Protein functionality as a potential bottleneck for somatic revertant variants
Authors: Fabian M.P. Kaiser, Ismail Reisli, Ingrid Pico-Knijnenburg, Anton W. Langerak, François G. Kavelaars, Hasibe Artac, Hanna IJspeert, Mirjam van der Burg
Journal: Journal of Allergy and Clinical Immunology (2020)

Somatic genome stability has been considered the rule, rather than the exception; yet, somatic mutations are driving factors in many diseases with a genetic background. Conversely, somatic reversion of germline mutations has been shown to mitigate pathogenicity by partially or completely restoring gene function. Such somatic revertant variants have been observed across a broad spectrum of immunodeficiencies and often result in atypical phenotypes that present a diagnostic and therapeutic challenge.1Mensa-Vilaro A. Bravo Garcia-Morato M. de la Calle-Martin O. Franco-Jarava C. Martinez-Saavedra M.T. Gonzalez-Granado L.I. et al.Unexpected relevant role of gene mosaicism in patients with primary immunodeficiency diseases.J Allergy Clin Immunol. 2019; 143: 359-368Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar In the majority of cases, one or a few reverting somatic variants have been identified per patient and the limitations of this "natural form of gene therapy" are unknown.1Mensa-Vilaro A. Bravo Garcia-Morato M. de la Calle-Martin O. Franco-Jarava C. Martinez-Saavedra M.T. Gonzalez-Granado L.I. et al.Unexpected relevant role of gene mosaicism in patients with primary immunodeficiency diseases.J Allergy Clin Immunol. 2019; 143: 359-368Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar We describe a 1-year-10-month-old patient with a germline-encoded, homozygous 2 bp deletion in the CD247 gene (CD247 c.43_44delCA; p.Gln15ValfsTer72) (Fig 1, A and B), which encodes for the CD3ζ chain, and dramatically reduced T-cell numbers (Fig 1, C and D; see Fig E1 and Table E1 in this article's Online Repository at www.jacionline.org) combined with a relatively mild clinical phenotype (see the Case Report in this article's Online Repository at www.jacionline.org). The mutation maps to the signal peptide of the protein and results in a premature stop codon at amino acid position 72. CD3ζ is required for assembly of T-cell receptor (TCR) complexes and the subsequent shuttling to the cell membrane.2Call M.E. Wucherpfennig K.W. The T cell receptor: critical role of the membrane environment in receptor assembly and function.Annu Rev Immunol. 2005; 23: 101-125Crossref PubMed Scopus (145) Google Scholar Given the essential role in TCR expression, CD3ζ deficiency presents as a classic T−/B+/NK+ severe combined immunodeficiency with reduced peripheral T lymphocytes devoid of TCR/CD3 surface coexpression.3Rieux-Laucat F. Hivroz C. Lim A. Mateo V. Pellier I. Selz F. et al.Inherited and somatic CD3zeta mutations in a patient with T-cell deficiency.N Engl J Med. 2006; 354: 1913-1921Crossref PubMed Scopus (91) Google Scholar,4Roberts J.L. Lauritsen J.P. Cooney M. Parrott R.E. Sajaroff E.O. Win C.M. et al.T-B+NK+ severe combined immunodeficiency caused by complete deficiency of the CD3zeta subunit of the T-cell antigen receptor complex.Blood. 2007; 109: 3198-3206Crossref PubMed Scopus (53) Google Scholar Unexpectedly, we identified a small population of T cells with TCRαβ/CD3 coexpression in the patient (Fig 1, E and F). We hypothesized that the mild clinical phenotype was caused by a somatic reversion of the mutation in this population and therefore performed deep sequencing of the first exon of the CD247 gene in CD3+, TCRαβ+ T cells to identify potential revertant variants. We discovered 52 unique somatic variants of which the majority of 49 restored the reading frame of the gene and thus prevented the premature stop during translation (see Fig E2 and Table E2 in this article's Online Repository at www.jacionline.org). The variants were mostly located