Routine use of clinical exome-based next-generation sequencing for evaluation of patients with thrombotic microangiopathies

Abstract

Next-generation sequencing is increasingly used for clinical evaluation of patients presenting with thrombotic microangiopathies because it allows for simultaneous interrogation of multiple complement and coagulation pathway genes known to be associated with disease. However, the diagnostic yield is undefined in routine clinical practice. Historic studies relied on case-control cohorts, did not apply current guidelines for variant pathogenicity assessment, and used targeted gene enrichment combined with next-generation sequencing. A clinically enhanced exome, targeting ~54 Mb, was sequenced for 73 patients. Variant analysis and interpretation were performed on genes with biological relevance in thrombotic microangiopathy (C3,CD46, CFB, CFH, CFI, DGKE, and THBD). CFHR3-CFHR1 deletion status was also assessed using multiplex ligation-dependent probe amplification. Variants were classified using American College of Medical Genetics and Genomics guidelines. We identified 5 unique novel and 14 unique rare variants in 25% (18/73) of patients, including a total of 5 pathogenic, 4 likely pathogenic, and 15 variants of uncertain clinical significance. Nine patients had homozygous deletions in CFHR3-CFHR1. The diagnostic yield, defined as the presence of a pathogenic variant, likely pathogenic variant or homozygous deletion of CFHR3-CFHR1, was 25% for all patients tested. Variants of uncertain clinical significance were identified in 21% (15/73) of patients.These results illustrate the expected diagnositic yield in the setting of thrombotic microangiopathies through the application of standardized variant interpretation, and highlight the utility of such an approach. Sequencing a clinically enhanced exome to enable targeted, disease-specific variant analysis is a viable approach. The moderate rate of variants of uncertain clinical significance highlights the paucity of data surrounding the variants in our cohort and illustrates the need for expanded variant curation resources to aid in thrombotic microangiopathy-related disease variant classification.

Figure 1

Flow chart depicting the thrombotic microangiopathy genetic testing results by numbers of patients with variants, variant interpretation and variant distribution among complement and coagulation pathway genes.

Figure 2

Summary of thrombotic microangiopathy -genetic testing results. Patients are grouped according to clinical diagnosis. Genes are grouped according to function. Age represents the age at the time of testing. AA – African American, CA – Caucasian, NA – Native American, H – Hispanic, A – Asian, Unk – Unknown, aHUS – atypical hemolytic uremic syndrome, HUS – hemolytic uremic syndrome, TMA – thrombotic microangiopathy, TTP – thrombotic thrombocytopenic purpura, VUS – variant of uncertain clinical significance.