MOLECULAR MARKERS PREDICTING THE EFFICACY OF INTERFERON-BASED THERAPIES IN PATIENTS WITH CHRONIC HEPATITIS C

MOLECULAR MARKERS PREDICTING THE EFFICACY OF INTERFERON-BASED THERAPIES IN PATIENTS WITH CHRONIC HEPATITIS C

Olfat G. Shaker*^A ; Gamal El Din Essmat^B and

Heba Allah M. N. Karmy^C

^A Medical Biochemistry and Molecular Biology Department, Faculty of Medicine, Cairo University, Cairo, Egypt

^B Endemic Medicine and Hepatology Department, Faculty of Medicine, Cairo University, Cairo, Egypt

^C Biomedical Chemistry Unit, Chemistry Dept., Animal Health Research Institute, Giza, 12618 Egypt

*Corresponding author:olfatshaker@yahoo.com

Key Words: Hepatitis C virus; Interferon; Pegylated interferon; Ribavirin; MxA; Myxovirus resistance protein; Polymerase chain reaction.

ABSTRACT

Detection of the SNPs (Single nucleotide polymorphisms) in the promoter region (-88,-123) of the MxA in the blood of chronic liver disease patients under treatment with interferon was investigated. The association between these polymorphisms and the response to IFN-α therapy was conducted on 125 HCV-infected Egyptian patients with chronic hepatitis C and 20 individuals with healthy livers (controls). Quantitation for HCV was performed using PCR for all patients to detect the viral load. MxA (positions -88 and -123) genotypes were determined using a polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) assay. Detection of HCV-specific antibodies in the patients serum were done using ELISA techniques. A significant difference in the frequencies of distribution of MxA promoter genotypes in responders as compared to non-responders was recorded. The frequency of the homozygous GG genotype was significantly higher in non-responders in comparison with responders; while the heterozygous GT genotype was highly significant in responders in relation to non-responders. Furthermore, sustained responders had the mutant T allele more frequently than non-responders with a statistical significance. According to position –123, Inheritance of the heterozygous CA genotype was significantly associated with responders to therapy. Moreover, the presence of the A allele was significantly correlated with response to IFN-α treatment.

1. INTRODUCTION:

The hepatitis C virus (HCV) infection is one of the main causes of chronic liver disease worldwide [1]. There are approximately 71 million chronically infected individuals worldwide with this virus [1, 2]. Global and region-specific estimates of HCV prevalence vary greatly, but the highest prevalence (15– 20%) has been reported from Egypt [3]. The surge in morbidity and mortality associated with the HCV virus is likely due to the widespread prevalence and availability of injectable treatments and the illegal use of injection drugs [4].Major known modes of transmission include intra-familial transmission, direct blood contact, maternal infant transmission, cosmetic services and folk medicines, medical and dental procedures, injection drug use [5].Thus the prevention of primary HCV infection is a public health issue of major importance [6].The primary goal of therapy in patient with HCV is to reach a sustained viral response (SVR) defined as the inability to detect HCV-RNA by polymerase chain reaction six months after completion of antiviral therapy where the secondary goals are: slow disease progression, improve underlying histologic findings, prevent the development of HCC and improve quality of life after therapy [7]. Thus, it is highly important to identify factors that can help predict the chance of each patient to respond to treatment before initiating therapy [8].Several viral and host factors have been implicated in response to therapy. Numerous studies have also indicated that specific single-nucleotide polymorphisms (SNPs) of certain host genes are probably involved [2, 9]. The interferon system is a crucial component of the innate immune response to infectious agents [10]; its biological activity is mediated by the induction of intracellular antiviral proteins, such as the myxovirus resistance A (MxA) protein, 20–50 oligoadenylate synthetase-1 (OAS-1), and the double-stranded RNA (dsRNA)-dependent protein kinase (PKR). Moreover, the MxA protein is assumed to be the most specific surrogate parameter for IFN action. Some reports have shown that increasing the MxA protein or mRNA levels were related to response to IFN-a therapy. However, the levels of the MxA protein or mRNA during IFN-a therapy differ between individuals. One cause of these differences may be associated with genomic factors [11]. The current study aims to investigate the association of MxA polymorphisms at positions -88 and -123, with the response to IFN therapy in HCV-infected Egyptian patients. The combination of these genetic polymorphisms is based on the link between the production of MxA protein as interferon-mediated antiviral response [12]

2.MATERIAL AND METHOD:

2.1.Experimental design:

The study included 145 individuals classified into two groups:

2.1.1.Group (A):

One hundred and twenty five hepatitis C patients were selected from Kasr el-Aini Hospital as naive patients, the age of patients varied from 20 to 54 years with a mean of 38.7±7.5 years. The hepatitis C group included 49 females and 76 males.

