Annals of Clinical & Laboratory Science 34:314-318 (2004)
© 2004 Association of Clinical Scientists
Two Prevalent h Alleles in para-Bombay Haplotypes among 250,000 Taiwanese
Ding-Ping Chen1,
Ching-Ping Tseng2,
Wei-Ting Wang1,
Chien-Ting Peng1,
Kuo-Chien Tsao1,
Tsu-Lan Wu1,
Kuan-Tsou Lin3 and
Chien-Feng Sun1
1 Department of Clinical Pathology, Chang Gung Memorial Hospital, Taoyuan, Taiwan 2 Graduate Institute of Medical Biotechnology and School of Medical Technology, Chang Gung University, Taoyuan, Taiwan and 3 Kaohsiung Blood Donation Center, Kaohsiung, Taiwan
Address correspondence to Chien-Feng Sun MD, Department of Clinical Pathology, Linkou Medical Center, Chang Gung Memorial Hospital, 5 Fu-Shin Street, Kweishan, Taoyuan, 333, Taiwan, ROC.; tel 886 3 328 1200 x2554; fax 886 3 397 1827; e-mail: suncgj{at}adm.cgmh.org.tw.
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Abstract
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(1,2)-Fucosyltransferase catalyzes the transfer of fucose to the C-2 position of galactose on type II precursor substrate Galß1-4GlcNAcß1-R. It plays an important biological role in the formation of H antigen, a precursor oligosaccharide for both A and B antigens on red blood cells. Aberration of (1,2)-fucosyltransferase activity by gene mutations results in decreased synthesis of H antigen, leading to the para-Bombay phenotype. In this study, we collected about 250,000 blood samples in Taiwan during 5 yr and identified the subjects with para-Bombay phenotype. Then we analyzed the sequence of the (1,2)-fucosyltransferase gene by direct sequencing and gene cloning methods, using the blood samples of 30 para-Bombay individuals and 30 control subjects who were randomly selected. The goals of this study were to search for new h alleles, to determine the h allele frequencies, and to test whether the sporadic theory is applicable in Taiwan. Six different h alleles (ha, 547~548 AG-del; hb, 880~881 TT-del; hc, R220C; hd, R220H; he, F174L; and hf, N327T) were observed. Two h alleles, he and hf, were newly discovered in Taiwan. The he allele has a nucleotide 522C>A point mutation, predicting the amino acid 174 substitution of Phe to Leu; the hf allele has missense mutation of nucleotide 980A>C, predicting the amino acid 327 substitution of Asn to Thr. Frequencies of the 6 alleles are ha 46.67%, hb 38.33%, hc 5.00%, hd 1.67%, he 3.33%, and hf 5.00%, respectively. These findings in the Taiwanese population confirm previous observations in other populations that the Bombay and para-Bombay phenotypes are due to diverse, sporadic, nonfunctional alleles, predominantly ha and hb, leading to H deficiency of red blood cells. In contrast to previous reports of non-prevalent associations of h alleles with para-Bombay phenotype, our results suggest a regional allele preference associated with para-Bombay individuals in Taiwan.
(received 16 April 2004; accepted 1 May 2004)
Keywords: para-Bombay phenotype, h alleles, ABO blood groups, blood transfusion
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Introduction
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(1,2)-Fucosyltransferase catalyzes transfer of fucose to the C-2 position of galactose on type II precursor substrate Galß1-4GlcNAcß1-R. Two types of (1,2)-fucosyl-transferase are found in human tissues, encoded by the H and SE genes, respectively. The H type of (1,2)-fucosyltransferase (FUT1) is crucial for the generation of a precursor oligo-saccharide H antigen that is processed to form both A and B antigens on red blood cells (RBC). The SE type of (1, 2)-fucosyltransferase (FUT2) mediates the production of H antigen and the generation of ABH antigens in secreting epithelia of the salivary glands. Homozygosity for non-functional h alleles with 1 or 2 active SE alleles results in a para-Bombay phenotype that has the ABH antigens in secreting epithelia but not on the RBC. In contrast, aberrancy of both H and SE genes elicits a Bombay phenotype that has weak or undetectable ABH antigens in the secreting epithelia and on the RBC.
