Altered B-cell, plasma cell and antibody immune profiles in blood of systemic mastocytosis.

BACKGROUND: Systemic mastocytosis (SM) is a heterogeneous disease characterised by an expansion of KIT-mutated constitutively activated mast cells (MC) which release MC mediators that might act on the tumour microenvironment including other immune cells. OBJECTIVE: Here we investigated the blood distribution of B-cell, plasma cell (PC) and antibody-isotype compartments in SM. METHODS: We used spectral flow cytometry and the EuroFlow Immunomonitoring panel and Lymphocyte Screening Tube to quantify B-cells, PC and their subsets in blood of 108 SM patients - 35 bone marrow mastocytosis (BMM), 64 indolent SM (ISM), 9 aggressive SM (ASM)- vs 117 age-matched healthy donors (HD) and paired bone marrow (BM) samples of 31 SM vs 17 controls, respectively. In parallel, immunoglobulin (Ig) M, IgD, IgG, IgA and IgE plasma levels of were measured. RESULTS: Compared to HD, SM patients showed an increased immature B-cell production in BM (P=0.003) associated with a greater release of pre-germinal center immature (P<0.001) and naive CD5+ B-lymphocytes (P<0.001) to blood, but a pronounced decrease in PC counts of all different IgH-isotypes and subclasses (P≤0.001) together with overall increased IgM (P=0.001) and IgD (P<0.001) plasma levels. Of note, different immune profiles were found per diagnostic subtype of the disease with progressively greater counts in blood of immature B-lymphocytes together with decreased IgMD+, IgG2+, IgA1+ and IgA2+ MBC (P≤0.032) and elevated IgM (P=0.017) plasma levels in ASM cases, increased IgM (P=0.001) and IgD (P=0.001) plasma levels in ISM patients and exacerbated IgE (P<0.001) with decreased IgG (P=0.008) plasma levels in BMM cases. CONCLUSION: Our results reveal a significant dysregulation of the B-cell and PC compartments in blood of SM patients, consistent with distinctly altered antibody-isotype profiles in plasma of BMM vs ISM vs ASM patients.

Genome-wide association study identifies novel susceptibility loci for KIT D816V positive mastocytosis.

Mastocytosis is a rare myeloid neoplasm characterized by uncontrolled expansion of mast cells, driven in >80% of affected individuals by acquisition of the KIT D816V mutation. To explore the hypothesis that inherited variation predisposes to mastocytosis, we performed a two-stage genome-wide association study, analyzing 1,035 individuals with KIT D816V positive disease and 17,960 healthy control individuals from five European populations. After quality control, we tested 592,007 SNPs at stage 1 and 75 SNPs at stage 2 for association by using logistic regression and performed a fixed effects meta-analysis to combine evidence across the two stages. From the meta-analysis, we identified three intergenic SNPs associated with mastocytosis that achieved genome-wide significance without heterogeneity between cohorts: rs4616402 (pmeta = 1.37 × 10-15, OR = 1.52), rs4662380 (pmeta = 2.11 × 10-12, OR = 1.46), and rs13077541 (pmeta = 2.10 × 10-9, OR = 1.33). Expression quantitative trait analyses demonstrated that rs4616402 is associated with the expression of CEBPA (peQTL = 2.3 × 10-14), a gene encoding a transcription factor known to play a critical role in myelopoiesis. The role of the other two SNPs is less clear: rs4662380 is associated with expression of the long non-coding RNA gene TEX41 (peQTL = 2.55 × 10-11), whereas rs13077541 is associated with the expression of TBL1XR1, which encodes transducin (ß)-like 1 X-linked receptor 1 (peQTL = 5.70 × 10-8). In individuals with available data and non-advanced disease, rs4616402 was associated with age at presentation (p = 0.009; beta = 4.41; n = 422). Additional focused analysis identified suggestive associations between mastocytosis and genetic variation at TERT, TPSAB1/TPSB2, and IL13. These findings demonstrate that multiple germline variants predispose to KIT D816V positive mastocytosis and provide novel avenues for functional investigation.

Frequency and prognostic impact of blood-circulating tumor mast cells in mastocytosis.

