Presentation is loading. Please wait.

Presentation is loading. Please wait.

HLA TYPING D Middleton MDSC175: Transplantation Science for Transplant Clinicians (Online) POSTGRADUATE SCHOOL OF MEDICINE A MEMBER OF THE RUSSELL GROUP.

Similar presentations


Presentation on theme: "HLA TYPING D Middleton MDSC175: Transplantation Science for Transplant Clinicians (Online) POSTGRADUATE SCHOOL OF MEDICINE A MEMBER OF THE RUSSELL GROUP."— Presentation transcript:

1 HLA TYPING D Middleton MDSC175: Transplantation Science for Transplant Clinicians (Online) POSTGRADUATE SCHOOL OF MEDICINE A MEMBER OF THE RUSSELL GROUP CONTINUING PROFESSIONAL DEVELOPMENT

2 2 HLA TYPING Genes and Chromosomes The MHC is a cluster of genes located on the short arm of chromosome 6. Class I genes (HLA-A,-B & -Cw) encode antigens which present peptide to CD8 + T-cells. Class II genes (HLA-DR,-DQ & -DP) encode antigens which present peptide to CD4 + T-cells. Many Class III genes (e.g TNF-alpha, C2 & C4 complement genes) are involved in aspects of the immune response.

3 3 HLA TYPING

4 4 Major Histocompatibility Complex Chromosome 6 TelLong armCenShort armTel 6p Class IIClass IIIClass I DPDMDQDRC4C2Hsp70TNFBCEAGF HLA Region Gene map of the human leukocyte antigen (HLA) region Expert Reviews in Molecular Medicine © 2003 Cambridge University Press Bf

5 5 HLA TYPING HLA Polymorphisms Most polymorphic system in the genome Related to function of peptide presentation Most polymorphism in peptide binding region driven by natural selection High level of coding (non-synonymous) mutations in PBR Some alleles common (gene freq >0.1%), others rare Ethnic variations

6 6 HLA TYPING HLA Class I Gene (A,B,C) 456 5’UT SS 33 TM C1 C2 C3 2 1 22 378 11 polymorphism in exons 2 & 3 encoding  1 and  2 domains molecular domains α2α2 β 2 - microglobulin α3α3 α1α1 Peptide binding cleft

7 7 HLA TYPING HLA Class II B Gene (of the Class II A genes, only DPA and DQA are significantly polymorphic) 345 5’UT SS 2 2 TM/C C polymorphism in exon 2 encoding  1 domain 21 11 3’UT molecular domains β2β2 α1α1 Peptide binding cleft β1β1 α2α2

8 8 HLA TYPING Linkage Disequilibrium The observation that two or more alleles at two are more loci in a population are associated more or less frequently than would be predicted from their individual frequencies. The non-random association of MHC alleles in a population.

9 9 HLA TYPING Structure HLA Class I  polypeptide chain with 3 domains +  2 microglobulin α2α2 β 2 - microglobulin α3α3 α1α1 Peptide binding cleft

10 10 HLA TYPING Structure Class II β2β2 α1α1 Peptide binding cleft β1β1 α2α2

11 11 HLA TYPING HLA Nomenclature HLA-A*02::: N HLA Prefix Gene Field 1; allele group Field 3; used to show a synonymous DNA Substitution within the coding region Field 3; used to show a synonymous DNA Substitution within the coding region Field 2; specific HLA protein Field 4; used to show differences in a non-coding region Separator Field Separators Hyphen used to separate gene name from HLA prefix Suffix used to denote changes in expression © SGE Marsh 04/10

12 12 HLA TYPING Why HLA Type?

13 13 HLA TYPING HLA A+B+DR Mismatches First Cadaver Kidney Transplants Collaborative Transplant Study

14 14 HLA TYPING HLA A+B+DR Mismatches Deceased Donor Kidney Transplants Collaborative Transplant Study

15 15 HLA TYPING DNA Typing HLA-DRB Mismatches Cadaver Kidney Transplants Mismatch HLA-DR Serology Collaborative Transplant Study

16 16 HLA TYPING HLA A+B+DR Mismatches First Orthotopic Heart Transplants Collaborative Transplant Study

17 17 HLA TYPING HLA-A+B+DR Mismatches First Liver Transplants Collaborative Transplant Study

18 18 HLA TYPING HLA A+B+DR Mismatches First Cadaver Kidney Transplants Cold Ischemia ≤6 Hours Collaborative Transplant Study

19 19 HLA TYPING Cold Ischemia Time – Shared/Local First Cadaver Kidney Transplants and 6 HLA-A+B+DR Mismatches Collaborative Transplant Study

20 20 HLA TYPING Inheritance Chart

21 21 HLA TYPING Kidney Transplants First Grafts Collaborative Transplant Study

22 22 HLA TYPING Unrelated Living Donors HLA-A+B+DR Mismatches Europe or North America Collaborative Transplant Study

23 23 HLA TYPING HLA-A+B+DR Mismatches Deceased Donor Kidney Transplants Collaborative Transplant Study