2.1.2. Group (B):

Twenty normal healthy individuals were included in this work as control. Their socioeconomic level was similar to that of hepatitis C patients. Their age ranged from 19 to 48 years with a mean of 35.6 ±7.9 years. The control group included 9 males and 11 females.

2.2. Biochemical protocol:

1- Full case history taking.

2- A complete blood picture was performed.

3- A liver biochemical profile was performed.

4- For all hepatitis C patients, full history and clinical data were recorded before and during the therapy. Patients were treated with PEG-IFN-a 2b, at a dose of 1.5 µg/kg subcutaneously every week, plus ribavirin at a dose of 1,000–1,200 mg per day, according to the patient’s body weight for 24 weeks. Patients were classified as ‘‘sustained responders’’ (SR) if they showed normalization of serum alanine transaminase (ALT) levels, and had undetectable plasma-viral RNA 6 months after treatment has stopped, while all the other patients who did not achieve sustained response were considered ‘‘non-responders’’ (NR).

5- Twenty normal healthy individuals were included in this work as control.

6- Quantitation for HCV was performed using PCR for all patients to detect the viral load.

2.3 Serological Tests and HCV-RNA Quantitation and Genotyping

At base time before any treatment with interferon, 5 ml venous blood samples were withdrawn and divided into two tubes under aseptic conditions from patients and controls after obtaining their written consent as follows: Three milliliters of blood were left for 10 min to clot and then centrifuged at 2,0009g for 5 min. The serum was then separated for determination of serological tests, as well as HCV RNA quantification. Serum HCV-RNA was extracted using a Qiagen Viral RNA kit (Hilden, Germany). HCVRNA was quantified in all patients by Real-time PCR using a Light Cycler system (Roche Diagnostics GmbH, Mannheim, Germany), and was classified into low, moderate, or high levels. The line probe assay was used to assess HCV genotypes using kits provided by INNOGENETICS, N.V. The 5-UTR region was amplified using nested PCR with biotinylated primers.

Two milliliters blood was collected on EDTA using a sterile tube; the EDTA samples were stored at -80 _C to be used for detection of MxA genes.

2.4 Genotyping of Allelic Variants:

The MxA SNPs was determined by restriction fragment length polymorphism (RFLP) technique. The DNA was extracted from whole blood using Qia-amplification extraction kit (Qiagen Inc., Valencia, CA, USA) and stored at -80 _C in aliquots until required. Amplification of MxA genes was carried out by polymerase chain reaction (PCR) using Taq polymerase enzyme and T-Gradient thermal cycler (Biometra, Germany). Identification of the two alleles at each polymorphic site was performed by incubating the PCR product with a restriction enzyme, followed by electrophoresis on agarose gels.The sequences of primer used for amplification are shown in Table (1). The MxA SNPs -88(G/T) and the -123(C/A) biallelic MxA promoter polymorphisms were detected by PCR using primers that amplified a fragment of DNA containing both polymorphisms. Amplification of the 599 bp fragment was performed in a volume of 50 µl containing 0.5 ng of template DNA, 5 µl of 10X reaction buffer with MgCl2 (Amersham Pharmacia Biotech, Piscataway, NJ, USA), 0.5 µM of each primer, 0.2 mML dNTPs (Perkin-Elmer Corporation, Foster City, CA, USA), and two units Taq DNA polymerase (Amersham Pharmacia Biotech, Piscataway, NJ, USA). The cycling conditions were as follows: denaturation at 95˚C for 10 min, followed by 35 cycles of denaturation at 95˚C for 10 s; annealing at 65˚C for 10 s; and extension at 72˚C for 1 min. Then final extension cycle was carried out at 72˚C for 7 min. Identification of the two alleles at position -88(G/T) was performed by incubating 10 µl of the 599 bp PCR product in a 20 µl reaction mixture containing 0.4 µl HhaI restriction enzyme (Promega Corporation, Madison, WI, USA) for 16 h at 55˚C. The presence of the T allele resulted in no cleavage indicated by the presence of a 599 bp fragment, while the presence of the G allele led to separation of 482 and 117 bp fragments after gel separation (Fig. 1). The SNP -123(C/ A) was detected by digestion of the PCR product with restriction endonucleases PstI (Promega Corporation, Madison, WI, USA) according to the manufacturer’s instructions. The presence of the A allele resulted in no cleavage resulting in the presence of a 599 bp fragment, whereas individuals homozygous for the C allele yielded two fragments of 464 and 153 bp. The DNA fragments were separated by electrophoresis on 2 % agarose gel andvisualized by ethidium bromide staining (Fig. 2).