Circulating tumor mast cells (CTMCs) have been identified in the blood of a small number of patients with advanced systemic mastocytosis (SM). However, data are limited about their frequency and prognostic impact in patients with MC activation syndrome (MCAS), cutaneous mastocytosis (CM) and nonadvanced SM. We investigated the presence of CTMCs and MC-committed CD34+ precursors in the blood of 214 patients with MCAS, CM, or SM using highly sensitive next-generation flow cytometry. CTMCs were detected at progressively lower counts in almost all patients with advanced SM (96%) and smoldering SM (SSM; 100%), nearly half of the patients (45%) with indolent SM (ISM), and a few patients (7%) with bone marrow (BM) mastocytosis but were systematically absent in patients with CM and MCAS (P < .0001). In contrast to CTMC counts, the number of MC-committed CD34+ precursors progressively decreased from MCAS, CM, and BM mastocytosis to ISM, SSM, and advanced SM (P < .0001). Clinically, the presence (and number) of CTMCs in blood of patients with SM in general and nonadvanced SM (ISM and BM mastocytosis) in particular was associated with more adverse features of the disease, poorer-risk prognostic subgroups as defined by the International Prognostic Scoring System for advanced SM (P < .0001) and the Global Prognostic Score for mastocytosis (P < .0001), and a significantly shortened progression-free survival (P < .0001) and overall survival (P = .01). On the basis of our results, CTMCs emerge as a novel candidate biomarker of disseminated disease in SM that is strongly associated with advanced SM and poorer prognosis in patients with ISM.

Clinical impact of the TPSAB1 genotype in mast cell diseases: A REMA study in a cohort of 959 individuals.

BACKGROUND: A close association between hereditary alpha-tryptasemia (HAT) and mast cell (MC) disorders has been previously reported. However, the relationship between HAT and the diagnostic subtypes and clinical features of MC disorders still remains to be established. OBJECTIVE: To determine the prevalence of HAT in healthy donors (HD) vs patients with different diagnostic subtypes of MC activation syndromes (MCAS) and mastocytosis, and its relationship with the clinical behavior of the disease. METHODS: A total of 959 subjects were studied including 346 healthy donors (HD), 464 mastocytosis, and 149 non-clonal MCAS patients. Molecular studies to assess the TPSAB1 genotype were performed, and data on serum baseline tryptase (sBT) and basal MC-mediator release episodes and triggers of anaphylaxis were collected. RESULTS: HAT was detected in 15/346 (4%) HD versus 43/149 (29%) non-clonal MCAS and 84/464 (18%) mastocytosis cases. Among mastocytosis, HAT was more frequently found in patients with MC-restricted KITD816V (21% vs. 10% among multilineage KITD816V patients; p = .008). Overall, median sBT was higher in cases presenting with HAT (28.9 vs. 24.5 ng/mL; p = .008), while no significant differences in sBT were observed among HAT+ mastocytosis patients depending on the presence of 1 vs. ≥2 extra copies of the α-tryptase gene (44.1 vs. 35.2 ng/mL, p > .05). In turn, anaphylaxis was more frequently observed in HAT+ versus HAT- mastocytosis patients (76% vs. 65%; p = .018), while HAT+ and HAT- patients who did not refer anaphylaxis as the presenting symptom (n = 308) showed a similar prevalence of subsequent anaphylaxis (35% vs. 36%, respectively). CONCLUSION: The frequency of HAT in MC disorders varies according to the diagnostic subtype of the disease. HAT does not imply a higher risk (and severity) of anaphylaxis in mastocytosis patients in whom anaphylaxis is not part of the presenting symptoms of the disease.

T-cell immune profile in blood of systemic mastocytosis: Association with disease features.

