A novel Werner Syndrome mutation: pharmacological treatment by read-through of nonsense mutations and epigenetic therapies

Abstract

Werner Syndrome (WS) is a rare inherited disease characterized by premature aging and increased propensity for cancer. Mutations in the WRN gene can be of several types, including nonsense mutations, leading to a truncated protein form. WRN is a RecQ family member with both helicase and exonuclease activities, and it participates in several cell metabolic pathways, including DNA replication, DNA repair, and telomere maintenance. Here, we reported a novel homozygous WS mutation (c.3767 C > G) in 2 Argentinian brothers, which resulted in a stop codon and a truncated protein (p.S1256X). We also observed increased WRN promoter methylation in the cells of patients and decreased messenger WRN RNA (WRN mRNA) expression. Finally, we showed that the read-through of nonsense mutation pharmacologic treatment with both aminoglycosides (AGs) and ataluren (PTC-124) in these cells restores full-length protein expression and WRN functionality.

Introduction

Werner syndrome (WS) is an autosomal recessive disease characterized by premature aging.^1,2 The clinical symptoms include premature hair greying, alopecia, skin atrophy, sclerodermiform skin changes with ulceration, typical habitus (a bird-like face and growth retardation), ocular cataracts, type II diabetes, hypogonadism, and several forms of atherosclerosis.^1,2 It has been suggested that WS is more than a segmental progeroid syndrome.³ WS patients also have a high incidence of cancers.^4-6 The median age of death is 46 years old due to neoplastic or atherosclerotic cardiovascular disease.^1-3 WS cells exhibit chromosomal instability characterized by chromosomal rearrangements and deletions.^7-10 These cells are sensitive to several DNA-damaging agents, including topoisomerase I inhibitors (camptothecin) and 4 nitroquinoline-1-oxide (4NQO).^11-14

The WRN gene encodes a protein that is a member of the RecQ family of DNA helicases,¹⁵ which are thought to be essential caretakers of the genome. The family consists of 5 members, 3 of which, WRN, RecQ4, and BLM, are associated with Bloom and Rothmund-Thompson and Werner syndromes.^16,17 WRN possesses 3′>5′ helicase and exonucleaseactivities,^18-21 and it participates in diverse pathways, including DNA repair, replication, telomere metabolism, and p53-mediated pathways.^17,22,23 The protein, which is mainly localized at nucleoli and relocates to nuclear repair foci upon DNA damage,²⁴ possesses nuclear localization signals that reside between aminoacids 1,369 and 1,402 at the C terminus and between residues 949 and 1,092 (nucleolar).^24-26 WRN mutations largely result in truncations of the WRN protein.^27-30 Some mutations are nonsense or premature termination codons (PTCs), and the mRNA is generally unstable and present in reduced levels.^31,32 Importantly, the WRN gene has also been found to be inactivated by CpG island promoter hypermethylation in a wide variety of tumors of mesenchymal and epithelial origin.³³

WS therapy remains elusive to date. However, for some inherited diseases resulting from nonsense mutations, AGs and PTC-124 have been shown to suppress translation termination and have merged as an important therapeutic option.³⁴ In this manuscript, we reported a new PTC mutation in 2 previously reported Argentinian brothers with WS.³⁵ The c.3767 C > G mutation (p.S1256X) was found to result in a stop codon that generates a truncated WRN protein.

We observed increased WRN promoter methylation and nonsense-mediated decay (NMD) as 2 potential mechanisms that may contribute to the decreased amount of WRN mRNA observed in the patients’ cells. The decreased WRN mRNA was rescued by the use of DNA-demethylating agents or NMD inhibitors. We also showed that aminoglycoside- and PTC-124-induced read-through of PTC mutations in the WRN gene results in a full-length product in cells from patients with p.S1256X and p.R369X mutations. Moreover, cell lines harboring the p.S1256X mutation were functionally rescued from 4NQO-induced apoptosis and DNA damage. Chromosome damage and impaired DNA replication were also rescued. These findings may provide a new therapeutic avenue for WS patients harboring PTC mutations.

Results

Identification of a novel WS mutation

Two patients with clinical diagnoses of WS have been recently described.³⁵ These patients presented the characteristic features of this syndrome, such as a bird-like face, a high-pitched voice, baldness, and leg ulcers.^36,37 To confirm the WS clinical diagnosis by mutational analysis, we first generated lymphoblastoid cell lines through lymphocyte immortalization using Epstein Barr Virus (EBV). The WRN cDNA was cloned into the pGEMT vector, and all coding exons of the WRN gene were sequenced for the 2 index cases, the patients’ mother and one healthy control. The mutation analysis revealed that the patients had a yet undescribed PTC homozygous mutation in exon 32 (c3767 C > G) (Fig. 1A), which generates a premature termination codon. As a result, a truncated protein (p.S1256X) lacking the nuclear localization signal (NLS) is generated, which is unable to exert its function in the nucleus (Fig. 1A and B).The mutation was confirmed by amplification and sequencing of genomic DNA from the 2 patients, a heterozygote carrier and a healthy donor. Interestingly, the patients’ parents are first cousins. Thus, it is not surprising that the same mutation was found in both brothers (Fig. 1B and C).