downstream of the CD247 c.43_44delCA mutation within the region that encodes the signal peptide and mainly encompassed in-frame additions of 2 or 5 nucleotides (Fig 2, A, Fig E2). To follow up, we repeated the analysis with a sample that had been obtained 1 year later at the age of 2 years and 10 months. At this time point, we detected 23 somatic variants, including 9 novel variants that had not been present earlier (Fig 2, A, Fig E2, Table E2). Twenty variants restored the disrupted reading frame of the protein. The disease-causing germline mutation was found in approximately one half of the reads of the CD3+ population at both time points (Table E2), suggesting that most CD3-expressing T cells were compound heterozygous for the germline mutation and a somatic variant, which is also reflected by the reduced CD3 and TCR expression (Fig 1, G and H). Of note, we only detected a few rare somatic variants in the CD3− T-cell population and none reverted the germline mutation (see Table E3 in this article's Online Repository at www.jacionline.org). Furthermore, we could only identify the germline mutation in the natural killer cells of the patient and no variants in T cells of healthy controls (see Table E4 in this article's Online Repository at www.jacionline.org).Fig 2Complex spectrum of somatic reverting variants in CD3+, TCRαβ+ T cells. A, Distribution of insertions and deletions in CD3+, TCRαβ+ T cells at both time points, referred to as T1 (n = 52) and T2 (n = 23). B, Identical somatic revertant variants based on biochemical characteristics of amino acids with indicated frequency of recurrence for both time points. Amino acids are symbolized as indicated in the legend. C, Somatic variants that restore the reading frame of the protein at both time points with likelihood of signal peptide function and relative frequency at given time point. D, Percentage of somatic variants that restore the reading frame of the protein and relative distribution at both time points. Upward and downward pointing arrows indicate relative increase or decrease between both time points, respectively. The number of each variant is based on Fig E2.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Signal peptides serve to shuttle proteins to the secretory pathway, membranous cellular compartments, or the plasma membrane prior to their cleavage from the mature protein.5von Heijne G. The signal peptide.J Membr Biol. 1990; 115: 195-201Crossref PubMed Scopus (827) Google Scholar,6Harter C. Wieland F. The secretory pathway: mechanisms of protein sorting and transport.Biochim Biophys Acta. 1996; 1286: 75-93Crossref PubMed Scopus (48) Google Scholar Interestingly, signal peptides usually do not have a fixed primary sequence, but begin with a few positively charged amino acids followed by a long stretch of hydrophobic residues. Most somatic variants retained such a characteristic distribution of amino acids despite a difference in the primary structure and several variants were found recurrently (Fig 2, B, Fig E2). Bioinformatic analysis of each somatic variant confirmed that the majority of variants conferred adequate signal peptide function, based on which we consider them somatic reverting variants (Fig 2, C, Fig E2).7Almagro Armenteros J.J. Tsirigos K.D. Sonderby C.K. Petersen T.N. Winther O. Brunak S. et al.SignalP 5.0 improves signal peptide predictions using deep neural networks.Nat Biotechnol. 