Prediction of Fibrosis Progression Rate in Patients with Chronic Hepatitis C Genotype 4: Role of Cirrhosis Risk Score and Host Factors

Abstract

The rate of liver fibrosis progression in chronic hepatitis C (CHC) patients is highly variable and affected by different factors. This study aimed to assess the role of cirrhosis risk score (CRS) based on 7 genetic variants (7 single-nucleotide polymorphisms [SNPs]) and host factors (age and sex) in the prediction of the rate of fibrosis progression in CHC. Duration of infection was determined in 115 patients. The fibrosis progression rate (FPR) per year was calculated as the ratio between fibrosis stage and the duration of infection. SNP genotyping were performed and CRS was determined based on it. FPR was significantly elevated in patients who acquired infection at age >40 years versus those who acquired infection at 30–40 years and those who acquired infection at <30 years. Median FPR was significantly higher in males than females (0.17 vs. 0.15) with P = 0.001. CRS value ≥0.8 is predictive of patients with high risk for cirrhosis, and CRS value <0.5 is predictive of patients with low risk for cirrhosis. There was significant positive correlation between CRS and FPR (P ≤ 0.001). CRS based on 7 SNPs at cutoff value ≥0.8, age at infection >40 years, and male sex are predictors of higher FPR.

Introduction

Hepatitis C virus (HCV) infection with a prevalence of about 130–150 million chronic infections worldwide is one of the principal causes of cirrhosis, hepatocellular carcinoma, and liver transplantation (Shepard and others 2005; WHO 2016).The world distribution of HCV infection shows high regional variability (Lavanchy 2011). Hepatitis C is the most pressing public health challenge in Egypt where HCV prevalence is the highest worldwide. In 2015, Egypt Health Issues Survey showed that 10% of Egyptians aged 15–59 years had HCV infection with percentage higher in males than females (12.4%, 8.1%), respectively (Ministry of Health and Population and others 2015).

About 50%–85% of patients infected with hepatitis C display chronic hepatitis C (CHC) and about 15%–30% will develop cirrhosis over decades (Vergniol and Lédinghen 2014). Progression to advanced disease is, however, difficult to predict on an individual basis (Marcellin and others 2002). Prediction of patients who will develop severe progressive disease and reasons for progression are not well defined. Viral factors such as viral load or HCV genotype have a minor influence on progression to cirrhosis and other severe complications, while host factors such as older age at the time of infection, male sex, obesity, and heavy alcohol consumption were found to accelerate fibrosis progression (Poynard and others 1997; Seeff 2009). However, altogether, these factors are poor predictors of fibrosis progression (Seeff and others 2001).

Identifying candidate genes involved in hepatic fibrogenesis had gained special interest considering that hepatic fibrosis is a dynamic process in which multiple genes interact with environmental factors (Marcolongo and others 2009). Association between liver fibrosis progression in HCV patients and multiple single-nucleotide polymorphisms (SNPs) had been described such as single-nucleotide mutations in the genes encoding for tumor necrosis factor α, interferon γ, inerleukin-10, low-density lipoprotein, and monocyte chemotactic protein 2 (Bataller and others 2003; Missiha and others 2008). Huang and others (2007) identified a 7-gene variant signature associated with fibrosis progression, which was termed cirrhosis risk score (CRS7). This score, which was calculated based on the genotypes of 7 SNPs in each patient, yielded an area under the receiver operating characteristic curve (AUROC) of 0.75 in detecting patients with bridging fibrosis/cirrhosis in the training set and an AUROC of 0.73 in the validation set. An Italian study showed that the mean CRS7 was significantly higher in fibrosis progression patients (Marcolongo and others 2009), also validated in HALT-C trial (Curto and others 2011).

Therefore, in this study, we evaluated the role of CRS and host factors (age and sex) for prediction of liver fibrosis progression in patients with CHC genotype 4.

Patients and Methods

Ethics statements

All patients provided written informed consent, and the institutional review board of our Faculty of Medicine approved this intervention study.

Patients

This study included 330 adult patients aged 18–75 years with HCV RNA positive and had a liver biopsy before, if any, treatment for HCV, all recruited at Tropical Medicine Department, Mansoura University Hospital, from the period of October 2012 to December 2015. Patients with chronic hepatitis C with positive viremia were included, while those with any other coexisting chronic liver diseases were excluded. All patients had routine liver function tests, polymerase chain reaction for HCV, HCV genotyping, CRS value, and liver biopsy.

