Susceptibility locus for inflammatory bowel disease on chromosome 16 has a role in Crohn's disease, but not in ulcerative colitis
Susceptibility locus for inflammatory bowel disease on chromosome 16 has a role in Crohn's disease, but not in ulcerative colitisJeffrey D. Ohmen1,+, Hui-Ying Yang2,+, Karen K. Yamamoto1, Hong-Yu Zhao3, Yuanhong Ma1, L. Gordon Bentley1, Zhihan Huang2, Scott Gerwehr1, Sheila Pressman2, Colleen McElree2, Stephan Targan4, Jerome I. Rotter2 and Nathan Fischel-Ghodsian1,2,*
1GenoMed Pharmaceuticals, Inc., Suite 360, 150 N. Robertson Blvd, Beverly Hills, CA 90211, USA, 2Departments of Medical Genetics and Pediatrics, Cedars-Sinai Research Institute, Cedars-Sinai Medical Center, 8700 Beverly Blvd, Los Angeles, CA 90048, USA, 3Department of Biostatistics, UCLA School of Public Health, 10833 LeConte Avenue, Los Angeles, CA 90095, USA and 4Department of Medicine, Division of Gastroenterology, Cedars-Sinai Research Institute, Cedars-Sinai Medical Center, 8700 Beverly Blvd, Los Angeles, CA 90048, USA
Received June 3, 1996;Revised and Accepted July 24, 1996
In the Western world, chronic inflammatory bowel disease (IBD) presents as two major clinical forms, Crohn's disease (CD) and ulcerative colitis (UC) [Targan,S.R. and Shanahan,F. (1994) In Retford,D.C (ed.), Inflammatory Bowel Disease: From Bench to Bedside. Williams and Wilkins, Baltimore]. Genetic epidemiological studies, the occurrence of rare syndromes associated with IBD, and animal models suggest that inherited factors play significant roles in the susceptibility to both forms of IBD [Yang,H.-Y. and Rotter,J.I. (1995) In Kirsner,J.B. and Shorter,R.G. (eds), Genetic Aspects of Idiopathic inflammatory Bowel Disease. Williams and Wilkins, Baltimore, pp. 301-331]. Recently, a genome-wide search on European families with multiple affected members with CD identified a putative susceptibility locus in the centromeric region of chromosome 16 [Hugot,J.-P. et al. (1996) Nature, 379, 821-823]. We have now tested this region in an independent set of US families, confirmed that this region is likely to contain a gene predisposing to CD, and further refined the chromosomal location of this gene. Most importantly with respect to this locus, our data also seem to indicate that there is heterogeneity both within the CD group, and between the CD and UC groups with respect to this locus. The susceptibility locus appears to be involved only in non-Jewish CD sibpairs and not in our Ashkenazi Jewish CD sibpairs. Additionally, we have tested sibpairs having either only UC or both UC and CD for involvement of this locus, and have found no evidence that this region predisposes to IBD in these patients.
Inflammatory bowel disease (IBD) is a complex disease of unknown etiology. The differences in disease frequency between geographic areas and racial/ethnic groups suggest that genetic factors play a role in etiology (2 ,4 ). However, data from migrant studies and the increasing incidence of Crohn's disease (CD) seen in many parts of the world during the past 30 years suggest that environmental agents are also important in disease etiology (5 ). The high toll of this disease combined with the lack of specific therapeutics has spurred a search for genes that contribute to disease susceptibility.
Ulcerative colitis (UC), like CD, is also characterized by inflammatory lesions in the bowel. It is not uncommon for families with an individual afflicted with one form of IBD to have additional siblings afflicted with the other form, and in fact this occurrence is substantially greater than expected from the relative frequency of the two diseases (6 ). It can be postulated, therefore, that IBD may involve genes in a pathway common to bowel inflammation, and that the phenotypic differences seen between the two diseases result from the expression of additional genes specific to CD or UC.
