Genotype Analysis Of 5488 Cases Of Common Thalassemia And Identification Of Rare Hongkongαα Thalassemia Thalassemia

Objective:Thalassemia(thal)is the common autosomal single-gene hereditary diseases in the world.The different genotypes may cause different clinical manifestations.Thalassemia minor can be small cell low pigmented mild anemia.Thalassemia intermedia(TI)and thalassemia major(TM)can cause moderate to severe hemolytic anemia,jaundice,hepatosplenomegaly,and stunted growth.Children with TM can even die before or after birth.At present,it can only be cured by hematopoietic stem cell transplantation or gene editing,but it is expensive and difficult to match human leukocyte antigen(HLA).It can not be widely used in clinic.In China,thalassemia occurs in areas around or south of the Yangtze River,such as Guangxi,Guangdong,Hainan,Yunnan,Fujian,Sichuan,Chongqing and other regions,and the carrying rates vary greatly in different regions from 3.3%to 24.07%.Different thalassaemia gene mutation spectrums exist in different regions and different populations.At present,the conventional thalassaemia gene detection method can only 24 kinds of Chinese common mutations.However,there are remaining a 3%risk in other mutations which are not in the detection range.HongKongαα thalassemia(HKαα)is a rare type of α-thal,which contains-α3.7 deletion and αααanti-4.2 triplication on the same chromosome.Due to the lack of detection of αααanti-4.2 triplication in the current detection kits,laboratory technicians may confuse-α3.7/αααanti-4.2 and HKaa thal as-α3.7/αα.When the spouse of-α3.7/αα carriers is--SEA/αα,their offspring have a 1/4 probability of-α3.7/--SEA,which is hemoglobin H disease(HbH).Hb H disease has different clinical manifestations.A few can be small cell hypopigmented mild anemia,most of which are moderate hemolytic anemia,can cause severe anemia,jaundice,and hepatosplenomegaly.In severe cases,the development is retarded and the splenomegaly is obvious.It can be complicated by infection and lead to aggravated condition.Therefore,it is necessary for pregnant women to perform prenatal diagnosis to determine the genotype.However,HKaa is neither a-α3.7 deletion nor αααanti-4.2 triploid,and HKaa/--SEA mainly manifests as mild α-thal.When HKaa is diagnosed as a-α3.7 deletion thalassemia carrier,and their spouse is--SEA/αα,pregnant need unnecessary invasive examinations for a clear diagnosis,which increases the risk of miscarriage and economic burden,and brings mental stress to pregnant women.When the patient’s genotype is-α3.7/αααati-4.2,regardless of whether the spouse is a thalassemia patient,the thalassemia gene will be inherited to the next generation.In addition,when combined with β-thal,because αααanti-4.2 can increase the synthesis of α-globin chain and aggravate the phenotype of β-thal.Because the clinical manifestations,genetic counseling,and prenatal diagnosis of-α3.7/αα are not the same for both genotypes,it is imminent to detect the specific genotypes ofαααanri-4.2 and HKaa carriers.The current classic detection method of HKαα,nested PCR,can not distinguish between HKaa/aa,HKαα/-α3.7,HKαα/αααanri-4.2,HKαα/αααanri-3.7,HKαα/HKαα,etc.Genotype is related to clinical manifestations,genetic probability,etc.,so it is necessary to identify the genotype of HKαα thalassemia.This study mainly used thalassemia gene testing in Sichuan population to understand the gene distribution of thalassemia in the region,and provided genetic counseling and prenatal diagnosis data for clinicians.At the same time,a method for identifyingαααanti-4.2 and HKaa genotypes of-α3.7 carriers was established.Methods:Samples were collected from the Sichuan Provincial People’s Hospital and tested for thalassaemia genotype from July 2017 to October 2019.Conventional thalassaemia gene detection uses automatic