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The Journal of Immunology, 1998, 161: 1406-1413.
Copyright © 1998 by The American Association of Immunologists

Sequence-Based Typing Provides a New Look at HLA-C Diversity1

Seán Turner*, Mary E. Ellexson*, Heather D. Hickman*, David A. Sidebottom*, Marcelo Fernández-Viña{dagger}, Dennis L. Confer{ddagger} and William H. Hildebrand2,*

* Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73190; {dagger} American Red Cross, National Histocompatibility Laboratory, Baltimore, MD 21201; and {ddagger} National Marrow Donor Program, Minneapolis, MN 55413


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
Although extensive HLA-A and HLA-B polymorphism is evident, the true diversity of HLA-C has remained hidden due to poor resolution of HLA-C Ags. To better understand the polymorphic nature of HLA-C molecules, 1823 samples from the National Marrow Donor Program research repository in North America have been typed by DNA sequencing and interpreted in terms of HLA-C diversification. Results show that HLA-Cw*0701 was the most common allele with a frequency of 16%, whereas 28% of the alleles typed as Cw12-18 (serologic blanks). The frequency of homozygotes was 9.8% as compared with previous studies of 18% for sequence-specific primers and 50% for serology. Most startling was the frequency at which new alleles were detected; 19 new HLA-C alleles were detected, representing a rate of ~1 in 100 samples typed. These new HLA-C alleles result from 29 nucleotide substitutions of which 4 are silent, such that coding substitutions concentrated about the Ag-binding groove predominate. Polymorphism at the HLA-C locus therefore resembles that at the HLA-A and HLA-B loci more than previously believed, indicating that antigenic stress is driving HLA-C evolution. However, sequence conservation in the {alpha}-helix of the first domain and a clustering of unique amino acids around the B pocket indicate that HLA-C alleles respond to antigenic pressures differently than HLA-A and HLA-B. Finally, because the samples characterized were predominantly from Caucasians, we hypothesize that HLA-C polymorphism will equal or exceed that of the HLA-A and -B loci as DNA sequence-based typing is extended to include more non-Caucasian individuals.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
Class I alleles of the MHC are among the most polymorphic genes in mammals, and class I polymorphism is driven by the intricate battle raging between class I molecules and intracellular pathogens that attempt to evade immune recognition through their own antigenic polymorphism (1, 2, 3). For the human population, HLA-A and -B class I polymorphism is extensive and is believed to act as a barrier that prevents pathogens such as viruses from escaping an entire population’s cellular immune response; a virus that escapes presentation to CTLs by one individual’s class I MHC molecules has not necessarily conquered Ag presentation by a second individual’s different class I molecules. Class I polymorphism therefore presents intracellular pathogens with a "moving target" that protects the population as a whole but not necessarily individuals within the population. However, while advantageous in keeping the population fit through reproductive age, class I polymorphism acts like a double-edged sword in that particular class I alleles are associated with the onset of autoimmune disorders and because allogeneic class I molecules stimulate the rejection of transplanted organs, tissue, and bone marrow (4, 5, 6, 7).

The extent and characteristics of HLA-A and -B polymorphism support the hypothesis that antigenic stress drives class I HLA diversification. For example, comparative analysis of the ~200 HLA-B alleles now recognized demonstrates that nonsynonymous nucleotide substitutions predominate in the evolution of HLA-B alleles, that HLA-B polymorphism is focused about the Ag-binding groove, and that particular alleles such as HLA-B*5301 are found at a high frequency in regions where these alleles convey disease resistance (3, 8, 9). The HLA-C locus, on the other hand, has remained more of an enigma largely because reagents capable of distinguishing HLA-C molecules from one another and from HLA-A and -B molecules are difficult to obtain (10, 11, 12, 13). Indeed, the inability to accurately establish the HLA-C type of patients with disease resistances, autoimmune disorders, or in need of a transplant has resulted in the functional contribution of HLA-C molecules being comparatively unexplored. Less cell surface expression of HLA-C compared with HLA-A and -B has led to speculation that the HLA-C molecules are deteriorating or lack function, while apparent stimulation of allograft rejection and their recognition by NK cells indicate that HLA-C molecules play an important role in stimulating cellular immune responses (14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25). However, without the ability to determine accurately the HLA-C type of a transplant donor/recipient pair or of an individual in which cellular immune responses induce disease susceptibility/resistance the enigma of HLA-C remains unresolved.

