Inflamm. Res. (2014) 63:799–801 DOI 10.1007/s00011-014-0754-0
Inflammation Research
SHORT COMMUNICATION
Sorcin antibody as a possible predictive factor in conversion from radiologically isolated syndrome to multiple sclerosis: a preliminary study Elc¸in S¸ ehitog˘lu • Filiz C ¸ avus¸ • Canan Ulusoy • Melike Ku¨c¸u¨kerden ¨ • Arda Orc¸en Deniz Akbas¸ -Demir • Arzu C ¸ oban • Burc¸ak Vural • • Erdem Tu¨zu¨n Recai Tu¨rkog˘lu
•
Received: 5 May 2014 / Revised: 13 June 2014 / Accepted: 18 June 2014 / Published online: 8 July 2014 Ó Springer Basel 2014
Abstract Objective To identify an antibody biomarker for prediction of conversion from radiologically isolated syndrome (RIS) to relapsing remitting multiple sclerosis (RRMS). Methods Sera of 13 RIS patients were screened by a protein macroarray derived from human fetal brain cDNA library. Results Sequencing of a clone with the highest signal intensity revealed sorcin as a potential target autoantigen in RIS patients. ELISA studies showed high-titer sorcin-antibodies in 3 of 4 RIS patients who converted to RRMS in a 5-year follow-up period and 13 of 23 control RRMS patients. Conclusion The value of sorcin antibody as a predictor of conversion from RIS to RRMS requires to be tested in larger prospective studies. Keywords Anti-neuronal antibody Autoantibody Multiple sclerosis Radiologically isolated syndrome Protein macroarray
Introduction The increasing use of magnetic resonance imaging (MRI) in diagnosis of neurological disorders has led to the detection of asymptomatic brain lesions, including those that are highly suggestive of multiple sclerosis (MS). These lesions might occur in the absence of any MS symptoms and signs, a condition named as radiologically isolated syndrome (RIS) [1, 2]. The 5-year conversion rate to the first clinical event in RIS is around 30 % [2], prompting characterization of laboratory methods for accurate selection of patients for early treatment. Improved understanding of the role of antibodies in MS pathogenesis [3] has brought forward identification of anti-neuronal antibodies that can serve as prognostic biomarkers. To identify a biomarker that would predict progression from RIS to MS, sera of RIS patients were screened with protein macroarray.
Materials and methods Responsible Editor: Ji Zhang. E. S¸ ehitog˘lu F. C¸avus¸ B. Vural Department of Genetics, Institute for Experimental Medicine (DETAE), Istanbul University, Istanbul, Turkey ¨ rc¸en A. C¸oban C. Ulusoy M. Ku¨c¸u¨kerden A. O E. Tu¨zu¨n Department of Neuroscience, Institute for Experimental Medicine (DETAE), Istanbul University, Istanbul, Turkey E. Tu¨zu¨n e-mail: [email protected] D. Akbas¸ -Demir R. Tu¨rkog˘lu (&) Department of Neurology, Haydarpasa Numune Education and Research Hospital, Istanbul, Turkey e-mail: [email protected]
Patients Among patients admitted to multiple sclerosis outpatient clinic, 13 consecutive, newly diagnosed patients (10 women, 3 men; mean age ± standard error, 37.2 ± 3.1) fulfilling the criteria for RIS [1] and with no symptoms suggestive of MS were enrolled. None of the subjects had previously experienced any other disease, had vascular risk factors or family history of MS. All patients had normal routine blood chemistry, complete blood count, sedimentation rate and mini mental status examination tests. Reason for first brain MRI was migraine in nine, nonmigraine headache in two and dizziness in two patients.
