Original Article
309
Granzyme B Gene Polymorphism Associated with Subacute Sclerosing Panencephalitis Sibel P. Yentur1 Hatice Nur Aydin1 Candan Gurses2 Veysi Demirbilek3 Umit Kuru4 Serap Uysal5 Zuhal Yapici2 Safa Baris6 Gülden Yilmaz7 Ozlem Cokar8 Emel Onal9 Ayşen Gokyigit2 Güher Saruhan-Direskeneli1
University, Istanbul, Turkey 2 Department of Neurology, Istanbul Medical Faculty, Istanbul University, Istanbul, Turkey 3 Department of Neurology, Cerrahpasa Medical Faculty, Istanbul University, Istanbul, Turkey 4 Department of Pediatrics, Bayrampaşa Hospital, Istanbul, Turkey 5 Department of Pediatrics, Cerrahpasa Medical Faculty, Istanbul University, Istanbul, Turkey 6 Department of Pediatrics, Marmara School of Medicine, Istanbul, Turkey 7 Department of Microbiology, Istanbul Medical Faculty, Istanbul, Turkey 8 Department of Neurology, Haseki Hospital, Istanbul, Turkey 9 Department of Public Health, Istanbul Medical Faculty, Istanbul University, Istanbul, Turkey
Address for correspondence Dr. Güher Saruhan-Direskeneli, MD, Istanbul Medical Faculty, Istanbul University, Fizyoloji AD, 34093 Çapa, Istanbul, Turkey (e-mail: [email protected]).
Neuropediatrics 2014;45:309–313.
Abstract
Keywords
► subacute sclerosing panencephalitis ► granzyme B ► single nucleotide polymorphism ► function
Background Subacute sclerosing panencephalitis (SSPE) is a late complication of measles infection. Immune dysfunction related to genetic susceptibility has been considered in disease pathogenesis. A functional single nucleotide polymorphism (SNP) of granzyme B gene (GZMB) reported in several pathologies may also be involved in susceptibility to SSPE. Patients and Methods An SNP (rs8192917, G ! A, R!Q) was screened in 118 SSPE patients and 221 healthy controls (HC) by polymerase chain reaction-restriction fragment length polymorphism. Frequencies were compared between groups. In vitro production of GZMB was measured in controls with different genotypes. Results The SNP had a minor allele (G) frequency of 0.22 in patients and 0.31 in controls. GG genotype was significantly less frequent in patients (odds ratio, 0.23). G allele carriers produced relatively higher levels of GZMB, when stimulated in vitro. Conclusion These findings implicate possible effect of this genetic polymorphism in susceptibility to SSPE which needs to be confirmed in bigger populations.
Introduction Subacute sclerosing panencephalitis (SSPE) is a fatal progressive disease of the central nervous system (CNS) caused by a persistent measles virus (MV). The cause of persistence or of
received December 17, 2013 accepted after revision March 30, 2014 published online May 29, 2014
the late reactivation of MV have been attributed to transcriptional defects of MV or variants which had undergone hypermutation events.1,2 Propagation of mutated, assembly-defective MV has been suggested in SSPE brains.3 However, host factors can also be involved in persistence
© 2014 Georg Thieme Verlag KG Stuttgart · New York
DOI http://dx.doi.org/ 10.1055/s-0034-1378129. ISSN 0174-304X.
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1 Department of Physiology, Istanbul Medical Faculty, Istanbul
GZMB Polymorphism in SSPE
Yentur et al.
