ORIGINAL RESEARCH published: 01 October 2019 doi: 10.3389/fimmu.2019.02325 Expanding the Clinical and Genetic Spectra of Primary Immunodeficiency-Related Disorders With Clinical Exome Sequencing: Expected and Unexpected Findings Francesc Rudilla 1,2† , Clara Franco-Jarava 3,4† , Mónica Martínez-Gallo 3,4 , Edited by: Marina Garcia-Prat 4,5 , Andrea Martín-Nalda 4,5 , Jacques Rivière 4,5 , Aina Aguiló-Cucurull 3,4 , Sudhir Gupta, Laura Mongay 1 , Francisco Vidal 1,2,6 , Xavier Solanich 7 , Iñaki Irastorza 8 , University of California, Irvine, United States Juan Luis Santos-Pérez 9 , Jesús Tercedor Sánchez 10 , Ivon Cuscó 11 , Clara Serra 11 , Noelia Baz-Redón 12 , Mónica Fernández-Cancio 12,13 , Carmen Carreras 14 , Reviewed by: José Manuel Vagace 15 , Vicenç Garcia-Patos 16 , Ricardo Pujol-Borrell 3,4 , Lennart Hammarström, Pere Soler-Palacín 4,5 and Roger Colobran 3,4,11* Karolinska Institute (KI), Sweden Bertrand Boisson, 1 Immunogenetics and Histocompatibility Laboratory, Banc de Sang i Teixits, Barcelona, Spain, 2 Transfusional Medicine The Rockefeller University, Group, Vall d’Hebron Research Institute, Autonomous University of Barcelona, Barcelona, Spain, 3 Immunology Division, United States Department of Cell Biology, Physiology and Immunology, Vall d’Hebron Research Institute, Hospital Universitari Vall d’Hebron, *Correspondence: Autonomous University of Barcelona, Barcelona, Spain, 4 Jeffrey Model Foundation Excellence Center, Barcelona, Spain, 5 Roger Colobran Pediatric Infectious Diseases and Immunodeficiencies Unit (UPIIP), Hospital Universitari Vall d’Hebron, Vall d’Hebron rcolobran@vhebron.net Research Institute, Autonomous University of Barcelona, Barcelona, Spain, 6 CIBER on Cardiovascular Diseases (CIBERCV), Instituto de Salud Carlos III (ISCIII), Valencia, Spain, 7 Adult Immunodeficiencies Unit (UFIPA), Internal Medicine Department, † These authors have contributed Institut d’Investigació Biomèdica de Bellvitge, Hospital Universitari de Bellvitge, L’Hospitalet de Llobregat, Barcelona, Spain, equally to this work 8 Pediatric Gastroenterology, Cruces University Hospital, Basque Country University, Bilbao, Spain, 9 Immunodeficiencies and Infectious Disease Unit, Universitary Hospital Virgen de las Nieves, Granada, Spain, 10 Unidad de Dermatología Pediátrica y Specialty section: Anomalías Vasculares, Servicio de Dermatología, Instituto de Investigación Biosanitaria IBS, Hospital Universitario Virgen de This article was submitted to las Nieves, Granada, Spain, 11 Genetics Department, Hospital Universitari Vall d’Hebron, Barcelona, Spain, 12 Growth and Primary Immunodeficiencies, Development Group, Vall d’Hebron Research Institute, Hospital Universitari Vall d’Hebron, Autonomous University of a section of the journal Barcelona, Barcelona, Spain, 13 CIBER Rare Diseases (CIBERER), Instituto de Salud Carlos III (ISCIII), Madrid, Spain, 14 Frontiers in Immunology Pediatric Hematology and Immunodeficiencies Unit, Hospital Universitario y Politécnico La Fe, Valencia, Spain, 15 Hematology Department, Complejo Hospitalario Universitario de Badajoz, Badajoz, Spain, 16 Dermatology Department, Received: 26 July 2019 Hospital Universitari Vall d’Hebron, Barcelona, Spain Accepted: 16 September 2019 Published: 01 October 2019 Citation: Primary immunodeficiencies (PIDs) refer to a clinically, immunologically, and genetically Rudilla F, Franco-Jarava C, heterogeneous group of over 350 disorders affecting development or function of the Martínez-Gallo M, Garcia-Prat M, Martín-Nalda A, Rivière J, immune system. The increasing use of next-generation sequencing (NGS) technology Aguiló-Cucurull A, Mongay L, Vidal F, has greatly facilitated identification of genetic defects in PID patients in daily clinical Solanich X, Irastorza I, practice. Several NGS approaches are available, from the unbiased whole exome Santos-Pérez JL, Tercedor Sánchez J, Cuscó I, Serra C, Baz-Redón N, sequencing (WES) to specific gene panels. Here, we report on a 3-year experience with Fernández-Cancio M, Carreras C, clinical exome sequencing (CES) for genetic diagnosis of PIDs. We used the TruSight Vagace JM, Garcia-Patos V, Pujol-Borrell R, Soler-Palacín P and One sequencing panel, which includes 4,813 disease-associated genes, in 61 unrelated Colobran R (2019) Expanding the patients (pediatric and adults). The analysis was done in 2 steps: first, we focused on a Clinical and Genetic Spectra of virtual PID panel and then, we expanded the analysis to the remaining genes. A molecular Primary Immunodeficiency-Related Disorders With Clinical Exome diagnosis was achieved in 19 (31%) patients: 12 (20%) with mutations in genes included Sequencing: Expected and in the virtual PID panel and 7 (11%) with mutations in other genes. These latter cases Unexpected Findings. Front. Immunol. 10:2325. provided interesting and somewhat unexpected findings that expand the clinical and doi: 10.3389/fimmu.2019.02325 genetic spectra of PID-related disorders, and are useful to consider in the differential Frontiers in Immunology | www.frontiersin.org 1 October 2019 | Volume 10 | Article 2325 Rudilla et al. Clinical Exome Sequencing in Primary Immunodeficiencies diagnosis. We also discuss 5 patients (8%) with incomplete genotypes or variants of uncertain significance. Finally, we address the limitations of CES exemplified by 7 patients (11%) with negative results on CES who were later diagnosed by other approaches (more specific PID panels, WES, and comparative genomic hybridization array). In summary, the genetic diagnosis rate using CES was 31% (including a description of 12 novel mutations), which rose to 42% after including diagnoses achieved by later use of other techniques. The description of patients with mutations in genes not included in the PID classification illustrates the heterogeneity and complexity of PID-related disorders. Keywords: primary immunodeficiencies, next generation sequencing, clinical exome sequencing, TruSight one sequencing panel, mutations, genetic variants INTRODUCTION 50% in the various cohorts reported (7–11). Several factors may explain this relatively low percentage of success, including the Primary immunodeficiencies (PIDs) are a phenotypically existence of complex models of inheritance (e.g., oligogenic, and genetically heterogeneous group of inborn errors of polygenic, epigenetic) which are just beginning to be addressed immunity leading to a predisposition to infections, autoimmune, by the scientific community (12, 13). Moreover, the patients or autoinflammatory diseases, lymphoproliferation, and included in these studies often show highly heterogeneous malignancies. Typically considered as rare diseases (PID PID symptoms and many are complex cases that remain prevalence ranges from 1:10,000 to 1:100,000 population) recent genetically unresolved after using traditional sequential Sanger studies indicate that they are more common than was formerly sequencing procedures. The other side of the coin of investigating believed (1). The diagnostic workup for PIDs has advanced from challenging patients is that novel PID-associated genes have been clinical evaluation with a detailed personal and family history discovered and unusual phenotypes caused by mutations in genes to a more recent series of complex laboratory assays, including known to cause PID have been identified (6). Hence, the use extensive flow cytometry studies, cell culture, or western blotting. of NGS has expanded our knowledge of the complex clinical As most described PIDs have a monogenic cause, molecular phenotypes associated with PID and has refined phenotype- genetic testing is usually the key to providing a definite diagnosis genotype correlations in many cases. Certain studies with more (2). A positive genetic diagnosis can direct the patient toward defined cohorts in terms of PID suspicion have obtained higher suitable prevention, monitoring, and treatment options. In percentages of positive diagnoses. An example is the recent addition, the genetic results can help patients make choices study by Yu et al. focused on molecular analysis of infants regarding their future, especially in terms of genetic counseling with abnormal T-cell receptor excision circles (TRECs), positive about having children. In the case of inherited mutations, testing newborn screening results, or a positive family history of PID. of relatives is also important to expand medical care, and genetic Using a customized NGS-based multigene-targeted panel for counseling to all affected family members and carriers. However, SCID and other severe PIDs, these authors identified disease- achieving a definite genetic diagnosis in a suspected case of PID causing mutations in 14 of 20 patients (70%) (14). can be a complex and laborious process that sometimes fails to Clinical exome sequencing (CES) represents an intermediate yield positive results. step between analysis by specific gene panels and WES. WES Identification of the numerous monogenic defects underlying relies on investigating all protein-coding regions of the genome PIDs has continuously increased over time. Evidence of this is (i.e., exons + flanking regions), and because most known disease- seen in the current IUIS report, which, as of February 2017, causing mutations occur in exons, WES is considered an effective included 354 distinct disorders with 344 different gene defects method to identify them. However, while hundreds of genetic listed (3). Remarkably, in the previous 2 years (2015–2016), conditions have been fully characterized at the clinical and 85 new causal genes were identified (4). This