Changes in hematological parameters in α-thalassemia individuals co-inherited with erythroid Krüppel-like factor mutations

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1 Clin Genet 2015: 88: Printed in Singapore. All rights reserved Short Report 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd CLINICAL GENETICS doi: /cge Changes in hematological parameters in α-thalassemia individuals co-inherited with erythroid Krüppel-like factor mutations Yu L-H, Liu D, Cai R, Shang X, Zhang X-H, Ma X-X, Yan S-H, Fang P, Zheng C-G, Wei X-F, Liu Y-H, Zhou T-B, Xu X-M. Changes in hematological parameters in α-thalassemia individuals co-inherited with erythroid Krüppel-like factor mutations. Clin Genet 2015: 88: John Wiley & Sons A/S. Published by John Wiley & Sons Ltd, 2014 Phenotypic variations in α-thalassemia mainly depend on the defective α-globin gene number. Genetic modifiers of the phenotype of Hemoglobin H (HbH) disease were poorly reported, apart from β-thalassemia allele that was identified ameliorating the severity of α-thalassemia. Because erythroid Krüppel-like factor (KLF1) mutations can modulate the red blood phenotype, we evaluated its effect on the α-thalassemia phenotype. Overall, we identified 72 subjects with five different KLF1 heterozygous mutations in 1468 individuals, including 65 out of 432 α-thalassemia carriers with fetal hemoglobin (HbF) levels 1%, 0 out of 310 carriers with HbF levels <1% and 7 out of 726 HbH disease patients. We firstly established the link between KLF1 mutations and relatively elevated hemoglobin A 2 (HbA 2 )and HbF levels, along with lower mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) values in a group of α-thalassemia carriers. However, we concluded that KLF1 mutations were not significantly linked to HbH disease severity. On the basis of HBA or HBB genotype and gender, clinical severity of patients with HbH disease was correctly predicted in 73.3% cases. It may improve the screening and diagnostic assessment of α-thalassemia. Conflict of interest The authors report no potential conflicts of interest. L.-H. Yu a,d.liu a,r.cai b, X. Shang a,x.-h.zhang c, X.-X. Ma a,s.-h.yan d,p.fang a, C.-G. Zheng e,x.-f.wei a, Y.-H. Liu f, T.-B. Zhou g and X.-M. Xu a a Department of Medical Genetics, School of Basic Medical Sciences, Southern Medical University, Guangzhou, Guangdong, P.R.China, b Department of Birth Health and Heredity, Liuzhou Women and Children Care Hospital, Liuzhou, Guangxi, P.R.China, c Department of Hematology, 303rd Hospital of the People s Liberation Army, Nanning, Guangxi, P.R.China, d Laboratory of Medical Genetics, Maternal and Child Health Care Hospital of Qinzhou, Qinzhou, Guangxi, P.R.China, e Prenatal Diagnostic Center, Guangxi Zhuang Autonomous Region Women and Children Care Hospital, Nanning, Guangxi, P.R.China, f Prenatal Diagnosis Center, Maternal and Child Health Hospital, Dongguan, Guangdong, P.R.China, and g Clinical Laboratory, Yunnan Women and Children Care Hospital, Kunming, Yunnan, P.R.China These authors contributed equally to this study. Key words: α-thalassemia clinical severity erythroid Krüppel-like factor genetic modifier red blood phenotype Correspondence author: Xiang-Min Xu, Department of Medical Genetics, School of Basic Medical Sciences, Southern Medical University, Guangzhou , Guangdong, P.R.China. Tel.: ; fax: ; gzxuxm@pub.guangzhou.gd.cn Received 6 April 2014, revised and accepted for publication 11 June

2 KLF1 mutations and α-thalassemia α-thalassemia is an inherited hemoglobin disorder with high carrier rates, characterized by defective synthesis of α-globin chains (1). The disease encompasses a wide phenotypic spectrum, spanning from asymptomatic to embryonic lethality, mainly depending on the number of defective α-globin genes and possibly influenced by the mutant allele type and unlinked modifying genes (2, 3). Unlike β-thalassemia, limited progress has been made in the identification of genetic modifiers of α-thalassemia. To date, only β-thalassemia allele that clearly ameliorates the severity of α-thalassemia has been identified (3, 4). Because individuals with identical α-and β-globin genotypes, phenotypic variations are frequently observed. For example, silent α-thalassemia carriers ( α 3.7 /αα, α 4.2 /αα and α WS α/αα) usually present with normal hematological manifestations, but sometimes display hypochromic and/or microcytic anemia (1, 5). Moreover, the severity of Hemoglobin H (HbH) disease may be modulated by several potential genetic modifiers, such