Contribution of genes of the major histocompatibility complex to susceptibility and disease phenotype in inflammatory bowel disease

Contribution of genes of the major histocompatibility complex to susceptibility and disease phenotype in inflammatory bowel disease Jack

Satsangi,

Ken I Welsh, Mike Bunce, Cecile Julier, J Mark Farrant, John I Bell, Derek P Jewell

Summary Background Despite strong evidence implicating immune dysfunction and genetic predisposition in the pathogenesis of the chronic inflammatory bowel diseases Crohn’s disease and ulcerative colitis, the importance of the genes of the major histocompatibility complex remains uncertain. We have investigated the contribution of HLA DRB1 and DQB genes by the strategies of non-parametric linkage analysis (affected sibling pair method) as well as association study. The relation between genotype and phenotype was examined in detail. Methods For

linkage analysis 74 families in whom two or siblings had inflammatory bowel disease were studied. A total of 83 affected sibling pairs were involved: in 42 pairs both siblings had Crohn’s disease; in 29 both had ulcerative colitis; in 12 one sibling had Crohn’s

more

disease, the other ulcerative colitis. For the association study there were 175 patients with ulcerative colitis, 173 with Crohn’s disease, and 472 controls. Details of sex, age of onset, disease extent, and family history were analysed. 24 patients with ulcerative colitis and 92 with Crohn’s disease required surgery for refractory disease. HLA DRB1 and DQB1 gene-typing was performed by polymerase chain reaction with sequence-specific primers. In ulcerative colitis, the sharing of alleles among affected sibling pairs provided evidence for linkage with DRB1 locus (p=0·017, X2=5·32). Of 29 affected sibling pairs studied, only one pair shared no DRB1 DQB haplotypes. 15 shared two DRB DQB haplotypes. In contrast, no linkage was noted for Crohn’s disease (42 sibling pairs; p=0·30, &KHgr;2=0·16) or for inflammatory bowel disease overall (83 sibling pairs, p=0·16, &KHgr;2=2·28). In the association study the rare DRB1*103 (8·3% vs 3·2% in controls) and DRB1*12 (8·6% vs 2·1% in controls) alleles were associated with ulcerative colitis (p=0·0074, &KHgr;2=7·22, odds ratio OR=2·9 [95% Cl 1·3-6·4] and p=0·0056, No OR=4·33 [1·8-11·0] respectively). &KHgr;2=12·63, association with alleles representing DR2 (p=0·55, &KHgr;2=0·34) was noted. No overall association was seen in Crohn’s disease. In ulcerative colitis, the frequency of DRB1*0301 DQB*0201 (DR3 DQ2) was reduced in females (9·8% vs 26·3% in controls, p=0·037, &KHgr;2=8·39 OR=0·34 [0·15-0·71]), particularly in those with distal disease

Findings

(2·3%,

p=0·001

vs

controls,

&KHgr;2=11·35

OR=0·07

Gastroenterology Unit (J Satsangl MRCP, J M Farrant MRCP, D P Jewell FRCP), Nuffield Departments of Medicine (J I Bell FRCP) and Surgery (K I Welsh PhD, M Bunce), Oxford Radcliffe Hospitals, Oxford; and Wellcome Trust Centre for Human Genetics (C Julier PhD), Oxford, UK

Correspondence to: Dr Jack Satsangi and Dr Derek P Jewell, Gastroenterology Unit, Radcliffe Infirmary, Woodstock Road, Oxford OX2 6HE, UK

1212

[0·00-0·39]). In both males and females, the DR3 DQ2 haplotype was predictive of extensive ulcerative colitis (32·9% vs 10·7% in distal disease, p<0·01, &KHgr;2=10·94, OR 4·09 [1·70-10·6]) but not of need for surgery (p=0·93, &KHgr;2=0·01). Interpretation These data provide strong evidence for genetic heterogeneity in inflammatory bowel disease. Genes of the major histocompatibility complex are implicated as important inherited determinants of susceptibility to ulcerative colitis and may also influence the pattern of disease. In Crohn’s disease, important susceptibility genes are likely to exist outside the HLA region. Lancet 1996; 347: 1212-17

