Annals of Clinical & Laboratory Science 35:265-269 (2005)
© 2005 Association of Clinical Scientists
Identification of a Novel B Variant Allele at the ABO Locus in Chinese Han Individuals with B Subgroup
Zhi-Hui Deng,
Qiong Yu,
Yan-Lian Lian,
Guo-Guang Wu,
Yu-Qing Su and
Xuan Zhang
Shen-Zhen Blood Center, Shen-Zhen Institute of Transfusion Medicine, Guang-Dong Province, China
Address correspondence to Dr. Zhi-Hui Deng, Shen-Zhen Institute of Transfusion Medicine, Ni-Gang Xi Road, Mei-Gang Nan Street, Shen-Zhen, Guang-Dong Province, 518035, People’s Republic of China; tel 86 755 8324 2567; fax 86 755 8322 1000; e-mail zhihui_deng{at}yahoo.com.cn.
 |
Abstract
|
|---|
We studied the molecular genetic background of the B subgroup in the Chinese Han population and identified a novel allele at the ABO locus. Ten control samples from randomly selected blood donors of normal B phenotype and 6 samples from individuals diagnosed as B subgroup by serological tests were genotyped by PCR-SSP and direct DNA sequencing at exons 6 and 7 of the ABO gene. Exons 6 and 7 and the intervening intron 6 of B alleles from the 6 B subgroup samples were analyzed by cloning and haplotype-sequencing. A novel B variant allele was identified in 2 individuals who were serologically-determined as members of the Bx and Bw subgroups, respectively. The novel B allele differs from allele B101 by a single 695T>C missense mutation in exon 7. The family of the individual with Bx subgroup was studied; among 8 family members tested, 4 had the novel B variant allele. No mutation at exon 6 or 7 of the ABO gene was detected in the 10 control samples or in the other 4 B subgroup samples. Mutation at position 695 where T is replaced by C results in an amino acid change from Leu to Pro, which is predicted to diminish B transferase activity. This indicates that alteration of the amino acid at position 232 is critical to the activity of glycosyltransferases.
(received 8 December 2004; accepted 5 February 2005)
Keywords: ABO blood group, B subgroup, cloning and DNA sequencing, novel B allele, glycosyltransferases
|
Introduction
|
|---|
The ABO blood group is beyond doubt the most important blood group system in transplantation, transfusion medicine, and paternity testing. It was discovered by Landsteiner in 1901. In 1990, the molecular backgrounds of the 3 main alleles A1, B, and O, were identified by Yamamoto et al [1], who cloned and sequenced the cDNA of the ABO gene. In addition to the common ABO phenotypes, many ABO subgroups with weak expression of the A or B antigens on red blood cells (RBCs) have been found. The subgroup phenotypes result from a variety of molecular changes in the ABO allele [2]. Sequencing the ABO alleles elucidates the molecular basis of the ABO subgroups and helps to delineate the influence of amino acid variations on serologic specificity and glycosyltransferase activity. Many ABO alleles have been identified in various populations [3,4].
To determine the molecular basis of ABO serologic subgroups in the Chinese Han population, we have previously studied individuals with the A2 subgroup using cloning and sequence analysis. We found that the A2 allele frequency and distribution in the Chinese Han population are quite different from other populations. A2-5 is the predominant allele of A2 in the Chinese Han population [5]. We have also identified a novel O1 variant allele in an A2 subgroup individual (genotyped as A2O1). This novel O1 allele (GenBank accession number: AY374123
[GenBank]
) is identical to the ABO*O101 allele except for A-deletion at position 496 in exon 7 [6].
In this study, we report a novel B allele, identified in two B-subgroup individuals, which differs from the allele B101 by single 695T>C missense mutation in exon 7. A family study demonstrated that this novel allele is inheritable and rules out a possible artifact or somatic mutation as the cause of nucleotide (nt)695T>C.
|
Materials and Methods
|
|---|
Samples.
Ten randomly-selected blood samples from blood donors with common B phenotype were used as controls. Six B subtype samples, which had previously been serologically typed, were obtained from our blood group reference laboratory. The blood samples all came from healthy blood donors of the Chinese Han population. Additionally, blood samples were obtained from 8 relatives of a Bx subgroup donor; these family members were all recruited with informed consent. Venous blood samples (3 ml) were drawn into EDTA tubes.
Serological tests.
Routine serological testing (eg, RBC and serum blood grouping procedures; adsorption and elution with anti-B and A, B, and H substance by saliva test) was performed by standard serological methods. The antisera included monoclonal anti-A and anti-B (Ortho, Raritan, NJ, USA), polyclonal anti-B (blend of human serum), and monoclonal anti-A,B (Immucor, Norcross, GA, USA). Lectins from Ulex europaeus were used for anti-H (Dominion, Nova Scotia, Canada).