BACKGROUND: Systemic mastocytosis (SM) is a heterogeneous disease characterized by an expansion of KIT-mutated mast cells (MC). KIT-mutated MC display activated features and release MC mediators that might act on the tumour microenvironment and other immune cells. Here, we investigated the distribution of lymphocyte subsets in blood of patients with distinct subtypes of SM and determined its association with other disease features. METHODS: We studied the distribution of TCD4+ and TCD4- cytotoxic cells and their subsets, as well as total NK- and B cells, in blood of 115 SM patients-38 bone marrow mastocytosis (BMM), 67 indolent SM (ISM), 10 aggressive SM (ASM)- and 83 age-matched healthy donors (HD), using spectral flow cytometry and the EuroFlow Immunomonitoring panel, and correlated it with multilineage KITD816V, the alpha-tryptasemia genotype (HαT) and the clinical manifestations of the disease. RESULTS: SM patients showed decreased counts (vs. HD) of TCD4- cytotoxic cells, NK cells and several functional subsets of TCD4+ cells (total Th1, Th2-effector memory, Th22-terminal effector and Th1-like Tregs), together with increased T-follicular-helper and Th1/Th17-like Treg counts, associated with different immune profiles per diagnostic subtype of SM, in multilineal versus MC-restricted KITD816V and in cases with a HαT+ versus HαT- genotype. Unique immune profiles were found among BMM and ISM patients with MC-restricted KITD816V who displayed HαT, anaphylaxis, hymenoptera venom allergy, bone disease, pruritus, flushing and GI symptoms. CONCLUSION: Our results reveal altered T- and NK-cell immune profiles in blood of SM, which vary per disease subtype, the pattern of involvement of haematopoiesis by KITD816V, the HαT genotype and specific clinical manifestations of the disease.

KITD816V mutation in blood for the diagnostic screening of systemic mastocytosis and mast cell activation syndromes.

BACKGROUND: Current diagnostic algorithms for systemic mastocytosis (SM) rely on the detection of KITD816V in blood to trigger subsequent bone marrow (BM) investigations. METHODS: Here, we correlated the KITD816V mutational status of paired blood and BM samples from 368 adults diagnosed with mast cell activation syndrome (MCAS) and mastocytosis and determined the potential utility of investigating KITD816V in genomic DNA from blood-purified myeloid cell populations to increase diagnostic sensitivity. In a subset of 69 patients, we further evaluated the kinetics of the KITD816V cell burden during follow-up and its association with disease outcome. RESULTS: Our results showed a high correlation (P < .0001) between the KITD816V mutation burden in blood and BM (74% concordant samples), but with a lower mean of KITD816V-mutated cells in blood (P = .0004) and a high rate of discordant BM+ /blood- samples particularly among clonal MCAS (73%) and BM mastocytosis (51%), but also in cutaneous mastocytosis (9%), indolent SM (15%), and well-differentiated variants of indolent SM (7%). Purification of different compartments of blood-derived myeloid cells was done in 28 patients who were BM mast cell (MC)+ /blood- for KITD816V, revealing KITD816V-mutated eosinophils (56%), basophils (25%), neutrophils (29%), and/or monocytes (31%) in most (61%) patients. Prognostically, the presence of ≥3.5% KITD816V-mutated cells (P < .0001) and an unstable KITD816V mutation cell burden (P < .0001) in blood and/or BM were both associated with a significantly shortened progression-free survival (PFS). CONCLUSIONS: These results confirm the high specificity but limited sensitivity of KITD816V analysis in whole blood for the diagnostic screening of SM and other primary MCAS, which might be overcome by assessing the mutation in blood-purified myeloid cell populations.

Frequency and prognostic impact of KIT and other genetic variants in indolent systemic mastocytosis.