See Commentary page ii98

Introduction The aetiology of Crohn’s disease and ulcerative colitis remains unknown, but strong evidence has accumulated from studies of different ethnic groups, families of patients, and twin pairs to implicate genetic susceptibility in pathogenesis.1,2 For Crohn’s disease in particular, the coefficient of heritability calculated from concordance rates in twin pairs is greater than for insulin-dependent

diabetes, asthma,

or

3

schizophrenia.

The mode of inheritance is unknown, but there is very limited support for a simple mendelian inheritance pattern in either disease. Complex segregation analysis suggests that simple models of inheritance are, at best, pertinent to only a small fraction of patients.4,5 It is more likely that inflammatory bowel disease is multifactorial in aetiology, and, in view of the variability of clinical phenotypes, the term inflammatory bowel disease may several related represent polygenic conditions. or environmental stimuli, may be Susceptibility genes, shared between subtypes, accounting for the concurrence of different phenotypes within one family. However, at present, the extent of heterogeneity and the relation between genotype and phenotype is uncertain. Previous attempts to identify susceptibility genes in inflammatory bowel disease by means of linkage analysis or population-based studies have generally been inconclusive. Few linkage studies involving multiply affected families have been reported and all have serious methodological defects-small numbers of families investigated, reliance on parametric linkage analysis, or The assumptions regarding disease homogeneity.1,4-<> complexity of inflammatory bowel disease demands large studies involving robust non-parametric methods of analysis such as the affected sibling pair method.7,s Several investigators have conducted association studies, usually focused on genes involved in the

regulation

of the immune response, in

particular

the

major histocompatibility complex (in man, the HLA human leucocyte antigen region).9,lo With the exception of the small subgroups of patients with primary sclerosing cholangitis9 or ankylosing spondylitis,9 results of previous HLA association studies have been confusing and inconsistent. Even molecular genotyping has produced conflicting results. Particular controversies concern putative class II gene associations in ulcerative colitis with the alleles representing the serological DR2 antigens9,lo and in Crohn’s disease with the DRI DQ5 haplotype.""’ A repository of clinical material (DNA, plasma, and frozen lymphocytes) from more than 400 European patients with non-familial inflammatory bowel disease and from members of 250 multiply affected families has been established in Oxford. Details of clinical history have

For this purpose, patients with no family history of inflammatory bowel disease were selected at random from adult outpatients attending the gastroenterology clinics at the John Radcliffe Hospital, Oxford, between January and November, 1994. Patients having affected first-degree relatives were selected from the Crohn’s and Colitis Gene bank database in Oxford, which now contains details of 250 multiply affected families throughout the UK. No two members from one family were included in the association study. In any family the affected individual chosen was the first in the family to develop disease. For both Crohn’s disease and ulcerative colitis, disease phenotype was defined by sex, age at onset of symptoms, familial disease, disease extent, and need for surgery (see below). Ethnic biases have been minimised by exclusion of all Asian and South European patients. The proportion of Jewish persons among the patient groups and controls is about 5%.

also been obtained and are stored on a computer database. This resource, the Crohn’s and Colitis Gene Bank, will allow investigation of the importance of genetic factors in disease susceptibility. We report here a detailed study of the contribution of HLA DRB1and DQB genes to the pathogenesis of inflammatory bowel disease.

