Primary Biliary Cirrhosis in a genetically homogeneous population: Disease associations and familial occurrence rates

Background Primary biliary cirrhosis (PBC) is a disease with genetic and environmental pathogenetic background. Chemicals, infectious agents, hormone therapy, reproductive history and surgical interventions have been implicated in the induction of PBC. Familial PBC has been documented in first degree relatives (FDR). Most cohort studies are genetically heterogeneous. Our study aimed to determine eventual lifestyle or disease associations and familial occurrence rates in a genetically homogeneous and geographically defined population of PBC patients. Methods 111 consenting PBC patients, were compared with 115 FDR and 149 controls matched for age, sex, Cretan origin and residence. All participants completed a questionnaire regarding demographics, lifestyle, medical, surgical and reproductive history. Significant variables on the univariate analysis were analyzed by multivariate analysis using a forward step-wise logistic regression model. Results Dyslipidaemia was found in 69.4% of patients, 60% of FDR and 40.9% of controls (p < 0.0001 and p = 0.003 respectively), autoimmune diseases in 36.9% of patients, 30.4% of FDR and 13.4% of controls (p < 0.0001 and p = 0.011 respectively). Hashimoto’s disease (p = 0.003), Raynaud syndrome (p = 0.023) and Sjögren syndrome (p = 0.044) were significantly associated with PBC. On multivariate analysis statistically significant associations were found with primary educational level (AOR 2.304, 95% CI 1.024-5.181), cholecystectomy (AOR 2.927, 95% CI 1.347-6.362) and the presence of at least another autoimmune disease (AOR 3.318, 95% CI 1.177-6.22). Cancer history was more frequent in patients than in controls (p = 0.033). Familial PBC was found to be 9.9%. Conclusions Dyslipidaemia and autoimmune diseases were significantly increased not only in patients as expected but also in their FDR. An increased prevalence of malignancies was found in patients. Primary educational level, cholecystectomy and the presence of at least another autoimmune disease were found as putative risk factors for PBC. No association was found with smoking, urinary tract infection or reproductive history. The reported high familial occurrence of PBC could imply screening with AMA of FDR with at least another autoimmune disease.


Background
Primary biliary cirrhosis (PBC) is a cholestatic disease of unknown aetiology primarily affecting middle-aged women. It is characterized by progressive destruction of the small intrahepatic bile ducts that leads to ductopenia, fibrosis and ultimately liver cirrhosis. The serologic hallmark of PBC in 95-98% of the patients are the M2 anti-mitochondrial autoantibodies (AMA), directed against the E2 sub-unit of the pyruvate dehydrogenase multi-enzyme complex (PDC) located in the inner membrane of the mitochondria. [1] It is considered an autoimmune disease resulting from the interaction of multiple environmental factors, the immune system and the liver of genetically susceptible individuals [2].
The role of the genetic factors in PBC is strongly supported by the 63% concordance rate in monozygotic twins, the second highest reported in autoimmunity after celiac disease [3]; as well as the high familial PBC occurrence rates reported 4% [4] to 9% [5] in first-degree relatives (FDR) of PBC patients [6]. Moreover the presence of AMA with no other clinical evidence of disease is reported in 13.1% [5]. The significant coexistence of other autoimmune diseases and/or other autoantibodies in both patients and their FDR, also indicates the genetic background of PBC [7].
The age at first pregnancy, the frequency of pregnancies, abortions, hormone replacement therapy (HRT) and a previous history of obstetric cholestasis, as well as urinary tract infections (UTI), tonsillectomy, appendectomy and cholecystectomy have also been reported as possible risk factors [4,[24][25][26].
Many studies include patients with ethnic variations that may influence the results. Studies in populations that share the same genetic background, common environment and low migration rates make it more plausible to identify environmental and/or genetic factors playing a role in a disease's induction.
Therefore we analyzed the demographics, lifestyle, medical and surgical history in a genetically and ethnically homogeneous group of Cretan PBC patients and their FDR compared with a control group matched for age, gender and residence with the patients in order to identify familial occurrence rates, associated lifestyle factors and/or comorbidities.

Methods
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)(26)(27)(28)(29)(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), γglutamine 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 cutoff 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.