Indolent systemic mastocytosis (ISM) patients have a normal life expectancy, except in the 5% to 10% of cases that progress to more advanced SM (advSM), which has a significantly poorer outcome. Mutations in genes other than KIT frequently found in myeloid neoplasms have been associated with a poorer outcome among advSM, whereas limited information exists about their frequency and prognostic impact in ISM. We investigated the frequency and prognostic impact of variants in 18 genes, found to be altered in advSM, in 322 ISM patients (median follow-up, 5.7 years) divided into discovery (n = 200) and validation (n = 122) cohorts. Overall, 71 genetic variants were detected in 55 of 322 (17%) patients. Mutated ISM cases, particularly those carrying ASXL1, RUNX1, and/or DNMT3A (A/R/D) pathogenic variant allele frequencies (VAFs) ≥ 30%, exhibited significantly shortened (P < .001) progression-free survival (PFS) and overall survival (OS). Multivariate analysis showed that serum ß2-microglobulin (sß2M) levels > 2.5 µg/mL (hazard ratio [HR], 9.8; P = .001), together with a KIT D816V VAF ≥ 1% in bone marrow (BM) (HR, 10.1; P = .02) and pathogenic variants of A/R/D VAFs ≥ 30% (HR, 4.2; P = .02), were the best combination of independent predictors for PFS. In turn, A/R/D gene pathogenic VAF ≥ 30% was the only independent predictor for OS (HR, 51.8; P < .001). Based on these variables, 2 scoring systems were constructed for risk stratification of ISM at diagnosis with significantly different 10-year PFS (100%, 91%, 0% for scores of 0, 1, ≥2, respectively) and OS (100% and 50% for scores of 0 and 1) rates.

Pathogenic and diagnostic relevance of KIT in primary mast cell activation disorders.

OBJECTIVE: Mast cell (MC) activation (MCA) defines the mechanism by which certain patients have symptoms owing to the effect of a wide range of mediators released from MCs upon their activation, when triggered by different stimuli. When these symptoms are severe and recurrent, the diagnosis of MCA syndrome (MCAS) might be considered. Here, we review the relevant aspects related to the pathogenesis of MCAS, with special emphasis on the prevalence and diagnostic relevance of KIT mutations. DATA SOURCES: PubMed was searched between 1980 and 2021 using the following terms: mast cell activation syndromes, mast cell activation, anaphylaxis, KIT mutations, KIT D816V, indolent systemic mastocytosis, bone marrow mastocytosis, cutaneous mastocytosis, IgE anaphylaxis, and idiopathic anaphylaxis. STUDY SELECTIONS: Only articles published in English were selected based on their relevance to MCAS or severe and recurrent anaphylaxis. RESULTS: MCAS can be classified as clonal MCAS and nonclonal MCAS depending on the presence vs absence of an underlying KIT mutation (mostly KIT D816V), respectively. In contrast to clonal MCAS in which MCA is associated with a primary MC disorder (ie, primary MCAS) such as mastocytosis or monoclonal MCAS, nonclonal MCAS can be secondary to known or unidentified triggers (ie, secondary and idiopathic MCAS, respectively). CONCLUSION: The clinical heterogeneity and complexity of the molecular assays needed for the study of patients with MCAS might lead to misdiagnosis, particularly when patients are evaluated at nonspecialized centers. Thus, referral of patients having clinical manifestations suggestive of MCAS to reference centers on mastocytosis and MC diseases is strongly recommended.

Genetic association between CD96 locus and immunogenicity to anti-TNF therapy in Crohn's disease.

The production of antibodies to anti-tumor necrosis factor alpha (TNF) agents is one of the main causes of treatment failure in Crohn's disease (CD). To date, however, the contribution of genetics to anti-TNF immunogenicity in CD is still unknown. The objective of the present study was to identify genetic variation associated with anti-TNF immunogenicity in CD. We performed a two-stage genome-wide association study in a cohort of 96 and 123 adalimumab-treated patients, respectively. In the discovery stage, we identified a genome-wide significant association between the CD96 locus and the production of antibodies to anti-TNF treatment (P = 1.88e-09). This association was validated in the replication stage (P < 0.05). The risk allele for anti-TNF immunogenicity was found to be also associated with a lack of response to anti-TNF therapy (P = 0.019). These findings represent an important step toward the understanding of the immunogenicity-based mechanisms that underlie anti-TNF response in CD.

Genetic variation at the glycosaminoglycan metabolism pathway contributes to the risk of psoriatic arthritis but not psoriasis.

