Clinical and genetic heterogeneity in Mexican patients with ulcerative colitis

Clinical and Genetic Heterogeneity in Mexican Patients With Ulcerative Colitis Jesu´s K. Yamamoto-Furusho, Luis F. Uscanga, Gilberto Vargas-Alarco´n, Jorge A. Ruiz-Morales, Lorena Higuera, Teresa Cutin˜o, Jose´ Manuel Rodrı´guez-Pe´rez, Cynthia Villarreal-Garza, and Julio Granados ABSTRACT: Ulcerative colitis (UC) is an inflammatory bowel disease of unknown etiology. Genetic factors implied on its onset and severity may include genes located within the class II major histocompatibility complex (MHC) region. The aim of this study was to determine the relationship between human leukocyte antigen (HLA)DRB1 alleles with the clinical disease patterns of UC in Mexican Mestizo patients. High-resolution HLA typing was performed by polymerase chain reaction-sequence specific oligonucleotide (PCR)-SSO reverse dot blot and PCR-single-strand polymorphism in 67 patients with UC and 99 ethnically matched healthy controls. UC patients overall showed an increased frequency of HLA-DR1 as compared with healthy controls (17.1% versus 5%, [pC ⫽ 0.003, OR ⫽ 3.9]). Patients with extensive colitis showed increased frequencies of HLA-DR1 (pC ⫽ 1 ⫻ 10⫺10, OR ⫽ 13.9), HLA-DRB1*0103 (pC ⫽ 1 ⫻ 10⫺3, OR ⫽ 21.7), HLA-DRB1*0102 (pC ⫽ 0.007, OR ⫽ undetermined), and HLA-DR15 (pC ⫽ 1 ⫻ 10⫺3, OR ⫽ 8.5) when compared with healthy controls. We also found a statistically increased frequency of HLA-DR15 in UC ABBREVIATIONS UC ulcerative colitis MHC major histocompatibility complex

patients with extensive colitis compared with UC patients with only distal colitis (18.7% versus 1.8%, pC ⫽ 0.03; OR ⫽ 12.2). When patients who underwent proctocolectomy were compared with those who did not, an increased frequency of HLA-DRB1*0103 was observed (21.8% versus 4.9%; pC ⫽ 0.03; OR ⫽ 5.4; 95% confidence interval, 1.39 –21.93). Also, patients with proctocolectomy showed increased frequencies of HLA-DR1 (pC ⫽ 1 ⫻ 10⫺3, OR ⫽ 24.2) and HLA-DRB1*0103 (pC ⫽ 1 ⫻ 10⫺3, OR ⫽ 50.6) when compared with healthy controls. We concluded that HLA-DR1 is associated with genetic susceptibility to UC in the Mexican Mestizo population. HLA-DR15 distinguishes a subgroup of patients with extensive colitis and the HLA-DRB1*0103 allele distinguishes a subgroup of severe form of disease that might require surgical management. Human Immunology 64, 119 –123 (2003). © American Society for Histocompatibility and Immunogenetics, 2003. Published by Elsevier Science Inc. KEYWORDS: HLA; ulcerative colitis; MHC; Mexicans

HLA EIMs

human leukocyte antigen extraintestinal manifestations

INTRODUCTION Ulcerative colitis (UC) is an inflammatory bowel disease of unknown etiology. However, the importance of genetic susceptibility has been clearly shown by epidemiologic information from family and twin studies [1]. The

data supporting this fact include ethnic differences in disease frequency, familial aggregation, higher concordance in monozygotic twins, and increased occurrence of the disease in certain genetic syndromes [2, 3].

From the Department of Gastroenterology (J.K.Y.-F., L.F.U.), Instituto Nacional de Ciencias Me´dicas y Nutricio´n Salvador Zubiran, Tlalpan, Mexico, Department of Physiology (G.V.A., J.M.R-P.), Instituto Nacional de Cardiologı´a Ignacio Cha´vez, Mexico City, Mexico, and Department of Immunology and Rheumatology (J.A.R.-M., L.H., T.C., C.V.-G., J.G.), Instituto Nacional de Ciencias Me´dicas y Nutricio´n Salvador Zubiran, Tlalpan, Mexico.

