Description of a novel splice site variant in UBA1 gene causing VEXAS syndrome.

OBJECTIVE: The vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic (VEXAS) syndrome is a complex immune disorder consequence of somatic UBA1 variants. Most reported pathogenic UBA1 variants are missense or splice site mutations directly impairing the translational start site at p. Met41, with recent studies showing that these variants are frequent causes of recurrent inflammation in older individuals. Here we aimed to characterize a novel UBA1 variant found in two patients clinically presenting with VEXAS syndrome. METHODS: Patients' data were collected from direct assessments and from their medical charts. Genomics analyses were performed by both Sanger and amplicon-based deep sequencing, mRNA studies were performed by both cDNA subcloning and mRNA sequencing. RESULTS: We report a novel, somatic variant in a canonical splice site of the UBA1 gene (c.346-2A>G), which was identified in two unrelated adult male patients with late-onset, unexplained inflammatory manifestations including recurrent fever, Sweet syndrome-like neutrophilic dermatosis, and lung inflammation responsive only to glucocorticoids. RNA analysis from patients' samples demonstrated aberrant mRNA splicing leading to multiple in-frame transcripts, including a transcript retaining the full sequence of intron 4 and a different transcript with the deletion of the first 15 nucleotides of exon 5. CONCLUSION: Here we describe the abnormal UBA1 transcription as a consequence of the novel c.346-2A>G variant identified in two patients with clinical features compatible with VEXAS syndrome. Overall, these results further demonstrate the expanding spectrum of variants in UBA1 leading to pathology and support for a complete gene evaluation in those candidate patients for VEXAS syndrome.

Spanish cohort of VEXAS syndrome: clinical manifestations, outcome of treatments and novel evidences about UBA1 mosaicism.

BACKGROUND: The vacuoles, E1-enzyme, X linked, autoinflammatory and somatic (VEXAS) syndrome is an adult-onset autoinflammatory disease (AID) due to postzygotic UBA1 variants. OBJECTIVES: To investigate the presence of VEXAS syndrome among patients with adult-onset undiagnosed AID. Additional studies evaluated the mosaicism distribution and the circulating cytokines. METHODS: Gene analyses were performed by both Sanger and amplicon-based deep sequencing. Patients' data were collected from their medical charts. Cytokines were quantified by Luminex. RESULTS: Genetic analyses of enrolled patients (n=42) identified 30 patients carrying UBA1 pathogenic variants, with frequencies compatible for postzygotic variants. All patients were male individuals who presented with a late-onset disease (mean 67.5 years; median 67.0 years) characterised by cutaneous lesions (90%), fever (66.7%), pulmonary manifestations (66.7%) and arthritis (53.3%). Macrocytic anaemia and increased erythrocyte sedimentation rate and ferritin were the most relevant analytical abnormalities. Glucocorticoids ameliorated the inflammatory manifestations, but most patients became glucocorticoid-dependent. Positive responses were obtained when targeting the haematopoietic component of the disease with either decitabine or allogeneic haematopoietic stem cell transplantation. Additional analyses detected the UBA1 variants in both haematopoietic and non-haematopoietic tissues. Finally, analysis of circulating cytokines did not identify inflammatory mediators of the disease. CONCLUSION: Thirty patients with adult-onset AID were definitively diagnosed with VEXAS syndrome through genetic analyses. Despite minor interindividual differences, their main characteristics were in concordance with previous reports. We detected for the first time the UBA1 mosaicism in non-haematopoietic tissue, which questions the previous concept of myeloid-restricted mosaicism and may have conceptual consequences for the disease mechanisms.

Paradigm shift in monogenic autoinflammatory diseases and systemic vasculitis: The VEXAS syndrome. / Cambio de paradigma en las enfermedades autoinflamatorias monogénicas y las vasculitis sistémicas: el síndrome VEXAS.