OBJECTIVE: Psoriatic arthritis (PsA) is a chronic inflammatory arthritis affecting up to 30% of patients with psoriasis (Ps). To date, most of the known risk loci for PsA are shared with Ps, and identifying disease-specific variation has proven very challenging. The objective of the present study was to identify genetic variation specific for PsA. METHODS: We performed a genome-wide association study in a cohort of 835 patients with PsA and 1558 controls from Spain. Genetic association was tested at the single marker level and at the pathway level. Meta-analysis was performed with a case-control cohort of 2847 individuals from North America. To confirm the specificity of the genetic associations with PsA, we tested the associated variation using a purely cutaneous psoriasis cohort (PsC, n=614) and a rheumatoid arthritis cohort (RA, n=1191). Using network and drug-repurposing analyses, we further investigated the potential of the PsA-specific associations to guide the development of new drugs in PsA. RESULTS: We identified a new PsA risk single-nucleotide polymorphism at B3GNT2 locus (p=1.10e-08). At the pathway level, we found 14 genetic pathways significantly associated with PsA (pFDR<0.05). From these, the glycosaminoglycan (GAG) metabolism pathway was confirmed to be disease-specific after comparing the PsA cohort with the cohorts of patients with PsC and RA. Finally, we identified candidate drug targets in the GAG metabolism pathway as well as new PsA indications for approved drugs. CONCLUSION: These findings provide insights into the biological mechanisms that are specific for PsA and could contribute to develop more effective therapies.

Systemic mastocytosis associated with myelodysplastic/myeloproliferative neoplasms with ring sideroblasts and thrombocytosis: Report of three cases.

The association of systemic mastocytosis with another hematologic neoplasia of myeloid or lymphoid origin is recognized as an advanced subvariant of mastocytosis. Here, we report the association of indolent or smoldering systemic mastocytosis with three cases of myelodysplastic/myeloproliferative neoplasms with ring sideroblasts and thrombocytosis, a recently recognized disease characterized by SF3B1 mutations. The hierarchical pattern of KIT, SF3B1, JAK2, and additional mutations was studied in whole and fractionated subpopulations of peripheral blood cells and whole bone marrow. In two cases, we could demonstrate a multilineage D816V KIT mutation, involving all myeloid lineages in one patient and also the lymphoid series in the other. Two patients displaying both SF3B1 and V617F JAK2 mutations had a very poor prognosis. Another patient bearing SF3B1, but not V617F JAK2 mutation, had a favorable response to erythropoietin treatment and long survival.

Bone Marrow Mast Cell Antibody-Targetable Cell Surface Protein Expression Profiles in Systemic Mastocytosis.

Despite recent therapeutic advances, systemic mastocytosis (SM) remains an incurable disease due to limited complete remission (CR) rates even after novel therapies. To date, no study has evaluated the expression on SM bone marrow mast cells (BMMC) of large panel of cell surface suitable for antibody-targeted therapy. In this study, we analyzed the expression profile of six cell-surface proteins for which antibody-based therapies are available, on BMMC from 166 SM patients vs. 40 controls. Overall, variable patterns of expression for the markers evaluated were observed among SM BMMC. Thus, CD22, CD30, and CD123, while expressed on BMMC from patients within every subtype of SM, showed highly variable patterns with a significant fraction of negative cases among advanced SM (aggressive SM (ASM), ASM with an associated clonal non-MC lineage disease (ASM-AHN) and MC leukemia (MCL)), 36%, 46%, and 39%, respectively. In turn, CD25 and FcεRI were found to be expressed in most cases (89% and 92%) in virtually all BMMC (median: 92% and 95%) from both indolent and advanced SM, but with lower/absent levels in a significant fraction of MC leukemia (MCL) and both in MCL and well-differentiated SM (WDSM) patients, respectively. In contrast, CD33 was the only marker expressed on all BMMC from every SM patient. Thus, CD33 emerges as the best potentially targetable cell-surface membrane marker in SM, particularly in advanced SM.

KIT D816V-mutated bone marrow mesenchymal stem cells in indolent systemic mastocytosis are associated with disease progression.

