ORIGINAL RESEARCH published: 31 January 2020 doi: 10.3389/fimmu.2020.00107 FHLdb: A Comprehensive Database on the Molecular Basis of Familial Hemophagocytic Lymphohistiocytosis Laura Viñas-Giménez 1,2† , Natàlia Padilla 3† , Laura Batlle-Masó 4,5 , Ferran Casals 4 , Jacques G. Rivière 6,2 , Mónica Martínez-Gallo 1,2 , Xavier de la Cruz 3,7 and Roger Colobran 1,2,8* 1 Immunology Division, Hospital Universitari Vall d’Hebron (HUVH), Vall d’Hebron Research Institute (VHIR), Department of Cell Biology, Physiology and Immunology, Autonomous University of Barcelona (UAB), Barcelona, Spain, 2 Jeffrey Model Foundation Excellence Center, Barcelona, Spain, 3 Research Unit in Clinical and Translational Bioinformatics, Vall d’Hebron Research Institute (VHIR), Barcelona, Spain, 4 Servei de Genòmica, Departament de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra, Parc de Recerca Biomèdica de Barcelona, Barcelona, Spain, 5 Departament de Ciències Experimentals i de la Salut, Institute of Evolutionary Biology (UPF-CSIC), Universitat Pompeu Fabra, Parc de Recerca Edited by: Biomèdica de Barcelona, Barcelona, Spain, 6 Pediatric Infectious Diseases and Immunodeficiencies Unit (UPIIP), Hospital Jordan Orange, Universitari Vall d’Hebron (HUVH), Vall d’Hebron Research Institute (VHIR), Universitat Autònoma de Barcelona (UAB), Columbia University, United States Barcelona, Spain, 7 Institut Catala per la Recerca i Estudis Avançats (ICREA), Barcelona, Spain, 8 Genetics Department, Reviewed by: Hospital Universitari Vall d’Hebron (HUVH), Barcelona, Spain Udo Zur Stadt, University Medical Center Hamburg-Eppendorf, Germany Background: Primary immunodeficiencies (PIDs) are a heterogeneous group of Samuel Cern Cher Chiang, disorders. The lack of comprehensive disease-specific mutation databases may hinder Cincinnati Children’s Hospital Medical Center, United States or delay classification of the genetic variants found in samples from these patients. This *Correspondence: is especially true for familial hemophagocytic lymphohistiocytosis (FHL), a life-threatening Roger Colobran PID classically considered an autosomal recessive condition, but with increasingly rcolobran@vhebron.net demonstrated genetic heterogeneity. † These authors have contributed Objective: The aim of this study was to build an open-access repository to collect equally to this work detailed information on the known genetic variants reported in FHL. Specialty section: Methods: We manually reviewed more than 120 articles to identify all reported variants This article was submitted to Primary Immunodeficiencies, related to FHL. We retrieved relevant information about the allelic status, the number of a section of the journal patients with the same variant, and whether functional assays were done. We stored all Frontiers in Immunology the data retrieved in a PostgreSQL database and then built a website on top of it, using Received: 17 October 2019 the Django framework. Accepted: 15 January 2020 Published: 31 January 2020 Results: The database designed (FHLdb) (https://www.biotoclin.org/FHLdb) contains Citation: comprehensive information on reported variants in the 4 genes related to FHL (PRF1, Viñas-Giménez L, Padilla N, Batlle-Masó L, Casals F, Rivière JG, UNC13D, STXBP2, STX11). It comprises 240 missense, 69 frameshift, 51 nonsense, 51 Martínez-Gallo M, de la Cruz X and splicing, 10 in-frame indel, 7 deep intronic, and 5 large rearrangement variants together Colobran R (2020) FHLdb: A with their allelic status, carrier(s) information, and functional evidence. All genetic variants Comprehensive Database on the Molecular Basis of Familial have been classified as pathogenic, likely pathogenic, uncertain significance, likely Hemophagocytic benign or benign, according to the American College of Medical Genetics guidelines. Lymphohistiocytosis. Front. Immunol. 