HLA-C polymorphisms and PCR dropout in exons 2 and 3 of the Cw*0706 allele in sequence-based typing for unrelated Chinese marrow donors

Human Immunology 71 (2010) 577–581

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HLA-C polymorphisms and PCR dropout in exons 2 and 3 of the Cw*0706 allele in sequence-based typing for unrelated Chinese marrow donors Zhihui Deng*, Daming Wang, Yunping Xu, Suqing Gao, Hongyan Zhou, Qiong Yu, Baocheng Yang Immunogenetics Laboratory, Shenzhen Blood Center, Shenzhen, Guangdong 518035, China

A R T I C L E

I N F O

Article history: Received 5 January 2010 Accepted 5 March 2010 Available online 26 March 2010

Keywords: HLA-C locus Polymorphism Allele dropout Cw*0706 Sequence-based typing

A B S T R A C T

To evaluate the accuracy of SBT protocols for HLA-C and to better understand the HLA-C polymorphism in Chinese, 1795 unrelated CMDP donors were typed at exons 2, 3, and 4 of the HLA-C gene using the Atria commercial kit. Of the study subjects, 1768 showed conclusive typing results, whereas the other 27 showed inconclusive results. Subsequent full-length cloning and haplotype sequencing showed that 11 of the 27 inconclusive results could be explained by the presence of nine novel alleles identified: Cw*0130, 0624, 070206, 075602, 0766, 0767, 0820, 0821, and 0827. These novel alleles were generated by a total of 10 coding-region substitutions, eight of them being located in the antigen-binding groove. Cw*0766 and Cw*075602 were detected three and two times, respectively, in the 1795 donors. The other 16 inconclusive samples were retested by SBT using our in-house PCR primers; all of them were found to carry Cw*0706, which dropped out in exons 2 and 3 in the initial PCR using the commercial primers amplifying from 5= UTR to intron 3. Our results showed the importance of the full-length genomic sequence and intronic SNPs for the development of more accurate SBT. The allele distribution and novel alleles detected in this study also provide further insights into the HLA-C polymorphism in the Chinese Han population. 䉷 2010 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

1. Introduction In the past, the importance of the classical HLA-C gene has been underestimated in part because of its low surface expression relative to HLA-A and -B. It had been speculated that HLA-C molecules are deteriorating or lack function [1,2]. In fact, HLA-C molecules are known to be able to present antigenic peptides to CD8⫹ T cells to induce adaptive cellular immune responses like other classical class I HLA molecules. More recent studies showed that HLA-C interacts with killer-cell immunoglobulinlike receptors (KIR) and play an important role in regulating inhibition and activation of natural killer (NK) cells [3]. In recent years, HLA-C has attracted revived research interests on its biologic functions, influence on marrow transplantation [4 –9], and allelic diversity in human populations [10,11]. High-resolution typing for HLA-C has important applications for unrelated hematopoietic stem cell transplantation (UR-HSCT). Studies from Fred Hutchinson Cancer Research Center (FHCRC) and National Marrow Donor Program (NMDP) have demonstrated that HLA-C mismatching is associated with a strong adverse effect on the outcome of transplantation and may result in a decrease in survival [4 – 6]. NMDP recommended the optimal criteria for donor/ receipt match: whenever possible, donors matched for high-

* Corresponding author. E-mail address: [email protected] (Z. Deng).

resolution HLA-A, -B, -C, and -DRB1 should be sought [7]. However, early studies from the Japan Marrow Donor Program (JMDP) indicated that HLA-C mismatch appeared permissible [8]. In a more recent report, Kawase et al. [9] analyzed 5210 JMDP donor/receipt pairs and found that seven of 15 nonpermissive mismatching allele combinations involved HLA-C were significant factors for severe aGVHD (III–IV) and should be avoided in the donor selection for UR-HSCT. In Chinese Marrow Donor Program (CMDP), HLA-A, -B, and -DRB1 are routinely genotyped for all voluntary stem-cell donors. Sequencing-based typing (SBT) for HLA-C is applied as a confirmatory test only if the unrelated donor/receipt pair matched at HLA-A, -B, and -DRB1. There have been many reports on HLA-A, -B, and -DRB1 about their allelic diversity and novel polymorphisms in Chinese Han populations [12–14], but very few studies have been focused on HLA-C on a large sample size (e.g., more than 1000 samples). Compared with other HLA loci our knowledge on HLA-C in Chinese populations is rather limited. Currently, the AlleleSEQR HLA-C plus SBT Sequencing Kit (Atria Genetics, San Francisco, CA) is used by the five CMDP confirmatory laboratories as well as many other clinical tissue typing laboratories worldwide. This study aims to evaluate the suitability and typing accuracy of the commercial kit for Chinese Han populations. Meanwhile, the HLA-C allelic polymorphism, including novel alleles in the Chinese Han populations, is characterized.