Figure 1.

Novel WS mutation (A) The representation of the mutation localization shows the DNA sequence with codons in alternate colors as well as the protein sequence. (B) Sequencing results detecting the homozygous substitution c.3767 C > G in WRN in both patients (WS1 and WS2) and the same heterozygous mutation in the mother (HT). The two peaks in the chromatogram show the normal and mutated allele. No mutation was found in the wild type control (CTRL). (C) Photographs of the 2 patients showing the typical WS premature appearance. Their mother, who is heterozygous for the mutation, did not show the WS phenotype.

Lack of WRN protein expression in the patients’ cells but not in control cells

We studied the level of WRN protein expression in the patients and controls. Importantly, the antibody used, 4H12, recognizes a protein sequence after the NLS at the C terminus resulting in the inability to recognize a truncated form of WRN (Fig. 2A).³⁸ First, we showed by western blotting that patients carrying the p.S1256X PTC mutation lack the WRN protein in contrast to the heterozygous control(HT-WRN±) and healthy control(CTRL-WRN+/+). The protein extract obtained from a WS patient cell line carrying the p.R369X PTC mutation was used as a negative control. As expected, no signal was observed in the patient sample, but it was observed in the positive controls (Fig. 2B). We did not detect WRN protein using immunofluorescence in the patient cells (WRN−/−). In contrast, a strong nuclear signal was observed in the HT-WRN+/− cells (Fig. 2C). In heterozygous individuals, wild type WRN properly localizes in the nucleus and exerts its function. As a consequence, no WS phenotype was observed in the mother of the 2 patients (Fig. 1C).

Figure 2 (See previous page).

WRN is not expressed in WS cells harboring the p.S1256X mutation: Methylation analysis of the WRN promoter in patients and controls. (A) The illustration shows the WRN protein domains, including the nuclear localization signal (NLS) and the positions of the p.S1256X and p.R369X (the most common mutation in Caucasians) mutations. The N-terminal epitope recognized by the 8H3 antibody and the C-terminal epitope recognized by the 4H12 antibody are shown. The 4H12 antibody only detects full-length WRN, whereas the 8H3 antibody recognizes both the full-length and truncated protein forms. (B) Western analysis of the WRN protein using the 4H12 antibody in the control (CTRL-WRN+/+), heterozygous (HT-WRN+/−), p.S1256X(WRN−/−), and p.R369X (WRN−/−) cell lines. Actin was used as a loading control. (C) Immunofluorescence using the 4H12 antibody (which only detects full-length WRN) in cell lines showing WRN expression and nuclear localization in HT (WRN+/+) and p.S1256X (WRN−/−) showing no WRN expression. Blue indicates DAPI staining (first panel), and green indicates WRN (second panel). The third panel includes the merged images. (D) Methylation analysis of the WRN promoter. A schematic description of the WRN CpG islands around the transcription start site is shown (long black arrow). CpG dinucleotides are represented as short vertical lines. Locations of bisulphite genomic sequencing PCR primers and methylation-specific PCR primers are indicated as white and gray arrows, respectively. The bisulphite genomic sequencing results are shown for 5 individual clones in B lymphocytes (HT, WS1 and WS2 for p.S1256X mutation and CTRL). (E) WRN mRNA expression levels for (HT, WS1 and WS2 for p.S1256X mutation and CTRL) in naïve B lymphocytes assessed by RT-PCR.GAPDH was used as a loading control. (F) Treatment with a demethylating agent (5-aza) reactivated WRN gene expression in the WS2 lymphoblastoid cell line. (G) Treatment with emetine increased WRN mRNA transcript levels in the WS2 lymphoblastoid cell line by inhibiting NMD.