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Fig. 1 ELISA detection of IgG antibodies directed against purified sorcin, in sera of multiple sclerosis (MS) patients, radiologically isolated syndrome (RIS) patients who have converted to MS (RIS?MS?), RIS patients who have not converted to MS (RIS?MS-) and healthy controls (HC). Note that none of the sera reacted with an irrelevant protein cysteine dioxygenase type 1
(CDT1) purified from the E. coli strain also used to express sorcin and E. coli lysate with no human protein expression. The dashed lines represent 2 standard deviations above the mean of the HC (cut-off values for positivity). Horizontal lines indicate the mean value of each group. ***p \ 0.0001 by ANOVA
All RIS patients underwent cranial MRI with the same 3-T scanner and followed by the same neurologist for 5 years. Patients were divided in two groups as RIS patients that converted to relapsing remitting MS (RRMS) within the 5-year follow-up period (RIS?MS?) as per the revised version of the 2005 McDonald criteria [4] and as RIS patients that did not convert to RRMS (RIS?MS-). Age/gender matched 23 relapsing remitting multiple sclerosis (RRMS) patients diagnosed by McDonald’s criteria [4] and 50 healthy individuals were enrolled as controls. The study protocol was approved by the local ethics committee and all subjects gave informed consent.
Results
Protein macroarray and ELISA Pooled sera of RIS patients obtained during the first MRI were screened using a high-density protein macroarray derived from human fetal brain cDNA expression library (hEX1) (ImaGenes, Berlin, Germany), as described previously [5]. The signal intensity was graded between 0 and 3. Plasmid DNAs from selected clones were isolated and nucleotide and translated amino acid sequences were compared with known sequences using BLAST algorithms. Histagged proteins were recombinantly expressed in Escherichia coli, purified by affinity chromatography and the purity of the proteins was documented by gel electrophoresis and immunoblotting performed with a commercial antibody [5]. ELISA studies were performed as previously reported to reveal serum antibodies to the purified proteins [5].
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Four of 13 (31 %) RIS patients developed clinically definite MS (at least two attacks and objective clinical evidence for at least two lesions) in a follow-up period of 1–3 years. All RIS?MS? patients were women, their ages at first MRI ranged between 28 and 53 and reason for first MRI was migraine in all patients. One clone with the highest signal intensity (3) and number of duplicates (4) in protein macroarray analysis was selected for further investigation. Sequencing studies demonstrated a single clone that matched sorcin (NCBI Accession No: NM_003130). In immunoblotting experiments performed with the protein purified from this clone, the commercial sorcin antibody (Abcam, Cambridge, MA, USA) bound a single band at *20 kDa, as predicted (not shown). ELISA studies showed high-titer serum antibodies to sorcin in 3/4 (75 %) of RIS?MS? and 13/23 (56 %) of RRMS patients but in none of RIS?MS- patients or healthy controls (Fig. 1). The mean signal ratios of RIS?MS? and RRMS patients were significantly higher than other groups by ANOVA (p \ 0.0001, Fig. 1).
Discussion Our preliminary study has implicated sorcin antibody as a potential biomarker for prediction of progression of RIS to MS. Although MS is not a classical antibody mediated
Antibody and MS
disease, anti-neuronal antibodies are crucially involved in its pathogenesis [3]. Potassium channel KIR4.1-antibody has recently been discovered in MS patients’ sera and been shown to exert astrocyte toxicity [6]. Like KIR4.1, sorcin is a membrane protein and regulates intracellular calcium homeostasis by modulating ryanodine receptor functions [7]. Ryanodine-dependent intracellular calcium increase has been implicated in activation of autoreactive T cells and oligodendrocyte death [8, 9]. During progression from RIS to MS, enhanced utilization of ryanodine receptors and consequent increased expression of sorcin might plausibly facilitate antibody formation against this modulator protein. An important limitation of our study was the small sample size, which should be dealt with using larger cohorts in future studies. Secondly, RIS is a recently proposed and currently evolving concept. Despite efforts for establishing universal criteria, RIS represents a heterogeneous group of patients with diverse clinical and imaging features. For instance some patients that strictly fulfill the RIS criteria might display mild cognitive impairment, whereas others show normal results in neuropsychological tests [2]. It is therefore important to approach cautiously to any biomarker study for RIS, until a more concrete understanding of this syndrome is established.
