A Novel cis-ab Allele Derived from the A Transferase Gene by Nucleotide Substitution C796A

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1 Annals of Clinical & Laboratory Science, vol. 34, no. 4, A Novel cis-ab Allele Derived from the A Transferase Gene by Nucleotide Substitution C796A Ding-Ping Chen, 1 Ching-Ping Tseng, 2 Wei-Ting Wang, 1 Mei-Chia Wang, 1 Kuo-Chien Tsao, 1 Tsu-Lan Wu, 1 and Chien-Feng Sun 1 1 Department of Clinical Pathology, Chang-Gung Memorial Hospital, Taoyuan County, Taiwan; 2 Graduate Institute of Medical Biotechnology and School of Medical Technology, Chang Gung University, Taoyuan County, Taiwan, ROC Abstract. The cis-ab is a very rare phenotype in the ABO blood group system. It corresponds to a special ABO allele encoding a glycosyltransferase that is capable of synthesizing both A and B antigens. Until now, gene sequences of only 3 cis-ab alleles were characterized. One was the A 1v allele with a nucleotide substitution G803C at codon 268; the second was the B allele with a nucleotide substitution A796C at codon 266; and the third arose from a point mutation C700T at codon 234 in exon 7 of the B transferase gene. In this study, we found a novel cis-ab allele when performing paternity tests in Chang Gung Memorial Hospital in Taiwan. Although his father was O blood type, a serologically AB blood type child was confirmed as being his father s offspring on the basis of 16 microsatellite markers (99.97% plausibility for the child and father). Exons 6 and 7 of the child s ABO alleles were characterized by direct sequencing and gene cloning. The results showed that the child has one O 1 allele and the second allele is almost identical to A 1*02 allele except for a single point mutation at nucleotide position 796, where an A replaces a C and leads to a change of leucine to methionine at amino acid 266. This implies that the child s O 1 allele was inherited from his father and the other allele was inherited from his mother. In conclusion, the novel cis-ab allele reported here is derived from the A transferase gene through a nucleotide substitution C796A, which differs from the 3 previously reported cis-ab alleles. (received 8 July 2003, accepted 2 August 2003) Keywords: blood typing, ABO blood group, glycosyltransferase, paternity testing, rare phenotype Introduction The cis-ab is a very rare phenotype in the ABO blood group system [1]. The phenotype was first reported by Seyfried in 1964 [2]. Serologically, it is characterized by the presence of A, weakened B, and elevated H antigens on the red blood cells (RBCs). Individuals with cis-ab phenotype have A and B transmitted as a single allele, referred to as cis-ab. In 1980, Yoshida et al [3,4] reported 2 possible genetic mechanisms of cis-ab inheritance, unequal chromosomal crossing-over [3] and structural Address correspondence to Chien-Feng Sun, M.D., Department of Clinical Pathology, Linkou Medical Center, Chang Gung Memorial Hospital, 5 Fu-Shin Street, Kweishan, Taoyuan 333, Taiwan, ROC; tel ext.2554; fax ; suncgj@adm.cgmh.org.tw. mutation of blood group glycosyltransferase [4]. The first genetic description in 1993 of cis-ab demonstrated that the cis-ab*01 sequence differs from A 1 *02 at codon 268 [5]. The change of amino acid 268 in the A 1 *02 transferase (alanine instead of glycine) was sufficient to shift the specificity of the transferase into a cis-ab enzyme. In 2000, Mifsud et al [6] reported another cis-ab (cis-ab var ), which differed from B 1 *01 by a single substitution A796C at codon 266 (Met266Leu) in exon 7. Recently, Roubinet et al [7] reported a novel cis-ab (cis- AB.tlse*01) that arose from a point mutation C700T at codon 234 in exon 7 of a B transferase gene. The nucleotide sequences of the novel cis-ab allele reported in this paper have been submitted to the GenBank with accession number AY /04/ $ by the Association of Clinical Scientists, Inc. 437_

