Modifying Effect Of AHSP Gene On β-thalassemia And The Role Of BCL11A Gene In The Persistence Of Fetal Hemoglobin In β-thalassemia Patient

PartⅠModifying effect of AHSP gene onβ-thalassemiaBackgound and Objectiveβ-thalassemia is an inherited hemoglobin disorder characterized byβ-globin deficiency and excessα-hemoglobin. Excessα-hemoglobin aggregates in red cells, resulting in ineffective erythropoiesis and shortened lifespan of circulating erythrocyte. Therefore, excessα-hemoglobin is regarded as a major determinant of the pathophysiology and the disease severity ofβ-thalassemia. Althoughβ-thalassemia is a classical monogenic disease, its clinical phenotype varies greatly even among the individuals with identical gene mutations. Some genetic modifiers that neutralize the deleterious effect of freeα-hemoglobin can explain a part of clinical heterogeneity ofβ-thalassemia, such as co-inheritance of hereditary persistence of fetal hemoglobin orα-thalassemia. However, the phenotypic variability of manyβ-thalassemia subjects cannot be explained by known determinants. Thus, additional genetic factors are believed to be contributing to the clinical outcomes ofβ-thalassemia.α-hemoglobin-stabilizing protein(AHSP) is a molecular chaperon that binds specifically to freeα-hemoglobin and prevent their precipitation, providing a potential compensatory mechanism to neutralize the deleterious effect of freeα-hemoglobin. AHSP knock-out mice show abnormal red cell production, which is also observed inβ-thalassemia. Moreover, the hematological abnormalities inβ-thalassemia mice are exacerbated by concomitant loss of AHSP gene. Based on these findings, it was proposed that AHSP gene might affect the clinical phenotype of the individuals withβ-thalassemia.Several studies have been done to assess the modifying effect of AHSP gene on phenotype ofβ-thalassemia patients, but no conclusive proof has been found yet. Lai et al reported that AHSP gene was a quantitative trait gene modifying the phenotype ofβ-thalassemia. Galanello et al reported that variation in AHSP expression was associated withβ-thalassemia phenotype.Vipraksit et al genotyped AHSP gene in 120 Thai patients with varying severities of HbE/β-thalassemia, neither mutation in AHSP gene nor specific relationship between AHSP haplotypes and disease severity was discovered. Southern China is one of the major regions with high prevalence ofβ-thalassemia, but no study of the modification effect of AHSP gene to the disease severity ofβ-thalassemia has been conducted in this region by now. Knowing the high prevalence and the great variations in the genetic characters ofβ-thalassemia in different geographic regions, it is highly significant to study the relationship between AHSP gene and theβ-thalassemia in Southern China.Samples and Methods1. Samples5789 consecutive blood samples were obtained from students in Guangxi province, which is the most major region with high incidence ofβ-thalassemia in Southern China. Total 370β-thalassemia carriers had been identified among them by routine hematological analysis and DNA analysis, but only 365β-thalassemia carriers were included in the study for five DNA samples being run out. The five majorβ-globin mutations in theseβ-thalassemia carriers are CD41-42(-CTTT) (42.90%), CD17(A→T)(24.60%), IVS-Ⅱ-654(C→T)(9.02%),-28(A→G)(9.02%) and CD71/72(+A)(6.28%). In the 365 individuals withβ-thalassemia,364 subjects were heterozygous carriers, one subject was compound heterozygous carrier. A total 71 out of the 365β-thalassemia carriers co-inherited a-thalassemia.2. Methods(1) Hematological studiesThe hematological phenotypes of thalassemic traits were determined based on full blood counts (FBCs) and Hb electrophoresis analysis. Red blood counts and indices were determined using a Model Sysmex F-820 Hematology Analyzer (Sysmex Co Ltd, Kobe, Japan). HbA, A2, F, and any abnormal Hb were quantitated with aⅡVARIAN T automatic Hemoglobin Analyzer (BIO-RAD, USA).The criteria that indicated the possibility of heterozygosity forβ-thalassemia included:MCV< 80 fL, MCH< 27 pg and HbA2 levels> 3.5%.(2) Mutation analysis of a-thalassemia andβ-thalassemiaGenomic DNA was prepared from peripheral blood leukocytes using standard protocols. Samples suggested to be positive for hemoglobinopathy by the hematological analysis were genotyped to identify the causative mutation. The molecular basis ofβ-thalassemia was determined using reverse dot-blot (RDB) hybridization, which simultaneously identifies 11 previously characterizedβ-thalassemia mutations in Chinese. The positive phenotypic samples with negative RDB results were subsequently analyzed by direct genomic sequencing of the entireβ-globin gene. The common a-thalassemia deletional mutations (—SEA/,-a3.7/and-a4.2/) were typed by gap polymerase chain reaction (Gap-PCR), six non-deletional mutations (αwsα,αCSα/,αQSα/,αCD30α/,αCD31α/, andαCD59α) were defined by RDB assay. All individuals withβ-thalassaemia were analysed to determine whether they had co-inherited one of the nine a-thalassaemia defects (—SEA/,-α3.7/,-α4.2/,αWSα/,αcsα/,αQSα/,αCD30α,αCD31α/, andαCD59α/) that are common among Chinese.