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Volume 54, Issue 1, January-March 2008 Print ISSN 0022-3859 E-ISSN 0972-2823

President, Staff Society M. E. Yeolekar Contents Editor Sandeep Bavdekar EDITORIAL Associate Editors Clinical Trial Registry - India (CTR-I): A meaningful initiative. How to take it Nithya Gogtay forward? D. Muzumdar Bavdekar SB 1 Consulting Editors Vinita Salvi ORIGINAL ARTICLES Pradip Vaideeswar Detection of Rh using two low ionic diluents: Extension of the Manjula Sarkar incubation time and the number of Rh antibodies detected Ombudsman Skaik YA 4 Sunil Pandya Immunophenotypic characterisation of peripheral T lymphocytes in pulmonary tuberculosis Managing Editor Al Majid FM, Abba AA 7 D. K. Sahu Relationship between N-terminal pro-B type natriuretic peptide and extensive Members echocardiographic parameters in mild to moderate aortic stenosis Amita Mehta Cemri M, Arslan U, Kocaman SA, Çengel A 12 Anil Patwardhan Atul Goel Relative efficiency of polymerase chain reaction and enzyme-linked Avinash Supe immunosorbant assay in determination of viral etiology in congenital cataract Keya Lahiri in infants Prafulla Kerkar Shyamala G, Sowmya P, Madhavan HN, Malathi J 17 Pritha Bhuiyan Stomaplasty—anterior advancement flap and lateral splaying of trachea, Rajeev Satoskar a simple and effective technique Sucheta Dandekar Trivedi NP, Patel D, Thankappan K, Iyer S, Kuriakose MA 21 Uday Khopkar CASE REPORTS Advisory Board Thomas B. Ferguson, USA Rhodotorula mucilaginosa as a cause of persistent femoral nonunion Nobuo Hashimoto, Japan Goyal R, Das S, Arora A, Aggarwal A 25 Laurence Klotz, Canada Repeated fracture of pacemaker leads with migration into the pulmonary Nora Noni MacDonald, Canada circulation and temporary pacemaker wire insertion via the azygous vein Anand Malaviya, India Udyavar AR, Pandurangi UM, Latchumanadhas K, Mullasari AS 28 Ana Marusic, Croatia V. Mohan, India Recurrent respiratory papillomatosis complicated by aspergillosis: A case Dan J. Ncayiyana, South Africa report with review of literature G. B. Parulkar, India Kuruvilla S, Saldanha R, Joseph LD 32 David J. Pierson, USA Citrobacter freundii infection in glutaric aciduria type 1: Adding insult to injury Andrew P. Schachat, USA Mukhopadhyay C, Dey A, Bairy I 35 Shirish S. Sheth, India Michael Swash, UK IMAGES IN RADIOLOGY P. N. Tandon, India Jim Thornton, UK Chordoma: A rare presentation as solitary ivory vertebra Jean-Louis Vincent, Belgium Kumar S, Hasan R 37 IMAGES IN PATHOLOGY Intracystic papillary carcinoma associated with ductal carcinoma in situ in a male breast Dragoumis DM, Tsiftsoglou AP 39

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! 68 ii J Postgrad Med January 2008 Vol 54 Issue 1 ! Review Article www.jpgmonline.com

Implications of HLA sequence-based typing in transplantation

Shankarkumar U, Pawar A, Ghosh K

Institute of ABSTRACT Immunohaematology, Serology-based conventional microlymphocytotoxicity HLA typing method, which has been regarded as the gold th 13 Floor, K.E.M standard in organ and hematopoietic stem cell transplantation, has been replaced now by DNA-based typing. Hospital, Parel, Mumbai, Maharashtra, India Many laboratories all over the world have already switched over to molecular methods. Microlymphocytotoxicity- based tissue typing was done using commercial sera, while the molecular typing by genomic DNA based. DNA CCorrespondence:orrespondence: quality and its quantity obtained using various DNA extraction protocols was found to be an important factor in U Shankarkumar the molecular method of tissue typing in transplant outcome. Many polymerase chain reaction-based molecular E-mail: shankarkumar16@ techniques have been adopted with far reaching clinical outcome. The sequence-based typing (SBT) has been hotmail.com the ultimate technique, which has been of the highest reliability in defining the HLA alleles. The nonavailability of specific HLA antisera from native populations, large number of blank alleles yet to be defined and comparable low resolution of HLA alleles in SSP or SSOP technique, suggests that highly refined DNA-based methods like Received : 31-10-06 SBT should be used as an adjunct to HLA serology and/or low/intermediate/high resolution HLA typing in order Review completed : 29-10-07 to achieve a better transplant outcome. Accepted : 13-11-07 PubMed ID : J Postgrad Med 2008;54:41-4 KEY WORDS: HLA-A antigens, sequence analysis, transplantation

