837 Braz J Med Biol Res 38(6) 2005 HLA-A typing by PCR-RFLP Typing class I HLA-A gene using a nested PCR-RFLP procedure 1TOXICAN, Departamento de Patologia, Faculdade de Medicina, Universidade Estadual Paulista, Botucatu, SP, Brasil 2Departamento de Genética, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brasil E.C. Castelli1, D.S. Gil1, L.C.S. Veiga2 and J.L.V. de Camargo1 Abstract In order to detect several new HLA-A class I alleles that have been described since 1998, the original PCR-RFLP method developed to identify the 78 alleles recognized at that time at high resolution level was adapted by us for low and medium resolution levels using a nested PCR-RFLP approach. The results obtained from blood samples of 23 subjects using both the PCR-RFLP method and a commercial kit (MicroSSP1A®, One Lambda Inc.) showed an agreement higher than 95%. The PCR-RFLP adapted method was effective in low and medium resolution histocompatibility evaluations. Correspondence E.C. Castelli TOXICAN Departamento de Patologia FMB, UNESP 18618-000 Botucatu, SP Brasil E-mail: erickcastelli@terra.com.br Research supported by FAPESP (Nos. 01/08139-0 and 01/9448-7), and CNPq (No. 302361/2003-0). Received April 30, 2004 Accepted February 10, 2005 Key words • Human leukocyte antigen-A • HLA-A • Typing • RFLP • Polymorphism • Major histocompatibility complex The human major histocompatibility com- plex, also known as the human leukocyte antigen (HLA) complex, is a 4-Mb high- density and polymorphic region located at 6p21.3 with more than 200 genes and which represents about 2.5% of the human chromo- some 6. These genes belong to three main groups, class I, II and III, which are structur- ally and functionally different (1-3). The HLA class I molecules are glycoproteins expressed in almost all nucleated cells that collect intracellular peptide fragments and transport them to the cell surface, where the HLA-peptide combination is presented to CD8+ T cells. Class II molecules are ex- pressed in antigen-presenting cells such as B lymphocytes and dendritic cells, and present peptides to CD4+ T cells. Class III genes encode soluble proteins of the complement system and cytokines like tumor necrosis factor (TNA-α and LTA-α) (1-3). High polymorphism is an important char- acteristic of the HLA complex. By Septem- ber 2004, 1114 alleles had been described for class I genes: 325 for HLA-A, 592 for HLA-B, 175 for HLA-C, 6 for HLA-E, 2 for HLA-F, and 15 for HLA-G. For class II, 707 alleles had been described: 3 for DRA, 458 for DRB, 28 for DQA1, 56 for DQB1, 20 for DPA1, 110 for DPB1, 4 for DMA, 6 for DMB, 8 for DOA, and 8 for DOB (4; http:// www.ebi.ac.uk/imgt/hla/stats.html). The compatibility of these polymorphisms be- tween transplant recipients and organ donors is necessary for graft acceptance and, there- fore, detailed histocompatibility evaluation is required to reduce or avoid tissue rejection (5). The homology among class I genes is a limiting factor for several HLA typing meth- ods and justifies molecular instead of sero- logical approaches (6). Sequence-based meth- ods involving sequence-specific primers (6- 8), sequence-specific oligonucleotide probes (9) and sequencing-based techniques (10) are powerful tools used to assign HLA poly- Brazilian Journal of Medical and Biological Research (2005) 38: 837-842 ISSN 0100-879X Short Communication 838 Braz J Med Biol Res 38(6) 2005 E.C. Castelli et al. morphisms. Their specific uses depend on individual case needs, resolution level, the number of analyses to be performed, and available funds. Laboratories that do not have sequencing facilities or sufficient fi- nancial support for expensive kits may ben- efit from alternative tools for HLA typing. We present here an alternative procedure for HLA-A polymorphism assignment based on a PCR-RFLP methodology originally used by Mitsunaga et al. (11) to analyze 78 HLA- A class I alleles. In order to validate this alternative procedure at low (allele family) and medium (allele family/allele) resolution levels, the present study was carried out to detect all of the 274 alleles described in the literature up to January 2003 (12). The Hos- pital Ethics Committee at this Medical School approved this study protocol. A nested PCR approach with generic primers was the first step for specific ampli- fication of exons 2 and 3, where the poly- morphisms within the HLA class I genes are mainly located. However, due to the great similarity of class I genes and the presence of pseudogenes in the HLA complex, the PCR primer used in the