CD is an ideal disease to utilize a genetic approach towards understanding its cellular basis. First, the genetic component is well documented by: (i) the monozygotic twin concordance rates (7 -9 ), (ii) the rarity of IBD concordance in spouses (10 ) and (iii) the numerous instances of family members with onset separated greatly in time (11 -13 ). Interestingly, there is an increased rate of CD among Ashkenazi Jews, suggesting that within this ethnic group, there may be less genetic heterogeneity for IBD predisposing loci and thus a potentially limited number of genes involved (14 -17 ).
Recently, Hugot et al. reported the results of a genome scan for CD using linkage analysis (3 ). Twenty-five families and ~41 sibpairs were included in the analysis resulting in the identification of three polymorphic markers on chromosome 16 and one on chromosome 1 as having P values of <0.01. The authors then used a separate panel of 53 families and 71 sibpairs to test further for the involvement of these two loci in IBD. The results from the second panel of families did not provide sufficient evidence for involvement of the chromosome 1 locus, but supported the presence of an IBD predisposing locus (IBD1) near D16S409. Interestingly, no other significant loci were found in their genome-wide search for IBD genes. We report here the results of sibpair analysis of IBD1 in a separate patient population with CD, as well as in UC and UC/CD sibpairs.
To identify genes that contribute to CD we studied 48 families with 75 sibpairs diagnosed with CD. Furthermore, 23 additional sibpairs from 16 families with UC and 19 sibpairs from 15 families with one sib having CD and the other having UC were analyzed to test whether the IBD1 locus contributes to UC. Unlike the primarily French and British population that constitutes the sample population used by Hugot et al. (3 ), our sample population is composed of North Americans, one-third being of Ashkenazi Jewish origin. Sixty percent of the non-Jewish subjects are of German, Italian and Eastern European origin.
To test the involvement of IBD1 in our CD patient population, we screened a chromosomal region of ~84 centimorgans (cM) around the putative susceptibility locus described by Hugot et al. on chromosome 16 (3 ). Seventeen microsatellite markers were genotyped in this region and analyzed by SIBPAL (18 ). As shown in Table 1 , there is a 26 cM region on chromosome 16 between markers D16S403 and D16S419 where sharing between affected sibpairs is greater than expected by chance (with maximum sharing at D16S411). We also tested the chromosome 1 marker D1S236 described by Hugot et al. (3 ), as well as two flanking markers D1S424 and D1S206 for linkage. Sharing of alleles between affected sibpairs was <50% for D1S236 and D1S424 and 53% for D1S206 (data not shown), indicating a lack of involvement of this locus in IBD.
Multipoint analysis (19 ) generated a maximum LOD score for linkage at marker D16S411 (Fig. 1 ). The location of this peak corresponds to the chromosomal region identified by Hugot et al. Our data differs significantly from Hugot et al. (3 ), in that we fail to detect any allele sharing at D16S503 as can be seen in Figure 1 . Combining the previously reported data with our data allows us to refine the localization of the susceptibility gene to be in the vicinity of markers D16S409 and D16S411.
When our sample population was stratified by ethnic origin (non-Jewish versus Jewish), sharing of alleles at D16S409 and D16S411 for non-Jews increased to 0.60 and 0.62 respectively, while allele sharing for Jews decreased to 0.48 and 0.50 (data not shown). Multipoint analysis showed a dramatic difference between Jewish and non-Jewish families (Fig. 1 ). The LOD score increased to 2.41 for the non-Jewish population, with the Jewish population showing a significant lack of evidence for linkage. When D16S411-D16S409 haplotypes were used to increase informativeness, heterogeneity between Jewish and non-Jewish families in the linkage of CD within this region was strongly suggested (Z = 1.60, P = 0.055).
