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HLA-DRB1 * intron-primed sequencing for haploid genotyping.

The HLA genes of the MHC on chromosome 6p manifest extensive allelic polymorphism and haplotype diversity comprising sequence-related genes and pseudogenes. Class II HLA DR molecules consist of noncovalent heteroduplexes derived from paired genes; a variable [beta]-chain with a constant [alpha]-chain. Currently, more than 300 different alleles are recognized for the DRB1* locus (1). Exon 2 of this [beta]-chain gene codes for many of the residues impinging on the peptide binding groove and has known nucleotide variants at more than one-third of 270 base positions. Except in rare homozygous individuals, genotyping of DRB1* produces frequent ambiguity because of shared sequence motifs among alleles and cis-trans uncertainty between polymorphisms.

For histocompatibility typing, DRB1* alleles are traditionally partitioned into groups that reflect serologic lineages. High-resolution genotyping often requires haplotype isolation using group-specific PCR (2, 3) or other methods (4, 5) to exclude some alleles and reduce complexity. Primer sites for group-specific amplification have traditionally involved the hypervariable region at the 5' end of exon 2. Blasczyk et al. (6) and Bergstrom et al. (7) have shown that sequence diversity within introns adjacent to exon 2 is also group-specific and can be used to separate alleles for unequivocal typing (8). Priming from intron sites increases the available region for primer design and expands the number of DRB group and subgroup sets. In addition, intron-primed amplicons include exon 2 in its entirety, defining polymorphisms obscured by, or external to, exon primers.

We assessed a DRB1* typing strategy based on group-level resolution by sequence-specific oligonucleotide (SSO) hybridization, followed by sequencing of intron-primed amplicons, a combination that provides rapid identification of potential DRB1* alleles and subsequent separation of haplotypes for definitive typing. Sequencing results using intron primers were evaluated for resolution of ambiguities and genotyping accuracy.

DNA was extracted from whole blood samples submitted for routine typing or from proficiency surveys. Initial DRB1* typing was performed using a commercial SSO assay based on multiplexed oligonucleotide probes attached to fluorescent microspheres (One Lambda). Intron oligonucleotides as described by Kotsch et al. (8) were synthesized on a Perseptive Biosystems 8900 Synthesizer (ABI) and purified by reversed-phase HPLC (BioCad Sprint-ABI). Sense primers RB1 and RB6 (for DR 1 and DR 4 groups, respectively) were replaced with the alternative primers RB1a (5'-gga agt gtt cac agg gtg aag-3) and RB6a (5'-ggc tgc gtg ttg tcg gg-3'). Table 1 shows the 14 different primer pairs used for DRB1* intron-primed amplification.

Template for sequence-based typing was obtained by adding 50 ng of genomic DNA to PCR buffer [50 mM Tris-HCl, 10 mM KCl, 5.0 mM [(N[H.sub.4]).sub.2]S[O.sub.4], 2.0 mM Mg[Cl.sub.2], pH 8.3] with purified nucleotides, 0.5x GC-rich solution, FastStart Taq polymerase (PCR reagents from Roche), and 20 pmol each of the forward and reverse primers. PCR conditions were the same for all primer pairs and consisted of 10 min of denaturation at 94[degrees]C; 10 two-temperature cycles between 94[degrees]C (30 s) and 65[degrees]C (50 s); and 20 three-temperature cycles of 94[degrees]C (30 s), 62[degrees]C (50 s), and 72[degrees]C (30 s). A 3-mL aliquot was electrophoresed on a 2% agarose gel to confirm the appropriate size band and estimate the concentration by comparison with a molecular mass ruler (Bio-Rad). After PCR, each reaction was digested (15 min at 37[degrees]C) with shrimp alkaline phosphatase (10 U; USB) and exonuclease I (50 U; USB) to remove residual nucleotides and primers. After digestion the reactions were incubated for 15 min at 80[degrees]C to denature the enzymes.

