Alice Le Clech

Noémie Simon-Tillaux

François Provôt

Yahsou Delmas

Paula Vieira-Martins

Sophie Limou

Jean-Michel Halimi

Moglie Le Quintrec

Ludivine Lebourg

Steven Grangé

Alexandre Karras

David Ribes

Noémie Jourde-Chiche

Eric Rondeau

Véronique Frémeaux- Bacchi

Fadi Fakhouri
email: fadi.fakhouri@univ-nantes.fr

Atypical and secondary hemolytic uremic syndromes have a distinct presentation and no common genetic risk factors

Keywords: haemolytic uremic syndrome, thrombotic microangiopathy, complement, eculizumab

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction:

The term hemolytic uremic syndrome (HUS) encompasses several forms of thrombotic microangiopathy affecting predominantly the kidney: shiga toxin-producing Escherichia coli. (STEC)-HUS, cobalamin C-defect HUS, Diacyl Glycerol Kinase epsilon-HUS, atypical HUS due to a dysregulation of the complement alternative pathway, and HUS associated with coexisting diseases and conditions (malignancy, drugs, transplantation, systemic diseases and infections) [START_REF] Fakhouri | Haemolytic uraemic syndrome[END_REF], usually termed "secondary HUS". These various forms of hemolytic uremic syndrome (HUS) have distinct mechanisms of initial endothelial cell injury but a common final phenotype of activated and prothrombotic endothelial cell [START_REF] Fakhouri | Haemolytic uraemic syndrome[END_REF]. Several classifications of HUS have been previously proposed, each carrying caveats [START_REF] George | Syndromes of thrombotic microangiopathy[END_REF][START_REF] Scully | How I treat thrombotic thrombocytopenic purpura and atypical haemolytic uraemic syndrome[END_REF][START_REF] Cataland | How I treat: the clinical differentiation and initial treatment of adult patients with atypical hemolytic uremic syndrome[END_REF], and the terminology of HUS is still evolving.

The group of secondary HUS is heterogeneous particularly in terms of mechanisms of endothelial cell injury [START_REF] Fakhouri | Haemolytic uraemic syndrome[END_REF]. It has also to date attracted a more limited number of studies as compared to atypical HUS or STEC-HUS and data regarding clinical presentation, response to treatments and outcome of secondary HUS, generated through large cohorts and registries, are lacking.

Moreover, the identification of the dysregulation of the complement alternative pathway as a major risk factor for atypical HUS [START_REF] Fremeaux-Bacchi | Genetics and outcome of atypical hemolytic uremic syndrome: a nationwide French series comparing children and adults[END_REF][START_REF] Noris | Atypical hemolytic-uremic syndrome[END_REF], and the clinical availability of the first complement inhibitor eculizumab [START_REF] Greenbaum | Eculizumab is a safe and effective treatment in pediatric patients with atypical hemolytic uremic syndrome[END_REF][START_REF] Legendre | Terminal complement inhibitor eculizumab in atypical hemolyticuremic syndrome[END_REF][START_REF] Licht | Efficacy and safety of eculizumab in atypical hemolytic uremic syndrome from 2-year extensions of phase 2 studies[END_REF][START_REF] Fakhouri | Terminal Complement Inhibitor Eculizumab in Adult Patients With Atypical Hemolytic Uremic Syndrome: A Single-Arm, Open-Label Trial[END_REF] have transformed the approach to all types of HUS. Currently, one of the most debated questions is whether, beyond atypical HUS, complement dysregulation/overactivation is involved in the pathogenesis of other types of HUS. This is particularly true for the group of secondary HUS for which natural history and therapeutic options remain ill-defined. Thus, we analyzed the presentation and frequency of complement gene variants in a large cohort of secondary HUS included in the nationwide French HUS registry.

Results

Presentation at diagnosis

One hundred and ten patients with secondary HUS diagnosed between 1999 and 2017 were included in the study. Thirty (27%) patients were included before 2010 and 80 (73%) between 2011 and 2017.

Mean age was 44 years . At diagnosis, mean serum creatinine was 3.9 mg/dL (0.5-25) and 45 (40%) of patients required dialysis. Mean platelet count was 94 G/L (10-450) and 11 (10%) patients had normal platelet count at presentation. Thirty-one (28%) patients presented with extra-renal manifestations, mainly (n=20, 65%) neurological involvement (confusion, seizure, paresthesia). A kidney biopsy was performed in 51 (46%) patients and disclosed signs of active TMA in all patients.

Main clinical and biological characteristics of these patients are shown in Tables 1 and2 and in Supplemental Figure S1 and Table S1.

Diseases/conditions associated to secondary HUS

Secondary HUS in our series was associated to drugs (n=32, 29%), autoimmune diseases (n=26, 24%), bacterial and viral infections (n=18, 17%), malignancies (n=11, 10%), glomerulopathies (n=10, 9%), extra-renal organ transplantation (n=9, 8%), and pancreatitis (n= 4, 3%) (Table 1 and Supplemental Table S1).

Complement work-up.

C3 serum level measured during HUS active phase was decreased in 17 (15%) patients, of whom nine had a systemic lupus erythematous-associated HUS. Eight patients had a low C3 and C4 plasma levels as a result of an activation of the classical pathway and 9 had only a low C3 level suggestive of an activation of the alternative pathway. Six (5%) patients had rare variants with MAF<0.1% in one of the 6 tested complement genes. These variants are located in CFH (n=3), CFI (n=1) and THBD (n=2) genes (Tables 3 and4). Out of the six rare variants, two are classified as pathogenic, the remaining four variants being classified as of undetermined significance. Characteristics of the 6 patients with complement gene variants are shown in Table 4. One patient had anti-FH antibodies with moderate titre (< 1000 arbitrary unit) and did not carry the homozygous CFHR1-3 deletion. None had a family history suggestive of HUS.

The frequency of complement gene rare variants in secondary HUS patients did not differ from the one found in healthy donors (5 % (n=6/110) vs 6% (=5/80) and 8% (n=42/503) in French and European controls, respectively) (p=1/0.4) (Table 3 and Supplemental Tables S2, S3 and S4). Altogether, the frequency of rare variants per gene was not significantly different between secondary HUS patients and controls. A rare (MAF <0.1%) pathogenic complement gene variant was identified in 2/111 (2%) secondary HUS patients versus 0/80 (0%) French controls (p=0.5) and 3/503 (1%) European controls (p=0.2). In contrast, the frequency of complement gene rare variants significantly differed between aHUS and secondary HUS patients (Table 3).

The frequency of at-risk polymorphisms tgtgt (CFH) and of CFHR1-3 deletion did not significantly differ between secondary HUS patients and healthy donors (Supplemental Tables S2 andS4). The frequency of at-risk polymorphisms ggaac (MCP) was increased in secondary HUS patients compared to French healthy donors (17% vs 6%; p=0.03)

Treatment

Fifty-five (50%) patients underwent plasma exchanges, ten (9%) received plasma infusions, thirty-one (27%) received corticosteroids and thirteen (27%) cyclophosphamide (used as a treatment of the underlying cause in all cases) (Table 1). Eculizumab was used in 38 (35%) cases. Treatment with eculizumab (regimen similar to the one used in atypical HUS [START_REF] Fakhouri | Terminal Complement Inhibitor Eculizumab in Adult Patients With Atypical Hemolytic Uremic Syndrome: A Single-Arm, Open-Label Trial[END_REF]) was started as a second-line therapy in 28 (26%) patients after plasma exchanges and treatment of the underlying cause failed to induce HUS remission. All eculizumab-treated patients received anti-meningococcal vaccines and antibioprophylaxis with methylpenicillin.

Outcome

Eight (7%) patients were lost to follow-up. Mean follow-up was 21 months (0.3-107) (Tables 1 and2).

Haematological remission was achieved in 75/95 (80%) patients. At 3 months of diagnosis, complete renal remission was obtained in 20/102 (19%) patients, 45/102 (45%) patients had CKD stages 3-4 and 34/102 (33%) reached end-stage renal disease (ESRD). At last follow-up, complete renal remission was obtained in 24/102 (24%) patients, 40/102 (39%) patients had CKD stages 3-4 and 38/102 (37%) reached end-stage renal disease (ESRD). Over time, the incidence of ESRD did not significantly vary between patients diagnosed with secondary HUS between 2009 and 2010 (54%) and those diagnosed after 2011 (32%) (Supplemental Table S5). Six patients underwent renal transplantation (IgA nephropathy (n=2), systemic lupus erythematous (n=2), Still's disease (n=1), heart transplantation (n=1)) and none experienced a HUS recurrence. Eleven (11%) patients died during follow-up and death rate did not vary over time (Table 1 and Supplemental tables S1 and S5). Death was related to complications of chemotherapy (n=2), malignancy (n=4), extra-renal transplantation (n=2), infection (n=1), auto-immune disease (n=1), and pancreatitis (n=1). Mean time between diagnosis and death was 254 days . Death occurred within three months after HUS onset in three patients (pancreatitis, n=1; cancer, n=1; infection, n=1).

Among the 73 patients who were not lost to follow-up and who did not die or reach ESRD within 3 months of HUS onset, one patient (1%) experienced a relapse of HUS. This 32-year female patient with a metastatic melanoma (skin, liver, lung) treated with vemurafenib and cobemitinib presented with severe HUS (acute kidney injury requiring dialysis, liver and heart involvement) nine months after the diagnosis of melanoma and six months after the start of treatment. Chemotherapy was stopped, the patient received eleven PE and her renal function partially recovered (SCr 1.5-2 mg/dL). One month after PE discontinuation, HUS recurred (acute kidney injury, SCr 6 mg/dL, mechanical hemolysis), eculizumab was started and, subsequently, SCr decreased to 2 mg/dL. Eculizumab was stopped after six months. One month later, SCr increased to 2.7 mg/dL, eculizumab was resumed and SCr decreased and stabilized at 1.1 mg/dL (eGFR 57 ml/min/1.73m 2 ). She is still treated with eculizumab and receives nivolumab for her metastatic melanoma. No complement gene variant was detected.

