Red Blood Cell Genotyping in China

Y. L. Ji1 & C. E. van der Schoot2 1Institute of Clinical Blood Transfusion, Guangzhou Blood Center, Guangzhou, China 2Sanquin Research and Landsteiner Laboratory, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands

The common antigens of ABO, Rh, MNS, Duffy, Kidd, Diego, Yt and Dombrock blood group systems have a polymorphic distribution in the Chinese population. The Mur antigen and anti-Mur are more common in the Chinese Han and some minority groups in the southern region of China. Except for the common ABO alleles (A101, A102, B101, O01 and O02), more than 100 new alleles have been reported in the Chinese population, in which the effect of many critical mutations on the variant expression of A and B antigens still needs to be investigated. For RHD genotyping, only 6% of Chinese D+ people were heterozygous for the RHD gene with Dd genotype. Compared with the complete deletion of the RHD gene accounting for D- phenotype in the Caucasian population, 20–40% of the Chinese individuals with D- phenotype carry RHD variant alleles. Among them, ‘Asian type’ DEL allele (RHD*DEL1 defined by 1227G>A, Lys409Lys) is the most common variant and is classified as D-, and it can only be identified by absorption/elution testing, which is not routinely conducted. Several studies indicated that the Chinese pregnant women with ‘Asian type’ DEL allele were not immu- nized by the incompatible pregnancy, which suggested that the ‘Asian type’ DEL recipient could receive D+ blood safely. Weak D type 15 and DVI type 3 are the most common RHD variant alleles, accounting for more than 70% of RhD variants in the Chinese population. In addition, the genotyping analysis for the rare

null phenotypes, including the para-Bombay, Jk(a-b-), Lu(a-b-), p, Rhnull and KELnull phenotypes, are also summarized. Key words: Red blood cell, blood group, genotyping, the Chinese population

The distribution study of blood group systems most of blood group antigens has been determined. The in the Chinese population distribution of the antigens which could not be tested by the serological methods (Yt [2–4], Scianna [3, 4], Dom- In the last century (especially in 1980s), the serological brock [2, 4], Colton [3, 4], LW [4, 5], Gerbich [6], Cromer typing for the common red cell antigens of ABO, MNS, [6], Knops [7], Indian [6], Ok [6], Chido-Rodgers [6] and Rh, Kidd, Duffy and Lewis blood groups has been con- Gil [6]) were analysed using the genotyping methods ducted on a large scale in the Chinese Han and many (majority by PCR-SSP) in China. minority groups in the mainland of China [1]. The distri- In summary, the common antigens of ABO, Rh, MNS, bution of the antigens in other blood group systems could Duffy, Kidd, Diego, Yt and Dombrock blood groups have not be determined in the Chinese population because of a polymorphic distribution in the Chinese population. In the lack of speciﬁc antibodies. In the 21st century, with contrast to the Caucasians, the antigens of the Lutheran the development of the blood group molecular genetics, (Lua), Kell (K), Colton (Cob), LW (LWb) and Knops (Knb) the majority of the genes encoding the blood group sys- blood group systems are not found in the Chinese popula- tems have been cloned and the molecular basis for the tion. In contrast, Dia antigen of Diego blood group and Mur and Sta antigens of MNS blood group are found with Á – – Á – Á Correspondence: Y. L. Ji, 31 LuYuan road, Guangzhou Blood Center, a higher frequency of 4 54 7% [4, 8 12], 0 5 24 7% Guangzhou, 510095, China. [13–21] and 2Á6% (26/1004, Z Wang, YN Liang, JZ Wen, E-mail: [email protected] L Wei, GP Luo, YL Ji our unpublished data), respectively,

