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Characterization of the Major Susceptibility Region for Psoriasis at Chromosome 6p21.3

1999; Elsevier BV; Volume: 113; Issue: 3 Linguagem: Inglês

10.1046/j.1523-1747.1999.00710.x

1523-1747

Nalini Balendran, R. Lee Clough, J. Rafael Argüello, Ruth Barber, Colin Veal, Andrew B. Jones, Jane L. Rosbotham, Anne-Margaret Little, J. Alejandro Madrigal, Juliet N. Barker, Stephen H. Powis, Richard C. Trembath,

T-cell and B-cell Immunology

Psoriasis is a common inflammatory skin condition caused by genetic and environmental factors. Recent genome-wide linkage analyses have identified a locus encoding susceptibility to psoriasis and placed this gene in the 12 cM interval between markers D6S426 and D6S276 on chromosome 6p21.3. This is a broad region and encompasses the human major histocompatibility complex. We have sought to localize the susceptibility gene more precisely by exploiting the linkage, haplotype, and linkage disequilibrium information available through genotyping 118 affected sib pairs, their parents and other affected family members. A total of 14 highly polymorphic markers were genotyped, combining anonymous loci with the class I genes HLA-B and -C distributed across a genetic interval of approximately 14 cM including the entire major histocompatibility complex. Through the application of higher density mapping within the major histocompatibility complex, we identified those regions most commonly shared identical by descent in patients with psoriasis. Using the transmission–disequilibrium test, we found significant evidence of linkage and allelic association across an interval defined by the markers tn62 (p = 1.0 × 10–7), HLA-B (p = 4.0 × 10–7), and HLA-C (p = 2.7 × 10–9), a region encompassed within a 285 kb genomic DNA fragment. Hence these studies contribute to the refinement of the localization of a major psoriasis susceptibility gene and place the critical region near to HLA-C. Psoriasis is a common inflammatory skin condition caused by genetic and environmental factors. Recent genome-wide linkage analyses have identified a locus encoding susceptibility to psoriasis and placed this gene in the 12 cM interval between markers D6S426 and D6S276 on chromosome 6p21.3. This is a broad region and encompasses the human major histocompatibility complex. We have sought to localize the susceptibility gene more precisely by exploiting the linkage, haplotype, and linkage disequilibrium information available through genotyping 118 affected sib pairs, their parents and other affected family members. A total of 14 highly polymorphic markers were genotyped, combining anonymous loci with the class I genes HLA-B and -C distributed across a genetic interval of approximately 14 cM including the entire major histocompatibility complex. Through the application of higher density mapping within the major histocompatibility complex, we identified those regions most commonly shared identical by descent in patients with psoriasis. Using the transmission–disequilibrium test, we found significant evidence of linkage and allelic association across an interval defined by the markers tn62 (p = 1.0 × 10–7), HLA-B (p = 4.0 × 10–7), and HLA-C (p = 2.7 × 10–9), a region encompassed within a 285 kb genomic DNA fragment. Hence these studies contribute to the refinement of the localization of a major psoriasis susceptibility gene and place the critical region near to HLA-C. linkage disequilibrium transmission-disequilibrium test Psoriasis is a relapsing chronic inflammatory skin disease affecting all racial groups with a peak prevalence of 3% in northern European and Scandinavian caucasians (Lomholt, 1963Lomholt G. Psoriasis: Prevalence, Spontaneous Course and Genetics. GEC