24 24 HLA TYPING Donor Age Related Donor Kidneys, First Grafts Collaborative Transplant Study

25 25 HLA TYPING Donor Age Deceased Donors, First Grafts Collaborative Transplant Study

26 26 HLA TYPING HLA-A+B+DR Mismatches Deceased Donor Kidneys Transplants Donor Age <40 Collaborative Transplant Study

27 27 HLA TYPING How to HLA Type

28 28 HLA TYPING SSP Primer matchPrimer mis-match 5’3’ 5’ 3’ 5’ Amplification No Amplification Positive control Specific products Agarose gel visualisation

29 29 HLA TYPING PCR-SSP Phenotype

30 30 HLA TYPING HLA Typing by PCR-SSOP Single generic PCR (using conserved sequence for primers) Hybridisation with multiple oligonucleotide probes on solid support (tray/membrane/bead) Positive/negative hybridisation identifies presence/absence of allele or allele group

31 31 HLA TYPING Principle of PCR-SSOP (using HLA-A as an example) EXON 2EXON 3 A*01 A*02 A*03 A*11 A*24 allele-specific probe sequences conserved sequence ( = 1 generic PCR per locus) intron 2

32 32 HLA TYPING HLA Typing by PCR-SSOP 1. patient DNA locus specific PCR (e.g. one primer is biotin labelled) 2. single stranded PCR product (NaOH or asymmetric PCR) 3. hybridise with specific probes bound to tray/membrane/ Luminex beads 4. detection of bound DNA (e.g. streptavidin)

33 33 HLA TYPING PCR-SSOP by Luminex Polystyrene beads of uniform size are used as the solid phase Each bead is dyed with two different fluorochromes – ratio Gives 100 distinctively coloured bead populations SSO’s attached to Luminex beads, multiplex reactions

34 34 HLA TYPING

35 35 HLA TYPING 2 nd Generation Sequencing High seq capacity allowing parallel analysis of amplicons for all relevant exons 250bp read spans most of relevant regions Pooling of amplicons from different individuals (48) needed to make cost effective Software needed for phasing of the amplicons and to filter out related sequence reads- coamplified Tissue Antigens 74,

36 36 HLA TYPING Which Method to Use Clinical Urgency Clinical Resolution Sample numbers Budget Equipment availability Staff skills Combination of methods

37 37 HLA TYPING

38 38 HLA TYPING Matching 1 Difference in AA. What is important? Quantity Position = Function Which Loci Resolution required By epitopes not by current nomenclature Rationalisation of alleles we test for

39 39 HLA TYPING Matching 2 Different approach for different patients Difference immunogenicity of HLA mismatches Genetic factors influencing immune response? (including innate response and immunosuppressive sensitivity) Intelligent mismatching Acceptable mismatch program

40 40 HLA TYPING Number of HLA Antigens and Alleles

41 41 HLA TYPING AllelePopulation Phenotype Frequency (%) Allele Frequency (3 decimals) Sample Size A*0201American Samoa A*0201Argentina Gran Chaco Eastern Toba A*0201Argentina Gran Chaco Mataco Wichi A*0201Argentina Gran Chaco Western Toba Pilaga A*0201Argentina Toba Rosario A*0201Australia New South Wales A*0201Australian Aborigine Cape York Peninsula A*0201Australian Aborigine Groote Eylandt A*0201Australian Aborigine Kimberly

42 42 HLA TYPING Allele: A*0201 Frequency Distribution

43 43 HLA TYPING HLA Alleles In N. Ireland Population (n=1000) 1994 HLA-A30/9033.3% HLA-B50/ % HLA-C23/7829.5% HLA-DRB133/ % /7334.1% 50/ % 23/3925.9% 33/6085.4%

44 44 HLA TYPING Summary of Results Very Rare (%)Rare (%)Frequent (%) Locus Number0 1, 2, 3 (inc. seq. confirmation) >3 All A B C DRB DQB DQA DPA DPB

45 45 HLA TYPING NMDP Analysis Single MM (low or high res) at HLA-A,B,C,DRB1 associated with higher mortality Single MM at HLA-B and C better tolerated MM at 2 or more loci compounded the problem MM at DQ or DP not associated with survival

46 46 HLA TYPING Months after transplant Probability of Overall Survival by HLA Matching for Early Disease Stage Curtesy of Stephanie Lee (2008 BMT Tandem Meetings) Log-rank p-value = < /8 HLA Matched (n=835) 7/8 HLA Matched (n=379) 50% 39% 28% 6/8 HLA Matched (n=241) Survival

47 A MEMBER OF THE RUSSELL GROUP FACULTY OF HEALTH & LIFE SCIENCES – CPD Institute for Learning & Teaching Faculty of Health & Life Sciences Room 2.16A, 4th Floor Thompson Yates Building Brownlow Hill Liverpool L69 3GB


Download ppt "HLA TYPING D Middleton MDSC175: Transplantation Science for Transplant Clinicians (Online) POSTGRADUATE SCHOOL OF MEDICINE A MEMBER OF THE RUSSELL GROUP."

Similar presentations


Ads by Google