FRA2A is a CGG repeat expansion associated with silencing of AFF3

Folate-sensitive fragile sites (FSFS) are a rare cytogenetically visible subset of dynamic mutations. Of the eight molecularly characterized FSFS, four are associated with intellectual disability (ID). Cytogenetic expression results from CGG tri-nucleotide-repeat expansion mutation associated with local CpG hypermethylation and transcriptional silencing. The best studied is the FRAXA site in the FMR1 gene, where large expansions cause fragile X syndrome, the most common inherited ID syndrome. Here we studied three families with FRA2A expression at 2q11 associated with a wide spectrum of neurodevelopmental phenotypes. We identified a polymorphic CGG repeat in a conserved, brain-active alternative promoter of the AFF3 gene, an autosomal homolog of the X-linked AFF2/FMR2 gene: Expansion of the AFF2 CGG repeat causes FRAXE ID. We found that FRA2A-expressing individuals have mosaic expansions of the AFF3 CGG repeat in the range of several hundred repeat units. Moreover, bisulfite sequencing and pyrosequencing both suggest AFF3 promoter hypermethylation. cSNP-analysis demonstrates monoallelic expression of the AFF3 gene in FRA2A carriers thus predicting that FRA2A expression results in functional haploinsufficiency for AFF3 at least in a subset of tissues. By whole-mount in situ hybridization the mouse AFF3 ortholog shows strong regional expression in the developing brain, somites and limb buds in 9.5-12.5dpc mouse embryos. Our data suggest that there may be an association between FRA2A and a delay in the acquisition of motor and language skills in the families studied here. However, additional cases are required to firmly establish a causal relationship

Loss of the BMP Antagonist, SMOC-1, Causes Ophthalmo-Acromelic (Waardenburg Anophthalmia) Syndrome in Humans and Mice

Ophthalmo-acromelic syndrome (OAS), also known as Waardenburg Anophthalmia syndrome, is defined by the combination of eye malformations, most commonly bilateral anophthalmia, with post-axial oligosyndactyly. Homozygosity mapping and subsequent targeted mutation analysis of a locus on 14q24.2 identified homozygous mutations in SMOC1 (SPARC-related modular calcium binding 1) in eight unrelated families. Four of these mutations are nonsense, two frame-shift, and two missense. The missense mutations are both in the second Thyroglobulin Type-1 (Tg1) domain of the protein. The orthologous gene in the mouse, Smoc1, shows site- and stage-specific expression during eye, limb, craniofacial, and somite development. We also report a targeted pre-conditional gene-trap mutation of Smoc1 (Smoc1tm1a) that reduces mRNA to ∼10% of wild-type levels. This gene-trap results in highly penetrant hindlimb post-axial oligosyndactyly in homozygous mutant animals (Smoc1tm1a/tm1a). Eye malformations, most commonly coloboma, and cleft palate occur in a significant proportion of Smoc1tm1a/tm1a embryos and pups. Thus partial loss of Smoc-1 results in a convincing phenocopy of the human disease. SMOC-1 is one of the two mammalian paralogs of Drosophila Pentagone, an inhibitor of decapentaplegic. The orthologous gene in Xenopus laevis, Smoc-1, also functions as a Bone Morphogenic Protein (BMP) antagonist in early embryogenesis. Loss of BMP antagonism during mammalian development provides a plausible explanation for both the limb and eye phenotype in humans and mice

Heterozygous loss-of-function mutations in YAP1 cause both isolated and syndromic optic fissure closure defects.

