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Objective: Our pharmacogenomic study evaluated the influence of the presence/absence of genetic variants of psoriasis-risk loci on the clinical response to secukinumab. Differences in the single-nucleotide polymorphism (SNP) pattern characterizing HLA-Cw6+ or HLA-Cw6- patient subpopulations, showing high or low responses to secukinumab, were also analyzed. Methods: 417 SNPs were analyzed by Next-Generation Sequencing technology, in a cohort of 62 psoriatic patients and undergone secukinumab treatment. Univariate regression analysis was employed to examine the association between SNP and clinical response to secukinumab. Multivariate analysis was also performed to assess multivariate differences in SNP pattern of HLA-Cw6+ or HLA-Cw6- patients showing high or low responses to secukinumab. Results: Eight SNPs in HLA-C and upstream region (rs13207315, rs6900444, rs12189871, rs12191877, rs4406273, and rs10484554), including HLA-Cw6 classical allele (rs1131118), and three in MICB-DT (rs9267325), DDX58 (rs34085293) and TYK2 (rs2304255) genes, associating with excellent response to secukinumab were identified. Importantly, rs34085293 or rs2304255 SNP status defined a subgroup of super-responder patients. We also found that HLA-Cw6+ and HLA-Cw6- patients carried out specific patterns of SNPs associating with different responses to secukinumab. Conclusion: Assessment of HLA-Cw6, together with other allelic variants of genes, could be helpful to define patients which better benefit from anti-IL-17 therapy. Abbreviations: PASI: Psoriasis Area and Severity Index; SNP: Single-Nucleotide Polymorphism Rs: Reference SNP; PASI75: 75% reduction in Psoriasis Area and Severity Index; PASI90: 90% reduction in Psoriasis Area and Severity Index; PASI100: 100% reduction in Psoriasis Area and Severity Index; NGS: Next-Generation Sequencing; OR: Odds Ratio; CAP: Canonical Analysis of Principal coordinates; BMI: Body Mass Index; LD: Linkage Disequilibrium.

Immunomodulation with anti‐TNF‐α is highly effective in the treatment of various immune‐mediated inflammatory diseases, including hidradenitis suppurativa (HS). However, this may be responsible for unexpected paradoxical psoriasiform reactions. The pathogenic mechanisms underlying the induction of these events are not clear, even though the involvement of innate immune responses driven by plasmacytoid dendritic cells (pDC) has been described. In addition, the genetic predisposition to psoriasis of patients could be determinant. In this study, we investigated the immunological and genetic profiles of three HS patients without psoriasis who developed paradoxical psoriasiform reactions following anti‐TNF‐α therapy with adalimumab. We found that paradoxical psoriasiform skin reactions show immunological features common to the early phases of psoriasis development, characterized by cellular players of innate immunity, such as pDC, neutrophils, mast cells, macrophages, and monocytes. In addition, IFN‐β and IFN‐α2a, two type I IFNs typical of early psoriasis, were highly expressed in paradoxical skin reactions. Concomitantly, other innate immunity molecules, such as the catheledicin LL37 and lymphotoxin (LT)‐α and LT‐β were overproduced. Interestingly, these innate immunity molecules were abundantly expressed by keratinocytes, in addition to the inflammatory infiltrate. In contrast to classical psoriasis, psoriasiform lesions of HS patients showed a reduced number of IFN‐γ and TNF‐α‐releasing T lymphocytes. On the contrary, IL‐22 immunoreactivity was significantly augmented together with the IL‐36γ staining in leukocytes infiltrating the dermis. Finally, we found that all HS patients with paradoxical reactions carried allelic variants in genes predisposing to psoriasis. Among them, SNPs in ERAP1, NFKBIZ, and TNFAIP genes and in the HLA‐C genomic region were found.

The partner and localizer of BRCA2 (PALB2) is a major BRCA2 binding partner that participates in homologous recombination repair in response to DNA double-strand breaks. Germline alterations of the PALB2 gene have recently been associated with a high risk of developing breast cancer. We investigated a 37-year-old Caucasian woman with breast cancer and family history of breast cancer using targeted next generation sequencing. A novel heterozygous deletion involving exons 5 and 6 was found in the PALB2 gene, and resulted in the production of a truncated PALB2 protein. These findings expand the mutational spectra of PALB2-associated breast cancer, and may improve the mutation-based screening and genetic diagnosis of breast cancer.