11:107. Additionally, it integrates information from other relevant databases: clinical evidence doi: 10.3389/fimmu.2020.00107 from ClinVar and UniProt, population allele frequency from ExAC and gnomAD, and Frontiers in Immunology | www.frontiersin.org 1 January 2020 | Volume 11 | Article 107 Viñas-Giménez et al. FHLdb: Familial Hemophagocytic Lymphohistiocytosis Database pathogenicity predictions from well-recognized tools (e.g., PolyPhen-2, SIFT). Finally, a diagram depicts the location of the variant relative to the gene exon and protein domain structures. Conclusion: FHLdb includes a broad range of data on the reported genetic variants in familial HLH genes. It is a free-access and easy-to-use resource that will facilitate the interpretation of molecular results of FHL patients, and it illustrates the potential value of disease-specific databases for other PIDs. Keywords: primary immunodeficiency, hemophagocytic lymphohistiocytosis, database, genetics, mutation, genetic variant INTRODUCTION ineffective immune response that typically produces a massive proinflammatory cytokine storm resulting in fever, pancytopenia, Primary immunodeficiencies (PIDs) are a heterogeneous group hemophagocytosis, central nervous system (CNS) dysfunction, of disorders affecting the immune system. Most PIDs are and multiorgan failure (7). It most frequently affects infants at considered to have a monogenic cause, but incomplete early ages, but the disease is also observed in children and adults penetrance, variable expressivity, and interactions between of all ages. If left untreated, the disease may rapidly progress genetic and environmental factors can contribute to their and lead to death in a few weeks. Treatment for this condition phenotypic diversity. The diagnostic workup for PIDs is based includes immunosuppressive and immune-modulating therapy on the patients’ clinical manifestations and the results of complex to control the inflammation and organ damage, but in the most laboratory techniques to guide selection of the candidate gene severe cases, hematopoietic stem cell transplantation (HSCT) is or genes to be tested (1). However, because of the diverse the definite cure (8). nature of these conditions, candidate gene selection is not HLH presents in a wide spectrum of clinical contexts, usually straightforward. Hence, the development of massive including fever of unknown origin, acute liver failure, sepsis- parallel sequencing or next-generation sequencing (NGS) is like, Kawasaki-like, and neurologic abnormalities (8). Although rapidly replacing direct sequencing (Sanger method) as the first- a distinctive constellation of clinical and laboratory features choice method for genetic diagnosis of PIDs (2). Several NGS- has been described for HLH, diagnosis remains challenging based approaches, from whole-genome sequencing to specific as patients may have very different clinical manifestations PID panels, are currently in use, and they have led to major associated with a variety of triggers (9). Atypical presentations breakthroughs in the diagnosis of these disorders (3–6). involving mainly CNS or chronic pathologic inflammation Nonetheless, the huge amount of data obtained by NGS manifestations are examples of such complexity (10–12). technologies makes analysis and interpretation of the results HLH patients are often categorized as having either a cumbersome process compared with conventional genetic primary/familial or secondary/sporadic HLH. Familial HLH testing. The clinical significance of a potentially relevant variant (FHL) is caused by biallelic mutations in genes involved in the may be difficult to ascertain if there is no easily accessible granule-dependent exocytosis pathway, which lead to impaired information to consult. Review of the literature is an option, but natural killer (NK) and T-cell cytotoxic activity. To date, 4 it can be time-consuming and sometimes, exhausting. The lack genes (PRF1, UNC13D, STXBP2, and STX11) and 1 genomic of uniform criteria across studies to establish the pathogenicity of region (9q21.3–22) have been identified as candidate causes genetic variants further complicates this task. of FHL2 (13), FHL3 (14), FHL4 (15), FHL5 (16), and FHL1 Disease-specific databases, mainly known as locus-specific (17), respectively (18). Other monogenic diseases that produce databases (LSDBs), may be the best tool to help professionals HLH are Chédiak-Higashi syndrome (LYST), Griscelli syndrome involved in genetic analysis. LSDBs must be comprehensive in type 2 (RAB27A), Hermansky-Pudlak syndrome (AP3B1), X- collecting genetic variants relevant for a disease and must be linked lymphoproliferative syndrome (XLP)-1 (SH2D1A), and rigorous in providing evidence that supports the role of the XLP-2 (XIAP) (19). HLH can also develop in the absence of variants. Ideally, LSDBs should interact with other established familial recurrence and without biallelic