Molecular Diagnosis Of Rare Thalassemia Gene Mutations

Background and ObjectiveThalassemia,also known as"marine anemia",hereinafter referred to as"thalassemia",is one of the earliest human single gene diseases in the world that have elucidated the molecular basis.In the Mediterranean,the Middle East,India and Southeast Asia and other regions along the high incidence,carrier rate of thalassemia gene in the world is about 1.5%-2%.In the southern region of China,especially Guangdong,Guangxi and Hainan,the incidence rate is higher,and the carrier rate of thalassemia gene is between 3%and 24%.The clinical phenotype of thalassemia is diverse,the light person needs to be transfusions repeatedly,the heavy person is aborted dead even the stillborn,bring tremendous mental pressure and economic burden to the family and society.At present,there is no radical cure for severe thalassemia,the most effective treatment is to carry out bone marrow stem cell transplantation.However,bone marrow transplantation is affected by many factors,such as:it is difficult to find a matching donor;transplant surgery is expensive,the ordinary families are unbearable;it also has the risk of recurrence,immunotherapy and ongoing monitoring is required after surgery and so on.So only a few patients are able to carry out a bone marrow transplant,and most of those with severe poverty are still unable to get effective treatment.At present,the control and reduction of the birth rate of children with severe thalassemia through thalassemia genetic diagnosis and prenatal diagnosis is the focus of thalassemia prevention and control work.Thalassemia is an autosomal recessive genetic disease,and the pathogenic gene is human globin gene.According to-the pathogenic,genes are mainly divided into two types of α and β.The α-globin gene is located at 16p13.3,and the β-globin gene is located at chromosome 11 p15.3.There are currently more than 500 species of thalassemia gene mutations in the world.On the commercial α-thalassemia gene detection kit mainly includes three kinds of deletion mutations(-SEA,-α3.7 and-α4.2)and three point mutations(CS,WS and QS).β-thalassemia gene detection kit mainly contains 17 kinds of point mutations(CD41-42,CD43,IVSII-654,-28,-29,-30,-32,CD71-72,βE,CD17,CD31,CD14-15,CD27-28,IVS-I-1,IVS-I-5,CAP + 1 and IntM).These two types of kits cover about 95%of the Chinese population of a-andβ-thalassemia pathogenic gene defects,still 5%rare or unknown mutations may be missed,it is necessary to detect rare thalassemia gene mutations in order to clear or exclude the causes.The method of detecting mutations in rare thalassemia is different and depends on the type of mutation,α-and β-globin gene sequencing analysis is mainly used to detect point mutations and small fragments of the deletion or repeated mutations;Gap-PCR can be used for the deletion of Southeast Asia-deficient HPFH(hereditary fetal hemoglobin persistent syndrome),Chinese-type Gγ+(Aγδβ)0thalassemia,Thai and Philippine a-thalassemia and Hong Kong-type a-thalassemia to detect;In addition,MLPA(multiplexed probe amplification technology),DHPLC(denaturing high performance liquid chromatography)and other methods have also been used for some rare missing thalassemia gene detection.To carry out rare thalassemia genetic testing,and used it in prenatal diagnosis,which can effectively reduce the birth rate of children with severe poverty;at the same time,not only we can find some new mutant types that enrich the thalassemia gene mutations spectrum but also we can provide data for the improvement of clinical diagnostic kitsthat can help to improve the accuracy of the kit detection and reduce the rate of missed diagnosis.Materials and Methods1.Research objectFrom 2011 to 2016 in the Southern Medical University,Nan Fang Hospital,prenatal diagnosis center for common cases of thalassemia gene detection.We screened out cases that phenotypic and genotypic are inconsistent.2.MethodsFor the rare cases of unmatched genotype and phenotype,we do the corresponding rare thalassemia gene detection based on their different phenotypic data.2.1 α-globin gene sequencing is mainly for the cases:they have the phenotype of small cell low pigment,hemoglobin(Hb)electrophoresis results show HbA2<2.5%,they aren’t iron deficiency anemia and common α-thalassemia gene detection is normal;or phenotype is hemoglobin H(HbH)disease,and the genotype is only the standard type.