0198-8859/10/$32.00 - see front matter 䉷 2010 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.humimm.2010.03.001

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2. Subjects and methods 2.1. Samples A total of 1795 peripheral blood samples were obtained with written consent from unrelated marrow donors during the period of September 2008 through October 2009 in Shenzhen, China. Each whole-blood sample (3–5 ml) was anticoagulated with 5% ethylenediaminetetraacetic acid (EDTA) and stored at ⫺80⬚C. Of the 1795 recruits, 1098 and 567 were Hans from southern and northern China, respectively. The remaining 130 had mixed ethnic and geographic backgrounds. 2.2. DNA extraction Genomic DNA was extracted from the peripheral blood with a TECAN DNA workstation (M×lndal, Sweden). DNA purity and concentration were tested using an Eppendorf spectral photometer. 2.3. HLA-C SBT at exons 2, 3 and 4 Extracted genomic DNA was used for SBT with the AlleleSEQR HLA-C Plus kit (Atria Genetics, San Francisco, CA) according manufacturer’s instructions. Two fragments of the HLA-C gene from the 5=-UTR to intron 3 and from intron 3 to intron 7 were co-amplified in a single polymerase chain reaction (PCR) and purified with ExoSAP-IT (Atria Genetics). Sequencing of exons 2, 3, and 4 was performed in both orientations on an ABI 3730 DNA Sequencer (Applied Biosystems, Foster City, CA). HLA-C alleles were assigned in four-digit level with the help of the ASSIGN 3.5 software (Conexio Genomics, Applecross, Australia). Synonymous mutations were not taken into account. Alleles that differed in nonsynonymous mutations outside exons 2– 4 were characterized with the letter “g” appended to the first allele according to Waller et al. [15]. Samples with inconclusive results were subjected to cloning and haplotype sequencing. 2.4. Full-length HLA-C cloning and haplotype sequencing The full-length of 4.5 kb of the HLA-C gene (nt -962–nt 3576) encompassing the 5=-promoter through the 3=-UTR was amplified by long-range PCR using Pfu high-fidelity polymerase (Stratagene Pfuultra II Fusion HS DNA polymerase) and our in-house forward (5=-CGCAACTTTGAGGTGATGACT-3=) and reverse (5=-TTGTCTCAGAAAGCACAGGGA-3=) primers. The PCR reaction was carried out in a volume of 50 ␮l containing 25.0 ␮l of 2 ⫻ GC buffer, 4.0 ␮l of each dNTP (25 mmol/l), 2 ␮l of each primer (10 ␮mol/l), 2.5 U of Pfu polymerase, 100 ng of genomic DNA. PCR was carried out in a GeneAmp 9700 thermal cycler (Applied Biosystems, Foster City, CA) under the following conditions: initial denaturation at 95⬚C for 3 minutes followed by 35 cycles of 30 seconds at 94⬚C, 30 seconds at 58⬚C, and 4 minutes at 72⬚C, and a final extension at 72⬚C of 15 minutes. We conducted “adding A” reactions in a cocktail of 20 ␮l containing the 4.5-kb PCR products (17 ␮l), 25 mmol/l dATP (0.5 ␮l), 10⫻ Taq Buffer (2.0 ␮l), and rTaq polymerase (0.5 ␮l, 5 U/␮l, Takara, Japan). After incubation at 37⬚C for 30 minutes, the PCR fragments conjugated with dATP at the 3= end were then purified using the Gel Extraction Kit (Axygen, USA). Based on the TA cloning method, three separate PCR products for each sample were pooled and ligated into the pGEM-T Easy Vector (Promega, Madison, WI). The ligated PCR fragments were transformed into the JM109 competent cells and cultivated overnight in the 2 YT medium containing 50 mg/ml ampicillin. For each sample, 10 positive clones were harvested to ensure that each allelic haplotype had at least three clones. The recombinant plasmid DNA was isolated with the Plasmid Miniprep Kit (Axygen, USA) and sequenced in both directions using the ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit with our in-house sequencing primers (Supplemen-