Dysregulated cross-talk between immune cells and epithelial compartments is responsible for the onset and amplification of pathogenic auto-inflammatory circuits occurring in psoriasis. NAMPT-mediated NAD salvage pathway has been recently described as an immunometabolic route having inflammatory function in several disorders, including arthritis and inflammatory bowel diseases. To date, the role of NAD salvage pathway has not been explored in the skin of patients affected by psoriasis. Here, we show that NAD content is enhanced in lesional skin of psoriatic patients and is associated to high NAMPT transcriptional levels. The latter are drastically reduced in psoriatic skin following treatment with the anti-IL-17A biologics secukinumab. We provide evidence that NAMPT-mediated NAD+ metabolism fuels the immune responses executed by resident skin cells in psoriatic skin. In particular, intracellular NAMPT, strongly induced by Th1/Th17-cytokines, acts on keratinocytes by inducing hyper-proliferation and impairing their terminal differentiation. Furthermore, NAMPT-mediated NAD+ boosting synergizes with psoriasis-related cytokines in the upregulation of inflammatory chemokines important for neutrophil and Th1/Th17 cell recruitment. In addition, extracellular NAMPT, abundantly released by keratinocytes and dermal fibroblasts, acts in a paracrine manner on endothelial cells by inducing their proliferation and migration, as well as the expression of ICAM-1 membrane molecule and chemokines important for leukocyte recruitment into inflamed skin. In conclusion, our results showed that NAMPT-mediated NAD salvage pathway contributes to psoriasis pathogenic processes by amplifying epithelial auto-inflammatory responses in psoriasis.

The ST18 ˗497-65050 T > C polymorphisms (rs17315309) exhibit a very strong association in the pathogenesis of Pemphigus Vulgaris (PV) and could represent a new potential molecular target for the treatment of disease. The present study aimed to establish a low-cost, sensitive and reliable assay using high-resolution melting curve analysis (HRMA) on magnetic induction rotor-based platform, the Magnetic Induction Cycler (MIC) (Bio molecular Systems). HRMA assay was able to identify easily and unambiguously the c.-497-65050 T > C genotypes evaluating melting curve shape and melting temperature (Tm). The results of HRMA were validated by direct DNA sequencing. The HRMA is rapid, sensitive, low-cost and high-throughput assay to screen the rs17315309 variant and could be used in clinical diagnostic laboratories.

Background Significant alterations of the cutaneous microbiota (CM) have been recently demonstrated in bullous pemphigoid (BP). Microbiome data of both oral cavity (OM) and gut (GM) from patients affected by bullous disease are not available yet and, further consistent studies focused on the role of such microbial populations are still missing. Objective Objective: In this pilot study we characterized and compared GM, OM and CM of patients affected by pemphigus vulgaris (PV) and BP to investigate a distinctive microbiome composition in this two rare dermatological disorders. Methods High-throughput sequencing of the V1-V3 hyper-variable regions of 16S rRNA was used to compare the bacterial community composition of stool, skin and oral mucosae swabs in a cohort of PV and BP patients. A dedicated bioinformatics software coupled with in-house pipeline was implemented to analyse and compare diseases dataset. Results GM samples of both PV and BP patients were principally characterized by Firmicutes and Bacteroidetes phyla. Interestingly, the Firmicutes phylum and Staphylococcus genus were mainly represented in cutaneous samples. The diversity of phyla in oral mucosae was higher than those of gut and skin samples and, Bacteroidetes phylum was significantly underrepresented in all PV samples. Conclusion Firmicutes phylum and Staphilococcus genus were the most represented in OM and CM swabs of PV and BP microbial populations. Moreover, we argue the quantitative imbalance linked to the decrease of Bacteriodetes in the oral cavity of PV patients might be associated to disease typical fetor. To shed light on this peculiar feature further studies are still required.