of MV in the CNS. The pathogenesis of SSPE is still not explained; however, both viral and host factors seem to be involved.4,5 Probable defects in host cell–mediated immune responses have also been considered in SSPE.6 Immune reactions toward MV are unique, as the infection induces both antiviral effector mechanisms as well as significant immunosuppression.7,8 Impairment of MV-specific cellmediated immunity has been observed in SSPE.9 Interleukin 12 (IL-12) production in response to MV was lower in SSPE patients than controls. Proliferation, as well as IFN-γ, IL-12, and IL-10 production of peripheral blood mononuclear cells (PBMC) in response to purified protein derivate of mycobacterium was impaired in SSPE patients.10 However, anti-MV antibodies are present in serum and cerebrospinal fluid (CSF). Th1 is generally downregulated and Th2 function is well preserved.11 On the contrary, genetic susceptibility to SSPE has been implicated in previous reports and associations with IL-4, interferon regulatory factor 1, MxA, IL-18, TLR3, as well as PD-1 gene polymorphisms are reported.12–17 The cytotoxic activity of T as well as NK cells is mediated partially by the serine protease, granzyme B (GZMB). On contact with a target cell, cytotoxic lymphocytes release perforin, granzymes, and death ligands from secretory granules. One way of death for target cells is the uptake of granzyme in a perforin dependent manner leading to caspase activation and intracellular proteolysis. When single nucleotide polymorphisms (SNP) of GZMB were screened, a common allele encoding a triple-mutated protein (R48A88H245, numbering with reference to the bovine chymotrypsinogen A sequence) has been identified. The “triple-mutated” variant protein of GZMB has shown to lose its proapoptotic capacity in vivo when compared with the wild type Q48P88Y245 form and the three polymorphisms (A/G, C/G, and T/C) are in strong linkage disequilibrium with each other.18 Identical proapoptotic activity of the variant protein has also been reported subsequently.19 The first SNP of this variant protein (rs8192917 R!Q at 55. amino acid, numbering with reference to the human chymotrypsinogen A sequence) has also been demonstrated to be functional.20 The presence of this variant of GZMB can also predict the outcome of unrelated bone marrow transplantation or graft survival in kidney transplantation.21,22 Recently, several SNPs of GZMB gene were screened in rheumatoid arthritis patients and another SNP was found to be associated with disease progression23 suggesting probable effect of the variant protein in several other diseases. On the contrary, the effect of GZMB is also implicated in the pathogenesis of MV infection. MV-infected monocytes are shown to induce apoptosis even in uninfected CD3þ T cells via activation of GZMB and caspase cascade.24 This finding provides evidence making GZMB a candidate gene for susceptibility to SSPE. Polymorphic variants of this gene may have potential effects which cause functional defects of T cells in the elimination of MV in SSPE patients. In this study, the nonsynonymous, amino acid changing GZMB exon 2 (rs8192917, G!A, R!Q) polymorphism is screened as susceptibility marker to SSPE comparing with HC. To provide a functional basis for the genetic association, Neuropediatrics
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GZMB production is measured in donors with different genotypes of this SNP.
Patients and Methods Patients and Controls A total of 118 SSPE patients were included in the study. The ages of SSPE patients ranged between 1 and 30 with a median of 8 years and 83 (70%) were boys. All of the SSPE patients fulfilled the diagnostic criteria as clinical features, anti-MV antibodies in CSF, and typical electroencephalography showing slow-wave complexes.25 Among 88 patients with known clinical staging, 83 patients had diseases at stage 2 and 5 patients were at stage 3.26 The median age at disease onset was 72 months. Totally 80 of 107 patients had natural measles occurring between the ages of 4 to 312 months (median, 12 months). Measles vaccination has been applied once to 61 of 81 patients and the measles and information on vaccination history was not accessible the other patients. All of the patients had antibodies against MV in the CSF and sera, and oligoclonal immunoglobulin G bands were present in the CSF of all patients. The HC were randomly selected from 65 healthy children between ages of 1 and 15 (median, 6 years) and 156 healthy adults (median age, 39 years). All parents and donors had given their informed consent to donate blood for the study. The Ethical Review Board of Istanbul Medical Faculty approved the study.
Genotyping GZMB exon 2 (rs8192917, G!A) nonsynonymous coding SNP (R!Q) was screened by polymerase chain reactionrestriction fragment length polymorphism (PCR-RFLP) method using the forward primer 5′-gaaattgaagccccttcctc-3′ and reverse primer 5′-agtgtttccaggagggtgtg-3′ selected by using a freely available software.27 Amplified PCR products of 331 base pair were digested by a restriction enzyme (Esp3 I, Fermentas Life Sciences) at 37°C with overnight incubation (http://www. restrictionmapper.org). The samples carrying G allele were cut into two fragments of 246-bp and 84-bp. Products were monitored by agarose gel electrophoresis. Allele and genotype frequencies were determined by counting.
Granzyme Production PBMC were freshly isolated from venous blood of HC with density gradient centrifugation (Ficoll-Histopaque). CD4þ T cells were isolated by magnetic bead separation (MACS, Miltenyi Biotec, Bergisch Gladbach, Germany). Cells were seeded on plates coated with anti-CD3 (10 γg/mL, Diaclone, France) and anti-CD28 (5 µg/mL, Ancell, US) with or without anti-CD46 (5 µg/mL, kind gift from C. Kemper) or with antiIgG1 isotype control (5 µg/mL, Diaclone, France) at 2 105 cells/well and incubated at 37°C in humidified air with 5% CO2.28 After 72 hours, culture supernatants were collected and kept at –80°C until used. GZMB levels in supernatants were detected by enzyme-linked immunosorbent assay (ELISA) (Diaclone, France) according to manufacturer’s instructions and absorbance values measured in the supernatants are presented.