55 56 Y. L. Ji & C. E. van der Schoot in the population from the mainland of China. These expression analysis in K562 cells in vitro confirmed its distribution studies indicate that antigen positive individ- effect on the weak expression of A antigen [29]. The uals following transfusion are different in the Chinese molecular basis for the rare A3, Ax, Aint, Am and Aw population compared with the Caucasians, which could phenotypes are more diverse without the dominant alleles produce different clinically significant alloantibodies. [27, 30, 31]. Therefore, the potential neglection of alloantibodies, such Besides the reported B subgroup and variant alleles, 4 as anti-Dia, anti-Mur and anti-Sta, should be noted when B(A) alleles (B(A)02, B(A)04 to B(A)06), 9 B3 alleles screening reagent cells designed for the Caucasian (B303 to B305, B307, B309, B310 and B313 to B315), populations are used for routine antibody screening in 6 Bel alleles (Bel03, Bel05 to Bel09), 1 Bm alleles the Chinese population. (Bm03), 16 Bw alleles (Bw11, Bw12, Bw14, Bw22, Bw24, Bw27, Bw28, Bw30, Bw31 and Bw33 to Bw39), ABO blood group genotyping 12 Bx alleles (Bx02 to Bx13) and 5 CisAB alleles (cis- AB01, cis-AB05 to cis-AB08) were firstly identified in ABO blood groups are genetically complex with more the Chinese population. B3 is the most common B sub- than 360 alleles described in NCBI dbRBC(http:// group in the Chinese population with a frequency of www.ncbi.nlm.nih.gov/projects/gv/mhc/xslcgi.cgi?cmd=bg 1:900 [32]. For the most common B303 (intron mut/systems_alleles&system=abo) to date. More than one- 3+5G>A) [27, 33, 34] and B305 (425T>C, Met142Thr) third (38Á4%, 140/365) of known ABO alleles were firstly alleles found in the Chinese [27, 35] and Korean popula- reported in the Chinese population. The five common tions [36], the expression studies in vitro were conducted ABO alleles (A101, A102, B101, O01 and O02), described in K562 cells [33] and Hela cells [36], respectively. The in other ethnic groups, are also most frequent in the Chi- decreased B glycosyltransferase activity and variant nese population with an allele frequency of 1Á6%, 19Á8%, expression of B antigen in vitro expression studies con- 20Á6%, 33Á7% and 22Á3%, respectively [22]. A101 is more firmed their effect on the B variant expression. Bel03 common than A102 in the Caucasian population, but it is with the unique mutation (502C>T, Arg168Trp) is the just opposite in Asian populations including the Chinese common allele in the probands with Bel phenotype from Han individuals. O01 and O02 alleles are distributed with Taiwan [37, 38] and the mainland of China [27], while an allele frequency of 47% and 49Á5%, respectively, the different amino acid change in the same position which accounts for more than 97% of Chinese individuals (502C>G, Arg168Gly) is reported to relate with Bw phe- with O phenotype [23]. Besides, at least ten novel rare O notype in one Chinese proband [39]. B(A)02 (700C>G, variant alleles with inactive products have been identified Pro234Ala) and B(A)04 (640A>G, Met214Val) alleles are (O09, O10, O12, O13, O56, O61, O74, O76, O77 and the common alleles accounting for B(A) phenotype in O80) in the Chinese population. Among them, two alleles the Chinese probands [27]. Cis-AB01 (803G>C, without an O-specific 261delG mutation but with extra Gly268Ala) allele is the dominating allele for the Chi- deletion (O74: 505delCAG, Gln169del [24]) or missense nese individuals with cis-AB phenotype [27]. Neverthe- mutation (O77: 563G>A, Arg188His, submitted to Gen- less, the molecular basis for the Bw phenotype is more Bank with accession number KM892859) were found on diverse without the dominating alleles [27]. the B101 backbone. DNA-based analysis for ABO genotyping has not been Except for the known A subgroup alleles, 14 A2 alleles widely used as serological typing is fast and reliable for (A206, A207, A208, A209, A210, A211, A213, A217, routine typing, and due to the complexity of setting up A218, A219, A220, A221, A222 and A223), 8 A3 alleles the molecular platform for so many alleles identified in (A303, A305, A306, A307, A308, A309, A311 and ABO blood group system. In the current situation, only A312), 9 Ael alleles (Ael04 to Ael12), 2 Aint alleles (Ain- few high-throughput genotyping platforms conduct ABO t01and Aint02), 3 Am alleles (Am02, Am03 and Am04), genotyping [40]. However, molecular methods could be 5 Aw alleles (Aw12, Aw30, Aw35, Aw36 and Aw37) and very useful to resolve the discrepancies for ABO serologi- 18 Ax alleles (Ax09, Ax10 to Ax26) have been firstly cal forward and back typing due to a genetic variation, reported in the Chinese population. Compared with the especially in the reference laboratory. It is often difficult common distribution of A2 phenotype (20%) in the Cau- to give an exact name to A or B variants (such as A3, Ax casian population, A2 is rare in the Chinese population, and Ael) using the serological testing for it is not very with a frequency of 0–1Á19% in the Chinese Han [25, 26]. standardized and depends very much on reagents used. In A205, A201 and A208 alleles are the most common alle- this situation, genotyping analysis could provide a con- les accounting for the A2 phenotype [25, 27]. In Tai- clusive result for the samples through matching the well- wanese, Ael04 is the common Ael allele, which is found defined ABO alleles in dbRBC without having to perform in all six probands with Ael or AelB phenotype [28] and laborious family studies.

