METHOD FOR DETERMINING P1/P2 BLOOD TYPE AND DETECTION KIT THEREOF

Description

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a method for determining P₁/P₂ blood type and a detection kit thereof, and more particularly to a method with a single nucleotide polymorphism for determining P₁/P₂ blood type and a detection kit thereof.

2. Description of Related Art

In 1927, Austrian Biologist Karl Landsteiner and American immunologist Philip Levine discovered a new antigen from the rabbits transfused human blood during research on hemolytic disease of the newborns. These antigens can cause clotting of the red blood cells in some people while other groups of people are unaffected. They namely these two blood types as P₁ (+) and P₁ (−), later, the P1 (+)- and P1 (−) type are referred to as P1 and P2 type. This P₁/P₂ blood type belongs to the third system among the present 33 human blood type.

P₁/P₂ blood type is a system composed of glycolipid antigens; however the molecular genetic basis is still not clear. P₁ blood type and P₂ blood type are correlated to P₁ and P^k blood antigens expressed on the red blood cell. The P₁ and P^k are carbohydrate antigens and are synthesized via different biosynthetic routes, starting from a common glycosphingolipid precursor, lactosylceramide. The P₁ and P^k antigens are determined by the enzymatic activity of α-1,4-galactosyltransferase (hereinafter referred to as A4GALT). P₁/P₂ blood type is a common phenotypic polymorphism and the distribution of which varies among different human populations. The frequency of P₁ type in Caucasians and Africans are 80% and 90-95%, respectively, while the frequency of P₁ type is relatively low in Asians. The frequency of P₁ blood type is about 30% to 40% in Taiwan.

Using DNA genotype as a method for detecting blood type will be a trend in the future. For instance, Progenika Biopharma, S.A. (Spain) has developed a blood chip and such blood chip contains various genotype variations of different blood type. However, in the past, the molecular genetic mechanism of P₁/P₂ blood type has not been verified, such that the current DNA test including Progenika blood chip could not contain this common P₁/P₂ blood type polymorphism to perform DNA analysis. Therefore, there still is a need for determining the P₁/P₂ blood type.

Thuresson et al. (Thuresson B, Westman J S, Olsson M L, Identification of a novel A4GALT exon reveals the genetic basis of the P₁/P₂ histo-blood groups. Blood. 2011; 117(2): 678-687) has reported the association between SNP rs8138197 and P₁/P₂ phenotypes, and proposed a novel molecular model of the formation of P₁/P₂ blood group.

The present invention utilizes a genetic association study among different ethnic populations to prove single nucleotide polymorphism (hereinafter referred to as SNP) rs2143918 and rs5751348 in A4GALT gene are completely associated with P₁/P₂ blood type. The result of the present invention provides the molecular genetic mechanism of P₁/P₂ blood type and further provides a marker for determining P₁/P₂ blood type by using the genotypes of SNPs rs2143918 and rs5751348.

SUMMARY OF THE INVENTION

The present invention aims to conduct a pilot investigation among four ethnic groups, and the results from analysis of P₁ type of Africans and a Taiwanese family pedigree labeled M3 reveals that a genotype of SNP rs8138197 is not associated with P₁/P₂ blood type.

The present invention provides a method for determining P₁/P₂ blood type, comprising steps of: providing a biological sample of a subject; detecting a genotype for a single nucleotide polymorphism at rs2143918 or rs5751348 in A4GALT gene of the biological sample; and determining a phenotype of the subject based on the genotype.

In one embodiment of the present invention, the genotype of the single nucleotide polymorphism at rs2143918 is selected from the group consisting of T/T, T/G and GIG.

In one embodiment of the present invention, T/T or T/G genotype of the single nucleotide polymorphism at rs2143918 represents P₁ phenotype in the subject.

In one embodiment of the present invention, G/G genotype of the single nucleotide polymorphism at rs2143918 represents P₂ phenotype in the subject.

In one embodiment of the present invention, the genotype of the single nucleotide polymorphism at rs5751348 is selected from the group consisting of G/G, G/T and T/T.

