Use of single cell DNA sequencing to characterize somatic mosaicism in a patient with two signal transduction and activator of transcription 1 (STAT1) gain-of-function variants

Abstract

There is increasing recognition that immune defects caused by somatic mosaicism are often clinically indistinguishable from classical germline inborn errors of immunity. Here we demonstrate the utility of single cell DNA sequencing in defining the cellular genotypes and specific cellular populations involved in the pathogenesis of immune dysregulation in a patient with two novel STAT1 variants and a clinical presentation consistent with STAT1 gain-of-function.A 5-month-old male presented with Pneumocystis jiroveci pneumonia, CMV viremia, and immune-mediated hepatitis. Lymphocyte subset analysis was not consistent with a combined immunodeficiency. Sequencing of genomic DNA using targeted panel of 407 inborn error of immunity genes revealed two heterozygous variants in the coiled-coil region of STAT1 (c.496_504del, p.Leu166_Asp168del; c.489_497del, p. Glu164_Leu166del). Functional analysis was consistent with STAT1 gain-of-function – patient peripheral blood mononuclear cells displayed increased IFN-alpha and IFN-beta-mediated STAT1 phosphorylation, and increased expression of downstream IFN-stimulated genes including IFI44, ISG15, and Ly6E.Further examination of the patient’s STAT1 sequencing data revealed that the two in-frame deletion variants were present on distinct alleles, and surprisingly, that a WT allele was also detected. Based on the presence of three distinct alleles, somatic mosaicism was suspected. Parental testing failed to detect either variant, suggesting that the variants were de novo. Sequencing of DNA extracted from a buccal swab of the patient revealed only wild type STAT1 sequence.Single cell DNA sequencing analysis demonstrated that the two in-frame deletions comprised three different genotypes –a compound heterozygous population comprising 9% of leukocytes, a heterozygous p. Leu166_Asp168del/wild type population comprising of 82% of leukocytes, and a WT population comprising 9% of leukocytes. This analysis additionally revealed that all three genotypes were uniformly distributed among both myeloid and lymphoid populations. Functional analysis of the two in-frame deletions demonstrated that both promote increased STAT1 activity.That both STAT1 variants occur at nearly identical locations, and the uniform distribution among leukocytes, suggests a still undefined genetic event occurring prior to the divergence of common myeloid and lymphoid progenitor cells in hematopoietic stem cell ontogeny. This case demonstrates the power of single cell DNA sequencing in precisely defining disease pathogenesis in patients with somatic mosaicism.