TYPE Original Research PUBLISHED 18 November 2022 DOI 10.3389/fimmu.2022.1014984 Detection and evolutionary OPEN ACCESS dynamics of somatic FAS EDITED BY Eduardo Lopez-Granados, University Hospital La Paz, Spain variants in autoimmune REVIEWED BY Masatoshi Takagi, lymphoproliferative syndrome: Tokyo Medical and Dental University, Japan Ana V. Marin, Diagnostic implications 12 de Octubre Health Research Institute (imas12), Spain Laura Batlle-Masó 1,2,3, Marina Garcia-Prat 1,2,3, *CORRESPONDENCE Roger Colobran Alba Parra-Martı́nez 1,2,3, Clara Franco-Jarava 3,4,5, roger.colobran@vallhebron.cat Aina Aguiló-Cucurull 3,4,5, Pablo Velasco 6, Marı́a Antolı́n 7, SPECIALTY SECTION Jacques G. Rivière 1,2,3, Andrea Martı́n-Nalda 1,2,3, This article was submitted to Primary Immunodeficiencies, Pere Soler-Palacı́n 1,2,3, Mónica Martı́nez-Gallo 3,4,5,8 a section of the journal Frontiers in Immunology and Roger Colobran 3,4,5,7,8* RECEIVED 09 August 2022 1 Infection in Immunocompromised Pediatric Patients Research Group, Vall d’Hebron Research ACCEPTED 24 October 2022 Institute (VHIR), Barcelona, Spain, 2 Pediatric Infectious Diseases and Immunodeficiencies Unit, Vall PUBLISHED 18 November 2022 d’Hebron University Hospital (HUVH), Barcelona, Spain, 3 Jeffrey Modell Diagnostic and Research Center for Primary Immunodeficiencies, Barcelona, Spain, 4 Translational Immunology Group, Vall CITATION d’Hebron Research Institute (VHIR), Barcelona, Spain, 5 Immunology Division, Vall d’Hebron Batlle-Masó L, Garcia-Prat M, University Hospital (HUVH), Barcelona, Spain, 6 Pediatric Oncology and Hematology Department, Parra-Martı´nez A, Franco-Jarava C, Vall d’Hebron University Hospital (HUVH), Barcelona, Spain, 7 Department of Clinical and Molecular Aguiló-Cucurull A, Velasco P, Genetics, Vall d’Hebron University Hospital (HUVH), Barcelona, Spain, 8 Department of Cell Biology, Antolı´n M, Rivière JG, Martı´n-Nalda A, Autonomous University of Barcelona (UAB), Physiology and Immunology, Bellaterra, Spain Soler-Palacı´n P, Martı´nez-Gallo M and Colobran R (2022) Detection and evolutionary dynamics of somatic FAS variants in autoimmune lymphoproliferative syndrome: Diagnostic implications. Autoimmune lymphoproliferative syndrome (ALPS) is a rare primary immune Front. Immunol. 13:1014984. doi: 10.3389/fimmu.2022.1014984 disorder characterized by impaired apoptotic homeostasis. The clinical COPYRIGHT characteristics include lymphoproliferation, autoimmunity (mainly cytopenia), © 2022 Batlle-Masó, Garcia-Prat, and an increased risk of lymphoma. A distinctive biological feature is Parra-Martı´nez, Franco-Jarava, accumulation (>2.5%) of an abnormal cell subset composed of TCRab+ CD4- Aguiló-Cucurull, Velasco, Antolı´n, Rivière, Martı´n-Nalda, Soler-Palacı´n, CD8 - T cells (DNTs). The most common genetic causes of ALPS are Martı´nez-Gallo and Colobran. This is an monoallelic pathogenic variants in the FAS gene followed by somatic FAS open-access article distributed under the terms of the Creative Commons variants, mainly restricted to DNTs. Identification of somatic FAS variants has Attribution License (CC BY). The use, been typically addressed by Sanger sequencing in isolated DNTs. However, this distribution or reproduction in other approach can be costly and technically challenging, and may not be successful forums is permitted, provided the original author(s) and the copyright in patients with normal DNT counts receiving immunosuppressive treatment. owner(s) are credited and that the In this study, we identified a novel somatic mutation in FAS (c.718_719insGTCG) original publication in this journal is cited, in accordance with accepted by Sanger sequencing on purified CD3 + cells. We then followed the academic practice. No use, evolutionary dynamics of the variant along time with an NGS-based distribution or reproduction is approach involving deep amplicon sequencing (DAS) at high coverage permitted which does not comply with these terms. (20,000-30,000x). Over five years of clinical follow-up, we obtained six blood samples for molecular study from the pre-treatment (DNTs>7%) and treatment (DNTs<2%) periods. DAS enabled detection of the somatic variant in all samples, even the one obtained after five years of immunosuppressive Frontiers in Immunology 01 frontiersin.org Batlle-Masó et al. 10.3389/fimmu.2022.1014984 treatment (DNTs: 0.89%). The variant allele frequency (VAF) range was 4%-5% in pre-treatment samples and <1.5% in treatment samples, and there was a strong positive correlation between DNT counts and VAF (Pearson’s R: 0.98, p=0.0003). We then explored whether the same approach could be used in a discovery setting. In the last follow-up sample (DNT: 0.89%) we performed somatic variant calling on the FAS exon 9 DAS data from whole blood and purified CD3+ cells using VarScan 2. The c.718_719insGTCG variant was identified in both samples and showed the highest VAF (0.67% blood, 1.58% CD3+ cells) among >400 variants called. In summary, our study illustrates the evolutionary dynamics of a somatic FAS mutation before and during immunosuppressive treatment. The results show that pathogenic somatic FAS variants can be identified with the use of DAS in whole blood of ALPS patients regardless of their DNT counts. KEYWORDS primary immunodeficiencies, inborn errors of immunity, autoimmune lymphoproliferative syndrome, FAS, somatic