Introduction Loss-of-function mutations in cytochrome P450 family 24 subfamily A member 1 (CYP24A1) gene are associated with Idiopathic Infantile Hypercalcemia (IIH) and adult kidney stone disease. The enzyme deficiency leads to an impaired vitamin D catabolism pathway, resulting in a syndrome characterized by recurrent hypercalcemia, hypercalciuria and suppressed parathyroid hormone (PTH) levels. In these patients, the genetic evaluation of CYP24A1 is an important diagnostic tool, allowing the definitive diagnosis of IIH. Methods A rapid CYP24A1 gene testing based on High Resolution Melting Analysis (HRMA) was designed in order to detect the CYP24A1 c.428_430delAAG (p.Glu143del), a recurrent IIH-associated variant. Results HRMA method was able to identify c.428_430delAAG genotypes evaluating melting curve shape and melting temperature (Tm). Heterozygous samples exhibited a typical melting profile while homozygous samples showed a specific Tm shift. Conclusions We provide evidence about application of HRMA in unambiguous genotyping of the CYP24A1 c.428_430delAAG variant, making this method useful in clinical molecular diagnostics. This approach opens the way to a helpful molecular analysis of CYP24A1 gene in IIH diagnosis, to an improved pharmacological treatment strategy and to a reduced risk of recurrent stones and worsening nephrocalcinosis.

To explore the role of asymmetric-dimethyl-L-arginine (ADMA), an endogenous nitric oxide synthetases (NOS) inhibitor, and nitrite/nitrate (NOx) in the pathogenesis of oxidative stress in early stages of type 1 diabetes mellitus.We measured in 99 female subjects with uncomplicated type 1 diabetes (duration disease10 years) and in 44 sex-matched controls (comparable for age, smoking habit, diet and physical activity) plasma levels of NOx, glycosylated haemoglobin (HbA1c), glucose, uric acid, total cholesterol, high density lipoprotein (HDL) cholesterol, triglycerides and serum ADMA.Type 1 diabetic subjects have higher levels of glycemia, HbA1c, LDL cholesterol and NOx, but lower ADMA and serum uric acid (UA), compared with the control group; no further differences were found. A significant linear and inverse correlation was found between NOx and ADMA levels (R(2)=0.237, p0.001).This study suggests a reduced ADMA inhibition of NOS as possible mechanism involved in the pathogenesis of oxidative stress in female subjects with a short duration and uncomplicated type 1 diabetes.

Objective: Massive parallel sequencing (MPS) is the new frontier for molecular diagnostics. Twenty-four papers regarding BRCA analysis were considered for reviewing all pipelines evaluated in this field. Methods: Proposed here is an integrated MPS workflow able to successfully identify BRCA1/2 mutational status on 212 Italian ovarian cancer patients. The review of literature data is reported. Result: The pipeline can be routinely used as robust molecular diagnostic strategy, being highly sensitive and specific. Conclusion: Literature data report that efforts are being made in order to fully translate MPS-based BRCA1/2 gene assay into routine clinical diagnostics. However, this study highlights the need of an integrated MPS BRCA1/2 molecular workflow fulfilling the standardized requirements needed in the routine clinical laboratory practice. Keywords: BRCA1/2 genes; bioinformatics NGS analysis; hereditary breast and ovarian cancer syndrome; massive parallel sequencing; next-generation sequencing.

Cystic fibrosis (CF; OMIM number 219700) is an autosomal recessive disease caused by mutations in theCFTR(cystic fibrosis transmembrane conductance regulator) gene, which results in abnormal viscous mucoid secretions in multiple organs and whose main clinical features are pancreatic insufficiency, chronic endobronchial infection, and male infertility. We report the case of a 47-year-old apparently normal male resulting in homozygosity for the mutation p.M348K from nonconsanguineous parents. The proband was screened using a standard panel of 70 different tested on NanoChip 400 platform. The massive parallel pyrosequencing on 454 JS machine allowed the second level analysis. The patient was firstly screened with two different platforms available in our laboratory, obtaining an ambiguous signal for the p.R347P mutation. For this reason we decided to clarify the discordant result ofCFTRstatus by Next Generation Sequencing (NGS) using 454 Junior instrument. The patient is resulted no carrier of the p.R347P mutation, but NGS highlighted a homozygous substitution from T>A at position 1043 in the coding region, causing an amino acid substitution from methionine to lysine (p.M348K). Casual finding of p.M348K homozygote mutation in an individual, without any feature of classical or nonclassical CF form, allowed us to confirm that p.M348K is a benign rare polymorphism.

