Genetic Analysis of Some Rheumatological Conditions <ul><li>Chris Amos, U.T. M.D.A.C.C. </li></ul><ul><li>Exper Clin Immun...
Some Goals in Genetic Epidemiology <ul><ul><li>Identify Genetic Factors for Complex Diseases </li></ul></ul><ul><ul><ul><l...
&quot;Alice laughed: &quot;There's no use trying,&quot; she said; &quot;one can't believe impossible things.&quot; &quot;I...
 
Genetic Analysis of Familial Periodic Fever Syndromes <ul><li>Rare! Autosomal Dominant Syndromes, including Familial Hiber...
 
Genetic Linkage analysis of 3 Familial Hibernian Fever Families
Salient Developments in Mapping Familial Periodic Fevers <ul><li>Lack of linkage in one family to TNF receptor 1 (p55), bu...
Molecular characterization of  TNFR1-associated periodic syndromes (TRAPS)  <ul><li>Mutations at any of several Cysteine r...
Treatment of TRAPs <ul><li>P75 (TNFR2) had previously been approved for use and effective treatment in a subset of RA pati...
Rheumatoid Arthritis (RA) <ul><li>Affects peripheral synovial joints </li></ul><ul><li>~1% of the adult world population h...
“ Diagnosis” of RA <ul><li>No single diagnostic test </li></ul><ul><li>RA is a heterogeneous disease </li></ul><ul><li>Oth...
 
The 1987 ACR Criteria for RA <ul><li>Morning stiffness (at least 1 hr) </li></ul><ul><li>Arthritis of at least 3 joint are...
Etiology of RA <ul><li>Etiology unknown </li></ul><ul><li>Genetic and environmental factors implicated in disease suscepti...
Study Population <ul><li>257 RA multicase families with at least 2 siblings affected with RA </li></ul><ul><ul><li>At leas...
Estimating the Genetic Contribution in RA Prevalence of RA in sibs of affected probands  (2 - 3%) Prevalence of RA in gene...
Chromosomal regions showing “ suggestive”  linkage at the level of  p<0.005   Jawaheer  et al  (2001) Am. J. Hum. Genet. 6...
HLA – Human Leucocyte Antigen <ul><li>Human equivalent of the MHC  (Major Histocompatibility Complex) </li></ul><ul><li>Ma...
Genetic Associations With RA <ul><li>1974 - The first genetic association with RA was discovered  (Stastny, 1974) </li></u...
Role of HLA in RA <ul><li>2001…  The nature of the HLA/RA association is still not understood! </li></ul><ul><ul><li>HLA m...
The Shared Epitope Hypothesis <ul><li>The RA-associated HLA-DRB1 alleles encode a common sequence of amino acids in the 3 ...
The RA Shared Epitope HLA-DRB1* Amino acid position 70 71 72 73 74 0101 Q R R A A 0401 Q K R A A 0404 Q R R A A 0405 Q R R...
Incomplete HLA-DRB1*04 Association <ul><li>(Different alleles of HLA-DRB1*04 identified at DNA level  (Gregersen  et al , ...
Other HLA-DRB1 associations <ul><li>Population RA-associated DRB1 allele </li></ul><ul><li>Ashkenazi Jewish *0101 </li></u...
The HLA Contribution in RA <ul><li>HLA probably makes the  largest  genetic contribution in RA –  1/3 to1/2 of the total r...
 
Susceptibility or Severity? <ul><li>Shared epitope (SE) -  “susceptibility epitope” </li></ul><ul><li>But… </li></ul><ul><...
Dissecting the Genetic Complexity of the HLA/RA Association <ul><li>Identify genetic markers throughout HLA complex </li><...
Microsatellite Markers <ul><li>Di-, tri- or tetra-nucleotide repeats </li></ul><ul><li>AGTTACCGAT   (CA) n   GTCCAGTACTA <...
“ Binning” of Alleles for MN6S1874
0 Kb 4000 Kb Class I Class III Class II HLA complex Chromosome 6 6p21.3 0 Kb 1000 Kb DP DQ DR Class II
Map of the HLA Complex on Chromosome 6 HLA-DRB1
Statistical Analysis <ul><li>Pedigree Disequilibrium Test (PDT) </li></ul><ul><ul><li>Association test to determine whethe...
Transmission Disequilibrium Testing 1 1 2 3 3 4 Marker NOT associated with disease P(T|D) = P(NT|D) Marker IS associated w...
Allelic Associations (PDT)
Class I Class III Class II DRB1 M6S163 M6S204 M6S125 M6S166 M6S140
 
Allelic Associations (Case-Control)
Joint TDT analysis of TNF and HLA region loci <ul><li>Two Locus Conditional Logistic Regression </li></ul><ul><li>Effect o...
