Transcription factor TCF4 (alias ITF2, SEF2 or E2-2) is a broadly expressed basic helix-loop-helix (bHLH) protein that functions as a homo- or heterodimer. Missense, nonsense, frame-shift and splice-site mutations as well as translocations and large deletions encompassing TCF4 gene cause Pitt-Hopkins syndrome (PTHS), a rare developmental disorder characterized by severe motor and mental retardation, typical facial features and breathing anomalies. Irrespective of the mutation, TCF4 haploinsufficiency has been proposed as an underlying mechanism for PTHS. We have recently demonstrated that human TCF4 gene is transcribed using numerous 5' exons. Here, we re-evaluated the impact of all the published PTHS-associated mutations, taking into account the diversity of TCF4 isoforms, and assessed how the reading frame elongating and missense mutations affect TCF4 functions. Our analysis revealed that not all deletions and truncating mutations in TCF4 result in complete loss-of-function and the impact of reading frame elongating and missense mutations ranges from subtle deficiencies to dominant-negative effects. We show that (i) missense mutations in TCF4 bHLH domain and the reading frame elongating mutation damage DNA-binding and transactivation ability in a manner dependent on dimer context (homodimer versus heterodimer with ASCL1 or NEUROD2); (ii) the elongating mutation and the missense mutation at the dimer interface of the HLH domain destabilize the protein; and (iii) missense mutations outside of the bHLH domain cause no major functional deficiencies. We conclude that different PTHS-associated mutations impair the functions of TCF4 by diverse mechanisms and to a varying extent, possibly contributing to the phenotypic variability of PTHS patients.