As compared to 125 atypical HUS patients (adult-onset of the disease) from the same registry, patients with secondary HUS required less frequently dialysis at presentation (41% vs 81%; p < 0.001), progressed less frequently to ESRD (37% vs 71%; p < 0.001) and experienced fewer relapses (1% vs 35%; p < 0.001) (Table 2). In contrast, patients with secondary HUS had a higher frequency neurological involvement (18% vs 8%; p=0.03) and death rates (11% vs 2%; p=0.007) compared to atypical HUS patients.

Impact of eculizumab on clinical outcome.

Characteristics and outcome of patients treated or not with eculiuzmab are shown in Table 5. The mean time between secondary HUS diagnosis and eculizumab initiation was 24 days (0.5-120), and in 11/38 (28%) patients eculizumab was started within 7 days of diagnosis. The mean duration of eculizumab treatment was 7 months (0.25-68) and the mean number of treatment doses was 20 .

The evolution of SCr, dialysis status, and platelet count at 3 months of eculizumab initiation is depicted in the Figure . No eculizumab-specific side effects (meningococcal infections) occurred. Eculizumabtreated patients had a more severe HUS at presentation with a more frequent need for dialysis (56% vs 32%; p=0.01) and more frequent neurological manifestations (28% vs 13%; p=0.04), as compared to patients not treated with eculizumab. In all, rates of haematological remission (69% vs 87%; p =0.06) of CKD stages 3-4 (51% vs 33%; p=0.09) of ESRD (36% vs 38%; p=1) and of death (10% vs 11%, p=1) rates were similar in in patients treated and those not treated with eculizumab. When the 38 patients treated with eculizumab were compared to 38 matched patients (based on age and SCr first, and platelet count and haemoglobin level second), renal outcome at 3 months of eculizumab start was not statistically different (Supplemental Table S6). When the comparison was restricted to the most severe patients who required dialysis, the renal outcome was similar in patients treated (n=22) or not (n=23) with eculizumab (Supplement Table S7). Median time between secondary HUS diagnosis and eculizumab initiation tended to be shorter in patients who had complete renal remission, CKD stages 3-4 and ESRD, but the difference did not reach statistical significance (Supplemental Table S8). Response to eculizumab according to the underlying condition associated to secondary HUS is depicted in Supplemental Table S9, but the low number of patients in some groups precluded any relevant statistical analysis.

At last follow-up, only four (10%) patients remained on eculizumab. One patient (melanoma/chemotherapy) had a HUS relapse after eculizumab discontinuation and was restarted on treatment. Two patients, one with a pulmonary carcinoid tumour and a pathogenic CFH variant (c.3047A>G; p.Tyr1016Cys) and one with systemic lupus erythematous had severe cardiac involvement (heart failure) and ESRD and were maintained on eculizumab awaiting renal or renal and cardiac transplantation. In the remaining patient, the reason for eculizumab continuation was the physician preference.

Discussion

This large retrospective series provides new clinical and genetic insights into secondary HUS.

The dissection of the genetic risk factors for atypical HUS and the availability of the first complement inhibitor, eculizumab, have undoubtedly fuelled the interest of clinicians for secondary HUS. Currently, one of the most debated question is whether secondary HUS belongs or not to the spectrum of complement-mediated atypical HUS.

The present series of 110 patients with secondary HUS and a complete complement work-up is the largest to date. It included a large variety of conditions associated to secondary HUS, with however a predominance of drug, auto-immune disease and infection-related HUS, in keeping with previously published series [START_REF] Terrell | The incidence of thrombotic thrombocytopenic purpurahemolytic uremic syndrome: all patients, idiopathic patients, and patients with severe ADAMTS-13 deficiency[END_REF][START_REF] Cavero | Eculizumab in secondary atypical haemolytic uraemic syndrome[END_REF].

Our results indicate that atypical HUS and secondary HUS have no common genetic risk factors distinct presentation and outcome. We showed that the frequency of rare (MAF < 0.1%) genetic variants in complement genes was similar in secondary HUS patients (5%) and healthy donors (6% and 8% of the French of the European controls, respectively). More interestingly, the frequency of pathogenic variants that impair complement regulatory activity was extremely low and did not differ between secondary HUS cases and healthy French and European donors (1% vs 0% and 1%, respectively). This is in sharp contrast to atypical HUS that has been linked in a significant number of large studies to a high incidence (40-70%) of rare and mostly pathogenic complement genes variants and is thus assumed to be mediated by uncontrolled complement activation [START_REF] George | Syndromes of thrombotic microangiopathy[END_REF][START_REF] Fremeaux-Bacchi | Genetics and outcome of atypical hemolytic uremic syndrome: a nationwide French series comparing children and adults[END_REF][START_REF] Noris | Relative role of genetic complement abnormalities in sporadic and familial aHUS and their impact on clinical phenotype[END_REF]. However, homozygous MCP haplotype ggaac, a known risk factor for atypical HUS [START_REF] Fremeaux-Bacchi | Genetics and outcome of atypical hemolytic uremic syndrome: a nationwide French series comparing children and adults[END_REF], was more frequently found in secondary HUS patients as compared to controls (Supplemental Table S2). This haplotype is probably associated to a decreased transcriptional activity of the MCP gene promotor [START_REF] Esparza-Gordillo | Predisposition to atypical hemolytic uremic syndrome involves the concurrence of different susceptibility alleles in the regulators of complement activation gene cluster in 1q32[END_REF] and its implication in the pathogenesis of secondary HUS warrants further assessment in larger cohorts. Moreover, one cannot exclude that transient complement activation may occur in some patients with secondary HUS and thus promotes TMA process, as suggested in pneumococcal or anti-VEGF drug-induced HUS [START_REF] Gilbert | Does dysregulated complement activation contribute to haemolytic uraemic syndrome secondary to Streptococcus pneumoniae?[END_REF][START_REF] Keir | VEGF regulates local inhibitory complement proteins in the eye and kidney[END_REF]. In the present series, 15% of included patients presented at onset with a low C3 serum level secondary to classical or alternative pathway consumption. Thus, even if complement activation does not seem to be the initial trigger in secondary HUS cases, such activation may occur in a subset of patients as a "second-hit" and perpetuates thrombotic microangiopathy. This hypothesis warrants additional ex vivo assays at the level of the endothelial cell to document a potential inadequate complement regulation at the endothelium surface [START_REF] Noris | Dynamics of complement activation in aHUS and how to monitor eculizumab therapy[END_REF]. However, to date, in clinical practice, systematic screening for complement gene variants is probably not warranted in patients with secondary HUS.

Secondary HUS is an acute, non-relapsing disease. Only one patient (1%) included in the present series experienced secondary HUS relapse in the setting of metastatic melanoma and chemotherapy, whereas, even in the eculizumab era, atypical HUS relapses rate remains high (31-50%) in the absence of specific treatment or after treatment discontinuation in patients with pathogenic complement gene variants [START_REF] Fakhouri | Haemolytic uraemic syndrome[END_REF][START_REF] Ardissino | Discontinuation of eculizumab treatment in atypical hemolytic uremic syndrome: an update[END_REF][START_REF] Fakhouri | Pathogenic Variants in Complement Genes and Risk of Atypical Hemolytic Uremic Syndrome Relapse after Eculizumab Discontinuation[END_REF][START_REF] Wetzels | Discontinuation of eculizumab maintenance treatment for atypical hemolytic uremic syndrome[END_REF]. The withdrawal or the treatment of the triggering factor or condition usually prevents secondary HUS relapse. Interestingly, patients included in the present study had been treated for a longer mean period of time (8 weeks versus 7 months) and had received a higher mean number of doses (6 versus 20) as compared to the previous study from Spain [START_REF] Cavero | Eculizumab in secondary atypical haemolytic uraemic syndrome[END_REF]. This may be due to the uncertainties about the potential overlap between atypical HUS and secondary HUS prior to the completion of the present study.

In the present series, the risk of ESRD in secondary HUS patients from the present series was high (37%) but lower than previously reported in atypical HUS in the pre-eculizumab era (64-67%) [START_REF] Fremeaux-Bacchi | Genetics and outcome of atypical hemolytic uremic syndrome: a nationwide French series comparing children and adults[END_REF][START_REF] Noris | Relative role of genetic complement abnormalities in sporadic and familial aHUS and their impact on clinical phenotype[END_REF]. Besides, a significant proportion (39%) of secondary HUS patients progressed to CKD stages 3-4.

However, progression of CKD in some patients may not be entirely related to HUS per se but to the underlying cause of HUS -autoimmune diseases, cancer, chemotherapy, glomerulopathy, transplantation -that may negatively impact renal function regardless of thrombotic microangiopathy.

Another remarkable difference with atypical HUS is the significant mortality rate in secondary HUS (11% vs 2%). Nevertheless, death was mainly related to the underlying condition rather than to HUS complications per se.

Even in the absence of a definite proven link between complement activation and secondary HUS, eculizumab is increasingly used in patients with this type of HUS. In the present series, more than one third of included patients who were diagnosed with secondary HUS after the approval of eculizumab for atypical HUS were treated with this C5 blocker. A growing number of case reports suggest potential benefit of eculizumab but carry the bias of preferential publication of positive results [START_REF] Ustwani | Eculizumab therapy for gemcitabine induced hemolytic uremic syndrome: case series and concise review[END_REF][START_REF] Chandran | Eculizumab for the treatment of de novo thrombotic microangiopathy post simultaneous pancreas-kidney transplantation--a case report[END_REF][START_REF] El-Husseini | Thrombotic microangiopathy in systemic lupus erythematosus: efficacy of eculizumab[END_REF][START_REF] Faguer | Eculizumab and drug-induced haemolytic-uraemic syndrome[END_REF][START_REF] Favre | Malignancy and thrombotic microangiopathy or atypical haemolytic and uraemic syndrome?[END_REF][START_REF] Merrill | Eculizumab cessation in atypical hemolytic uremic syndrome[END_REF]. A recent retrospective study reviewed 29 cases of secondary HUS treated with eculizumab in eleven Spanish nephrology centers [START_REF] Cavero | Eculizumab in secondary atypical haemolytic uraemic syndrome[END_REF]. HUS was mainly drug-induced (n=15) or related to systemic diseases (n=8) and was severe as 14 (52%) patients required dialysis and 11 (38%) presented with extrarenal manifestations (mainly neurological). In 24 (83%) patients, plasmapheresis failed to control TMA leading to the use of eculizumab. C5 blockade was associated with a rapid (within 1 month of start of treatment) improvement of renal and haematological features of TMA in 20 (69%) patients. In six (21%) additional patients, hematological remission occurred without any renal improvement and in the three (10%) remaining patients no benefit of eculizumab was noted. However, a comparison with historical controls not treated with eculizumab was not performed in this retrospective study.

