Marshall-Smith syndrome (MSS) is a congenital disorder characterised by developmental delay, failure to thrive and skeletal abnormalities such as accelerated osseous development, osteopenia, bullet-shaped middle phalanges and kyphoscoliosis. MSS is caused by truncating or frameshift mutations of the nuclear factor I/X (NFIX) gene, which encodes a ubiquitously expressed transcription factor that regulates expression of viral and cellular genes, including Bobby sox (BBX) and glial fibrillary acidic protein (GFAP). To further elucidate the role of NFIX mutations in MSS, we studied their effects on BBX and GFAP expression using fibroblast cell lines obtained from 5 MSS patients and 3 unaffected individuals. Informed consent was obtained using protocols approved by local ethics committees. The 5 MSS fibroblast cell lines were confirmed to have NFIX mutations in exons 68 by Sanger DNA sequencing and multiplex ligation-dependent probe amplification analyses, and these comprised 3 deletions (c.819-732_1079-948del, c.819-471_1079-687del, c.819-592_1079-808del), an insertion (c.1037_1038insT) and a duplication (c.1090dupG) that were all predicted to result in premature truncations. Transient transfection of N-terminal-FLAG tagged wild-type and MSS-mutant NFIX cDNA constructs in monkey kidney fibroblast (COS-7) cells followed by Western blot analysis confirmed that the MSS-associated NFIX mutants resulted in truncated proteins. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis using RNA from the human MSS fibroblasts showed no difference in NFIX transcript levels when compared to controls, indicating that the mutant transcripts were not cleared by nonsense-mediated-decay mechanisms. qRT-PCR and Western blot analyses using RNA and protein extracted from MSS fibroblasts and control fibroblasts showed no differences in BBX expression, but the NFIX c.1090dupG mutant was found to result in a 24-fold (P<0.05, n=4) decrease in GFAP protein. Thus, our results suggest that the majority of MSS-associated NFIX mutations are not acting via BBX or GFAP, but via other downstream target genes that remain to be identified.