The molecular basis of Bombay and Para-Bombay phenotypes was reported in 1994 by Kelly et al [1]. They found that a missense mutation of 491T>A, and nonsense mutations of 826C>T and 948C>G are responsible for the H blood group deficiencies in Bombay and para-Bombay individuals. To date, 25 types of H (FUT1) gene defects that lead to impaired (1,2)-fucosyltransferase function have been reported in persons with either para-Bombay or Bombay phenotype [1–11]. These defects include missense mutations, nonsense mutations, and short deletions of DNA base-pairs. In populations of Europe [8] and North America [1], the occurrence of the nonfunctional alleles is diverse and sporadic with no prevalence of any specific h allele. The population of Reunion Island seems to be an exception, since 349C>T and 725T>G are the prevalent mutations [4].
In Taiwan, the incidence of para-Bombay phenotype was estimated to be 1 in 8000 residents based on serological analysis [12]. To analyze para-Bombay genotypes and to determine if any prevalent allele is present in the Taiwanese population, we performed a large-scale population screening to identify individuals with para-Bombay phenotypes. Sequencing analysis revealed 6 different para-Bombay-associated h alleles with deletions or missense mutations in the coding region of the H gene. Frequencies of the para-Bombay-associated h alleles are reported and discussed in this paper.
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Materials and Methods
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Sample preparation.
Fresh blood and saliva samples were collected from 30 healthy and unrelated Taiwanese subjects with the para-Bombay phenotype and 30 randomly selected control subjects, based upon the screening of 249,779 individuals in this study. RBC ABH antigens and serum antibodies were detected by standard hemagglutination, absorption, and elution tests. Salivary ABH substances were determined by a salivary inhibition study [13]. The genomic DNA was extracted from fresh blood samples by the QIAamp DNA mini-kit (QIAGEN, GmbH, Hilden, Germany).
PCR amplification.
The PCR was setup in a reaction volume of 50 µl containing 1x reaction buffer (1.5 nmol/µl, 50 nmol/µl KCl, and 10 nmol/µl Tris-HCl, pH 9.0 at 25°C), 10 nmol of dNTP, 6 pmol of forward and reverse primers (Table 1
), 300 ng of genomic DNA, and 1 U AmpliTaq Gold (Applied Biosystems, Foster City, CA). The reaction was performed in the GeneAmp PCR system 9600 (Applied Biosystems) with the following cycle conditions: 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 was fractionated on 1.5% agarose gel containing 0.56 µg/ml ethidium bromide. The other 40 µl of PCR products was used for direct sequencing.
Direct sequencing of PCR products.
The PCR products were purified using QIAquick DNA Purification Kit (QIAGEN) according to the manufacturer’s instructions. The purified PCR products were sequenced using the Big Dye Terminator Cycle Sequencing kit (Applied Biosystems) and analyzed by an ABI PRISM 377 Genetic Analyzer (Applied Biosystems) according to the manufacturer’s instructions.
Cloning of PCR product.
The gel-purified PCR product was cloned into the pCRII-TOPO vector by a TOPO TA Cloning kit (Invitrogen, Groningen, Netherlands). When a new h allele was discovered, the gene cloning technique was used to confirm this finding. The PCR product of the new allele was cloned into the pCRII-TOPO vector by a TOPO TA Cloning kit (Invitrogen) for sequencing. Then DNA sequences were determined as described.
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Results
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For comprehensive analysis of the H gene coding sequences in para-Bombay individuals, a large-scale population screening was performed during a 5 yr period (1999–2003). Serological study revealed 30 subjects with para-Bombay phenotype in a total of 249,779 individuals, consistent with the previous estimate that the incidence of para-Bombay phenotype is 1 in 8000 in the Taiwanese population [12].
The coding sequences of the H gene in 30 normal ABH phenotype and 30 para-Bombay individuals were PCR amplified with f and r primers. The PCR product was cloned into the pTOPO cloning vector for sequence analysis with the primers f, r, midf, and midr, respectively. The sequences of the oligonucleotide primers used for PCR amplification and sequencing of FUT1 are shown in Table 1
. In the normal ABH individuals, the coding sequences of the wild type H allele were identical to those reported by Larsen et al [14].
