Clinical Presentation and Genetic Variants in Autoin�ammatory Diseases: Results From the German anti-IL1 Registry for Orphan Diseases (GARROD)

Background: To investigate the clinical presentation and genetic variants in patients of the German anti-IL1 registry for autoin�ammatory orphan diseases (GARROD) between 2013-2019. Methods: Multicenter, retrospective analysis of demographic, clinical and genetic data of 231 patients with autoin�ammatory diseases (AID) who received anti-IL1 targeted therapy. Results: In�ammatory attacks started before the age of 18 years in 44% of the patients. Symptom onset was on average 17.8 years prior to the initiation of anti-IL1 targeted therapy. AID spectrum comprised familial Mediterranean fever (FMF; n=71; 30.7%), cryopyrin-associated periodic syndromes (CAPS; n=43; 18.6%), adult-onset Still’s disease (AOSD, n=32; 13.8%) or other AID (n=85; 36.8%). Monogenetic AID were diagnosed by clinical symptoms. Genetic analyses con�rmed the diagnosis in 65% of patients with FMF, 14% with CAPS and 47% with TNF receptor-associated periodic syndrome (TRAPS). Heterozygous MEFV variants and variants of unknown signi�cance (VUS) were detected in 23% of patients with FMF, 51% with CAPS and 47% with TRAPS. Patients with VUS were older at disease onset, indicating a milder phenotype. Twenty-nine patients (12.5%) had secondary AA amyloidosis at initiation of anti-IL1 therapy. Primary Diseases were FMF (n=22), TRAPS (n=1) and idiopathic AA amyloidosis (n=6). Conclusions: Molecular genetic analyses might substantiate the clinical diagnosis of a monogenetic AID. In the absence of a known pathogenic variant, the functional role of VUS can be discussed. Our data support the concept of a variable penetrance of VUS, leading to late-onset AID. Anti-IL1 targeted drugs are used to control systemic in�ammation, prevent disease �ares and AA amyloidosis.


Introduction
The increasing knowledge about autoin ammatory diseases (AID) has changed our view of systemic in ammation over the recent decades.FMF is the most prevalent AID world-wide and colchicine is the standard therapy to prevent FMF attacks and AA amyloidosis.Classi cation criteria were developed for familial mediterranean fever (FMF) [1], Cryopyrin associated periodic syndrome (CAPS) [2] and TNF Receptor associated periodic syndrome (TRAPS) [3].Although genetic variants for FMF, CAPS and TRAPS are known, these variants are neither essential for AID diagnosis nor have they been included in the classi cation criteria.
Patients with systemic onset of juvenile idiopathic arthritis (soJIA, classic Still's disease) and adult onset Still's disease (AOSD) are considered to be polygenetic diseases representing two manifestations within a disease spectrum [4].The demonstration of genetic variants in a subgroup of patients with AOSD suggests a genetic overlap between monogenetic and polygenetic disease with similar clinical features [5].The clinical and laboratory features of AOSD were classi ed previously [6,7].Recently, AOSD was characterized as a disease comprising a broad spectrum of manifestations and potentially lifethreatening complications [8].
Investigations on the pathophysiological role of the in ammasome led to the recognition of interleukin-1beta (IL-1β) as the key cytokine in monogenetic and polygenetic AID.Blocking of IL-1β is an effective treatment option for monogenetic and polygenetic AID [9,10].The IL1-receptor antagonist anakinra (AKN) inhibits the binding of IL-1β to the IL1-receptor.The monoclonal antibody canakinumab (CAN) inhibits IL-1β activity by binding to IL-1β.Recently, AKN [11] and CAN [12] showed e cacy for AOSD treatment with and without macrophage activation syndrome.
The German Anti-IL1 treatment RegistRy in Orphan Diseases (GARROD) was initiated in 2013 when it became evident that blocking IL-1β is effective in AID [6,7].The GARROD registry is a multicentric retrospective analysis of adult patients with autoin ammatory diseases (AID) which were treated with AKN or CAN on an individual basis.The aim of the registry was to provide a research platform to gather and analyze data on the health care of patients with AID as well as to collect information on the safety and e cacy of anti-IL1β treatment.
We compared the clinical diagnoses with the results of genetic analyses in patients with monogenetic and polygenetic AID.

Patient data:
The GARROD registry was initiated in October 2013 at the University of Heidelberg.It was established as a multi-center registry and until December 2018 various institutions in Germany contributed depersonalized data to GARROD.The registry included a consecutive cohort of patients with AID who were treated with AKN or CAN for longer than two months.The purpose of GARROD was to gather and analyze data about diagnoses, safety and e cacy of anti-IL1β treatment in patients with AID.The project was approved by the ethical review committee at the University of Heidelberg (S-103/2013) and by the local ethical committees at the participating centers.