Gad, Copenhagen1963Google Scholar;Camp et al., 1998Camp R.D.R. Psoriasis.in: Champion RH Burton JL Burns DA Breathnach SM Textbook of Dermatology. Blackwell Science, Oxford1998: 1589-1649Google Scholar). Phenotypic expression is mainly confined to the skin, with well circumscribed erythematous scaling plaques in a symmetrical distribution. Characteristic features include epidermal keratinocyte hyperproliferation, altered epidermal maturation, vascular proliferation, and inflammatory cell accumulation (Camp et al., 1998Camp R.D.R. Psoriasis.in: Champion RH Burton JL Burns DA Breathnach SM Textbook of Dermatology. Blackwell Science, Oxford1998: 1589-1649Google Scholar). Whereas the pathogenesis of psoriasis remains uncertain, current evidence suggests a central role for T lymphocytes in modulating the disease process (Barker, 1991Barker J.N. The pathophysiology of psoriasis.Lancet. 1991; 338: 227-230Abstract PubMed Scopus (201) Google Scholar). Epidemiologic studies have implicated a genetic component to psoriasis, including a monozygotic twin concordance rate of 70% compared with a dizygotic twin concordance rate of 20% (Brandrup et al., 1982Brandrup F. Holm N. Grunnet N. Henningsen K. Hansen H.E. Psoriasis in monozygotic twins: variations in expression in individuals with identical genetic constitution.Acta Derm Venereol (Stockh). 1982; 62: 229-236PubMed Google Scholar), a 5–10-fold increased risk in first-degree relatives (Theeuwes and Leder, 1993Theeuwes M. Leder R. Hereditary insights in psoriasis.Eur J Dermatol. 1993; 3: 335-341Google Scholar) and large multiple case pedigrees. Previous case–control studies have demonstrated associations with multiple major histocompatibility complex (MHC) class I and II alleles. Interpretation of such studies, however, has been potentially confounded by patient ascertainment bias, population stratification effects, and reliance on serologically defined HLA typing. Genome-wide linkage analyses have identified putative susceptibility loci on chromosomes 1q, 2p, 4q, 6p, 8q, 16q, 17q, and 20p (Tomfohrde et al., 1994Tomfohrde J. Silverman A. Barnes R. et al.Gene for familial psoriasis susceptibility mapped to the distal end of human chromosome 17q.Science. 1994; 264: 1141-1145Crossref PubMed Scopus (363) Google Scholar;Matthews et al., 1996Matthews D. Fry L. Powles A. et al.Evidence that a locus for familial psoriasis maps to chromosome 4q.Nat Genet. 1996; 14: 231-233Crossref PubMed Scopus (183) Google Scholar;Nair et al., 1997Nair R.P. Henseler T. Jenisch S. et al.Evidence for two psoriasis susceptibility loci (HLA and 17q) and two novel candidate regions (16q and 20p) by genome-wide scan.Hum Mol Genet. 1997; 6: 1349-1356Crossref PubMed Scopus (357) Google Scholar;Trembath et al., 1997Trembath R.C. Clough R.L. Rosbotham J.L. et al.Identification of a major susceptibility locus on chromosome 6p and evidence for further disease loci revealed by a two stage genome-wide search in psoriasis.Hum Mol Genet. 1997; 6: 813-820Crossref PubMed Scopus (440) Google Scholar;Capon et al., 1999Capon F. Novelli G. Semprini S. et al.Searching for psoriasis susceptibility genes in Italy: genome scan and evidence for a new locus on chromosome 1.J Invest Dermatol. 1999; 112: 32-35Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). Recently, two genome wide linkage scans (Nair et al., 1997Nair R.P. Henseler T. Jenisch S. et al.Evidence for two psoriasis susceptibility loci (HLA and 17q) and two novel candidate regions (16q and 20p) by genome-wide scan.Hum Mol Genet. 1997; 6: 1349-1356Crossref PubMed Scopus (357) Google Scholar;Trembath et al., 1997Trembath R.C. Clough R.L. Rosbotham J.L. et al.Identification of a major susceptibility locus on chromosome 6p and evidence for further disease loci revealed by a two stage genome-wide search in psoriasis.Hum Mol Genet. 