Functional analysis of novel TBX5 T-box mutations associated with Holt-Oram syndrome.
OBJECTIVE
Holt-Oram syndrome (HOS) is a heart/hand syndrome clinically characterized by upper limb and cardiac malformations. Mutations in T-box transcription factor 5 (TBX5) underlie this syndrome, the majority of which lead to premature stops. In this study, we present our functional analyses of five (novel) missense TBX5 mutations identified in HOS patients, most of whom presented with severe cardiac malformations.


RESULTS
Functional characterization of mutant proteins shows a dramatic loss of DNA-binding capacity, as well as diminished binding to known cardiac interaction partners NKX2-5 and GATA4. The disturbance of these interactions leads to a loss of function, as measured by the reduced activation of Nppa and FGF10 in rat heart derived cells, although with variable severity. Two out of the five mutations are peculiar: one, p.H220del, is associated with additional extra-cardiac defects, perhaps by interfering with other T-box dependant pathways, and another, p.I106V, leads to limb defects only, which is supported by its normal interaction with cardiac-specific interaction partners.


CONCLUSIONS
Overall, our data are consistent with the hypothesis that these novel missense mutations in TBX5 lead to functional haploinsufficiency and result in a reduced transcriptional activation of target genes, which is likely central to the pathogenesis of HOS.
Boogerd
Cornelis J J
CJ
Heart Failure Research Center, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.
Dooijes
Dennis
D
Ilgun
Aho
A
Mathijssen
Inge B
IB
Hordijk
Roel
R
van de Laar
Ingrid M B H
IM
Rump
Patrick
P
Veenstra-Knol
Hermine E
HE
Moorman
Antoon F M
AF
Barnett
Phil
P
Postma
Alex V
AV
D000015:Abnormalities, Multiple / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D009320:Atrial Natriuretic Factor / Q000235:genetics; D001665:Binding Sites; D016022:Case-Control Studies; D002460:Cell Line; D004252:DNA Mutational Analysis; D024202:Electrophoretic Mobility Shift Assay; D051526:Fibroblast Growth Factor 10 / Q000235:genetics; D050991:GATA4 Transcription Factor / Q000235:genetics / Q000378:metabolism; D005838:Genotype; D006330:Heart Defects, Congenital / Q000235:genetics* / Q000378:metabolism; D006344:Heart Septal Defects, Atrial / Q000235:genetics / Q000378:metabolism; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D038061:Lower Extremity Deformities, Congenital / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D010641:Phenotype; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011487:Protein Conformation; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020825:T-Box Domain Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D014162:Transfection; D038062:Upper Extremity Deformities, Congenital / Q000235:genetics* / Q000378:metabolism
D051526:Fibroblast Growth Factor 10; D050991:GATA4 Transcription Factor; D018398:Homeodomain Proteins; D011993:Recombinant Fusion Proteins; D020825:T-Box Domain Proteins; C103172:T-box transcription factor 5; D009320:Atrial Natriuretic Factor