HLA AND DISEASE M. Toungouz Nevessignsky Université Libre de Bruxelles (ULB) Erasme Hospital Brussels, Belgium
HLA and disease: pioneer studies 1972: Falcuk et al HLA B8 and coeliac disease 1972: Russell et al HLA B27 and psoriasis 1973: Brewerton et al, Schlochtein et al HLA B27 ankylosing spondylitis
Methods Family data (linkage studies) Populations (association studies) Comparison of HLA typing between patients and healthy subjects Comparison of alleles frequencies between patient cohort and healthy controls Most data come from association studies
To keep in mind HLA associated diseases are most of the time multifactorial: HLA: one/multiple alleles/genes Non HLA Environmental factors The disease has to be acurately diagnosed (homogeneity)
Methods Ensure that there is a major genetic component in the disease Concordance rates in identical twins compared with non identical twins: risk ratio (λ)( = risk to sibs/population prevalence λ= = 15 for type 1 diabetes; λ= = 50000 for hemophilia λ<10 small genetic contribution Identify a chromosomal region or interval that may play a role in i disease etiology Idendify the gene involved Two critical sources of information: The ability to distinguish individuals from one another A large source of information about DNA sequences
HEMOCHROMATOSIS AND CLASSICAL HLA 1975 Simon et al (Rennes) (a) HLA-A3 A3 : Disease : 75%; Healthy control: 28% (b) Demonstration by the study of informative families (2 cases): (a) (b) the hypothetical gene is located within MHC this gene is recessive: homozygosity for the gene (HLA identity betweeen affected subjects) is a necessary condition for the expression of the disease. Possibility to predict brother or sister who will be affected by the disease.
HEMOCHROMATOSIS STUDY USING CLASSICAL HLA TYPING Phenotypes of parents and children haplotypes genotypes in order to follow the transmission of the still hypothetical gene "H" by the indirect method of linkage: control case identity reveals homozygosity Phenotype : Parents : P HLA-A2 A2, -A10, -B12, -B5 M HLA-A3 A3, -A1, -B7, -B8 Children : 1 (patient) HLA-A2 A2, -A3, -B12, -B7 = Control case 2 -A2, -A1, -B12, -B8 3 -A10, -A3, -B5, -B7 4 -A10, -A1, -B5, -B8 5 -A2, -A3, -B12, -B7 = HLA identity
DEDUCED GENOTYPE From the control case *a *c, one identifies in the parents the haplotypes *a et *c encoding the involved gene. Parents : F * a * A2 B12 b A10 B5 M * c * A3 B7 d A1 B8 Children : 1 *a*c Hom 2 *ad Het 3 *bc Het 4 bd 5 *a*c Hom Het Het HLA identity means homozygosity for the «H» gene *H gene
HEMOCHROMATOSIS AND HFE 1996 Feder : discovery of a candidate gene for hemochromatosis 4,5 Mégabases from HLA-A in telomeric position (HLA( HLA-H) (a) Corresponding protein: HFE = HLA class I like molecule (= non classical) involved in the intestinal absorption of iron by association with the transferin receptor (TfR). (b) the mutation HFE C282Y is present in 85 to 90% of hemochromatosis in Northern Europe (type I Hemochromatosis) Ineffective [HFE TfR] complex
Association studies Comparison of the frequency of an allele in a sample of affected individuals with its frequency in a case-control control population Major problem: the control population which is the most common source of false-association association data careful selection for ethnicity, age, gender and environment Statistics: Significance: chi-square test (Fischer exact test if expected frequencies are too small) Strength: relative risk (odds ratio with 95% confidence interval)
Bonferoni correction In case on performing a χ square test, several antigens may show evidence of an association Statistical significance? probability that a result would occur if there were no association on P=0.05 means that 1 out of 20 analyses could produce the oberved result by chance Bonferoni correction: calculation of the p value for each test and then mutliplication of the uncorrected p-value p by the number of (anti)gen(es)) tested Example: Analysis of one locus one statistical test/antigen If 20 HLA-DR types considered AND the p-value p for ONE DR type is 0.001 the reported value for the study should be 0.02 (= 20x0.001) If p-value p still <0.05 indication of a HLA-disease association
Relative risk (RR) expressed as OR (Odds Ratio) RR measures the strength of the association (risk of disease among those positive for he antigen as compared to those without the antigen) RR = 1 no increased risk for those carrying the antigen RR>1 increased risk (positive association) RR< decreased risk (negative association) HLA and disease Association Allele Present Patient a b Controls c d RR= ad/bc Absent
HLA B27 and ankylosing spondylitis AS : 88% Control : 8% X² = 236 p < 0.0001 FD (1-FC) RR = = 90 FC (1-FD) (Schlosstein and Terasaki, 1973) Significant: increase of the frequency of the HLA antigen in the disease as compared to the healthy population (ethnic background +++) concept based on retrospective epidemiological studies (accuracy of diagnosis ). Prospective applications? Armor criteria: HLA = 2 points out of 6 necessary for the diagnosis Combination of genetic and environmental factors