1997; 6: 813-820Crossref PubMed Scopus (440) Google Scholar) and a region specific analysis (Burden et al., 1998Burden A.D. Javed S. Bailey M. Hodgins M. Connor M. Tillman D. Genetics of psoriasis: paternal inheritance and a locus on chromosome 6p.J Invest Dermatol. 1998; 110: 958-960Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar) have mapped a major locus for psoriasis susceptibility to a 12 cM region at chromosome 6p21.3. As this interval contains the 4 Mb MHC, these studies support prior case–control studies in strongly suggesting that a MHC gene confers the major genetic component to psoriasis. The human MHC (HLA region) was first identified because it contains genes encoding the major transplantation antigens. The physiologic role for these cell surface molecules, the class I and II antigens, were subsequently shown to be presentation of processed antigen to T lymphocytes (Browning and McMichael, 1996Browning M. McMichael A. HLA and MHC. Genes Molecules and Functions. Oxford, BIOS Scientific1996Google Scholar). The MHC is now known to contain over 100 other genes, some of which play additional important parts in the immune response. These observations are of particular interest because immune mediators have been implicated in the pathogenesis of psoriasis (Barker, 1991Barker J.N. The pathophysiology of psoriasis.Lancet. 1991; 338: 227-230Abstract PubMed Scopus (201) Google Scholar). As the majority of MHC genes do not have immune function, however, it remains possible that the MHC encoded psoriasis susceptibility gene is not an immune system gene. Linkage disequilibrium (LD) mapping offers an approach to narrow the MHC candidate region (Spielman and Ewens, 1996Spielman R.S. Ewens W.J. The TDT and other family-based tests for linkage disequilibrium and association.Am J Hum Genet. 1996; 59: 983-989PubMed Google Scholar). When a mutation or polymorphism responsible for a specific disease is first introduced into a population, it resides on a background haplotype of linked markers in LD with one another. Recombination through many generations will tend to dissipate LD for all but those markers in very close proximity to the disease locus. We now describe the refinement of the MHC candidate region by typing both microsatellite and HLA markers at intermediate density in a British Caucasian psoriasis population. Using allele sharing and family-based linkage disequilibrium analytical methods, we show that a 285 kb region around markers tn62, HLA-B, and HLA-C is most likely to contain the MHC-encoded psoriasis susceptibility gene. Individuals were studied from the caucasoid cohort reported byTrembath et al., 1997Trembath R.C. Clough R.L. Rosbotham J.L. et al.Identification of a major susceptibility locus on chromosome 6p and evidence for further disease loci revealed by a two stage genome-wide search in psoriasis.Hum Mol Genet. 1997; 6: 813-820Crossref PubMed Scopus (440) Google Scholar. In brief a total of 78 north European families, ranging from nuclear pedigrees to extended kindreds, all with a minimum of two affected members with psoriasis, were investigated (Table 1). A diagnosis of psoriasis was made using standard clinical criteria (Camp et al., 1998Camp R.D.R. Psoriasis.in: Champion RH Burton JL Burns DA Breathnach SM Textbook of Dermatology. Blackwell Science, Oxford1998: 1589-1649Google Scholar). All affected and unaffected individuals were assessed by one of two experienced dermatologists to ensure consistency of application of diagnostic criteria. All probands had chronic plaque type psoriasis and any doubtful cases were excluded. The population consisted of 238 affected individuals, 45% male, 55% female, with a mean age of onset of 16.1 y (range 1–59). Affected individuals were investigated for both allele sharing and linkage disequilibrium. Genomic DNA was extracted from peripheral blood by standard procedures and stored at –70°C until genotyped. Ethical approval was obtained for this study.Table 1Characteristics of the study cohortaIn the statistical analyses dependence of multiple affected sibling pairs was taken into account as previously described (Satsangi et al.1996).Affected relations and familiesNumberTotal number of independent families78Total affected sibling pairs118 2 siblings54 3 siblings14 4 siblings2 5 siblings1 2 parents available83 1 parent available25 0 parents available10Total affected relative pairs (non sib)18Total number of trios with both parents available146Total number of "trios" with one parent available38Number of fully independent trios77Total number of affected individuals238Total number of unaffected individuals187a In the statistical analyses dependence of multiple affected sibling pairs was taken into account as previously described (Satsangi et al., 1996Satsangi J. Parkes M. Louis E. et al.Two stage genome-wide search in inflammatory bowel disease provides evidence for susceptibility loci on chromosomes 3, 7 and 12.Nat Genet. 1996; 14: 199-202Crossref PubMed Scopus (659) Google Scholar). Open table in a new tab HLA-B genotypes were initially determined by sequence specific oligonucleotide typing as previously described (Yoshida et al., 1992Yoshida M. Kimura A. Numano F. Sasazuki T. Polymerase-chain-reaction-based analysis of polymorphism in the HLA-B gene.Hum Immunol. 1992; 34: 257-266Crossref PubMed Scopus (97) Google Scholar). Briefly, four primers (supplied by Oswel DNA Services, Southampton, U.K.) were used for locus-specific amplification of all of exon 2 and all of exon 3. PCR reactions were performed with 1600 ng DNA in an 80 μl reaction containing 8 μl 10 × PCR buffer (Bioline, London, U.K.), 0.195 mM of each primer, 0.2 mM of each dNTP, 2 mM of magnesium chloride, and 1.6 units of Taq polymerase (Bioline). Polymerase chain reaction (PCR) amplification was performed as follows: 96°C for 5 min; 96°C for 1 min, 65°C for 1 min, 72°C for 2 min, for 5 cycles; 96°C for 1 min, 55°C for 1 min, 72°C for 2 min, for 25 cycles; 72°C for 8 min (using a 9600 thermal cycler, Perkin-Elmer, Warrington, U.K.). The PCR products were dot blotted, cross-linked on to a nylon membrane and hybridized with 35 digoxigenin labeled sequence-specific oligonucleotide probes (obtained from the British Society for Histocompatibility and Immunogenetics). Following stringent washing, detection was achieved using anti-digoxigenin alkaline phosphatase Fab fragments (Boehringer Mannheim GmbH, Germany). HLA-C typing was performed as previously described (Trembath et al., 1997Trembath R.C. Clough R.L. Rosbotham J.L. et al.Identification of a major susceptibility locus on chromosome 6p and evidence for further disease loci revealed by a two stage genome-wide search in psoriasis.Hum Mol Genet. 1997; 6: 813-820Crossref PubMed Scopus (440) Google Scholar). The nomenclature used for genotyped alleles is the name of the locus followed by an asterisk and the number of the allele (e.g., B*5701); the nomenclature for serologic assignment is the name of the locus followed by the specificity (e.g., B57). Allele-specific high resolution HLA-B genotyping was determined by reference strand mediated conformational analysis (previously described as double strand conformational analysis;Arguello et al., 1998Arguello J.R. Little A.M. Pay A.L. et al.Mutation detection and typing of polymorphic loci through double-strand conformation analysis.Nat Genet. 