The analysis of the impact of eculizumab on the outcome of secondary HUS is not straightforward. Firstly, underlying conditions and pathogenic mechanisms are heterogeneous, several triggering factors may coexist particularly in patients with malignancies treated with chemotherapy and it is hard to ascertain the respective role of each condition in triggering HUS. Moreover, the exact mechanism of TMA is illusive in some subtypes of secondary HUS (pancreatitis-associated HUS, for example). Thus, response to treatments may vary accordingly. Secondly, management of patients with secondary HUS usually includes the withdrawal or treatment of the underlying disease, and thus the removal or control of the initial main trigger of TMA. Such approach may be sufficient in some instances to halt the TMA process. Finally, in contrast to patients with atypical HUS (excluding those with anti-CFH antibodies), eculizumab is not frequently the sole treatment used in patients with secondary HUS.

In the present series, 13% of eculizumab-treated patients had a complete renal and haematological remission. An additional two-thirds had a haematological remission and an improvement or stabilization of their renal function. However, a similar haematological and renal outcome was noted in patients not treated with eculizumab (38% of whom received plasmatherapy).

Nevertheless, compared with patients not treated with eculizumab, patients who received eculizumab had a more severe disease, as exemplified by a more frequent need for dialysis and more frequent neurological manifestations. The finding that eculizumab-treated patients shared with patients not treated with eculizumab a similar haematological and renal outcome, despite a more severe presentation, may suggest a potential benefit of complement blockade in this subset of patients. Nevertheless, when the eculizumab-treated patients were compared to matched patients or when the comparison (eculizumab versus no eculizumab) was restricted to the most severe cases requiring dialysis, the renal outcome was not significantly altered by the use of the complement inhibitor. Noteworthy, in contrast to secondary HUS, the beneficial impact of eculizumab on the renal outcome of patients with atypical HUS was suggested by a similar retrospective analysis of even a smaller series from the same French registry [START_REF] Fakhouri | Insights from the use in clinical practice of eculizumab in adult patients with atypical hemolytic uremic syndrome affecting the native kidneys: an analysis of 19 cases[END_REF].

No firm conclusion can be drawn regarding the efficacy of eculizumab in secondary HUS.

However, treatment was well tolerated and short-term complement blockade may represent a reasonable therapeutic option in patients with severe renal and/or extra-renal manifestations of HUS. Early use of eculizumab in secondary HUS may be associated to a better renal recovery as suggested, but not fully proven, by the present series (supplemental table S8) and by the previous one from Spain [START_REF] Cavero | Eculizumab in secondary atypical haemolytic uraemic syndrome[END_REF]. The rapid achievement of at least a haematological remission with eculizumab may also facilitate the use of chemotherapy and/or cytotoxic agents for the treatment of the underlying cause of HUS. Prospective controlled studies with eculizumab in distinct subtypes of secondary HUS are obviously needed but several already outlined issues (wide heterogeneity of secondary HUS, effect of the treatment/withdrawal of the underlying cause/condition) may be an obstacle to the optimal design of such studies.

Our study has limitations. It was retrospective and included a heterogeneous cohort of secondary HUS. Patients with hematopoietic stem cell transplantation-TMA from the French HUS registry were excluded, as their cases have been previously reported [START_REF] De Fontbrune | Use of Eculizumab in Patients With Allogeneic Stem Cell Transplant-Associated Thrombotic Microangiopathy: A Study From the SFGM-TC[END_REF]. However, hematopoietic stem cell transplantation-TMA is a peculiar type of secondary TMA that has attracted a substantial number of specific studies [START_REF] Jodele | Eculizumab therapy in children with severe hematopoietic stem cell transplantation-associated thrombotic microangiopathy[END_REF][START_REF] Jodele | A new paradigm: Diagnosis and management of HSCT-associated thrombotic microangiopathy as multi-system endothelial injury[END_REF][START_REF] Jodele | Abnormalities in the alternative pathway of complement in children with hematopoietic stem cell transplant-associated thrombotic microangiopathy[END_REF][START_REF] Jodele | The genetic fingerprint of susceptibility for transplant-associated thrombotic microangiopathy[END_REF], and in which complement involvement has been assessed with discrepant results [START_REF] De Fontbrune | Use of Eculizumab in Patients With Allogeneic Stem Cell Transplant-Associated Thrombotic Microangiopathy: A Study From the SFGM-TC[END_REF][START_REF] Jodele | Abnormalities in the alternative pathway of complement in children with hematopoietic stem cell transplant-associated thrombotic microangiopathy[END_REF]. Nevertheless, our study is the first large study to provide a global clinical and genetic picture of secondary HUS.

In all, secondary HUS appears as an acute, non-relapsing form of HUS, not related to an autoimmune or constitutional dysregulation of the CAP. It is nevertheless a severe form of HUS with a high morbidity and mortality. Complement blockade may represent an empirical therapeutic option in severe forms of HUS with life-threatening manifestations. However, its efficacy in this setting is, so far, not established.

Methods

Study population

In this academic non industry-sponsored study, we retrospectively identified through a computerized database all patients with secondary HUS diagnosed between 1999 and 2017 and included in the French national registry of HUS patients referred for complement analysis. This registry is based in the Laboratory of Immunology at Hôpital Européen Georges Pompidou, a national reference centre for complement analysis in France. Blood samples were collected from 80 French controls (healthy adult blood donors at Hôpital Européen Georges Pompidou where the French HUS registry is based), to establish normal values of complement factors and the frequency of complement variants in the French population. Previously published clinical, biological and genetic data of 125 patients with atypical HUS from the same registry were also included in the analysis [START_REF] Fremeaux-Bacchi | Genetics and outcome of atypical hemolytic uremic syndrome: a nationwide French series comparing children and adults[END_REF].

Definitions

All patients with a diagnosis of secondary HUS and available clinical data were included in the study.

Their medical records were reviewed and relevant clinical and biological features were collected. HUS was defined by the association of at least three of the following criteria: mechanical hemolytic anemia (hemoglobin < 10g/dl, lactate dehydrogenase level > upper limit of normal, undetectable haptoglobin, presence of schistocytes on blood smear), thrombocytopenia (platelets count < 150 G/L), acute kidney injury or typical features of thrombotic microangiopathy in a kidney biopsy (fibrin/platelet thrombi, endothelial cells swelling and detachment from the basement membrane, double contours). Secondary HUS was defined as a HUS associated with an active disease or condition or to an ongoing treatment including: an uncontrolled autoimmune disease, an ongoing bacterial or viral infection (excluding postdiarrheal STEC-HUS), a progressing (or not in full remission) malignancy in the last 6 months preceding HUS, the use of medications previously reported to be associated with HUS, a glomerulopathy documented by a kidney biopsy, an extra-renal transplantation or a pancreatitis. In patients with malignancies, HUS was considered to be "malignancy-associated" if it had occurred before the start of chemotherapy and "drug-induced" if it had occurred after the start of chemotherapy.

HUS relapse was defined by the association of at least two of the biological criteria of HUS, occurring after at least 6 weeks of haematological remission. All included patients diagnosed with secondary HUS after 2000 had detectable ADAMTS 13 activity (> 10%). Cases of HUS occurring de novo after renal transplantation [START_REF] Quintrec | Complement mutation-associated de novo thrombotic microangiopathy following kidney transplantation[END_REF], following hematopoietic stem cell transplantation [START_REF] De Fontbrune | Use of Eculizumab in Patients With Allogeneic Stem Cell Transplant-Associated Thrombotic Microangiopathy: A Study From the SFGM-TC[END_REF] or overlapping cases of HUS and C3 glomerulopathy [START_REF] Servais | Acquired and genetic complement abnormalities play a critical role in dense deposit disease and other C3 glomerulopathies[END_REF] have been reported previously and are not included in the present series. A previously published series [START_REF] Fremeaux-Bacchi | Genetics and outcome of atypical hemolytic uremic syndrome: a nationwide French series comparing children and adults[END_REF] of hundred and twenty-five patients with adult-onset atypical HUS (without coexisting conditions/diseases) from the same French HUS registry was included in the analysis as historical controls.

Haematological remission of HUS was defined by a normalization of platelet count and a LDH level < 1.5 upper limit of normal for at least 8 consecutive weeks. Complete renal remission was defined by a glomerular filtration rate estimated using the Modification of Diet in Renal Diseases formula > 60 ml/min/1.73m 2 and proteinuria/creatininuria ratio < 0.05 g/mmol. Chronic kidney disease (CKD) was defined by an estimated glomerular filtration rate < 60 ml/min/1.73m 2 and end-stage renal disease (CKD stage 5) was defined by an estimated glomerular filtration rate < 15 ml/min/1.73m 2 or the need to start chronic dialysis. In patients who required dialysis, the initial SCr refers to the highest value measured prior to the start of dialysis. Normalization of proteinuria was defined by a proteinuria/creatininuria ratio < 0.05 g/mmol. Stable SCr was defined by a SCr value unchanged (±10%) on at least two measurements three months apart. Unless specified, outcomes were assessed at the last visit for living patients who had not reached CKD stage 5 or ESRD.

Complement analysis

Complement work-up and genetic analysis were performed in the usual clinical management of the patients. All patients gave informed consent for genetic analysis according to the Declaration of Helsinki. Measurement of plasma concentrations of C3, C4, complement Factor H (FH) and complement Factor I (FI), membrane cofactor protein (MCP) expression on granulocytes, and test for anti-FH antibodies were performed as previously described [START_REF] Roumenina | Alternative complement pathway assessment in patients with atypical HUS[END_REF].

All coding sequences for CFH, CFI, MCP, C3, Factor B (FB) and thrombomodulin (THBD) genes were sequenced using next-generation sequencing (NGS). Multiplex ligation-dependent probe amplification was performed to detect CFH hybrid genes and CFH-related protein 1-3 (CFHR1-CFHR3) genes deletion. The Minor allele frequency (MAF) of the genetic changes was obtained from the Exome aggregation consortium database (http://exac.broadinstitute.org) [START_REF] Lek | Analysis of protein-coding genetic variation in 60,706 humans[END_REF].