tary Table 1) on an ABI 3730 DNA sequencer. The strategy of HLA-C haplotype sequencing is described in Supplementary Fig. 1. Assignment of each haplotype was accomplished using the NCBI SBT Interface software (http://www.ncbi.nlm.nih.gov/gv/mhc/sbt). 2.5. PCR-SBT retest using in-house PCR primers To investigate the cause of the inconclusive SBT results, we designed in-house forward (5=-CCAATCAGCGTCTCCGCAGTC-3=) and reverse (5=-ACCCCYCATYCCCCTCCTTAC-3=) primers based on the full-length genomic HLA-C sequences available on the IMGT/ HLA Database (http://www.ebi.ac.uk/imgt/hla/, Version Report2.28.0) and our 35 HLA-C, including Cw*0706 genomic sequences (data not shown), to amplify a ⬃2000-bp fragment spanning exons 1– 4. PCR was conducted in a volume of 20 ␮l containing 10.0 ␮l of 2 ⫻ GC buffer, 0.2 ␮l of each dNTP (25 mmol/l), 1 ␮l of each primer (10 ␮mol/l), 50 ng of genomic DNA, and 2.5 U of Pfu high-fidelity polymerase. PCR conditions were 95⬚C for 3 minutes, followed by 30 cycles of 30 seconds at 95⬚C, 30 seconds at 62⬚C, and 2 minutes at 72⬚C, and a final extension at 72⬚C for 15 minutes. Purification of PCR products and sequencing reaction at exons 2, 3, and 4 were conducted using the Atria AlleleSEQR HLA-C plus kit. Except for the initial PCR procedure, the downstream SBT procedures exactly followed the manufacturer’s instructions. To resolve the Cw*070101/070102/0706/0718 ambiguity, exons 5 and 6 were examined using our in-house SBT protocol. A fragment of 1050-bp sequence spanning exons 5– 8 was amplified using our in-house forward (5=-TTM TCA GRG AAA GCA GAA GTC3=) and reverse (5=-AAT CCT GCA TCT CAG TCC CAC-3=) primers. PCR was carried out in a volume of 25 ␮l containing 12.5 ␮l of 2 ⫻ GC buffer, 1.5 ␮l of MgCl2 (25 mmol/l), 1.0 ␮l of each dNTP (25 mM), 1 ␮l of each PCR primer (10 ␮mol/l), 100 ng of genomic DNA, and 2.5 U of LA Taq polymerase (Takara, China). PCR conditions were 95⬚C for 3 minutes followed by 30 cycles of 30 seconds at 95⬚C, 30 seconds at 62⬚C, and 2 minutes at 72⬚C, and a final extension at 72⬚C for 15 minutes. PCR products were purified with ExoSAP-IT. Sequencing was performed using the ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit and our in-house sequencing primers (exon 5: forward 5=-GTCAGGGCTGAGGCTTG3=, reverse 5=-GATGGTGCTTCCAGTAAC-3=; exon 6: forward 5=TCCAAGACTAGGAGGTTC-3=). 3. Results 3.1. .HLA-C SBT results Of the 1795 samples typed for HLA-C using the AlleleSEQR HLA-C plus SBT commercial kit, 1768 showed full match results, whereas the other 27 showed inconclusive results, as their consensus sequences did not match any of the known alleles. 3.2. Cloning and haplotype sequencing results Cloning and haplotype sequencing for the inclusive 27 samples identified nine novel HLA-C variant alleles in 11 individuals. These novel alleles have now been officially designated by the World Health Organization Nomenclature Committee for Factors of HLA System [16] as Cw*0130, 0624, 070206, 075602, 0766, 0767, 0820, 0821 and 0827 as shown in Table 1. Cw*0766 and Cw*075602 were detected in three and two individuals, respectively. These novel HLA-C alleles accounted for 0.5% (9/1795) of all samples typed in the study. Haplotype sequencing performed for the other 16 samples with inconclusive SBT results confirmed that all these samples carried Cw*0706 rather than an novel variant allele. Retrospectively analysis of their initial SBT patterns using the Atria kit revealed that their SBT results did not match any of the known genotypes. Compared with their closest genotypes, they all had one nucleotide mismatch at varied positions. In addition, heterozygosity could be