1998; 18: 192-194Crossref PubMed Scopus (93) Google Scholar). Briefly, fragments containing exon 2, intron 2, and exon 3 of HLA-B were amplified with locus specific primers. Labeled reference strands were prepared from homozygous B-LCLs SP0010 (B*4402) and RSH (B*4201) using 5′ fluorochrome Cy-5 (Pharmacia Biotech, Uppsala, Sweden). Duplexes were formed by the addition of 1 μl of labeled reference PCR product to 3 μl of sample PCR product followed by denaturation at 95°C for 4 min; 55°C for 5 min and addition of an aliquot of each marker mix (2 μl). Duplexes (2 μl) were separated by electrophoresis for 530 min at 40°C through a nondenaturing 6% polyacrylamide gel (Long Ranger Gel Solution, JT Baker, Lichfield, U.K.) in an ALFexpress automated sequencer (Pharmacia) at 30 W constant power. The mobilities of each fluorescent duplex were analyzed using Fragment Manager software (Pharmacia). Genotyping was performed for 12 microsatellite polymorphic markers from chromosome 6p21.3. Oligonucleotide primers for microsatellites lh1, d3a, f91, tn821, and tn62 (Lako et al., 1999Lako M. Ramsden S. Campbell R.D. Strachan T. Mutation screening in British 21-hydroxylase deficiency families and development of novel microsatellite based approaches to prenatal diagnosis.J Med Genet. 1999; 36: 119-124PubMed Google Scholar) were kindly provided by Dr R.D. Campbell and amplified as described byHsieh et al., 1997Hsieh S.L. March R.E. Khanna A. Cross S.J. Campbell R.D. Mapping of 10 novel microsatellites in the MHC class III region: application to the study of autoimmune disease.J Rheumatol. 1997; 24: 220-222PubMed Google Scholar. Other microsatellites were chosen form published genetic maps (Reed et al., 1994Reed P.W. Davies J.L. Copeman J.B. et al.Chromosome-specific microsatellite sets for fluorescence-based, semi-automated genome mapping.Nat Genet. 1994; 7: 390-395Crossref PubMed Scopus (300) Google Scholar;Dib et al., 1996Dib C. Faure S. Fizames C. et al.A comprehensive genetic map of the human genome based on 5,264 microsatellites.Nature. 1996; 380: 152-154Crossref PubMed Scopus (2668) Google Scholar;Martin et al., 1998Martin M.P. Harding A. Chadwick R. et al.Characterization of 12 microsatellite loci of the human MHC in a panel of reference cell.Immunogenetics. 1998; 47: 131-138Crossref PubMed Scopus (25) Google Scholar). The CEPH-Genethon map was used to establish intermarker distances for some markers, and the rest were integrated using published physical distances assuming 1 cM ≈ 1 Mb. The mean heterozygosity of markers was 75.1%. Genotypes at each marker were determined using fluorescence labeled 5′ primers and semiautomated techniques as described previously (Trembath et al., 1997Trembath R.C. Clough R.L. Rosbotham J.L. et al.Identification of a major susceptibility locus on chromosome 6p and evidence for further disease loci revealed by a two stage genome-wide search in psoriasis.Hum Mol Genet. 1997; 6: 813-820Crossref PubMed Scopus (440) Google Scholar). Briefly, pooled amplified DNA was electrophoresed on 6% acrylamide gels, which were run for 3 h at 900 V in an ABI 373A automated sequencer (Perkin-Elmer). Semi-automated fragment sizing was performed using GENESCAN 672 (version 2.1) software (Perkin-Elmer), and genotyping carried out using GENOTYPER (version 2.0) software (Perkin-Elmer). The microsatellite RING3CA was genotyped by radionucleotide inclusion according to standard protocols. All genetic marker data were maintained in the Cyrillic database system (Cherwell Scientific, Oxford, U.K.). Haplotypes were constructed through the application of the Cyrillic pedigree data program and by visual inspection, assuming the minimum number of recombination events observed in each family. If required to resolve apparent multiple recombination events, markers were re-genotyped. All paternity issues had previously been identified and excluded from the study. At each marker locus, an ancestral allele was defined as that allele occurring in greatest excess frequency in the affected cohort compared with the unaffected cohort. The assumed ancestral haplotype, presumed to carry the disease mutation, was then constructed using these alleles (Feder et al., 1996Feder J.N. Gnirke A. Thomas W. et al.A novel MHC class I-like gene is mutated in patients with hereditary haemochromatosis.Nat Genet. 