Autoimmune lymphoproliferative syndrome (ALPS, OMIM #601859) is characterized by a lack of control over lymphocyte apoptosis. This results in an elevated number of lymphocytes, which can accumulate and lead to enlarged lymph nodes, liver, and spleen. The main clinical features in ALPS are lymphadenopathy, hepatomegaly, splenomegaly, and autoimmune cytopenia. There is also an increased risk for developing severe health conditions such as autoimmune diseases and hematological malignancies (Hodgkin and non-Hodgkin lymphoma) (1).

The diagnosis of ALPS is based on two major criteria: elevated (>2.5%) CD3⁺ TCRαβ⁺ CD4^-CD8^- T cells (hereafter referred to as double-negative T cells, [DNTs]), and chronic nonmalignant/noninfectious lymphoproliferation. The accessory criteria include defective lymphocyte apoptosis, presence of pathogenic genetic variants, elevated levels of soluble molecules such as FAS ligand, IL10 and vitamin B12, typical immunohistological findings, autoimmune cytopenia, and a positive family history (2). Immunosuppressive drugs, such as steroids, mycophenolate and the mTOR inhibitor rapamycin (sirolimus), are good treatment options for ALPS patients (3), as they effectively reduce cytopenia and lymphoproliferation. However, patients should be monitored to control drug-related toxicity. Hematopoietic stem cell transplantation (HSCT) has been successful for curing severe ALPS, but it is a complex procedure and the risk/benefit balance must be carefully evaluated in each patient (4).

In most cases, the molecular origin of ALPS is associated with pathogenic germline variants in the FAS gene, which encodes the FAS cell surface death receptor (5, 6). Most of these variants are monoallelic (autosomal dominant), affect the intracellular death domain (DD) region, and exert a dominant-negative effect related to the trimeric nature of the FAS protein (7). Heterozygous loss-of-function variants leading to FAS haploinsufficiency without a dominant negative effect are a less frequent cause. These variants are usually located in the FAS extracellular region and are associated with milder clinical manifestations and lower penetrance (8). In addition, biallelic germline variants in FAS have been reported in severe early-onset ALPS patients (6, 9). In these cases, heterozygous carriers were asymptomatic, indicating that some pathogenic FAS variants behave as recessive alleles whereas others are dominant (1).

Pathogenic germline variants in other genes affecting the FAS-related apoptosis pathway have been reported as a cause of ALPS in a minor fraction of patients: FASLG (FAS ligand) (10), FADD (FAS-associated death domain protein) (11), CASP8 (caspase 8) (12), and CASP10 (caspase 10) (13). However, the role of CASP10 variants as a causative versus predisposing factor in ALPS is still controversial (14).

After germline FAS mutations, heterozygous somatic FAS mutations are the second most common genetic etiology of ALPS, accounting for up to 15% of patients (15). Several features of somatic FAS variants have been reported to date: 1) They appear in hematopoietic progenitors and are predominantly detected in DNTs, although they are also present in smaller proportions in other lymphoid and myeloid lineages (15); 2) They are mainly located in exons 7, 8 and 9 of the FAS gene, encoding the intracellular domain (16); and 3) They cause clinical phenotypes indistinguishable from those caused by germline FAS variants (17).

To further complicate the molecular basis of ALPS, other inheritance patterns have been described, including the double-hit hypothesis involving “second-hit” variants in the FAS gene itself or in other disease-modifying genes (18).

New sequencing technologies, such as deep amplicon sequencing, have enabled better and more comprehensive detection of true pathogenic somatic variants in next-generation sequencing (NGS) data (19). Optimized callers for somatic variation are being developed and improved (20), and although somatic variants have been traditionally investigated in 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 in whom a novel pathogenic somatic variant was identified and studied over five years. We evaluated the dynamics of the variant, the DNT population, and the patient’s clinical course, focusing on future application of this strategy in a discovery setting for patients with no available pretreatment samples. In addition, we performed a literature review to gather all pathogenic somatic variants associated with ALPS to facilitate identification of future novel variants. Our results provide additional information on the genetic bases of the disease and clues to improve diagnostic rates and validate the effectiveness of treatment at the molecular level.

