Genome-wide association study reveals novel genetic loci:a new polygenic risk score for mitral valve prolapse

AIMS: Mitral valve prolapse (MVP) is a common valvular heart disease with a prevalence of >2% in the general adult population. Despite this high incidence, there is a limited understanding of the molecular mechanism of this disease, and no medical therapy is available for this disease. We aimed to elucidate the genetic basis of MVP in order to better understand this complex disorder. METHODS AND RESULTS: We performed a meta-analysis of six genome-wide association studies that included 4884 cases and 434 649 controls. We identified 14 loci associated with MVP in our primary analysis and 2 additional loci associated with a subset of the samples that additionally underwent mitral valve surgery. Integration of epigenetic, transcriptional, and proteomic data identified candidate MVP genes including LMCD1, SPTBN1, LTBP2, TGFB2, NMB, and ALPK3. We created a polygenic risk score (PRS) for MVP and showed an improved MVP risk prediction beyond age, sex, and clinical risk factors. CONCLUSION: We identified 14 genetic loci that are associated with MVP. Multiple analyses identified candidate genes including two transforming growth factor-beta signalling molecules and spectrin beta. We present the first PRS for MVP that could eventually aid risk stratification of patients for MVP screening in a clinical setting. These findings advance our understanding of this common valvular heart disease and may reveal novel therapeutic targets for intervention. KEY QUESTION: Expand our understanding of the genetic basis for mitral valve prolapse (MVP). Uncover relevant pathways and target genes for MVP pathophysiology. Leverage genetic data for MVP risk prediction. KEY FINDING: Sixteen genetic loci were significantly associated with MVP, including 13 novel loci. Interesting target genes at these loci included LTBP2, TGFB2, ALKP3, BAG3, RBM20, and SPTBN1. A risk score including clinical factors and a polygenic risk score, performed best at predicting MVP, with an area under the receiver operating characteristics curve of 0.677. TAKE-HOME MESSAGE: Mitral valve prolapse has a polygenic basis: many genetic variants cumulatively influence pre-disposition for disease. Disease risk may be modulated via changes to transforming growth factor-beta signalling, the cytoskeleton, as well as cardiomyopathy pathways. Polygenic risk scores could enhance the MVP risk prediction

Influence of protein-micelle ratios and cysteine residues on the kinetic stability and unfolding rates of human mitochondrial VDAC-2.

Delineating the kinetic and thermodynamic factors which contribute to the stability of transmembrane β-barrels is critical to gain an in-depth understanding of membrane protein behavior. Human mitochondrial voltage-dependent anion channel isoform 2 (hVDAC-2), one of the key anti-apoptotic eukaryotic β-barrel proteins, is of paramount importance, owing to its indispensable role in cell survival. We demonstrate here that the stability of hVDAC-2 bears a strong kinetic contribution that is dependent on the absolute micellar concentration used for barrel folding. The refolding efficiency and ensuing stability is sensitive to the lipid-to-protein (LPR) ratio, and displays a non-linear relationship, with both low and high micellar amounts being detrimental to hVDAC-2 structure. Unfolding and aggregation process are sequential events and show strong temperature dependence. We demonstrate that an optimal lipid-to-protein ratio of 2600∶1 - 13,000∶1 offers the highest protection against thermal denaturation. Activation energies derived only for lower LPRs are ∼17 kcal mol(-1) for full-length hVDAC-2 and ∼23 kcal mol(-1) for the Cys-less mutant, suggesting that the nine cysteine residues of hVDAC-2 impart additional malleability to the barrel scaffold. Our studies reveal that cysteine residues play a key role in the kinetic stability of the protein, determine barrel rigidity and thereby give rise to strong micellar association of hVDAC-2. Non-linearity of the Arrhenius plot at high LPRs coupled with observation of protein aggregation upon thermal denaturation indicates that contributions from both kinetic and thermodynamic components stabilize the 19-stranded β-barrel. Lipid-protein interaction and the linked kinetic contribution to free energy of the folded protein are together expected to play a key role in hVDAC-2 recycling and the functional switch at the onset of apoptosis