original RFLP methodol- ogy (11), ASP5 - GCCCCGAACCCTCSTC CTGCTA/ASP3 - CCGTGGCCCCTGGT ACCCGT, frequently also amplify other class I genes. The HLA-B and -C loci have only one or two mismatches in the region of the original ASP5, depending on the polymor- phism of each individual. As a consequence, these loci usually provide PCR products of the same size (not distinguished by common electrophoresis) and provide the same RFLP patterns for most endonucleases applied, confusing the analysis. Therefore, a new forward primer (ASPTBE) with 3 terminal mismatches was developed in our laboratory to be used with the original ASP3 primer. Initially, amplifi- cation of genomic DNA with generic prim- ers (ASPTBE, CAGACSCCGAGGATGG CC/ASP3 - CCGTGGCCCCTGGTACCC GT) was performed to obtain a 1017-bp sequence including exon 1 to intron 3 of the HLA-A gene. This PCR product was diluted 1:1000 in TE buffer (10 mM Tris-HCl, pH 7.5, and 1 mM EDTA) and used as template for two further PCR procedures to amplify separately exon 2 (NA23 - GKCCTCGCTCT GGTTGTAGTAGC/NA25 - CAGGCTCY CACTCCATGAGGTATTTC primers) (11) and exon 3 (NA33 - CGTCTCCTTCCCGTT CTCCAGGT/NA35 - GTCSGGGCCAGGT TCTCACAC primers) (11). DMSO was in- cluded in all reactions to increase PCR speci- ficity. Restriction analyses were performed for exons 2 and 3 with endonucleases that were chosen on the basis of the resolution level needed (see below). In order to obtain a low resolution level, the PCR-RFLP methodology was adapted to permit the separate evaluation of each HLA- A allele family. After amplification of exons 2 and 3, RFLP analyses were performed. Endonucleases Bsp1286I, BsrI, BstNI, MnlI, HinfI, MspI, SacII, and PstI were used for exon 2, and endonucleases BslI, Fnu4HI, BsrI, Bsp1286I, HaeIII, NlaIII, HhaI, and MspI were used for exon 3, for a total of 16 RFLP analyses (with 13 different endonu- cleases). Tables with the RFLP patterns for each allele family are available from the corresponding author. To obtain a level of medium resolution, the endonucleases originally proposed (11), BsaJI, Bsp1286I, BsrI, BstNI, HinfI, MspI, MnlI, and SacII for exon 2, and BsaAI, BslI, BsoFI, Bsp1286I, BsrI, HaeIII, HgaI, HhaI, MspI, MnlI, and NlaIII for exon 3, are not suitable because they do not permit the de- tection of the recently described and specific high frequency HLA-A alleles. Therefore, other restriction endonucleases were added in order to increase the resolution and/or to facilitate the detection of heterozygous com- binations. For exon 2 analysis, 12 enzymes were added: PstI and NlaIV for standard digestion and TspRI, AvaII, BslI, HhaI, NlaIII, HgaI, BsaHI, HaeIII, FokI, and HphI for additional digestion. For exon 3, seven en- 839 Braz J Med Biol Res 38(6) 2005 HLA-A typing by PCR-RFLP donucleases were added: TspRI and NlaIV for standard digestion and MspA1, BsaJI, BstNI, SacII, and HphI for additional diges- tion. The RFLP pattern for each HLA-A allele and the endonucleases proposed for the medium resolution level are available in PDF format (contact the corresponding au- thor). Currently, 325 alleles for the HLA-A locus are known (4) and new ones are ex- pected to be continuously described; all of these new alleles can be adequately inserted into the technical flowchart presented. The procedure is as follows: genomic DNA was obtained using the GFX® Genom- ic Blood DNA Purification kit (Amersham Pharmacia Biotech Inc., Piscataway, NJ, USA) following manufacturer recommen- dations. The genomic DNA was amplified by PCR using the ASPTBE/ASP3 primers. PCR was performed in a final volume of 20 µl containing 4% DMSO, 0.3 mM of each dNTP, 2.0 mM magnesium chloride, 0.30 µM of each primer, and 1 unit of DNA polymerase (Invitrogen Corporation, Carls- bad, CA, USA). The mixture was heated to 94ºC for 6 min for DNA denaturation fol- lowed by 34 cycles of 94ºC (1 min), 64ºC (1 min), and 72ºC (1 min) each. An 8-µl aliquot of each reaction was checked on 2% agarose gel stained with ethidium bromide. The PCR product was diluted in TE buffer or ultrapure water at 1:1000 and used as template (usu- ally 2 µl) to amplify the HLA-A gene exons 2 (NA23/NA25) and 3 (NA33/NA35). Re- actions for exons 2 and 3 were performed in 100 µl and 120 µl final volumes, respective- ly, containing 3.5% DMSO, 0.25 mM of each dNTP, 2.0 mM magnesium chloride, 0.30 µM of each primer, and 1.5 unit of DNA polymerase (Invitrogen). The reaction mixtures were heated to 64ºC for 3 min for DNA denaturation followed by 34 cycles of 94ºC (1 min), 65ºC (1 min), and 72ºC (1 min) each. After