as HS-40, BRG1, PIP4KIIA and AHSP, which have only been reported in sporadic patients and murine models (6 9). Erythroid Krüppel-like factor (KLF1) gene regulates the development of erythroid cells as activators of a diverse set of genes and controls fetal-to-adult globin switching (10, 11). Recent studies have shown that variations in the KLF1 gene trigger a series of benign human red blood phenotypes, such as increase in hemoglobin A 2 (HbA 2 ) and fetal hemoglobin (HbF) levels, in non-thalassemics and sporadic subjects with α 3.7 /αα (12 14). Therefore, KLF1 mutations may relate to the phenotype of α-thalassemia. In this investigation, we examined a large group of individuals with α-thalassemia to evaluate whether KLF1 mutations play a role in modulation of α-thalassemia phenotypes. Materials and methods Study subjects We performed a retrospective study on 1468 unrelated individuals with α-thalassemia from South China (Fig. 1) consisting of 742 adult α-thalassemia carriers with no β-thalassemia co-inheritence (Groups A and B; age: years) and 726 consecutive HbH disease patients (Group C; age: 1 70 years). Among the 742 carriers, 432 (Group A; Table S1, Supporting Information) with elevated HbF levels ( 1%) were enrolled, with the aim of establishing the link between KLF1 mutations and HbF levels. In addition, 310 other carriers with normal HbF levels were collected as the control group (Group B). Patients in Group C were classified as relatively mild form (n = 243) or relatively severe form (n = 483) phenotype, based on earlier criteria (3, 15). The relatively mild phenotype included patients maintaining hemoglobin (Hb) levels above 90 g/l without the need for regular transfusions while the relatively severe phenotype included patients with Hb levels 90 g/l and/or needing regular transfusions. The hematological characteristics of HbH disease patients were examined before transfusion (Table S2). Our study was approved by the local ethical committee at Nanfang Hospital, and informed consent obtained from participants. Hematological and molecular genetic tests Hematological parameters were determined using automatic cell counter and high-performance liquid chromatography. Genomic DNA was extracted from peripheral blood using standard protocols. Deletions and point mutations in HBA and HBB loci were genotyped according to previously documented methods (1). All participants were genotyped for KLF1 variations using high resolution melting (HRM) analysis, based on our recently described assay (16). DNA sequencing was used to determine the variations in the KLF1 gene. SIFT and PolyPhen-2 were applied to predict the effects of mutations on protein structure and function. We defined those variations both predicted to affect protein structure and function (SIFT and PolyPhen-2) or ablated the zinc finger domains and leading to reduced CD44 expression in erythrocytes, assessed by flow cytometric analysis, as functionally effective mutations of the KLF1 gene. Statistical analysis Statistical comparisons were conducted using spss 19.0 statistical software, with a p value <0.05 considered statistically significant. Independent t-test and chi-squared test were used to assess significant differences in hematological parameters and qualitative data, respectively. Logistic regression analysis was conducted to evaluate the phenotypes of HbH disease patients associated with four factors, including HBA genotype, HBB genotype, KLF1 genotype and gender. Odds ratios (OR) were estimated and presented with 95% confidence interval. Factors were removed from the model when the p value was greater than 0.05, and the predictive accuracy of the logistic regression model was estimated. Results Identification of KLF1 mutations In total, 72 subjects were identified with a single loss-of-function KLF1 mutation from 1468 α-thalassemia individuals in South China, consisted of 65 carriers in Group A, 0 in Group B and 7 patients in Group C (Fig. 1). A total of 18 variations, including five functionally effective mutations, four common SNPs and nine neutral variations, in the KLF1 gene were screened using the HRM assay (Fig. S1). The five functionally effective mutations included four missense mutations (p.ala298pro, p.his299asp, p.thr334arg and p.pro338ser) and one frameshift mutation (p.gly176alafsx179), shown in Table S3. Missense mutations were located in the zinc finger domains and highly conserved across various mammalian species, while the frameshift mutation led to ablation of the zinc finger domains (Fig. S2). Two novel silent variations were detected, specifically, c.-23_-21delc in the 5 untranslated region (UTR) and c.*119c > Tin3 UTR. 57