Address reprint requests to: Dr. Julio Granados, Department of Immunology and Rheumatology, Instituto Nacional de Ciencias Me´dicas y Nutricio´n Salvador Zubiran, Vasco de Quiroga 15 Colonia Seccio´n XVI, Tlalpan 14000, Mexico; Tel: (525) 5731200, ext. 2601; E-mail: kazuofurusho@ hotmail.com. Received June 25, 2002; revised September 13, 2002; accepted September 30, 2002.

Human Immunology 64, 119 –123 (2003) © American Society for Histocompatibility and Immunogenetics, 2003 Published by Elsevier Science Inc.

0198-8859/03/$–see front matter PII S0198-8859(02)00772-3

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Putative associations of UC with the polymorphic genes of the major histocompatibility complex (MHC) located on the short arm of the chromosome 6 suggest a role in the genetic susceptibility to develop UC in several populations of different ethnic and geographic background. Class II genes are primary candidate genes for the genetic study of UC because of their crucial role in antigen recognition, importance in immune response, and their association with various autoimmune diseases. Previous studies describing different associations of class II genes with UC suggest a genetic heterogeneity for disease susceptibility. Human leukocyte antigen (HLA)-DRB1*0103 has been associated with extraintestinal manifestations (EIMs), larger extension, and the need for colectomy in various Caucasian populations, including Jewish [4] and non-Jewish European patients [5–7]. Other studies have shown that the HLADRB1*1502 alleles is associated with UC in the Japanese and Spanish populations [8, 9]. The purpose of the present study was to determine the association between the HLA-DRB1 class II alleles of the MHC and UC genetic susceptibility and the clinical presentation of the disease in Mexican patients with UC. PATIENTS AND METHODS This study was approved by the facility’s Research Ethics Committee in December 2000. All patients participated in the study after giving their informed consent. Sixtyseven Mexican Mestizo patients with a diagnosis of UC confirmed by histology were studied. Details of demographic and clinical characteristics of UC were obtained by a questionnaire, review of records, and personal interview. No patient had family history for UC. Disease extension was defined by colonoscopy. Thus disease was classified either as extensive colitis (inflammation proximal to the splenic flexure) or distal colitis. Controls Blood samples were obtained from 99 Mexican Mestizo healthy, unrelated individuals with no family history of UC who were ethnically matched to patients with UC. Mexican Mestizo individuals included in this study have a proportion of 56% Native American Indian genes, 40% white genes, and 4% black genes [10]. Both patients and controls were living in Mexico City, and therefore were considered representative of most of Mexico inhabitants because Mexico City has been an important immigration geographic site. All individuals were asked the location of their birthplace and that of their parents and maternal and paternal grandparents. We considered as Mexican Mestizos only those individuals who for two generations, including their own, had been born in Mexico. A Mexican Mestizo is defined as