In one embodiment of the present invention, G/G or G/T genotype of the single nucleotide polymorphism at rs5751348 represents P₁ phenotype in the subject.

In one embodiment of the present invention, T/T genotype of the single nucleotide polymorphism at rs5751348 represents P₂ phenotype in the subject.

In one embodiment of the present invention, the biological samples are blood or saliva.

In one embodiment of the present invention, a nucleic acid in the biological sample is determined by polymerase chain reaction (hereinafter referred to as PCR).

In one embodiment of the present invention, a primer pair of SEQ ID Nos: 1 and 2 are used in the PCR for determining the nucleic acids of the biological samples.

In one embodiment of the present invention, a primer pair of SEQ ID Nos: 3 and 4 are used in the PCR for determining the nucleic acids of the biological samples.

The present invention also provides a detection kit for determining P₁/P₂ blood type, comprising: a primer pair for determining a genotype of a single nucleotide polymorphism at rs2143918 or rs5751348 of A4GALT gene in a nucleic acid sample of a subject.

In one embodiment of the present invention, the primer pair has sequences of SEQ ID Nos: 1 and 2.

In one embodiment of the present invention, the primer pair has sequences of SEQ ID Nos: 3 and 4.

In one embodiment of the present invention, the genotype of the single nucleotide polymorphism at rs2143918 is selected from the group consisting of T/T, T/G and GIG.

In one embodiment of the present invention, T/T or T/G genotype of the single nucleotide polymorphism at rs2143918 represents P₁ phenotype in the subject.

In one embodiment of the present invention, G/G genotype of the single nucleotide polymorphism at rs2143918 represents P₂ phenotype in the subject.

In one embodiment of the present invention, the genotype of the single nucleotide polymorphism at rs5751348 is selected from the group consisting of G/G, G/T and T/T.

In one embodiment of the present invention, G/G or G/T genotype of the single nucleotide polymorphism at rs5751348 represents P₁ phenotype in the subject. In one embodiment of the present invention, T/T genotype of the single nucleotide polymorphism at rs5751348 represents P₂ phenotype in the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows distributions of the most likely haplotype pairs in P₁/P₂ individuals across 4 ethnic populations; and

FIG. 2 shows P₁/P₂ phenotypes and the most likely haplotype pairs of M3 family.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following specific examples are used for illustrating the present invention. A person skilled in the art can easily conceive the other advantages and effects of the present invention. The present invention can also be implemented by different specific cases be enacted or application, the details of the instructions can also be based on different perspectives and applications in various modifications and changes do not depart from the spirit of the creation.

The present invention provides a method for determining P₁/P₂ blood type, comprising steps of providing a biological sample of a subject; determining a genotype for a single nucleotide polymorphism (SNP) of A4GALT gene from the biological samples, wherein the SNP is located at SNP rs2143918 or SNP rs5751348; and discriminating the subject has P₁ or P₂ phenotype according to the genotype of SNP rs2143918 or SNP rs5751348.

The present invention also provides a kit for determining P₁/P₂ blood type, comprising a PCR primer pair, which is used to amplify nucleic acid fragments of a single nucleotide polymorphism of A4GALT gene by using the nucleic acid of the subject to be determined as a template. The single nucleotide polymorphism is located at SNP rs2143918 or SNP rs5751348, wherein the PCR primer pair can perform an extension reaction of the primer so as to determine the genotype of SNP rs2143918 or SNP rs5751348 and discriminate the subject has P₁ or P₂ phenotype.

Example 1

Method for Determining P₁/P₂ Phenotype

1. Sample Preparation and P₁/P₂ Phenotype

A total of 338 non-related subjects across four ethnic populations, including 227 Taiwanese, 32 Indians, 46 Caucasians, and 33 Africans (Blacks, including 2 African-Americans) were enrolled in the present invention. Peripheral blood samples were collected from each subject. The genomic DNAs were purified from the peripheral blood samples using QIAamp DNA Blood Mini Kit (Qiagen GmbH, Hilden, Germany), and then the P₁/P₂ phenotype of each sample was determined by a standard hemagglutination test using monoclonal anti-P1 reagent (Immucor Inc., Houston, Tex.).

