Establishing disease causality for a novel gene variant in familial dilated cardiomyopathy using a functional in-vitro assay of regulated thin filaments and human cardiac myosin.
BACKGROUND
As next generation sequencing for the genetic diagnosis of cardiovascular disorders becomes more widely used, establishing causality for putative disease causing variants becomes increasingly relevant. Diseases of the cardiac sarcomere provide a particular challenge in this regard because of the complexity of assaying the effect of genetic variants in human cardiac contractile proteins.


RESULTS
In this study we identified a novel variant R205Q in the cardiac troponin T gene (TNNT2). Carriers of the variant allele exhibited increased chamber volumes associated with decreased left ventricular ejection fraction. To clarify the causal role of this variant, we generated recombinant variant human protein and examined its calcium kinetics as well as the maximally activated ADP release of human β-cardiac myosin with regulated thin filaments containing the mutant troponin T. We found that the R205Q mutation significantly decreased the calcium sensitivity of the thin filament by altering the effective calcium dissociation kinetics.


CONCLUSIONS
The development of moderate throughput post-genomic assays is an essential step in the realization of the potential of next generation sequencing. Although technically challenging, biochemical and functional assays of human cardiac contractile proteins of the thin filament can be achieved and provide an orthogonal source of information to inform the question of causality for individual variants.
Leon H. Charney Division of Cardiology, NYU Langone Medical Center, New York, NY, USA. Stephen.Pan@nyumc.org.
Stephen
S
Pan
Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, MN, USA. rsommese@umn.edu.
Ruth F
RF
Sommese
Departments of Medicine (Cardiovascular Medicine), Stanford University School of Medicine, Stanford, CA, USA. sallam@stanford.edu.
Karim I
KI
Sallam
Department of Biochemistry, Stanford University School of Medicine, Stanford, CA, USA. sumannag@stanford.edu.
Suman
S
Nag
Department of Biochemistry, Stanford University School of Medicine, Stanford, CA, USA. scsutton@stanford.edu.
Shirley
S
Sutton
Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, CA, USA. Susan.MillerPhD@ucsf.edu.
Susan M
SM
Miller
Department of Biochemistry, Stanford University School of Medicine, Stanford, CA, USA. jspudich@stanford.edu.
James A
JA
Spudich
Department of Biochemistry, Stanford University School of Medicine, Stanford, CA, USA. kruppel@stanford.edu.|Department of Pediatrics (Cardiology), Stanford University School of Medicine, Stanford, CA, USA. kruppel@stanford.edu.
Kathleen M
KM
Ruppel
Departments of Medicine (Cardiovascular Medicine), Stanford University School of Medicine, Stanford, CA, USA. euan@stanford.edu.
Euan A
EA
Ashley
D000328:Adult; D002118:Calcium / Q000378:metabolism*; D024722:Cardiac Myosins / Q000235:genetics / Q000378:metabolism; D002311:Cardiomyopathy, Dilated / Q000235:genetics* / Q000503:physiopathology*; D002648:Child; D002675:Child, Preschool; D005260:Female; D020022:Genetic Predisposition to Disease; D006801:Humans; D066298:In Vitro Techniques; D008297:Male; D009154:Mutation; D018995:Myosin Heavy Chains / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013318:Stroke Volume; D020107:Troponin T / Q000235:genetics* / Q000378:metabolism*
C501641:MYH7 protein, human; D011994:Recombinant Proteins; C545568:TNNT2 protein, human; D020107:Troponin T; D024722:Cardiac Myosins; D018995:Myosin Heavy Chains; D002118:Calcium