mutation, genetics, next-generation sequencing Introduction monoallelic (autosomal dominant), affect the intracellular death domain (DD) region, and exert a dominant-negative effect related Autoimmune lymphoproliferative syndrome (ALPS, OMIM to the trimeric nature of the FAS protein (7). Heterozygous loss- #601859) is characterized by a lack of control over lymphocyte of-function variants leading to FAS haploinsufficiency without a apoptosis. This results in an elevated number of lymphocytes, which dominant negative effect are a less frequent cause. These variants can accumulate and lead to enlarged lymph nodes, liver, and spleen. are usually located in the FAS extracellular region and are The main clinical features in ALPS are lymphadenopathy, associated with milder clinical manifestations and lower hepatomegaly, splenomegaly, and autoimmune cytopenia. There penetrance (8). In addition, biallelic germline variants in FAS is also an increased risk for developing severe health conditions such have been reported in severe early-onset ALPS patients (6, 9). In as autoimmune diseases and hematological malignancies (Hodgkin these cases, heterozygous carriers were asymptomatic, indicating and non-Hodgkin lymphoma) (1). that some pathogenic FAS variants behave as recessive alleles The diagnosis of ALPS is based on two major criteria: elevated whereas others are dominant (1). (>2.5%) CD3+ TCRab+ CD4-CD8- T cells (hereafter referred to as Pathogenic germline variants in other genes affecting the double-negative T cells, [DNTs]), and chronic nonmalignant/ FAS-related apoptosis pathway have been reported as a cause of noninfectious lymphoproliferation. The accessory criteria include ALPS in a minor fraction of patients: FASLG (FAS ligand) (10), defective lymphocyte apoptosis, presence of pathogenic genetic FADD (FAS-associated death domain protein) (11), CASP8 variants, elevated levels of soluble molecules such as FAS ligand, (caspase 8) (12), and CASP10 (caspase 10) (13). However, the IL10 and vitamin B12, typical immunohistological findings, role of CASP10 variants as a causative versus predisposing factor autoimmune cytopenia, and a positive family history (2). in ALPS is still controversial (14). Immunosuppressive drugs, such as steroids, mycophenolate and After germline FAS mutations, heterozygous somatic FAS the mTOR inhibitor rapamycin (sirolimus), are good treatment mutations are the second most common genetic etiology of options for ALPS patients (3), as they effectively reduce cytopenia ALPS, accounting for up to 15% of patients (15). Several features and lymphoproliferation. However, patients should be monitored of somatic FAS variants have been reported to date: 1) They to control drug-related toxicity. Hematopoietic stem cell appear in hematopoietic progenitors and are predominantly transplantation (HSCT) has been successful for curing severe detected in DNTs, although they are also present in smaller ALPS, but it is a complex procedure and the risk/benefit balance proportions in other lymphoid and myeloid lineages (15); 2) must be carefully evaluated in each patient (4). They are mainly located in exons 7, 8 and 9 of the FAS gene, In most cases, the molecular origin of ALPS is associated with encoding the intracellular domain (16); and 3) They cause pathogenic germline variants in the FAS gene, which encodes the clinical phenotypes indistinguishable from those caused by FAS cell surface death receptor (5, 6). Most of these variants are germline FAS variants (17). Frontiers in Immunology 02 frontiersin.org Batlle-Masó et al. 10.3389/fimmu.2022.1014984 To further complicate the molecular basis of ALPS, other CD3+ cell enrichment inheritance patterns have been described, including the double- hit hypothesis involving “second-hit” variants in the FAS gene Human CD3+ cells were isolated from 8 mL of heparinized itself or in other disease-modifying genes (18). whole blood by negative selection using the EasySep Human T New sequencing technologies, such as deep amplicon Cell Isolation Kit (#17951, Stemcell Technologies, Vancouver, sequencing, have enabled better and more comprehensive BC, Canada) in 2017, and the RosetteSep Human T Cell detection of true pathogenic somatic variants in next- Enrichment Cocktail Kit (#15021, Stemcell Technologies) in generation sequencing (NGS) data (19). Optimized callers for 2021. Purification was confirmed by flow cytometry, obtaining somatic variation are being developed and improved (20), and >95% purity in both cases. CD3+ pellets were stored at -80°C although somatic variants have been traditionally investigated in until use. the context of cancer, their role in immune-mediated disorders is increasingly recognized (18, 19, 21, 22). Here, we present an analysis of data from a pediatric patient Genetic analysis by Sanger sequencing in whom a novel pathogenic somatic variant was identified and studied over five years. We evaluated the dynamics of the All exons and flanking regions of the FAS, CASP8, variant, the DNT population, and the patient’s clinical course, and CASP10 genes were sequenced by Sanger method focusing on future application of this strategy in a discovery (Supplementary Table 1). Genomic DNA was extracted from setting for patients with no available pretreatment samples. In EDTA-containing whole-blood samples using the QIAamp addition, we performed