Vitamin D Levels and Liver Fibrosis Severity in Hepatitis C

Association of Vitamin D Serum Levels and Its Common Genetic Determinants, With Severity of Liver Fibrosis in Genotype 1 Chronic Hepatitis C Patients

S. Petta, S. Grimaudo, V. D. Marco, C. Scazzone, F. S. Macaluso, C. Cammà, D. Cabibi, R. Pipitone, A. Craxì

J Viral Hepat. 2013;20(7):486-493.

Abstract and Introduction
Abstract
Lower 25-hydroxyvitamin D [25(OH)D] serum levels have been associated with the severity of liver fibrosis in genotype 1 chronic hepatitis C patients (G1CHC). In addition, a recent genome-wide study identified genetic variants (rs12785878, near dehydrocholesterol reductase, DHCR7; rs10741657, near CYP2R1; and rs7041, near vitamin D-binding protein, GC) affecting 25(OH)D serum levels in healthy populations. We aimed to assess the association between vitamin D serum levels and its genetic determinants, with the severity of liver fibrosis. Two hundred and sixty patients with biopsy-proven G1CHC were consecutively evaluated. The 25(OH)D serum levels were measured by high-pressure liquid chromatography. All patients were genotyped for DHCR7 rs12785878, CYP2R1 rs10741657 and GC rs7041 single nucleotide polymorphisms. DHCR7 GG genotype (P = 0.003) and the severity of fibrosis (P = 0.03) were independent factors associated with lower 25(OH)D serum levels in multiple linear regression analysis. Interestingly, 53.8% (7/13) of patients with DHCR7 GG genotype had severe liver fibrosis, compared to 27.1% (67/247) of those with DHCR7 TT/TG genotype (P = 0.03). By multivariate logistic regression analysis, severe fibrosis was independently associated with older age (OR, 1.056; 95% CI, 1.023–1.089, P = 0.001), low cholesterol (OR, 0.984; 95% CI, 0.974–0.994, P = 0.002), high triglycerides (OR, 1.008; 95% CI, 1.002–1.015, P = 0.01), low 25(OH)D (OR, 0.958; 95% CI, 0.919–0.999, P = 0.04), DHCR7 GG genotype (OR, 4.222; 95% CI, 1.106–16.120; P = 0.03), moderate–severe steatosis (OR, 2.588; 95% CI, 1.355–4.943; P = 0.004) and moderate–severe necroinflammatory activity (grading) (OR, 2.437; 95% CI, 1.307–4.763; P = 0.001). No associations were found between liver fibrosis and both CYP2R1 and GC genotypes. In patients with G1CHC, GG homozygosis for DHCR7 gene and lower 25(OH)D levels are independently associated with the severity of liver fibrosis.

Introduction
The key issue in patients with chronic hepatitis C (CHC) is the progression of liver fibrosis as a consequence of various mechanisms of tissue damage caused by viral infection,^[1] with the ultimate development of cirrhosis and its complications.

Other than well known risk factors for fibrosis severity, like liver necroinflammation, older age, consumption of alcohol, duration of infection and viral coinfections,^[2] metabolic alterations, namely steatosis,^[3] insulin resistance (IR)^[4] and menopause (in females)^[5] can affect the degree of liver fibrosis.

In this complex and interesting interplay between liver and metabolic factors, growing evidence also suggest a role of vitamin D status on liver disease severity in patients with chronic hepatitis C. In particular, we firstly reported that fully compensated genotype 1 (G1) CHC patients are characterized by a higher prevalence of 25-hydroxyvitamin D [25(OH)D] deficiency compared to a control population, also showing in this clinical setting an independent inverse relationship between 25(OH)D serum levels and liver fibrosis severity.^[6] These clinical data not only were further confirmed by other groups,^[7,8] but also their strength was supported by experimental studies showing that vitamin D, acting via its nuclear vitamin D receptor, exerts its protective effect by inhibiting stellate cell proliferation and their profibrogenic activation.^[9]
                       
