Epidemiological Investigation Of Thalassemia In Dehong And Xishuangbanna Regions And Research On New Types Of Thalassemia Mutations

Objective:To analyze the gene carrying rate,mutation type and frequency of thalassemia in the childbearing age population of Dai people in Dehong prefecture,Xishuangbanna prefecture,Yunnan province,and children aged 0-7 in Yingjiang and Mengla.Report one newly discovered case ofα-thalassaemia,two newly discovered cases ofβ-thalassemia and two cases of large fragment deletionβ-thalassemia,and their hematological characteristics were analyzed.Provide a theoretical basis for the prevention and control of birth defects.Methods:1614 Dai people of childbearing age in Dehong prefecture and Xishuangbanna prefecture of Yunnan province and 590 0-7-year-old children in Yingjiang and Mengla were subjected to routine blood testing,capillary electrophoresis,and next generation sequencing.SPSS 17.0 was used to analyze the experimental results,and the independent sample t test and ROC curve analysis were used in the analysis process.Next-generation sequencing technology（N GS）was used to detect new globin mutations in three individuals,and verified by Sanger sequencing of the globin gene.Use routine blood testing and capillary electrophoresis to analyze its hematological characteristics.Biological software analyzes the isoelectric point（p I）,molecular weight,conservation and pathogenicity of protein mutations.Two cases of large fragment deletionβ-thalassemia were subjected to routine blood testing,capillary electrophoresis,and next generation sequencing of the globin gene.MLPA technology was used to detect the newly discovered deletion typeβ-thalassemia gene mutation and verified by Sanger sequencing of the globin gene.Results:1.Epidemiological survey of thalassemia in two areas of Yunnan province.（1）A total of 1,614 cases of Dai people of childbearing age were examined in Dehong and Xishuangbanna prefectures,and a total of 809 cases of thalassaemia carriers were detected,with a carrier rate of 50.13%（809/1614）,of which the carrier rate ofα-thalassaemia was 33.09%（534/1614）,the carrying rate ofβ-thalassaemia was 10.66%（172/1614）,and the carrying rate of compositeαandβthalassemia was6.38%（103/1614）.In Yingjiang and Mengla,a total of 590 children aged 0-7 were examined,and a total of 211 carriers of thalassaemia were detected,with a carrier rate of 35.76%（211/590）,of which the carrier rate ofα-thalassaemia was 21.69%（128/1614）,the carrying rate ofβ-thalassaemia was 10.51%（62/590）,and the carrying rate of compositeαandβthalassemia was 3.56%（103/590）.The most common mutation type ofα-thalassaemia among the Dai people of childbearing age in Dehong and Xishuangbanna prefectures is-α^3.7,followed by--^SEA;the most common mutation type ofβ-thalassaemia is CD26（G>A）,followed by CD17（A>T）.The most common gene mutation type ofα-thalassaemia among children aged 0-7 in Yingjiang and Mengla was-α^3.7,followed by--^SEA;β-thalassaemia was most common with CD26（G>A）,followed by CD41/42（-TTCT）.（2）Comparing the hematological parameters of patients with confirmed adult thalassaemia,the MCV ofαα/--^SEA was relatively low and the Hb A₂ofαα/α^CSαwas relatively low inα-thalassaemia,and MCV of CD41/42（-TTCT）was relatively low inβ-thalassaemia.Hb A ofαα/-α^3.7combined with CD26（G>A）was relatively low in compositeαandβthalassemia.Comparing the hematological parameters of children with thalassaemia diagnosed 0-7 years old,Hb A₂ofαα/--^SEA was relatively low inα-thalassaemia.The hematological characteristics of the adult male sample and the child male sample are compared.The Hb of the child is lower than that of the adult,and the hematological characteristic of the adult female sample is compared with the child female sample.The child MCH is lower than the adult.（3）The best cut-off value for the diagnosis of adult thalassaemia MCV is 84.15 fl,the sensitivity is 81.1%,and the specificity is 85.8%;the best cut-off value of MCH is27.95 pg,the sensitivity is 86.8%,and the specificity is 84.3%.The best cut-off value of thalassaemia MCV for children aged 0-7 years is 76.95 fl,the sensitivity is 79.2%,and the specificity is 81.4%;the best cut-off value of MCH is 25.36 pg,the sensitivity is 89.8%,and the specificity is 78.1%.（4）In adults and children,when MCV<80 fl or MC H<27 pg or Hb A₂abnormality is used as the screening positive criterion,the specificity is the lowest;MCV<80 fl or MCH<27 pg and Hb A₂ abnormality is the screening positive When determining the criteria,the specificity of thalassaemia screening is higher.2.One newly discovered case ofα-thalassaemia and two newly discovered cases ofβ-thalassaemia.（1）The proband of family 1 is a 26-year-old female with HBA2:c.54del C,whose hematological parameters are small cell hypochromic anemia,which is not found in her mother and sister,and has been sequenced by Sanger of the globin gene authenticating.（2）Family 2 is a heterozygous mutation of HBB:c.373C>A,resulting in amino acid changes（Pro>Thr）,and its hematological phenotype is normal.The mother of the proband was also proved to be a carrier of the HBB:c.373C>A mutation,while his father and younger brother were normal individuals.A ll samples were verified by Sanger sequencing.Through online bioinformatics analysis of the mutation influence on globin function and protein structure and function,the Pro amino acids in CD124（Pro124,HBB:c.373C>A）were conserved in all 10 species,and the biological software suggested that HBB:c.373C>A mutation was harmful.（3）Family 3 has a heterozygous mutation of HBB:c.40G>A,a point mutation of c.40G>A in CD13,resulting in amino acid changes（Ala>Thr）,its hematological phenotype is normal,and her mother and sister are normal individuals.All samples were verified by Sanger sequencing.Through online bioinformatics analysis of the mutation influence on globin function and protein structure and function,the Ala amino acid in CD13（Ala13,HBB:c.40G>A）is only conserved among the five species,and the biological software prompts HBB:c.40G>A mutation is benign.3.Two cases of large fragment deletionβ-thalassemia.（1）The proband in Family 4 is a 6-year-old female with moderate to severe anemia.The next generation sequencing of globin revealed that there were large fragment deletions and CD41/42（-TTCT）mutations.The MLPA test results showed the range of HBB exon 1（148nt）-HBB downstream gene（173nt）deletions.The proband’s large fragment deletion was inherited from his father,and the C D41/42（-TTCT）mutation was inherited from his mother.（2）The proband of case 5 is a 2-year-old female with small cell hypochromic anemia.The next generation sequencing of globin revealed that there was a deletion of large fragments.The results of MLPA test showed that the range of HBG1 exon 3（436nt）-OR51V1（486nt）was missing.The proband’s large fragment deletion was inherited from his mother.Conclusions:1.Dehong and Xishuangbanna areas in Yunnan province are areas with a high incidence of thalassaemia,and the Dai people belong to the ethnic group with a high incidence of thalassaemia.2.The carrying rate ofα-thalassaemia is greater than that ofβ-thalassaemia.Most of the clinical manifestations ofα-thalassaemia orβ-thalassaemia are asymptomatic or mild.3.Static thalassaemia mutations cannot be screened by conventional methods.NGS is a more effective screening method for static thalassaemia;4.Some rare large fragment deletion thalassaemias cannot be detected by conventional kits,which may lead to missed diagnosis,to give birth to children with moderate to severe anemia,using NGS and combined with blood routine,capillary electrophoresis results for comprehensive analysis can avoid misdiagnosis.