a literature review to gather all DNA blood mini kit (Qiagen; Hilden, Germany) according to pathogenic somatic variants associated with ALPS to facilitate the manufacturer’s instructions. A polymerase chain reaction identification of future novel variants. Our results provide (PCR) technique was carried out to amplify exons and flanking additional information on the genetic bases of the disease and regions. A 40-ng amount of genomic DNA was used in each PCR clues to improve diagnostic rates and validate the effectiveness of reaction. FAS exon 9 was amplified using the following treatment at the molecular level. oligonucleotides: forward 5’-TCTTGTTTCTGTATTCCCCTA GTCAGC-3’, reverse 5’-AAACCAAGCAGTATTTACAGC CAGC-3’ (amplimer size: 570bp). An internal oligonucleotide Materials and methods was used for sequencing: 5’-AGACCTTTAGGACTTAGCTAT- 3’. PCR conditions (35 cycles) were as follows: 95°C for 30 s, 62°C Patient and samples for 30 s and 72°C for 40 s. Purified PCR products were sequenced on an ABI 3100 DNA sequencer using the BigDye Terminator The patient included in this study was attended during a sequencing kit, 3.1 (Applied Biosystems; Foster, VA, USA). five-year period (2017–2021) at Hospital Universitari Vall d’Hebron (HUVH) (Barcelona, Catalonia, Spain). Written informed consent for the studies reported here and for Genetic analysis by NGS-based deep publication was obtained from t he patient’s le gal amplicon sequencing (DAS) representatives, according to the procedures of the Ethics Review Board of Hospital Universitari Vall d’Hebron [code: FAS exon 9 amplimers containing the whole exon and PR(AG)69/2016]. flanking regions were PCR-generated using 40-ng of DNA from whole blood or CD3+ cells. Two different sets of primers were used to ensure unbiased amplification, obtaining equivalent Evaluation of DNTs by flow cytometry DAS results. Primer pair 1 was the same as that used for Sanger sequencing and primer pair 2 was the following: forward 5’- Whole blood collected in EDTA was stained with the TGGGTATATGGCAGGATTTGGA-3’, reverse 5’-CCTTG following antibodies—TCRab-FITC, CD4-PE, CD3-APC, GAGGCAGAATCATGAG-3’ (amplimer size: 695bp). PCR CD8-PerCP-Cy5—and analyzed with a FACSCanto II flow conditions for primer pair 2 (35 cycles) were 95°C for 30 s, cytometer (Becton Dickinson). Data were processed with 58°C for 30 s and 72°C for 45 s. PCR products were quantified Kaluza analysis software (Beckman Coulter) using the with a Qubit 2.0 Fluorometer (Thermo Fisher Scientific, MA, following gating strategy: FSC vs SSC (lymphocyte USA). A 300- to 900-ng amount of DNA was fragmented using selection), CD3 + T cell selection, TCRab selection, and NEBNext dsDNA Fragmentase (New England Biolabs, Ipswich, finally CD4 and CD8 dot plot selecting DNT lymphocytes. MA, USA) to obtain fragments of approximately 200bp in size. The double-negative population was reported over the End repair, ligation of the adapter for Illumina, and sample CD3+ population. indexing by a small amplification were then carried out using the Frontiers in Immunology 03 frontiersin.org Batlle-Masó et al. 10.3389/fimmu.2022.1014984 NEBNext Ultra DNA library prep kit for Illumina and the Literature review NEBNext MultiplexOligos for Illumina Dual Index Primers Set 1 (New England Biolabs, Ipswich, MA, USA). All necessary A comprehensive literature search in PubMed, Google Scholar, purifications and size selections to recover only the fragments of SNPdb, ClinVar, HGMD, and Ensembl was conducted to gather all interest were done using AMPure XP beads (Beckman Coulter). FAS gene somatic variants described in ALPS patients. To retrieve Libraries were quality-evaluated using QIAxcel (Qiagen) and the maximum number of peer-reviewed papers, we combined quantified with a Qubit 2.0 Fluorometer. The DNA sample (among others) the following keywords: ALPS, autoimmune libraries were then mixed in equimolecular amounts and lymphoproliferative syndrome, somatic, FAS, post-zygotic, sequenced in a MiSeq instrument (Illumina, San Diego, CA, TNFRSF6, mutation, variant, and genetic variant. Each article was USA) using the 500-cycle MiSeq reagent kit v2 with a paired-end manually curated, and additional information on each variant was run of 2×250-bp reads. All procedures were performed annotated to the final table using UniProt, gnomAD, and Varsome. according to the manufacturer’s instructions. ProteinPaint (23), was used to create a base figure which was then adapted to our final version. Bioinformatic analysis Results Results were aligned to the reference genome, hg38, using BWA (https://github.com/lh3/bwa). Duplicate reads were Discovery of a novel pathogenic marked using Picard MarkDuplicates (version 2.18.6, https:// somatic variant underlying ALPS in github.com/broadinstitute/picard). Next, GATK 4.1.8.1(https:// a pediatric patient github.com/broadinstitute/gatk) was used to calibrate and calculate BQSR scores (BaseRecalibrator, ApplyBQSR). The patient under study was an infant boy who presented with Filtering was then done using SAMtools (https://github.com/ recurrent nonspecific fever, splenomegaly, diarrhea, and upper samtools/samtools) to discard low-quality reads (mapping respiratory tract infections at 11 months of age (Table 1). He was quality < 30, samtools view -bSq 30) and secondary born in Bolivia to non-consanguineous parents, and there was no alignments (samtools view -F 256). Each allele was counted family history of immune disorders. Laboratory analysis showed using the Pysam Python package, version 0.15.2 (https://github. thrombocytopenia (platelet count: 67x109/L; reference range: 150- com/pysam-developers/pysam), using the fetch function as 500x109/L), anemia (hemoglobin: 7.9 g/dL; reference range: 11.5- indicated by the package documentation (https://pysam. 