Ulcerative colitis-175 patients with definitive ulcerative colitis studied (92 male), chosen as described above. 52 had one or more affected first-degree relative; 123 patients had no family history. Disease extent was defined by the most proximal extent of disease at the most recent investigation performed (barium enema or colonoscopy). If macroscopic extent differed from the microscopic assessment, the microscopic extent was recorded. Thus, disease was classified as extensive (inflammation proximal to the splenic flexure) in 85 patients (37 female, 48 male; 65 non-familial, 20 familial) and as distal in 84 patients (40 male, 44 female; 56 sporadic, 28 familial). In six patients, the disease extent was uncertain. 24 patients (ten female; six familial) had required colectomy for severe disease refractory to medical therapy. Five patients (three extensive, two distal) had primary sclerosing cholangitis; one had primary biliary cirrhosis; all others had normal liver biochemistry. One patient had vitiligo and one had multiple sclerosis. The median age of onset in ulcerative colitis was 35-0 years (interquartile range [IQR] 25-0-48-0 years). Median age of onset was lower in familial disease (28-5 years [IQR 23-0-42-0] vs 37-5 years [26-0-38-0] in patients with non-familial disease, p<0-02, Kruskal-Wallis analysis) and in patients with extensive disease (31-5 years [IQR 22-0-48-0] vs 36 years [28’0-47.0] in patients with distal disease, not significant).

Methods This investigation was approved by the Central Oxford Research Ethics Committee and all participants gave informed consent. Clinical data were obtained by questionnaire, by personal interview, and from case records.

Linkage analysis: affected sibling pair method families, all European caucasoid and resident in the UK, in which two or more siblings had inflammatory bowel disease, were identified with the help of the National Association for Crohn’s and Colitis (NACC) and cooperating physicians. Advertisements were placed in the NACC newsletter asking suitable families to contact the investigators. Interviews (either in person or by telephone) and collection of blood from affected siblings and all willing healthy first-degree relatives (parents, offspring, siblings) were arranged. Clinical details were obtained from hospital physicians or family doctors. 74

Crohn’s disease families--In 30 families, two siblings had Crohn’s disease; in a further four families, three siblings were affected (12 sibling pairs). Hence, a total of 42 affected sibling pairs with Crohn’s disease, together with their healthy firstdegree relatives, were studied. In 18 pairs both affected siblings were female, in five both were male, and in 19 pairs one sibling with Crohn’s disease was male, the other female. Clinical details of disease extent were available for 41 pairs. In 27 both siblings had ileo-colonic disease; in 2 pairs both siblings had exclusively colonic disease; and in one pair both siblings had jejunal disease only. In the other 11 pairs for whom clinical data were available, one sibling had colonic and one ileo-colonic Crohn’s disease.

Ulcerative colitis-In 27 families,

two siblings had ulcerative pairs of siblings in different generations were affected. 29 pairs of affected siblings together with their healthy relatives were therefore studied. In 11 pairs both affected siblings were female, and in 12 pairs both were

colitis; and in

one

family

two

male. "Mixed disease" pairs-In twelve sibling pairs one sibling had Crohn’s disease, the other ulcerative colitis. In two pairs both were male; in three pairs both were female. Of note, seven of the siblings with Crohn’s disease had exclusively colonic disease, with no small-bowel involvement. Fuller clinical details and pedigrees are available from the authors.

Association

study

An allelic association study was performed, involving 348 unrelated patients (175 ulcerative colitis, 173 Crohn’s disease).

were

Crohn’s disease--The association study included 173 unrelated patients with definite Crohn’s disease. 68 were male. 67 patients had a positive family history of inflammatory bowel disease; 106 had