Vitamin D has therefore a relevant role in patients with CHC, and its metabolism is regulated by several environmental factors, in particular sunlight and diet. In addition, a recent genome-wide association study (GWAS), in a large screening population of about 30 000 European descent individuals divided in a training and a validation set demonstrated that serum concentrations of 25(OH)-vitamin D are influenced by variants near genes involved in cholesterol synthesis (7-dehydrocholesterol reductase -DHCR7), vitamin D hydroxylation (CYP2R1) and vitamin D transport (vitamin D-binding protein–GC).^[10]
                       
With this in mind, in a cohort of biopsy-proven G1 CHC patients, we aimed to assess the association between vitamin serum levels and its genetic determinants, with the severity of liver fibrosis.

Materials and Methods
Patients

Two hundred and sixty consecutive patients with G1 CHC, recruited at the Gastrointestinal and Liver Unit at the University Hospital in Palermo and fulfilling all inclusion and exclusion criteria detailed below, were assessed. Patients were included if they had a histological diagnosis of CHC (any degree of fibrosis, including cirrhosis) on a liver biopsy performed within 6 months prior to enrolment. G1 CHC patients were characterized by the presence of anti-HCV and HCV RNA, with persistently abnormal alanine aminotransferase (ALT) levels, and by alcohol consumption of <20 g/day in the last year or more, evaluated by a specific questionnaire. Exclusion criteria were (i) advanced cirrhosis (Child-Pugh B and C); (ii) hepatocellular carcinoma; (iii) other causes of liver disease of mixed aetiologies (excessive alcohol consumption, hepatitis B, autoimmune liver disease, Wilson's disease, hemochromatosis, α1-antitrypsin deficiency); (iv) HIV infection; (v) previous treatment with antiviral therapy, immunosuppressive drug and/or regular use of steatosis-inducing drugs (corticosteroids, valproic acid, tamoxifen, amiodarone); (vi) therapy with medications known to affect vitamin D3 metabolism, including vitamin/mineral supplements; and (vii) active IV drug addiction.

The study was performed in accordance with the principles of the Declaration of Helsinki and its appendices, and with local and national laws. Approval was obtained from the hospital's Institutional Review Board and Ethics Committee, and written informed consent was obtained from all patients.

Clinical and Laboratory Assessment
Clinical and anthropometric data were collected at the time of liver biopsy. BMI was calculated on the basis of weight in kilograms and height (in metres), and patients were classified as normal weight (BMI, 18.5–24.9 kg/m²), overweight (BMI, 25–29.9) or obese (BMI ≥ 30). The diagnosis of arterial hypertension was based on the following criteria: systolic blood pressure ≥135 mm Hg and/or diastolic blood pressure ≥85 mm Hg (measured three times within 30 min, in the sitting position and using a brachial sphygmomanometer) or use of blood pressure–lowering agents. The diagnosis of type 2 diabetes was based on the revised criteria of the American Diabetes Association, using a value of fasting blood glucose ≥126 mg/dL on at least two occasions.^[11] In patients with a previous diagnosis of type 2 diabetes, current therapy with insulin or oral hypoglycaemic agents was documented.

A 12-hour overnight fasting blood sample was drawn at the time of biopsy to determine serum levels of ALT, total cholesterol, HDL and LDL cholesterol, triglycerides, plasma glucose concentration, insulin and platelet count. Insulin resistance (IR) was determined with the homoeostasis model assessment (HOMA), using the following equation:^[12] insulin resistance (HOMA-IR) = fasting insulin (μU/mL) × fasting glucose (mm)/22.5. HOMA-IR has been validated in comparison with the euglycemic/hyperinsulinemic clamp technique in both diabetic and nondiabetic patients.^[13]
The analysis of serum 25(OH) D was performed using a Chromosystem reagent kit and a chromatographic system equipped with a Waters 1525 Binary high-pressure liquid chromatography pump connected to a photo diode array detector, and detection was carried out at 265 nm. 25(OH)D serum levels <10 μg/L, from 10 to 30 μg/L, and >30 μg/L, were considered the threshold values for identifying deficiency, insufficiency and normality of vitamin D levels, respectively.
All patients were tested at the time of biopsy for HCV RNA (RT-PCR homemade; limit of detection: 12 IU/mL). Genotyping was performed by INNO-LiPA, HCV II, Bayer.

