| Background and Objectiveβ-thalassemia is one of the most common monogenic disorders in the world. The incidence for this disease is high in areas of the tropics and subtropics including southern China. In southern China, the carrier rate ofβ-thalassemia is 2.54% in Guangdong and 6.78% in Guangxi where are two provinces of the most frequently thalassemia occoured. According to the clinical phenotypes,β-thalassemia can be divided into three main types: thalassemia major (TM), thalassemia trait (TT) and thalassemia intermedia (TI). TM is a severe form that requires transfusions from infancy for survival, whereas TT is usually asymptomatic. TI is used to indicate a clinical condition of intermediate gravity between TT and TM, which encompasses a wide phenotypic spectrum spanning from mild anemia to more severe anemia with required occasional transfusions.Corresponding to the phenotypic diversity, the molecular basis of TI is also variable. Thein has reviewed the major genetic modifiers ofβ-thalassemia: genotypes ofβ- andα-globin and expression ofγ-globin. Some genotypic factors have been reported to affect synthesis of theγ-globin chain, such as the 3'HS1 (+179 C→T) polymorphism, the (AT)xNy(AT)z motif in the 5 'HS2 site, and the (AT)x(T)y motif in the -540 region of theβ-globin gene. Variation of rs11886868 (T→C) in the BCL11A gene has also been shown to correlate with increased HbF in European TI patients. In addition, those factors that can moderate globin imbalances indirectly or cause theβ-thalassemia-like phenotype, such as GATA-1,AHSP, and heme-regulated initiation factor 2 alpha kinase ( HRI),are also thought to contribute to the phenotypic diversity of TI.Researchers have described molecular characterization of TI in Iranian, Indian, Italian, and other populations. However, to date, the genetic basis of TI in Chinese patients is poorly understood. In this study, we performed a comprehensive analysis of the molecular basis underlying TI in southern China. Genotypes ofβ-globin and other known modifiers linked toα/βimbalance were investigated in 117 patients withβ-thalassemia intermedia phenotypes. Their clinical, hematological, and molecular data were analyzed systematically with the aim of creating a genotype-phenotype correlation.Samples and MethodsSamplesWe recruited the TI patients in this study according to previously described criteria, in whom the classical clinical diagnosis of TI patients, such as the steady state Hb level of 60-105g/L, age at onset over two years old, and transfusion independence were emphasized for all our patients. A total of 117 patients from 109families with TI phenotypes were recruited for this study.MethodsClinical events analysis: Complete blood counts and red cell indices were determined by automated cell counting (Model Sysmex F-820; Sysmex Co Ltd, Kobe, Japan); the levels of HbA, HbA2 and HbF were analyzed on the Bio-Rad Variantâ…¡HPLC system (HPLC, VARIANTM, Bio-Rad, Hercules, CA, USA). The information about blood transfusions, thalassemia appearance, age at onset, hepatosplenomegaly and splenectomy was obtained by retrospective clinical data.DNA analysis: We collected the blood with EDTA anticoagulation of 117 Chinese TI patients. Genomic DNA was extracted from peripheral blood by standard phenol/chloroform methods. The 11 knownβ-thalassemia mutations, the two common deletions including Chinese Gγ+(Aγδβ)0 thalassemia and Southeast Asian hereditary persistence of fetal hemoglobin (SEA-HPFH) , the three commonα-thalassemia deletions (--SEA,-α3.7 and -α4.2),the six non-deletional mutations (αCD30,αCD31,αCD59,αQS,αCS andαWS), theαααanti3.7 orαααanti4.2 triplication and Xmn1 site -158 of the Gγ-globin gene were analyzed. Further sequence analysis was applied on bothβ0/β0 andβ+/βN orβ0/βN samples, analyzed targets for the former (β0/β0) include both 3'HS1 and 5'HS2 core region, the promoters of the Gγ- and Aγ-globin genes, the (AT)x(T)y sequence variations at the position -540 of theβ-globin gene, SNP polymorphism of rs11886868 in the BCL11A gene, as well as the wholeα2- andα1-globin genes; and those for the latter (β+/βN orβ0/βN) include the core regions of both 5'HS2 and 5'HS3,the wholeβ-globin gene and AHSP gene, and the cDNA of GATA-1 generated by RT-PCR from mRNA.Since HRI has been shown to modify the phenotypic severity ofβ-thalassemia in murine models, we also sequenced the HRI cDNA inβ+/βN orβ0/βN samples by RT-PCR.RNA analysis: Total cellular RNA was isolated from fresh peripheral blood using Gentra Purescript RNA Kit (Gentra, Americia). The cDNA synthesis was performed using the ExScript RT reagent Kit (TaKaRa Biotechnology, China). The expression levels ofβ-globin (target gene) andβ-actin (control) were measured by SYBR Green-based relative quantitative RT-PCR assays. Five heterozygous subjects carrying the Cap+39(C→T) mutation and three heterozygous subjects carrying the Term CD+32(A→C) mutation were selected respectively, as two patient groups, while six normal subjects served as the control group.