Development of molecular diagnostic platform for α0 -thalassemia 44.6 kb (Chiang Rai, --CR ) deletion in individuals with microcytic red blood cells across Thailand.

INTRODUCTION: The α0 -thalassemia 44.6 kb or Chiang Rai (--CR ) deletion has been reported in northern Thailand and is capable of causing hemoglobin (Hb) H disease and a lethal α-thalassemia genotype, Hb Bart's hydrops fetalis, in this region. However, there are no current data regarding the frequency of --CR nationwide due to a lack of effective diagnostic assay. Therefore, this study aimed to develop a reliable platform for simultaneous genotyping of --CR and two common α0 -thalassemias in Thailand (--SEA and --THAI ) and investigate the frequency of --CR across Thailand. METHODS: Multiplex gap-PCR assay and five renewable plasmid DNA controls for --CR , --SEA , --THAI , α2-globin (HBA2), and ß-actin (ACTB) were newly developed and validated with reference methods. The developed assay was further tested on 1046 unrelated individuals with a reduced mean corpuscular volume (MCV) of less than 75 fl for investigating genotypic and allelic spectrum of --CR . RESULTS: Our developed assay showed 100% concordance with reference methods. The results were valid and reproducible throughout hundreds of reactions. Comparison of the genotypic and allelic spectra revealed that heterozygous --SEA (--SEA /αα) and --SEA alleles were dominant with the frequency of 22.85% (239/1046) and 13.34% (279/2092), respectively. Of these, --THAI and --CR were relatively rare in this population and comparable to each other with the allelic frequency of 0.14% (3/2092). CONCLUSION: This study successfully established a reliable molecular diagnostic platform for genotyping of --CR , --SEA , and --THAI in a single reaction. Additionally, we demonstrated the frequency of --CR in Thailand for the first time and provided knowledge basis for the planning of severe α-thalassemia prevention and control programs in Thailand, where thalassemia is endemic.

Thalassemia in Thailand.

Thailand has a population of 66.2 million with 30.0-40.0% of them carrying thalassemia genes. Interaction of these thalassemia genes lead to more than 60 genotypes with a wide spectrum of clinical severity from asymptomatic to lethal. Estimation based on gene frequencies and number of babies born each year, there will be about 1.2% babies born with severe cases of thalassemia each year. Further estimation revealed that 1.0% of the Thai population have thalassemia disease, which is a big health problem for the country. Thalassemia prevention and control programs were introduced using post conception screening in couples and prenatal diagnosis (PND) for the prevention of new thalassemic births. Moreover, the majority of existing cases are undergoing supportive treatment with regular blood transfusions and iron chelation. Curative treatment by hematopoietic stem cell transplantation (HSCT) is available but is limited to a minority of the patients.

Community participation for thalassemia prevention initiated by village health volunteers in northeastern Thailand.

The study was conducted to assess the achievement of a thalassemia screening program at a community level supported by village health volunteers (VHVs) of 2 subdistricts in the northeast of Thailand. One subdistrict served as the intervention and the other as the control area. A training program was organized for the village health volunteers from the intervention area. Essential information about the risk and danger of thalassemia was given to the participants who wanted to have children in the community as well. Of the 206 individuals who wanted to have children living in the intervention area, 190 (92.2%) agreed to undergo screening. Of the 196 individuals within the control area, only 26 (13.3%) voluntarily participated in the screening tests. Attitude toward prevention and knowledge about the disease improved significantly in both areas, but the differences between the scores were statistically significantly higher for individuals living in the intervention area.

Hemoglobin Pyrgos with hemoglobin H disease: new triple heterozygosity.

A 19-year old Thai male presented to the hospital with fever, acute hemolysis, pallor and jaundice without hepatosplenomegaly. On admission his hematocrit was 17.4% and a blood smear showed moderate hypochromia with mild anisopoikilocytosis. Hemoglobin (Hb) electrophoresis revealed Hb A2ABart's Hb H with an abnormal band, which on PCR proved to be Hb Pyrgos (beta83, glycine --> aspartic acid). The patient inherited beta(Pyrgos) globin from his mother and alpha-thalassemia-1 from his father. He was diagnosed as having Hb H (alpha-thalassemia-1/alpha-thalssemia-2) heterozygous Hb Pyrgos. He was treated with a transfusion of packed red blood cells. During follow-up his hematocrits ranged from 31 to 34%. The Hb Pyrgos did not add any deleterious effect to his Hb H disease.

False positive rates of thalassemia screening in rural clinical setting: 10-year experience in Thailand.

This retrospective study aimed to describe the magnitude of false positive screening for thalassemia in a primary care setting of Thailand. The study was conducted from 1999 to 2008 and analyzed 13,745 positive cases. It involved a combination of one tube osmotic fragility (OF) and dichlorophenol indophenol (DCIP) precipitation tests. The number of cases increased over the ten-year period, corresponding well to an exponential model. Based on hemoglobin and DNA analysis, cases with alpha-thalassemia 1, beta-thalassemia, and hemoglobin E were defined as true positive cases, and the remaining were considered as false positives. The false positive rate was in the range of 20.1-36.1%. The proportion of false positive cases of thalassemia from the screening tests was associated with a trend which was statistically significant (p < 0.001). The estimated cost of further hemoglobin analysis resulting from false positive cases was approximately 40.2-72.2 million THB/year for an estimated 800,000 annual births. The combination of the OF and DCIP test, which has been the strategy for screening of thalassemia and HbE in pregnant women throughout this country, resulted in a large economic burden in terms of high cost and workload associated with further hemoglobin and DNA analyses of false positive samples. Measures to reduce false positive should be developed and implemented.