2 438 Annals of Clinical & Laboratory Science, vol. 34, no. 4, 2004 Table 1. The blood typing results for the child and the father in this study. Forward Typing Results Reverse Typing Results Anti-A Anti-B Anti-H A1 cells B cells Child Father In this study, we found a novel cis-ab allele through paternity tests at Chang Gung Memorial Hospital in Taiwan. The novel nucleotide sequence differs from all ABO alleles so far reported, including cis-ab*01, cis-ab var, and cis-ab.tlse*01. This specific allele is nominated as cis-ab*02. Materials and Methods Blood typing. A child s blood and his father s blood were collected into EDTA anti-coagulant tubes for blood typing and molecular analyses. The blood typing was performed by standard hemagglutination tests, including forward and reverse typing. For forward typing, monoclonal anti-a, anti-b, and anti- H reagents were used. Then A 1 cell and B cell reagents were used for reverse typing. STR-amplification for paternity test. Genomic DNA was extracted from blood using the QIAamp DNA Mini Kit (QIAGEN, Hilden, Germany). The AmpFlSTR Identifier PCR amplification kit (Applied Biosystems, Foster City, CA, USA) was used to perform STR-PCR, which can provide paternity information. The tetranucleotide STR loci that were amplified in this reaction included: D8S1179, D21S11, D7S820; CSF1PO (all labeled with 6-FAM); D3S1358, TH01, D13S317, D16S539, D2S1338 (all labeled with VIC); D19S433, vwa, TPOX, D18S51 (all labeled with NED); and amelogenin, D5S818, FGA (all labeled with NED). For each PCR reaction, 1 ng of genomic DNA was amplified in the final reaction volume of 12.5 µl. The cycle conditions were: 1 cycle of 95 C for 11 min, followed by 28 cycles of 94 C for 1 min, 59 C for 1 min and 72 C for 1 min. The final elongation step was 45 min at 60 C. The PCR products were analyzed by an ABI PRISM 377 Genetic Analyzer (Applied Biosystems) according to the manufacturer s protocol. PCR amplification and direct sequencing for ABO gene. Because 91% of the ABO coding sequences lies in exons 6 and 7 [2], PCR-based gene analysis was performed for these 2 exons. Briefly, PCR was set up in a reaction volume of 50 µl containing 1 x reaction buffer (10 mm Tris-HCl, ph 8.3, 50 mm KCl and 1.5 mm MgCl 2 ), 10 nmol of dntp, 6 pmol of forward and reverse primers (Table 1), 300 ng of genomic DNA, and 1 U AmpliTaq Gold DNA polymerase (Applied Biosystems). The reaction was performed in the GeneAmp PCR system 9600 (Applied Biosystems) with the following cycle condition: 1 cycle of 95 C for 10 min, 35 cycles of 94 C for 20 sec, 62 C for 30 sec, and 1 cycle of 72 C for 1 min. The final elongation step was 10 min at 72 C. Subsequently, 10 µl of PCR products were fractionated on 1.5% agarose gel and visualized by ethidium bromide staining. The remaining PCR product was used to carry out direct sequencing using the Big Dye Terminator Cycle Sequencing kit (Applied Biosystems) and an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems) according to the manufacturer s protocols. Cloning of PCR product from ABO gene. The gelpurified PCR product was cloned into the pcrii- TOPO vector by a Zero Blunt TOPO PCR Cloning kit (Invitrogen, Groningen, Netherlands). Briefly, 4 µl of fresh PCR product was mixed with 1 µl of TOPO vector for 5 min. Of the reaction mixture, 2 µl was used to transform the E. coli competent cells as described by the manufacturer. The DNA sequences of the PCR inserts were then determined using the Sequencing Kit (Applied Biosystems). 437_

3 A novel cis-ab allele 439 Fig. 1. The nucleotide sequences from nt 629 to nt 820 of the novel cis-ab allele. The red arrows indicate the positions for nt 657, nt 703, nt 796, and nt 803, respectively. 437_

4 440 Annals of Clinical & Laboratory Science, vol. 34, no. 4, 2004 Table 2. The paternity test for identification of the relationship between a child with AB blood type and a father with O blood type, based on STR typing results, as discussed in this study. Markers Child Father Parentage index ABO blood groups AB/O O/O D3S vwa D16S D2S D8S D21S D18S D19S THO FGA TPOX CSF1PO D5S D13S D7S Cumulative parentage index Plausibility percentage Results Serology. In a paternity case at our hospital, the serological findings of the child and his father (Table 1) suggested the child has AB blood type, although his father has a normal O phenotype. This conclusion was based on our finding that the child s red cells show a normal hemagglutination result of 4+ titer for anti-a antibody, but they react weakly with anti-b antibody as 3+ titer. Nonetheless, serologic tests showed elevated H antigens on the RBCs. These results suggest that the child is likely to have a cis-ab genotype. STR and blood typing of the child and the father. To clarify the paternal relationship between the father and child, we obtained the STR typing results (Table 2) and calculated them after completing the STR PCR and electrophoretic studies. The parentage indexes of the D3S1358, vwa, D16S539, D2S1338, D8S1179, D21S11, D18S51, D19S433, THO1, FGA, TPOX, CSF1PO, D5S818, D13S317, and D7S820 between the child and the father were , , , , , , , , , , , , , , and , respectively. The cumulative parentage index for the child and the father is This means that the probability that the father contributed the obligatory haplotypes is times higher in comparison to a random Chinese. In other words, the likelihood for the putative father to be the real father is %. Although the parentage of the putative father as the real father is practically proven by these markers, it is interesting to note that the blood typing results of the child and father are AB group and O group, respectively. These blood typing results appeared contrary to the Mendelian mono-factorial mode of ABO inheritance theory. We thus hypothesized the cis-ab allele in the child was inherited from the child s mother. Genotyping, cloning, and sequencing. To test our hypothesis, the genotype of the child was determined by amplification and direct sequencing of exons 6 and 7 of the ABO allele (data not shown). We found that one of the 2 alleles displayed a point deletion at 437_

