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Prevalence of autoantibodies in the course of Gaucher disease type 1: A multicenter study comparing Gaucher disease patients to healthy subjects
Accepted Manuscript Title: Prevalence of autoantibodies in the course of Gaucher disease type 1: a multicenter study comparing Gaucher disease patients to healthy subjects Author: id="aut0005" author-id="S1297319X16302391e428fee597da77273ac31bf409984be4"> Christine Serratrice id="aut0010" author-id="S1297319X16302391ceba0be65fad629ab591c74cf0ebfd4d"> Nesma Bensalah id="aut0015" author-id="S1297319X163023912dc692e817ccff2e877a509a7a43f837"> Guillaume Penaranda id="aut0020" author-id="S1297319X16302391b9bd51e3a9cf4e8162d4bef712e687d1"> Nathalie Bardin id="aut0025" author-id="S1297319X1630239168fcf2353eaca004715ae4686846371b"> Nadia Belmatoug id="aut0030" author-id="S1297319X16302391177e8f87843857f91ccd0e81bd36a683"> Agathe Masseau id="aut0035" author-id="S1297319X163023910c134f652f43b947084a693f9f13dacf"> Christian Rose id="aut0040" author-id="S1297319X163023918a51cb38de00b8abdb7c34377861bdc9"> Olivier Lidove id="aut0045" author-id="S1297319X16302391d45fa1d712617295a36c54283737693b"> Fabrice Camou id="aut0050" author-id="S1297319X163023919a514c442fa644bc50c5d8e34af5278a"> Franc¸ois Maillot id="aut0055" author-id="S1297319X16302391e46ed47515ac36f48ff0b6ea294c7328"> Vanessa Leguy id="aut0060" author-id="S1297319X1630239164d9df9478145347a8ff8bbe8a9f3f41"> Nadine Magy-Bertrand id="aut0065" author-id="S1297319X16302391e2edb99477dcfa86fcd8570eef3cb476"> Isabelle Marie id="aut0070" author-id="S1297319X16302391d609fe3fd259152e1c6a727d44aeea03"> Patrick Cherin id="aut0075" author-id="S1297319X163023913312f3e5035c18a9fb88843138732c27"> Monia Bengherbia id="aut0080" author-id="S1297319X16302391be26162de90bc17f5facfe9a6f6aa1c6"> Sebastian Carballo id="aut0085" author-id="S1297319X16302391d996605abfd307e1eb9e309a74ac8833"> Jos´e Boucraut id="aut0090" author-id="S1297319X16302391-
8d8cc5357e5ba736f1b963c9bd799f5f"> Jacques Serratrice id="aut0095" author-id="S1297319X16302391275622992fa076b08480e12812bb469e"> Marc Berger id="aut0100" author-id="S1297319X16302391d38c0ab15cb058360ce113c0f87c5fce"> Denis Verrot PII: DOI: Reference:
S1297-319X(16)30239-1 http://dx.doi.org/doi:10.1016/j.jbspin.2016.12.002 BONSOI 4513
To appear in: Received date: Accepted date:
14-10-2016 7-12-2016
Please cite this article as: Serratrice C, Bensalah N, Penaranda G, Bardin N, Belmatoug N, Masseau A, Rose C, Lidove O, Camou F, Maillot F, Leguy V, Magy-Bertrand N, Marie I, Cherin P, Bengherbia M, Carballo S, Boucraut J, Serratrice J, Berger M, Verrot D, Prevalence of autoantibodies in the course of Gaucher disease type 1: a multicenter study comparing Gaucher disease patients to healthy subjects, Joint Bone Spine (2016), http://dx.doi.org/10.1016/j.jbspin.2016.12.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Prevalence of autoantibodies in the course of Gaucher disease type 1: a multicenter study comparing Gaucher disease patients to healthy subjects
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Christine Serratricea,b, Nesma Bensalahc, Guillaume Penarandad, Nathalie Bardinc, Nadia Belmatouge, Agathe Masseauf, Christian Roseg, Olivier Lidoveh, Fabrice Camoui, François Maillotj, Vanessa Leguyk, Nadine Magy-Bertrandl, Isabelle Mariem, Patrick Cherinn, Monia Bengherbiae; Sebastian Carballob, José Boucrautc , Jacques Serratriceb, Marc Bergero, Denis Verrota . a
Internal Medicine Department, Hôpital Saint Joseph, 26 bd de Louvain, 13008 Marseille, France Internal Medicine Department, University Hospital of Geneva, Rue Gabrielle-Perret-Gentil 4, 1205 Geneva, Switzerland c Immunology laboratory, University Hôpital La Conception, 147, boulevard Baille, 13005 Marseille d Alphabio Laboratory, Hôpital Européen, 6 Rue Désirée Clary, 13003 Marseille e Internal Medicine Department, Hôpital Beaujon, 100 Boulevard du Général Leclerc, 92110 Clichy f Internal Medicine Department, CHU, 44093 Nantes cedex1, France g Internal Medicine Department, Catholic University, 60 Boulevard Vauban, 59800 Lille, France h Internal Medicine and Rheumatology Department, Hôpital La Croix Saint Simon, 75020 Paris, France i Intensive Care Department, Hôpital Pellegrin, Place Amélie Raba Léon, 33076 Bordeaux, France j CHRU, Université François Rabelais, INSERM 1069, 10 boulevard Tonnellé, 37032 Tours Cedex, France k Internal Medicine and Clinical Immunology Department, CHU, 4 rue Paul Gaffarel BP 77908 , 21079 Dijon, France l Internal Medicine Department, Hôpital Minjoz, 3 Boulevard A. Fleming, 25000 Besançon, France m Internal Medicine Department, CHU, 1 rue de Germont, 76031 Rouen, France n Internal Medicine Department, CHU la Pitié Salpêtrière, 47-83 Boulevard de l'Hôpital, 75013 Paris, France o Hematology Biology, CHU Estaing, 1 Rue Lucie Aubrac, 63100 Clermont Ferrand, France
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Corresponding author : Christine C Serratrice, Internal Medicine Department Hôpital Saint Joseph, 26 bd de Louvain 13008 Marseille, France [email protected] Tél.: 041798731474
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Abstract Objectives. Type 1 Gaucher disease may be related to the presence of autoantibodies. Their clinical significance is questioned. Primary Endpoint was to compare the prevalence of autoantibodies in type 1 Gaucher disease
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patients with healthy subjects, seeking correlations with autoimmune characteristics. Secondary endpoints were to determine whether patients with autoantibodies reported autoimmunity-related symptoms and if genotype, splenectomy or treatment influenced autoantibodies presence.
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Methods. Type 1 Gaucher disease patients and healthy volunteers were included in this national
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multicenter exploratory study. Autoantibodies presence was compared in both groups and assessed regarding to genotype, splenectomy, Gaucher disease treatment and autoimmunity-related symptoms.
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Results. Twenty healthy subjects and 40 type 1 Gaucher disease patients were included. Of the studied group: 15 patients undergone splenectomy, 37 were treated either with enzyme replacement therapy (34) or with substrate reduction therapy (3), 25 were homozygous/heterozygous for the N370S
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mutation.
In type 1 Gaucher disease group (studied group), 52% had positive autoantibodies versus 26% in
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control group. Antiphospholipid antibodies were more frequent in the studied group (30% vs 5%), but without correlation to thrombosis, osteonecrosis or bone infarcts. In the studied group, antinuclear
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antibodies were more frequent (25% vs 16%). None of the patients with autoantibodies had clinical manifestations of autoimmune diseases. Autoantibodies were not correlated with treatment, genotype, or splenectomy, except for anticardiolipid, more frequent in splenectomized patients. Conclusions. In type 1 Gaucher disease, autoantibodies were more frequent compared to a healthy population. However, they were not associated with an increased prevalence of clinical active
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autoimmune diseases.
Keywords: Gaucher disease, Auto immunity, Antibodies, Antinuclear, Antiphospholipid.
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1. Introduction Gaucher disease (GD) is a rare genetic lysosomal storage disorder inherited in an autosomal recessive pattern [1]. GD is due to the deficiency of a lysosomal enzyme, acid beta-glucosidase (or
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glucocerebrosidase) or in rare cases of its activator, saposin C. Prevalence worldwide is 1/60000 and 1/6000 in Israel. In the French Gaucher registry, prevalence is estimated at 1/140000 [2]. GD
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diagnosis is confirmed by the detection of low glucocerebrosidase activity in peripheral leucocytes [3] .
