DNA Repair Gene MLHJ - NCBI

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Dec 13, 1996 - products that could incite a localized or systemic auto- immune response.'2 The ... whether the DNA mismatch repair gene MLHI was associated with the ..... class II genes with inflammatory bowel disease. Gastroenterology.
ANNALS OF SURGERY Vol. 225, No. 6, 718-725 © 1997 Lippincott-Raven Publishers

Crohn's Disease and Ulcerative Colitis Are Associated With the DNA Repair Gene MLHJ Richard M. Pokorny, M.D.,* Annegret Hofmeister, M.D.,* Susan Galandiuk, M.D.,* Allan B. Dietz, Ph.D.Jt Noah D. Cohen, V.M.D., Ph.D. 4 and Holly L. Neibergs, Ph.D.*

From the Department of Surgery,* Price Institute of Surgical Research, University of Louisville School of Medicine, Louisville, Kentucky; The Human Gene Therapy Research Institute, t Des Moines, Iowa; Department of Large Animal Medicine and Surgery, X College of Veterinary Medicine, Texas A & M University, College Station, Texas

Objective The purpose of this study was to determine whether there is an association between Crohn's disease and ulcerative colitis with MLH1.

Summary Background Data Identification of genes involved in the etiology of inflammatory bowel disease may lead to the development of markers that objectively can define disease and permit therapy. The treatment of Crohn's disease of the colon and ulcerative colitis also is complicated by difficulties in differentiating the two conditions.

Methods The DNA and clinical data were obtained on 126 unrelated individuals (45 Crohn's disease, 36 ulcerative colitis, and 45 control subjects without intestinal disease). Polymerase chain reaction products were analyzed by single-strand conformation polymorphisms (MLH1 exons 9, 11, 14, 15, and 16) and polyacrylamide gel electrophoresis (markers D3S1 61 1 and D3S1 768). All comparisons were analyzed by chi square test. The association between single haplotypes and disease was expressed as relative odds.

Results MLH1 exons 9, 11, 14, and 16 were monomorphic. Two, four, and six alleles were detected in MLH1 exon 15, D3S1 611, and D3S1 768, respectively. Significant associations were observed for MLH1 exon 1 5/D3S1 61 1 haplotypes AB (OR = 5.5; p = 0.007) and BA (p = 0.002) with Crohn's disease and for haplotypes AB (OR = 4.0; p = 0.042), BA (p = 0.035), and BC (OR = 6.1; p = 0.016) with ulcerative colitis. Family history of inflammatory bowel disease was associated with D3S1 768/D3S1611 (p = 0.05) and MLH1 exon 1 5/D3S1611 haplotypes (p = 0.03). D3S1 61 1/D3S1 768 haplotype CD (OR = 1 1.3; p = 0.03) was associated with disease, whereas MLH1 exon 1 5/D3S1 611 haplotype AA (OR = 0.25; p = 0.02) was protective. Comparisons of MLH1 exon 1 5/D3S1611 haplotypes of Crohn's colitis and patients with ulcerative colitis were significant (p = 0.037).

Conclusions This study identifies a novel genetic and clinical association between MLH1 and inflammatory bowel disease.