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310
GZMB Polymorphism in SSPE Statistical Analysis
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Results The distribution of the GZMB (rs8192917, G!A) alleles in HC was in Hardy–Weinberg equilibrium. The screened SNP of GZMB was highly polymorphic with a minor allele frequency of 0.22 in patients and 0.31 in controls. The distribution of the GZMB genotypes showed a significant difference between SSPE and control groups. The homozygous polymorphic genotype (GG, the absence of wild type A allele) was significantly less frequent in SSPE patients than in HC (►Table 1) conferring a resistance for susceptibility to SSPE (p ¼ 0.023, odds ratio [OR], 0.26, 95% confidence interval [CI], 0.07–0.9). In SSPE group, the AA, AG, and GG genotypes were detected in 57.6, 39.8, and 2.5% of the patients. In the HC group, including healthy children and adults, AA, AG, and GG genotypes were found as 47.1, 43.9, and 9.0%, respectively. To assign a functional outcome to this association, we have measured GZMB production of healthy donors with different GZMB rs8192917 genotypes in response to T cell stimulation. Only one donor with GG genotype, but six with AG, and eight donors with AA genotypes were included. When isolated CD4þ T cells of these 15 HC are stimulated with anti-CD3 and anti-CD28 antibodies, GZMB secretion levels were higher in G carriers (R amino acid residue) than noncarriers (Gþ [GG þ AG] vs. G [AA]: 1.67 vs. 0.8, not significant). Although this difference was also observed with the addition of anti-CD46 stimulation (Gþ [GG þ AG] vs. G- [AA]: 1.46 vs. 0.3), it did not reach the statistical significance level in the small number of donors included in this part of the study.
SSPE is considered as a rare, probably preventable disease and has mostly disappeared in countries with effective vaccination programs. Although the vaccination was not effectively applied until recently, it has not a very high incidence in Turkey either29 implicating other factors’ contribution to susceptibility to disease. Considering ethnical differences, we have compared a genetic polymorphism that may have a potential role in antiviral immune response in a matched control population with SSPE patients. As a susceptibility gene, we have investigated GZMB, which was reported to be polymorphic with possible functional effects. We provide evidence for an association of a GZMB polymorphism with SSPE in Turkey that may have predisposing effect in the production of this cytotoxic enzyme and influence combating the virus in the patients. An association between GZMB and SSPE is demonstrated suggesting that the polymorphism of GZMB may be a host factor that contributes to a predisposition to SSPE. Cytotoxic T or NK cell-mediated apoptosis of virus-infected cells represents a central mechanism of immune system control of viral pathogenesis. MV gains access to the CNS at the primary infection according to the findings of MV genome in CSF samples of acute measles patients.30 In eliminating MV during CNS infection, T cell response plays a dominant role. Individuals with A allele might be more susceptible to MV persistence in the CNS because the proapoptotic activity of GZMB gene is not as high as the healthy population which may help the persistence of the infected cells in CNS. As viral persistence is essential in the pathogenesis of SSPE, it is likely that this GZMB polymorphism confers host genetic susceptibility to SSPE. Recent evidence suggested that GZMB may also directly target mediators of viral replication and inhibit viral spread without causing apoptosis of infected cells.24 An alteration in this function may also contribute to the inefficient immune response in the SSPE. The triple-mutated variant of GZMB protein encoded by a common allele was shown to be unable to induce apoptosis in vivo, although the enzymatic activity of the variant form
Table 1 Genotype and allele frequencies of GZMB rs8192917, R!Q polymorphism in SSPE patients and HC SSPE
p
HC
118
%
221
%
AA
68
57.6
104
47.1
AG
47
39.8
97
43.9
GG
3
2.5
20
9
OR
95% CI
1.53
(0.97–2.4)
0.26
(0.07–0.9)
1.55
(1.1–2.2)
0.64
(0.4–0.9)
Genotype
A G
Af 183 53
0.775 0.224
0.023a
Af 305 137
0.69 0.31
0.019
b
Abbreviations: Af, allele frequency; CI, confidence interval; HC, healthy controls; OR, odds ratio; SSPE, subacute sclerosing panencephalitis. a p value refers to the comparison of GG versus AG þ AA genotypes. b p value refers to the comparison of A allele frequency differences. Neuropediatrics
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Discussion
The distribution of the polymorphism was tested in the control population to confirm Hardy–Weinberg equilibrium. Allele and genotype frequencies were compared between patient and HC groups by chi-square and Fisher exact tests, as appropriate. Cytokine secretion was analyzed with Mann– Whitney U-test. The results are presented as median of absorbance values. p values less than 0.05 were considered statistically significant.