Genetic Analyses. DNA was purified using the QIAmp blood Mini Kit (Qiagen, Mainz, Germany), and DNA samples were quantified using spectrophotometric determination.

Genotyping for IL28B (rs12979860), PNPLA3 (rs738409), CYP2R1 (rs 10741657), NADSYN1(rs 12785878) and GC (rs 2282679) was carried out using the TaqMan SNP genotyping allelic discrimination method (Applied Biosystems, Foster City, CA, USA). Commercial genotyping assays were available for the SNPs: rs738409 (cat. C_7241_10), rs 10741657 (cat. C_2958430_10); rs 12785878 (cat. C_32063037_10); rs 2282679 (cat. C_26407519_10). Instead, a custom assay has been created by AB for rs12979860.

The genotyping call was performed with SDS software v.1.3.0 (ABI Prism 7500, Foster City, CA, USA). Genotyping was conducted in a blinded fashion relative to patient characteristics.

Histology
Slides were coded and read by one pathologist (D.C.), who was unaware of the patient's identity and history. A minimum length of 15 mm of biopsy specimen or the presence of at least 10 complete portal tracts was required.^[14] Biopsies were classified according to the Scheuer numerical scoring system.^[15] The percentage of hepatocytes containing macrovescicular fat was determined for each 10× field. An average percentage of steatosis was then determined for the entire specimen. Steatosis was assessed as the percentage of hepatocytes containing fat droplets (minimum 5%) and evaluated as a continuous variable. Steatosis was classified as absent-mild at <20% or moderate–severe at ≥20%.

Statistics. Continuous variables were summarized as mean ± standard deviation and categorical variables as frequency and percentage. The Student's t-test and analysis of variance were used when appropriate. Multiple linear regression analysis was performed to identify independent predictors of 25(OH)D serum levels as a continuous dependent variable. As candidate risk factors for low serum levels of 25(OH)D, we selected age, sex, body mass index, baseline ALT, platelet count, total cholesterol, high-density lipoprotein cholesterol, triglycerides, blood glucose, insulin, HOMA score, diabetes, arterial hypertension, DHCR7,CYP2R1 and CG SNPs, HCV RNA levels, steatosis and activity score.

Multiple logistic regression models were used to assess the relationship of fibrosis to the demographic, metabolic, genetic and histological characteristics of patients. In the model, the dependent variable was severe fibrosis coded as 1 = F3–F4 in the fibrosis score versus 0 = F1–F2. As candidate risk factors, we selected the same independent variables included in the 25(OH)D model and added 25(OH)D serum levels as an additional independent variable.

In all analyses, DHCR7 SNP was evaluated as TT/TG vs GG, CYP2R1 as AA/AG vs GG, and CG as TT/TG vs GG, according to published data on genotypes associated with lower vitamin D levels.^[10]
Variables associated with the dependent variable at univariate analyses (probability threshold, P < 0.10) were included in the multivariate regression models. Regression analyses were performed by SAS.^[16]

Results
Patient Features and Histology
The baseline features of the 260 patients are shown in Table 1. Most of our patients were in the overweight to obesity range. One patient in four had fibrosis of at least three by Scheuer score, with a high prevalence of moderate/severe necroinflammation (grading 2–3). One patient in three had histological evidence of steatosis of moderate/severe grade.
Mean serum values of 25(OH)D were 24.5 ± 8.4 μg/L. Accordingly, vitamin D deficiency, insufficiency and normality were observed in 0.4%, 75% and 24.6% of G1 CHC patients, respectively.

