A First Step for the Molecular Characterization of Neurological Involvement of Behçet Syndrome: an Italian Pivotal Study

Behçet syndrome (BS) is a vasculitis characterized by several clinical manifestations including the rare neurological involvement (neuro-BS, NBS). The aim of our pivotal study was to investigate the mutational status of several inflammation-related genes in a cohort of Italian patients with and without the neurological involvement (20 NBS vs 40 no-NBS patients). The preliminary in silico single nucleotide polymorphism (SNP) selection and primer design were performed by NCBI Primer-Blast tool. Genomic DNA was isolated and amplified using PCR. PCR amplicons were sequenced and bioinformatically analysed. Twelve tagSNPs were selected and genotyped: ERAP1 rs30187, rs17482078, and rs27044; IL10 rs1800872 and rs1518111, IL12A rs17810546, IL23R rs17375018, IL23R-IL12RB2 rs924080, STAT4 rs7572482, CCR1 rs7616215, KLRC4 rs2617170, and UBAC2 rs3825427. ERAP1 and IL23R SNPs showed statistically significant higher frequencies in NBS group than no-NBS. ERAP1 rs30187 AA was more common in no-NBS patients (20.0% NBS vs 47.5% no-NBS; p < 0.05), while rs17482078 GA frequency was higher in NBS patients (55.0% NBS vs 22.5% no-NBS; p < 0.05, OR: 4.21). IL23R rs17375018 GG was more frequent in NBS group (65.0% NBS vs 40.0% no-NBS; p < 0.05), according to a previous finding. No other statistically significant differences were found. In conclusion, ERAP1 and IL23R SNPs were found associated with neurological involvement of BS. Additional and larger analyses were required to verify our preliminary findings

FRI0569 SERUM AMYLOID A: ASSESSMENT OF REFERENCE VALUE AND COMPARISON OF SERUM CONCENTRATION IN HEALTHY SUBJECTS AND PATIENTS WITH BEHÇET SYNDROME

Background:Serum amyloid A (SAA) is a family of acute-phase reactants. The rise of SAA concentration in blood circulation is a clinical marker of active inflammation in several auto-inflammatory diseases, including Behçet syndrome (BS). Despite its practical and analytical advantages, SAA measurement by ELISA has been mainly used as a research tool rather than for the routine laboratory testing due to the lack of a robust reference data in the literature.Objectives:Using the recommended procedures of the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC), we aimed to develop the SAA reference interval for a well-defined Italian healthy population (HC). Secondly, we compared the SAA serum concentration between HC and patients with BS.Methods:Sera specimens were collected from adult healthy blood donors after rule out the exclusion criteria (inflammatory disorders, ongoing infections, pregnancy and breastfeeding, obesity, using oral contraceptives, use of any medication, or consumed of alcohol), and from unselected BS patients fulfilling the International Study Group (ISG) classification criteria. Serum SAA concentrations were detected and quantified with a commercial solid phase sandwich enzyme-linked immunosorbent assay (Human SAA ELISA kit, IBL International GmbH, Hamburg, Germany) used on automated analyzer (Immunomat, SERION Diagnostic, Alifax, Polverara (PD), Italy) according to the manufacturer's protocol. Statistical analysis and data normalization of HC SAA values were carried out to determine the reference cut off. In the second step of the study, HC and BS patients were stratified in two groups according to the cut-off value.Results:We recruited 141 HC (84 M and 57 F; mean age, 44.5±13.2 years) and 63 BS patients (39 M and 24 F mean age, 45.3±13.2 years) assayed for SAA. The reference cut-off was calculated as 225 ng/ml. No statistically significant differences were found between males and females when SAA means were compared, suggesting that not gender-partitioned reference range is recommended for this analyte. After the stratification according to the cut-off value (group 1: 225 ng/ml), we found 53/63 (84.1%) BS patients and 133/141 (94.3%) HC with concentration less than cut-off value, respectively. We identified 10/63 (15.9%) BS patients and 8/141 (5.7%) HC within the second group. The difference was statistically significant (p=0.0177; OR: 3.14, 95% CI: 1.17-3.38).Conclusion:This study allowed to define a widely accepted reference cut-off for the SAA detected by ELISA, responding to an unmet need of laboratory medicine. We found a statistically significant higher frequency of BS patients compared with HC when SAA values is higher than cut-off (225 ng/ml). This preliminary data could add significant information for better clarify the role of SAA as biomarker of inflammation and in guidance of clinical practice. Further studies will be required to stratify SAA values in relation to disease activity of BS.Disclosure of Interests:Teresa Carbone: None declared, Maria Carmela Padula: None declared, Vito Pafundi: None declared, Carlo Schievano: None declared, Nancy Lascaro: None declared, Angela Padula: None declared, Pietro Leccese: None declared, Salvatore D'Angelo Consultant of: AbbVie, Biogen, BMS, Celgene, Eli Lilly, MSD, Novartis, and UCB, Speakers bureau: AbbVie, BMS, Celgene, Eli Lilly, Novartis, Pfizer, and Sanof

