Rapid Screening of Multiple β-Globin Gene Mutations by Real-Time PCR on the LightCycler: Application to Carrier Screening and Prenatal Diagnosis of Thalassemia Syndromes

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Abstract
Background: Hemoglobinopathies are priority genetic diseases for prevention programs. Rapid genotype characterization is fundamental in the diagnostic laboratory, especially when offering prenatal diagnosis for carrier couples. Methods: As a model, we designed a protocol based on the LightCycler™ technology to screen for a spectrum of β-globin gene mutations in the Greek population. Design was facilitated by dual fluorochrome detection and close proximity of many mutations. Three probe sets were capable of screening 95% of β-globin gene mutations in the Greek population, including IVSII-745C→G, HbS, Cd5-CT, Cd6-A, Cd8-AA, IVSI-1G→A, IVSI-5G→A, IVSI-6T→C, IVSI-110G→A, and Cd39 C→T. Results: The protocol, standardized by analysis of 100 β-thalassemia heterozygotes with known mutations, was 100% reliable in distinguishing wild-type from mutant alleles. Subsequent screening of 100 Greek β-thalassemia heterozygotes with unknown mutations found 96 of 100 samples heterozygous for 1 of the 10 mutations, although melting curves were indistinguishable for mutations ΗbS/Cd6 and IVSI-5/IVSI-1, indicating a need of alternative methods for definitive diagnosis. One sample demonstrating a unique melting curve was characterized by sequencing as Cd8/9+G. Three samples carried mutations outside the gene region covered by the probes. The protocol was 100% accurate in 25 prenatal diagnosis samples, with 14 different genotype combinations diagnosed. The protocol was also flexible, detecting five β-globin gene mutations from other population groups (IVSI-1G→T, IVSI-5G→C, IVSI-116T→G, Cd37 TGG→TGA, and Cd41/42 −TCTT). Conclusions: The described LightCycler system protocol can rapidly screen for many β-globin gene mutations. It is appropriate for use in many populations for directing definitive mutation diagnosis and is suited for rapid prenatal diagnosis with low cost per assay.