801
References 1. Okuda DT, Mowry EM, Beheshtian A, Waubant E, Baranzini SE, Goodin DS, Hauser SL, Pelletier D. Incidental MRI anomalies suggestive of multiple sclerosis: the radiologically isolated syndrome. Neurology. 2009;72:800–5. 2. Siva A. Asymptomatic MS. Clin Neurol Neurosurg. 2013;115: S1–5. 3. Krumbholz M, Derfuss T, Hohlfeld R, Meinl E. B cells and antibodies in multiple sclerosis pathogenesis and therapy. Nat Rev Neurol. 2012;8:613–23. 4. Polman CH, Reingold SC, Edan G, et al. Diagnostic criteria for multiple sclerosis: 2005 revisions to the ‘‘McDonald Criteria’’. Ann Neurol. 2005;58:840–6. 5. Vural B, Ug˘urel E, Tu¨zu¨n E, et al. Anti-neuronal and stressinduced-phosphoprotein 1 antibodies in neuro-Behc¸et’s disease. J Neuroimmunol. 2011;239:91–7. 6. Srivastava R, Aslam M, Kalluri SR, et al. Potassium channel KIR4.1 as an immune target in multiple sclerosis. N Engl J Med. 2012;367:115–23. 7. Pickel VM, Clarke CL, Meyers MB. Ultrastructural localization of sorcin, a 22 kDa calcium binding protein, in the rat caudateputamen nucleus: association with ryanodine receptors and intracellular calcium release. J Comp Neurol. 1997;386:625–34. 8. Cordiglieri C, Odoardi F, Zhang B, et al. Nicotinic acid adenine dinucleotide phosphate-mediated calcium signalling in effector T cells regulates autoimmunity of the central nervous system. Brain. 2010;133:1930–43. 9. Ruiz A, Matute C, Alberdi E. Intracellular Ca2? release through ryanodine receptors contributes to AMPA receptor-mediated mitochondrial dysfunction and ER stress in oligodendrocytes. Cell Death Dis. 2010;1:e54.
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Inflammation Research
SHORT COMMUNICATION
Sorcin antibody as a possible predictive factor in conversion from radiologically isolated syndrome to multiple sclerosis: a preliminary study Elc¸in S¸ ehitog˘lu • Filiz C ¸ avus¸ • Canan Ulusoy • Melike Ku¨c¸u¨kerden ¨ • Arda Orc¸en Deniz Akbas¸ -Demir • Arzu C ¸ oban • Burc¸ak Vural • • Erdem Tu¨zu¨n Recai Tu¨rkog˘lu
•
Received: 5 May 2014 / Revised: 13 June 2014 / Accepted: 18 June 2014 / Published online: 8 July 2014 Ó Springer Basel 2014
Abstract Objective To identify an antibody biomarker for prediction of conversion from radiologically isolated syndrome (RIS) to relapsing remitting multiple sclerosis (RRMS). Methods Sera of 13 RIS patients were screened by a protein macroarray derived from human fetal brain cDNA library. Results Sequencing of a clone with the highest signal intensity revealed sorcin as a potential target autoantigen in RIS patients. ELISA studies showed high-titer sorcin-antibodies in 3 of 4 RIS patients who converted to RRMS in a 5-year follow-up period and 13 of 23 control RRMS patients. Conclusion The value of sorcin antibody as a predictor of conversion from RIS to RRMS requires to be tested in larger prospective studies. Keywords Anti-neuronal antibody Autoantibody Multiple sclerosis Radiologically isolated syndrome Protein macroarray
Introduction The increasing use of magnetic resonance imaging (MRI) in diagnosis of neurological disorders has led to the detection of asymptomatic brain lesions, including those that are highly suggestive of multiple sclerosis (MS). These lesions might occur in the absence of any MS symptoms and signs, a condition named as radiologically isolated syndrome (RIS) [1, 2]. The 5-year conversion rate to the first clinical event in RIS is around 30 % [2], prompting characterization of laboratory methods for accurate selection of patients for early treatment. Improved understanding of the role of antibodies in MS pathogenesis [3] has brought forward identification of anti-neuronal antibodies that can serve as prognostic biomarkers. To identify a biomarker that would predict progression from RIS to MS, sera of RIS patients were screened with protein macroarray.
