1071 CLINICAL REPORT Acquired Cold Urticaria vs. Autoinflammatory Diseases, Genetic ActaDV and Clinical Profile and Differential Diagnosis: Study of a Cohort of Patients in a Tertiary Reference Centre Gustavo DEZA1, Anna MENSA-VILARÓ2,3, Alvaro MARCH-RODRIGUEZ1, Silvia SÁNCHEZ1, Ramon M. PUJOL1, Juan I. ARÓSTEGUI2–4# and Ana M. GIMÉNEZ-ARNAU1# 1 Department of Dermatology, Hospital del Mar – Institut Mar d’Investigacions Mèdiques, Universitat Autònoma de Barcelona (UAB), 2Department of Immunology, Hospital Clínic, 3Institut d’Investigacions Biomèdiques August Pi i Sunyer, and 4University of Barcelona, Barcelona, Spain # These authors contributed equally to this work. Acta Dermato-Venereologica Acquired cold urticaria (ACU) is characterized by the SIGNIFICANCE development of itchy wheals after cold exposure. Ge- neralized urticarial skin rashes triggered by cold ex- Acquired cold urticaria represents a subtype of inducible posure characterize certain monogenic autoinflamma- urticaria characterized by the development of itchy wheals tory diseases (AIDs). The objective of this study is to after cold exposure. Generalized cold-induced urticarial investigate the presence of variants in genes causing rashes are also seen in certain monogenic autoinflamma- AIDs that present with cold-induced urticarial skin tory diseases. In the present study, we demonstrated that rashes in patients clinically diagnosed with ACU, in or- acquired cold urticaria is not related to the presence of der to look for susceptibility factors for the disease. germline and post-zygotic pathogenic variants on genes Fifty patients with primary ACU were studied. Germ- causing autoinflammatory diseases that present with cold- line and post-zygotic variants on the NLRP3, NLRP12, induced urticarial skin rashes (i.e. NLRP3, NLRP12, NLRC4 NLRC4 and PLCG2 genes were investigated using next- and PLCG2 genes). However, the presence of cold urticaria generation sequencing technology. Seven patients in addition to systemic manifestations, family history and/ (14%) carried 8 heterozygous germline variants in the or laboratory abnormalities should alert physicians to the following genes: NLRP3 (n = 1), NLRP12 (n = 3), NLRC4 potential diagnosis of a monogenic autoinflammatory di- (n = 1), PLCG2 (n = 3). No pathogenic or likely pathoge- sease. ActaDV nic variants were detected, and deep analyses of the sequences obtained did not identify any post-zygotic variant. In conclusion, ACU is not related to post-zy- is characterized by the negative responses after CSTs, gotic or germline pathogenic variants in the NLRP3, and therefore, its diagnosis is established mainly on the NLRP12, NLRC4 and PLCG2 genes. basis of a detailed clinical history (3). ACU most fre- quently affects young adults, although up to 15–25% of Key words: autoinflammatory diseases; cold urticaria; genetic variant; urticaria; urticarial skin rash. patients may show an onset of symptoms before the age of 18 years (2, 4), with recent evidence suggesting that Accepted Aug 12, 2019; E-published Aug 13, 2019 paediatric-onset patients might exhibit distinctive clinical features (2). Its symptoms are usually limited to cold- Advances in dermatology and venereology Acta Derm Venereol 2019; 99: 1071–1077. exposed skin areas and typically appear a few minutes Corr: Ana M. Giménez-Arnau, Department of Dermatology, Hospital del Mar – Institut Mar d’Investigacions Mèdiques, Universitat Autònoma de after exposure to cold air, liquids and/or solids. Exten- Barcelona (UAB), Passeig Marítim, 25–29, ES-08003 Barcelona, Spain. E-mail: anamariagimenezarnau@gmail.com, 22505aga@comb.cat sive cold contact may occasionally result in generalized symptoms, including headache, dyspnoea, hypotension or loss of consciousness (1, 5). It is known that ACU is C old urticaria is a physical urticaria characterized by the development of itchy wheals and/or angioedema after direct contact between the skin and cold air, liquids caused by the release of histamine, leukotrienes, platelet activating factor and other proinflammatory mast-cell mediators (3–6). However, the complete pathophysiology and/or objects (1–3). Its diagnosis is usually