To test the potential role of the IBD1 locus in UC, we used markers D16S403, D16S420, D16S409, D16S411, D16S419, D16S408 and D16S503 to genotype UC and UC/CD sibpairs (Table 2 ). The mean value for alleles shared identity by descent (i.b.d.) never exceeded 0.503 (P = 0.477) at any of the six loci tested. When the observed mean values of alleles shared identical by descent in CD sibpairs were compared with those observed in UC and UC/CD sibpairs, the difference reached statistical significance at the P = 0.005 level (Z = 2.608). The chromosome 16 locus therefore appears to contribute specifically to CD, and is unlikely to be involved in a common pathway of IBD.
Each of the markers used to test UC and UC/CD sibpairs is shown in Table 2. The corresponding Z score and resulting P value are also shown. For the map position of each marker, refer to Figure 1.
In this report, we have used linkage analysis to confirm and refine the findings of Hugot et al. (3 ), regarding the presence of a region on chromosome 16 that appears to encode a CD predisposing factor. Our results define a more narrow region in the neighborhood of markers D16S409 and D16S411 in which to begin the search for IBD1. Interestingly, our data indicate that IBD1 appears to predispose only in non-Jewish families with CD, while not playing a role in Jewish CD families. These data are in accordance with the emerging model of IBD being clinically, serologically, and molecularly more heterogeneous than the existing grouping into CD or UC suggests (2 ). People of Ashkenazi Jewish ethnicity are two to four times more susceptible than Caucasians to the development of IBD, and the genetic factors responsible for this increased frequency will need to be identified by additional analyses with families enriched for Ashkenazi Jews.
It is interesting, given the importance of the genetic component in CD (7 -13 ), that Hugot et al. (3 ) found only one susceptibility locus for CD. Several lines of evidence suggest that there are other loci involved in CD. Perhaps most importantly, the amount of allele sharing seen by Hugot et al. and ourselves at IBD1 is quite small (0.64 and 0.58 respectively), indicating only a modest role for IBD1. Secondly, the data regarding the lack of involvement of IBD1 among Ashkenazi Jews also indicate that additional CD predisposing loci must be present. One possible reason that Hugot et al. didn't find more loci that contribute to CD within their families may be due to the small number of sibpairs included in their screening panel. The lack of sufficient sample size results in type II errors. Clearly, additional searches will be required to find other loci involved in CD.
There are a number of interesting candidate genes described by Hugot et al. in the region of IBD1 (3 ). Most of these genes are involved in normal immune function. However, these genes all map >10 cM away from D16S411. We found only two genes that map within 10 cM of D16S411, and neither appears to be an obvious candidate. Cholesteryl ester transfer protein (CETP; GenBank accession no. G11210) and carboxylesterase 1 (CES1; GenBank accession no. G06045) both map closely to D16S411 (20 ). Further refinement of the chromosomal location of IBD1 will require identification of linkage disequilibrium, which in turn will allow positional cloning of IBD1 through a general positional cloning approach. Identification of the CD susceptibility gene on chromosome 16 will provide a starting point for understanding the underlying genetic and biochemical factors that contribute to this disease, and may ultimately lead to therapeutic modalities for the treatment of CD.
Patients diagnosed with CD or UC were ascertained from the IBD programs at Cedars-Sinai Medical Center (CSMC), the IBD Clinical Research Program at the University of Chicago, or referred by the Crohn's and Colitis Foundation of America. The study protocols were approved by the CSMC Human Subject Review Committee. The diagnosis of CD or UC was documented by conventional endoscopic, histopathological and clinical criteria, as previously described (1 ,22 ). Patients were excluded if they had evidence for bacterial or parasitic infections. Families with a total of two or more siblings affected with CD or UC or both were selected for the current study. The medical records of affected relatives were obtained to confirm the diagnosis of CD or UC, using standard criteria (1 ,22 ). All families were nuclear families consisting of one or two parents, the majority of whom were not affected with CD or UC.