Table (1): The sequence of primers used for amplification of MxA.

Fig (1): SNPs in the promoter region of the MxA gene. The sites of polymorphism are indicated by double-underlines and nucleotide positions. The –123 SNP is involved in the PstI recognition site (shadowed), an RFLP assay is available, as was for the –88 SNP with HhaI. ISRE = Interferon-stimulated response element.

A

B                

Fig. ( 2):  Representative agarose gel electrophoresis illustrating PCR products for the MxA polymorphisms.

(A) MxA –88 polymorphism: lane 1, bp marker; lanes 2 and 3, homozygous GG subject, G allele doesn’t cut with HhaI; lane 4, homozygous TT subjects, T allele cuts with HhaI to generate 482- and 117- bp fragments; lane 5, heterozygous GT subject.

(B) MxA –123 polymorphism: lane 1, bp marker; lane 2, homozygous CC subject, C allele doesn’t cut with PstI; lane 4, heterozygous CA subject; lane 5, homozygous AA subject, A allele cuts with PstI to generate 464- and 153- bp fragments.

2.5. Statistical Methods:

Data management and analysis were performed using Statistical Package for Social Sciences (SPSS) vs. 17.

Numerical data were summarized using means and standard deviations. Categorical data were summarized as percentages. Comparisons between cases and controls with respect to numeric variables were done using the Student t-test. The chi-square test was used to compare between the groups with respect to categorical data. To measure the strength of the association between respondents and nonrespondents and all other parameters, the odds ratio and the 95% confidence intervals were calculated [13].

All p-values are two-sided. P-values < 0.05 were considered significant.

3. RESULTS:

Characteristics of Patients with Chronic HCV Infection Compared with Healthy Individuals. Comparison of the demographic and laboratory data of HCV patients and the control group are shown in Table 2. Analysis of Predictive Factors for INF Therapy Compared Between responders and Non-responders. The clinical characteristics of the SR and NR are compared in Table 3. The stage of fibrosis 0–2 was predictive of responder status to IFN (51.9 vs. 3.0 %, P = 0.001). A low level of viremia was also significantly associated with response to treatment (48.1 vs. 19.6 %; P = 0.005). No significant associations were found between response to treatment and gender. Comparison of the MxA promoter Allele and Genotypes Freuencies between HCV patients and control group are shown in Table 4. As regards to the distribution of the MxA gene at position (–88), 54 (43.2%) of HCV patients were homozygous (GG), 62 (49.6%) were heterozygous (GT) and 9 (7.2%) were homozygous (TT); as for the twenty control subjects, 10 (50%) were homozygous (GG), 9 (45%) were heterozygous (GT) and 1 (5%) was homozygous (TT). Moreover, at position (–123) of the MxA gene, 66 (52.9%) of the HCV patients were homozygous (CC), 52 (41.2%) were heterozygous (AC) and 7 (5.9%) were homozygous (AA). Among the control subjects, 14 (70%) were homozygous (CC), 6 (30%) were heterozygous (AC), while none (0%) were found to be homozygous (AA). No significant differences in the genotype frequencies could be demonstrated between the HCV patients and the control population for MxA gene. According to the present study, loss of HCV RNA was reported in 79 patients (responders) after completing six months of therapy. Table 5 shows the MxA allele and genotype distribution between SR and NR. The frequency of the homozygous G/G genotype was significantly higher in non-responders in comparison with responders (78.3% vs. 22.8%, p=0.001); while the heterozygous G/T genotype was highly significant in responders in relation to non-responders (68.4% vs. 17.4%, p=0.001). Furthermore, sustained responders had the mutant T allele more frequently than non-responders with a statistical significance (p=0.001). According to position –123, Inheritance of the heterozygous C/A genotype was significantly associated with responders to therapy (49.4% vs. 26.1%, p=0.014). Moreover, the presence of the A allele was significantly correlated with response to IFN-α treatment (p=0.003).