Nucleotide sequencing represents an accurate means of determining an individual’s HLA-C type, and we have adapted DNA sequencing such that it can be applied to HLA-C typing in a high throughput, robust, and cost-efficient manner. By applying this class I HLA-C DNA sequence-based typing (SBT)3 protocol to ~2000 bone marrow donor/recipient pairs in the National Marrow Donor Program’s (NMDP) research repository, we accurately establish parameters such as HLA-C homozygosity rates, HLA-C allele frequencies, and the frequency of serologic blanks in the samples typed. In terms of HLA-C polymorphism, positions both conserved and polymorphic within the class I Ag-binding groove become apparent while the pool of nucleotide sequence provided identifies synonymous and nonsynonymous nucleotide substitution rates. The data interpretation herein is significantly different from that of other studies due to the large number of new HLA-C alleles we have contributed for comparative analyses, and the HLA-C SBT data presented here provide a step forward in unraveling the polymorphic nature of HLA-C molecules and how these HLA-C polymorphisms might impact cellular immune responses.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
DNA samples

Genomic DNA from 1823 individuals were prepared from 200 µl of frozen whole blood (citrate/EDTA), PBLs, granulocytes, or EBV cell lines, provided by the NMDP repository in North America, using the commercially available Qiagen QIAamp (Qiagen, Valencia, CA) blood kit according to manufacturer’s protocol.

PCR amplification

Exons 2 and 3 of HLA-C loci were amplified as previously described by Cereb et al. (26) using primers 5'CI1 and 3'BCI3 (Table IGo). This first round PCR, C1, was diluted 1:100 and used as template for nested PCR reactions that amplify exons 2 and 3 separately (Fig. 1Go). The four nested PCR reactions, two for each exon, include 10 pM concentrations of each HLA class I generic primer mix (Table IGo): C2 (biotin-5'I1E2C + 3'I2E2ABC-U); C3 (5'I1E2C-U + biotin-3'I2E2ABC); C4 (biotin-5'I2E3C + 3'BCI3-U); and C5 (5'I2E3C-U + biotin-3'BCI3); 1.5 mM MgCl2; 50 mM KCl; 10 mM Tris-HCl (pH 8.3); 200 mM concentrations each of dATP, dTTP, dGTP, and dCTP; and 0.5 U of Taq polymerase in a final volume of 60 µl. Amplifications were accomplished on an MJ Research Tetrad (MJ Research, Watertown, MA), PTC-225 (heated bonnet) thermocycler using the following cycling conditions: 30 cycles of 95°C for 1 min; 54°C for 1 min; and 72°C for 1 min; and a final cycle of 72°C for 5 min.


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  		Table I. HLA-C SBT PCR primers

 


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  		FIGURE 1. SBT strategy for direct heterozygous sequencing of HLA-C exons 2 and 3. Genomic DNA, represented here as a linear diagram of exons 2 and 3 flanked by introns 1, 2, and 3, acts as template for the primary (1°) PCR reaction. HLA-C locus-specific oligonucleotide primers, 5'CI1 and 3'BCI3, hybridize in introns 1 and 3 resulting in a primary HLA-C amplicon ~914 bp long. This primary HLA-C PCR product then serves as template for four nested and heminested secondary (2°) PCR reactions, shown below the linear diagram of exons 2 and 3. Oligonucleotide primer mixes C2, C3, C4, and C5 generate separate exon 2 and 3 HLA-C amplicons ~340 bp long with an M13 universal primer site on one end and biotinylated on the other. The HLA-C exon 2 and 3 biotinylated PCR products are then bound to a streptavidin-coated support on which bidirectional DNA sequencing reactions are performed.

 
Direct sequencing of PCR products

Bidirectional sequencing of each exon was performed. A Cy5 dye-labeled -21-mer M13 Universal primer and an AutoLoad Kit (Amersham Pharmacia Biotech, Piscataway, NJ) were used for the sequencing of each nested PCR product (45 µl). Sequencing reactions were then loaded onto a 6% Page Plus (Amresco, Solon, OH) gel and run on a Pharmacia ALFexpress automated DNA sequencer. Data were analyzed using the Pharmacia HLA SequiTyper software (version 2.0).