Multilineage involvement of bone marrow (BM) hematopoiesis by the somatic KIT D816V mutation is present in a subset of adult indolent systemic mastocytosis (ISM) patients in association with a poorer prognosis. Here, we investigated the potential involvement of BM mesenchymal stem cells (MSCs) from ISM patients by the KIT D816V mutation and its potential impact on disease progression and outcome. This mutation was investigated in highly purified BM MSCs and other BM cell populations from 83 ISM patients followed for a median of 116 months. KIT D816V-mutated MSCs were detected in 22 of 83 cases. All MSC-mutated patients had multilineage KIT mutation (100% vs 30%, P = .0001) and they more frequently showed involvement of lymphoid plus myeloid BM cells (59% vs 22%; P = .03) and a polyclonal pattern of inactivation of the X-chromosome of KIT-mutated BM mast cells (64% vs 0%; P = .01) vs other multilineage ISM cases. Moreover, presence of KIT-mutated MSCs was associated with more advanced disease features, a greater rate of disease progression (50% vs 17%; P = .04), and a shorter progression-free survival (P ≤ .003). Overall, these results support the notion that ISM patients with mutated MSCs may have acquired the KIT mutation in a common pluripotent progenitor cell, prior to differentiation into MSCs and hematopoietic precursor cells, before the X-chromosome inactivation process occurs. From a clinical point of view, acquisition of the KIT mutation in an earlier BM precursor cell confers a significantly greater risk for disease progression and a poorer outcome.

Clinical, immunophenotypic, and molecular characteristics of well-differentiated systemic mastocytosis.

BACKGROUND: Well-differentiated systemic mastocytosis (WDSM) is a rare variant of systemic mastocytosis (SM) characterized by bone marrow (BM) infiltration by mature-appearing mast cells (MCs) often lacking exon 17 KIT mutations. Because of its rarity, the clinical and biological features of WDSM remain poorly defined. OBJECTIVE: We sought to determine the clinical, biological, and molecular features of a cohort of 33 patients with mastocytosis in the skin in association with BM infiltration by well-differentiated MCs and to establish potential diagnostic criteria for WDSM. METHODS: Thirty-three patients with mastocytosis in the skin plus BM aggregates of round, fully granulated MCs lacking strong CD25 and CD2 expression in association with clonal MC features were studied. RESULTS: Our cohort of patients showed female predominance (female/male ratio, 4:1) and childhood onset of the disease (91%) with frequent familial aggregation (39%). Skin involvement was heterogeneous, including maculopapular (82%), nodular (6%), and diffuse cutaneous (12%) mastocytosis. KIT mutations were detected in only 10 (30%) of 33 patients, including the KIT D816V (n = 5), K509I (n = 3), N819Y (n = 1), and I817V (n = 1) mutations. BM MCs displayed a unique immunophenotypic pattern consisting of increased light scatter features, overexpression of cytoplasmic carboxypeptidase, and aberrant expression of CD30, together with absent (79%) or low (21%) positivity for CD25, CD2, or both. Despite only 9 (27%) of 33 patients fulfilling the World Health Organization criteria for SM, our findings allowed us to establish the systemic nature of the disease, which fit with the definition of WDSM. CONCLUSIONS: WDSM represents a rare clinically and molecularly heterogeneous variant of SM that requires unique diagnostic criteria to avoid a misdiagnosis of cutaneous mastocytosis per current World Health Organization criteria.

A genome-wide association study identifies a novel locus at 6q22.1 associated with ulcerative colitis.

The genetic analysis of ulcerative colitis (UC) has provided new insights into the etiology of this prevalent inflammatory bowel disease. However, most of the heritability of UC (>70%) has still not been characterized. To identify new risk loci for UC we have performed the first genome-wide association study (GWAS) in a Southern European population and undertaken a meta-analysis study combining the newly genotyped 825 UC patients and 1525 healthy controls from Spain with the six previously published GWAS comprising 6687 cases and 19 718 controls from Northern-European ancestry. We identified a novel locus with genome-wide significance at 6q22.1 [rs2858829, P = 8.97 × 10(-9), odds ratio (OR) (95% confidence interval, CI] = 1.12 (1.08-1.16)] that was validated with genotype data from a replication cohort of the same Southern European ancestry consisting in 1073 cases and 1279 controls [combined P = 7.59 × 10(-10), OR (95% CI) = 1.12 (1.08-1.16)]. Furthermore, we confirmed the association of 33 reported associations with UC and we nominally validated the GWAS results of nine new risk loci (P < 0.05, same direction of effect). SNP rs2858829 lies in an intergenic region and is a strong cis-eQTL for FAM26F gene, a gene that is shown to be selectively upregulated in UC colonic mucosa with active inflammation. Our results provide new insight into the genetic risk background of UC, confirming that there is a genetic risk component that differentiates from Crohn's Disease, the other major form of inflammatory bowel disease.

