Vitamin D Profile : Can it Affect the Response to Standard Hepatitis C Treatment in Egyptian Patients ?

95% CI= 2.9-21.3, P<0.005 respectively). Serum levels of vitamin D showed statistically significant negative correlations with the activity and fibrosis of the liver in both responders and non responders. Also, there was negative correlation between vitamin D level and viral load in non responder patients (r= -.232, P=0.01). As regard the value of serum vitamin D level in discriminating responders from non responders; area under the ROC curve was 0.708 (95% CI 0.643-0.774). At a cutoff value of 19 ng/dL of serum vitamin D yielded sensitivity 79%, specificity 58%, positive predictive value (PPV) 65%, and negative predictive value (NPV) 73%. Conclusion: Vitamin D serum level and CYB27B1 -1260 genotype could be used as a predictor to anti HCV treatment response in our locality.


INTRODUCTION
Egypt reports the highest prevalence of hepatitis C virus (HCV) world wide, ranging from 60% to more than 40% among regions and demographic group [1].The recommended therapy for chronic hepatitis C, is pegylated interferon and ribavirin for 24 or 48 weeks [2].Sustained virological response (SVR), defined as undetection of HCV RNA in patient's serum for 6 months after end of treatment, is ranging from 42.9% to 69% in patients with genotype 4 Vitamin D was initially identified as a calcium homeostatic hormone.Vitamin D is now known to have pleiotropic functions, dealing with both innate and adaptative immunity.Calcitriol mediates its biological effects by binding to the vitamin D receptor (VDR), which is expressed not only by intestine, bone and kidney but also on cell membranes of T lymphocytes, B lymphocytes, dendritic cells and macrophages responsiveness.
Immunomodulatory actions of vitamin D are elicited through its direct action on T-cell antigen-presenting cell function [8].

Moreover vitamin D improves insulin sensitivity, suppresses proinflammatory cytokines, increases anti-inflammatory cytokines, and improves CD4 T cell hyper-responsiveness [9,10].
Vitamin D deficiency is very common among patients with chronic liver disease (92%), and at least one-third suffer from severe vitamin D deficiency (<12 ng/ml) [11].Serum vitamin D deficiency and the CYP27B1-1260 promoter polymorphism are more prevalent in patients with chronic hepatitis C and related to more fibrosis, and that they are associated with a lower response rate to interferon-alfa based therapy in genotype 1 chronic hepatitis C (CHC) [12].
Our aim is to evaluate the relationship between vitamin D metabolism-related genes and vitamin D level and the response to standard care of treatment for chronic hepatitis C infection in our locality, as genotype 4 is the predominant.

PATIENTS AND METHODS
This cross-sectional study was carried out in Tropical Medicine Department and Medical Biochemistry Department, Zagazig University Hospital, 245 Egyptian patients with compensated liver function out of 270 patients with chronic HCV, their ages ranged from 18 to 65 years, were enrolled in this study during the period from January 2013 to January 2014.All patients were receiving Peg interferon-á-2b (1.5 ug/kg per week) plus ribavirin (1000-1200 mg/d).the studied population included 123 patients with sustained virological response defined as undetectable HCV RNA at 24 weeks post treatment and another 122 patients with treatment failure," a non-responder is someone who does not have disappearance of the HCV RNA, does not ever have a 2-log drop in hepatitis C viral load at 12 weeks, and if HCV RNA was still detectable at week 24 in those patients in whom HCV RNA dropped more than 2 log at 12 week [13].Both groups were matched for age, sex and body mass index.

Exclusion criteria :
The patients were excluded from the study if their WBCs less than 4000/mm 3 , absolute neutrophil count of <1500 per mm 3 , a platelet count of <90 000 per mm 3 , hemoglobin level was abnormal, or if they had increased serum bilirubin more than 2 mg/dl [14].Also patients with hepatitis B, auto immune hepatitis, metabolic liver disease, hepato-cellular carcinoma, renal failure and heart failure, decompansated chronic liver disease, or those who had any problem necessities stoppage or interruption of treatment were excluded [15].In addition to those who are previously treated or those were receiving adjuvant medication with immunemodulatory effect.
An informed consent was taken from each patient.All patients were subjected to complete history taking, full clinical examination, ultrasonographic and histopathological evaluation according to Metavair score [16].

Laboratory investigation :
All subjects were subjected to routine laboratory investigation including complete blood picture, liver function test and renal function test.Viral markers including hepatitis C virus antibodies (HCV Abs), hepatitis B surface antigen (HBsAg) and hepatitis B core antibodies (HBcAbs) were tested using ELISA.HCV RNA levels were determined by using the Real time PCR (Step One Real-time PCR System, Applied Biosystem), performed strictly in accordance with the manufacturer's instructions.Serum alpha-feto protein, thyroid hormone (triiodothyronine (T3), thyroxine (T4) and thyroid stimulating hormone (TSH), anti nuclear antibodies (ANA Abs) and 25-OH vitamin D level in serum were measured using ELISA kits (kits provided by Biosource Europe S.A, Belguim).