Joint Logistic Regression Analysis of TNF and HLA region
Conclusions <ul><li>HLA-DRB1 association with RA confirmed </li></ul><ul><li>Evidence for an independent effect of TNF fro...
Acknowledgements NS-LIJ Research Institute Univ. of Michigan Peter K. Gregersen Dr. J.T. Elder (Chief, Div of Bio & Hum Ge...
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  • Chronic, systemic, inflammatory disease. Although disease onset can be at any age, mean age at onset is mid to late 40’s.
  • Heterogeneous – acute onset and mild disease - mild onset, steady progression into severe RA… crippled - 65% periods of exacerbations and remissions IBD, SLE – joint involvement Psoriasis – worse erosions than in RA (characteristically, PIP affected – not in RA!!)
  • Synovium is inflamed Thickens into mass of tissue called pannus Pannus invades entire joint space Pannus releases enzymes that erode cartilage Osteoclasts (formed from synoviocytes) chew away bone
  • Env. Factors – Suggested microbial infection (mycobacterial, human Parvovirus) – RA does NOT occur in epidemics – argues against theory of infection Other env factors – smoking, blood transfusion, obesity Basically, need to have the genetic predisposition + be exposed to the env. trigger
  • Genetic component definitely present, though small
  • 27 years of research on this ….
  • Caucasians- &gt;90% SE+ in RA 40-50% in controls
  • Conservative substitutions among SE (basic -&gt; basic) Non-conservative subst. In others
  • Diff ethnic groups, diff. Populations… At this stage.. Very confusing – DR4 association Other DRB1 associations What is the relationship between them all?
  • HLA cannot account for all of the genetic risk Non-HLA loci identified have modest contribution
  • SE in alpha-helical portion of DRB1 chain. Some residues face into peptide groove, others face up towards TCR
  • Assumed that SE involved in onset No. of observations suggesting otherwise… RA not involved in onset – maybe previous associations observed in pop’s of patients with well-established RA HLA seems to be assoc. with progression/severity Low freq of SE -&gt; mild RA … assoc with progression/severity Still debatable – need stronger evidence
  • Pool all data for 1 marker and verify that it “looks ok” e.g. DNR should have alleles 2 bp apart as above. Then do inheritance check to verify there are no inheritance errors
  • Markers developed in collaboration with Dr JT Elder (univ. of Michigan) &amp; Dr Tim Behrens (Univ of Minnesota)
  • Montreal 2002 slides

    1. 1. Genetic Analysis of Some Rheumatological Conditions <ul><li>Chris Amos, U.T. M.D.A.C.C. </li></ul><ul><li>Exper Clin Immunogenetics 12:141-155, 1995. </li></ul><ul><li>Cell 97:133-144, 1999 </li></ul><ul><ul><li>AJHG 68:927-936, 2001 </li></ul></ul><ul><li>AJHG Sept 2002 </li></ul>
    2. 2. Some Goals in Genetic Epidemiology <ul><ul><li>Identify Genetic Factors for Complex Diseases </li></ul></ul><ul><ul><ul><li>Some genetic factors may permit modulation </li></ul></ul></ul><ul><ul><ul><li>Identifying and conditioning on genetic factors may assist in identifying environmental triggers </li></ul></ul></ul><ul><ul><ul><li>Identifying individuals at genetically determined high risk permits the development of early intervention strategies </li></ul></ul></ul><ul><ul><li>Characterize the prevalence of genetic factors </li></ul></ul>
    3. 3. &quot;Alice laughed: &quot;There's no use trying,&quot; she said; &quot;one can't believe impossible things.&quot; &quot;I daresay you haven't had much practice,&quot; said the Queen. &quot;When I was younger, I always did it for half an hour a day. Why, sometimes I've believed as many as six impossible things before breakfast.&quot;
    4. 5. Genetic Analysis of Familial Periodic Fever Syndromes <ul><li>Rare! Autosomal Dominant Syndromes, including Familial Hibernian Fever </li></ul><ul><li>Variable phenotype consisting of episodic/periodic high fevers of (previously) idiopathic etiology, sometimes requiring long-term treatment with steroids </li></ul><ul><li>Some patients also have amyloidosis </li></ul><ul><li>Not responsive to colchicine (like FMF) </li></ul>
    5. 7. Genetic Linkage analysis of 3 Familial Hibernian Fever Families
