Mapping and Phenotyping Genes in the Del(13)Svea36H Region of Mouse Chromosome 13

Del(13)Svea36H (also known as Del36H) is a 12.7Mb gene-rich region that has been deleted in mouse chromosome 13. It contains numerous disease loci as well as showing conserved synteny to two regions of human chromosome 6p which can be missing in some deletion syndromes. The mouse deletion causes homozygous embryonic lethality but is hemizygously viable. It has therefore been used as a tool to screen for lethal recessive mutations that could help to identify developmental genes that are responsible for the symptoms of the associated human diseases. A Physical map of the region was constructed in order to facilitate the mapping of mutations arising from the lethal screen. Eleven lethal lines have been mapped to varying degrees of accuracy, including one lethal mutation that exhibited circulation defects which localised to Sox4. The forkhead transcription factor Foxf2 was considered a potential candidate for embryonic lethal mutations within the interval. To complement the lethal recessive screen, an attempt was made to identify an allelic series of mutations in the gene. ENU mutagenised mouse archives were screened for novel Foxf2 mutations. Three potential functional mutations were discovered - two coding mutations (Foxf2W174R and Foxf2V412F), and a splice site mutation. Foxf2W174R heterozygotes exhibited thinning of the iris stroma, alongside hypoplasia of the trabecular meshwork and canal of Schlemm and a reduction in the iridocorneal angle. Homozygote eyes at E18.5 show no signs of the developing ciliary body which were seen in wildtype littermates. Homozygote carriers survive for several days, significantly longer than the 18hr survival time observed in the knockout, suggesting a hypomorphic mutation. In contrast to Foxf2 knockouts, homozygotes for the W174R mutation do not possess a cleft palate or malformed tongue

Foxf2: A Novel Locus for Anterior Segment Dysgenesis Adjacent to the Foxc1 Gene

Anterior segment dysgenesis (ASD) is characterised by an abnormal migration of neural crest cells or an aberrant differentiation of the mesenchymal cells during the formation of the eye's anterior segment. These abnormalities result in multiple tissue defects affecting the iris, cornea and drainage structures of the iridocorneal angle including the ciliary body, trabecular meshwork and Schlemm's canal. In some cases, abnormal ASD development leads to glaucoma, which is usually associated with increased intraocular pressure. Haploinsufficiency through mutation or chromosomal deletion of the human FOXC1 transcription factor gene or duplications of the 6p25 region is associated with a spectrum of ocular abnormalities including ASD. However, mapping data and phenotype analysis of human deletions suggests that an additional locus for this condition may be present in the same chromosomal region as FOXC1. DHPLC screening of ENU mutagenised mouse archival tissue revealed five novel mouse Foxf2 mutations. Re-derivation of one of these (the Foxf2W174R mouse lineage) resulted in heterozygote mice that exhibited thinning of the iris stroma, hyperplasia of the trabecular meshwork, small or absent Schlemm's canal and a reduction in the iridocorneal angle. Homozygous E18.5 mice showed absence of ciliary body projections, demonstrating a critical role for Foxf2 in the developing eye. These data provide evidence that the Foxf2 gene, separated from Foxc1 by less than 70 kb of genomic sequence (250 kb in human DNA), may explain human abnormalities in some cases of ASD where FOXC1 has been excluded genetically

Analysis of disease-linked rhodopsin mutations based on structure, function, and protein stability calculations

Retinitis pigmentosa (RP) refers to a heterogeneous group of inherited diseases that result in progressive retinal degeneration, characterized by visual field constriction and night blindness. A total of 103 mutations in rhodopsin are linked to RP to date, and the phenotypes range from severe to asymptomatic. To study the relation between phenotype and rhodopsin stability in disease mutants, we used a structure-based approach. For 12 of the mutants located at the protein lipid interphase, we used the von Heijne water membrane transfer scale, and we find that 9 of the mutations could affect membrane insertion. For 91 mutants, we used the protein design algorithm FoldX. The 3 asymptomatic mutations had no significant reduced stability, 2 were unsuitable for FoldX analysis since the structure was incorrect in this region, 63 mutations had a significant change in protein stability (>1.6 kcal/mol), and 23 mutations had energy change values under the prediction error threshold (<1.6 kcal/mol). Out of these 23, the disease-causing effect could be explained by the involvement in other functions (e.g., glycosylation motifs, the interface with arrestin and transducin, and the cilia-binding motif) for 19 mutants. The remaining 4 mutants were probably incorrectly associated with RP or have functionalities not discovered yet. For destabilizing mutations where clinical data were available, we found a highly significant correlation between FoldX energy changes and the average age of night blindness and between FoldX energy changes and daytime vision loss onset. Our detailed structural, functional, and energetic analysis provides a complete picture of the rhodopsin mutations and can guide mutation-specific therapies. (C) 2010 Elsevier Ltd. All rights reserved

Primers used for sequencing mouse <i>Foxc</i>1.

