Vrettou Christina, Traeger-Synodinos Joanne, Tzetis Maria, Malamis George, Kanavakis Emmanuel
Medical Genetics, Athens University, St Sophia's Children's Hospital, Athens 11527, Greece.
Clin Chem. 2003 May;49(5):769-76. doi: 10.1373/49.5.769.
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.
As a model, we designed a protocol based on the LightCycler technology to screen for a spectrum of beta-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 beta-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.
The protocol, standardized by analysis of 100 beta-thalassemia heterozygotes with known mutations, was 100% reliable in distinguishing wild-type from mutant alleles. Subsequent screening of 100 Greek beta-thalassemia heterozygotes with unknown mutations found 96 of 100 samples heterozygous for 1 of the 10 mutations, although melting curves were indistinguishable for mutations HbS/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 beta-globin gene mutations from other population groups (IVSI-1G-->T, IVSI-5G-->C, IVSI-116T-->G, Cd37 TGG-->TGA, and Cd41/42 -TCTT).
The described LightCycler system protocol can rapidly screen for many beta-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.
血红蛋白病是预防计划中的重点遗传疾病。快速进行基因型鉴定在诊断实验室中至关重要，尤其是为携带致病基因的夫妇提供产前诊断时。
作为一个模型，我们设计了一种基于LightCycler技术的方案，用于筛查希腊人群中的一系列β-珠蛋白基因突变。双荧光染料检测以及许多突变位置的紧密相邻有助于方案的设计。三个探针组能够筛查希腊人群中95%的β-珠蛋白基因突变，包括IVSII-745C→G、HbS、Cd5-CT、Cd6-A、Cd8-AA、IVSI-1G→A、IVSI-5G→A、IVSI-6T→C、IVSI-110G→A以及Cd39 C→T。
通过对100例已知突变的β地中海贫血杂合子进行分析，该方案得以标准化，在区分野生型和突变型等位基因方面100%可靠。随后对100例未知突变的希腊β地中海贫血杂合子进行筛查，发现100个样本中有96个样本为10种突变中的一种杂合子，不过HbS/Cd6和IVSI-5/IVSI-1突变的熔解曲线无法区分，这表明需要其他方法进行明确诊断。一个呈现独特熔解曲线的样本经测序鉴定为Cd8/9+G。三个样本携带的突变位于探针覆盖的基因区域之外。该方案在25例产前诊断样本中100%准确，诊断出了14种不同的基因型组合。该方案也具有灵活性，能够检测来自其他人群组的5种β-珠蛋白基因突变（IVSI-1G→T、IVSI-5G→C、IVSI-116T→G、Cd37 TGG→TGA以及Cd41/42 -TCTT）。
所描述的LightCycler系统方案能够快速筛查多种β-珠蛋白基因突变。它适用于许多人群，用于指导明确的突变诊断，并且适合低成本的快速产前诊断。