2. Analysis of the Single Nucleotide Polymorphism

57 DNA fragments were amplified by polymerase chain reaction (hereinafter referred to as PCR), and these encompassed all SNP sites distribute from −7.0 kb to +17.3 kb of the A4GALT gene. The sequences of PCR primer pairs locations thereof are listed in Table 1. Then, the nucleotides at the SNP sites were determined by direct sequencing the amplified DNA fragments using Sanger's method.

TABLE 1
PCR primers for SNP analysis

primer	sequence(5′→3′)	locationa

930R	GTGCATGCCTGTAATCACAG	−7085
930F	GTGTCACAGTGACTGCTGTG	−6765
217F	ATTGACAAGGGCGAGCCAC	−6800
217R	AAGCAAACACTCCTCCCTCC	−6618
928R	ATGGGTGGAGTGGAAAGTG	−6564
928F	AAGACAGAGTCTCCCTGTC	−6236
931F	TGTGGCTCTGACTACTGAG	−5713
931R	ACACACCTAGAAGCCATCC	−5983
678F	GCAGTGGCTCCCTTGACATAAGTAACTCC	−5265
677R	TTATGTCAAGGGAGCCACTG	−5285
677F	GTCAACATCTAGACCACTGC	−4950
584F	ACCAGAAAGTTGGAGCTAGG	−5129
584R	AACTGGAGCTGGTCATCTGG	−4521
331R	ATCTGCTTTGGAGCATGGGC	−4834
331F	CTGGGATTACAGGCGTGAGC	−4437
853R	CAGCTCCAGTTTACTGATGG	−4532
853F	GTCAGGCTGGTCTTGATCAC	−4056
690F1	CAAGACCAGCCTGACCAACG	−4070
690R2	GGTTGAGCACACAGGCTCTG	−3480
628R2	CTCTCAGAGCCTGTGTGCTC	−3503
628F1	AGCCCCAGCAGCCTTTGAG	−3193
644F	AATGATCCTACTGCCTCAGC	−3078
676F	TATTCAAGGCAGGCCAGCAC	−2182
676R	TCGCTCTGTCGCCATACTGG	−2684
676F	TATTCAAGGCAGGCCAGCAC	−2182
795F	CTCAGCCTCCCACAGTGTGG	−1607
795R	GACGGAGTCTCACTCTGTCG	−2138
326R	GTTGCCTTCTAGTCTCTGAG	−1783
326F	GAAGAGACTCACTCTGTTACC	−1383
035R	TTAGCCATGGTTGTGCATGCCTGTAGTCC	−1524
035F	AGGCCAGGCAAGCACTCACGCCTGTAATC	−1056
369R	ATCTCAGGTGACCTGCTTGC	−1125
369F	GGGTGACTTCTTGAGTGCTG	−680
898R	ACCCATTGAGTGCCAGGCTC	−799
898F	ACCGTGCTAAGGGCATTGCC	−373
A4GTFa	CACCAGGACTGTGACATGCTGGAAACATGG	−626
PKRL	CTGCCTCGAACCCGGCTTCT	+430
469F3	GGAGAAGCCGGGTTCGAGGCAGGCTCTGC	+409
469R4	GCTTTAACCCACTCAGGGCCAGAGGCTCAGC	+939
845R	GTGAGGATTTGGACCTGCCC	+596
927F	ACTACAGTGAGCTGCCACAG	+1472
573R	CAACGTCGAGTGATTGTTCC	+1002
573F	GGTTGCAGCTATGGAAACAC	+1303
927R	ATAGCAGAGTGCGGATGGAG	+1188
927F	ACTACAGTGAGCTGCCACAG	+1472
672R	TGTAGTCTCAAACTGCTAGG	+1467
672F	CAATCCTGCTCCACACTGG	+1783
920R	GCTGCTAGGTTAATGGGTCC	+1710
920F	TTCAGGAAGCACCTGCTAGG	+2186
522R	AAGTGCACCTCCTCTCACTC	+1985
522F	CTCACTGCACTCTCTGCCTC	+2538
175F	TCTAGCTTTCCCATCAGC	+3239