We collected the genotypes in 503 European individuals from the 1000 Genomes project [START_REF] Auton | A global reference for human genetic variation[END_REF] for 6 genes of interest: CFH, CFI, CFB, MCP, C3 and THBD. The 1000 Genomes project was an international study that applied whole-genome sequencing to a large cohort of individuals from multiple populations (2,504 individuals from 26 populations), including 503 individuals with European ancestry (Northern and Western European, Finnish, British, Spanish and Tuscan). The variant call format files located on the 1000 Genomes server (ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/release/20130502/) were parsed using the Ferret tool [START_REF] Limou | Ferret: a user-friendly Java tool to extract data from the 1000 Genomes Project[END_REF] to extract the genetic information of rare coding variants (minor allele frequency (MAF) <1%). From the genotypes, we computed the occurrence of rare coding variants in each individual.

In this study, a variant is defined as rare when its MAF in the general population is < 0.1%. A variant was classified as pathogenic when the genetic change affects the protein function (well-established in vitro functional studies supportive of a damaging effect on the gene product), and/or the genetic change is found in a disease-related functional domain or affects the protein expression (nonsense, frameshift, canonical ±1 or 2 splice sites variants, or well demonstrated lack of in vitro synthesis, or quantitative deficiency in the patient's plasma). The others variants were classified as Variants of Uncertain Significance (VUS) when no available functional data are available (definitions adapted from).

Statistical analysis

Data are presented as percentages or means (ranges). The Wilcoxon test was performed for quantitative variables, and Fischer's exact test for qualitative data. All analyses with p value <0.05 were considered statistically significant. *Heart failure (n=8), digestive system (colitis, ischemic cholangitis and jejunitis) (n=3). ** Used for the treatment of the underlying cause of HUS (lupus (n=9), myositis (n=1), ANCA-associated vasculitis (n=1), heart transplantation (n=1), drug (bevacizumab, n=1)). *** Eight patients were lost to follow-up. € Among 73 patients who were not lost to follow-up and who did die or reach end-stage renal disease within 3 months of HUS onset. # Among the 11 patients who died, one had a complete remission, six had chronic kidney disease (stages 3-4) and four had reached end-stage renal disease. * Among the 4 eculizumab-treated patients who died, two had chronic kidney disease and 2 had reached end-stage renal disease. Among the 7 patients not treated with eculizumab who died, one had a complete remission, four had chronic kidney disease, and two had reached end-stage renal disease.

  c) Allele frequency given by Exome aggregation consortium * This patient had a partial renal recovery under eculizumab (SCr, 1.2 mg/dL) but developed severe hypertension, uncontrolled despite the use of six antihypertensive drugs. She also had severe heart failure (left ventricular ejection fraction 15-20%) due to HUS but also to the intractable hypertension and she underwent binephrectomy. Subsequently, hypertension improved and ejection fraction increased to 55%. # This patient died 2 weeks after the onset of HUS in the setting of sepsis metastatic cancer.

Table 1 :

 1 Main characteristics of 110 patients with secondary HUS included in the study.

Table 2 :

 2 Presentation and outcome of 110 patients with secondary HUS and 125 patients with adult-onset atypical HUS in the pre-eculizumab era (from the French HUS registry). Abbreviations: HUS, haemolytic and uremic syndrome.

Table 3 :

 3 Frequency of complement gene rare variants in 110 secondary HUS cases, 125 adult-onset atypical HUS cases included in the French HUS Registry, 80 French healthy donors and 503 European healthy individuals from the 1000 Genomes project. All donors were screened for variants in the 6 tested complement genes. Abbreviations: aHUS, atypical haemolytic uremic syndrome. CFH, complement factor H. CFI, complement factor I. MCP, membrane cofactor-protein. FB, factor B. THBD, thrombomodulin.

	Pt Gender	Associated	Plt	SCr	Hb	Treatment	Outcome	Complement	Variant	MAF c	Polyphen 2	Functional	Variant
		/Age	disease/	(G/L)	(mg/dL)	(g/dL)			gene		(%)	prediction	consequences	categorization
			condition
	1	F/26	SLE	40	4.3	8.5	PE/Ecu/Cs/C	ESRD	THBD	c.707C>G	Novel	Benign	NA	VUS
							YP			p.Ala236Gly
	2	F/80	Breast cancer	129	1.3	10.6	PI	CKD / Death #	THBD	c.91G>A	Novel	Benign	NA	VUS
										p.Val31Ile
	3 a	F/39	Drug (IFN)	54	4	8.1	Drug		CFI	c.11T>A	0.0033	Benign	NA	VUS
							withdrawal/	CKD		p.L4H
							PE/Cs
	4	M/59	Infection	27	ND/HD	ND	Antibiotics	CKD	CFH	c.643G>A	Novel	Benign	NA	VUS
			(E.Coli)							p.Val215Ile
	5	F/43	Pulmonary	80	6.3/HD	7.2	PE/Ecu	ESRD*	CFH	c.3047A>G	Novel	Probably	Located in	Pathogenic
			carcinoid							p.Tyr1016Cys		damaging	disease-related
			tumor										functional
													domain
	6 b	F/32	(Vemurafenib) Drug	27	5.7	7.8	withdrawal/ Drug	after Ecu CKD / Relapse	CFH	p.Phe1199Ser c.3596T>C	Novel	damaging Probably	disease-related Located in	Pathogenic
							PE/Ecu	discontinuation					functional
													domain

Table 4 :

 4 Main characteristics of the 6 patients with secondary HUS and complement gene rare variants. Abbreviations: Pt, patient. F, female. M, male. SLE, systemic lupus erythematous. VEGF, vascular endothelial growth factor. APS, antiphospholipid syndrome. IFN, interferon. Plt, platelet count. SCr, serum creatinine. HD, hemodialysis. NA, not available. PE, plasma exchanges. Ecu, eculizumab. Cs, corticosteroids. CYP, cyclophosphamide. ESRD, endstage renal disease. CR, complete remission. CKD, chronic kidney disease. Hb, haemoglobin. MAF, minor allele frequency. CFH, complement factor H. CFI, complement factor I. . THBD, thrombomodulin.. VUS, variant of unknown significance. NA: not available a) This patient also carried a VUS in CFI gene with a minor allele frequency of 0.8 (c.1642G>C p.E548Q). b) This patient carried a C3 p.Lys155Gln (c.463A>C) pathogenic variant with a minor allele frequency of 0.33 in control populations.

Acknowledgments:

We would like to thank Pr Christine Piétrement (CHU de Reims), Dr Sophie Taque (CHU de Rennes) and Dr Laurent Guillaume (CH de Perpignan) for their help.

This study was supported by research grants from La Société Francophone de Néphrologie et de Dialyse (to FF) (unrestricted funding by Alexion Pharmaceuticals who was not involved at any stage in the study design, analysis of the data and writing of the manuscript), the Programme Hospitalier de Recherche Clinique (grant AOM08198) (to VFB), the EU FP7 program 2012-305608 (EURenOmics) (to VFB), the Fondation du rein (FRM, Prix 2012 FDR) (to VFB) and the Association pour l'Information et la Recherche dans les maladies Rénales Génétiques (AIRG France).

Disclosures: François Provôt, Moglie Le Quintrec, Yahsou Delmas, Jean-Michel Halimi, Steven Grangé, Eric Rondeau, Véronique Frémeaux-Bacchi and Fadi Fakhouri have received consultancy and/or lecture fees and/or travel support from Alexion Pharmaceuticals. Yahsou Delmas, Jean-Michel Halimi and Steven Grangé have received consultancy fees from Ablynx. Noémie Joude-Chiche has received a research grant from Genzyme.

Authorship contributions:

ALC, VF-B and FF designed the study, and collected and analyzed the data. VF-B and P V-M performed the complement work-up and genetic screening. All authors were involved in the clinical management of the patients. FF and VFB drafted the manuscript and all authors reviewed, amended and approved the manuscript. FF had full access to the data in the study and final responsibility for the decision to submit for publication.

Supplementary Material:

Supplementary Figure S1: Flow chart of 110 patients with secondary hemolytic uremic syndrome (HUS) included in the study. * Among 73 patients who were not lost to follow-up and who did not die or reach end-stage renal disease within 3 months of HUS onset. S1: Characteristics of 110 secondary HUS patients according to the associated conditions/diseases. Abbreviations: VEGF, vascular endothelial growth factor. CMV, cytomegalovirus. HIV, human immunodeficiency virus. HSV, herpes simplex virus. GBM, glomerular basement membrane. ANCA, anti-neutrophil cytoplasm antibodies. *, Interferon-β (n=9), interferon-α (n=1), ** including lupus with APS (n=4). *** sacroidosis (n=1), undetermined connective disease (n=1). a, the two patients had chronic kidney disease (stages 3-4). b, the patient had reached end-stage renal disease. c, the patient had chronic kidney disease (stages 3-4). d, one patient had a complete remission, two had chronic kidney disease (stages 3-4) and one had reached end-stage renal disease. e, both patients had chronic kidney disease (stages 3-4). f, the patient had reached end-stage renal disease. S2

Supplementary Table

Supplementary Table

Patient Selection

Presentation of secondary HUS 40% of patients required dialysis 18% had neurological manifestations.

Treatment

Plasmatherapy (50%) Eculizumab (35%)..

Outcome

Haematological remission: 80% Complete renal remission: 24% CKD (stages 3-4): 39% ESRD: 37% HUS relapse (<1%).

Benefit of eculizumab unclear

Frequency of complement gene rare variants similar in secondary HUS patients (5%) and 2 cohorts of healthy individuals (6-8%).