(3) SNP of AHSP gene discovery and genotypingAHSP gene polymorphic sites were identified by denaturing high-performance liquid chromatography (DHPLC) in combination with sequencing. Six polymerase chain action (PCR) primer pairs were used to amplify a 1.5kb of the AHSP gene from genomic DNA. These examined 451 bp upsteam of the transcription initiation site, the entire mRNA coding region, intervening intronic sequence and 167 bp of downstream sequences. PCR conditions are available on request. Prior to DHPLC analysis, PCR products were denatured for 5 min at 95℃and then cooled to 49℃at a rate of 1℃/min to allow for heteroduplex formation. Denatured products were analyzed with the WAVE DNA Fragment Analysis System (Transgenomic).8μL of denatured PCR products was injected into the mobile phase (buffer A,0.1 M TEAA; buffer B,0.1 M TEAA/25% acetonitrile) using a flow rate of 0.9 mL/min. The gradient start and end points were adjusted according to the size of the PCR amplicon. Column temperature for screening of input DNA fragment was recommended by the DHPLC melt program software available at the website www.insertion.stanford.edu/melt.html.(4) Statistical analysis:Chi-square test was carried out to analyze the distribution of AHSP haplotypes and the co-inheritance ofα-thalassemia among the groups with different phenotypes ofβ-thalassemia. Hematological parameters were analyzed with one-way analysis of variance between the groups ofβ-thalassemia carriers with T18/T18, T18/T15 and T15/T15 genotypes. A P value less than 0.05 was considered significant. Statistical analyses were conducted with SPSS software, version 13.0(SPSS Inc, Chicago, USA). ResultsWe have identified six common SNPs, two rare SNPs and two missense mutations in 365 individuals withβ-thalassemia. Six common SNPs-two in upstream of exon 1, another two in intron 1, and one synonymous coding SNP in exon 3, as well as one in the 3'-untranslated region. All these 6 common polymorphic sites have been reported previously. Four sequence variants of them (rs4499252, rs5816533, rs4296276 and rs17677) are in near-complete linkage disequilibrium. By analyzing SNP associations, five different AHSP haplotypes were determined among the 365β-thalassemia individuals, which resolved into two main haplotype clades (haplotype A and haplotype B).The frequency of the five haplotypes is 22.8%,41.0%,14.9%, 0.1% and 21.2% respectively. We also identified two rare SNPs which were never published in public databases. For the two rare SNPs, each was only detected in one subject. One of them is located in the 5'flanking region (11810, G>A), and the other one (12802,C>T) causes synonymous substitution of the amino acid position 47. One novel missense mutation was also detected. An A to T transversion at gene position 12750 which substitutes aspartic acid for valine at amino acid position 29(AHSP D29V) was detected in threeβ-thalassemia carriers. A rare AHSP missense mutation, 12831 T→G, which substitutes valine for glycine at amino acid position 56 (AHSP V56G), was identified in only one sample.By analyzing the hematological parameters among theβ-thalassemia carriers with AHSP D29V or AHSP V56G mutation, no obvious modifying effect of these AHSP mutations on the phenotype ofβ-thalassemia carriers was observed. Moreover, a family study of the proband 4 was also performed. A total of 18 members were collected from this family, three members among them wereβ-thalassemia carriers co-inheriting AHSP D29V mutation. Still, it was found that AHSP D29V mutation did not lead to significant phenotypic change of the twoβ-thalassemia carriers. In the 365β-thalassemia carriers,40 of them had lower hemoglobin level (Hb≤105 g/dL), and the others all had higher hemoglobin level (Hb>105 g/dL). The study demonstrated significant difference of co-inheritance ofα-thalassemia in the two groups (χ2=4.096,P=0.043), which is a known modifying factor ofβ-thalassemia. However, the frequencies of the haplotype A and haplotype B showed no statistical difference in the two groups(χ2=2.016, P=0.365). Previous study suggested that AHSP gene was a quantitative trait modifier ofβ-thalassemia. On the contrary to longer T homopolymer(T18), shorter T homopolymer (T15) in promoter region of AHSP gene results in lower expression of AHSP, which was generally known as a deleterious factor ofβ-thalassemia. We analyzed the hematological parameters of 14β-thalassemia carriers who were all T15 homozygous,223β-thalassemia carriers who were T18 homozygous and 128β-thalassemia carriers who were T18/T15 heterozygous. The results showed no significant difference among these three groups. After excluding the effect of co-inheritance ofα-thalassemia, we still found similar hematological level in these