13.5 g/dL), monocytosis (absolute monocyte numbers: 1.6x109/L; readthedocs.io/en/latest/api.html). Next we manually validated reference range: 0.1-0.7x109/L), hypergammaglobulinemia (IgG: using the integrative genomics viewer (IGV) software (https:// 1882 mg/dL; reference range: 196-1046 mg/dL), and elevated software.broadinstitute.org/software/igv/). Additional somatic IL10 (132 pg/mL; reference range 0-7.8 pg/mL) and vitamin B12 variant-calling was performed with VarScan 2, version 2.4.340 (>2000 pg/mL; reference range: 211-911 pg/mL) levels. DNTs were (https://sourceforge.net/projects/varscan/files/) to compare the evaluated to investigate suspected ALPS, and the value obtained was causal genetic variant with other hits found by the software; off- clearly elevated (7.4%; reference range <2.5%) (Figure 1A). target reads and those of low quality (less than 500 total counts) Direct sequencing (Sanger method) of the FAS, CASP8, and were excluded. CASP10 genes ruled out pathogenic germline variants in these TABLE 1 Information on the clinical diagnosis, laboratory tests, and treatment of the index patient starting from the initial visit (May 2017) to the latest follow up (March 2021). 2017 (May) 2017 (August) 2017 (November) 2018 (May) 2018 (December) 2021 (March) Age 12 months 15 months 17 months 2 years 2.5 years 5 years Clinical Erythema, cough, Respiratory infection, Respiratory infection, mild Clinically Clinically asymptomatic Clinically data nonspecific fever splenomegaly, fever splenomegaly, without fever asymptomatic asymptomatic Laboratory DNTs elevated DNTs elevated (7.8%) DNTs within normal (1.9%) DNTs within DNTs within normal DNTs within data (7.4%) normal (1.4%) (1.76%) normal (0.89%) Treatment None None Sirolimusa Sirolimus Sirolimus Sirolimus At each time point, a blood sample was obtained for molecular analysis. DNTs, TCRab+ CD4-CD8- T cells a Sirolimus treatment was started at 15 months, just after the second follow up (August 2017). Frontiers in Immunology 04 frontiersin.org Batlle-Masó et al. 10.3389/fimmu.2022.1014984 A B FIGURE 1 Cellular and molecular characterization of the patient’s samples. (A) Evaluation of TCRab+ CD4-CD8- T cells (DNTs) by flow cytometry. The index patient and a representative healthy donor are shown. Gating strategy: on CD3+ T cells, TCRab positive cells were first selected; next, CD4 and CD8 dot-plot analysis was performed. The lower-left quadrant contains DNT data (percentage of DNTs) is indicated. (B) Sanger sequencing results from the initial sample (2017). The upper panel corresponds to DNA extracted from whole blood where no somatic variant was detected. The lower panel shows the CD3+ enriched sample, where a double peak pattern appeared, indicating the presence of a 4-bp insertion (c.718_719insGTCG). genes. As somatic mutations in FAS (mainly limited to DNTs) are day), which significantly improved the clinical symptoms and the second most common genetic cause of ALPS, we addressed this markedly reduced the number of DNTs. During the next five possibility by purifying and sequencing (Sanger method) CD3+ cells years, the patient remained asymptomatic and sirolimus dose from whole blood. Although the gold standard is to isolate DNTs by was periodically adjusted according to weight. During this cell sorting, we performed a more accessible method: CD3+ cell clinical follow-up (2017–2021), six blood samples were purification by magnetic beads. In our sample, DNTs represented obtained and stored for molecular studies: two in the pre- 7.4% of all CD3+ cells; therefore, somatic variants were enriched in treatment period and four while receiving treatment (Table 1). this cell population compared with that of whole blood. We DNT counts were assessed in each sample. Using NGS-based observed a small double peak pattern in exon 9 of FAS, deep amplicon sequencing (DAS) at high coverage (20,000- consisting of a 4-bp insertion (c.718_719insGTCG), which could 30,000x), we aimed to detect and track the c.718_719insGTCG have corresponded to a somatic variant. Sequencing was repeated somatic variant in all samples (whole blood). First, we confirmed twice with the same results, and the small double peak pattern was the presence of the variant in the initial sample at a variant allele not visible in DNA from whole blood (Figure 1B). The size of the frequency (VAF) of 4.3% (Figure 2). Second, we detected the peaks corresponding to the somatic variant was concordant with somatic variant in all samples, even in the one obtained after five the 7.4% of DNTs where the variant was expected to be present. years of treatment. At that time, DNT counts were within the This novel insertion was predicted to have a frameshift effect and to reference range (0.89%) and the variant was still detectable, cause a premature STOP codon (p.Met240SerfsX8). It is located at although at a low VAF (0.67%). Third, we observed a strong an evolutionarily highly conserved