no

affected relatives. Disease

extent

was

defined from

clinical, radiological, endoscopic, and histological data. 107 patients (40 male) had both small-bowel and large-bowel involvement, predominantly ileo-caecal disease. Patients with limited terminal ileal disease were included in this group.12 42 patients (18 male) had exclusively colonic disease (including perianal disease alone). 18 patients (ten male) had exclusively jejunal or proximal ileal disease. In six patients (all female) disease extent was uncertain. 13 patients had predominantly fistulising disease, 31 had stricturing disease, and the remainder were judged to have predominantly inflammatory disease. 92 patients had required surgery for refractory disease (excluding perianal sepsis alone). In 13 of these laparotomy had revealed perforating disease. Two patients had developed carcinomas (one adenocarcinoma of the small bowel, one adenocarcinoma of the rectum). One patient with extensive Crohn’s colitis had primary sclerosing cholangitis. The median age of onset of Crohn’s disease was 24-2 years (IQR 20-0-33-0 years; p<0-00001 vs ulcerative colitis patients). Patients with colonic disease had a higher median age of onset (32-0 years [IQR 24-5-475] vs 22-0 Kruskalyears [19’0-30.0] in ileo-colonic disease, p<0-001, Wallis analysis). Controls-During 1992-94, 472 unrelated European caucasoid individuals were HLA genotyped by molecular methods. These controls were recruited from hospital workers and prospective blood and organ donors. No one in the control group had a disease with known genetic predisposition. All were Oxfordshire residents. 1213

The gels were run for 18 min at 15 V per cm in 0-5XTBE buffer. About 5% of DNA samples were typed on two occasions to establish reproducibility (no discrepancies were present). All the results were checked independently by two of the investigators.

Data

Table 1: DRB1

DQB haplotype sharing in affected sibling pairs

Laboratory techniques extracted from 10 mL venous blood by a modified and was resuspended in sterile distilled water at a final concentration of 0·1-1· 0 µg/µL before use. HLA DRB and DQB genotyping was performed by polymerase chain reaction amplification with sequence-specific primers (PCR-SSP). Methodological details have been published previously.14-16 Briefly, each group of alleles or individual allele making up a serological defined specificity was amplified by a primer pair specific for the template. Stringent conditions for PCR avoided non-specific amplification. Details of the amplification primers, with annealing sites and alleles detected, are available from the authors. Amplification control primers giving rise to a 796 base-pair fragment from the third intron of HLA DRBwere included in each reaction. 13 (JbL amplification reaction mixtures consisted of 67 mmol/L Tris base pH 8-8, 16-6 mmol[L ammonium sulphate, 2 mmol/L magnesium chloride, 0-01% (vol/vol) Tween-20, 200 mmol/L of each dNTP, 1-3 mmol/L allele-specific primers, 0-1 mmol/L DRB11 control primers, and 0-025 units taq polymerase (Advanced Biotechnology, London). Amplifications were done with Gene Amp PCR system 9600 (Perkin-Elmer Corporation) or MJ Research 96 V machines. The cycling parameters for 13 )iL reactions in rapid cycling PCR machines with a heated lid were: 1 min at 96°C followed by five cycles of 20 s at 96°C, 45 s at 70°C, 25 s at 72°C, followed by 20 cycles of 20 s at 96°C, 50 s at 65°C, 30 s at 72°C, followed by five cycles of 20 s at 96°C, 60 s at 55°C, 120 s at 72°C. PCR products were separated by electrophoresis in 1 % agarose gels containing ethidium bromide. DNA

was

"salting-out" technique13

1214

analysis

and statistics

between susceptibility to Linkage analysis-Linkage class II bowel disease and HLA inflammatory genes was assessed by comparing the sharing of alleles among affected siblings. Complete information regarding parental haplotypes was available for 56 affected sibling pairs, allowing attribution of haplotypes and alleles identical by descent. To utilise information from all 83 affected sibling pairs, sharing of haplotypes identical by state was also assessed," with corrections derived from the DRB1and DQB allele frequency in the control population. The numbers of affected sibling pairs sharing no haplotypes and two haplotypes were compared by X2 testing. Probability values of were taken as evidence of significant linkage. In four p<0-05 families with Crohn’s disease, three siblings all were affected. In the results presented, these siblings have been included as three independent pairs. Association

study-Interim analysis was performed in May, patients with ulcerative colitis had been genotyped. On interim analysis, significant differences in the frequency of the DRB 1 *03011 DQB*02011 haplotype (representative of the serologically defined DR3 DQ2 haplotype) were apparent between subgroups of patients with ulcerative colitis (male vs female; distal vs extensive disease). The frequencies of the 1994. 74

