Between March and October 2007 we mailed study invitation letters to 196 PBC patients and their FDR (parents and children), who were regularly followed at the Department of Gastroenterology and Hepatology of the University Hospital of Heraklion (Crete, Greece), referral centre for liver disease in the island. The letter explained in detail the scientific data concerning the genetic and environmental factors in the pathogenesis of the disease, and pointed out the goals and the procedures of the study for both patients and their FDR. A hundred-eleven patients consented (56.6%), 40 patients declined and 45 patients did not respond. A hundred-fifteen FDR (75 females, 40 males) also consented to be enrolled (55% of the living FDR). A hundred-forty-nine unrelated controls matched to PBC patient by age (±2.5
years), gender, Cretan origin and residence were also enrolled at the study. The unrelated control group was enrolled among the visitors of the hospital.
All study participants, after signing an informed consent, completed a questionnaire through an interview performed by the same doctor. The questionnaire included information regarding demographics (age, gender, place of origin and residence), socioeconomics (profession, educational status), lifestyle (body mass index (BMI) grouped as ‘high’ (>30), ‘medium’ (25–30), and ‘low’(<25), smoking status in 20≤, >20 packs/year including past smoking, weekly alcohol consumption analyzed as “no use” (no consumption), “use” (less than 14 units of alcohol per week for men and 7 units women) or “misuse” (more than 14 units of alcohol per week for men and 7 units for women) counting 1 unit equal to 12gr of alcohol. ‘Hair dye use’ is referred as at least once a month per year, whereas ‘no hair dye use’ as never used. ‘N;ail polish use’ is referred as ≥10 times per year, whereas ‘no nail polish use’ is referred as occasional use or never used. Medical and surgical history questions referred at the period prior to PBC diagnosis. Moreover detailed questions concerning the frequency of vaginal and UTI, thyroid gland dysfunction, chronic diseases (hypertension (HT), diabetes mellitus (DM), dyslipidaemia, coronary artery disease (CAD), peripheral vasculopathy, asthma/chronic obstructive pulmonary disease (COPD)), allergies, other possible liver diseases and other autoimmune diseases were recorded. Diagnosis of Hashimoto’s disease was established by the combination of hypothyroidism and elevated thyroperoxidase (TPO) and thyroglobulin (TG) antibodies. For the female participants a detailed reproductive history prior to PBC diagnosis was assessed, which included the date of menarche, date of first pregnancy, number of pregnancies, childbirths, abortions or miscarriages; menopausal status, use of oral contraceptives or HRT and gynecological surgical history.
Collection of data lasted from January 2008 to December 2010. Medical files of the PBC patients were retrieved and reviewed and data concerning their clinical parameters, liver biopsies, Mayo risk score at diagnosis and recent laboratory tests were annotated. FDR and controls after the interview were also clinically examined and tested for: alanine aminotransferase (ALT), aspartate aminotranferase (AST), alkaline phosphatase (ALP), γ-glutamine transferase (γ-GT), bilirubin, glucose, urea, cholesterol, triglycerides, high density lipoprotein (HDL), low density lipoprotein (LDL), fT3, fT4, TSH, anti-TPO and anti-TG, immunoglobulins IgA, IgG, IgM, rheumatoid factor (RF). Viral hepatitis B and C markers were assessed by ELISA.
Antinuclear antibodies (ANA) were tested by indirect immunofluorescence on Hep-2 substrate with 1/80 cut-off of positivity. Anti-mitochodrial antibodies (AMA) and anti-smooth muscle antibodies (SMA) were tested by an indirect immunofluorescence (IIFL) assay of Nova Lite TM (IFA) on Mouse Kidney & Stomach substrate (Inova Diagnostics, San Diego CA, Inc) and a titre of ≥1/40 was considered positive, according to the manufacture’ s instructions. Anti-M2 antibodies were assessed by qualitative and quantitative ELISA (AESKULISA, German). Negative was 1-12U/ml, grey zone 12-18U/ml and positive >18U/ml.
The study protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki (6th revision, 2008) and was approved by the Hospital’s ethical committee.
All data were evaluated in two sets of comparisons: patients with controls, FDR with controls. Comparisons were made by Student’sT
-test for continuous variables, Fisher’s exact probability test and the χ
2 test for the analysis of categorical variables. All variables found to be significant in the univariate analyses for PBC patients were entered into the multivariate analyses using a forward step-wise logistic regression model (0.05 for entry and 0.10 for removal probability). A p
-value of <0.05 was considered statistically significant. Statistical analyses were performed, using the SPSS software package (version 18, SPSS Inc. Chicago, IL, USA).