position (CADD v1.6 = 23) in positive correlation between DNT counts and the VAF the intracellular FAS region where most somatic variants have been (Pearson’s R: 0.98, p=0.0003). These results confirmed that the reported (16). The variant was not found in the main population somatic variant was mainly restricted to DNTs and that cells databases (ExAC, gnomAD) and has not been described in the derived from the mutated clone were present in peripheral blood literature. We classified the variant as pathogenic, reported it to at very low but detectable levels even after years of ClinVar (accession number: SCV002568438), and considered it to immunosuppressive treatment. be the genetic cause of the patient’s clinical phenotype. Detection of somatic FAS variants under Evolutionary dynamics of the somatic immunosuppressive treatment: FAS variant and DNTs along time Diagnostic implications At the age of 15 months, the patient was started on Detection of pathogenic somatic FAS variants in ALPS can immunosuppressive treatment with sirolimus (2 mg/m2 per be relatively straightforward when the patient has elevated DNTs Frontiers in Immunology 05 frontiersin.org Batlle-Masó et al. 10.3389/fimmu.2022.1014984 FIGURE 2 Evolutionary dynamics of the somatic FAS variant and correlation with DNT percentages during a five-year follow up. Each time point shows the TCRab+ CD4-CD8- T cells (DNTs) (in blue) and variant allele frequency (VAF) of the pathogenic somatic FAS variant (c.718_719insGTCG) (in red). The yellow shading indicates the sirolimus treatment period. The bottom panels show the absolute values and percentage of read counts supporting each allele in the position of the variant c.718_719insGTCG (values were extracted from IGV data after quality filtering). Total RD, Total read depth. that can be isolated by cell sorting and Sanger sequenced. that only a small percentage of somatic calls obtained with this Nevertheless, sometimes patients are already under immuno type of software are true somatic variants (24); therefore, we suppressive treatment when the search for somatic FAS variants used the VAF and p-values to differentiate between background is considered. In this situation, the number of DNTs is likely noise and true variants. The background noise was similar in the within the reference range and the process of isolation by cell two samples, with most variants detected showing a VAF <0.4% sorting and Sanger sequencing may be unsuccessful. (Figure 3C) and a p-value <1x10-4 (Supplementary Figure 1). Over the patient’s five-year follow-up, the pathogenic somatic The c.718_719insGTCG variant was identified in both samples variant was detected by DAS in all samples regardless of the DNT and showed the highest VAF (0.67% in whole blood and 1.58% count. However, we had the advantage of knowing a priori the in CD3+ cells) and lowest p-value (1x10-7 in whole blood and candidate variant. We then aimed to explore whether the same 1x10-20 in CD3+ cells) of all variants identified (Figure 3C and approach could be used in a discovery setting. For that purpose, Supplementary Figure 1). Remarkably, the simple CD3 we focused on the last follow-up, when the patient had been enrichment step clearly improved the process of detecting true receiving immunosuppressive treatment for five years and DNTs somatic variants, increasing the VAF and p-value by more than were present at only 0.89% (Table 1, 2021). Whole blood and 2-fold each. purified CD3+ cells were obtained using a simple method based on standard density gradient centrifugation with RosetteSep. We sequenced the entire exon 9 of FAS from blood and CD3+ cells Review of somatic FAS variants reported by Sanger and DAS (coverage 20,000x). Sanger sequencing in ALPS patients showed no trace of the somatic variant in either whole blood or CD3+ cells (Figure 3A). This was expected because the percentage Identification of somatic FAS mutations is becoming more of DNTs in the two samples was below the sensitivity of the accessible with the advent of new deep sequencing technologies. technique. In contrast, with the use of DAS, the somatic variant However, their interpretation remains challenging because of the was detected in both blood and CD3+ cells (Figure 3B). complex link between genetic variants and their functional effects. As was mentioned above, we were aware of the candidate We performed a literature search to gather and describe all and this greatly facilitated analysis of the DAS data. However, we pathogenic and likely pathogenic somatic FAS variants wanted to go one step further and determine whether the associated with ALPS. We manually inspected all indexed pathogenic somatic variant could have been identified with no papers describing this type of variation and found 40 variants a priori knowledge. To that end, we performed somatic variant (including the one described in this study): 18 frameshift, 12 calling on the raw BAM files from the DAS analysis using missense, 7 nonsense, 1 in-frame deletion, and 2 splice variants VarScan 2. The software called 470 candidate variants from (Figure 4 and Table 2). Most were located in the death domain of whole blood and 450 from CD3+ cells (Figure 3C). It is known exon 9 (15 of 40, 37.5%), including the novel frameshift variant Frontiers in Immunology 06 frontiersin.org Batlle-Masó et al. 10.3389/fimmu.2022.1014984 A B C