Table (2): Demographic and laboratory data of HCV patients and the control group (mean ± S.D.).

• Data were expressed as mean ± standard deviation or number (%). 
• Significant difference from control group using independent t test (**p< 0.01= highly significant.)
• (ALT) alanine aminotransferase, (AST) aspartate aminotransferase, (T.bilirubin) total bilirubin, (D. bilirubin) direct bilirubin, (ALP) alkaline phosphatase, (AFP) alpha-feto protein as well as (PT)  prothrombin time was significantly higher in HCV patients in comparison with control group, while the mean serum level of albumin was significantly lower.

Table (3): Factors associated with response to treatment with IFN-α.

• Data were expressed as mean ± standard deviation or number (%).
• P > 0.05= not significant; P < 0.01= highly significant.

Fig (3): Relation between gender and responder, non-responder.

Fig (4): Relation between stage of fibrosis and responder, non-responder.

Fig (5): Relation between viremia and responder, non-responder.

Table (4): Frequency of MxA promoter genotypes in HCV patients and control group.

• Data were expressed as number (%).

Fig (6): Relation between the distribution of the MxA gene at position (–88) and control group.

Fig (7): Relation between the distribution of the MxA gene at position (123) and control group.

Table (5): MxA promoter genotype and allele frequencies compared between interferon responders and non-responders.

• Data were expressed as mean ± standard deviation or number (%).
• P > 0.05= not significant.

4. DISCUSSION:

            The standard of care (SOC) for HCV infection consists in the combination of pegylated interferon (PEG-IFN) plus ribavirin. We studied the relationship between MxA promoter polymorphisms with response to IFN therapy in 125 Egyptian patients infected with HCV genotype 4. Our study showed that the sustained virological response (SVR) in patients with HCV genotype 4 reaches 65%. These response rates for genotype 4 are higher than those for genotype 1 (50%), but lower than those for genotypes 2 or 3 (80%). These findings are in agreement with those reported previously [14–15]. Furthermore, our results indicate that inheritance of particular MxA promoter genotypes has a significant role in determining the outcome of HCV infection to IFN.

            As regard gender in the present study, there was no significant statistical difference between males and females regarding end of treatment response.

The current study aims to investigate the association MxA polymorphisms at positions–88 and–123, with the response to IFN-α therapy in HCV-infected Egyptian patients; and also searched for potential interactions of different factors associated with response to treatment with IFN-α.

            According to the present study, the genotype and allele frequencies at both positions–88 and–123 of the MxA promoter were insignificant in HCV patients as compared to the control group. As regards to the MxA gene at position–88, the current results showed that the rate of G/G homozygosity was significantly lower in sustained responders to IFN-α therapy than in non-responders. Conversely, heterozygotes (G/T) were more likely to have sustained response with high significance. Inheritance of the T allele was significantly correlated with sustained responders than non-responders to IFN-α therapy. The results of the current study were similar to a Japanese study [16,17],where the evidence that patients with MxA–88 T/T or G/T genotype infection have better therapeutic effectiveness than those with G/G genotype when treated with IFN-α was justified.

It is quite possible that the SNP of the MxA promoter might influence the expression of MxA, and that carriers of MxA-T-positive might express the protein more efficiently when treated with IFN-a than those who are MxA-T-negative [8]. This polymorphic site (MxA promoter at nucleotide -88) is involved in a genetic element with high homology to the ISRE consensus sequence, and the G-to-T change at this position increases this similarity [18, 19]. This suggests that the SNP of the MxA promoter might affect the expression of MxA. Consequently, patients who possess the G/G genotype at position -88 may produce a suboptimal MxA response when given IFN-a [10]. The apparent effect of the T-allele has also been shown to play a role in regulation of a downstream reporter gene by a luciferase reporter assay, suggesting that this variant has higher transcriptional activity than the G allele when stimulated with IFN-a [18,20].

Regarding position–123, the frequency of the C/A genotype was significantly higher in the responder group of patients than in the non-responder group. Furthermore, the frequency of the A allele was significantly higher in the responder group of patients than in the non-responder group. The present work was in accordance with study performed by Hijikata et al. [16], which involved demonstration of the –123 SNP showed a strong association with treatment outcomes in patients with HCV genotypes 2a or 2b, where the frequency of the C/C homozygotes was significantly higher in non-responders than in sustained responders to IFN-α. Moreover, the relation between the MxA promoter polymorphism at positions–88 and–123 with the decrease in serum ALT after IFN-α therapy was investigated. The decrease in serum ALT was significantly associated with carriage of genotypes G/T or T/T at position–88; and with all the genotypes at position–123.