RHD genotyping Compared with the African population, around 20-40% Chinese individuals with D- phenotype carry RHD variant RHD genotyping in D+ individuals alleles [47, 48] and most carry the ‘Asian type’ DEL allele (RHD*DEL1 defined by 1227G>A, Lys409Lys). In the Serologically typed RhD-positive individuals account for Chinese truly D- population, the most common non- Á more than 99 6% of the Chinese population. The sequence functional RHD alleles are RHD*01N.03 (hybrid + of the RHD gene in Chinese D individuals is in complete RHD*D-CE(2–9)-D) and RHD*01N.16 (RHD*711delC), concordance with other populations. RHD zygosity analy- whereby there is no D antigen expression [43, 45, 46, 49, + sis in Chinese D individuals shows that only 6% have a 50]. Besides these, other rare non-functional RHD alleles Dd genotype, while others are DD genotype (our unpub- have also been reported in few Chinese probands with lished data analysed by blood group multiplex ligation- truly D- phenotype (Table 1). dependent probe amplification assay [41], n = 200). Based on this data, a truly D- Chinese pregnant women with a D+ husband (with 6% chance to be heterozygous Dd) has DEL genotyping only a 3% of probability to have a D- baby. Considering Á – that 17Á6–30% [42–48] of serologically D- pregnant Chi- About 17 6 30% Chinese individuals with a serological nese women are actually ‘Asian Type’ DEL, they do not D- phenotype are actually DEL individuals with a Ce (r’) produce anti-D. Thus, the proportion of Chinese D- haplotype in most of cases. Using classical serology typ- women who require anti-D prophylaxis during pregnancy ing methods, DEL could only be identified by the absorp- – is markedly lower than for Caucasian D- women. tion elution testing for a few numbers of D antigens expressions on the surface of red cells. Consequently, the very common DEL individuals have been typed as D- D- phenotype with non-functional RHD alleles individual for a long time in China due to lack of routine The D- phenotype is rare in the Chinese population use of the absorption–elution testing. (3–4%), although it is very common in Caucasians (15– In recent years, the molecular genetic background and 17%) and less common in African (3–5%). The D- pheno- mechanistic studies of DEL have been investigated sys- type results from the deletion of the complete RHD gene tematically in the Chinese population. Two comprehensive in most of Caucasian D- individuals, whereas it is due to genetic studies with a large number of samples revealed a 37-bp insertion in the RHD gene and a consequent pre- that 95Á4% (436/457) Chinese individuals with a DEL phe- mature stop codon in 66% of the African D- individuals. notype resulted from the ‘Asian type’ DEL mutation

Table 1 Non-functional RHD alleles identiﬁed in the Chinese D- individuals

Shenzhen Shanghai Handan Sichuan [43, 125] Jinan [45] [49, 50] Xi’an [46] [126] [127] Total Non-functional RHD alleles (n = 13) (n = 6) (n = 130) (n = 184) (n = 21) (n = 15) (n = 369)

1 RHD*D-CE(2-9)-D 8 5 109 163 20 13 318 (86Á2%) 2 RHD*711delC 1 – 12 19 1 1 34 (9Á2%) 3 RHD*D-CE(2-10) 1 – 1 ––– 2(0Á5%) 4 RHD*D-CE(4-9)-D –– 2 ––– 2(0Á5%) 5 RHD*78delC –– – 1 –– 1(0Á3%) 6 RHD*270A 1 –––––1(0Á3%) 7 RHD*325delA –– 1 ––– 1(0Á3%) 8 RHD*520A, 1080-1089delTCCAGGTCCT –– – 1 –– 1(0Á3%) 9 RHD*933A – 1 ––––1(0Á3%) 10 RHD*970delCGC, 976delTCCATCATGGGCTACA –– 1 ––– 1(0Á3%) 11 RHD*IVS2+1G>A - – 1 ––– 1(0Á3%) 12 RHD*IVS2-1G>A 1 –– 1(0Á3%) 13 RHD*IVS6+1-4delGTAA, 904-905insGGCTT 1 –––––1(0Á3%) 14 RHD*D-CE(2-7)-D - – 1 ––– 1(0Á3%) 15 RHD*D-CE(3-9)-D –– 1 ––– 1(0Á3%) 16 RHD*D-CE(8-9)-D –– – ––11(0Á3%) 17 RHD*CE-D(6)-CE 1 –––––1(0Á3%)

(1227G>A, Lys409Lys) in exon 9 of the RHD gene, while and rapid genotyping method, such as specific sequence the remainder of 4Á6% (21/457) of DEL individuals carried primer–polymerase chain reaction (SSP-PCR) [48] or real- the rare RHD variant alleles [RHD*3A (n = 12), RHD*28T time PCR [54], rather than the classical absorption–elution (n = 1), RHD*53C (n = 1), RHD*251C (n = 1), RHD*410A testing. (n = 1), RHD*D-CE(4-9)-D (n = 2), RHD*D-CE(2-5)-D (n = 2) and one suspected RHD*D(1-9)-CE hybrid allele D variants genotyping (n = 1)] [42, 51]. Additionally, a series of studies also confirmed that ‘Asian type’ DEL was highly prevalent in In the Chinese population, D variants are rare compared the Chinese DEL individuals [43–47]. The aberrant splic- with the Caucasian population (around 1% [55]) and some ing resulting from this synonymous mutation, including specific ethnic groups, such as in the Indian population three transcripts (skipping of exons 8-9, exon 9 or exon 9 [56]. There are at least three reports about the distribution with an inserted 170-bp cryptic exon located between of D variants in the Chinese Han population from Shang- exons 7 and 8) and a series of aberrant mRNAs except hai (0Á015%, 189/1 274 540 [57]), Xian (0Á012%, 108/ for normal RHD mRNA, has been identified in the ‘Asian 890 403 [46]) and Zhejiang (0Á012%, 37/305 572 [58]). type’ DEL probands [52, 53]. Thus, RHD_1227A can be To date, at least 27 weak D alleles [43, 46, 47, 58–63] used as an important and useful genetic marker for Chi- and 14 partial D alleles [46, 57, 58, 61] have been identi- nese DEL individuals [48]. This single nucleotide mutation fied in the Chinese population (Tables 2 and 3), which are can be easily detected for DEL identification by a simple low compared with the hundreds found in the Caucasian