926R	GACAGAGAGATAGAGAGGAG	+3132
926F	TATGTGGACACTGGTCTGG	+3800
991R	CCTTCCAGACCAGTGTCCAC	+3778
991F	AACAGAGCATGGTGGCTGAG	+4136
181R	CCCTGATCATCTGTGACC	+4018
181F	AAAAGCTCACTGTCAGGC	+4501
181Fs	CTATTAAACCACACAGCTCC	+4462
860F	ATTAGCAGGGAATGGTGG	+5307
183F	GAGTGCACTGGTGCAATCATG	+5189
183R	CTTCATACCATAAATTCCAAG	+5674
894R	GTCCTTCAGCAGCTCTCAAG	+5750
892R	TGGCTCCCTCCTGTAATTC	+6553
176F	GGAGTGCGGTGGTATGAG	+6627
858R	ATCTGGCAAAACCCCACCTC	+7245
245F	CCTAACCTCAGGTGATCCACC	+7267
245R	CTCCCCCAGCATCACCTAC	+7915
718F	CCCCTTCTCAGGCAGTATCC	+7843
718R	CACCTTCGCTCTGGACAC	+8550
291F	TTGCATCTCTGGGATCTCTG	+8429
291R	AGTACCTGACTACTTGCCAG	+8760
888F	TGAGAAGCCAGCCCCACC	+8637
888R	CCGAGTCTCGCTCTGTTGCC	+9215
185F	GAGGTTGTAGTGAGCCAAG	+9153
185R	CTGTGAGCAAACAGGCATG	+9533
306F	TGTGGTAGGATCTGTGCTGG	+9272
306R	TAGCTACTCAGGAGGCTGAG	+9942
887R	TGCAGCAAGCCAAGATGGTG	+10216
887F	AGCCTCCTGAGTAGCTAGGG	+9926
081F	CATCTTGGCTTGCTGCAGCC	+10200
081R	ACAGTTTCATACCTGGGCAC	+10779
347R	GCTCAAGCAATCTGGCTGCC	+10368
347F	ACACAGAAGCCAGGAACCAG	+10825
793F	TGGTGGTGGCAGCATCTGTG	+10719
793R	GGCAGGTGGATCACTTGAGG	+11293
653F	GATTACAGTTGTGCCCCACC	+11179
479R	CAAGTACATGATCCTCCCAC	+11697
616F	TGGGAGCCTAGGAATTCAAG	+11563
616R	GGATCCCAGAAGACATAGC	+11988
993F	GTAGATTAGCTATGTCTTCTGGG	+11962
993R	ATCGCGCCATTGCATTCTAG	+12521
227F	CTGGCACTGCAGGTACACAC	+12422
227R	ACACAGAAACATGGCTGGTC	+13012
634F	ATTGTTACATACACTGGTGG	+13082
634R	ACTCTCTACCCTAGTGATAG	+13708
280F	GCACAGTATCTATCACTAGG	+13680
280R	GAGACATACCTTAAACGAAG	+14387
712R	ATCCTGGCTAACACGGTG	+14693
036F	TCAGCCTCCCGAGTAACTG	+14594
036R	TCGCTTGAACCTGGGAAGTG	+14884
903F	CCTCGGCCTATTAAAGTGC	+15079
903R	TGAGGAGCACAAATACTCGC	+15590
557F	AGCGAGTATTTGTGCTCCTC	+15570
557R	AAGACCCCAGAAAAGGCC	+15934
881F	AAAGGCTCCCTCCTCTGTTG	+15731
881R	GAAGCCAGGAATCAAGCAGG	+16230
436F	TCTGTTTGTAACGTCCACCC	+16177
436R	CCTTCACTGCTTTGTCCATC	+16889
193F	GCAGGGTTTGGAAGCTCTGG	+16787
193R	TGTGCCCGGCCTGCAATAAG	+17308
aTranscription initiation nucleotide of A4GALT exon 1 of RefSeq Transcript

3. Primer Pairs for Amplification and Identification of the Genotypes of rs2143918 and rs5751348

• • The forward primer for amplification and identification the genotype of rs2143918 has the sequence of SEQ ID No. 1, while the reverse primer has the sequence of SEQ ID No. 2.