HLA CLASS I and disease D Ct RR (%) 1. Idiopathic hemochromatosis HLA-A3 75 28 10 2. «Birdshot» retinopathy A29 90 8 90 3. Ankylosing spondilytis B27 > 90 9 90 -Reiter B27 75 9 40 - Anterior uveitis B27 50 9 10 4. Behcet B51 50 à 70 6 à 30 10 5. Psoriasis (type I, < 30 yrs) Cw6 75 16 15
HLA CLASS II and disease D Ct RR (%) 1. DR15 (2) - DQ6 (1) - Multiple sclerosis DR15 70 30 ~ 10 - Narcolepsy DR15 > 90 30 ~ 90 - Goodpasture DR15 85 30 ~ 15 2. DR3 - DQ2 - Type I diabetes DR3 50 25 ~ 10 NB DR4 75 34 ~ 10 DR3-DR4 30 0 ~ 30 - Coeliac disease DR3 75 25 ~ 10 - Dermatitis herpetiformis DR3 85 25 ~ 10 3. DR4 - DQ8 - Rheumatoïdarhtitis DR4 70 34 ~ 10 - Pemphigus vulgaris DR4 85 34 ~ 10
HLA and disease Functional involvement of HLA? Linkage : gene location close to the locus involved Hemochromatosis Behcet? Psoriasis? HLA = accompanying marker Largely hypotetical for numerous HLA associated diseases (absence e of known autoantigen, no confirming functional analysis): Ankylosing spondilytis Narcolepsy Rheumatoid arthritis Better understanding in some diseases (autoantigen(s) as well as HLA associated allele(s) better characterized): Coeliac disease Type I diabetes Recent review: Caillat-Zucman, Tissue Antigens 2008
BEHCET DISEASE High Prevalence in subjects from mediterranean and oriental origin («silk trading route») : Japan 90/10 5 ; Saudi Arabia 20/10 5 ; Marocco 15/10 5 ; Turkey 14/10 5 ; Iran 17/10 5 ; Palestine-Israel 10/10 5. Unkown etiology Immunosuppressive treatment sometimes necessary Difficult diagnosis genetic test? HLA-B51
HLA-B51: World distribution and homo sapiens migration between 300,000 and 10,000 years ago
BEHCET and HLA-B51 Patient (%) Control (%) RR Ohno 1976 (Japan) 75 31 6 Muftueglu 1981 (Turkey) 77 33 7 Baricordi 1986 (Italy) 81 21 16 Chajek 1987 (Israel) 72 13 20 Kilmartin 1997 (Ireland) 25 0 6
NARCOLEPSY (S. de Gelineau 1880) - Rare (0.02 à 0.06%) - Difficult diagnosis despite polysomnography - Etiology: autoimmunity? - Search for genetic marker - Association: -Initially: DR2(DRB1*1501) -Then: DQ6=DQB1*0602DQB1*0602 (linkage disequilibrium not only with DR2 but also DR6): -healthy controls: 18 to 35 % -narcolepsy +cataplexy: 95% -Narcolepsy without cataplexy: 40%
PHYSOPATHOLOGY OF NARCOLEPSY? (Peyron, Mignot 2000) Hypothesis: Autoimmune disease (?) leading to the destruction of hypothalamic nuclei (fornix) secreting an hormone, the hypocretin (HCT) (absence of HCT in CSF in narcoleptic patients) Until now, not supported by studies of the cellular and humoral immune responses Crystal structure studies show that the volume of the P4 pocket of the DQ6 molecule is central to susceptibility (Siebold et al 2004)
Rheumatoïd d arhtitis and DR4
Citrulline hypothesis (Hill) Loss of tolerance for to posttranslationally modified (citrullinated) antigens (Hill et al, J Exp Med 2008) Presence in RA of a perinculear antibody directed against proteins rich in citrullin (filaggrin, kératin,vimentin,fibrinogen) k Presence in synovial cells of an enzyme (PAD=peptidylarginine deiminase) responsible for the conversion of arginine to citrullin increasing the affinity for a DR4 shared epitope inflammatory lesions and production of anticitrullin autoantibodies (Hill et al, JI 2003)
DR4 alleles Only found: DRB1*0401,0404, 0405 sharing one epitope (QKRAA) in position 70-74
COELIAC DISEASE Autoimmune (lymphoïd d intraepithelial hyperplasia) enteropathy (diarrhea, malabsorption) Strong genetic component: >90% DQ2 molecule encoded by DQA1*0501-DQB1*0201 DQB1*0201 Most remaining patients are DQ8 encoded by DQA1*0301-DQB1*0302 DQB1*0302 Absence of DQ2 and DQ8 has a negative predictive value for CD close to 100% Central role of (gliadin speficic) CD4 T cells Serologically: Antigliadine antibodies(g (G) Anti TTG (tissue transglutaminase) autoantibodies Prevalence: improved diagnosis 1/1000 (80 ) 1/300 (90 ) 1/150
COELIAC DISEASE TTG provokes the deamidation of glutamine (Q) in glutamic acid (E)( which leads to the appearance of negative charges affinity (anchoring) of gluten derived peptides for HLA DQ2 and DQ8 molecules (which are positively charged) T lymphocytes activation destruction of intestinal cells B lymphocytes activation production of anti G et anti TTG antibodies
HLA DISEASE HLA DRUG TOXICITY
HLA-B*5701 Screening for Hypersensitivity to Abacavir Mallal S et al. N Engl J Med 2008;358:568-579 Hypersensitivity reactions to abacavir are tightly associated with HLA-B*5701 In this global, multicenter, prospective, randomized study, 1956 patients with HIV-1 1 infection who had not previously received abacavir were randomly assigned to undergo HLA- B*5701 screening or to receive the standard of care Screening eliminated immunologically confirmed hypersensitivity reaction to abacavir in this predominantly white population In this case, a pharmacogenetic test can prevent the toxic effects of this drug
Incidence of Hypersensitivity Reaction to Abacavir Mallal S et al. N Engl J Med 2008;358:568-579