Genotyping by PCR-SSP.
Four pairs of sequence-specific primers (SSP) were synthesized. The first primer pair, designed by us (1sense/2antisense):
5'-gga agg atg tcc tcg tgg tg-3'; 5'-tga gga tgt gga tgt tga at-3') was used to amply the common A allele. Three other pairs, designed by Seltsam [7], were (27sense /33 antisense primers):
5'-cat tgt ctg gga ggg cac g-3'; 5'-ctt gat ggc aaa cac agt taa c-3'; (21sense/33 antisense primers):
5'-gga agg atg tcc tcg tgg ta-3'; 5'-ctt gat ggc aaa cac agt tac c-3'; (41sense/46 antisense primers):
5'-acg tgg ctt tcc tga agc tg-3'; 5'-gcg ggg cac cgc ggc ca-3') were used to amplify the common B, O1, and A102 alleles respectively.
Polymerase chain reaction (PCR) amplification was carried out in a reaction volume of 10 µl containing 1 x PCR buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl, and 1.5 mM MgCl2), primer mix, 80 µM of each dNTP, 1U Taq polymerase, and 100 ng of genomic DNA. The reaction was performed in a GeneAmp PCR system 9700 using the following PCR cycling parameters: 1 cycle at 95°C for 5 min; 30 cycles at 95°C for 30 sec, 60°C for 30 sec, and 72°C for 90 sec; followed by a final extension for 72°C for 5 min. PCR products were electrophoresed on 4% agarose gels containing 0.5 ug/ml ethidium bromide and visualized by UV transillumination.
Direct sequencing of exons 6 and 7 of ABO gene.
Because 91% of the ABO coding sequences lie in exons 6 and 7 [8], PCR-based gene analyses were performed on the 2 exons. Primer pairs mol-46/mol-57 and mol-71/mol-101 described in a previous study [9] were used to amplify exons 6 and 7. The PCR fragment sizes for exons 6 and 7 were 252 bp (251 bp for O1) and 843 bp, respectively. PCR amplification was carried out in a reaction volume of 50 µl containing 1 x PCR buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl, and 1.5 mM MgCl2 ), 400 µM each of dNTP, 0.1µM of each primer pair, 300–500 ng of genomic DNA, and 2.5 U of Taq DNA polymerase. Amplification was carried out under the following conditions: 95°C for 10 min; 10 cycles: 94°C for 60 sec, 63°C for 90 sec, and 72°C for 60 sec; 25 cycles: 94°C for 60 sec, 61°C for 90 sec, and 72°C for 60 sec; followed by a final elongation at 72°C for 10 min.The PCR products were purified using Takara DNA Fragment Purification Kit (TaKaRa, Dalian, China) according to the manufacturer’s instruction. The purified PCR products were directly sequenced using BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems) and analyzed by an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems).
Cloning and haplotype sequencing of exon 6, intron 6, and exon 7 at the ABO locus.
To determine the haploid type of ABO gene, a fragment of 2170 bp spanning exon 6, intron 6, and exon 7 was amplified using the following primer pair, designed by us:
5'-cgt gaa ggg tgg tca gag ga -3'; 5'-gtt act cac aac agg acg gac -3'. The PCR amplification was carried out in a volume of 50 µl containing 2 µl of 2 x GC buffer I/II,100 µM each of dNTPs, 0.1 µM each of 2 primers, 300 to 500 ng of genomic DNA, and 2 U of LA Taq polymerase (Takara). The reaction was performed in a GeneAmp PCR system 9600 using the following PCR conditions: 1 cycle of 95°C for 10 min; 30 cycles of 94°C for 30 sec, 60°C for 30 sec, and 72°C for 150 sec; and finally 72°C for 10 min. The gel-purified PCR product was cloned into the pCRII vector using TOPO cloning kit (Invitrogen, Groningen, Netherlands). Isolated clones containing the B allele were screened using the 27s/33as primer pair by the PCR-SSP method. The haplotype sequencing reaction was performed in a final volume of 10 µl using the following 5 forward sequencing primers:
| Name |
Location |
Sequences |
|
| AF1 |
Intron 5 |
5'-GGC GGC CGT GTG CCA GA-3' |
| AF2 |
Intron 6 |
5'-TTG TCC TCC CAG AGG GTA GA-3' |
| AF3 |
Intron 6 |
5'-CAA CCG CAG ACA CAT ACT TGA-3' |
| AF4 |
Exon 7 |
5'-CAG GAC GGG CCT CCT GCA-3' |
| AF5 |
Exon 7 |
5'-CCA GTC CCA GGC CTA CAT-3'. |
|
Results
|
|---|
Serologic phenotypes and respective genotypes.