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Surgical management of patients with inflammatory bowel disease (IBD) often is challenging, due in part to the difficulty in differentiating ulcerative colitis from Crohn's disease affecting the colon. This may result in significant treatment complications, particularly the development of active small bowel Crohn's disease in patients who have undergone ileopouch-anal anastomosis with an initial diagnosis of ulcerative colitis. These clinical dilemmas underscore the need to identify genetic markers that would aid in correct diagnosis and patient management. More important, the identification of such markers also may provide insight into the etiology of IBD. Substantial evidence supports a genetically mediated mechanism in the pathogenesis of IBD.1 Epidemiologic studies show a greater disease prevalence among Jews and whites compared to Asians and blacks.2 These conditions, however, are not inherited in a simple Mendelian pattern, suggesting that IBD consists of several etiologically and genetically distinct diseases presenting a similar clinical picture.2 This supports a multigenic mode of inheritance. Studies of ulcerative colitis-associated dysplasias and cancers have shown genetic abnormalities in proto-oncogenes and tumor suppressor genes, as well as genomic or microsatellite instability.35 This phenomenon also has been observed in DNA from patients with colorectal cancer as part of the hereditary nonpolyposis colorectal carcinoma syndrome.6 The DNA mismatch repair genes implicated in hereditary nonpolyposis colorectal carcinoma include MSH2 on chromosome 2p, MLHI on 3p, PMSJ on 2q, and PMS2 on 7p.7"-1 Mutations in the DNA mismatch repair genes impart a mutator phenotype to the affected cells in ulcerative colitis so that increased genomic instability is seen. The lack of efficient DNA mismatch repair can result in genomic consequences leading to carcinoma, as well as the production of aberrant protein products that could incite a localized or systemic autoimmune response.'2 The clinical aspects of IBD and the predisposition to colon carcinoma may, therefore, be consistent with a defect in a DNA mismatch repair gene. The purpose of this study was to determine whether the DNA mismatch repair gene MLHI was associated with the incidence of ulcerative colitis or Crohn's disease using an epidemiologic approach. Presented at the 108th Annual Meeting of the Southern Surgical Association, December 1-4, 1996, Palm Beach, Florida. Supported in part by the American Cancer Society Fellowship Award SC95-54-1 (Richard Pokorny) and the John W. Price, Jr., M.D., and Barbara Thruston Atwood Price Trust. Address reprint requests to Holly L. Neibergs, Ph.D., Department of Surgery, University of Louisville, Louisville, KY 40292. Accepted for publication December 13, 1996.

MLH1 in Crohn's Disease and Ulcerative Colitis

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PATIENTS AND METHODS Patients DNA was prepared from peripheral lymphocytes or surgical specimens of 126 unrelated individuals (45 patients with Crohn's disease, 36 patients with ulcerative colitis, and 45 consecutive hospital patients without intestinal disease). Ethnicity, race, and gender were similar among groups. Disease diagnoses were based on conventional clinical, radiologic, endoscopic, and pathologic criteria. Clinical data regarding age of onset, anatomic location, presence of family history of IBD, extraintestinal manifestations, and fistulas were recorded specifically. Age of onset of each disease was divided into three groups (0-17, 18-30, 30 years of age or older for Crohn's disease; 0-17, 18-40, 40 years of age or older for ulcerative colitis) based on observed age of onset among patients. The study protocol was approved by the University of Louisville Institutional Review Board.

DNA Markers Exons 9, 11, 14, 15, and 16 of MLHI were chosen for single-strand conformation polymorphism analysis, since previous studies had shown an association between MLHI mutations and the incidence of hereditary nonpolyposis colorectal carcinoma in families.7" Polymerase chain reaction (PCR) primers for MLHJ were chosen from the available sequence to amplify the intron-exon splice sites as well as each exon.9 MLHI PCR products ranged in size from 169 to 216 bases. Single-strand conformation polymorphism analysis was performed as described previously'3 with the exception of MLHI exon 15, which required some modifications as described below. An intragenic (D3S 1611) and flanking marker (D3S 1768) also were analyzed. The PCR primers were obtained from published oligonucleotide sequences to amplify a 278nucleotide fragment for D3S 1611 and a 197-nucleotide fragment for D3S1768.'4"5 The PCR reactions were carried out in a 10-,uL volume containing 100 ng of genomic DNA, 70 MM of each dNTP, 1.0 yM of each primer, 0.5 to 1 unit Taq DNA polymerase, and 1 ,iCi of [32P]a-dCTP (3000 Ci/mmol). Samples were denatured at 94 C for 5 minutes followed by 30 cycles of 94 C for 20 seconds, 55 C (except for D3S1611 at 59 C) for 30 seconds, 72 C for 1 minute, and a final extension at 72 C for 5 minutes. Exon 15 of MLHI was amplified subsequently by asymmetric PCRsingle-strand conformation polymorphism. 16 Singlestrand conformation polymorphism and microsatellite PCR products were electrophoresed on a 6% Long Ranger polyacrylamide gel with 5% glycerol at room temperature in 0.6X Tris-Borate EDTA buffer at 6 W for 15 to 18 hours and 60 W for 3 to 5 hours, respectively. Autoradio-

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grams were scored for band shifts by two observers who were unaware of the identity of the individuals or group scored. Seventy PCR products of MLHI exon 15 either were sequenced directly or subcloned with the TA Cloning Kit (Invitrogen, San Diego, CA) and sequenced using SP6 and T7 primers and an automated fluorescent sequencer (373; Applied Biosystems, Foster City, CA) to identify the polymorphism in each exon.