Diagnosis and classification of mastocytosis in non-specialized versus reference centres: a Spanish Network on Mastocytosis (REMA) study on 122 patients.

The diagnosis of 'rare diseases', such as mastocytosis, remains a challenge. Despite this, the precise benefits of referral of mastocytosis patients to highly specialized reference centres are poorly defined and whether patients should be managed at non-specialized versus reference centres remains a matter of debate. To evaluate the quality and efficiency of diagnostic procedures performed at the reference centres for mastocytosis in Spain (REMA) versus other non-reference centres, we retrospectively analysed a series of 122 patients, for the overall degree of agreement obtained for the World Health Organization (WHO) diagnostic and classification criteria betwen the referring and REMA centres. Our results showed that not all WHO diagnostic criteria were frequently investigated at the referring centres. Among the five WHO diagnostic criteria, the highest degree of agreement was obtained for serum tryptase levels [median 90% (95% confidence interval 84-96%)]; in turn, the overall agreement was significantly lower for the major histopathological criterion [80% (72-89%)], and the other three minor criteria: cytomorphology [68% (56-80%)] immunophenotyping of BM mast cells [75% (62-87%)] and detection of the KIT mutation [34% (8-60%)]. Referral of patients with diagnostic suspicion of mastocytosis to a multidisciplinary reference centre improves diagnostic efficiency and quality.

Identification of risk loci for Crohn's disease phenotypes using a genome-wide association study.

BACKGROUND & AIMS: Crohn's disease is a highly heterogeneous inflammatory bowel disease comprising multiple clinical phenotypes. Genome-wide association studies (GWASs) have associated a large number of loci with disease risk but have not associated any specific genetic variants with clinical phenotypes. We performed a GWAS of clinical phenotypes in Crohn's disease. METHODS: We genotyped 576,818 single-nucleotide polymorphisms in a well-characterized cohort of 1090 Crohn's disease patients of European ancestry. We assessed their association with 17 phenotypes of Crohn's disease (based on disease location, disease behavior, disease course, age at onset, and extraintestinal manifestations). A total of 57 markers with strong associations to Crohn's disease phenotypes (P < 2 × 10(-4)) were subsequently analyzed in an independent replication cohort of 1296 patients of European ancestry. RESULTS: We replicated the association of 4 loci with different Crohn's disease phenotypes. Variants in MAGI1, CLCA2, 2q24.1, and LY75 loci were associated with a complicated stricturing disease course (Pcombined = 2.01 × 10(-8)), disease location (Pcombined = 1.3 × 10(-6)), mild disease course (Pcombined = 5.94 × 10(-7)), and erythema nodosum (Pcombined = 2.27 × 10(-6)), respectively. CONCLUSIONS: In a GWAS, we associated 4 loci with clinical phenotypes of Crohn's disease. These findings indicate a genetic basis for the clinical heterogeneity observed for this inflammatory bowel disease.

A genome-wide association study identifies SLC8A3 as a susceptibility locus for ACPA-positive rheumatoid arthritis.

