Linkage analysis of candidate regions for coeliac disease genes
Linkage analysis of candidate regions for coeliac disease genesR. S. Houlston1,*, I. P. M. Tomlinson1, D. Ford1, S. Seal1, A. M. Marossy1, A. Ferguson2, G. K. T. Holmes3, K. B. Hosie4, P. D. Howdle5, D. P. Jewell6, A. Godkin6, G. D. Kerr7, P. Kumar8, R. F. A. Logan9, A. H. G. Love10, S. Johnston10, M. N. Marsh11, S. Mitton12, D. O'Donoghue13, A. Roberts14, J. A. Walker-Smith15 and M. F. Stratton1
1Institute of Cancer Research, Sutton, Surrey SM2 5NG, UK, 2Department of Medicine, University of Edinburgh Western General Hospital, Edinburgh, UK, 3Department of Medicine, Derbyshire Royal Infirmary, Derby, UK, 4University Surgical Unit, Northern General Hospital, Sheffield, UK, 5Division of Medicine, St. James's University Hospital, Leeds, UK, 6Gastroenterology Unit, The Radcliffe Infirmary, Oxford, UK, 7Gastroenterology Unit, Royal Shrewsbury Hospital, Shrewsbury, UK, 8Digestive Diseases Research Centre, St. Bartholomews's and The Royal London School of Medicine and Dentistry, London, UK, 9Department of Public Health and Epidemiology, University Hospital Queen's Medical Centre, Nottingham, UK, 10Department of Medicine, Institute of Clinical Science, The Queen's University of Belfast, Belfast, UK, 11Section of Gastroenterology, University Department of Medicine, Hope Hospital, Salford, Manchester, UK, 12Department of Paediatrics, St. George's Hospital, London, UK, 13Gastroenterology and Liver Unit, St. Vincent's Hospital, Dublin, Ireland, 14Department of Clinical Genetics, Churchill Hospital, Oxford, UK and 15University Department of Paediatric Gastroenterology, Royal Free Hospital, London, UK
Received March 26, 1997;Revised and Accepted May 15, 1997
A strong HLA association is seen in coeliac disease [specifically to the DQ([alpha]1*0501,[beta]1*0201 heterodimer], but this cannot entirely account for the increased risk seen in relatives of affected cases. One or more genes at HLA-unlinked loci also predispose to coeliac disease and are probably stronger determinants of disease susceptibility than HLA. A recent study has proposed a number of candidate regions on chromosomes 6p23 (distinct from HLA), 6p12, 3q27, 5q33.3, 7q31.3, 11p11, 15q26, 19p13.3, 19q13.1, 19q13.4 and 22cen for the location of a non-HLA linked susceptibility gene. We have examined these regions in 28 coeliac disease families by linkage analysis. There was excess sharing of chromosome 6p markers, but no support for a predisposition locus telomeric to HLA. No significant evidence in favour of linkage to coeliac disease was obtained for chromosomes 3q27, 5q33.3, 7q31.3, 11p11, 19p13.3, 19q13.1, 19q13.4 or 22cen. There was, however, excess sharing close to D15S642. The maximum non-parametric linkage score was 1.99 (P = 0.03). Although the evidence for linkage of coeliac disease to chromosome 15q26 is not strong, the well established association between coeliac disease and insulin dependent diabetes mellitus, together with the mapping of an IDDM susceptibilty locus (IDDM3) to chromosome 15q26, provide indirect support for this as a candidate locus conferring susceptibilty to coeliac disease in some families.
Gluten sensitivity, or coeliac disease, is due to T cell sensitisation and results in a range of mucosal abnormalities that may lead to malabsorption (1 ,2 ). Estimates of prevalence in Europe have ranged from a high of 1 in 300 in Western Ireland to between 1 in 1000 and 1 in 2000 in other regions (1 ,3 ). However, the true prevalence of coeliac disease in some studies is likely to have been underestimated, because a substantial number of individuals are asymptomatic or only have mild symptoms. Recent studies using antibodies for screening have shown that the frequency of coeliac disease among some symptom-free individuals is high (~1 in 200) (4 -8 ).
Studies of small bowel biopsy specimens from first degree relatives of patients with coeliac disease have provided compelling evidence that genetic factors influence susceptibility to this disease (9 ,10 ). The risk of coeliac disease among first-degree relatives of patients is ~10%. Additional support for an inherited predisposition to develop coeliac disease comes from twin studies in which the concordance rate of coeliac disease in monozygotic twins is ~70% (9 ,10 ).