striking increase molecular level, most human genes are not actually related to in the number of disorders recognized in the last years is human diseases. CES focuses on known disease-associated genes undoubtedly driven by the increasingly more extensive use of and provides a rapid and cost-effective sequencing, analysis, and next-generation sequencing (NGS) technology. NGS has greatly interpretation of the results. accelerated the discovery of novel disease-causing genes and Here, we report our experience using CES for the genetic facilitated the genetic diagnosis of patients with monogenic diagnosis of PIDs. The study included 61 children and adults, inborn errors of immunity (5). Several NGS approaches, ranging and the molecular diagnosis was reached in 19 of them (31%). from the unbiased whole exome sequencing (WES) and whole The results are presented after dividing patients into those with genome sequencing (WGS) to the more restricted targeted gene mutations in PID-causing genes (based on the IUIS classification) panel sequencing have been successfully used for the diagnosis and those with mutations in genes not included in the PID of PIDs (6). The efficacy of NGS in terms of the percentage of classification. These latter cases have yielded interesting and positive diagnoses is difficult to establish, as it is highly dependent somewhat unexpected findings that expand the clinical and of the type of PID suspected, but in general it does not reach genetic spectra of PID and PID-related disorders. We also discuss Frontiers in Immunology | www.frontiersin.org 2 October 2019 | Volume 10 | Article 2325 Rudilla et al. Clinical Exome Sequencing in Primary Immunodeficiencies the limitations of CES, exemplified by patients with negatives TABLE 1 | Epidemiology and results summary. results on this test, but later diagnosed by other methods, such as Total number of patients studied 61 more specific PID panels, WES, and array comparative genomic hybridization (aCGH). Female 25 (41%) Male 36 (59%) MATERIALS AND METHODS Mean age in years (range) 11 (0–57) Pediatric (<18 years) 50 (82%) Patients and Samples Adult (>18 years) 11 (18%) This study included 61 unrelated patients attended during a 3- 1. Patients with a genetic defect identified by CES 19 (31%) year period (2015–2017). Most patients were attended at Hospital Pediatric (% success among pediatric patients) 14 (28%) Universitari Vall d’Hebron (HUVH) Barcelona (Spain), but as Adult (% success among adult patients) 5 (45%) HUVH is a reference center for the diagnosis of PID in Spain, 2. Incomplete genotypes/VUS 5 (8%) samples from other regions of the country were also included. 3. Patients with a genetic defect identified after CES 7 (11%) All patients underwent a clinical history and laboratory analyses Because gene was not included in TSO 4 (including immunophenotyping and functional tests), which Other reasons 3 pointed to suspected PID. Clinical data were obtained from 4. Patients without relevant genetic findings 30 (49%) the patient’s medical chart. In some patients, direct sequencing (Sanger method) of suspected genes had been performed CES, clinical exome sequencing; TSO, TruSight One Sequencing Panel; VUS, variant of previously with negative results. DNA was isolated using different uncertain significance. methods, but ultimately, all samples were precipitated and resuspended with pure water to homogenize all DNAs. obtained using the quality parameters established by the filter Written informed consent for the studies reported here pass in the above-mentioned software. Variants considered in and for the publication was obtained from the patients or this analysis were single nucleotide changes and short insertions their legal representatives, according to the procedures of the or deletions. PolyPhen-2 and SIFT were used to predict the Ethics Review Board of Hospital Universitari Vall d’Hebron pathogenicity of the missense variants detected (17, 18). All [code: PR(AG)69/2016]. pathogenic variants were confirmed by Sanger sequencing. Clinical Exome Sequencing and Data Analysis RESULTS AND DISCUSSION CES was carried out using the TruSight One (TSO) Sequencing Panel (Illumina, San Diego, CA, USA) according to the Overall Sequencing Data and Results manufacturer’s instructions. The panel covers 4,813 disease- Summary associated genes. Targeted exonic regions underwent paired- In this study, we report the CES results in 61 unrelated end sequencing on an Illumina platform using a MiSeq or patients with a clinically suspected PID. DNA samples were NextSeq 500 sequencing system (NextSeq High Output Kit, 300 sequenced using the TSO sequencing panel, which covers the cycles). The MSR:Enrichment v2.4.60.8 and BWA Enrichment exons and flanking regions of 4,813 disease-associated genes. v2.1.1-v2.1.2 packages (BaseSpace, Illumina) were used to align The mean target coverage depth was 81 ± 28X, with a mean the resulting sequence reads to a reference genome (GRCh37, 20X target coverage of 89 ± 4% (Supplementary Table 2). hg19) (Burrows-Wheeler Aligner, BWA) and to carry out variant Biological data analysis involved 2 steps: First, we analyzed the calling (Genome Analysis Toolkit, GATK) (15, 16). The Isaac variants in a virtual PID panel of 260 genes that contained Enrichment v1.0 and Enrichment v2.1.0–v3.0.0 tools were also the TSO genes included in the current IUIS classification used for alignment and variant calling (BaseSpace, Illumina). (Supplementary Table 3) (3), and then, we extended the analysis The sequencing quality parameters obtained from the MiSeq to the full TSO gene panel in patients with negative results in the sequencing platform are shown in Supplementary Table 1. virtual PID panel. Considering the average value of a set of 20 runs with 3 samples Sixty-one patients were included in the study, 36 males (59%) in each sequencing run, more than 23 million mapped reads and 25 females (41%), with a mean age of 11 years at enrollment per run were obtained in a cluster density of 1,183 K/mm2 , (range, 0–57 years); 82% were pediatric patients (50/61). Eighteen with the cluster PF being 88.6%. In total, 89.7% of bases percent of patients were non-Caucasian, and 23% had a family showed a desirable quality score of ≥Q30. Two sequencing history of consanguinity. The patients’ epidemiologic data are runs of 36 samples each were performed using the NexSeq 500 summarized in Table 1. A genetic cause explaining the patient’s platform (Supplementary Table 1). The cluster density was 151 phenotype was found in 31% of patients (19/61), a percentage K/mm2 , the cluster PF was 86.5%, and there were 93 million comparable to the rates reported in other NGS-based studies targeted reads. The percentage of bases with ≥Q30 were within (8–10, 19–21). Among these positive genetic results, 12 were specifications in both runs, with a mean of 80.5%. associated with genes included in the latest IUIS classification The data analysis of relevant disease variants was carried (3), and 7 involved genes that are not typically associated out with the Illumina Variant Studio software v3.0 and with classical PID phenotypes. Additionally, 5 patients showed BaseSpaceVariant Interpreter Beta (Illumina). All variants were variants of uncertain significance (VUS) or incomplete genotypes Frontiers in Immunology | www.frontiersin.org 3 October 2019 | Volume 10 | Article 2325 Rudilla et al. Clinical Exome Sequencing in Primary Immunodeficiencies in genes related to their clinical phenotype. Of the 39 patients 10% of CVID patients bear a mutation in TACI (compared to without a positive genetic diagnosis by CES, 7 (11%) were approximately 1% of the healthy population), which supports successfully diagnosed later using other approaches. CES had a role of TACI mutations in CVID or CVID-like disorders. failed for the following reasons: (1) the genes were not included Therefore, in P2 and P3 heterozygous TACI mutations cannot in the CES panel, (2) large genomic rearrangements were not be considered causal by itself, but a predisposing factor in successfully detected by CES, and (3) mutations occurred in combination with other genetic and/or environmental factors. non-coding regions, which are beyond the scope of CES. In P4 is an 18-year-old male who, since the age of 14, the following sections, these results are described in detail had experienced severe Crohn’s-like gut inflammation and and discussed. The clinical and laboratory data of the patients arthritis. He had a maternal family history of autoimmunity, included in the next sections are summarized in Tables 2, 3. The and Crohn’s disease on the paternal side. Mutations in XIAP clinical and laboratory data of all patients (n = 61) included in and FOXP3 had been ruled out prior to this study. CES the study are summarized in Supplementary Tables 4, 5. revealed a hemizygous missense mutation in IKBKG (the gene that codes for NEMO), c.169G>A/p.Glu57Lys. IKBKG hypomorphic mutations, which reduce but do not abolish Patients With Mutations in PID Genes (2017 NF-κB activation, have been identified in male patients with IUIS Classification) a spectrum of X-linked clinical phenotypes, ranging from The filtering strategy for PID genes included in the current IUIS anhidrotic ectodermal dysplasia (EDA) with immunodeficiency classification led to a molecular diagnosis in 12 cases (Table 4). (EDA-ID) to immunodeficiency without EDA. IKBKG loss-of- Mean age in this group was 14.8 years (range, 1–57), and the function mutations are lethal in males and cause incontinentia male-to-female ratio was 1.4. pigmenti (IP) in females (39). Our