groups. Even the hematological parameters were compared according to genders, the result still showed no significant difference.DiscussionThe mutation spectrum ofβ-thalassemia in Southern China is wide. In order to comprehensively evaluate the modifying effect of AHSP gene on the phenotype ofβ-thalassemia in Southern China, we must have high quality samples and big enough sample size at the same time. Therefore, we collected 5789 consecutive blood samples from Southern China, and identified 365β-thalassemia carriers from them.Two missense mutations (AHSP D29V and AHSP V56G) were identified from theβ-thalassemia carriers. It was reported that the major interface of AHSP for a-Hb binding included the C terminus of helix 1, the N-terminal part of helix 2 and the loop connecting helices 1 and 2. AHSP D29V mutation is in the loop, but previous study has demonstrated that mutations of AHSP 29 have no impact on the interaction between AHSP and a-Hb. AHSP V56G mutation is not in this interface.One study had demonstrated that the mutation did not perturb the association of AHSP with a-hemoglobin. However, the stability of AHSPV56G would decrease, and would lead degradation of AHSPV56G. Neither of the two missense mutations leads to significant phenotypic variability ofβ-thalassemia carriers. We did not detect the AHSP 12888 A>T, which causes a isoleucine to asparagines and leads to less effectivity in protecting erythroid cells from the oxidative effects of free a-Hb. Our study indicates that it is not likely that AHSP gene acts as a common modifier ofβ-thalassemia by mutations in Southern China.An association study between AHSP gene and the severity ofβ-thalassemia was also conducted. No significant association was found between AHSP gene and the disease severity ofβ-thalassemia, although ameliorating effect of co-inheritance of a-thalassemia was observed in the P-thalassemia subjects. Lai MI et al reported that the shorter homopolymer (T15) in promoter led to lower AHSP expression compared with the longer homopolymer (T18). In a survey of nine thalassemia intermedia patients who wereβ-thalassemia heterozygous and at the same time co-inheritance of an extra copy of normal a-globin gene (aaa/aa), much higher frequency of T15 was reported. Therefore, AHSP gene was regarded as a quantitative trait modifier ofβ-thalassemia. Here we also identified three thalassemia intermedia patients with an extra copy normal a-globin gene. However, the frequency of T15 was not higher than expected. Furthermore, we analyzed the hematological parameters ofβ-thalassemia carriers homozygous for T15, homozygous for T18 and heterozygous for T18/T15. The hematological parameters had no significant difference among the three groups. Considering the fact that 71β-thalassemia carriers were co-inheritance of a-thalassemia in the study, and it may affect the phenotype of P-thalassemia which might cover the modifying effect of AHSP, we analyzed the hematological parameters again after excluding theβ-thalassemia carriers co-inheriting a-thalassemia. Still, the same result was observed. It was also reported that the common 12391 A allele in AHSP gene can impair AHSP expression by interfering binding to Oct-1, which was considered to be able to modify the severity ofβ-thalassemia. In the 365β-thalassemia carriers, the 12391 A and 12020 T15 alleles are in complete linkage disequilibrium.The AHSP 12391 A allele was tested to also have no association with the severity of P-thalassemia. The studies suggest that AHSP gene is not a quantitative trait modifier of the phenotype ofβ-thalassemia in Southern China.AHSP knock-out mice show abnormal pathological features which are also observed inβ-thalassemia carriers, concomitant loss of AHSP (AHSP-/-) exacerbates the hematological abnormalities inβ-thalassemia mice. It suggests that altered concentration or function of AHSP might account for some of the clinical variability inβ-thalassemia carriers. Although AHSP 12391 A and 12020 T15 alleles were reported to cause lower expression of AHSP, our study showed that the two alleles had no modifying effect on the phenotype ofβ-thalassemia carriers. We tried to explain it from the following aspects. AHSP protein acts as a molecular chaperone which can detoxify the excessα-hemoglobin and stabilize nascentα-hemoglobin to form HbA. Inβ-thalassemia carriers orβ-thalassemia mice, the amount of excessα-hemoglobin is much more than AHSP. Therefore, the reduction of detoxifying capability of AHSP does not exacerbate the clinical outcomes ofβ-thalassemia. However, the wholly loss of AHSP (AHSP-/-) inβ-thalassemia mice would lead to no AHSP protein expression in erythrocytes, which would impair HbA assembly, resulting in moreα-Hb precipitation and evenβ-globin precipitation. The hematological abnormalities are exacerbated finally. But AHSP 12391 A and 12020 T15 alleles only lead to a part of reduction in AHSP expression. Meanwhile, the production