Functional Studies and In Silico Analyses to Evaluate Non-Coding Variants in Inherited Cardiomyopathies.
Point mutations are the most common cause of inherited diseases. Bioinformatics tools can help to predict the pathogenicity of mutations found during genetic screening, but they may work less well in determining the effect of point mutations in non-coding regions. In silico analysis of intronic variants can reveal their impact on the splicing process, but the consequence of a given substitution is generally not predictable. The aim of this study was to functionally test five intronic variants (MYBPC3-c.506-2A>C,MYBPC3-c.906-7G>T,MYBPC3-c.2308+3G>C,SCN5A-c.393-5C>A, andACTC1-c.617-7T>C) found in five patients affected by inherited cardiomyopathies in the attempt to verify their pathogenic role. Analysis of theMYBPC3-c.506-2A>C mutation in mRNA from the peripheral blood of one of the patients affected by hypertrophic cardiac myopathy revealed the loss of the canonical splice site and the use of an alternative splicing site, which caused the loss of the first seven nucleotides of exon 5 (MYBPC3-G169AfsX14). In the other four patients, we generated minigene constructs and transfected them in HEK-293 cells. This minigene approach showed thatMYBPC3-c.2308+3G>C andSCN5A-c.393-5C>A altered pre-mRNA processing, thus resulting in the skipping of one exon. No alterations were found in eitherMYBPC3-c.906-7G>T orACTC1-c.617-7T>C. In conclusion, functional in vitro analysis of the effects of potential splicing mutations can confirm or otherwise the putative pathogenicity of non-coding mutations, and thus help to guide the patient's clinical management and improve genetic counseling in affected families.
CEINGE-Biotecnologie Avanzate s.c.a r.l., 80145 Napoli, Italy. gfrisso@unina.it.|Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università di Napoli Federico II, 80131 Napoli, Italy. gfrisso@unina.it.
Giulia
G
Frisso
CEINGE-Biotecnologie Avanzate s.c.a r.l., 80145 Napoli, Italy. detta@ceinge.unina.it.
Nicola
N
Detta
CEINGE-Biotecnologie Avanzate s.c.a r.l., 80145 Napoli, Italy. coppolap@ceinge.unina.it.|Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università di Napoli Federico II, 80131 Napoli, Italy. coppolap@ceinge.unina.it.
Pamela
P
Coppola
CEINGE-Biotecnologie Avanzate s.c.a r.l., 80145 Napoli, Italy. cristina.mazzaccara@unina.it.|Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università di Napoli Federico II, 80131 Napoli, Italy. cristina.mazzaccara@unina.it.
Cristina
C
Mazzaccara
Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università di Napoli Federico II, 80131 Napoli, Italy. mariarosaria.pricolo@unina.it.
Maria Rosaria
MR
Pricolo
Dipartimento di Cardiologia, Seconda Università degli Studi di Napoli, A.O. Monaldi, Azienda dei Colli, 80131 Napoli, Italy. donofrioant@iol.it.
Antonio
A
D'Onofrio
Dipartimento di Cardiologia, Seconda Università degli Studi di Napoli, A.O. Monaldi, Azienda dei Colli, 80131 Napoli, Italy. limongelligiuseppe@libero.it.
Giuseppe
G
Limongelli
Dipartimento di Cardiologia, Seconda Università degli Studi di Napoli, A.O. Monaldi, Azienda dei Colli, 80131 Napoli, Italy. raffaele.calabro@unina2.it.
Raffaele
R
Calabrò
CEINGE-Biotecnologie Avanzate s.c.a r.l., 80145 Napoli, Italy. salvator@unina.it.|IRCCS-Fondazione SDN, 80143 Napoli, Italy. salvator@unina.it.
Francesco
F
Salvatore
D000199:Actins / Q000235:genetics; D001483:Base Sequence; D009202:Cardiomyopathies / Q000235:genetics*; D002312:Cardiomyopathy, Hypertrophic / Q000235:genetics; D002352:Carrier Proteins / Q000235:genetics; D003198:Computer Simulation; D005192:Family Health; D005260:Female; D020022:Genetic Predisposition to Disease / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D007438:Introns / Q000235:genetics*; D008297:Male; D008957:Models, Genetic; D062554:NAV1.5 Voltage-Gated Sodium Channel / Q000235:genetics; D010375:Pedigree; D017354:Point Mutation; D022821:RNA Splice Sites / Q000235:genetics; D012326:RNA Splicing; D000099163:Myosin Binding Protein C
D000199:Actins; D002352:Carrier Proteins; D062554:NAV1.5 Voltage-Gated Sodium Channel; D022821:RNA Splice Sites; D000099163:Myosin Binding Protein C; C526913:ACTC1 protein, human; C568320:SCN5A protein, human
ACTC1; MYBPC3; SCN5A; inherited cardiomyopathies; intronic mutations; minigene; non-coding variations; splicing analysis