supported of the disease, particularly the atypical ACU, remains by cold-contact stimulation tests (CSTs), in which an undetermined (2). immediate wheal is induced after the application of a Autoinflammatory diseases (AIDs) are a group of cold stimulus to the skin (1). Cold urticaria syndromes inherited conditions of innate immunity characterized are very heterogeneous and can be classified into acqui- by seemingly unprovoked and recurrent episodes of red and familial disorders (3). Acquired cold urticaria sterile inflammation. The main subgroups among AIDs (ACU) are further divided into 2 subgroups: (i) primary are the inflammasomopathies, which are characterized (“idiopathic”) and (ii) secondary ACU, depending on by a dysfunction of the inflammasome, a cytosolic the presence of an underlying disease or factor associa- multiprotein complex regulating the pyroptosis and the ted with the induction of the cold urticaria symptoms. release of caspase-1 activation-dependent inflammatory A particular subtype of ACU, known as atypical ACU, cytokines (7). From a clinical point of view, patients with This is an open access article under the CC BY-NC license. www.medicaljournals.se/acta doi: 10.2340/00015555-3292 Journal Compilation © 2019 Acta Dermato-Venereologica. Acta Derm Venereol 2019; 99: 1071–1077 1072 G. Deza et al. AIDs usually display fever and inflammation at different organs, including the skin, joints, central nervous system ActaDV and gastrointestinal tract, and its clinical course may be occasionally complicated with AA-type amyloidosis as a consequence of the long-term systemic inflammation (7). In some conditions, cutaneous manifestations may represent the earliest and most prominent symptoms that often help clinicians to identify AIDs in their early stages (8, 9). This is the case of the generalized urticarial skin rash triggered and/or exacerbated by cold expo- Acta Dermato-Venereologica sure, which is a characteristic feature of certain AIDs including cryopyrin-associated periodic syndromes (CAPS), NLRP12-associated familial cold-induced au- toinflammatory syndrome (FCAS2), NLRC4-associated autoinflammatory syndrome and PLCγ2-associated antibody deficiency and immune dysregulation (PLAID) syndrome (8–11). These disorders are typically charac- terized by recurrent episodes of systemic inflammation, cold urticaria and multi-organ involvement with onset of symptoms during early infancy. However, atypical vari- ants of the diseases may present with non-conventional clinical manifestations, disease onset during adulthood, Fig. 1. Study design. CSTs: cold-contact stimulation tests. absence of urticarial rash at the onset or throughout the course of the disease, or milder disease phenotypes tein (CRP), erythrocyte sedimentation rate (ESR), levels of IgE, with prominent and isolated cutaneous manifestations serum chemistry and serum electrophoresis. Disease severity was (12–14). It has been postulated that these atypical cases categorized into 3 types based on the classification suggested by might be related to the presence of low-penetrance or Wanderer et al. (16): type I, localized urticaria and/or angioedema; post-zygotic mutations in known AIDs genes (12–15), type II, generalized urticaria and/or angioedema without hypoten- ActaDV sive or respiratory symptoms; and type III, severe systemic reac- and the diagnosis in such patients may be extremely tions with ≥ 1 episodes suggestive of hypotension (i.e. dizziness, difficult, often leading to a remarkable diagnostic delay. disorientation or shock) or respiratory distress (e.g. shortness of Therefore, we hypothesized that the presence of vari- breath or wheezing). Other systemic manifestations occurring ants on genes causing AIDs with urticarial skin rashes as during the acute phase of the disease were also registered. Disease the most prominent cutaneous feature (NLRP3, NLRP12, duration was defined as the time from symptom onset to the last follow-up visit, and disease control as an improvement of the signs NLRC4 and PLCG2) may constitute genetic suscepti- and symptoms referred by the patient using the Urticaria Control bility factors of the disease in patients