| α-thalassemia is the most common human monogenic hereditary diseases in the world. Generally,α-thalassemia is mainly resulted fromα-globin gene defects which located in 16p13.3.α-thalassemia is classified as deletional or non-deletional according to the mutational mechanism involved. The deletional types comprise the majority of cases ofα-thalassemia. The Southeast Asian deletion (--SEA), rightward deletion (-α3.7), and leftward deletion (-α4.2) are the most common causes of this disorder in the Guangdong population, accounting for more than 96% of allα-thalassemia. Populations in southern China have such high prevalence rates ofα-thalassemia that they present a public health concern. The screen ofα-thalassemia carrier, antenatal gene diagnosis and selective abortion is the only choice to controlα-thalassemia due to the lack of ideal treatment forα-thalassemia in clinic. The prerequisites for the purpose are the detailed genetic epidemiology of a defined population and the correspondent optimized gene analysis strategy.At present, various molecular diagnosis technologies have been applied to theα-thalassemia assay. Gap-PCR is widely applied to the clinical diagnosis and molecular screening of deletionalα-thalassemia carriers. But the method has limits on assay for the old genomic DNA. The DNA microarray technology which has been applied to diagnosis and research of various genetic diseases could provide the further improvement for the diagnosis of deletionalα-thalassemia. Thus, a simple, fast, and highly reproducible protocol for detection of deletionalα-thalassemia using an oligonucleotide microarray was developed.Specific PCR primers for the three deletionalα-thalassemia and normalα2 gene were designed through consulting and contrasting the domestic and foreign literatures. A single tube quadruple PCR reaction system was set up and the conditions were optimized. A series of probes specified to different PCR products were designed. The microarray with 70mer oligonucleotide probes was prepared using the optimized technological conditions. PCR products were directly hybridized to the microarrays with different specific probes. Genotypes were determined by quantitative analysis of the fluorescent signals detected by fluorescence scanning. The detection criteria are as follows: (a) spots, with signal-to-background ratios greater than 10.0 and intensities subtracted local background signals more than 1000, were considered positive; (b) spots forα2 were considered positive only when signal of spots forα2 was more than half of that for -α3.7 when the spots for-α3.7 are positive. The preparation protocols and the detection criteria were further optimized based on the analysis of 32 DNA samples.As far as the epidemiology of thalassemia is concerned, the epidemiology study of hemoglobinpathies in China mainland in the middle of 80's of last century played critical poles in controlling thalassemia. But a new round epidemiology investigation all over the country is imperative because previous study emphasized on abnormal hemoglobin diseases rather than thalassemia, and the epidemiology of thalassemia may be changed due to massive and long standing migration across the country with the fast social and economic development of China. This will be a tremendous task. A small scale investigation on thalassemia in representative population will be of certain guiding significance for the new round epidemiology. The prevalence and spectrum of thalassemia in Shenzhen is representative for the whole China because its population comes from all of the country. Thus, the molecular epidemiology of thalassemia in Shenzhen populations was carried out by using the home-made microarray.In the molecular epidemiological study of thalassemia from Shenzhen population by using the home-made microarray, we found a special case that was positive for the ?α3.7 junction fragment, --SEA junction fragment andα2. Phenotypic analysis revealed that the proband presented a typicalα-thalassemic trait. The molecular analysis identified that the genotype of proband was compound heterozygosity for HKααand --SEA (HKαα/--SEA). To our knowledge, this is the first report on heterozygosity for HKααand --SEA. Thus, the current case provided a chance to investigate the hematological and clinical impact of the HKαα. Asαα/--SEA, HKαα/--SEA presented a typicalα-thalassemic trait and there is no evident haematological and clinical difference between the HKαα/--SEA andαα/--SEAWe have obtained the prevalence and spectrum ofα- andβ-thalassemia mutations in Shenzhn by screening clinical blood samples. Of total 3721 samples, 241 (6.49%) were carriers of thalassemia, of which 161 (4.34%) hadα-thalassemia, 74 (1.99%) hadβ-thalassemia, and 6 (0.16%) had bothα- andβ-thalassemia. Thus, the prevalence of thalassemia mutations in the Shenzhen population is 6.49%. We identified three deletionalα-thalassemia mutations but only one nondeletional point mutation (–αCS). Of theseα-thalassemia mutations, the Southeast Asian deletion accounted for about 80% ofα-thalassemia chromosomes. More than 90% of theβ-mutations were accounted for by codon 41/42 (–CTTT), IVS-II-654 (C→T), codon 17 (A→T), and–28 (A→G).Compared with other areas in Guangdong Province, the prevalence of thalassemia in Shenzhen was lower, while there was no evident difference for the spectrum of mutations.To sum up, an oligonucleotide microarray for detection of the three most frequently observed deletionalα-thalassemia (--SEA, -α3.7, -α4.2) was developed. Purification, fragmentation, and nested PCR are not needed, which makes it possible to complete the entire protocol in a work day. A novel genotype was discovered while the prevalence and spectrum of thalassemia mutations in Shenzhen was investigated by using the home-made microarray. These results showed that the simple, fast, and highly reproducible protocol may be suitable for routine clinical use and population screening for deletionalα-thalassemia.
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