Haplotype Analyses Genotypes were used to construct haplotypes of each individual. Haplotypes represent the combination of two separate alleles on a chromosome. MLHI exon 15/ D3S1611, D3S1611/D3S1768, and the combination of MLHI exon 15/D3S 1768 represented the haplotypes. Every individual contributed two haplotypes to the analysis; one haplotype contributed by each parent. Thus, each haplotype represented an independent observation. Individuals who were homozygous at one or both loci were fully informative for analysis. For individuals who were heterozygous at both loci, haplotypes were based on the haplotype frequencies obtained from homozygous individuals.'7 For clinical comparisons, only individuals with uninferred haplotypes were used. Exclusion of inferred haplotypes resulted in smaller numbers per group, but reflected the most accurate genetic comparisons possible using an epidemologic approach. Haplotypes that were found to be different globally between groups were analyzed individually.

Statistical Analyses The distributions of haplotypes were compared between the control group and each of the disease groups. The relation between the dichotomous outcome of disease (presence of ulcerative colitis or Crohn's disease) and individual haplotypes was examined using two-way contingency comparisons. The chi square and Fisher's exact tests were used to compare proportions.'8 Clinical data of IBD patients with uninferred haplotypes of MLHI exon 15, D3S1611, and D3S1768 also were compared by chi square test. The crude measure of association between single haplotypes and disease or clinical manifestation was expressed as the relative odds. Relative odds were obtained from the cross-products ratio of contingency ta-

bles.'8 Confidence limits for the univariate odds ratios were derived using the test-based method. The odds ratio for a given haplotype is the ratio of the odds of control subjects having the haplotype, to the odds of patients with disease having the haplotype. For rare diseases, assuming that cases and control subjects are representative of the general population, the estimated odds ratio will approach

Ann. Surg. * June 1997

Table 1. CLINICAL DATA OF INFLAMMATORY BOWEL DISEASE PATIENTS

Age (yr) Range Mean ± SD Median Age of onset (yr) Range Mean ± SD Median Gender (no. of patients) Male Female Site of disease (% of patients) Small bowel Ileocolic Left colon Pancolitis Perianal Multiple locations Fistula (% of patients) Family history (% of patients) Extraintestinal disease (% of patients) Surgery (% of patients)

Crohn's Disease (n = 45)

Ulcerative Colitis (n = 36)

17-72 40 ± 15 34

14-80 43 ± 19 43

9-69 28 ± 16 24

12-76 31 ± 16 30

20 25

18 18

18 49 27 29 29 49 54 46 46 75

34 63

24

24 88

SD = standard deviation.

the estimated relative risk. Consequently, odds ratios for haplotypes may be interpreted as the magnitude of risk of disease associated with having a given haplotype. An odds ratio > 1 indicates a positive association of a haplotype with disease (increased risk of disease with that haplotype), whereas an odds ratio of 1 means there is no association of the haplotype with disease. A significance level of 0.05 was used.

RESULTS A summary of the Crohn' s disease and ulcerative colitis patients' clinical data is listed in Table 1.

Allele and Genotype Frequencies MLHI exons 9, 11, 14, and 16 were monomorphic. Two alleles were observed in exon 15 of MLHJ. The A allele of MLHI exon 15 corresponded to a thiamine 17 bases 5' to the initial nucleotide of exon 15. The B allele resulted from a cytosine at that position. D3S1611 had four alleles and D3S1768 had six alleles in the tested

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A ~ ~ ~ ~ AMPff~ ._-~~-

B AWA

~

~

~

~

E

~

A Figure 1. Representative autoradiographs of (A) D3S1611, (B) D3S1 768, and (C) MLH1 exon 15 alleles. Names of alleles are designated to the right.

population (Fig. 1). The distributions of alleles were the same for control, ulcerative colitis, and Crohn's disease groups (Table 2). Genotype frequencies for MLHJ exon 15, D3S 161 1, and D3S 1768 were not different between control, ulcerative colitis, and Crohn's disease groups (data not shown). Genotype frequencies of MLHI exon 15, D3S1611, and D3S1768 were in Hardy'9 and Weinberg20 equilibrium for all groups.

