REPORT OF NEW ALLELES Two novel KEL alleles encoding K0 phenotypes in Brazilians Carine Prisco Arnoni,1 Diana Gazito,1 Janaína Guilhem Muniz,1 Rosangela de Medeiros Person,1 Frederico Brandão,1 Maria Goretti de Araújo Marques,1 José Augusto Barreto,1 Lilian Castilho,2 and Flavia Roche Moreira Latini1
K0
(null) is a rare phenotype in which no Kell antigens are expressed on the red blood cell (RBC) membrane.1 Anti-Ku seen in K0 individuals has previously been shown to cause hemolytic disease of newborn and severe hemolytic transfusion reactions.2,3 Several molecular mechanisms associated with the K0 phenotype have already been described, including those that lead to alternative RNA splicing, premature stop codons, and amino acid changes that affect protein trafficking.4,5 We herein report two novel KEL alleles encoding K0 phenotypes in Brazilians. We analyzed genomic DNA of two unrelated Caucasian females referred to our reference laboratory. Both patients were phenotyped as K−, k−, Kp(a−), Kp(b−), Js(a−), Js(b−) and presented an antibody reactive 2+ in gel indirect antiglobulin test with all RBCs except their own.
BRIEF METHODS Genomic DNA was isolated from peripheral blood with a commercially available purification kit (QIAamp, blood mini kit, Qiagen, Inc., Valencia, CA). KEL genotyping was performed by polymerase chain reaction–restriction fragment length polymorphism and the HEA BeadChip according to manufacturer’s instructions. Sequence analysis was performed on all 19 exons of KEL and on three exons of XK genes. Coding and intron-exon splice regions were sequenced as previously described.6
RESULTS Proband 1 KEL genotyping showed KEL*02/02, KEL*04/04, and KEL*07/07 genotypes. Sequence analysis revealed a new homozygous change in KEL Exon 2, a guanine to an adenine substitution at Position 71 (c.71G>A, NCBI_ss#: 831878329) leading to a premature stop codon (Trp24Stop; Fig. S1A, available as supporting information in the online version of this paper). No other changes were identified and no mutations were found on XK gene.
Proband 2 Molecular testing showed KEL*02/02, KEL*04/04, and KEL*07/07 genotypes. Analysis of coding and intron-exon splice regions of KEL of this K0 individual showed a change of a guanine to an adenine at the first nucleotide of Intron 16 (IVS16+1g>a, NCBI_ss#: 831878330). The mutation changes the conserved gt sequence at the donor splice site to at, altering the splice donor site, and preventing the removal of the intron. This event creates a premature stop codon, two codons downstream from the SNP. Unfortunately, the patient sample was not available to perform further RNA analysis. The changes are summarized in Table 1 and the eletropherograms are included in Fig. S1.
CONFLICT OF INTEREST The authors report no conflicts of interest or funding sources.
From 1Colsan–Associação Beneficente de Coleta de Sangue, São Paulo, SP, Brazil; and 2INCTs–Hemocentro-Unicamp, Campinas, SP, Brazil. Address reprint requests to: Carine Prisco Arnoni, Colsan–Associação Beneficente de Coleta de Sangue, Avenida Jandira 1260, Indianópolis–CEP 04080-006, Brazil; e-mail: [email protected] Received for publication September 26, 2013; revision received November 29, 2013, and accepted December 6, 2013. doi: 10.1111/trf.12564 © 2014 AABB TRANSFUSION 2014;54:2128-2129. 2128
TRANSFUSION Volume 54, August 2014
TABLE 1. Phenotype, nucleotide and amino acid changes, GenBank database submitting numbers, and ISBT allele numbers Proband 1
2
Phenotype K−, k−, Kp(a−), Kp(b−), Js(a−), Js(b−) K−, k−, Kp(a−), Kp(b−), Js(a−), Js(b−)
Nucleotide change
Amino acid change
NCBI_ss#
ISBT allele
71G>A
Trp24Stop
831878329
KEL*02N.30
IVS16+ 1g>a
Alternative Splicing
831878330
KEL*02N.31
TWO NOVEL KEL ALLELES ENCODING K0 PHENOTYPES IN BRAZILIANS
REFERENCES
6. Yang MH, Li L, Kuo YF, et al. Genetic and functional analyses describe a novel 730delG mutation in the KEL gene
1. Lee S, Russo D, Redman C. Functional and structural aspects of the Kell blood group system. Transfus Med Rev 2000;14:93-103.
causing K0 phenotype in a Taiwanese blood donor. Transfus Med 2011;21:318-24.
2. Nunn HD, Giles CM, Dormandy KM. A second example of anti-Ku in a patient who has the rare Kell phenotype, Ko. Vox Sang 1966;11:611-9. 3. Chown B, Lewis M, Kaita K. A new Kell blood-group phenotype. Nature 1957;180:711. 4. Yu LC, Twu YC, Chang CY, et al. Molecular basis of the Kell-null phenotype: a mutation at the splice site of human KEL gene abolishes the expression of Kell blood group antigens. J Biol Chem 2001;276:10247-52. 5. Lee S, Russo DC, Reiner AP, et al. Molecular defects underlying the Kell null phenotype. J Biol Chem 2001;276: 27281-9.
SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article at the publisher’s Web-site: Fig. S1. Electropherogram of the 2 K0 samples studied. Control samples are on the top of the figure and probands are bellow. (A) Proband 1 is homozygous for a c.71G>A in Exon 2 that leads to a premature stop codon (Trp24Stop). (B) Proband 2 is homozygous for the mutation g>a at position +1 of Intron 16, that alters splice donor site, following the coding region until the premature stop codon (TAA).