1996; 13: 399-408Crossref PubMed Scopus (3203) Google Scholar). Sib pair analysis, requiring no prior assumptions of the mode of inheritance of susceptibility alleles in psoriasis, was performed for the whole cohort using the SIBPAIR program (version 2.1) within the ANALYZE package and as previously described (Trembath et al., 1997Trembath R.C. Clough R.L. Rosbotham J.L. et al.Identification of a major susceptibility locus on chromosome 6p and evidence for further disease loci revealed by a two stage genome-wide search in psoriasis.Hum Mol Genet. 1997; 6: 813-820Crossref PubMed Scopus (440) Google Scholar). The transmission of alleles from heterozygous parents to affected offspring was assessed by the use of the transmission-disequilibrium test (TDT) as implemented in the program TDTLIKE (version 2.1), generating a TDT-like likelihood ratio statistic, based on the algorithm byTerwilliger, 1995Terwilliger J.D. A powerful likelihood method for the analysis of linkage disequilibrium between trait loci and one or more polymorphic marker loci.Am J Hum Genet. 1995; 56: 777-787PubMed Google Scholar. Multipoint nonparametric analysis was performed using GENEHUNTER version 1.1. in the nonparametric mode without partitioning for heterogeneity (Kruglyak et al., 1996Kruglyak L. Daly M.J. Reeve-Daly M.P. Lander E.S. Parametric and nonparametric linkage analysis: a unified multipoint approach.Am J Hum Genet. 1996; 58: 1347-1363PubMed Google Scholar). The program was configured for scoring of all possible affected relative pairs (settings were skip large = off, analysis = nonparametric linkage, score = all, and increment = 5). As psoriasis is a multifactorial disease with an ambiguous mode of inheritance, nonparametric methods of data analysis, which identify regions of excess allele sharing compared with that under independent assortment, have advantages over conventional parametric linkage analysis. To determine the extent of allele sharing at chromosome 6p21.3, we genotyped 78 independent psoriatic families, containing 118 affected sib pairs and 18 non-sib relative pairs (Table 1). We analyzed a total of 14 markers (average intermarker distance 0.6 cM) to maximize the information content for the region. LOD scores suggestive of linkage (> 2.4) were maintained across the interval flanked by D6S291 and D6S265 (7.5 cM centromeric to telomeric). Within this interval, evidence of linkage was identified at marker loci tn821 (LOD = 3.42), TNFA (LOD = 5.70), tn62 (LOD = 6.47), HLA-B (LOD = 6.16) and HLA-C (LOD = 3.65). These results are summarized in Table 2.Table 2Linkage results for the study cohortSIBPAIR /ASPaASP, affected sib pair. linkage valuesMarkerNo. of alleles observed in cohortHeterozygosityLODpNPLbNPL, non parametric linkage values obtained from the GENEHUNTER analysis. scoreD6S29160.701.209.4 × 10–31.78Ring3ca100.772.861.4 × 10–41.88lh1130.713.157.0 × 10–52.51d3a130.762.071.0 × 10–32.75f9n190.531.633.0 × 10–33.06D6S27380.752.453.9 × 10–43.28tn821130.813.423.6 × 10–53.29TNFA150.765.701.5 × 10–73.09tn62120.826.472.4 × 10–83.21HLA B40c223 B alleles defined to date.6.165.0 × 10–82.94HLA C20d63 C alleles defined to date.3.652.0 × 10–42.24D6S26580.782.179.9 × 10–52.19D6S105100.790.211.7 × 10–11.29D6S276140.831.841.25 × 10–31.48a ASP, affected sib pair.b NPL, non parametric linkage