DHCR7 rs12785878 TT genotype was present in 114 (43.8%) patients, compared to 133 (51.2%) and 13 (5%) with TG and GG variants, respectively. The CYP2R1 rs10741657 AA genotype was observed in 29 patients (11.1%) compared with 109 (41.9%) and 122 (46.9%) with AG and GG variants, respectively. Finally, GC rs7041 TT genotype was present in 149 patients (57.3%) compared with 94 (36.2%) and 17 (6.5%) with TG and GG variants, respectively.

Serum 25(OH)D Levels
Older age (P = 0.002), female sex (P = 0.03), DHCR7 GG (P = 0.003), GC GG (P = 0.05) and the severity of fibrosis (P = 0.001) were associated with lower 25(OH)D levels in G1 CHC, although only DHCR7 GG (P = 0.003) (P = 0.008) and the severity of fibrosis (P = 0.03) were independent factors in multiple linear regression analysis (Table 2). Figure 1 shows the distribution of serum 25(OH)D levels in relation to DHCR7 genotype.

Figure 1.

25(OH)D serum levels according to DHCR7 genotype, in 260 patients with genotype 1 chronic hepatitis C.

Accordingly, all patients with DHCR7 GG genotype had vitamin D insufficiency/deficiency, compared to 74% with DHCR7 TT/TG genotype (P = 0.03).

Variables Related to Severe Fibrosis
The univariate and multivariate comparison of variables between patients with and without severe fibrosis (F3–F4) are reported in Table 3. Older age, male sex, high baseline values of ALT, low cholesterol, high triglycerides, high blood glucose, high HOMA, diabetes, low 25(OH)D, vitamin D insufficiency/deficiency, DHCR7 GG genotype, moderate–severe steatosis and moderate–severe necroinflammatory activity were associated with severe fibrosis (P < 0.10). Multivariate logistic regression analysis showed that the following features were independently linked to severe fibrosis (Scheuer score ≥3): older age (OR, 1.056; 95% CI, 1.023–1.089, P = 0.001), low cholesterol (OR, 0.984; 95% CI, 0.974–0.994, P = 0.002), high triglycerides (OR, 1.008; 95% CI, 1.002–1.015, P = 0.01), low 25(OH)D (OR, 0.958; 95% CI, 0.919–0.999, P = 0.04), DHCR7 GG genotype (OR, 4.222; 95% CI, 1.106–16.120; P = 0.03), moderate–severe steatosis (OR, 2.588; 95% CI, 1.355–4.943; P = 0.004) and moderate–severe necroinflammatory activity (grading) (OR, 2.437; 95% CI, 1.307–4.763; P = 0.001). The overall area under the curve (AUC) of this model was good (AUC, 0.870). Figure 2 showed the prevalence of severe fibrosis, according to DHCR7 genotype

Figure 2.

Prevalence of severe liver fibrosis according to DHCR7 genotype, in 260 patients with genotype 1 chronic hepatitis C.

Discussion
In this study, we have shown that, in a cohort of patients with biopsy-proven G1 CHC, DHCR7 GG genotype, other than being associated with lower vitamin D serum levels, was also independently linked to the severity of liver fibrosis, together with well known risk factors for fibrosis, including lower vitamin D serum levels.

Different lines of evidence showed a relevant role of vitamin D status in patients with CHC patients, and with neoplastic and cardiometabolic disorders,^[6,17] prompting genetic, clinical and experimental research on vitamin D metabolism and actions.

In our study we showed that, in G1 CHC patients, lower levels of serum 25(OH)D were independently linked to the DHCR7 GG genotype. Our data are in agreement with the GWAS study of Wong and colleagues on a cohort of about 30 000 subjects that identified the DHCR7 gene as able to affect vitamin D serum levels, with the lowest values in GG patients.^[10] In addition, the presence of lower vitamin D serum levels in patients with the DHCR7 GG genotype was also recently reported in a large cohort of Caucasian patients with chronic liver diseases due to different aetiologies.^[18] In our study, we did not identify a link between CYP2R1 and GC SNPs, also linked to vitamin D deficiency in the above quoted GWAS study,^[10] and vitamin D serum levels. This issue could be related to the demographic, clinical and biochemical characteristics of our studied population, like the very high prevalence of vitamin D deficiency, as well as to the relative low number of included patients.