Allele
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was intact.18 Using a similar approach, identical activity of both variants of GZMB is also demonstrated in vitro and claimed that polymorphic allele represents a neutral polymorphism. The frequency of polymorphic GZMB allele was reported to be 25 to 30% in another population,19 which is replicated in this population with a frequency of 31%. As in both previous studies, the three polymorphisms were in strong linkage disequilibrium, we have investigated one of the SNPs (R!Q) that could be related to functional alterations of T cells in SSPE. The relatively high frequency of this SNP in this studied group provided an informative polymorphic marker for this gene. In the SSPE patient group, the polymorphic homozygous GZMB genotype (GG or R only) was significantly less frequent compared with HC (OR: 0.26). Although the study group is relatively small for genetic studies, we still could demonstrate an association, which needs further replication to be considered causative in SSPE. We consider this study on a rare disease as a screening study and interpret our results with caution. MV infection-induced activation of GZMB in PBMC which is postulated to be one important contact-dependent mechanism used by cytotoxic T cells to eliminate virus-infected cells.31 Alterations in GZMB activity could impair cytotoxic T cell-mediated mechanisms rendering the host more susceptible to virus persistence. A previous study has shown that after mitogen stimulation, in vitro expression of GZMB was significantly increased in the GG carriers compared with AA carriers.20 A similar tendency of higher production due to the same polymorphism is implicated by the presented data, although the numbers are too small to be convincing. Considering the multitude of the factors influencing the cytotoxic response in patients, this finding is only supportive for the association. Although the association of GZMB polymorphism with SSPE is not very strong, the absence of an association with another disease in the same population is implicating its functional and specific nature in SSPE.32 Our results indicate that GZMB polymorphism is associated with SSPE in Turkey and this association can have contributed to lower responses of cytotoxic cells to the MV causing ineffective elimination and persistence of it. Future studies on genetics in SSPE may facilitate the understanding of the disease development in some of the children without effective immunization against MV.
2 Breschkin AM, Morgan EM, McKimm J, Rapp F. SSPE-BIKEN: a
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8 9
10
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12
13
14
15
16
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Acknowledgment The study is supported by Istanbul University Research Fund.
20
21
Disclosure No competing financial interests exist. 22
References 1 Baczko K, Lampe J, Liebert UG, et al. Clonal expansion of hyper-
mutated measles virus in a SSPE brain. Virology 1993;197(1): 188–195
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naturally arising hemagglutination-defective mutant of measles virus. J Med Virol 1979;4(1):67–80 Cattaneo R, Schmid A, Spielhofer P, et al. Mutated and hypermutated genes of persistent measles viruses which caused lethal human brain diseases. Virology 1989;173(2):415–425 Dyken PR. Neuroprogressive disease of post-infectious origin: a review of a resurging subacute sclerosing panencephalitis (SSPE). Ment Retard Dev Disabil Res Rev 2001;7(3):217–225 Graves MC. Subacute sclerosing panencephalitis. Neurol Clin 1984;2(2):267–280 Schneider-Schaulies J, Meulen Vt, Schneider-Schaulies S. Measles infection of the central nervous system. J Neurovirol 2003;9(2): 247–252 Schneider-Schaulies J, ter Meulen V, Schneider-Schaulies S. Measles virus interactions with cellular receptors: consequences for viral pathogenesis. J Neurovirol 2001;7(5):391–399 van Els CA, Nanan R. T cell responses in acute measles. Viral Immunol 2002;15(3):435–450 Dhib-Jalbut S, Jacobson S, McFarlin DE, McFarland HF. Impaired measles-specific cytotoxic T-cell response in subacute sclerosing panencephalitis. Ann N Y Acad Sci 1988;540:645–648 Yentür SP, Gürses C, Demirbilek V, et al. Alterations in cellmediated immune response in subacute sclerosing panencephalitis. J Neuroimmunol 2005;170(1-2):179–185 Hara T, Yamashita S, Aiba H, et al. Measles virus-specific T helper 1/T helper 2-cytokine production in subacute sclerosing panencephalitis. J Neurovirol 2000;6(2):121–126 Inoue T, Kira R, Nakao F, et al. Contribution of the interleukin 4 gene to susceptibility to subacute sclerosing panencephalitis. Arch Neurol 2002;59(5):822–827 Torisu H, Kusuhara K, Kira R, et al. Functional MxA promoter polymorphism associated