Materials and methods Responsible Editor: Ji Zhang. E. S¸ ehitog˘lu F. C¸avus¸ B. Vural Department of Genetics, Institute for Experimental Medicine (DETAE), Istanbul University, Istanbul, Turkey ¨ rc¸en A. C¸oban C. Ulusoy M. Ku¨c¸u¨kerden A. O E. Tu¨zu¨n Department of Neuroscience, Institute for Experimental Medicine (DETAE), Istanbul University, Istanbul, Turkey E. Tu¨zu¨n e-mail: [email protected] D. Akbas¸ -Demir R. Tu¨rkog˘lu (&) Department of Neurology, Haydarpasa Numune Education and Research Hospital, Istanbul, Turkey e-mail: [email protected]
Patients Among patients admitted to multiple sclerosis outpatient clinic, 13 consecutive, newly diagnosed patients (10 women, 3 men; mean age ± standard error, 37.2 ± 3.1) fulfilling the criteria for RIS [1] and with no symptoms suggestive of MS were enrolled. None of the subjects had previously experienced any other disease, had vascular risk factors or family history of MS. All patients had normal routine blood chemistry, complete blood count, sedimentation rate and mini mental status examination tests. Reason for first brain MRI was migraine in nine, nonmigraine headache in two and dizziness in two patients.
123
E. S¸ ehitog˘lu et al.
800
Fig. 1 ELISA detection of IgG antibodies directed against purified sorcin, in sera of multiple sclerosis (MS) patients, radiologically isolated syndrome (RIS) patients who have converted to MS (RIS?MS?), RIS patients who have not converted to MS (RIS?MS-) and healthy controls (HC). Note that none of the sera reacted with an irrelevant protein cysteine dioxygenase type 1
(CDT1) purified from the E. coli strain also used to express sorcin and E. coli lysate with no human protein expression. The dashed lines represent 2 standard deviations above the mean of the HC (cut-off values for positivity). Horizontal lines indicate the mean value of each group. ***p \ 0.0001 by ANOVA
All RIS patients underwent cranial MRI with the same 3-T scanner and followed by the same neurologist for 5 years. Patients were divided in two groups as RIS patients that converted to relapsing remitting MS (RRMS) within the 5-year follow-up period (RIS?MS?) as per the revised version of the 2005 McDonald criteria [4] and as RIS patients that did not convert to RRMS (RIS?MS-). Age/gender matched 23 relapsing remitting multiple sclerosis (RRMS) patients diagnosed by McDonald’s criteria [4] and 50 healthy individuals were enrolled as controls. The study protocol was approved by the local ethics committee and all subjects gave informed consent.
Results
Protein macroarray and ELISA Pooled sera of RIS patients obtained during the first MRI were screened using a high-density protein macroarray derived from human fetal brain cDNA expression library (hEX1) (ImaGenes, Berlin, Germany), as described previously [5]. The signal intensity was graded between 0 and 3. Plasmid DNAs from selected clones were isolated and nucleotide and translated amino acid sequences were compared with known sequences using BLAST algorithms. Histagged proteins were recombinantly expressed in Escherichia coli, purified by affinity chromatography and the purity of the proteins was documented by gel electrophoresis and immunoblotting performed with a commercial antibody [5]. ELISA studies were performed as previously reported to reveal serum antibodies to the purified proteins [5].