Identification of a de novo NLRP3 gene variation in an Italian Behçet syndrome patient

A novel nonsynonymous variation of NLRP3 was identified in an Italian patient with Behçet syndrome using both bioinformatics and molecular methods. This variation was a thymine to guanine polymorphism responsible for the isoleucine to serine amino acid change at position 348. The novel variation was predicted to be a pathogenic allele

Genotyping of Italian patients with Behçet syndrome identified two novel ERAP1 polymorphisms using sequencing-based approach

The endoplasmic reticulum aminopeptidase protein 1 gene (ERAP1) is related to several human diseases, including Behçet syndrome (BS), a multisystemic disorder with unknown etiology. ERAP1 is involved in immune response and its role can be influenced by gene single nucleotide variations (SNVs). We genotyped the ERAP1 whole structure in 50 consecutive BS patients and 50 ethnically-matched healthy controls using both bioinformatics and molecular methodologies. We identified two novel heterozygous missense SNVs of ERAP1 exon3 responsible for the p.Glu183Val and p.Phe199Ser changes. The first variation was recognized in 7/50 (14%) BS patients and involved the substrate binding site (p.Glu183) required for the anchorage of the peptide N-terminal group. The SNV was predicted to be a damaging variation, as well as the p.Phe199Ser substitution (PolyPhen-2 and SIFT on line software). 3D protein structure prediction showed a change in energy score when the wild-type and the variant states were compared, probably influencing the substrate binding and the protein folding. The first variation was associated to a more stable protein chain, while the second polymorphism was related to a less stable protein chain. Our data need to be tested in larger genetic studies

From structure to function for the characterization of ERAP1 active site in Behçet syndrome. A novel polymorphism associated with known gene variations

Introduction: ERAP1 has been recently proposed as risk marker of Behçet syndrome (BS). Gene single nucleotide polymorphisms (SNPs) could affect the enzymatic activity and the conserved active site is pivotal for the aminopeptidase function. This study aims to characterize the ERAP1 active site in a cohort of BS patients vs healthy controls (HC) integrating genomics, transcriptomics and bioinformatics approach. Materials and methods: We recruited 109 consecutive Italian BS patients (63M:46 F; mean age: 45.07 ± 12.28 years) and 106 matched HC (55M:51 F; mean age: 42.57 ± 12.29 years). DNA was isolated and amplified using PCR with home made-primer pairs. PCR products were directly sequenced and computational analyses were performed to search active site SNPs (NCBI-BlastN tool), to predict SNPs functional effect (PolyPhen-2 software) and to obtain protein 3D modelling (Protean3D software). In a second phase of analysis, RNA was extracted and reverse transcribed. Quantitative Real-Time PCR (qPCR) was performed to assess ERAP1 mRNA level in presence (target) and in absence (control) of gene polymorphisms. The Fold change was calculated for the relative quantification of gene expression. Results: A novel coding variation (NG_027839.1:g.25637 T > G; NP_057526.3:p.Phe360Cys, HGSV nomenclature) was found in heterozygosity state in 5/109 BS patients (4.59 % of cases) and none of HC. It was recognized in association with rs2287987, rs30187, rs17482078, and rs27044 BS-related polymorphisms for 4 out of 5 patients. All patients carrying the novel SNP were HLA-B*51-positive. The novel SNP was released in GenBank database with MK140632.1 ID. The SNP was predicted to be damaging and resides within the Zn-binding HEXXH(X)18E region of the active site, changing the structurally conserved region for the amminopeptidase function. In fact, the change in energy (ΔE) score between wild-type and SNP-containing protein showed a less stable protein in presence of p.Cys360 (ΔE:3.584) (Protean3D prediction). Preliminary qPCR results underlined a significant difference in fold change value when target and control values were compared (p < 0.05), suggesting a reduced expression of ERAP1 mRNA in presence of the novel SNP. Conclusions: Our study strengthens the association between ERAP1 and BS. The most significant point was the localization of the novel p.Phe360Cys SNP within the Zn-binding region of protein active site that was predicted to affect its function, causing protein destabilization. Our findings need to be tested in larger genetic studies