Aliquots of the amplified DNA (30-50 ng) were sequenced in both directions with ABI BigDye terminator mixture (10 [micro]L/reaction) and 3.2 pmol of forward or reverse sequencing primer. Four different forward primers were used: three as described previously (8) and one additional primer [2a(7); 5'-aga ccg ccc ctg tga cc-3'] for DR7 forward sequencing. This primer differs from Seq 2 primer (8) by one nucleotide substitution at the third position (A instead of G) and produces a stronger sequencing signal for DR7 alleles. The reverse primers were the same as the reverse PCR primers except that RB28 replaced RB39 in the sequencing reactions. Cycle sequencing was performed for 25 three-temperature cycles of 96[degrees]C (10 s), 50[degrees]C (5 s), and 60[degrees]C (4 min). The extension products were precipitated by centrifugation in isopropanol (600 mL/L) and run on an ABI Prism 377 automated sequencer with ABI Prism Sequence Analysis software. Sequence Navigator and Factura-HLA were used for editing and allele assignments.

More than 1300 samples were typed for DRB1* by SSO analysis, which completely resolved 13% of alleles to a single genotype. Assignments for the remaining alleles were ambiguous, with probe hybridization patterns matching multiple allele combinations; however, most samples were equivocal between only two DR groups. Failure to differentiate silent or intron polymorphisms was not considered an ambiguity for this assessment.

We reamplified 172 of the samples, using group-specific intron primers selected according to the SSO results. Fifty percent of sequenced alleles were from DR groups 1-4 and 25% were from groups 11 and/or 13. The remaining alleles were distributed among the other DR groups (Table 1). Sequencing produced allele-level resolution in 96% of samples. For 12% of samples, both alleles were assigned to the same DRB1* group, precluding separation with intron primers, but 7% of these were homozygous at DRB1* (exon 2), as confirmed by sequencing, and only seven samples (4%) remained ambiguous. Six of these involved codon 86 Gly/Val dimorphisms, for which specific exon primers were used for subsequent haplotyping (9).

Sequencing of DRB1* genes with intron primers enabled separation of 14 different allele groups or intragroup subsets and, with codon 86 primers, completely resolved 99% of samples. Twenty-five percent of the samples were obtained from proficiency surveys and were in complete agreement with the consensus genotype compiled from participating laboratories.

DNA methods for HLA-DRB typing provide the highest resolution and most complete information of current typing techniques. DRB1* exon 2 genotyping is essential for bone marrow transplantation from unrelated donors and is being performed with increasing frequency for routine histocompatibility testing. The large number of closely related DRB alleles leads to frequent ambiguities that are resolved only by analyzing haplotypes separately.

Our typing strategy used an initial, intermediate-resolution SSO to provide DRB1* group identity for subsequent sequencing, as opposed to first amplifying with all DR group primers and sequencing positive reactions (3). This approach limits the number of secondary PCR reactions required, and only those samples requiring further, allele-level resolution are reamplified.

We have confirmed the use of intron primers for group-specific PCR and DNA sequencing as originally reported by Kotsch et al. (8) to resolve most DRB1* alleles and provide the complete exon 2 sequence. The specificity within intron sequence also extends to DRB3*, -4*, and -5* genes (6, 8), which are coexpressed with certain DRB1* alleles. As a result, confusion resulting from amplification of these or from nonexpressed pseudogenes is avoided. Because sequence data for DRB introns are limited, the possibility of failed amplification because of primer mismatch adds a degree of uncertainty that both DRB1* alleles have been amplified. Previous SSO with generic DRB primers provides some assurance in this regard, whereas positive reactions from intron priming serve to confirm SSO results.

SSO can be performed after allele separation as well, but sequences not covered by oligonucleotide probes remain undefined, and assumptions are made regarding common probe combinations rather than unequivocal linkage. The sequencing of the complete DRB exon 2 and a partial intron sequence could serve to establish the frequency and locations of polymorphisms in this area and validate intron-primed amplicons as suitable templates for HLA typing.

References

(1.) Robinson J, Waller MJ, Parham P, Bodmer JG, Marsh SG. IMGT/HLA Database--a sequence database for the human major histocompatibility complex. Nucleic Acids Res 2001;29:210-3.