he major complex (MHC), a group of different HLA haplotypes and billions of possible diploid T closely linked genes on chromosome 6, encodes the combinations. class I (HLA-A, -B, -C) and class II (HLA-DR,-DQ,-DP) HLA molecules, which, in concert with T-cell receptors, make possible HLA Null Allele the immune recognition of foreign antigens. HLA molecules are also alloantigens that can trigger immune recognition and graft The occurrence of null alleles has important implications in the rejection in unmatched transplant recipients.[1] An enormous strategies that a laboratory adopts to select HLA typing methods effort to define and characterize new HLA alleles has culminated to be used for routine testing. Most of these expression variants in a large number of alleles and continues to grow at a sustained were identified by discrepant results between serologic and pace, making the polymorphism of the linked genes of the HLA DNA-based testing methodologies and serendipitously in other complex truly astounding. As of October 2007, a total of 2941 instances by the identification of novel DNA polymorphisms in alleles 2009 (of class I), 932 (of Class II) and 97 (other loci) the HLA genes. It must be stressed that there may be important comprising of 617 HLA-A, 960 HLA-B, 335 HLA-C, 9 HLA E, biological differences between low-expression (L) and null (N) 21 HLA F, 28 HLA G, 12 HLA H, 9 HLA J, 6 HLA K, 5 HLA alleles. Examples of low expression and null alleles have been L, of class I while, 626 HLA-DRB1, 34 HLA-DQA1, 87 HLA- identified in the A*24 group (A*2402102L, A*2409N, A*2411N DQB1, 23 HLA-DPA1, 127 HLA-DPB1, 4 DMA, 7 DMB, 12 A*2436N, A*2440N, A*2445N, A*2448N, A*2460N, and DOA, 9 DOB, of Class II alleles have been described.[2] Apart A*2483N). While the low-expression alleles may be poor targets form this, 61 MICA and 30 MICB alleles have been described. for and T-cell recognition, these alleles probably have HLA Null alleles have been described in HLA A (43), HLA B sufficient expression levels in the thymus and periphery to result (32), HLA C (7), HLA G (1) in class I while DRB1 (8), DQA1 in tolerance to wild-type alleles (with higher levels of expression (1), DQB1 (1), DPB1 (2), DOA (1) in class II and in MICB (2). and with identical or similar amino acid sequence as one of Further 21 different HLA-class I expression variant alleles; 12 the low-expression alleles). In contrast, individuals carrying a are HLA-A locus alleles and 9 are HLA-B locus alleles have been null allele are able to mount an immune response against the identified. These alleles have high sequence homology with expressed alleles with similar nucleotide sequence. wild-type alleles, but because of mutations in their nucleotide sequence are not expressed on the cell surface (null alleles) or Serology—Molecular Technique a Comparison are expressed at levels that make them undetectable by routine serologic (and possibly cellular) methods. Theoretically, the The usefulness of conventional serologic assays for HLA typing alleles of different loci can combine to produce over 11 million has been limited by the availability of allele-specific sera [Figure

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1]. As antibodies identify structural differences on the surface diagnostic methods. For clinical procedures such as bone marrow of HLA molecules, protein structure differences caused by transplants, detailed genotype information on both the recipient single or limited nucleotide polymorphism particularly within and the donor is required. The SBT is the most comprehensive the peptide-binding groove of the HLA heavy chain are not method for characterizing gene detectable by these techniques. However, these differences are polymorphisms. The SBT involves PCR amplification of specific of functional significance as they determine the specificity and coding regions of HLA genes and sequencing of the amplicons. affinity of peptide binding[3,4] and therefore, T-cell recognition A detailed interpretation of HLA alleles is possible by comparing of self as well as allogeneic target cells.[5,6] For this reason, nucleotide sequences of the HLA gene to an online database functionally significant high-resolution typing of HLA is only of possible allelic combinations[12] (http://www.ncbi.nlm.nih. achievable through molecular methods. gov/mhc/sbt.cgi?cmd=main).