Cloning and sequencing

Putative new HLA-C alleles were reamplified from genomic DNA as previously described using HLA class I locus-specific primers, C1 mix (Table IGo) and cloned into the blunt-end TA vector using the TA cloning kit (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Ten white colonies from each sample were picked and grown overnight in 10 ml of Luria-Bertani medium containing 50 mg/ml ampicillin. Plasmid DNA was isolated using the Promega Wizard kit (Promega, Madison, WI) according to the manufacturer’s instructions. Each clone underwent EcoRI digestion and was run on a 0.8% agarose gel to screen for insert. Screen sequencing of clones containing the insert was achieved with -21-mer M13 Universal primer and a Thermosequenase cycle sequencing kit (Amersham Pharmacia Biotech, Piscataway, NJ). Bidirectional sequencing of clones from the desired populations was performed as before using M13 reverse primer. Sequencing reactions were loaded onto a 5% Page Plus (Amresco, Solon, OH) gel run on a Pharmacia ALFexpress automated DNA sequencer. Sequence data were analyzed using the Wisconsin GCG (Genetics Computer Group, Madison, WI) sequence analysis system on a Digital Equipment Corporation VAX 6610.

Accession numbers and nomenclature

The new HLA-C alleles detected here have been submitted to GenBank and assigned the accession numbers detailed in Table IIGo, which also contains the names officially assigned by the World Health Organization (WHO) Nomenclature Committee (27). This follows the agreed policy that, subject to the conditions stated in the Nomenclature Report, names will be assigned to new sequences as they are identified. Lists of such new names will be published in the next WHO Nomenclature Report.


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  		Table II. GenBank accession numbers

 

   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
Each month 540 samples were processed through a class I HLA SBT methodology with each HLA-C sample sequenced in the forward and reverse directions for exons 2 and 3 (Fig. 1Go). Bidirectional sequencing was implemented to ensure that heterozygous positions were not overlooked due to compressions or loss of resolution near the end of a reading (Fig. 2Go). Each sequence-based type was analyzed by two independent operators using Pharmacia HLA SequiTyper software (28). Each HLA-C type was independently confirmed at the American Red Cross HLA typing laboratory using sequence-specific oligonucleotide primers (29, 30).



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  		FIGURE 2. Heterozygous class I HLA SBT detects new and previously reported polymorphisms. Depicted are overlapping bidirectional sequencing chromatograms of the new allele HLA-Cw*16041 and the previously reported Cw*0303 in exon 3 (the top panel shows the forward sequencing chromatogram and the bottom panel shows the reverse direction). Heterozygous positions are denoted by letters other than A, C, G, or T; a Y at 538 represents the occurrence of a "C" and "T", with the "T" contributing to the new tryptophan at amino acid 156 in Cw*16041.

 
DNA aspects of new alleles

The most surprising aspect of this study was the number of new alleles that were uncovered, in total 19, all of which were confirmed by traditional cloning (31, 32). This number of new alleles gives a rate of ~1 new HLA-C allele in every 100 samples typed. Because HLA-C has historically been characterized as less polymorphic than HLA-A and -B (33), we analyzed the nature of these previously undetected HLA-C polymorphisms to learn more about HLA-C diversification. A total of 29 nucleotide substitutions give rise to the 19 new alleles with only 4 of the new alleles not resulting from a coding substitution; coding substitutions predominate in HLA-C. Twelve of the new HLA-C alleles differ from their closest HLA-C relative by 1 nucleotide while the remaining 7 new alleles differ from their closest relative by >=2 nucleotides (Table IIIGo). On the surface, these single nucleotide polymorphisms indicate that point mutations predominate over recombinations in the generation of HLA-C diversification. However, few (3 of 12) of the point mutations creating new alleles are unique to a particular HLA-C allele such that most point substitutions can be explained through gene conversion with DNA sequences present in another HLA-C molecule. As with HLA-A and -B, intralocus gene conversion in exons 2 and 3 therefore appears to be the primary mechanism driving HLA-C diversification.


