Human platelet antigen-3 polymorphism as a risk factor for rheumatological manifestations in hepatitis C

Abstract INTRODUCTION: Hepatitis C virus (HCV) infection is involved in the pathogenesis of autoimmune and rheumatic disorders. Although the human platelet antigens (HPA) polymorphism are associated with HCV persistence, they have not been investigated in rheumatological manifestations (RM). This study focused on verifying associations between allele and genotype HPA and RM in patients with chronic hepatitis C. METHODS: Patients (159) with chronic hepatitis C of both genders were analyzed. RESULTS: Women showed association between HPA-3 polymorphisms and RM. CONCLUSIONS: An unprecedented strong association between rheumatological manifestations and HPA-3 polymorphism, possibly predisposing women to complications during the disease course, was observed.

Chronic hepatitis C virus (HCV) infection is a worldwide public health problem with a global prevalence of 2-3% 1 . In addition to being a frequent cause of chronic liver diseases such as hepatitis, cirrhosis, and hepatocellular carcinoma, it is also involved in the pathogenesis of various autoimmune and rheumatic disorders such as arthritis, vasculitis, sicca syndrome, porphyria cutanea tarda, lichen planus, nephropathies, thyroid diseases, lung fibrosis, among others 1,2,3 . Even though the rheumatic disorders are common among the extrahepatic manifestations, the mechanisms involved in the onset of these symptoms as well as the associated genetic factors are yet to be understood completely.
Host genetic factors such as the the human platelet antigens (HPA) polymorphism are also known to be associated with the infection and persistence of HCV 4,5 . However, there is no evidence of its relation to rheumatological manifestations.
HPAs result from the polymorphisms in the genes encoding surface glycoproteins of platelets, endothelial cells, and fibroblasts 6,7 , and are commonly involved in rheumatological diseases. Considering that the fibroblasts express HPA, these proteins could be involved in rheumatological manifestations. Thus, the aim of this study was to verify associations between allele and genotype HPA-1, -3, and -5 polymorphisms and rheumatological manifestations in patients with chronic hepatitis C.
A total of 159 individuals aged between 18 and 80 years, of both genders and affected by chronic hepatitis C, assisted at the Viral Hepatitis Outpatient Clinic of Botucatu Medical School, Unesp, Brazil, were included in this study. We only considered detectable HCV-RNA cases, with identification of HCV genotype, no previous hepatitis C treatment (naïve patients), with known fibrosis stage or clinical diagnosis of cirrhosis by image. Patients with HBV/HIV co-infection, chronic renal insufficiency, liver or renal transplantation, liver diseases, and other diffuse connective tissue diseases, including rheumatoid arthritis, according to Rheumatoid Arthritis Classification Criteria (ACR-EULAR 2010), were excluded 8 .
Clinical symptoms, such as presence of paresthesia sensations, Raynaud's phenomenon, cutaneous alterations, subcutaneous nodule, myalgia, muscle weakness, non-mechanical low back pain, arthralgia, arthritis, and other rheumatological manifestations were considered as rheumatological manifestations in this study. Laboratorial parameters evaluated were rheumatoid factor (qualitative and semi-quantitative) and anti-CCP (semi-quantitative) using Reumalatex kit (Labtest Diagnostica S/A, Lagoa Santa, MG, Deoxyribonucleic acid (DNA) was isolated from the total blood using the Wizard ® Minipreps DNA Purification System and used to genotype HPA-1 and HPA-3 with polymerase chain reaction-sequencespecific primers (PCR-SSP), as described by Klüter et al 9 . HPA-5 was genotyped using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), as described by Kalb et al 10 .
The association analysis between the categorical variables was performed using the χ 2 or Fisher's exact test. Student's t-test was used for comparing the mean ages. Logistic regression was used to categorize the risk of the association among the groups. Odds ratio values with 95% confidence interval were also calculated. P ≤ 0.05 was considered statistically significant.  1975, 1983, 1989, 1996, and 2000. All the participants of this study signed the individually informed consent forms.
Rheumatological manifestations were present in 72.3% of the patients. Table 1 shows the demographic and clinical characteristics related to rheumatic involvement. An association between female gender and development of rheumatological manifestations (P = 0.0201) was observed. This association was maintained when the data was subjected to multivariate logistic regression analysis (P = 0.0381). It is well-known that the rheumatic diseases are more prevalent in women, regardless of other concomitant clinical conditions 11 . The correlation between rheumatic manifestation and female gender was already observed in a research conducted with Egyptian population affected by chronic hepatitis C 12 . In addition, Cacoub et al 13 showed that more than 70% of the HCV-infected patients showed extrahepatic manifestations involving primarily joints, muscles, and skin, which as per our findings, were also associated to female gender.
In this context, it is noteworthy that HPA-1 and HPA-3 are located in the same glycoprotein complex (GPIIb-IIIa) expressed in both endothelial cells and fibroblasts 7 , which are the cells commonly involved in rheumatological diseases. However, additional studies involving other populations are necessary to confirm these data and to improve the understanding of the mechanisms involved in rheumatic manifestations in chronic HCV infection. Similar to the well-established association of human leukocyte antigens (HLA) and diseases, studies involving HPA may also contribute towards the identification of clinically important molecular markers, thereby aiding in understanding the pathophysiological mechanisms involved in the diseases.
Our study is the first report of a strong association between rheumatological manifestations and HPA-3 polymorphism, which may explain the possible predisposition of women to complications during the course of chronic hepatitis C.

ACKNOWLEDGMENTS
We would like to thank all the patients who participated in the study and all the employees of the institution, who in some way, collaborated with the execution of the research. Fisher's exact test; OR: odds ratio; CI: confidence interval; P ≤ 0.05 is considered a statistically significant relation. Fisher's exact test; OR: odds ratio; CI: confidence interval; P ≤ 0.05 is considered a statistically significant relation.