Table 2 Weak D alleles identiﬁed in the Chinese D variant individuals

Shanghai Zhejiang Shenzhen Handan Guangzhou Weak D alleles or [60, 63] [58, 61] Xi’an [46] [43] Taiwan [59] Anhui [47] [62] [128] Total genotypes (n = 9) (n = 46) (n = 72) (n = 3) (n = 7) (n = 3) (n = 1) (n = 1) (n = 142)

1 RHD*weak partial 15 – 22 62 1 4 2 –– 91 (64Á1%) 2 RHD*DEL1 – 11 – 2 ––––13 (9Á2%) 3 RHD*weak D type 33 – 32 – 1 –––6(4Á2%) 4 RHD*weak partial 15/ – 12 –– 1 –– 4(2Á8%) RHD*DEL1 5 RHD*weak D type 80 3 –– –– – –– 3(2Á1%) 6 RHD*101G – 11 –– – –– 2(1Á4%) 7 RHD*670G – 2 ––– – –– 2(1Á4%) 8 RHD*weak D type 25 ––1 –– – –12(1Á4%) 9 RHD*weak D type 1 ––– –1 –––1(0Á7%) 10 RHD*weak D type 6 ––– –1 –––1(0Á7%) 11 RHD*weak D type 12 ––– –– – 1 – 1(0Á7%) 12 RHD*weak partial 15/ – 1 ––– – –– 1(0Á7%) RHD*weak D type 17 13 RHD*weak D type 24 ––1 –– – –– 1(0Á7%) 14 RHD*weak D type 51 – 1 ––– – –– 1(0Á7%) 15 RHD*weak D type 52 – 1 ––– – –– 1(0Á7%) 16 RHD*weak D type 53 – 1 ––– – –– 1(0Á7%) 17 RHD*weak D type 71 1 –– –– – –– 1(0Á7%) 18 RHD*weak D type 72 1 –– –– – –– 1(0Á7%) 19 RHD*weak D type 73 1 –– –– – –– 1(0Á7%) 20 RHD*weak D type 81 1 –– –– – –– 1(0Á7%) 21 RHD*weak D type 82 1 –– –– – –– 1(0Á7%) 22 RHD*weak D type 83 1 –– –– – –– 1(0Á7%) 23 RHD*95A/RHD*DEL1 – 1 ––– – –– 1(0Á7%) 24 RHD*357C ––1 –– – –– 1(0Á7%) 25 RHD*436A ––1 –– – –– 1(0Á7%) 26 RHD*438C ––1 –– – –– 1(0Á7%) 27 RHD*779G – 1 ––– – –– 1(0Á7%)