MxA protein expression is a sensentive biological marker for ongoing virus replication and presence of type 1 IFN. These results highlight the important value of MxA level at the start of therapy as a predictable factor for assessing the likelihood of HCV genotype 4 SVR to treatment with interferon α 2 in combination with ribavirin.

MxA gene as pretreatment virological factor is considering an important influence on the way patients respond to interferon IFN α therapy.

5. CONCLUSION:

It is well-known that there are many factors that affect a successful treatment outcome like polymorphism of the MxA gene. When trying to make a decision about the therapy, it is important to take many of these predictors of treatment response into consideration.  In summary, our observations imply that heterozygosity for both -88 and -123 polymorphisms of the MxA gene may be important determinants of sustained response to IFN therapy. However, these polymorphisms can explain only a small part of genetic susceptibility to response to IFN therapy. More studies and larger number of participants are needed to characterize the molecular mechanisms by which MxA are involved in IFN therapeutic effectiveness.

6. REFERENCE:

1- Polaris Observatory HCV Collaborators (2017). Global prevalence and genotype distribution of hepatitis C virus infection in 2015: a modelling study. Lancet Gastroenterol Hepatol; 2:161–176.

2- European Union HCV Collaborators (2017). Hepatitis C virus prevalence and level of intervention required to achieve the WHO targets for elimination in the European Union by 2030: a modelling study.Lancet Gastroenterol Hepatol; 2:325–336.

3- Eassa, S. ; M. Eissa ; S.M. Sharaf ; M.H. Ibrahim and O.M. Hassanein, et al. (2007). Prevalence of Hepatitis C Virus Infection and Evaluation of a Health Education Program in El-Ghar Village in Zagazig, Egypt.J Egypt Public Health Assoc; 82:379–404.

4- Alter, M. J., et al. (2007). Epidemiology of hepatitis C virus infection.World J Gastroenterol; 13:2436

5- Centers for Disease Control and Prevention (2011). Hepatitis C information for health professionals. Atlanta, GA: CDC.Available at: http://www.cdc.gov/hepatitis/HCV/index.htm (accessed 2013).

6- Centers for Disease Control and Prevention (2009). Morbidity and mortality weekly report. Atlanta, GA: CDC.Available at: http://www.cdc.gov/mmwr/ (accessed 2013).

7- Wohnsland, A. ; W.P. Hofmann and C. Sarrazin, et al. (2007) Viral determinations of resistance to patients with hepatitis C,Clin Microbiol Rev., 20(1): 23-38.

8- Asselah, T. ; E. Estrabaud ; I. Bieche ; M. Lapalus ; S. De Muynck and M. Vidaud (2010). Hepatitis C: viral and host factors associated with non-response to pegylated interferon plus ribavirin.Liver International, 30: 1259–1269.

9- Yeh, S.H. ; D.S. Chen and P.J. Chen, et al. (2003). A prospect for pharmacogenomics in the interferon therapy of chronic viral hepatitis.Journal of Antimicrobial Chemotherapy, 52: 149–151.

10-Knapp, S. ; L.J. Yee ; A.J. Frodsham ; B.J.W. Hennig ; S. Hellier and L. Zhang, et al. (2003). Polymorphisms in interferon-induced genes and the outcome of hepatitis C virus infection: Roles of MxA, OAS-1 and PKR. Genes and Immunity, 4: 411–419.

11- Suzuki, F. ; Y. Arase ; Y. Suzuki ; A. Tsubota ; N. Akuta and T. Hosaka, et al. (2004). Single nucleotide polymorphism of the MxA gene promoter influences the response to interferon monotherapy in patients with hepatitis C viral infection.Journal of Viral Hepatitis, 11(3): 271–276.

12- Sadler, A., & Williams, B., et al. (2008). Interferon-inducible antiviral effectors. Nature Reviews Immunology, 8, 559–568.

13- Dawson, B. and R.G. Trapp, et al. (2001). Basic & Clinical Biostatistics, Lange Medical Book/ McGraw-Hill, Medical Publishing Division. 3rd ed.,Ch., 7-9: 161-218.

