Chang-Qing Zheng, Gang-Zheng Hu, Lian-Jie Lin, Gin-Ge Gu, Department of Gastroenterology, the Second Affiliated Clinical College of China Medical University, Shenyang 110001, Liaoning Province, China
Zhao-Shu Zeng, Department of Serology, College of Forensic Medicine, China Medical University, Shenyang 110001, Liaoning Province, China
Correspondence to: Professor Chang-Qing Zheng, Department of Internal Medicine, the Second Affiliated Clinical College of China Medical University, Shenyang 110001, Liaoning Province, China. zhengchangqing88@163.com
Telephone: +86-24-83956682    Fax: +86-24-83956682
Received: 2003-03-03    Accepted: 2003-04-11

Abstract
Inflammatory bowel disease (IBD) includes two clinical subtypes: Crohn disease (CD) and ulcerative colitis (UC). The general prevalence is about 1.0-2.0 % in Western countries. It is predominantly regarded as a multifactorial disorder involving environmental factors and polygenic defects. The view was confirmed by a lot of evidences from clinical attributions and animal models, especially from epidemiological investigations. So the etiological study of IBD has been focused on searching for susceptibility genes by positional cloning, which consists of two steps: linkage analysis and association analysis. Linkage analysis has been an important method of searching for susceptibility genes to polygenic diseases as well as single-gene disorders. IBD, as a polygenic disease, has been widely investigated by linkage analysis for susceptibility gene since 1996. The paper reviewed 38 articles, which covered almost all original researches in relation to IBD and linkage analysis. So far, several loci, such as 16q, 12q, 6p and 3p, have been identified by the studies. The most striking is 16q12 (IBD1), which linked only with CD not UC in the majority of studies. Association analysis, as one essential step for positional cloning, is usually carried out by genotyping candidate genes selected by means of linkage analysis or other methods, for figuring out the frequencies of alleles and comparing the frequencies between IBD group and healthy control group to identify the specific allele. It has been established that IBD is implicated in immune disorder. So the studies were centered on the genes of NOD2/CARD15, HLA-II, cytokine, cytokine receptor and adhesion molecule. This paper reviewed 14 original articles on association between NOD2 and IBD that have been published since 2001. All results, with the exception of one report from a Japanese group, provide evidences that the three kinds of variants of NOD2 are susceptibility factors for IBD. This article also comprehensively analyzed 18 original researches of HLA gene polymorphism in IBD. We found extensive discrepancy among the conclusions and a novel hypothesis was put forward to explain the discordance. Most studies published recently on association between IBD and cytokine gene polymorphism were reviewed.

Zheng CQ, Hu GZ, Zeng ZS, Lin LJ, Gu GG. Progress in searching for susceptibility gene for inflammatory bowel disease by positional cloning. World J Gastroenterol  2003; 9(8): 1646-1656
http://www.wjgnet.com/1007-9327/9/1646.asp

INTRODUCTION
Inflammatory bowel disease (IBD) is composed of two clinical subtypes: Crohn disease (CD) and ulcerative colitis (UC). Its general prevalence is 0.1-0.2 % in Western countries^[1]. There has been no epidemiological investigation of large scale for the prevalence or incidence of IBD in China so far, despite the facts that UC is common in China and CD has been more frequently diagnosed by clinical physicians in recent years^[2]. The pathogenesis of IBD has not been clearly identified. Today, the most generally accepted pathogenesis of IBD is that IBD is resulted from abnormal immune response to enteric bacteria in individuals with susceptibility due to genetically polygenic defects. Therefore, investigators have searched human genome for the loci of susceptibility genes by linkage analysis and have achieved great success. On the other hand, association analysis, as one of the essential steps for positional cloning, was carried out by many investigators to identify the specific allele. It has established that IBD is implicated in immune disorder. Biochemical substances involved in immunoregulation are very rich and the corresponding genes are widely distributed in genome. Genome-wide linkage analysis has suggested multiple candidate regions in several chromosomes for IBD, therefore, considerable numbers of candidate genes should be selected for association analysis. In recent years, these studies were centered on the genes of NOD2/CARD15, HLA-II, cytokine, cytokine receptor and adhesion molecule. These studies were summarized in this review.

IBD IS A MULTIFACTORIAL DISEASE
There have been a number of hypotheses about the pathogenesis of IBD, but neither environmental factors, such as habit of diet and behavior, infection of microorganisms and contact of chemical or physical pathogenic agents, nor single-gene genetic disorder alone can fully explain its complex phenotypes. Thereby, it is thought to be a multifactorial disease. The view was supported by a larger amount of evidences from clinical attributions and animal models, especially epidemiologic investigations and linkage analyses.

