The basic helix-loop-helix protein Twist, a transcriptional repressor, is 
essential for embryogenesis in both invertebrates and vertebrates. 
Haploinsufficiency of the human TWIST1 gene, which causes the craniosynostosis 
disorder Saethre-Chotzen syndrome (SCS), is related to failure to repress 
transcription of CDKN1A (which encodes p21/WAF1/CIP1), promoting osteoblast 
differentiation. We have examined the functional significance of natural TWIST1 
variants present in craniosynostosis patients and in their healthy relatives. 
Both deletion and duplication variants of the glycine-rich tract Gly5AlaGly5 
inhibited E2A (E12/E47)-dependent transcription of CDKN1A to a similar degree as 
wild-type protein, indicating that the length of this glycine tract is not 
critical for efficient transcriptional repression. We also evaluated a newly 
identified heterozygous TWIST1 variant (c.115C>G, encoding p.Arg39Gly), located 
within a putative nuclear localization signal (NLS), that was present in a child 
with mild SCS and her clinically unaffected father and grandmother. Unlike 
wild-type protein, this mutant required cotransfected E12 to localize to the 
nucleus, indicating that the NLS, including amino acid 39, is essential for 
nuclear localization; inhibition of E2A-dependent transcription of CDKN1A 
occurred normally. This analysis further dissects the structure-function 
relationships of TWIST and corroborates with phenotypic observations of disease 
expressivity.