Clinical diagnoses of AID and genetic analysis
FMF [1], CAPS [2], TRAPS [3] and AOSD [4,5] were diagnosed by clinical AID experts, according to the previously published classi cation criteria.Patients with non-classi ed AID, treatment-refractory gout, pleuro-pericarditis, Schnitzler's syndrome, idiopathic AA amyloidosis and other rare diseases according to expert opinion were also included in this registry.Patients with the clinical diagnosis of a monogenetic AID were screened for variants in MEFV (FMF), NLRP3 (CAPS) and TNFRSF1A (TRAPS).Genetic analyses were conducted at several commercial laboratories.
Single nucleotide polymorphisms (SNP) with a minor allele frequency (MAF) of ≥ 1% in the general population were considered as a variant of unknown signi cance (VUS) or as benign variant.The Infevers database [13, last access 04 Jan 2021] served as a reference to evaluate the functional role of single variants.The nomenclature of genetic variants followed the current guidelines of the Human Genome Organisation (HUGO), Human Genome Nomenclature Committee (HGNC), Human Genome Variation Society (HGVS) and the Infevers database [13].The MAF was derived from the National Center for Biotechnology Information (NCBI) SNP data base (https://www.ncbi.nlm.nih.gov/pubmed).

Anti-IL1 targeted medication:
AKN was initiated using 100mg sc daily.Patients with renal failure started with AKN 100mg sc 3 times a week.Patients with severe local reactions to AKN reduced their schedule to AKN every 2 to 3 days until local reactions disappeared.Patients with partial control of the attacks used AKN 100mg bid during an attack and 100mg every day for maintenance.CAN was initiated using 150mg sc.every 8 weeks.Patients with a transient response for 4 to 8 weeks reduced the intervals to 150mg every 4 weeks.Patients with a partial response to 150mg used CAN 300mg every 4 to 8 weeks to maximize the response and to achieve remission.Some patients who had an inadequate response or intolerance to AKN were switched to CAN.

Concomitant medication:
Concomitant medication was continued if the patient reported at least partial bene ts and additional effectivity compared IL-1β inhibitory monotherapy.FMF patients continued with colchicine if they tolerated at least 0.5mg per day or more.The majority of FMF patients took 2 mg colchicine per day.
Patients with other AID had concomitant colchicine if they reported any bene t before the addition of AKN.Patients with AOSD, TRAPS and pleuro-pericarditis used pulses of prednisolone 40mg and tapering to control in ammatory attacks.

Diagnosis of amyloidosis:
All patients were screened for renal insu ciency, proteinuria and myocardial hypertrophy as potential indicators for amyloidosis [14].If functional organ abnormalities were detected, the affected organs were biopsied.The diagnosis of amyloidosis was based on a positive Congo red staining and a yellow-green birefringence on polarization microscopy.The type of AA amyloid was con rmed by using immunohistochemistry [15].

Results
Two hundred and thirty-one adult patients with AID were analyzed in the GARROD registry.After previous anti-in ammatory treatment options such as for instance NSAIDS, colchicine or glucocorticoids failed to control the systemic in ammation anti-IL-1β treatment was initiated and patients could be enrolled in the GARROD registry immediately or within the following 12 months.Demographic characteristics are indicated in Table 1.The mean age of patients with AID at symptom onset was 25.1 years.The time between the rst AID symptoms and recruitment to the GARROD registry was 17.8 years.Mean age at baseline was 42.9 years.Twenty-nine patients with AID (13%) had primary or secondary AA amyloidosis at the time of initiation of the anti-IL1β treatment [Table 1].
One-hundred and twenty-nine patients (56%) had an onset of AID symptoms after the age of 18 years (Table 2).Patients with monogenetic AID (FMF, CAPS, TRAPS) tend to report a disease onset before the age of eighteen (63%), whereas only 37% of polygenetic or unclassi ed AID symptoms started before the age of 18 years.Patients with unclassi ed AID were evenly distributed between juvenile and adult-onset.Polygenetic AID disease onset was more common in adults (Table 2).

Clinical diagnosis and genetic testing for AID
Molecular for monogenetic AIDs were performed in 128 of 133 patients (96%) and compared with the clinical diagnosis of FMF (n = 71), CAPS (n = 43) and TRAPS (n = 19).Genetic variants were considered to be either pathogenic (p), variants of unknown signi cance (v), benign polymorphisms or wild type (w) (Table 3).The autosomal recessive type of FMF with two pathogenic MEFV variants, either homozygous or compound heterozygous, was genetically con rmed in 65% of patients with FMF.By including an autosomal dominant type of FMF with at least one FMF variant (pv, pw, vv or vw), the rate of molecular genetic con rmation increased to 88% of patients with the clinical diagnosis of FMF (Table 3).Nine patients with a clinical FMF diagnosis (13%) had no molecular genetic con rmation by MEFV analysis.
CAPS and TRAPS are rare diseases with an autosomal dominant trait.However, pathogenic variants were con rmed only in 14% of NLRP3 and 47% of TNFRSF1A analyses.The detection of VUS (51% in NLRP3, 47% in TNFRSF1A, Table 3) might support the clinical diagnosis of a monogenetic AID which was su cient to initiate anti-IL1β therapy.
Further analyses for the identi cation and characterization of pathogenic variants and VUS will be necessary for the interpretation of molecular genetic results in the future.

