Truncating plakophilin-2 mutations in arrhythmogenic cardiomyopathy are associated with protein haploinsufficiency in both myocardium and epidermis.
BACKGROUND
Arrhythmogenic cardiomyopathy (AC) is a hereditary cardiac condition associated with ventricular arrhythmias, heart failure, and sudden death. The disease is most often caused by mutations in the desmosomal gene for plakophilin-2 (PKP2), which is expressed in both myocardial and epidermal tissue. This study aimed to investigate protein expression in myocardial tissue of patients with AC carrying PKP2 mutations and elucidate whether keratinocytes of the same individuals exhibited a similar pattern of protein expression.


RESULTS
Direct sequencing of 5 AC genes in 71 unrelated patients with AC identified 10 different PKP2 mutations in 12 index patients. One patient, heterozygous for a PKP2 nonsense mutation, developed severe heart failure and underwent cardiac transplantation. Western blotting and immunohistochemistry of the explanted heart showed a significant decrease in PKP2 protein expression without detectable amounts of truncated PKP2 protein. Cultured keratinocytes of the patient showed a similar reduction in PKP2 protein expression. Nine additional PKP2 mutations were investigated in both cultured keratinocytes and endomyocardial biopsies from affected individuals. It was evident that PKP2 mutations introducing a premature termination codon in the reading frame were associated with PKP2 transcript and protein levels reduced to ≈50%, whereas a missense variant did not seem to affect the amount of PKP2 protein.


CONCLUSIONS
The results of this study showed that truncating PKP2 mutations in AC are associated with low expression of the mutant allele and that the myocardial protein expression of PKP2 is mirrored in keratinocytes. These findings indicate that PKP2 haploinsufficiency contributes to pathogenesis in AC.
From the Department of Cardiology (T.B.R., W.Y.K., H.M., H.K.J., J.M.), Research Unit for Molecular Medicine (T.B.R., J.P., P.B.), Department of Clinical Biochemistry (P.H.N., L.H.), Institute of Pathology (S.D.), Department of Clinical Genetics (U.B.J.), and MR Centre (W.Y.K.), Aarhus University Hospital, Aarhus, Denmark; Clinical Research Center, Vendsyssel Hospital, Aalborg University, Hjørring, Denmark (U.T.B.); Division of Cardiovascular Medicine, Radcliffe Department of Medicine, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom (K.G.); and Department of Cardiology, Odense University Hospital, Odense, Denmark (J.M.).
Torsten B
TB
Rasmussen
From the Department of Cardiology (T.B.R., W.Y.K., H.M., H.K.J., J.M.), Research Unit for Molecular Medicine (T.B.R., J.P., P.B.), Department of Clinical Biochemistry (P.H.N., L.H.), Institute of Pathology (S.D.), Department of Clinical Genetics (U.B.J.), and MR Centre (W.Y.K.), Aarhus University Hospital, Aarhus, Denmark; Clinical Research Center, Vendsyssel Hospital, Aalborg University, Hjørring, Denmark (U.T.B.); Division of Cardiovascular Medicine, Radcliffe Department of Medicine, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom (K.G.); and Department of Cardiology, Odense University Hospital, Odense, Denmark (J.M.).
Peter H
PH
Nissen
From the Department of Cardiology (T.B.R., W.Y.K., H.M., H.K.J., J.M.), Research Unit for Molecular Medicine (T.B.R., J.P., P.B.), Department of Clinical Biochemistry (P.H.N., L.H.), Institute of Pathology (S.D.), Department of Clinical Genetics (U.B.J.), and MR Centre (W.Y.K.), Aarhus University Hospital, Aarhus, Denmark; Clinical Research Center, Vendsyssel Hospital, Aalborg University, Hjørring, Denmark (U.T.B.); Division of Cardiovascular Medicine, Radcliffe Department of Medicine, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom (K.G.); and Department of Cardiology, Odense University Hospital, Odense, Denmark (J.M.).