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alleles of D3S1611 and D3S1768 (data not shown). The haplotype distribution for MLHJ exon 15/D3S 1611 markedly was different between the control group and both ulcerative colitis (p = 0.004) and Crohn's disease (p < 0.001) patients by chi square analysis (Fig. 2). Frequency distributions were not different between ulcerative colitis and Crohn's disease groups (p = 0.3). Significant associations were observed for haplotype AB (p = 0.007) and BA (p = 0.002) with Crohn's disease and for haplotypes AB (p = 0.042), BA (p = 0.035), and BC (p = 0.016) with ulcerative colitis. Although the estimated odds ratio for Crohn's disease with haplotype BC was 2.1, this did not reach statistical significance (Table 3). There were no associations with the D3S 17681MLHI exon 15 haplotypes or D3S1611/D3S1768 haplotypes. No association was found for MLHI exon 15, D3S 161 1, or D3S 1768 haplotypes and the presence of fistulas, small bowel, ileocolic, or perianal Crohn's disease. MLHJ exon 15/D3S1611 haplotypes were associated with Crohn's colitis as compared to ulcerative colitis (p = 0.037). No haplotypes were associated with age of onset, Crohn's involvement of the colon, or presence of extraintestinal manifestations. Family history of Crohn's disease or ulcerative colitis was associated with D3S1611/D3S1768 (p = 0.05) and MLHI exon 15/D3S 1611 (p = 0.03), but not MLHI exon 15/D3S1768 haplotypes (p = 0.70). The CD haplotype of D3S1611/D3S1768 was associated with a family history of IBD (OR = 11.3, p = 0.03) whereas the MLHI exon 15/D3S1611 haplotype AA (OR = 0.25; p = 0.02) was protective.

DISCUSSION

Haplotype Frequencies Eight haplotypes of MLHI exon 15/D3S 1611 resulted from the two MLHI exon 15 alleles and four D3S1611 alleles. Twelve haplotypes were observed from the combination of the 2 MLHI exon 15 alleles and 6 D3S1768 alleles. Twenty-four haplotypes were observed with the

Complex decisions are required in establishing the cordiagnosis and choosing the optimal treatment for patients with IBD. The differentiation between Crohn's disease of the colon and ulcerative colitis may be extremely difficult and is critical because major treatment decisions rect

Table 2. ALLELE FREQUENCIES OF MLH1 EXON 15, D3S1611, AND D3S1768 D3S1768

D3S1611

MLH1 Exon 15

Allele

Controls

Crohn's Disease

Ulcerative Colitis

A B C D E F

0.57 0.43

0.53 0.47

0.58 0.42

Controls

Crohn's Disease

Ulcerative Colitis

0.37 0.43 0.19 0.01

0.31 0.48 0.14 0.07

0.38 0.35 0.26 0.01

Controls

Crohn's Disease

Ulcerative Colitis

0.11 0.11 0.20 0.48 0.06 0.04

0.04 0.08 0.27 0.51 0.04 0.06

0.07 0.07 0.28 0.44 0.04 0.10

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Ann. Surg. * June 1997

A. 0.4

0.3U 0

3

0.2

LU.

0.1

0.0 M

AC

BB

AB

BC

AD

BD

BA

Haplotypes B.

0U4-

*

0.1

0.0 AA

AC

BB

AB

BC

AD

BD

BA

Haplotypes

Figure 2. Haplotype distributions of MLH1 exon 15/D3S1611 in (A) control and Crohn's disease groups and (B) control and ulcerative colitis groups. Control subjects are designated by white bars and the disease groups by black bars. Haplotypes listed represent the MLH1 exon 15 allele, followed by the D3S1611 allele present on a single chromosome of an individual. Haplotypes with significant associations (OR > 1.0; p < 0.05) are noted (*).