	AAGTGCACCTCCTCTCACTC	(SEQ ID NO. 1)
	TCTAGCTTTCCCATCAGC	(SEQ ID NO. 2)

• • The forward primer for amplification and identification the genotype of rs5751347 has the sequence of SEQ ID No. 3, while the reverse primer has the sequence of SEQ ID No. 4.

	TCACGAGCATTCCTCATC	(SEQ ID NO: 3)
	CTCCTCTCTATCTCTCTGTC	(SEQ ID NO: 4)

4. Result

1) A Pilot Investigation for Identification of SNPs in A4GALT Gene Associated with the P₁/P₂ Phenotype.

In order to explore the molecular genetic basis of the P₁/P₂ blood groups, the present invention conducted a pilot investigation, which involved the detailed and stepwise screening of SNPs in A4GALT gene from four Taiwanese with the P₁ phenotype and four Taiwanese with the P₂ phenotype. The screening of SNP started from the 5′ promoter region and then extended stepwise to the 5′ and 3′ region of the gene to identify any polymorphic nucleotide positions that may be related to P₁/P₂ phenotypes. The PCR, which was used to amplify the DNA fragments encompassing each SNP in the A4GALT gene, has been recorded in the SNP database of the National Center for Biotechnology Information (NCBI). Eventually, 57 DNA fragments were amplified respectively by PCR. The amplified DNA fragments contain a total of 416 different SNP sites which were distributed over 24.3 kb region of the A4GALT gene including 7.0 kb of the 5′ promoter region, exon 1 (74 bp), and 17.3 kb of the 5′ part of intron 1. The nucleotides at each SNP over the eight samples were determined and the results are shown in Table 2. These show that 11 SNP sits of 8 subjects exhibited an association with the P₁/P₂ phenotype. These 11 SNPs are distributed from the +1.3 kb to +11.5 kb region in the intron 1 of the A4GALT gene, and they are denoted as SNP1 to SNP11 from 5′ to 3′.

TABLE 2
SNPs in the A4GALT gene that show an association with the
P1/P2 blood group phenotypes in the pilot investigation

	SNP1	SNP2	SNP3	SNP4	SNPS	SNP6

Locationa	+1352	+2326	+2414	+2837	+2857	+3084
SNPID	rs67162484b	rs8138197	rs10713068	rs2143919	rs2143918	rs5751348
	rs66781836b
Nucleotide	AGAA/—	C/T	C/—	C/G	T/G	G/T
Polymorphismsc

Phenotype	Sample	Genotypec

P1	M8	+/+	C/C	C/C	C/C	T/T	G/G
	M5	+/+	C/C	C/C	C/C	T/T	G/G
	M33	+/−	C/T	C/—	C/G	T/G	G/T
	HSC	+/−	C/T	C/—	C/G	T/G	G/T
P2	M12	−/−	T/T	—/—	G/G	G/G	T/T
	T9	−/−	T/T	—/—	G/G	G/G	T/T
	J1	−/−	T/T	—/—	G/G	G/G	T/T
	Y1	−/−	T/T	—/—	G/G	G/G	T/T

	SNP7	SNP8	SNP9	SNP10	SNP11

	Locationa	+3443	+5012	+6762	+7038	+11507
	SNPID	rs9623671	rs111626860	rs5758891	rs8139674	rs66463955
	Nucleotide	G/A	ACTCCA/—	T/C	G/T	G/—
	Polymorphismsc

	Phenotype	Sample	Genotypec

	P1	M8	G/G	+/+	T/T	G/G	G/G
		M5	G/G	+/+	T/T	G/G	G/G
		M33	G/A	+/−	T/C	G/T	G/—
		HSC	G/A	+/−	T/C	G/T	G/—
	P2	M12	A/A	−/−	C/C	T/T	—/—
		T9	A/A	−/−	C/C	T/T	—/—
		J1	A/A	−/−	C/C	T/T	—/—
		Y1	A/A	−/−	C/C	T/T	—/—
	aTranscription initiation nucleotide of A4GALT exon 1 (74 bp) of RefSeq Transcript NM_017436.4 in the NCBI database as +1
	bSNPs rs67162484 and rs66781836 yield identical sequence polymorphisms.
	c“+” and “−” indicate the presence and the absence of the nucleotide(s), respectively.

