Introduction
The H antigen is the precursor of the A and B antigens on red blood cells. The ABO locus determines the A and B antigens, whereas α-(1,2)-fucosyltransferase genes FUT1 (or H) and FUT2 (or Se) determine the H antigen, the precursor of A and B antigens. In the para-Bombay blood group, ABH antigens are not expressed on red blood cells, but ABH substances are present in saliva. The para-Bombay phenotype results either from an inactive FUT1 gene present together with a normal FUT2 gene, or from a mutated FUT1 gene present with or without an active FUT2 gene. In this study, we reported 17 para-Bombay genotypes, among which two novel FUT1 mutations were identified. Thus, as of February 2011, 39 FUT1 non-functional alleles have been found to affect the enzyme's activity1,2 including the two reported here.
Materials and Methods
Blood samples
Venous blood specimens of 17 unrelated Chinese para-Bombay individuals and 100 randomly selected Chinese individuals were collected into dried tubes and tubes containing 3.2% trisodium citrate.
Blood group serological studies
ABO serology was performed with standard serological techniques. An adsorption-elution test3 was used to detect trace amounts of A/B antigens on red blood cells or H antigens in sera. The presence or absence of ABH antigens in saliva was determined by a haemagglutination inhibition test3. Lewis blood group was tested to deduce secretory type in the condition of lack of saliva.
Molecular biology analysis
Genomic DNA was extracted from leucocytes of citrated peripheral blood. The ABO genotypes were determined using a Ready Gene ABO SSP kit (Inno-Train GmbH, Kronberg, Germany). The primer design and polymerase chain reaction (PCR) amplification of FUT1 and FUT2 were performed as described in the literature4. The PCR products were sequenced directly or sequenced after TA cloning (TaKaRa, Dalian, China) by the ABI 377 sequencer (Applied Biosystems, CA, USA). The same region of the FUT1 gene from 100 random Chinese individuals with the common ABO phenotype was also sequenced. The allele nomenclature used here follows the terminology according to the Blood Group Antigen Gene Mutation Database (BGMUT)1.
Results and discussion
In this study, 17 unrelated individuals were diagnosed as having the para-Bombay phenotype (Table I) because of the following serological characteristics: firstly, H antigen on red cells could not be detected and A/B antigens on red cells significantly decreased (A/B antigens on most individuals' red blood cells were detected only by the absorption-elution method); secondly, they were all demonstrated to be secretors either by their Le (a−b+) blood group or the ABH substances present in the saliva; thirdly, all of the para-Bombay individuals' sera contained anti-H. The individuals' serological ABO types correlated with their ABO genotypes. Direct DNA sequencing of the whole FUT1 coding region and TA cloning revealed two novel alleles, FUT1 649T (649G>T, V217F, GenBank N. GQ336988) and FUT1 35T, 423A (35C>T, A12V; 423G>A, W141X, GenBank N. HQ699894), and four previously reported defective FUT1 alleles FUT1 547delAG, FUT1 880delTT, FUT1 658T, FUT1 293T were also identified1. The two novel mutations were not identified in 100 random Chinese individuals with the common ABO phenotype as analysed by DNA sequencing, suggesting they are mutations instead of common single nucleotide polymorphisms in Chinese persons. Four FUT2 alleles were detected in our study: Se, Se357, Se357,716 and se357,385. Both Se357 and se357,385 alleles are very common in the Asian population. The Se357,716 allele was found only with the FUT1 880delTT allele in our study, which was also observed in para-Bombay individuals in Southeast China5. FUT2 gene analysis results were consistent with the subjects' secretor status.
Table I.
Phenotypes and genotypes of 17 Chinese para-Bombay individuals.