mutations in the causal databases and computational tools to depict an overall view of genes, usually in the context of infections, malignancies, and each variant. Unfortunately, LSDBs for PIDs are scarce, often autoinflammatory or metabolic diseases. These are considered outdated, and not user-friendly. This study was developed when “secondary” or “sporadic” HLH (sHLH) (20). In recent years we realized that there was no LSDB for familial hemophagocytic and coinciding with the rapid evolution of NGS technology, lymphohistiocytosis (FHL), an interesting and complex PID, the number of studies reporting new HLH-related variants has whose genetic basis is currently under discussion (evolving from significantly increased (21, 22), and these have elucidated that classical autosomal recessive inheritance to other models). a proportion of sHLH patients with or without a functional Hemophagocytic lymphohistiocytosis (HLH) comprises a defect harbor monoallelic variants in one of the FHL-related group of rare disorders characterized by a highly stimulated but genes (23, 24). In addition, novel types of inheritance have Frontiers in Immunology | www.frontiersin.org 2 January 2020 | Volume 11 | Article 107 Viñas-Giménez et al. FHLdb: Familial Hemophagocytic Lymphohistiocytosis Database been proposed for HLH, including polygenic and dominant transmission models (25–28). A percentage of the reported HLH variants can be found in generalist databases such as ClinVar, OMIM (Online Mendelian Inheritance in Man), LOVD (Leiden Open Variation Database), and HGMD (Human Gene Mutation Database). However, these databases may be incomplete, outdated, or not manually curated. As was mentioned, there is no dedicated LSDB for FHL genes, and this may lead to delays in classifying the genetic variants found in FHL patients and, consequently, in their diagnosis and treatment. Here, we present FHLdb, a comprehensive database on the molecular basis of familial HLH. FHLdb is a web-based open- access repository of reported variants in the PRF1, UNC13D, FIGURE 1 | Distribution of genetic variants according to their effect at the STXBP2, and STX11 genes. FHLdb provides detailed information protein level. Histograms show the distribution of STX11, STXBP2, PRF1, and UNC13D variants included in FHLdb. The total number of variants reported in on each variant, including functional evidence of pathogenicity each gene is indicated within parentheses. when available, and enables links with other widely used databases (e.g., ClinVar, dbSNP, ExAC, gnomAD) to provide users with the most easily accessible and complete genetic information related to this condition. follows: 240 missense, 69 frameshift, 51 nonsense, 51 splicing, 10 in-frame indel, 7 deep intronic, and 5 large rearrangements METHODS (Figure 1). UNC13D showed the largest number of variants (189), followed by PRF1 (157), STXBP2 (66), and STX11 (21) Bibliographic Data (Figure 2). A systematic search in the medical literature retrieved more Each variant was manually curated, and we followed the than 120 related articles, which were manually reviewed to Human Genome Variation Society (HGVS) guidelines for collect all reported variants in FHL patients. Literature search variant nomenclature (http://varnomen.hgvs.org) to obtain a was mainly performed using the Medline database from the harmonized, interchangeable, clear dataset. We found that the National Library of Medicine through the PubMed search nomenclature of 29% of the variants was incomplete or did engine. PubMed is a free resource supporting the search and not follow these guidelines in their original description; hence, retrieval of peer-reviewed scientific literature. Combinations these variants were renamed (Supplementary Table 1). In this of the following keywords have been used for the search manuscript, both “mutation” and “variant” terms have been used. procedure: HLH, FHL, hemophagocytic lymphohistiocytosis, While both terms indicate a change in the nucleotide sequence, familial hemophagocytic lymphohistiocytosis, hemophagocytic in medicine “mutation” is used to indicate a sequence variant syndrome, PRF1, UNC13D, STXBP2, STX11, mutation, associated with a disease phenotype. On the other hand, the variant, genetic variant, biallelic, monoallelic, heterozygous, current guidelines of authoritative organizations recommend homozygous, etc. using neutral