with subacute sclerosing panencephalitis. Neurology 2004;62(3):457–460 Ishizaki Y, Takemoto M, Kira R, et al. Association of toll-like receptor 3 gene polymorphism with subacute sclerosing panencephalitis. J Neurovirol 2008;14(6):486–491 Ishizaki Y, Yukaya N, Kusuhara K, et al. PD1 as a common candidate susceptibility gene of subacute sclerosing panencephalitis. Hum Genet 2010;127(4):411–419 Piskin IE, Calık M, Abuhandan M, Kolsal E, Celik SK, Iscan A. PD-1 gene polymorphism in children with subacute sclerosing panencephalitis. Neuropediatrics 2013;44(4):187–190 Piskin IE, Karakas-Celik S, Calik M, et al. Association of interleukin 18, interleukin 2, and tumor necrosis factor polymorphisms with subacute sclerosing panencephalitis. DNA Cell Biol 2013;32(6): 336–340 McIlroy D, Cartron PF, Tuffery P, et al. A triple-mutated allele of granzyme B incapable of inducing apoptosis. Proc Natl Acad Sci U S A 2003;100(5):2562–2567 Sun J, Bird CH, Thia KY, Matthews AY, Trapani JA, Bird PI. Granzyme B encoded by the commonly occurring human RAH allele retains pro-apoptotic activity. J Biol Chem 2004;279(17):16907–16911 Girnita DM, Webber SA, Brooks MM, et al. Genotypic variation and phenotypic characterization of granzyme B gene polymorphisms. Transplantation 2009;87(12):1801–1806 Corrales-Tellez E, Vu D, Shah T, Hutchinson I, Min DI. Association between granzyme B and perforin I polymorphisms and allograft outcomes in Hispanic kidney transplant recipients. Clin Transplant 2013;27(3):E308–E315 Espinoza LJ, Takami A, Nakata K, et al; Japan Marrow Donor Program. Genetic variants of human granzyme B predict transplant outcomes after HLA matched unrelated bone marrow transplantation for myeloid malignancies. PLoS ONE 2011;6(8):e23827 Knevel R, Krabben A, Wilson AG, et al. A genetic variant in granzyme B is associated with progression of joint destruction in rheumatoid arthritis. Arthritis Rheum 2013;65(3):582–589
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in uninfected bystander T cells and leads to granzyme B and caspase activation in peripheral blood mononuclear cell cultures. Eur J Clin Invest 2003;33(5):434–442 Gascon GG. Subacute sclerosing panencephalitis. Semin Pediatr Neurol 1996;3(4):260–269 Oztürk A, Gürses C, Baykan B, Gökyigit A, Eraksoy M. Subacute sclerosing panencephalitis: clinical and magnetic resonance imaging evaluation of 36 patients. J Child Neurol 2002;17(1): 25–29 Untergasser A, Cutcutache I, Koressaar T, et al. Primer3— new capabilities and interfaces. Nucleic Acids Res 2012; 40(15):e115 Kemper C, Chan AC, Green JM, Brett KA, Murphy KM, Atkinson JP. Activation of human CD4þ cells with CD3 and CD46
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induces a T-regulatory cell 1 phenotype. Nature 2003; 421(6921):388–392 Onal AE, Gurses C, Direskeneli GS, et al. Subacute sclerosing panencephalitis surveillance study in Istanbul. Brain Dev 2006; 28(3):183–189 Nakayama T, Mori T, Yamaguchi S, et al. Detection of measles virus genome directly from clinical samples by reverse transcriptasepolymerase chain reaction and genetic variability. Virus Res 1995; 35(1):1–16 Trapani JA, Sutton VR, Smyth MJ. CTL granules: evolution of vesicles essential for combating virus infections. Immunol Today 1999;20(8):351–356 Kucuksezer UC, Duymaz-Tozkir J, Gül A, Saruhan-Direskeneli G. No association of granzyme B gene polymorphism with Behçet’s disease. Clin Exp Rheumatol 2009;27(2, Suppl 53):S102
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24 Vuorinen T, Peri P, Vainionpää R. Measles virus induces apoptosis
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309
Granzyme B Gene Polymorphism Associated with Subacute Sclerosing Panencephalitis Sibel P. Yentur1 Hatice Nur Aydin1 Candan Gurses2 Veysi Demirbilek3 Umit Kuru4 Serap Uysal5 Zuhal Yapici2 Safa Baris6 Gülden Yilmaz7 Ozlem Cokar8 Emel Onal9 Ayşen Gokyigit2 Güher Saruhan-Direskeneli1
University, Istanbul, Turkey 2 Department of Neurology, Istanbul Medical Faculty, Istanbul University, Istanbul, Turkey 3 Department of Neurology, Cerrahpasa Medical Faculty, Istanbul University, Istanbul, Turkey 4 Department of Pediatrics, Bayrampaşa Hospital, Istanbul, Turkey 5 Department of Pediatrics, Cerrahpasa Medical Faculty, Istanbul University, Istanbul, Turkey 6 Department of Pediatrics, Marmara School of Medicine, Istanbul, Turkey 7 Department of Microbiology, Istanbul Medical Faculty, Istanbul, Turkey 8 Department of Neurology, Haseki Hospital, Istanbul, Turkey 9 Department of Public Health, Istanbul Medical Faculty, Istanbul University, Istanbul, Turkey
Address for correspondence Dr. Güher Saruhan-Direskeneli, MD, Istanbul Medical Faculty, Istanbul University, Fizyoloji AD, 34093 Çapa, Istanbul, Turkey (e-mail: [email protected]).
Neuropediatrics 2014;45:309–313.