Genetics


Alejandro Von Chong
email: alejandro.von-chong@ensea.fr

Mehdi Terosiet

Aymeric Histace

Olivier Romain

A Von Chong

Towards a novel single-LED pulse oximeter based on a multispectral sensor for IoT applications

Keywords: Pulse oximetry, multiwavelength oximetry, BQJ, blood plethysmography

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Pulse oximeters have become an essential tool for patient monitoring, ranging from intensive care units and operating rooms in hospitals, to sleep disorder supervision in the patient's home. They rely on the photoplethysmography technique for non-invasively estimating the saturation of arterial blood with oxygen (SpO2). This is a major indicator of a patient's cardiorespiratory status since it provides the percentage of hemoglobin bound with oxygen in the arterial blood. Indeed, for healthy patients, the majority of molecular oxygen (O2) is bound to hemoglobin and only a small fraction is dissolved in plasma. However, patients suffering from respiratory problems or certain metabolic and genetic disorders, can reach a dangerously low amount of oxygenated hemoglobin [START_REF] Haymond | Laboratory Assessment of Oxygenation in Methemoglobinemia[END_REF]. Beyond these applications, additional information other than oxygenation can be derived with the photopleghysmography technique, i.e. the plethysmogram waveform (PPG). This additional information include the heart rate, tissue perfusion and arterial stiffness [START_REF] Cannesson | Relation between respiratory variations in pulse oximetry plethysmographic waveform amplitude and arterial pulse pressure in ventilated patients[END_REF].

With classic pulse oximeters, the arterial blood saturation (SpO2) is estimated with an optical setup working on well perfused tissue such as a finger or earlobe. This setup is composed of two light emitting diodes (LED), usually red at 660 nm and infrared at 940 nm, alternatively shining through the tissue and, on the opposite side, a detector measures the transmitted light, which was not absorbed by other light absorbents in finger, such as blood, skin, nails and bone, see Fig. 1. These wavelengths are widely used because of the hemoglobin's optical properties: at 660 nm, deoxygenated hemoglobin (deoxyhemoglobin or Hb) absorbs more light than oxygenated hemoglobin (oxyhemoglobin or HbO2) while at 940 nm, oxyhemoglobin absorbs more light than deoxyhemoglobin. The ratio between the two absorption coefficients is approximately tenfold [START_REF] Nitzan | The measurement of oxygen saturation in arterial and venous blood[END_REF]. From there, and with some electronic circuitry, the PPG signals at 660 nm and 940 nm are acquired. Fig. 2 illustrates their typical waveform. The AC/DC ratio of their respective components is blood oxygenationdependent. The relationship between this AC/DC ratio and SpO2 is then empirically calibrated [START_REF] Nitzan | Pulse oximetry: fundamentals and technology update[END_REF]. However, this approach exhibits some limitations.

Problem statement

Classical oximeters are subjected to several sources of inaccuracies. The fact is that there are discrepancies in accuracy between commercial pulse oximeters, in part, as a result of the differences in their calibration processes and algorithms employed for their signal processing. One of them is observed when oxygen saturation drops below 70%, the scattering difference between these two wavelengths affect the SpO2 estimation [START_REF] Schmitt | Simple photon diffusion analysis of the effects of multiple scattering on pulse oximetry[END_REF]. Calibration-free pulse oximetry addresses this problematic, but its practical implementation still poses some technological limitations [START_REF] Nitzan | Calibration-Free Pulse Oximetry Based on Two Wavelengths in the Infrared -A Preliminary Study[END_REF]. Another important source of erroneousness is related to the wavelength displacement of the incident wave from its typical value. A led's peak emission wavelength might be shifted due to a change in the led's temperature, ageing or fabrication processes. A change of just 4 nm in one of the led's peak emission (with the other led's peak emission wavelength remaining constant) produces an error of 2% at 97% SpO2, 4% at 90% SpO2 and 7% at 80% SpO2 [START_REF] Milner | An assessment of the accuracy of pulse oximeters[END_REF].

Considering these limitations, recently manufactured oximeters use up to 8 wavelengths to improve accuracy, increase performance at low saturations and enhance robustness against noise and motion artifacts. Moreover, the wavelength addition enables the estimation of other types of hemoglobins such as carboxyhemoglobin and methemoglobin. This is the case for the Masimo SET® pulse co-oximeters very often used as gold-standards in literature [START_REF] Barker | Measurement of carboxyhemoglobin and methemoglobin by pulse oximetry: a human volunteer study[END_REF][START_REF] Kost | Continuous noninvasive hemoglobin monitoring: the standard of care and future impact[END_REF][START_REF] Goldman | Masimo Signal Extraction Pulse Oximetry[END_REF].

In the field of Internet-of-Things for biomedical applications, almost everything remains to be done as regards to these last considerations. Even if a remarkable work was published by Theodor et al.,[START_REF] Theodor | Implantable pulse oximetry on subcutaneous tissue[END_REF], with a pulse oximeter implanted in subcutaneous tissues, the deviations of their results are above the accepted limit range defined by the ISO 80601-2-61 standard [START_REF] Milner | An assessment of the accuracy of pulse oximeters[END_REF]. Another work, in accordance with the paradigm of N-wavelength oximetry (N=1, 2, 3, ...), show a real interest for connected wearable pulse oximeters [START_REF] Leboeuf | Custom multi-wavelength monolithic optical source applied to sensing of physiological and biometrical parameters[END_REF]. This study examines the possibility to design a multi-wavelength LED by using rare-earth excited by a commercial blue LED. On this basis, the authors aim to evaluate a pulse oximeter embedded in a Valencell earbud. However, there is not enough results to conclude. Hence, at this stage, the open-ended question remains while the direct implementation of an N-LED system runs completely counter to the system miniaturization needed for connected objects.

In this study, we address the emphasized lack of actual devices. For that, we introduce a new paradigm for the instrumentation by substituting the multiwavelength light source and its associated photodetector by a multi-spectral optical sensor and a single LED. This approach could reduce most of the imprecisions related to the scattering phenomena, and led deterioration. Furthermore, in the perspective of a silicon implementation, it could significantly alleviate the electrical power consumption of the pulse oximeter.

This original idea is laid out into the six following parts. Firstly, the Buried Quad Junction photodetector (BQJ), which is the substrate of the proposed approach is described. Once these elements of understanding are provided, the fundamentals of the new sensing method is given in the fourth section. The experimental setup comprising the electronic acquisition circuit associated to its driving software and the testing protocol are depicted, in the fifth section. Finally, before discussion, conclusion and future perspectives of this work, the experimental proof of concept is provided in the sixth part.

Buried Quad Junction -BQJ -Photodetector

Connected devices take advantages of advances in CMOS technology fabrication process. Downscaling has allowed embedding more and more electronic functionalities and the maturity of fabrication process enables the integration of MEMS elements (Micro-Electro-Mechanical Systems) for actuators and sensors [START_REF] Dong | MEMS based Smart Gas Metering for Internet of Things[END_REF]. There is now the possibility to design multiple buried-junction photodetectors for applications requiring spectral analysis. The operation of these optical sensors draws on the strong dependence of silicon absorption depth on the incident light wavelength. The exponential decrease in depth of generated photo-carriers is distinctly related to the wavelength. A first CMOS design including two junctions was introduced in 1996 with [START_REF] Lu | Colour detection using a buried double p-n junction structure implemented in the CMOS process[END_REF] and was the subject to several studies [START_REF] Feruglio | Dark Current and Signal-to-Noise Ratio in BDJ Image Sensors[END_REF][START_REF] Feruglio | A general model of the CMOS buried double junction photodetector[END_REF]. One year later, a BiCMOS implementation stacking three buried junctions was presented for colour detection like the previous one [START_REF] Chouikha | Buried triple p-n junction structure in a BiCMOS technology for color detection[END_REF]. To achieve an highest spectral discrimination ability, authors of these research works have gone one step further and are now proposing the BQJ (Buried Quad Junction) photodetector for which important characterization efforts can be found in [START_REF] Feruglio | A CMOS Buried Quad p-n Junction Photodetector Model[END_REF][START_REF] Richard | A CMOS Buried Quad p-n Junction Photodetector Model[END_REF].

This last upgrade is designed by Teledyne-DALSA with a 0.8 μm High Voltage CMOS process. As depicted by Fig. 3, for 200 μm square area, it implements four vertically-stacked p-n junctions with four different spectral responses sensing from near UV to near IR. We take advantage of this particular feature for the single-LED pulse oximeter implementation. Each junction drives an output current proportional to the incident light intensity while the normalized current distribution over the four junctions, as a function of the wavelength, is depicted on Fig. 4. This characterization was obtained by measuring current outputs when the sensor was illuminated by a SPEX 270M monochromator.

It is worth noting that, for a given wavelength, a specific and unique current distribution through the four channels is exhibited by the BQJ. By extension, the BQJ's response to an incident light beam, necessarily respects a specific distribution of output currents in accordance with the Fig. 4. The concept to be understood for the next section is: a light beam, with a specific optical spectrum, implies a corresponding signature and thus can be seen as an "equivalent wavelength" from the BQJ's perspective. By exploiting these unique properties, a novel sensing method was developed. It benefits from the spectral discrimination ability of this compact and smart sensor so as to estimate the blood oxygenation.

New sensing approach

Blood signature extract from the BQJ responses

The optical spectrum of the light transmitted through the finger depends, on one hand, on the light source's spectrum and on the other hand, on the extinction coefficient of blood. Both parameters directly influence the light intensity and spectrum received by the photodetector. Concerning the blood light absorption, the Fig. 5 illustrates its absorption functions related to oxyhemoglobin (HbO2) and deoxyhemoglobin (Hb) with respect to wavelength.

Oxygenated and deoxygenated blood have different absorption spectrums, meaning that a change in the oxygenation level applies a different optical filter to the led's incident light. So, due to the fact that the BQJ can distinguish changes in the incident's light spectrum, we can use it in order to detect a change of a led's incident light spectrum as blood's oxygenation levels change. The most important change would be seen where there's the biggest slope difference for the two hemoglobin types. In order to determine the best possible wavelength range to exploit this, the derivative of the oxyhemoglobin's and deoxyhemoglobin's absorption spectrum was calculated. The absolute value of the ratio between these derivatives ([dHb(λ)/dλ] / [dHbO2(λ)/dλ]) is shown in Fig. 5.

Thus, for the proposed approach, one can see that the range from 660 nm to 700 nm has the biggest slope difference. Due to the commercial availability, a 680 nm (with a spectral line half width of 30 nm) was chosen.