Shear stress triggers conformational stretching of von Willebrand factor (VWF), which is responsible for its self-association and binding to the platelet receptor glycoprotein (GP)Ibα. This phenomenon supports primary hemostasis under flow. Type 2B VWF natural mutants are considered to have increased affinity for platelet GPIbα.To assess the mechanism responsible for the enhanced interaction of the p.R1306W VWF mutant with the platelet receptor.The interaction of GPIbα with wild-type (WT) and p.R1306W VWF multimers and A1-A2-A3 constructs was investigated with surface plasmon resonance spectroscopy. Analysis of the static VWF conformation in solution was performed with dynamic light scattering spectroscopy. The shear stress-induced self-association of VWF multimers was investigated with atomic force microscopy (AFM) over a 0-60 dyn cm(-2) range.WT VWF did not interact with GPIbα under static conditions, whereas the mutant at ~ 2 μg mL(-1) already bound to the receptor. By contrast, the WT and p.R1306W-A1-A2-A3 constructs showed comparable affinities for GPIbα (Kd ~ 20 nm). The hydrodynamic diameter of resting R1306W VWF multimers was significantly greater than that of the wild type (210 ± 60 nm vs. 87 ± 22 nm). At shear forces of 14 dyn cm(-2) , the p.R1306W multimers rapidly changed conformation, entering a regime of self-aggregation, which, in contrast, was induced for WT VWF by shear forces of 30 dyn cm(-2) . Mechanical stretching AFM experiments showed that p.R1306W multimers needed less energy per length unit (~ 10 pN) to be stretched than the WT protein.The increased affinity of p.R1306W VWF for GPIbα arises mostly from higher sensitivity to shear stress, which facilitates exposure of GPIbα binding sites.

Abstract 3306 Background. Von Willebrand factor (vWF) is involved in relevant biological functions such as i) platelet adhesion to the vascular endothelium, through interaction with the platelet membrane glycoprotein 1b-alpha (GpIbα), ii) transport of factor VIII in the circulation, preventing its proteolytic degradation by protein C and iii) regulation of angiogenesis and megakaryocytopoiesis Under the effect of hydrodynamic stress vWF changes its conformation from a globular to an elongated shape, which allows self-aggregation of vWF multimers and the formation of sticky grid, where blood platelets can adhere under high shear flow. Natural type 2B vW mutants (T2B vWD) are considered to have both an increased affinity for platelet GpIb and accelerated hydrolysis by ADAMTS-13. Methods. In this study, the ability of recombinant WT and R1306W vWF mutant to self associate and bind to platelets was investigated in a flow-chamber system under controlled shear stress ranging from 5 to 60 dyn/cm2. The recombinant proteins were produced in HEK-293 cells and purified by affinity and size-exclusion chromatography. The analysis of vWF self-association was performed by atomic force microscopy and dynamic light scattering spectroscopy. The interaction between immobilized recombinant GpIb and WT and R1306W vWF was studied with Surface Plasmon Resonance spectroscopy (SPR). Results. The SPR measurements showed that WT and mutant R1306W A1-A2-A3 domains bind to platelet GpIb with comparable affinity (Kd ≈ 20 nM). Full length WT vWF does not significantly interact with GpIb under static conditions, whereas the R1306W mutant showed a significant binding to GpIb. The process of vWF self-association evolved differently as a function of shear stress, whereby at values Conclusions. These findings showed that R1306W vWF does not have an increased intrinsic affinity for GpIb. Instead, its increased avidity for platelet receptors arises from an increased sensitivity to hydrodynamic stress, which more easily exposes the binding sites for GpIb. Disclosures: No relevant conflicts of interest to declare.