Genomic DNA was isolated from EBV-transformed lymphoblastoid cell lines using Qiagen Inc. (Chatsworth, CA 91311) QIAamp Tissue columns following the manufacturer's instructions. The polymorphic markers used in this study are shown in Table 1 . Many of these markers are part of the ABI Prism linkage mapping panels (Perkin Elmer/Applied Biosystems Division, Foster City CA 94404), with the remainder selected using information from the Genome Data Base (Johns Hopkins University, Baltimore, MD) and purchased prelabeled from Research Genetics (Huntsville, AL 35801). Genotyping for each of the selected markers was performed by PCR amplification using 75 ng of genomic DNA and 25 ng of each oligonucleotide primer in 15 [mu]l amplification reactions consisting of 10 mM Tris-HCl pH 9.0, 50 mM KCl, 2.5 mM MgCl2, 0.1% Triton X-100, 0.01% (w/v) gelatin, 250 mM of each dNTP and 0.6 U of Ampli-Taq DNA polymerase (Perkin Elmer/Applied Biosystems Division, Foster City CA 94404). Amplification conditions consisted of an initial denaturation at 95oC for 5 min, 10 cycles of denaturation at 94oC for 15 s, annealing at 55oC for 15 s and extension at 72oC for 30 s, followed by 20 cycles with parameters the same as the first 10 cycles, with the exception that the denaturation temperature is reduced to 89oC. The resulting PCR products were pooled and diluted ~20-fold and denatured before loading on a 36 cM long, 0.2 mM thick, 4.25% (6 M urea) denaturing polyacrylamide gel. Data were collected with an ABI 377 DNA sequencer using ABI PrismTM Data Collection software (version 1.1). Initial analyses of the gels were accomplished with ABI GeneScan (version 2.0.2) software. Allele sizes were determined with ABI Genotyper software (version 1.1). Fluorescently labeled oligonucleotide primers were purchased from either ABI or from Research Genetics (Huntsville, AL 35801). The polymorphic markers used in this study are shown in Table 1 .
Two point affected sibpair analysis with SIBPAL (version 2.7) was used to calculate the allele sharing between sibs (18 ). The allele frequencies for each marker were estimated from 73 parents in the CD families and 60 parents in the UC and UC/CD families, with all of the parents being unrelated to each other. The mean proportion of alleles shared i.b.d. was estimated and tested against the null hypothesis with the mean allele sharing among sibs assumed to be P = 0.5. The multipoint LOD scores were calculated using MAPMAKER/SIBS (19 ) for the Jewish and non-Jewish families. The allele frequencies were estimated as in the two point analysis. Genetic distances between the markers were from published Genethon maps (21 ). All independent sibpairs were used in the analysis. To test for heterogeneity of linkage with D16S411 between Jewish and non-Jewish families and between CD only and UC-UC or UC-CD sibpairs, we generated haplotypes with two closely linked markers, i.e. D16S409-D16S411 to increase the informativeness of the studied families. Mean proportion of haplotype (p) shared i.b.d. were calculated as the ratio of the number of haplotypes shared i.b.d. (I) and counts of the total number of informative haplotypes (N), i.e. P = I/N. Z statistic for a binomial proportion (4 ) was used to test differences between the two groups.
We wish to thank the physicians and patients who have contributed to this study. This research was supported by grants from the IBD Program Project Grant (NIH-NIDDK DK46763), the Crohn's and Colitis Foundation of America, GenoMed Inc., the Feintech Family Chair in IBD (SRT) and the Board of Governors Chair in Medical Genetics (JIR). S.A.G.E. programs are supported by grant 1 P41 RR03655.
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20 NCBI Genome Data Base. Version 3.7 Update 4/9/96.
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*To whom correspondence should be addressed at: Department of Pediatrics, Cedars-Sinai Research Institute, Cedars-Sinai Medical Center, 8700 Beverly Blvd, Los Angeles, CA 90048, USA
+Both authors contributed equally to this work
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