Variants of Unknown Significance in Genes Associated with Heritable Thoracic Aortic Disease Can Be Low Penetrant "Risk Variants"

Abstract

Thoracic aortic aneurysms leading to acute aortic dissections are a preventable cause of premature deaths if individuals at risk can be identified. Individuals with early-onset aortic dissections without a family history or syndromic features have an increased burden of rare genetic variants of unknown significance (VUSs) in genes with pathogenic variants for heritable thoracic aortic disease (HTAD). We assessed the role of VUSs in the development of disease using both in vitro enzymatic assays and mouse models. VUSs in LOX and MYLK identified in individuals with acute aortic dissections were assayed to determine whether they disrupted enzymatic activity. A subset of VUSs reduced enzymatic activity compared to the wild-type proteins but less than pathogenic variants. Additionally, a Myh11 variant, p.Arg247Cys, which does not cause aortic disease in either humans or mice, was crossed with the Acta2^-/- mouse, which has aortic enlargement with age while Acta2^+/- mice do not. Acta2^+/-Myh11^R247C/R247C mice have aortic dilation by 3 months of age without medial degeneration, indicating that two variants not known to cause disease do lead to aortic enlargement in combination. Furthermore, the addition of Myh11^R247C/R247C to the Acta2^-/- mouse model accelerates aortic enlargement and increases medial degeneration. Therefore, our results emphasize the need for a classification system for variants in Mendelian genes that goes beyond the 5-tier system of pathogenic, likely pathogenic, VUS, likely benign, and benign, and includes a designation for low-penetrant "risk variants" that trigger disease either in combination with other risk factors or in a stochastic manner.

Keywords: aortic dissection; heritable thoracic aortic diseases; variant classification; variants of unknown significance.

Figure 1

Enzymatic Activity in Recombinant LOX and MYLK Proteins Carrying VUSs Identified in ESTAD Case Subjects (A) Lysyl oxidase (LOX). Top: Diagram depicting the domains of lysyl oxidase, with the cleaved propeptide in light blue and the mature enzymatic domain in yellow. The positions of amino acid substitutions are shown for pathogenic variants (p.Met298Arg, p.Ser348Arg, p.Ser280Arg) in black and ESTAD VUSs analyzed in this study and found to be benign (p.Met233Val) in green and mildly dysfunctional (p.Ser247Arg) in blue. Asterisks indicate the location of amino acid substitutions in variants identified in ESP-EA. Below, left: Time course data for LOX enzymatic activity assays (product formation in relative fluorescence units, RFU). Data were normalized to the activity of the WT LOX. Below, right: Immunoblot analyses of HeLa lysates transfected with plasmids containing WT or variant LOX, showing comparable expression of active LOX with all plasmids. (B) Myosin light chain kinase (MLCK). Top: Diagram depicting the MLCK protein domains, and the positions of the variants. Pathogenic variant p.Ser1759Pro is shown in black, variants analyzed in this study and found to be benign are shown in gray, and variants analyzed in this study and found to be risk variants are shown in blue (p.Tyr1635Cys) and red (p.Glu1446Lys). Asterisks indicate the location of amino acid substitutions in variants identified in ESP-EA. Bottom: Dependence of kinase activity of WT MLCK and three variants on concentrations of substrate (RLC, left side) and activator (calmodulin, CaM, right side). Each point represents the mean ± standard error from three experiments. The lines represent the nonlinear least-squares fits of individual datasets to the Michaelis-Menten equation.

Figure 2

Aortic Disease in Acta2^+/−Myh11^R247C/R247C Mice (A and B) Aortic diameter measured by echocardiography at 3 months of age shows increased growth of the aortic root (A) and the ascending aorta (B) in Acta2^+/−Myh11^R247C/R247C mice compared with Acta2^+/−Myh11^+/+ mice. (C) Representative images of MOVAT stain performed on ascending aortic tissue collected at 6 months of age. (D and E) Quantification of proteoglycan deposition (blue color, D) and fragmentation of elastin (black color, E) from the MOVAT stains done at 6 months of age. For all graphs, error bars represent standard error.

Figure 3

Increased Aortic Diameter in Acta2^−/−Myh11^R247C/R247C Mice Is Not Associated with Changes in Blood Pressure or Contractility (A and B) Aortic diameter measured by echocardiography shows that growth of the aortic root (A) and the ascending aorta (B) is increased at 6 months of age in Acta2^−/−Myh11^R247C/R247C mice compared with Acta2^−/−Myh11^+/+ mice. (C) Mean arterial blood pressure measured by tail cuff shows that hypotension in Acta2^−/− mice is not affected by addition of Myh11^R247C/R247C. (D and E) Maximal force development of aortic rings was assessed ex vivo in response to KCl (D) or phenylephrine (E). Both heterozygous loss of Acta2 and homozygous Myh11^R247C/R247C cause significant decreases in aortic contractility, but the combination of the two genetic changes does not further decrease aortic contractility. For all graphs, error bars represent standard error.

Figure 4

Aortic Pathology in Acta2^−/−Myh11^R247C/R247C Mice (A) Representative images of MOVAT stain performed on ascending aortic tissue collected at 6 months of age. (B) Cells counted from H&E slides of ascending aortic tissue from 12-week-old mice shows no changes in cell density between any of the six genotypes assessed. (C and D) Quantification of fragmentation of elastin (black color, C) and proteoglycan deposition (blue color, D) from the MOVAT stains done at 6 months of age shows increased proteoglycan deposition in Acta2^−/−Myh11^R247C/R247C aortas. For all graphs, error bars represent standard error.