The WRN promoter is methylated in WS patients carrying the p.S1256X mutation

Tumor suppressor genes (TSGs) usually gain promoter methylation during aging.³⁹ Therefore, because WRN is a TSG and has dual roles in aging and cancer, we hypothesized that WRN may have an increased level of promoter methylation, which could correlate to the decreased amount of WRN mRNA found in WS cells. This phenomenon is frequently found in WS cells and is thought to be caused by promoter downregulation or a lack of mRNA stability due to NMD or another decay mechanism.^31,32 Therefore, the methylation status of the WRN promoter was evaluated in patients (WRN−/−) carrying the reported mutation, an heterozygote (HT-WRN±) for this mutation and a healthy control (CTRL-WRN+/+) using both naïve B lymphocytes and lymphoblastoid cell lines. Surprisingly, increased promoter methylation was found in both patients, being more important in one of the patients (WS2) when they were compared with the heterozygous (HT-WRN±) and the CTRL-WRN+/+ samples (Fig. 2D). Due to the increased WRN promoter methylation, we assessed a possible correlation between this epigenetic mark and the expression of the WRN gene at the RNA level. Both patients exhibited a reduced amount of WRN RNA (assessed by RT-PCR) when compared to the levels of HT-WRN± and CTRL-WRN+/+. One patient in particular (WS2) showed WRN promoter methylation with minimum expression (Fig. 2E). Interestingly, the treatment of the WS cell lines with the demethylating drug, 5-aza-2′-deoxycytidine (5-aza), at a 1 μM concentration for 3 days, increased the expression of WRN mRNA transcript (Fig. 2F). We next studied the possibility that the NMD mechanism may be involved in the reduced amount of WRN mRNA transcript. We treated the mutant cell lines with emetine, which is an antibiotic that inhibits translation acting as a mechanism of NMD inhibition. Importantly, the treatment of the WS cell lines with 100 μg/ml emetine for 10 h moderately increased the amount of WRN mRNA transcript (assessed by RT-PCR) (Fig. 2G). We concluded that both mechanisms (methylation and NMD inhibition) may potentially contribute to the reduced WRN mRNA levels observed in these cells.

Aminoglycoside- and PTC-124-induced read-through of PTC mutations in the WRN gene: Restoration of full-length WRN

Using the anti-WRN antibody 8H3 (Fig. 2A), which detects an amino-terminal epitope of the protein³⁸, we observed the accumulation of the truncated WRN protein in the cytoplasm of the patient's cells (Fig. 3A).

Figure 3.

PTC read-through treatment in WS cells harboring the p.S1256X mutation restores the full-length WRN protein. (A) Immunofluorescence of WRN in cells harboring the p.S1256X mutation shows cytoplasmic localization of the truncated protein detected with the 8H3 antibody indicating that the protein is stable in the cytosol. (B) Western analysis of WRN protein with the 8H3 antibody, which recognizes the truncated forms of WRN, as follows: CTRL (WRN+/+), HT (WRN+/−), p.S1256X (WRN−/−), p.S1256X (WRN−/−) + PTC-124, and p.S1256X(WRN−/−) + gentamicin. In lane one (CTRL), only full-length WRN was detected. In the HT, both full-length WRN and the truncated protein forms were detected. In p.S1256X (WRN−/−), only the truncated form was detected. Upon treatment with PTC-124 or gentamicin, restoration of full-length WRN (upper band) was observed together with a reduction of truncated WRN (lower band). (C) Immunofluorescence using the 4H12 antibody in p.S1256X (WRN−/−) untreated cells (in which no WRN expression was detected) or p.S1256X (WRN−/−) cells treated with PTC-124, gentamicin, streptomycin, or G418 showing restored nuclear WRN expression. Plots of the percentage (%) of cells harboring the p.S1256X and p.R369X mutations and showing nuclear WRN expression after being treated with the 3 different AGs and PTC-124 by read-through therapy of PTC mutations are shown.

The ability of AGs or PTC-124 to restore a full-length protein by inducing read-through of PTC mutations is a promising therapeutic resource. Of note, these agents can distinguish between premature and functional stop codons. Therefore, we evaluated the capacity of these drugs to restore full-length WRN in cells from the patients. Total protein extracts derived from the patient's cells harboring the p.S1256X mutation were obtained after treating the cells with 400 μg/ml gentamicin or 3.3 μM PTC-124 for 24 h, which are the drugs with the most potential for use in the clinical setting.⁴⁰ We found a strong increase in the expression of full-length WRN and a reduction in the truncated protein in the protein extracts from the cells treated with both drugs. The increase in full-length WRN was evident when compared to the untreated sample, which only had the truncated form (lower band) (Fig. 3B). The CTRL-WRN+/+ sample only showed the upper band corresponding to the full-length WRN. Interestingly, both bands were observed with equal intensity in the HT-WRN+/− sample (the patients’ mother) (Fig. 3B).

