| Objectives:Elevated fetal hemoglobin ameliorates the clinical severity ofβ-thalassemia.In this study,the rs368698783(G>A)and rs7482144(C>T)polymorphic loci were analyzed for association with Hb F expression.To explore the genetic modifier gene polymorphism loci involved in regulating Hb F expression levels inβ-thalassaemia patients.To analyse the impact of mutant loci polymorphisms in related genes on Hb F expression.To search for the presence of previously unreported mutant loci,to identify genetic associations and to explore the association between these mutant loci on Hb F levels and on the alleviation of clinical symptoms in patients withβ-thalassaemia.Methods:(1)Samples ofβ-thalassaemia pure congeners(β~0/β~0,β~+/β~+),β-thalassaemia compound heterozygous samples(β~0/β~+),Hb E/β-thalassaemia samples(β~E/β~0,β~E/β~+)and Hb E purex samples(β~E/β~E)were collected by the thalassaemia genetic test.Hb F>5%samples were collected by haemoglobin electrophoresis test.The samples were divided into Hb F>5%(β~N/β~N)samples and Hb F>5%(β~M/β~N)samples according to the results of the gene test for thalassaemia.The unrelated samples with normal haemoglobin electrophoresis results and the results of the gene test for thalassaemia were collected as controls.Genomic DNA was extracted from the collected samples and the rs368698783 and rs7482144 loci were examined by sanger sequencing.The relationship between rs36868698783 and Hb F levels was analysed by comparing haematological parameters,mutant gene detection rates and MAF.Randomly selected samples were subjected to RNA extraction.γ-globin transcripts were analysed for ~Gγ/~Aγm RNA expression content,and the functions brought about by the linkage and non-linkage of the rs368698783 locus to the rs7482144 locus were analysed Impact.(2)Seventy-six samples ofβ-thalassaemia patients were collected and genomic DNA extracted by the Thalassaemia Genetic Test.A custom gene microarray was designed for the modifier genes involved in the regulation of Hb F expression,and a gene panel was designed to detect the relevant modifier genes using high-throughput sequencing technology.Genome-wide association analysis was performed to compare the polymorphisms in Hb F expression levels between individuals withβ-thalassaemia and to analyse the correlation between mutant loci and Hb F expression.Results:(1)The detection rate of mutant alleles at the rs368698783 and rs7482144 loci in non-transfusion-dependentβ-thalassaemia patients was significantly different compared to normal controls(P<0.05),while transfusion-dependentβ-thalassaemia was not significantly different compared to controls(P>0.05).There was a significant difference in the detection rate of mutant alleles in transfusion-dependentβ-thalassaemia compared to non-transfusion-dependentβ-thalassaemia(P<0.05).The detection rate of mutations at rs368698783 and rs7482144 loci was significantly different in Hb E/β-geoblastic patients and Hb E pure congeners compared to controls(P<0.05),whereas no significant differences were found inβ-thalassaemia patients compared to controls(P>0.05).(2)Hb F>5%(β~N/β~N)and Hb F>5%(β~M/β~N)samples were significantly different from the control rs368698783 and rs7482144 loci mutation detection rate(P<0.05).The association between SNPs polymorphisms and Hb F levels was not significant between the groups of patients withβ-lesion.(3)The mutant allele at the rs368698783 locus resulted in a ratio of ~Gγ/~Aγm RNA expression(P<0.05).There was one case where rs368698783 was wild-type and rs7482144 was heterozygous,and the Gγ/Aγm RNA ratio was within the wild-type range of rs368698783,indicating that the modification effect of rs368698783 was stronger than that of the rs7482144 locus.(4)NGS of the modifier genes involved in regulating Hb F expression inβ-thalassaemia patients resulted in the detection of 17,072 mutations.Includes structural variants such as SNPs,insertions,deletions,and indels.A total of 135mutant loci associated with Hb F levels were detected(P<0.05).Fifteen mutant loci with mutations associated with the expression of Hb F were identified on chromosomes 2,6,9,11,14,15,16 and 19(P<0.001).These included two missense variant,two synonymous variant,one 3’UTR variant,eight intron variant and two intergenic variant.Conclusions:(1)The rs368698783 and rs7482144 mutation is associated with elevated Hb F levels and correlates with Hb E mutations.The A allele and T allele can reduce the degree of anaemia and alleviate the clinical symptoms ofβ-thalassemia.(2)Fifteen mutant loci were found to be associated with high Hb F levels inβ-thalassaemia patients.This study further confirms and enriches the genetic modification loci in thalassaemia,providing an experimental basis and theoretical foundation for elucidating the molecular pathological mechanism of thalassaemia,screening drug targets and formulating treatment protocols. |
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