β-globin gene haplotype analysis: Theβ-globin haplotypes associated with the two novel mutations were analyzed with PCR amplification followed by restrictionenzyme digestion. Seven classical polymorphic restriction enzyme sites selected for haplotype analysis were Hincâ…¡-5'ε,Hindâ…¢-Gγ,Hindâ…¢-Aγ,Hincâ…¡-ψβ,Hincâ…¡-3'ψβ, Avaâ…¡-βand Bam HI-3'β. Each individual was scored for the presence (+) or absence (-) of each of the seven RFLP sites.Statistical methods: Statistical analysis was performed using SPSS software (Version 13.0, SPSS inc, USA). The difference of the relative mean mRNA concentration between mutation carriers and normal individuals were analyzed by the independent samples t-test. Ap-value < 0.05 was considered statistically significant.Results and DiscussionIn this study,117 Chinese individuals between 2 and 61 years old were enrolled to characterize the molecular basis ofβ-thalassemia intermedia in southern China. The level of Hb is between 60 and 105g/L. The age at onset is between 1.5 and 27 years old. Seventy-five TI patients have thalassemia-like appearance. Sixty-two ones have no transfusion, fifteen ones have single time transfusion, and thirty-one TI patients need occasional transfusion. Sixty-nine ones are of hepatosplenomegaly, twenty-three ones are of splenectomy, and thirty-six ones have normal liver and spleen.According to their genotype ofβ-globin, we divided 117 TI patients into two types: Typeâ… Î²-thalassemia homozygotes (n=97) who inherited two deficientβ-globin alleles and TypeⅡβ-thalassemia heterozygotes (n=20) who had only a singleβ-thalassemia allele. In total, we detected 18β-thalassemia alterations including two novel ones which were termed as Term CD+32(A→C) and Cap+39(C→T) respectively,β-globin variant HbE, the two deletional mutations which can lead to SEA-HPFH or (δβ)0-thalassemia Chinese Gγ+(Aγδβ)0.Typeâ… Î²-thalassemia homozygosity was detected in 97 TI patients (82.9%) as follows: (i) Forty-four (37.6%) had homozygous or compound heterozygousβ-thalassemia alleles and normalα-globin genes,(ii) Twenty-seven (23.1%) were HbE/β0 compound heterozygotes. No HbE/β+ heterozygote or co-incidentα-globin mutations were found in this group,(iii) Fifteen (12.8%) had twoβ-thalassemiamutations, and in addition carried theα-thalassemia alterations including the -α3.7, -α4.2,--SEA alleles orαCS.We observed two particular patient cases. One was caused by co-existence of Hb H disease (--SEA/-α4.2) andβ-thalassemia compound heterozygosityβCD17(A→T)/βIVS-2-654(C→T),while the other was caused byαα/αααanti3.7andβ-thalassemia homozygosity of the -28 (A→G) mutation, (iv) Eleven (9.4%) carried SEA-HPFH (7 cases) or Chinese Gγ+(Aγδβ)0 (4 cases) deletions in addition to having oneβ-thalassemia mutation. Among them, two were IVS-2-654 (C→T) / Chinese Gγ+(Aγδβ)0 and -28 (A→G) / Chinese Gγ+(Aγδβ)0,respectively, both plus one --SEA deletion.TypeⅡβ-thalassemia heterozygosity was detected in 20 TI patients (17.1%) as follows: (i) Fourteen (12.0%) carried a singleβ-thalassemia allele and normalα-globin genes.(ii) Five (4.2%) had a singleβ-thalassemia allele and co-inheritedαααanti3.7 triplication. Noαααanti4.2 triplication was detected.(iii) One patient (0.9%), in a Miao family, was a heterozygote for a frameshiftβ-thalassemia mutation at CD 53 (-T). There were threeβ0/β0 TI cases whose genotypes wereβCD17(A→T)/βIVS- 2-654(C→T),βIVS-1-1(G→T)/βCD41-42(-CTTT) andβCD17(A→T)/βCD17(A→T).We detectedα-globingene and other modifiers which could increase the production of HbF including the promoter region of bothβ- andγ-globin gene, the core regions of both HS2 and HS3, 3'HS1,and the SNP rs11886868 in BCL11A gene. The analysis of XmnI site -158 of the Gγ-globin gene showed that two patients were +/- and one case was -/-.The three patients all had C/C genotypes of both at +179 of 3'HS1 and rs11886868. However, we could not consider that C/C genotypes of rs11886868 contributed to the high HbF level based on two facts. One is that the frequency of C/C genotypes was 0.867 in Chinese people from the HapMap data (http://www.hapmap.org/).The second is that an additional 115 samples were analyzed, including 30 TI patients ( 25β0/β+,5β+/β+) and 85 normal individuals, in which all were found to be the C/C genotype. As to other modifiers, the results of analysis showed that they were normal.It was notable that we found 14 patients with heterozygous forβ-thalassemia (1 case with the genotype ofβ-28(A→G)/βN,7 cases with the genotype ofβIVS-2-654(C→T) /βN,3 cases with the genotype ofβCD17(A→T)/Î’N,2 cases with the genotype ofβCD71-72(+A)/Î’N and 1 case with the genotype ofβCD41-42(-CTTT)/Î’N) among our 117 TI patient cohort, in whom theβ-globin gene was found to be structurally intact by sequence analysis and excluded theα-globin gene