International Mammalian Genome Society

The 13th International Mouse Genome Conference
October 31-November 3, 1999

Table of Contents * Structure * Bioinformatics * Sequence * Mapping * New Tools * Gene Discovery * Developmental * Mutagenesis * Functional Genomics

G12 Positional Cloning of the exma Locus, an X-Linked Insertional Mutation Causing Exencephaly and Microphthalmia/Anophthalmia

David Cunningham, Qiang Xiao, Aurobindo Chatterjee1, Kathleen Sulik2, Diana Juriloff3, Frederick Elder4, Wilbur Harrison4, Gabriele Schuster5, Paul Overbeek5, Gail E. Herman. Division of Human and Molecular Genetics, Dept. of Pediatrics and Children's Hospital Research Foundation, Ohio State University, Columbus, OH; 1Parke-Davis, Alameda, CA; 2Dept. of Cell Biology and Anatomy, University of North Carolina, Chapel Hill, NC; 3Dept. of Medical Genetics, University of British Columbia, Vancouver, Canada; 4Dept. of Pathology and Laboratory Medicine, University of Texas, Houston TX; 5Dept. of Cell Biology, Baylor College of Medicine, Houston, TX

Neural tube defects (NTDs) comprise a group of common, severe congenital malformations, including spina bifida and anencephaly. The etiology of most NTDs in humans is poorly understood, but appears to be multifactoral, involving multiple genes as well as environmental factors. In order to identify genes directly involved in neural tube development, we are attempting to clone the exma locus in the mouse.

exma is an X-linked, male perinatal lethal mutation that results in exencephaly, anopthalmia and craniofacial abnormalities in affected males and reduced viability and microphthalmia in heterozygous females. Abnormal development is evident by embryonic day 9.5 in affected males, at which time the neural tube has failed to close in the region of the developing forebrain, and optic vesicles are absent. The mutation arose from the random integration of a transgene carrying a tyrosinase minigene. FISH mapping using known markers localized the insertion site to the interval between Dmd and Zfx on the mouse X chromosome. Sequences flanking the transgene were isolated from l clones of mutant genomic DNA and were used to screen for normal BACs from the region. The site of transgene insertion has been localized to within approximately 100 kb of the 3' end of the Pola gene. A contig of BAC clones is being assembled which will be used to further map the region, search for nearby genes and characterize possible large-scale deletions or rearrangements in the mutant DNA.

By analyzing human genomic sequence from the interval between DMD and ZFX available in the Baylor College of Medicine Human Genome Project database, we were able to localize the previously cloned mouse Arx gene to the interval between the site of transgene insertion and the 3' end of Pola. Arx represents an attractive candidate gene for exma since it encodes a highly conserved homeobox protein that has been shown by others to be expressed in the developing forebrain of the mouse. Data from Southern blots of normal and transgenic DNA suggest that a segment of the mouse X chromosome including Arx has been duplicated as a result of the transgene insertion, raising the possibility that the mutant phenotype arises from over-expression of Arx or another gene within the duplicated interval. Additionally, we have identified a unique 1.2 kb sequence that lies with 1 kb of the transgene and shows 93% identity between the mouse and human. The sequence shows no apparent coding potential or significant identity to sequences within the databases. We are currently determining whether it is part of a transcription unit that may have been disrupted by integration of the transgene.


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