However, serologic techniques and reagents cannot reveal all Most SBT typing strategies currently employed use the exons currently known HLA molecular variants. Whole families of 2 and 3 sequences for HLA class I analysis and exon 2 alone for alleles whose HLA products share serologic markers have been HLA class II analysis. Sequencing strategies used in SBT differ found to encode distinct molecular variants. They can be between laboratories and can generate either heterozygous distinguished by high-resolution techniques, including direct sequences, haploid sequences (after allele separation of sequencing of the corresponding alleles and other sequence- the sample) or a combination of heterozygous and haploid based methods.[7] Current DNA-based methods that are in use sequences for each typed sample [Figure 3]. While SBT permits for HLA typing are polymerase chain reaction-sequence-specific the highest resolution of genotypes, it has its limitations. One priming (PCR-SSP).[8,9] Polymerase chain reaction-sequence- of the problems with SBT is interpretation of ambiguous allele specific oligo hybridization (PCR-SSO),[10] and sequence-based combinations that can occur due to several reasons.[13] typing (SBT).[11] The PCR-SSO and -SSP are powerful methods for detecting genetic variability by identifying sequence motifs Since both the alleles are amplified and sequenced, it is [Figure 2]. However, to maintain the high accuracy of these difficult to determine which two alleles were responsible for methods, the number of probes and primers has to keep up the sequence results. Two or more different allele combinations with the rapidly increasing allelic diversity. Direct sequencing combine to produce identical sequences due to the of genomic DNA or cDNA is being used increasingly in routine heterozygous base pair combinations. For example, in the class I region HLA B*070201, 3503 would have the same nucleotide sequence as HLA B*0724, 3533 in positions 559 and 560 as a result the interpretation of the high-resolution typing cannot be made because it is not known which allele combination is correct. Ambiguity may result from a nucleotide difference outside of the region amplified or when the entire sequence of the amplified region is not known. Resolving the ambiguity can be difficult and laborious. When two alleles differ by a single nucleotide, it may or may not be necessary to resolve the ambiguity because many single nucleotide changes do not affect the function of the HLA molecule. If the ambiguity is a result of an identical heterozygous sequence, a group specific primary amplification,[13] cloning,[14] reference strand conformational analysis (RSCA),[15] pyrosequencing[16] and denaturing high performance liquid chromatography (DHPLC)[17] to produce an unambiguous homozygous sequence can be followed. Some ambiguous allele combinations due to heterozygosity can Figure 1: Serological typing results of modified NIH two-color be resolved by sequence-specific primers (SSP) or sequence fl uorescence technique specific oligonucleotide probes (SSOP) and may be a more viable approach for laboratories already performing these technologies.

Transplantation Outcome

High-resolution HLA typing is increasingly in demand in clinical and experimental settings. In allogeneic transplantation, it is important to know if HLA antigens are expressed or not thus necessitating the need for accurate HLA typing for donor and recipient matching.[18] The HLA matching between the donor and the recipient improves the success of unrelated hematopoietic stem cell transplantation (HSCT). The HSCT Figure 2: PCR-SSOP technique results with phenotypically matched unrelated donors is associated