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Table 1 The characteristics of nine novel alleles at HLA-C locus identified in 1795 CMDP samples Sample no.

New allele

Accession no.

Most homologous HLA-C allele

Codon/nucleotide change

Nucleotide change position

Residue change

HLA-C genotype

D#0216 D#0383

Cw*0820 Cw*0766 Cw*0767 Cw*0821 Cw*075602 Cw*075602 Cw*0624 Cw*070206 Cw*0827

FJ785725 FJ785729 FJ785730 FJ785726 GU352936 GU352936 GU376470 GU376471 GU352835

Cw*080101 Cw*07020101 Cw*07020103 Cw*080101 Cw*075601 Cw*075601 Cw*06020101 Cw*07020101 Cw*0802

194:GTC ⬎ATC 206:CTG⬎ATG 161:GAG⬎GTG 73:ACT⬎GCT 91:AGG⬎CGG 91:AGG⬎CGG 159:TAC⬎CAC 143:ACC⬎ACT Genomic nt 503 A⬎C 138:AAG⬎ACG 156:CGG⬎TGG Genomic nt 1137T⬎C Genomic nt 1338A⬎G 171:TAC⬎CAC 206:CTG⬎ATG 206:CTG⬎ATG

Exon 4 Exon 4 Exon 3 Exon 2 Exon 2 Exon 2 Exon 3 Exon 3 Intron 2 Exon 3 Exon 3 Intron 3 Intron 3 Exon 3 Exon 4 Exon 4

Val194Ile Leu206Met Glu161Val Thr73Ala Silent Silent Tyr159His Silent

Cw*080101, 0820 Cw*0766, 0767

D#0423 D#0033 D#8961 D#8943 D#0655 D#0270

D#1170 D#1397 D#1646

Cw*0130 Cw*0766 Cw*0766

GU376469 FJ785729 FJ785729

Cw*010201 Cw*07020101 Cw*07020101

Cw*070401, 0821 Cw*030301, 075602 Cw*150201, 075602 Cw*0802, 0624 Cw*0602, 070206 Cw*030301, 0827

Lys138Thr Arg156Trp Tyr171His Leu206Met Leu206Met

Cw*010201, 0130 Cw*060201, 0766 Cw*0766, 120202

Note: Sample D#0383 carried the Cw*0766 and Cw*0767 novel alleles.

