Julio Garcia-Paredes

Positive and negative associations of distinct HLA-DR2 subtypes with ulcerative colitis (UC)

In UC, as in many other diseases with a suspected autoimmune etiology or pathogenesis, an association with certain HLA class II specificities has been investigated. In the Japanese, several studies have shown a positive association with DR2, but in Caucasian populations the results are conflicting. Therefore we undertook a study of HLA class II gene association with UC in a large group of white Madrid patients and ethnically matched controls, using molecular biology techniques to investigate whether any allelic subspecificity within the HLA‐DR2 group is associated with susceptibility to or protection against UC. Patients with ulcerative colitis (n=107) and 200 controls were typed using molecular, DNA‐based techniques for HLA‐DRB1, DQA1 and DQB1 alleles. Those HLA‐DR2+ were then specifically typed for the individual alleles within the HLA‐DR2 group. We observed a positive association with HLA‐DR15 (P=0.021) and its subtype DRB1*1501 (P=0.018). HLA‐DRB1*1502 was also increased, although its frequency both in patients and controls was very low. When the HLA‐DR2+ population was studied, HLA‐DRB1*1601 was significantly decreased in patients (P=0.026). Both HLA‐DR3 (P=0.002) and HLA‐DQB1*02 (P=0.001) were also negatively associated with the disease, the latter especially with pancolitis. Therefore, HLA class II association with UC is complex, and separate alleles confer either susceptibility or resistance. Conflicting results with HLA‐DR2 appear to be due to the presence in this group of both positively associated (HLA‐DRB1*1501 and DRB1*1502) and negatively associated (HLA‐DRB1*1601) subspecificities. Moreover, HLA‐DR3 and HLA‐DQB1*02 are associated negatively.

IBD1 and IBD3 determine location of Crohn's Disease in the Spanish population

Background:Crohn’s disease is a heterogeneous disease from both genetic and clinical points of view. Aims:To look for associations between distinct genetic polymorphisms and clinical subgroups of the disease. Subjects:A total of 210 patients and 343 healthy control subjects, all adult, unrelated, white, Spanish individuals. Methods:DNA was purified from peripheral blood samples and was typed by sequence-specific oligonucleotide polymerase chain reaction (PCR) method for human leukocyte antigen (HLA)-DRB1 alleles (IBD3) and by allele-specific PCR for NOD2/CARD15 (IBD1) polymorphisms. Results:NOD2/CARD15 mutations and HLA-DRB1*07 confer susceptibility only to the ileal location of the disease, whereas HLA-DRB1*0103 is associated only with the colonic location of the disease. The IBD3 effect was overshadowed by IBD1 mutations when present. Conclusion:The studied genetic polymorphisms of Crohn’s disease basically determine the location of the disease and, only secondarily, the clinical form of the disease. This appears to be true for both inflammatory bowel diseases as HLA-DRB1*0103 is associated both with colonic Crohn’s disease and ulcerative colitis.

Interleukin-10 polymorphisms in Spanish patients with IBD

Background: Inflammatory bowel disease (IBD) is considered a heterogeneous, complex polygenic disease where both genetic and environmental factors are involved in the development of the disease. Interleukin‐10 (IL‐10) is a regulatory cytokine that might play an important role in disease pathogenesis. IL‐10 contains single nucleotide polymorphisms and 2 polymorphic microsatellites in the 5′‐flanking region. Our aim was to ascertain if any of these polymorphic markers is associated with IBD among Spanish patients. Methods: We genotyped 470 patients with IBD, 242 with ulcerative colitis and 228 with Crohns disease (CD), and 572 ethnically matched controls for microsatellites IL‐10R and IL‐10G and 2 single nucleotide polymorphisms at positions ‐1082 and ‐819 in the proximal promoter of the gene. Results: IL‐10G14 microsatellite allele as well as ‐1082G allele were significantly increased in patients with CD. The combined presence of both alleles in 1 individual notably increased the risk to develop CD (P = 0.00006, odds ratio = 3.18). Conclusion: IL‐10 polymorphisms contribute to susceptibility to CD in Spanish population.

Amino Acid Polymorphism at Residue 71 in HLA-DR Beta Chain Plays a Critical Role in Susceptibility to Ulcerative Colitis

The association of ulcerative colitis withdistinct HLA-DRB1 alleles has not been easy toascertain. Recent studies show that among HLA-DR2alleles, DRB1*15 but not DRB1*16, is associated with thedisease. Similarly, in the HLA-DR1 group, only DRB1*0103is increased in ulcerative colitis patients. The aim ofour study was to identify critical DRB1 residues thatmight account for these differences. We typed 121 patients with ulcerative colitis and 275controls using gene amplification and sequence-specificoligonucleotide probing for HLA-DRB1 and DRB3. Weobserved a strong negative association between HLA-DRB1 alleles that encode lysine at position 71 intheir β-chain and susceptibility to ulcerativecolitis. Differences in the prevalence among otheralleles differing only in the third hypervariable region suggested a hierarchy of susceptible andprotective class II alleles related to the presence ofan acidic, neutral, or basic amino acid residue atposition 71. These data implicate most strongly theamino acid residues in the third hypervariable regionof the DRβ chain, especially DRβ71, in theassociation between ulcerative colitis and HLA. However,this does not exclude the contribution of other parts of the molecule and otherimmunoregulatory genes.

Debrisoquine Oxidation Polymorphism in Patients with Chronic Inflammatory Bowel Disease

Polymorphic hydroxylation of debrisoquine (DBQ) is a Mendelian genetic trait related to the risk of suffering some spontaneous disorders. To elucidate whether such a relation exists between this polymorphism and chronic inflammatory bowel disease (CIBD), 67 (39 males) patients with ulcerative colitis (UC), 52 (35 males) patients with Crohns disease (CD) and 837 healthy controls (391 males) received 10 mg debrisoquine. DBQ and its metabolite, 4-OH-DBQ, were measured in urine to calculate metabolic ratio. Subjects with MR < 12.6 (log 10 MR < 1.1) were extensive metabolizers (EM) of DBQ, whereas those with MR < 12.6 were poor metabolizers (PM). Four UC (5.97%), 1 CD (1.92%) patients and 42 controls (5.03%) were PM of DBQ (nonsignificant difference). When analysing the EM subjects separately, log10 MR were lower in controls (mean = -0.295, SD 0.427, P = 0.0015)) and in Crohns disease patients (man = -0.281, SD 0.495, P = 0.03) than in ulcerative colitis patients (mean = -0.085, SD = 0.495). There is no relationship between oxidative phenotype of DBQ and the risk for CIBD. Nevertheless, the EM phenotype includes both homo- and heterozygote genotypes for functioning alleles exerting a gene-dose effect that gives a higher oxidative capability to homozygote EMs, reflected in a lower MR value. Genotyping studies are needed to disclose whether heterozygote EMs are over-represented among UC patients and to identify any nonfunctioning allele possibly linked to the risk of developing this disease.