J. K. Yamamoto-Furusho et al.

someone born in Mexico who is a descendant of the original autochthonous inhabitants of the region and of individuals, mainly Spaniards, of Caucasian or black origin, who came to America during the 16th century. HLA Typing Genomic DNA was isolated from peripheral venous blood by a modified “salting out” technique [11], precipitated with ethanol, and resuspended in sterile distilled water at a final concentration 0.1 to 1.0 ␮g/␮l before use. Generic HLA-DRB1 and HLA-DQB1 typing was performed by polymerase chain reaction-sequence specific oligonucleotide (PCR)-SSO reverse dot blot hybridization (Amplicor, Hoffmann La Roche, Basel, Switzerland). High-resolution HLA typing was performed by dot blot hybridization of amplified DNA with sequencespecific oligonucleotide probes labeled with digoxigenin di-deoxy-uridine-triphosphate. Information about DRB1 sequence was obtained from the 12th International Histocompatibility Workshop [12]. Statistical Analysis Gene frequencies were compared using a 2 ⫻ 2 contingency table and ␹2 analysis. Odds ratio (OR) have been calculated for the disease in carriers of specific alleles. Comparisons of allele frequencies between subgroups were performed using the EPIINFO statistical package (Version 5.0; USD Inc., Stone Mountain, GA). All p values quoted were corrected by Bonferroni test for multiple comparisons, taking into account the number of alleles studied. Statistical significance was considered as p ⬍ 0.05. HLA-DR-DQB haplotypes were deduced from: (1) known patterns of linkage disequilibrium in the Mexican population [13, unpublished results]; (2) the previously described haplotypes in other populations [14]; and (3) haplotypes if they appeared in two or more individuals and the alternative haplotype was well defined. RESULTS Clinical Features Of the 67 patients, 34 (50.7%) were female and 33 (49.3%) male, with ages ranging from 20 to 40 (mean, 30 y). Disease duration ranged between 1 and 15.6 years (mean, 8.1 y). Extensive colitis was present in 40 patients (59.7%) and distal colitis in 27 (40.3%). Twenty-seven patients presented EIMs (40.3%) that included arthritis (22.4%), primary sclerosing cholangitis (10.4%), sacroiliitis (6%), erythema nodosum (6%), ankylosing spondilytis (1.5%), and aphthous ulceration (1.5%). In regard to surgical treatment, 16 patients (23.9%) underwent radical colectomy resulting from refractory medical therapy (Table 1).

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TABLE 1 Demographic characteristics in Mexican patients with UC Gender (male/female)

TABLE 3 Association between HLA-DR alleles and extension of colitis in UC patients

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Extent of UC (%) Extensive Distal Extraintestinal manifestations (%) With Without Colectomy (%) Yes No Age at presentation (y), mean Duration of disease (y), mean Smoking habits (%) Nonsmokers Ex-smokers Smokers

40 (59.7%) 27 (40.3%) 27 (40.3%) 40 (59.7%) 16 (23.9%) 51 (76.1%) 30.3 ⫾ 11 8.1 ⫾ 7.5 62.7 31.3 6.0

Abbreviation: UC ⫽ ulcerative colitis.

HLA-Gene Frequencies As can be seen in Table 2, there is an increased frequency of HLA-DR1 in the group of UC patients compared with healthy controls (17.1% vs 5%, corrected p [pC] ⫽ 0.003; odds ratio [OR] ⫽ 3.9; 95% confidence intervals [CI 95%] 1.69 –9.14). Also, a nonsignificant increased frequency of HLA-DR2 was found in the UC patients group (18.6% vs 9%, pC ⫽ 0.1; OR ⫽ 2.29; CI 95%, 1.14 – 4.62) (see Table 2). The distribution of HLA-DQB alleles in patients and controls was similar (data not shown). However, UC patients with extensive colitis showed a statistically increased frequency of HLA-DR15 as compared with UC patients with distal colitis (18.7% vs 1.8%; pC ⫽ 0.03; OR ⫽ 12.2; CI 95%, 1.6 –256.2]

DR1 *0103 *0102 *0101 DR15 DR16

Extensive n ⫽ 40

Distal n ⫽ 27

Controls n ⫽ 99

0.212a 0.125b 0.050c 0.037 0.187d,e 0.062

0.111 0.055 0.036 0.018 0.037 0.148

0.050 0.015 0.000 0.035 0.065 0.025

Extensive colitis versus controls: a pC ⬍ 1 ⫻ 10⫺3; OR ⫽ 13.9; CI 95%: 5.25–37.51. b pC ⬍ 1 ⫻ 10⫺3; OR ⫽ 21.7: CI 95%: 5.1–106.1. c pC ⫽ 0.007; OR and CI 95%: undetermined. d pC ⬍ 1 ⫻ 10⫺3; OR ⫽ 8.54; CI 95%: 2.72–7.45. Extensive versus distal colitis: e pC ⫽ 0.03, OR ⫽ 12.23; CI 95%: 1.6 –256.2. Abbreviations: HLA ⫽ human leukocyte antigen; UC, ulcerative colitis; pC ⫽ p corrected; OR ⫽ odds ratio; CI ⫽ confidence interval.