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  		Table III. New HLA-C alleles

 
While intralocus conversion events between different class I alleles frequently account for the generation of new alleles, interlocus events between alleles are not common. Two of the new HLA-C alleles reported here, Cw*0203 and Cw*0305, differ by a tight cluster of nucleotides from their closest HLA-C relative, yet no HLA-C allele contains the converted DNA sequences; no apparent HLA-C donor appears to exist. Rather, for Cw*0305 the Thr94/Leu95/Ser97 motif appears to have been donated by an HLA-B allele in a gene conversion event because the Ser97 is only reported among HLA-B molecules. In the case of Cw*0203 the Val152/Leu156 motif does not appear in any HLA-C locus alleles but is found in HLA-A and -B alleles. Allele Cw*0203 therefore appears to have arisen in a conversion event between Cw*02022 and an HLA-A or -B allele. The only previous reports of interlocus conversion events involving HLA-C alleles involve the formation of B*4601 and B*5401, and in these two cases HLA-C sequence motifs were transferred onto HLA-B backbones (34, 35). Here we show that for Cw*0203 and Cw*0305 the HLA-B, and possibly HLA-A, molecules have reciprocated in the interlocus generation of HLA-C polymorphism.

Location and nature of new HLA-C polymorphisms

A predominance of coding substitutions indicates that HLA-C genes are under selective pressure, and the fact that a majority of polymorphisms that result in new HLA-C alleles are focused in the peptide-binding groove further substantiates the functional significance of HLA-C. Twelve of the 19 new HLA-C alleles shown in Table IIIGo are polymorphic at amino acids with side chains positioned to influence peptide presentation (36, 37, 38). Polymorphisms at residues 9 and 66 in the B specificity pocket distinguish the two new alleles Cw*0308 and Cw*0603, and because positions 9 and 66 lie in the B specificity pocket with the HLA-C unique glycine-45, it is likely that peptides bound by Cw*0308 and Cw*0603 are unique at their N termini (36, 39). In a similar fashion, 9 of the new HLA-C alleles listed in Table IIIGo are polymorphic in the E specificity pocket while 6 new alleles have modified F specificity pockets (36, 39). Thus, a majority of the new HLA-C polymorphisms reported here are positioned to preferentially alter the anchoring of peptides near their N and C termini.

While a majority (10 of 14) of the HLA-C amino acid polymorphisms leading to new alleles represent polymorphisms also seen in HLA-A and -B alleles, it is noteworthy that 4 of the new polymorphisms detected in this study are unique to the HLA-C locus (Table IIIGo; Fig. 3Go). For example, while positions 80 and 114 are polymorphic across all three HLA class I loci, three of the new HLA-C alleles (Cw*0306, Cw*0709, and Cw*12042) detected here have amino acids previously unseen at one of these two positions. Furthermore, the new alleles Cw*1206 and Cw*0405 have the unique amino acids Val120 and Leu28, respectively; these positions are conserved across all other HLA class I alleles (Table IIIGo). Polymorphisms unique to the HLA-C locus therefore continue to be propagated both inside (positions 80 and 114) and outside (positions 28 and 120) of the Ag-binding groove.



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  		FIGURE 3. A ribbon diagram of the HLA class I heavy chain illustrating that polymorphic positions detected in 19 new HLA-C alleles are focused about the Ag-binding groove. Positions of polymorphism are colored black, and positions with residues unique to HLA-C molecules are numbered.

 
HLA-C comparative analysis

The detection of 19 new alleles demonstrates that class I DNA SBT is capable of unveiling characteristics of HLA-C not appreciated with lower resolution typing methods. Indeed, SBT provides more precise data concerning the distribution of existing HLA-C subtypes. For example, from the SBT typing data, it was found that Cw*0701 was the most common allele with an overall frequency of 15.66%, and the most prevalent genotype was Cw*0701, Cw*0702. Contrasting the high incidence of Cw*0701 was the less frequent Cw*0704 at a frequency of 1.73% and the complete absence of Cw*0703 and Cw*0705 in this sample set. Perhaps one of the most interesting findings was that 28% of the samples contained "serologic blanks," Cw12-18, which would be undefined by serology due to a lack of suitable reagents. That 28% of the samples typed here contained at least one serologic Cw12-18 blank suggests many previously typed homozygous samples may well be heterozygotes. Indeed, previous studies have shown 18% homozygosity by PCR sequence-specific primers and up to 50% homozygosity by serology (40, 41). In comparison, we found the rate of HLA-C homozygosity to be 9.8% in the NMDP research repository samples, suggesting that like the HLA-A and -B loci, a heterozygous state of ~90% is favored at the HLA-C locus.