VEXAS syndrome was described by the end of 2020 as an autoinflammatory disease caused by post-zygotic variants in the UBA1 gene. VEXAS syndrome occurs in adult males with recurrent fever, arthralgia/arthritis, ear/nose chondritis, neutrophilic dermatosis, lung inflammation, venous thrombosis, and different types of vasculitis. Common laboratory changes include raised acute phase reactants and macrocytic anemia. The coexistence of myelodysplasia is frequent, and bone marrow vacuolization of myeloid and erythroid precursors is characteristic. Glucocorticoids are effective at medium-high doses, but the remaining immunosuppressive drugs, either conventional or biological, have showed limited or absent efficacy. Azacitidine has been associated with a good response, especially in patients with accompanying myelodysplastic syndrome. Allogeneic hematopoietic stem cell transplantation appears to be the only curative therapy by now. VEXAS syndrome has become a paradigm shift in the diagnosis and treatment of autoinflammatory diseases and systemic vasculitis.

Vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic syndrome (VEXAS syndrome) with prominent supraglottic larynx involvement: a case-based review.

Vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic syndrome (VEXAS syndrome) is a recently described genetic disorder that gathers autoinflammatory symptoms and myeloid dysplasia. The first description was reported in 2020, and subsequently, a growing number of cases have been described worldwide. Herein, we describe a case of a 72-year-old male patient with VEXAS syndrome with p.Met41Val mutation of the UBA1 gene, prominent supraglottic larynx involvement, and costochondritis. To our knowledge, this is the first report of VEXAS syndrome in Colombia and South America. This disease could present features of relapsing polychondritis, polyarteritis nodosa, giant cell arteritis, and Sweet syndrome, associated with hematologic involvement, including cytopenias, myelodysplastic syndrome, or thromboembolic disease. Supraglottic larynx chondritis and costochondritis are atypical manifestations. These features were proposed previously to differentiate relapsing polychondritis from VEXAS syndrome but are not entirely reliable like in the case described. A diagnosis of VEXAS should be considered in male patients with incomplete or complete features of the previously described conditions, refractory to treatment, requiring high-dose glucocorticoids, and associated progressive hematologic abnormalities. Key Points • VEXAS syndrome is a recently described genetic (somatic mutations in UBA1 gene) disorder that gathers autoinflammatory and hematologic manifestations. • VEXAS syndrome should be considered in male patients with incomplete or complete features of relapsing polychondritis, polyarteritis nodosa, giant cell arteritis, and Sweet syndrome, refractory to treatment, associated with hematologic involvement, including cytopenias, myelodysplastic syndrome, or thromboembolic disease. • Glucocorticoids ameliorate symptoms effectively. However, other treatment options are limited due to a lack of evidence. Traditional immunosuppressants and biological therapy have been used empirically with limited efficacy and a transient effect. Bone marrow transplant offers a curative approach, but it has high morbidity and mortality.

Cytokine profile and brain biopsy in a case of childhood-onset central nervous system vasculitis in Noonan syndrome-like disorder due to a novel CBL variant.

The authors describe a 5-year-old girl who developed a Noonan syndrome-like disorder as a result of the CBL c.1194C>G/p.His398Gln variant, including headache, papilledema, intracranial hypertension, hyperproteinorrhachia, leucorrhachia, and brain inflammation and vasculitis with CD3 positive lymphocyte infiltration. The patient responded partially to corticosteroids, acetazolamide, and ventriculoperitoneal valve placement. The serum cytokine profile revealed persistently elevated levels of IL-1 RA, IL-2R alpha, IL-6, IL-18, MCP-1, and MCP-3. Cyclophosphamide was used as a bridge to allogeneic hematopoietic stem cell transplantation in this case.

First Description of Late-Onset Autoinflammatory Disease Due to Somatic NLRC4 Mosaicism.