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Figure 3: HLA A SBT typing results with an increased rate of post-transplant complications mainly These remarkable observations suggest new strategies for the due to serologically undisclosed HLA incompatibilities.[18] selection of bone marrow donors that could improve the odds Retrospective studies have also shown that HLA disparities were of engraftment by categorizing single HLA class I mismatches not necessarily associated with post-transplant complications, according to their implications for the risk of rejection. First, although an overall beneficial effect of HLA classes I and II if the recipient is homozygous for an HLA allele, donors with compatibility is determined by DNA typing methodology.[19] It a single HLA mismatch who are also homozygous for the same has been shown that the use of high-resolution tissue typing allele are preferable. Second, when the single mismatch involves to obtain the best possible HLA match has resulted in an only an allele (e.g., patient type, A*0202, donor type, A*0203), improved transplant outcome.[20] The HPCT involving partially the mismatch predicts no increase in the odds of rejection and mismatched or unrelated donor-recipient pairs require a high- this donor could be accepted as if fully matched. By contrast, resolution typing, but those involving HLA identical siblings a single antigen mismatch (e.g., patient type, A*0202, donor may not. Recently, multivariate analysis in 334 patients from 12 type, A*0302) does increase the risk of rejection and a donor French transplant centers for HLA A, B, C, DRB1, and DQB1 with no antigen mismatches should be sought. loci mismatches revealed that the number of mismatches is strongly associated with overall survival.[21] Due to the important In the allogeneic HPCT settings that utilize unrelated donors role that HLA molecules play in antigen presentation and the or donors and recipients that are not identical by descent, stringency of the relationship between and associated distinguishing null alleles from the expressed alleles (rather than HLA allele, high-resolution typing is increasingly requested for those of low-expression alleles) is of great importance. If only appropriate enrollment of patients into immunization protocols DNA typing methods are used to type donors and recipients aimed at the enhancement of T-cell responses.[22] Molecular and the methods applied cannot distinguish the expressed vs. HLA identity between unrelated donors and recipients of the the null alleles (ambiguities), it is possible to mis-classify donors HPCT, approaching that, which exists between HLA-identical and recipients that are mismatched in the expressed alleles. If siblings, seems to provide the best chance for avoiding graft the donor carries a null allele and the recipient an expressed rejection and other serious complications of the HPCT.[23,24] one, then the transplanted immune system of donor origin will The number of molecular mismatches may be associated be able to mount a response against the recipient’s tissues and with different degrees of risk of graft rejection. In one study, organs (graft vs. host disease). In cases in which the recipient recipients mismatched for a single antigen and homozygous carries a null allele and the donor an expressed one, the recipient at that locus had a greater probability of graft failure than may have preformed antibodies against the donor expressed heterozygous recipients.[25] Homozygosity reduces the number HLA antigens; the presence of donor-specific alloantibodies in of the recipient’s class I molecular targets against which the the patient’s serum may increase the risk for rejection of the donor’s immune cells can react. Fewer class I targets in the transplanted marrow. HLA Class I sequence-based typing (HLA- recipient would then tip the balance toward rejection and away SBT) is intended to identify HLA allelic polymorphisms at the from graft-vs.-host disease as the immune cells of the donor and level of individual nucleotides using genomic DNA amplified the recipient react against one another.[26] by PCR. HLA Class I SBT should be used as an adjunct to HLA