Next, we used immunofluorescence to detect if the cells derived from the patients carrying the p.S1256X mutation lacked WRN expression (Fig 3C). We used the 4H12 antibody, which recognizes the C-terminal epitope of WRN (Fig. 2A). This antibody recognizes the full-length WRN but not the truncated form.³⁸ Cells from the WS patients were left untreated or treated for 48 h with gentamicin, PTC-124, streptomycin, or G418. Nuclear expression of full-length WRN (green) was found in more than 90% of the cells treated with PTC-124 or gentamicin in comparison with the untreated control cells, which showed no expression. Moreover, for the cells treated with streptomycin or G418, approximately 70% and 80%, respectively, expressed WRN. Streptomycin was the least efficient of all the AGs assayed (Fig. 3C). Similar results were obtained in cells harboring the p.R369X mutation with approximately 70% of the cells treated with gentamicin, PTC-124, or G418 and 60% of the cells treated with streptomycin expressing WRN. Thus, AGs and PTC-124 read-through of WRN were more efficient in cells carrying the p.S1256X mutation than in cells with the p.R369X mutation. Streptomycin was confirmed to be the least efficient AG in both cell lines.

Treated cells show nucleolar WRN localization after PTC read-through treatment

Because WRN typically has a nucleolar localization,²⁵ WRN localization was assayed by immunofluorescence in the patient's cells after treatment with the AGs mentioned above or PTC-124. In cells carrying the p.S1256X mutation, approximately 80% of WRN colocalized with nucleolin (a constitutive nucleolar protein). For cells carrying the p.R369X mutation, we observed colocalization in approximately 60% of the cells treated with gentamicin or PTC-124, and we also observed colocalization in approximately 50% and 40% of the cells treated with streptomycin and G418, respectively (Fig. 4A and B). These results clearly showed that the WRN protein is correctly localized in the cell nucleus with a defined nucleolar pattern after its induced expression in the cell lines assayed.

Figure 4 (See previous page).

Restoration of nuclear localization and functionality of WRN as measured by induced apoptosis andγ-H2AX foci upon read-through treatment in WS cells harboring the pS1256X mutation. (A) After PTC-124 or AGs treatment of WS cells harboring the p.S1256X or p.R369X mutation, WRN was localized in the nucleolus, which is its main localization. Immunofluorescence shows the colocalization of WRN (red) and nucleolin (green) in p.S1256X(WRN−/−) cells treated with PTC-124 or gentamicin. (B) Plot representing the percentage (%) of WRN and nucleolin colocalization against treatment with PTC-124 and 3 different AGs in cells harboring the p.S1256X or p.R369Xmutation. (C) WS cells are sensitive to the 4NQO DNA-damaging agent. Decreased apoptosis was measured by the TUNEL assay in WS cells harboring the p.S1256X mutation and treated with PTC-124 or AGs. Upon treatment, the apoptosis levels decreased to l for some drugs, which was close to the levels in control cells (WRN+/+). Untreated WRN(−/−) was compared to WRN(+/+) and AGs or PTC-124 treated (WRN−/−). Error bars represent the standard deviation (n = 9); **P < 0.01. P-values were determined by Student's t-test. (D) The apoptotic cells are shown in green for the different conditions. (E) Statistical representation of γ-H2AX foci in WS cells harboring the p.S1256X mutation left untreated or treated with PTC-124 or AGs and control cells (WRN+/+ cells) upon 4NQO treatment. Error bars represent the standard deviation (n = 9); *P < 0.05. P-values were determined by Student's t-test. (F) Representative images showing γ-H2AX foci in WS cells left untreated or treated with PTC-124 or AGs.

PTC read-through treatment rescues WS cells harboring the p.S1256X mutation from 4NQO-induced apoptosis and DNA damage

It is well known that lymphoblastoid cells established from WS patients are hypersensitive to 4NQO-induced apoptosis and DNA damage.¹²

Using a TUNEL assay, we observed increased 4NQO-induced apoptosis in the WS cell lines harboring the p.S1256X mutation when compared with a cell line derived from a healthy control (WRN+/+). In contrast, the WS cell line was markedly resistant to 4NQO-induced apoptosis when it was treated with PTC-124, gentamicin, streptomycin, or G418 (Fig. 4C). Consistently, streptomycin was the least efficient of all AGs tested (Fig. 4C–D). Next, we observed the rescue of 4NQO-induced DNA damage by visualizing γ-H2AX focus formation. When cells are exposed to DNA-damaging agents, such as 4NQO, double-stranded breaks (DSBs) are rapidly generated resulting in phosphorylation of the histone H2A variant, H2AX. Because phosphorylation of H2AX at Ser139 (γ-H2AX) correlates well with each DSB, it is the most sensitive marker known to examine the DNA damage produced and the subsequent repair of the DNA lesion.

We detected an increased number of γ-H2AX foci in the WS cell lines carrying the p.S1256X mutation as compared to the healthy donor control cell line (WRN+/+). Importantly, the number of γ-H2AX foci was greatly reduced after PTC read-through treatment of WS cells indicating that the DNA damage in the PTC-treated WS cells was reduced nearly to that of the control cells with gentamicin and PTC-124 being the most effective treatments (Fig. 4E–F). To summarize, PTC read-through treatment of WS cell lines rescued 4NQO-induced apoptosis and DNA damage when the cells were treated with the aforementioned AGs or PTC-124.

