Application Of Third-generation Sequencing Technology In Thalassemia Gene Detectio

Objective:To explore the value of third-generation sequencing（TGS）for genetic diagnosis and prenatal genetic screening of thalassemia genes;To understand the gene carriage rate,mutation types and constituent ratio of thalassemia in the Buyi population in Qiannan District,Guizhou Province,to construct a more detailed globin gene mutation map of the Buyi population in Qiannan Prefecture,Guizhou Province,and to provide a new investigation basis for prevention,control and treatment of thalassemia in the Buyi population in Qiannan Prefecture.Methods:1.Peripheral blood samples were collected from 176 subjects who visited the Guizhou Provincial People’s Hospital from January 2020 to December 2021 and were assessed to have a positive thalassemia phenotype.Thalassemia genes were detected simultaneously in each sample using both the Pac Bio TGS platform based on single-molecule real-time（SMRT）technology and the conventional PCR-reverse dot blot（PCR-RDB）.Sanger sequencing was used for validation when results were discordant between the two methods.2.Peripheral blood samples were collected from 53 couples who attended Guizhou Provincial People’s Hospital between January 2022 and June 2022 to undergo genetic screening for thalassemia in preparation for pregnancy or during the first trimester,with at least one member of each couple having a positive thalassemia phenotype.Thalassemia genetic testing was performed using third-generation sequencing technology on the 53 couples,and they were screened for homotypic thalassemia variants by TGS,and the risk of pregnancies with babies presenting with severe thalassemia,was assessed.3.1003 Buyi subjects who received gene screening for thalassemia in 5 areas of Qiannan Prefecture of Guizhou Province from January 2021 to November 2022 were selected as subjects.Hematological phenotypic analysis was first performed on 1003samples,followed by thalassemia gene testing using third-generation sequencing technology,and the carrier rate,gene mutation type and composition ratio of thalassemia in Buyi population in this area were calculated.Results:1.Of the 176 subjects,175 had concordant genotypes between the two methods,including 63 normal subjects and 112α-and/orβ-thalassemia gene carriers,with a concordance rate of 99.43%.TGS detected a rareβ-thalassemia gene variant-50（G>A）that was not detected by conventional PCR-RDB.2.TGS identified seven of the 53 couples as homotypic thalassemia gene carriers,five of whom were at risk of pregnancies with severe thalassemia.3.The results of thalassemia gene detection in 1003 Buyi people in Qiannan Prefecture are as follows.（1）229 cases of thalassemia gene carriers were detected among 1003 Buyi population in Qiannan Prefecture,with a gene carrier rate of22.83%（229/1003）for thalassemia,and that ofα-thalassemia was 15.15%（152/1003）,including 10 cases of structural variant triplicatedα-globin genes,with a population carrying rate of 1.00%（10/1003）.57 cases ofβ-thalassemia,carrying rate for 5.68%（57/1003）,8 cases ofα-compoundβ-thalassemia with a carriage rate of 0.80%（8/1003）,and a total of 12 cases ofδ-thalassemia andδ-thalassemia combined withα-or/andβ-thalassemia with a carriage rate of 1.2%（12/1003）.The constituent ratios of the above four types of thalassemia are 66.38%,24.89%,3.49%and 5.24%,respectively.（2）According to the detection results of the above four types of thalassemia genes,the total carrying rates ofα-thalassemia,β-thalassemia andδ-thalassemia were 16.15%（162/1003）,6.68%（67/1003）and 1.20%（12/1003）,respectively.（3）A total of 13 genotypes were detected inα-thalassemia,including 11common and 2 rare variant types.Among them,the most common deletion type genotypes were-α^3.7/ααand--^SEA/αα,with constituent ratios of 32.89%and 25.66%,respectively.And the common point mutation genotypeα^CSα/ααaccounted for17.76%.The structural variants includedααα^anti3.7/αα,ααα^anti4.2/αα,whose constituent ratios were 5.26%and 1.32%,respectively.（4）A total of six genotypes ofβCD17（AAG>TAG）/βN、βCD41/42（-CTTT）/βN、βCD43（G-T）/βN、βCD71/72（+A）/βN、βCD26（G-A）/βN、β^{IVS-II-654（C-T）}/β^Nwere detected inβ-thalassemia.The constituent ratios of the two most common genotypesβ^{CD17（AAG>TAG）}/β^Nandβ^{CD41/42（-CTTT）}/β^Nwere 45.61%and 43.86%respectively,andβ⁰accounted for 96.49%inβ-thalassemia.（5）A total of 4combinational types ofαandβcompound thalassemia were detected,and theαα/-α^3.7compoundβ^{CD17（AAG>TAG）}/β^Nwas the most common type,accounting for 50%.（6）According to the types of thalassemia gene mutations detected,there were sevenα-thalassemia gene mutations,sixβ-thalassemia gene mutations and oneδ-thalassemia gene mutation（HBD:c.-127T>C）.Among them,the three most common types ofα-thalassemia gene mutations were-α^3.7,--^SEAandα^CSα,accounting for 80.59%（137/170）of the total allelic variation.The constituent ratios of structural variationααα^anti3.7andααα^anti4.2were 4.71%and 1.18%,respectively.CD17 and CD41-42 were the most common types ofβ-thalassemia gene mutations,with a total constituent ratio of 89.55%（60/67）.Conclusion:1.TGS can effectively detect common and rare thalassemia gene variants,with high accuracy and comprehensive test sites,and is suitable for thalassemia gene screening in populations with high thalassemia prevalence and high-risk couples,providing a reliable guarantee for genetic diagnosis of thalassemia.2.Buyi people in Qiannan Prefecture of Guizhou Province have a high incidence of thalassemia.The carrying rate of thalassemia gene mutation is 22.83%.The total carrying rates ofα-thalassemia,β-thalassemia andδ-thalassemia are 16.15%,6.68%and 1.20%,respectively.The most common mutation type ofα-thalassemia was-α^3.7/αα,and the most common mutation type ofβ-thalassemia wasβ^{CD17（AAG>TAG）}/β^N.The variation types of thalassemia in Buyi population in Qiannan Prefecture are complex and diverse,and the mutation spectrum of globin gene has ethnic and regional characteristics.