FIGURE 3 Detection of the somatic FAS variant in blood and CD3+ cells. (A) Sanger sequencing of whole blood and CD3+ cells (sample obtained in 2021). DNT count at that time accounted for 0.89% of the total CD3+ lymphocytes. There is no evidence of the somatic variant in chromatograms of the two samples. (B) Details of the deep amplicon sequencing results for the two samples. IGV screenshot and total read counts are shown. (C) Chromosome position and variant allele frequency (VAF) of all presumed somatic variants called by VarScan 2 in exon 9 of the FAS gene (hg38). Each dot is a variant: 470 variants were obtained from whole blood (dots in red) and 450 from CD3+ cells (dots in blue). The pathogenic somatic variant (chr10:89,014,160; c.718_719insGTCG) is indicated with an arrow. described here, thus supporting the relevance of the FAS protein molecular screening methods such as Sanger is challenging. intracellular domain in the development of ALPS. The variant may go unnoticed or the variant peak on the chromatogram may be mistaken for background noise. In our case, we were fortunate that the variant was a frameshift. The Discussion double peak pattern, although the height was small, was highly suspicious for a somatic variant. Moreover, it was specific to Study of somatic variants underlying inborn errors of CD3+ cells and not present in whole blood. However, the single immunity (IEI) is gaining increasing interest due to biological, small peak of a single nucleotide variant could be easily technological, and clinical reasons (30). In 2004, ALPS was the misinterpreted as background noise. Hence, we believe that first IEI in which somatic mosaicism was reported as the cause of Sanger sequencing on purified CD3+ cells may not be sensitive the disease (15). Since then, other IEIs caused by somatic enough for use as a routine diagnostic method in these cases. mutations have been described, with autoinflammatory NGS-based methods are highly suitable for detecting disorders having the highest incidence (19, 30). Many IEIs somatic variants, although their precision and robustness relies resulting from mosaicism are also germline disorders, as in the on the read depth. Whole-exome sequencing (WES) and whole- case of most autoinflammatory disorders and ALPS, whereas few genome sequencing (WGS) are intended to identify germline IEIs are predominantly caused by somatic mutations (eg, variants, as their mean coverage is usually <200x and <100x, VEXAS syndrome, TLR8 gain-of-function, and Ras-associated respectively. They can also be used for somatic variant autoimmune leukoproliferative disorder) (31–33). identification, but typically cannot easily detect those at <10% In this study, we identified a novel somatic mutation in FAS VAF. Targeted gene panels usually have a higher overall read by Sanger sequencing of purified CD3+ cells and followed the depth (200x-500x). Recently, Ló pez-Nevado et al. used a targeted evolutionary dynamics of the variant over time with DAS. gene panel in whole blood samples to detect germline and Detection of somatic variants at low VAF using classic somatic FAS mutations in ALPS patients (28). These authors Frontiers in Immunology 07 frontiersin.org Batlle-Masó et al. 10.3389/fimmu.2022.1014984 FIGURE 4 Somatic variants reported in ALPS patients. A diagram of the FAS protein is shown with the locations of somatic variants. Exons are separated by dashed lines and indicated with grey numbers. The transmembrane and death domains of the protein are highlighted in grey and amino acid numbers are indicated. Details of all variants are shown in Table 2. identified a pathogenic somatic FAS variant in a patient who had may not be available in all centers. Ló pez-Nevado et al. no pathogenic germline variants in the panel. To validate the demonstrated that somatic FAS variants can be detected by somatic variant call, they used blood DNA from seven other NGS-based targeted gene panel analysis in blood of patients with ALPS patients previously diagnosed with somatic FAS variants. elevated DNTs (28). However, their results showed that the depth In all patients but one, they were able to detect somatic variants of coverage with this approach may not suffice to disclose variants in a VAF range of 1.9% to 11.5% with a variable read depth in the for which only a few reads support the alternative allele (low VAF). specific position (101x to 399x). Interestingly, the single patient We found that NGS-based DAS can detect somatic variants in with a previously identified somatic FAS variant that went ALPS patients even when DNTs are within the reference range. In undetected with this method had DNT counts within the our opinion, a targeted gene panel would be an appropriate initial reference range. In the present study, the higher-depth NGS genetic approach for patients with ALPS suspicion, since it has the approach (DAS coverage >20,000x) enabled detection of the advantage of covering germinal mutations in FAS (and other somatic variant in blood samples containing a very low ALPS-related genes) and it may detect somatic variants in some percentage of DNTs (<1%). The most striking example is cases. Nevertheless, their limited sensitivity may not detect somatic detection of the somatic variant at 0.67% VAF in a sample variants at low VAF. Therefore, in case of negative results in a obtained after five