3 Yu et al. Fig. 1. Profile of study subjects. A total of 1468 participants, including 742 phenotype-oriented α-thalassemia carriers and 726 consecutive Hemoglobin H (HbH) disease patients in South China, were screened to detect KLF1 mutations using high resolution melting assay. Among them, 72 subjects that co-inherited a functionally effective KLF1 heterozygous mutation were identified, including 65 individuals from Group A, 0 from B, and 7 from C. Seven subjects homozygous for α + -thalassemia in Group A included three cases with α 3.7 / α 3.7, two with α 3.7 /α CS α, one with α 3.7 / α 4.2 and one with α 4.2 /α CS α. Changes in hematological parameters of α-thalassemia carriers Among the 432 carriers in Group A, 16, including 7 with homozygous for α + -thalassemia alleles and 9 with α QS α/αα, were excluded from further analysis, since we identified only one subject in the homozygous group and none in the α QS α/αα group with a KLF1 mutation. Hematological parameters were compared between carriers with wild-type KLF1 and those with identical HBA genotype co-inherited KLF1 mutations. Significantly, lower MCV and MCH values, along with higher HbA 2 levels were observed in KLF1 mutation-positive α-thalassemia carriers (Fig. 2). The hematological parameters of the KLF1 mutation-positive carriers were presented in Table S4. Additionally, we detected a marked increase in HbF levels in α-thalassemia carriers with KLF1 mutations, except in those with α 3.7 /αα. Significant changes in Hb levels were only evident in female subjects with α 3.7 /αα (p = 0.027; Table S5). Determinants associated with severity of Hb H disease In total, 243 HbH disease patients displaying relatively mild phenotype and 483 with relatively severe phenotype in Group C were typed into four factors (HBA genotype, HBB genotype, KLF1 genotype and gender), and the frequencies of ameliorative factors for each phenotype were calculated (Table 1). We observed a significant association between the relatively mild phenotype of HbH disease and ameliorative factors in HBA genotype [OR = ( ), p < 0.001], HBB genotype [OR = ( ), p < 0.001] and gender [OR = ( ), p < 0.001] (Table 1). Notably, the HBA genotype had strongest effect on severity of HbH disease, followed by HBB genotype and subsequently, gender. The KLF1 genotype did not show significant association (p = 0.194) and was consequently removed from the logistic regression model. The hematological parameters of KLF1 mutation-positive HbH disease patients were shown in Table S6. Discussion In order to systematically determine whether KLF1 mutations might play a role in modulation of α-thalassemia phenotypes in South China, we examined 1468 individuals with α-thalassemia. A total of 18 variations, including five functionally effective mutations, were identified in this study. Among them, the most common KLF1 mutation, identified in 66 subjects (accounted for 91.67% cases), was the frameshift mutation p.gly176alafsx179. This finding is consistent with our research data on the mutation spectrum of KLF1 in South China (16). However, further functional studies are required to determine whether the nine neutral variations contribute to the phenotypes of α-thalassemia. 58

4 KLF1 mutations and α-thalassemia Fig. 2. Hematological parameter analysis of KLF1 heterozygotes. Comparisons of MCV (a), MCH (b), hemoglobin A 2 (HbA 2 )(c), and fetal hemoglobin (HbF) (d) levels between α-thalassemia carriers with wild-type KLF1 (triangle) and heterozygous KLF1 mutations (square) according to the HBA genotype. Data are presented as mean ± standard deviation (SD). Symbols represent p values showing significant differences: ***p < 0.001; **p < 0.01; *p < 0.05; NS, not significant. Reference range values are shown as a dotted line: MCV, fl; MCH, pg; HbA 2, %; HbF, <1%. 59

