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A novel 519_525dup mutation of KLF1 gene identified in a Chinese blood donor with Lu(a–b–) phenotype

The Lutheran blood group system consists of 20 antigens encoded by LU gene and includes four pairs of antithetical antigens. The remainders are high-frequency antigens. Lu(a–b–)/Lunull is a rare phenotype first described by Crawford and colleagues in 1961 characterized by the absence of all Lutheran system antigens, which distributed with a frequency of 1:3000 in South East England and 1:5000 in South Wales blood donors. Three molecular backgrounds account for this rare phenotype. First, the rare recessive Lunull phenotype arises from homozygosity for inactivating mutations in the LU gene, which is generally ascertained through the presence of anti-Lu3. Second, a mutation of erythroid transcription factors GATA1 (GATA1-binding protein 1) has been shown to be responsible for X-linked Lu(a–b–) phenotype in one family. Third, the majority of probands with dominant inherited Lu(a–b–)/In(Lu) phenotype result from inactivating heterozygous mutations in KLF1. The red blood cells (RBCs) with this phenotype express traces of Lutheran antigen that could only be detected by absorption/ elution technique; therefore, their blood has been used to satisfy the requirement of patients with anti-Lu3 since they are relatively common. So far, four inactive mutations of LU gene accounting for Lunull phenotype and 11 KLF1 mutations accounting for In(lu) phenotype have been described (see Web Resource). In our study, one male donor with Lu(a–b–) phenotype (20 years old and without transfusion history) was identified accidentally while high-throughput genotyping and corresponding serologic typing were conducted in 90 Chinese donors. Anti-Lu and anti-Lu (Sanquin Reagents, Amsterdam, the Netherlands) were used to type Lu(a–b–) phenotype. Absorption/elution testing was conducted by using the anti-Lu serum. The donor’s RBCs were also tested with anti-AnWj and anti-INFI since the highincidence AnWj (901009) antigen and Indian blood group system antigens including INFI (IN3) are also known to be weakened in the In(Lu) phenotype. HbF level was also tested by using high-performance liquid chromatography (Variant II hemoglobin testing system, Bio-Rad, Hercules, CA). Unfortunately, the samples of the other family members were not available. Furthermore, both LU and KLF1 genes were analyzed by direct sequencing to clarify the molecular background. Genomic DNA was extracted from peripheral blood of Lu(a–b–) proband and 90 random individuals as controls. Exons 1 to 15 of LU gene, the promoter and Exons 1 to 3 of KLF1 gene, and the exon– intron boundaries were amplified by polymerase chain reaction (PCR). Amplification primers, annealing temperature, and PCR condition have been described previously, except that GC Buffer II (Takara, Dalian, China) was used to amplify the Exon_2 of KLF1. PCR products were sequenced using a sequencing kit on a genetic analyzer (ABI BigDye Terminator, Version 3.1, and ABI 3130XL, respectively, Applied Biosystems, Foster City, CA). Heterozygous mutations identified were further examined by TOPO TA cloning (Invitrogen, Carlsbad, CA). The novel mutation identified was tested in 90 random blood donors by direct sequencing. Negative agglutination with anti-Anwj and anti-INFI serum and negative antibody screening results (absence of anti-Lu3) were obtained in this donor. Absorption/ elution testing showed very weak expression of lu antigen compared with normal controls. No mutations were identified in all 15 exons of LU gene. However, a novel heterozygous seven-base duplicated insertion in Exon_2 of KLF1 gene (c.519_525dupCGGCGCC) was detected (Fig. 1A), which resulted in frameshift mutations from Amino Acid 176 (p.Gly176Argfs*179) and created a truncated protein with a premature stop codon (Fig. 1B). This heterozygous mutation was further confirmed by cloning analysis (registered in GenBank as Number JX877554) and was not found in 90 random donors. EKLF encoded by KLF1 gene is an essential transcription factor in the process of erythroid differentiation. The duplicated insertion will disrupt the EKLF transactivation domain in the proline-rich N-terminal region (Amino Acids 1-275) and three zinc finger domain in the C-terminal region (Amino Acids 276-358) that maybe influence the erythroid-specific genes expression resulting in In(lu) phenotype. Based on the weak expression of lu antigen, the absent expression of highfrequency Anwj and INFI antigens, and the novel mutation identified in KLF1 gene, In(lu) phenotype is confirmed in this Chinese donor. Besides, KLF1 mutations are also responsible for persistent hyper– hemoglobin F syndrome. In this donor, mean 0.8% HbF level was obtained compared with 0.7% in controls that indicate the effects of this mutation may be additive for this syndrome. Recently, the heterozygous mutation and compound heterozygous with L51R missense mutation were found in one Korean patient with 1.7% HbF level and one Vietnamese patient with 9.5% HbF level respectively, but the In(lu) phenotype was not tested.