Abstract
Keywords
► subacute sclerosing panencephalitis ► granzyme B ► single nucleotide polymorphism ► function
Background Subacute sclerosing panencephalitis (SSPE) is a late complication of measles infection. Immune dysfunction related to genetic susceptibility has been considered in disease pathogenesis. A functional single nucleotide polymorphism (SNP) of granzyme B gene (GZMB) reported in several pathologies may also be involved in susceptibility to SSPE. Patients and Methods An SNP (rs8192917, G ! A, R!Q) was screened in 118 SSPE patients and 221 healthy controls (HC) by polymerase chain reaction-restriction fragment length polymorphism. Frequencies were compared between groups. In vitro production of GZMB was measured in controls with different genotypes. Results The SNP had a minor allele (G) frequency of 0.22 in patients and 0.31 in controls. GG genotype was significantly less frequent in patients (odds ratio, 0.23). G allele carriers produced relatively higher levels of GZMB, when stimulated in vitro. Conclusion These findings implicate possible effect of this genetic polymorphism in susceptibility to SSPE which needs to be confirmed in bigger populations.
Introduction Subacute sclerosing panencephalitis (SSPE) is a fatal progressive disease of the central nervous system (CNS) caused by a persistent measles virus (MV). The cause of persistence or of
received December 17, 2013 accepted after revision March 30, 2014 published online May 29, 2014
the late reactivation of MV have been attributed to transcriptional defects of MV or variants which had undergone hypermutation events.1,2 Propagation of mutated, assembly-defective MV has been suggested in SSPE brains.3 However, host factors can also be involved in persistence
© 2014 Georg Thieme Verlag KG Stuttgart · New York
DOI http://dx.doi.org/ 10.1055/s-0034-1378129. ISSN 0174-304X.
This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.
1 Department of Physiology, Istanbul Medical Faculty, Istanbul
GZMB Polymorphism in SSPE
Yentur et al.
of MV in the CNS. The pathogenesis of SSPE is still not explained; however, both viral and host factors seem to be involved.4,5 Probable defects in host cell–mediated immune responses have also been considered in SSPE.6 Immune reactions toward MV are unique, as the infection induces both antiviral effector mechanisms as well as significant immunosuppression.7,8 Impairment of MV-specific cellmediated immunity has been observed in SSPE.9 Interleukin 12 (IL-12) production in response to MV was lower in SSPE patients than controls. Proliferation, as well as IFN-γ, IL-12, and IL-10 production of peripheral blood mononuclear cells (PBMC) in response to purified protein derivate of mycobacterium was impaired in SSPE patients.10 However, anti-MV antibodies are present in serum and cerebrospinal fluid (CSF). Th1 is generally downregulated and Th2 function is well preserved.11 On the contrary, genetic susceptibility to SSPE has been implicated in previous reports and associations with IL-4, interferon regulatory factor 1, MxA, IL-18, TLR3, as well as PD-1 gene polymorphisms are reported.12–17 The cytotoxic activity of T as well as NK cells is mediated partially by the serine protease, granzyme B (GZMB). On contact with a target cell, cytotoxic lymphocytes release perforin, granzymes, and death ligands from secretory granules. One way of death for target cells is the uptake of granzyme in a perforin dependent manner leading to caspase activation and intracellular proteolysis. When single nucleotide polymorphisms (SNP) of GZMB were screened, a common allele encoding a triple-mutated protein (R48A88H245, numbering with reference to the bovine chymotrypsinogen A sequence) has been identified. The “triple-mutated” variant protein of GZMB has shown to lose its proapoptotic capacity in vivo when compared with the wild type Q48P88Y245 form and the three polymorphisms (A/G, C/G, and T/C) are in strong linkage disequilibrium with each other.18 Identical proapoptotic activity of the variant protein has also been reported subsequently.19 The first SNP of this variant protein (rs8192917 R!Q at 55. amino acid, numbering with reference to the human chymotrypsinogen A sequence) has also been demonstrated to be functional.20 The presence of this variant of GZMB can also predict the outcome of unrelated bone marrow transplantation or graft survival in kidney transplantation.21,22 Recently, several SNPs of GZMB gene were screened in rheumatoid arthritis patients and another SNP was found to be associated with disease progression23 suggesting probable effect of the variant protein in several other diseases. On the contrary, the effect of GZMB is also implicated in the pathogenesis of MV infection. MV-infected monocytes are shown to induce apoptosis even in uninfected CD3þ T cells via activation of GZMB and caspase cascade.24 This finding provides evidence making GZMB a candidate gene for susceptibility to SSPE. Polymorphic variants of this gene may have potential effects which cause functional defects of T cells in the elimination of MV in SSPE patients. In this study, the nonsynonymous, amino acid changing GZMB exon 2 (rs8192917, G!A, R!Q) polymorphism is screened as susceptibility marker to SSPE comparing with HC. To provide a functional basis for the genetic association, Neuropediatrics
Vol. 45
No. 5/2014
GZMB production is measured in donors with different genotypes of this SNP.
