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Prioritizing causal disease genes using unbiased genomic features.

Deo RC, Musso G, Tasan M, Tang P, Poon A, Yuan C, Felix JF, Vasan RS, Beroukhim R, De Marco T, Kwok PY, MacRae CA, Roth FP - Genome Biol. (2014)

Bottom Line: In practice, however, identification of the actual genes contributing to disease pathogenesis has lagged behind identification of associated loci, thus limiting the clinical benefits.Using a zebrafish model, we experimentally validate FLNC and identify a novel FLNC splice-site mutation in a patient with severe DCM.Our approach stands to assist interpretation of large-scale genetic studies without compromising their fundamentally unbiased nature.

View Article: PubMed Central - PubMed

ABSTRACT

Background: Cardiovascular disease (CVD) is the leading cause of death in the developed world. Human genetic studies, including genome-wide sequencing and SNP-array approaches, promise to reveal disease genes and mechanisms representing new therapeutic targets. In practice, however, identification of the actual genes contributing to disease pathogenesis has lagged behind identification of associated loci, thus limiting the clinical benefits.

Results: To aid in localizing causal genes, we develop a machine learning approach, Objective Prioritization for Enhanced Novelty (OPEN), which quantitatively prioritizes gene-disease associations based on a diverse group of genomic features. This approach uses only unbiased predictive features and thus is not hampered by a preference towards previously well-characterized genes. We demonstrate success in identifying genetic determinants for CVD-related traits, including cholesterol levels, blood pressure, and conduction system and cardiomyopathy phenotypes. Using OPEN, we prioritize genes, including FLNC, for association with increased left ventricular diameter, which is a defining feature of a prevalent cardiovascular disorder, dilated cardiomyopathy or DCM. Using a zebrafish model, we experimentally validate FLNC and identify a novel FLNC splice-site mutation in a patient with severe DCM.

Conclusion: Our approach stands to assist interpretation of large-scale genetic studies without compromising their fundamentally unbiased nature.

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Related in: MedlinePlus

OPEN prioritized genes contribute to cardiac phenotypes in zebrafish. (A) Knockdown of USP13 caused a dose-dependent decrease in cardiac output, due to both a decrease in heart rate and ventricular stroke volume. (B) Injection of a morpholino (MO) targeting a specific splicing event in FLNCb (see Materials and methods) caused apparent cardiac-specific defects. Images on the right show embryos at 48 hours post-fertilization (hpf) with decreasing injected morpholino concentration. Optical mapping confirmed a significant decrease in cardiac conduction velocity in isolated hearts following FLNCb splice inhibition (top right: isochronal maps on right, red box indicates measured region of interest, isochrones are 5 ms apart). Conduction velocity was unaltered in other regions of the heart (middle right: bar graph, regions examined were atrial inner curvature (AIC), atrial outer curvature (AOC), AV node (AV), ventricular inner curvature (VIC), and ventricular outer curvature (VOC)). Additionally, FLNCb splice inhibition resulted in increased atrial cardiomyocyte size (bottom left: beta-catenin stained in green, DAPI in blue, V and A denote ventricle and atria, respectively). RT-PCR confirmed inhibition of the predicted splicing event in FLNCb (bottom right). (C) Knockdown of SVIL causes cardiac edema as well as noticeable spinal curvature at higher morhpolino doses, with only cardiac edema notable at lower doses. Images on left again show decreasing morpholino dose at 48 hpf. Optical mapping (right) confirmed a significant decrease in atrial conduction velocity following SVIL knockdown. ***P < 0.001, **P < 0.01, *P < 0.05.
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Fig5: OPEN prioritized genes contribute to cardiac phenotypes in zebrafish. (A) Knockdown of USP13 caused a dose-dependent decrease in cardiac output, due to both a decrease in heart rate and ventricular stroke volume. (B) Injection of a morpholino (MO) targeting a specific splicing event in FLNCb (see Materials and methods) caused apparent cardiac-specific defects. Images on the right show embryos at 48 hours post-fertilization (hpf) with decreasing injected morpholino concentration. Optical mapping confirmed a significant decrease in cardiac conduction velocity in isolated hearts following FLNCb splice inhibition (top right: isochronal maps on right, red box indicates measured region of interest, isochrones are 5 ms apart). Conduction velocity was unaltered in other regions of the heart (middle right: bar graph, regions examined were atrial inner curvature (AIC), atrial outer curvature (AOC), AV node (AV), ventricular inner curvature (VIC), and ventricular outer curvature (VOC)). Additionally, FLNCb splice inhibition resulted in increased atrial cardiomyocyte size (bottom left: beta-catenin stained in green, DAPI in blue, V and A denote ventricle and atria, respectively). RT-PCR confirmed inhibition of the predicted splicing event in FLNCb (bottom right). (C) Knockdown of SVIL causes cardiac edema as well as noticeable spinal curvature at higher morhpolino doses, with only cardiac edema notable at lower doses. Images on left again show decreasing morpholino dose at 48 hpf. Optical mapping (right) confirmed a significant decrease in atrial conduction velocity following SVIL knockdown. ***P < 0.001, **P < 0.01, *P < 0.05.

