A New Variant in CTLA4 Highlights the Heterogeneous Phenotype of CTLA4 Haploinsufficiency

Abstract

Haploinsufficiency of cytotoxic T-lymphocyte associated protein 4 (CTLA4), a known cause of inborn errors of immunity, can lead to autoimmunity, inflammation, neoplasia and infections. A previously undescribed CTLA4 variant was identified in a patient who presented with life-threatening cutaneous infection caused by Pseudomonas aeruginosa, severe VZV infection, and Evans syndrome. Our aim was to assess the pathogenicity of the previously undescribed c.379T > G variant in the CTLA4 gene and explore its phenotypic presentation in relatives. We employed genetic and protein-based in silico analyses to evaluate the potential role of the c.379T > G variant in the CTLA4 gene. We subsequently studied CTLA4 expression ex vivo, analyzed the clinical presentation of affected carriers and compared the biological status across affected individuals, healthy carriers and noncarriers. In silico analyses revealed that the c.379T > G mutation, which is located within the ligand binding area of CTLA4, is highly pathogenic. Its clinical manifestations, which are sometimes fatal, include multiple infections, autoimmunity, inflammation of the central nervous system, lymphoproliferation and thymoma with Good's syndrome. Compared with its expression in healthy donors, the expression of CTLA4 following stimulation in memory regulatory T cells was decreased in affected individuals. Immunophenotyping revealed an increased proportion of immature and double-negative B cells in affected patients compared with their nonmutated relatives. Additionally, a reduction in naive T cells and an increase in CD4 + CD25-Foxp3 + cells were observed in carriers compared with controls. The c.379T > G variant of CTLA4, resulting in a p.Tyr127Asp substitution, is pathogenic and contributes to the development of a CTLA4 haploinsufficiency phenotype. This finding highlights the heterogeneous presentations of the disease, including oncological and neurological manifestations.

Keywords: Abatacept; Autoimmunity; Common variable immunodeficiency; Cytotoxic t-lymphocyte antigen 4; Genetics; Hypogammaglobulinemia; Immune dysregulation; Malignancy; Primary immunodeficiency.

Fig. 1

The pedigree and genetics of the family carrying the c. 379 T > G CTLA4 variant. A. Family pedigree. Squares: males; circles: females; black: affected mutation carriers; gray: unaffected mutation carriers; gray cross line: deceased patient; arrow: index case; * patient III.3: presented with lymphoid proliferation, central nervous system inflammation (transverse myelitis and extensive disseminated encephalomyelitis), epilepsy, chronic kidney disease with one transplantation (benign polyclonal B-cell infiltration, interstitial fibrosis), interstitial pneumopathy, splenomegaly, hepatic abnormality with diffuse nodular hyperplasia, rectocolitis, extensive varicella zoster virus infection (VZV), viral encephalitis without documentation, Epstein–Barr virus (EBV) chronic viremia, Clostridium difficile severe colitis. Abbreviations: ARDS, acute respiratory distress syndrome; CMV, cytomegalovirus; ENT, ear-nose-throat; D, disease; IPF, interstitial pulmonary fibrosis; Sd, syndrome; SpA, spondyloarthritis. B. Linear protein model showing the location of c.379T > G in the CTLA4 variant with the results of the in silico analysis. AF, allele frequency; CADD, combined annotation-dependent depletion; TM, transmembrane region. (C) Multiple alignments of CTLA4 orthologs, paralogs (CD28) from different species, abatacept, a recombinant CTLA4 (rCTLA4) protein and ipilimumab, an anti-CTLA4 antibody (aCTLA4 ab), using Clustal Omega software. The highly conserved and affected amino acid Y127D is highlighted in yellow. (D) Chromatogram of patient II.1. The CTLA4 sequence from a gDNA extract of a skin biopsy sample showing the heterozygotic c.379T > G mutation

Fig. 2

Clinical and radiological hallmarks of patients II.1 and III.3. (A) Confluent erythematous-squamous lesions on the back of patient II.1. (B) Heterogenicity of the skin lesions with squamous, nonconfluent, patchy lesions and plaque psoriasis on the knees of patient II.1, simultaneously with back lesions. (C) and (D) Chest computed tomography scan of patient II.1 showing bronchiectasis, honeycombing and multiple condensations suggesting acute interstitial lung disease. (E) Sagittal dorso-lumbar magnetic resonance imaging (T1 gadolinium) of the spine of patient III.3 showing a medullar lesion (red arrow) on the Th12 level suggesting transverse myelitis. (F) and (G) Axial cerebral magnetic resonance imaging (T2 FLAIR) revealing a large hypersignal of the white matter of the left frontotemporal lobe. Contralateral nodular lesions of the subcortical and profound white matter

Fig. 3

Reduced CTLA4 expression in memory regulatory T cells. (A) Relative CTLA4 expression in healthy controls and patients III.7 and II.3 with the heterozygous CTLA4 c.379T > G mutation (results of two different experiments). Relative expression was calculated as the fold change in the mean fluorescent intensity (MFI) of CTLA4 between naive conventional T cells (nTconv) and memory regulatory T cells (mTreg). (B) Relative CTLA4 expression in healthy controls and patients III.7 and II.3 (the results of two different experiments) after stimulation. The relative expression was calculated as described in Fig. 1.A. (C) Comparison of the percentage of Foxp3 + cells among stimulated CD4 + T cells between CTLA4-mutated patients (II.3 and III.7) and healthy controls (results of two different experiments)