Estimating the duration of infection and FPR

Meticulous history was taken from the patients to trace their risk factors for acquiring HCV infection and suspected year of infection. When a history of a particular incident was elicited, it was presumed to be the time of acquiring HCV infection considering finding only a single incident that can lead to the infection. Of the 330 patients, only 132 (40%) had a history of a specific single event that led to acquiring HCV infection. Patients with an uncertain or equivocal date of infection or more than one potential source of infection were not included. The duration of infection was estimated from the year of infection to the year of liver biopsy. When the duration of infection could be estimated, fibrosis progression rate (FPR) was defined as the ratio between the fibrosis stage in METAVIR units and the estimated duration of infection in years and it was expressed as fibrosis units per year. For example, for a patient with stage F1 fibrosis and length of infection estimated at 20 years, the FPR would be 1/20 = 0.05 fibrosis units per year.

Liver biopsy

Samples of liver biopsy of adequate length were taken and evaluated by double pathologists. For each liver biopsy sample, a grade of activity and a stage of fibrosis were determined according to Metavir score (Bedossa and Poynard 1996). Patients were included in the study when the stage of fibrosis was F1, F2, F3, or F4 and they were excluded if the stage of fibrosis was F0 as it is not applicable to measure FPR with F0 (as FPR will be zero).

Cirrhosis risk score

For each patient, the results of the 7 SNPs composing CRS were determined. The genomic DNA (gDNA) was extracted by DNeasy Blood and Tissue kit (Qiagen, Valencia, CA) according to manufacturer instructions. The TaqMan^® SNP Genotyping Assays (life technologies, Carlsbad, CA) use TaqMan 5′-nuclease chemistry for assay of rs17740066 A/G, rs62522600 A/G, rs4986791 C/T, rs2878771 C/G, rs4290029 C/G, rs2290351 A/G, and rs886277 C/T for amplifying and detecting specific polymorphisms in purified gDNA samples. Each assay allows genotyping of individuals for an SNP. These assays use TaqMan minor groove-binding (MGB) probes for superior allelic discrimination, improved signal-to-noise ratios, and design flexibility. SNP genotyping assays contain VIC^® dye-labeled probe, FAMTM dye-labeled probe, and 2 target-specific primers. TaqMan probes incorporate MGB technology at the 3′ end to deliver superior allelic discrimination. The MGB molecule binds to the DNA helix minor groove, improving hybridization-based assays by stabilizing the MGB probe–template complex. This increased binding stability permits the use of probes as short as 13 bases for improved mismatch discrimination and greater flexibility when designing assays for difficult or variable sequences. All MGB probes also include a nonfluorescent quencher that virtually eliminates the background fluorescence and provides excellent signal-to-noise ratio for superior assay sensitivity.

The value of CRS was calculated for each patient from the results of the 7 SNPs using the original Naïve Bayes formula as described in detail by Huang and others (2007). The Table 1 shows the SNPs composing CRS signature.

Table 1.

Single-Nucleotide Polymorphisms Composing Cirrhosis Risk Score Signature (HapMap)

SNP	Gene	Name	Chromosome
rs62522600	AZIN1	Antizyme inhibitor 1	8
rs4986791	TLR4	Toll-like receptor 4	9
rs886277	TRPM5	Transient receptor potential cation channel subfamily M member 5	11
rs2290351	AP3S2	Adaptor-related protein complex 3 sigma 2 subunit	15
rs4290029	DEGS1	Degenerative spermatocyte homolog 1 lipid desaturase	1
rs17740066	STXBP5L	Syntaxin binding-protein 5-like	3
rs2878771	AQP2	Aquaporin 2	12

SNP, single-nucleotide polymorphism.

CRS7 scores were derived as described previously (Huang and others 2007). The scores range from 0 to 1 with higher scores indicating higher risk of cirrhosis. Patients were classified as having high risk (CRS7 > 0.7), intermediate risk (CRS7 = 0.5–0.7), or low risk (CRS7 < 0.5) of cirrhosis using cutoffs based on a receiver operating characteristic curve in the study in which CRS7 was developed (Huang and others 2007).