Persuasive evidences of genetic contribution to IBD ^[3-12]
A. The first-degree relatives of affected individuals show about 20-50-fold increased risk of developing the disease compared with the general population for CD, and 10-20-fold increased risks for UC. Moreover, the affected siblings frequently present at similar ages and concordance rates reach up to 80 % for disease site, behavior and presence of extraintestinal manifestation. B. Twin studies have shown that the concordance rate of CD is about 20-44 % for monozygotic twins, and 3.8-6.5 % for dizygotic twins; the concordance rate of UC is about 6-16 % and 3 % respectively^[6,7]. C. There are significant ethnic differences in disease frequency. For instance, the prevalence in Ashkenazi Jews is much higher than that in other races, even though they share similar living environment in the same community^[8,9]. D. All genome-scanning linkage analyses detected some linkage loci, certain of which were subsequently confirmed by replication studies only involving certain chromosomes; NOD2 was consistently identified as the susceptibility gene for CD in recent years. E. Simulation studies on animal models have showed that transgenic mice or gene-knockout mice are subject to colitis similar to human IBD, and that spontaneous colitis or hapten-induced colitis manifests fairly different in different strains of mice^[10-12].

Evidence of environmental contribution
A. The concordance rate of IBD for monozygotic twins is much less than 100 %. The identical genotype with different phenotypes means that environmental factors take part in the pathogenesis of the disease^[6,7]. B. Intestinal bacteria are suggested as the main environmental contributions demonstrated by many evidences: antibiotic therapy can usually induce temporary remission for most IBD cases^[13], diversion of faeces stream can make distal improvement in patients with CD^[14], some studies suggested that certain strains of intestinal bacteria were associated with IBD^[15,16], colonization with normal enteric bacterial flora was required for the occurrence of disease in animals with CD irrespective of the underlying defect^[10-12]. C. smoking is likely to be associated with the progress of IBD^[17,18]. D. Migrant epidemiological studies demonstrated that population of identical ethnic background, when lived in different communities, showed discordant incidence^[8,9,19,20].

IBD is not a disorder of simple mendelian inheritance ^[3,21-25]
Genetic disease of classic Mendelian model, which consists of Mendelian dominant and recessive genetic disorders, is a phenotype of single-gene defect and called single-gene disorder. IBD has previously been interpreted as genetic disease of Mendelian recessive model. But segregation analyses offered counter-evidence that IBD followed the principle of Mendelian inheritance. Parents of most IBD probands were healthy, frequency of siblings or children of the patients was much less than 50 %, the decline in frequency of affected second-degree relatives compared with first-degree relatives was greater than that predicted by autosomal dominant inheritance, in which the frequency was expected to decrease by 1/2 with each step. Incidence of IBD in children of affected spouses was sharply less than 100 % and a similar proportion of affected siblings and children of affected probands was inconsistent with autosomal recessive inheritance. Linkage analysis has detected several linkage loci that are distributed on a number of chromosomes.

