Molecular HLA Typing


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Molecular HLA Typing: An Overview

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Molecular HLA Typing

  1. 1. Molecular HLA TypingSalwa Hassan TeamaMolecular Biology Department/ Medical Research CenterAin Shams University/ Cairo/ Egypt
  2. 2. Contents Major Histocompatibility Complex MHC Genetics Human Leukocyte Antigens Inheritance HLA and Transplantation HLA Polymorphism and Nomenclature Molecular HLA Typing
  3. 3. Human Leukocyte Antigen (HLA)The term HLA refers to Human Leukocyte Antigens.HLA proteins found in the membranes (outer coating) ofnearly every cell in the body (all cells that have anucleus). These antigens are in especially highconcentration on the surface of white blood cells(leukocytes).The HLA loci are part of the genetic region known as themajor histocompatibility complex.
  4. 4. Major Histocompatibility ComplexThe MHC system in humans was subsequentlydiscovered in early 1950s.The MHC has genes (including the HLA) that form partof the normal function of the immune system.The MHC is an extreme gene-dense region of thegenome, and it can be divided into three sub-regions; theclass I, the class and the class III regions. All encoded bya gene complex located on the short arm of 6.
  5. 5. MHC GeneticsThe class I region of approximately 2000 kilobases include;the polymorphic HLA-A, B, C loci; non classical class HLA-E, F, G,..The class II region of approximately 1000 kilobases include;HLA-DR,DQ an DP loci, ……….and non classical class HLAclass II HLA-DM, DO,.The class III region of approximately 1000 kilobases encodegenes with diverse functions and does not contain any HLAgenes. Contain loci responsible for the complement,hormones,…
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  7. 7. Expression of MHC Genes
  8. 8. Histocompatibility Antigens (HLA)Histocompatibility antigens: A set of alloantigens, i.e.tissue surface antigens that differ among members of thesame species, which were significant as targets for theallograft reaction.Histocompatibility antigens serve as self-recognitionmolecules, they help to trigger the T cell dependentimmune response and actively participate in the immuneelimination of cells infested with foreign substance.
  9. 9. Histocompatibility AntigensThe genes encoding histocompatibility antigens arelocated within the MHC class I and class II.The class III region contains genes encoding moleculesinvolved in immune function that are not targets forallorecognition.
  10. 10. StructureThe class I molecules are heterodimers composed of twononcovalently, linked polypeptide chains, a polymorphic heavychain subunit encoded by genes in the MHC complex onchromosome 6 with a nonvariable light chain subunit ( 2-microglobulin) that is encoded by a gene located outside theMHC (chromosome 15).The Class II molecules are heterodimer as well and arecomposed of two transmembrane proteins, the alpha and the betachains.
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  12. 12. Antigen Processing and Presentation
  13. 13. HLA Polymorphism and NomenclatureHLA is extremely polymorphic. It is one of the mostpolymorphic genetic system in man. Associated witheach of the loci is a set of alleles, each of which giverises to the production of a unique antigenic specificitythat is expressed on cell surface and that can bedetected by specific antibodies or immunologicallyactivated T cells.
  14. 14. InheritanceMost individuals inherit a set of nonrecombined HLA alleles fromeach parent.These genes are codominantly expressed. Thus if the HLA typesof family member are determined, segregation of HLA typeswithin the family can be used to construct the HLA types fromeach chromosome.The set of HLA alleles found on one chromosome is called ahaplotype. Determination of haplotype is important foridentification of HLA identical siblings because sharing ofantigen from different haplotypes is common.
  15. 15. HLA typing plays an important role in medicine: The primary target of immune responses to allogenic transplants. Critical for response to antigenic stimuli. Implicated in genetic susceptibility to autoimmune disease.
  16. 16. Typing Resolutionand Techniques
  17. 17. Source: Wikipedia
  18. 18. Typing Resolution and TechniquesLow resolution or generic, typing that definesgroups of alleles usually approximating serologicspecificities (HLA-DRB1-04).High resolution, typing methods that resolve allknown alleles (HLA-DRB1*0401).Intermediate resolution, typing that resolves HLAtypes beyond serologic specificities but does notachieve the allele level.
  19. 19. Selection of Histocompatibility TestsU, usually V, variable R, rarely I, increasing use