5 Yu et al. Table 1. Analysis of the association of HbH disease severity and genetic factors Factors (ameliorative factors) Frequencies of ameliorative factors in HbH disease patients Relatively mild phenotype (n = 243) Multivariate analysis Relatively severe phenotype (n = 483) OR 95% CI p value HBA genotypes a 75.31% 28.99% <0.001 HBB genotypes (β WT/MU ) 9.88% 2.48% <0.001 Gender (male) 58.03% 41.82% <0.001 KLF1 genotypes (KLF1 WT/MU ) b 1.65% 0.62% CI, confidence interval; HbH, Hemoglobin H; OR, odds ratio. a Ameliorative factors of HBA genotypes include -- SEA / α 3.7,-- SEA / α 4.2 and -- SEA /α WS α. b KLF1 was removed from the multivariate analysis model. Recent studies have reported that increased levels of HbA 2 and HbF induced by KLF1 mutations were observed in normal individuals and sporadic subjects with α 3.7 /αα (12 14). On the basis of our results, these findings can be extended to α 0 -andα + -thalassemia carriers. The prevalence of KLF1 mutations in Group A is significantly higher, compared with that in Group B (15.05% vs 0%), further indicating an association between KLF1 mutations and HbF levels. Our previous study have showed that mutations of KLF1 result in the MCV and MCH values at the lower end of the normal range (82.45 ± 5.39 fl and ± 2.11 pg, respectively) in non-thalassemic subjects (16). Interestingly, in this similar study, we found that lower MCV and MCH values are present in the individuals with co-inherited α-thalassemia and KLF1 mutations, thus providing another evidence that a loss-of-function mutation on one allele of KLF1 is responsible for the relatively low MCV and MCH values and implying additive effects of these two mutations on red cell phenotypes (Fig. 2). Our study design of effects on hematologic parameters is mainly aimed at analyzing one of various genetic factors, a KLF1 mutation and not involving the iron deficiency anemia (IDA) that could yet affect changes in red cell indices. We think that it will be necessary to fundamentally examine iron status in those KLF1 mutation-positive α-thalassemia carriers in the future study. As an higher prevalence of the KLF1 mutations are observed in thalassemia endemic regions in this study and our epidemiological investigation (16). We hypothesize that the frequent occurrence of the KLF1 mutations (like thalassemia) may arise due to selection pressure from malaria because of conferring resistance to malaria by such mutations resulting in the decreased MCV and MCH. It is noteworthy that silent α-thalassemia ( α 3.7 /αα, α 4.2 /αα and α WS α/αα) carriers with mutant KLF1 alleles showed reduced MCV and MCH, borderline or slightly elevated HbA 2 levels (3.25 ± 0.20%, range: %, Table S4) and increased HbF levels (2.77 ± 0.63%, range: %, Table S4). A part of them are more probably to be misdiagnosed as β + -thalassemia or the co-existence of a β- and δ-thalassemia because of the overlapping features of hematological parameters (17 19). Furthermore, our study indicates that a distinct group of α-thalassemia co-existed a KLF1 mutation can similarly lead to elevation of HbF although in general people regards this to be a variant of β-thalassemia disorder when having elevated HbF. In China, individuals with the above hematological manifestations may only undergo molecular screening for β-thalassemia, which would result in increased risk of their offspring inheriting HbH disease, especially in high-incidence regions of α-thalassemia when the partner carries an α 0 -thalassemia allele. Therefore, particular attention should be paid to prevent overlooking the screening of the undetected α-thalassemia mutant allele owing to misdiagnosis. Investigation of 726 HbH disease patients for nearly 2 years revealed only seven individuals with a single KLF1 mutation. Thus, it was difficult to compare the clinical severity and hematological parameters between patients with and without KLF1 mutations who co-inherited the same HBA and HBB genotypes. Furthermore, we observed no significant differences in the carrier rates of KLF1 mutations between HbH patients and the general population in South China (0.96% vs 1.25%, p = 0.688) (16). Large-scale evaluation of the role of KLF1 mutations in HbH disease severity by increasing sample sizes in various populations is required to resolve this issue. To our knowledge, this is the first study to measure the contribution of HBA and HBB genotypes and gender to HbH disease severity, although similar studies on β-thalassemia severity have been performed (20 22). On the basis of these three positive factors, clinical severity of patients with HbH disease is correctly predicted in 73.6% cases. The unexplained 26.4% cases may be attributed to different genetic backgrounds and environments. Supporting Information Additional supporting information may be found in the online version of this article at the publisher s web-site. Acknowledgements This study was supported by a grant from National Natural Science Foundation of China (NSFC)-Guangdong Joint Fund (No. 60

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