14- Barnes, E. ; G. Webster ; S. Whalley and G. Dusheiko, et al. (1999). Predictors of a favorable response to alpha interferon therapy forhepatitis C. Clinics in Liver Disease, 3(4): 775–791.

15- Yee, L.J. ; J. Tang ; A.W. Gibson ; R. Kimberly ; D.J. Van Leeuwen and R.A. Kaslow, et al. (2001). Interleukin-10 polymorphisms aspredictors of sustained response in antiviral therapy for chronichepatitis C infection.Hepatology, 33: 708–712.

16- Hijikata, M. ; S. Mishiro ; C. Miyamoto ; Y. Furuichi ; M. Hashimoto and Y. Ohta, et al. (2001). Genetic polymorphism of the MxA gene promoter and interferon responsiveness of hepatitis C patients: Revisited by analyzing two SNP sites (-123 and -88) in vivo and in vitro.Intervirology, 44: 379–382.

17-Huang, Y.X. ; L.N. Ma ; X.Y. Chen ; Y.L. Huang ; C.L. Shen and B. Ma, et al. (2008). Genetic polymorphisms of MxA protein and eIF-2a-reg2 and their responses to interferon treatment. ZHGZ, 15: 187–191.

18- Hijikata, M. ; Y. Ohta  and S. Mishiro, et al. (2000). Identification of a single nucleotide polymorphism in the MxA gene promoter (G/T at nt -88) correlated with the response of hepatitis C patients to interferon.Intervirology, 43: 124–127.

19- Nakade, K. ; H. Honda and K. Nagata, et al. (1997). Promoter structure of the MxA gene that confers resistance to influenza virus. FEBS Letters, 418: 315–318.

20-Ferna´ndez-Arca´s, N. ; A. Blanco ; M.J. Gaita´n ; M. Nyqvist ; A. Alonso and A. Reyes-Engel, et al. (2004). Differential transcriptional expression of the polymorphic myxovirus resistance protein A in response to interferon alpha treatment.Pharmacogenetics, 14(3): 189–193.

العلامات الجزیئه التى تنبىء  بفاعلیه العلاج المعتمد على الانترفیرون الخاص بمرضى فیروس التهاب الکبد الوبائى سى

الفت جمیل شاکر⁽¹⁾ – جمال الدین عصمت⁽²⁾ – هبة الله محمد محمد⁽³⁾

1- قسم الکمیاء الطبیة والحیویة کلیة الطب جامعة القاهرة

2- قسم الامراض المتوطنة – کلیة الطب جامعة القاهرة

3- قسم الکمیاء الحیوى – کلیة العلوم جامعة القاهرة

من المعروف أن فیروس اللالتهاب الکبدی سی  یحث على إنتاج النوع الاول  من الانترفیرونات ومنتجات جین الانترفیرونات الذی تم استثارته شاملة بروتین MxA. لذلک یعتقد أن الانترفیرون المحدث یلعب دور مفتاحی مضاد فیروسی ضد فیروس الالتهاب الکبدی سی.

ولقد أجریت هذه الدراسة على 125 مریضا مصابین بالالتهاب الکبدی المزمن سی و20 فردا أصحاء و کبدهم سلیم (کمجموعة ضابطة). لقد تم اختیار المرضی  من عیادة أمراض الکبد بالقصرالعینی.

تعرض جمیع المرضى إلى:

1-      معرفة تاریخهم المرضی بالکامل.

2-      إجراء تحلیل صورة دم کاملة.

3-      إجراء تحلیل کیمیائی حیوی للکبد.

4-      تم علاج 125 من هؤلاء المرضى بـ PEG-IFN-α-2b، بجرعة 1.5 میکروجرام / کجم تحت الجلد کل أسبوع، بالإضافة إلى الریبافیرین بجرعة 1000-1200 مجم/یوم، طبقا لوزن جسم المریض لمدة 24 أسبوع.

5-      تم اشتمال عشرین فردا أصحاء فی هذا العمل کمجموعة ضابطة (کنترول). کان مستواهم الاجتماعی الاقتصادی مشابه لمرضى التهاب الکبد الوبائی سی.

6-      أجریت المعایرة بفیروس التهاب الکبد الوبائی باستخدام اختبار pcr لکل المرضى لتعیین الحمولة الفیروسیة.