The NMDP research pool contains ethnically diverse samples of which the vast majority are Caucasian at an overall frequency of 66.85%. Of the 61 HLA-C sequences now in the database, our SBT detected 48 in the NMDP research repository samples. The 12 HLA-C alleles not detected here were primarily of restricted ethnic origin (Table IVGo). Construction of an evolutionary tree shows that alleles not detected in this study, as well as new alleles detected here, were dispersed among all of the HLA-C families such that no apparent SBT bias exists in the detection of HLA-C alleles (Fig. 4Go). The Figure 4Go dendrogram illustrates that new alleles were detected in the Cw2, Cw3, Cw4, Cw7, Cw8, and Cw12, 14, 15, 16 families, while the alleles not detected were dispersed in a similar fashion. Thus, the SBT approach applied herein did not favor the detection of alleles in any particular HLA-C family, nor do alleles in any given HLA-C family represent SBT "blanks."


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  		Table IV. Alleles not detected in study group

 


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  		FIGURE 4. New and undetected HLA-C alleles are evenly dispersed among the HLA-C families. An unrooted HLA-C phylogenetic tree generated by the neighbor joining/UPGMA method (50) using exons 2 and 3 demonstrates that new HLA-C alleles detected in this study (bold) and HLA-C alleles not detected in this study (shadowed) are not clustered together, therefore demonstrating that the typing of HLA-C alleles herein was unbiased.

 
Alternative typing approaches detect new HLA class I alleles with differing degrees of success, and we report the rate at which DNA SBT detects new HLA-C alleles as 1 new allele in every 100 individuals typed. Three factors indicate that a 1/100 rate of finding new HLA-C alleles is a conservative estimate of the actual number of new alleles present in the population: 1) the samples analyzed here were predominantly Caucasian whereas new HLA-A, -B, and -C alleles tend to be found at a greater frequency in non-Caucasians (42, 43); 2) the SBT approach described herein does not detect new polymorphisms outside of exons 2 and 3 such that some new alleles may escape detection; 3) a small percentage of our samples generate an ambiguous HLA-C type (heterozygous SBT of an Cw*0802/1602 individual produces the same SBT as an Cw*0501/1601 individual) such that some heterozygous individuals with a new allele could produce a SBT identical with a combination of known HLA-C alleles. The detection of 1 new HLA-C allele in every 100 samples is therefore a conservative estimate of the frequency of new alleles in the population.

Given a Caucasian ethnic disposition and a precise means of typing, it was anticipated that a majority of the samples exhibiting new alleles would stem from the Caucasian population. This is indeed the case as 13 of 19 new alleles were found in Caucasian individuals. A most interesting observation, however, was that new alleles found in non-Caucasians were often observed in multiple samples, whereas new alleles detected in samples of Caucasian origin were generally observed in single samples. For example, in Caucasians a new allele was seen in no more than 3 samples (Cw*02024 and Cw*16041), while Cw*03042 was detected in 6 African American samples and 1 Asian Pacific Islander and Cw*02024 was detected in 10 African Americans and 2 Hispanics. This suggests that new alleles are "hidden" in the Caucasian population although common in ethnic minorities. On the basis of this observation, we hypothesize that the SBT of non-Caucasian populations will reveal many more new alleles, ultimately demonstrating that HLA-C polymorphism is equivalent to that of the HLA-A and HLA-B loci.

