KLF1 mutations are relatively more common in a thalassemia endemic region and ameliorate the severity of β-thalassemia

Abstract

Mutations in human Krüppel-like factor 1 (KLF1) have recently been reported to be responsible for increased fetal hemoglobin (HbF) and hemoglobin A2 (HbA2). Because increased HbF and HbA2 levels are important features of β-thalassemia, we examined whether there is any relationship between KLF1 mutation and β-thalassemia in China. To do this, we first studied the incidence of KLF1 mutations in 2 Chinese populations: 3839 individuals from a thalassemia endemic region in south China and 1190 individuals from a non-thalassemia endemic region in north China. Interestingly, we found that the prevalence of KLF1 mutations is significantly higher in the thalassemia endemic region than that in non-thalassemia endemic region (1.25% vs 0.08%). Furthermore, we identified 7 functional variants including 4 previously reported (p.Gly176AlafsX179, p.Ala298Pro, p.Thr334Arg, and c.913+1G>A) and 3 novel variants (p.His299Asp, p.Cys341Tyr, and p.Glu5Lys) in southern China. The 2 most common mutations, p.Gly176AlafsX179 and p.His299Asp, accounted for 90.6% of the total. We found that zinc-finger mutations in KLF1 were selectively represented in 12 β-thalassemia intermedia patients and resulted in significantly different transfusion-free survival curves. Our findings suggest that KLF1 mutations occur selectively in the presence of β-thalassemia to increase the production of HbF, which in turn ameliorates the clinical severity of β-thalassemia.

Figure 1

The prevalence and spectrum of KLF1 mutations in the Chinese population. (A) Sampling design and study outcomes. Southern Chinese population: Three consecutive cohorts (A-C) of 3839 subjects from 2 thalassemia endemic regions (Guangxi and Guangdong provinces) were designed to investigate the incidence of KLF1 mutations; 79 phenotype-oriented subjects (cohort D) selected from the same region were recruited for enrichment of KLF1 mutation–positive subjects. The β-globin gene in all 79 subjects was completely analyzed; those patients positive for β-thalassemia mutations were excluded from analysis. All mutant alleles (n = 64) detected in these 2 approaches were used to assess the mutation spectrum of KLF1. ^#HBB genotype categories of 11 KLF1 mutation–positive subjects in cohort B were as follows: 1 β⁺/β^N, 1 β^E/β^N, 7 β⁰/β^N, and 2 β⁰/β^N coinherited with α-thalassemia. Northern Chinese population: 1190 nonthalassemics were recruited from a nonthalassemia endemic region (Shandong Province). (B) Exons, introns, and domains are shown with the untranslated regions (white), proline-rich regions (blue), and zinc fingers (ZFs; purple). The positions of the HRM primers are noted above the physical map, and 4 novel and 4 previously reported functional KLF1 mutations in south (red) and north (gray) China are shown below the map. Allelic distribution of KLF1 mutations in the southern Chinese population are indicated in parentheses.

Figure 2

Characteristic hematological analyses of KLF1 heterozygotes in cohorts A and B. (A) MCV and MCH values in normal individuals (n = 1946 from cohort A), nonthalassemics (n = 41; 25 from cohort A and 16 from cohort D), and 7 KLF1–heterozygous mutations, β⁰-thalassemia heterozygotes (n = 217 from cohort B), and β⁰-thalassemia heterozygotes coinherited with KLF1 mutations (n = 14; 7 β⁰/β^N of 11 from cohort B and another 7 β⁰/β^N from 6 β-thalassemia families with KLF1 mutations; supplemental Figure 2). The α-thalassemia deletions or point mutations were excluded in all nonthalassemic and heterozygous β⁰-thalassemia individuals with and without KLF1 mutations. The data shown are expressed as mean ± SD. P value was determined using the Mann-Whitney U test. (B) HbA₂ and HbF levels in normal individuals (left) and β-thalassemia heterozygotes (right) with or without KLF1 mutations (all have normal α globin genotypes). (Left) Solid circles represent samples with KLF1 mutations, including 25 from cohort A (blue), 16 from cohort D (green), and 50 without KLF1 mutations (black; HbA₂, 2.81 ± 0.20%; range, 2.50% to 3.20%; HbF, 0.23 ± 0.22%, range, 0.00% to 0.80%). (Right) Solid circles represent 14 samples with KLF1 mutations (red) and 50 without KLF1 mutations (black; HbA₂, 4.96 ± 0.49%, range, 3.70% to 6.10%; HbF, 0.93 ± 0.70%, range, 0.20% to 4.10%). Genotype symbols of subjects are shown on the bottom right of the chart.

Figure 3

Kaplan-Meier survival curves for β-thalassemia (β-thal) patients with or without KLF1 mutations for needing transfusion in cohort C (log-rank test P value). Patients had not received a transfusion by the end of the study, which was recorded as censored data. The 2 sample-matched pairs, 7 vs 362 β-thalassemia cases with or without KLF1 mutations and 12 vs 342 TI cases with or without KLF1 mutations, are shown at the top of the chart. The 4 colored lines represent each of the 4 groups, respectively. Selection of well-matched 7 vs 362 subjects is described in our “Statistical analysis” section. To compare the median time to the first transfusion in the 2 groups of TI patients, all 12 TI cases with KLF1 mutations vs all 342 TI cases without KLF1 mutations were used for the Kaplan-Meier survival curve analysis.