RESEARCH ARTICLE Absence of pathogenic mutations in CD59 in chronic inflammatory demyelinating polyradiculoneuropathy Lena Duchateau1☯, Lorena Martı́n-Aguilar ID2☯, Cinta Lleixà2, Andrea Cortese3, Oriol Dols- Icardo1,4, Laura Cervera-Carles ID1,4, Elba Pascual-Goñi2, Jordi Diaz-Manera2,5, Ilaria Calegari3, Diego Franciotta3, Ricard Rojas-Garcia2,5, Isabel Illa2,5, Jordi Clarimon1,4, Luis Querol ID2,5* 1 Memory Unit, Hospital de la Santa Creu i Sant Pau, Universitat Autònoma de Barcelona, Barcelona, Spain, 2 Neuromuscular Diseases Unit, Hospital de la Santa Creu i Sant Pau, Universitat Autònoma de Barcelona, a1111111111 Barcelona, Spain, 3 IRCCS Mondino Foundation, Pavia, Italy, 4 Centro para la Investigación Biomédica en a1111111111 Red en Enfermedades Neurodegenerativas (CIBERNED), Madrid, Spain, 5 Centro para la Investigación Biomédica en Red en Enfermedades Raras (CIBERER), Madrid, Spain a1111111111 a1111111111 ☯ These authors contributed equally to this work. a1111111111 * lquerol@santpau.cat Abstract OPEN ACCESS Citation: Duchateau L, Martı́n-Aguilar L, Lleixà C, Objective Cortese A, Dols-Icardo O, Cervera-Carles L, et al. (2019) Absence of pathogenic mutations in CD59 Mutations in CD59 cause CIDP-like polyneuropathy in children with inherited chronic hemo- in chronic inflammatory demyelinating lysis. We hypothesized that mutations in CD59 might be found in a subset of sporadic CIDP polyradiculoneuropathy. PLoS ONE 14(2): patients. e0212647. https://doi.org/10.1371/journal. pone.0212647 Editor: Alfred S. Lewin, University of Florida, Methods UNITED STATES 35 patients from two centers, fulfilling the EFNS/PNS diagnostic criteria for CIDP were Received: November 5, 2018 included. CD59 coding region was amplified by PCR and Sanger sequenced. Accepted: February 6, 2019 Results Published: February 22, 2019 One rare variant was detected in a patient which resulted in a synonymous change and pre- Copyright: © 2019 Duchateau et al. This is an open dicted to be neutral. Pathogenic variants were absent in our cohort. access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and Interpretation reproduction in any medium, provided the original author and source are credited. Our pilot study suggests that mutations in CD59 are absent in adult-onset sporadic CIDP. Data Availability Statement: Data cannot be shared publicly because they include pseudo anonymized clinical and epidemiological information that could lead to patient identification, considering the rarity of this disease. This Introduction restriction is imposed by the Ethics’ Committee of Hospital de la Santa Creu i Sant Pau. Specific parts Chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) is a rare and heteroge- of the dataset are available under request neous neurological disorder that is diagnosed according to clinical and electrophysiological (malonsoma@santpau.cat). diagnostic criteria. Its pathogenesis is largely unknown although an autoimmune origin is PLOS ONE | https://doi.org/10.1371/journal.pone.0212647 February 22, 2019 1/6 Absence of pathogenic mutations in CD59 in chronic inflammatory demyelinating polyradiculoneuropathy Funding: This project was supported by Fondo de widely accepted. The response to immunomodulatory therapy, including intravenous immu- Investigaciones Sanitarias (FIS), Instituto de Salud noglobulins (IVIg), glucocorticosteroids and plasma exchange, supports the autoimmune Carlos III, Spain and FEDER under grant FIS16/ hypothesis and the role of humoral factors, including autoantibodies, in its pathogenesis. Tra- 00627, and a personal grant to LQ SLT006/17/ 00131 of the Pla Estratègic de Recerca i Innovació ditional CIDP pathogenic models describe the presence of combined cell-mediated and en Salut (PERIS) 2016-2020 of the Departament de humoral immunity that result in an aberrant immune response targeting myelinated fibres of Salut of the Generalitat de Catalunya, pricipal peripheral nerves [1]. However, the relative contribution of each component of the immune investigator LQ. response is unknown. The recent discovery of disease-specific antibodies, such as anti-neuro- Competing interests: LQ has provided expert fascin 155 (anti-NF155), anti-contactin-1, anti-contactin-associated protein 1 and nodal neu- testimony for Grifols, Genzyme and CSL Behring, rofascin antibodies, that are only present in 5–10% of patients [2–4], suggests the existence of received speaking honoraria from Biogen Spain small but homogeneous subgroups of CIDP patients in which specific effector mechanisms and Roche and received research funds from drive the disease. This model may explain better patient heterogeneity within the CIDP spec- Grifols (Spin Award) and LFB. This does not alter the authors’ adherence to PLOS ONE policies on trum. Indeed the clinical, pathological and