HLA class I comparative analysis

The ribbon diagram of Figure 3Go illustrates that four of the new HLA-C alleles detected here result from unique polymorphisms (i.e., either location or amino acid residue) as compared with HLA-A and -B molecules. A more encompassing comparison of all HLA-C amino acid sequences demonstrates that this locus is distinguished by several unique characteristics (Fig. 5Go). In building the HLA-C database to 61 alleles, we were able to revisit an earlier observation that the {alpha}1 {alpha} helix of the HLA-C molecules is relatively conserved as is the glycine residue at amino acid 45 (33). Indeed, the distribution of polymorphic residues around the HLA-C peptide-binding groove is not uniform in that the {alpha}1 {alpha} helix remains highly conserved in comparison with other regions of the molecule: within the {alpha}1 and {alpha}2 {alpha} helices, 16 polymorphisms were noted with 5 of these located on the {alpha}1 {alpha} helix (Table VGo; Fig. 5Go). Conservation of the {alpha}1 {alpha} helix is positioned to affect Ag presentation and receptor engagement. For example, conservation of the {alpha}1 {alpha} helix might allow NK receptors specific for HLA-C to position themselves on the conserved portion of the {alpha}1 {alpha} helix for examination of the polymorphic HLA-C NK epitope at amino acids 77 and 80 (44). Alternatively, it has been proposed that polymorphisms in the {alpha}1 {alpha} helix induce major changes in the peptides bound by class I HLA-B molecules, and a conserved {alpha}1 {alpha} helix might therefore conserve a particular HLA-C peptide presentation property (45).



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  		FIGURE 5. A ribbon diagram of the HLA class I heavy chain illustrating polymorphic positions located within all HLA-C molecules. Positions of polymorphism are colored black, and residues unique to the HLA-C molecules are numbered.

 

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  		Table V. Number of polymorphic positions of exons 2 and 3 of HLA-C1

 
When considering the peptides presented by HLA-C molecules, an HLA-C unique glycine at 45 is conserved among the 61 HLA-C molecules compared. Also apparent in Figure 5Go is the clustering of HLA-C-unique amino acids at the end of the groove where the N termini of bound peptides are found: on the floor of the groove positions 6, 28, 45, 49, and 50 are HLA-C-unique while another cluster of HLA-C unique residues at 170, 173, 175, and 177 distinguish the end of the {alpha}2 {alpha} helix from HLA-A and -B loci. The functional implication of these HLA-C-specific characteristics is difficult to decipher because comparatively little is known pertaining to the presentation of peptides and the receptor ligands that recognize HLA-C molecules and their peptide cargo (46, 47, 48, 49). For example, it is unclear how the unique cluster of amino acids on the floor of the groove near the 45 pocket relates to the observation that many HLA-C ligands do not have a dominant P2 anchor, and it is also unclear how the HLA-C-unique residues near the end of the {alpha}2 {alpha} helix affect the positioning of peptides in the 45 pocket. However, the observation that dominant and strong anchors are more prevalent in the middle (P4–P6) of HLA-C ligands than for HLA-A and HLA-B suggests that these HLA-C-unique residues impact how the N termini of peptide ligands lie in the groove (44).

In summary, application of HLA-C DNA SBT provides structural, and therefore functional, insights into HLA-C that were previously unrealized. The 19-allele increase in HLA-C diversity reported here demonstrates that the polymorphic nature of the HLA-C locus has been underestimated and that greater HLA-C diversity will become apparent as ethnic minorities are typed via DNA sequencing. That HLA-C polymorphism, the generation of this polymorphism, and the frequency of heterozygotes resemble the HLA-A and HLA-B loci more than previously realized suggests that the pressures driving HLA-A and HLA-B evolution also act on HLA-C. However, specific characteristics such as a conserved {alpha}1 {alpha} helix, a conserved Gly45, and clustering of unique polymorphisms around the 45 pocket demonstrate that some pressures faced by HLA-C molecules are unique to that locus. Although the precise role that HLA-C plays in the adaptive immune response, transplant compatibility, and autoimmunity is unclear, the results presented here illustrate that understanding the function of particular HLA-C alleles will first require a means to distinguish among varied HLA-C subtypes.


   Acknowledgments
 
We thank Tara Bennett for manuscript preparation.


   Footnotes
 
1 This study was funded by National Marrow Donor Program Contract 7105 (W.H. and M.F.-V.) and by Department of Defense Office of Naval Research Grant N00014-95-0074 (W.H. and D.C.). D.S. is supported by an Amersham Pharmacia Biotech Postdoctoral Award. Back

2 Address correspondence and reprint requests to Dr. William Hildebrand, Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, P.O. Box 26901, Oklahoma City, OK 73190. Back

3 Abbreviations used in this paper: SBT, sequence-based typing; NMDP, National Marrow Donor Program. Back

Received for publication December 22, 1997. Accepted for publication April 6, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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