Distribution of rs17482078 and rs27044 ERAP1 polymorphisms in a group of Italian Behçet’s syndrome patients: a preliminary case–control study

The Endoplasmic reticulum aminopeptidase protein 1 (ERAP1) trims N-terminal amino acids from epitope precursors for Major Histocompatibility Complex class I presentation. Genome-wide association studies demonstrated that ERAP1 gene single nucleotide polymorphisms (SNPs) are associated with Behçet’s syndrome (BS). This study was conducted on the two most consistently BS-associated ERAP1 polymorphisms, rs17482078 (NG_027839.1:g.35983G>A) and rs27044 (NG_027839.1:g.35997C>G) to analyse their distribution in 55 Italian BS patients and 65 ethnically matched controls (healthy controls, HC) and to test their association with BS risk. SNPs were detected by isolation, amplification of genomic DNA and direct sequencing. SNPs functional effects were predicted by bioinformatics software. The odds ratio (OR) with 95% confidence intervals was calculated to assess the strength of BS association for genotypes and alleles, also validated by logistic regression (LR). LR was used to test the association between both SNPs and patients HLA genetic data. Bonferroni correction was also applied. Comparing patients and controls, we found a significant higher frequency of rs17482078 A allele (32.73% BS vs 17.69% HC, p = 0.007) and AA genotype (18.18% BS vs 0% HC; p = 0.0003) and rs27044 G allele (63.64% BS vs 46.92% HC; p = 0.0096) in BS group after Bonferroni correction. No association was found between HLA-B*51 and both ERAP1 SNPs. Although preliminary, our data show a stronger association of rs17482078 with BS compared to rs27044 by means of case–control genetic analysis and bioinformatics prediction of protein structure change. A larger series of patients and controls is required to confirm our preliminary findings

Real-world effectiveness of apremilast in multirefractory mucosal involvement of Beh\ue7et\u2019s disease

Relapsing oral and genital ulcers (OGUs) represent the stigmata of Beh\ue7et\u2019s disease (BD) and may be very painful, affecting both quality of life and relationships. A wide number of topical and immunosuppressive drugs can be used to treat ulcers [1], but failures are commonly reported. The efficacy of the phosphodiesterase-4 inhibitor apremilast has been proven in OGUs of BD in two randomized clinical trials (RCT) [2, 3], whereas only two case reports are available until now [4, 5]. We aimed at evaluating the real-world effectiveness of apremilast in BD patients with OGUs refractory to conventional and/or biologic treatments. We retrospectively evaluated patients classified as BD, according to International Criteria for BD [6] and International Study Group [7] criteria, who underwent apremilast (30 mg twice daily) for multirefractory OGUs from November 2017 to January 2019. The number of OGUs was assessed at baseline and either at 3 and 6 months. Pain from ulcers and BD activity were evaluated via 100-mm visual-analogue scale (VAS) and BD Current Activity Form (BDCAF). We also recorded the number of oral and genital ulcer flares both in the 4 weeks prior to apremilast start and throughout the observation period (Table 1 and Supplementary Table 2). The occurrence of adverse events was also reported. Paired t-test or Wilcoxon matched-pair signed rank test were used for statistical analysis. The off-label use of apremilast was approved by the Hospital Ethics Committee in compliance with the Declaration of Helsinki. All patients provided a written informed consent. Thirteen patients (females 9/13) with disease duration (mean \ub1 SD) of 154 \ub1 167 months were analysed (Table 1). At 3 months, (data from 12/13 patients) active OGUs were significantly less (p=0.02 for both) than baseline (Table 2). Three patients stopped the treatment due to diarrhoea. At 6 months, active oral ulcers and oral relapses were still lower than baseline (p=0.03 for both), whereas only a positive trend (p=0.07) for genital ulcers was seen (data from 8/13 patients) (Table 2). Ulcer VAS pain was 67 \ub1 16 at baseline, and a prompt amelioration was observed at 3 months (29 \ub1 32, p=0.002), and confirmed at 6 months (20 \ub1 19, p=0.005) (Table 2). Likewise, BDCAF dropped from 4.5 \ub1 2.9 of baseline to 3.2 \ub1 3.4 at 3 months (p=0.01), and was persistently low up to 6 months (2.3 \ub1 3.7, p=0.01) (Table 2). Serious adverse events were not observed. Our findings are consistent with a recent RCT on 111 BD patients [2], which showed the efficacy of apremilast in reducing both number and pain of oral ulcers [2]. Preliminary results from another study confirm the significant decrease of total number of oral ulcers and resolution of genital ulcers over 12 weeks in the apremilast group [3]. Similarly, in our study the mean number of oral relapses during therapy was significantly lower than that in the 4 weeks prior to apremilast. Interestingly, an appreciable reduction of VAS pain and BDCAF was already seen at 3 months and persisted up to 6 months. Of note, the overall beneficial effect of apremilast also on joint symptoms should be highlighted, as emerged by the BDCAF evaluations. Apremilast was safe and no serious adverse events were observed during the time span of our study. The main limitations of our study were the small sample size and the short-term follow-up. In addition, patients had been referred to our tertiary care centres since they were difficult-to-treat or refractory to therapy, configuring a possible selection bias. Nevertheless we provide evidence that apremilast may induce a meaningful and early benefit in BD patients with multirefractory OGUs also in real-life settings

