Identification of cellular retinoic acid binding protein 2 (CRABP2) as downstream target of nuclear factor I/X (NFIX): implications for skeletal dysplasia syndromes.

Nuclear factor I/X ( NFIX ) mutations are associated with 2 skeletal dysplasias, Marshall-Smith (MSS) and Malan (MAL) syndromes. NFIX encodes a transcription factor that regulates expression of genes, including Bobby sox ( BBX ) and glial fibrillary acidic protein ( GFAP ) in neural progenitor cells and astrocytes, respectively. To elucidate the role of NFIX mutations in MSS, we studied their effects in fibroblast cell lines obtained from 5 MSS unrelated patients and 3 unaffected individuals. The 5 MSS NFIX frameshift mutations in exons 6-8 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). Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot analyses using MSS and unrelated control fibroblasts and in vitro expression studies in monkey kidney fibroblast (COS-7) cells showed that frameshift mutations in NFIX exons 6-8 generated mutant transcripts that were not cleared by nonsense-mediated-decay mechanisms and encoded truncated NFIX proteins. Moreover, BBX or GFAP expression was unaffected in the majority of MSS fibroblasts. To identify novel NFIX downstream target genes, RNA sequencing and proteomics analyses were performed on mouse embryonic fibroblast (MEF) cells derived from control Nfix +/+ , Nfix +/Del2 , Nfix +/Del24 , Nfix Del24/Del24 , Nfix +/Del140 , and Nfix Del140/Del140 mice, compared with Nfix Del2/Del2 mice which had developmental, skeletal, and neural abnormalities. This identified 191 transcripts and 815 proteins misregulated in Nfix Del2/Del2 MEFs with ≥2-fold-change ( P  <0 .05). Validation studies using qRT-PCR and western blot analyses confirmed that 2 genes, cellular retinoic acid binding protein 2 ( Crabp2 ) and vascular cell adhesion molecule 1 ( Vcam1 ), were misregulated at the RNA and protein levels in Nfix Del2/Del2 MEFs, and that CRABP2 and VCAM1 expressions were altered in 60%-100% of MSS fibroblast cells. Furthermore, in vitro luciferase reporter assays confirmed that NFIX directly regulates CRABP2 promoter activity. Thus, these altered genes and pathways may represent possible targets for drugs as potential treatments and therapies for MSS.