years of immunosuppressive treatment. This targeted gene panel, DAS should be performed since this technique is a proof of concept that pathogenic somatic FAS variants can be increases the likelihood of finding the somatic variants, especially at detected in whole blood of ALPS patients regardless of their low VAF. In both targeted gene panel analysis and DAS analysis, DNT counts with the use of DAS. For that purpose, high the PCR steps may introduce sequence errors that confound coverage of the target gene or exons is needed to ensure identification of somatic variants with a very low VAF (<1%- identification of variants having a low percentage of 2%). To overcome this limitation in our DAS technique, we applied supporting reads. In addition, specific bioinformatic processing the quality scores provided by VarScan 2, to identify variants and of the data is required to avoid filtering of true somatic variants distinguish true somatic variants from background noise. It is likely by conventional germline-focused strategies. Various somatic that use of CD3+-enriched samples and more affordable coverage variant callers have been created and compared in this regard (5000x) will enable identification of somatic variants in a (20, 34, 35). These are especially useful when the number of diagnostic setting. In addition, the method could be useful for candidate genes or exons is not suitable for manual analysis. patients who have started immunosuppressive treatment without a One helpful finding obtained here is that a simple CD3+ molecular diagnosis. This opens the door to revisiting previously enrichment step may be useful to enhance the sensitivity and unsolved cases and may increase the genetic diagnosis rate in reliability of NGS for somatic FAS variant detection. The VAF of pediatric ALPS. the somatic variant in our patient was 2.5-fold higher in the To our knowledge, this is the first reported description of the CD3+ sample than in whole blood, whereas the background evolutionary dynamics of a somatic FAS mutation in ALPS. We noise (artefactual variants) was similar in the two samples. Thus, detected the pathogenic somatic variant in all six blood samples the probability that true somatic variants would be detected collected over a five-year period, including those obtained during increased after enrichment. sirolimus treatment, which had normal DNT counts. Our results Traditionally, the genetic diagnosis of somatic variants in indicate that with a proper approach, genetic assessment of ALPS has been done by sorting and sequencing DNTs (15, 36). somatic FAS variants in ALPS patients can be performed at any Nonetheless, this is a time- and resource-consuming strategy that time over the clinical course, as immunosuppressive treatment did Frontiers in Immunology 08 frontiersin.org Batlle-Masó et al. 10.3389/fimmu.2022.1014984 TABLE 2 Somatic FAS variants associated with ALPS. Exon Region Position (hg38) Nucleotide change Amino acid change Reference 2 Extracellular chr10:89003098 c.100G>T p.G34X (25) 3 Extracellular chr10:89007700 c.197G>A p.G66D (26) 3 Extracellular chr10:89007835 c.332A>G p.H111Ra (17) 5 Extracellular chr10:89010566 c.471C>A p.C157X (26) 6 Transmembrane n.a. n.a. (donor splice-site) p.W189fs (stop at 225)a (15) 6 Transmembrane n.a. n.a. p.W189fs (stop at 220) (16) 7 Intracellular chr10:89012061 c.631G>T p.E211Xa (17) 7 Intracellular n.a. n.a. p.P217fs (stop at 220) (16) 8 Intracellular chr10:89012083 c.651+2T>C p.P217fsa (15) 8 Intracellular chr10:89013368 c.676+1G>A p.E218MfsX4a (17) 8 Intracellular chr10:89013334 c.652-9_653del p.E218MfsX4a (17) a 8 Intracellular chr10:89013342 c.652-1G>T p.E218MfsX4 (17) 8 Intracellular chr10:89013367 c.676G>T p.E218MfsX4a (17) 8 Intracellular chr10:89013343 c.652G>T p.E218Xa (17) 8 Intracellular chr10:89013348 c.657_658del p.V220GfsX6 (17) 8 Intracellular chr10:89013348 c.657_658delAG p.V220GfsX6 (27) 8 Intracellular chr10:89013359 c.668_675del p.N223RfsX3 (17) 8 Intracellular chr10:89013362 c.671T>G p.L224Xa (17) 8 Intracellular chr10:89013365 c.674C>A p.S225Yb (26) 8 Intracellular n.a. n.a. p.D226fs (stop at 277)b (16) 8 Intracellular chr10:89013341 c.652-2A>T Splice (27) 8 Intracellular chr10:89013342 c.652-1G>A Splice (27) 9 Intracellular chr10:89014121 c.679delG p.V227LfsX3 (28) 9 Intracellular chr10:89014122 c.682_686del p.D228EfsX2 (17) 9 Intracellular chr10:89014128 c.686T>A p.L229X (29) 9 Intracellular (DD) n.a. n.a. (deletion of 8 bp) p.S230fs (stop at 243)a (15) 9 Intracellular (DD) n.a. n.a. p.S230fs (stop at 240) (16) 9 Intracellular (DD) chr10:89014151 c.709G>C p.A237P (25) 9 Intracellular (DD) chr10:89014154 c.712G>T p.G238X (27) 9 Intracellular (DD) chr10:89014161 c.718_719insGTCG p.M240SfsX8 This study 9 Intracellular (DD) chr10:89014191 c.749G>A p.R250Q (16) 9 Intracellular (DD) chr10:89014190 c.768C>G p.R250G (27) 9 Intracellular (DD) chr10:89014221 c.779A>T p.D260Va (15) 9 Intracellular (DD) chr10:89014220 c.778G>A p.D260Na (17) 9 Intracellular (DD) chr10:89014221 c.779A>G p.D260G (29) 9 Intracellular (DD) chr10:89014220 c.778G>T p.D260Y (27) 9 Intracellular (DD) chr10:89014251 c.812_814del p.A271del (27) 9 Intracellular (DD) chr10:89014257 c.815A>G p.E272G (27) 9 Intracellular (DD) chr10:89014259 c.817C>A p.Q273K (16) 9 Intracellular (DD) n.a. n.a. (1 bp insertion) p.V275fs (stop at 280) (25) DD, death domain; n.a., value not