7-      تم تعیین طرز الـ  MxAالجینیة (عند النقطتین -88 و -123) باستخدام تحلیل تفاعل إنزیم بلمرة السلسلة (pcr) ثم القطع بالانزیم.

أثناء العلاج:

• تم قیاس مستویات إنزیم أمینو ترانسفیریز، بروتین ألفا فیتو وPT کل 4 أسابیع.
• تم قیاس PCR کل 3 شهور أثناء العلاج.
• تم قیاس کمیة الفیروس کل 3-6 شهور أثناء العلاج.

تم إجراء المعایرة للـ HCV بواسطة الـ PCR قبل بدایة العلاج وبعد 3 أشهر من العلاج بالانترفیرون.

• تم تنفیذ تعیین التعدد الشکلی فی جین MxA باستخدام إنزیم بلمرة السلسلة.

تم الأخذ فی الإعتبار الجنس فی الدراسة الحالیة، لم یوجد اختلاف احصائی بین الذکور والإناث فیما یتعلق بنهایة الاستجابة للعلاج.

تهدف الدراسة الحالیة لبحث ارتباط تعدد شکل MxA فی المواقع -88 و-123، مع الاستجابة للعلاج بالانترفیرون ألفا فی المرضى المصریین المصابین بالـHCV؛ وأیضا البحث عن التفاعلات المحتملة للعوامل المختلفة المصاحبة للاستجابة للعلاج بالانترفیرون ألفا.

أوضحت هذه الدراسة أن البیانات المعملیة قبل العلاج شاملة متوسط مستویات المصل من إنزیمات الکبد (ALT، (AST، البیلیروبین (إجمالی، ومباشر)، کذلک الـ ALP، AFP وPT کانت أعلى بکثیر فی مجموعة مرضى الـHCV مقارنة بالمجموعة الضابطة، بینما متوسط مستوى المصل من الألبومین کان أقل.

طبقا للدراسة الحالیة، کانت تکرارات الطراز الجینی والألیلات فی کلا الموقعین -88 و -123 لبادئ الـ  MxAلایستهان بها فی مرضى الـHCV مقارنة بالمجموعة الضابطة.

بالنظر إلى جین MxA فی الموقع -88، أوضحت النتائج الحالیة أن معدل التجانس الزیجوتی G/G کان أقل بکثیر فی المستجیبین المعرضین للعلاج بالانترفیرون ألفا من غیر المستجیبین. وعلى العکس کانت الزیجوتات الغیر متجانسة (G/T) أکثر ترجیحا لأن یکون لها استجابة محتملة بمعنویة عالیة. ارتبطت وراثة الألیل T بالمستجیبین المحتملین أکثر من غیر المستجیبین للعلاج بالانترفیرون ألفا.

بالنظر إلى الموقع -123، کان تکرار الطراز الجینی C/A أعلى بکثیر فی المجموعة المستجیبة من المرضى منه فی المجموعة الغیر مستجیبة. علاوة على ذلک، کان تکرار الألیل A أعلى فی المجموعة المستجیبة من المرضى منه فی المجموعة الغیر مستجیبة.

فی الدراسة الحالیة، تم بحث العلاقة بین تعدد شکل بادئ الـ  MxAفی المواقع -88 و-123 مع انخفاض الـ ALT فی المصل بعد العلاج بالانترفیرون ألفا. وجد ارتباط کبیر بین انخفاض نزیم الکبد  ALTفی المصل وحمل الطرز الجینیة G/T أو T/T فی المواقع -88؛ ومع کل الطرز الجینیة فی الموقع -123.

یعتبر التعبیر عن بروتین الـ  MxAأحد الدلالات الحیویة الحساسة لتضاعف الفیروس المستمر ووجود النوع 1 من الانترفیرون. تسلط هذه النتائج الضوء على القیمة المهمة لمستوى الـ  MxAعند بدایة العلاج کعامل تنبؤی لتقدیر ترجیح الطراز الجینی الرابع للالتهاب الکبدی الوبائی سی للعلاج بالانترفیرون ألفا 2 فی اتحاد مع الریبافیرین.

یعتبر جین MxA عامل له تأثیر مهم قبل العلاج على الطریقة التی یستجیب بها المرضى للعلاج بالانترفیرون لذلک نستطیع أن نستنتج أن الجین MxA له قدرة تنبؤیة جیدة للاستجابة للعلاج بالانترفیرون ألفا.