patient did not have Three patients (P1, P2, and P3) had mutations in genes EDA and the main clinical manifestation was gastrointestinal predominantly causing antibody deficiencies. inflammation. He did not show hypogammaglobulinemia or P1 is a 15-year-old male referred to our center at the age of 4 relevant infections, 2 clinical features usually seen in typical EDA- due to recurrent viral infections (rubella, EBV, cytomegalovirus), ID patients (39). The IKBKG p.Glu57Lys mutation has been growth delay, lymphadenopathies, and chronic ear infections. previously described in 2 unrelated patients, 1 with EDA-ID The patient further developed a large bowel lymphoma, and (40), and 1 with immunodeficiency without EDA (28). The latter presented an ALPS-like phenotype. Previous Sanger sequencing study demonstrated that IKBKG p.Glu57Lys leads to specific studies of the FAS and XIAP genes were negative. CES revealed immunological defects in vitro (28). Interestingly, this mutation a heterozygous mutation in the PIK3R1 gene, responsible for has also been associated with a mild form of IP (29, 41). Given the activated PI3K delta syndrome type 2 (APDS2). The mutation, relatively high frequency of p.Glu57Lys in the general population c.1425+1G>A, is the one most often described in this syndrome (MAF = 0.001), this mutation if pathogenic should be considered (22, 23). Identification of this mutation allowed the patient to to have low penetrance. enter a clinical trial for a selective PI3K delta inhibitor, but he had P5 is 10-year-old girl who experienced recurrent atypical to discontinue after several weeks due to severe adverse effects. mycobacterial infections mainly in skin and gut since the age Unfortunately, he ultimately died at the age of 18 years. of 4. She also had hypertelorism, craniosynostosis, and scoliosis. Patients P2 and P3 had common variable immune deficiency Low Th17 cell counts and increased IgE levels pointed to (CVID)-like antibody deficiencies. P2 is a 57-year-old woman mutations in genes responsible for hyper IgE syndrome, and who reported recurrent upper respiratory tract infections indeed, CES revealed a heterozygous missense mutation in STAT3 since the age of 14 years, which continued despite intravenous (c.1144C>T/p.Arg382Trp). This mutation, located in the DNA- immunoglobulin (IVIG) treatment. She also experienced binding domain of the protein, is one of the most frequent acute diarrhea due to Aeromonas Hydrophila, granulomatous STAT3-dominant negative mutations described (30, 31). lymphadenopathies, hepatosplenomegaly, long periods of fever P6, a 2-year-old boy born of consanguineous parents, with elevated acute phase reactants, hypogammaglobulinemia, presented with enteropathy, bacterial infections, and growth central pancytopenia, and granulomatous-lymphocytic delay at the age of 10 months. Based on a strong suspicion interstitial lung disease (GLILD). P3 showed the first symptoms of PID, the attending pediatricians sent us a sample for at the age of 3. She had episodes of EBV infection, autoimmune inclusion in our genetic study, with no previous laboratory pancytopenia, hypogammaglobulinemia, pathological axillary determinations. We found a novel hemizygous truncating lymphadenopathies, and hepatosplenomegaly. Previous genetic mutation in XIAP (c.888_892del/p.Lys299LeufsX9), causing type studies ruled out FAS and PIK3R1mutations. In both patients, 2 X-linked proliferation syndrome (XLP-2) (42), which perfectly heterozygous mutations were found in TNFRSF13B (the gene fit the patient’s clinical phenotype. that codes for TACI). P2 had one of the most common missense P7 is a boy referred to our hospital at the age of 4 months mutations associated with CVID, c.310T>C/p.Cys104Arg due to severe osteomyelitis caused by Salmonella spp. Functional (24, 25), and P3 had the c.260T>A/p.Ile87Asn mutation (26, 27). tests revealed a normal respiratory burst test, but in vitro IFNγ It has been demonstrated that both these mutations impair and IL-12 production was impaired. Surprisingly, CES revealed the capacity of TACI ligation to activate the NFκB pathway a hemizygous mutation in G6PD, c.934G>C/p.Asp312His, (27). TACI mutations are not considered to be fully-penetrant reported to be responsible for class III G6PD deficiency. causal mutations, as most carriers show no disease, but up to G6PD deficiency is the most common genetic cause of chronic Frontiers in Immunology | www.frontiersin.org 4 October 2019 | Volume 10 | Article 2325 TABLE 2 | Main clinical features of patients with relevant genetic findings. Frontiers in Immunology | www.frontiersin.org Rudilla et al. ID Sex Age at Gene Family Consanguinity Age of Syndromic Infections Autoimmunity Lymphoproliferation Inflammation Main clinical features, key Treatment study (y) history onset (y) features words PATIENTS WITH MUTATIONS IN PID GENES (2017 IUIS CLASSIFICATION) P1 M 15 PIK3R1 – – 7 – V – Malign Gut IgA deficiency, immune IVIG, immunosuppression dysregulation, growth delay, (rapamycin and rituximab) enteropathy, intestinal large bowel lymphoma P2 F 57 TNFRSF13B – – 14 – B – Benign Gut, lung CVID, infections, GLILD, IVIG, immunosuppression granulomas, adenopathies, (rituximab, corticoids, pancytopenia azathioprine), antibiotics √ √ P3 F 8 TNFRSF13B – 3 – B, V Benign – EBV, pancytopenia, IVIG, rapamycin hepatosplenomegaly, hypogammaglobulinemia, ALPS-like, CVID-like √ P4 M 18 IKBKG – 14 – – – – Gut, joints Crohn’s disease, joint affectation, Infliximab CGD, NEMO deficiency √ P5 F 10 STAT3 – – 4 B, F, Myc – – – Hyper IgE, atypical mycobacteria Antifungal prophylaxis, vitamin D, IVIG, antibiotics √ √ P6 M 2 XIAP – 0.8 B – – Gut Enteropathy, bacterial infection, IVIG, patient died growth delay P7 M 1 G6PD – – 0.3 – B – – Bone Osteomyelitis, Salmonella spp. - √ P8 M 30 STAT1 – – 0.0 – F – Skin, mucosa Dermatophytosis, oral candidiasis Antifungal prophylaxis, 5 P9 F 4 STAT1 – – 0.8 – F – – Joints CMC, polyarthritis, episcleritis, Ruxolitinib, HSCT bronchiectasis √ √ P10 M 6 STAT1 4 – F – – – Familial CMC Antifungal prophylaxis √ P11 F 12 PLCG2 – – 0.8 B – – Skin, lungs Agammaglobulinemia, severe IVIG, antibiotics, corticosteroids, cutaneous inflammation, etanercept, anakinra bronchiectasis, B cell lymphopenia, growth delay P12 M 15 ADA – – 8 – B – Malign – Hodgkin lymphoma, B cell Chemotherapy Clinical Exome Sequencing in Primary Immunodeficiencies deficiency PATIENTS WITH MUTATIONS IN GENES NOT INCLUDED IN THE PID CLASSIFICATION √ P13 M 5 SKIV2L – – 0.8 B – – Gut Inflammatory enteropathy, growth Antibiotics, IVIG, adalimumab, delay immunosuppression (azathioprine, prednisone, October 2019 | Volume 10 | Article 2325 rapamycin) √ P14 M 0.25 MMACHC – 0.1 – – – – – fHLH, XLP Died before diagnosis √ P15 F 44 SLC27A4 – n.a. – – – – Skin Netherton syndrome - P16 F 0.3 DSG1 – – 0.3 – B – – – Erythroderma, Netherton syndrome, Infliximab, adalimumab, hyper IgE, eosinophilia cyclosporine √ √ √ P17 F 1 DNAI2 0.1 – V, F Malign – Recurrent bronchitis, biphenotypic HSCT (due to leukemia) leukemia, growth delay √ √ P18 M 38 SIX6 n.a. – B – – – CID, Low IgA IVIG √ √ P19 M 5 RECQL4 – 1 V, F – – – CVID, growth delay IVIG, antibiotic prophylaxis (Continued) Frontiers in Immunology | www.frontiersin.org Rudilla et al. TABLE 2 | Continued ID Sex Age at Gene Family Consanguinity Age of Syndromic Infections Autoimmunity Lymphoproliferation Inflammation Main clinical features, key Treatment study (y) history onset (y) features words PATIENTS WITH INCOMPLETE GENOTYPES/VARIANTS OF UNCERTAIN SIGNIFICATE (VUS) √ P20 M 11 UNC13D – 8 – V – Malign Gut Pancytopenia, HSCT (due to T cell lymphoma) hepatosplenomegaly, hemophagocytosis, panniculitic T cell lymphoma √ √ P21 M 13 RAG2 – 6 – – Benign Systemic Persistent fever, intermittent Immunosuppression abdominal pain, granulomatous (methotrexate + colchicine + hepatitis corticosteroids) √ √ P22 M 4 PLCG2 1 – V – – Skin Periodic fever, skin rash - P23 F 11 TRAF3 – – 11 – V – – – Herpes Zoster, VZV - meningoencephalitis P24 M 2 NOD2 – – 0.6 – – – – Gut Early-onset colitis - PATIENTS WITH GENETIC FINDINGS POST-CLINICAL EXOME √ √ P25 F 8 LRBA – 0.7 – B, V, F Malign Gut EBV, lymphoproliferation, IVIG, rapamycin, antibiotic autoimmunity, infections, prophylaxis, HSCT dysregulation, enteropathy, autoimmune cytopenia √ P26 F 11 LRBA – – 2 – V Benign – ALPS IVIG, abatacept √ P27 F 7 LRBA – – 7 – B Benign – Antibody deficiency, autoimmunity, IVIG, abatacept 6 lymphoproliferative syndrome √ √ P28 F 14 IKZF1 – 5 B – – – Agammaglobulinemia, neurological IVIG delay √ √ √ P29 M 9 13 Mb del cr.6 0.0 B, V – – – Neutrophilic dermatosis, oral and Etanercept genital aphthae, growth delay P30 M 6 BTK – – 4 – B – – – Pneumonia, IVIG hypogammaglobulinemia, absence of B cells Clinical Exome Sequencing in Primary Immunodeficiencies √ P31 M 25 Gorham- – – 21 B – – Joints Osteopenia, chylothorax, - Staut lymphopenia, S.Aureus bacteremia, disease septic arthritis n.a., not available; –, no; V, virus; B, bacteria; F, fungi; Myc, mycobacteria; ALPS, autoimmune lymphoproliferative syndrome; CID, combined immunodeficiency; CGD, chronic granulomatous disease; CMC, chronic mucocutaneous candidiasis; CVID, common variable immunodeficiency; EBV, Epstein-Barr virus; fHLH, familial hemophagocytic lymphohistiocytosis; GLILD, granulomatous and lymphocytic interstitial lung disease; HSCT, hematopoietic stem cell October 2019 | Volume 10 | Article 2325 √ transplant; IVIG, intravenous immunoglobulin; VZV, varicella-zoster virus; XLP, X-linked lymphoproliferation. , The patient presents this feature. Frontiers in Immunology | www.frontiersin.org Rudilla et al. TABLE 3 | Laboratory data of patients with relevant genetic findings. ID Sex Age at Gene Hypogammaglobulinemia Neutropenia Lymphopenia Thrombocytopenia Immunophenotype Functional tests study (y) PATIENTS WITH MUTATIONS IN PID GENES (2017 IUIS CLASSIFICATION) P1 M 15 PIK3R1 – – – – Inverse CD4/CD8. No switched Low proliferation with anti-CD3, memory B cells altered in vivo response to Haemophilus influenzae √ √ √ √ P2 F 57 TNFRSF13B Low CD4 naïve T cells, low Tregs, n.a. low pre-switched and switched B cells √ √ √ √ P3 F 8 TNFRSF13B Low CD4 naïve T cells, low Normal proliferation, cytotoxicity pre-switched and switched B cells and degranulation P4 M 18 IKBKG – – – – Increased DN gamma delta T cells Normal respiratory burst test and (22%) cytotoxicity/degranulation assays P5 F 10 STAT3 – – – – Low Tregs, low Th17 Very low proliferation with anti-CD3 P6 M 2 XIAP – – – – n.a. n.a. P7 M 1 G6PD – – – – Inverse CD4/CD8 ratio. Increased Very low proliferation with effector HLA-DR+ CD8+ T cells, anti-CD3 and ConA, very low low switched B cells IFNγ and IL12 production P8 M 30 STAT1 – – – – Myeloid DC >>> plasmacytoid Negative in vivo response to DC Candidin. 