DRB1*0103 allele and the DRB1*12 allele were both increased in patients with ulcerative colitis. Hence, preliminary hypotheses All comparisons of allele frequency between made by use of a 2 X 2 contingency table and X= groups statistics. Corrections were made where necessary for small sample numbers (Fisher’s exact test). All comparisons and derived probability values were subject to correction for multiple testing, except for the comparisons made on the basis of preexisting hypotheses.10 Subgroup analyses were done with the Knowledge Seeker data analysis package.

were

generated. were

Results results Ulcerative colitis-Of 29 affected sibling pairs with ulcerative colitis, 15 shared two HLA DR]311 DQB haplotypes and only one pair of siblings shared zero haplotypes (table 1). Comparison of the number of sibling pairs with ulcerative colitis sharing zero and two haplotypes identical by state (29 sibling pairs) provided strong evidence of linkage in ulcerative colitis with the DRB1 DQB haplotype (X2=5’27, p=0.016, 1 df), and the DRB1 locus (=5-32, p=0-017, 1 df). Only one pair of siblings were discordant for both haplotypes. This method of analysis, which takes into account control frequencies of DRB1and DQB alleles, is conservative. Of the 19 affected sibling pairs with ulcerative colitis for whom full parental information was available, ten shared two DRB1 DQB haplotypes identical by descent, and only one pair shared none. By use of these data and previously reported relative risks in siblings and twins, it is possible to estimate the contribution of the HLA class II region to overall genetic susceptibility in ulcerative colitis. By either the method of Risch" or that of Rotter and Landaw," it that appears important genetic determinants of to ulcerative colitis are linked to the HLA susceptibility class II region. On the assumption of an empirical risk of ulcerative colitis in siblings of between 2% and 4% and a monozygotic twin concordance rate of 6-3%/ the derived coefficent of genetic contribution" for the HLA genes is between 64% and 100%. This estimated value is reliant

Linkage

In the association study, DRB1*0301 was m linkage disequilibrium with DQB1*0201. Probability values shown (those with p<0.05) have been corrected for multiple analyses. The DRB1*0301 DQB1*0201 haplotype frequency was lower m females with ulcerative colitis than in controls (9.8% vs 26.3%, p=0-0037, X’=8.39, OR=0.34

[0,15-0.71]). Table 3: HLA DRB1 allele

frequency (%) genotype-phenotype analysis

in ulcerative colitis:

the accuracy of the twin concordance rate and on the sibling relative risk data.’8 Of the 15 pairs of affected siblings with ulcerative colitis sharing two haplotypes, 13 were of the same sex (eight both male, five both female). on

Crohn’s disease--In ulcerative colitis, there

with the findings in was no evidence for linkage with DRBI, DQB loci, or the DRB1DQB haplotype when allele sharing among sibling pairs with Crohn’s disease was compared. In total, 42 sibling pairs were available for identity by state analysis, and 28 for identity by descent analysis. Formal identity by state analysis showed no significant linkage with the HLA DRB1 DQB loci or HLA DRB11 DQB haplotype with Crohn’s disease (p=0-30, =0-15, 1 df). The contribution of the HLA region to overall genetic susceptibility in Crohn’s disease derived from the allele sharing in affected sibling pairs is no more than 10%.18 Of the 12 pairs of affected siblings sharing two haplotypes, eight were of the same sex (two male; six female). Four families in which three siblings each had Crohn’s disease were studied. In all four families, two affected siblings were identical for both HLA types, with the other sibling sharing only one of his contrast

siblings’ haplotypes. Association study The association study involved 175 patients with ulcerative colitis, 173 patients with Crohn’s disease, and 472 controls. Interim analysis was conducted in May, 1994, at which time 74 patients with ulcerative colitis and 75 patients with Crohn’s disease had been genotyped. On the basis of this analysis, interim hypotheses were generated: these were tested at final analysis. At the final analysis in November, 1994, all comparisons and probability values were corrected for multiple testing, except for specific comparisons made on the basis of pre-