Patients and Methods Patients and Controls A total of 118 SSPE patients were included in the study. The ages of SSPE patients ranged between 1 and 30 with a median of 8 years and 83 (70%) were boys. All of the SSPE patients fulfilled the diagnostic criteria as clinical features, anti-MV antibodies in CSF, and typical electroencephalography showing slow-wave complexes.25 Among 88 patients with known clinical staging, 83 patients had diseases at stage 2 and 5 patients were at stage 3.26 The median age at disease onset was 72 months. Totally 80 of 107 patients had natural measles occurring between the ages of 4 to 312 months (median, 12 months). Measles vaccination has been applied once to 61 of 81 patients and the measles and information on vaccination history was not accessible the other patients. All of the patients had antibodies against MV in the CSF and sera, and oligoclonal immunoglobulin G bands were present in the CSF of all patients. The HC were randomly selected from 65 healthy children between ages of 1 and 15 (median, 6 years) and 156 healthy adults (median age, 39 years). All parents and donors had given their informed consent to donate blood for the study. The Ethical Review Board of Istanbul Medical Faculty approved the study.
Genotyping GZMB exon 2 (rs8192917, G!A) nonsynonymous coding SNP (R!Q) was screened by polymerase chain reactionrestriction fragment length polymorphism (PCR-RFLP) method using the forward primer 5′-gaaattgaagccccttcctc-3′ and reverse primer 5′-agtgtttccaggagggtgtg-3′ selected by using a freely available software.27 Amplified PCR products of 331 base pair were digested by a restriction enzyme (Esp3 I, Fermentas Life Sciences) at 37°C with overnight incubation (http://www. restrictionmapper.org). The samples carrying G allele were cut into two fragments of 246-bp and 84-bp. Products were monitored by agarose gel electrophoresis. Allele and genotype frequencies were determined by counting.
Granzyme Production PBMC were freshly isolated from venous blood of HC with density gradient centrifugation (Ficoll-Histopaque). CD4þ T cells were isolated by magnetic bead separation (MACS, Miltenyi Biotec, Bergisch Gladbach, Germany). Cells were seeded on plates coated with anti-CD3 (10 γg/mL, Diaclone, France) and anti-CD28 (5 µg/mL, Ancell, US) with or without anti-CD46 (5 µg/mL, kind gift from C. Kemper) or with antiIgG1 isotype control (5 µg/mL, Diaclone, France) at 2 105 cells/well and incubated at 37°C in humidified air with 5% CO2.28 After 72 hours, culture supernatants were collected and kept at –80°C until used. GZMB levels in supernatants were detected by enzyme-linked immunosorbent assay (ELISA) (Diaclone, France) according to manufacturer’s instructions and absorbance values measured in the supernatants are presented.
This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.
310
GZMB Polymorphism in SSPE Statistical Analysis
Yentur et al.
Results The distribution of the GZMB (rs8192917, G!A) alleles in HC was in Hardy–Weinberg equilibrium. The screened SNP of GZMB was highly polymorphic with a minor allele frequency of 0.22 in patients and 0.31 in controls. The distribution of the GZMB genotypes showed a significant difference between SSPE and control groups. The homozygous polymorphic genotype (GG, the absence of wild type A allele) was significantly less frequent in SSPE patients than in HC (►Table 1) conferring a resistance for susceptibility to SSPE (p ¼ 0.023, odds ratio [OR], 0.26, 95% confidence interval [CI], 0.07–0.9). In SSPE group, the AA, AG, and GG genotypes were detected in 57.6, 39.8, and 2.5% of the patients. In the HC group, including healthy children and adults, AA, AG, and GG genotypes were found as 47.1, 43.9, and 9.0%, respectively. To assign a functional outcome to this association, we have measured GZMB production of healthy donors with different GZMB rs8192917 genotypes in response to T cell stimulation. Only one donor with GG genotype, but six with AG, and eight donors with AA genotypes were included. When isolated CD4þ T cells of these 15 HC are stimulated with anti-CD3 and anti-CD28 antibodies, GZMB secretion levels were higher in G carriers (R amino acid residue) than noncarriers (Gþ [GG þ AG] vs. G [AA]: 1.67 vs. 0.8, not significant). Although this difference was also observed with the addition of anti-CD46 stimulation (Gþ [GG þ AG] vs. G- [AA]: 1.46 vs. 0.3), it did not reach the statistical significance level in the small number of donors included in this part of the study.