genetic heterogeneity disappears when patients are sharing data and materials. stratified according to highly specific biomarkers, such as autoantibodies [5–7]. The recent description of a significantly increased frequency of the HLA DRB1� 15 allele in anti-NF155 antibody-positive patients in comparison with those anti-NF155 antibody-negative, an associ- ation that remained hidden before the description of these antibodies, supports this hypothesis [7]. These findings strongly suggest that, even though CIDP has an autoimmune pathogenesis, genetic factors could be essential in the development of CIDP in a subset of patients. Unfortu- nately, due to the rarity of the disease and the difficulty to recruit biologically homogeneous series of patients, research on the genetic factors related to CIDP is scarce [8]. A non-synonymous homozygous mutation (p.Cys58Tyr) in CD59, a complement inhibitor present in the surface of red blood cells, was discovered in five Jewish children from North- Africa with chronic haemolysis and childhood relapsing immune-mediated polyneuropathy [9]. Interestingly, their symptoms were very similar to those of patients suffering from CIDP and the children had a partial response to IVIg and corticosteroid treatment. We then hypothesized that de novo mutations in CD59 might account for a fraction of adult-onset sporadic CIDP patients. With the aim of evaluating the possible role of CD59 in CIDP, its coding region was fully sequenced in a homogeneous series of patients who did not present detectable anti-NF155, anti-contactin-1, NF140/186 or CASPR1 autoantibodies. Material and methods Patients, samples, protocol approvals and patient consents Patients diagnosed with CIDP according to the European Federation of Neurological Socie- ties/Peripheral Nerve Society (EFNS/PNS) diagnostic criteria[10] provided written informed consent to participate and were included in the study according to a protocol approved by the Institution’s Ethics’ Committee of Hospital de la Santa Creu i Sant Pau. Patients were recruited between January and December of 2016. Whole blood was drawn in EDTA tubes and DNA extracted following standard protocols and stored until needed. Genetic studies The entire coding region of CD59 gene (transcript variant CD59-211 ENST00000642928.1) was amplified by polymerase chain reaction (PCR) and Sanger sequenced on an ABI 3100 automatic sequencer (Applied Biosystems, Foster City, CA, USA). Resulting electropherograms were visually analyzed using Sequencher software (Gene Codes Corp. Ann Arbor, MI, USA). Primer pair sequences and PCR conditions are available under request. In silico evaluation of potential deleterious effects of CD59 genetic variants was performed with the CADD-score (http://cadd.gs.washington.edu/), Mutation Taster (http:// PLOS ONE | https://doi.org/10.1371/journal.pone.0212647 February 22, 2019 2/6 Absence of pathogenic mutations in CD59 in chronic inflammatory demyelinating polyradiculoneuropathy www.mutationtaster.org/), SIFT (http://sift.jcvi.org/), and Human Splicing Finder tool (http:// www.umd.be/HSF3/index.html). Results A total of 35 patients (57% male, mean age at inclusion 61 years old) were included in the study. Direct sequencing of all coding exons of CD59 was performed. Only one variant was detected in one patient (Fig 1), a heterozygous guanine to alanine substitution (c.18G>A) which resulted in the synonymous change rs111771149 (p.Gly6Gly). According to the genome aggregation database (http://gnomad.broadinstitute.org/) this is a rare variant with an allele frequency of 0.003 in ExAC database and 0.001899 in gnomAD database in non-Finnish Euro- peans. In silico analysis of possible damaging consequences did not reveal any potential delete- rious effect related to this genetic variant. The previously reported pathogenic p.Cys58Tyr mutation was not present in our patient cohort. Discussion Our pilot study failed to identify functionally-relevant CD59 mutations in sporadic adult- onset CIDP patients suggesting that genetic dysfunction of CD59 is not a frequent cause of CIDP. A rare variant was found in one patient but was predicted to be functionally irrelevant. CD59 (protectin) encodes a glycosylphosphatidylinositol (GPI)-anchored cell surface mem- brane glycoprotein, which inhibits polymerization of complement molecule C9, the final step of membrane attack complex (MAC) formation, to protect host cells from complement-medi- ated lysis [9]. Loss of function of CD59 thus might make cells, such as Schwann cells, susceptible to MAC-mediated lysis, possibly causing demyelination. In fact, complement deposits have been observed in areas of demyelination in sural nerve biopsies of patients with CIDP [11,12]. Also, CD59-deficient mice show MAC deposits in the perivascular tissue in the areas