| No. | Haemagglutination | Absorption- elution | Anti-H in serum | Phenotypes * | Genotypes | ||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| A | B | H | Lewis | A | B | ABO | FUT 1 | FUT2 | |||
| 1 | − | − | − | a−b− | − | + | + | OhB-secretor# | O1B | FUT1 547delAG/FUT1 547delAG | Se357Se357 |
| 2 | − | − | − | a−b+ | − | + | + | OhB-secretor | O1B | FUT1 547delAG/FUT1 547delAG | Se357Se357 |
| 3 | − | − | − | a−b+ | + | − | + | OhA-secretor | O1A | FUT1 547delAG/FUT1 547delAG | Se357Se357 |
| 4 | − | − | a−b+ | − | + | + | OhB-secretor | O1B | FUT1 547delAG/FUT1 547delAG | Se357Se357 | |
| 5 | − | − | − | a−b+ | − | + | + | OhB-secretor | O1B | FUT1 547delAG/FUT1 880delTT | Se357,Se357,716 |
| 6 | − | − | − | a−b+ | + | − | + | OhA-secretor | AA | FUT1 547delAG/FUT1 880delTT | Se357Se357,716 |
| 7 | − | − | − | a−b+ | − | + | + | OhB-secretor | O1B | FUT1 547delAG/FUT1 880delTT | Se357Se357,716 |
| 8 | − | − | − | a−b+ | + | − | + | OhA-secretor | O1A | FUT1 547delAG/FUT1 880delTT | Se357,Se357,716 |
| 9 | − | − | − | a−b+ | + | − | + | OhA-secretor | O1A | FUT1 547delAG/FUT1 880delTT | Se357,Se357,716 |
| 10 | − | − | a−b+ | − | + | + | OhB-secretor | O1B | FUT1 547delAG/FUT1 880delTT | Se357Se357,716 | |
| 11 | − | − | − | a−b+ | − | + | + | OhB-secretor | O1B | FUT1 547delAG/FUT1 658T | Se357Se357 |
| 12 | 1+ | 2+ | − | a−b+ | + | + | + | OhAB-secretor | AB | FUT1 547delAG/FUT1 649T | Se357Se |
| 13 | − | − | − | a−b+ | + | + | + | OhAB-secretor | AB | FUT1 547delAG/FUT1 35T, 423A | Se357Se357,385 |
| 14 | − | − | a−b+ | + | − | + | OhA-secretor | O1A | FUT1 880delTT/FUT1 880delTT | Se357,716Se357,716 | |
| 15 | − | − | − | a−b+ | + | − | + | OhA-secretor | O1A | FUT1 880delTT/FUT1 880delTT | Se357,716Se357,716 |
| 16 | − | − | − | a−b+ | − | + | + | OhB-secretor | O1B | FUT1 658T/FUT1 658T | Se357Se357 |
| 17 | 1+ | − | − | a−b+ | + | − | + | OhA-secretor | O1A | FUT1 649T/FUT1 293 | Se357Se357,385 |
−: absent; +: present; 1+ to 4+: present according to forward typing agglutination strength with monoclonal antisera;
ABO phenotypes were determined by adsorption and elution tests;
B/H substances were detected in the saliva sample of n. 410.
The G649T mutation causes the substitution Val217Phe. Mutations introducing Phe disturb the structure of the polypeptide chain by causing a difference in bulk between the normal and the mutated amino acid6. Val217 is highly conserved in FUT1 enzymes of different species (Figure 1). This may imply that Val217 is essential for the expression and/or activity of the FUT1 enzyme. It is, however, worth noting that AB antigens on red blood cells were detected by direct agglutination only when the FUT1 649T allele was found, perhaps suggesting that the V217F replacement in FUT1 649T affected the H enzyme less than the R220C substitution in FUT1 658T did. In the FUT1 35T, 423A allele the 35C>T substitution is a single nucleotide polymorphism in the Chinese population7, and the 423G>A mutation might lead to a deficiency of H by promoting the selective degradation of the corresponding mRNA8,9. A termination codon was previously reported at position 1412 except that the G>A mutation occurred at nucleotide 422. We speculate that the position around nucleotides 422–423 may be a hot spot for H gene mutation.
Figure 1.
Amino acid sequence alignment for FUT1 enzymes. The affected amino acid in the FUT1 649T allele is underlined.
In summary, two novel non-functional FUT1 alleles (FUT1 649T and FUT1 35T, 423A) were identified in para-Bombay individuals. The 423G>A mutation silences the H gene and the 649G>T mutation affects the efficiency of the encoded 2-α-fucosyltransferase through the amino acid substitution V217F.
Acknowledgements
This work was supported by grants from the Shanghai Municipal Natural Science Foundation (10ZR1428500) to C-XH and Youth Foundation of Shanghai Municipal Public Health Bureau (2009Y098) to C-XH.
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