123
Four of 13 (31 %) RIS patients developed clinically definite MS (at least two attacks and objective clinical evidence for at least two lesions) in a follow-up period of 1–3 years. All RIS?MS? patients were women, their ages at first MRI ranged between 28 and 53 and reason for first MRI was migraine in all patients. One clone with the highest signal intensity (3) and number of duplicates (4) in protein macroarray analysis was selected for further investigation. Sequencing studies demonstrated a single clone that matched sorcin (NCBI Accession No: NM_003130). In immunoblotting experiments performed with the protein purified from this clone, the commercial sorcin antibody (Abcam, Cambridge, MA, USA) bound a single band at *20 kDa, as predicted (not shown). ELISA studies showed high-titer serum antibodies to sorcin in 3/4 (75 %) of RIS?MS? and 13/23 (56 %) of RRMS patients but in none of RIS?MS- patients or healthy controls (Fig. 1). The mean signal ratios of RIS?MS? and RRMS patients were significantly higher than other groups by ANOVA (p \ 0.0001, Fig. 1).
Discussion Our preliminary study has implicated sorcin antibody as a potential biomarker for prediction of progression of RIS to MS. Although MS is not a classical antibody mediated
Antibody and MS
disease, anti-neuronal antibodies are crucially involved in its pathogenesis [3]. Potassium channel KIR4.1-antibody has recently been discovered in MS patients’ sera and been shown to exert astrocyte toxicity [6]. Like KIR4.1, sorcin is a membrane protein and regulates intracellular calcium homeostasis by modulating ryanodine receptor functions [7]. Ryanodine-dependent intracellular calcium increase has been implicated in activation of autoreactive T cells and oligodendrocyte death [8, 9]. During progression from RIS to MS, enhanced utilization of ryanodine receptors and consequent increased expression of sorcin might plausibly facilitate antibody formation against this modulator protein. An important limitation of our study was the small sample size, which should be dealt with using larger cohorts in future studies. Secondly, RIS is a recently proposed and currently evolving concept. Despite efforts for establishing universal criteria, RIS represents a heterogeneous group of patients with diverse clinical and imaging features. For instance some patients that strictly fulfill the RIS criteria might display mild cognitive impairment, whereas others show normal results in neuropsychological tests [2]. It is therefore important to approach cautiously to any biomarker study for RIS, until a more concrete understanding of this syndrome is established.
801
References 1. Okuda DT, Mowry EM, Beheshtian A, Waubant E, Baranzini SE, Goodin DS, Hauser SL, Pelletier D. Incidental MRI anomalies suggestive of multiple sclerosis: the radiologically isolated syndrome. Neurology. 2009;72:800–5. 2. Siva A. Asymptomatic MS. Clin Neurol Neurosurg. 2013;115: S1–5. 3. Krumbholz M, Derfuss T, Hohlfeld R, Meinl E. B cells and antibodies in multiple sclerosis pathogenesis and therapy. Nat Rev Neurol. 2012;8:613–23. 4. Polman CH, Reingold SC, Edan G, et al. Diagnostic criteria for multiple sclerosis: 2005 revisions to the ‘‘McDonald Criteria’’. Ann Neurol. 2005;58:840–6. 5. Vural B, Ug˘urel E, Tu¨zu¨n E, et al. Anti-neuronal and stressinduced-phosphoprotein 1 antibodies in neuro-Behc¸et’s disease. J Neuroimmunol. 2011;239:91–7. 6. Srivastava R, Aslam M, Kalluri SR, et al. Potassium channel KIR4.1 as an immune target in multiple sclerosis. N Engl J Med. 2012;367:115–23. 7. Pickel VM, Clarke CL, Meyers MB. Ultrastructural localization of sorcin, a 22 kDa calcium binding protein, in the rat caudateputamen nucleus: association with ryanodine receptors and intracellular calcium release. J Comp Neurol. 1997;386:625–34. 8. Cordiglieri C, Odoardi F, Zhang B, et al. Nicotinic acid adenine dinucleotide phosphate-mediated calcium signalling in effector T cells regulates autoimmunity of the central nervous system. Brain. 2010;133:1930–43. 9. Ruiz A, Matute C, Alberdi E. Intracellular Ca2? release through ryanodine receptors contributes to AMPA receptor-mediated mitochondrial dysfunction and ER stress in oligodendrocytes. Cell Death Dis. 2010;1:e54.
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