7 P9 F 4 STAT1 – – – – Low CD4 naïve T cells, low Th17, Low proliferation with PHA low pre-switched B cells P10 M 6 STAT1 – – – – 3.4% DN TCR αβ T cells, increased Low IL12 and IFNγ production Th1, low Th17, low Tregs P11 F 12 PLCG2 – – – – T+ B- NK+ Normal proliferation, normal respiratory burst test √ P12 M 15 ADA – – – T+ B- NK- Normal respiratory burst test PATIENTS WITH MUTATIONS IN GENES NOT INCLUDED IN THE PID CLASSIFICATION Clinical Exome Sequencing in Primary Immunodeficiencies √ P13 M 5 SKIV2L – – – Normal Low proliferation with PHA (normal to anti-CD3 and ConA), normal respiratory burst test √ √ P14 M 0.25 MMACHC – – n.a. Absent degranulation and cytotoxicity October 2019 | Volume 10 | Article 2325 P15 F 44 SLC27A4 – – – – Normal Normal P16 F 0.3 DSG1 – – – – Very low effector and memory T n.a. cells, low Th2, low Th1/Th17 P17 F 1 DNAI2 – – – – Normal Low proliferation with PWM and PHA √ P18 M 38 SIX6 – – – Low switched memory B cells Low proliferation with anti-CD3 √ √ √ P19 M 5 RECQL4 – Increased CD4/CD8 ration Low proliferation with PWM, anti-CD3, and ConA. (Continued) Frontiers in Immunology | www.frontiersin.org Rudilla et al. TABLE 3 | Continued ID Sex Age at Gene Hypogammaglobulinemia Neutropenia Lymphopenia Thrombocytopenia Immunophenotype Functional tests study (y) PATIENTS WITH INCOMPLETE GENOTYPES/VARIANTS OF UNCERTAIN SIGNIFICANCE (VUS) √ √ P20 M 11 UNC13D – – Normal Alternate low/normal degranulation and cytotoxicity √ √ P21 M 13 RAG2 – – Low T cells, low NK cells. Normal proliferation, normal respiratory burst test √ P22 M 4 PLCG2 – – – Normal n.a. P23 F 11 TRAF3 – – – – Normal Absent IL-12 production, normal IFNγ production P24 M 2.0 NOD2 – – – – Normal Normal proliferation, normal respiratory burst test PATIENTS WITH GENETIC FINDINGS POST-CLINICAL EXOME √ √ √ P25 F 8 LRBA – T+ B- NK- Low/absent degranulation and cytotoxicity P26 F 11 LRBA – – – – 4.2% DN TCR αβ T cells, low Low CD69 and CD40L expression effector and memory T cells, low Tregs √ P27 F 7 LRBA – – – Low pre-switched and switched B Low pneumococcal response cells √ P28 F 14 IKZF1 – – – T+ B- NK+, low pre-switched and Negative ASLO 8 switched B cells P29 M 9 13 Mb del – – – – Normal Low proliferation with anti-CD3, cr.6 normal TNFα production in response to LPS, normal degranulation and cytotoxicity √ P30 M 6 BTK – – – T+ B- NK+ n.a. √ P31 M 25 Gorham- – – – Low CD4 T cells, increased NK Severe defect in IL12 production, Staut cells altered respiratory burst test Clinical Exome Sequencing in Primary Immunodeficiencies disease √ n.a., not available; -, no; ASLO, anti-streptolysin O; ConA, concanavalin A; DC, dendritic cell; DN, double negative; PHA, phytohemagglutinin; PMA, phorbol 12-myristate 13-acetate; PWM, pokeweed mitogen. , The patient presents this feature. October 2019 | Volume 10 | Article 2325 Frontiers in Immunology | www.frontiersin.org Rudilla et al. TABLE 4 | Relevant genetic findings in patients included in this study. Patient Gene Chr OMIM cDNA Protein Consequence Zygosity Inheritance dbSNP ID ExAc PolyPhen2 SIFT Previous report MAF of variant MUTATIONS IN PID GENES (2017 IUIS CLASSIFICATION) P1 PIK3R1 5 616005 c.1425+1G>A Splicing defect Heterozygous AD rs587777709 (22, 23) P2 TNFRSF13B 17 240500 c.310T>C p.Cys104Arg Missense Heterozygous AR, AD rs34557412 0.003212 Deleterious (0) probably damaging (1) (24, 25) P3 TNFRSF13B 17 240500 c.260T>A p.Ile87Asn Missense Heterozygous AR, AD rs72553877 0.000461 Deleterious (0) probably_damaging (0.924) (26, 27) P4 IKBKG X 300291 c.169G>A p.Glu57Lys Missense Hemizygous X-linked rs148695964 0.001190 Deleterious probably_damaging (0.995) (28, 29) (0.01) P5 STAT3 17 147060 c.1144C>T p.Arg382Trp Missense Heterozygous AD rs113994135 Deleterious (0) probably_damaging (0.999) (30, 31) P6 XIAP X 300635 c.888_892del p.Lys299LeufsX9 Frameshift Hemizygous X-linked Novel P7 G6PD X 300908 c.934G>C p.Asp312His Missense Hemizygous X-linked rs137852318 0.000840 Deleterious (0) probably_damaging (0.911) (32) P8 STAT1 2 614162 c.1060C>G p.Leu354Val Missense Heterozygous AD Tolerated (0.19) benign (0.363) Novel P9 STAT1 2 614162 c.1030A>G p.Lys344Glu Missense Heterozygous AD Deleterious (0) probably_damaging (0.977) Novel P10 STAT1 2 614162 c.397A>G p.Thr133Ala Missense Heterozygous AD rs1397948697 Tolerated (0.8) benign (0.002) Novel P11 PLCG2 16 614878 c.2534_2545del p.Leu845_Leu848del In frame Heterozygous AD Novel deletion P12 ADA 20 102700 c.320T>C p.Leu107Pro Missense Compound Het AR rs121908739 0.000074 Deleterious (0) probably_damaging (0.999) (33) P12 ADA 20 102700 c.1A>G p.Met1Val Start loss Compound Het AR rs1363043396 Novel MUTATIONS IN GENES NOT INCLUDED IN THE PID CLASSIFICATION (UNEXPECTED FINDINGS) 9 P13 SKIV2L 6 614602 c.2203-1G>A Splicing defect Compound Het AR rs144714933 0.000008 (34) P13 SKIV2L 6 614602 c.3187C>T p.Arg1063X Stop gained Compound Het AR rs138923214 0.000129 (35, 36) P14 MMACHC 1 277400 c.271dupA p.Arg91LysfsX14 Frameshift Homozygous AR (37) P15 SLC27A4 9 608649 c.1320_1321insCp.Gly442ArgfsX2 Frameshift Homozygous AR Novel P16 DSG1 18 615508 c.1A>G p.Met1Val Start loss Compound Het AR rs1388337318 Novel P16 DSG1 18 615508 c.2569C>T p.Arg857X Non-sense Compound Het AR rs756245217 Novel P17 DNAI2 17 612444 c.546C>A p.Tyr182X Non-sense Homozygous AR rs770946088 Novel Clinical Exome Sequencing in Primary Immunodeficiencies P18 SIX6 14 212550 c.86G>C p.Arg29Pro Missense Homozygous AR Deleterious Benign (0.387) Novel (0.02) P19 RECQL4 8 268400 c.2789_2812del p.His930_Leu937del In frame Homozygous AR Novel deletion October 2019 | Volume 10 | Article 2325 INCOMPLETE GENOTYPES/VARIANTS OF UNCERTAIN SIGNIFICANCE (VUS) P20 UNC13D 17 608898 c.3224G>A p.Arg1075Gln Missense Heterozygous AR rs377594755 0.000197 Deleterious Possibly_damaging (0.892) Novel (0.01) P21 RAG2 11 603554 c.1324G>A p.Ala442Thr Missense Heterozygous AR rs763513886 0.000008 Deleterious (0) Probably_damaging (0.998) Novel P22 PLCG2 16 614878 c.2393A>G p.Asn798Ser Missense Heterozygous AD rs117077093 0.000676 Deleterious Probably_damaging (0.998) Novel (0.05) P23 TRAF3 14 614849 c.718G>A p.Val240Ile Missense Heterozygous AD rs200118821 0.000082 Tolerated (0.15) Benign (0.01) Novel P24 NOD2 16 266600 c.2753C>A p.Ala918Asp Missense Heterozygous AR, AD rs104895452 0.000395 Deleterious (0) Probably_damaging (0.997) (38) AD, autosomal dominant; AR, autosomal recessive; Chr, chromosome; IUIS, international union of immunological societies; MAF, minor allele frequency; SIFT, sorting intolerant from tolerant. Rudilla et al. Clinical Exome Sequencing in Primary Immunodeficiencies hemolytic anemia (43). To date, nearly 200 G6PD mutations have 2 different syndromes: PLAID (phospholipase Cγ2-associated been identified (44, 45), all of them causing a more or less marked antibody deficiency and immune dysregulation) and APLAID G6PD deficiency, but preserving some residual G6PD activity in (autoinflammation and phospholipase Cγ2-associated antibody red cells (complete absence of G6PD activity would be lethal) deficiency and immune dysregulation) (52, 53). Both syndromes (43). In the original descriptions, each G6PD variant was assigned are autoinflammatory-related disorders that typically show to a class defined on the basis of residual enzymatic activity and sinopulmonary infections caused by a humoral defect. The clinical manifestations. The p.Asp312His mutation, also known main difference between them is that APLAID patients do as the Seattle-like variant, is a class III variant in which enzymatic not have cold-induced urticaria, as occurs in patients with activity is about 15% of normal (activity associated with class PLAID syndrome (54). The 2 phenotypes seem to be related III ranges from 10 to 60% of normal activity) (46). Our patient to the type of mutation: PLAID is a consequence of large did not have anemia or other cytopenias, although he showed genomic PLCG2 deletions (52), and the only 2 families reported reduced G6PD activity in red cells. Therefore, he was currently with APLAID carried missense mutations (S707Y and L848P) asymptomatic in terms of hemolytic anemia, which is not (53, 55). However, since very few families with PLAID or surprising as class III variants are considered to have incomplete APLAID have been described so far, this distinction must be penetrance (47). Furthermore, although severe G6PD deficiency considered with caution. In fact, P11 had a clinical phenotype can be a phenocopy