in controls, p=0-0074, x’=7-22, OR 2-86 [95% CI 1-27-6-42]) and the DRBI*12 allele (serological equivalent DR12, 8-6% vs 2-1%, p=0-0056, Xz=12-63, OR 4-33 [1-77-10-98]). The DRB1*04 alleles (serological equivalent DR4) were modestly reduced (27-4% vs 35-8% in controls, p=0-046, x’=4-01, p=0-045, OR 0-68 [0-45-1-01]) at interim and final analysis. In subgroup analysis with the Knowledge Seeker package, which incorporates a partial Bonferroni correction for multiple testing, several strong associations were observed at both interim and final analyses (table 3). Thus, the DR3-DQ2 haplotype was reduced in females with ulcerative colitis (9-8% vs 32-6% in males, p=0-008, X2 12-22, 1 df). This difference was most pronounced in females with limited distal disease (only one of 44 had this haplotype, 2-3%, p=0-001 compared with control population, X2=11·35, OR 0-07 [0-00-0-39]). For both males and females, the DR3 DQ2 haplotype was predictive of extensive rather than distal disease (32-9 vs 10-7%, p
(stricturing vs fistulising, perforating vs non-perforating).

existing hypotheses. Table 2 shows the class II HLA allele

frequencies

in

patients with Crohn’s disease and ulcerative colitis.

Comparisons

have been made with the control population. The expected linkage disequilibrium in white people between the DRBand DQB loci was noted in patients and in controls.

Ulcerative colitis-In ulcerative colitis, no overall association was present with the alleles corresponding to the DR2 antigen (32·0% vs 29·2% in controls, p>0·5). Associations were noted, both at interim and at final analyses, with the rare DRB1*0103 allele (8·6% vs 3·2%

Discussion The results of this investigation provide strong evidence for genetic heterogeneity within inflammatory bowel disease. The comparisons of haplotype sharing in affected sibling pairs offer evidence of linkage between HLA class II genes and ulcerative colitis, but not Crohn’s disease or inflammatory bowel disease overall. In view of the estimated relative risks for siblings and concordance rates in twin pairs, these data suggest that genes of the major histocompatibility complex encode important determinants of suscpeptibility in ulcerative colitis. 1215

no individual allele or haplotype is implicated the primary disease susceptibility gene in ulcerative colitis. Overall associations were noted only with two uncommon alleles (DRBI*0103 and DRB1*12), for of patients with ulcerative colitis. 17-2% accounting The contrast between the very strong evidence for the presence of a susceptibility gene in the HLA region (from the affected sibling pair analysis) and the size of the associations noted is intriguing, and may signify that the primary disease susceptibility gene(s) lie not in the class II region but in linked genes in the adjacent class I or class III regions. The association with the DRB 1 *O 103 allele was most pronounced in male patients with extensive disease. This allele was also associated with an increased risk of colectomy for severe disease. These results are consistent with previous work in which this allele was associated not only with severe colitis necessitating colectomy but also with recurrent inflammation of the ileo-anal pouch