2) SNPs rs2143918 and rs5751348 Show a Definite Association with the P₁/P₂ Phenotypes Across Different Ethnic Populations.

In order to verify the association of these 11 SNPs (SNP1 to SNP11) and the phenotypes of P₁/P₂ phenotypes, a large scale association study using various ethnic populations was carried out There were 227 Taiwanese (including 8 subjects analyzed in the previous pilot study), 32 Indians, 46 Caucasians and 33 Africans (Black) enrolled in this study. The P₁/P₂ phenotypes and the genotypes at SNP1 to SNP11 of these 338 subjects were determined. The distributions of the genotypes at the 11 SNPs in the P₁ and P₂ individuals of each population are shown in Table 3. The haplotypes of the 11 SNPs in the 4 populations were reconstructed and the most likely haplotype was assigned for each subject using PHASE program (version 2.1). FIG. 1 shows the distribution of the most likely haplotype pairs in the P₁ and P₂ subjects among each ethnic population.

The two haplotypes identified by using 8 Taiwanese in the pilot study were found to be major haplotypes present in the wider study and were also associated with the P₁ and P₂ phenotypes across all four populations. In addition to the two haplotypes found in the four populations, haplotypes with recombination between the 11 SNPs were identified and were found to be more frequent in Caucasians and Africans than in Taiwanese and Indians. The correlation of SNP1, SNP3, SNP4, and SNP7 to SNP11 could be easily excluded while multiple cases distributed across the different ethnic populations were identified as recombinant haplotypes between these SNPs, as shown in Table 3 and FIG. 1. Thus, it creates inconsistencies in the genotype-phenotype correlation observed across the majority of other enrolled subjects.

Among the 338 enrolled subjects, only one subject (P₁ Africans, denoted as ** in Table 3 and * in FIG. 1) showed the genotype-phenotype inconsistency of SNP rs8138197. In the previous analysis for the P₁ and P₂ phenotype from 208 Swedish subjects, there was one case found the genotype-phenotype inconsistency of SNP rs8138197. In addition to such P₁ African case, a P₁ Taiwanese (denoted as * in FIG. 1) labeled as M3 family was pointed out as having an allele which is involved recombination between SNP3 and SNP4. As shown in FIG. 2, the M3 family was then enrolled to perform the pedigree analysis and the recombinant allele identified in M3 was found to be present in M3's father and brother who were labeled with the P₂ phenotype. The results obtained from this pedigree analysis suggest that SNP2 (i.e. rs8138197) is nor involved in defining the P₁ and P₂ phenotypes.

When the above results were taken together, the SNPs that show definite association with the P₁/P₂ phenotypes could be narrowed down to two, namely SNP5 (rs2143918) and SNP6 (rs5751348), using this pilot study across different ethnic populations. The genotypes at the SNPs distributed along the +1.3 kb through +3.7 kb region of the A4GALT gene, which encompasses the SNP1 to SNP7 sites, of the enrolled 338 subjects were thoroughly examined, and no other SNP was found an association with the P₁ and P₂ phenotypes. The genotypes at SNPs rs2143918 and rs5751348 were consistently associated with the P₁ and P₂ phenotypic polymorphisms while 338 subjects across the four ethnic populations have considerable genetic distances.