terms like “variant” followed by a classification term From these studies, we also collected other relevant like pathogenic, benign, etc. (see HGVS website for extended information to include in FHLdb, such as the reported status of discussion about terminology). Therefore, we preferably used the variant (biallelic and/or monoallelic), whether the variant “variant” and restricted the use of “mutation” in some specific was found in more than one patient, and whether a functional cases to indicate disease-causing variants. assay had been carried out to determine the consequences of The allelic status of each variant is reported in the following the variant. terms: biallelic, when it was reported in homozygous or compound heterozygous state, and monoallelic. Thus, a variant Database Set-Up can be reported as biallelic, monoallelic, or both. Although most At the technical level, to guarantee user-friendly access to the variants are found in biallelic state, because of the recessive data, we stored all information in a PostgreSQL database and inheritance of these 4 genes, a significant percentage (ranging then built a website on top of that, using the Django framework. from 12 to 36%) were reported as monoallelic (Figure 3), which Finally, we completed the information on each variant by indicates the increasingly recognized role of monoallelic variants providing links to other databases of interest. in FHL (22, 23, 25, 28). However, caution must be exercised in evaluating the role of monoallelic variants: FHL is essentially RESULTS an autosomal recessive disease and there is only one well- described example of monoallelic variants as disease causing FHLdb (https://www.biotoclin.org/FHLdb) is a comprehensive mutations through a dominant-negative mechanism (28). The collection of reported variants in the 4 genes known to cause other reported monoallelic variants in FHL are not proven to FHL: PRF1, UNC13D, STXBP2, and STX11. The literature search be disease causing by itself but may represent susceptibility/risk yielded 433 different variants in these genes, distributed as factors in genetic predisposition to FHL (23, 30). Frontiers in Immunology | www.frontiersin.org 3 January 2020 | Volume 11 | Article 107 Viñas-Giménez et al. FHLdb: Familial Hemophagocytic Lymphohistiocytosis Database FIGURE 2 | FHLdb genetic variants in the UNC13D, PRF1, STXBP2, and STX11 genes. Linear representation of UNC13D, PRF1, STXBP2, and STX11 at the protein level. The diagram shows the distribution and location of all variants reported in FHLdb, except for large genomic rearrangements. Dotted lines delimitate each exon. Functional domains are indicated above each gray-scale colored square. MHD1 and MHD2, Munc13 homology domains 1 and 2; SP, signal peptide; MACPF, perforin membrane attack complex; EGF, epidermal growth factor-like; C2, calcium binding domain. The figure was designed using the Protein Paint free software included in the St. Jude PeCan Data Portal (https://pecan.stjude.cloud) (29). We also reviewed whether functional assays had been carried from PolyPhen-2, CADD, PON-P2, and SIFT. We found out to decipher the functional consequences of the variant. that 91% of the missense variants were predicted to be This is one of the main challenges in the interpretation of pathogenic by at least one in silico predictor, and 96% of genetic variants, especially those that are not clearly related to the variants had an allele frequency below 1% in the ExAC loss of function (e.g., missense variants). Variants with reported database. Remarkably, only 20% of variants were included functional studies are indicated and the corresponding reference in the ClinVar database, the largest resource to support is provided. clinical variant interpretation. This finding indicates that To help the interpretation, we manually classified all genetic there is still little available information in ClinVar about variants according to the American College of Medical Genetics FHL genetic variants, and it underscores the need for (ACMG) guidelines. ACMG recommends a five-tier system of disease-specific databases. classification using a specific standard terminology: pathogenic, All the information obtained was compiled in an open, user- likely pathogenic, uncertain significance, likely benign and friendly website (https://www.biotoclin.org/FHLdb), divided benign (31). into three main