2019; 37: 420-423Crossref PubMed Scopus (595) Google Scholar These observations raise the question at which stage the revertant variants had arisen. Given that the spectrum of variants decreased after 1 year (Fig 2, D, Fig E2), one could speculate that dominant clones with the potential for self-renewal had been selected. However, the spectrum of variants alternated between the 2 time points and all except for 1 were found at a lower frequency in the second sample (Fig 2, D, Fig E2), suggesting that the generation of T cells with revertant variants was an ongoing process that occurred at later stages of development. In addition, varying antigen exposure could have shaped the distribution of functional T-cell clones with somatic revertants. Somatic mosaicism caused by revertant variants has been observed in many cases of severe combined immunodeficiency—including several reports on CD247 deficiency—but never to this extreme extent, which may be explained by the lack of a deep-sequencing–based approach.3Rieux-Laucat F. Hivroz C. Lim A. Mateo V. Pellier I. Selz F. et al.Inherited and somatic CD3zeta mutations in a patient with T-cell deficiency.N Engl J Med. 2006; 354: 1913-1921Crossref PubMed Scopus (91) Google Scholar,8Blazquez-Moreno A. Perez-Portilla A. Agundez-Llaca M. Dukovska D. Vales-Gomez M. Aydogmus C. et al.Analysis of the recovery of CD247 expression in a PID patient: insights into the spontaneous repair of defective genes.Blood. 2017; 130: 1205-1208Crossref PubMed Scopus (5) Google Scholar,9Marin A.V. Jimenez-Reinoso A. Briones A.C. Munoz-Ruiz M. Aydogmus C. Pasick L.J. et al.Primary T-cell immunodeficiency with functional revertant somatic mosaicism in CD247.J Allergy Clin Immunol. 2017; 139: 347-349.e8Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar We believe that the unique location of the disease-causing mutation in the signal peptide sequence is integral to understanding the pathogenicity of this case. Disease-causing germline mutations are mostly located in regions that are integral to protein function. In order to revert the deleterious effects of such a mutation, somatic reverting variants must therefore either directly restore the primary sequence of the protein, encode for an amino acid with similar properties, or remove an adjacent nonsense or frameshift mutation without affecting the functionality of the domain. Because signal peptides are cleaved from most mature proteins and lack a highly conserved motif,5von Heijne G. The signal peptide.J Membr Biol. 1990; 115: 195-201Crossref PubMed Scopus (827) Google Scholar they may tolerate a certain degree of mutations as long as the reading frame of the mature protein remains unaffected, which we have observed in our case for most revertant variants. We therefore postulate that the unique location of the germline mutation is key to unmasking the striking spectrum of revertant mutations within the T-cell pool. The true degree of mosaicism may be difficult to assess as the majority of variants in coding regions could potentially alter protein function and would therefore subject the mutated cell to positive or negative selection, thereby affecting its frequency within the cell pool. However, if the affected domain can buffer a certain degree of genetic variability—such as signal peptides—the true spectrum of mosaicism may be unmasked. We hypothesize that somatic revertant variants in highly proliferative tissues—such as the hematopoietic system—are common and suggest that somatic revertant variants occur frequently in the slipstream of pathogenic germline mutations, but are limited by the necessity for protein functionality, which acts as the bottleneck. We thank the patient and the family for their participation in the study. We are indebted to Barbara Barendregt and Peter Valk for technical support. The research for this manuscript was performed within the framework of the Erasmus Postgraduate School Molecular Medicine. Peripheral blood and clinical data were obtained according to the guidelines of the Medical Ethics Committees of Meram Medical Faculty at Selçuk University in Konya and the Erasmus Medical Center in Rotterdam. The family gave written informed consent to the study. Detailed information regarding the methodology follows. Flow cytometric analysis and fluorescence-activated cell sorting (FACS) was