Chromosomal damage is reduced by PTC read-through treatment of WS cells harboring the p.S1256X mutation

WS cells are characterized by what is known as variegated translocation mosaicism, a phenomenon that reflects the genome instability characteristic of these cells.^7-10 Therefore, we assessed if the PTC read-through treatment could rescue this genome instability by analyzing chromosomal aberrations.

Three independent experiments were performed. A control cell line (WRN+/+) and the WS p.S1256X cell line were left untreated or treated with the 3 AGs and PTC-124. One hundred metaphases were analyzed per condition in each experiment. We also checked for the common chromosomal aberrations frequently found in WS-like chromosome breaks and the characteristic quadriradial forms^7-10 (Fig 5A). We found that the number of metaphases with aberrations in the WS cells was reduced almost to the levels of that in the control cells after AGs or PTC-124 treatment suggesting that PTC treatment of WS p.S1256X cell lines reduces chromosomal aberrations and chromosomal instability.

Figure 5 (See previous page).

Chromosomal damage and impaired DNA replication are reduced by PTC read-through therapy in WS cells harboring the p.S1256X mutation. (A) Chromosomal breakage measured by cytogenetic analysis of metaphase chromosomes. Untreated WS cells harboring the p.S1256X mutation had increased fragility when compared to control (WRN +/+) cells. Fragility was decreased to the control cell level in mutant cells after cell treatment with PTC-124 or AGs. The metaphase spreads of control WS cells harboring the p.S1256X mutation left untreated or treated with PTC-124 and gentamicin are shown. Error bars represent the s.standard deviation (n = 9); *P < 0.05. P-values were determined by Student's t-test. (B) Analysis of DNA replication by fiber assays of control (WRN +/+) and WS cells harboring the p.S1256X mutation left untreated or treated with PTC-124 or gentamicin. The average fiber length for the control was 6–9 μm, and the average fiber length was 3–6 μm for the WS cells harboring the p.S1256X mutation. Upon WS cell treatment with PTC-124 or gentamicin, the average fiber was 6–9 μM, which was similar to the fiber length in the control. Two plots of one representative experiment out of 3 biological replicas are shown (n = 200 for each condition). The DNA fiber assays were performed with the control cells and WS cells harboring the p.S1256X mutation left untreated or treated with PTC-124 or gentamicin. BrdU-labeled fibers are stained in green.

PTC-read through treatment of WS cells harboring the p.S1256X mutation rescues impaired DNA replication, a characteristic of WS

Replication fork progression was analyzed by DNA fiber assays.⁴¹ On average, the WS lymphocytes with the described mutation exhibited shorter replication fibers (3–6 μM) when compared with the control lymphocytes (6–9 μM). These data were in accordance with the fork progression defects characteristic of WS cells.⁴¹ For the WS lymphocytes treated with AGs and PTC-124, the average fiber length after treatment with both PTC-124 and gentamicin was similar to that in the control (6–9 μM) (Fig. 5B). These results indicated that functional replication defects are corrected when cells are treated with these compounds.

Discussion

We described a new nonsense homozygous mutation (p.S1256X) in 2 brothers whose parents are first cousins. In these patients, clinical diagnosis was previously performed based on all the characteristic symptoms of this disease.³⁵ As mentioned above, this mutation generates a truncated protein lacking the NLS (Fig. 2A).

Patients with WS display a remarkable number of clinical signs and symptoms associated with premature aging, including greying of the hair, cataracts, osteoporosis, diabetes, and atherosclerosis, starting as early as the second or third decade of life.³⁶ Many mutations spanning the entire gene have been described as follows: insertions or deletions leading to a frameshift and subsequent termination of protein translation; substitutions at the splice junctions that cause the skipping of exons and a subsequent frameshift; missense mutations that lead to amino acid changes in the protein affecting all different protein domains; and PTC mutations, which change an amino acid codon to a stop codon leading to the termination of protein translation, thereby generating a truncated protein lacking the NLS that is unable to exert its function in the nucleus.

The most common mutation in Caucasians (25%) is PTC (p.R369X), which is the second most common mutation in Japanese patients (19%). Moreover, several other PTC mutations have been found. Importantly PTC mutations account for more than 10%^27-30 of WRN mutations, and they lead to a faster degradation of the mRNA template and the production of a truncated, non-functional protein. Mutant products truncated N-terminal to the helicase domain are less stable than products truncated C-terminal to the helicase domain²⁷ However, the function of mutant WRN proteins in the cytosol is unknown. Consistent with the literature, we found accumulation of the truncated p.1256X protein in the cytosol of the patients’ cells²⁴ (Fig 3A).