Statistical analysis

Data were analyzed using SPSS version 20 (SPSS, Inc., Chicago, IL). Variables were described as number and percent or mean (standard deviation), as appropriate. FPR was found to be nonparametric in distribution by Kolmogorov–Smirnov test. So, it is presented as median (minimum–maximum). Mann–Whitney test was used to compare FPR between 2 groups and Kruskal–Wallis test was used for more than 2 group comparison. Spearman correlation coefficient was used to calculate correlation between FPR and CRS in different age and sex groups. P ≤ 0.05 was considered statistically significant.

Results

Of 330 patients, a suspected year of infection and, subsequently, duration of infection were determined in 132 patients; from them, 17 patients were excluded (7 patients were F0, and in 10 patients, CRS was not determined due to technical problems, either insufficient DNA in blood samples or the 7 SNPs were not complete). The characteristics of the 115 patients included in this study are shown in Table 2. The median age of acquiring infection was 31 years and the median duration of infection was 19 years. The median FPR was 0.16 fibrosis units per year (range of 0.09–0.33). At this rate of fibrosis progression, the median duration of developing cirrhosis was 25 years (range of 12–44 years). The median CRS was 0.79 with a range of 0.25–0.94.

Table 2.

Demographic and Laboratory Data of the Studied Patients

	Median (min-max)
Sex: male/female	73/42
Current age (years)	49.5 (24.0–65.0)
Age at infection (years)	31.0 (4.0–56.0)
Duration of infection (years)	19.0 (3.5–45.0)
FPR	0.16 (0.09–0.33)
CRS	0.79 (0.25–0.94)

	Mean ± SD
ALT (IU/L)	55.95 ± 35.9
AST (IU/L)	53.5 ± 30.6
HCV PCR	620900 ± 838820
HB	12.7 ± 2.0
WBC, ×1000/mm³	5.9 ± 2.2
Platelet count ×1000/mm³	161.6 ± 60.9
Total bilirubin (mg/dL)	1.1 ± 1.04
Serum albumin (g/dL)	3.9 ± 0.7
INR	1.2 ± 0.2
AFP	8.2 ± 10.7
Activity
1	31 (27.0%)
2	66 (57.4%)
3	18 (15.6%)
Fibrosis
1	23 (20%)
2	24 (20.9%)
3	31 (27%)
4	37 (32.1%)

ALT, alanine transaminase; AFP, alpha-fetoprotein; AST, aspartate transaminase; CRS, cirrhosis risk score; FPR, fibrosis progression rate; HB, hemoglobin; HCV, hepatitis C virus; INR, international normalized ratio; PCR, polymerase chain reaction; WBC, white blood cell.

In this study, the patients were classified according to age at acquiring HCV infection into 3 groups as shown in Table 3. Patients who acquired infection after the age of 40 years had FPR 0.25 with a median duration for developing cirrhosis of 16 years. For those who acquired infection from the age of 30 years to 40 years, their FPR was 0.17 with a median duration for developing cirrhosis of 23.5 years. On the other hand, those who acquired infection before the age of 30 years had FPR 0.12 with a median duration for developing cirrhosis of 33 years. The FPR was significantly affected by the age of acquiring HCV infection.

Table 3.

Fibrosis Progression Rate of the Different Groups

	Number (n = 115)	FPR Median (min-max)	Significance
Age at infection (years)
<30	51 (44.3%)	0.12 (0.09–0.15)^A,B	KWχ² = 93.2
30–40	34 (29.6%)	0.17 (0.13–0.22)^A,C	P ≤ 0.001
More than 40	30 (26.1%)	0.25 (0.18–0.33)^B,C
Sex
Male	73 (63.5%)	0.17 (0.09–0.33)	Z = 3.3
Female	42 (36.5%)	0.15 (0.09–0.28)	P = 0.001
CRS
<0.5	10 (8.7%)	0.1 (0.09–0.18)^A–C
0.5–0.7	17 (14.8%)	0.16 (0.11–0.2)^A,D	KWχ² = 30.6,
>0.7 to <0.8	34 (29.5%)	0.125 (0.11–0.25)^B,E	P ≤ 0.001
0.8 and more	54 (47%)	0.2 (0.12–0.33)^C–E

A–E

Significant differences between the corresponding group by Mann–Whitney test.