Mentions: We focused on the remaining candidates (USP13, FLNC, SVIL), which, at the time of our study, had no known link to cardiac function but were attractive given that: 1) they were found in loci that had been associated with LVD; and 2) they had high OPEN scores for DCM despite having a small number of genes at their locus. All three ranked highly on our overall genome-wide ranking of DCM candidates with FLNC being the number 2 candidate overall, USP13 number 105, and SVIL number 298. We knocked each of these down in embryonic zebrafish and looked for evidence of phenotypic change. All three demonstrated abnormalities in cardiac morphology or function (Figure 5). Specifically, microinjection of a morpholino targeting USP13 caused a dose-dependent decrease in cardiac output. Notably, this decrease was due to both decreased ventricular stroke volume and heart rate (Figure 5A). Next, although previous efforts have described skeletal muscle-specific defects following morpholino-based introduction of a premature stop codon in flncb (one of two zebrafish orthologs of human FLNC [43]), cardiac defects related to FLNC have not been explicitly described. Following injection of a splice-blocking morpholino we noted obvious distention of the atrium and backflow upon cardiac contraction (Additional file 42). Immunological staining confirmed hypertrophy of atrial cardiomyocytes (Figure 5B). Additionally, optical mapping showed a decrease in ventricular conduction velocity in isolated hearts following flncb splice inhibition compared with sham-injected controls, suggesting alterations in junctional remodeling and cell-cell coupling (Figure 5B). Finally, knockdown of our third candidate, SVIL, caused obvious pericardial edema at low morpholino doses (Figure 5C) with optical mapping confirming an underlying decrease in atrial conduction velocity.Figure 5


Prioritizing causal disease genes using unbiased genomic features.

Deo RC, Musso G, Tasan M, Tang P, Poon A, Yuan C, Felix JF, Vasan RS, Beroukhim R, De Marco T, Kwok PY, MacRae CA, Roth FP - Genome Biol. (2014)