parts. First, there is a welcome page presenting FHLdb integrates information from other widely used the database, where the user finds the 4 genes typically associated databases, such as clinical evidence from ClinVar and UniProt, with FHL (PRF1, UNC13D, STXBP2, STX11), a description of and population allele frequency from ExAC and gnomAD, the information available for each variant, and other general and it provides computational pathogenicity predictions information. Second, after selecting the gene of interest, the user Frontiers in Immunology | www.frontiersin.org 4 January 2020 | Volume 11 | Article 107 Viñas-Giménez et al. FHLdb: Familial Hemophagocytic Lymphohistiocytosis Database is transferred to the gene view page where all variants are listed CONCLUDING REMARKS and the main characteristics are shown (Figure 4). Third, a link to more detailed information located in the last column allows To our knowledge, this is the first LSDB for FHL syndrome. the user to access a section about the specific variant, which A prototypical definition of LSDB is “a collection of sequence includes the following: (1) a summary of the references (at least variants in a specific gene(s) that causes a Mendelian disorder the first) describing the variant, whether the variant has been or change in phenotype” (32). FHLdb is intended to collect all reported as biallelic and/or monoallelic, whether it was found known variants in the 4 genes typically associated with familial in more than 1 patient, and whether a functional assay was HLH published in peer-reviewed literature. Beyond simply listing carried out; (2) the in silico pathogenicity predictions (CADD, all reported variants in the PRF1, UNC13D, STXBP2, and STX11 PolyPhen-2, PON-P2, and SIFT); (3) links to other databases genes, FHLdb includes other relevant data, such as the variant of interest regarding the specific variant (ClinVar, UniProt, frequency in the general population, computational prediction of dbSNP, Ensembl, ExAC, Gnomad) and databases containing pathogenicity, and functional evidence. Additionally, it provided general knowledge about the disease, the gene, and the protein links to external reference databases to enable users to easily (e.g., Decipher, GeneReviews, OMIM, GeneCards, NCBI); and access all available related resources. (4) a diagram showing the location of the variant in the To build FHLdb, we followed general consensus protein (Figure 5). recommendations to enable broad and easy to manage use (32, 33). FHLdb is an open-access resource, licensed under the Creative Commons Attribution-ShareAlike (4.0) International License, which allows users to adapt, remix, transform, and build upon the material for any purpose, even commercial intents, under the terms specified in the full-text license (https:// creativecommons.org/licenses/by-sa/4.0/legalcode). All existing data in FHLdb can be downloaded in a user-friendly format (txt) and in other programming languages (e.g., Python, Curl, or Wget). Curation is one of the greatest strengths of LSDBs and is likely the aspect that most differentiates them from general databases (34). Each variant in FHLdb was manually curated and almost one third of entries were renamed to adapt the nomenclature to the HGVS. We decided to include only genetic variants supported by peer-reviewed, indexed articles. Therefore, each variant has FIGURE 3 | Distribution of genetic variants according their allelic status. at least one associated article where the user can find further Histograms show the distribution of variants in the STX11, STXBP2, PRF1, information beyond that included in FHLdb. and UNC13D genes included in FHLdb. The total number of variants reported It is important that LSDB creators accept a long commitment in each gene is indicated within parentheses. and continued updating of the database. In our case, FHL/HLH FIGURE 4 | Example of the gene view page where all variants are listed and their main characteristics described. The PRF1 gene view page is shown. The variant indicated by the pointer is highlighted in gray (in this example, c.445G>A). Frontiers in Immunology | www.frontiersin.org 5 January 2020 | Volume 11 | Article 107 Viñas-Giménez et al. FHLdb: Familial Hemophagocytic Lymphohistiocytosis Database FIGURE 5 | Example of the detailed information page, containing specific information about a variant. A missense variant