Validity of Machine Learning in Predicting Giant Cell Arteritis Flare After Glucocorticoids Tapering

Background: Inferential statistical methods failed in identifying reliable biomarkers and risk factors for relapsing giant cell arteritis (GCA) after glucocorticoids (GCs) tapering. A ML approach allows to handle complex non-linear relationships between patient attributes that are hard to model with traditional statistical methods, merging them to output a forecast or a probability for a given outcome. Objective: The objective of the study was to assess whether ML algorithms can predict GCA relapse after GCs tapering. Methods: GCA patients who underwent GCs therapy and regular follow-up visits for at least 12 months, were retrospectively analyzed and used for implementing 3 ML algorithms, namely, Logistic Regression (LR), Decision Tree (DT), and Random Forest (RF). The outcome of interest was disease relapse within 3 months during GCs tapering. After a ML variable selection method, based on a XGBoost wrapper, an attribute core set was used to train and test each algorithm using 5-fold cross-validation. The performance of each algorithm in both phases was assessed in terms of accuracy and area under receiver operating characteristic curve (AUROC). Results: The dataset consisted of 107 GCA patients (73 women, 68.2%) with mean age (± SD) 74.1 (± 8.5) years at presentation. GCA flare occurred in 40/107 patients (37.4%) within 3 months after GCs tapering. As a result of ML wrapper, the attribute core set with the least number of variables used for algorithm training included presence/absence of diabetes mellitus and concomitant polymyalgia rheumatica as well as erythrocyte sedimentation rate level at GCs baseline. RF showed the best performance, being significantly superior to other algorithms in accuracy (RF 71.4% vs LR 70.4% vs DT 62.9%). Consistently, RF precision (72.1%) was significantly greater than those of LR (62.6%) and DT (50.8%). Conversely, LR was superior to RF and DT in recall (RF 60% vs LR 62.5% vs DT 47.5%). Moreover, RF AUROC (0.76) was more significant compared to LR (0.73) and DT (0.65). Conclusions: RF algorithm can predict GCA relapse after GCs tapering with sufficient accuracy. To date, this is one of the most accurate predictive modelings for such outcome. This ML method represents a reproducible tool, capable of supporting clinicians in GCA patient management

HCV infection is a risk factor for gallstone disease in liver cirrhosis: An Italian epidemiological survey

We assessed the prevalence of gallbladder disease (i.e. gallstones plus cholecystectomy) among patients with liver disease and its association with the severity and aetiology of hepatic injury. Subjects, referred to 79 Italian hospitals, were enrolled in a 6-month period. The independent effect of the severity and aetiology of liver disease on gallstone disease prevalence was assessed by multiple logistic regression analysis. Overall, 4867 subjects tested anti-hepatitis C virus (HCV) positive alone, 839 were hepatitis B virus surface antigen (HBsAg) alone, and 652 had an excessive alcohol intake. The prevalence of gallstone disease was 23.3% in anti-HCV-positive patients, 12.4% in HBsAg positive and 24.2% in subjects reporting excessive alcohol intake, respectively. Gallstone disease prevalence increased by age in each aetiological category. The proportion of patients with gallstone disease who had a cholecystectomy was the highest in HCV+ subjects. After adjusting for the confounding effect of age and body mass index, compared with patients with less severe liver disease, subjects with HCV-related cirrhosis, but not those with alcohol-related cirrhosis, were more likely to have gallstone disease. Subjects with HCV-related cirrhosis (OR 2.13, 95% CI: 1.38-3.26) were more likely to have gallstone disease when compared with those with HBV-related cirrhosis. HCV infection is a risk factor for gallstone disease. In Italy, the high prevalence of HCV infection among cirrhotic patients has important implications, as cholecystectomy in these subjects is associated with high risk of morbidity and mortality. \uc2\ua9 2007 The Authors