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Three different types of GD have been described: type 1 (GD1) is characterized by thrombocytopenia, anemia, an enlarged spleen and liver as well as bone complications (Erlenmeyer flask deformity,
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osteoporosis, lytic lesions, pathological and vertebral fractures, bone infarcts and avascular necrosis leading to degenerative arthropathy). GD1 represents 90% of all cases. Type 2 Gaucher disease is an
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acute lethal neuronopathic form resulting in death within the first or second year of life, often occurring after aspiration pneumonia and/or apnea or laryngospasm [4]. Type 3, or chronic neuronopathic,
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Gaucher disease is a “catch all” encompassing patients who survived infancy but had some forms of neurologic involvement. Often the neurologic manifestations are subtle, such as the slowing of the horizontal saccadic eye movements, but other patients develop neurodegeneration, myoclonic
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epilepsy, or psychiatric manifestations. It is now recognized that there is an overlap among these phenotypes and GD is considered as a continuum between these 3 entities [5].
Since 1991 GD1 can be effectively treated by enzyme replacement therapy (ERT). Three recombinant glucocerebrosidase are now available: imiglucerase (Sanofi/Genzyme Corporation); velaglucerase
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(Shire HGT); taliglucerase, (Pfizer). Two treatments by substrate reduction therapy obtained a marketing authorization: miglustat, (Actelion Pharmaceuticals) and eliglustat (Sanofi/Genzyme Corporation). Mutations in the gene coding glucocerebrosidase (GBA) lead to an accumulation of the sphingolipid glucocerebroside within macrophages [3]. The accumulation of lipids in GD cells is associated with an inflammatory state, an activation of macrophages, and cytokine secretion [6,7]. Some of these interleukins, as Il-6, Il-10 or Il-2 R, are associated with the hyperactivity of the immune system and
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related to the development of monoclonal or polyclonal gammopathies [8,9]. Moreover, lysosome plays a key role for antigenic presentation through major histocompatibility complex (MHC) molecules I and II, and CD1 (cluster of differentiation 1) molecules. In 2005 Balreira and coll suggested a link between sphingolipid metabolism and abnormalities in expression of MHC family [10]. They first demonstrated an increased expression of CD1d and MHC class II in GD monocytes. More recently,
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Nair and coll, have reported that β -glucosylceramide 22:0 and glucosylsphingosine can be recognized by a distinct subset of CDd1-restricted human and murine type II NKT (Natural Killer T) cells which
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could lead to a dysregulation of humoral immunity in metabolic lipid disorders [11]. On the other hand,
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ERT induce a decrease in MHC class II expression which may interfere with humoral response. Thus, this biological context may explain the high frequency of polyclonal and monoclonal gammopathies,
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associated with a high risk of multiple myeloma. Polyclonal gammopathy can favour the emergence of autoantibodies but there is little data published about autoimmunity during the course GD1. One of the first reports of immunoglobulin abnormalities was published in 1968 [12]. Thereafter, several studies
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analyzed the link between GD and monoclonal gammopathies, and the risk of myeloma [9,13].
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Shoenfeld has shown that in GD there was a chronic stimulation of the humoral immune system [14]. Later he searched for autoantibodies reactive to 14 autoantigens in a cohort of 43 patients with GD
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[15]. The sera of 5 patients reacted with 1 autoantigen, the sera of 17 patients reacted with 2 to 6 antigens, and 11 patients reacted with 8 to 12 antigens. No correlation was found between autoreactivity and serum immunoglobulin concentration. Moreover these antibodies are “natural antibodies” and don’t lead to any immune manifestations. This hypothesis was later confirmed by several reports on the presence of anticardiolipid (aCL) and/or lupus anticoagulant in GD, without
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clinical manifestations [16].
However, some rare cases of autoimmune diseases in patients with GD1 have been reported as autoimmune hemolytic anemia (Burroughs S PR, Bartolomeo A, Greenstein R. Hemolytic anemia in Gaucher Disease. Blood Cells Molecular Diseases 1994:589A) and [17], antiphospholipid syndrome
[18], and immune thrombocytopenia [19]. Among 84 consecutive patients, Rosenbaum et al. identified 3.5% of patients with immune thrombocytopenia, 2.3% with immune hemolytic anemia, but also the presence of aCL and lupus anticoagulant in 5.9% (one had antiphospholipid syndrome [APLS] with
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recurrent abortions), anti-nuclear and anti DNA in 8.3% and anti-parietal cells antibodies in 3.5% [20]. In a preliminary limited personal experience, autoantibodies have been detected in 62.5% of GD1 patients, and we have noted that for a few patients, antibody rate decreased after treatment [21]. Curiously, to our knowledge, with the exception of immune hemolytic anemia, immune thrombocytopenia, and APLS, no significant association of GD1 with other autoimmune diseases,
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such as Grave's disease, Hashimoto disease or systemic sclerosis have been reported. Some rare case reports mentioned coexistence of GD1 and diabetes mellitus (only one case published) [22],
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recent report described the coexistence of lupus nephritis and GD1 [26].
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rheumatoid arthritis (one case reported) [23], sicca syndrome (two cases reported) [24,25]. One
In our study, the primary endpoint was to compare the prevalence of autoantibodies in GD1 patients
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with healthy subjects. Secondary endpoints were to determine whether patients with auto-antibodies reported autoimmunity-related symptoms and if genotype, splenectomy or treatment had an impact on
2.1.
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2. Methods
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the presence or absence of autoantibodies.
Investigators and centers
All investigators of this study belonged to the "Internal Medicine and Lysosomal Disease" group created in 2007 and the Committee for evaluation and treatment of Gaucher disease led by Pr Marc
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Berger and approved in 2004. All investigators were involved in the management of Gaucher disease. The principal investigator was Dr. Christine Serratrice, Internal Medicine department, Saint Joseph Hospital (Marseille). All other investigators were affiliated to university hospitals or general hospital centers. Patients were included in 12 centers: Hôpital Saint Joseph-Marseille, Hôpital Beaujon-Clichy, CHUNantes, Catholic University-Lille, CHU Hôpital Brabois-Vandoeuvre les Nancy, Hôpital La Croix Saint Simon-Paris, Hôpital Pellegrin-Bordeaux, CHRU-Tours, CHU-Dijon, Hôpital Minjoz-Besançon, CHU Bois Guillaume-Rouen, CHU Pitié Salpêtrière-Paris.
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2.2.
Patients
Forty consecutive patients with GD1 from the 12 centers and 20 healthy volunteers (control group) were included in the study.
2.2.1. Details of ethics approval
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This national multicenter exploratory study obtained agreements from the French Ethics Committee
(CPP Aix Marseille I), and from the French National Agency for Medicines and Health Products Safety
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was registered in ClinicalTrial.gov under the number NCT02650219.
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(ANSM). The study received approval from the French Data Protection Authority (CNIL). The study
2.2.2. Patient consent statement
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All investigations were conducted according to the declaration of Helsinki.
Written informed consents were obtained after the nature and possible consequences of the studies
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had been fully explained. All patients and healthy subjects gave specific written consent for blood sample collection and blood samples were collected during a routine monitoring of the disease.
Data collection
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2.3.
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Inclusion criteria of GD1 patients are presented in table 1.
For all GD1 patients and healthy subjects the following elements were collected: diagnosis characteristics, splenectomy, bone events, pulmonary hypertension, specific treatment and nonspecific therapies. Taking into account clinical American College of Rheumatology (ACR) and
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Systemic Lupus International Collaborating Clinics (SLICC) classification criterias [27-29] clinical features for systemic lupus (SLE), sicca syndrome (SS), [30] Antiphospholipid syndrome (APLS), [31] and ACR /EULAR Rheumatoid arthritis (RA) clinical criterias [32] as well as diagnosis of thyroiditis, were also systematically evaluated in patients and controls. Furthermore, sera from 40 GD1 patients and from the 20 healthy subjects were collected from January 2010 to April 2011. Blood samples were collected during a routine monitoring of the disease in GD1 group. Blood samples were forwarded to a single specialized immunology laboratory and were tested
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during the same period of time to avoid technical bias. GD diagnosis was confirmed by demonstration of low β-glucosidase acid activity and/or molecular diagnosis. Thus, antinuclear, anti-SSa, anti-SSb, anti-RNP, anti-DNA, anti-Sm, anticardiolipid, anti β2Gp1, antiganglioside autoantibodies were
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systematically screened in every patient.
Biological analyses
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2.4.1. Determination of AntiNuclear Antibodies (aNA) and anti Extractable Nuclear
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Antigen antibodies (ENA)
Total aNA were detected by indirect immunofluorescence on HEp-2 cells (Bio-Rad, Hercules, CA,
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USA) with a screening dilution of 1:100. ENA and anti-dsDNA antibodies were detected by ELIA™ test (ImmunoCAP 250, Thermofisher, FREIBURG, Germany). DNA were measured with dsDNA ELIA™
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test (Phadia GmbH). Antigangliosides were detected by enzyme-linked immunosorbent assay (ELISA).
