The genetic basis of Turner syndrome aortopathy

Abstract

Our goal is to identify the genetic underpinnings of bicuspid aortic valve and aortopathy in Turner syndrome. We performed whole exome sequencing on 188 Turner syndrome study subjects from the GenTAC registry. A gene-based burden test, SKAT-O, was used to evaluate the data using bicuspid aortic valve (BAV) and aortic dimension z-scores as covariates. This revealed that TIMP3 was associated with BAV and increased aortic dimensions at exome-wide significance. It had been previously shown that genes on chromosome Xp contribute to aortopathy when hemizygous. Our analysis of Xp genes revealed that hemizygosity for TIMP1, a functionally redundant paralogue of TIMP3, increased the odds of having BAV aortopathy compared to individuals with more than one TIMP1 copy. The combinatorial effect of a single copy of TIMP1 and TIMP3 risk alleles synergistically increased the risk for BAV aortopathy to nearly 13-fold. TIMP1 and TIMP3 are tissue inhibitors of matrix metalloproteinases (TIMPs) which are involved in development of the aortic valve and protection from thoracic aneurysms. We propose that the combination of TIMP1 haploinsufficiency and deleterious variants in TIMP3 significantly increases the risk of BAV aortopathy in Turner syndrome, and suggest that TIMP1 hemizygosity may play a role in euploid male aortic disease.

Keywords: aortic disease; bicuspid aortic valve; congenital cardiovascular disease; polygenic trait.

Figure 1.. Disease threshold model to illustrate polygenic disease and the role of effect size of risk variants.

The horizontal dotted line represents a theoretical threshold of disease. Factors, such as genetic variants are represented by boxes forming vertical columns. Columns that cross the dotted line breach the disease threshold indicating that a sufficient burden of risk factors exists to cause the disease, in this case BAV. In Turner syndrome the loss of the second sex chromosome is a risk factor of very large effect size. However, it is not sufficient alone to breach the disease threshold for BAV as not everyone with Turner syndrome has BAV. However, it is likely that fewer risk factors are required to cause BAV compared to the euploid population, which has an inherently low risk.

Figure 2.. SKAT-O Analysis Shows that TIMP3 Variants are Associated with BAV/TAD.

Manhattan plots showing the exome-wide significant finding that TIMP3 variants are associated with BAV, and with AR enlargement as an indicator of TAD. The horizontal line is the threshold for exome- wide significance (based on testing 19,392 genes, the exome-wide significance p-value=2.578×10–6). TIMP3 is the only gene that exceeds exome-wide significance. It is notable that no other genes approach the significance line. A) Shows the association with BAV as the sole predictor (p=1.58×10–6). B) Shows the association results for BAV and aortic root (AR) z-scores as covariates (p=2.27×10–7). The significance level for TIMP3 increases nearly 10-fold when AR z-scores were added. (From Corbitt H et al. (2018) TIMP3 and TIMP1 are risk genes for bicuspid aortic valve and aortopathy in Turner syndrome. PLoS Genetics, 2018 Oct 3;14(10):e1007692. doi: 10.1371/journal.pgen.1007692.)

Figure 3.. Quantile-Quantile (Q-Q) Plots for the SKAT-O Analyses.

Q-Q plots for SKAT-O analysis of BAV and BAV with AR Z-scores, which shows no significant deviation from the normal distribution.

Figure 4.. TIMP3 variants decrease expression.

Western blot of secreted protein from TIMP3 constructs, in duplicates. Lanes 1–2 His83/Ser87 double variant; Lanes 3–4, His83; Lanes 5–6 Ser87; Lanes 7–8 wild type; Lane 9, empty vector. GAPDH loading control is superimposed. Note that the His83 and Ser87 variants significantly reduce secreted TIMP3 expression, particularly the glycosylated form.

Figure 5.. TIMP1 copy number is associated with the risk of BAV and BAV with TAD.

Bar graph showing the frequency of BAV with or without TAD when only one copy of TIMP1 is present compared to the frequency when there is more than one copy of TIMP1. A) The dark blue bars represent individuals with a diagnosis of BAV and the light blue bars represent individuals without a BAV. B) The dark blue bars represent individuals with a diagnosis of BAV with TAD and the light blue bars represent individuals without a BAV with TAD. For both a logistic regression model was run, with TIMP1 copy number as the categorical predictor and the phenotype as the response variable. Odds ratio and p-value were calculated and showed that having a single copy of TIMP1 significantly increased the odds of having a BAV and a BAV with TAD. (From Corbitt H et al. (2018) TIMP3 and TIMP1 are risk genes for bicuspid aortic valve and aortopathy in Turner syndrome. PLoS Genetics, 2018 Oct 3;14(10):e1007692. doi: 10.1371/journal.pgen.1007692.)

Figure 6.. TIMP1 protein levels in plasma.

Expression of TIMP1 levels in plasma as measured by ELISA plotted by the number of TIMP1 gene copy number. Note that TIMP1 copy number is significantly correlated with TIMP1 protein levels in plasma. The samples with only one copy of TIMP1 are tightly clustered with little variability, whereas the samples with more than 1 copy are highly variable, as has been previously described.

Figure 7.. TIMP1 copy number vrs aortic z-scores.

Panel A shows a box plot representing study subject aortic root z-scores plotted against TIMP1 copy number. Panel B shows the box plot for ascending aorta z-scores plotted against TIMP1 copy number. Note that for both aortic root and ascending aorta z-scores there is a significant difference in aortic diameters comparing individuals with only one copy of TIMP1 to those with greater than one copy of TIMP1.

Figure 8.. TS aorta CTGF and extracellular matrix staining.

Analysis of the same aortic tissue from a young woman with TS who died from a dissection of the ascending aorta to visualize extracellular matrix components. The top two panels (A, B) are stained with an antibody to connective tissue growth factor (CTGF). Panel A is from an age and gender matched normal control aorta. Note the lack of staining for CTGF. Panel B is from a non-aneurysm section of aorta from the TS patient. Note the significantly increased level of CTGF indicating an activation of connective tissue production. Panels C and D are stained with trichrome, staining collagens blue. Note the substantial increase in staining in the TS aorta (D) compared to the control (C), showing increased collagen deposition in the TS aorta (fibrosis), which is thought to be compensatory for aortic wall instability.

Figure 9.. TS aorta SMAD staining.

Immunohistochemical analysis the same aortas shown in figure 6 to assess levels of TGF-β signaling. The two left panels (A, C) are from control aortas. The two right panels are sections from the TS aorta. All are stained with p-SMAD antibody. SMADs are downstream products of TGF-β signaling. Note the increase in staining (reddish-brown) in the TS aorta (Panels B and D) compared to a normal control (Panels A and C), indicating an increase in p-SMAD production as a result of increased TGF-β signaling. Excessive TGF-β signaling is seen in aortas from individuals with Marfan syndrome and is thought to contribute to aortic wall instability. This is the first demonstration of excessive TGF-β signaling in TS.

Figure 10.. Theoretical model of aortic disease pathogenesis in TS.

This model depicts the proposed pathogenic events underlying aortopathy in Turner syndrome. We propose that there is an inherent imbalance in the TIMP/MMP ratio that leads to the degradation of the ECM, including fibrillin-1 microfibrils. As in Marfan syndrome this releases active TGFβ resulting in an increase in TGFβ signaling. One of the consequences of TGFβ signaling is an increase in MMP expression, which feeds into the TIMP/MMP imbalance. There is also a feedback loop through which increased MMP activity proteolytically cleaves latent TGFβ producing the active form.