The promoter of some tumor suppressor genes that are hypermethylated in cancer show increased methylation during aging (e.g., p14, MGMT, or hMLH1).^42,43 In addition, WRN is a tumor suppressor gene⁴⁴ that is hypermethylated in cancer^33,45 and has been found to have altered methylation in different tissues according to age.⁴⁶

Importantly, the methylation changes found in aging cells is sometimes subtle and less frequent than in cancer.^47-51 In addition, these changes are quite specific^47-50

We hypothesized that if these patients have accelerated aging features, they may have increased WRN promoter methylation, a phenomenon that is well known to silence WRN in different types of cancer.^33,45 This situation was the case in this study at least for naïve B-lymphocytes and lymphoblastoid cell lines derived from the patients, which had increased WRN promoter methylation (Fig 2D).

In this study, we identified a novel homozygous PTC mutation, and we also found that the WRN promoter in the cells of these patients had increased methylation.

The fact that one patient had acquired more WRN promoter methylation than his brother might be due to environmental factors or different lifestyles as suggested by a previous report studying methylation changes in monozygotic twins⁵² and another recent study.⁵³ It will be interesting in the future to evaluate whether different WS patients’ tissues exhibit increased WRN promoter methylation similar to the naïve B cells and derived immortalized lymphoblastoid cell lines. Moreover, WRN mRNA expression was increased upon treatment of the cells with the demethylating agent 5-aza (Fig. 2F), suggesting that the low level of mRNA generally found in the cells of WS patients might be partially related to a downregulation of the WRN promoter, as previously reported.³²

Through the inhibition of NMD by the translation inhibitor emetine, we identified NMD as a mechanism that reduced WRN mRNA transcript in the cells of these patients (Fig 2G). Both mechanisms, namely NMD and increased methylation, might contribute to the low mRNA levels observed in the cells of the patients.

Finally, we demonstrated that aminoglycoside- and PTC-124-induced read-through of PTC mutations in the WRN gene resulted in a full-length product with functional properties in WS cells from patients with the p.1256X (described here for the first time) or p.R369X (the most common mutation in Caucasians) mutation.

AG or PTC-124 -induced read-through of human PTC mutations has therapeutic potential due to its unique ability to suppress translation termination by nonsense mutations. Xeroderma Pigmentosum, Rett Syndrome, p53 mutations, or adenomatous polyposis coli are several examples in which this approach has shown enormous efficiency.^54-57 The paradigmatic example is Duchene dystrophy, for which great success has been achieved using this type of therapy in both preclinical and clinical pilot studies.^58,59

AGs have the ability to suppress translation termination induced by nonsense mutations and correction of the phenotype by promoting otherwise defective protein synthesis. AGs suppress codons with different efficiencies (UGA>UAG>UAA), and their suppression activity is further dependent on the fourth nucleotide immediately downstream of the stop codon (C>U>A>G) and the local sequence around the stop codon.⁶⁰ According to some reports, the presence of a cytosine in the +4 position promotes higher basal and gentamicin-induced read-through than other nucleotides.⁶⁰ Importantly, a U in the -1 position is a key determinant of gentamicin read-through⁶⁰ (Fig. 1A).

Interestingly, the mutation described here fulfilled all the criteria mentioned above for optimal gentamicin read-through. In addition, the p.R369X mutation also generates an UGA stop codon, but with 2 As in +4 and -1 positions.

We also observed PTC-124-induced read-through in cells harboring both mutations. This drug may be preferable in the clinical setting as its use is not associated with the toxicity observed with the prolonged administration of AGs, such as gentamicin. Indeed, PTC-124 has been used with great success to suppress PTC in different pathologies.^61-64 We observed similar results with both drugs, namely gentamicin and PTC-124, the latter of which is more amenable to use in clinical practice. Of note, nonsense-mediated mRNA decay affecting nonsense transcript levels influences the response of cystic fibrosis patients to gentamicin.⁶⁵ However, as previously mentioned, many WS patients have decreased mRNA either by promoter downregulation, unstable mRNA or both.^31,32,66 We found that increased promoter methylation and NMD correlate to reduced levels of WRN mRNA in the cells of these patients. Importantly, the concomitant contribution of these 2 mechanisms in regulating the mRNA expression has been observed for other genes.⁶⁷ Accordingly, considering the case of WS, increasing mRNA concentrations by 5-aza might have therapeutic value in compensating for the effect of nonsense-mediated decay if combined with PTC read-through therapy.^68-72

Finally, more research is needed to explore these therapeutic venues for WS patients carrying PTC mutations for whom PTC read-through therapy alone or combined with 5-aza (in patients with increased WRN promoter methylation) may be feasible in the future.