5 A novel cis-ab allele 441 Table 3. Comparison of key amino acids of cis AB*02 with other ABO alleles. Allele nt 467 nt 526 nt 700 nt 703 nt 796 nt 803 A 1 *01 CCG CGC CCC GGC CTG GGG Pro Arg Pro Gly Leu Gly A 1 *02 CTG Leu B 1 * GGC AGC ATG GCG Gly Ser Met Ala cis-ab*01 CTG GCG cis-ab*02 CTG ATG cis-ab var GGC AGC GCG cis-ab.tlse* GGC TCC AGC ATG GCG Ser B(A)* GGC ATG GCG B(A) 700 [10] GGC GCC AGC ATG GCG Ala nucleotide 261 in exon 6, which is characteristic of the most frequent O allele and produces a frameshift mutation with a premature stop codon. This result indicates that the child had only one functional ABO glycosyltransferase that was able to synthesize A and B at the RBC surfaces and thus confirmed that the child had cis-ab. Exon 6 and exon 7 sequencing from clones of the child s alleles confirmed the presence of a classic O*01 and showed that the other allele corresponded to an A 1 *02 designated as cis-ab*02 (Fig. 1). The cis AB*02 allele was identical to A 1 *02 except at position 796, where a C was replaced by an A nucleotide (Table 3). This nucleotide substitution led to an amino acid replacement at position 266, where the leucine found in A 1 was replaced by a methionine. Discussion Because of the confidentiality of data for paternity testing, we were unable to obtain a DNA specimen from the child s mother to establish the pedigree directly. Instead, the paternity identification method was used to confirm the paternal relationship between the father and child. This is the first published report of using this method to identify a new cis-ab allele. The data suggest that the cis-ab allele described in this study arose from a point mutation at nt 796 of a seemingly normal A allele. In this case, the mutation involved the substitution of the A- transferase-specific cytosine residue with a B- transferase-specific adenosine residue. A C796A substitution results in the amino acid substitution Leu266 to Met, which changes the sugar donor specificity at that site to a B-transferase and could give rise to a bifunctional transferase, as suggested by the serological findings in this study. Based on the results from 16 combinational AB transferase chimera constructions at 4 amino acid substitutions and DNA transfection experiments by Yamamoto et al [8], the cis-ab*02 is of the type AABA at codons 176, 235, 266, and 268, differing 437_

6 442 Annals of Clinical & Laboratory Science, vol. 34, no. 4, 2004 from the 3 previously published cis-ab or B(A), which are of type AAAB (cis-ab*01), BBAB (cis- AB var ), and BABB (B(A)*01) [9], respectively. Molecular recombination between A and B alleles may provide an explanation for the generation of this cis-ab genotype. However, occurrence of a double recombination within such a short nucleotide span is less plausible. We believe that these cis-ab alleles arose through a point mutation during evolution of ABO genes rather than the recombination between A and B alleles. In conclusion, this study provides molecular evidence that the novel cis- AB*02 phenotype arises from genetic aberrations of the A transferase gene. Acknowledgements This work was supported in part by Grant CMRP- 797 from Chang Gung Memorial Hospital and by Grant NSC B from the National Science Council oftaiwan. References 1. Daniels G. Human Blood Groups. Blackwell, Oxford, 1995; pp Seyfried H, Walewska B. Unusual inheritance of ABO group in a family with weak B antigens. Vox Sang 1964; 9: Yoshida A, Yamaguchi H, Okubo Y. Genetic mechanism of cis-ab inheritance. I. A case associated with unequal chromosomal crossing over. Am J Hum Genet 1980;32: Yoshida A, Yamaguchi H, Okubo Y. Genetic mechanism of cis-ab inheritance. II. Cases associated with structural mutation of blood group glycosyltransferase. Am J Hum Genet 1980;32: Yamamoto F, McNeill PD, Kominato Y, Yamamoto M, Hakomori S, Ishimoto S, Nishida S, Shima M, Fujimura Y. Molecular genetic analysis of the ABO blood group system: 2. cis AB alleles. Vox Sang 1993;64: Mifsud NA, Watt JM, Condon JA, Haddad AP, Sparrow RL. A novel cis AB variant allele arising from a nucleotide substitution A796C in the B transferase gene. Transfusion 2000;40: Roubinet F, Janvier D, Blancher A. A novel cis AB allele derived from a B allele through a single point mutation. Transfusion 2002;42: Yamamoto F, Hakomori S. Sugarinucleotide donor specificity of histo-blood group A and B transferase is based on amino acid substitutions. J Biol Chem 1990; 265: Yamamoto F, McNeill PD, Yamamoto M, Hakomori S, Harris T. Molecular genetic analysis of the ABO blood group system: 3. A x and B (A) alleles. Vox Sang 1993;64: Yu LC, Lee HL, Chan YS, Lin M. The molecular basis for the B(A) allele: an amino acid alteration in the human histoblood group B α-(1,3)-galactosyl- transferase increases its intrinsic α-(1,3)-n-acetylgalactosaminyltransferase activity. Biochem Biophys Res Commun 1999; 262: _