Cysteine Residues Impact the Stability and Micelle Interaction Dynamics of the Human Mitochondrial β-Barrel Anion Channel hVDAC-2

<div><p>The anti-apoptotic 19-stranded transmembrane human voltage dependent anion channel isoform 2 (hVDAC-2) β-barrel stability is crucial for anion transport in mitochondria. The role of the unusually high number of cysteine residues in this isoform is poorly understood. Using a Cys-less construct of hVDAC-2, we haveinvestigated the contribution of cysteines to channel function, barrel stability and its influence on the strength of protein-micelle interactions. We observe that despite the overall preservation in barrel structure upon cysteine mutation, subtle local variations in the mode of interaction of the barrel with its refolded micellar environment arise, which may manifest itself in the channel activity of both the proteins.Fluorescence measurements of the Trp residues in hVDAC-2 point to possible differences in the association of the barrel with lauryldimethylamine oxide (LDAO) micelles. Upon replacement of cysteines in hVDAC-2, our data suggests greater barrel rigidity by way of intra-protein interactions. This, in turn, lowers the equilibrium barrel thermodynamic parameters in LDAOby perturbingthe stability of the protein-micelle complex. In addition to this, we also find a difference in the cooperativity of unfolding upon increasing the LDAO concentration, implying the importance of micelle concentration and micelle-protein ratios on the stability of this barrel. Our results indicate that the nine cysteine residues of hVDAC-2 are the key in establishing strong(er) barrel interactions with its environment and also impart additional malleability to the barrel scaffold.</p></div

Cartoon representations of hVDAC-2 WT (left) and C0 (right).

<p>The structures were modeled using I-TASSER <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087701#pone.0087701-Roy1" target="_blank">[55]</a> using the crystal (2JK4 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087701#pone.0087701-Hiller1" target="_blank">[29]</a>) and NMR (2K4T <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087701#pone.0087701-Bayrhuber1" target="_blank">[56]</a>) structure of hVDAC-1 as the template. The cysteine residues have been highlighted as yellow spheres for the WT protein and they are largely oriented towards the intermembrane space in the 19-stranded model <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087701#pone.0087701-Maurya1" target="_blank">[22]</a>. The corresponding mutated residues in C0 have been represented as spheres and colored according to the chemical characteristics of the amino acid.</p

Thermal denaturation (T-scans) of refolded hVDAC-2 in various LDAO concentrations.

<p>Representative profiles of refolded hVDAC-2 WT (left) and C0 (right) in 5 mM (<b>○</b>, black), 13 mM (<b>□</b>, red), 30 mM (<b>⋄</b>, green), 65 mM (<b>▵</b>, blue), 80 mM (<b>▿</b>, dark pink) and 100 mM (<b>▹</b>, dark yellow) are shown here as unfolded fractions obtained at the various temperatures. The solid lines represent fits to a two-state equation. Note that hVDAC-2 C0 (right) shows substantial increase (∼30%) in the unfolded fraction by ∼80°C in 80 mM and 100 mM LDAO, even before commencement of the unfolding transition. Insets show the fits for samples directly refolded in 5 mM LDAO, to achieve an LPR of 2600∶1 (purple) or 1000∶1 (brown), and are compared with the 5 mM LDAO sample prepared by dilution (black) of the refolding stock to also achieve an LPR of 2600∶1 (see text for details). Actual data points have been omitted for clarity. The directly refolded samples, despite exhibiting a similar <i>T_m</i>, show a distinct loss in unfolding cooperativity, when compared with samples prepared by dilution.</p

Comparison of the rate of unfolding of hVDAC-2 WT and C0.

<p>Rate of unfolding of refolded hVDAC-2 WT and C0 as a function of LDAO concentrations (A) and temperature (B) derived from isothermal kinetics experiments. Panel (A) highlights the decrease in unfolding rate with increasing LDAO concentration. Panel (B) highlights the increase in the unfolding rate with increase in temperature. Values for the solid symbols have been derived from the <i>k</i>_u1 of the double exponential fits whereas the hollow symbols are for the single exponential fits. Figure legends in panel (B) are distributed in the left and right graphs. Error bars denote the goodness of the fit obtained by fitting the mean data of three independent experiments.</p

Representative far-UV circular dichroism profiles of refolded hVDAC-2 WT and C0 with increasing LDAOconcentrations.