Johan
J
Palmfeldt
From the Department of Cardiology (T.B.R., W.Y.K., H.M., H.K.J., J.M.), Research Unit for Molecular Medicine (T.B.R., J.P., P.B.), Department of Clinical Biochemistry (P.H.N., L.H.), Institute of Pathology (S.D.), Department of Clinical Genetics (U.B.J.), and MR Centre (W.Y.K.), Aarhus University Hospital, Aarhus, Denmark; Clinical Research Center, Vendsyssel Hospital, Aalborg University, Hjørring, Denmark (U.T.B.); Division of Cardiovascular Medicine, Radcliffe Department of Medicine, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom (K.G.); and Department of Cardiology, Odense University Hospital, Odense, Denmark (J.M.).
Katja
K
Gehmlich
From the Department of Cardiology (T.B.R., W.Y.K., H.M., H.K.J., J.M.), Research Unit for Molecular Medicine (T.B.R., J.P., P.B.), Department of Clinical Biochemistry (P.H.N., L.H.), Institute of Pathology (S.D.), Department of Clinical Genetics (U.B.J.), and MR Centre (W.Y.K.), Aarhus University Hospital, Aarhus, Denmark; Clinical Research Center, Vendsyssel Hospital, Aalborg University, Hjørring, Denmark (U.T.B.); Division of Cardiovascular Medicine, Radcliffe Department of Medicine, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom (K.G.); and Department of Cardiology, Odense University Hospital, Odense, Denmark (J.M.).
Søren
S
Dalager
From the Department of Cardiology (T.B.R., W.Y.K., H.M., H.K.J., J.M.), Research Unit for Molecular Medicine (T.B.R., J.P., P.B.), Department of Clinical Biochemistry (P.H.N., L.H.), Institute of Pathology (S.D.), Department of Clinical Genetics (U.B.J.), and MR Centre (W.Y.K.), Aarhus University Hospital, Aarhus, Denmark; Clinical Research Center, Vendsyssel Hospital, Aalborg University, Hjørring, Denmark (U.T.B.); Division of Cardiovascular Medicine, Radcliffe Department of Medicine, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom (K.G.); and Department of Cardiology, Odense University Hospital, Odense, Denmark (J.M.).
Uffe B
UB
Jensen
From the Department of Cardiology (T.B.R., W.Y.K., H.M., H.K.J., J.M.), Research Unit for Molecular Medicine (T.B.R., J.P., P.B.), Department of Clinical Biochemistry (P.H.N., L.H.), Institute of Pathology (S.D.), Department of Clinical Genetics (U.B.J.), and MR Centre (W.Y.K.), Aarhus University Hospital, Aarhus, Denmark; Clinical Research Center, Vendsyssel Hospital, Aalborg University, Hjørring, Denmark (U.T.B.); Division of Cardiovascular Medicine, Radcliffe Department of Medicine, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom (K.G.); and Department of Cardiology, Odense University Hospital, Odense, Denmark (J.M.).
Won Y
WY
Kim
From the Department of Cardiology (T.B.R., W.Y.K., H.M., H.K.J., J.M.), Research Unit for Molecular Medicine (T.B.R., J.P., P.B.), Department of Clinical Biochemistry (P.H.N., L.H.), Institute of Pathology (S.D.), Department of Clinical Genetics (U.B.J.), and MR Centre (W.Y.K.), Aarhus University Hospital, Aarhus, Denmark; Clinical Research Center, Vendsyssel Hospital, Aalborg University, Hjørring, Denmark (U.T.B.); Division of Cardiovascular Medicine, Radcliffe Department of Medicine, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom (K.G.); and Department of Cardiology, Odense University Hospital, Odense, Denmark (J.M.).
Lene
L
Heickendorff
From the Department of Cardiology (T.B.R., W.Y.K., H.M., H.K.J., J.M.), Research Unit for Molecular Medicine (T.B.R., J.P., P.B.), Department of Clinical Biochemistry (P.H.N., L.H.), Institute of Pathology (S.D.), Department of Clinical Genetics (U.B.J.), and MR Centre (W.Y.K.), Aarhus University Hospital, Aarhus, Denmark; Clinical Research Center, Vendsyssel Hospital, Aalborg University, Hjørring, Denmark (U.T.B.); Division of Cardiovascular Medicine, Radcliffe Department of Medicine, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom (K.G.); and Department of Cardiology, Odense University Hospital, Odense, Denmark (J.M.).