OBJECTIVE: RA patients with serum ACPA have a strong and specific genetic background. The objective of the study was to identify new susceptibility genes for ACPA-positive RA using a genome-wide association approach. METHODS: A total of 924 ACPA-positive RA patients with joint damage in hands and/or feet, and 1524 healthy controls were genotyped in 582 591 single-nucleotide polymorphisms (SNPs) in the discovery phase. In the validation phase, the most significant SNPs in the genome-wide association study representing new candidate loci for RA were tested in an independent cohort of 863 ACPA-positive patients with joint damage and 1152 healthy controls. All individuals from the discovery and validation cohorts were Caucasian and of Southern European ancestry. RESULTS: In the discovery phase, 60 loci not previously associated with RA risk showed evidence for association at P < 5×10(-4) and were tested for replication in the validation cohort. A total of 12 loci were replicated at the nominal level (P < 0.05, same direction of effect as in the discovery phase). When combining the discovery and validation cohorts, an intronic SNP in the Solute Carrier family 8 gene (SLC8A3) was found to be associated with ACPA-positive RA at a genome-wide level of significance RA [odds ratio (95% CI): 1.42 (1.25, 1.6), Pcombined = 3.19×10(-8)]. CONCLUSIONS: SLC8A3 was identified as a new risk locus for ACPA-positive RA. This study demonstrates the advantage of analysing relevant subsets of RA patients to identify new genetic risk variants.

Detection of the KIT D816V mutation in peripheral blood of systemic mastocytosis: diagnostic implications.

Recent studies have found the KIT D816V mutation in peripheral blood of virtually all adult systemic mastocytosis patients once highly sensitive PCR techniques were used; thus, detection of the KIT D816V mutation in peripheral blood has been proposed to be included in the diagnostic work-up of systemic mastocytosis algorithms. However, the precise frequency of the mutation, the biological significance of peripheral blood-mutated cells and their potential association with involvement of bone marrow hematopoietic cells other than mast cells still remain to be investigated. Here, we determined the frequency of peripheral blood involvement by the KIT D816V mutation, as assessed by two highly sensitive PCR methods, and investigated its relationship with multilineage involvement of bone marrow hematopoiesis. Overall, our results confirmed the presence of the KIT D816V mutation in peripheral blood of most systemic mastocytosis cases (161/190; 85%)--with an increasing frequency from indolent systemic mastocytosis without skin lesions (29/44; 66%) to indolent systemic mastocytosis with skin involvement (124/135; 92%), and more aggressive disease subtypes (11/11; 100%)--as assessed by the allele-specific oligonucleotide-qPCR method, which was more sensitive (P<.0001) than the peptide nucleic acid-mediated PCR approach (84/190; 44%). Although the presence of the KIT mutation in peripheral blood, as assessed by the allele-specific oligonucleotide-qPCR technique, did not accurately predict for multilineage bone marrow involvement of hematopoiesis, the allele-specific oligonucleotide-qPCR allele burden and the peptide nucleic acid-mediated-PCR approach did. These results suggest that both methods provide clinically useful and complementary information through the identification and/or quantification of the KIT D816V mutation in peripheral blood of patients suspected of systemic mastocytosis.

A deletion at ADAMTS9-MAGI1 locus is associated with psoriatic arthritis risk.

OBJECTIVE: Copy number variants (CNVs) have been associated with the risk to develop multiple autoimmune diseases. Our objective was to identify CNVs associated with the risk to develop psoriatic arthritis (PsA) using a genome-wide analysis approach. METHODS: A total of 835 patients with PsA and 1498 healthy controls were genotyped for CNVs using the Illumina HumanHap610 BeadChip genotyping platform. Genomic CNVs were characterised using CNstream analysis software and analysed for association using the χ(2) test. The most significant genomic CNV associations with PsA risk were independently tested in a validation sample of 1133 patients with PsA and 1831 healthy controls. In order to test for the specificity of the variants with PsA aetiology, we also analysed the association to a cohort of 822 patients with purely cutaneous psoriasis (PsC). RESULTS: A total of 165 common CNVs were identified in the genome-wide analysis. We found a highly significant association of an intergenic deletion between ADAMTS9 and MAGI1 genes on chromosome 3p14.1 (p=0.00014). Using the independent patient and control cohort, we validated the association between ADAMTS9-MAGI1 deletion and PsA risk (p=0.032). Using next-generation sequencing, we characterised the 26 kb associated deletion. Finally, analysing the PsC cohort we found a lower frequency of the deletion compared with the PsA cohort (p=0.0088) and a similar frequency to that of healthy controls (p>0.3). CONCLUSIONS: The present genome-wide scan for CNVs associated with PsA risk has identified a new deletion associated with disease risk and which is also differential from PsC risk.