the reaction, 5 µl of each reaction mixture was checked on 2% aga- rose gel stained with ethidium bromide. Aliquots of 7 µl of each PCR product were digested with the restriction endonu- cleases used for the medium resolution level according to supplier recommendations. The cleavage products were subjected to 7% poly- acrylamide gel electrophoresis stained with silver nitrate (Figure 1). The fragment pat- terns generated by the RFLP procedures were compared using an RAS software (RFLP Analysis System) developed in our labora- tory. This software is free with a controlled distribution that uses a database containing all of the patterns of HLA-A allele for each endonuclease proposed in order to calculate which allele may be present in a specific sample. Profiles for HLA-A and HLA-G are currently available (contact the correspond- ing author). The PCR-RFLP approach at the low reso- lution level was able to discriminate amongst all allele families and, in a few cases, a specific allele. This typing tool could be very useful for screening the distribution tenden- cies of HLA-A antigens in diseases. At the medium resolution level the updated nested PCR-RFLP approach permitted the discrimi- nation of heterozygous or homozygous HLA- A allele family combinations with a small group of alleles each, and in some cases, specific alleles. 1 2 3 4 5 6 7 8 9 Non-digested (272 bp) RFLP results (in bp)RFLP results (in bp)RFLP results (in bp)RFLP results (in bp)RFLP results (in bp) 1: 125 + 101 + 87 + 43 2: 125 + 101 + 43 3: 125 + 101 + 43 4: 226 + 125 + 101 + 43 5: 125 + 101 + 43 6: 125 + 101 + 43 7: 125 + 101 + 87 + 43 8: 125 + 101 + 43 9: 125 + 101 + 43 Exon 2Exon 2Exon 2Exon 2Exon 2 MspI endonuclease 10 bp DNA ladder 120 - 100 - 80 - Figure 1. Restriction fragment length polymorphism (RFLP) banding patterns of HLA-A exon 2 digested with endonuclease MspI (lanes 1-9). The results (in bp) are indicated on the right side (7% polyacrylamide gel stained with silver nitrate). 840 Braz J Med Biol Res 38(6) 2005 E.C. Castelli et al. To test the efficiency of the nested PCR- RFLP methodology at a medium resolution level, DNA from blood samples of 23 volun- teers was evaluated using the proposed method and a commercial kit (MicroSSP1A®, One Lambda Inc., Canoga Park, CA, USA), widely employed in histocompatibility tests for organ transplantation. All analyses per- formed using the nested PCR-RFLP meth- odology and the commercial kit were com- patible, assigning either homozygous or het- erozygous combinations (Table 1). Two evaluations using the commercial kit did not detect one of the alleles, probably due to the use of samples under conditions which dif- fered from the kit recommendations, leading Table 1. Comparison between nested PCR-RFLP and a commercial kit (MicroSSP1A®): results of 23 blood samples from normal subjects. The results of the analyses agreed, except for 2 cases for which only one allele family was detected by the commercial kit (weak amplification probably due to low quality DNA). 841 Braz J Med Biol Res 38(6) 2005 HLA-A typing by PCR-RFLP to weak amplifications. However, the RFLP methodology detected both alleles (allele families) in these two samples, in which the single allele assigned by the kit was also detected by RFLP. Although only 23 indi- viduals were tested, the more than 95% agree- ment obtained between the methodologies suggests that the PCR-RFLP technique for HLA-A typing is as sensitive as the commer- cial kit and shows a high level of reliability. Rare alleles are theoretically distinguishable using this approach since RFLP is based on specific endonucleases that recognize spe- cific DNA sequences, but this could not be confirmed due to the absence of samples carrying these alleles. In addition, blood samples from 70 ran- domly chosen individuals were evaluated by the PCR-RFLP methodology to compare their HLA-A distribution with the previously re- ported distribution within the Brazilian popu- lation (13-15). The most frequent HLA-A allele family in Brazil is the A2 group, but representative frequencies also occur for the A1, A3, A24, and A68 groups (13-15). In previous reports of the HLA-A frequency in 3 geographically separate Brazilian subpopu- lations, Paraná (Southern Brazil), Pernam- buco (Northeastern Brazil) and Minas Gerais (Southeastern Brazil), the frequencies were around 8-10% for HLA-A1, 26-29% for HLA-A2, 8-14% for HLA-A3, 10-14% for HLA-A24, and 5-9% for HLA-A68. In the present study, the frequencies of these alle- les were 10% for A1, 27% for A2, 8.6% for