(2.) Gao XJ, Fernandez-Vina M, Shumway W, Stastny P. DNA typing for class II HLA antigens with allele-specific or group-specific amplification. I. Typing for subsets of HLA-DR4. Hum Immunol 1990;27:40-50.

(3.) McGinnis MD, Conrad MP, Bouwens AG, Tilanus MG, Kronick MN. Automated, solid-phase sequencing of DRB region genes using T7 sequencing chemistry and dye-labeled primers. Tissue Antigens 1995;46:173-9.

(4.) Olerup O, Zetterquist H. HLA-DR typing by PCR amplification with sequence-specific primers (PCR-SSP) in 2 hours: an alternative to serological DR typing in clinical practice including donor-recipient matching in cadaveric transplantation. Tissue Antigens 1992;39:225-35.

(5.) Paul P, Thomas D, Kawczak P, Good D, Cook DJ, Ball EJ. Resolution of cis-trans ambiguities between HLA-DRB1 alleles using single-strand conformation polymorphisms and sequencing. Tissue Antigens 2001;57:300-7.

(6.) Blasczyk R, Kotsch K, Wehling J. The nature of polymorphism of the HLA-DRB intron sequences is lineage specific. Tissue Antigens 1998;52:19-26.

(7.) Bergstrom TF, Josefsson A, Erlich HA, Gyllensten U. Recent origin of HLA-DRB1 alleles and implications for human evolution. Nat Genet 1998; 18:237-42.

(8.) Kotsch K, Wehling J, Blasczyk R. Sequencing of HLA class II genes based on the conserved diversity of the non-coding regions: sequencing based typing of HLA-DRB genes. Tissue Antigens 1999;53:486-97.

(9.) Shaffer AL, Falk-Wade JA, Tortorelli V, Cigan A, Carter C, Hassan K, et al. HLA-DRw52-associated DRB1 alleles: identification using polymerase chain reaction-amplified DNA, sequence-specific oligonucleotide probes, and a chemiluminescent detection system. Tissue Antigens 1992;39:84-90.

Philip Paul, * Josh Apgar, and Edward J. Ball (Allogen Laboratories, The Cleveland Clinic Foundation, Cleveland, OH 44195; * author for correspondence: fax 216-444-8261, e-mail pp@tt.ccf.org)
Table 1. HLA-DRB1 intron primers. (a)

 DR3, -11,
DRB1 * group specificity DR1 DR2 DR3 DR4 -13, -14

Forward primer, intron 1 RB1a RB3 RB5 RB6a RB9
Reverse primer, intron 2 RB2 RB28 RB28 RB7 RB28
Amplicon size, bp 748 720 760 415 465
Frequency of alleles 0.1 0.14 0.13 0.15 0.01
 sequenced

 DR11, DR3,
DRB1 * group specificity -13, -14 -11, -13 DR12 DR1301/02

Forward primer, intron 1 RB40 RB41 RB8 RB10
Reverse primer, intron 2 RB39 RB39 RB28 RB28
Amplicon size, bp 1018 793 586 463
Frequency of alleles 0.09 0.04 <0.01 0.10
 sequenced

DRB1 * group specificity DR14 DR7 DR8 DR9 DR10

Forward primer, intron 1 RB11 RB12 RB14 RB15 RB22
Reverse primer, intron 2 RB28 RB13 RB28 RB28 RB35
Amplicon size, bp 535 384 784 442 459
Frequency of alleles 0.03 0.12 0.06 <0.01 0.02
 sequenced

(a) Primer sequences are as per Kotsch et al. (8), except for
alternative intron 1 primers RB1a (nucleotide positions 48-68)
and RB6a (nucleotide positions 340-356), which are numbered
according to Blasczyk et al. (6).
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Title Annotation:Technical Briefs
Author:Paul, Philip; Apgar, Josh; Ball, Edward J.
Publication:Clinical Chemistry
Date:Apr 1, 2003
Words:1789
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