detected in exon 4 indicating the presence of two alleles, whereas exons 2 and 3 showed homozygosity of a single allele (Supplementary Fig. 2). 3.3. SBT retest using in-house PCR primers Using our in-house PCR primers, confirmatory SBT of both strands detected heterozygous sequences in exons 2 and 3 as well as exon 4 (Supplementary Fig. 2). The fully conclusive results also matched the results of haplotype sequencing. The confirmatory SBT demonstrated that the Atria PCR primer set for amplifying fragment from the 5= UTR to intron 3 is unable to amplify Cw*0706. Cw*0706 was first identified by cDNA cloning in 1996 with only the coding sequence published [17]. In this study, we obtained the full-length (4551 bp) genomic sequence of Cw*0706 and submitted it to GenBank (accession number FJ785732, released in IMGT/HLA Database in November 2009, Version 2.27.1) [18]. Alignment of the sequence of Cw*0706 with its closest allele Cw*070101, except for two exonic substitutions at genomic nt 2076 T⬎A in exon 5 and nt 2566 C⬎T in exon 6, two intronic substitutions at nt1280 C⬎T in intron 3 and nt 1971 C⬎T in intron 4, and two mutations in the 3=-UTR of Cw*0706, nt 2987 G⬎A and nt 3005 T⬎C, were identified for the first time in this study. These SNPs can provide valuable information for the design of primers and probes for more accurate HLA-C molecular typing. 3.4. Distribution of HLA-C alleles in Chinese Hans Table 2 shows the allele frequencies of HLA-C in southern and northern Han populations. A total of 41 HLA-C alleles and 155 genotypes were detected in the southern Han population; 31 alleles and 161 genotypes being detected in the northern Han population. The observed homozygoty was 10.2% and 7.4%, respectively, in the southern and northern Han populations. The most common alleles with a frequency of more than 10% were Cw*0102, Cw*0702, and Cw*0304 in the southern Han, and Cw*0702, Cw*0602, and Cw*0102 in the northern Han populations. The number of common HLA-C alleles (allele frequency ⬎1%) in the southern and northern Hans was 13 and 17, respectively. Cw*0706 was detected with frequencies of 0.27% and 0.88%, respectively in southern and northern Hans. In both Han populations Cw*0706 are in strong linkage disequilibrium with B*4403, all the 16 Cw*0706-carrying individuals being also positive for B*4403. 4. Discussion In previous studies, HLA-C typing by SBT was restricted to analyze polymorphisms at exons 2 and 3 [10,11,19,20]. The HLA-C

diversity and novel polymorphisms had been reported for NMDP samples of which the majority are of Caucasian origin, but the allelic diversity of HLA-C in non-Caucasians is still underestimated [10]. In the present experiments, we performed for, the first time, high-resolution HLA-C typing by SBT at exons 2, 3, and 4 using the widely available Atria HLA-C SBT kit in a large sample of 1795 unrelated CMDP donors and addressed three major issues, discussed below. First, using the PCR-SBT technique, previously unknown HLA-C variant alleles were identified in the Chinese Han population. In 1998, Turner et al. [10] reported 19 novel alleles in a sample of 1823 NMDP donors, representing a novel allele rate of ⬃1 in 100 samples typed. Of 19 new alleles, 13 were found in Caucasian individuals. Six years later, Lebedeva et al. [11] identified 20 novel alleles in Table 2 HLA-C gene frequencies in the southern and northern Chinese Han populations Frequency Allele

Cw*0102 Cw*0103 Cw*0106 Cw*0108 Cw*0130 Cw*0202 Cw*0302 Cw*0303g Cw*0304 Cw*0307 Cw*0317 Cw*0401g Cw*0403 Cw*0406 Cw*0415 Cw*0501 Cw*0602 Cw*0624 Cw*0701 Cw*0702 Cw*0704g Cw*0706 Cw*0743

Frequency

Southern Chinese Han (N ⫽ 1098)

Northern Chinese Han (N ⫽ 567)

0.2017 0.0027 0.0005 0.0005 0.0005 0.0041 0.0929 0.0551 0.1207 0.0005 0.0009 0.0478 0.0132 0.0005 0.0005 0.0018 0.0310 0.0005 0.0023 0.1776 0.0050 0.0027 —

0.1041 0.0053 0.0009 — — 0.0150 0.0556 0.0882 0.0882 — — 0.0670 0.0018 — — 0.0106 0.1155 — 0.0079 0.1243 0.0106 0.0088 0.0009

Allele

Cw*0756 Cw*0766 Cw*0767 Cw*0801 Cw*0802 Cw*0803 Cw*0820 Cw*0821 Cw*0827 Cw*1202 Cw*1203 Cw*1402 Cw*1403 Cw*1502 Cw*1505 Cw*1507 Cw*1513 Cw*1517 Cw*1601 Cw*1602 Cw*1604 Cw*1701

Southern Chinese Han (N ⫽ 1098)

Northern Chinese Han (N ⫽ 567)