KW, Kruskal–Wallis test Z of Mann–Whitney.

Male patients had higher FPR than female (0.17 vs. 0.15 with P value of 0.001) and consequently the time for development for cirrhosis is significantly less in males than in females.

In this study, patients were classified into 4 groups according to their CRS values as shown in Table 3. In patients who had CRS <0.5, the median FPR was 0.1 fibrosis units per year with a median duration to develop cirrhosis of 40 years. Patients who had CRS ranging from 0.5 to 0.7 had a median FPR of 0.16 and a median duration to develop cirrhosis of 25 years. In patients with CRS more than 0.7 to <0.8, the median FPR was 0.125 fibrosis units per year with a median duration to develop cirrhosis of 32 years. In patients with CRS ≥0.8, the median FPR was 0.2 fibrosis units per year with a median duration to develop cirrhosis of 20 years.

There was a statistically significant difference between FPR of the group with CRS <0.5 and the group with CRS of 0.5–0.7 and the group with CRS of more than 0.7, but no difference between the group with CRS 0.5–0.7 and the group of CRS more than 0.7. The group with CRS >0.7 had a median FPR similar to the group with CRS 0.5–0.7, but with a faster range. There were statistically significant differences between FPR of the group with CRS of <0.5 and FPR of the other 3 groups. In addition, there were statistically significant differences between FPR of the group with CRS ≥0.8 and FPR of the other 3 groups, but no statistically significant difference between FPR of the group with CRS <0.5 and the group of CRS more than 0.7 to <0.8.

Table 4 shows a significant positive correlation between CRS and FPR in the 3 age groups of patients after fixation of age at infection and the sex of patients.

Table 4.

Correlation Between Fibrosis Progression Rate and Cirrhosis Risk Score in Relation to Age and Sex

	FPR
	r	P
CRS
Age: <30
Male	0.965	≤0.001
Female	0.958	≤0.001
Age: 30–40
Male	0.927	≤0.001
Female	0.928	≤0.001
Age: >40
Male	0.850	≤0.001
Female	0.969	≤0.001

r, Spearman correlation coefficient.

Discussion

Host genetics was suggested to play a role in the natural history of CHC (Pradat and others 2007). Identifying patients at risk of disease progression is required to treat patients in earlier stages of the disease with potentially curative antiviral therapy that would be most cost-effective (Ghany and others 2009). There are multiple factors found to affect the FPR. This study evaluated the role of CRS, host factors (age and sex) that affect the FPR in patients with CHC genotype 4.

In this study, the median rate of fibrosis progression was 0.16 fibrosis units per year and, at this rate, the median duration of developing cirrhosis was 25 years. This median duration of developing cirrhosis was not in accordance with Poynard and others (1997) and a UK study done by Ryder and others (2004) who reported that the median duration of developing cirrhosis was 30 years, while studies in the United States by Ghany and others (2003) and Hoefs and others (2011) found a median duration of developing cirrhosis of 50 years. In Spain, Fernández-Rodriguez and others (2004) found a median duration of developing cirrhosis of 44 years. Matsumura and others (2000) found a median duration of developing cirrhosis of 33 years in Japan. The median duration of developing cirrhosis was shorter in studies done in India by Amarapurkar (2000) and Hissar and others (2009) that showed a duration of 20 and 16 years, respectively; this observation in this current study could be attributed to genotype 4 predominance.

The age at which HCV infection was acquired was found by most studies to affect the rate of progression of liver fibrosis. Our results showed significant differences in the rate of liver fibrosis progression in 3 age groups: those that acquired HCV infection before 30 years, from 30 to 40 years, and after the age of 40 years with a median FPR of 0.12 versus 0.17 versus 0.25 fibrosis units per year with a median duration to develop cirrhosis of 33 years versus 23.5 years versus 16 years. So, the older the patient when he acquires HCV infection, the more rapid the rate of fibrosis progression and the lesser the time to develop liver cirrhosis. In some studies, age of infection more than 40 years was found to accelerate liver fibrosis (Deuffic and others 1999; Poynarda and others 2001; Svirtlih and others 2007). In a study in India by Hissar and others (2009) and also a study in Japan by Matsumura and others (2000), age of infection of more than 30 years was found to be associated with accelerated rate of liver fibrosis. On the other hand, in a study by Amarapurkar (2000) in India, age of infection of more than 35 years was found to be associated with accelerated rate of liver fibrosis. The role of aging in affecting fibrosis progression could be linked to a higher vulnerability to environmental factors (especially oxidative stress), to decrease in blood flow, mitochondria capacity, or in diminishing immune capacities (Poynarda and others 2001). In addition, it has been postulated that antioxidant defenses decline with aging, thereby being more easily overcome by oxidative stress (Fernández-Rodriguez and others 2004).