Materials and Methods

Cells and cell lines from normal individuals and patients and DNA extraction

Blood samples from 2 brothers with clinical WS diagnosis, their mother (expected to be heterozygous for the mutation), and a healthy individual were obtained. All patients gave their informed consent before inclusion in this study and were approved by the ethics committee. Cell lines were obtained from Coriell Cell Repositories. The samples consisted of 1 WRN−/−(AG14426) and 1 control WRN+/+ (AG19614) collected and anonymized by the National Institute of General Medical Science (NIGMS). All subjects provided written consent for experimental use of their samples.

PBLs from patients and normal individuals were extracted using a lymphocyte Ficoll separation medium and were transformed by EBV propagated in the B95-8 marmoset cell line.^73,74 To separate CD19-positive cells, microbeads (Miltenyi Biotec) were applied following the manufacturer's instructions. The cell lines were cultured as described.^73,74

For methylation analysis, cell lines were maintained in appropriate media and treated with 1 μM 5-aza-2′-deoxycytidine (5-aza) (Sigma) for 3 days to achieve demethylation.

For the ribosomal read-through analysis of premature termination codons, cells were left untreated or treated for 24 h with 400 μg/ml streptomycin sulfate (Life Technologies), 400 μg/ml gentamicin sulfate (Santa Cruz), 3.3 μM ataluren (PTC-124; Selleckchem) or 50 μg/ml G418 Geneticin® (Life Technologies).

To study NMD inhibition, cells were grown and treated with 100 μg/ml emetine dihydrochloride (Santa Cruz), which blocks protein synthesis, for 10 h, as previously described.⁷⁵ DNA was extracted using thephenol/chloroform/isoamylalcohol procedure (Sigma).

Cloning and mutational analysis of WRN

WRN cDNA from patients and controls was amplified by Phusion® High-Fidelity DNA Polymerase (New England Biolabs) and cloned into the pGEM-T vector. Eleven pairs of primers were designed to cover the entire WRN cDNA sequence. When a mutation was identified by RT-PCR sequencing, the PCR products of the mutated exon were sequenced in the genomic DNA to confirm the mutation. The GenBank reference sequences used for the analysis are NG_008870.1, NM_000553.4, and ENSG_00000165392.

Methylation analysis of the WRN gene

Bisulphite transformation of DNA was performed using the EpiTect Bisulphite Kit (Qiagen).

WRN CpG island methylation status was established by PCR analysis of bisulphite-modified genomic DNA. The methylation status was analyzed by bisulphite genomic sequencing of both strands of the CpG island. The following previously described primers³³ were used: 5′-AGG TTT TTA GTY GGY GGG TAT TTA-3′ (sense) and 5′-AAC CCC CTC TTC CCC TCA-3′ (antisense), which are located at −209 bp and +164 bp from the transcription start site. For methylation-specific PCR (MSP), we used primers specific for either the methylated or modified unmethylated DNA. The primers used have been previously described.³³ The primer sequences for the unmethylated reaction were 5′-GTA GTT GGG TAG GGG TAT TGT TTG T-3′ (sense) and 5′-AAA CAA AAT CCA CCA CCC ACC CC-3′ (antisense), and the primer sequences for the methylated reaction were 5′-CGG GTA GGG GTA TCG TTC GC-3′ (sense) and 5′-AAC GAA ATC CAC CGC CCG CC-3′ (antisense). The primers are located at −36 (sense) and +129 (antisense) from the transcription start site.

WRN RNA and protein analysis

Total cell extracts were prepared with radioimmunoprecipitation assay buffer.⁷⁶ Cell extracts and protein gel blots were performed as previously described.⁷⁶ The following antibodies were used: mouse anti-WRN [8H3] amino-terminal antibody (1:200; ab66601, Abcam); and mouse anti-WRN [4H12] antibody (1:200; ab66606, Abcam). RNA was isolated using TRIzol (Life Technologies). RNA (2 μg) was reverse-transcribed using SuperScript II reverse transcriptase (Life Technologies) and amplified using specific primers for WRN described elsewhere.³³

Immunofluorescence analysis

Cells were attached to adhesion slides (Marilienfeld) and immunostaining was performed as previously described.⁷⁶ Briefly, cells were fixed for 10 min with 4% paraformaldehyde in phosphate-buffered saline (PBS), permeabilized for 5 min with 0.1% Na-citrate/0.5% Triton X-100 and blocked for 30 min with PBS containing 5% bovine serum albumin and 0.1% Tween-20. The antibodies and dilutions used for the analysis were as follows: mouse anti-WRN [8H3] amino-terminal antibody (1:200; ab66601, Abcam); mouse anti-WRN [4H12] antibody (1:200; ab66606, Abcam); rabbit anti-WRN [H-300] antibody (1:100; sc-5629, Santa Cruz); mouse anti-nucleolin [C23] antibody (1:300; MS-3; sc-8031, Santa Cruz), and rabbit anti-nucleolin [C23] antibody (1:1000; ab22758, Abcam) .The secondary antibodies used have been previously described.⁷⁶ Vectashield (Vector Laboratories) was used as the imaging medium. DNA was stained with 40, 60-diamidino-2-phenylindole (DAPI). Images were captured either with an OLYMPUS IX81 fluorescence microscope (Olympus) or a LEICA TCS-SP5-DMI6000 confocal microscope (Leica Microsystems) and then analyzed using Image-Pro Plus software (Media Cybernetics, Inc.) or LAS AF (Lite Leica Microsystems), respectively.