A3, 7% for A24, and 8% for A68, showing orders of magnitude similar to those of the frequencies reported (13-15). Therefore, the more representative alleles reported for a highly heterogeneous population like the Brazilian one demonstrated a good resolu- tion with the nested PCR-RFLP methodol- ogy and a high reliability using the typing procedures reported in the present study. The alternative methodology for HLA-A polymorphism assignment proposed here was shown to be adequate for low or medium resolution levels. Furthermore, it also per- mitted the positive discrimination of het- erozygous combinations, as do other typing methods (7,16). This may be very useful since the use of commercial kits is relatively expensive for each individual sample. Al- though the nested PCR-RFLP methodology is more laborious, with a large initial invest- ment in endonucleases, HLA-A typing using this procedure is cheaper since the endonu- cleases can be used for several analyses, and are at least as sensitive, if not more so, than generic commercial kits. Acknowledgments The authors would like to thank the blood donors, Drs. Elida Benquique Ojopi and Maria Inês de Campos Pardini for helpful suggestions and discussions, Maria Luiza Andanaz Falaguera for blood collection, and Bruna Liboni for invaluable technical help. References 1. Doherty PC & Zinkernagel RM (1975). A biological role for the major histocompatibility antigens. Lancet, 1: 1406-1409. 2. Klein J & Sato A (2000). The HLA system. First of two parts. New England Journal of Medicine, 343: 702-709. 3. Undlien DE, Lie BA & Thorsby E (2001). HLA complex genes in type 1 diabetes and other autoimmune diseases. Which genes are in- volved? Trends in Genetics, 17: 93-100. 4. Robinson J, Waller MJ, Parham P, de Groot N, Bontrop R, Kennedy LJ, Stoehr P & Marsh SGE (2003). IMGT/HLA and IMGT/MHC: sequence databases for the study of the major histocompatibility complex. Nucleic Acids Research, 31: 311-314. 5. Fleischhauer K, Kernan NA, O’Reilly RJ, Dupont B & Yang SY (1990). Bone marrow-allograft rejection by T lymphocytes recognizing a single amino acid difference in HLA-B44. New England Journal of Medicine, 323: 1818-1822. 6. Schaffer M & Olerup O (2001). HLA-AB typing by polymerase-chain reaction with sequence-specific primers: more accurate, less er- rors, and increased resolution compared to serological typing. Tis- sue Antigens, 58: 299-307. 7. Mytilineos J, Lempert M, Scherer S, Schwarz V & Opelz G (1998). 842 Braz J Med Biol Res 38(6) 2005 E.C. Castelli et al. Comparison of serological and DNA PCR-SSP typing results for HLA-A and HLA-B in 421 Black individuals: a Collaborative Trans- plant Study report. Human Immunology, 59: 512-517. 8. Chen DF, Seibert I, Chen HY, Herbst-Kiene I, Pastucha LT & Stangel W (1998). Improvement of HLA class I and class II PCR-SSP typing by using timed-release activity of DNA polymerase. Tissue Anti- gens, 51: 645-648. 9. Williams F, Meenagh A, Maxwell AP & Middleton D (1999). Allele resolution of HLA-A using oligonucleotide probes in a two-stage typing strategy. Tissue Antigens, 54: 59-68. 10. Scheltinga SA, Johnston-Dow LA, White CB, van der ZwA, Bakema JE, Rozemuller EH, van den TwJ, Kronick MN & Tilanus MG (1997). A generic sequencing based typing approach for the identification of HLA-A diversity. Human Immunology, 57: 120-128. 11. Mitsunaga S, Tokunaga K, Kashiwase K, Akaza T, Tadokoro K & Juji T (1998). A nested PCR-RFLP method for high-resolution typing of HLA-A alleles. European Journal of Immunogenetics, 25: 15-27. 12. Marsh SG, Albert ED, Bodmer WF et al. (2002). Nomenclature for factors of the HLA system. Tissue Antigens, 60: 407-464. 13. Middleton D, Williams F, Meenagh A, Daar AS, Gorodezky C, Hammond M, Nascimento E, Briceno I & Perez MP (2000). Analysis of the distribution of HLA-A alleles in populations from five conti- nents. Human Immunology, 61: 1048-1052. 14. Nigam P, Dellalibera E, Maurício-da-Silva L, Donadi EA & Silva RS (2004). Polymorphism of HLA class I genes in the Brazilian popula- tion from the Northeastern State of Pernambuco corroborates an- thropological evidence of its origin. Tissue Antigens, 64: 204-209. 15. Braun-Prado K, Mion ALV, Pereira NF, Culpi L & Petzl-Erler M (2000). HLA class I polymorphism, as characterized by PCR-SSOP, in a Brazilian exogamic population. Tissue Antigens, 56: 417-427. 16. Moribe T, Kaneshige T & Inoko H (1997). Complete HLA-A DNA typing using the PCR-RFLP method combined with allele group- and sequence-specific amplification. Tissue Antigens, 50: 535-545.