ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Endocrine Abstracts (2015) 38 P2 | DOI: 10.1530/endoabs.38.P2

Exploring the N-ethyl-N-nitrosourea mutagenesis DNA archive for mutations in nuclear factor I/X to derive mouse models for Marshall-Smith syndrome

Kreepa Kooblall1, Mark Stevenson1, Sian Piret1, Paul Potter2, Roger Cox2, Steve Brown2, Raoul Hennekam3 & Rajesh Thakker1

1University of Oxford, Oxford, UK; 2MRC Harwell, Harwell, UK; 3University of Amsterdam, Amsterdam, The Netherlands.

Marshall-Smith syndrome (MSS) is a congenital disorder affecting skeletal and neural development due to mutations in the nuclear factor I/X (NFIX) gene. Of these mutations, 61% are small insertions/deletions, 12% are splice site mutations and 27% are large exonic deletions clustered in exons 6–10 of the NFIX gene. In order to derive a MSS mouse model, the N-ethyl-N-nitrosourea (ENU) mutagenesis DNA archive was screened for mutations in NFIX. Three point mutations were identified. The first mutation caused a T to A transversion 8 nucleotides prior to exon 5 (IVS4-8t>a). The second mutation caused a T to G transversion in exon 4 (Trp214Gly). The third mutation caused a G to T transversion in exon 8 (Ala356Ser). The three mutations were characterised using in vitro minigene and expression assays to investigate whether the IVS4-8t>a mutation affected splicing and whether the Trp214Gly and Ala356Ser mutations affected NFIX cellular localisation and function respectively. RT-PCR using RNA from the NFIX minigene assay showed that the IVS4-8t>a mutation did not affect splicing. In vitro expression assays using NFIX cDNA with the Trp214Gly and Ala356Ser mutations showed that these mutations did not affect NFIX cellular localisation. As the mutations are found within the C-terminal transactivation/repression domain they might affect the expression of downstream target genes. Western blot analysis showed an increase in NFIX protein level in the Trp214Gly mutant and a reduction in NFIX protein level in the Ala356Ser mutant. Reporter assays under the control of NFIX binding sites showed that only the Ala356Ser mutation reduced NFIX transactivation activity at the glial fibrillary acidic protein (GFAP) locus while repression activity at the Bobby sox (BBX) locus was unaffected, consistent with in vivo data from MSS patients cell lines. ENU induced Ala356Ser mutant mice may therefore provide a representative model for MSS.