triplication. As for the underlying defective targets, we focused on primary genetic modifiers that could modulate the imbalance ofα/βfor investigation in ten TI patients. We evaluated the effects of some potential modifiers involving one linked to theβ-globin gene cluster, the LCR resides in the 5'HS2 and 5'HS3 regions, and three ones not to be linked to theβ-globin cluster, AHSP, GATA-1 and HRI.Sequence analysis of the core regions of 5'HS2 and 5'HS3 showed wild type sequences, thus excluding mutations in these control regions in reducing expression ofβ-globin gene. The sequence of AHSP gene was normal. No mutations in the cDNA sequences of GATA-1 have been found. In the case of HRI, the polymorphism of rs2639 and rs2640 were detected. The rs2639 (A→G) is a synonymous mutation and the rs2640 (A→G) is a missense mutation resulting in a K558R substitution. However, the affect on the function of this substitution is unknown since Lys (K) and Arg(R) are similar in amino acid properties. These results indicate the existence of causative genetic determinants not yet molecularly defined.Two novel mutations ofβglobin were analyzed. The proband of Term CD +32(A→C) mutation was a compound heterozygote ofβTerm CD+32(A→C)/βCD 27-28(+C) with XmnI (+/+) homozygosity. By using PCR-based RFLP method and family linkage study, the haplotypes linked to the TermCD+32(A→C) mutation and CD27-28(+C) were identified as "- -+++-+" and "--++++-", respectively. The proband of Cap+39 (C→T) mutation was a compound heterozygote ofβCap+39(C→T) /βCD41-42(-CTTT) with Xmn I(-/-) homozygosity. Interestingly, his father and four other family members were all heterozygotes of Cap+39(C→T) mutations. Since they do not have any evident hematologic phenotype, we regard this mutation asβ++ (silentβthalassemia).We used real-time PCR to analyze the mRNA level. Two standard curves were generated: y =-3.672x +19.787 (R2=0.999) forβ-globin mRNA and y= -3.466x+24.000 (R2=0.999) for p-actin mRNA. The mean relative mRNA concentrations were 0.835±0.048 (n=3) for the Term CD+32(A→C) group, 1.093±0.118 (n=5) for the Cap+39(C→T) group, and 1.016±0.098 (n=6) for the normal control group after a normalization procedure using linear regression equations. Statistical analysis showed that there was a significant difference (P=0.021) of mean relativeβ-globin mRNA concentration between the Term CD+32(A→C) group and the control group. The decreased level ofβ-globin mRNA in the patient group compared with that of the normal control group was calculated to be 17.8%. In contrast, no significant difference (P=0.270) was found between the Cap+39(C→T) group and the control group. Sequence alignment showed that Term CD+32(A) and Cap+39(C) were conserved sites during evolution. These two novel mutations weren't detected in 156 random samples from southern Chinese individuals.ConclusionsChinese TI patients showed a high degree of heterogeneity in both phenotypic and genotypic aspects. According to their molecular basis,117 TI patients can divide into two types which are 97β-thalassemia homozygotes who inherited two deficientβ-globin alleles and 20β-thalassemia heterozygotes who had only a singleβ-thalassemia allele. The former includes five sub-types: Forty-four had homozygous or compound heterozygousβ-thalassemia alleles and normalα-globin genes; Twenty-seven were HbE/β0 compound heterozygotes; Fifteen had twoβ-thalassemia mutations, and in addition carried theα-thalassemia alterations including the -α3.7,-α4.2,--SEA alleles orαCS;Eleven carried SEA-HPFH (7 cases) or Chinese Gγ+(Aγδβ)0 (4 cases) deletions in addition to having oneβ-thalassemia mutation. Among them, two wereβIVS-2-654(C→T)/ Chinese Gγ+(Aγδβ)0 andβ-28(A→G)/Chinese Gγ+(Aγδβ)0,respectively, both plus one --SEA deletion.The latter includes three sub-types: Fourteen carried a singleβ-thalassemia allele and normalα-globin genes; Five had a singleβ-thalassemia allele and co-inheritedαααanti3.7 triplication; One patient was a heterozygote for a frameshiftβ-thalassemia mutation at CD 53 (-T).In a word, we have basically clarified the molecular basis of the 117 TI patients.The molecular basis of 14 TI patients (13β0/βN and 1β+/βN cases) with known heterozygous mutations ofβ-thalassemia and three ones with homozygousβ-thalassemia (β0/β0) were uncertain, the existence of other causative genetic determinants are remaining to be molecularly defined.We have detected two novel mutations which are Term CD+32(A→C) and Cap+39(C→T).After assessing them, we think that the former mutation can decrease the level ofβ-globin mRNA and the latter has no influence upon the level ofβ-globin mRNA. In addition to the pedigrees data of the two novel mutations, we regard Cap+39(C→T) mutation asβ++ (silentβ-thalassemia) and Term CD +32(A→C) mutation asβ+.The haplotypes associated with Term CD+32(A→C) mutation is"--+++-+".
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