Exome sequence analysis of affected individuals from two families with autosomal-dominant inheritance of coloboma identified two different cosegregating heterozygous nonsense mutations (c.370C>T [p.Arg124*] and c. 1066G>T [p.Glu356*]) in YAP1. The phenotypes of the affected families differed in that one included no extraocular features and the other manifested with highly variable multisystem involvement, including hearing loss, intellectual disability, hematuria, and orofacial clefting. A combined LOD score of 4.2 was obtained for the association between YAP1 loss-of-function mutations and the phenotype in these families. YAP1 encodes an effector of the HIPPO-pathway-induced growth response, and whole-mount in situ hybridization in mouse embryos has shown that Yap1 is strongly expressed in the eye, brain, and fusing facial processes. RT-PCR showed that an alternative transcription start site (TSS) in intron 1 of YAP1 and Yap1 is widely used in human and mouse development, respectively. Transcripts from the alternative TSS are predicted to initiate at codon Met179 relative to the canonical transcript (RefSeq NM_001130145). In these alternative transcripts, the c.370C>T mutation in family 1305 is within the 5' UTR and cannot result in nonsense-mediated decay (NMD). The c. 1066G>T mutation in family 132 should result in NMD in transcripts from either TSS. Amelioration of the phenotype by the alternative transcripts provides a plausible explanation for the phenotypic differences between the families

Heterozygous loss-of-function mutations in YAP1 cause both isolated and syndromic optic fissure closure defects

Exome sequence analysis of affected individuals from two families with autosomal-dominant inheritance of coloboma identified two different cosegregating heterozygous nonsense mutations (c.370C>T [p.Arg124∗] and c. 1066G>T [p.Glu356∗]) in YAP1. The phenotypes of the affected families differed in that one included no extraocular features and the other manifested with highly variable multisystem involvement, including hearing loss, intellectual disability, hematuria, and orofacial clefting. A combined LOD score of 4.2 was obtained for the association between YAP1 loss-of-function mutations and the phenotype in these families. YAP1 encodes an effector of the HIPPO-pathway-induced growth response, and whole-mount in situ hybridization in mouse embryos has shown that Yap1 is strongly expressed in the eye, brain, and fusing facial processes. RT-PCR showed that an alternative transcription start site (TSS) in intron 1 of YAP1 and Yap1 is widely used in human and mouse development, respectively. Transcripts from the alternative TSS are predicted to initiate at codon Met179 relative to the canonical transcript (RefSeq NM_001130145). In these alternative transcripts, the c.370C>T mutation in family 1305 is within the 5′ UTR and cannot result in nonsense-mediated decay (NMD). The c. 1066G>T mutation in family 132 should result in NMD in transcripts from either TSS. Amelioration of the phenotype by the alternative transcripts provides a plausible explanation for the phenotypic differences between the families

<i>Mp</i> eyes displayed structural defects and abnormal ciliary development.

<p>Histological comparison of eye tissues in Wt (A), <i>Mp/+</i>(B), and <i>Mp/Mp</i> (C) at P21 revealed severe pan-ocular defects. Mutant eyes displayed microphthalmia and retinal rosetting (arrowheads), together with loss of vitreous in the anterior chamber and between lens and retina. Lens size was also reduced in both mutant genotypes. Scale bars = 500 µm; sections are oriented in the sagittal plane. (D) Enlarged view of iris and the anterior region of retinas revealed the absence of ciliary body structures in both mutant types. (E–F) Genetic crosses of <i>Mp</i> with the Wnt-signalling reporter mouse line BAT-gal, revealed a significant reduction in galactosidase-positive cells in E14.5 <i>Mp</i> retinas compared to Wt, specifically in the dorsal and temporal regions of the anterior retina (<i>n = 8</i> per genotype). In contrast, non-ocular tissue displayed slightly increased staining in <i>Mp:B-gal</i> compared to Wt:<i>B-gal</i>, due to the slight increase in staining time in these samples. Ventral regions had low expression in both genotypes and were used as reference for quantitative comparison (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003998#pgen.1003998.s002" target="_blank">Figure S2</a>). (G–H) Histological analysis of anterior retinas at E15.5 revealed a reduction in non-pigmented ciliary body tissue (arrows) in <i>Mp/Mp</i> compared to Wt. (I–J) Immunohistochemical staining of anterior retinal structures with antibodies specific for Pax6 and Sox2 at E15.5 revealed reduction in the Pax6-positive and Sox2-negative non-pigmented ciliary epithelial region in <i>Mp</i>. Scale bars, 50 µm. The asterisk in C indicates artefactual disruption to the corneal epithelium during sample processing. Abbreviations: ac, anterior chamber; c, cornea; cb, ciliary body; ir, iris; ln, lens; nr, neural retina; rpe, retinal pigmented epithelium; vb, vitreous body.</p

Fibrillin-2^Mp aggregated into large intracellular inclusions within the rough endoplasmic reticulum.