of chronic granulomatous disease (48), that compatible with APLAID syndrome, whereas the mutation would not be the case of the p.Asp312His mutation associated was a small in-frame deletion, a type of mutation that has not with a mild form of G6PD deficiency. As was mentioned, our yet been reported in APLAID. Immunological and functional patient had a normal respiratory burst test and G6PD activity studies are being conducted to investigate the pathogenicity in granulocytes was within the normal range. In summary, this of the PLCG2 p.Leu845_Leu848del mutation and determine patient has a G6PD mutation that does not fully explain the the functional abnormalities related with activation of the clinical phenotype, and he remains under study. NLRP3 inflammasome. In patients P8, P9 and P10, all showing a predominant clinical P12 is a 15-year old boy who presented with Hodgkin phenotype of chronic fungal infections since early ages, we lymphoma and hypogammaglobulinemia (with low B and NK found heterozygous mutations in STAT1. Heterozygous gain- cell counts) at the age of 8 years. He experienced recurrent of-function (GOF) mutations in STAT1 are the most common bacterial respiratory tract infections, including pneumonia. At genetic cause of chronic mucocutaneous candidiasis (CMC) the time of the Hodgkin lymphoma diagnosis, concomitant EBV (49, 50). Almost all reported patients present with fungal infection was detected. This led us to perform direct sequencing infections (mainly candida), but also with a wide range of of the SH2D1A (SAP), XIAP, and PIK3R1 genes, with negative other clinical features, including bacterial infections primarily results. CES revealed a compound heterozygous mutation in the affecting the respiratory tract (51). The STAT1 mutations in ADA gene, consisting of a previously reported missense mutation P8 (c.1060C>G/p.Leu354Val) and P9 (c.1030A>G/p.Lys344Glu) (c.320T>C/p.Leu107Pro) and a novel mutation affecting the were novel and had occurred de novo in the patients. Both start codon (c.1A>G/p.Met1Val). It has been demonstrated mutations are located in the DNA-binding domain of the that p.Leu107Pro is a loss-of-function mutation that retains protein, which, together with the coiled-coil domain (CCD), <0.01% of wild-type activity (56). Complete ADA deficiency are the 2 main STAT1 domains where GOF mutations are is a well-known cause of severe combined immunodeficiency located (49, 51). A mutation affecting the same residue of (SCID) but, clearly, our patient did not have a SCID phenotype. STAT1 (p.Leu354Met) as in P8 has been described in a Hypomorphic mutations in the ADA gene can lead to a different patient with CMC (51). Patient P10 also showed a novel immunodeficiency with a variable phenotype including signs STAT1 mutation (c.397A>G/p.Thr133Ala) inherited from his of immune dysregulation (56, 57). As our patient fit in the asymptomatic mother. The Thr133Ala variant is located outside category of late-onset ADA deficiency, we hypothesize that the of, but very close to the CCD, which spans residues 136 to 317. novel p.Met1Val is a hypomorphic mutation allowing some ADA The patient has 2 siblings who are also carriers of the mutation activity and thereby, rescuing our patient from a more severe and who have also had CMC episodes since an early age. The phenotype (the functional effect of the p.Met1Val mutation is CMC episodes of all 3 affected children resolved with the oral under study). antifungal fluconazole, and all of them remain symptom-free to date. P11 is a 12-year old girl with very early-onset severe skin Patients With Mutations in Genes Not and eye inflammation and recurrent respiratory infections Included in the PID Classification: (due to profound hypogammaglobulinemia), leading to the Unexpected Findings development of multiple bronchiectasis. CES revealed a After the first step in the analytical strategy, focused on PID novel, de novo heterozygous mutation in PLCG2, consisting genes included in the IUIS classification, all patients with of a 12-bp in-frame deletion in exon 24, leading to the loss negative findings underwent a second analysis including the of 4 amino acids (c.2534_2545del/p.Leu845_Leu848del). remaining genes of the clinical exome. Seven additional patients The deletion was located in the protein region encoding were diagnosed in this second analysis. As all patients were autoinhibitory domains. Heterozygous GOF mutations affecting included in this study based on a suspicion of PID, most of the the autoinhibitory domains of PLCG2 have been associated with following findings were unexpected and interesting to consider Frontiers in Immunology | www.frontiersin.org 10 October 2019 | Volume 10 | Article 2325 Rudilla et al. Clinical Exome Sequencing in Primary Immunodeficiencies in the differential diagnosis of suspected PID. The clinical and he was referred to our center at the age of 1 month laboratory data of all patients described are summarized in with a clinical phenotype of pancytopenia, bone marrow Tables 3, 4. hemophagocytosis, and increased ferritin levels, familial HLH and X-linked lymphoproliferative syndrome (XLP-1, XLP-2) SKIV2L—Trichohepatoenteric Syndrome 2 (OMIM were both suspected. The patient showed no cytotoxicity or #614602) degranulation, and perforin expression was normal on flow P13 is a 5-year-old boy who presented with acute gastroenteritis, cytometry. Following CES, we carefully analyzed genes related Enterococcus faecalis bacteremia, and dehydration at the age with HLH and XLP (PRF1, UNC13D, STX11, STXBP2, SH2D1A, of 8 months. Thereafter, he had episodes of viral disease XIAP), but no mutations were found. Other genes in the that severely affected his enteropathy. He also experienced virtual PID panel were also normal. Finally, in the overall CES catheter-associated bacteremia caused by Staphylococcus analysis, a homozygous insertion was detected in one nucleotide epidermidis. The patient had short stature and psychomotor of the MMACHC gene (c.271dupA), leading to a truncated delay. Immunological laboratory tests disclosed profound protein (p.Arg91LysfsX14). The non-consanguineous parents hypogammaglobulinemia, absence of a vaccine response, were heterozygous carriers. Biallelic mutations in MMACHC and low lymphocyte proliferation after PHA stimulation. cause a disease referred to as methylmalonic aciduria and Based on these findings, IVIG treatment was initiated. The homocystinuria, which is a genetically heterogeneous disorder immunodysregulation polyendocrinopathy enteropathy X- of cobalamin (vitamin B12) metabolism (61). Two distinct linked (IPEX) syndrome was initially suspected, but direct phenotypes of MMACHC (cobalamin C) deficiency have been sequencing of FOXP3 showed no mutations. A second CES defined in terms of age of onset. Early-onset patients present analysis revealed a compound heterozygous mutation in SKIV2L, in the first year of life with non-specific systemic, neurological, consisting of one mutation affecting the intron 18 consensus and hematological abnormalities, and have a poor outcome. acceptor splice site (c.2203-1G>A) and another introducing a Late-onset patients usually show acute neurological deterioration premature stop codon in exon 26 (c.3187C>T/p.Arg1063X). after the age of 4 years, with a better outcome after treatment The parents were heterozygous carriers. Both these mutations (62, 63). Our patient clearly fit in the early-onset group, which is have been recently reported in several patients (34–36). concordant with his genotype, as c.271dupA is the most common Homozygous or compound heterozygous mutations in SKIV2L related mutation in Europeans and is associated with severe lead to type-2 trichohepatoenteric syndrome (THES2), first early-onset disease (37, 64). described in 2012 as a rare congenital bowel disorder mainly Although uncommon, HLH can be the initial presentation of characterized by intractable diarrhea (58). Fewer than 40 patients MMACHC deficiency. Wu and collaborators reported on a 4- have been reported so far with this condition [excellently month-old patient initially diagnosed with HLH, who was later reviewed in Bourgeois et al. (36)], which is characterized by diagnosed with cobalamin C disease (cblC) (65). Therefore, early- 9 main clinical signs: intractable diarrhea, hair abnormalities, onset cblC should be considered in the differential diagnosis of a facial dysmorphism, intrauterine growth restriction (IUGR), patient with a clinical presentation of very early-onset infantile immunodeficiency, skin abnormalities, liver disease, congenital HLH. Our patient ultimately died despite the start of intravenous heart defects, and platelet anomalies. Accordingly, our patient of vitamin B12 treatment. Tandem mass spectrometry-based presented a similar phenotype with low stature, neonatal newborn screening should include the detection of congenital enteropathy, absence of a vaccination response, intellectual cobalamin defects, including MMACHC deficiency, to allow the disability, and extraordinarily brittle hair. early diagnosis prior to the onset of symptoms. Generically, trichohepatoenteric syndrome can be caused by mutations in either SKIV2L (THES2) or TTC37 (THES1), SLC27A4—Ichthyosis Prematurity Syndrome (OMIM with very similar clinical manifestations (although patients with #608649) SKIV2L mutations seem more severely affected than those with P15 is a 44-year-old woman from a consanguineous family. TTC37). Whereas, TTC37 deficiency is included in the IUIS She was referred to the Dermatology Department due to classification of PIDs in the category