For the experimental analyses, primary skin fibroblasts from nine patients diagnosed with the classic form of Hutchinson-Gilford Progeria Syndrome, as well as six unaffected controls (see Table 10), were obtained. The group of controls contained fibroblasts from four age-matched, i.e., young donors, and fibroblasts from two genetically matched individuals, i.e., parents of one of the HGPS patients. The HGPS samples were divided into an HGPS young (<8 years) and an HGPS old (>8 years) subgroup whenever appropriate. Furthermore, all cell lines were initially tested for the presence of the classic HGPS mutation (1824C>T), characteristic nuclear morphology changes and Progerin expression at the transcript and protein levels.
3.1.1. Verification of mutational status and Progerin expression
Using Sanger sequencing with primers amplifying the region of interest in the LMNA gene, all cell lines were analyzed for the presence of the classic HGPS mutation (1824C>T). As expected, HGPS samples revealed mixed cytosine/thymine signals at position 1824 (Figure S34), indicative of the heterozygous presence of the mutation. Control cell lines, in contrast, exhibited uniform cytosine-specific signal, verifying the absence of the mutation (Figure S34).
Subsequently, expression of the mutant Δ150 LMNA mRNA and Progerin protein was quantified in all cell lines. Using primers designed to specifically amplify either Δ150 LMNA or a control LMNA mRNA (Figure 7A), strong expression of the progerin-specific transcript was detected in HGPS cells from both young and old patients (Figure 7B and C). Interestingly, the Δ150 LMNA transcript was also detected in some of the control samples, albeit at considerably lower levels (Figure 7B). However, it did not result in the expression of detectable amounts of Progerin protein, which was restricted to HGPS samples only (Figure 8).
3. Results 3.1 Characterization of the fibroblast model system
Figure 7: Δ150 LMNA mRNA expression in the sample set. (A) Schematic representation of the LMNA gene, exons 3-12. The location of the 1824C>T mutation (in dark grey) and those of the oligos used for the amplification of the Δ150 LMNA mRNA, as well as an unaffected control mRNA, are indicated. (B) Detection of Δ150 LMNA and control mRNA in sample set using RT-PCR. (C) qRT-PCR-based quantification of Δ150 LMNA and control mRNA levels relative to SRSF4 and TBP levels in the sample set.
3. Results 3.1 Characterization of the fibroblast model system
3.1.1. HGPS fibroblasts reveal characteristic nuclear morphology changes
Alterations in the structure of the nuclear lamina resulting from the expression of Progerin are one of the most characteristic and commonly used phenotypic markers of HGPS cells (Erikssonet al., 2003; Liu et al., 2011; Zhang et al., 2011; Shimi, Butin-Israeli and Goldman, 2012; Miller et al., 2013). To verify their presence or absence in the primary fibroblasts, immunofluorescence
experiments using an α-Lamin A/C antibody were performed. As shown in Figure 9A and Figure S35, HGPS nuclei were characterized by a wide range of malformations including characteristic
Figure 8: Progerin protein expression in the sample set. Immunoblot of total protein extracts (20 µg) from all samples. Lamin A, Progerin and Lamin C were detected using a mouse α-Lamin A/C antibody. Lamin C signal was used as a loading control.
Figure 9: Nuclear malformation in HGPS fibroblasts. (A) Immunofluorescence of Lamin A/C confirms characteristic nuclear malformations in HGPS nuclei. The range of nuclear morphologies scored as ‘malformed’ is shown in Figure S35. Scale bar = 10 µm. Magnification: 10x. (B) Quantification of (A) in fibroblast samples. Bars represent the mean of three technical replicates with 100 cells counted per replicate. *P<0.01, unpaired t-test.
3. Results 3.1 Characterization of the fibroblast model system
wrinkling and lobulation of the nuclear lamina. These morphologies were substantially more frequent in patient-derived cells: In HGPS samples, the fraction of cells with dysmorphic nuclei ranged from 30-89 %, whereas only 3-15 % of control cells exhibited similar changes (P<0.01, unpaired t-test; Figure 9B). Additionally, despite a slight trend towards higher levels of malformation in samples from older patients, no correlation between patient age and the fraction of dysmorphic nuclei was found (Figure 9B and Table 10). These results confirm that the reported nuclear alterations, while mostly absent in control samples, are indeed present in the obtained HGPS fibroblasts.
3.1.2. HGPS cells exhibit minimal cell cycle changes
In addition to the distinctive nuclear phenotype, HGPS cells are characterized by premature cellular senescence and persistent DNA damage foci (Allsopp et al., 1992; Liu et al., 2005; Scaffidi and Misteli, 2006). However, early-passage HGPS fibroblasts have been reported to divide normally, without obvious defects (Bridger and Kill, 2004; Goldman et al., 2004; Paradisi
et al., 2005). This raises the question whether cell cycle dynamics are noticeably altered in
HGPS fibroblasts. To answer this question, HGPS and control samples were subjected to a Propidium Iodide (PI) staining, followed by ‘Fluorescence-activated Cell Sorting’ (FACS) analysis of DNA content. In brief, despite minor variations in the proportion of cells in G1, S or G2/M phase, respectively (Figure 10A), no significant differences were found when comparing cell cycle populations from two HGPS and two control samples (Figure 10B, unpaired t-test for all). The large majority of HGPS fibroblasts contained DNA content indicative G1 phase, a minority exhibited intermediate DNA content reflecting ongoing S phase, and a slightly larger fraction revealed DNA content characteristic of G2/M phase (Figure 10A and B). However, control cells were characterized by a highly similar distribution, thus indicating that population- scale cell cycle differences in the obtained HGPS samples are limited and should not constitute a decisive factor in the analysis of epigenetic alterations.