<p>Both WT (left) and C0 (right) display CD spectra corresponding to an extended conformation, with a negative maximum at ∼215 nm. Both proteins exhibit comparable secondary structure content, despite a 20-fold change in the LDAO concentration from 5 mM to 100 mM. The inset shows the CD spectra of control samples prepared by ‘direct’ refolding in LPRs of 2600∶1 (5 mM D2) and 1000∶1 (5 mM D5), and are compared with ‘refolded’ protein in 5 mM LDAO, also having an LPR of 2600:1. Molar ellipticity (ME) values are lower for the control samples, suggesting the importance of absolute LDAO concentrations to mediate optimal refolding.</p

Monitoring the susceptibility of refolded hVDAC-2 to trypsinization with increasing LDAO.

<p>Representative silver stained SDS-PAGE gels of refolded WT (top) and C0 (bottom) subjected to a 10 min exposure to trypsin (+) and arrested by the addition of 5 mM PMSF are compared with undigested samples (–). The LDAO concentrations in each sample are indicated above each lane and the refolding method employed is also indicated (samples 0D, 2D and 5D are directly refolded and labeled ‘Direct’; those labeled ‘Refolded’ were generated by dilution of the refolded stock; see Materials and Methods for details of sample preparation). Note that D2, 5 and 13 samples possess a similar LPR of 2600:1. The observed distortion in protein migration, particularly in high LPR, is due to the presence of excess LDAO in the sample, which interferes in proper gel running. Relevant molecular weight standards (M) are indicated on either side of each gel. Dashed lines are used to separate different gels that are presented together.</p

Summary of Trp fluorescence lifetime and anisotropy values of WT and C0 refolded in various LPRs.

a<p>Changes observed in Trp lifetime and anisotropy of the denatured samples can be explained by taking into account the change in viscosity of the sample; <i>^b</i> Average tryptophan lifetimes obtained from fits to a three exponential function. The values provided are in ns; <i>^c</i> Goodness of fit for the fluorescence lifetime measurements; <i>^d</i> Samples have trace amounts of GdnHCl due to direct (D) refolding; 5 mM D2 corresponds to 2 μM protein directly refolded in 5 mM LDAO; <i>^e</i> Samples have been diluted from the refolded stock (RF) solution; <i>^f</i> ND – Not determined, due to scattering by aggregated protein.</p

Equilibrium unfolding studies of refolded hVDAC-2 using GdnHCl.

<p>(A) Representative Trp fluorescence wavelength scans of refolded hVDAC-2 WT (left) and C0 (right) in 13 mM LDAO in a GdnHCl gradient, recorded after 1 h incubation at 25°C. Note the decrease in intensity as well as the red shifted λ_em-max in GdnHCl, indicating near-complete protein unfolding. (B) Refolded hVDAC-2 WT (left) and C0 (right) in different LDAO concentrations were subjected to an increasing GdnHCl gradient at 25°C. The plots represent unfolded fractions derived from the change in Trp fluorescence intensity at 340 nm, as the protein unfolds. Solid lines denote fits to a two-state equation, except in the case of 80 mM and 100 mM experiments, wherein a sigmoidal fit was used to illustrate the trend in the dataset. Also shown, as insets, are the unfolding curves obtained for aggregated protein (protein in buffer; 0 D) and hVDAC-2 refolded directly in 5 mM LDAO (5 D; LPR of 1000:1). Note the shift in <i>C_m</i> for the former and/or loss in cooperativity in the latter. The color scheme used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092183#pone-0092183-g005" target="_blank">Figure 5</a> for the various samples is also retained here. Legends are distributed in the left and right panel. Data points shown here are the mean of three datasets and error bars are omitted for clarity.</p