0.0005 0.0009 0.0005 0.0993 0.0018 0.0032 0.0005 — 0.0005 0.0301 0.0214 0.0383 0.0032 0.0287 0.0059 0.0005 — 0.0005 — 0.0009 0.0005 0.0005

0.0009 — — 0.0891 0.0071 0.0115 — 0.0009 — 0.0317 0.0282 0.0467 0.0212 0.0397 0.0097 — 0.0009 — 0.0009 — 0.0018 0.0053

Note: Alleles cannot be distinguished within exons 2, 3, and 4 were assigned with an abbreviation for each allele group according to Waller et al. [15]. Cw*0303g is the abbreviation for Cw*030301/0320N, Cw*0401g for Cw*04010101/04010102/0409N, Cw*0704g for Cw*070401/0711.

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3500 NMDP donors. The frequency of emerging new alleles still remains high, and some new alleles Cw*030304, 0413 detected four and three times in African Americans, respectively, suggesting that HLA-C common to African Americans and probably other minorities is still not adequately characterized [11]. In this study, we examined 1795 Chinese Han donors by SBT and identified nine novel HLA-C alleles with a new allele emerging rate of 0.5% (9/1795), which is comparable to that reported by Lebedeva et al. (0.57%, 20/3500). Two of the novel alleles, Cw*0766 and Cw*075602, were detected in three and two individuals, respectively, indicating that these two alleles are common and welldocumented alleles and should not be ruled out as rare alleles in HLA-C high resolution typing for clinical transplantation. These nine novel alleles were generated by a total of 10 coding-region substitutions. Except for two substitutions in exon 4, all other mutations are located in the peptide binding groove indicating a possible driving force by selection. The full-length genomic sequences of the nine novel alleles obtained in this study can be used to design appropriate primers/probes and improve the accuracy and allele coverage of the routine HLA-C DNA typing. Second, Cw*0706 dropout in exons 2 and 3 was observed in 16 cases with unusual SBT patterns using the Atria commercial kit. Comparing the initial and in-house SBT results, we concluded that the Atria primer set amplifying fragment from the 5= UTR to intron 3 does not fully match the Cw*0706 sequence and therefore caused the Cw*0706 allele dropout. Alignment of the full-length Cw*0706 sequence characterized in this study with its closest allele Cw*070101, except for two known substitutions in the coding sequence, four previously unknown substitutions in introns 3 and 4 as well as in the 3=-UTR were identified by us. The nt1280 C⬎T substitution detected in Cw*0706 is unique in the 48 HLA-C genomic sequences released in the IMGT/HLA Database (http:// www.ebi.ac.uk/imgt/hla/docs/release.html, Version Report 2.28.0). As the sequence and location of the Atria primers were not available, we speculate that the nt1280 C⬎T substitution in intron 3 of Cw*0706 may prevent correct annealing step, and thus the target fragment from 5= UTR to intron 3 failed to be amplified using the Atria kit. In the absence of the exon 4 sequence, the ASSIGN 3.5 software would call a fully matched non-Cw*0706 homozygous genotype for Cw*0706-positive heterozygous samples. Therefore, some of the previously typed HLA-C homozygous samples might actually be heterozygous with Cw*0706 dropped out. Allele dropout has also been described in previous studies. Delfino et al. [21] reported that a reverse primer DLREX5 designed to sequence exon 5 didn’t match Cw*07 because of a nt 79 A⬎G polymorphism in intron 5. Heinod et al. [22] reported that the PCR primer mix 2 of the Protrans S3 HLA-A sequencing kit (Protrans Medizinische Diagnostische Produkte, Ketsch am Rhein, Germany), designed to be A*02 specific, failed to amplify A*02 in certain A*02-positive samples, which led to the identification of A*02010103, an allele with three nucleotide mismatches in intron 1 relative to its closest match A*02010101. Both these cases and our present study showed the importance of the intronic sequence in the development of more accurate SBT protocols. Cw*0701g is the abbreviation for Cw*070101/070102/0706/ 0718; these alleles are identical in exons 2 and 3. Many HLA-C population studies presented the typing results with a solution to the 4-digital allele level within the antigen binding domain [23,24], thus the Cw*0701g carrier was not subtyped. According to the frequency data released by NMDP (http://bioinformatics.nmdp. org/), Cw*0701g was detected with allelic frequencies of more than 10% in Caucasian (16.658%), African-American (12.401%) and Hispanic race (10.355%). In Asian/Pacific Islander Cw*0701g showed a relatively lower frequency (3.894%). As for the exact allele frequency of Cw*0706, Turner et al. [10] reported that Cw*0706 was not detected in a sample of 1823 NMDP donors. In another report,