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2.4.2. Determination of antiphospholipid antibodies In-house ELISAs were used to determine aCL (IgM, IgG, IgA) and anti β2GPI IgA, as previously
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described [33–35]. Determination of antiβ2GPI (IgM, IgG) were obtained by using Orgentec ELISA kit. In each ELISA assay, each sample was tested in three wells, two with antigen, and one without. The latter corresponding to a sample background was subtracted from the specific binding. One negative and two positive controls were included in each run. Positive controls were from patients with
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antiphospholipid syndrome and the negative control from a healthy blood donor. For each antiphospholipid antibody (aPL)-ELISA, the cut-off level was determined by the analysis of the samples of 100 blood donors (control group) and was calculated at the 99th percentile. The results were expressed in GPLU and MPLU for IgG, IgM-aCL and antiβ2GPI, in delta optical density for IgA isotype of aCL and antiβ2GPI. The cut-off values were the following: 1/aCL-ELISA: IgG = 20 GPLU; IgM = 8 MPLU; IgA = 0.25; 2/anti β2GPI-ELISA: IgG = 8 B2GU; IgM = 8 B2MU; IgA = 0.26 (“GPLU, MPLU and B2GU” are arbitrary units for respectively IgG IgM Phospholipids and IgG β2glycoprotein I antibodies).
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Electrophoresis, immunoelectrophoresis and determination of serum free light chain System Capillary®2 were used. Serum immunofixation was obtained by using Hydrasis System. Free light chains were measured by nephelometry.
2.5.
Statistical analyses
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Categorical data were expressed as proportions and compared using χ² or Fisher tests. Continuous data were expressed as mean ± standard deviation and median [interquartile range Q1-Q3][95%
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confidence interval], and were compared using nonparametric Wilcoxon test or Kruskal-Wallis test when three or more items were compared. A significance criterion of α=0.05 was used as a threshold
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to determine the statistical significance. Median 95% confidence interval was also used to confirm significance if results were considered to be unreliable. All analyses were performed using the SAS
Role of the funding source
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2.6.
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statistical software version 9.1 (SAS Institute Inc, Cary, NC).
The funding of the study was supported by the patients’ association "Vaincre les Maladies
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Lysosomales” helping families and patients affected by lysosomal storage disorders. The funder was not involved in the study design; in the collection, analysis, or interpretation of data; in the writing of
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the report; or in the decision to submit the paper for publication.
3. Results
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3.1.
Population characteristics
Forty patients with GD1 were included: 22 females, 18 men; mean age: 52.4±13.4 years (min-max 2679). Twenty healthy subjects were analyzed as well: 13 females and 7 men; mean age 47.3±15.4 (min-max 25-77). There were no significant statistical differences between the 2 groups for either gender or age (p=0.5814 and p=0.2833 respectively). Fifteen GD1 patients were splenectomized (38%). One had partial splenectomy and was associated with non splenectomized patients. Most of the patients were treated (92%, 37/40), with enzyme replacement therapy (91.8%, 34/37) and subtract reduction therapy (8.2%, 3/37). Genotype analysis was made in 32 patients: 10 were homozygote
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N370S, 15 were heterozygote for N370S with L444P for 5, and 7 had other mutations (W312S/W393S, L444P/R463C, C16W/unknown). No L444P homozygote mutation was retrieved (table 2). Twelve patients had osteonecrosis (30%) and 5 had bone infarcts (12%). None of the patients presented clinical symptoms matching with ARC/SLICC LES, SS, APLS or ACR/EULAR RA clinical criteria. None had thyroiditis. Twenty percent of the patients had arthralgia without arthritis and
Autoantibodies
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3.2.
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7% had thrombosis (2 phlebitis and 1 pulmonary embolism).
In the GD1 group, 21 patients (52%) had autoantibodies, compared to 5 volunteers (26%) in the
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control group (p=0.0918) (figure 1). Three patients in GD1 group had several autoantibodies (2 or 3).
3.2.1. Antinuclear antibodies (figures 1 & 2)
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These autoantibodies were antinuclear, aCL, anti β2 Gp1, antigangliosides.
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In the GD1 group, 10 patients (25%) had aNA, compared to 3 (16%) in the control group (p=0.51) (this prevalence is in the range of what is commonly admitted in the general population, [36]). Three GD1
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patients had anti SSa autoantibodies; for one of them, aNA was SSa 52 (Trim 21), and for the 2 other patients aNA was SSa 60 and SSa 52. None of them were associated with SSb antibodies. Only one
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female patient had sicca syndrome, but she had no secondary salivary gland biopsy that could confirm Gougerot Sjögren syndrome and she was being treated with antidepressant medication. Moreover sicca syndrome resolved itself several months despite the persistence of anti SSa. The 2 other patients with anti SSa autoantibodies had no symptoms related to the presence of anti SSa (no arthralgia, no sicca syndrome, no muscular deficiency, no photosensitivity and no Raynaud
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phenomenon).
In the GD1 group, aNAs were strongly positive in 2 patients at 800 IU and 400 IU and moderate in 4 patients at 200 IU. Among patients with aNA, none had criteria for autoimmune disease, even those with a high aNA rate. Two patients had arthralgia but without arthritis which was inconsistent with a diagnosis of any autoimmune disease. None of them had osteonecrosis or bone infarct. Thirty three percent of splenectomized GD1 patients had positive aNA whereas 21% of intact spleen patients had positive aNA (p=0.4633).
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Only 3 GD1 patients were not treated, and these 3 patients had no autoantibodies. However the number of patients included was too small to be conclusive on the impact of GD treatment on the onset of autoantibodies (p=0.5597). Genotype did not influence either the presence or the absence of aNA (p=1.000).
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3.2.2. Antiphospholipid antibodies (figures 1 & 3) APL rate was significantly higher in the GD1 population than in the healthy control group, affecting
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respectively 30% (n=12) of the GD1 group and 5% (n=1) of the control group (p=0.0438). In detail,
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these aPL were anti β2 Gp1 (1 IgG ,2 IgM, 3 IgA) for 5, and CLA IgG for 7. One patient had a high rate of anti β2Gp1 (IgG: 50.16 B2GU, IgM: 85.98 B2MU) and another one had a high rate of IgA anti β2Gp1 (0.32 B2GU). For aCL, we did not find any significant high rates (mean [min-max]: 10.34 [9.07-
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35.6]).
None of the patients with IgG and IgM aPL antibodies had thromboembolic events nor did any female
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experience miscarriage. Only one patient had multiple superficial phlebitis with IgA anti β2Gp1. The presence of aPL antibodies was associated with neither osteonecrosis (p= 0.4526, p=0.4096,
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p=0.6266 respectively for aPL, aCL and anti beta β2Gp1), nor with bone infarcts (p=1.000) (figure 3).
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There was a tendency to observe more aCL antibodies in GD1 patients with splenectomy than in those not splenectomized (33% versus 8%, p=0,0846). The isotype of these aCL were IgA. However, we didn’t find this tendency in anti β2Gp1.
There was no influence of treatment on the presence of aPL antibodies but it was impossible to conclude because there were only 3 non-treated patients.
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3.2.3. Antiganglioside antibodies (figures 1 & 4) Twelve percent of patients (n=5) with GD1 had antiganglioside antibodies vs 15% in the control group (p=1,000). All these 5 GD1 patients had IgM antigangliosides, and none had IgG, whereas in the control group we found either IgG or IgM. None of the patients, in GD1 group or in control group, had neuropathy, which is usually associated with antiganglioside antibodies. Moreover neither splenectomy nor treatment, nor genotype were correlated with the presence of antigangliosides
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3.2.4. Protein electrophoresis and immunoelectrophoresis Sixty five percent (n=26) of GD1 patients had electrophoresis abnormalities, compared to 5% (n=1) in the control group (p<0.0001). Seven patients (12%) had monoclonal gammopathy: 3 IgG lambda, 2 IgG Kappa and IgM lambda, but only one patient had an elevated kappa/lambda ratio associated with
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a monoclonal IgG kappa. We did not have any data about bone marrow smears for this patient. This patient was not splenectomised, his genotype was N370S/N370S and he was treated with enzyme
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replacement therapy.
Among the other electrophoresis results, the following abnormalities were noted: 7 heterogeneity
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restriction, 8 oligoclonalities, 2 hypogammaglobulinemia, 1 isolated hypergammaglobulinemia, and 1 beta/gamma block. Statistical analysis showed no significant link between the presence of
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autoantibodies and presence of monoclonal or polyclonal gammopathy.