Study of somatic variants underlying inborn errors of immunity (IEI) is gaining increasing interest due to biological, technological, and clinical reasons (30). In 2004, ALPS was the first IEI in which somatic mosaicism was reported as the cause of the disease (15). Since then, other IEIs caused by somatic mutations have been described, with autoinflammatory disorders having the highest incidence (19, 30). Many IEIs resulting from mosaicism are also germline disorders, as in the case of most autoinflammatory disorders and ALPS, whereas few IEIs are predominantly caused by somatic mutations (eg, VEXAS syndrome, TLR8 gain-of-function, and Ras-associated autoimmune leukoproliferative disorder) (31–33).

In this study, we identified a novel somatic mutation in FAS by Sanger sequencing of purified CD3⁺ cells and followed the evolutionary dynamics of the variant over time with DAS. Detection of somatic variants at low VAF using classic molecular screening methods such as Sanger is challenging. 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 double peak pattern, although the height was small, was highly suspicious for a somatic variant. Moreover, it was specific to CD3⁺ cells and not present in whole blood. However, the single small peak of a single nucleotide variant could be easily misinterpreted as background noise. Hence, we believe that Sanger sequencing on purified CD3⁺ cells may not be sensitive enough for use as a routine diagnostic method in these cases.

NGS-based methods are highly suitable for detecting somatic variants, although their precision and robustness relies on the read depth. Whole-exome sequencing (WES) and whole-genome sequencing (WGS) are intended to identify germline variants, as their mean coverage is usually <200x and <100x, respectively. They can also be used for somatic variant identification, but typically cannot easily detect those at <10% VAF. Targeted gene panels usually have a higher overall read depth (200x-500x). Recently, López-Nevado et al. used a targeted gene panel in whole blood samples to detect germline and somatic FAS mutations in ALPS patients (28). These authors identified a pathogenic somatic FAS variant in a patient who had no pathogenic germline variants in the panel. To validate the somatic variant call, they used blood DNA from seven other ALPS patients previously diagnosed with somatic FAS variants. In all patients but one, they were able to detect somatic variants in a VAF range of 1.9% to 11.5% with a variable read depth in the specific position (101x to 399x). Interestingly, the single patient with a previously identified somatic FAS variant that went undetected with this method had DNT counts within the reference range. In the present study, the higher-depth NGS approach (DAS coverage >20,000x) enabled detection of the somatic variant in blood samples containing a very low percentage of DNTs (<1%). The most striking example is detection of the somatic variant at 0.67% VAF in a sample obtained after five years of immunosuppressive treatment. This is a proof of concept that pathogenic somatic FAS variants can be detected in whole blood of ALPS patients regardless of their DNT counts with the use of DAS. For that purpose, high coverage of the target gene or exons is needed to ensure identification of variants having a low percentage of supporting reads. In addition, specific bioinformatic processing of the data is required to avoid filtering of true somatic variants by conventional germline-focused strategies. Various somatic variant callers have been created and compared in this regard (20, 34, 35). These are especially useful when the number of candidate genes or exons is not suitable for manual analysis.

One helpful finding obtained here is that a simple CD3⁺ enrichment step may be useful to enhance the sensitivity and reliability of NGS for somatic FAS variant detection. The VAF of the somatic variant in our patient was 2.5-fold higher in the CD3⁺ sample than in whole blood, whereas the background noise (artefactual variants) was similar in the two samples. Thus, the probability that true somatic variants would be detected increased after enrichment.