  20. 20. Molecular HLA TypingDNA based procedures has increased the accuracy ofHLA typing and lead to the identification ofserologically undetected alleles and of many subtypesof serological specificities.DNA based methods to type for HLA alleles havetherefore focused in the analysis of nucleotide variationoccurring in both exon 2 and 3 of class I genes andexon-2 of class II genes.
  21. 21. HLA specificities identified by serology versus Molecularmethods (February 2007)
  22. 22. PCR Based Methods / Three CategoriesPolymorphism is identified directly as part of the PCRprocess, although there are post amplification steps e.g.(SSP).Product containing internally located polymorphism thatcan be identified by a second technique e.g. PCR sequencespecific oligonucleotide probing (SSOP), PCR-RFLP, PCRfollowed by sequencing.Conformational analysis
  23. 23. Steps of Molecular Typing Extraction of genomic DNA Amplification of the genes of interest and Detection of sequence polymorphism that define the alleles.
  24. 24. DNA Extraction Genomic DNA extracted from nucleated cells. The purity of the DNA extracted may be an important factor for successful results.
  25. 25. Gene Amplification/ PCR The specificity of the amplification can be locus specific e.g. (HLA-A, HLA-B, HLA-DRB1), group specific. e.g.(DRB1- 01 , DRB1-02) or allele specific (DRB1-0401 ,DRB1*0402). The primers are selected to amplify a single allele or a group of alleles. For PCR, the specificity is determined by the sequence of the primers and amplification condition. Most typing scheme require conditions that avoid the coamplification of pseudogene.
  26. 26. Gene Amplification/ PCR Amplification of exon 2 (Approximately 270 bp) of HLA class II is sufficient to achieve the highest resolution level. For typing HLA class I, both exon 2 and 3 and the intervening intron (fragment longer than 900 bp) are amplified by single pair of primers.
  27. 27. Detection of the sequence polymorphism that define the alleles Sequence specific primers (SSP) (group and alleles specific primers). Hybridization with Sequence specific oligonucleotide probes (SSOP). Sequence based typing (SSP)
  28. 28. Sequence Specific PrimingSSP is a rapid method of typing that uses sets of primerpairs to amplify specific region of genomic DNA.The efficiency of the amplification reaction iscontrolled by the primers that amplify conservedsequences of a selected gene.
  29. 29. SSP SSP
  30. 30. Sequence Specific Oligonucleotide ProbeSSOP: this method involves selective amplification oftarget followed by hybridization to a panel ofoligonucleotide probes.Specificity for a particular HLA locus was achievedby selecting PCR primers specific for a sequence inthe conserved region of the second exon.
  31. 31. SSOPAfter the PCR amplification process, the amplicons are chemically denatured to form single-stranded DNA, these are added to a nylon membrane whichcontains an array of immobilized, sequence-specific oligonucleotide (SSO) probes. The biotin-labelled amplicons then bind (hybridise) to those SSOprobes that contain a complementary target sequence and thus are "captured" onto the membranestrip.
  32. 32. Sequence Based HLA TypingSequence based HLA typing involvesdetermining the nucleotide sequence of anamplified segment of an HLA gene. SBT presents the advantages over the otherprocedure because of the relatively fast (24-48)hrs, high level resolution.
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  34. 34. Conformational AnalysisDifferent mutation generates specific conformationalchanges in PCR products. These are identified byelectrophoresis analysis. e.g. heteroduplex analysis,single strand conformation polymorphism (SSCP),denaturing gradient gel electrophoresis (DGGE) andtemperature gradient gel electrophoresis (TGGE).
  35. 35. Comparison ofDNA TypingMethods
  36. 36. Advantages of the DNA Based Typing Methods Greater accuracy High level of resolution compared with serological typing.
  37. 37. Greater extent of HLA matching results in better graft survival, less GvHD, andrequire less immunosuppression following transplantation
  38. 38. References and Online Further Reading Lawlor DA, Zemmour J, Ennis PD, ET AL. Evolution of Class I MHC genes and proteins; from natural selection to thymic selection. Annu Rev Immunol 1990;8:23-63. So A. Genetic polymorphism and regulation of expression of HLA region genes. In: Lecher A. HLA and Disease. San Diego; Academic Press. Inc., 1994: 1-34. Daniel P. Stites,Abba T. Terr. Basic Human Immunology: ISBN. 0838505430. Dyer P, Middleton D Histocompatibility testing .A practical Approach. IRL Press,1993. Annia Ferrer et al., Genomic structure of the human MHC. Biotechnologia Applicada. 2005,vol.22,No.2
  39. 39. Thank You