of predominantly antibody excessive skin inflammation since infancy. Dermatological deficiencies (59), SKIV2L deficiency is not present. We propose findings lead to a suspicion of Netherton syndrome, an autosomal that SKIVL2 deficiency be included in the next IUIS classification. recessive disease caused by mutations in the SPINK5 gene The above-mentioned features and the defects in the B and and characterized by congenital ichthyosis, bamboo hair, and T cell compartments seen in our patient and other reported atopic diathesis. Although mainly a skin disorder, Netherton THE patients (60), indicate that both TTC37 and SKIV2L syndrome was defined as a PID in 2009, when the group of deficiency should be included in the category of combined Professor H. Ochs evaluated the immune system in a cohort of immunodeficiencies with associated or syndromic features. Netherton patients and found that almost all had reductions in memory B cell counts, a defective vaccine response, increased MMACHC—Methylmalonic Aciduria and inflammatory cytokines, and low NK cytotoxicity (66). The initial Homocystinuria (OMIM #277400) CES analysis focused on the SPINK5 gene, which was found to P14 had a family history of an older brother who died at be completely normal. The differential diagnosis of Netherton the age of 4 months with a diagnosis of hemophagocytic syndrome includes genes causing inherited ichthyoses, PIDs lymphohistiocytosis (HLH) and pancytopenia. Hence, when with hyper IgE, atopic dermatitis, and seborrheic dermatitis, Frontiers in Immunology | www.frontiersin.org 11 October 2019 | Volume 10 | Article 2325 Rudilla et al. Clinical Exome Sequencing in Primary Immunodeficiencies among other conditions (67, 68). The virtual PID panel carriers. SAM syndrome closely resembles Netherton syndrome, analysis was negative, but analysis of the other related genes including congenital ichthyosis, erythroderma, and severe atopic identified a homozygous truncating mutation in the SLC27A4 dermatitis (73). As occurred in our patient and is reported in gene (c.1320_1321insC/p.Gly442ArgfsX2). Biallelic mutations in Netherton syndrome, elevated serum IgE and absolute eosinophil SLC27A4 (mainly known as FATP4) cause ichthyosis prematurity counts are found in DSG1-deficient patients (74–76). Other syndrome (IPS), an autosomal recessive disorder characterized laboratory immune defects have not been described to date. It by premature birth and neonatal respiratory complications, was recently demonstrated that DSG1 inhibits skin inflammation followed by lifelong ichthyosis with atopic manifestations (69). by inhibiting the NF-kB signaling pathway (77). Consequently, In patients with autosomal recessive congenital ichthyosis, IPS is DSG1 deficiency is linked to inflammation and points to a crucial a rare condition, occurring in <4% of cases (70). Although they link between loss of epithelial barrier integrity and immunologic are very similar conditions, there are some differences between dysregulation (77). Netherton syndrome and IPS, mainly related with the skin manifestations. Relevant to immunity, Netherton syndrome may involve recurrent infections whereas IPS does not. This and the DNAI2—Primary Ciliary Dyskinesia (OMIM #612444) immune defects initially reported by Professor H. Ochs indicate P17 is a 1-year old girl born of consanguineous parents. She had a that immunological laboratory tests are important to confirm or sister who died in the first year of life due to an unspecified heart rule out suspected Netherton syndrome. disease, and a 9-year-old brother with asthma, growth delay, and multiple respiratory infections. At the age of 1 month, the DSG1—Erythroderma, Congenital, With Palmoplantar patient was referred to our hospital due to malignant pertussis. Keratoderma, Hypotrichosis, and Hyper IgE (OMIM During the following 3 months she experienced 4 episodes of #615508) acute bronchitis, requiring 3 hospitalizations. At the age of 6 P16 is a female born of non-consanguineous Spanish parents months she was admitted again to our hospital to study failure who, since the first 48 h of life, presented with erythrodermic to thrive. At 18 months of age, she developed biphenotypic skin lesions co-occurring with bacterial infections, mainly skin acute leukemia (i.e., acute leukemia with a single population infections due to Staphylococcus aureus and sepsis caused of blasts co-expressing markers of 2 different lineages), which by S. epidermidis. The lesions increased in extension and was treated with allogeneic cord blood hematopoietic stem predominantly affected the distal limbs and the perioral and cell transplantation. PID was suspected based on the family periocular regions, causing uncontrollable itching. Laboratory history, recurrent respiratory tract infections, and malignancy. findings showed marked eosinophilia, reaching a value of No pathogenic variants were found in the virtual PID panel, but 18% and 3.3 × 109 cells/L (normal range: 6–6.5%; 0–0.7 analysis of the other genes identified a homozygous non-sense × 109 /L). Immunoglobulin levels were normal except for mutation in the DNAI2 gene (c.546C>A/p.Tyr182X). This gene increased IgE (>2,000 KU/L). Immunophenotyping showed codes for the dynein axonemal intermediate chain 2 (DNAI2), an abnormal T cell phenotype, with a predominance of a protein belonging to the dynein intermediate chain family. memory/effector T cells. All these findings led to a differential DNAI2 is highly expressed in trachea and testis and is involved diagnosis that initially included hyper IgE syndrome (TYK2, in the dynein regulatory complex of respiratory cilia and sperm STAT3, DOCK8), Netherton syndrome (SPINK5), and Omenn flagella motility (78). Biallelic mutations in DNAI2 have been syndrome (RAG1, RAG2). Previous Sanger sequencing of described as causing primary ciliary dyskinesia (PCD) (79). RAG1 and RAG2 showed no mutations, and the patient was PCD comprises a group of rare and genetically heterogeneous included in the CES project. The first analysis of sequencing disorders characterized by defective ciliary motility. PCD is data (virtual PID panel) showed a heterozygous missense caused by biallelic mutations in more than 30 genes (the variant in SPINK5 (c.2773G>C/p.Asp925His). This variant number is rapidly increasing) related with the structure and was not reported in the main databases (dnSNP, ExAC, function of the cilia and flagella (80). Patients with these gnomAD), and the computational predictions were discordant disorders have a history of neonatal respiratory distress and (Supplementary Table 6). The variant was classified as a VUS, later, recurrent and chronic infections of the upper and lower and no other low-frequency variants were found in SPINK5. respiratory tracts that can lead to bronchiectasis and a progressive Netherton syndrome is caused by biallelic mutations in SPINK5, decline in lung function. Other manifestations include organ and all those reported are loss-of-function mutations (non-sense, laterality defects (situs inversus), congenital heart disease, and indels, and splicing mutations) (71). Therefore, the diagnosis male infertility (81). As it can be seen, some clinical features of of Netherton syndrome was not supported by the genetic data. PCD can overlap with other conditions, such as cystic fibrosis A subsequent analysis revealed 2 heterozygous mutations in and primary immunodeficiencies. Therefore, the differential DSG1: one was a start loss mutation (c.1A>G/p.Met1Val) and diagnosis between PCD and PID may be difficult, especially the other a non-sense mutation (c.2569C>T/p.Arg857X). DSG1 in children. The non-sense homozygous mutation found in encodes desmoglein 1, a major constituent of desmosomes, DNAI2 perfectly explains the clinical phenotype of our patient. which have a crucial role in maintaining epidermal integrity Family study showed that the parents were heterozygous carriers and barrier function. Biallelic mutations in DSG1 cause SAM and the older brother was homozygous, in accordance with (severe dermatitis, multiple allergies, and metabolic wasting) his clinical symptoms (asthma, growth delay, and multiple syndrome (OMIM#615508) (72). Both mutations found in respiratory infections). It is likely that this mutation was the cause our patient were private and her parents were heterozygous of their sister’s early death due to a congenital heart disease. Frontiers in Immunology | www.frontiersin.org 12 October 2019 | Volume 10 | Article 2325 Rudilla et al. Clinical Exome Sequencing in Primary Immunodeficiencies SIX6—Optic Disc Anomalies With Retinal and/or The patient was then included in the CES study because Macular Dystrophy (OMIM #212550) of suspected combined immunodeficiency (CID). None of P18 is a 43-year old man born of consanguineous parents, the genes responsible for CID included in the panel carried who presented with a dual phenotype showing both ocular pathogenic mutations. At that time, a diagnosis of Rothmund- and immunological manifestations. The patient’s brother had Thomson syndrome (RTS) was suggested by the clinical died at the age of 7 years due to complications related to geneticists and, indeed, a homozygous mutation was found in an immunodeficiency phenotype (more specific information the RECQL4 gene. It consisted of an in-frame deletion of 24 was not available). The pedigree revealed a high degree of bp (c.2789_2812del), leading to the loss of 8 amino acids of the consanguinity in the family, and 2 cousins (a girl and a boy) had protein (p.His930_Leu937del). This mutation, along with other the same ocular