The median FPR was found to be significantly higher in males than in females (0.17 vs. 0.15 with P value of 0.001). This result matches with other studies evaluating the role of sex in liver fibrosis progression (Poynard and others 1997; Thomas and others 2000; Harris and others 2002; Kryczka and others 2003), which demonstrated that males develop cirrhosis in a lesser duration than females do. A study done in women who received HCV-contaminated Rh immune globulin also suggested an important role of sex in progression of fibrosis (Freeman and others 2001). On the other hand, Hissar and others (2009) found no statistically significant differences in FPR between males and females. In addition, in a study done in the United States by Hu and others (2009), which was done to evaluate parameters for their association with stage 3 and 4 fibrosis, fibrosis progression was not associated with sex. It has been suggested that long-term exposure of females to estrogen may reduce liver fibrosis progression, whereas menopause may be associated with accelerated fibrosis progression in patients with CHC (Wu and others 2014).

Current study classified the patients into 4 groups according to the CRS level: <0.5, 0.5–0.7, > 0.7 to <0.8, and ≥ to 0.8. There were statistically significant differences between FPR of the group with CRS of <0.5 and FPR of the other 3 groups, also FPR of the group with CRS ≥0.8 and the other 3 groups. On the other hand, there was no statistically significant difference between FPR of the group with CRS 0.5–0.7 and the group of CRS >0.7 to <0.8.

The group with CRS >0.7 to <0.8 showed that 20 patients of 34 patients of this group (about 59%) had infection before the age of 30 years, while the group with CRS 0.5–0.7 showed nearly even distribution of age at infection.

There was positive significant correlation between CRS and FPR in each of the 3 age groups after fixation of age at infection and sex. Our findings are in agreement with Trepo and others (2011) who found that CRS predicts fibrosis progression in initially mild CHC and also in agreement with (HALT-C) Trial, in which CRS7 was validated as a predictor of fibrosis progression and cirrhosis (Curto and others 2011), but unlike Marcolongo and others (2009) who showed that the mean CRS7 was significantly higher in male patients with fibrosis progression than in those who did not have fibrosis progression.

Current study found that 76.5% of the patients had CRS of more than 0.7, the level that was associated with high risk for cirrhosis according to Huang and others (2007). According to results of our study, we can consider that the patients with high risk for cirrhosis are those with value equal to or more than 0.8, which represented 47% of the patients with a median FPR of 0.2 and a median time to develop cirrhosis of 20 years.

In conclusion, the median FPR in CHC patients with genotype 4 was 0.16 fibrosis units per year with a median time to develop cirrhosis of 25 years. CRS based on 7 SNPs at cutoff value ≥0.8, age at infection >40 years, and male sex are predictors of higher FPR.

There are some limitations of this study. First, to be ideal, it should be a longitudinal study with repeated biopsies from patients instead of a cross-sectional one, but this is not practical because a small percentage of patients only underwent repeated biopsies before the antiviral therapy. Second, depending on calculating FPR from identifying the suspected year of infection from the history is not ideal due to the difficulty in identifying the potential incident that led to HCV infection, but we tried to make the results better by excluding patients with a doubtful or equivocal date of infection or more than one potential source of infection.

Footnotes

Acknowledgments

The authors would like to thank the Clinical Pathology Department and Pathology Department-Mansoura Faculty of Medicine for their kind help and support to complete routine laboratory work. This research was funded by the Science and Technology Development Foundation (STDF), Project NO. 3457 (TC/4/Health/2010/hep-1.6).

Author Disclosure Statement

The authors in this study declared that they do not have anything to disclose regarding funding or conflicts of interest with respect to this article.

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