Statistical analysis
All data were evaluated in two sets of comparisons: patients with controls, FDR with controls. Comparisons were made by Student's T-test for continuous variables, Fisher's exact probability test and the χ2 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).

Results
The diagnosis of all PBC patients was based on compatible clinical, immunological and histological parameters and all patients were on ursodeoxycholic acid 15 mg/kg since diagnosis. The mean age and the mean Mayo risk score at the time of diagnosis was 56.87 ± 11.42 years and 4.7 ± 1.6, respectively. A hundred and one patients (86 females) were AMA positive in a titre ≥1/40 on IIFL with M2 higher than 18 U/ml on ELISA, while 10/111 (8 females) were AMA negative. Fourty-three of the 111 PBC patients were ANA positive, 16 with MND pattern, 5 MND and peri-nuclear, 4 peri-nuclear, 1 peri-nuclear and anti-nucleolar, 12 speckled, 1 speckled and MND, 1 speckled and anti-nucleolar and 3 diffuse.
Fourteen patients were at stage IV at diagnosis (9 on liver biopsy, and 5 AMA positive with clinical evidence of portal hypertenstion that did not underwent liver biopsy). Of those to the end of the study, two died after diagnosis of HCC, one with HCC is still alive and 3 are alive with de-compensated cirrhosis.
Seventeen more patients were at stage III (2 AMA negative), 3 of those at stage IV alive at the end of the study, one de-compensated. Two dead (one AMA negative) of liver related causes, one within 30 days of OLT.
According to liver biopsy results at diagnosis, 79 (8 AMA negative) PBC patients were at an early stage (Ludwig I-II). During the study period three died of non liver related causes. Five are at stage IV (1 de-compensated) at the end of the study.
One female AMA positive patient refused to undergo biopsy for staging.
Mild piecemeal necrosis was present in 42.35% of the biopsies, moderate in 30.6% and no piece-meal in 27.05%.
Mean follow-up from diagnosis to interview was 91.7 ± 61 months. The mean age and the mean Mayo risk score at 2010 was 64.8 ± 11.9 and 4.99 ± 1.46, respectively.

Sociodemographic and lifestyle characteristics
The results of the sociodemographic at interview and lifestyle variables of the three study groups are seen in Table 1.
More PBC patients than controls had an elementary education (p = 0.01) and more FDR had a University degree (p < 0.0001). Among lifestyle factors, alcohol consumption and smoking were more frequent in FDR than controls (p < 0.0001 and p = 0.006, respectively), whereas mean BMI was found significantly lower in FDR (p = 0.003). The distribution of autoimmune diseases in PBC patients and controls are shown in Table 2. Among other autoimmune diseases diagnosed in PBC patients were: autoimmune thrombocytopenia in 2/111, multiple sclerosis in 1/111, celiac disease in 1/111, vitiligo in 1/111, autoimmune gastritis in 3/111, Guillain Barre syndrome in 1/111, BOOP in 1/111, vasculitis of autoimmune origin in 1/111, antiphospholipid syndrome in 1/111. Most patients had more than one autoimmune disease. There was a patient who had simultaneously rheumatoid arthritis, Sjögren syndrome, Raynaud syndrome, psoriasis, Hashimoto's disease and eczema.

FDR vs Controls
Four PBC patients were FDR of other patients resulting in a known familial history of 3.6%. Occurrence of PBC was diagnosed in another 7 FDR (6.3%) ( Table 3). Five AMA positive among them underwent liver biopsy that  Diagnosis of HT, DM, CAD, peripheral vasculopathy, asthma/COPD and allergies did not differ significantly between the two groups. UTIs did not differ between groups (p = 0.065) and more than 10 UTI/lifetime were reported by 9.7% patients vs. 6.9% of the controls (p = 0.187).

Reproductive history of PBC patients, FDR and controls
Results and comparisons among the three groups are shown in Table 4. All parameters with significant differences between FDR and controls can be explained by the difference between the menopausal women in the two groups (i.e. 18 menopausal FDR vs. 108 controls) as expected by the age difference.

