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

This article was downloaded by: [University of Nebraska, Lincoln] On: 06 April 2015, At: 10:25 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK

Epigenetics Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/kepi20

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

a

b

a

Ruben Agrelo , Miguel Arocena Sutz , Fernando Setien , Fabian Aldunate , Manel b

a

c

Esteller , Valeria Da Costa & Ricardo Achenbach a

Epigenetics of Cancer and Aging Laboratory Institut Pasteur de Montevideo(IPMON), Mataojo 2020, 1140 Montevideo, Uruguay b

Cancer Epigenetics and Biology Program (PEBC), Bellvitge Biomedical Research Institute, L'Hospitalet, Barcelona, Catalonia 08908, Spain c

Click for updates

Hospital General de AgudosDr. I. Pirovano. Unidad de Dermatología. Av. Monroe 3551 (1430). Ciudad Autónoma de la Ciudad de Buenos Aires. Argentina. Accepted author version posted online: 01 Apr 2015.

To cite this article: Ruben Agrelo, Miguel Arocena Sutz, Fernando Setien, Fabian Aldunate, Manel Esteller, Valeria Da Costa & Ricardo Achenbach (2015): A novel Werner Syndrome mutation: Pharmacological treatment by read-through of nonsense mutations and epigenetic therapies, Epigenetics To link to this article: http://dx.doi.org/10.1080/15592294.2015.1027853

Disclaimer: This is a version of an unedited manuscript that has been accepted for publication. As a service to authors and researchers we are providing this version of the accepted manuscript (AM). Copyediting, typesetting, and review of the resulting proof will be undertaken on this manuscript before final publication of the Version of Record (VoR). During production and pre-press, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal relate to this version also.

PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions

A novel Werner Syndrome mutation: Pharmacological treatment by read-through of nonsense mutations and epigenetic therapies Ruben Agreloa*, Miguel Arocena Sutza, Fernando Setienb, Fabian Aldunatea, Manel Estellerb,

a

ip t

Valeria Da Costaa, Ricardo Achenbachc Epigenetics of Cancer and Aging Laboratory Institut Pasteur de Montevideo(IPMON), Mataojo

cr

b

us

Cancer Epigenetics and Biology Program (PEBC), Bellvitge Biomedical Research Institute,

c

Hospital

General

de

an

L’Hospitalet, Barcelona,Catalonia 08908, Spain

AgudosDr.

I.

Pirovano.

Unidad

de

Dermatología.

M

Av. Monroe 3551 (1430). Ciudad Autónoma de la Ciudad de Buenos Aires. Argentina.

ed

* Corresponding author: Dr. Ruben Agrelo, Epigenetics of Cancer and Aging Laboratory, Institut Pasteur de Montevideo (IPMON), Mataojo 2020, 1140, Montevideo, Uruguay; E-

pt

Mail: [email protected]

ce

Keywords: Werner Syndrome, Mutation, Methylation, PTC read-through therapy, Epigenetics Abstract

Ac

Downloaded by [University of Nebraska, Lincoln] at 10:25 06 April 2015

2020, 1140 Montevideo, Uruguay

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, 1

including DNA replication, DNA repair, and telomere maintenance. Here, we reported a novel homozygous WS mutation (c.3767 C>G) in two 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)

ip t

expression. Finally, we showed that the read-through of nonsense mutation pharmacologic

ce

pt

ed

M

cr

an

us

length protein expression and WRN functionality.

Ac


treatment with both aminoglycosides (AGs) and ataluren (PTC-124) in these cells restores full-

2

Introduction Werner syndrome (WS) is an autosomal recessive disease characterized by premature aging1,2. The clinical symptoms include premature hair greying, alopecia, skin atrophy, sclerodermiform

ip t

skin changes with ulceration, typical habitus (a bird-like face and growth retardation), ocular cataracts, type II diabetes, hypogonadism, and several forms of atherosclerosis1,2. It has been

cr

atherosclerotic

cardiovascular

disease1,2,3.