<p>(A–B) Whole Mount <i>In Situ Hybridisation</i> to <i>Fbn2</i> using antisense 3′-UTR riboprobes of <i>Fbn2</i> for Wt and <i>Isoc1</i> for <i>Mp/Mp</i> in early Wt and <i>Mp/Mp</i> embryos (E11.5) revealed the spaciotemporal expression of the variant <i>Fbn2</i> alleles appeared unaffected by the genomic inversion and that <i>cis</i>-regulation of the genes was unchanged. <i>Fbn2</i> was expressed in the developing eyes (arrows), limbs (arrowheads), and tail (asterisks), and expression was also observed in the somites. (C–D) Section <i>In Situ Hybridisation</i> for <i>Fbn2</i> at E13.5 again showed that mutant <i>Fbn2</i> expression was comparable to Wt in the eyes, with <i>Fbn2</i> identified in the periocular (m) and corneal (c) mesenchyme, and faintly in the anterior retina (arrows). No expression was identified in the lens (ln). Scale bars in, 100 µm. (E–F) Immunohistochemical analysis of the anterior region of E13.5 Wt eyes illustrated that Fibrillin-2 was localised to extracellular regions in the corneal mesenchyme and in the region of apposition between the lens and neural retina (arrowheads). In contrast, Fibrillin-2^Mp in <i>Mp/Mp</i> eyes was not observed in these extracellular locations but instead appeared to be retained within cells throughout the developing eye, with the anterior neural retina (arrows) and adjacent RPE displaying the most numbers of positive-cells. Scale bars, 50 µm.</p

Fibrillin-2^Mp inclusions resulted in ER-stress and cell death in the developing <i>Mp</i> eye.

<p>(A) TEM micrograph of <i>Mp/Mp</i> ocular scleral cells revealed multiple intracellular inclusions located within the enlarged rough endoplasmic reticulum (rER) membrane (arrows). Scale bar, 2 µm. (B) Enlarged micrograph illustrating the structural periodicity of the inclusions with regular banding, possibly representing heterotypic fibrils. Arrowheads indicate rER membrane. Scale bar, 500 nm. (C) Immuno-gold labelling of the inclusions in <i>Mp/Mp</i> scleral cells with polyclonal anti-Fibrillin-2 antibody pAb868 and beads conjugated to secondary antibody revealed the inclusions to be composed of Fibrillin-2. (D) Fibrillin-2 immunofluorescence in <i>Mp</i> MEF cultures illustrated the perinuclear localisation and bundle-like organisation of the mutant protein aggregates. Scale bar, 10 µm. (E) Co-immunofluorescence with anti-ER marker PDI (red) confirmed the Fibrillin-2^Mp (green) inclusions colocalised within the ER (yellow stain; arrow). (F) Staining with the golgi-specific marker GM130 (green staining; arrowhead) showed that Fibrillin-2^Mp (red staining, arrows) was not localised within the Golgi. (G) Non-overlapping staining of Fibrillin-2 (green) and phalloidin (red) indicated that the inclusions were not located in the cytoplasm. Scale bars in E–G, 100 µm. (H) The ratio of <i>Xbp-1s</i> to <i>Xbp-1u</i> was significantly increased in <i>Mp/Mp</i> compared to Wt in RNA samples collected from MEFs and from <i>Mp/Mp</i> eyes at E13.5 and E16.5. (I) Section <i>In Situ</i> for ER-stress marker <i>Hspa5</i> mRNA revealed no staining in Wt retinas at E16.5, (J) however there was clear signal in the non-pigmented ciliary epithelium (arrows) in <i>Mp/Mp</i>. Scale bars, 50 µm. (K) (J) Activated-Caspase-3 stained cells from E13.5 and E16.5 Wt and <i>Mp/Mp</i> retinas were quantified and revealed significant increase in mutant eyes. Error bars are s.d. for H & K (**<i>P</i><0.005; *** <i>P</i><0.001).</p