| Background and PurposeHemoglobin is a protein responsible for carrying oxygen in higher organisms, consists of two a-globin chains and two β~-globin chains. The human β~-globin gene cluster, spanning a 70-kb region, expressing five types of β~-like globin in development:ε-globin in embryonic period, ~Gγ-and Ay-globin in fetal period,δ-and P-globin in adulthood. β~-thalassemia is a common single-gene disorder that leads to a quantitative defect in the synthesis of β~-globin chains. The vast majority of β~-thalassemia mutations are point mutations. The locations of P-thalassemia mutations in southern Chinese population are distributing in the open reading frame and the promoter region of β~-globin gene. The pathogenic mutations could be divided into three types according to their different functional affects:β++ mutations (silent type), β+ mutations (intermedia type) or β~0 mutations (major type). β~-thalassemia would not have clinical symptoms of severe anemia for β~-globin gene is turned off in the fetal period until Hb F expression silenced and Hb A becomes the majority type of hemoglobin after birth. The phenotypic diversity of β~-thalassemia reflects the underlying a marked genotypic heterogeneity of β~-globin gene mutations. Clinical manifestations of β~-thalassemia mainly depend on the disease-causing mutation in the β~-globin gene. However, the same genotype of β~-thalassemia can manifest variable clinical severity. Several genetic modulators and cis-regulatory elements involved in the regulation of human HbF have been identified, and concomitant a-thalassemia is an ameliorator of β~-thalassemia. Many genetic polymorphisms exhibiting association with fetal globin induction have been validated for the prediction of β~-thalassemia severity, including large deletions in δβ~-thalassemia/~Aγδβ~-thalassemia, several genetic polymorphisms which cause hereditary persistence of fetal hemoglobin in the γ-globin gene promoter, an intergenic region between HBS1L and MYB, and a transcription factor BCL11A, as well as mutations in the erythroid transcriptional KLF1. These studies provide candidate molecular targets for the treatment of β~-thalassemia major.The molecular mechanism of y-globin gene reactivation in adulthood has been a hot topic since high expression of Hb F is beneficial in attenuating the severity of the symptoms of P-thalassemia. About 300bp promoter region upstream of γ-globin gene 5’UTR has been extensively studied, about 20 transcriptional regulatory factor binding sites were found. Majority of point mutations which causes hereditary persistence of fetal hemoglobin are located in the γ-globin gene promoter region, and within the transcription factor binding sites. These findings make the genetic polymorphisms and quantitative trait loci become the key mechanism of fetal hemoglobin hereditary persistence in adulthood. Moreover, researchers performed genome-wide association study, pedigrees analysis and gene function studies, some transcription factors that negatively regulates y-globin gene were found, such as BCL11A, MYB, HBS1L, KLF1 etc. The single-nucleotide polymorphisms or mutations located in these negative regulators lead to Hb F levels increasing and alleviating the clinical phenotype severity of β~-thalassemia. The molecular mechanisms of these genetic variants have been revealed gradually. In 2014, LYAR, a broad-spectrum transcription factor was found binding with GGTTAT sequence in 5’UTR of y-globin gene. LYAR can raise histone methyltransferase PRMT5 and methyl transferases DNMT3A, leading silencing of y-globin gene in adulthood. To explore the potential genetic variants that ameliorate the severity of β~-thalassemia, we performed capture-based next-generation sequencing of an 80-kb region spanning the β~-globin cluster with samples from 1142 β~-thalassemia patients and indentified 271 common SNPs. We evaluated the potential regulatory effects β~-thalassemia of the variants and noticed a high-frequency SNP Ay+25 (G>A, rs368698783) which almost completely linked with ~Gγ-158 (C>T, XmnI polymorphism) and within the binding site (GGTTAT) of LYAR. We suspected that Ay+25 variant was a motif-disrupting SNP which weakened