Clonal haematopoiesis across the age spectrum of vasculitis patients with Takayasu's arteritis, ANCA-associated vasculitis and giant cell arteritis

Abstract

Objectives: Ageing and inflammation are associated with clonal haematopoiesis (CH), the emergence of somatic mutations in haematopoietic cells. This study details CH in patients with systemic vasculitis in association with clinical, haematological and immunological parameters.

Methods: Patients with three forms of vasculitis were screened for CH in peripheral blood by error-corrected sequencing. Relative contributions of age and vasculitis on CH prevalence were calculated using multivariable logistic regression. Clonal hierarchies were assessed by proteogenomic single-cell DNA sequencing, and functional experiments were performed in association with CH status.

Results: Patients with Takayasu's arteritis (TAK; n=70; mean age=33.2 years), antineutrophil cytoplasmic antibody-associated vasculitis (AAV; n=47; mean age=55.3 years) and giant cell arteritis (GCA; n=59; mean age=71.2 years) were studied. CH, most commonly in DNMT3A and TET2, was detected in 34% (60/176) of patients versus 18% (28/151) of age-matched controls (p<0.01). Prevalence of CH was independently associated with age (standardised B=0.96, p<0.01) and vasculitis (standardised B=0.46, p<0.01), occurring in 61%, 32% and 13% of patients with GCA, AAV and TAK, respectively. Both branched and linear clonal trajectories showed myeloid-lineage bias, and CH was associated with markers of cellular activation. In GCA, mutations were detected in temporal artery biopsies, and clinical relapse correlated with CH in a dose-dependent relationship with clone size.

Conclusions: Age was more strongly associated with CH prevalence than inflammation in systemic vasculitis. Clonal profile was dominated by DNMT3A mutations which were associated with relapse in GCA. CH is not likely a primary causal factor in systemic vasculitis but may contribute to inflammation.

Keywords: Giant Cell Arteritis; Polymorphism, Genetic; Vasculitis.

Figure 1.. Clonal landscape of systemic vasculitis.

A) Oncoprint with somatic mutations identified in 176 patients with systemic vasculitis according to their age. Patients with Takayasu’s arteritis (TAK; n = 70), ANCA-associated vasculitis (AAV; n = 47), and giant cell arteritis (GCA; n= 59) are colored in the graph. The mutated gene and range of variants allele frequency (VAF), as well as their frequencies, are shown in the figure. B) Frequency of patients with clonal hematopoiesis according to their vasculitis type and variants VAFs. C) Variant allele frequency (VAF) of somatic mutations identified in peripheral blood (PB). The median VAFs and ranges are shown in the figure according to the mutated gene. D) Frequency of patients with vasculitis (TAK, AAV, and GCA) and healthy controls (HC) with multiple variants (>2 somatic mutations), or with both DNMT3A and TET2 mutations. E) CH frequency in TAK, AAV, GCA, and healthy controls (HC) according to age range and the two most commonly mutated genes, DNMT3A and TET2.

Figure 2.. Clonal dynamics and trajectories in systemic vasculitis.

A) Longitudinal analysis of clonal hematopoiesis. Somatic mutations in DNMT3A, TET2, and other genes identified in 17 patients at first visit were tracked over a median follow-up of 3.1 (± 2.1) years; all variants were mostly stable. Variants at VAFs> 5% are colored and their location is shown in the graph. B) Clonal trajectories of two patients (V053 and V639) with GCA with multiple somatic mutations in DNMT3A/TET2. Clonal hierarches, both linear and branched, of variants detected by bulk error-corrected sequencing (ECS) were assessed by single-cell proteogenomic analysis (scDNA). The frequency of different cell clusters based on the expression of protein surface markers are shown in figure according to cells’ genotype. Representative data from peripheral blood cells derived from a healthy control (HC) were used as a reference for analysis. Single cells were labeled according to their genotypes; wild-type (WT) cells were labeled as either from the healthy control (WT-HC) or V053 and V639 patients. Cells clusters were characterized according to their differentially expressed protein surface markers (normalized reads) when stained with the cocktail of 45 antibodies targeting common blood protein surface markers (TotalSeq-D Heme Oncology Cocktail antibody-oligo conjugate) and processed with the Tapestry protocol (Mission Bio). Cell populations were characterized based on a reference data from healthy samples and available Mission Bio datasets as: myeloid progenitor cells (MP; CD34lowCD38+CD117+CD123+CD45RA-CD141+CD71+CD7+CD33+CD64+), neutrophils (Net; CD16+CD62L+Lin-) with high or lower CD11b expression, monocytes (Mono; CD14+CD16+/−), plasmacytoid dendritic cells (pDC; CD14⁻CD123⁺FcεRIa⁺), and conventional DC (DC; CD14⁻CD141⁺CD11c⁺CD11b⁺), T lymphocytes (CD3+CD8+ or CD4+), B lymphocytes (CD19+), natural killer (NK; CD3-CD56+CD7). C) scDNA profiles and relative expression of specific protein markers in blood subpopulations. The Uniform Manifold Approximation and Projection (UMAP) scDNA profiles for healthy and vasculitis samples are shown in the graph. Cells from healthy and V421 were all wild-type while a subset of cells from V053 and V639 were DNMT3A/TET2 mutated. Cells’ genotypes according to their UMAP projection are shown in the figure. Bottom panels show the relative expression of markers linked to activation of monocytes, NK and T cells in cells from controls (HC), V421, V053, and V639 according to their genotype, wild-type (WT) or with CH mutations (CH).

Figure 3.. Clinical and functional impact of clonal hematopoiesis.

A) CD14+ cell stimulation experiments. Protein levels of MCP-1, MIP1α, and MIP1β were increased in supernatants of CD14+ cells from GCA patients with CH stimulated with INF-ɣ in comparison to stimulated non-mutated cells. MIP1α levels were also increased in supernatants of CD14+ cells from healthy controls with CH in comparison to non-mutated cells stimulated under different experimental conditions. B) Dose response association of CH variant allele frequency (VAF) and relapse risk of GCA patients. Higher mutations VAFs correlated with relapse in GCA. All GCA patients with CH at VAF ≥ 5% experienced relapse.