MEFV variants correlate with FMF
FMF is the most abundant AID in this cohort.The MEFV variants p.Met680Ile/Val and p.Met694Val/Ile are well known pathogenic variants that are associated with a more severe FMF disease.Patients with two high penetrance variants are younger at FMF onset, have a higher FMF activity score and are more likely to develop secondary AA amyloidosis (Table 4).Patients with FMF + AA had a mean age of 17.3 years at FMF onset and 45.6 years at the diagnosis of AA amyloidosis (Table 5).Patients with idiopathic AA amyloidosis seem to have no symptomatic primary in ammatory disease and develop AA amyloidosis at the same age of 44.2 years compared to patients with FMF + AA (Table 5).

Discussion
This is a multicenter retrospective analysis of 231 patients with AID who were treated with IL-1β blocking medication between 2013 and 2018.The mean age at baseline was 42.9 years which is consistent with the data from previously published registries [11,16,17].In 56% of the patients the rst in ammatory attacks occurred after the age of 18 years.The mean disease duration was 17.8 years between the symptom onset and the initiation of anti-IL-1β medication, highlighting an urgent need for diagnostic and therapeutic improvement.
The most prevalent diagnoses were FMF, CAPS and AOSD.When the GARROD registry was established in 2013, CAN and AKN were both approved only for CAPS.In the past years, AKN was widely used for rare AID in Germany without approval, probably because of the advantage of a short half-life of AKN, and to avoid the high costs of CAN.Only recently, CAN and AKN were both approved for sJIA and AOSD.A randomized controlled trial of CAN for patients with colchicine-resistant FMF, TRAPS and HIDS nally resulted in the approval of CAN for these rare diseases [18].AKN has been investigated in an RCT in patients with crFMF [19].Several investigators report the use of AKN in crFMF [20,21].Our data con rm the observations from previous reports and suggest that AKN can be used for patients with crFMF.Finally, AKN was approved for crFMF by the European Medical Agency (EMA) in April 2020.
Genetic analyses were performed in 129 patients with the clinical diagnoses of FMF, CAPS and TRAPS.Analysis of classi ed pathogenic variants led to genetic con rmation in FMF (65%), CAPS (14%) and TRAPS (47%).
There is an ongoing controversy regarding the relevance of heterozygous variants in FMF, pathogenic variants and variants of unknown signi cance (VUS) in all monogenetic AID.
If we consider VUS as low penetrance variants that might contribute to the in ammatory attacks, the rate of genetic con rmation increased by + 23% in FMF (88%), + 51% in CAPS (65%) and + 47% in TRAPS (94%).These data show a signi cant discrepancy between clinical diagnosis and molecular genetic con rmation of an AID, especially for rare diseases like CAPS and TRAPS.
The NLRP3 variants V198M and Q703K are considered as VUS or benign polymorphism [13; 22; 23] and as low penetrance variants by others [24; 25; 26].There is a controversy on the importance of the most prevalent TNFRSF1A variant R92Q.Some authors consider it as VUS [13] and others as low penetrance variant [27; 28].However, the clinical phenotype should determine the diagnosis and if necessary, the treatment initiation of anti-IL1β drugs.If anti-IL1β treatment is effective to control clinical symptoms and systemic in ammation, the treatment regime should be continued even if the genetic test does not con rm the clinical diagnosis.
Our data con rm a dose dependent genotype phenotype correlation of high penetrance MEFV variants and a more severe FMF disease.These results are consistent with other reports in cohorts with pediatric [29] and adult patients [21,30].Our observations suggest the AID comprise a spectrum of disease which can present at any age.Therefore, pediatric and adult rheumatologists should be familiar with symptoms and differential diagnoses of various AIDs.
In our cohort FMF patients with AA amyloidosis had a mean disease duration of 28.3 years until the diagnosis of AA amyloidosis was established.Lack of information, limited compliance and incomplete control of the in ammatory attacks probably contribute to the risk of AA amyloidosis.However, even if we would be able to prevent AA amyloidosis in all AID patients, we would continue to observe patients with idiopathic AA amyloidosis [21].
Limitations of our study were the retrospective analysis which was partially dependent on the patients recall of their rst attacks and the heterogeneous cohort with monogenetic and polygenetic AID with and without AA-amyloidosis.
Our data underline the importance for clinicians to learn the features of clinical presentation, differential diagnosis and treatment of AID.Our ndings suggest that patients with AID should be guided by pediatric and adult rheumatologists, nephrologists and dermatologists.Networks of pediatric and adult AID specialists could help to increase the knowledge about the presentation of AID in small children, adolescents and adults.
Statistics:Statistical analyses were performed using the Statistical Package for the Social Sciences (IBM SPSS Version 19) and the R statistical program (The R Foundation for Statistical Computing Platform, version 3.3.2.).Results are presented as mean and standard deviation (SD), median and interquartile range (IQR) or range as indicated.Categorical variables were compared using Fishers exact test.Numerical variables were compared with unpaired, two-tailed Student's T-test.Statistical signi cance is considered if p was below 0.05.