The serologic phenotypes and genotypes are shown in Table 1
. Six B-subgroup samples (Samples 1 to 6) were typed as Bw, ABel, Bm, Bx, Bw, and B3 by adsorption-elution and saliva testing, and were also genotyped by PCR-SSP. Consistent with the serologic phenotypes, the genotypes were all B1O1 except sample 2, which was typed as A102B. A family study was performed in 7 relatives of a Bx subgroup individual (Fig. 1). Of the 8 family members, 4 (including the propositus, her mother, uncle, and sister) were identified as Bx by serologic testing. Both grandparents of the propositus had died, so they were unavailable for testing
Directing sequencing and cloning analysis.
In this study, each B allelic clone was selected for haplotype sequencing analysis, which allowed us to identify the nucleotide sequence of B allele at exons 6 and 7 for all of the B subgroup samples. According to each sample’s direct sequencing results, which were the heterozygous and haplotype sequence of B allele, we deduced the nucleotide sequence of another ABO haplotype belonging to the A allele or O allele. We defined each ABO allele using the method described by Chester and Olsson [10]. As a result, 4 common B samples were defined as B101O1; the other 6 common B samples were B101O1v; no novel mutation was found at exons 6 or 7 in the control group. For the 6 B-subgroup samples, sample 1 to 3 and sample 6 were defined as B101O1v, A102B101, B101O1, and B101O1, respectively. The single nucleotide polymorphism (SNP) sites of sample 4 at one haplotype were determined as 261G deletion, 297A, 467C, 526C, 657C, 695T, 703G, 796C, 803G, 930G, and 1096G; the sample can be defined as wild-type O101. However, other haplotype SNP sites of sample 4 were determined as 261G, 297G, 467C, 526G, 657T, 695C, 703A, 797A, 803C, 930A, 1096A, and a novel SNP site mutation at (nt)695 (695T>C) in exon 7 was identified; we define this as a novel variant B allele. The family study showed that this novel B allele is present in 4 family members. The finding that this novel B allele is inheritable rules out a possible artifact or somatic mutation as the cause of the (nt)695T>C missense (Figs. 2,3). The sequencing results of sample 5 (Bw subgroup) were identical to sample 4 and the (nt)695T>C mutation was also determined as a B allelic haplotype.
View larger version (36K):
[in this window]
[in a new window]
|
Fig. 2. Sequencing result at position 695 in wild-type B allele. The 2 nucleotides indicated by dashes are G and C, respectively.
|
|
|
Discussion
|
|---|
The weak B phenotype is more frequent than the weak A subgroup in the Chinese Han population. The subgroup B is characterized by no agglutination, or only weak hemagglutination, of RBCs with anti-B and anti-A-anti-B antibody. Without excellent operating technique and a proper titer of antiserum, the weak B phenotype is often misrecognized as O phenotype. Although the B antigen expression of weak B individuals is very weak, its clinical effect is clear. In this report, we identified the molecular basis of the B subgroup polymorphism. Based on serological findings, the B subgroups were classified into 5 categories [11]: B3, Bx, Bm, Bel, and Bw. We adopt the designation Bw for this antigen for those B variants that do not fit in the other 4 categories [12].
Until now, many novel ABO alleles have been identified and characterized. According to the Blood Group Antigen Gene Mutation Database [13], more than 20 B alleles have been previously reported and have been classified in 6 categories: B1, B3, Bel, Bw, CisAB, and B(A). Compared to the reference A101 and B101 allelic sequence, the B variant alleles have 3 highly-conserved mutations at nt703, nt796, and nt803; the other mutation sites vary from B allele to B allele. Take alleles of B3, Bel, and Bw subgroups for instance: mutations at nt703, nt796, and nt803 are the same for all these alleles, and one or more of an additional 13 new and rare nucleotide variations (eg, C502T, G539A, A548G, T641G, G669T, C721T, T863G, G871A, C873G, C1036G, A1037T, C1054T, and G1055A) are also observed.
Molecular cloning analysis of the ABO gene allows the elucidation of the molecular basis of its various alleles. Lin et al [14] identified a unique 502C>T mutation in a Bel-subgroup individual in Taiwan; Lung-Chih Yu et al [15] found a 247G>T mutation in a B3 subgroup individual; Estalote et al [16] found a novel 556A>G missense mutation at exon 7 in a Caucasian family.
In the present study, we singled out B allelic clones and performed sequencing analysis at exons 6 and 7 and the interval intron 6. A novel B allele was found in 2 individuals who were serologically-identified as Bx and Bw, respectively. The novel B allele shared high sequence homology with B101 and differed from B101 by single 695T>C missense mutation in exon 7. We define it as a new variant allele. To confirm our findings, we obtained blood samples from relatives of the Bx propositus. The family study showed that 4 of 8 relatives carry this novel B allele. These findings indicate that this novel B allele is inheritable and rule out a possible artifact or somatic mutation.