phenotype. The patient had congenital cataracts, in-frame RECQL4 deletions/duplications, have been described as strabismus, and color blindness, although the most severe defect pathogenic and a cause of RTS (85). RTS is an autosomal recessive was coloboma. He reported a history of multiple infections and genodermatosis presenting in infancy with a characteristic facial had a low platelet count and IgA deficiency. Despite normal rash (poikiloderma) and other manifestations, including short numbers of T, B and NK cells, the patient’s B cell subpopulations stature, skeletal abnormalities, ocular defects, premature aging, were imbalanced, with increased numbers of naïve B cells and and a predisposition to cancer (86). Although immune defects reduced class-switch memory B cells. Immunodeficiency with are not among the classical features of this syndrome, a few RTS coloboma has been reported in CHARGE syndrome (82), but patients with immunological abnormalities have been reported. our patient did not show most of the typical manifestations of In the 1990s, 2 RTS patients with humoral immune deficiencies this syndrome (OMIM#214800). aCGH showed no abnormalities (involving hypogammaglobulinemia) were described (87, 88). and the patient was included in the CES study. Results of the In 2006, Broom et al. described a patient with CID and a virtual PID panel analysis were negative, but in the subsequent classic RTS phenotype who experienced successful immune analysis, a rare homozygous variant was identified in the reconstitution following umbilical cord blood transplantation SIX6 gene (c.86G>C/p.Arg29Pro). This variant had not been (89). Later, in 2010, De Somer at al presented a patient with previously described in general population. Biallelic mutations RTS and immune deficiency who developed granulomatous skin in the SIX6 gene cause optic disc anomalies with retinal or lesions after primary varicella-zoster virus infection (90). In a macular dystrophy (OMIM #212550), an autosomal recessive more recent study, Smeets et al. shed light on the question of condition characterized by iris coloboma, decreased or absent why mutations in RECQL4 can lead to these immunological vision, retinal dystrophy, and a colobomatous optic disc, among abnormalities. These authors found that Recql4 loss causes rapid other manifestations (83, 84). Segregation studies in the family bone marrow failure in mice, leading to profound disruption showed that both parents were heterozygous carriers of the of immune development, including impaired B and T cell p.Arg29Pro mutation. However, functional studies should be development (91). performed to confirm the pathogenicity of this variant. Since the Collectively, these data suggest a role of the DNA helicase defects reported in patients with SIX6 deficiency are restricted RECQL4 in the development of the immune system, and indicate to ocular anomalies and 2 relatives have the ocular phenotype that screening for immune deficiency should be considered in but not the immunodeficiency, we conclude that our patient’s patients with RTS. immunological changes were caused by other genetic factors, monogenic, oligogenic, or involving other more complex models (as has been proposed in CVID and in selective IgA deficiency). Incomplete Genotypes and Variants of Uncertain Significance RECQL4—Rothmund-Thomson Syndrome (OMIM An inherent feature of NGS is generation of large amounts of data #268400) and identification of a myriad of genetic variants. The clinical P19 is a 5-year old boy from a consanguineous Pakistani significance of a variable fraction of these variants may be difficult family. At the age of 6 months, he experienced severe to ascertain based on current knowledge; thus, they are referred diarrhea that lasted for 1 month. He had growth delay and to as variants of uncertain/unknown significance (VUS). a polymalformative syndrome with skeletal malformations and In all patients undergoing CES in this study and excluding the poikiloderma. He developed systemic cytomegalovirus infection causal mutations described above (Table 4), we found a varying affecting the lung, and invasive aspergillosis that was difficult number of low-frequency (MAF>0.01) variants in the virtual to control. Laboratory data showed hypogammaglobulinemia, PID panel, considered not to be a cause of the clinical phenotype lymphopenia, and thrombocytopenia. The CD4/CD8 ratio was (Supplementary Table 6, which does not include variants in increased, with a predominance of memory cells within the CD8 Table 4). Most of these variants are rare polymorphisms with compartment (59% of CD8+ CD45RO+ T cells). The in vitro no clear functional impact, but others are monoallelic loss-of- lymphoproliferation capacity was impaired after stimulation function variants in recessive genes that are clearly unrelated to with pokeweed mitogen, anti-CD3 (also supplemented with IL- the patients’ disease. Nonetheless, a potential role of some of 2), or PHA. However, lymphoproliferation was normal with these variants as modulators of the clinical phenotype cannot PMA-ionomycin, thus indicating an upstream signaling defect. be excluded. Based on the patient’s syndromic features, the first genetic A few of these variants, located in genes related to the patients’ test was aCGH, which showed no significant abnormalities. phenotypes, drew our attention (Table 4). Frontiers in Immunology | www.frontiersin.org 13 October 2019 | Volume 10 | Article 2325 Rudilla et al. Clinical Exome Sequencing in Primary Immunodeficiencies A heterozygous missense variant in UNC13D (p.Arg1075Gln) analyzed by other methods, and some of them ultimately was identified in P20, who presented with pancytopenia, received a genetic diagnosis. In P25, P26, and P27, clinically hepatosplenomegaly, hemophagocytosis, and subcutaneous diagnosed as having CVID, biallelic mutations were identified panniculitis-like T cell lymphoma at the age of 8 years. Elevated in the LRBA gene. In 2012, LRBA deficiency was described HLH biomarkers and absent cytotoxicity and degranulation as a monogenic cause of CVID associated with autoimmunity supported the diagnosis of fHLH. However, the functional tests (100). LRBA is not included in the TruSight One Sequencing normalized after the episode. The role of monoallelic variants in Panel, likely because it was discovered as a cause of CVID HLH is currently a topic of great interest and debate (92). We after the panel was designed (Illumina announced launch of also sequenced UNC13D intron 1 by Sanger because pathogenic the TSO panel in October 2013). This is one of the main mutations have been reported in several HLH patients with limitations of CES: it can lack relevant genes described after monoallelic mutations in the coding regions (93–95), but it was designed. Samples from P26 and P27 were included in no relevant variants were found. The role of the UNC13D a targeted panel of CVID genes and P25 underwent WES. Of p.Arg1075Gln variant is under study. note, in P25, we reported the first case of LRBA deficiency due P21 is a male with failure to thrive and a family to uniparental disomy (101). A similar situation occurred in history of a sister who died at the age of 6 months due P28, clinically diagnosed as having CVID, but later tested with to unknown reasons. The patient had a clinical phenotype a more specific PID panel, which detected a heterozygous loss- of multiple lymphadenopathies, intermittent abdominal pain, of-function mutation in IKZF1. Monoallelic mutations in IKZF1 persistent fever, and granulomatous hepatitis. Analyses showed (encoding IKAROS) were described as a monogenic cause of lymphopenia and low platelet counts. We found a heterozygous CVID in 2016 (102), but earlier, in 2012, a single patient with missense variant at very low frequency in the RAG2 gene IKZF1 mutation was reported to have congenital pancytopenia (p.Ala442Thr), classified as likely pathogenic following the (103). This gene is also absent from the TSO. P29 is a ACMG rules (96). The clinical phenotype of the patient fits well patient with autoinflammatory manifestations, and additionally, with those observed in some patients with biallelic RAG1 or psychomotor and growth delay. Following CES, we performed RAG2 mutations (97), but in this case we found only 1 mutation. aCGH, which revealed a deletion of 13Mb on chromosome 6 P22 presented with recurrent fevers seriously affecting his including the TNFAIP3 gene. TNFAIP3 haploinsufficiency causes quality of life, with no response to colchicine or IL-1 inhibitors. an autoinflammatory syndrome (Behcet-like) (104), which in our Since the age of 6 years, he has also experienced episodes of patient occurred together with other developmental impairments sudden loss of muscle tone and consciousness. We identified a due to this large deletion (105). CES does not perform well for heterozygous missense variant in the PLCG2 gene (p.Asn798Ser), detecting large genomic rearrangements and, indeed, this defect predicted to be pathogenic by SIFT and PolyPhen2. The was not detected in the CES data analysis. P30, with a strong prevalence of this variant population is low (but not very low), clinical suspicion of X-linked agammaglobulinemia (XLA), was and it was inherited from his mother. included in CES because we found no mutations in BTK by In P23, a girl with herpes simplex encephalitis, we found a low- direct sequencing. CES was also negative. Since suspicion of XLA frequency heterozygous missense variant in TRAF3 (p.Val240Ile). persisted, we performed western blot analysis of the BTK protein, TRAF3 deficiency (autosomal