Isolation of DNA :
Genomic DNA was extracted from EDTA whole blood using a spin column method according to the protocol (QI Aamp Blood Kit; Qiagen GmbH, Hilden, Germany).

Genetic polymorphism detection of the CYP27B1 gene :
The -1260C>A polymorphism (rs10877012) of the CYP27B1 gene was analyzed by restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR) method described by Jennings et al. [17].The 1260 C was amplified in a single product using the primers forward 5-GTGTTCCCTAAGTGTTGTCTC-3 and reverse 5-GCTGACTCGGTCTCCTCTG-3. Fragments were amplified in 50μl reaction mixtures containing 10 μl genomic DNA, 30 µl one step PCR mixture (1 unit Taq polymerase, 10 mM KcL, 10 mM (NH 4 ) 2 SO 4 , 20mM Tris Hcl (PH 8.75), 0.1% Triton X-100, 0.1 mg/ml BSA and 200 µm dTNPs) and 2 μl of each primer (BioBasic Inc., Ontario, Canada) and 8 µl DdH 2 O. Reaction conditions used with the thermal cycler (Biometra, Göttingem, Germany) were as follows: an initial incubation at 94ºC for 5 minutes followed by 30 cycles of incubation at 94ºC for 45 seconds, 58ºC for 45 seconds and 72ºC for 45 seconds with a final extension at 72ºC for 7 minutes.Subsequently, it was subjected to digestion with TfiI enzyme, which cleaved the A allele into two fragments of 195 and 103 bp.

Statistical analysis :
Data were analyzed with SPSS for version 15.0 (statistical package for the Social Science, Chicago, IL).Quantitative data were expressed as mean  standard deviation (SD), data were analyzed by independent sample t and One Way Analysis Of Variance (ANOVA).While qualitative data were expressed as number and percentage and were analyzed by Chi square (X 2 ) test.Correlation was done using Pearson correlation test.The receiver operating characteristic (ROC) curve and 95% confidence interval (CI) was performed to determine cutoff values for serum level of Vitamin D. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were determined.Odds ratios (ORs) and confidence intervals (CI) were calculated.Pvalue was considered significant if <0.05 and highly significant if <0.001.

RESULTS
The study included 245 Egyptian patients (123 patients with sustained virological response and 122 patients with treatment failure), their clinical characteristics is shown in table (1).Serum levels of vitamin D showed statistically significant increase in responders in comparison with the non responders.
Serum levels of vitamin D showed statistically significant decrease among those with advanced grading and staging, with a statistically significant negative correlation between vitamin D level and the activity and fibrosis of the liver in both responders and non responders.Also, there was negative correlation between vitamin D level and viral load in non responder patients (r= -.232, P=0.01, data not shown).As regard the value of serum vitamin D level in discriminating responders from non responders; area under the ROC curve was 0.708 (95% CI 0.643-0.774),(Fig. 1).At a cutoff value of 19 ng/dl of serum vitamin D yielded sensitivity 79%, specificity 58%, positive predictive value (PPV) 65%, and negative predictive value (NPV) 73%.

[ 3 - 4 ].
Several factors are associated with treatment failure including host and viral predictors such as body weight, ethnicity, liver histology, genotype, viral load and metabolic factors such as elevated fasting glucose [5,6,7].

Table ( 5 ) :
Correlation between vitamin D level and histopathological findings.
[33]egard the value of serum vitamin D level in discriminating responders from non responders; we analyzed the receiving operating curve (ROC) and found that area under the ROC curve was 0.708 (95% CI 0.643-0.774).At a cutoff value of 19 ng/dL of serum vitamin D yielded sensitivity 79%, specificity 58%, positive predictive value (PPV) 65%, and negative predictive value (NPV) 73%.Future studies can combine vitamin D with other predictors to improve its validity in prediction of treatment response.Vitamin D insufficiency (defined by a 25-hydroxyvitamin D [25(OH)D 3 ] serum concentration <20 ng/mL) has been proposed as a predictor of failure of treatment of chronic hepatitis C with PEG-IFN-α and ribavirin in others genotypes[12].These findings may have important implications for the management of chronic hepatitis C, as vitamin D status is a potentially modifiable determinant of treatment outcome[33].So, we concluded that vitamin D levels were decreased with the increase in disease severity.Vitamin D serum level concentration and CYB27B1 -1260 genotype could be used as a predictors to HCV treatment response in our locality.