β-globin gene sequencing is mainly for the cases:they-have the phenotype of small cell low pigment,Hb electrophoresis results show HbA2>3.5%,common β-thalassemia gene detection is normal;or phenotype is heavy β-thalassemia,but the genotype is only carriers.2.2 The detection of Southeast Asia missing type HPFH and Chinese type Gγ+(Aγδβ)0 thalassemia gene is to detect the cases of the phenotype of small cell low pigment,Hb electrophoresis HbF increased(usually greater than 8%),and commonβ-thalassemia gene detection of normal cases;or children genotype is β-thalassemia homozygous,but β-thalassemia gene detection inone of the parentsis in normal situation.2.3 Thai-type and Philppine-type β-thalassemia gene detection is mainly for these cases:they have the phenotype of small cell low pigment,hemoglobin(Hb)electrophoresis results show HbA2<2.5%,they aren’t deficiency anemia,and commonα-thalassemia gene detection is normal;or the situation of Bart’s hydrops fetalis,butα-thalassemia gene detection in one of the parents is normal.2.4 Hong Kong-type α-thalassemia gene detection is mainly for the case which detection of common missing α-thalassemia gene by agarose gel electroph-oresis were three bands(3.7 bands,normal bands,SEA bands),or those with two bands(weak 3.7 band and normal band).2.5 Excluding Thai and Philippine type α-thalassemia,fertility in children with HbH or Bart’s edema,but a-thalassemia gene detection in one of the parents is normal,we need todo the MLPA detection of rare missing a-thalassemia.excluding Southeast Asia missing type of HPFH and the Chinese type Gγ+(Aγδβ)0 thalassemia,fertile homozygous heavy thalassemia in children,but β-thalassemia gene detection in one of the parents is normal,-we need to do MLPA Detection of rare absent β-thalassemia genes.Results1.A total of 23 rare alpha-thalassemia gene mutations and 11 mutant types were found in 45 suspected cases via a-globin gene sequencing.A total of 79 cases of rare(3-thalassemia gene mutations and 35 mutant types were found in 108 suspected rare cases via β-globim gene sequencing.2.A total of 85 suspected Southeast Asia-deficient HPFH and Chinese type Gγ+(Aγδβ)0 cases to do mutation detection.A total of 23 cases of exogenous HPFH mutation in Southeast Asia were detected,and 40 cases of Chinese Gγ+(Aγδβ)0mutation were detected.3.For 28 suspected cases of the Philippines and Thailand type we do a-type thalassemia genetic testing,found five Thailand α-thalassemia mutation type cases,and no Philippines a-thalassemia positive cases.4.For 7 cases of suspected cases of Hong Kong-type we do a-thalassemia gene detection,7 cases were Hong-type a-thalassemia gene mutation Carriers.5.For one suspected of rare a-thalassemia deletion and 5 suspected rare β-thalassemia deletion we do MLPA detection,6 cases are rare deletion thalassemia eariers.ConclusionsA total of 46 rare mutation types were found by sequencing analysis of a-and β-globin gene,four types of mutations(IVS-2-2(-T),CD30-32(+ CGCTG),IVS-2-672(A→C),IVS-2-806(G→C))were first reported in Domestic and foreign which enriched the Chinese population thalassemia mutation spectrum and determined the patient’s genotype.Five mutations(aaCD29(T→C),αCD30(-GAG)α,CD37(GA),-90(C→T),IVS-2-5(G→C))have a higher frequency,we suggest to add these high frequency mutations into common point mutation detection range,which can improve the kits accuracy and reduce the missed diagnosis rate.Except the globin gene sequencing analyses,according to its different phenotypic data,we also carried out other rare mutations detection.A large number of positive cases were found,and we found the pathogenic gene loci which provide data to support the rare thalassemia gene detection kit in the future.Each rare gene mutation types has its corresponding clinical phenotypes,according to different phenotypic data we select the corresponding rare thalassemia gene detection method.By analyzing the relationship between genotype and phenotype of rare mutations in various cases,we determined the various rare thalassemia detection range and crowd,which can help clinicians to have a better understanding of rare gene mutations and to strengthen differential diagnosis.Thalassemia is a serious cause of death and disability,and has a great impact on family and society.In this study,we conducted prenatal diagnosis of dozens of rare thalassemia families,defined the fetal genotype,provided strong evidence for the clinical outcome of the fetus,and reduced the birth rate of children with thalassemia.