Traditionally, the genetic diagnosis of somatic variants in ALPS has been done by sorting and sequencing DNTs (15, 36). Nonetheless, this is a time- and resource-consuming strategy that may not be available in all centers. López-Nevado et al. demonstrated that somatic FAS variants can be detected by NGS-based targeted gene panel analysis in blood of patients with elevated DNTs (28). However, their results showed that the depth of coverage with this approach may not suffice to disclose variants for which only a few reads support the alternative allele (low VAF). We found that NGS-based DAS can detect somatic variants in ALPS patients even when DNTs are within the reference range. In our opinion, a targeted gene panel would be an appropriate initial genetic approach for patients with ALPS suspicion, since it has the advantage of covering germinal mutations in FAS (and other ALPS-related genes) and it may detect somatic variants in some cases. Nevertheless, their limited sensitivity may not detect somatic variants at low VAF. Therefore, in case of negative results in a targeted gene panel, DAS should be performed since this technique increases the likelihood of finding the somatic variants, especially at low VAF. In both targeted gene panel analysis and DAS analysis, the PCR steps may introduce sequence errors that confound identification of somatic variants with a very low VAF (<1%-2%). To overcome this limitation in our DAS technique, we applied the quality scores provided by VarScan 2, to identify variants and distinguish true somatic variants from background noise. It is likely that use of CD3⁺-enriched samples and more affordable coverage (5000x) will enable identification of somatic variants in a diagnostic setting. In addition, the method could be useful for patients who have started immunosuppressive treatment without a molecular diagnosis. This opens the door to revisiting previously unsolved cases and may increase the genetic diagnosis rate in pediatric ALPS.

To our knowledge, this is the first reported description of the evolutionary dynamics of a somatic FAS mutation in ALPS. We detected the pathogenic somatic variant in all six blood samples collected over a five-year period, including those obtained during sirolimus treatment, which had normal DNT counts. Our results indicate that with a proper approach, genetic assessment of somatic FAS variants in ALPS patients can be performed at any time over the clinical course, as immunosuppressive treatment did not eliminate all mutated cells, although their numbers in peripheral blood were considerably reduced. This is consistent with the fact that somatic mutations initially occur in hematopoietic progenitor cells (HPCs) and become enriched in DNTs due to a selective survival advantage (15). HPCs provide a constant supply of mutated descendant cells that are impossible to eliminate with immune suppression (the only known cure is HSCT); thus, the need for chronic treatment in ALPS patients (4, 37). We observed a strong positive correlation between DNT count and the VAF. This finding confirms that somatic FAS mutations are mainly restricted to DNTs, although they can be found in varying percentages in other leukocyte subsets (15).

The pathogenic somatic variant reported in this study (c.718_719insGTCG, p.Met240SerfsX8) is located in the intracellular region, within the DD. This domain is essential for formation of the death-inducing signaling complex (DISC), which triggers the extrinsic apoptosis pathway through stimulation by death ligands (38). We reviewed all reported somatic FAS mutations and found 40 variants. The majority (34 of 40, 85%) were located in the intracellular domain of the protein and 15 (37.5% of the total) specifically in the DD. Few variants were found in the extracellular or transmembrane domains (4 and 2 respectively, 10% and 5% of the total). The clinical penetrance of intracellular mutations is higher than that of mutations located in the extracellular domain, as intracellular mutant proteins exert a dominant-negative effect, interfering 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 missense mutations show highest penetrance (16).

In summary, our study illustrates the evolutionary dynamics of a somatic FAS mutation before and during immunosuppressive treatment. We found that the use of high-coverage DAS enabled 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 receiving treatment. We demonstrated that the VAF strongly correlated with DNT counts and proposed a simple CD3+ enrichment step to increase the probability of detecting true somatic variants in patients with DNTs within the reference 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.

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=NM_000043.6(FAS):c.718_719insGTCG%20(p.Met240fs) / accession number: SCV002568438.

The studies involving human participants were reviewed and 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 guardian/next of kin. Written informed consent was obtained from the minor(s)’ legal guardian/next of kin for the publication of any potentially identifiable images or data included in this article.

LB-M performed the bioinformatics analysis of NGS data, somatic variant calling, and the literature review. MG-P, AP-M, and CF-J performed laboratory analyses and CD3⁺ purification. MM-G performed flow cytometry studies. AA-C performed sample collection and Sanger sequencing. PV, JR, AM-N, and PS-P provided patient care, and collected and provided clinical data. MA was responsible for the NGS technical process, including library preparation and sequencing. LB-M and RC analyzed and interpreted the data, and wrote the manuscript. RC designed and supervised the project, provided resources, and edited the manuscript. All co-authors reviewed, commented on, and approved the final version of the manuscript.

This study was funded by Instituto de Salud Carlos III, grant PI20/00761, cofinanced by the European Regional Development Fund (ERDF).

This research is supported by the European Reference Network for Rare Immunodeficiency, Autoinflammatory and Autoimmune Diseases (ERN-RITA).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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