clinically diag- Test (UCT) until they did not cause interferences in daily life. nosed with ACU, and may have relevant consequences When the patient report outcome UCT scores > 12 the disease is Advances in dermatology and venereology regarding patients’ definitive diagnosis and therapeutic considered well controlled (1). approaches. According to current guidelines, the diagnosis of ACU was sup- ported by the ice cube challenge test (1). The standard protocol consisted of the application of the cold stimulus over the patient’s forearm for 5 min followed by 10 min of re-warming (1). The test PATIENTS AND METHODS was considered positive if a coalescent wheal was elicited over Study design, participants and data collected the application site. In addition, all patients underwent thresholds assessment using the TempTest® 3.0 at the baseline evaluation (1, This prospective study included patients with ACU referred to the 17). The cold stimulation time threshold and the critical tempe- Urticaria Clinic of the Department of Dermatology of Hospital rature threshold were defined as the shortest time and the highest del Mar, Barcelona, Spain, during the period from November temperature at which a wheal appears through cold provocation, 2015 to June 2018 (Fig. 1). The diagnosis of ACU was based on respectively. Subjects without wheal formation after the CSTs a patient’s clinical history of wheals, angioedema, or both, after were labelled as atypical ACU. cold exposure. Patients with positive evidence of an underlying In addition, whole peripheral blood was collected from enrol- condition associated with the induction of cold urticaria rashes led subjects for genetic testing after obtaining written informed (i.e. secondary ACU, such as cryoglobulinaemia, cold agglu- consent from patients (> 18 years) or from their parents/guardians tinins, cryofibrinogenaemia, leukocytoclastic vasculitis, drugs or (< 18 years). Genetic variants were classified according to the infectious diseases) were excluded. The clinical research ethics joint consensus recommendation of the American College of committee from Hospital del Mar granted ethical approval for the Medical Genetics and Genomics (ACMG) and the Association for study (reference number: 2017/7469/I). Molecular Pathology (18), and a genetic report of each enrolled Following a systematized protocol, data regarding patients’ patient was generated. Briefly, these recommendations classify epidemiological and clinical features were collected at their the sequence variants into 5 categories (benign, likely benign, initial evaluation. Baseline blood tests from enrolled subjects variant of uncertain significance (VUS), likely pathogenic, and included haemogram, liver and renal functions, C-reactive pro- pathogenic) according to the results of different criteria including: www.medicaljournals.se/acta Acquired cold urticaria and autoinflammatory diseases 1073 (i) allele frequency in different public databases, (ii) previous by in-house designed PCR, purified with Illustra ExoStar 1-Step reports of the variant in the medical literature, (iii) registration of kit (GE Healthcare, Chicago, IL, USA), bidirectional fluorescence ActaDV the concrete sequence variant in phenotype-genotype registries sequenced using ABI BigDye® Terminator v3.1 Cycle Sequencing (i.e. ClinVar, INFEVERS, Human Gene Mutation Database), (iv) Kit (Applied Biosystems, Foster City, CA, USA) and run on an au- structural features of the sequence variant, (v) results of functional tomated ABI 3730XL DNA analyser (Applied Biosystems, USA). studies, (vi) results of intrafamilial segregation analyses, (vii) de novo nature of the sequence variant, (viii) results of different bio- informatic analyses. In specific cases, genetic analysis of patient’s RESULTS relatives was requested in order to improve the clinical relevance and re-classification of detected variants. A total of 50 patients fulfilling the inclusion criteria were enrolled in the study. Clinical and demographic Sequencing methods characteristics of such patients are summarized in Table Acta Dermato-Venereologica I. The median (range) age at disease onset was 27 (1–74) Genomic