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

RESULTS AND DISCUSSION

The vast majority of subjects referred to the laboratory were heterozygous carriers of hemoglobinopathy mutations and non carriers referred by their medical specialist or general practitioner to exclude a hemoglobinopathy. Our diagnostic algorithm is presented in Figure 1. When a case is referred to the laboratory for hemoglobinopathy examination, we initially review the patient data including standard blood test results and family history and run a second tier of hematology tests, namely HPLC quantification of normal and abnormal Hb variants and assessment of red cell osmotic fragility. If a Hb variant, e.g., the relatively common Hb LBW, is present in a sample it is identified on the HPLC profile at this stage by virtue of its specific retention time, proportion of total Hb and peak characteristics. Samples identified as Hb LBW heterozygotes or homozygotes by HPLC are tested by the Lepore PCR assay to confirm the presence of the Hb LBW chromosome. This approach warrants unequivocal identification of Hb LBW cases at an early stage of the procedure. These samples are then excluded from further analysis unless a severe clinical picture suggests compound heterozygosity (see below). Thalassemia is diagnosed based on red blood cell indices combined with the results of the osmotic fragility test and the HPLC analysis. The main diagnostic parameters pointing to probable b-thal trait are: elevated Hb A2, low total Hb level, low mean corpuscular volume (MCV), low mean corpuscular Hb (MCH), elevated Hb F, decreased osmotic fragility. b-Thalassemia symptoms can vary between carriers, e.g., an individual could feature most if not all of these indicators to almost none or borderline values. Using several independent parameters minimizes the risk of missing b-thal carriers in the initial screen. The clinical picture for homozygotes and compound heterozygotes is largely clearer and there is a much lower risk of misdiagnosing these cases. Based on the results of these analyses. we assign cases for molecular detection of b-thal trait. These samples are first tested for the presence of the eight most common b-thal mutations by the multiplex single-nucleotide primer extension assay. In the past, our protocol for molecular characterization of the HBB gene was prohibitively time-consuming, forcing us to apply relatively stringent inclusion criteria. The introduction of the new multiplex assay allows us to test the majority of the subjects referred to the laboratory. In particular, children are tested for the most common mutations by default even if their blood test results are compatible with a normal genotype. Parents are also invited to provide blood samples so that the diagnosis is cross-checked independently. It has to be pointed out that the presence of high levels of Hb F in children younger than 1 year of age can mask the manifestation of b-thal. It is therefore important to apply definitive DNA tests to eliminate false negative results. Collectively, the most common b-thal mutations detected by the multiplex assay account for approximately 90.0% of all hemoglobinopathy cases in the Republic of Macedonia [7,8]. In case the multiplex assay yields a normal genotype while the hematology data points to the presence of a b-thal mutation, the sample is assigned for sequencing of the HBB gene in order to reveal genetic variations not tested in the multiplex assay. Since we incorporated the multiplex assay and the Lepore PCR assay into the routine hemoglobinopathy work-up in our laboratory, we have processed a total of 186 patient samples. For 83 cases, the data from the various hematological tests were concordant and compatible with a normal genotype and further testing was not necessary. Nevertheless, these samples were assayed by the multiplex assay partly to corroborate the absence of common mutations, partly to assess the assay reproducibility. Hemoglobinopathies were thus excluded in these cases. For the remaining 103 cases, there were indications for genetic abnormalities affecting the HBB gene. Three of these patients were confirmed to be Hb LBW heterozygotes (Table 1) and were not tested further. We applied the multiplex thalassemia assay to the remaining 100 samples and identified 95 b-thal heterozygotes, each carrying one of the b-thal mutations included in the assay (Table 1). The multiplex assay failed to identify any mutations in five samples. These were subjected to direct sequencing of the HBB gene and were found to carry other b-thal mutations (Table 1) in unison with the hematology data. Thus, a conclusive diagnosis was reached for every case. Importantly, the newly developed procedures have significantly reduced the time and cost necessary to complete the analyses.

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