Unexpected relevant role of gene mosaicism in patients with primary immunodeficiency diseases.

BACKGROUND: Postzygotic de novo mutations lead to the phenomenon of gene mosaicism. The 3 main types are called somatic, gonadal, and gonosomal mosaicism, which differ in terms of the body distribution of postzygotic mutations. Mosaicism has been reported occasionally in patients with primary immunodeficiency diseases (PIDs) since the early 1990s, but its real involvement has not been systematically addressed. OBJECTIVE: We sought to investigate the incidence of gene mosaicism in patients with PIDs. METHODS: The amplicon-based deep sequencing method was used in the 3 parts of the study that establish (1) the allele frequency of germline variants (n = 100), (2) the incidence of parental gonosomal mosaicism in families with PIDs with de novo mutations (n = 92), and (3) the incidence of mosaicism in families with PIDs with moderate-to-high suspicion of gene mosaicism (n = 36). Additional investigations evaluated body distribution of postzygotic mutations, their stability over time, and their characteristics. RESULTS: The range of allele frequency (44.1% to 55.6%) was established for germline variants. Those with minor allele frequencies of less than 44.1% were assumed to be postzygotic. Mosaicism was detected in 30 (23.4%) of 128 families with PIDs, with a variable minor allele frequency (0.8% to 40.5%). Parental gonosomal mosaicism was detected in 6 (6.5%) of 92 families with de novo mutations, and a high incidence of mosaicism (63.9%) was detected among families with moderate-to-high suspicion of gene mosaicism. In most analyzed cases mosaicism was found to be both uniformly distributed and stable over time. CONCLUSION: This study represents the largest performed to date to investigate mosaicism in patients with PIDs, revealing that it affects approximately 25% of enrolled families. Our results might have serious consequences regarding treatment and genetic counseling and reinforce the use of next-generation sequencing-based methods in the routine analyses of PIDs.

Brief Report: First Identification of Intrafamilial Recurrence of Blau Syndrome due to Gonosomal NOD2 Mosaicism.

OBJECTIVE: Blau syndrome is characterized by noncaseating granulomatous arthritis, dermatitis, and uveitis, and results from gain-of-function NOD2 mutations. This study was undertaken to identify the genetic cause of the disease in a family with 3 members with Blau syndrome. METHODS: We studied a family with 3 affected members across 2 consecutive generations. The children's symptoms started early (at 6 and 7 months of age) and included polyarthritis, dermatitis, uveitis, and fever. In contrast, the father's symptoms started later (at 22 years of age) and included noncaseating granulomatous dermatitis and uveitis. We analyzed the NOD2 gene in all patients by both the Sanger method of DNA sequencing and amplicon-based deep sequencing using an Ion Torrent PGM platform. RESULTS: Sanger chromatograms revealed the heterozygous c.1001G>A transition in both children, which resulted in the p.Arg334Gln mutation that causes Blau syndrome. In contrast, the father's chromatograms revealed a small peak of adenine at the c.1001 position, suggesting the presence of a somatic NOD2 mutation. To evaluate this hypothesis, we performed amplicon-based deep sequencing using DNA from different tissues, which confirmed a variable degree (0.9-12.9%) of somatic NOD2 mosaicism. The previous detection of the NOD2 mutation in his daughters strongly suggests the presence of gonosomal (somatic plus gonadal) NOD2 mosaicism in the father. Comparative analyses with Blau syndrome patients carrying the germline p.Arg334Gln NOD2 mutation revealed late onset of the disease, a mild inflammatory phenotype, and an absence of complications in patients with NOD2 mosaicism. CONCLUSION: This is the first description of gonosomal NOD2 mosaicism as the cause of intrafamilial recurrence of Blau syndrome. Our findings also indicate that Blau syndrome includes more diverse and milder phenotypes than previously described.

Comparison of blood and synovial fluid th17 and novel peptidase inhibitor 16 Treg cell subsets in juvenile idiopathic arthritis.

OBJECTIVE: Early recognition and treatment of juvenile idiopathic arthritis (JIA) can prevent joint damage and minimize side effects of medication. The balance between proinflammatory and antiinflammatory mechanisms is known to be important in JIA, and we therefore investigated T cell subsets including Th cells, autoaggressive Th17 cells, and regulatory T cells (Treg), including a novel Treg subset in peripheral blood (PB) and synovial fluid (SF) of patients with JIA. METHODS: Fifty children with JIA were enrolled in our study. Frequency, phenotype, and function of T lymphocytes in PB and SF were characterized using flow cytometry. Migration capabilities of PB and SF cells were compared. RESULTS: Synovial T cells showed different phenotype and function compared with PB T cells, with an increased proportion of memory T cells, expression of CCR4, CCR5, CXCR3, interleukin 23R, and an increased ratio of Th17 to Treg. Although Treg were increased in SF compared with the PB, we found a significant decrease in the numbers of peptidase inhibitor 16 (PI16)+ Treg in active joints compared with peripheral blood. Coexpression of CCR4 and CCR6 was reduced on PI16+ Treg in PB and SF of patients with JIA compared with healthy children, however the ability of these cells to migrate toward their ligands was unaffected. CONCLUSION: This is a comprehensive characterization of novel PI16+ Treg and Th17 cells in matched blood and synovial fluid samples of patients with JIA. Despite an increased number of Treg within the inflamed joint, lower numbers of PI16+ Treg but high numbers of Th17 cells might contribute to the inability to control disease.