http://informahealthcare.com/hem ISSN: 0363-0269 (print), 1532-432X (electronic) Hemoglobin, Early Online: 1–6 ! 2015 Informa Healthcare USA, Inc. DOI: 10.3109/03630269.2015.1008702 ORIGINAL ARTICLE A New Kru ¨ ppel-Like Factor 1 Mutation (c.947G4A or p.C316Y) in Humans Causes b-Thalassemia Minor Takenori Nitta 1 , Fumio Kawano 2 , Yasuhiro Yamashiro 1 , Fumiya Takagi 3 , Tomoaki Murata 4 , Tatehiko Tanaka 1 , Mella Ferania 1 , Chris Adhiyanto 5 , and Yukio Hattori 6 1 Department of Health Science, Yamaguchi University Graduate School of Medicine, Ube, Japan, 2 Department of Hematology, National Hospital Organization, Kumamoto Medical Center, Kumamoto, Japan, 3 Department of Clinical Laboratory, Fukuyama Medical Center, Fukuyama, Japan, 4 Animal Experimental Department, Comprehensive Scientific Research Center, Yamaguchi University, 5 Faculty of Medicine and Health Science, Universitas Islam Negeri Syarif Hidayatullah, Jakarta, Indonesia, and 6 Department of Clinical Laboratory, Saiseikai Yamaguchi General Hospital, Yamaguchi, Japan Abstract Here we describe a Japanese patient with mild b-thalassemia (b-thal) with an intact b-globin gene but a new missense mutation of c.947G4A or p.C316Y in the erythroid Kru ¨ppel-Like Factor (KLF1) gene which is strongly associated with the expression of the b-globin gene. The association of the KLF1 mutation with b-thal, is here described. The p.C316Y mutation occurred at one of the cysteines that constitute the second zinc finger motif of KLF1, and would have changed the zinc finger conformation to impair the DNA binding properties or the promoter function of the b-globin gene. Our expression study found that the mutant KLF1 gene had a markedly negative effect on the b-globin gene expression, or 7.0% of that of its normal counterpart. A presumed heterozygous state, or equimolar presence of the mutant and normal KLF1s reduced the expression rate to 70.0% of the normal alone. This degree of the decrease may explain the very mild phenotype of the patient’s b-thal. Furthermore, the patient’s whole- exome analysis using next-generation sequencing revealed that the b-thal defect is caused by only this KLF1 gene mutation. The Hb A 2 and Hb F levels that are frequently elevated in KLF1 mutations were elevated by 4.1 and 1.3%, respectively, in this case. The contribution to their elevation by KLF1: p.C316Y is uncertain. Keywords b-Globin expression, b-thalassemia (b-thal), Kru ¨ ppel-Like Factor 1 (KLF1), next-generation sequencing History Received 27 July 2014 Revised 9 September 2014 Accepted 9 September 2014 Published online 18 February 2015 Introduction b-Thalassemia (b-thal) is one of the most prevalent congenital hemolytic disorders in the world. It occurs because of impaired production of b-globin mutating the b-globin gene; mostly these are point or frameshift mutations of a few base deletions/insertions. Mutation of the distal and proximal CACCC boxes within the b-globin gene promoter cause b + -thal. The mutations that occur in the CACCC box affect the binding and responsiveness to erythroid Kru ¨ppel-like factor (EKLF, KLF1) (1), which is essential for expression of the b-globin gene. Furthermore, a number of KLF1 mutations have been reported (2). The KLF1 has three C2H2 zinc finger (ZF) domains that bind to the CACCC box and increase the expression of the b-globin gene (Figure 1). Moreover, KLF1 works one of the key regulators of the g- to b-globin gene switching (3), and its mutation gives rise to increase in the level of Hb F and Hb A 2 (4–6). One of the mutations causes a congenital dyserythropoietic anemia (CDA) type (7) and hemolytic anemia. Thus, KLF1 mutations exert diverse functional effects. In this study we describe a Japanese patient suspected of carrying a mild b-thal. However, no mutations were found from 1 kb upstream to 1 kb downstream of the b-globin gene that includes the promoter region, all exons and introns and the 5 0 and 3 0 non coding regions. In addition, no b-globin gene deletion was found. Therefore, we focused on the KLF1 that was known to cause b-thal in a KO model mouse (8). In addition, it was suspected that some of the undefined b-thalassemias have abnormally unlinked to the b-globin gene (9). As expected, the mutation of the KLF1: c.947G4A coding for KLF1: p.C316Y was discovered in our patient. This has as yet not been reported in the literature, and we studied the association of the KLF1: p.C316Y with b-globin gene expression in humans by analysis of transient transfec- tion assay. We used the next-generation sequencing (NGS) analysis to see whether other mutations relevant to hemoglo- binopathies were present anywhere in the patient’s whole exons. Address correspondence to Mr. Takenori Nitta, Department of Health Science, Yamaguchi University Graduate School of Medicine, 1-1-1 Minami-Kogushi, Ube, Yamaguchi, Ube 755-8505, Japan. Tel./Fax: +81-836-222-863; E-mail: s002ur@yamaguchi-u.ac.jp Hemoglobin Downloaded from informahealthcare.com by York University Libraries on 03/01/15 For personal use only.