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4. Discussion
The present study showed that autoantibodies tend to be more frequent in GD1 patients as compared to healthy control subjects (p=0.0918). Sera from 40 patients with GD1, and 20 sera from healthy
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individuals were analyzed. Fifty two percent of the GD1 patients had serum autoantibodies, almost twice as many as in the control group. The most frequent autoantibodies found in GD1 were aNA with a rate between 200 to 800 IU, and among them anti SSa (Ro 50 or 60). In the control group, no antiSSa antibodies have been found. Despite the presence of such autoantibodies, even with high titers,
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no patients displayed any symptoms of Sjögren syndrome or systemic lupus. In the report by Dayan, ANA were determined in 61/65 patients, and 9 sera (5 females, 4 males) were positive (14.8%). Only 2/21 patients with xerostomia had positive ANA, compared to 7/40 patients with normal salivary output; this difference was not statistically significant (p= 0.61) confirming ANA wasn't associated with autoimmune disease [24]. Unlike other autoantibodies, we did not find a higher prevalence of antiganglioside antibodies in the GD1 patient group compared to the control group. Antiphospholipid antibodies were significantly more present in GD1 patients than in the control group (p=0.0438), and were mainly represented by IgG aCL. No IgA aCL were found. Nine patients had IgG
Page 11 of 25
or IgM, and 3 of them had IgA anti β2Gp1 antibodies. Among patients with IgA antiβ2Gp1, one had a past history of multiple instances of superficial phlebitis. In a recent study Despierres et al. have confirmed the value of IgA anti β2GP1 in the investigation of APLS and have suggested that identification of target domains of anti β2GP1 IgA may be useful in the evaluation of thrombotic risk in lupic patients [35]. IgA anti β2Gp1 are now recognized as an element of the APLS.
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We found more IgG aCL among splenectomized GD1 patients in our population. In the literature,
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results are discordant about the presence of autoantibodies in splenectomized patients [17,37]. Anticardiolipin antibodies can persist after splenectomy done for immune thrombocytopenia [38].
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However as the presence of aCL antibodies has never been demonstrated in the splenectomized population, we hypothesize that they might be specific to GD1 patients. From our point of view, this
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must be carefully considered although our study included only a small number of patients. A recent study, evidenced in patients with GD and splenectomized, significantly fewer CD27(+)/IgM(+) B-cells
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but more CD4(+)/CD45RO(+) and CD8(+)/CD45RO(+) T-cells. These patients had also an absence of circulating dendritic cells and more MGUS [39]. We also searched for a link between the presence of
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aPL antibodies and the occurrence of osteonecrosis or bone infarct, but did not identify more bone events among our patients with aPL antibodies. Thus, this result suggests that the presence of
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antiphospholipid aPL antibodies may not be a risk factor for bone events in GD1 patients. To our knowledge, this is the first exploratory study, comparing GD1 patients (treated or not) to a healthy control group, showing an increased prevalence of autoantibodies in GD1 population. The sera were analyzed in the same laboratory to limit technical bias. However our study has some limitations mainly due to the small size of the groups. Influence of treatment or splenectomy can’t be
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clearly established. A prospective confirmatory study analyzing autoantibodies before and after treatment would be useful to ascertain the impact of treatment on autoimmunity in GD. Moreover, antibodies were tested at only one time point. Multiple measurements might be informative, especially for aPL antibodies. The results of this study confirmed that the mechanisms leading to autoimmunity in GD still remain complex and have not been completely clarified yet.
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Acknowledgments: We dedicate this publication to the memory of Doctor Denis Verrot and Professor Pierre Kaminsky for their significant contributions. We thank Professor Gilles Kaplanski for helpful discussion. We thank all patients and healthy volunteers for accepting to take part in this study, the working group “Médecine Interne et Maladies Lysosomales”, the patients association «Vaincre les Maladies lysosomales» for its
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financial support, Laurence Curel and the Clinical Research department of Saint Joseph Hospital for their support, Terri Galli for the English translation, Genzyme Sanofi for their logistic support, and Dr
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Denis Arniaud for his collaboration. We thank the specialists in GD and the Committee of Evaluation
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and Treatment of Gaucher disease (CETG).
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Disclosure of interest
The authors have no conflicts of interest to disclose that might have a bearing on the work reported in
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the manuscript.
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[18] Sherer Y, Dulitzki M, Levy Y, Livneh A, Shoenfeld Y, Langevitz P. Successful pregnancy outcome in a patient with Gaucher’s disease and antiphospholipid syndrome. Ann Hematol 2002;81:161–3. [19] Lester TJ, Grabowski GA, Goldblatt J, Leiderman IZ, Zaroulis CG. Immune thrombocytopenia and Gaucher’s disease. Am J Med 1984;77:569–71.
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[20] Rosenbaum H, Hoffman R, Brenner B, Rowe J. Immune and autoimmune phenomena in type 1 Gaucher disease. Blood 2002:3623. [21] De Roux-Serratrice C, Serratrice J, San Marco M, Ené N, Ben Amri A, Ben Sahla Thalet H, et al. Devenir des auto-anticorps au cours du traitement de la Maladie de Gaucher.
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[24] Dayan B, Elstein D, Zimran A, Nesher G. Decreased salivary output in patients with Gaucher disease. QJM 2003;96:53-6. QJM Mon J Assoc Physicians 2003:53–6.
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[27] Tan EM, Cohen AS, Fries JF, Masi AT, McShane DJ, Rothfield NF, et al. The 1982 revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum 1982;25:1271–7. [28] Hochberg MC. Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum 1997;40:1725.
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[29] Petri M, Orbai A-M, Alarcón GS, Gordon C, Merrill JT, Fortin PR, et al. Derivation and validation of the Systemic Lupus International Collaborating Clinics classification criteria for systemic lupus erythematosus. Arthritis Rheum 2012;64:2677–86. [30] Shiboski SC, Shiboski CH, Criswell LA, Baer AN, Challacombe S, Lanfranchi H, et al.
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[31] Miyakis S, Lockshin MD, Atsumi T, Branch DW, Brey RL, Cervera R, et al.
International consensus statement on an update of the classification criteria for definite
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antiphospholipid syndrome (APS). J Thromb Haemost JTH 2006;4:295–306. [32] Aletaha D, Neogi T, Silman AJ, Funovits J, Felson DT, Bingham CO, et al. 2010
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[33] Sanmarco M, Bardin N, Camoin L, Beziane A, Dignat-George F, Gamerre M, et al.
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Antigenic profile, prevalence, and clinical significance of antiphospholipid antibodies in women referred for in vitro fertilization. Ann N Y Acad Sci 2007;1108:457–65. [34] Sanmarco M. Les autoanticorps anti-phospholipides sont devenus bien hétérogènes. Immuno Anal Biol Spéc 2011;26:47–54.
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[35] Despierres L, Beziane A, Kaplanski G, Granel B, Serratrice J, Cohen W, et al. Contribution of anti-β2glycoprotein I IgA antibodies to the diagnosis of antiphospholipid syndrome: potential interest of target domains to discriminate thrombotic and non-thrombotic patients. Rheumatol Oxf Engl 2014;53:1215–8. [36] Selmi C, Ceribelli A, Generali E, Scirè CA, Alborghetti F, Colloredo G, et al. Serum antinuclear and extractable nuclear antigen antibody prevalence and associated
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morbidity and mortality in the general population over 15 years. Autoimmun Rev. 2016;15(2):162-6. [37] Balsalobre B, Hernández-Godoy J, Planelles D. Autoantibodies in splenectomized patients as a consequence of abdominal trauma. J Investig Allergol Clin Immunol
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1992;2:91–5. [38] Aaberge IS, Gaarder PI. Autoantibodies in individuals splenectomized because of
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trauma. Scand J Haematol 1986;37:296–300.
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[39] Sønder SU, Limgala RP, Ivanova MM, Ioanou C, Plassmeyer M, Marti GE, et al. Persistent immune alterations and comorbidities in splenectomized patients with
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Gaucher disease. Blood Cells Mol Dis 2016;59:8–15.
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Page 18 of 25
Table 1: Inclusion and exclusion criteria for Gaucher Disease type 1 group Exclusion criteria:
Adult patients >= 18 years old
Under 18 years old
Gaucher disease type 1, confirmed by low
Pregnant or breast-feeding
betaglucosidase, with or without treatment
Patients under administrative control
Patients having read, understood and signed
Prisoners
informed consent.
Patients without social rights
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Emergency hospitalization
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Inclusion criteria:
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Table 2: Patients’ characteristics Characteristics
Gaucher Disease type 1
Control group
group (n=40)
(n=20)
Age
P-Value (Fisher) 0.2833*
Median [Q1Q3][IC95]
50 [43-63][45-57]
49 [36-60][37-59]
Mean ±Sd [min-max]
52.4±13.4 [26-79]
47.3±15.4[25-77]
22/40 (55%)
13/20 (65%)
Gender
Genetic mutations 10/32 (31.2%)
Heterozygote for N370S.– n/total (%)
15/32 (46.9%)
Heterozygote for N370S with L444P.