Apoptosis analysis

WRN-deficient and control lymphocytes were untreated or treated for 24 h with PTC-124 or the AGs described above. Briefly, cells were first treated with 0.8 μg/ml 4-nitroquinoline N-oxide (4NQO; Sigma) for 1 h, washed, and then allowed to grow for 48 h without antibiotics, as previously described.¹² β-terminal deoxynucleotidyl-transferase dUTP nick-end labeling staining was performed using the in situ cell death detection kit (Roche Diagnostics). Nuclei were counterstained with DAPI. Three independent experiments (n₁ = 3) with 3 replications per experiment (n₂ = 9) were performed. Three hundred nuclei were counted per condition in each experiment. The P-values were determined by Student's t-test.

Detection of DNA damage by γ-H2AX staining after 4NQO treatment

WRN-deficient and control lymphocytes were left untreated or treated for 24 h with PTC-124 and the AGs described above.

Subsequently, the cells were treated with 0.8 μg/ml 4-nitroquinoline N-oxide (4NQO;Sigma) for 1 h, washed, and allowed to grow for 48 h in normal medium with or without antibiotics as previously described.¹²

The cells were incubated with the primary mouse monoclonal anti-γ-H2AX antibody, H2A.X (phosphoS139; ab2893, Abcam), followed by incubation with the secondary Alexa-488-conjugated anti-mouse IgG (Molecular Probes). Nuclei were counterstained with DAPI. Three independent experiments (n₁ = 3) with 3 replications per experiment (n₂ = 9) were performed. Three hundred nuclei were counted per condition in each experiment. The P-values were determined by Student's t-test.

Analysis of metaphase chromosomes

Metaphase spreads were prepared from WRN-deficient and control lymphocytes either untreated or treated for 24 h with antibiotics as described above. Briefly, cells were treated with the Colcemid™ Invitrogen/KaryoMAX® solution (Life Technologies) in HBSS for 30 min, harvested and incubated in 75 mM KCl for 25 min at 37ºC followed by fixation in Carnoy's fixative. Metaphase spreads were then made by placing the cells onto a glass slide and staining with DAPI. Images were captured with an OLYMPUS IX81 epifluorescence microscope (Olympus) and then analyzed using Image-Pro Plus (Media Cybernetics, Inc..) software. Three independent experiments were performed (n₁ = 3) with 3 replications per experiment (n₂ = 9). One hundred metaphases were analyzed per condition in each experiment. The P-values were determined by Student's t-test.

DNA fiber assays

DNA fibers were prepared from WRN-deficient and control lymphocytes either untreated or treated for 24 h with antibiotics as described above. The cells were exposed to 50 μM BrdU for 30 min and immediately washed with cold phosphate-buffered saline (PBS). The DNA spreads were air-dried and fixed in Carnoy's Fixative for 15 min. The slides were subsequently treated with 2.5 M HCl at room temperature for 1 h, neutralized in Tris-buffer (pH 7.5) for 10 min and subsequently blocked for 1 h with 2% bovine serum albumin (BSA) and 10% goat serum in PBST. Antibodies were diluted in blocking buffer as follows: mouse monoclonal antibody for BrdU (BD Biosciences), 1:20; and anti-mouse Alexa Fluor® 488 goat anti-mouse IgG (H⁺L) antibody (Invitrogen). The incubations with antibodies were at 37°C for 2 h (for primary antibodies) or1 h (for secondary antibodies). DNA was stained with SYTOX orange (Invitrogen) for 5 min and washed with PBS. The slides were mounted in Vectashield mounting medium (Vector Laboratories). Images were captured with an OLYMPUS IX81 epifluorescence microscope (Olympus) and then analyzed using Image-ProPlus (Media Cybernetics, Inc.). The number of fibers was counted and plotted against the fiber length (μm). Three different experiments (n₁ = 3) with 3 replications per experiment (n₂ = 9) were performed. Two hundred fibers were counted for each condition for all 3 experiments.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgment

We thank Cecilia Portela and Gonzalo Greif for their technical advice.

Funding

This work was supported by the AgenciaNacional de Investigacion e Inovacion (Program INNOVA URUGUAY-DCI-ALA/2007/19.040 URU-UE) and partially funded by FOCEM (MERCOSUR Structural Convergence Fund; COF 03/11).