the negative regulation of LYAR and reactivated the expression of y-globin gene. We would perform a systemic genotype-phenotype analysis using 2738 individuals including normal controls, β~-thalassemia heterozygous and β~-thalassemia patients in southern China as well as HbEE patients in southeastern Thailand, moreover, exploring the functional effects in mRNA level.Design and Methods1. Cohort design and phenotypic analysis(1) Cohort designWe designed a large population-based study that consisted of 2738 consecutive subjects divided into 4 cohorts based on β~-globin genotypes. The cohorts comprised 513 non-thalassemia controls (αα/αα, βN/βN, cohort A),515 β~-thalassemia heterozygotes (αα/αα, βM/βN, cohort B),1142 β~-thalassemia homozygotes or compound heterozygotes (βM/βM, cohort C), and 568 Thai HbEE patients (βE/βE, cohort D), who were used in the validation study.(2) Phenotypic analysisThe main testing indicators are as follows:hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, red blood cell distribution width, Hb F, Hb A2. Definition of thalassemia major or thalassemia intermedia in this study is based on the following 4 clinical indications:onset of anemia,transfusion before 4 years of age, steady-state hemoglobin levels and growth and development conditions.2. Molecular genetic testing(1) HBA and HBB genotypingWe detected three common α-thalassemia deletions of Chinese participants by multiple Gap-PCR. We detected point mutantions of α-and β~-thalassemia by PCR-RDB technology.(2) SNPs genotyping of β~-globin gene cluster by target capture-based NGSTarget fragments of the β~-globin cluster region were enriched using high density probes designed by Agilent SureDesign platform and then sequenced on a HiSeq2000 instrument and perform the bioinformatics analysis in BGI company.(3) Genetic variation detection of HBG promoterThe proximal promoter regions of the two y-globin genes, spanning from 610 bp upstream to 120 bp downstream of the transcription start site, were further sequenced with specific primers. ~Gγ-158 variants were either detected using PCR-RFLP technology.(4) Gy/Ay mRNA expression analysisThe HBG:c.410G>C polymorphism in exon 3 of the HBG mRNA was used as a marker to quantify the ratio of Gy/Ay-globin mRNA expression and the allelic expression of Ay-rs368698783 based on the G (in Gy mRNA only) and C (in Ay mRNA only) allele peaks on the sequencing chromatographs of reverse-transcribed PCR products using the BioEdit Sequence Alignment Editor.3. Statistical AnalysisThe association study and conditional analysis were carried out using PLINK v1.07. We considered HbF Z-scores as the dependent variable in the above analysis. Tag-SNPs selection and alleles captured were based on Paul de Bakker’s Tagger algorithm of the software HaploView. Multivariate and univariate analyses, as well as stepwise Cox analysis, were performed using SPSS v20.0 to evaluate the effects of genetic variations on the clinical features of cohorts C and D. Significant differences in HbF levels or in mRNA or protein expression between different genotype cohorts or controls were examined with Student’s t-test, the Mann-Whitney U test, or the Kruskal-Wallis test as appropriate. The survival curves were generated using the Kaplan-Meier log rank test in SPSS.Results1. We identified ~Aγ+25 as a genetic modifier by excluding other SNPs in β~-globin clusterTo explore the potential genetic cis-variants that ameliorate β~-thalassemia severity, we performed capture-based next-generation sequencing (NGS) analysis of an 80-kb region spanning the β~-globin cluster from the DNA of 1142 Chinese β~-thalassemia patients. Using NGS data from these 1142 β~-thalassemia patients, we discovered271 common SNPs (MAF>0.01) in the β~-globin clusterand the SNPs with r2>0.8 were distributed into 162 LD blocks. We identified 53 significant LDs by association study of HbF levels as well as 53 tag-SNPs (minimum of P-value) of each LD. We performed stepwise Cox proportional hazards analysis of these 53 tag-SNPs, along with 10 known modifiers, measured by the non-transfusion survival time, identified 7 LDs which include 66 SNPs had the significant strong effect on the clinicalseverity of