values obtained from the GENEHUNTER analysis.c 223 B alleles defined to date.d 63 C alleles defined to date. Open table in a new tab Linkage of psoriasis to chromosome 6p21.3 was also demonstrated by analysis of the genotype data with a nonparametric multipoint method, GENEHUNTER, which supported excess allele sharing for the region encompassed by d3a to HLA-B (Figure 1) with a maximum nonparametric linkage value of 3.29 for the microsatellite number tn821. For the multipoint analysis we based marker order on the Genethon map and where necessary on known physical intervals, using sex averaged recombination fractions as follows: tel-D6S276–1.0–D6S105–0.5–D6S265–0.4–HLA-C-0.10–HLA-B–0.1–tn62–0.05–TNFA–0.05– tn82–0.1–D6S273–0.1–f9n1–0.35–d3a–0.3–lh1–0.5–Ring3ca–5.45–D6S291–cen. To reduce the misleading effects of population stratification on association, non-transmitted parental genotypes were used as internal controls in the TDT as a test of allelic association. For each locus, the alleles with the most significant TDT values (using the multiallelic statistic corrected for multiple allele testing) are represented in Table 3. Ancestral alleles, as defined as the allele in greatest excess in the patient group compared with the control group, were D6S291–4, Ring3ca–3, lh1–8, d3a–5, f9n1–4, D6S273–2, tn821–11, TNFA-4, tn62–3, HLA-B*5701, HLA-Cw*0602, D6S265–2, D6S105–4, and D6S276–2. Alleles at contiguous loci tn62, HLA-B, and HLA-C, encompassing an ≈ 285 kb region, show the strongest linkage disequilibrium with the disease phenotype, all at a significance level of p < 2 × 10–7 using the TDT test. Parent to offspring transmission of alleles HLA-Cw*0602 and HLA-B*5701 occurred on 92 and 60 occasions, respectively, compared with nontransmissions 28 and 16 times, respectively. Immediately flanking markers on the centromeric side of this peak also demonstrate significant allelic association (TNFA, tn821, f9n1), but support for allelic association declines rapidly for more telometric markers (D6S265, D6S105; see Figure 1). These data strongly suggest the localization of the susceptibility locus to the proximal part of the MHC in the 285 kb region flanked by markers tn62 and HLA-C.Table 3Association results for the study cohortaThe most significant alleles at each locus detected via the multiallelic TDT linkage disequilibrium test (corrected for multiple testing;Spielman & Ewens 1996) are shown.Most significant alleles detected via the multiallelic TDTMarkerAllele bHLA-B and-C alleles are defined according to the XII Histocompatibility Workshop nomenclature (Bodmer et al.1996); all other alleles are defined on the basis of their mobility score (Reed et al.1994).Allele size cExpressed as mobility units on an ABI 373.HRR (p value)Overall TDT (max likelihood estimate) (p value)AlleleAllele-specific p valueD6S29142072.2 × 10–39.7 × 10–244.90 × 10–230532.86 × 102Ring3ca3–3.8 × 10–33.7 × 10–331.10 × 10–2lh18937.0 × 10–18.3 × 10–282.15 × 10–1d3a51256.5 × 10–13.5 × 10–153.37 × 10–1f9n14982.1 × 10–36.8 × 10–542.29 × 104610263.30 × 101D6S27321324.7 × 1011.9 × 10–121.53 × 10–1113011.70 × 10–1513657.20 × 10–1tn821111105.0 × 10–37.2 × 10–5112.28 × 10–439438.57 × 10–4TNFA41045.6 × 10–32.0 × 10–348,52 × 10–3711077.28 × 10–1tn6231522.0 × 10–61.0 × 10–735.07 × 10–7716072.37 × 10–1HLA-B5701–1.0 10–64.0 × 10–757013.08 × 10–65001–50018.97 × 10–2HLA-C0602–2.0 × 10–52.7 × 10–906022.07 × 10–81201,12021,12022–1201,12021,12022D6S26521831.4 × 10–16.0 × 10–221.49 × 10–1619162.86 × 10–1D6S10541536.2 × 10–11.9 × 10–12.86 × 10–14.73 × 10–1615768.94 × 10–1D6S27622065.0 × 10–15.0 × 10–329.42 × 10–3521457.55 × 10–1a The most significant alleles at each locus detected via the multiallelic TDT linkage disequilibrium test (corrected for multiple testing;Spielman and Ewens, 1996Spielman R.S. Ewens W.J. The TDT and other family-based tests for linkage disequilibrium and association.Am J Hum Genet. 