nucleic acid extraction instrument to extract DNA,αααanri-4.2 and HKaa gene detection uses Kangwei Century and Qiagen DNA extraction kits to extract DNA.Primer 3 was used to design the primers required for HKaa thalassemia gene detection,and the primers were synthesized by Shanghai Biotech Co.,Ltd.Twenty-four common types of Chinese thalassemia mutation were screened by gap-polymerase chain reaction(Gap-PCR)and reverse dot blot(RDB).Anti-4.2 multiplex-PCR was used to confirm carriers of the αααanti-4.2 duplication with-α3.7 deletion.Two-round nested PCR and multiplex ligation-dependent probe amplification(MLPA)were applied to accurately identify and confirm their genotypes.For data analysis,we used descriptive statistics.Results:1.Among 5,488 subjects in Sichuan Province,a total of 2544 cases of thalassaemia carriers and patients were detected,with 38 types of thalassaemia genotypes.Among them,there were 1190 cases of α-thal(46.78%,1190/2544),1286 cases of β-thal(50.55%,1286/2544),and 68 cases of αβcompound thal(2.67%,68/2544).Among them,there are 9 kinds of α-globin gene mutations,with--SEA mutation being the most common,accounting for 69.77%of all a-globin gene mutations.There are 13 kinds of β-globin gene mutations,with CD 17(A>T),The 42(-TTCT)and IVS-II-654(C>T)mutant alleles were dominant,accounting for 32.92%,28.88%,24.12%of allβ-globin gene mutations.In 1190 cases of α-thal,19 genotypes were detected.--SEA/aa was the dominant(71.76)%,accounting for more than 2/3 of α-thal.Among 1,286 cases of β-thalassemia,18 genotypes were detected.It is mainlyβcd17/βN(33.28%),βcd41-42/βN(28.46%),βIVS-2-654/βN(23.79%),which together account for 85.53%of β-thal.In 68 cases of a complex β thal,the first three were-α3.7/αα complex βcd17/βN(19.12%),-α3.7/aa complex βcd41-42/βN(17.65%),-a3.7/aa complex βIVS-2-654/βN(11.76%).2.After Gap-PCR testing,a total of 227 samples from thalassemia patients were identified as-α3.7/αα by Gap-PCR,and the genotypes of two samples were suspected to HKαα thalassemia.The combined use of nested PCR and MLPA for HKaa and αααanti-4.2 genotype analysis showed that including 15 patients of HKaa/aa,2 patients of HKaa/aa and βIVS-2-654/βN coinheritance,1 patients of HKaa/aa and βcd41-42/βN coinheritance 1 patient of HKaa/--SEA,1 patient of HKaa/-a4.2 and βIVS-2-654/βN coinheritance,1 patient of-α3.7/αααanti-4.2 and βIVS-2-654/βN coinheritance.The frequency of HKαα allele was as high as 7.93%(18/227)among-α3.7/αα.It is suggested that the error rate of diagnosis is 9.17%(21/229)by Gap-PCR in-α3.7 deletion.In addition,HKaa mutations accounted for 1.5%of a-globin gene mutations.HKaa composite β thal was higher,accounting for 5.88%(4/68)of a composite β3 thal.There were no significant abnormalities in the hematological parameters of HKaa/aa genotypes.Conclusions:1.The deletional α-thal in Sichuan were mainly--SEA/aa,-α3.7/αα,-α3.7/--SEA,and the nondeletional α-thal were mainly ααQS/αα.β-thal were dominated by βcd17/βN,βcd41-42/βN,βIVS-2-654/βN.And β-thal was slightly more than a-thal.2.At the same time,the ratio of HKaa(7.93%)is relatively high in-α3.7/αα.The use of Gap-PCR technology commonly used in clinical practice will be misdiagnosed as-α3.7,which will increase unnecessary prenatal genetic diagnosis of pregnant women and increase the risk of abortion.Attention should be paid to the possibility of HKαα/αα,HKαα/-α3.7,HKαα/αααanti-4.2,HKαα/αααanti-3.7 if Gap-PCR only obtains α2 and-α3.7 positive fragments.It is necessary to use nested PCR combined with MLPA forαααanti-4.2 and HKaa genotype identification,and at the same time-α3.7/αααanti-4.2 can be diagnosed.This strategy can reduce the diagnostic error rate of-α3.7 carriers,identify the-α3.7/αααanti-4.2 and HKaa genotypes,and provide clinicians with more accurate genetic counseling and prenatal diagnosis.