OBJECTIVE: Autoinflammatory diseases are inherited disorders of innate immunity that usually start during childhood. However, several recent reports have described an increasing number of patients with autoinflammatory disease starting in adulthood. This study was undertaken to investigate the underlying cause of a case of late-onset uncharacterized autoinflammatory disease. METHODS: Genetics studies were performed using Sanger sequencing and next-generation sequencing (NGS) methods. In silico, in vitro, and ex vivo analyses were performed to determine the functional consequences of the detected variant. RESULTS: We studied a 57-year-old woman who at the age of 47 years began to have recurrent episodes of fever, myalgias, arthralgias, diffuse abdominal pain, diarrhea, adenopathies, and systemic inflammation, which were relatively well controlled with anti-interleukin-1 (anti-IL-1) drugs. NGS analyses did not detect germline variants in any of the known autoinflammatory disease-associated genes, but they identified the p.Ser171Phe NLRC4 variant in unfractionated blood, with an allele fraction (2-4%) compatible with gene mosaicism. Structural modeling analyses suggested that this missense variant might favor the open, active conformation of the NLRC4 protein, and in vitro and ex vivo analyses confirmed its propensity to oligomerize and activate the NLRC4 inflammasome, with subsequent overproduction of IL-18. CONCLUSION: Our findings indicate that the postzygotic p.Ser171Phe NLRC4 variant is a plausible cause of the disease in the enrolled patient. Functional and structural studies clearly support, for the first time, its gain-of-function behavior, consistent with previously reported NLRC4 pathogenic variants. These novel findings should be considered in the diagnostic evaluation of patients with adult-onset uncharacterized autoinflammatory disease.

Assessment of the gene mosaicism burden in blood and its implications for immune disorders.

There are increasing evidences showing the contribution of somatic genetic variants to non-cancer diseases. However, their detection using massive parallel sequencing methods still has important limitations. In addition, the relative importance and dynamics of somatic variation in healthy tissues are not fully understood. We performed high-depth whole-exome sequencing in 16 samples from patients with a previously determined pathogenic somatic variant for a primary immunodeficiency and tested different variant callers detection ability. Subsequently, we explored the load of somatic variants in the whole blood of these individuals and validated it by amplicon-based deep sequencing. Variant callers allowing low frequency read thresholds were able to detect most of the variants, even at very low frequencies in the tissue. The genetic load of somatic coding variants detectable in whole blood is low, ranging from 1 to 2 variants in our dataset, except for one case with 17 variants compatible with clonal haematopoiesis under genetic drift. Because of the ability we demonstrated to detect this type of genetic variation, and its relevant role in disorders such as primary immunodeficiencies, we suggest considering this model of gene mosaicism in future genetic studies and considering revisiting previous massive parallel sequencing data in patients with negative results.

ISSAID/EMQN Best Practice Guidelines for the Genetic Diagnosis of Monogenic Autoinflammatory Diseases in the Next-Generation Sequencing Era.

BACKGROUND: Monogenic autoinflammatory diseases are caused by pathogenic variants in genes that regulate innate immune responses, and are characterized by sterile systemic inflammatory episodes. Since symptoms can overlap within this rapidly expanding disease category, accurate genetic diagnosis is of the utmost importance to initiate early inflammation-targeted treatment and prevent clinically significant or life-threatening complications. Initial recommendations for the genetic diagnosis of autoinflammatory diseases were limited to a gene-by-gene diagnosis strategy based on the Sanger method, and restricted to the 4 prototypic recurrent fevers (MEFV, MVK, TNFRSF1A, and NLRP3 genes). The development of best practices guidelines integrating critical recent discoveries has become essential. METHODS: The preparatory steps included 2 online surveys and pathogenicity annotation of newly recommended genes. The current guidelines were drafted by European Molecular Genetics Quality Network members, then discussed by a panel of experts of the International Society for Systemic Autoinflammatory Diseases during a consensus meeting. RESULTS: In these guidelines, we combine the diagnostic strength of next-generation sequencing and recommendations to 4 more recently identified genes (ADA2, NOD2, PSTPIP1, and TNFAIP3), nonclassical pathogenic genetic alterations, and atypical phenotypes. We present a referral-based decision tree for test scope and method (Sanger versus next-generation sequencing) and recommend on complementary explorations for mosaicism, copy-number variants, and gene dose. A genotype table based on the 5-category variant pathogenicity classification provides the clinical significance of prototypic genotypes per gene and disease. CONCLUSIONS: These guidelines will orient and assist geneticists and health practitioners in providing up-to-date and appropriate diagnosis to their patients.