ofβ-globin inβ-thalassemia subjects obviously decreases, which would lead to notable decrease of HbA assembly. It is natural that the need of AHSP in β-thalassemia carriers would also decrease accordingly. Therefore, the reduction of AHSP expression caused by 12391 A and 12020 T15 alleles does not exacerbate the hematological features ofβ-thalassemia. It is similar to the observation that there is no significant difference of erythrocyte indices between the AHS+/+ and AHSP+/-β-thalassemia mice. If the expression or function of AHSP inβ-thalassemia carriers completely lost or be less than the need for HbA assemble, hematological parameters would get worse. However, such carrier in which the expression or function of AHSP completely lost or be less than the need for HbA assemble may be too rare to be detected in the 365 individuals withβ-thalassemia. Our study suggested that AHSP gene is not a genetic modifier of theβ-thalassemia carriers in Southern China. At the same time, we speculate that AHSP gene also is not genetic modifi er ofβ-thalassemia in other region if there is not common polymorphisms that affect expression or function of AHSP.ConclusionsAHSP gene is not a genetic modifier to the phenotype ofβ-thalassemia in Southern China. PartⅡThe role of BCL11A gene in the persistence of fetal hemoglobin inβ-thalassemia patientBackgound and ObjectivePeople have noticed the phenomenon thatβ-thalassemia patients had high expression of fetal hemoglobin(HbF)for a long time. However, the molecular mechanism still has not been elucidated. It has been discovered that some genes and gene variations are associated with the persistent expression of HbF. Nevertheless, a reasonable explanation to the phenomenon still can not be got from these genes and gene variations.It was confirmed recently that BCL11A gene was a gene involved in HbF expression. At the same time, the correlation between BCL11A gene and HbF expression inβ-thalassemia patients was also oberserved. Therefore, we give a speculation that the reduction or deficiency of BCL11A causes the persistence of HbF inβ-thalassemia patients.In the view of the significant difference of HbF among normal individuals,β-thalassemia carriers andβ-thalassemia patients, we try to reveal the role of BCL11A gene in the persistence of HbF inβ-thalassemia patient by comparing the expression of BCL11A gene among the former three kinds of individuals.Samples and Methods1. Peripheral blood samples:blood samples of normal individuals came from healthy volunteers, blood samples ofβ-thalassemia patients came from the 303 hospital in Manning.2. Test of BCL11A gene expression in reticulocytes:take 1 ml of peripheral blood, lyse red blood cells with lysis solution and collect cells after centrifugation, then extract RNA according the protocol of Trizol reagent, at last detect the expression of BCL11A gene by RT-PCR. 3. Test of BCL11A gene expression in erythroblasts cultured in vitro:collecte 50 ml blood from normal individual, or collecte 10 ml blood fromβ-thalassemia patient, isolate mononuclear cells from blood by density gradient centrifugation, inoculate the mononuclear cells into culture medium, collect cells after being cultured for 19 days and extract RNA according the protocol of Trizol reagent, at last detect the expression of BCL11A gene by RT-PCR.Results and DiscussionConsidering the reticulocytes in peripheral blood still remain a part of mRNA, and the fact that it is easier for us to obtain blood samples than bone marrow samples, we first want to directly use peripheral blood as experimental materials. However, no product of BCL11A gene was found in blood from a number of normal individuals, which indicated that we can not clarify the role of BCL11A gene in the perisistence of HbF inβ-thalassemia patient by comparing the expression of BCL11A gene in peripheral blood cells from normal individuals, individuals withβ-thalassemia andβ-thalassemia patients.Erythroid cells cultured in vitro can simulate the differenration and maturation of erythroid cells in vivo. Therefore, we try to know about whether BCL11A gene is the reason causing the persistence of HbF inβ-thalassemia patients by detecting the BCL11A expression in erythroblasts culutured in vitro. However, the result of RT-PCR showed that there was no BCL11A expression in erythroblasts culutured in vitro, which may be caused by culture in vitro. The result showed that the erythroblasts culutured in vitro was not the suitable experimental materials for this study.Erythroblasts in bone marrow have intact nucleus, and do not be cultured in vitro. For the above reasons, erythroblasts from bone marrow should be the suitable experimental materials for the study. Therefore, we attempt to reveal the role of BCL11A gene in the persistence of HbF inβ-thalassemia patients by comparing the BCL11A expression in erythroblasts from bone marrow among normal individuals,β-thalassemia carriers andβ-thalassemia patients.