-
5/15
-
7/32 (21.9)
Treatment– n/n total (%)
37/40 (92%)
With ERT n/total (%)
34/37 (91.8%)
With SRT n/total (%)
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Other mutations
N/A
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Homozygote for N370S – n/total (%)
0.5814
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Women – n/total (%)
-
3/37 (8.2%)
Splenectomy
16/40 (40%)
Total splenectomy – n/total (%)
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15/40 (38%)
Partial splenectomy– n/total (%)
1/40 (3%)
Bone infarcts– n/n total (%)
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0/38 (0%)
0
0/38 (0%)
0
Photosensitivity– n/total (%)
0/38 (0%)
0
Serositis
0/38 (0%)
0
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Malar or discoid Rash
-
12/40 (30%)
ACR Clinical classification criteria Oral ulcer
-
5 (12%)
Osteonecrosis– n/total (%)
Neurologic Disorder
0/38 (0%)
Arthralgia– n/total (%)
8/38 (20%)
0
Arthritis – n/total (%)
0/38 (0%)
0
Sicca syndrome– n/total (%)
1/38 (2%)
0
1.0000
Thrombosis– n/total (%)
3/38 (7%)
0
0.1969**
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Phlebitis – n/total
2/3
Pulmonary embolism – n/total
1/3
0.0413
Values are expressed as n and % or as Median [Q1Q3][IC95] and Mean±SD [min-max] *Wilcoxon test ** χ² test
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Figure legends Figure 1: % of positive results of autoantibodies in the two studied groups
Figure 2: % of positive Antinuclear antibodies in Gaucher’s disease group regarding: splenectomy,
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treatment and genotype mutations
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Figure 3: % of positive Antiphospholipid antibodies in Gaucher’s disease group regarding:
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osteonecrosis and bone infarcts
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Figure 4: % of positive Antiganglioside antibodies in Gaucher’s disease group regarding: splenectomy,
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treatment and genotype mutations
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8d8cc5357e5ba736f1b963c9bd799f5f"> Jacques Serratrice id="aut0095" author-id="S1297319X16302391275622992fa076b08480e12812bb469e"> Marc Berger id="aut0100" author-id="S1297319X16302391d38c0ab15cb058360ce113c0f87c5fce"> Denis Verrot PII: DOI: Reference:
S1297-319X(16)30239-1 http://dx.doi.org/doi:10.1016/j.jbspin.2016.12.002 BONSOI 4513
To appear in: Received date: Accepted date:
14-10-2016 7-12-2016
Please cite this article as: Serratrice C, Bensalah N, Penaranda G, Bardin N, Belmatoug N, Masseau A, Rose C, Lidove O, Camou F, Maillot F, Leguy V, Magy-Bertrand N, Marie I, Cherin P, Bengherbia M, Carballo S, Boucraut J, Serratrice J, Berger M, Verrot D, Prevalence of autoantibodies in the course of Gaucher disease type 1: a multicenter study comparing Gaucher disease patients to healthy subjects, Joint Bone Spine (2016), http://dx.doi.org/10.1016/j.jbspin.2016.12.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Prevalence of autoantibodies in the course of Gaucher disease type 1: a multicenter study comparing Gaucher disease patients to healthy subjects
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Christine Serratricea,b, Nesma Bensalahc, Guillaume Penarandad, Nathalie Bardinc, Nadia Belmatouge, Agathe Masseauf, Christian Roseg, Olivier Lidoveh, Fabrice Camoui, François Maillotj, Vanessa Leguyk, Nadine Magy-Bertrandl, Isabelle Mariem, Patrick Cherinn, Monia Bengherbiae; Sebastian Carballob, José Boucrautc , Jacques Serratriceb, Marc Bergero, Denis Verrota . a
Internal Medicine Department, Hôpital Saint Joseph, 26 bd de Louvain, 13008 Marseille, France Internal Medicine Department, University Hospital of Geneva, Rue Gabrielle-Perret-Gentil 4, 1205 Geneva, Switzerland c Immunology laboratory, University Hôpital La Conception, 147, boulevard Baille, 13005 Marseille d Alphabio Laboratory, Hôpital Européen, 6 Rue Désirée Clary, 13003 Marseille e Internal Medicine Department, Hôpital Beaujon, 100 Boulevard du Général Leclerc, 92110 Clichy f Internal Medicine Department, CHU, 44093 Nantes cedex1, France g Internal Medicine Department, Catholic University, 60 Boulevard Vauban, 59800 Lille, France h Internal Medicine and Rheumatology Department, Hôpital La Croix Saint Simon, 75020 Paris, France i Intensive Care Department, Hôpital Pellegrin, Place Amélie Raba Léon, 33076 Bordeaux, France j CHRU, Université François Rabelais, INSERM 1069, 10 boulevard Tonnellé, 37032 Tours Cedex, France k Internal Medicine and Clinical Immunology Department, CHU, 4 rue Paul Gaffarel BP 77908 , 21079 Dijon, France l Internal Medicine Department, Hôpital Minjoz, 3 Boulevard A. Fleming, 25000 Besançon, France m Internal Medicine Department, CHU, 1 rue de Germont, 76031 Rouen, France n Internal Medicine Department, CHU la Pitié Salpêtrière, 47-83 Boulevard de l'Hôpital, 75013 Paris, France o Hematology Biology, CHU Estaing, 1 Rue Lucie Aubrac, 63100 Clermont Ferrand, France
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Corresponding author : Christine C Serratrice, Internal Medicine Department Hôpital Saint Joseph, 26 bd de Louvain 13008 Marseille, France [email protected] Tél.: 041798731474
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Abstract Objectives. Type 1 Gaucher disease may be related to the presence of autoantibodies. Their clinical significance is questioned. Primary Endpoint was to compare the prevalence of autoantibodies in type 1 Gaucher disease
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patients with healthy subjects, seeking correlations with autoimmune characteristics. Secondary endpoints were to determine whether patients with autoantibodies reported autoimmunity-related symptoms and if genotype, splenectomy or treatment influenced autoantibodies presence.
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Methods. Type 1 Gaucher disease patients and healthy volunteers were included in this national
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multicenter exploratory study. Autoantibodies presence was compared in both groups and assessed regarding to genotype, splenectomy, Gaucher disease treatment and autoimmunity-related symptoms.
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Results. Twenty healthy subjects and 40 type 1 Gaucher disease patients were included. Of the studied group: 15 patients undergone splenectomy, 37 were treated either with enzyme replacement therapy (34) or with substrate reduction therapy (3), 25 were homozygous/heterozygous for the N370S
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mutation.
In type 1 Gaucher disease group (studied group), 52% had positive autoantibodies versus 26% in
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control group. Antiphospholipid antibodies were more frequent in the studied group (30% vs 5%), but without correlation to thrombosis, osteonecrosis or bone infarcts. In the studied group, antinuclear
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antibodies were more frequent (25% vs 16%). None of the patients with autoantibodies had clinical manifestations of autoimmune diseases. Autoantibodies were not correlated with treatment, genotype, or splenectomy, except for anticardiolipid, more frequent in splenectomized patients. Conclusions. In type 1 Gaucher disease, autoantibodies were more frequent compared to a healthy population. However, they were not associated with an increased prevalence of clinical active
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autoimmune diseases.
Keywords: Gaucher disease, Auto immunity, Antibodies, Antinuclear, Antiphospholipid.
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1. Introduction Gaucher disease (GD) is a rare genetic lysosomal storage disorder inherited in an autosomal recessive pattern [1]. GD is due to the deficiency of a lysosomal enzyme, acid beta-glucosidase (or
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glucocerebrosidase) or in rare cases of its activator, saposin C. Prevalence worldwide is 1/60000 and 1/6000 in Israel. In the French Gaucher registry, prevalence is estimated at 1/140000 [2]. GD
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diagnosis is confirmed by the detection of low glucocerebrosidase activity in peripheral leucocytes [3] .