<p>Primers used for sequencing mouse <i>Foxc</i>1.</p

Iridocorneal phenotypic features in <i>Foxf2</i>^W174R mice.

<p>(<b>i</b>), in wildtype mice Schlemm's canal extends between the two white arrows. The trabecular meshwork is indicated by an asterisk. The ciliary body (CB) and ciliary body process (CBP) extend into the anterior chamber. The ciliary muscle is indicated by the black arrow. Iris Normal iridocorneal angle (<b>A</b>) indicated by double-headed arrow. (<b>B–E</b>), phenotype in heterozygous <i>Foxf2</i>^W174R mice. All mice had shortened or absent Schlemm's canal and a hypoplastic CB or CBP. (<b>C</b>), Trabecular meshwork is hypoplastic and the iris is parallel to the cornea (no angle). (<b>E</b>), Trabecular meshwork is hypoplastic and iridocorneal adhesion (arrowhead) is present. Scale bar  = 50 µm.</p

Foxf2 structural organization.

<p><b>A</b>, Protein domains of Foxf2 with mouse mutations identified in the DHPLC screen. The domain structure is shown as described for mouse <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025489#pone.0025489-Miura1" target="_blank">[23]</a> including two activation domains at the 5′ end, but overlayed by the activation domain structure that was described for the human gene <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025489#pone.0025489-Hellqvist1" target="_blank">[49]</a> with AD2 synergistic sub-domains (ovals) and AD1 domain (circle). <b>B</b>, The tryptophan codon at position 174 is conserved in the forkhead domain of all Fox proteins, of which several examples are shown. Foxf2 is highlighted by the grey box. Conserved residues are highlighted in black.</p

Phenotype of homozygous <i>Foxf2</i>^W174R pup compared with wildtype.

<p><b>A</b>, A noticeable difference in gross size can be seen by 3 days of age in mutant pup. <b>B&C</b>, In contrast to the homozygous knockout, the homozygous <i>Foxf2</i>^W174R individuals appear normal at birth, although the amount of milk consumed (arrow) is reduced compared to wildtype littermates.</p

Iris phenotype of <i>Foxf2</i>^W174R mice.

<p>The iris in wildtype mice (+/+) consists of a robust upper stromal layer (arrowhead) and a lower pigmented epithelium (arrow). The stroma in three heterozygous (m/+) mice is flattened or atrophic. The bottom image shows variable thickness across the iris tissue in both stromal and epithelial layers. Scale bar  = 40 µm.</p

Representative images of eye phenotype of aged heterozygous <i>Foxf2</i>^W174R mutant mice.

<p><b>A</b>, 6 month old <i>Foxf2</i>^W174R eye (m+/) showing normal retinal termination at the edge of the optic nerve fibre layer (black arrows). <b>B&C,</b> sections through two other 6 month old <i>Foxf2</i>^W174R eyes showing abnormal bulge in the nerve fibre layer. The outer nuclear layer of the retina can be seen to continue at the surface of this bulging tissue (white arrows). <b>D&E</b>, retinal sections in 6 month eyes. <b>F&G</b>, corneal sections in 6 month eyes. <b>H–J</b>, 12 month <i>Foxf2</i>^W174R optic nerves that do not show signs of glaucomatous change. v, vein; p, photoreceptors; onl, outer nuclear layer; inl, inner nuclear layer; ce, corneal epithelium; cs, corneal stroma. Scale bar  = 50 µm.</p

Primers used for the gene driven screen and mouse <i>Foxf2</i> sequencing.

<p>Primer sets Exon1a-1e were used to screen the first exon and flanking regions, primer set Exon2a was used to screen exon 2. * Primer set Exon1e replaced Exon1d for the screen of the MRC archive.</p