The difference in concordance rates between monozygotic twins and HLA identical siblings (70% versus 30%) implicates non-HLA genes in genetic predisposition to coeliac disease (9 ,10 ). The overall relative risk in siblings (due to HLA and other loci/environmental effects) is at least 20, and is therefore 4-fold higher than that attributable to HLA alone under any postulated genetic model of inheritance. A second gene, unlinked to HLA, is therefore likely to be a stronger determinant of disease susceptibility than the HLA-linked locus (10 ) and the familial risks of coeliac disease seen in siblings and monozygotic twins are most parsimonious with a multiplicative model (10 ).
Using family data, Pena et al. originally proposed the involvement of two distinct unlinked genes in the aetiology of coeliac disease (12 ). A prerequisite for developing coeliac disease was homozygosity at the HLA-unlinked locus and participation of an independently inherited gene or genes located in or tightly linked to the major histocompatibility system acting in a dominant fashion. This proposal has been supported by some but not all other studies (13 ,14 ). Most agreed on recessivity at the HLA-unlinked locus, but differed with respect to dominance or recessivity at the HLA-linked disease susceptibility locus.
Recently a study of 15 coeliac disease families has proposed a number of candidate regions on chromosomes 6p23 (distinct from HLA), 6p12, 3q27, 5q33.3, 7q31.3, 11p11, 15q26, 19p13.3, 19q13.1, 19q13.4 and 22cen for the location of non-HLA linked genes causing coeliac disease (15 ). We report a study of 28 families for linkage between coeliac disease and these putative candidate regions.
Twenty families were used in the linkage study of candidate regions on chromosomes 3, 5, 7, 11, 19 and 22, but all 28 families were used in the analysis of candidate regions on chromosomes 6 and 15.
Non-parametric linkage (NPL) scores are presented in Tables 0 , 0 and 0 for markers at the candidate loci. These scores are model-independent and effectively measure the extent to which marker allele sharing by descent between affected individuals is greater than expected under random segregation. Table 1 shows the results for markers on chromosomes 3, 5, 7, 11, 19 and 22. There was no evidence in family members of excess sharing at any of these markers.
Seventy three of the 78 (94%) affected individuals in the families studied had HLA class II genotypes compatible with possession of the DQ([alpha]1*0501,[beta]1*0201) alleles. Direct evidence that the HLA association in coeliac disease is solely due to this heterodimer was seen in Families 4 and 20. In Family 4 the affected members 602, 603 201 and 202 possess the heterodimer, and whilst individuals 602, 603 and 201 share at HLA, individual 202 does not have the common haplotype. In Family 20, individuals 103 and 201 both possess the heterodimer but individual 101 does not possess DQ[alpha]1*0501 and 102 does not possess DQ[beta]1*0201; hence individual 201 has inherited the heterodimer in trans.
Table 2 shows the multipoint NPL scores for the region on chromosome 6. As expected there is evidence for excess sharing at HLA and at close flanking markers. The markers at D6S273 and D6S276 are within 1 cM of each other and lie within the HLA system. D6S465 is 30 cM centromeric to HLA and so lies well away from the candidate locus on chromosome 6p. This locus was examined because there it also showed some evidence for linkage in the study of Michalski et al. (15 ).
Table 3 shows the NPL scores for the chromosome 15 markers. In our initial screen of candidate loci we typed markers at D15S642 and D15S207. Following suggestive evidence for excess sharing at D15S642 we typed a further five markers. There is weak evidence for excess sharing distal to D15S657. Under both recessive models the LOD score maximises at D15S642. Assuming a rare recessive gene, 36% of families are estimated as linked and the maximum LOD score achieved is 1.39. Alternatively, if the gene is common, 53% of the families are estimated as linked (LOD = 1.23). If the disease is rare and dominantly acting, the best position for the locus is at D15S1034 with 27% of the families linked (LOD = 1.013). Under a common dominant disease gene the best position for the locus is at D15S642 with 44% of families linked (LOD = 0.84).
The study by Michalski et al. found linkage between coeliac disease and a number of chromosomes: HLA, 6p23 (telomeric to the HLA system), 6p12, 3q27, 5q33.3, 7q31.3, 11p11, 15q26, 19p13.3, 19q13.1, 19q13.4 and 22cen (15 ). Regions having a multipoint maximum LOD score >2.0 were 6p23 (outside the HLA system), 7q31.3, 11p11, 15q26 and 22cen. The most significant result was between D6S259 and D6S4696. These findings were based on 15 nuclear families ascertained from the west of Ireland, an area known for a high prevalence of coeliac disease.