are based on these differential diagnoses. Identification of molecular markers for IBD, which could identify individuals who are susceptible to disease or which could distinguish between these two disorders, singularly could be beneficial to the physician and patient. Genetic association studies can lead to confounding results when the groups studied are not representative of the populations they are taken from. To control for this, the control group consisted of the same proportion of individuals of differing races, ethnicity, and gender as that of the IBD groups. When individuals of different ethnic backgrounds were compared jointly, or as single groups, the genetic associations remained unchanged. Epidemiologic or linkage studies can be used to examine whether a gene is associated with a specific disease. The epidemiologic approach determines whether an association is present throughout a cross-section of the popula-

tion. Family or linkage studies are limited in this respect, because only the founders of the families and individuals who marry into the family represent independent genetic observations. In family studies, however, it is always apparent which alleles are inherited from each parent, because a child receives one of each pair of chromosomes from each parent. This results in an unambiguous assignment of haplotypes. With an epidemiologic approach, haplotypes are only known if one of two loci in the haplotype is homozygous. If both loci are heterozygous, the haplotype must be inferred by determining the proportion of known haplotypes and inferring the unknown haplotypes to similar proportions. Depending on the number of homozygous individuals, the number of haplotypes that must be inferred may be a small or a large proportion of the studied population. The number of homozygous individuals is dependent on the presence of multiple or rare alleles. In this study, approximately half of the individual haplotypes were inferred. Although this is an accepted method of determining haplotypes, it is a potential weakness of epidemiologic studies that can be overcome by analyzing the data without using the inferred haplotypes. In this study, all significant associations remained when inferred haplotypes were excluded, even though the sample sizes were reduced substantially. The MLHI gene has been localized to human chromosome 3p217122 Recently, this region of the genome has been identified as an inflammatory bowel disease locus by Satsangi and coworkers using a linkage analysis approach.23 In linkage studies, an association of a marker with a second marker or a trait is often measured in lod Table 3. SIGNIFICANT ASSOCIATIONS OF INDIVIDUAL MLH1 EXON 15/D3S1611 HAPLOTYPES WITH INFLAMMATORY BOWEL DISEASE

Haplotype AB Controls Crohn's disease Ulcerative colitis BA Controls Crohn's disease Ulcerative colitis BC

Controls Crohn's disease Ulcerative colitis

95% Cl for Odds p Ratio Value

No. (%) with Haplotype

Odds Ratio

3 (6.7) 13 (28.3) 8 (22.2)

1 5.5 4.0

1.6, 19.1 1.1, 15.4

0.007 0.042

0 (0) 9 (19.6) 4 (11.1)

1 IND IND

IND IND

0.002 0.035

2 (4.4) 4 (8.7) 8 (22.2)

1 2.1 6.1

0.4, 11.5 1.4, 27.0

0.416 0.016

IND = odds ratio was indeterminable because the number of controls with the haplotype equaled 0; Cl = confidence interval.

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(logarithm of odds) scores, where a score of 3 or greater is evidence of linkage (the marker is 1000 times more likely to be associated than not associated). The value of theta is an estimate of the percent recombination (and thereby the distance in centiMorgans) between markers on the chromosome. The D3S 1573 marker that Satsangi et al. found to be associated with inflammatory bowel disease is very closely linked to the flanking (D3S1768, lod score 10.2, theta 0.02) and intragenic markers (D3S1611, lod score 16.3, theta 0.11) reported in our study.24 The confirmation of our data by these investigators underscores the validity of our methods and findings in this exciting and unfolding area of research. It is now imperative to investigate and identify the causative mutations responsible for the etiology of inflammatory bowel disease. To determine whether MLHI is involved in the etiology of inflammatory bowel disease, additional studies are ongoing to sequence the coding region of the gene. The conclusions of this study are as follows: 1. Novel associations exist between IBD and two genetic markers within, and one nearby the DNA mismatch repair gene MLHJ. 2. Two categories of haplotypes were associated with the family history of Crohn's disease or ulcerative colitis. 3. The significant association of MLHJ exon 15/ D3S1611 haplotype with colonic Crohn's disease, as opposed to ulcerative colitis, may prove to be useful in distinguishing ulcerative colitis from Crohn's disease of the colon. 4. This information may be helpful in the identification of individuals who are at high risk of developing inflammatory bowel disease.

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Discussion DR. SAMUEL A. WELLS, JR. (St. Louis, Missouri): I would like to ask three questions pertaining to this work. The use of polymorphic DNA markers, such as those described in the present study, have been useful in the haplotyping of kindreds with an inherited disease to establish in individual family members the probability of their inheriting a genetic mutation. In the present study, some of the patients had familial disease.