dominant) has been associated which revealed an absence of BTK. Finally, a large deletion with a clinical phenotype limited to herpes simplex encephalitis affecting the BTK 5′ UTR was identified. As CES is focused resulting from impairment of the TLR3 response. Only 1 patient on the coding regions, it was unable to detect this deletion. with this condition has been reported (98). Our variant shows This limitation is inherent to all the approaches based on a low frequency in the general population, SIFT and PolyPhen2 sequencing only the coding regions (usually targeted gene panels classified it as benign, and it was inherited from the patient’s and WES) and can only be overcome by using WGS (or other mother. DNA-sensing pathway responsiveness is under study to specific techniques to detect CNVs). Finally P31 presented with evaluate the functional impact of this TRAF3 variant. monostotic fibrous dysplasia of the lumbar spine and episodes P24 presented with early-onset inflammatory bowel disease at of bacterial infections with a complicated course. After CES had the age of 7 months, together with hypogammaglobulinemia. We yielded negative results, we performed aCGH, which provided identified a low-frequency missense variant in the NOD2 gene no relevant findings. Ultimately, the patient was diagnosed with (p.Ala918Asp). NOD2 variants have been repeatedly associated Gorham-Stout Disease (GSD), a rare disorder characterized by with Crohn’s disease (99), and p.Ala918Asp is among the ones angiogenesis, lymphangiomatosis, and severe bone resorption most strongly associated (38). It seems clear that this variant is (106). The exact cause of GSD is unknown and no environmental, not the cause of the full clinical phenotype of P24, but it may play immunological or genetic risk factors have been identified. a role in the patient’s gastrointestinal manifestations. Bone loss in GSD is accompanied by uncontrolled growth (proliferation) of lymphatic tissue. Lymphatic abnormalities Genetic Findings Post-clinical Exome in could be related to the recurrent infections in P31. Patients With Negative Results Typically, patients with negative results on genetic testing CONCLUDING REMARKS remain as “genetically undiagnosed,” and if suspicion of a genetic defect persists, they are usually included in other In this study, CES was used to analyze samples from 61 genetic testing approaches over time. During the 3 years of patients with clinically suspected PID. Aside from detection this study, most patients with negative CES results have been of mutations in genes typically causing PID, CES enabled Frontiers in Immunology | www.frontiersin.org 14 October 2019 | Volume 10 | Article 2325 Rudilla et al. Clinical Exome Sequencing in Primary Immunodeficiencies molecular identification of defects in genes that are not present PIDs. The cases described illustrate the heterogeneity and in the IUIS classification or in specific PID panels, as well complexity encountered by professionals involved in the clinical as identification of unsuspected molecular etiologies. These management and genetic diagnosis of these disorders. findings indicate that some cases of clinically suspected PID may ultimately not correspond to a “genuine” PID, as a number DATA AVAILABILITY STATEMENT of rare diseases include immune-related symptoms in their typical or atypical presentations. Furthermore, they illustrate All relevant data from datasets generated for this study are one of the advantages of CES over the use of more specific included in the manuscript/Supplementary Files. PID panels. In contrast, the main limitation of CES for genetic diagnosis of PIDs is the lack of a considerable number of ETHICS STATEMENT PID-causing genes. There is a simple reason for this: most of the absent genes were discovered after the clinical exome The studies involving human participants were reviewed and panel was designed. Typically, clinical exome panels provided by approved by Ethics Review Board of Hospital Universitari companies are not easily customized. In contrast, specific tailor- Vall d’Hebron. Written informed consent was obtained from made PID panels can be periodically updated and may be more the patients or their legal representatives, according to the comprehensive in terms of PID-causing genes. Nonetheless, WES procedures of the Ethics Review Board of Hospital Universitari is becoming the gold-standard technique for the diagnosis of Vall d’Hebron. genetic diseases, and it is replacing CES as an unbiased approach covering all coding regions of the genome. However, despite AUTHOR CONTRIBUTIONS continuing decreases in the cost of this technique, it remains expensive and the data analysis is difficult to manage for some FR performed the clinical exome sequencing and bioinformatics centers. Hence, CES may still be a good practical option for data analysis, and wrote a part of the manuscript. CF-J performed this purpose. the immunological analyses, collected data from all patients, and Overall, we obtained a positive genetic finding in 42% wrote a part of the manuscript. MM-G and MG-P performed of patients including CES approach and the other diagnoses immunological analyses and collected patient data. AA-C and achieved by later use of other techniques. Several reasons may LM provided technical support for sample collection and the explain the lack of genetic diagnosis in 58% of patients; the most sequencing process. FV made substantial contributions to the intuitive one is the limited number of genes included in CES. study design and provided technical support in the sequencing Probably the use of WES and WGS would increase diagnostic process. IC, CS, NB-R, and MF-C contributed to the data analysis rate but still a percentage of patients would remain undiagnosed. and interpretation of variants. AM-N, JR, VG-P, XS, II, JS-P, JT, Very recently, Yska and collaborators systematically reviewed CC, JV, RP-B, and PS-P clinicians in charge of patient care, were the diagnostic yield of NGS in genetically undiagnosed patients involved in management of the patients and collecting clinical with PIDs and they found a broad range among the different data. RC performed the genetic analysis and was responsible for studies (15–79%) (107). Several factors may explain this designing the study, writing the manuscript, and approving the high variability, including methodological differences and the final draft. All authors reviewed the manuscript and contributed selected study populations. Other biological reasons like the to the final draft. existence of complex models of inheritance (e.g., oligogenic, polygenic, epigenetic) may difficult to achieve a definitive FUNDING genetic diagnosis. The number of VUS obtained in CES, WES and especially in This study was funded by Instituto de Salud Carlos III, WGS is also a challenging aspect inherent of NGS approaches. grants PI14/00405 and PI17/00660, cofinanced by the European The ACMG guidelines suggest the management of VUS Regional Development Fund (ERDF). as follows: “A variant of uncertain significance should not be used in clinical decision making. Efforts to resolve the ACKNOWLEDGMENTS classification of the variant as pathogenic or benign should be undertaken. While this effort to reclassify the variant is We are deeply grateful to the affected individuals who underway, additional monitoring of the patient for the disorder participated in this study and their families. We thank in question may be prudent” (96). However, how to deal with the Barcelona PID Foundation for patient support and for VUS in a clinical setting is a continuous matter of debate (108, funding MG-P. We acknowledge Celine Cavallo for English 109). language support. In summary, this study describes our experience in using CES as a tool for the genetic diagnosis of PIDs and discusses SUPPLEMENTARY MATERIAL the expected and unexpected findings obtained. We believe it is of clinical interest to provide descriptions of patients The Supplementary Material for this article can be found with mutations in genes that are not included in the PID online at: https://www.frontiersin.org/articles/10.3389/fimmu. classification to aid in the differential diagnosis of some suspected 2019.02325/full#supplementary-material Frontiers in Immunology | www.frontiersin.org 15 October 2019 | Volume 10 | Article 2325 Rudilla et al. Clinical Exome Sequencing in Primary Immunodeficiencies REFERENCES 17. Adzhubei IA, Schmidt S, Peshkin L, Ramensky VE, Gerasimova A, Bork P, et al. A method and server for predicting damaging missense mutations. Nat 1. 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Clin Conflict of Interest: The authors declare that the research was conducted in the Rev Allergy Immunol. (2017) 52:125–32. doi: 10.1007/s12016-016-8594-z absence of any commercial or financial relationships that could be construed as a 107. Yska HAF, Elsink K, Kuijpers TW, Frederix GWJ, van Gijn ME, van potential conflict of interest. Montfrans JM. Diagnostic yield of next generation sequencing in genetically undiagnosed patients with primary immunodeficiencies: a systematic Copyright © 2019 Rudilla, Franco-Jarava, Martínez-Gallo, Garcia-Prat, Martín- review. J Clin Immunol. (2019) 39:577–91. doi: 10.1007/s10875-019-0 Nalda, Rivière, Aguiló-Cucurull, Mongay, Vidal, Solanich, Irastorza, Santos-Pérez, 0656-x Tercedor Sánchez, Cuscó, Serra, Baz-Redón, Fernández-Cancio, Carreras, Vagace, 108. Vears DF, Sénécal K, Borry P. Reporting practices for variants of Garcia-Patos, Pujol-Borrell, Soler-Palacín and Colobran. 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