Multivariate analysis
Variables entered in the multivariate analysis for all patients were rural residence, primary educational level, RA, Sjogren syndrome, Raynaud syndrome, Hashimoto, the presence of at least another autoimmune disease, thyroid gland dysfunction, cholecystectomy and cancer. Primary educational level (AOR 2.304, 95% CI 1.024-5.181), cholecystectomy (AOR 2.927, 95% CI 1.347-6.362) and the presence of at least another autoimmune disease (AOR 3.318, 95% CI 1.177-6.22) were a genderindependent risk factor associated with PBC.

Discussion
In this case control study we report the prevalence of autoimmune and other co morbidities as well as the sociodemographic and lifestyle factors associated with PBC in an ethnically homogeneous and geographically defined group of PBC patients. The definite advantages of population isolates, such as the Cretan population, referring to more uniform environment, genetic homogeneity and low migration rates give an added value at the study.
The limitation of the study, as with all studies that use questionnaires, is that it comprehends the risk of putative reports that lead to bias. We lowered this risk by interviewing all participants by the same doctor and validated the collected data for the PBC patients, by patient record review.
The potentially low percentage of patients that agreed to participate in the study (56.6%), could be justified by Cretan cultural ethics and prejudices, the advanced age at diagnosis, rural residence (almost half of our patients) and their low educational level. The same reasons could also explain the less frequent use of hair dye and nail polish in our patients. Indeed our findings do not support previous findings in other populations indicating nail polish or hair dye [4,25,27] as a putative risk for PBC.
The educational level of our PBC patients was lower than the controls and of that reported in other populations [4,25] and was found to be an independent predictor for the disease. Previous American and French studies reported lower BMI of PBC patients compared with their controls [4,25] but this was not found in the Cretan population. Most patients in this study although not different from controls were overweight. Current or previous smoking did not differ between PBC and controls but the passive exposure to tobacco smoke was not investigated.
Similar prevalence of autoimmune diseases in our controls with those of the American study (13.4% vs 13%) was found. However our patients had an even higher prevalence of at least one autoimmune disease compared to theirs (36.9% vs 32%) [7]. As in previous studies [7,25] the most frequent autoimmune diseases with very high OR in univariate analysis were autoimmune thyroiditis, Raynaud and Sjögren syndrome, all found to be significantly associated with PBC while the presence of at least another autoimmune disease was found to be an independent risk factor for the disease.
FDR also had a significant higher prevalence of autoimmune diseases (30.4%). The most frequent autoimmune disease found in FDR was Hashimoto (13.9%), followed by PBC (9.9%). Indeed the known prevalence of PBC in FDR in Crete prior to the study was 3.6% similar to the 6% reported in the US study, the 5% in UK patients [7], the 4% in France and the 5.1% in Japan [28]. Nonetheless the familial screening of FDR during the study raised the PBC prevalence in FDR to 9.9%, a figure closer to the 9% reported by Lazaridis et al [5].
Although most patients were overweight and with elevated cholesterol, DM, HT, and CAD did not differ between patients and controls in accordance with the hypothesis that PBC patients may be protected against the metabolic syndrome, due to the prevention of LDL oxidation by lipoprotein X, the antioxidant effects of bilirubin and/or the elevated levels of adiponectin [29][30][31].
Similarly FDR as expected by the age factor had lower prevalence of HT, DM and CAD nonetheless hyperlipidemia was significantly increased compared to controls.
We did not confirm UTI prevalence in patients compared to controls, but the limitation of self-reporting, might interfere, creating misinterpretations. Tonsillectomy was not associated with PBC. By contrast cholecystectomy was, in accordance with the French findings [4].
More patients than controls reported malignancies, one third of which were HCC, giving an OR 4.29 (95%CI 1. 13-16.13). This is in agreement with the increased risk for malignancies found in 212 Greek patients previously reported [32]. Non significant difference in reproductive history of PBC patients was identified.

Conclusions
In conclusion, this study has demonstrated that hyperlipidaemia and autoimmune diseases were significantly increased not only in PBC patients as expected, but also in their younger FDR compared to controls. Primary educational level, cholecystectomy and the presence of at least another autoimmune disease were found to be putative risk factors for PBC in our Greek population. The increased prevalence of malignancies previously reported was also confirmed in our study.
Given the high occurrence of familial PBC, the screening of PBC family members with AMA, especially those with at least another autoimmune disease, could be suggested in our population in order to diagnose and eventually treat the disease at an earlier stage.