    6. 8. Salient Developments in Mapping Familial Periodic Fevers <ul><li>Lack of linkage in one family to TNF receptor 1 (p55), but strong evidence for linkage in other families </li></ul><ul><li>Increased levels of (borderline significance) of p55 in serum from patients with FHF compared to family controls </li></ul>
    7. 9. Molecular characterization of TNFR1-associated periodic syndromes (TRAPS) <ul><li>Mutations at any of several Cysteine residues leads to decreased cytokine clearance of P55 - increased binding to cellular membrane </li></ul><ul><li>Inter- and Intrafamilial analysis of variance shows significant interindividual and intrafamilial variability of serum p55 among normal individuals, and little variability among TRAPS affected </li></ul>
    8. 10. Treatment of TRAPs <ul><li>P75 (TNFR2) had previously been approved for use and effective treatment in a subset of RA patients (Entanercept) </li></ul><ul><li>IV administration of P75 leads to a reversion of most clinical symptoms of TRAPs (Drenth JPH, van der Meer JWM NEJM, 345:1748-1757). </li></ul><ul><li>Long-term efficacy of P75 treatment unknown (but better than other treatments) </li></ul>
    9. 11. Rheumatoid Arthritis (RA) <ul><li>Affects peripheral synovial joints </li></ul><ul><li>~1% of the adult world population has RA </li></ul><ul><li>Female-predominant disease (female : male = 3 : 1) </li></ul><ul><li>Mean age at onset – mid- to late-40’s </li></ul>
    10. 12. “ Diagnosis” of RA <ul><li>No single diagnostic test </li></ul><ul><li>RA is a heterogeneous disease </li></ul><ul><li>Other rheumatic diseases (IBD, SLE, psoriasis) can mimic symptoms of RA </li></ul><ul><li>Classification of disease status based on a defined set of clinical criteria </li></ul>
    11. 14. The 1987 ACR Criteria for RA <ul><li>Morning stiffness (at least 1 hr) </li></ul><ul><li>Arthritis of at least 3 joint areas (soft-tissue swelling) </li></ul><ul><li>Arthritis of hands (wrist, MCP or PIP) </li></ul><ul><li>Symmetric arthritis </li></ul><ul><li>Rheumatoid nodules </li></ul><ul><li>Serum rheumatoid factor </li></ul><ul><li>Radiologic changes </li></ul>
    12. 15. Etiology of RA <ul><li>Etiology unknown </li></ul><ul><li>Genetic and environmental factors implicated in disease susceptibility </li></ul><ul><li>Twin studies: </li></ul><ul><li>RA concordance among MZ twins 12-15% </li></ul><ul><li>RA concordance among DZ twins 5% </li></ul><ul><li>Modest familial aggregation </li></ul>
    13. 16. Study Population <ul><li>257 RA multicase families with at least 2 siblings affected with RA </li></ul><ul><ul><li>At least one sibling had erosive disease </li></ul></ul><ul><ul><li>At least one sibling had disease onset between the ages of 18 and 60 </li></ul></ul><ul><li>229 Caucasian Probands, 97 Caucasian Controls </li></ul>
    14. 17. Estimating the Genetic Contribution in RA Prevalence of RA in sibs of affected probands (2 - 3%) Prevalence of RA in general population (0.25 – 1%)  s = <ul><ul><li>In RA,  s ranges from 2 – 12 </li></ul></ul>
    15. 18. Chromosomal regions showing “ suggestive” linkage at the level of p<0.005 Jawaheer et al (2001) Am. J. Hum. Genet. 68: 927-936
    16. 19. HLA – Human Leucocyte Antigen <ul><li>Human equivalent of the MHC (Major Histocompatibility Complex) </li></ul><ul><li>Mapped to short arm of chromosome 6 </li></ul><ul><li>Consists of a large gene cluster (~4 x 10 6 bp) in the distal portion of the 6p21.3 band </li></ul>
    17. 20. Genetic Associations With RA <ul><li>1974 - The first genetic association with RA was discovered (Stastny, 1974) </li></ul><ul><li>Increased frequency of HLA-DRB1*04 among RA patients (65-80%) compared to controls (25-34%) </li></ul><ul><li>Observed in most Caucasian populations studied – North American, British, Austrian, Venezuelan </li></ul>
    18. 21. Role of HLA in RA <ul><li>2001… The nature of the HLA/RA association is still not understood! </li></ul><ul><ul><li>HLA may be directly involved in RA pathogenesis/progression </li></ul></ul><ul><ul><li>Possible that a gene(s) close to HLA-DRB1 on chromosome 6 are involved in RA </li></ul></ul>
    19. 22. The Shared Epitope Hypothesis <ul><li>The RA-associated HLA-DRB1 alleles encode a common sequence of amino acids in the 3 rd HVR of the DR β1 chain – the “shared epitope” (Gregersen et al. , 1987) </li></ul>
