title: Eosinophil associated genes in the inflammatory bowel disease 4 region: correlation to inflammatory bowel disease revealed.
abstract: OBJECTIVE
  To study the association between inflammatory bowel disease (IBD) and genetic variations in eosinophil protein X (EPX) and eosinophil cationic protein (ECP).
  
  
  METHODS
  DNA was extracted from ethylene diamine tetraacetic acid blood of 587 patients with Crohn's disease (CD), 592 with ulcerative colitis (UC) and 300 healthy subjects. The EPX405 (G > C, rs2013109), ECP434 (G > C, rs2073342) and ECP562 (G > C, rs2233860) gene polymorphisms were analysed, by the 5'-nuclease allelic discrimination assay. For determination of intracellular content of EPX and ECP in granulocytes, 39 blood samples was collected and extracted with a buffer containing cetyltrimethylammonium bromide. The intracellular content of EPX was analysed using an enzyme-linked immunosorbent assay. The intracellular content of ECP was analysed with the UniCAP(®) system as described by the manufacturer. Statistical tests for calculations of results were χ(2) test, Fisher's exact test, ANOVA, Student-Newman-Keuls test, and Kaplan-Meier survival curve with Log-rank test for trend, the probability values of P < 0.05 were considered statistically significant.
  
  
  RESULTS
  The genotype frequency for males with UC and with an age of disease onset of ≥ 45 years (n = 57) was for ECP434 and ECP562, GG = 37%, GC = 60%, CC = 4% and GG = 51%, GC = 49%, CC = 0% respectively. This was significantly different from the healthy subject's genotype frequencies of ECP434 (GG = 57%, GC = 38%, CC = 5%; P = 0.010) and ECP562 (GG = 68%, GC = 29%,CC = 3%; P = 0.009). The genotype frequencies for females, with an age of disease onset of ≥ 45 years with CD (n = 62), was for the ECP434 and ECP562 genotypes GG = 37%, GC = 52%, CC = 11% and GG = 48%, GC = 47% and CC = 5% respectively. This was also statistically different from healthy controls for both ECP434 (P = 0.010) and ECP562 (P = 0.013). The intracellular protein concentration of EPX and ECP was calculated in μg/10(6) eosinophils and then correlated to the EPX 405 genotypes. The protein content of EPX was highest in the patients with the CC genotype of EPX405 (GG = 4.65, GC = 5.93, and CC = 6.57) and for ECP in the patients with the GG genotype of EPX405 (GG = 2.70, GC = 2.47 and CC = 1.90). ANOVA test demonstrated a difference in intracellular protein content for EPX (P = 0.009) and ECP (P = 0.022). The age of disease onset was linked to haplotypes of the EPX405, ECP434 and ECP562 genotypes. Kaplan Maier curve showed a difference between haplotype distributions for the females with CD (P = 0.003). The highest age of disease onset was seen in females with the EPX405CC, ECP434GC, ECP562CC haplotype (34 years) and the lowest in females with the EPX405GC, ECP434GC, ECP562GG haplotype (21 years). For males with UC there was also a difference between the highest and lowest age of the disease onset (EPX405CC, ECP434CC, ECP562CC, mean 24 years vs EPX405GC, ECP434GC, ECP562GG, mean 34 years, P = 0.0009). The relative risk for UC patients with ECP434 or ECP562-GC/CC genotypes to develop dysplasia/cancer was 2.5 (95%CI: 1.2-5.4, P = 0.01) and 2.5 (95%CI: 1.1-5.4, P = 0.02) respectively, compared to patients carrying the GG-genotypes.
  
  
  CONCLUSIONS
  Polymorphisms of EPX and ECP are associated to IBD in an age and gender dependent manner, suggesting an essential role of eosinophils in the pathophysiology of IBD.
authors.affiliation: Department of Medical Sciences, Clinical Chemistry, Uppsala University Hospital, SE-75185 Uppsala, Sweden. kristin.blom@medsci.uu.se
authors.forename: Kristin
authors.initials: K
authors.lastname: Blom
authors.forename: Jenny
authors.initials: J
authors.lastname: Rubin
authors.forename: Jonas
authors.initials: J
authors.lastname: Halfvarson
authors.forename: Leif
authors.initials: L
authors.lastname: Törkvist
authors.forename: Anders
authors.initials: A
authors.lastname: Rönnblom
authors.forename: Per
authors.initials: P
authors.lastname: Sangfelt
authors.forename: Mikael
authors.initials: M
authors.lastname: Lördal
authors.forename: Ulla-Britt
authors.initials: UB
authors.lastname: Jönsson
authors.forename: Urban
authors.initials: U
authors.lastname: Sjöqvist
authors.forename: Lena Douhan
authors.initials: LD
authors.lastname: Håkansson
authors.forename: Per
authors.initials: P
authors.lastname: Venge
authors.forename: Marie
authors.initials: M
authors.lastname: Carlson
mesh_terms: D000293:Adolescent; D000328:Adult; D017668:Age of Onset; D000368:Aged; D000369:Aged, 80 and over; D000704:Analysis of Variance; D016022:Case-Control Studies; D016009:Chi-Square Distribution; D003093:Colitis, Ulcerative / Q000097:blood / Q000201:enzymology / Q000235:genetics* / Q000276:immunology; D003424:Crohn Disease / Q000097:blood / Q000201:enzymology / Q000235:genetics* / Q000276:immunology; D047150:Eosinophil Cationic Protein / Q000097:blood / Q000235:genetics*; D047208:Eosinophil-Derived Neurotoxin / Q000097:blood / Q000235:genetics*; D004804:Eosinophils / Q000201:enzymology* / Q000276:immunology; D005260:Female; D005787:Gene Frequency; D020022:Genetic Predisposition to Disease; D006239:Haplotypes; D006801:Humans; D053208:Kaplan-Meier Estimate; D008297:Male; D008875:Middle Aged; D010641:Phenotype; D011110:Polymorphism, Genetic; D012737:Sex Factors; D013548:Sweden; D055815:Young Adult
chemical_list: D047208:Eosinophil-Derived Neurotoxin; D047150:Eosinophil Cationic Protein; C479220:RNASE3 protein, human