Molecular genetic investigation, clinical features, and response to treatment in 21 patients with Schnitzler syndrome.

To date, the pathogenic mechanisms underlying Schnitzler syndrome remain obscure, in particular, the interplay between the monoclonal protein and increased interleukin-1ß (IL-1ß) production, although interest in the contribution of genetic factors has been fueled by detection of somatic NLRP3 mosaicism in 2 patients with the variant-type Schnitzler syndrome. At 2 specialist UK centers, we have identified 21 patients who fulfilled diagnostic criteria for Schnitzler syndrome with urticarial rash, fever, arthralgia, and bone pain; 47% reported weight loss, 40% fatigue, and 21% lymphadenopathy. An immunoglobulin M (IgM) κ paraprotein was detected in 86%; the remainder had IgM λ or IgG κ. Patients underwent searches for germ line and somatic mutations using next-generation sequencing technology. Moreover, we designed a panel consisting of 32 autoinflammatory genes to explore genetic susceptibility factor(s) to Schnitzler syndrome. Genetic analysis revealed neither germ line nor somatic NLRP3, TNFRSF1A, NLRC4, or NOD2 mutations, apart from 1 patient with a germ line NLRP3 p.V198M substitution. The proinflammatory cytokines and extracellular apoptosis-associated speck-like protein with caspase recruitment domain (ASC) measured in the serum of Schnitzler syndrome patients during active disease were significantly higher than healthy controls. Ninety-five percent of our cohort achieved a complete response to recombinant IL-1 receptor antagonist (anakinra). Our findings do not support a role for somatic NLRP3 mosaicism in disease pathogenesis; although elevated levels of ASC, IL-6, and IL-18 in patients' serum, and the response to anakinra, suggest that Schnitzler syndrome is associated with upregulated inflammasome activation. Despite its rarity, Schnitzler syndrome is an important diagnosis as treatment with IL-1 antagonists dramatically improves quality of life for patients.

Late-Onset Cryopyrin-Associated Periodic Syndromes Caused by Somatic NLRP3 Mosaicism-UK Single Center Experience.

Cryopyrin-associated periodic syndrome (CAPS) is caused by gain-of-function NLRP3 mutations. Recently, somatic NLRP3 mosaicism has been reported in some CAPS patients who were previously classified as "mutation-negative." We describe here the clinical and laboratory findings in eight British adult patients who presented with symptoms typical of CAPS other than an onset in mid-late adulthood. All patients underwent comprehensive clinical and laboratory investigations, including analysis of the NLRP3 gene using Sanger and amplicon-based deep sequencing (ADS) along with measurements of extracellular apoptosis-associated speck-like protein with CARD domain (ASC) aggregates. The clinical phenotype in all subjects was consistent with mid-spectrum CAPS, except a median age at disease onset of 50 years. Sanger sequencing of NLRP3 was non-diagnostic but ADS detected a somatic NLRP3 mutation in each case. In one patient, DNA isolated from blood demonstrated an increase in the mutant allele from 5 to 45% over 12 years. ASC aggregates in patients' serum measured during active disease were significantly higher than healthy controls. This series represents 8% of CAPS patients diagnosed in a single center, suggesting that acquired NLRP3 mutations may not be an uncommon cause of the syndrome and should be sought in all patients with late-onset symptoms otherwise compatible with CAPS. Steadily worsening CAPS symptoms in one patient were associated with clonal expansion of the mutant allele predominantly affecting myeloid cells. Two patients developed AA amyloidosis, which previously has only been reported in CAPS in association with life-long germline NLRP3 mutations.

Cryopyrin-associated Periodic Syndromes in Italian Patients: Evaluation of the Rate of Somatic NLRP3 Mosaicism and Phenotypic Characterization.