OPEN prioritized genes contribute to cardiac phenotypes in zebrafish. (A) Knockdown of USP13 caused a dose-dependent decrease in cardiac output, due to both a decrease in heart rate and ventricular stroke volume. (B) Injection of a morpholino (MO) targeting a specific splicing event in FLNCb (see Materials and methods) caused apparent cardiac-specific defects. Images on the right show embryos at 48 hours post-fertilization (hpf) with decreasing injected morpholino concentration. Optical mapping confirmed a significant decrease in cardiac conduction velocity in isolated hearts following FLNCb splice inhibition (top right: isochronal maps on right, red box indicates measured region of interest, isochrones are 5 ms apart). Conduction velocity was unaltered in other regions of the heart (middle right: bar graph, regions examined were atrial inner curvature (AIC), atrial outer curvature (AOC), AV node (AV), ventricular inner curvature (VIC), and ventricular outer curvature (VOC)). Additionally, FLNCb splice inhibition resulted in increased atrial cardiomyocyte size (bottom left: beta-catenin stained in green, DAPI in blue, V and A denote ventricle and atria, respectively). RT-PCR confirmed inhibition of the predicted splicing event in FLNCb (bottom right). (C) Knockdown of SVIL causes cardiac edema as well as noticeable spinal curvature at higher morhpolino doses, with only cardiac edema notable at lower doses. Images on left again show decreasing morpholino dose at 48 hpf. Optical mapping (right) confirmed a significant decrease in atrial conduction velocity following SVIL knockdown. ***P < 0.001, **P < 0.01, *P < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig5: OPEN prioritized genes contribute to cardiac phenotypes in zebrafish. (A) Knockdown of USP13 caused a dose-dependent decrease in cardiac output, due to both a decrease in heart rate and ventricular stroke volume. (B) Injection of a morpholino (MO) targeting a specific splicing event in FLNCb (see Materials and methods) caused apparent cardiac-specific defects. Images on the right show embryos at 48 hours post-fertilization (hpf) with decreasing injected morpholino concentration. Optical mapping confirmed a significant decrease in cardiac conduction velocity in isolated hearts following FLNCb splice inhibition (top right: isochronal maps on right, red box indicates measured region of interest, isochrones are 5 ms apart). Conduction velocity was unaltered in other regions of the heart (middle right: bar graph, regions examined were atrial inner curvature (AIC), atrial outer curvature (AOC), AV node (AV), ventricular inner curvature (VIC), and ventricular outer curvature (VOC)). Additionally, FLNCb splice inhibition resulted in increased atrial cardiomyocyte size (bottom left: beta-catenin stained in green, DAPI in blue, V and A denote ventricle and atria, respectively). RT-PCR confirmed inhibition of the predicted splicing event in FLNCb (bottom right). (C) Knockdown of SVIL causes cardiac edema as well as noticeable spinal curvature at higher morhpolino doses, with only cardiac edema notable at lower doses. Images on left again show decreasing morpholino dose at 48 hpf. Optical mapping (right) confirmed a significant decrease in atrial conduction velocity following SVIL knockdown. ***P < 0.001, **P < 0.01, *P < 0.05.
Mentions: We focused on the remaining candidates (USP13, FLNC, SVIL), which, at the time of our study, had no known link to cardiac function but were attractive given that: 1) they were found in loci that had been associated with LVD; and 2) they had high OPEN scores for DCM despite having a small number of genes at their locus. All three ranked highly on our overall genome-wide ranking of DCM candidates with FLNC being the number 2 candidate overall, USP13 number 105, and SVIL number 298. We knocked each of these down in embryonic zebrafish and looked for evidence of phenotypic change. All three demonstrated abnormalities in cardiac morphology or function (Figure 5). Specifically, microinjection of a morpholino targeting USP13 caused a dose-dependent decrease in cardiac output. Notably, this decrease was due to both decreased ventricular stroke volume and heart rate (Figure 5A). Next, although previous efforts have described skeletal muscle-specific defects following morpholino-based introduction of a premature stop codon in flncb (one of two zebrafish orthologs of human FLNC [43]), cardiac defects related to FLNC have not been explicitly described. Following injection of a splice-blocking morpholino we noted obvious distention of the atrium and backflow upon cardiac contraction (Additional file 42). Immunological staining confirmed hypertrophy of atrial cardiomyocytes (Figure 5B). Additionally, optical mapping showed a decrease in ventricular conduction velocity in isolated hearts following flncb splice inhibition compared with sham-injected controls, suggesting alterations in junctional remodeling and cell-cell coupling (Figure 5B). Finally, knockdown of our third candidate, SVIL, caused obvious pericardial edema at low morpholino doses (Figure 5C) with optical mapping confirming an underlying decrease in atrial conduction velocity.Figure 5

Bottom Line: In practice, however, identification of the actual genes contributing to disease pathogenesis has lagged behind identification of associated loci, thus limiting the clinical benefits.Using a zebrafish model, we experimentally validate FLNC and identify a novel FLNC splice-site mutation in a patient with severe DCM.Our approach stands to assist interpretation of large-scale genetic studies without compromising their fundamentally unbiased nature.

View Article: PubMed Central - PubMed

ABSTRACT

Background: Cardiovascular disease (CVD) is the leading cause of death in the developed world. Human genetic studies, including genome-wide sequencing and SNP-array approaches, promise to reveal disease genes and mechanisms representing new therapeutic targets. In practice, however, identification of the actual genes contributing to disease pathogenesis has lagged behind identification of associated loci, thus limiting the clinical benefits.

Results: To aid in localizing causal genes, we develop a machine learning approach, Objective Prioritization for Enhanced Novelty (OPEN), which quantitatively prioritizes gene-disease associations based on a diverse group of genomic features. This approach uses only unbiased predictive features and thus is not hampered by a preference towards previously well-characterized genes. We demonstrate success in identifying genetic determinants for CVD-related traits, including cholesterol levels, blood pressure, and conduction system and cardiomyopathy phenotypes. Using OPEN, we prioritize genes, including FLNC, for association with increased left ventricular diameter, which is a defining feature of a prevalent cardiovascular disorder, dilated cardiomyopathy or DCM. Using a zebrafish model, we experimentally validate FLNC and identify a novel FLNC splice-site mutation in a patient with severe DCM.

Conclusion: Our approach stands to assist interpretation of large-scale genetic studies without compromising their fundamentally unbiased nature.

Show MeSH
Related in: MedlinePlus