WS

us

incidence of cancers4,5,6. The median age of death is 46 years old due to neoplastic or cells

exhibit

chromosomal

instability

an

characterized by chromosomal rearrangements and deletions7,8,9,10. These cells are sensitive to several DNA-damaging agents, including topoisomerase I inhibitors (camptothecin) and 4

M

nitroquinoline-1-oxide (4NQO)11,12,13,14.

ed

The WRN gene encodes a protein that is a member of the RecQ family of DNA helicases15, which are thought to be essential caretakers of the genome. The family consists of five members,

Thompson

pt

and three of which, WRN RecQ4, and BLM, are associated with Bloom and Rothmundand Werner syndromes, respectively16,17.WRN possesses 3’-5’ helicase and

ce

exonucleaseactivities18,19,20,21, and it participates in diverse pathways, including DNA repair, replication, telomere metabolism, and p53-mediated pathways17,22,23. The protein, which is mainly localized at nucleoli and relocates to nuclear repair foci upon DNA damage24, possesses

Ac


suggested that WS is more than a segmental progeroid syndrome3. WS patients also have a high

nuclear localization signals that reside between aminoacids 1369 and 1402 at the C terminus and between residues 949 and 1092 (nucleolar)24,25,26. WRN mutations largely result in truncations of

the WRN protein27,28,29,30. Some mutations are nonsense or premature termination codons (PTCs), and the mRNA is generally unstable and present in reduced levels31, 32. Importantly, the 3

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

ip t

nonsense mutations, AGs and PTC-124 have been shown to suppress translation termination and have merged as an important therapeutic option34. In this manuscript, we reported a new PTC

cr

us

(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 two

an

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

M

agents or NMD inhibitors. We also showed that aminoglycoside- and PTC-124-induced read-

ed

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

pt

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

ce

therapeutic avenue for WS patients harboring PTT mutations.

Results

Ac


mutation in two previously reported Argentinian brothers with WS35. The c.3767 C>G mutation

Identification of a novel WS mutation Two patients with clinical diagnoses of WS have been recently described35. These patients presented the characteristic features of this syndrome, such as a bird-like face, a high-pitched voice, baldness, and leg ulcers36, 37. To confirm the WS clinical diagnosis by mutational analysis, 4

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 two index cases (the patients’ mother and one healthy control). The mutation analysis revealed that the patients had a yet undescribed PTC

ip t

homozygous mutation in exon 32 (c3767 C>G) (Fig. 1A), which generates a premature

cr

signal (NLS) is generated, which is unable to exert its function in the nucleus (Fig. 1A and

us

1B).The mutation was confirmed by amplification and sequencing of genomic DNA from the two patients, a heterozygote carrier and a healthy donor. Interestingly, the patients’ parents are

an

first cousins. Thus, it is not surprising that the same mutation was found in both brothers (Fig. 1B

M

and 1C).

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

ed

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

pt

the inability to recognize a truncated form of WRN (Fig. 2A)

38

. First, we showed by western

ce

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

Ac


termination codon. As a result, a truncated protein (p.S1256X) lacking the nuclear localization

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

5

and exerts its function. As a consequence, no WS phenotype was observed in the mother of the two patients (Fig. 1C). The WRN promoter is methylated in WS patients carrying the p.S1256X mutation

ip t

Tumor suppressor genes (TSGs) usually gain promoter methylation during aging39. Therefore,

because WRN is a TSG and has dual roles in aging and cancer, we hypothesized that WRN may

cr

us

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 32

. Therefore, the methylation status of the WRN promoter was

an

another decay mechanism31,

evaluated in patients (WRN-/-) carrying the reported mutation, an heterozygote (HT-WRN+/-)

M

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

ed

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

pt

WRN promoter methylation, we assessed a possible correlation between this epigenetic mark and

ce

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 CTRLWRN+/+. One patient in particular (WS2) showed WRN promoter methylation with minimum

Ac


have an increased level of promoter methylation, which could correlate to the decreased amount

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 three 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 6

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 RTPCR) (Fig. 2G). We concluded that both mechanisms (methylation and NMD inhibition) may

ip t

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:

cr

us

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

an

patient’s cells (Fig. 3A).

M

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

ed

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

pt

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

ce

use in the clinical setting40. 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

Ac


Restoration of full-length WRN

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).