Henning
H
Mølgaard
From the Department of Cardiology (T.B.R., W.Y.K., H.M., H.K.J., J.M.), Research Unit for Molecular Medicine (T.B.R., J.P., P.B.), Department of Clinical Biochemistry (P.H.N., L.H.), Institute of Pathology (S.D.), Department of Clinical Genetics (U.B.J.), and MR Centre (W.Y.K.), Aarhus University Hospital, Aarhus, Denmark; Clinical Research Center, Vendsyssel Hospital, Aalborg University, Hjørring, Denmark (U.T.B.); Division of Cardiovascular Medicine, Radcliffe Department of Medicine, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom (K.G.); and Department of Cardiology, Odense University Hospital, Odense, Denmark (J.M.).
Henrik K
HK
Jensen
From the Department of Cardiology (T.B.R., W.Y.K., H.M., H.K.J., J.M.), Research Unit for Molecular Medicine (T.B.R., J.P., P.B.), Department of Clinical Biochemistry (P.H.N., L.H.), Institute of Pathology (S.D.), Department of Clinical Genetics (U.B.J.), and MR Centre (W.Y.K.), Aarhus University Hospital, Aarhus, Denmark; Clinical Research Center, Vendsyssel Hospital, Aalborg University, Hjørring, Denmark (U.T.B.); Division of Cardiovascular Medicine, Radcliffe Department of Medicine, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom (K.G.); and Department of Cardiology, Odense University Hospital, Odense, Denmark (J.M.).
Ulrik T
UT
Baandrup
From the Department of Cardiology (T.B.R., W.Y.K., H.M., H.K.J., J.M.), Research Unit for Molecular Medicine (T.B.R., J.P., P.B.), Department of Clinical Biochemistry (P.H.N., L.H.), Institute of Pathology (S.D.), Department of Clinical Genetics (U.B.J.), and MR Centre (W.Y.K.), Aarhus University Hospital, Aarhus, Denmark; Clinical Research Center, Vendsyssel Hospital, Aalborg University, Hjørring, Denmark (U.T.B.); Division of Cardiovascular Medicine, Radcliffe Department of Medicine, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom (K.G.); and Department of Cardiology, Odense University Hospital, Odense, Denmark (J.M.).
Peter
P
Bross
From the Department of Cardiology (T.B.R., W.Y.K., H.M., H.K.J., J.M.), Research Unit for Molecular Medicine (T.B.R., J.P., P.B.), Department of Clinical Biochemistry (P.H.N., L.H.), Institute of Pathology (S.D.), Department of Clinical Genetics (U.B.J.), and MR Centre (W.Y.K.), Aarhus University Hospital, Aarhus, Denmark; Clinical Research Center, Vendsyssel Hospital, Aalborg University, Hjørring, Denmark (U.T.B.); Division of Cardiovascular Medicine, Radcliffe Department of Medicine, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom (K.G.); and Department of Cardiology, Odense University Hospital, Odense, Denmark (J.M.). jens.mogensen@dadlnet.dk.
Jens
J
Mogensen
D000293:Adolescent; D000328:Adult; D019571:Arrhythmogenic Right Ventricular Dysplasia / Q000235:genetics* / Q000378:metabolism; D004817:Epidermis / Q000378:metabolism*; D005260:Female; D057895:Haploinsufficiency; D006579:Heterozygote; D006801:Humans; D008297:Male; D008875:Middle Aged; D009206:Myocardium / Q000378:metabolism*; D010375:Pedigree; D051191:Plakophilins / Q000235:genetics* / Q000378:metabolism; D017384:Sequence Deletion; D055815:Young Adult
C495337:PKP2 protein, human; D051191:Plakophilins
arrhythmogenic right ventricular dysplasia; genetics; keratinocytes; plakophilins; proteomics