DNA samples were prepared using a QIAmp DNA Blood Mini Kit (QIAgen, Germany). For simultaneous gene analyses, years, and 34 (68.0%) patients were female. None of the amplicons covering all coding exons and adjacent intronic boun- 50 patients referred family history of similar symptoms daries of NLRP3 (RefSeq NM_001243133.1), NLRP12 (RefSeq (urticarial skin rashes after cold exposure). Thirty-nine NM_144687.2), NLRC4 (RefSeq NM_001199139.1) and PLCG2 (78.0%) patients experienced generalized urticaria af- (RefSeq NM_002661.4) genes were generated by in-house desig- ter cold exposure (types II and III reactions), with 12 ned PCR amplification in an Access Array System 48.48 platform (Fluidigm, South San Francisco, CA, USA). Library preparation, (24.0%) showing severe reactions with ≥ 1 episode sug- control quality and quantification were performed according to gestive of hypotension or respiratory distress. Other re- the manufacturers’ instructions. Emulsion PCR was performed ported systemic symptoms during urticarial flares include on a One Touch2 platform, and sequencing was performed on arthralgias (n = 5), abdominal pain (n = 2) and myalgias an IonTorrent PGM platform using the IonTorrent PGM 400 bp (n = 1). Fourteen (28.0%) patients were diagnosed with Sequencing kit. Reads were mapped against the human reference genome build hg19 using the Burrows-Wheeler Aligner software, atypical ACU due to the negative results of the CSTs. and bam and bai files were obtained. The obtained sequences were Regarding response to treatment, which was evaluated analysed using the Torrent Server and the Ion Reporter softwares after performing the respective genetic analyses, 38 (ThermoFisher Scientific, Waltham, MA, USA). High sequencing (76.0%) and 12 (24.0%) patients showed a satisfactory coverage was obtained for all genes (mean depth per amplicon was and a poor response to non-sedating H1-antihistamines × > 2,000), and thus allowed the detection of somatic mosaicism as low as 2%. (doses ranging from 1 to 4 times the recommended dose ActaDV All detected candidate gene variants were re-sequenced using depending on the patient’s symptoms response), respec- Sanger method, in which concrete gene regions were amplified tively. From these 12 non-responders to antihistamines, Table I. Clinical and demographic characteristics of the study population All patients ACU patients with positive CSTs Atypical ACU patients (n = 50) (n = 36) (n = 14) Patients carrying germline variants on AIDs genes, n (%) 7 (14.0) 2 (5.6) 5 (35.7) Female sex, n (%) 34 (68.0) 27 (75.0) 7 (50.0) Age, years, median (range) 39 (5–79) 39 (9–79) 38 (5–70) Advances in dermatology and venereology Age at disease onset, years, median (range) 27 (1–74) 27 (1–74) 26 (1–60) Atopy, n (%) 11 (22.0) 10 (27.8) 1 (7.1) Family history of cold urticaria symptoms, n (%) 0 (0) 0 (0) 0 (0) Angioedema, n (%) 8 (16.0) 6 (16.7) 2 (14.3) Recurrent sinopulmonary infections, n (%) 5 (10.0) 4 (11.1) 1 (7.1) Associated autoimmune conditions, n (%)a 2 (4) 0 (0) 2 (14.3) Positive antinuclear antibodies, n (%)b 8 (16.7) 5 (14.3) 3 (23.1) Time from cold exposure to symptoms onset, min, median (range) 5 (0.5–180) 5 (0.5–30) 10 (1–180) Disease severity, n (%) I 11 (22.0) 7 (19.4) 4 (28.6) II 27 (54.0) 19 (52.8) 8 (57.1) III 12 (24.0) 10 (27.8) 2 (14.3) Fever or other systemic symptoms, n (%)c 8 (16.0) 5 (13.9) 3 (21.4) Cold triggers, n (%) Water 46 (92.0) 33 (91.7) 13 (92.9) Solids 24 (48.0) 23 (63.9) 1 (7.1) Air 41 (82.0) 30 (83.3) 11 (78.6) Critical stimulation time threshold, min, median (range) 3 (1–5) 3 (1–5) – Critical temperature threshold, ºC, median (range) 14 (4–26) 14 (4–26) – Total serum IgE, kU/l, median (range) 69.5 (5–4,700) 71 (5–4,700) 61 (5–1,689) Elevated inflammatory markers during active disease, n (%)d 7 (14.0) 6 (16.7) 1 (7.1) Normal pattern of serum electrophoresis, n (%) 48 (96.0) 35 (97.2) 13 (92.9) e Disease control, n (%) 45 (90.0) 32 (88.9) 13 (92.9) Disease duration, years, median (range) 7 (1–26) 8 (1–26) 4 (1–15) a Registered autoimmune conditions included 2 patients diagnosed with autoimmune thyroid disease. bAntinuclear antibodies were tested in 48 patients with