OBJECTIVE: To evaluate the rate of somatic NLRP3 mosaicism in an Italian cohort of mutation-negative patients with cryopyrin-associated periodic syndrome (CAPS). METHODS: The study enrolled 14 patients with a clinical phenotype consistent with CAPS in whom Sanger sequencing of the NLRP3 gene yielded negative results. Patients' DNA were subjected to amplicon-based NLRP3 deep sequencing. RESULTS: Low-level somatic NLRP3 mosaicism has been detected in 4 patients, 3 affected with chronic infantile neurological cutaneous and articular syndrome and 1 with Muckle-Wells syndrome. Identified nucleotide substitutions encode for 4 different amino acid exchanges, with 2 of them being novel (p.Y563C and p.G564S). In vitro functional studies confirmed the deleterious behavior of the 4 somatic NLRP3 mutations. Among the different neurological manifestations detected, 1 patient displayed mild loss of white matter volume on brain magnetic resonance imaging. CONCLUSION: The allele frequency of somatic NLRP3 mutations occurs generally under 15%, considered the threshold of detectability using the Sanger method of DNA sequencing. Consequently, routine genetic diagnostic of CAPS should be currently performed by next-generation techniques ensuring high coverage to identify also low-level mosaicism, whose actual frequency is yet unknown and probably underestimated.

Novel evidences of atypical manifestations in cryopyrin-associated periodic syndromes.

OBJECTIVES: Cryopyrin-associated periodic syndromes (CAPS) usually start during infancy as an urticarial-like rash and a marked acute phase response, with additional manifestations appearing during its evolution. The aim of this study was to expand the clinical diversity of CAPS by the description of novel atypical features. METHODS: Clinical data were collected from patients' medical charts. Sanger sequencing analyzed NLRP3. Response to anti-IL-1 blockade was evaluated by clinical assessments and by measurements of laboratory parameters. RESULTS: Seventeen patients from two families (A and B), carrying the p.Ala439Thr and p.Arg260Trp NLRP3 mutations respectively, were enrolled. The disease was unexpectedly atypical in all members of Family A, with a 16-year-old asymptomatic carrier, and onset in adulthood associated with absence of skin lesions in four affected members. Surprisingly, one patient from each family suffered from severe haemorrhagic cystitis due to AA amyloidosis in the urinary bladder. Members of Family B displayed a classical phenotype, with two patients suffering from olfactive disorders. CONCLUSIONS: Our evidence suggests that CAPS may occasionally be presented as a late-onset, recurrent inflammatory disease without urticarial-like rash. In some patients, AA amyloidosis in strange locations like urinary bladder may complicate the clinical course. The response to IL-1 blockade in these atypical CAPS was similar to that described in classical forms. Consequently, we suggest that CAPS should be included in the differential diagnosis of adult patients with unexplained, recurrent inflammatory diseases, and once confirmed, the early initiation of anti-IL-1 blockade will probably prevent the development of life-threatening complications.

Brief Report: Late-Onset Cryopyrin-Associated Periodic Syndrome Due to Myeloid-Restricted Somatic NLRP3 Mosaicism.

OBJECTIVE: Gain-of-function NLRP3 mutations cause cryopyrin-associated periodic syndrome (CAPS), with gene mosaicism playing a relevant role in the pathogenesis. This study was undertaken to characterize the genetic cause underlying late-onset but otherwise typical CAPS. METHODS: We studied a 64-year-old patient who presented with recurrent episodes of urticaria-like rash, fever, conjunctivitis, and oligoarthritis at age 56 years. DNA was extracted from both unfractionated blood and isolated leukocyte and CD34+ subpopulations. Genetic studies were performed using both the Sanger method of DNA sequencing and next-generation sequencing (NGS) methods. In vitro and ex vivo analyses were performed to determine the consequences that the presence of the variant have in the normal structure or function of the protein of the detected variant. RESULTS: NGS analyses revealed the novel p.Gln636Glu NLRP3 variant in unfractionated blood, with an allele frequency (18.4%) compatible with gene mosaicism. Sanger sequence chromatograms revealed a small peak corresponding to the variant allele. Amplicon-based deep sequencing revealed somatic NLRP3 mosaicism restricted to myeloid cells (31.8% in monocytes, 24.6% in neutrophils, and 11.2% in circulating CD34+ common myeloid progenitor cells) and its complete absence in lymphoid cells. Functional analyses confirmed the gain-of-function behavior of the gene variant and hyperactivity of the NLRP3 inflammasome in the patient. Treatment with anakinra resulted in good control of the disease. CONCLUSION: We identified the novel gain-of-function p.Gln636Glu NLRP3 mutation, which was detected as a somatic mutation restricted to myeloid cells, as the cause of late-onset but otherwise typical CAPS. Our results expand the diversity of CAPS toward milder phenotypes than previously reported, including those starting during adulthood.