performed with antibodies against CD3 (FITC; clone SK7; BD Biosciences, San Jose, Calif), CD19 (PerCP-Cy5,5; clone SJ25C1; BD Biosciences); CD16 (PE; clone 3G8; BD Biosciences); CD56 (APC; N901 clone NKH1; Beckman Coulter Immunotech; Marseille, France); CD4 (BV510; clone OKT4; BioLegend, San Diego, Calif); CD8 (APC-AF750; clone B9.11; Beckman Coulter, Brea, Calif); TCRαβ (PE; clone IP26A; Beckman Coulter). Flow cytometric analysis and FACS were performed on a BD FACSCanto II cell analyzer and a BD FACSAria III sorter (BD, Franklin Lakes, NJ), respectively. Data analysis was performed with FlowJo (version 10; BD). Candidate genes (CD3D, CD3E, CD3G, and CD247) were amplified by PCR with AmpliTaq Gold DNA Polymerase (Thermo Fisher Scientific, Waltham, Mass) and subjected to Sanger sequencing as part of routine diagnostics. Sorted CD3+, TCRαβ+ T cells were centrifuged in a microcentrifuge tube and the cell pellet was resuspended in 20 μL lysis buffer (10 mmol/L Tris-HCl [pH 7.6], 50 mmol/L NaCl, 6.25 mmol/L MgCl2, 0.045% NP40, 0.45% Tween-20). A total volume of 1 μL proteinase K (20 mg/mL) was added and samples were incubated for 1 hour at 56°C before heat-inactivation for 15 minutes at 95°C. Exon 1 of CD247 was amplified by PCR and subjected to deep sequencing on either a 454 GS junior instrument (Roche, Branford, Conn) or the MiSeq System (Illumina, San Diego, Calif). For Roche 454 sequencing, exon 1 and flanking parts of the intron were amplified using primers that were adapted with Roche Lib-A adapters and sample-specific multiplex identifier (MID) tags: Forward: 5′-CAGACAGATACATACACACACCCCAA-3′ Reverse: 5′-AAGGAGACCCCAGCCCCTCAC-3′ PCR products were purified by means of gel extraction using the QIAgen Gel Extraction Kit (Qiagen, Hilden, Germany) and Agencourt AMPure XP beads (Beckman Coulter). Subsequently, DNA concentrations of the libraries were measured using the Quant-iT Picogreen dsDNA Assay Kit (Invitrogen, Thermo Fisher Scientific). For the MiSeq System, the coding region of CD247 was amplified by PCR over 30 cycles with the following primers: Forward: 5′-ACACTCTTTCCCTACACGACGCTCTTCCGATCTCAGCCTCTTTCTGAGGGAAA-3′ Reverse: 5′-TCGCGAGTTAATGCAACGATCGTCGAAATTCGCTCACTTGCCCATTGATTTGA-3′ Subsequently, PCR products were purified using Ampure XP beads (Beckman Coulter) followed by a nested PCR reaction (10 cycles) to include the sample-specific indices and Illumina sequencing adapters, using primers from the Truseq Custom Amplicon Index Kit (Illumina). The final concentrations of the libraries were measured using the Quant-it Picogreen dsDNA Assay Kit (Invitrogen). Libraries were paired-end sequenced (2 × 221 bps) on a MiSeq System with use of a MiSeq Reagent Kit v3, according to the manufacturer's protocol (Illumina). Paired sequences were merged using paired-end read merger (PEAR) to create a FASTQ file of each sample.E1Zhang J. Kobert K. Flouri T. Stamatakis A. PEAR: a fast and accurate Illumina Paired-End reAd mergeR.Bioinformatics. 2014; 30: 614-620Crossref PubMed Scopus (2151) Google Scholar Sequences were filtered and analysed using Microsoft Excel 2016. Only sequences with an exact match of the first and last 8 nucleotides of the coding region of exon 1 were included for analysis. Per variant, the frequency of variant reads and the average quality score per base were calculated. Only variant reads that were present in >0.1% of reads with a minimum average quality score above 20 were included for analysis. All detected variants were compared to the reference sequence and analyzed by 2 persons individually. Prediction of signal peptide function was performed with the software SignalP 5.0 according to the authors' instructions (http://www.cbs.dtu.dk/services/SignalP/).E2Almagro Armenteros J.J. Tsirigos K.D. Sonderby C.K. Petersen T.N. Winther O. Brunak S. et al.SignalP 5.0 improves signal peptide predictions using deep neural networks.Nat Biotechnol. 