Table 3 Partial D alleles identified in the Chinese D variant individuals produce alloanti-D [64]. For the remaining RhD variants identified in the Chinese population, there are still no ’ Shanghai Zhejiang Xi an reports about the alloanti-D immunization. Therefore, the Partial D alleles [57] [58, 61] [46] Total Chinese RhD variants, except for ‘Asian type’ DEL, should or genotypes (n = 44) (n = 17) (n = 36) (n = 97) be regarded as D- recipients when they receive blood 1 RHD*DVI.3 29 10 31 70 (72Á2%) transfusion and their donated blood should be labelled as 2 RHD*DVI 3 –– 3(3Á1%) D+ blood at this time. 3 RHD*DV.2 – 14 5(5Á2%) 4 RHD*DVI.3/ 4 –– 4(4Á1%) RHD*DEL1 MNS hybrid glycophorins – – Á 5 RHD*DVI.2 2 2(21%) MNS blood group system includes numerous hybrid gly- 6 RHD*DV.5 1 – 12(2Á1%) cophorins, which are rare in most of ethnic groups except 7 RHD*DV.1 – 1 – 1(1Á0%) for South-East Asian populations (such as GP.Mur with a 8 RHD*DV.8 – 1 – 1(1Á0%) – – 9 RHD*DBT1 1 –– 1(1Á0%) frequency of 2 10% [65 67]). The alloantibodies against 10 DFR 1 –– 1(1Á0%) the hybrid glycophorin antigens, such as anti-Mur, elicit (RHD*505C,514T) strong allo-immune responses and result in the severe 11 RHD*DFR4 1 –– 1(1Á0%) intravascular haemolytic transfusion reaction [68, 69] and 12 DLX 1 –– 1(1Á0%) haemolytic diseases of foetus and newborn (HDFN) [70, (RHD*CE(5-6)-D) 71] with fatal cases, and some case reports also emerge in 13 DCC (RHD*677A) 1 –– 1(1Á0%) the mainland of China [72–75]. – – Á 14 RHD*DCS1 1 1(10%) GP.Mur was reported with a mean frequency of 7Á3% –– Á 15 RHD*DCS2 1 1(10%) among the Taiwan Chinese ethnic group and with the 16 DCS-3 1 –– 1(1Á0%) highest reported frequency of 88Á4% in the Taiwanese (RHD*667G, indigenous ethnic groups [76, 77]. Sta is also reported 676C, 697C) Á – Á 17 RHD*130- – 1 – 1(1Á0%) with a frequency of 1 63 5 2% (with higher frequency of Á 132delTCT 5 2% in Chinese Hakka) [76, 78, 79] and GP.Hil with a frequency of 0Á38% [76] using the serological typing or the genotyping methods (such as PCR-RFLP [76]). A term ‘Mia’ is used as a collective term for most of the antigens population. RHD*weak partial 15 and RHD*DVI.3 are the associated with the variant hybrid glycophorins, which is most common D variant alleles, accounting for more than easily confused with the specific Mia antigen (MNS007) 70% of D variants in the Chinese population. Aside from recognized by ISBT [68]. The sera against the high fre- the most common RHD*weak partial 15 and RHD*DVI.3, quently distributed GP.Mur are often a mixture of alloan- RHD*weak D type 33 (6/141) and RHD*DV.2 (5/97) are tibodies including anti-Mia, anti-Mur, anti-MUT, anti-Hil the less common RHD variant alleles. In addition, at least and anti-MINY, which are difficult to isolate and there- 11 individuals from Zhejiang province [58, 61] and 2 fore have been collectively called as anti-’Mia’. Anti-’Mia’ from Shenzhen [43] with weak positive agglutination in is one of the most common antibodies in Taiwan [65, 80]. IAT testing were identified with RHD*DEL1 allele, which Therefore, ‘Mia’+ red cells (generally using GP.Mur cells) is mainly present in ‘Asian type’ DEL people. The quanti- have been included in the panels of reagent cells for the tative difference of D antigens expression in individuals routine antibody screening for more than 20 years in Tai- with RHD*DEL1 allele might account for this. For the rest wan [77]. In addition, the same strategy has also been of numerous RHD variant alleles, they are very rare with adopted in Hong Kong. only one proband being reported, except for RHD*DV.5 In the mainland of China, a series of distribution stud- [46, 57] with two cases being reported. Based on this, for ies for the Mur antigen have been reported in recent the high-throughput genotyping platforms to aim for the years, most of which were conducted using serological Chinese D variants to date, at least four variant alleles testing, and some by PCR-SSP method. The data show (RHD*weak partial 15, RHD*DVI.3, RHD*weak D type 33 that the Mur antigen distributes with an increasing inci- and RHD*DV.2) and very common RHD*DEL1 allele must dence from the north region to the south region of China. be involved. The Mur antigen is rarely encountered (0/78) in the Chi- Numerous cases with RHD*weak partial 15 and nese Han from Taiwan with an origin from the north RHD*DVI.3 allele have been found with alloanti-D in dif- region of the Yangtze River of mainland of China [77], a ferent ethnic groups, while the individuals with RHD*DV, low frequency of 0Á5–1Á87% in the Chinese Han from the RHD*DBT and DFR alleles have also been reported to central region along the Yangtze River (Shanghai [19, 81]