β~-thalassemia phenotype. After searching for sequence-matched motifs of the known erythroid transcription factors, we uncovered three potentially functional regulatory variants (rs368698783, rs10768737 and rs5010981) from 7 preliminary candidate LDs. Apart from rs368698783 comprehensively discussed in this paper, the other 2 SNPs will also be enrolled for our further exploration. The motif-disrupting variant of SNP rs368698783 had the strongest effect on the clinical severity of the β~-thalassemia phenotype (HR=0.341, P=7.68×10-13) among three potentially functional variants and show a stronger effect than rs7482144 (HR=0.352, P=2.84×10-12), suggested that the rSNP rs368698783 might be associated with ameliorating P-thalassemia severity.2. ~Aγ+25 variant has a wide spectrum of in southern China and Thailand HbEE crowdWe indentified 388 mutant allele (A, DAF=0.089) in 2170 cases of southern China (cohort A, B, C) including 338 heterozygotes and 25 homozygotes. We indentified 896 mutant allele (A, DAF=0.789) in 568 cases of southeastern Thailand HbEE patients (cohort D) including 180 heterozygotes and 358 homozygotes. Moreover, we noticed that ~Aγ+25 Variant had the linkage disequilibrium (D’=0.715, R2=0.210) with HbE (HBB:c.79G> A) mutation.3. Ay+25 variant ameliorates P-thalassemia severity by regulation of Hb F expressionWe then validated the effects of rs368698783 genotype on HbF levels in 2738 consecutive individuals from two ethnic subpopulations. The genotypes GA and AA of HBG1-rs368698783 exhibited a significantly elevated level of HbF compared with the genotype GG in each of three cohorts from China. The HBG1-rs368698783 AA genotype exhibited significantly elevated HbF levels compared with GA in thalassemia (cohorts B and C) and HbEE (cohort D) patients. After correction for HBB, HBA, KLF1, BCL11A and HBS1L-MYB genotypes, univariate analysis demonstrated that variant HBG1-rs368698783 could act as a modifier associated with elevated levels of HbF in β~-thalassemia (P<0.001) and HbEE (P=0.003) and could ameliorate the severity of β~-thalassemia in age at onset (p<0.001) and transfusion requirements (OR=0.165, P<0.001) and reduce the risk of developing TM (OR=0.147, P<0.001). Moreover, AA (P=0.002) and GA (P<0.001) genotypes had significantly increased survival time with β~-thalassemia according to the Kaplan-Meier log rank test.4. Ay+25 variant impacted the relative expression of Gy/AyWe observed that the A allele significantly increased the Gy/Ay ratio through an increase in the production of y-globin mRNA and peptide, as well as allele-biased RNA expression in P-thalassemia patients (βM/βM) with an A/G allelic ratio of 2.4 compared with non-thalassemic controls (βN/βN) or β~-thalassemic traits (βM/βN) with an A/G allelic ratio of 1.1. This evidence supports the conclusion that the HbF level in P-thalassemia major is elevated by a variant-dependent reactivation of the y-globin gene with an increasing Gy/Ay ratio.Main conclusions1. We evaluated the genetic modification of β~-thalassemia among 271 common SNP in β~-globin gene cluster and identified+25 variant (rs368698783) at 5’UTR of Ay-globin gene (HBG1) as a genetic modifier by excluding other SNPs in the β~-globin cluster. Thus, the study expands our knowledge of the regulation of hemoglobin switching and provides potential targets for the treatment of β~-hemoglobinopathy patients.2. We verify the modification of β~-thalassemia phenotype of Ay+25 Variant in two large populations. This variant accounted for 41.2% of patients with β~-thalassemia in this study and will be applicable to the prediction of β~-thalassemia severity worldwide. Hypothetically, bias of the variants in the Thai subpopulation might be related to the malaria-protective effects of thalassemia and, conversely, exhibited effects on amelioration of thalassemia severity.3. We identified ~Aγ+25 variant could impact the relative expression of ~Gγ/~Aγ as well as allele-biased RNA expression in β~-thalassemia patients. This evidence supports the conclusion that the HbF level in β~-thalassemia major is elevated by a variant-dependent reactivation of the γ-globin gene. |
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