1996; 59: 983-989PubMed Google Scholar) are shown.b HLA-B and-C alleles are defined according to the XII Histocompatibility Workshop nomenclature (Bodmer et al., 1996Bodmer J.G. Marsh S.G.E. Albert E.D. et al.Nomenclature for factors of the HLA system.Tissue Antigens. 1996; 49: 297-321Crossref Scopus (299) Google Scholar); all other alleles are defined on the basis of their mobility score (Reed et al., 1994Reed P.W. Davies J.L. Copeman J.B. et al.Chromosome-specific microsatellite sets for fluorescence-based, semi-automated genome mapping.Nat Genet. 1994; 7: 390-395Crossref PubMed Scopus (300) Google Scholar).c Expressed as mobility units on an ABI 373. Open table in a new tab Using the marker order shown above, haplotypes were constructed by inference from the genotypes of the affected, the parents and other family members. Phase assignment was such as to minimize the number of recombination events in each family. Both chromosomes of all affected and unaffected individuals were included in the construction of haplotype sets, giving a total of 422, 130, and 194 chromosomes analyzed for affected, unaffected sibs, and unaffected spouses, respectively. Within these sets, the most common haplotypes are represented in Table 4. In the affected individuals the most commonly occurring haplotype, tn62–3, HLA-B*5701, HLA-Cw*0602, includes 91 chromosomes. Although extending the haplotype reduces the total number of chromosomes for the common haplotype, the more extended haplotype f9n1–4, D6S273–5, tn821–11, TNFA-4, tn62–3, HLA-B*5701, HLA-Cw*0602, D6S265–2 more closely resembles the assumed ancestral haplotype. The "fractured" haplotypes are most likely to reflect the effects of recombination, thereby reducing the interval maintained under linkage disequilibrium. For the haplotype f9n1–4, D6S273–5, tn821–11, TNFA-4, tn62–3, HLA-B*5701, HLA-Cw*0602, D6S265–2, the most frequently occurring allele, HLA-Cw*0602, was found in 37% of chromosomes in affected individuals (typically in a heterozygous form) compared with 21% and 18% of chromosomes in unaffected sibs and spouses, respectively. The only other haplotype at greater frequency in the affected cohort also included HLA-Cw*0602. The extended haplotype D6S273–2, tn821–4, TNFA-9, tn62–7, HLA-B*1302, HLA-Cw*0602 occurred in 3%, 1.5%, and 1.3% of all chromosomes in affected individuals, unaffected sibs, and unaffected spouses, respectively. No other common haplotype was observed in the affected group, even for those alleles with significant TDT values as listed in Table 3.Table 4Haplotypes associated with susceptibility to psoriasisaAll unambiguous haplotypes occurring at greater frequency in affected than unaffected individuals are shown. Inferred haplotypes, where data for any of the markers was missing, were excluded from the haplotype subtotals. The most extended haplotypes more closely resemble the "founder" haplotype. The shorter haplotypes reflect the effects of recombination narrowing the region maintained under LD. Exclusive haplotypes represent chromosomes in which a shorter haplotype occurs and has not already been represented within a larger haplotype.(a)f9n1D6S273tn821TNFAtn62HLA-BHLA-CD6S265Affected (422 chromosomes)Unaffected sibs (130 chromosomes)Unaffected spouses (194 chromosomes)Cumulative haplotypesExclusive haplotypesCumulative haplotypesExclusive haplotypesCumulative haplotypesExclusive haplotypes451143570106022212133334511435701060230930635114357010602233124152511435701060257158510411435701060266910211143570106027261001433570106029119133173570106021081716322557011251716024206021787028124220(b)D6S273tn821TNFatn62HLA-BHLA-C