The data from the families we have studied provide no significant evidence in favour of linkage of coeliac disease to the putative candidate regions on chromosomes 3q27, 5q33.3, 7q31.3, 11p11, 19p13.3, 19q13.1, 19q13.4 or 22cen. The results from our study also provide no strong support for a predisposition locus on chromosome 6p distinct from HLA. The marker at D6S259 is ~20-30 cM telomeric from HLA and in the report by Michalski et al. (15 ), the LOD score between HLA and D6S259 fell below significance. This result was based on typings in 30 of the 45 affected sibling pairs at D6S259 and flanking markers within 1 cM showed no significant evidence for linkage. In our study, there was excess sharing at D6S259 but sharing was more pronounced at HLA and sharing at intervening markers largely reflected inheritance of an extended haplotype on 6p.
In our study, there was excess sharing at 15q26 and although the evidence for linkage of coeliac disease to this chromosome is not strong, there is indirect supporting evidence for this as a candidate locus conferring susceptibilty to coeliac disease in some families. There is a well established association between coeliac disease and insulin dependent diabetes mellitus (IDDM) and a susceptibilty locus termed IDDM3 on chromosome 15q26 has been mapped close to D15S107 (16 ,17 ). The markers D15S120 and D15S1034 flank D15S107 by 3 cM. The inheritance of coeliac disease in some of the families we have studied is more suggestive of a dominant mode of transmission for a non-HLA disease gene. However, the highest LOD scores were achieved under models based on a recessive mode of transmission. This is in keeping with the mode of inheritance of a non-HLA linked gene predicted from segregation analyses of coeliac disease families (12 -14 ). Under both recessive models the maximal LOD score was obtained at D15S642. The maximum multipoint linkage result on 15q reported by Michalski et al. was between D15S107 and D15S207 and there was also evidence for a recessive mode of inheritance (15 ). Whilst our findings at chromosome 15q are broadly concordant with that of Michalski et al. (15 ), this result needs validation in a larger series of families.
Twenty eight families with two or more members affected with coeliac disease were recruited for this study (Fig. 1 ). All the families resided in the British Isles. The diagnosis of coeliac disease was established in all cases by demonstration of severe enteropathy with villous atrophy on small bowel biopsy. All made a symptomatic recovery and the majority of patients had a follow up biopsy demonstrating mucosal recovery on a gluten-free diet. The age at diagnosis ranged from 6 months to 74 years with a median of 32 years.
Samples of peripheral blood (10 ml) were taken from appropriate individuals and DNA was extracted using standard methods. These individuals were genotyped at the following loci: D6S1721, D6S259, D6S429, D6S422, D6S276, D6S273, D6465, D5S498, D7S480, D22S247, D19S591, D19S418, D19S178, D3S1754, D3S1262, D11S871, D11S929, D15S1004, D15S207, D15S1014, D15S1034, D15S120, D15S642. These loci were those which gave the strongest evidence for linkage in the study by Michalski et al. (15 ). The markers chosen were either identical to or mapped very close to the original markers used.
Genomic DNA (50 ng) was amplified using PCR in 25 [mu]l of 75 mM Tris-HCl (pH 9), 0.01% (v/v) Tween, 1.5 mM MgCl2, 0.25 U Taq polymerase, 20 mM (NH4)2SO4, 0.1% (w/v) BSA, 0.2 mM each dNTP and each primer at 5 ng/[mu]l. One primer had previously been end-labelled with [[gamma]-32P]dATP using 3 U T4 polynucleotide kinase. Thirty five cycles of PCR were carried out at 94oC for 1 min, 50-55oC for 1 min and 72oC for 1 min with a final extension step at 72oC for 10 min. The products were electrophoresed through 6% denaturing polyacrylamide gels, dried and exposed to X-ray film for 24 h. All family members were typed for HLA-DQ[alpha]1*0501 and [beta]1*0201 alleles by PCR-SSP (details from O. Olerup, personal communication).
The program GENEHUNTER was used for NPL and for computation of multipoint LOD scores under four disease models (18 ). In the absence of a definitive model for the mode of inheritance of coeliac disease, models for the parametric analyses were chosen assuming that the sibling relative risk conferred by the disease gene was 3.3. This follows from the assumptions that the overall disease prevalence is 0.005, that the HLA-linked locus acts dominantly and there are at least two equal risk genes acting multiplicatively with HLA (10 ). Rates in non-carriers were estimated to achieve a prevalence of 0.005. The models were: (i) a rare recessive disease gene (P = 0.05) with penetrance of 29% and a disease risk of 0.43% in non-carriers; (ii) a common recessive disease gene (P = 0.2) with penetrance 6.9% and a disease risk of 0.23% in non-carriers; (iii) a rare dominant disease gene (P = 0.003) with a penetrance of 14% and a disease risk of 0.42% in non-carriers; and (iv) a common dominant disease gene (P = 0.05) with a penetrance of 3.8% and a disease risk of 0.15% in non-carriers. All unaffected individuals were classified as of unknown phenotype. This allows for the possibility that an apparently unaffected individual may actually be affected and ensures that false negative data are not generated. The HOMOG program was used to compute LOD scores under heterogeneity (19 ).