available in the original paper. a Nomenclature adjusted to the current Human Genome Variation Society (HGVS) guidelines. b Amino acid adjusted to the canonical transcript (NM_000043.6). not eliminate all mutated cells, although their numbers in eliminate with immune suppression (the only known cure is peripheral blood were considerably reduced. This is consistent HSCT); thus, the need for chronic treatment in ALPS patients with the fact that somatic mutations initially occur in (4, 37). We observed a strong positive correlation between DNT hematopoietic progenitor cells (HPCs) and become enriched in count and the VAF. This finding confirms that somatic FAS DNTs due to a selective survival advantage (15). HPCs provide a mutations are mainly restricted to DNTs, although they can be constant supply of mutated descendant cells that are impossible to found in varying percentages in other leukocyte subsets (15). Frontiers in Immunology 09 frontiersin.org Batlle-Masó et al. 10.3389/fimmu.2022.1014984 The pathogenic somatic variant reported in this study Author contributions (c.718_719insGTCG, p.Met240SerfsX8) is located in the intracellular region, within the DD. This domain is essential LB-M performed the bioinformatics analysis of NGS data, for formation of the death-inducing signaling complex (DISC), somatic variant calling, and the literature review. MG-P, AP-M, which triggers the extrinsic apoptosis pathway through and CF-J performed laboratory analyses and CD3+ purification. stimulation by death ligands (38). We reviewed all reported MM-G performed flow cytometry studies. AA-C performed somatic FAS mutations and found 40 variants. The majority (34 sample collection and Sanger sequencing. PV, JR, AM-N, and of 40, 85%) were located in the intracellular domain of the PS-P provided patient care, and collected and provided clinical protein and 15 (37.5% of the total) specifically in the DD. Few data. MA was responsible for the NGS technical process, variants were found in the extracellular or transmembrane including library preparation and sequencing. LB-M and RC domains (4 and 2 respectively, 10% and 5% of the total). The analyzed and interpreted the data, and wrote the manuscript. RC clinical penetrance of intracellular mutations is higher than that designed and supervised the project, provided resources, and of mutations located in the extracellular domain, as intracellular edited the manuscript. All co-authors reviewed, commented on, mutant proteins exert a dominant-negative effect, interfering and approved the final version of the manuscript. with assembly of FAS homotrimers within the cell membrane (39). Interestingly, most of the somatic missense variants reported (9 of 12, 75%) were located in the DD, where Funding missense mutations show highest penetrance (16). In summary, our study illustrates the evolutionary dynamics This study was funded by Instituto de Salud Carlos III, grant of a somatic FAS mutation before and during immunosuppressive PI20/00761, cofinanced by the European Regional Development treatment. We found that the use of high-coverage DAS enabled Fund (ERDF). detection of the somatic variant in whole blood samples regardless of the percentage of DNTs. This opens the possibility to perform genetic testing in patients with suspected somatic ALPS who are Acknowledgments receiving treatment. We demonstrated that the VAF strongly correlated with DNT counts and proposed a simple CD3+ This research is supported by the European Reference enrichment step to increase the probability of detecting true Network for Rare Immunodeficiency, Autoinflammatory and somatic variants in patients with DNTs within the reference Autoimmune Diseases (ERN-RITA). range. The capability of the proposed method will facilitate future identification of somatic FAS variants and help other researchers and clinicians on their way toward a genetic diagnosis. Conflict of interest The authors declare that the research was conducted in the Data availability statement absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The data presented in the study are deposited in the ClinVar repository. This data can be found here: Link: https://www.ncbi. nlm.nih.gov/clinvar/variation/1703254/?oq=SCV002568438&m= Publisher’s note NM_000043.6(FAS):c.718_719insGTCG%20(p.Met240fs) / accession number: SCV002568438. All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the Ethics statement reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed The studies involving human participants were reviewed and or endorsed by the publisher. approved by Ethics Review Board of Hospital Universitari Vall d’Hebron [code: PR(AG)69/2016]. Written informed consent to participate in this study was provided by the participants’ legal Supplementary material guardian/next of kin. Written informed consent was obtained from the minor(s)’ legal guardian/next of kin for the publication The Supplementary Material for this article can be found of any potentially identifiable images or data included in online at: https://www.frontiersin.org/articles/10.3389/ this article. fimmu.2022.1014984/full#supplementary-material Frontiers in Immunology 10 frontiersin.org Batlle-Masó et al. 10.3389/fimmu.2022.1014984 References 1. Rieux-Laucat F. What’s up in the ALPS. Curr Opin Immunol (2017) 49:79– 20. Xu C. 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