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

Endocrine Abstracts (2019) 65 OC3.1 | DOI: 10.1530/endoabs.65.OC3.1

A mouse model generated by CRISPR-Cas9 with a frameshift mutation in the nuclear factor 1/X (NFIX) gene has phenotypic features reported in Marshall-Smith Syndrome (MSS) patients

Kreepa Kooblall1, Mark Stevenson1, Michelle Stewart2, Zsombor Szoke-Kovacs2, Tertius Hough2, Houfu Leng3, Nicole Horwood3, Tonia Vincent3, Raoul Hennekam4, Paul Potter2, Roger Cox2, Stephen Brown2, Sara Wells2, Lydia Teboul2 & Rajesh Thakker1

1OCDEM, RDM, University of Oxford, Oxford, UK; 2MRC Harwell, Mary Lyon Centre, Oxford, UK; 3KIR, NDORMS, University of Oxford, Oxford, UK; 4Department of Pediatrics, University of Amsterdam, Amsterdam, Netherlands

Marshall-Smith syndrome (MSS) is a congenital disorder characterised by developmental delay, short stature, respiratory difficulties, distinctive facial features, skeletal abnormalities (such as kyphoscoliosis, dysostosis and osteopenia) and delayed neural development, and is due to heterozygous mutations that are clustered in exons 6–10 of the transcription factor nuclear factor I/X (NFIX) gene. These frameshift and splice-site NFIX variants result in the production of aberrant transcripts that escape nonsense mediated mRNA decay and lead to the production of dominant negative mutant NFIX protein. To elucidate the in vivo effects of mutant NFIX, CRISPR-Cas9 was used to generate a mutant mouse model with a frameshift deletion of 2 nucleotides (Nfix Del2) in Nfix exon 7. All animal studies were ethically performed under an approved UK Home Office Animal License. Nfix+/Del2 mice were viable, normal and fertile but NfixDel2/Del2 mice had significantly reduced viability (P<0.002), and died at 2–3 weeks of age. Phenotypic characterisation of the 2–3 weeks NfixDel2/Del2 mice showed that, compared to Nfix+/+ and Nfix+/Del2 mice, NfixDel2/Del2 mice had significantly reduced: growth rate (0.8-fold, P<0.05); tail length (0.9-fold, P<0.001); lean (0.8-fold, P<0.0001) and fat (0.4-fold, P<0.0001) mass; weight (0.8-fold, P<0.0001); and total tissue mass (0.8-fold, P<0.0001). Moreover, >30% (P<0.0001) of NfixDel2/Del2 mice had kyphosis compared to <10% Nfix+/+ and Nfix+/Del2 mice, and micro-CT scans of the lumbar and thoracic vertebrae revealed NfixDel2/Del2 mice to have osteopenia. In addition, plasma biochemistry analysis of NfixDel2/Del2 mice revealed that, compared to Nfix+/+ and Nfix+/Del2 mice, NfixDel2/Del2 mice had significantly: increased plasma urea (1.4-fold, P<0.0001) and total bilirubin (1.5-fold, P<0.0001) concentrations; and alkaline phosphatase activity (1.5-fold, P<0.0001); but decreased procollagen type 1 N-terminal propeptide (0.8-fold, P<0.01) concentrations. Thus, NfixDel2/Del2 mice provide a useful model for studying the skeletal, renal and hepatic effects of mutant NFIX and the mechanisms underlying the aetiology of MSS.