Macedonia and several neighboring countries [7-10]. Until recently, the primary method for the detection of b-thal mutations in our laboratory was allele-specific oligonucleotide hybridization, whereby consecutive rounds of hybridization with several mutation-specific probes were performed. We were looking to reduce the time necessary for reaching a definitive diagnosis by introducing a semi-automated technique allowing simultaneous detection of the most commonly occurring b-thal mutations. Unfortunately, published techniques, such as primer extension and melting curve analysis were suboptimal in terms of precision and multiplexing [11-13]. We therefore set out to develop a new assay for the identification of common Mediterranean mutations that occur at high frequencies in the Republic of Macedonia, namely HBB:c.93-21G>A, HBB:c.92+1G>A, HBB:c.92+6T>C, HBB:c.118C>T, HBB:c.316- 106C>G, HBB:c.17_18 del CT, HBB:c.20delA, HBB:c.25_26delAA and HBB:c.20 A>T [14,15]. Our protocol utilizes single-nucleotide primer extension to interrogate the whole panel of mutations in a single, internally controlled reaction. We also designed a complementary duplex polymerase chain reaction (PCR) assay for detection of the Hb LBW deletion NG_000007.3:g.63632_71046 del, the most common Hb variant in our geographic area [9]. Both assays have been thoroughly validated and evaluated as described elsewhere (Atanasovska et al., in preparation). Here we report the overall diagnostic strategy for the identification of b-thal mutations and Hb variants that includes the new assays.

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