TABLE 3
SNPs associated with the P1/P2 blood group phenotypes and distributions of
the SNP genotypes in the P1 and P2 subjects of various ethnic populations

	SNP1	SNP2	SNP3	SNP4	SNP5	SNP6

Locationa	+1352	+2326	+2414	+2837	+2857	+3084
SNP ID	rs67162484b	rs8138197	rs10713068	rs2143919	rs2143918	rs5751348
	rs66781836b
Nucleotide	AGAA	C	C	C	T	G
Polymorphismsc	(▪)/—(□)	(▪)/T(□)	(▪)/—(□)	(▪)/G(□)	(▪)/G(□)	(▪)/T(□)

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Taiwanese	P1 (n = 63)	5	58	0	5	58	0	5	58	0	4	59	0	4	59	0	4	59	0
	P2 (n = 164)	0	0	164	0	0	164	0	0	164	0	0	164	0	0	164	0	0	164
Indian	P1 (n = 20)	4	16	0	4	16	0	4	15	1	4	15	1	4	16	0	4	16	0
	P2 (n = 12)	0	0	12	0	0	12	0	0	12	0	0	12	0	0	12	0	0	12
Caucasian	P1 (n = 32)	11	21	0	12	20	0	9	17	6	9	17	6	12	20	0	12	20	0
	P2 (n = 14)	0	0	14	0	0	14	0	0	14	0	0	14	0	0	14	0	0	14
Africand	P1 (n = 31)	11	17	3	13	17	1*	5	16	10	10	20	1	13	18	0	13	18	0
	P2 (n = 2)	0	0	2	0	0	2	0	0	2	0	0	2	0	0	2	0	0	2
Swedishe	P1 (n = 151)				57	93	1
	P2 (n = 57)				0	0	57

	SNP7	SNP8	SNP9	SNP10	SNP11

	Locationa	+3443	+5012	+6762	+7038	+11507
	SNP ID	rs9623671	rs111626860	rs5758891	rs8139674	rs66463955
	Nucleotide	G	ACTCCA	T	G	G
	Polymorphismsc	(▪)/A(□)	(▪)/—(□)	(▪)/C(□)	(▪)/T(□)	(▪)/—(□)

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	Taiwanese	P1 (n = 63)	4	59	0	4	59	0	4	59	0	4	59	0	4	59	0
		P2 (n = 164)	0	0	164	0	0	164	0	1	163	0	5	159	0	2	162
	Indian	P1 (n = 20)	4	15	1	4	15	1	4	15	1	6	13	1	4	15	1
		P2 (n = 12)	0	0	12	0	0	12	0	0	12	0	0	12	0	0	12
	Caucasian	P1 (n = 32)	9	17	6	9	17	6	8	18	6	8	20	6	8	18	6
		P2 (n = 14)	0	0	14	0	0	14	0	0	14	0	2	10	0	1	13
	Africand	P1 (n = 31)	10	20	1	9	21	1	11	20	0	18	13	0	12	19	0
		P2 (n = 2)	0	0	2	0	0	2	0	0	2	0	0	2	0	0	2
	Swedishe	P1 (n = 151)
		P2 (n = 57)
	The numbers of the individuals that show genotype-phenotype inconsistency compared to the majority of others are typed in red.
	aTranscription initiation nucleotide of A4GALT exon 1 (74 bp) of RefSeq Transcript NM_017436.4 in the NCBI database as +1
	bSNPs rs67162484 and rs66781836 yield identical sequence polymorphisms.
	cThe two different nucleotide genotypes at each SNP are denoted by ▪ and □.
	dAn asterisk indicates a P1 African who shows a genotype-phenotype discrepancy at SNP2.
	eRef. 28. The association dataset for the other SNPs in this population is not available.

By a pilot investigation and stepwise SNP screening analysis in A4GALT gene followed by an association study using four ethnic populations, the present invention confirms that SNPs rs2143918 and rs5751348 are correlated with the P1 and P2 blood type. Accordingly, the T/T and T/G genotypes at the SNP rs2143918 are associated with P₁ phenotype while the G/G genotype at the SNP rs2143918 is associated with P₂ phenotype. The G/G and G/T genotypes at the SNP rs5751348 are associated with P₁ phenotype while the T/T genotype at the SNP rs5751348 is associated with P₂ phenotype.

While some of the embodiments of the present invention have been described in detail in the above, it is, however, possible for those of ordinary skill in the art to make various modifications and changes to the particular embodiments shown without substantially departing from the teaching and advantages of the present invention. Such modifications and changes are encompassed in the spirit and scope of the present invention as set forth in the appended claim.