2019; 37: 420-423Crossref PubMed Scopus (1295) Google Scholar We describe a girl of consanguineous parents with a history of recurrent infections and lymphopenia that was admitted to our hospital at the age of 1 year and 10 months with severe tachypnea, productive coughing, and fever. Prior to admission, the patient had suffered from recurring bouts of oral moniliasis, sinusitis, otitis media, and pneumonia. She regularly required intravenous antibiotic therapy for the control of her infections. After BCG vaccination, she developed suppurative lymphadenitis. Her mother and father had not shown any signs of immunodeficiency. Her paternal aunt had passed away as an infant due to unknown reasons. On physical examination, she was severely tachypneic (64 breaths/min) and showed diffuse bilateral crackles on pulmonary auscultation. Her tonsils and lymph nodes did not show any gross alterations in size. Her height was 84 cm (25%-50%), her weight was 11 kg (10%-25%), and her head circumference was 46.5 cm (50%-75%). The white blood cell count was 9.9 × 109/L [6.0-17.0 × 109/L] with 62% neutrophils (15%-46%), 32% lymphocytes (45%-76%), 6% monocytes (3%-6%), and a platelet count of 380 × 109/L [150-450 × 109/L]. T-cell counts were low, but B-cell and NK-cell counts were normal (Table E1). Her serum immunoglobulins were 10 g/L IgG (6.05-14.3 g/L), 1.56 g/L IgA (0.3-10.7 g/L), and 2.32 g/L IgM (0.6-2.2 g/L). Lymphoblastic transformation in response to phytohemagglutinin was normal, but she showed no reaction in the tuberculin skin test despite BCG vaccination. TCRβ spectratyping unveiled a restricted, yet polyclonal pattern (Fig E1).E3van Dongen J.J. Langerak A.W. Bruggemann M. Evans P.A. Hummel M. Lavender F.L. et al.Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 Concerted Action BMH4-CT98-3936.Leukemia. 2003; 17: 2257-2317Crossref PubMed Scopus (2371) Google Scholar A chest computed tomography scan showed bilateral pneumonic consolidation and a small thymus. On admission, she received intravenous antibiotic treatment and was supplemented with intravenous immunoglobulins, which substantially improved her condition. Afterward, she received prophylactic treatment with trimethoprim/sulfamethoxazole and did not develop additional severe infections. Whereas the majority of CD4+ and CD8+ T cells showed neither CD3 nor TCR expression, a small subset expressed reduced levels of the CD3 complex and TCRαβ. Given the essential role of the CD3 complex in TCR surface expression and signal transduction, we performed targeted DNA sequencing of potential candidate genes and identified a homozygous 2 bp deletion in CD247 (CD247 c.43_44delCA; p.Gln15ValfsTer72) that is located in the N-terminal signal peptide of the protein and results in a premature stop codon at amino acid position 72. Six months after admission, she developed a generalized maculopapular rash, neutropenia, and bilateral arthritis in the ankles. Her clinical presentation was accompanied by an elevated erythrocyte sedimentation rate (74 mm/h) and increased CRP levels (19.1 mg/dL). Antinuclear antibodies were not detected. Serology unveiled Borrelia burgdorferi–specific IgM. In the bone marrow, we detected a reduction of mature neutrophils. She was treated with penicillin for 2 weeks. Given that the symptoms did not improve after 6 weeks, she was diagnosed with juvenile rheumatoid arthritis and treated with ibuprofen. Three months after initiation of treatment, her symptoms waned. Shortly thereafter, she developed arthritis in the ankles, wrists, and fingers. She was treated with ibuprofen, corticosteroids, and cyclosporine, which substantially improved her condition. At the age of 7 years, the patient developed autoimmune hemolytic anemia and thrombocytopenia. She was diagnosed with Evans syndrome and successfully treated with corticosteroids, intravenous