acquired cold urticaria (ACU): 35 with positive cold-contact stimulation tests (CSTs) and 13 atypical ACU patients. cSystemic manifestations during urticarial flares different from hypotension or respiratory symptoms. dThe evaluated inflammatory markers were the C-reactive protein and the erythrocyte sedimentation rate. eDisease control with conventional treatments (counselling + antihistamines and/or omalizumab). Acta Derm Venereol 2019 Advances in dermatology and venereology ActaDV Acta Dermato-Venereologica ActaDV 1074 G. Deza et al. www.medicaljournals.se/acta post-zygotic gene variant. sess the clinical evolution. patients 2 and 3 was not possible. patients (14.0%) carried 8 heterozygous were detected in any of the patients included Regarding genetic studies, a total of 7 carrying heterozygous germline variants garding the genetic investigations performed for one, which was synonymous. Three patients carrying VUS (patients 2, 3 and Clinical features of the 7 patients carrying was detected. Relatives’ participation for third-line therapies (omalizumab [n = 4] and patient’s parents was possible in 4 families in the study. Furthermore, deep analysis of (Tables II and III). Sanger sequencing con- (n = 3), NLRC4 (n = 1), PLCG2 (n = 3). Ac- lower than 1% in public databases (1000 Ge- cyclosporine [n = 1]) and are pending to as- a well-controlled disease (UCT ≥ 12), and to CSTs. All 3 patients referred symptoms and 2 (66.7%) showed negative responses summarized in Table III. Regarding the 3 6), 2 (66.7%) exhibit an adult-onset ACU patient number 6, and no missense variant proband was also detected in at least one of a single relative (mother) was possible for in the patients’ relatives, genotyping of both variants classified as probably benign. Re- their healthy relatives. The participation of (patients 1, 4, 5 and 7). In all these cases, have higher predicted scores by Combined No pathogenic or likely pathogenic variants wing genes: NLRP3, NLRP12 and PLCG2 and were included one in each of the follo- except for 3, which were classified as VUS nomes Project, ExAC and gnomAD) in the being all of them previously described in triggered by the exposure of cold water and public databases are shown in Table II. All with allelic frequencies lower than 1% in Genetic characteristics of the 7 patients of them were classified as “likely benign” following genes: NLRP3 (n = 1), NLRP12 the remaining 5 patients have just initiated 7 were treated with omalizumab, achieving public databases. Notably, these 3 VUS variants (42.8%) were classified as VUS, the obtained sequences did not identify any germline variants with allelic frequencies the aforementioned germline variants are these rare variants were missense, except the concrete gene variant detected in the Annotation-Dependent Depletion than those cording to the ACMG recommendations, all firmed all 8 heterozygous germline variants. Table II. Summary of genetic characteristics from patients carrying germline variants with allelic frequencies lower than 1% in public databases (1000 Genomes Project, ExAC and gnomAD) Population genetics Bioinformatics Pat. Nucleotide Amino acid 1000 Genomes gnomAD Polyphen-2 (HumDiv Pathogenicity No. Gene exchangea exchange dbSNP Project (%; hom) (%; hom) score) SIFT(score) Mutation taster (score) CADD classificationb 1 NLRC4 c.2785G>T p.Ala929Ser rs61754192 0.30; 0 0.76; 9 Benign (0) Tolerated (0.78) Polymorphism (99) 5.770 Likely benign 2 NLRP3 c.2336G>A p.Gly779Asp rs768252357 0; 0 0.002; 0 Possibly damaging (0.623) Tolerated (0.65) Polymorphism (94) 21.3 VUS PLCG2 c.3125G>C p.Ser1042Thr rs114262189 0.82; 1 0.30; 14 Benign (0.033) Tolerated (0.4) Polymorphism (58) 15.82 Likely benign 3 NLRP12 c.857C>T p.Pro286Leu rs201940393 0; 0 0.014; 0 Possibly damaging (0.877) Deleterious (0) Polymorphism (98) 19.76 VUS 4 NLRP12 c.910C>T p.His304Tyr rs141245482 0.22; 0 0.45; 4 Probably damaging (0.958) Deleterious (0.01) Polymorphism (83) 22.9 Likely benign 5 NLRP12 c.2830C>A p.Arg944= rs104895570 0.28; 0 0.34; 3 – – Polymorphism 0.613 Likely benign 6 PLCG2 c.1274T>G p.Phe425Cys rs1166280990 0; 0 0.0008; 0 Possibly damaging (0.641) Deleterious (0) Disease causing (205) 22.9 VUS 7 PLCG2 c.1565C>G p.Pro522Arg rs72824905 0.28; 0 0.51; 8 Benign (0) Tolerated (0.21) Polymorphism (103) 17.37 Likely benign a NCBI RefSeq: NLRC4, NM_001199139.1; NLRP3, NM_001243133.1; NLRP12, NM_144687.2; PLCG2, NM_002661.4. bClassification of pathogenicity of sequence variants based on the joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology: (i) benign, (ii) likely benign, (iii) variant of uncertain significance, (iv) likely pathogenic and (v) pathogenic. CADD: combined annotation-dependent depletion; dbSNP: database for single nucleotide polymorphisms; gnomAD: genome aggregation database; hom: homocigotos; SIFT: sorting intolerant from tolerant; VUS: variant of uncertain significance. Table III. Clinical features of patients carrying germline variants with allelic frequencies lower than 1% in public databases (1000 Genomes Project, ExAC and gnomAD) Age at Recurrent Time from cold Elevated Disease Patient Gene disease onset sino-pulmonary exposure to symptoms Disease Fever or other Response inflammatory duration number/sex variant (years) infections onset (min) severity systemic symptoms Cold urticaria triggers to CSTs markers?a Disease control (years) 1/ F NLRC4 5 Yes 5 Type-II Arthralgias Water and air Negative No Antihistamines 6 2/ M NLRP3, PLCG2 35 No 5 Type-II Abdominal pain Water and air Negative No Antihistamines 12 3/ F NLRP12 54 No 10 Type-II – Water and air Positive No Antihistamines 13 4/ F NLRP12 44 No 10 Type-III Arthralgias Water and air Negative No Omalizumab 7 5/ F NLRP12 10 No 5 Type-II – Water, air and objects Negative No Antihistamines 4 6/ M PLCG2 5 No 10 Type-II – Water and air Negative No Antihistamines 4 7/ F PLCG2 14 No 5 Type-II – Water, air and objects Positive No Antihistamines 25 a The evaluated inflammatory markers during active disease were the C-reactive protein and the erythrocyte sedimentation rate. CSTs: cold-contact stimulation tests. Acquired cold urticaria and autoinflammatory diseases 1075 cold air, but not to cold objects, and they also experienced conventional CSTs (8, 9, 11, 22, 25, 26). Clinically, the generalized cold urticaria, with one subject (33.3%) also cold urticarial rash in AIDs has a broader spectrum of ActaDV referring abdominal pain during urticaria flares. The time lesions, occasionally with urticarial features, but more from cold exposure to onset of symptoms was less than commonly with erythematous and oedematous papules 10 min in all cases. None of these patients had associated and plaques. This rash is rather symmetrically distribu- autoimmune conditions or history of granulomatous skin ted on the trunk and/or extremities, usually sparing the disease or sinopulmonary infections during childhood. In head, rarely pruritic, and often described as tender and/ addition, blood tests (including haemogram, CRP, ESR or eliciting a burning sensation (8, 9). In contrast, ACU and serum electrophoresis) were within normal range is characterized by the typical itchy wheal-and-flare-type during active disease. Regarding response to treatment, skin reactions that are asymmetrically distributed on the Acta Dermato-Venereologica these 3 patients carrying VUS showed a satisfactory body (1, 2, 5). The duration of single lesions, on average, clinical response to antihistamines. is also longer in AIDs (up to 24 h) compared with ACU (minutes to hours) (25, 26). Finally, patients with AIDs do not respond to conventional urticaria therapies, such DISCUSSION as antihistamines, leukotriene antagonists and/or oma- AIDs may present with a myriad of cutaneous lesions, in- lizumab (8). cluding urticarial and maculopapular eruptions, pustules, The pathophysiology of cold urticaria is yet to be ulcerative lesions, and granulomatous and erysipelas-like fully elucidated, but the activation and degranulation lesions (19). Urticarial rash, usually triggered and/or of tissue-resident mast cells and circulating basophils exacerbated by generalized cold exposure, represents the with the subsequent release of inflammatory mediators most prominent cutaneous feature in patients with CAPS, have been shown to play key roles (6, 27). As yet, it is FCAS2, PLAID and