Marker allele frequencies were estimated from observed relative frequencies in founder individuals. (These were similar to the relative frequencies seen across the larger number of all typed individuals.) Heterozygosity for each marker was estimated from these frequencies. Distances between markers were obtained from the Marshfield (http://genetics.mfldclin.edu), GDB (http://www. hgmp.mrc.ac.uk) and Genethon (http://www.cephb.fr/ceph-genethon-map.html) databases.
This work was supported by the Cancer Research Campaign. We are grateful to Ole Olerup, for providing us with details of the PCR-SSP method for HLA typing, to Anne Lewis for help in the ascertainment of families and to Diane Chandley for secertarial assistance. Finally we would like to thank all members of the families who participated in the study.
5 Bode,S., GudmandHoyer,E. (1996) Incidence and prevalence of adult coeliac disease within a defined geographic area in Denmark. Scand. J. Gastroent.31, 694-699.
6 Catassi,C., Ratsch,I.M., Fabiani,E., Ricci,S., Bordicchia,F., Pierdomenico,R., Giorgi,P.L. (1995) High prevalence of undiagnosed coeliac disease in 5280 Italian students screened by antigliadin antibodies. Acta Paed. Int. J. Paed.84, 672-676.
7 Johnston,S.D., Watson,R.G.P., McMillan,S.A., McMaster,D., Evans,A. (1996) Preliminary results from follow-up of a large-scale population survey of antibodies to gliadin, reticulin and endomysium. Int. J. Paed.(Supplement), 85, 61-64.
8 Catassi,C., Fabiani,E., Ratsch,I.M. et al. (1996) The coeliac iceberg in Italy. A multicentre antigliadin antibodies screening for coeliac disease in school-age subjects. Int. J. Paed. (Supplement) 85, 29-35.
9 Sollid,L.M. and Thorsby,E. (1993) HLA susceptibility genes in celiac disease: genetic mapping and role in pathogenesis. Gastroeneterology105, 910-922.
10 Houlston,R.S. and Ford,D. (1996) Genetics of coeliac disease. Q. J. Med.89, 737-743.
11 Kagnoff,M.F., Harwood,J.I., Bugawan,T.L. et al. (1989) Structural analysis of the HLA-DR, -DQ and -DP alleles on the celiac disease associated HLA-DR3 (Drw17) haplotype. Proc. Natl. Acad. Sci. USA86, 6274-6278.MEDLINE Abstract
12 Pena,A.S., Mann,D.L., Hague,N.E. et al. (1978) Genetic basis of gluten-sensitive entropathy. Gastroenterology75, 230-235.MEDLINE Abstract
13 Greenberg,D.A., Hodge,S.E. and Rotter,J.I. (1982) Evidence for recessive and against dominant inheritance at the HLA-linked locus in coeliac disease. Am. J. Hum. Genet. 34, 263-277.MEDLINE Abstract
14 Hernandez,J.L., Michalski,J.P., McCombs,C.C. et al. (1991) Evidence for a dominant gene mechanism underlying coeliac disease in the West of Ireland. Genet. Epidem.8, 13-27.
15 Zhong,F., McCombs,C.C., Olson,J.M., Elston,R.C., Stevens,F.M., McCarthy,C.F., Michalski,J.P. (1996) An autosomal screen for genes that predispose to celiac disease in the western counties of Ireland. Nature Genet.14, 329-333. MEDLINE Abstract
16 Field,L.L., Tobias,R. and Magnus,T. (1994) A locus on chromosome 15q26 (IDDM) produces susceptibility to insulin-dependent diabetes mellitus. Nature Genet. 8, 189-194.MEDLINE Abstract
17 Zamani,M., Pocoit,F., Raeymaekers,P., Nereup,J. and Cassiman,J.J. (1996) Linkage of type 1 diabetes to 15q26 (IDDM3) in the Danish population. Hum. Genet.98, 491-496.MEDLINE Abstract
18 Kruglyyak,L., Daly,M.J., Reeve-Daly,M.P. and Lander,E.S. (1996) Parametric and non-parametric linkage analysis: A unified Multipoint approach. Am. J. Hum. Genet.58, 1347-1363.
19 Ott,J. (1985) Analysis of Human Genetic Linkage. Johns Hopkins University Press. Baltimore and London.
*To whom correspondence should be addressed. Tel: +44 181 643 8901; Fax: +44 181 770 7876; Email: r.houlston@icr.ac.uk
-->
This page is maintained by OUP admin. Last updated Tue Jul 15 11:13:42 BST 1997. Part of the OUP Journals World Wide Web service.
Copyright
Oxford University Press, 1996