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Class I serology and/or low/intermediate resolution molecular 1997;20:425-30. typing using either SSP or SSOP technology. For clinical 12. Robinson J, Malik A, Parham P, Bodmer JG, Marsh SG. IMGT/ HLA database: A sequence database for the human major purposes, this test should be used whenever possible to identify histocompatibility complex. Tissue Antigens 2000;55:28-37. unrelated allogeneic bone marrow donors who are HLA-A, B and 13. Rozemuller E. Collection and analysis of SBT results data. 13th IHWS C genotypic matches with the intended recipient. Technology Joint Report. In: Hansen JA, editors. Immunobiology of Human MHC, Vol. 1, 2006. p. 413-6. 14. Cox ST, McWhinnie AJ, Robinson J, Marsh SG, Parham P, Madrigal Conclusion JA, et al. Cloning and sequencing full length HLA-B and -C genes. Tissue Antigens 2003;61:20-48. Recent advances in technology have created the ability to 15. Xiangjun L, Kalve I, Larsen P, Parlow M, Ramon D, Wang L. Evaluation of the capability of RSCA to separate HLA-A alleles, which have only provide high-resolution SBT at a high throughput level in a a single base difference. Hum Immunol 2001;62:S151. [27] routine laboratory, but there are many aspects, which need 16. Wang Y, Ramon D, Branden M, Kalve I, Xiangjun L, Wang L. to be contemplated by a laboratory before a decision is made Pyrosequencing based genotyping of the HLA DRB1 locus. Hum to implement this technology. The implications of being able Immunol 2002;63:S98. 17. Etokebe GE, Opsahl M, Tveter AK, Lie BA, Thorsby E, Vartdal F, et al. to provide high-resolution HLA allele typings are far reaching Physical separation of HLA-A alleles by denaturing high performance - not only for the knowledge that will be provided to the HLA liquid chromatography. Tissue Antigens 2003;61:443-50. community, but also for the potential clinical benefits of such 18. Wagner JE, Barker JN, DeFor TE, Baker KS, Blazar BR, Eide C, et information. Due to the strong connection between HLA al. Transplantation of unrelated donor umbilical cord blood in 102 patients with malignant and non malignant diseases: Influence of and immunological response, high-resolution HLA typing is CD34 cell dose and HLA disparity on treatment related mortality and important for vaccine trials, unrelated bone marrow transplant survival. Blood 2002;100:1611-8. and many other areas of clinical interest. 19. Petersdorf EW, Kollman C, Hurley CK, Dupont B, Nademanee A, Begovich AB, et al. Effect of HLA class II gene disparity on clinical outcome in unrelated donor hematopoietic cell transplantation References for chronic myeloid leukemia: The US National Donor Program Experience. Blood 2001;98:2922-9. 1. Parham P. Virtual reality in the MHC. Immunol Rev 1999;167:5-15. 20. Kassar EL. High resolution HLA class I and class II typing and CTLp 2. Marsh SG, editor. IMGT/HLA sequence database. [Last accessed on frequency in unrelated donor transplantation: A single institution 2007 Oct 30]. Available from: http://www.ebi.ac.uk/imgt/hla retrospective study of 69 BMTs. Bone Marrow Transplant 2001;27:35- 3. Parker KC, Bednarek MA, Hull LK, Ultz U, Cunningham B, Zweerink HJ, 43. et al. Sequence motifs important for peptide binding to the human 21. Loiseau P, Busson M, Balore ML Dormony A, Bignon JD, Gagne K, MHC class I molecule, HLA-A2. J Immunol 1992;149:3580-7. et al. HLA association with heamtopoietic stem cell transplantation 4. Rotzschke O, Falk K, Stevanovic S, Jung G, Rammensee HG. outcome: The number of mismatches at HLA-A, -B, -C, -DRB1 and Peptide motifs of closely related HLA class I molecules encompass DQB1 is strongly associated with overall survival. Biol Blood Marrow substantial differences. Eur J Immunol 1992;22:2453-6. Transplant 2007;13:965-74. 5. Sette A, Vitiello A, Reherman B, Flower P, Nayersina R, Kast WM, et al. 22. Bettinoti MP, Marincola FM. HLA and cancer: Immunogenetics in The relationship between class I binding affinity and immunogenicity action. ASHI Quart 2001;25:7-10. of potential cytotoxic . J Immunol 1994;153:5586- 23. Petersdorf EW, Gooley TA, Anasetti C, Martin PJ, Smith AJ, Mickelson 92. EM, et al. Optimizing outcome after unrelated marrow transplantation 6. Rivoltini L, Loftus DJ, Barracchini K, Arienti F, Mazzocchi A, Biddison by comprehensive matching of HLA class I and II alleles in the donor WE, et al. Binding and presentation of peptides derived from and recipient. Blood 1998;92:3515-20. melanoma antigens MART-1 and gp100 by HLA-A2 subtypes: 24. Sasazuki T, Juji T, Morishima Y, Kinukawa N, Kashiwabara H, Inoko Implications for peptide-based immunotherapy. J Immunol H, et al. Effect of matching of class I HLA alleles on clinical outcome 1996;156:3882-91. after transplantation of hematopoietic stem cells from an unrelated 7. Marsh SG, Parham P, Barber LD. The HLA factsbook. Academic Press: donor. N Engl J Med 1998;339:1177-85. San Diego, Calif; 2000. 25. Anasetti C. Hematopoietic cell transplantation from HLA partially 8. Bunce M, O’Neill CM, Barnardo MC, Karusa P, Browning MJ, Morris matched related donors. In: Thomas ED, Blume KG, Forman SJ, PJ, et al. Phototyping. Comprehensive DNA typing for HLA-A, B, editors. Hematopoietic cell transplantation. 2nd ed. Blackwell Science: C, DRB1, DRB3, DRB4, DRB5 and DQB1 by PCR with 144 primer Boston; 1999. p. 904-14. mixes utilizing sequence-specific primers (PCR-SSP). Tissue Antigens 26. Petersdorf EW, Hansen JA, Martin PJ, Woolfrey A, Malkki M, Gooley 1995;46:355-67. T, et al. Major Histocompatibility Complex class I alleles and antigens 9. Krausa P, Browning MJ. A comprehensive PCR-SSP typing system for in hematopoietic-cell transplantation. N Engl J Med 2001;345:1794- identification of HLA-A locus alleles. Tissue Antigens 1996;47:237- 800. 44. 27. Adams SD, Barracchini KB, Simonis TS, Stroncek D, Marincola FM. 10. Ng J, Hurley K, Baxter-Lowe LA, Chopek M, Coppo PA, Hegland High throughput HLA sequence-based typing (SBT) utilizing the ABI J, et al. Large scale oligonucleotide typing for HLA-DRB1/3/4 and prism® 3700 DNA analyzer. Tumori 2001;87:S41-4. HLA-DQB1 is highly accurate, specific and reliable. Tissue Antigens 1993;41:473-9. 11. Bettinotti M, Mitsuishi Y, Bibee K, Lau M, Terasaki PI. Comprehensive method for the typing of HLA-A, B and C Alleles by direct sequencing Source of Support: Nil, Confl ict of Interest: None declared. of PCR products obtained from genomic DNA. J Immunother

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