This study offers the first evidence that the DHCR7 GG genotype, together with lower 25(OH)D serum levels, and with other known risk factors for fibrosis severity, such as older age, low cholesterol and high triglycerides levels, moderate–severe steatosis and high necroinflammatory activity, is independently associated with the presence of severe liver fibrosis in G1 CHC patients. Grünhage and colleagues,^[18] in a cohort of more than seven hundred patients with mostly clinically diagnosed chronic liver disease due to different aetiologies (60% HCV related), showed that, among subgroups of patients with liver stiffness measurement (LSM) lower than 7 kPa and lower than 9.5 kPa, the DHCR7 GG genotype was associated with higher LSM values. These data therefore suggested, with limits related to LSM use as surrogate marker of fibrosis, and to subgroups analyses, a potential association between severity of liver disease and the DHCR7 GG genotype. In this line, our study added further and relevant evidence about this issue, demonstrating the association between the DHCR7 GG genotype and severity of histological liver fibrosis, in a cohort of compensated, homogeneous and fully characterized biopsy-proven G1 CHC patients.

Another relevant finding of our study is that both the DHCR7 genotype and vitamin D levels were independently linked to the presence of severe liver fibrosis. This issue suggests that the association between the DHCR7 GG genotype and liver fibrosis is far complex. In fact, on the one hand, it is plausible that DHCR leads to fibrosis via lowering vitamin D serum levels.^[10] Experimental evidence in fact suggests that vitamin D, via interaction with VDR, is able to inhibit stellate cell proliferation and their profibrogenic activation.^[9] On the other hand, our results suggest that DHCR7 genotype could prompt fibrogenesis also via other direct/indirect mechanisms. However, literature data do not provide us with further help on this issue, and therefore, additional experimental work is needed.

The main limitation of this study lies in the low number of patients carrying the at-risk DHCR7 GG genotype. This issue could affect the interpretation of our results. However, the similar low prevalence of DHCR7 GG genotype reported in other studies,^[10,18] then our biologically plausible results^[9,10] and the presence in the literature of similar results^[10,18] makes us confident about the accuracy of our data, which obviously needs further validation in large cohort studies. Another limitation of our study is its cross-sectional nature and its inability to dissect the temporal relation between DHCR7 genotype, 25(OH)D and fibrosis. A further methodological drawback is the potentially limited external validity of the results for different populations and settings. Another limitation of this study is the lack of data on the potential confounders that may influence the levels of vitamin D, such as exposure to sunshine, dietary intake and the prevalence of osteoporosis. However, all of the subjects involved in this study lived in Sicily, where sunshine is abundant.

In conclusion, this study showed that in a homogeneous cohort of compensated biopsy-proven G1 CHC patients, DHCR7 GG genotype, other than leading to lower vitamin D serum levels, is also associated with the severity of liver fibrosis, suggesting a complex interplay between liver damage, vitamin D and genetic determinants of vitamin D deficiency.

Abstract and Introduction

Materials and Methods

Results 

Discussion

Abbreviations                
AUC, area under the curve; CHC, chronic hepatitis C; DHCR7, dehydrocholesterol reductase; G1, genotype 1; GC, vitamin Dbinding protein; GWAS, genome-wide association study; HCV, hepatitis C virus; HOMA, homoeostasis model assessment; IR, insulin resistance; LSM, liver stiffness measurement.

Contributors          
S. Petta designed the study, contributed to data acquisition, was responsible for writing the manuscript and participated in statistical analysis. C. Camma', V. Di Marco and A. Craxì (Director of the GI and Liver Unit) were responsible for the project and writing of the manuscript. D. Cabibi, S. Grimaudo, F.S. Macaluso, R. Pipitone and C. Scazzone participated in patient management and data collection. All authors have seen and approved the final version of the manuscript.
J Viral Hepat. 2013;20(7):486-493. © 2013  Blackwell Publishing

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