(Table 3). Patients with extensive colitis showed an increased frequencies of HLA-DR1 (pC ⫽ 1 ⫻ 10⫺3, OR ⫽ 13.9, CI 95%, 5.25–37.52), HLA-DRB1*0103 (pC ⫽ 1 ⫻ 10⫺3, OR ⫽ 21.7, CI 95%, 5.1–106.1), HLADRB1*0102 (pC ⫽ 0.007, OR ⫽ undetermined), and HLA-DR15 (pC ⫽ 1 ⫻ 10⫺3, OR ⫽ 8.5, CI 95%, 2.72–7.45) when compared with healthy controls. Similarly, when we compared patients with EIMs versus patients without them, we found an increased frequency of HLA-DR15 (38.1% vs 13.8%; pC ⫽ 0.9; OR ⫽ 3.85; CI 95%, 0.92–15.2) (data not shown). On the other hand, the HLA-DRB1*0103 alleles was increased in patients who underwent proctocolectomy compared with those who did not (21.8% vs 4.9%; pC ⫽ 0.03; OR ⫽ 5.4; CI 95%, 1.39 –21.93) (Table 4). Also, patients with

TABLE 2 HLA-DR gene frequencies (gf) in patients with UC and controls UC n ⫽ 67

Healthy individuals n ⫽ 99

HLA

n

gf

n

gf

pC

OR

CI 95%

DR2 DR1 DR4 DR8 DR14 DR7 DR13 DR11 DR3 DR10 DR12 DR9

25 23 23 15 11 11 10 9 5 3 0 0

0.186 0.171 0.171 0.111 0.082 0.082 0.074 0.067 0.037 0.03 0 0

14 10 47 33 21 22 10 20 11 1 2 3

0.090 0.050 0.237 0.165 0.109 0.111 0.05 0.100 0.055 0.005 0.010 0.015

0.10 0.003 0.19 0.22 0.46 0.38 0.36 0.28 0.44 0.15 0.51 0.27

2.29 3.9 0.67 0.63 0.75 0.81 1.52 0.64 0.66 4.51 0 0

1.14–4.62 1.69–9.14 0.38–1.16 0.33–1.21 0.33–1.71 0.31–1.61 0.56–4.08 0.26–1.54 0.19–2.11 0.41–113.8 0–6.03 0–3.3

Abbreviations: UC ⫽ ulcerative colitis; HLA ⫽ human leukocyte antigen; pC ⫽ p corrected; OR ⫽ odds ratio; CI ⫽ confidence interval.

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TABLE 4 Association between HLA-DR alleles and proctocolectomy in UC patients

DR1 *0103 *0102 *0101 DR15 DR16

Yes n ⫽ 16

No n ⫽ 51

Controls n ⫽ 99

0.281a 0.218b,c 0.031 0.031 0.156 0.031

0.137 0.049 0.049 0.039 0.107 0.078

0.050 0.015 0.000 0.035 0.065 0.025

Proctocolectomy present versus controls: a pC ⬍ 1 ⫻ 10⫺3; OR ⫽ 24.2; CI 95%: 6.5–93.2. b pC ⬍ 1 ⫻ 10⫺3; OR ⫽ 50.6; CI 95%: 9.46 –306.1. Proctocolectomy present versus absent: c pC ⫽ 0.03; OR ⫽ 5.4; CI 95%: 1.39 –21.93. Abbreviations: HLA ⫽ human leukocyte antigen; UC ⫽ ulcerative colitis; pC ⫽ p corrected; OR ⫽ odds ratio; CI ⫽ confidence interval.