NLRC4-inflammasomopathies unclear exactly what causes such activation and degra- (8–11, 20). In addition to skin lesions, patients with such nulation. The most supported hypothesis is that different conditions may have a great variety of systemic symp- autoantigens, the expression of which might be induced toms. Thus, patients with CAPS usually have arthralgias, by the appropriate environmental triggers, bind to IgE myalgias, recurrent fever, headache or conjunctivitis on the surface of mast cells and basophils through the (20), with the severest phenotypes also developing high-affinity IgE receptor (FcεRI), resulting in the acti- sensorineural hearing loss, central nervous symptoms vation of these cells and the release of the inflammatory ActaDV and/or deforming arthropathy (20). The phenotype of mediators (6, 27). In this sense, the FcεRI expression on NLRP12 and NLRC4-associated familial cold-induced effector cells has been found to be significantly upregu- autoinflammatory syndrome (FCAS) may resemble a lated in patients with ACU (28). In this complex process, mild form of CAPS (10, 21). Patients with PLAID exhibit nevertheless, no genetic susceptibility factors have yet long-standing cold urticaria, recurrent sinopulmonary been identified. Thus, to the best of our knowledge, infections, hypogammaglobulinaemia, autoimmunity this is the first study to investigate the presence of both and granulomatous skin disease (11, 22). Physicians germline and post-zygotic variants on genes previously should consider these diagnoses in patients presenting described as associated with AIDs in a large cohort of Advances in dermatology and venereology with a combination of the aforementioned symptoms patients with ACU. in addition to further hints, including a positive family Somatic gene mosaicism has been described as an history, laboratory abnormalities (e.g. elevated inflam- important disease-causing mechanism in many AIDs mation markers during active disease) and poor response (29–33). In particular, somatic NLRP3 mosaicism has to conventional therapies. A selected gene mutation ana- been shown to play an important role in the pathoge- lysis will confirm the diagnosis, and a prompt and early nesis of patients with a clinical diagnosis of CAPS, but treatment with specific drugs can dramatically improve mutation negative by conventional genetic studies (29, the clinical manifestations (10, 23, 24). 30). Some recent studies support the role of somatic The cold urticarial rash observed in patients with the mosaicisms in milder and/or later-onset of the disease aforementioned AIDs may be indistinguishable from (15, 34). The novel next-generation sequencing (NGS) that of ACU. However, it is convenient to remark that technologies have been crucial to identify low-level there are some features that may point to AIDs and somatic mosaicism and allowed achieving the definitive help to distinguish them from ACU (8, 11, 22, 25, 26). diagnosis and starting the appropriate anti-inflammatory In AIDs, cold-induced urticarial rash frequently starts treatment. In the present study, a deep search for somatic during infancy or childhood and is lifelong (25, 26). mosaicism using highly-sensitive NGS technology in the These symptoms are usually triggered by generalized 4 candidate genes did not identify any candidate variant. exposure to cold stimuli, such as cold air or cold water, On the other hand, germline variants on the NLRP3, rather than by contact with cold objects (11, 22). Their NLRP12, NLRC4 and PLCG2 genes with low allelic development is often delayed (> 1–2 h) after cold expo- frequencies (<1%) in public databases were found in 7 of sure, and is typically associated with negative results on our 50 patients with ACU. Nevertheless, no pathogenic Acta Derm Venereol 2019 1076 G. Deza et al. or likely pathogenic variants were detected, and 4 out of genetic studies in subjects without enough evidence to 7 subjects carried variants classified as probably benign suspect an AID, in order to avoid false-positive diagno- ActaDV according to ACMG recommendations. By contrast, VUS ses and the consequent overtreatment, given the high were detected in the