    20. 23. The RA Shared Epitope HLA-DRB1* Amino acid position 70 71 72 73 74 0101 Q R R A A 0401 Q K R A A 0404 Q R R A A 0405 Q R R A A 0408 Q R R A A 1001 R R R A A 1402 Q R R A A 0402 D E R A A 0403 Q R R A E
    21. 24. Incomplete HLA-DRB1*04 Association <ul><li>(Different alleles of HLA-DRB1*04 identified at DNA level (Gregersen et al , 1986)) </li></ul><ul><li>Association only with certain subtypes of HLA-DRB1*04 ( -DRB1*0401, *0404, *0408 and not *0402, *0403 or *0407) </li></ul><ul><li>20-35% of RA patients do NOT have HLA-DRB1*04 </li></ul>
    22. 25. Other HLA-DRB1 associations <ul><li>Population RA-associated DRB1 allele </li></ul><ul><li>Ashkenazi Jewish *0101 </li></ul><ul><li>Asian Indian *0101 </li></ul><ul><li>Spanish *1001 </li></ul><ul><li>Israeli *1001 </li></ul><ul><li>Yakima Indian *1402 </li></ul><ul><li>Japanese *0405 </li></ul>
    23. 26. The HLA Contribution in RA <ul><li>HLA probably makes the largest genetic contribution in RA – 1/3 to1/2 of the total risk </li></ul><ul><li>No other association as strong as HLA has been observed so far </li></ul><ul><li>Individually, non-HLA genes only make a modest contribution compared to HLA </li></ul>
    24. 28. Susceptibility or Severity? <ul><li>Shared epitope (SE) - “susceptibility epitope” </li></ul><ul><li>But… </li></ul><ul><ul><li>Absence of HLA-DRB1 associations in newly diagnosed cases of RA </li></ul></ul><ul><ul><li>Severe forms of RA associated with genotypes *0401/*0404 and *0401/*01 </li></ul></ul><ul><ul><li>In populations having low frequencies of SE (e.g. in Greece), RA is mild </li></ul></ul>
    25. 29. Dissecting the Genetic Complexity of the HLA/RA Association <ul><li>Identify genetic markers throughout HLA complex </li></ul><ul><li>Examine the inheritance of marker alleles in RA families </li></ul><ul><li>Determine which specific regions of the HLA complex are associated with RA </li></ul>
    26. 30. Microsatellite Markers <ul><li>Di-, tri- or tetra-nucleotide repeats </li></ul><ul><li>AGTTACCGAT (CA) n GTCCAGTACTA </li></ul><ul><li>TCAATGGCTA (GT) n CAGGTCATGAT </li></ul><ul><li>Unique flanking sequences </li></ul><ul><li>Easily typed by PCR </li></ul>
    27. 31. “ Binning” of Alleles for MN6S1874
    28. 32. 0 Kb 4000 Kb Class I Class III Class II HLA complex Chromosome 6 6p21.3 0 Kb 1000 Kb DP DQ DR Class II
    29. 33. Map of the HLA Complex on Chromosome 6 HLA-DRB1
    30. 34. Statistical Analysis <ul><li>Pedigree Disequilibrium Test (PDT) </li></ul><ul><ul><li>Association test to determine whether any marker alleles are preferentially transmitted from parent to affected offspring </li></ul></ul><ul><li>Case-control analysis </li></ul><ul><ul><li>Odds-ratios (95% confidence interval) </li></ul></ul>
    31. 35. Transmission Disequilibrium Testing 1 1 2 3 3 4 Marker NOT associated with disease P(T|D) = P(NT|D) Marker IS associated with disease P(T|D) > P(NT|D)
    32. 36. Allelic Associations (PDT)
    33. 37. Class I Class III Class II DRB1 M6S163 M6S204 M6S125 M6S166 M6S140
    34. 39. Allelic Associations (Case-Control)
    35. 40. Joint TDT analysis of TNF and HLA region loci <ul><li>Two Locus Conditional Logistic Regression </li></ul><ul><li>Effect of allele 12, M6S125 (near TNF) OR = 3.47, 95% CI - [1.65, 7.29] </li></ul><ul><li>Effect of allele 2, M6S118 (near DR) OR = 2.56, 95% CI - [1.25, 5.22] </li></ul>
    36. 41. Joint Logistic Regression Analysis of TNF and HLA region
    37. 42. Conclusions <ul><li>HLA-DRB1 association with RA confirmed </li></ul><ul><li>Evidence for an independent effect of TNF from both family and case-control studies, but further delineation of TNF region needed </li></ul><ul><li>Effects from class I </li></ul><ul><li>Large Sample sizes are needed because of LD across region </li></ul>
    38. 43. Acknowledgements NS-LIJ Research Institute Univ. of Michigan Peter K. Gregersen Dr. J.T. Elder (Chief, Div of Bio & Hum Genet) Univ of Minnesota Genotyping Dr. Tim Behrens Aarti Damle, Joanita Monteiro Rob Graham Oligonucleotide synthesis MD Anderson Cancer Center Dorothy Guzowski Dr. Chris Amos Wei Chen Statistical Analysis Dakai Zhu Nina Kohn The North American Rheumatoid Roche Molecular Systems Arthritis Consortium (NARAC) Ann Begovich
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