However,

as

("pouchitis")." The present study reveals no association between alleles representing DR2 and ulcerative colitis. These results are consistent with recent data from Pittsburgh,2O but contrast sharply with findings from Japanz’ and California." Ethnic differences between the populations studied probably account for these discrepancies (table 4). In Oxford and Pittsburgh, the populations were predominantly white non-Jewish, of European origin. In this ethnic group, DRB 1*1501 is the only common allele of DR2,22 and DRB1*1502 accounts for less than 5% of alleles. By contrast, DRB1*1502 is the most common allele representing DR2 in Japanese23 and Jewish2’ populations: it is this allele (or its extended haplotype) that seems to account for the association reported between DR2 and ulcerative colitis in California, where the population was mixed Jewish/non-Jewish. Strong associations were noted between class II genotypes and subgroups defined by phenotype. Both at interim analysis and at final analysis, the DR3 DQ2 haplotype (part of the extended HLA Al B8 DR3 DQ2 haplotype) was implicated as an important determinant of disease behaviour. This haplotype emerges as a potential marker of extensive disease, particularly in females. Only one of 44 female patients with distal disease possessed this haplotype, compared with 26-3% of the controls overall and 32-9% of patients with extensive disease. These results are consistent with and may even explain the well-accepted association between this haplotype and primary sclerosing cholangitis, a disease most common in male patients with extensive ulcerative colitis.25 The region of the Al B8 DR3 DQ2 haplotype primarily associated with disease remains unknown. In primary sclerosing cholangitis" and other immune-mediated diseases,27 investigators have concentrated on the structure of the cell surface class II molecules themselves and have tried to implicate specific aminoacid residues involved in antigen presentation. Before these molecules can be implicated in the pathogenesis of ulcerative colitis,

further association studies and linkage analyses are needed to assess the contribution of linked genes. Polymorphisms of genes of the class I and class III regions, including the genes encoding complement components and the tumour necrosis factor a, may be most

colectomy. with the results in ulcerative colitis, the no evidence of linkage between HLA class II genes and Crohn’s disease. The sibling pair method is relatively insensitive to disease heterogeneity; and it remains possible that these genes are important in only a small subgroup of patients. Nevertheless, in view of the strong evidence from concordance rates in twin pairs that genetic factors are important in the pathogenesis of Crohn’s disease, it is likely that important susceptibility genes exist outside the HLA region. Although the association study does not confirm previously described HLA associations in Crohn’s disease,’"" it does lend some weight to the concept of heterogeneity within Crohn’s disease. The clinical as well as immunogenetic data support the idea that Crohn’s disease of the colon is more closely related to ulcerative colitis than to small-bowel Crohn’s disease: median age at onset of disease in patients with colonic Crohn’s disease was higher than in patients with ileo-colonic or smallbowel disease. Furthermore, in patients with colonic Crohn’s disease the DR3 DQ2 haplotype was implicated as a marker of disease behaviour, as in ulcerative colitis. These results have important implications for future studies of the genetics of inflammatory bowel disease and also of other complex disorders of polygenic inheritance. Future association (or linkage) studies must be designed to permit assessment of the relation between phenotype and genotype. Subclinical as well as clinical markers should be considered. Non-HLA genes must be studied. In Crohn’s disease in particular, our investigation suggests that HLA genes are only weakly involved; other susceptibility genes must be present to explain the striking concordance rate in family members and twin pairs. In ulcerative colitis, further work is needed to define more closely the genes in the HLA region that are of primary importance in determining susceptibility and disease behaviour. In

ethnic groups 1216

frequency (%) of DR2 alleles in different

contrast

linkage analysis has provided

We

Table 4: Relative

pertinent.

The results reported here are consistent with findings in systemic sclerosis2g and rheumatoid disease29 which have implicated class II alleles as markers of disease severity (if not overall susceptibility). In both conditions, DRB1 genotyping has already become useful in the clinical assessment of newly diagnosed patients. The striking difference in frequency of the DR3 DQ2 haplotype between male and female patients with ulcerative colitis also warrants further attention. Although the present study has not confirmed the association with the DR2 specificity reported by Toyoda and colleagues,’° the results do confirm a reduction in the DR4 specificity in patients with ulcerative colitis. This effect is most pronounced in patients with extensive colitis and those requiring

are thank all the patients and physicians who have taken part in this study, the National Association for Crohn’s and Colitis, and the European Collection of Animal and Cell Cultures. Heather Holt organised the collection of blood samples and clinical data and was supported by Astro Draco. JS is a Medical Research Council training fellow. We thank G M Lathrop for his critical advice and Clare O’Neill for technical guidance.

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