Genetic and clinical features of cryopyrin-associated periodic syndromes in Turkish children.

OBJECTIVES: The aim of this study was to present the genetic and clinical data of the largest cohort of Turkish cryopyrin-associated periodic syndromes (CAPS) patients. METHODS: This is a two-centre descriptive study of Turkish children with clinical diagnosis of CAPS. NLRP3 analyses were performed by Sanger sequencing and by massively parallel sequencing. ASC dependent NF-κB activation and transfection-induced THP-1 cell death assays determined the functional consequences of the detected variants. Disease activity and response to anti interleukin 1 (anti-IL-1) treatment was also assessed. RESULTS: Heterozygous germline NLRP3 mutation was detected in 8 of 14 enrolled patients (57.1%). Two novel somatic mutations Y560H and G307D were found which induced both THP-1 cell death and ASC dependent NF-kB activation. With anti-IL-1 treatment the disease activity was improved in all patients except one. Except two patients with macrophage activation syndrome (MAS) attack, there were no serious adverse events requiring hospitalisation. CONCLUSIONS: CAPS should be considered in all patients with typical symptoms even if Sanger-based genetic analysis is negative, since a considerable number of patients have mosaicism. Treatment should be patient-tailored and MAS should be considered as a rare complication.

Brief Report: Association of Tumor Necrosis Factor Receptor-Associated Periodic Syndrome With Gonosomal Mosaicism of a Novel 24-Nucleotide TNFRSF1A Deletion.

OBJECTIVE: To investigate the molecular cause of persistent fevers in a patient returning from working overseas, in whom investigations for tropical diseases yielded negative results. METHODS: DNA was extracted from the patient's whole blood, leukocyte subpopulations, saliva, hair root, and sperm. The TNFRSF1A gene was analyzed by polymerase chain reaction (PCR), allele-specific PCR, Sanger sequencing, and next-generation sequencing. In silico molecular modeling was performed to predict the structural and functional consequences of the tumor necrosis factor receptor (TNFR) type I protein mutation in the extracellular domain. RESULTS: Sanger sequencing corroborated by allele-specific PCR detected a novel in-frame deletion of 24 nucleotides (c.255_278del) in the TNFRSF1A gene, and this was subsequently confirmed using next-generation sequencing methods (targeted sequencing and amplicon-based deep sequencing). Results of amplicon-based deep sequencing revealed variable frequency of the mutant allele among different cell lines, including sperm, thus supporting the presence of gonosomal TNFRSF1A mosaicism. The patient had a complete response to treatment with interleukin-1 (IL-1) blockade, with resolution of symptoms and normalization of acute-phase protein levels. CONCLUSION: We describe the first case of gonosomal TNFRSF1A mosaicism in a patient with TNFR-associated periodic syndrome (TRAPS), which was attributable to a novel, somatic 24-nucleotide in-frame deletion. The clinical picture in this patient, including the complete response to IL-1 blockade, was typical of that found in TRAPS. This case adds TRAPS to the list of dominantly inherited autoinflammatory diseases reported to be caused by somatic (or postzygotic) mutation.

Clinical and genetic characterization of the autoinflammatory diseases diagnosed in an adult reference center.