immunoglobulins, and cyclosporine. At the age of 9 years, she developed cervical lymphadenopathy and was diagnosed with stage 4 non-Hodgkin lymphoma. She was treated according to the LMB89 Group B protocol. Four months after therapy, she developed a cervical mass that was confirmed as a relapse. She received treatment with rituximab, ifosfamide, carboplatin, and etoposide (ICE protocol) for 2 months and underwent bone marrow transplantation at the age of 10 years, using a nonrelated, partially HLA-mismatched (9/10 matched) donor and carmustine, etoposide, and cytarabine as the conditioning regime. She received cyclosporine and methotrexate for graft-versus-host disease prophylaxis. Two weeks after transplantation, she developed cutaneous and intestinal graft-versus-host disease and was treated with tacrolimus and corticosteroids for 6 months. Two years post bone marrow transplantation, she has 100% donor chimerism and received all appropriate vaccinations. She has remained well and did not show any signs of relapse.Fig E2Somatic variants that restore the reading frame in CD3+, TCRαβ+ T cells. Spectrum of functional revertant variants that restore the reading frame of the protein with indicated frequency at each time point, amino acid sequence that differs from WT protein, and signal peptide analysis. Amino acids are symbolized as white circles for moderate hydropathy, blue circles for hydrophilic amino acids, and red circles for hydrophobic amino acids. Black outline denotes neutral charge, whereas blue and red outlines refer to positive and negative charges, respectively. Signal peptide analysis was performed as recommended for SignalP 5.0, including likelihood of signal peptide function, cleavage site, and probability of cleavage. Variants with signal peptide score <0.5 are not predicted to be signal peptides.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table E1WBC count and lymphocyte subsets at admissionVariablePatient (× 109/L)Normal range (× 109/L)WBCs7.96.0-17.0Neutrophils6.1 (77%)1.5-8.5 (15%-46%)Lymphocytes1.2 (15%)1.5-9.5 (45%-76%)Monocytes0.6 (7.6%)0.2-1.2 (3%-6%)Platelets380150-450T cells (CD3+)0.31.4-8.0T helper cells (CD3+, CD4+)0.250.9-5.5Cytotoxic T cells (CD3+, CD8+)0.010.4-2.3B cells (CD19+)0.50.6-3.1NK cells (CD16+, CD56+)0.30.1-1.4NK, Natural killer; WBC, white blood cell. Open table in a new tab Table E2Somatic variants that do not restore the reading frame in CD3+, TCRαβ+ T cellsNumberSequenceT1 (%)T2 (%)Position of stop codon1c.43_44delCA (germline mutation)54.1659.58722c.43_44delCA, c.50_51insC1.150.43513c.43_44delCA, c.51_52insG0.160.00514c.5A>G, c.43_44delCA0.180.00725c.43_44delCA, c.52_53insCGA0.000.21736c.43_44delCA, c.49_50insG0.000.1551 Open table in a new tab Table E3Somatic variants in CD3−, TCRαβ− T cellsNumberSequenceFrequency (%)1c.43_44delCA (germline mutation)98.272c.5A>G, c.43_44delCA0.203c.11A>G, c.43_44delCA0.194c.14C>A, c.43_44delCA0.145c.16C>T, c.43_44delCA0.246c.22A>G, c.43_44delCA0.107c.26C>T, c.43_44delCA0.328c.30C>T, c.43_44delCA0.319c.32T>C, c.43_44delCA0.1310c.38A>G, c.43_44delCA0.10 Open table in a new tab Table E4Overview of next-generation sequencing dataSampleCell populationSequencing platformTotal readsRead count after filteringNumber of variantsReads with c.43_44delCA variant (%)Reference sequence (%)PatientTCRαβ−, CD3− T cellsRoche 45422,41720,4141098.270PatientTCRαβ−, CD3− T cells at T1Roche 45424,80823,1895354.200PatientTCRαβ−, CD3− T cells at T2Roche 45423,23221,6872459.720PatientNK cellsIllumina102,11898,23901000Control 1T cellsIllumina32,84231,93500100Control 2T cellsIllumina45,42144,17700100Control 3T cellsIllumina33,73432,77400100Control 4T cellsIllumina33,31232,38000100Control 5T cellsIllumina34,72033,73600100Control 6T cellsIllumina31,68830,83000100Control 7T cellsIllumina26,58725,85100100 Open table in a new tab NK, Natural killer; WBC, white blood cell.