SSPE is considered as a rare, probably preventable disease and has mostly disappeared in countries with effective vaccination programs. Although the vaccination was not effectively applied until recently, it has not a very high incidence in Turkey either29 implicating other factors’ contribution to susceptibility to disease. Considering ethnical differences, we have compared a genetic polymorphism that may have a potential role in antiviral immune response in a matched control population with SSPE patients. As a susceptibility gene, we have investigated GZMB, which was reported to be polymorphic with possible functional effects. We provide evidence for an association of a GZMB polymorphism with SSPE in Turkey that may have predisposing effect in the production of this cytotoxic enzyme and influence combating the virus in the patients. An association between GZMB and SSPE is demonstrated suggesting that the polymorphism of GZMB may be a host factor that contributes to a predisposition to SSPE. Cytotoxic T or NK cell-mediated apoptosis of virus-infected cells represents a central mechanism of immune system control of viral pathogenesis. MV gains access to the CNS at the primary infection according to the findings of MV genome in CSF samples of acute measles patients.30 In eliminating MV during CNS infection, T cell response plays a dominant role. Individuals with A allele might be more susceptible to MV persistence in the CNS because the proapoptotic activity of GZMB gene is not as high as the healthy population which may help the persistence of the infected cells in CNS. As viral persistence is essential in the pathogenesis of SSPE, it is likely that this GZMB polymorphism confers host genetic susceptibility to SSPE. Recent evidence suggested that GZMB may also directly target mediators of viral replication and inhibit viral spread without causing apoptosis of infected cells.24 An alteration in this function may also contribute to the inefficient immune response in the SSPE. The triple-mutated variant of GZMB protein encoded by a common allele was shown to be unable to induce apoptosis in vivo, although the enzymatic activity of the variant form
Table 1 Genotype and allele frequencies of GZMB rs8192917, R!Q polymorphism in SSPE patients and HC SSPE
p
HC
118
%
221
%
AA
68
57.6
104
47.1
AG
47
39.8
97
43.9
GG
3
2.5
20
9
OR
95% CI
1.53
(0.97–2.4)
0.26
(0.07–0.9)
1.55
(1.1–2.2)
0.64
(0.4–0.9)
Genotype
A G
Af 183 53
0.775 0.224
0.023a
Af 305 137
0.69 0.31
0.019
b
Abbreviations: Af, allele frequency; CI, confidence interval; HC, healthy controls; OR, odds ratio; SSPE, subacute sclerosing panencephalitis. a p value refers to the comparison of GG versus AG þ AA genotypes. b p value refers to the comparison of A allele frequency differences. Neuropediatrics
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No. 5/2014
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Discussion
The distribution of the polymorphism was tested in the control population to confirm Hardy–Weinberg equilibrium. Allele and genotype frequencies were compared between patient and HC groups by chi-square and Fisher exact tests, as appropriate. Cytokine secretion was analyzed with Mann– Whitney U-test. The results are presented as median of absorbance values. p values less than 0.05 were considered statistically significant.
Allele
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was intact.18 Using a similar approach, identical activity of both variants of GZMB is also demonstrated in vitro and claimed that polymorphic allele represents a neutral polymorphism. The frequency of polymorphic GZMB allele was reported to be 25 to 30% in another population,19 which is replicated in this population with a frequency of 31%. As in both previous studies, the three polymorphisms were in strong linkage disequilibrium, we have investigated one of the SNPs (R!Q) that could be related to functional alterations of T cells in SSPE. The relatively high frequency of this SNP in this studied group provided an informative polymorphic marker for this gene. In the SSPE patient group, the polymorphic homozygous GZMB genotype (GG or R only) was significantly less frequent compared with HC (OR: 0.26). Although the study group is relatively small for genetic studies, we still could demonstrate an association, which needs further replication to be considered causative in SSPE. We consider this study on a rare disease as a screening study and interpret our results with caution. MV infection-induced activation of GZMB in PBMC which is postulated to be one important contact-dependent mechanism used by cytotoxic T cells to eliminate virus-infected cells.31 Alterations in GZMB activity could impair cytotoxic T cell-mediated mechanisms rendering the host more susceptible to virus persistence. A previous study has shown that after mitogen stimulation, in vitro expression of GZMB was significantly increased in the GG carriers compared with AA carriers.20 A similar tendency of higher production due to the same polymorphism is implicated by the presented data, although the numbers are too small to be convincing. Considering the multitude of the factors influencing the cytotoxic response in patients, this finding is only supportive for the association. Although the association of GZMB polymorphism with SSPE is not very strong, the absence of an association with another disease in the same population is implicating its functional and specific nature in SSPE.32 Our results indicate that GZMB polymorphism is associated with SSPE in Turkey and this association can have contributed to lower responses of cytotoxic cells to the MV causing ineffective elimination and persistence of it. Future studies on genetics in SSPE may facilitate the understanding of the disease development in some of the children without effective immunization against MV.
2 Breschkin AM, Morgan EM, McKimm J, Rapp F. SSPE-BIKEN: a
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Acknowledgment The study is supported by Istanbul University Research Fund.
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Disclosure No competing financial interests exist. 22
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24 Vuorinen T, Peri P, Vainionpää R. Measles virus induces apoptosis
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