We can conclude that for each SpO2 value, a signature or a distribution of BQJ's currents is Fig. 5. Left: Oxyhemoglobin (HbO2) and deoxyhemoglobin (Hb) extinction coefficients with respect to the wavelength. Data obtained from [START_REF] Omlc | Tabulated Molar Extinction Coefficient for Hemoglobin in Water[END_REF]. Right: Absolute value of the ratio of the Hb and HbO2 derivatives with respect to wavelength. associated. Their relation depends on the signature and their sensitivity slopes where at 680 nm the contrast is enhanced between oxygenated and deoxygenated blood. What differentiates this method from the classic approach, is that the latter measures the change in absorbance for two different wavelengths, while we measure the spectral change for a single wavelength using a multispectral photodetector.

Arterial saturation estimation

For the arterial oxygen saturation estimation, we use the AC amplitudes of the resulting PPG. Remembering that the BQJ's output currents are proportional to light intensity and that, for a specific wavelength, current distribution remains constant, we calculate a ratio between channels which we denote by , as in equation 1.

, = (1)
Where , stand for the AC output of the resulting plethysmogram for two different channels. Thus, an estimation of the oxygen saturation can be made by using one led.

In our case, the normalization of the DC components is not necessary in order to nullify the difference in intensities because the magnitude of all outputs are already linearly dependent.

Experimental Setup

So as to validate the proposed approach and the sensing method, we developed an acquisition system which associates mixed electronic circuits and its driving software shown in Fig. 6.

. Acquisition system

The acquisition system is illustrated in Fig. 7. BQJ's output currents pass through signal conditioning circuit in order to obtain voltages of resulting from the received beam light. These voltages are acquired by the 24 bit ΣΔ Analog-to-Digital converter ADS1220. Digital data are then read by a LPC1768 microcontroller (32-bits ARM Cortex-M3) and sent to a host computer using LabVIEW for monitoring and data storage in spreadsheet files. The Masimo Radical 7 was used as the measuring reference and data were recorded simultaneously with the ADC measurements by the host computer.

Signal conditioning circuitry

The AFE is designed in order to process BQJ's output currents, see Fig. 8. These outputs are converted from current to voltage with a Transimpedance-Amplifier built from an OPA4348 (Texas Instruments, 4 channel -single supply). A second order low-pass filter is set at a cut-off frequency of 10 Hz because all the useful information is contained within the 0-10 Hz band. An instrumentation amplifier, with INA2332 (Texas Instruments, CMRR 73 dB), is used to subtract the bias voltage, needed for the polarization of each of the BQJ's junctions, and to amplify this voltage difference by 5. The obtained measurements of received light are then digitized by an ADS1220. Finally, the described analog chain is implemented for each channel of the BQJ.

Light Source

The LED selected for this study is the MTE6800N2-UR, Marktech Optoelectronics, which has its peak emission at 681 nm. Its measured optical spectrum is given in Fig. 9 The spectrophotometric measurements were made with the Avantes AvaSpec-ULS2048L spectrometer for a LED forward current of 5 mA.

In order to hold the BQJ in position, a 3D printed finger probe was produced. The probe held the BQJ in one side and the leds in the opposite side. Careful attention was paid in order to align led beam with the BQJ. The probe had a compression spring in the rear end order to apply a constant pressure to the finger during tests. Silicone was applied to the surface in contact with the finger better contact and comfort. The probe can be seen in Fig. 11.

Testing Protocol

Measurements were done on 3 subjects by breath holding so as to stop their oxygen supply and thus decreasing their SpO2. Test were performed in a dark room. Subjects had their arms placed flat on a table at the height of their chests and performed the following protocol:

1. Deep breathing (hyperventilation) during 1 minute. 2. Breath holding. 2.1 Simultaneously with 2, begin data recording.

3. Subject breathes. 4. Continue to record data 1 minute after breathing. 5. Led turn-off (for offset calibration). 6. End test. Measurements for our system and the reference instrument, the Masimo Radical-7, were made with the left hand: index for our system and annular for the Masimo. For all tests performed, led intensity was unchanged.

In vivo Results

In order to test our led choice, we performed a first breath holding test with a volunteer for 3 different wavelengths. So as to determine which wavelength was more sensitive to the deoxygenation process through the whole finger (including venous blood), measurements were taken and a Matlab's peak detection algorithm was used. The ratio between peaks and valleys for each channel was calculated. Fig. 12. shows the ratio between channels 3 and 4 for the calculation done previously. Chosen leds had peak wavelengths and spectral line half widths at 640 ± 25 nm, 660 ± 25 nm and 680 ± 30 nm. This way we can conclude that our choice for a 680 ± 30 nm is the appropriate for this application. From the acquisition system described previously and following the developed protocol, we present in Fig. 10 a sample of the acquired data from a subject and for the four channels of the BQJ. Spreadsheet files containing acquired data were processed afterwards with Matlab where an algorithm for peak detection was used to extract the AC envelope.

After extraction of the peak's and valley's envelope, the AC is calculated by subtracting the former from the latter, results can be seen in Fig. 13.

In order to determine which channels are more sensitive to the spectral change due to the filter applied by the deoxygenation process for the arterial blood, we proceed to calculate the normalized responses of the AC voltages from Fig. 13. This represents the spectral 'signature' seen by the BQJ for the arterial blood. Filtered results are shown in Fig. 14. As expected, the sensibility changes and the ratio can decrease or increase depending on the chosen channels. Even though channels 3 and 4 are of the same form, we privileged channel 4 because of the higher linearity for a change in wavelength as seen in Fig. 4. From there, the relation between the SpO2 measured by the reference instrument and , can be established.

To do so, only the deoxygenation segment of Fig. 15 is considered, because during the breath holding tests, once the volunteer starts to breathe, signals are contaminated by motion artifacts, irregular respiration patterns and abrupt changes in heart rate who decrease the performance of the peak detection algorithm. Curves obtained by breath holding experiment with the three test subjects yielded different oxygenation levels. For comparison purposes, measurement results were truncated to the SpO2 range shared by the three subjects: 97% to 80% SpO2.

Finally, Fig. 16 demonstrates the good agreement for the SpO2 measurements with the three subjects. The commercial permissive tolerance established by the ISO 80601-2-61 standard is 3%. Two of the three tests fall below the tolerated error for the whole process, while the third one, goes above slightly at high saturation (4% error) as seen in Fig. 15b. We can see that there is a good correlation between , and SpO2. Tests 1, 2 and 3 had a correlation coefficient of r = 0.995, 0.980 and 0.990 respectively between , and SpO2.

Discussion -Possible extention to Venous Oxygen Saturation Estimation

Oxygen saturation in the peripheral venous blood (SvO2) also has physiological and clinical significance, as it enables an evaluation of the severity of shock or cardiac failure [START_REF] Ousmane | Utilisation de la SvO2[END_REF]. Its estimation by optical means was already reported by changing the venous blood volume with the application of a handcuff to the patient's hands and fingers. It permitted to calculate the absorption difference between a "relaxed" state and a "pressured" state. As done in classic pulse oximetry calculations, a ratio between two wavelengths in the "relaxed" and "pressured" state was obtained to estimate venous saturation. In contrast to arterial saturation, there is no accepted method for the SvO2 estimation [START_REF] Nitzan | The measurement of oxygen saturation in arterial and venous blood[END_REF].

With our methodology, we consider we can estimate SvO2 without occlusion. Indeed, the nonpulsatile portion of the plethysmogram represents all the other components met by the light beam all along its optical path including the venous blood. Assuming nail, skin and bone absorptions remain constant, an estimation of the SvO2 might also be made by relating the change in the "equivalent wavelength" seen by the BQJ, with the DC component of the PPG.

Conclusion

A novel method for estimating blood oxygen saturation has been introduced. For the first time, a multispectral photodiode has been utilised to estimate SpO2 with a single led. Two of the three in-vivo results from the SpO2 estimations fall within the accepted tolerance range of 3%, and among the others, a maximum error of 4% was found in the third one. Our estimations were solely calculated with the change in absorption between channels. So, precision and robustness can be increased by adding some of the already developed correction algorithms regularly used to fix common oximetry sources of errors such as motion artifacts [START_REF] Goldman | Masimo Signal Extraction Pulse Oximetry[END_REF]. It is worth stressing that only two of the four channels were used for this implementation. This is because it is preferable to use channels with highest and opposite sensitivity for the chosen wavelength. However, other wavelengths can be used for exploiting the other two channels. For instance, the estimation of other types of hemoglobins (e.g. carboxyhemoglobin and methemoglobin) or other physiological parameters.

The proposed approach can also be complementary to the classic or the multiwavelength pulse oximetry approach, as long as the BQJ is employed as the photodetector. Additionally, with this new methodology, we can quantify the changes through the whole finger, including venous blood, so a venous oxygen saturation technique might be developed in the future.

Additionally, a study regarding the influence of skin color and nail polish will be performed, as we estimate that the use of one led could reduce the errors introduced by an increased melanin or the presence of nail polish.

At the end, the proposed system is fully compatible with a monolithic integration for the development of future connected objects targeting well-being applications.

Fig. 1 .

 1 Fig. 1. Optical setup [4].

Fig. 2 .

 2 Fig. 2. Typical plethysmograph signal [4].

Fig. 3 .

 3 Fig. 3. Sectional view of the BQJ [21].

Fig. 4 .

 4 Fig. 4. Normalized response of the BQJ output currents as a function to the wavelength.

Fig. 6 .

 6 Fig. 6. Measuring setup comprised by the Masimo Radical 7 as the measuring reference, the analog front end, the printed finger probe, the Masimo's Radical 7 finger probe and the PC with the LabVIEW-based acquisition software.

Fig. 7 .

 7 Fig. 7. Block diagram of the utilized system

Fig. 8 a

 8 Fig. 8 a. Signal conditioning circuit schematic for each channel.

Fig. 8 b

 8 Fig. 8 b. Signal conditioning circuit.

Fig. 9 .

 9 Fig. 9. Measured optical spectrum of the 681 nm, MTE6800N2-UR, Marktech Optoelectronics.

Fig. 10

 10 Fig. 10 a. Signals digitized by the ADS1220 with temporal delimitations during the test. Region A represents the relatively constant oxygenation at the beginning the breath holding period. B represents the deoxygenation due to breath holding. C represents the oxygenation due to breathing and D represents the return to the normal oxygenation level.

Fig. 11 .

 11 Fig. 11. Printed finger probe.

Fig. 10 .