remaining 3 patients. Despite the fact frequency of healthy carriers. Further studies focusing that these patients exhibited certain clinical features that on the investigation of genetic susceptibility factors in could resemble the cold urticaria symptoms associated patients with chronic urticaria would help a better un- with AIDs (developing a generalized urticaria (no patient derstanding of the complex pathogenesis of the disease. was classified as type-I severity) triggered mostly by the exposure to cold water and/or cold air rather than cold objects, with a high proportion of atypical ACU), the ACKNOWLEDGEMENTS Acta Dermato-Venereologica exact causal role of such genetic variants is unknown. This work was conducted within the framework of the PhD in VUS have been described both in patients with recurrent medicine for Dr Gustavo Deza from the Department of Medicina inflammatory attacks and in healthy subjects, raising the of Universitat Autònoma de Barcelona (UAB). question of whether these variants are silent polymor­ This study was partially funded by PI17/00198 grant from Fondo phisms or low-penetrance disease-associated mutations de Investigación Sanitaria (FIS), Instituto de Salud Carlos III, Mi- nistry of Economy and Competitiveness co-financed by European (35–37). It has been also postulated that these genetic Regional Development Fund (AG-A), CERCA Programme/Gene- variations might function as susceptibility alleles to ralitat de Catalunya (JIA), SAF2015-68472-C2-1-R grant from the inflammation rather than disease-associated mutations, Spanish Ministry of Economy and Competitiveness co-financed causing an inflammatory phenotype in concomitance by European Regional Development Fund (JIA), and AC15/00027 with other eventual environmental and/or genetic factors grant from the Instituto de Salud Carlos III/Transnational Research Projects on Rare Diseases (JIA). (12). However, there are several reasons that suggest The funding organizations had no role in the design and conduct that the genetic variants found in our study do not play of the study; collection, management, analysis and interpretation a relevant role in these particular patients. First, all of the data; preparation, review or approval of the manuscript; and patients achieved control of the disease manifestations decision to submit the manuscript for publication. with conventional urticaria therapies (antihistamines and Conflicts of interest. GD has participated in clinical trials and omalizumab), which support the involvement of mast educational activities sponsored by Novartis. AMG-A is a medical cells and basophils in the pathophysiology of the disease, advisor for Uriach Pharma, Genentech, and Novartis; has received research grants from Intendis-Bayer, Uriach Pharma, and Novartis; in contrast to AIDs in which there is no evidence for his- ActaDV and has participated in educational activities sponsored by Uriach tamine release (25). In addition, the delayed cutaneous Pharma, Novartis, Genentech, Menarini, Glaxo Smith & Kline, response of hours after cold exposure, which is a typical Merck MSD, Almirall, and Leo Pharma. The other authors have feature of AIDs, was not seen in any of these patients. no relevant conflicts of interest to declare. The normal values of acute phase reactants during ac- tive disease and the absence of family history are also not characteristic of patients with AIDs. Finally, the REFERENCES presence of the same genetic variant in the – otherwise 1. Magerl M, Altrichter S, Borzova E, Giménez-Arnau A, Grattan CEH, Lawlor F, et al. The definition, diagnostic testing, and healthy – relatives of the analysed patients supports the management of chronic inducible urticarias – The EAACI/ non-pathogenicity of the findings. Taken together, these Advances in dermatology and venereology GA(2) LEN/EDF/UNEV consensus recommendations 2016 observations strongly support the fact that the detected update and revision. Allergy 2016; 71: 780–802. 2. Deza G, Brasileiro A, Bertolín-Colilla M, Curto-Barredo L, Pujol variants in genes causing AIDs with cold-induced urtica- RM, Giménez-Arnau AM. 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