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Three different types of GD have been described: type 1 (GD1) is characterized by thrombocytopenia, anemia, an enlarged spleen and liver as well as bone complications (Erlenmeyer flask deformity,
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osteoporosis, lytic lesions, pathological and vertebral fractures, bone infarcts and avascular necrosis leading to degenerative arthropathy). GD1 represents 90% of all cases. Type 2 Gaucher disease is an
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acute lethal neuronopathic form resulting in death within the first or second year of life, often occurring after aspiration pneumonia and/or apnea or laryngospasm [4]. Type 3, or chronic neuronopathic,
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Gaucher disease is a “catch all” encompassing patients who survived infancy but had some forms of neurologic involvement. Often the neurologic manifestations are subtle, such as the slowing of the horizontal saccadic eye movements, but other patients develop neurodegeneration, myoclonic
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epilepsy, or psychiatric manifestations. It is now recognized that there is an overlap among these phenotypes and GD is considered as a continuum between these 3 entities [5].
Since 1991 GD1 can be effectively treated by enzyme replacement therapy (ERT). Three recombinant glucocerebrosidase are now available: imiglucerase (Sanofi/Genzyme Corporation); velaglucerase
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(Shire HGT); taliglucerase, (Pfizer). Two treatments by substrate reduction therapy obtained a marketing authorization: miglustat, (Actelion Pharmaceuticals) and eliglustat (Sanofi/Genzyme Corporation). Mutations in the gene coding glucocerebrosidase (GBA) lead to an accumulation of the sphingolipid glucocerebroside within macrophages [3]. The accumulation of lipids in GD cells is associated with an inflammatory state, an activation of macrophages, and cytokine secretion [6,7]. Some of these interleukins, as Il-6, Il-10 or Il-2 R, are associated with the hyperactivity of the immune system and
Page 3 of 25
related to the development of monoclonal or polyclonal gammopathies [8,9]. Moreover, lysosome plays a key role for antigenic presentation through major histocompatibility complex (MHC) molecules I and II, and CD1 (cluster of differentiation 1) molecules. In 2005 Balreira and coll suggested a link between sphingolipid metabolism and abnormalities in expression of MHC family [10]. They first demonstrated an increased expression of CD1d and MHC class II in GD monocytes. More recently,
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Nair and coll, have reported that β -glucosylceramide 22:0 and glucosylsphingosine can be recognized by a distinct subset of CDd1-restricted human and murine type II NKT (Natural Killer T) cells which
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could lead to a dysregulation of humoral immunity in metabolic lipid disorders [11]. On the other hand,
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ERT induce a decrease in MHC class II expression which may interfere with humoral response. Thus, this biological context may explain the high frequency of polyclonal and monoclonal gammopathies,
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associated with a high risk of multiple myeloma. Polyclonal gammopathy can favour the emergence of autoantibodies but there is little data published about autoimmunity during the course GD1. One of the first reports of immunoglobulin abnormalities was published in 1968 [12]. Thereafter, several studies
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analyzed the link between GD and monoclonal gammopathies, and the risk of myeloma [9,13].
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Shoenfeld has shown that in GD there was a chronic stimulation of the humoral immune system [14]. Later he searched for autoantibodies reactive to 14 autoantigens in a cohort of 43 patients with GD
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[15]. The sera of 5 patients reacted with 1 autoantigen, the sera of 17 patients reacted with 2 to 6 antigens, and 11 patients reacted with 8 to 12 antigens. No correlation was found between autoreactivity and serum immunoglobulin concentration. Moreover these antibodies are “natural antibodies” and don’t lead to any immune manifestations. This hypothesis was later confirmed by several reports on the presence of anticardiolipid (aCL) and/or lupus anticoagulant in GD, without
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clinical manifestations [16].
However, some rare cases of autoimmune diseases in patients with GD1 have been reported as autoimmune hemolytic anemia (Burroughs S PR, Bartolomeo A, Greenstein R. Hemolytic anemia in Gaucher Disease. Blood Cells Molecular Diseases 1994:589A) and [17], antiphospholipid syndrome
[18], and immune thrombocytopenia [19]. Among 84 consecutive patients, Rosenbaum et al. identified 3.5% of patients with immune thrombocytopenia, 2.3% with immune hemolytic anemia, but also the presence of aCL and lupus anticoagulant in 5.9% (one had antiphospholipid syndrome [APLS] with
Page 4 of 25
recurrent abortions), anti-nuclear and anti DNA in 8.3% and anti-parietal cells antibodies in 3.5% [20]. In a preliminary limited personal experience, autoantibodies have been detected in 62.5% of GD1 patients, and we have noted that for a few patients, antibody rate decreased after treatment [21]. Curiously, to our knowledge, with the exception of immune hemolytic anemia, immune thrombocytopenia, and APLS, no significant association of GD1 with other autoimmune diseases,
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such as Grave's disease, Hashimoto disease or systemic sclerosis have been reported. Some rare case reports mentioned coexistence of GD1 and diabetes mellitus (only one case published) [22],
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recent report described the coexistence of lupus nephritis and GD1 [26].
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rheumatoid arthritis (one case reported) [23], sicca syndrome (two cases reported) [24,25]. One
In our study, the primary endpoint was to compare the prevalence of autoantibodies in GD1 patients
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with healthy subjects. Secondary endpoints were to determine whether patients with auto-antibodies reported autoimmunity-related symptoms and if genotype, splenectomy or treatment had an impact on
2.1.
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2. Methods
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the presence or absence of autoantibodies.
Investigators and centers
All investigators of this study belonged to the "Internal Medicine and Lysosomal Disease" group created in 2007 and the Committee for evaluation and treatment of Gaucher disease led by Pr Marc
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Berger and approved in 2004. All investigators were involved in the management of Gaucher disease. The principal investigator was Dr. Christine Serratrice, Internal Medicine department, Saint Joseph Hospital (Marseille). All other investigators were affiliated to university hospitals or general hospital centers. Patients were included in 12 centers: Hôpital Saint Joseph-Marseille, Hôpital Beaujon-Clichy, CHUNantes, Catholic University-Lille, CHU Hôpital Brabois-Vandoeuvre les Nancy, Hôpital La Croix Saint Simon-Paris, Hôpital Pellegrin-Bordeaux, CHRU-Tours, CHU-Dijon, Hôpital Minjoz-Besançon, CHU Bois Guillaume-Rouen, CHU Pitié Salpêtrière-Paris.
Page 5 of 25
2.2.
Patients
Forty consecutive patients with GD1 from the 12 centers and 20 healthy volunteers (control group) were included in the study.
2.2.1. Details of ethics approval
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This national multicenter exploratory study obtained agreements from the French Ethics Committee
(CPP Aix Marseille I), and from the French National Agency for Medicines and Health Products Safety
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was registered in ClinicalTrial.gov under the number NCT02650219.
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(ANSM). The study received approval from the French Data Protection Authority (CNIL). The study
2.2.2. Patient consent statement
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All investigations were conducted according to the declaration of Helsinki.
Written informed consents were obtained after the nature and possible consequences of the studies
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had been fully explained. All patients and healthy subjects gave specific written consent for blood sample collection and blood samples were collected during a routine monitoring of the disease.
Data collection
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2.3.
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Inclusion criteria of GD1 patients are presented in table 1.
For all GD1 patients and healthy subjects the following elements were collected: diagnosis characteristics, splenectomy, bone events, pulmonary hypertension, specific treatment and nonspecific therapies. Taking into account clinical American College of Rheumatology (ACR) and
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Systemic Lupus International Collaborating Clinics (SLICC) classification criterias [27-29] clinical features for systemic lupus (SLE), sicca syndrome (SS), [30] Antiphospholipid syndrome (APLS), [31] and ACR /EULAR Rheumatoid arthritis (RA) clinical criterias [32] as well as diagnosis of thyroiditis, were also systematically evaluated in patients and controls. Furthermore, sera from 40 GD1 patients and from the 20 healthy subjects were collected from January 2010 to April 2011. Blood samples were collected during a routine monitoring of the disease in GD1 group. Blood samples were forwarded to a single specialized immunology laboratory and were tested
Page 6 of 25
during the same period of time to avoid technical bias. GD diagnosis was confirmed by demonstration of low β-glucosidase acid activity and/or molecular diagnosis. Thus, antinuclear, anti-SSa, anti-SSb, anti-RNP, anti-DNA, anti-Sm, anticardiolipid, anti β2Gp1, antiganglioside autoantibodies were
2.4.
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systematically screened in every patient.
Biological analyses
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2.4.1. Determination of AntiNuclear Antibodies (aNA) and anti Extractable Nuclear
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Antigen antibodies (ENA)
Total aNA were detected by indirect immunofluorescence on HEp-2 cells (Bio-Rad, Hercules, CA,
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USA) with a screening dilution of 1:100. ENA and anti-dsDNA antibodies were detected by ELIA™ test (ImmunoCAP 250, Thermofisher, FREIBURG, Germany). DNA were measured with dsDNA ELIA™
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test (Phadia GmbH). Antigangliosides were detected by enzyme-linked immunosorbent assay (ELISA).