 10 Fig. 10.b. Zoomed output from channel 3 showing plethysmogram with its AC and DC components.

Fig. 14

 14 Fig. 14 a. Voltage distribution for the AC components of the PPG.

Fig. 14 b

 14 Fig. 14 b. Zoomed voltage distribution during oxygenation and deoxygenation for channels 1 and 2.

Fig. 13 .

 13 Fig. 13. AC magnitudes obtained for the four channels from the peak detection algorithm.

Fig. 12 .

 12 Fig. 12. Sensibility curves for different wavelengths

Fig. 15 .

 15 Fig. 15. β between channels 1 and 4 as a function of time.

Fig. 15 a

 15 Fig. 15 a. Relation between SpO2 and , .

Fig. 15 b

 15 Fig. 15 b. Percentual error curves for the three tests.

Acknowledgments

We would like to thank to Mr. Guo-Neng Lu, Mr. P. Pittet, Mr. T. Courcier and V. Amiez for the access to the BQJ technologies and sensor. We would also like to thank SENACYT, Panama for partial funding of this research.


PharmD Guilaine Boursier

MD Veronique Hentgen

MD Guillaume Sarrabay

MD Isabelle Koné- Paut

MD, PhD Isabelle Touitou
email: isabelle.touitou@inserm.fr

The Changing Concepts regarding the Mediterranean Fever Gene (MEFV): Toward a Spectrum of Pyrin-Associated Autoinflammatory Diseases (PAAD) with Variable Heredity

Keywords: FMF, autoinflammatory disorder, PAAD, PAAND Pyogenic arthritis, pyoderma gangrenosum and acne PFAPA: Periodic fever' apthous-stomatitis, cervical pharyngitis and adenitis SAID: Systemic autoinflammatory disease

WES: Whole-exome sequencing

Familial Mediterranean fever (FMF) is a genetic disease characterized by recurrent fever and serositic attacks. It is the oldest known hereditary recurrent fever 1, 2 and the first systemic autoinflammatory

2 disease (SAID) whose gene (MEFV for Mediterranean FeVer, NM_000243.2) has been identified. [START_REF] Thefrenchfmfconsortium | A candidate gene for familial Mediterranean fever[END_REF][START_REF] Theinternationalfmfconsortium | Ancient missense mutations in a new member of the RoRet gene family are likely to cause familial Mediterranean fever[END_REF] The two decades that followed this discovery have been fruitful in clinical, genetic and functional developments, making FMF a unique and fascinating model for understanding this disease, and the innate immune defense in general. FMF usually begins at pediatric age and can be seen by a wide variety of pediatricians: generalists, rheumatologists, infectious disease specialists, gastroenterologists or dermatologists.

We will retrace the impressive scientific and medical journey that has switched a little-known rare disease gene into a field of intense interest leading from basic research to personalized medicine. We will explain how a gene initially identified from individuals meeting well-defined criteria (FMF) is now recognized as being at the origin of a wide and unanticipated range of clinical phenotypes jointly referred to as pyrin-associated autoinflammatory disease (PAAD).

The evolution of knowledge has led to a better interpretation of the impact of mutations in the various clinical pictures associated with the MEFV gene, resulting in increasingly relevant diagnoses.

From benign paroxysmal peritonitis to FMF

The various names given to FMF since its description in the early 1900s reflect the main features of the disease. Historical cases of FMF were reviewed in a previous dedicated study. [START_REF] Touitou | New genetic interpretation of old diseases[END_REF] Figure 1 summarizes the key steps of the clinical and genetic discoveries in FMF. The first indisputable report of FMF was by Janeway, in 1908 (paroxysmal syndrome, with recurrent vomiting). [START_REF] Janeway | An unusual paroxysmal syndrome, probably allied with recurrent vomiting, with a study of the nitrogen metabolism[END_REF] Heller, in 1955, coined the current name "familial Mediterranean fever", [START_REF] Heller | Familial Mediterranean fever[END_REF] which was recently validated by an international consortium for taxonomy and definition of autoinflammatory diseases. [START_REF] Ben-Chetrit | Consensus proposal for taxonomy and definition of the autoinflammatory diseases (AIDs): a Delphi study[END_REF] From classical FMF to the clinical criteria for its diagnosis FMF is a recessive disease rare in Europe or USA, but relatively common in some populations of the Mediterranean basin, with prevalence of healthy carriers (ie, individuals carrying one pathogenic mutation) reaching up to one in five in Armenians . [START_REF] Touitou | The spectrum of Familial Mediterranean Fever (FMF) mutations[END_REF] The estimated worldwide prevalence of FMF is 120-150,000 patients. [START_REF] Ben-Chetrit | Familial Mediterranean Fever in the world[END_REF] The classical FMF phenotype is characterized by self-limited and shortduration episodes of fever lasting 2 to 3 days. [START_REF] Ben-Chetrit | Familial Mediterranean fever[END_REF] The mean age at disease onset is 4 years, but symptoms can occur as early as the first year of life or late in adulthood. Symptoms of acute inflammation include sterile peritonitis, pleural inflammation, arthritis, skin rashes, and elevated levels of acute phase reactants related to inappropriate secretion of interleukin 1 (IL)-1, IL-6 and tumor necrosis factor α (TNFα) reflecting the activation of the innate immune system. [START_REF] Yalcinkaya | The value of the levels of acute phase reactants for the prediction of familial Mediterranean fever associated amyloidosis: a case control study[END_REF] Absence of autoantibodies and auto-reactive T cells denotes the absence of abnormal stimulation of adaptive immunity and provides an additional argument for recurrent autoinflammatory fever. Colchicine at an adequate dose and full compliance prevents inflammatory attacks and occurrence of renal AA amyloidosis in approximately 95% of cases. IL-1 blockers can be effective in refractory cases. [START_REF] Benedetti | Canakinumab for the Treatment of Autoinflammatory Recurrent Fever Syndromes[END_REF] FMF is diagnosed on a clinical basis and can be confirmed by genetic testing (see below). The most commonly used clinical criteria are those of Tel Hashomer, [START_REF] Sohar | Tel Hashomer key to severity scores for FMF[END_REF] which are established in the Jewish adult population but also validated in children of other ethnicities. [START_REF] Kondi | Validation of the new paediatric criteria for the diagnosis of familial Mediterranean fever: data from a mixed population of 100 children from the French reference centre for auto-inflammatory disorders[END_REF] The Livneh criteria are also widely used, but they lack specificity in the pediatric population. [START_REF] Livneh | Criteria for the diagnosis of familial Mediterranean fever[END_REF] A Turkish group has proposed new criteria for FMF diagnosis in children, [START_REF] Yalcinkaya | A new set of criteria for the diagnosis of familial Mediterranean fever in childhood[END_REF] which were validated in several ethnic groups. [START_REF] Kondi | Validation of the new paediatric criteria for the diagnosis of familial Mediterranean fever: data from a mixed population of 100 children from the French reference centre for auto-inflammatory disorders[END_REF][START_REF] Demirkaya | Performance of Different Diagnostic Criteria for Familial Mediterranean Fever in Children with Periodic Fevers: Results from a Multicenter International Registry[END_REF] New, more comprehensive diagnostic criteria, are under development.

From the clinic to genetics

The observation of familial FMF cases fueled gene hunting in the early 1990s. The MEFV gene contains 10 exons and encodes the pyrin protein. Four founding mutations in exon 10 (p.M680I, p.M694I, p.M694V, and p.V726A) account for almost 80% of clinically and ethnically typical cases. [START_REF] Touitou | The spectrum of Familial Mediterranean Fever (FMF) mutations[END_REF] To date, nearly 100 times more sequence variants have been recorded in the dedicated online database in fevers (https://infevers.umai-montpellier.fr/). 17 Homozygosity for p.M694V confers the most severe phenotype (more joint and renal damage, earlier onset), [START_REF] Grossman | Familial Mediterranean fever (FMF) phenotype in patients homozygous to the MEFV M694V mutation[END_REF] whereas other low-penetrant variants such as p.E148Q in exon 2, are considered susceptibility factors for inflammation. [START_REF] Marek-Yagel | Is E148Q a benign polymorphism or a diseasecausing mutation?[END_REF]

From classical recessive inheritance to possible dominance and gene-dosage effect

Initial familial segregation analyses demonstrated autosomal recessive inheritance, exacerbated by consanguinity, a cultural habit in Mediterranean countries. [START_REF] Rogers | Familial Mediterranean fever in Armenians: autosomal recessive inheritance with high gene frequency[END_REF] However, in about one-third of typical clinical cases, only one mutation is detected. [START_REF] Jeru | The risk of familial Mediterranean fever in MEFV heterozygotes: a statistical approach[END_REF] The recognition of a large series of heterozygous individuals with a clinical picture of FMF supports that some single mutations (mainly in exon 10) may induce clinical signs. [START_REF] Hentgen | Familial Mediterranean fever in heterozygotes: are we able to accurately diagnose the disease in very young children?[END_REF] Many alternative explanations have been proposed, such as the existence of a second hit in another region of the gene or in another gene implicated in the regulatory pathways of the immune system, epigenetics or environmental factors, but none of these hypotheses have been definitely proven to date. The non-detection of a second mutation does not necessarily confirm a dominant transmission. True dominant transmission is rare in FMF and requires proof of segregation over several consecutive generations, i.e. that patients with overt disease do carry the pathogenic mutation, whereas asymptomatic individuals do not. Anecdotal cases of proven vertical transmission of p.M694I [START_REF] Booth | The genetic basis of autosomal dominant familial Mediterranean fever[END_REF] and p.M694del in clinically ascertained FMF have been described (Table ; available at www.jpeds.com), thus challenging the paradigm of pure recessive inheritance. 24, From gene identification to the understanding of the Pyrin function The apparently recessive nature of FMF has led to the postulation that the underlying molecular mechanism was a loss-of-function of the mutated pyrin. In asymptomatic carriers, the defective function of a mutated allele is compensated by the second wild-type allele and pathogenic mutations in each parental gene are classically necessary for the disease to be expressed. The demonstration that MEFV mutations are in fact gain-of-function reconverted this prevailing concept. [START_REF] Chae | Gain-of-function Pyrin mutations induce NLRP3 protein-independent interleukin-1beta activation and severe autoinflammation in mice[END_REF] The introduction of various exon 10 mutations into mouse pyrin (knock-in) induced a phenotype similar to human FMF, whereas deletion of mouse pyrin (knock-out) did not result in enhanced inflammation.

