Sizing the repeat in all available family members with gene specific repeat primed PCR.

<p>Alleles were sized by gene specific repeat primed PCR. As expansions containing over 300 repeated units cannot be reliably sized using this technique, we indicated these alleles as “>300”. Subject A.II.4 is marked with an asterisk as she is mosaic for a 134-bp deletion taking away the entire CGG repeat in combination with a largely expanded allele, and this in addition to a normal sized allele.</p

FISH analysis of the FRA2A fragile site.

<p><b>A.</b> The BAC-clone 549H5 (labelled green) spans the fragile site FRA2A. <b>B.</b> FISH analysis using the 10 kb L10K (labelled green; chr2: 100721983–100733233; hg19) and 18 kb L18K (labelled red; chr2: 100700447–100718834; hg19) PCR-generated probes, targeted to map either side of FRA2A. The additional telomeric FISH signal on the red channel (red arrow chr2: 110520380–110538822 and chr2:111347822–111366260; hg19) is the result of a 24 kb low copy repeat (24 kb LCR pink text) encompassing L18K. <b>C.</b> A schematic representation of the position of the LCR.</p

Pyrosequencing reliably quantified the methylation of four consecutive CpG dinucleotides per individual (chr2:100721843–100721885; hg19).

<p>A fifth CpG site is preceded by a 7 base pair T stretch (after bisulfite conversion), making it impossible to assess the exact percentage of cytosines compared to thymines for that particular site due to the limitations of the pyrosequencing technique. For each individual at least three separate analysis of different bisulfite conversions were performed. Average methylation percentages and confidence intervals are presented in this table. Methylation cut-off value was set at 10% according to the manufacturers guidelines.</p

Clinical findings in FRA2A carriers.

<p>Clinical findings in FRA2A carriers.</p

Site and stage specific expression of <i>Aff3</i> in mouse embryos.

<p><b>A.</b> photomicrograph of a lateral view of a 12.5<i>Aff3</i>. The only area of strong expression at this stage is in the hand plate. <b>B.</b> show a false coloured but unthresholded image of the brightfield optical projection tomography (OPT) scan of a 11.5 dpc embryo with digital sagittal and coronal sections from the same embryo shown in <b>C</b> and <b>D.</b> Strong expression is seen in the somites, upper limb bud, the fusing primary palate and the diencephalon and prosomere1 regions of the developing brain.</p

Southern blot analysis of the <i>AFF3</i> CGG repeat in all available members of the three families and two unrelated control individuals (C).

<p>DNA restriction fragments obtained from blood samples of all available members of family A (lanes 1–4), B (lanes 5–6) and C (lanes 7–9) where blotted with a specific 32P-labeled probe (chr2:100088460–100089451; hg19) after digestion with <i>Hind</i>III. Lanes 10 and 11 contain DNA restriction fragments from two unrelated control samples (represented as diamonds) after digestion with <i>Hind</i>III. In addition to a normal size allele fragment of 4.4 kb, individuals CII.1, CI.2, BII.1, BI.2, AII.4, AII.3 and AIII.1 show additional fragments of a larger size, indicating repeat expansion. These expanded fragments were not present in the controls and individuals CI.1 and AI.1. A 1 kb length marker is presented at the left of the figure.</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

Allele frequencies of the FRA2A associated CGG repeat in a population of 100 control individuals.

<p>PCR amplification of the repeat and subsequent sequencing in 200 control chromosomes revealed that it is highly polymorphic with a length ranging from 3 to 20 copies. The most commonly found allele contains eight repeated units.</p

<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