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K.M. Huang, J. Wu, M. Duncan, C. Moy, A. Dutra, J. Favor, D. Stambolian; Xcat, a Novel Mouse Model for Nance Horan Syndrome Inhibits Expression of the Cytoplasmic–Targeted Nhs1 Isoform . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2524.
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© ARVO (1962-2015); The Authors (2016-present)
Nance Horan Syndrome (NHS) is an X–linked disorder characterized by congenital cataracts, dental anomalies, dysmorphic features and mental retardation. A recent report suggests that the novel gene NHS1 is involved in this disorder due to the presence of point mutations in NHS patients. A possible mouse model for NHS, Xcat, was mapped to a 2.11 Mb interval on the X chromosome. The purpose of this study is to determine the genetic defect in Xcat.
The Xcat critical region was examined by making a BAC library from Xcat genomic DNA and then sequencing the BACs that mapped to the Xcat critical region. Chromsomes from Xcat mice were subjected to FISH analysis using labeled BACs as probes. The expression of mouse Nhs1 was analyzed by quantitative RT–PCR using RNA isolated from neonate eye, mouth or paw tissue. Antibodies were raised against the mouse Nhs1 protein and used for immunohistochemical analyses of normal and Xcat neonate lens. CHO cells were transiently transfected with vectors encoding Nhs1–GFP fusion proteins and then analyzed by fluorescence microscopy.
Sequence and FISH analysis of the X chromosome region containing the Xcat mutation reveals a large insertion between exons 1 and 2 of the mouse Nhs1 gene. The insertion inhibits the expression of the Nhs1 isoform containing exon 1 and results in exclusive expression of the alternative isoform containing exon 1A. Quantitative RT–PCR of Xcat cDNA shows reduced levels of Nhs1 transcripts. The Nhs1 protein is strongly expressed within the cytoplasm of elongating lens fiber cells from wildtype neonate lens, but is significantly reduced within the Xcat lens. Transient transfection studies of CHO cells with Nhs1–GFP fusion proteins revealed that amino acids encoded by exon 1 were critical for protein localization. We found that fusion proteins containing Nhs1 exon 1 localized to the cytoplasm, while fusion proteins lacking Nhs1 exon 1 are predominantly nuclear.
This study indicates that the Xcat mutation is a large insertion within the first intron of the Nhs1 gene. The insertion results in lack of expression of Nhs1 transcripts containing exon 1. In vitro studies indicate that Nhs1 exon 1 contains crucial information required for the proper expression and localization of Nhs1 protein. Inhibition of expression of the exon 1–containing Nhs1 isoform results in the abnormal phenotype of Xcat.
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