In para-Bombay individuals, 6 kinds of h alleles were found; 3 of the 6 h alleles, ha, hb, and hc, were identical to the h1, h2, and h3 alleles previously described by Yu et al [2]. The other 3 h alleles were the hd, he, and hf. The h4 allele, with 35C>T and 980A>C, and the h5 allele, with 460T>C mutation, reported by Yu et al [2], were not observed in the current study. Both ha and hb alleles were 2-base deletions, leading to frame shifts: 1 of the 3 AG repeats at nucleotides 547–552 was deleted in ha allele, and 2 of the 3 T bases at nucleotides 880–882 were deleted in hb allele. The hc allele has a nucleotide 658C>T mutation, predicting an amino acid substitution of Arg to Cys. The hd allele is a missense mutation 659G>A with an amino acid substitution of Arg to His that has been reported by Sun et al [14]. Another 2 new alleles, designated as he and hf, are newly identified in the Taiwanese population. The he allele is a 522C>A mutation with an amino acid Phe to Leu substitution and was previously described in another population [9]. The hf allele is the missense mutation 980A>C, which leads to an amino acid substitution of Asn to Thr (Fig.1).
Of the 30 para-Bombay individuals, the h allele has the following haplotype combinations: ha/ha (n = 10), ha/hb (n = 6), hb/hb (n = 6), ha/hf (n = 3), ha/hc (n =1), ha/he (n =1), hc/hd (n =1), hb/he (n=1), and hb/ hc (n =1). Accordingly, the percentages of h alleles are ha 46.67% (n = 28), hb 38.33% (n = 23), hc 5.00% (n = 3), hd 1.67% (n = 1), he 3.33% (n = 2), and hf 5.00% (n = 3), respectively. (Fig. 1) These results indicate that ha and hb alleles are prevalently associated with para-Bombay individuals in Taiwan.
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Discussion
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In this study, 6 different h alleles were observed in the 30 Taiwanese with para-Bombay phenotype, whereas the wild type H allele, as reported by Larsen et al [14], was associated with the normal ABH phenotype. Haplotypes of the para-Bombay individuals consisted of combinations of 6 mutated h alleles that were not found in any control donors with normal ABO blood groups. These h alleles are probably the inactivating mutations leading to the H deficiency of RBCs and are responsible for the inactivation of the FUT1-encoded enzyme activity. A definite association of these h mutant alleles with the lack of FUT1 enzymatic activity can be revealed by transient expression of the mutant allele in COS-7 cells and subsequently enzymatic assay, as in the study of the group A and B transferases mutant alleles [15].
The h alleles of para-Bombay phenotypes were associated with many different mutational types. To date, including this work, 26 h alleles have been reported (Table 2). Analysis of the mutation positions derived from this study and others reveal that most mutations are in regions nt 442–552, 658–882, and 944–1047 of the H gene. Although this conclusion has been published previously, it is noteworthy that both of the new alleles are consistent with this localization of mutations. One newly observed h allele has its mutation at the previously reported amino acid position 327.
The studies from North American and Europe suggest that h allele mutations occur in a sporadic manner without any allele prevalence and support the "sporadic theory" for the occurrence of h allele mutations [1,8]. In contrast, we report that ha and hb alleles account for more than 85% of all h alleles associated with para-Bombay individuals in Taiwan. The reason for the higher incidence of ha and hb alleles in the para-Bombay H gene of Taiwanese is unclear. It may reflect an early point mutation of the population or indicate that the sequences of the mutant alleles represent hot spots of genetic attack and damage. Consistent with our findings, different prevalent null alleles were seen in another regional population, ie, Reunion Island [4]. The allele prevalence may be region-specific. Alternatively, the limited numbers of para-Bombay subjects enrolled in various studies could account for the discrepancy of allele prevalence between studies. A large-scale population study may be needed to determine whether prevalent alleles also occur in Western countries.
In conclusion, this study indicates a preferential association of specific h alleles with the para-Bombay phenotypes in the Taiwanese population and suggests a way to develop molecular techniques for para-Bombay detection. Rapid molecular diagnostic methods, such as sequence-specific priming (SSP) and real-time PCR for detection of single nucleotide polymorphism, are under development in our laboratory to screen the prevalent ha and hb alleles. This approach expediently uses the large population databases that are available in transfusion medicine and enables the rapid acquisition of molecular genetics data that are relevant for many genes.
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Acknowledgements
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This work was partly supported by Grant CMRP-797 from Chang Gung Memorial Hospital and Grant NSC-91-2314-B-182-093 from the National Science Council, Taiwan.
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Abstract
Introduction