© 2016 International Society of Blood Transfusion, ISBT Science Series (2016) 11 (Suppl. 2), 55–68 60 Y. L. Ji & C. E. van der Schoot and Anhui [17, 21]) and a higher frequency of 6Á3–7Á55% being described before [89], which indicated the amino in the Chinese Han from the southern region of the acid at position 858 on AE1 protein was important to keep Yangtze River (Fujian [14], Guangdong [20], and Hong the structure or function of this protein. In addition, three Kong [18]). Moreover, it is most common in some minor- heterozygous synonymous mutations (2562G>A, Pro854- ity groups (7Á9% in Yunnan Yi [13], 11Á29% in Guangxi Pro; 2565C>T, Ala855Ala; and 2625G>C, Pro875Pro) were Zhuang [82], 15Á4% in Guangxi Dong [16], 22Á65% in identified in each Chinese donor (1/1053) respectively Yunnan Nu [15], 24Á7% in Yunnan Dai [13]) from the [11], which are close to the Dia/Dib polymorphism position southwest mountain region of China. The distribution of (2561C>T, Pro854Leu), and should be taken into account the Mur antigen is consistent with a prediction that the when establishing the DNA-based genotyping method for hybrid glycophorin is associated with malaria [68]. How- Dia/Dib typing in the Chinese population. ever, the exact molecular mechanism for the correlation of the common hybrid glycophorin (such as GP.Mur) with Genotyping studies for the null phenotypes malaria still needs further investigation. The detection of RBCs rate of anti-Mur from different regions of China also reflects the same trend. In the Chinese Han patients with For the null phenotypes of RBCs, the para-Bombay, JK(a-b- the alloantibodies from the central region of China ), and Lu(a-b-) phenotypes are comparatively common in (Shanghai and Hubei), 1Á36% (3/220) [83] and 1Á42% (3/ the Chinese populations. The molecular genetic studies 212) [84] of them were identified with anti-Mur. But in reveal the diverse genetic backgrounds accounting for the southern province, Guangxi, two reports about a mix- them. Rhnull and KELnull (Ko) and p phenotype are very rare ture population with the Chinese Han and minorities with only few cases reported in the Chinese populations. showed that 12% (5/42) [85] and 11Á4% (19/166) [86] patients with the alloantibodies were identified with anti- FUT1 and FUT2 genotyping in Para-Bombay Mur. This indicates that the alloantibodies against GP.Mur individuals are more common and clinically significant in the southern regions of China, especially in the regions where the Compared with 1:1000 frequency of Bombay phenotype minority groups live. ‘Mia’+ red cells should be included in the Indian population, Bombay phenotype is very rare in the panel of reagent cells for routine antibody screen- in the Chinese population but para-Bombay is more com- ing to avoid the neglection of anti-’Mia’ in the southern mon. The para-Bombay phenotype is less common in region of China. Dongguan from Guangdong province of China (1:30 000, For Sta, a total of six distinct alleles (Sta type A, B, C 6/159 515) [90], more common in Fujian province [91] of [87],D[88], E and F [79]) with the different crossover China (1:8539, 10/85 390), which is similar in Taiwan sites in intron 3 have been identified to date. Thirty-five [92] (1:8000), and most common in Chinese Lahu minor- Sta positive samples (3Á4%, 35/1027) identified by specific ity group (2Á2%, 7/324) [93]. At least 134 Chinese Han PCR in Taiwanese were analysed, in which 29 samples probands and 8 Chinese Lahu probands with the para- belong to the previous reported Sta type B, 4 to be a Bombay phenotype were reported in the Chinese popula- novel Sta type E and 2 to be a novel Sta type F [79]. In tions including the probands from Taiwan and Hong the mainland of China, a novel GYP.Bun allele (GenBank Kong (Table 4) [90, 94–98]. Among them, 20 FUT1 alleles accession number: KR363627) with a different recombina- have been identified (Table 4). The four FUT1 alleles, h1 tion site in intron 3 compared with the reported GYP.Bun (547delAG), h2 (880delTT), h3 (C658T) and h4 (C35T; sequence has been identified. A980C) are most prevalent with an allele frequency of 49Á3%, 24Á3%, 11Á3% and 3Á2%, respectively, which Variant alleles in Diego blood group system account for 88% of allele counts, while hG328A is more common in Lahu minority group with an allele frequency Besides the numerous studies of the distribution of DI*01/ of 4Á9%. Therefore, these five alleles are essential when DI*02 alleles in China, there are few reports about the developing a para-Bombay genotyping kit for the Chinese variant allele of DI gene. A variant DI*02(2572G>T, population. Among the 142 para-Bombay probands of Ala858Ser) was identified in one Chinese donor [11], and Chinese Han undergoing FUT1 genotyping, FUT2 geno- its effect on the Dib expression and the structure or func- typing analyses were only conducted in 85 probands [94, tion of the erythrocyte band 3 protein (Anion Exchanger 97, 98]. Compared with the diverse distributed alleles of 1, AE1) encoded by DI gene needs further investigation. FUT1, only four FUT2 alleles (Se357,Se357,716,Se357,385, The Ala858Asp replacement on kidney AE1 protein, Se) with frequency of 75Á9%, 14Á7%, 8Á2% and 1Á2%, located at the same amino acid position, is a dominant respectively, were identified in these para-Bombay indi- mutation that causes mild distal renal tubular acidosis viduals (Table 5).

Table 4 The distribution of the reported FUT1 genotypes in the Chinese individuals with para-Bombay phenotype

Southwestern Shanghai Fujian Xiamen Zhejiang Ningbo Wenzhou Dongguan Shenzhen Hongkong Taiwan China [93] Genotypes [129, 130] [91, 98, 131] [96] [132–137]* [97, 138] [95] Guangzhou [90] [139–141] [142] [92, 143] (Lahu minority) Total of FUT1 (n = 27) (n = 22) (n = 3) (n = 9) (n = 3) (n = 1) [94] (n = 9) (n = 6) (n = 11) (n = 5) (n = 38) (n = 8) (n = 142)

1 h1/h1 61222111 – 6 – 12 – 43 (30Á3%) 2 h1/h2 77 – 1 ––31138– 31 (21Á8%) 3 h2/h2 4 –––––––316– 14 (9Á9%) 4 h1/h3 1 ––21– 31––2 – 10 (7Á0%) 5 h2/h3 – 1 –– – – 11––2 – 5(3Á5%) 6 h3/h3 2 –––––11–––15(3Á5%) 7 h3/hG328A – 1 –– – – – – – – – 45(3Á5%) STSineSeries Science ISBT 8 h1/h4 –– 1 –––––––3 – 4(2Á8%) 9 hG328A/hG328A –– –––––––––33(2Á1%) 10 h2/h6 –– –––––––11– 2(1Á4%) 11 h4/h4 2 ––––––––––– 2(1Á4%) 12 h1/hC293T –– –2 ––– – – – –– 2(1Á4%) 13 h1/h6 – 1 –– – – – – – – 1 – 2(1Á4%) (2016) 14 h2/hG328A 1 ––––––1 –––– 2(1Á4%) 15 h2/h4 –– ––––––––1 – 1(0Á7%) 11 16 h1/h5 –– ––––––––1 – 1(0Á7%) Spl ) 55–68 2), (Suppl. 17 h1/h7 1 ––––––––––– 1(0Á7%) 18 h1/h8 1 ––––––––––– 1(0Á7%) 19 h3/hG659A –– ––––––––1 – 1(0Á7%) 20 h3/hC586T 1 ––––––––––– 1(0Á7%)