in the PRF1 gene is shown. Frontiers in Immunology | www.frontiersin.org 6 January 2020 | Volume 11 | Article 107 Viñas-Giménez et al. FHLdb: Familial Hemophagocytic Lymphohistiocytosis Database is a strategic research field in our laboratory. We are currently XC collaborated in the database design and literature search. working on two research projects focused on HLH, funded by RC was responsible for designing the study, supervising the Instituto de Salud Carlos III in Spain (see funding section), and literature search and database set-up, writing the manuscript, and we are determined to keep FHLdb updated in the future. To approving the final draft. All authors reviewed the manuscript accomplish that, we will be alert to new publications on the and contributed to the final draft. genetic basis of HLH and we encourage users to report on a new variant by sending us the article describing it. FUNDING In this first version of FHLdb, we focused on genes involved in the cytotoxic pathway. Other genes causing congenital This study was funded by Instituto de Salud Carlos III, immunodeficiency syndromes that are associated with HLH grants PI17/00660 and PI18/00346, cofinanced by the European (e.g., NLRC4, LYST, RAB27A, SH2D1A, XIAP) have not as yet Regional Development Fund (ERDF). LB-M is supported by been reported but it is planned to include them in FHLdb in a Formació de Personal Investigador (FPI) fellowship from the near future, expanding the data base to a compilation of Generalitat de Catalunya (2018_FI_B00072). FC was funded by genetic variants associated to HLH. The increasing use of NGS grants SAF2015-68472-C2-2-R from the Ministerio de Economía is rapidly expanding the number of genetic variants found in y Competitividad (Spain) and FEDER (EU) and RTI2018- HLH patients. Implementation of FHLdb aims to help scientists 096824-B-C22 from the Spanish Ministry of Science, Innovation and clinicians working in the field in their search for FHL- and Universities co-financed by ERDF (FC), and by Direcció related genetic variants to support their research and daily General de Recerca, Generalitat de Catalunya (2014SGR-866 clinical practice. and 2017SGR-702). DATA AVAILABILITY STATEMENT ACKNOWLEDGMENTS Publicly available datasets were analyzed in this study. All data is We acknowledge Celine Cavallo for the English available in http://www.biotoclin.org/FHLdb/. language support. AUTHOR CONTRIBUTIONS SUPPLEMENTARY MATERIAL LV-G performed the literature search, selection, and manual The Supplementary Material for this article can be found curation of the genetic variant data, and wrote part of the online at: https://www.frontiersin.org/articles/10.3389/fimmu. manuscript. NP adjusted the variants to the HGVS guidelines, 2020.00107/full#supplementary-material performed the technical database set-up and web development, Supplementary Table 1 | Variants renamed in FHLdb according the and wrote part of the manuscript. LB-M, FC, JR, MM-G, and HGVS guidelines. REFERENCES diagnosis and management. Clin Exp Immunol. (2011) 163:271–83. doi: 10.1111/j.1365-2249.2010.04302.x 1. Bousfiha A, Jeddane L, Picard C, Ailal F, Bobby Gaspar H, Al-Herz W, et al. 8. Jordan MB, Allen CE, Weitzman S, Filipovich AH, McClain KL. How The 2017 IUIS phenotypic classification for primary immunodeficiencies. J I treat hemophagocytic lymphohistiocytosis. Blood. (2011) 118:4041–52. 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Synergistic defects of different molecules in the cytotoxic pathway Gallo, de la Cruz and Colobran. This is an open-access article distributed under the lead to clinical familial hemophagocytic lymphohistiocytosis. Blood. (2014) terms of the Creative Commons Attribution License (CC BY). The use, distribution 124:1331–4. doi: 10.1182/blood-2014-05-573105 or reproduction in other forums is permitted, provided the original author(s) and 26. Gao L, Zhu L, Huang L, Zhou J. Synergistic defects of UNC13D and AP3B1 the copyright owner(s) are credited and that the original publication in this journal leading to adult hemophagocytic lymphohistiocytosis. Int J Hematol. (2015) is cited, in accordance with accepted academic practice. No use, distribution or 102:488–92. doi: 10.1007/s12185-015-1807-z reproduction is permitted which does not comply with these terms. Frontiers in Immunology | www.frontiersin.org 8 January 2020 | Volume 11 | Article 107