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2.4.2. Determination of antiphospholipid antibodies In-house ELISAs were used to determine aCL (IgM, IgG, IgA) and anti β2GPI IgA, as previously
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described [33–35]. Determination of antiβ2GPI (IgM, IgG) were obtained by using Orgentec ELISA kit. In each ELISA assay, each sample was tested in three wells, two with antigen, and one without. The latter corresponding to a sample background was subtracted from the specific binding. One negative and two positive controls were included in each run. Positive controls were from patients with
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antiphospholipid syndrome and the negative control from a healthy blood donor. For each antiphospholipid antibody (aPL)-ELISA, the cut-off level was determined by the analysis of the samples of 100 blood donors (control group) and was calculated at the 99th percentile. The results were expressed in GPLU and MPLU for IgG, IgM-aCL and antiβ2GPI, in delta optical density for IgA isotype of aCL and antiβ2GPI. The cut-off values were the following: 1/aCL-ELISA: IgG = 20 GPLU; IgM = 8 MPLU; IgA = 0.25; 2/anti β2GPI-ELISA: IgG = 8 B2GU; IgM = 8 B2MU; IgA = 0.26 (“GPLU, MPLU and B2GU” are arbitrary units for respectively IgG IgM Phospholipids and IgG β2glycoprotein I antibodies).
Page 7 of 25
Electrophoresis, immunoelectrophoresis and determination of serum free light chain System Capillary®2 were used. Serum immunofixation was obtained by using Hydrasis System. Free light chains were measured by nephelometry.
2.5.
Statistical analyses
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Categorical data were expressed as proportions and compared using χ² or Fisher tests. Continuous data were expressed as mean ± standard deviation and median [interquartile range Q1-Q3][95%
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confidence interval], and were compared using nonparametric Wilcoxon test or Kruskal-Wallis test when three or more items were compared. A significance criterion of α=0.05 was used as a threshold
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to determine the statistical significance. Median 95% confidence interval was also used to confirm significance if results were considered to be unreliable. All analyses were performed using the SAS
Role of the funding source
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2.6.
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statistical software version 9.1 (SAS Institute Inc, Cary, NC).
The funding of the study was supported by the patients’ association "Vaincre les Maladies
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Lysosomales” helping families and patients affected by lysosomal storage disorders. The funder was not involved in the study design; in the collection, analysis, or interpretation of data; in the writing of
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the report; or in the decision to submit the paper for publication.
3. Results
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3.1.
Population characteristics
Forty patients with GD1 were included: 22 females, 18 men; mean age: 52.4±13.4 years (min-max 2679). Twenty healthy subjects were analyzed as well: 13 females and 7 men; mean age 47.3±15.4 (min-max 25-77). There were no significant statistical differences between the 2 groups for either gender or age (p=0.5814 and p=0.2833 respectively). Fifteen GD1 patients were splenectomized (38%). One had partial splenectomy and was associated with non splenectomized patients. Most of the patients were treated (92%, 37/40), with enzyme replacement therapy (91.8%, 34/37) and subtract reduction therapy (8.2%, 3/37). Genotype analysis was made in 32 patients: 10 were homozygote
Page 8 of 25
N370S, 15 were heterozygote for N370S with L444P for 5, and 7 had other mutations (W312S/W393S, L444P/R463C, C16W/unknown). No L444P homozygote mutation was retrieved (table 2). Twelve patients had osteonecrosis (30%) and 5 had bone infarcts (12%). None of the patients presented clinical symptoms matching with ARC/SLICC LES, SS, APLS or ACR/EULAR RA clinical criteria. None had thyroiditis. Twenty percent of the patients had arthralgia without arthritis and
Autoantibodies
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3.2.
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7% had thrombosis (2 phlebitis and 1 pulmonary embolism).
In the GD1 group, 21 patients (52%) had autoantibodies, compared to 5 volunteers (26%) in the
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control group (p=0.0918) (figure 1). Three patients in GD1 group had several autoantibodies (2 or 3).
3.2.1. Antinuclear antibodies (figures 1 & 2)
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These autoantibodies were antinuclear, aCL, anti β2 Gp1, antigangliosides.
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In the GD1 group, 10 patients (25%) had aNA, compared to 3 (16%) in the control group (p=0.51) (this prevalence is in the range of what is commonly admitted in the general population, [36]). Three GD1
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patients had anti SSa autoantibodies; for one of them, aNA was SSa 52 (Trim 21), and for the 2 other patients aNA was SSa 60 and SSa 52. None of them were associated with SSb antibodies. Only one
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female patient had sicca syndrome, but she had no secondary salivary gland biopsy that could confirm Gougerot Sjögren syndrome and she was being treated with antidepressant medication. Moreover sicca syndrome resolved itself several months despite the persistence of anti SSa. The 2 other patients with anti SSa autoantibodies had no symptoms related to the presence of anti SSa (no arthralgia, no sicca syndrome, no muscular deficiency, no photosensitivity and no Raynaud
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phenomenon).
In the GD1 group, aNAs were strongly positive in 2 patients at 800 IU and 400 IU and moderate in 4 patients at 200 IU. Among patients with aNA, none had criteria for autoimmune disease, even those with a high aNA rate. Two patients had arthralgia but without arthritis which was inconsistent with a diagnosis of any autoimmune disease. None of them had osteonecrosis or bone infarct. Thirty three percent of splenectomized GD1 patients had positive aNA whereas 21% of intact spleen patients had positive aNA (p=0.4633).
Page 9 of 25
Only 3 GD1 patients were not treated, and these 3 patients had no autoantibodies. However the number of patients included was too small to be conclusive on the impact of GD treatment on the onset of autoantibodies (p=0.5597). Genotype did not influence either the presence or the absence of aNA (p=1.000).
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3.2.2. Antiphospholipid antibodies (figures 1 & 3) APL rate was significantly higher in the GD1 population than in the healthy control group, affecting
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respectively 30% (n=12) of the GD1 group and 5% (n=1) of the control group (p=0.0438). In detail,
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these aPL were anti β2 Gp1 (1 IgG ,2 IgM, 3 IgA) for 5, and CLA IgG for 7. One patient had a high rate of anti β2Gp1 (IgG: 50.16 B2GU, IgM: 85.98 B2MU) and another one had a high rate of IgA anti β2Gp1 (0.32 B2GU). For aCL, we did not find any significant high rates (mean [min-max]: 10.34 [9.07-
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35.6]).
None of the patients with IgG and IgM aPL antibodies had thromboembolic events nor did any female
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experience miscarriage. Only one patient had multiple superficial phlebitis with IgA anti β2Gp1. The presence of aPL antibodies was associated with neither osteonecrosis (p= 0.4526, p=0.4096,
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p=0.6266 respectively for aPL, aCL and anti beta β2Gp1), nor with bone infarcts (p=1.000) (figure 3).
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There was a tendency to observe more aCL antibodies in GD1 patients with splenectomy than in those not splenectomized (33% versus 8%, p=0,0846). The isotype of these aCL were IgA. However, we didn’t find this tendency in anti β2Gp1.
There was no influence of treatment on the presence of aPL antibodies but it was impossible to conclude because there were only 3 non-treated patients.
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3.2.3. Antiganglioside antibodies (figures 1 & 4) Twelve percent of patients (n=5) with GD1 had antiganglioside antibodies vs 15% in the control group (p=1,000). All these 5 GD1 patients had IgM antigangliosides, and none had IgG, whereas in the control group we found either IgG or IgM. None of the patients, in GD1 group or in control group, had neuropathy, which is usually associated with antiganglioside antibodies. Moreover neither splenectomy nor treatment, nor genotype were correlated with the presence of antigangliosides
Page 10 of 25
3.2.4. Protein electrophoresis and immunoelectrophoresis Sixty five percent (n=26) of GD1 patients had electrophoresis abnormalities, compared to 5% (n=1) in the control group (p<0.0001). Seven patients (12%) had monoclonal gammopathy: 3 IgG lambda, 2 IgG Kappa and IgM lambda, but only one patient had an elevated kappa/lambda ratio associated with
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a monoclonal IgG kappa. We did not have any data about bone marrow smears for this patient. This patient was not splenectomised, his genotype was N370S/N370S and he was treated with enzyme
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replacement therapy.
Among the other electrophoresis results, the following abnormalities were noted: 7 heterogeneity
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restriction, 8 oligoclonalities, 2 hypogammaglobulinemia, 1 isolated hypergammaglobulinemia, and 1 beta/gamma block. Statistical analysis showed no significant link between the presence of
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autoantibodies and presence of monoclonal or polyclonal gammopathy.