proctocolectomy showed increased frequencies of HLADR1 (pC ⫽ 1 ⫻ 10⫺3, OR ⫽ 24.2, CI 95%, 6.5–93.2) and HLA-DRB1*0103 (pC ⫽ 1 ⫻ 10⫺3, OR ⫽ 50.6, CI 95%, 9.46 –306.1) when compared with healthy controls. The most frequent HLA-DR phenotypes observed were DR4/DR8 and DR15/DR1; the former was not associated with EIMs, extensive colitis, or proctocolectomy; whereas the latter was more prevalent in patients with EIMs and extensive colitis. Haplotype analysis showed that DRB1*0103DQB1*0501 was significantly increased in patients with UC (p ⫽ 0.002; OR ⫽ 5.65; CI 95%, 1.3–27.6) as compared with healthy controls. DISCUSSION The results in this study provide further evidence of the important role of MHC genes class II in the genetic susceptibility to develop UC and also the potential role in predicting the clinical pattern of the disease. One of the relevant findings was the increased frequency of HLA-DR1 in UC patients compared with healthy controls. This finding has been previously reported in patients of Caucasian origin [5–7, 15]. The significant increased frequency of the HLADRB1*0103 in those patients who underwent proctocolectomy suggest that this could be a marker for a more aggressive type of disease. This confirms other studies in Caucasians that reported an increased frequency of this particular allele in UC patients with extensive colitis and EIMs [6, 14] and also patients who underwent proctocolectomy [7, 16], suggesting that this allele may be a prognostic genetic marker in UC patients. On the other hand, several studies have demonstrated

the association between HLA-DR2 and the susceptibility to UC [9, 17–19]. In the present study, although we only found a marginal association with DR2, this would suggest that in Mexicans the susceptibility to UC is also influenced by the admixture with Asians because the gene frequency of DR2 is relatively low in autochthonous populations of America [20]. Nevertheless, when we analyzed the subgroup of patients with extensive colitis, the HLA-DRB1*15 allele showed an increased frequency in these patients, which confirms previous reports on this regard, both in Caucasians [16] and Japanese [21]. High-resolution DNA typing on Mexican UC patients who are DRB1*15-positive is in progress to define if they are DRB1*1502-positive or not. Nevertheless, the gene frequency of the DRB1*1502 allele is extremely low in the normal Mexican population (Mestizos and Amerindians). When we compared the different types of EIMs (arthritis, uveitis, cholangitis, ankylosing spondilitis, and erythema nodosum), we were unable to confirm previous associations of HLA-DRB1*0103 with arthritis [22]. It is important to note that the frequency of HLADRB1*0103 and HLA-DRB1*15 is relatively low in the Mexican population (Mestizos and Ameridians) [23]. For instance in the Mexican Mazatecans HLA-DRB1*0103 was not detected at all and the frequency of DRB1*15 was only 4.9% [18, 21]. These facts and data from the present study suggest that DRB1*0103 and DRB1*15 were acquired by an admixture with Caucasians and Asians, respectively. It is important to mention that haplotype HLA-DRB1*1501-DQA1*0102-DQB1*0602 was acquired most likely during the 1500s and 1600s when Spaniards traveled to America via Asia as a result of the intensive commercial relationship between Europe and America. Indeed, the frequency of this haplotype in the normal Mexican Mestizo population is 4.5%, whereas it is absent in Mexican Amerindians [20, and unpublished results]. The HLA-DRB1*0103-DQB1*0501 haplotype previously has been found to be associated with UC in the Caucasian population [4]. In the present study this haplotype was also increased, reinforcing that Mexicans acquired this haplotype from Caucasian origin. In conclusion, HLA-DR1 is associated with the genetic susceptibility to UC in the Mexican Mestizo population, whereas HLA-DRB1*0103 seems to be associated with a more aggressive form of the disease. Likewise, HLA-DR15 is associated with extensive colitis. These findings suggest that both alleles DR1 and DR15 are involved in the pathophysiology of the disease and that Mexicans acquired the susceptibility haplotypes by admixture with Caucasians and Asians.

Mexican Patients With Ulcerative Colitis

ACKNOWLEDGMENTS

This project was supported in part by Consejo Nacional de Ciencia y Tecnologı´a CONACYT with register number 153237.

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