Among the 6 B-subgroup samples, 2 that were serologically-determined as Bx or Bw were found to carry the novel B variant allele. This suggests that the novel B allele may be frequent in the Chinese Han population, and indicates that different serologic subgroups may have a common molecular basis. Monoclonal reagents have been replacing traditional polyclonal antisera and may have been causing difficulties in serological classification of the subgroups. The novel mutation, which changes T to C at nt695 in exon 7, results in an amino acid change of Leu to Pro. The mutation hypothetically diminishes the 
-1,3-galactosyltransferase (B transferase) activity that is responsible for the formation of the B determinant by transfer of Gal from the UDP-Gal donor to the H precursor. In a normal adult, B antigen sites number about 600,000 to 800,000. While we observed that RBCs from the 2 individuals reacted weakly with anti-B sera, we think that some B subgroups may have a quantitative difference in their B antigens. We predict that the nucleotide at position 695 is critical for glycosyltransferase activity.
Although no new mutation was identified in the 4 other B-subgroup individuals, further study needs to be undertaken as to whether additional mutations exist in other exons or introns, or in the promoter region of the B subgroup gene, which could contribute to the B subgroup phenotype.
In summary, this study documents a novel B allele in the ABO blood group. Our findings suggest that the molecular determinants of the B subgroup in the Chinese Han population may differ from those reported in other populations.
|
Acknowledgments
|
|---|
The authors thank the Wu family for providing blood and DNA samples. This work was supported by the Research Fund of Guang-Dong Health Department (Project 2001641) and by the Research Fund of ShenZhen Bureau of Science & Technology (Project 200304217).
|
References
|
|---|
- Yamamoto F, Marken J, Tsuji T, White T, Clausen H, Hakomori S. Cloning and characterization of DNA complementary to human UDP-GalNac: fucal2GalNac transferase mRNA. J Biol Chem 1990;256:1146–1151.
- Yip SP. Sequence variation at the human ABO locus. Ann Hum Genet 2002;66:1–27[Medline]
- Seltsam A, Hallensleben M, Kollmann A, Blasczyk R. The nature of diversity and diversification at the ABO locus. Blood 2003;102:3035–3042.[Abstract/Free Full Text]
- Olsson ML, Irshaid NM, Hosseini-Maaf B, Hellberg A, Moulds MK, Sareneva H, Chester MA. Genomic analysis of clinical samples with serological ABO blood grouping discrepancies: identification of fifteen novel A and B subgroup alleles. Blood 2001;98:1585–1593.[Abstract/Free Full Text]
- Yu Q, Wu GG., Deng ZH, Su YQ, Lian YL, Wei TL. Study on the molecular genetic background of A2 subgroup in Chinese Han population. Chin J Blood Transfusion 2004;17:83–86.
- Deng ZH, Yu Q, Wu GG, Lian YL, Su YQ, Wei TL. Identification of a novel O1 variant allele at the ABO blood group locus. Chin J Lab Med 2004;27:405–407.
- Seltsam A, Hallensleben M, Kollmann A, Burkhart J, Blasczyk R. Systematic analysis of the ABO gene diversity within exon 6 and exon 7 by PCR screening reveals new ABO alleles. Transfusion 2003;43:428–439.[Medline]
- Yamatoto F, McNeil PD, Hakomori S. Genomic organization of human histo-blood group ABO gene. Glycobiology 1995;5:51–58.[Abstract/Free Full Text]
- Olsson ML, Chester MA: Polymorphisms at the ABO locus in subgroup A individuals. Transfusion 1996;36: 309–313.[Medline]
- Chester MA, Olsson ML. The ABO blood group gene: A locus of considerable genetic diversity. Transfus Med Rev 2001;15:177–200.[Medline]
- Vengelen-Tyler V. AABB Technical Manual. American Association of Blood Banks, Bethesda, MD, 1999.
- Daniels G. Human Blood Groups, Blackwell, Oxford, UK, 1995.
- Blood Group Antigen Gene Mutation Database, Dept of Biochemistry, Albert Einstein College of Medicine, 2004; http://www.aecom.yu.edu/biochemistry/bgmut/abo.htm.
- Lin.PH., Li L., Lin-Tsai SJ, Lin KT, Chen JM, .Chu DC. A unique 502C>T mutation in exon 7 of ABO gene associated with the Bel phenotype in Taiwan. Transfusion 2003;43:1254–1259.[Medline]
- Yu LC, Twu YC, Chou ML, Chang CY, Wu CY, Lin M. Molecular genetic analysis for the B3 allele. Blood 2002; 100:1490–1492.[Abstract/Free Full Text]
- Estalote AC, Palatnik M, Chester MA, Olsson ML, Sant’Anna Gomes BM. A novel blood group B subgroup: serological and genetic studies. Transfus Med 2004; 14:173–180.[Medline]
Top
Abstract
Introduction