Volume 54, August 2014 TRANSFUSION
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K0
(null) is a rare phenotype in which no Kell antigens are expressed on the red blood cell (RBC) membrane.1 Anti-Ku seen in K0 individuals has previously been shown to cause hemolytic disease of newborn and severe hemolytic transfusion reactions.2,3 Several molecular mechanisms associated with the K0 phenotype have already been described, including those that lead to alternative RNA splicing, premature stop codons, and amino acid changes that affect protein trafficking.4,5 We herein report two novel KEL alleles encoding K0 phenotypes in Brazilians. We analyzed genomic DNA of two unrelated Caucasian females referred to our reference laboratory. Both patients were phenotyped as K−, k−, Kp(a−), Kp(b−), Js(a−), Js(b−) and presented an antibody reactive 2+ in gel indirect antiglobulin test with all RBCs except their own.
BRIEF METHODS Genomic DNA was isolated from peripheral blood with a commercially available purification kit (QIAamp, blood mini kit, Qiagen, Inc., Valencia, CA). KEL genotyping was performed by polymerase chain reaction–restriction fragment length polymorphism and the HEA BeadChip according to manufacturer’s instructions. Sequence analysis was performed on all 19 exons of KEL and on three exons of XK genes. Coding and intron-exon splice regions were sequenced as previously described.6
RESULTS Proband 1 KEL genotyping showed KEL*02/02, KEL*04/04, and KEL*07/07 genotypes. Sequence analysis revealed a new homozygous change in KEL Exon 2, a guanine to an adenine substitution at Position 71 (c.71G>A, NCBI_ss#: 831878329) leading to a premature stop codon (Trp24Stop; Fig. S1A, available as supporting information in the online version of this paper). No other changes were identified and no mutations were found on XK gene.
Proband 2 Molecular testing showed KEL*02/02, KEL*04/04, and KEL*07/07 genotypes. Analysis of coding and intron-exon splice regions of KEL of this K0 individual showed a change of a guanine to an adenine at the first nucleotide of Intron 16 (IVS16+1g>a, NCBI_ss#: 831878330). The mutation changes the conserved gt sequence at the donor splice site to at, altering the splice donor site, and preventing the removal of the intron. This event creates a premature stop codon, two codons downstream from the SNP. Unfortunately, the patient sample was not available to perform further RNA analysis. The changes are summarized in Table 1 and the eletropherograms are included in Fig. S1.
CONFLICT OF INTEREST The authors report no conflicts of interest or funding sources.
From 1Colsan–Associação Beneficente de Coleta de Sangue, São Paulo, SP, Brazil; and 2INCTs–Hemocentro-Unicamp, Campinas, SP, Brazil. Address reprint requests to: Carine Prisco Arnoni, Colsan–Associação Beneficente de Coleta de Sangue, Avenida Jandira 1260, Indianópolis–CEP 04080-006, Brazil; e-mail: [email protected] Received for publication September 26, 2013; revision received November 29, 2013, and accepted December 6, 2013. doi: 10.1111/trf.12564 © 2014 AABB TRANSFUSION 2014;54:2128-2129. 2128
TRANSFUSION Volume 54, August 2014
TABLE 1. Phenotype, nucleotide and amino acid changes, GenBank database submitting numbers, and ISBT allele numbers Proband 1
2
Phenotype K−, k−, Kp(a−), Kp(b−), Js(a−), Js(b−) K−, k−, Kp(a−), Kp(b−), Js(a−), Js(b−)
Nucleotide change
Amino acid change
NCBI_ss#
ISBT allele
71G>A
Trp24Stop
831878329
KEL*02N.30
IVS16+ 1g>a
Alternative Splicing
831878330
KEL*02N.31
TWO NOVEL KEL ALLELES ENCODING K0 PHENOTYPES IN BRAZILIANS
REFERENCES
6. Yang MH, Li L, Kuo YF, et al. Genetic and functional analyses describe a novel 730delG mutation in the KEL gene
1. Lee S, Russo D, Redman C. Functional and structural aspects of the Kell blood group system. Transfus Med Rev 2000;14:93-103.
causing K0 phenotype in a Taiwanese blood donor. Transfus Med 2011;21:318-24.
2. Nunn HD, Giles CM, Dormandy KM. A second example of anti-Ku in a patient who has the rare Kell phenotype, Ko. Vox Sang 1966;11:611-9. 3. Chown B, Lewis M, Kaita K. A new Kell blood-group phenotype. Nature 1957;180:711. 4. Yu LC, Twu YC, Chang CY, et al. Molecular basis of the Kell-null phenotype: a mutation at the splice site of human KEL gene abolishes the expression of Kell blood group antigens. J Biol Chem 2001;276:10247-52. 5. Lee S, Russo DC, Reiner AP, et al. Molecular defects underlying the Kell null phenotype. J Biol Chem 2001;276: 27281-9.
SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article at the publisher’s Web-site: Fig. S1. Electropherogram of the 2 K0 samples studied. Control samples are on the top of the figure and probands are bellow. (A) Proband 1 is homozygous for a c.71G>A in Exon 2 that leads to a premature stop codon (Trp24Stop). (B) Proband 2 is homozygous for the mutation g>a at position +1 of Intron 16, that alters splice donor site, following the coding region until the premature stop codon (TAA).
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