Problems in determining thalassemia carrier status in a program for prevention and control of severe thalassemia syndromes: a lesson from Thailand.

BACKGROUND: Prevention and control of severe ß thalassemia by carrier detection and identification of couples at risk in developed countries is one of the most successful stories in modern medicine. Similar programs in developing countries especially Southeast Asia, are more problematic because both α and ß thalassemias are highly prevalent. In Thailand, there are limited data on whether we could determine, based on hematological phenotypes, the mutation severity and/or coinheritance of α thalassemia in ß thalassemia traits. METHODS: Comprehensive molecular, hematology and hemoglobin analyses of the α and ß globin genes were performed in 141 healthy individuals identified as ß thalassemia carriers. RESULTS: Seventeen different ß globin mutations were successfully identified out of all cases analyzed. Although the majority of the mutations identified were the ß° or severe ß⁺ thalassemia alleles, a high proportion of mild mutations (25%) was observed. Of these ß thalassemia traits, 22.3% were found to co-inherit the α thalassemias. Milder hematological phenotypes were noted in ß⁺ compared with ß° thalassemia traits when the α globin genes were intact. Although co-inheritance of α° thalassemia might be suspected in cases with skewed profiles, due to the overlapping values, it remains difficult to apply these parameters for reliable carrier determination. CONCLUSIONS: A combination of hemoglobin analysis and DNA testing seems to be the best way to confirm carrier status in a region with high frequency for both α and ß thalassemias. Underdiagnoses of carrier status could hamper the effectiveness of a thalassemia prevention and control program.

Simple, efficient, and cost-effective multiplex genotyping with matrix assisted laser desorption/ionization time-of-flight mass spectrometry of hemoglobin beta gene mutations.

A number of common mutations in the hemoglobin beta (HBB) gene cause beta-thalassemia, a monogenic disease with high prevalence in certain ethnic groups. As there are 30 HBB variants that cover more than 99.5% of HBB mutant alleles in the Thai population, an efficient and cost-effective screening method is required. Three panels of multiplex primer extensions, followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry were developed. The first panel simultaneously detected 21 of the most common HBB mutations, while the second panel screened nine additional mutations, plus seven of the first panel for confirmation; the third panel was used to confirm three HBB mutations, yielding a 9-Da mass difference that could not be clearly distinguished by the previous two panels. The protocol was both standardized using 40 samples of known genotypes and subsequently validated in 162 blind samples with 27 different genotypes (including a normal control), comprising heterozygous, compound heterozygous, and homozygous beta-thalassemia. Results were in complete agreement with those from the genotyping results, conducted using three different methods overall. The method developed here permitted the detection of mutations missed using a single genotyping procedure. The procedure should serve as the method of choice for HBB genotyping due to its accuracy, sensitivity, and cost-effectiveness, and can be applied to studies of other gene variants that are potential disease biomarkers.