21 h1/hA682G –– –1 ––– – – – –– 1(0Á7%) China in genotyping cell blood Red 22 hG328A/h366- –– ––––––1 ––– 1(0Á7%) 398del33 23 h7/hC293T 1 ––––––––––– 1(0Á7%) 24 hC35T,A682G/ –– –1 ––– – – – –– 1(0Á7%) hG235C 25 h1/hA896C –– –– 1 –– – – – – – 1(0Á7%) 26 hC35T, C748T/ –– ––––– 1 –––– 1(0Á7%) hG655C 61 62 Y. L. Ji & C. E. van der Schoot

Table 5 The distribution of the reported FUT2 genotypes in the Chinese individuals with para-Bombay phenotype

Zhejiang Shanghai Ninbo Fujian [132, Guangzhou Shenzhen Hongkong Southwestern Genotypes [129, 130] [97] [91, 98] 134–137] [94] [140, 141] [142] China (Lahu minority Total of FUT2 (n = 27) (n = 1) (n = 19) (n = 7) (n = 9) (n = 9) (n = 5) [93]) (n = 8) (n = 85)

1 Se357Se357 13 1 12 3 4 5 4 8 50 (58Á8%) 2 Se357Se357, 716 7 – 6 – 4 ––– 17 (20Á0%) 3 Se357Se357, 385 2 – 13 1 3 1 – 11 (12Á9%) 4 Se357,716Se357, 716 3 –– – – 1 –– 4(4Á7%) 5 Se357, 385Se357, 385 1 –– – – – – – 1(1Á2%) 6 SeSe357 1 –– – – – – – 1(1Á2%) 7 SeSe357, 385 –––1 –––– 1(1Á2%)

Shanghai [99]; 1:17000–1:20 000 (3/52 260 [102] to 2/ Genotyping analysis for JK(a-b-) phenotype 40 337 [103]) in Nanjing; not found (0/23 909) in Zhe-

Individuals with the JK(a-b-) (JKnull) phenotype are sus- jiang [104]) using the red blood cell urea lysis resistance ceptible to immunization against the high incidence Jk3 assay for screening. Several patients with anti-Jk3 were antigens and produce anti-Jk3 and could only receive JK also encountered in Guangzhou [105]. Therefore, the stor- (a-b-) matched blood in the situation with anti-Jk3. This age and supply of rare JK(a-b-) blood is necessary in this rare phenotype has been reported with the highest fre- region. Further, the urea lysis resistance method is a very quency in Polynesians (0Á1–1Á4%). In the Chinese popula- cheap and feasible method that could be used for screen- tions, the frequency of JK(a-b-) in the southern regions is ing the JK (a-b-) blood on a large scale. about 1:5000 (10/50 000 [99, 100] in Guangzhou), which At least 50 probands with JK(a-b-) phenotype were is higher than some other reported regions (1:20 000 in identiﬁed in the Chinese population for JK genotyping Changchun [101]; 1:25 000 (4/102 760 and 2/48 400) in analysis (Table 6) [99–102, 106–109]. Among them, nine

Table 6 The reported JK genotypes in the Chinese individuals with JK(a-b-) phenotype

Shanghai Dongguan Guangzhou ShenZhen Jiangsu Zhejiang Chengdu Changchun Taiwan [99] [99] [100] [144] [102, 145] [108] [106, 107] [101] [109] Total Genotypes of JK (n = 6) (n = 10) (n = 8) (n = 3) (n = 4) (n = 1) (n = 9) (n = 1) (n = 8) (n = 50)

1 JK*B(IVS5-1g>a)/ 762215– 3 26 (52Á0%) JK*B(IVS5-1g>a) 2 JK*B(IVS5-1g>a)/ 321––1 – 3 10 (20Á0%) JK*B(896G>A) 3 JK*B(896G>A)/ 2 ––––2 ––4(8Á0%) JK*B(896G>A) 4 JK*B(IVS5-1g>a)/ ––––1 – 23(6Á0%) JK*B(222C>A) – 5 JK*B(IVS5-1g>a)/ 1 –––––––1(2Á0%) JK*B(512G>A) 6 JK*B(IVS5-1g>a)/ 1 –––––––1(2Á0%) JK*B(437T>C) 7 JK*B(222C>A)/ 1 –––––––1(2Á0%) JK*B(536C>G) 8 JK*A(IVS8+5g>a)/ 1 –––––––1(2Á0%) JK*B(IVS5-1g>a) 9 JK*B(222C>A)/ ––1 –– – –1(2Á0%) JK*B(896G>A) – 10 JK*B(222C>A)/ ––1 –– – –1(2Á0%) JK*A(737T>G)) – 11 JK*B(IVS5-1g>a)/ –––––1 – 1(2Á0%) JK*B(IVS5-1g>c) –

© 2016 International Society of Blood Transfusion, ISBT Science Series (2016) 11 (Suppl. 2), 55–68 Red blood cell genotyping in China 63 mutations of JK gene were found, in which seven muta- antibody known as anti-Tja. A total of ﬁve Chinese Han tions occured on the JK*B allele and two on the JK*A probands with p phenotype were identiﬁed from Zhejiang allele. The most three common alleles are the Polynesian (n = 3, males) [117–119], Guangdong (n = 1, female with