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4. Discussion
The present study showed that autoantibodies tend to be more frequent in GD1 patients as compared to healthy control subjects (p=0.0918). Sera from 40 patients with GD1, and 20 sera from healthy
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individuals were analyzed. Fifty two percent of the GD1 patients had serum autoantibodies, almost twice as many as in the control group. The most frequent autoantibodies found in GD1 were aNA with a rate between 200 to 800 IU, and among them anti SSa (Ro 50 or 60). In the control group, no antiSSa antibodies have been found. Despite the presence of such autoantibodies, even with high titers,
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no patients displayed any symptoms of Sjögren syndrome or systemic lupus. In the report by Dayan, ANA were determined in 61/65 patients, and 9 sera (5 females, 4 males) were positive (14.8%). Only 2/21 patients with xerostomia had positive ANA, compared to 7/40 patients with normal salivary output; this difference was not statistically significant (p= 0.61) confirming ANA wasn't associated with autoimmune disease [24]. Unlike other autoantibodies, we did not find a higher prevalence of antiganglioside antibodies in the GD1 patient group compared to the control group. Antiphospholipid antibodies were significantly more present in GD1 patients than in the control group (p=0.0438), and were mainly represented by IgG aCL. No IgA aCL were found. Nine patients had IgG
Page 11 of 25
or IgM, and 3 of them had IgA anti β2Gp1 antibodies. Among patients with IgA antiβ2Gp1, one had a past history of multiple instances of superficial phlebitis. In a recent study Despierres et al. have confirmed the value of IgA anti β2GP1 in the investigation of APLS and have suggested that identification of target domains of anti β2GP1 IgA may be useful in the evaluation of thrombotic risk in lupic patients [35]. IgA anti β2Gp1 are now recognized as an element of the APLS.
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We found more IgG aCL among splenectomized GD1 patients in our population. In the literature,
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results are discordant about the presence of autoantibodies in splenectomized patients [17,37]. Anticardiolipin antibodies can persist after splenectomy done for immune thrombocytopenia [38].
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However as the presence of aCL antibodies has never been demonstrated in the splenectomized population, we hypothesize that they might be specific to GD1 patients. From our point of view, this
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must be carefully considered although our study included only a small number of patients. A recent study, evidenced in patients with GD and splenectomized, significantly fewer CD27(+)/IgM(+) B-cells
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but more CD4(+)/CD45RO(+) and CD8(+)/CD45RO(+) T-cells. These patients had also an absence of circulating dendritic cells and more MGUS [39]. We also searched for a link between the presence of
ed
aPL antibodies and the occurrence of osteonecrosis or bone infarct, but did not identify more bone events among our patients with aPL antibodies. Thus, this result suggests that the presence of
ce pt
antiphospholipid aPL antibodies may not be a risk factor for bone events in GD1 patients. To our knowledge, this is the first exploratory study, comparing GD1 patients (treated or not) to a healthy control group, showing an increased prevalence of autoantibodies in GD1 population. The sera were analyzed in the same laboratory to limit technical bias. However our study has some limitations mainly due to the small size of the groups. Influence of treatment or splenectomy can’t be
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clearly established. A prospective confirmatory study analyzing autoantibodies before and after treatment would be useful to ascertain the impact of treatment on autoimmunity in GD. Moreover, antibodies were tested at only one time point. Multiple measurements might be informative, especially for aPL antibodies. The results of this study confirmed that the mechanisms leading to autoimmunity in GD still remain complex and have not been completely clarified yet.
Page 12 of 25
Acknowledgments: We dedicate this publication to the memory of Doctor Denis Verrot and Professor Pierre Kaminsky for their significant contributions. We thank Professor Gilles Kaplanski for helpful discussion. We thank all patients and healthy volunteers for accepting to take part in this study, the working group “Médecine Interne et Maladies Lysosomales”, the patients association «Vaincre les Maladies lysosomales» for its
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financial support, Laurence Curel and the Clinical Research department of Saint Joseph Hospital for their support, Terri Galli for the English translation, Genzyme Sanofi for their logistic support, and Dr
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Denis Arniaud for his collaboration. We thank the specialists in GD and the Committee of Evaluation
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and Treatment of Gaucher disease (CETG).
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Disclosure of interest
The authors have no conflicts of interest to disclose that might have a bearing on the work reported in
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the manuscript.
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Page 13 of 25
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Antigenic profile, prevalence, and clinical significance of antiphospholipid antibodies in women referred for in vitro fertilization. Ann N Y Acad Sci 2007;1108:457–65. [34] Sanmarco M. Les autoanticorps anti-phospholipides sont devenus bien hétérogènes. Immuno Anal Biol Spéc 2011;26:47–54.
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[35] Despierres L, Beziane A, Kaplanski G, Granel B, Serratrice J, Cohen W, et al. Contribution of anti-β2glycoprotein I IgA antibodies to the diagnosis of antiphospholipid syndrome: potential interest of target domains to discriminate thrombotic and non-thrombotic patients. Rheumatol Oxf Engl 2014;53:1215–8. [36] Selmi C, Ceribelli A, Generali E, Scirè CA, Alborghetti F, Colloredo G, et al. Serum antinuclear and extractable nuclear antigen antibody prevalence and associated
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morbidity and mortality in the general population over 15 years. Autoimmun Rev. 2016;15(2):162-6. [37] Balsalobre B, Hernández-Godoy J, Planelles D. Autoantibodies in splenectomized patients as a consequence of abdominal trauma. J Investig Allergol Clin Immunol
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1992;2:91–5. [38] Aaberge IS, Gaarder PI. Autoantibodies in individuals splenectomized because of
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trauma. Scand J Haematol 1986;37:296–300.
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[39] Sønder SU, Limgala RP, Ivanova MM, Ioanou C, Plassmeyer M, Marti GE, et al. Persistent immune alterations and comorbidities in splenectomized patients with
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Gaucher disease. Blood Cells Mol Dis 2016;59:8–15.
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Table 1: Inclusion and exclusion criteria for Gaucher Disease type 1 group Exclusion criteria:
Adult patients >= 18 years old
Under 18 years old
Gaucher disease type 1, confirmed by low
Pregnant or breast-feeding
betaglucosidase, with or without treatment
Patients under administrative control
Patients having read, understood and signed
Prisoners
informed consent.
Patients without social rights
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Emergency hospitalization
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Inclusion criteria:
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Table 2: Patients’ characteristics Characteristics
Gaucher Disease type 1
Control group
group (n=40)
(n=20)
Age
P-Value (Fisher) 0.2833*
Median [Q1Q3][IC95]
50 [43-63][45-57]
49 [36-60][37-59]
Mean ±Sd [min-max]
52.4±13.4 [26-79]
47.3±15.4[25-77]
22/40 (55%)
13/20 (65%)
Gender
Genetic mutations 10/32 (31.2%)
Heterozygote for N370S.– n/total (%)
15/32 (46.9%)
Heterozygote for N370S with L444P.
-
5/15
-
7/32 (21.9)
Treatment– n/n total (%)
37/40 (92%)
With ERT n/total (%)
34/37 (91.8%)
With SRT n/total (%)
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Other mutations
N/A
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Homozygote for N370S – n/total (%)
0.5814
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Women – n/total (%)
-
3/37 (8.2%)
Splenectomy
16/40 (40%)
Total splenectomy – n/total (%)
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15/40 (38%)
Partial splenectomy– n/total (%)
1/40 (3%)
Bone infarcts– n/n total (%)
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0/38 (0%)
0
0/38 (0%)
0
Photosensitivity– n/total (%)
0/38 (0%)
0
Serositis
0/38 (0%)
0
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Malar or discoid Rash
-
12/40 (30%)
ACR Clinical classification criteria Oral ulcer
-
5 (12%)
Osteonecrosis– n/total (%)
Neurologic Disorder
0/38 (0%)
Arthralgia– n/total (%)
8/38 (20%)
0
Arthritis – n/total (%)
0/38 (0%)
0
Sicca syndrome– n/total (%)
1/38 (2%)
0
1.0000
Thrombosis– n/total (%)
3/38 (7%)
0
0.1969**
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Phlebitis – n/total
2/3
Pulmonary embolism – n/total
1/3
0.0413
Values are expressed as n and % or as Median [Q1Q3][IC95] and Mean±SD [min-max] *Wilcoxon test ** χ² test
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Figure legends Figure 1: % of positive results of autoantibodies in the two studied groups
Figure 2: % of positive Antinuclear antibodies in Gaucher’s disease group regarding: splenectomy,
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treatment and genotype mutations
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Figure 3: % of positive Antiphospholipid antibodies in Gaucher’s disease group regarding:
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osteonecrosis and bone infarcts
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Figure 4: % of positive Antiganglioside antibodies in Gaucher’s disease group regarding: splenectomy,
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treatment and genotype mutations
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