Pyrin is a key component of a specific inflammasome. [START_REF] Park | Pyrin inflammasome activation and RhoA signaling in the autoinflammatory diseases FMF and HIDS[END_REF] Inflammasomes are multiprotein platforms leading to activation of the pro-inflammatory IL-1 and IL-18 following a pro-inflammatory event. [START_REF] Harapas | An Update on Autoinflammatory Diseases: Inflammasomopathies[END_REF] RhoA GTPases regulate pyrin by activating serine-threonine kinases PKN1 and PKN2, which in turn, phosphorylate pyrin at two specific serines (S208 and S242 in humans). [START_REF] Gao | Site-specific phosphorylation and microtubule dynamics control Pyrin inflammasome activation[END_REF][START_REF] Xu | Innate immune sensing of bacterial modifications of Rho GTPases by the Pyrin inflammasome[END_REF] GTPase-mediated pyrin phosphorylation results in inhibitory binding by cellular 14-3-3 proteins (Figure 2). Upon toxin stimulation or bacterial infection, RhoA GTPases are inactivated, which leads to serine dephosphorylation, 14-3-3 dissociation, and subsequent pyrin inflammasome activation.

From FMF to PAAD

Patients with FMF criteria now benefit from the invaluable progress brought about by the discovery of the MEFV gene. However, sequencing of specific exons has sometimes revealed unusual phenotypes, variants and inheritance. For example, p.P369S and p.R408Q substitutions in exon 3, which most often come from the same parental chromosome (cis variant or complex allele), were associated with a highly variable phenotype, and were infrequently associated with typical FMF symptoms. [START_REF] Ryan | Clinical features and functional significance of the P369S/R408Q variant in pyrin, the familial Mediterranean fever protein[END_REF] The associated phenotypes included atypical FMF, [START_REF] Ryan | Clinical features and functional significance of the P369S/R408Q variant in pyrin, the familial Mediterranean fever protein[END_REF] PFAPA-like [START_REF] Yamagami | Familial Mediterranean fever with P369S/R408Q exon 3 variant in pyrin presenting as symptoms of PFAPA[END_REF] and entero-Behçet disease. [START_REF] Fujikawa | Mediterranean fever (MEFV) variant P369S/R408Q in a patient with entero-Behçet's disease who successfully responded to treatment with colchicine[END_REF] In general, the patients are colchicine-responders. In a three-generation Spanish kindred, five affected members fulfilling the Tel-Hashomer criteria presented with a severe periodic inflammatory disorder segregating with the rare p.H478Y MEFV variant located in exon 5. [START_REF] Aldea | A severe autosomaldominant periodic inflammatory disorder with renal AA amyloidosis and colchicine resistance associated to the MEFV H478Y variant in a Spanish kindred: an unusual familial Mediterranean fever phenotype or another MEFV-associated periodic inflammatory disorder?[END_REF] Thus, the authors questioned whether this familial phenotype represented a case of unusual FMF phenotype or another MEFV-associated periodic inflammatory disorder because of its dominant inheritance, the prolonged febrile episodes, and the resistance to colchicine. Recently, next generation sequencing of a panel of known SAID genes or whole-exome sequencing (WES) facilitated the identification of new mutations and SAID genes but also yielded unexpected results. Two such examples are worth noting (Table andFigure 2). In a family of British descent with a colchicine-responsive autosomal-dominant periodic fever syndrome, WES sequencing was conducted and revealed a c.1730C>A missense mutation in exon 8 resulting in the p.T577N substitution. [START_REF] Stoffels | MEFV mutations affecting pyrin amino acid 577 cause autosomal dominant autoinflammatory disease[END_REF] Mutations at the same codon were found in three other individuals of various ethnic backgrounds. Their disorder was similar to FMF (fever, systemic inflammation, response to colchicine or IL-1 blockade) but somewhat distinct to it (inheritance pattern, duration of attacks lasting one week, evanescent urticaria-like skin rash). The authors hypothesized that mutations at codon 577 lead to a structural change significantly affecting pyrin function or oligomerisation. Dominant inheritance of genetic variants leading to p.T577N was also observed in a Japanese family with PAAD. [START_REF] Thefrenchfmfconsortium | A candidate gene for familial Mediterranean fever[END_REF] 7 In individuals with severe neutrophilic dermatosis resembling pyogenic arthritis, pyoderma gangrenosum and acne (PAPA) syndrome WES investigations disclosed a dominant mutation within and in the immediate vicinity of the 14-3-3 protein pyrin binding domain (Table 1). [START_REF] Masters | Familial autoinflammation with neutrophilic dermatosis reveals a regulatory mechanism of pyrin activation[END_REF][START_REF] Moghaddas | A novel Pyrin-Associated Autoinflammation with Neutrophilic Dermatosis mutation further defines 14-3-3 binding of pyrin and distinction to Familial Mediterranean Fever[END_REF] This new pyrin-associated autoinflammatory disease with neutrophilic dermatosis was named PAAND. Only three PAAND families, all of European origin, have been reported so far: two harbored the p.S242R substitution and the third had the p.E244K substitution. Both mutations abolished the ability of 14-3-3 to bind pyrin, which resulted in a constitutive inflammasome formation.

Conclusion

Pan-genomic approaches have revolutionized the identification of the pathophysiological pathways involved in SAID, an understanding of their regulation but also their better nosological classification.

PAAD is an "umbrella" name chosen by the taxonomy committee to include all diseases caused by pyrin defects or MEFV mutations. [START_REF] Ben-Chetrit | Consensus proposal for taxonomy and definition of the autoinflammatory diseases (AIDs): a Delphi study[END_REF] This general term of PAAD was developed to link different subtypes of disorders of the same genetic origin. [START_REF] Ben-Chetrit | Consensus proposal for taxonomy and definition of the autoinflammatory diseases (AIDs): a Delphi study[END_REF] Butbul Aviel et al found that PFAPA (with and without FMF features) may be more common in patients from Mediterranean ancestry and suggested that the incidence and features of PFAPA may be influenced by the presence of the common MEFV variants. [START_REF] Aviel | Familial Mediterranean Fever Is Commonly Diagnosed in Children in Israel with Periodic Fever Aphthous Stomatitis, Pharyngitis, and Adenitis Syndrome[END_REF] Association of MEFV variants with multifactorial disorders has often been reported (see review). [START_REF] Touitou | Inheritance of autoinflammatory diseases: shifting paradigms and nomenclature[END_REF] However, these variants are thought to act as susceptibility factors with a weak individual effect, and cannot be considered as directly causative of the disease. Therefore, PFAPA does not belong to the PAAD family. In contrast, the molecular mechanism involving the MEFV gene as the cause of the disease is clearly established in PAAND which therefore represents a PAAD subtype. [START_REF] Masters | Familial autoinflammation with neutrophilic dermatosis reveals a regulatory mechanism of pyrin activation[END_REF] Comprehensive analysis of mutations in the MEFV gene revealed that symptom severity is associated with sequence variants in specific exons, at least in typical Armenian individuals. [START_REF] Moradian | Comprehensive analysis of mutations in the MEFV gene reveal that the location and not the substitution type determines symptom severity in FMF[END_REF] The exon in which the genetic variant is located also seems to affect the hereditary mode of transmission of the PAAD concerned, although the exact molecular mechanism underlying these observations remains elusive.

Variable modes of transmission need to be considered for genetic counseling.

We have depicted the steps that led to the elucidation of the role of defective pyrin and have pinpointed the importance of the interpretation of mutations in less commonly affected exons, specifically in non-Mediterranean individuals, or in those in whom only one MEFV mutation has been detected. As a result, retro-phenotyping, which involves the molecular geneticist verifying the clinical presentation of the patient after the mutations have been identified to check for consistency, is becoming increasingly challenging. The reason is that quite often, genetic variants predicted as pathogenic based on familial segregation, mutation type, frequency in the general population, or in silico tools are found in genes that do not seem relevant to the phenotype of the patient. However, effective and targeted treatment depends on the correct genetic diagnosis. Three main periods can be delineated. The first steps were clinical (in red), basically devoted to the disease description, with definitive naming and clinical criteria as key events. The second important period required both clinical and genetic investigations (in purple). Identification of the FMF gene and its protein pyrin was the "diamond" of 1997. The last decade (in blue) saw the advent of high throughput sequencing approaches pinpointing pyrin as being involved in many more phenotypes described under the "umbrella" pyrin-associated autoinflammatory disease (PAAD). The unraveling of the normal pyrin function as well as the demonstration that mutations are gain of function with gene dosage helped in understanding the clinical spectrum of PAAD. a c.1729A>T (p.T577S) mutation was also detected in a Dutch patient with an autoinflammatory phenotype but no familial nor clinical data was available. ** Sporadic patients described in this paper are not shown as dominant transmission is not ascertained *** E148Q was detected in some patients but is not mentioned as it is now recognized as a variant of unknown significance

Legends to figures

PAAND: pyrin-associated autoinflammatory disease with neutrophilic dermatosis

Figure 1 :

 1 Figure 1: Key milestones of the clinical and genetic discoveries in familial Mediterranean fever

Figure 2 :

 2 Figure 2: Schematic representation of the pyrin protein and mutations resulting in pyrin-associated

Table 1 . Families with proven dominant inheritance associated with pyrin mutation compared to classical recessive FMF

 1

	Recessive

A large study from an international database of patients with SAID (Eurofever) has provided clues to begin to understand this apparent contradiction.

The authors highlighted a qualitative (severe mutations vs polymorphisms) and quantitative (2, 1, 0 mutations) molecular gradient paralleling the clinical picture (i.e., ranging from typical FMF to periodic fever, aphthous-stomatitis, cervical pharyngitis and adenitis (PFAPA), a disease for which no causative gene has been pinpointed).[START_REF] Federici | Clinical impact of MEFV mutations in children with periodic fever in a prevalent western European Caucasian population[END_REF] The authors suggested a dose effect (genetic dosage) involved in the phenotype and hypothesized that, under certain circumstances (e.g. presence of variants of still unidentified modifier genes), high penetrance MEFV mutations could give rise to symptom onset even in the presence of one wild-type allele.

Acknowledgments

We thank Laura Smales (biomedediting.com) for editing of English language.