of demyelin- ation, and are more susceptible to experimental autoimmune encephalomyelitis, inflammation and axonal loss than wild type mice [13]. Congenital CD59 deficiency is an extremely rare and recently described disease character- ized by hemolysis, recurrent ischemic strokes and relapsing immune-mediated polyneuropa- thy [9,14]. After the description of the pathogenic CD59 mutation p.Cys58Tyr[9], other CD59 muta- tions have been described. In 2015, a homozygous missense mutation (p.Asp49Val) was reported to be pathogenic in three Turkish patients from a two-generation family with immune-mediated peripheral neuropathy, chronic hemolysis and strokes [15]. Recently, a Fig 1. Electropherogram of the variant c.18G>A (above) and without the variant (down). https://doi.org/10.1371/journal.pone.0212647.g001 PLOS ONE | https://doi.org/10.1371/journal.pone.0212647 February 22, 2019 3/6 Absence of pathogenic mutations in CD59 in chronic inflammatory demyelinating polyradiculoneuropathy homozygous frameshift deletion in CD59 (c.146delA, pAsp49Valfs� 31) has been described in a 7-year-old girl with a demyelinating polyneuropathy and cerebral vasculopathy [14]. These severe symptoms support the importance of CD59 as an essential complement regu- latory protein for protection of hematopoietic and Schwann cells against complement attack. Acquired CD59 deficiency is well known in paroxysmal nocturnal hemoglobinuria (PNH), where a clonal defect in GPI biosynthesis confined to the hematological system, results in hemolysis and prothrombotic tendency[16]. Eculizumab, a C5 inhibitor, is a treatment approved for PNH [17]. Therefore, treatment with eculizumab proved also effective in congen- ital CD59 deficiency; with a marked clinical improvement of these children, a reduction of hospitalizations and a reduction of IVIg and steroid doses [18]. Detection of CD59 mutations would have provided a novel therapeutic target in sporadic CIDP patients. To our knowledge, this is the first genetic study aimed at identifying CD59 alterations in CIDP. However, a study investigated the presence of antibodies targeting the complement inhibitors CD46, CD55 and CD59 and found no association of these antibodies with inflam- matory neuropathies [19]. In summary, our results suggest that CD59 mutations are not present in sporadic CIDP patients. We did not study somatic mutations restricted to nerve (mosaicism) or to regulatory regions of CD59 and, thus, the possibility that CD59 dysfunction plays a role in some CIDP patients has not been completely ruled out. Caution should be made in interpreting our data, since we used a limited series of patients and, therefore, we cannot exclude that rare CD59 mutations are the cause of sporadic CIDP. However, our study suggests that these mutations are not a frequent cause of CIDP. Since CIDP is a very rare disease and single-center case series are consequently small, further genetic and immunologic studies in larger, collaborative, CIDP cohorts [20] are needed to unravel the genetic basis of CIDP, improve pathogenetic models and find clinically useful biomarkers. Disclosures LQ has provided expert testimony for Grifols, Genzyme and CSL Behring, received speaking honoraria from Biogen Spain and Roche and received research funds from Grifols (Spin Award) and LFB. This does not alter our adherence to PLOS ONE policies on sharing data and materials. Author Contributions Conceptualization: Luis Querol. Formal analysis: Jordi Clarimon. Investigation: Lena Duchateau, Lorena Martı́n-Aguilar, Jordi Clarimon, Luis Querol. Methodology: Lena Duchateau, Oriol Dols-Icardo, Laura Cervera-Carles, Jordi Clarimon, Luis Querol. Project administration: Jordi Clarimon, Luis Querol. Supervision: Jordi Clarimon, Luis Querol. Validation: Luis Querol. Writing – original draft: Lena Duchateau, Lorena Martı́n-Aguilar. Writing – review & editing: Lena Duchateau, Lorena Martı́n-Aguilar, Cinta Lleixà, Andrea Cortese, Oriol Dols-Icardo, Laura Cervera-Carles, Elba Pascual-Goñi, Jordi Diaz-Manera, PLOS ONE | https://doi.org/10.1371/journal.pone.0212647 February 22, 2019 4/6 Absence of pathogenic mutations in CD59 in chronic inflammatory demyelinating polyradiculoneuropathy Ilaria Calegari, Diego Franciotta, Ricard Rojas-Garcia, Isabel Illa, Jordi Clarimon, Luis Querol. References 1. Mathey EK, Park SB, Hughes RAC, Pollard JD, Armati PJ, Barnett MH, et al. Chronic inflammatory demyelinating polyradiculoneuropathy: from pathology to phenotype. J Neurol Neurosurg Psychiatry. 2015; https://doi.org/10.1136/jnnp-2014-309697 PMID: 25677463 2. Querol L, Nogales-Gadea G, Rojas-Garcia R, Martinez-Hernandez E, Diaz-Manera J, Suárez-Calvet X, et al. Antibodies to contactin-1 in chronic inflammatory demyelinating polyneuropathy. 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