INTRODUCTION: Autoinflammatory diseases (AID) are usually diagnosed during the pediatric age. However, adult-onset disease or diagnosis during adulthood has been occasionally described. OBJECTIVES: To assess the clinical and genetic characteristics of adult patients diagnosed with an AID in an adult referral center for AID. METHODS: We retrospectively evaluated clinical and genetic features of adult patients (≥16 years) diagnosed with an AID or referred after AID diagnosis to the Clinical Unit of AID, at the Department of Autoimmune Diseases, Hospital Clínic of Barcelona, from 2008 to 2014. RESULTS: During the study period, a genetic study for suspected AID was requested to 90 patients at the Department of Autoimmune Diseases. A final diagnosis of monogenic AID was achieved in 17 patients (19% of patients tested). Five additional cases were diagnosed with periodic fever, aphthous stomatitis, pharyngitis and cervical adenitis (PFAPA) syndrome and 10 patients with AID were referred from other adult departments. Finally, a total of 32 patients with AID were finally diagnosed or monitored in our Clinical Unit. These included 12 (37.5%) familial Mediterranean fever, 6 (18.8%) tumour necrosis factor-receptor associated periodic syndrome, 8 (25%) cryopirin-associated periodic syndromes (Muckle-Wells syndrome [MWS] or overlap familial cold-associated periodic syndrome/MWS), 1 (3.1%) mevalonate kinase deficiency, and 5 (15.6%) PFAPA. Clinical evidence of disease-onset during childhood and adulthood was observed in 15 (47%) and 17 (53%) patients, respectively. Overall, the final diagnosis was obtained after a delay of a mean of 12 years (range 0-47 years). Compared to children, adult patients with AID in our series presented more frequently with non-severe manifestations and none of them developed amyloidosis during follow-up. Adult patients also carried higher proportion of low-penetrance mutations or polymorphisms and all genetic variants were presented in heterozygosis or as heterozygous compounds. CONCLUSIONS: Adult disease-onset or delayed diagnosis of AID during adulthood is associated with milder disease phenotypes, and seem to be driven by mild genotypes, with predominant presence of low-penetrance mutations or polymorphisms.

Brief Report: First Identification of Intrafamilial Recurrence of Blau Syndrome due to Gonosomal NOD2 Mosaicism.

OBJECTIVE: Blau syndrome is characterized by noncaseating granulomatous arthritis, dermatitis, and uveitis, and results from gain-of-function NOD2 mutations. This study was undertaken to identify the genetic cause of the disease in a family with 3 members with Blau syndrome. METHODS: We studied a family with 3 affected members across 2 consecutive generations. The children's symptoms started early (at 6 and 7 months of age) and included polyarthritis, dermatitis, uveitis, and fever. In contrast, the father's symptoms started later (at 22 years of age) and included noncaseating granulomatous dermatitis and uveitis. We analyzed the NOD2 gene in all patients by both the Sanger method of DNA sequencing and amplicon-based deep sequencing using an Ion Torrent PGM platform. RESULTS: Sanger chromatograms revealed the heterozygous c.1001G>A transition in both children, which resulted in the p.Arg334Gln mutation that causes Blau syndrome. In contrast, the father's chromatograms revealed a small peak of adenine at the c.1001 position, suggesting the presence of a somatic NOD2 mutation. To evaluate this hypothesis, we performed amplicon-based deep sequencing using DNA from different tissues, which confirmed a variable degree (0.9-12.9%) of somatic NOD2 mosaicism. The previous detection of the NOD2 mutation in his daughters strongly suggests the presence of gonosomal (somatic plus gonadal) NOD2 mosaicism in the father. Comparative analyses with Blau syndrome patients carrying the germline p.Arg334Gln NOD2 mutation revealed late onset of the disease, a mild inflammatory phenotype, and an absence of complications in patients with NOD2 mosaicism. CONCLUSION: This is the first description of gonosomal NOD2 mosaicism as the cause of intrafamilial recurrence of Blau syndrome. Our findings also indicate that Blau syndrome includes more diverse and milder phenotypes than previously described.