JKnull allele (JK*B(IVS5-1g>a)), JK*B(896G>A) and JK*B several spontaneous miscarriages and without successful (222C>A) with an allele frequency of 69%, 19% and 6%, pregnancy) [120] and Taiwan (n = 1, female) [121]. respectively, which accounts for 94% for the all JKnull Four different mutations (300_301delG, Ala101Profs*11 alleles indentiﬁed in the Chinese population. [117]; 418_428del11ins34, Gln139Trpfs*72 [120, 121]; 972_997del26, Thr324Thrfs*112 [118]; and 1029_ 1030insC, Thr344Hisfs*101 [119]) of the A4GALT gene, Genotyping analysis for Lu(a-b-) phenotype of which some previously described in USA and Japanese,

Lu(a–b–)/Lunull is a rare phenotype, which is distributed were identified. with a frequency of 1:3000 in South-East England, 1:5000 in South Wales blood donors, and around 1:5000 (10/44 331) in Chinese Han from Shanghai of China Genotyping analysis for the rare Rhnull and KELnull [110], but 1:206 (22/4527) in Guangxi Zhuang minority phenotype group in China [111]. Three molecular backgrounds For the rare Rhnull phenotype, a Chinese family was anal- account for this rare phenotype, including a recessive ysed and the proband with homozygous 672C>A (Ser224- Lunull phenotype with inactivating mutations in the LU Arg) mutation in exon 5 of the RHAG gene was gene and generally with anti-Lu3 [112], an X-linked Lu identified, in which the mutation was located in the – – (a b ) phenotype with the mutation in erythroid tran- transmembrane segment 7 of RhAG glycoprotein [122]. scription factors GATA1 (GATA1-binding protein 1) For the rare KELnull (Ko) phenotype, two unrelated Chi- – – [113], and a dominant inherited Lu(a b )/In(Lu) pheno- nese females with this phenotype were found in a large- type resulting from heterozygous inactivating mutations scale screening, and the frequency of KELnull (0Á00228%, 2/ in erythroid transcription factor gene EKLF/KLF1 [114]. 87 665) in the Chinese population was obtained [123]. The In the 33 Chinese probands identified with Lu(a-b-) phe- same genotype with two novel heterozygous KEL variant notype, all of them were confirmed or predicted with In alleles (KEL*02(185insT, Ser62Phefs*16)/KEL*02(715G>T, (lu) phenotype. RBCs with this phenotype express traces Glu239X)) was identified in the two probands. In addition, of Lutheran antigen that could only be detected by one proband with KELnull from Taiwan was identified with – absorption elution testing. Therefore, their blood could be a novel homozygous mutation (IVS3+1G>C) at the splice used to satisfy the requirement for patients with anti-Lu3, donor site, which destroys the conserved GT sequence of since In(lu) is relatively common compared with Lu(a-b-). the splice donor site and results in an aberrant transcript In the 33 Chinese probands reported with Lu(a-b-) with the exon 3 skipped, to predict an abolition of the phenotype, no mutations were found in the LU gene, translation of C-terminal segment [124]. while 8 heterozygous mutations (519_525dupCGGCGCC, Gly176Argfs*179 [110, 111, 115]; 519delC, Gly174A- lafsX63 [110]; 618_682insC, Gly228ArgfsX125 [110]; Acknowledgements IVS1+12delAAGGTGGGGTCTAG [110]; 1001C>A, Thr334Lys The National Natural Science Foundation of China (Grant [110]; 1012C>A, Pro338Thr [110]; 1046C>T, Ser349Leu 81500155), the Science and Technology Project of [110]; 895C>G, His299Asp [111]) in the EKLF/KLF1 gene Guangzhou City (Grant 201509010009), and the Key were identified. The 519_525dupCGGCGCC mutation in Medical Disciplines and Specialities Program of Guangz- EKLF/KLF1 gene was the most common one (24/33) in hou in China supported this work. the Chinese In(lu) individuals identified. This mutation has also been identified in the Korean and Vietnamese adults with an increased foetal haemoglobin level [116], References as the EKLF/KLF1 mutation is also responsible for 1 Zhao TM. Human Blood Group Genetics (in Chinese). Bei- persistent hyper-haemoglobin F syndrome. jing, Science Press, 1987. 2 Liu M, Jiang D, Liu S, et al.: Frequencies of the major alleles A4GALT genotyping in individuals with of the Diego, Dombrock,Yt, and Ok blood group systems in p phenotype the Chinese Han, Hui, and Tibetan nationalities. Immunohe- matology 2003; 19:22–25 For the rare p phenotype in P1Pk blood group system, P1 3 Yan L, Zhu F, Fu Q, et al.: ABO, Rh, MNS, Duffy, Kidd,Yt, and Pk antigens are absent from the surface of red cells, Scianna, and Colton blood group systems in indigenous Chi- and sometimes, the probands carry a naturally occurring nese. Immunohematology 2005; 21:10–14

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