| BACKGROUND AND OBJECTIVEβ-thalassemia is a autosomal recessive disorders, and is one of the most commonsingle gene disorders in the world. It is a result of malarial selection and that is nowgenerally accepted. Therefore, the main distribution regions ofβthalassemia are thetropics and subtropics with high incidence of malarial, extending from theMediterranean basin and parts of Africa, through the Middle East, the Indiansubcontinent, South China, South-east Asia and Melanesia and out into the Pacific. Inaddition, the USA and some countries in Central and South America that hadsubstantial immigration from Europe and Africa, also have a high incidence ofβthalassemia. In China, the provinces located in the south of the Yangtze River have arelatively high incidence ofβthalassemia, especially Guangdong, Guangxi, Hainan,Jiangxi, Taiwan and Hong Kong with a total population over two hundred millionpeople.Theβthalassemias are a heterogeneous group of the inherited hemolytic anemiamainly resulting from single base substitutions, minor deletions or insertions withintheβ-globin gene in the chromosome 11, and are classified intoβ~+ andβ~0 varieties,reflecting a reduced or an absent synthesis of theβ-globin chain of hemoglobin,respectively. More than 255 different mutations ofβthalassemia to date have been reported in the world, and each ethnic population has its own cluster of commonmutations. It is well known that the severe forms ofβthalassemia major is a fataldisorder, and the patients need regular blood transfusion and desferal therapy tosustain their life. Recently, the thalassemia still poses serious public health issues inthese areas. As for a country or a region with high prevalence of thalassemias, themost effective method to control this disorder is to establish a prevention programbased on the genetic epidemiology study, which is to implement a large-scalepopulation screening and prenatal diagnosis of high-risk couples to prevent the birthof infants with severe thalassemia.Gene diagnosis and prenatal diagnosis ofβthalassemia had experienced fromDNA dot blot, zymogram analysis of restriction enzyme, linkage analysis ofrestriction fragment length polymorphism, allele specific oligonucleotide to gene chip.At present, the main molecular biological method to diagnoseβthalassemia isReverse Dot Blot. But all above-mentioned methods are trivial and complicated. Theyneed synthesis and symbol of primer and probe, and moreover, the speed of thesemethods is slowly. False positive and false negative results will lead to misdiagnosisor missed diagnosis and so they are incompetence for screening.Denaturing high-performance liquid chromatography (DHPLC) proved to be areliable, rapid, and highly sensitive method for detecting point mutations based on thedetection of hetroduplexes in PCR products by ion pair reverse-phase HPLC underpartially denaturing conditions. Its sensitivity and specificity can reach 96～100%,which are higher than commonly used DGGE, CCM, CSGE and SSCP technologies(104～108). Only F2SSCP, which is on account of capillary electrophoresis, is on a parwith DHPLC in sensitivity and specificity (109). In addition, to select moretemperature condition at the same time, its mutation detection rate will be raised.The purpose of this study is to establish a rapid, highly sensitive, reliable and economic method for detectingβthalassemia, which can be used in rapid genotypingtest with highly accuracy and reproducibility suitable for the known mutations areneeded for pre-and post-natal screening and diagnosis of this disease in ClinicalGenetics Laboratories and in discovering new mutation site or single nucleotidepolymorphisms.MATERIAL AND METHOD3 pairs of high-specific polymerase chain reaction PCR amplification primerswere designed according to the sequences ofβ-globin gene, the three regionsencompass the sites of all known mutations occurred inβ-globin gene in the Chinesepopulation. The three PCR can be amplified in the same condition through optimizereaction conditions. The PCR products from the tested sample were analyzed on aDHPLC column via differential elution profiles under partially denaturing conditions.The methodologies gave accuracy and reproducible results from both each of theuniquely profiled patterns by heteroduplexe analysis and retention times of separateeluted peaks by homoduplexe analysis. Assays conditions were established based onthe analysis of 795 DNA samples comprise of the previously genotyped 407β-thalassemia DNA samples and 388 healthy individuals.The blinded validation was studied in 319 samples randomly selected from eachof genotype groups which were detected by DHPLC profiling, including 258 caseswithβ-thalassemia mutations or a complex state of mutations plus polymorphismsand 61 cases with polymorphisms. In addition, samples showing unique positivepatterns tested by DHPLC are sent to do DNA sequence analysis in order to find newmutation or SNP site.After the method is established and evaluated, we describe the application of thisnew method to the prenatal diagnosis in high-risk families forβ-thalassemia. Theresult contrast to RDB's to gain its practical utility. RESULTS AND DISCUSSIONAssays conditions were established based on the analysis of 795 DNA samplesfrom a group of various genotypes for the 20 disease-causing mutations and 8polymorphisms inβ-globin gene. A total of 68 amplicon-based DHPLC patterns weredistinctly categorized. The assays were blindly validated on 319 DNA samplespreviously genotyped by other methods, with complete concordance of results. Fourrare mutations (all of which, reported here for the first time) that standard mutationdetection methods failed to reveal, were also identified. At the same time, anothermutation of-98 is found, but it is still not clear that it whether or not could causeβ-thalassemia. It was used successfully for prenatal diagnosis ofβ-globin mutationsin nine Chinese families.CONCLUSIONSThe rapid and reliable method can meet the requirements of direct genotyping ofknownβ-thalassemia mutations and/or polymorphisms, and strongly suggests thisassay can be used as a useful tool in Clinical Genetics Laboratories.
|
PDF Full Text Request