only one of 42 Cw*0701/0706-positive individuals of mainly Dutch origin was detected as Cw*0706 positive, the other 41 being all Cw*0701 [19]. Cw*0706 was also found to be in strong linkage disequilibrium with B*4403 in the Bubi population from Equatorial Guinea [25]. Twenty B*44/Cw*07 positive individuals in this population were all typed as Cw*0706 positive, and 19 of them carried B*4403. In the present study, Cw*0706 was detected in southern and northern Chinese Hans with frequencies of 0.27% and 0.88%, respectively. A strong linkage disequilibrium of Cw*0706 with B*4403 was also detected as all of the 16 Cw*0706-positive Han donors carrying B*4403 as well. These results suggest that using the Atria SBT kit Cw*0706 dropout in exons 2 and 3 might present a bigger problem in northern Chinese than in Caucasians. Third, in the present study, we characterized the HLA-C polymorphism and allele distributions in the southern and northern Chinese Han populations and detected some unique Asian/Chinese features in these populations. Examples of unique alleles in Asian populations include Cw*0403 and Cw*1403 [26]. Cw*0103 is also an Asian-specific allele detected in Japanese (0.4%) [27], southern Chinese (0.27%), and northern Chinese (0.53%). Compared with previous population studies [26,27], the three most frequent alleles (Cw*0102, Cw*0702, and Cw*0304) observed in the southern Chinese Han also exhibit relatively high frequencies in other populations. By cloning and haplotype sequencing, we were able to identify all seven Cw*17-positive samples as Cw*17010102 (GenBank accession number GQ472844). It is noteworthy that, in the study of Chinese Han populations, Cw*0701 showed a frequency similar to that of Cw*0706. Because HLA-C typing is not routinely included for marrow registry donors, 31% NMDP HLA-A, -B, and -DRB1 six antigenmatched unrelated donor/receipt pairs mismatched at HLA-C locus [28]. Within CMDP, the ratio of HLA-C mismatches is even higher (36.8%) [29]. Therefore, a thorough understanding of the HLA-C polymorphism can help in searching for the optimal HLA-matched donors and can also provide valuable information for HLAassociated disease studies. In summary, in the present study, we identified nine novel HLA-C alleles, confirmed 16 Cw*0706 dropout events, and characterized the HLA-C polymorphism in southern and northern Chinese Han populations. Our results showed that inconclusive SBT results could result from both PCR dropout and the presence of novel variant alleles. Genomic full-length HLA-C sequence can provide valuable SNPs information to improve the accuracy of SBT for clinical HLA matching. Acknowledgments This study was supported by the research fund of Guangdong Science & Technology Department (Research Project Number 2008B030301277). We thank the marrow registry donors for generously providing blood samples for this study. We also thank Dr. Xiaojiang Gao and Dr. Mary N. Carrington, of the National Cancer Institute, Frederick, Maryland, for critical reading and revising of the manuscript. Appendix. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.humimm.2010.03.001. References [1] Snary D, Barnstable CJ, Bodmer WF, Crumpton MJ. Molecular structure of human histocompatibility antigens: The HLA-C series. Eur J Immunol 1977;7: 580 –5. [2] Gu¨ssow D, Rein RS, Meijer I, de Hoog W, Seemann GH, Hochstenbach FM, et al. Isolation, expression, and the primary structure of HLA-Cw1 and HLA-Cw2 genes: Evolutionary aspects. Immunogenetics 1987;25:313–22. [3] Colonna M, Brooks EG, Falco M, Ferrara GB, Strominger JL. Generation of allospecific natural killer cells by stimulation across a polymorphism of HLA-C. Science 1993;260:1121– 4.

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