LINKAGE ANALYSIS
It is very difficult to find the biochemical substances, which express qualitative difference between patients and healthy population by means of classical functional cloning. So linkage analysis, as the first step of positional cloning, may serve as a unique and practicable substitution for the time being. Figuring out genetic distance between marked loci and susceptibility gene by means of pedigree investigation and genotyping, then defining the approximate position of susceptibility gene in genomic map are the essential courses of linkage analysis. The dramatic progress of human genome project, which has located nearly 10 000 marker loci in genomic map, has greatly boosted positional cloning for complex genetic diseases. Epidemiological studies have identified striking genetic contributions to the etiology of IBD, but so far, studies with traditional biochemical methods have not yet identified the products with quantitative defects. Many investigators have turned to linkage analysis and have achieved great success. The important data from 38 original researches, which covered almost all articles in relation to IBD and linkage analysis that have been published since 1996, are listed in Table 1^[26-63], and some aspects were reviewed as follows.
      The common course of linkage analysis for IBD is: collecting families with affected sibling pairs (ASP) or affected relative pairs (ARP) ≥2 by strict ascertainment, genotyping of genome-wide or certain chromosomes according to microsatellite polymorphisms, figuring out multi-point maximal non-parametric LOD score (MLOD) and two-point LOD score by means of statistical software, inferring genetic distances of susceptibility genes to marker loci and locations in physical genome map, offering candidate genes for association analysis. The majority of investigations found certain suggestive linkage loci with various LOD score, but when defined according to different LOD thresholds, the locations or number of linkage loci were variable. In view of the traits of statistical software and quantity of subjects in most studies, we only displayed the results with MLOD ≥2.0 or 3.0, represented by  and +. The chromosomes, on which the linkage loci strongly supported (MLOD≥3.0) by at least one of 8 linkage analyses of genome-wide scanning located, include chromosomes 1, 3, 5, 6, 7, 12, 14, 16, 18 and 19, as well as chromosomes 4, 10, 17 and x with suggestive evidence (2.0≤MLOD<3.0). Although there was striking discrepancy among the genome-wide scans in respect of linkage loci, almost all studies detected more than 3 linkage loci. This shows that the pathogenesis of IBD is involved in multiple genes and manifests obvious genetic heterogeneity. Several loci were supported by relative more studies, such as 16q, 12q, 6p, and 3p. Because Hugot et al ^[26] and Satsangi et al ^[27] detected strong linkage evidences for chromosomes 16, 12, 6, 3 and 7 in 1996, subsequent studies mainly focused on these chromosomes. It can be seen from Table 1 that more evidences were offered for these loci, with the exception of 16q, simply because these loci were investigated by more studies. Some loci supported by certain genome-wide scans, such as 14q, 5q, 19p, likely to harbor susceptibility genes, were less investigated.
      Stratification studies demonstrated significant variances as to the degree and loci of linkage between families with severe IBD and those with only slight IBD, male patients and female patients, Jewish people and non-Jewish people, as well as between UC and CD. Some investigators examined families with CD patients only; others examined families with UC patients only, but most studies detected both families and those with mixed patients and compared the differences of linkage loci between the two groups. As shown in Table 1, there were some differences between UC and CD. The most striking is 16q12 (IBD1), which linked only with CD not UC in the majority of studies. This shows that CD and UC have some common susceptibility genes, as well as certain individual susceptibility genes. Three studies^[32,47,48] found that certain loci linked only with the families with early onset of CD. All subjects examined by the studies listed in Table 1 included Caucasian or Jewish patients from Europe, Australia and northern America, but no Mongolian patients. Three studies^[28,29,42] demonstrated significant differences between Jewish patients and non-Jewish patients. In respect of nationality of patients, it seems there are no remarkable differences among American, English, German, Australian, Canadian, Italian, Dutch and Belgian. But Paavola et al ^[40] examined chromosomes 1, 3, 7, 12, 14 and 16 in Finnish patients and did not find linkage loci. Fisher et al^[33] found that some loci on chromosomes 6p, 1, 14 and 18 linked only with IBD of male sufferer. These results confirm the extensive genetic heterogeneity of IBD.
      Linkage analysis is intended as an essential tactic to offer candidate genes for association analysis. We should focus our attention on the linkage loci containing some candidate genes, products of which have been suggested as pathogenic factors by other methods, as well as confirmed by subsequent replication studies. The loci meeting these conditions were briefly reviewed here.

Table 1  Data of linkage analysis

Note: CD, CD-only family; UC, UC-only family; IBD=UC+CD+mixed family; +, convincing linkage (LOD≥3.0);± , suggestive linkage (3.0>LOD≥2.0); - -, no suggestive linkage (LOD<2.0); ^a, including Jewish; ^b, family from English, German and Dutch; ^c, family from Northern American, European and Australian; ^d, family from French and Belgian; ^e, linkage only for Jewish; ^f, linkage only for IBD with early onset.

Chromosome 16  As shown in Table 1, 14 out of 25 related studies found linkage loci for CD with MLOD more than 2.0 on the chromosome, additionally, some loci with suggestive score (MLOD between 1.0 and 2.0) were detected. Only 3 studies found linkage loci with UC, furthermore, 2 of them also detected linkage with CD, and the other one merely examined UC families. It can be inferred from these studies that chromosome 16 contains susceptibility gene for CD rather than UC. Chromosome 16 is comparatively short, with 98 Mb of physical length, 130.8 cm of genetic distance, and has been spaced by about 200 microsatellite markers^[64]. The linkage loci suggested by the studies in Table 1 were distributed in most part of chromosome 16 (for instance, D16S409-419^[26], D16S748-764^[28], D16S411^[42,43], D16S3136^[50]), but only pericentromeric region on 16q was the most consistent linkage region. The important candidate genes in the region are NOD2, CD11integrins, CD19, Sialophorin, IL-4 receptor gene etc. NOD2 gene has been established as susceptibility gene to CD. It remains unanswered if there are other susceptibility genes in the chromosome. Hampe et al^[51] examined additional regions with high-density experiment using 39 microsatellite markers and found three-peak logarithm of odds (LOD) scores of 2.7, 3.2, and 3.1 on proximal 16p, proximal 16q, and central 16q, respectively. Taking account of the differences of suggestive markers, it is probable that there are other susceptibility genes for CD in the chromosome.

Table 2  Data of association analysis for NOD2

  Chromosome 12  Six out of 19 studies found suggestive linkage loci in the chromosome, 4 of them for CD, and one for UC. Though the studies with suggestive MLOD are rare, several studies found linkage loci with slightly suggestive significance (MLOD between 1.0 and 2.0) with IBD, especially with UC. This may result from the fact that the sample sizes in most studies were not large enough; furthermore, they were predominantly consisted of CD families. Parkes et al^[59] examined 581 affected relative pairs, of which 252 were from CD-only families, 138