Analysis of Pax6 Contiguous Gene Deletions in the Mouse,Mus musculus,Identifies Regions Distinct from Pax6 Responsible for Extreme Small-Eye and Belly-Spotting Phenotypes

In the mouse Pax6 function is critical in a dose-dependent manner for proper eye development. Pax6 contiguous gene deletions were shown to be homozygous lethal at an early embryonic stage. Heterozygotes express belly spotting and extreme microphthalmia. The eye phenotype is more severe than in heterozygous Pax6 intragenic null mutants, raising the possibility that deletions are functionally different from intragenic null mutations or that a region distinct from Pax6 included in the deletions affects eye phenotype. We recovered and identified the exact regions deleted in three new Pax6 deletions. All are homozygous lethal at an early embryonic stage. None express belly spotting. One expresses extreme microphthalmia and two express the milder eye phenotype similar to Pax6 intragenic null mutants. Analysis of Pax6 expression levels and the major isoforms excluded the hypothesis that the deletions expressing extreme microphthalmia are directly due to the action of Pax6 and functionally different from intragenic null mutations. A region distinct from Pax6 containing eight genes was identified for belly spotting. A second region containing one gene (Rcn1) was identified for the extreme microphthalmia phenotype. Rcn1 is a Ca⁺²-binding protein, resident in the endoplasmic reticulum, participates in the secretory pathway and expressed in the eye. Our results suggest that deletion of Rcn1 directly or indirectly contributes to the eye phenotype in Pax6 contiguous gene deletions.CONTIGUOUS gene deletions account for a significant portion of human genetic syndromes. The application of fluorescence in situ hybridization (FISH) cytogenetics and array comparative genome hybridization (array-CGH) technologies have enabled more accurate localization of deletion breakpoints. This deletion information combined with the annotation of the human genome structure provides critical information to identify genes responsible for particular phenotypes associated with a syndrome. For example, deletions of the 11p11p12 and 11p13 regions on the short arm of human chromosome (Chr) 11 have been identified in the Potocki–Shaffer syndrome (Shaffer et al. 1993; Bartsch et al. 1996; Potocki and Shaffer 1996) and the Wilm''s tumor- aniridia- genitourinary abnormalities- mental retardation (WAGR) syndrome (Riccardi et al. 1978; Francke et al. 1979; Hittner et al. 1979; Fryns et al. 1981), respectively. Deletion analyses were important in identifying genes associated with clinical features of the syndromes: EXT2 for multiple exostoses and ALX4 for parietal foramina in Potocki–Shaffer syndrome (Ligon et al. 1998; Wu et al. 2000; Wakui et al. 2005), WT1 for Wilm''s tumor, and PAX6 for aniridia in WAGR syndrome (van Heyningen et al. 1985; Glaser et al. 1986, 1992; Fantes et al. 1992). Deletion analyses have also defined the extent of the deleted region in patients with combined Potocki–Shaffer and WAGR syndromes (McGaughran et al. 1995; Brémond-Gignac et al. 2005) as well as microdeletions 3′ to PAX6, which prevent expression of PAX6 and cause aniridia (Lauderdale et al. 2000; D''elia et al. 2007; Davis et al. 2008).The mouse Chr 2 region homologous to the human WAGR region contains the genes Wt1, Rcn1, Pax6, and Elp4. An extensive allelic series at Pax6 has been identified (Bult et al. 2008). Heterozygote Pax6 intragenic null mutants express microphthalmia, iris anomalies, corneal opacities, lens opacities, and lens-corneal adhesions. Homozygotes are anophthalmic and die shortly after birth (Roberts 1967; Hogan et al. 1986). Five deletions in the region have been identified: Pax6^Sey-Dey, Pax6^Sey-H, Pax6^Sey-2H, Pax6^Sey-3H, Pax6^Sey-4H of which two, Pax6^Sey-H (Hogan et al. 1986; Kent et al. 1997; Kleinjan et al. 2002; Webb et al. 2008) and Pax6^Sey-Dey (Theiler et al. 1978; Hogan et al. 1987; Glaser et al. 1990), have been well characterized. Heterozygotes for both deletions express belly spotting and a more extreme eye phenotype than that observed for heterozygotes of intragenic Pax6 null mutations. Homozygotes for both deletions are lethal at an early embryonic stage.We were particularly interested in the extreme eye phenotype associated with the Pax6 deletions and considered two alternative hypotheses. Either Pax6 deletions are functionally different from Pax6 intragenic null mutations or deletion of a region linked to but distinct from the Pax6 structural gene affects the eye phenotype.In the present study we identify three new deletions encompassing the Pax6 region of the mouse. They have been assigned the mutant allele symbols Del(2)Pax6^11Neu/1Neu, Del(2)Pax6^12Neu/2Neu, and Del(2)Pax6^13Neu/3Neu and will be referred to throughout this publication as Pax6^11Neu, Pax6^12Neu, and Pax6^13Neu, respectively. All three deletions are homozygous lethal at an early embryonic stage. The deletions differentiate for the extent of the eye abnormality expressed: Pax6^11Neu heterozygotes express extreme microphthalmia similar to that observed in the Pax6^Sey-Dey and Pax6^Sey-H deletions. Pax6^12Neu and Pax6^13Neu heterozygotes express the milder eye abnormality seen in heterozygous intragenic null mutants. For all three deletions, heterozygotes do not express belly spotting. Genetic, phenotypic, and molecular characterization of the deletions allowed us to identify regions associated with the array of phenotypes in these contiguous gene deletions.