7

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 form38. Cells from the WS patients were left untreated or treated for

ip t

48 h with gentamicin, PTC-124, streptomycin, or G418. Nuclear expression of full-length WRN

cr

comparison with the untreated control cells, which showed no expression. Moreover, for the cells

us

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

an

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

M

WRN. Thus, AGs and PTC-124 read-through of WRN were more efficient in cells carrying the

ed

p.S1256X mutation than in cells with the p.R369X mutation. Streptomycin was confirmed to be the least efficient AG in both cell lines.

pt

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

ce

Because WRN typically has a nucleolar localisation25, WRN localization was assayed by immunofluorescence in the patient’s cells after treatment with the AGs mentioned above or PTC124. In cells carrying the p.S1256X mutation, approximately 80% of WRN colocalized with

Ac


(green) was found in more than 90% of the cells treated with PTC-124 or gentamicin in

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 4B). These results clearly showed that the

8

WRN protein is correctly localized in the cell nucleus with a defined nucleolar pattern after its induced expression in the cell lines assayed. PTC read-through treatment rescues WS cells harboring the p.S1256X mutation from

ip t

4NQO-induced apoptosis and DNA damage.

cr

4NQO-induced apoptosis and DNA damage 12.

us

Using a TUNEL assay, we observed increased 4NQO-induced apoptosis in the WS cell lines

an

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

M

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-4D). Next, we observed the rescue

ed

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

pt

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

ce

sensitive marker known to examine the DNA damage produced and the subsequent repair of the DNA lesion

Ac


It is well known that lymphoblastoid cells established from WS patients are hypersensitive to

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-4F). To summarize, 9

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

ip t

the p.S1256X mutation

cr

phenomenon that reflects the genome instability characteristic of these cells7, 8,9,10. Therefore, we

us

assessed if the PTC read-through treatment could rescue this genome instability by analyzing

an

chromosomal aberrations.

Three independent experiments were performed. A control cell line (WRN+/+) and the WS

M

p.S1256X cell line were left untreated or treated with the three AGs and PTC-124. One hundred metaphases were analyzed per condition in each experiment. We also checked for the common

ed

chromosomal aberrations frequently found in WS-like chromosome breaks and the characteristic quadriradial forms7,8,9,10 (Fig 5A). We found that the number of metaphases with aberrations in

pt

the WS cells was reduced almost to the levels of that in the control cells after AGs or PTC treatment suggesting that PTC treatment of WS p.S1256X cell lines reduces chromosomal

ce

aberrations and chromosomal instability. PTC-read through treatment of WS cells harboring the p.S1256X mutation rescues

Ac


WS cells are characterized by what is known as variegated translocation mosaicism, a

impaired DNA replication, a characteristic of WS. Replication fork progression was analyzed by DNA fiber assays41. 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 10

progression defects characteristic of WS cells41. 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

ip t

defects are corrected when cells are treated with these compounds. Discussion

cr

us

are first cousins. In these patients, clinical diagnosis was previously performed based on all the characteristic symptoms of this disease35. As mentioned above, this mutation generates a

an

truncated protein lacking the NLS (Fig. 2A).

M

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

ed

atherosclerosis, starting as early as the second or third decade of life36. Many mutations spanning the entire gene have been described as follows: insertions or deletions leading to a frameshift and

pt

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

ce

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

Ac


We described a new nonsense homozygous mutation (p.S1256X) in two brothers whose parents

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%27,28,29,30 of WRN mutations, and they lead to a faster degradation of the mRNA template and the production of a truncated, non11

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).

ip t

The promoter of some tumor suppressor genes that are hypermethylated in cancer show

cr

methylation in different tissues according to age46.

us

tumor suppressor gene44 that is hypermethylated in cancer33,45 and has been found to have altered

Importantly, the methylation changes found in aging cells is sometimes subtle and less frequent

an

than in cancer47, 48,49,50,51. In addition, these changes are quite specific47, 48, 49, 50. We hypothesized that if these patients have accelerated aging features, they may have increased

M

WRN promoter methylation, a phenomenon that is well known to silence WRN in different types

ed

of cancer33, 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

pt

methylation (Fig 2D).

In this study, we identified a novel homozygous PTC mutation, and we also found that the WRN

ce

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

Ac


increased methylation during aging (e.g., p14, MGMT, or hMLH1)42,43. In addition, WRN is a

methylation changes in monozygotic twins52 and another recent study53. 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 12

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

ip t

mechanism that reduced WRN mRNA transcript in the cells of these patients (Fig 2G). Both

cr us

observed in the cells of the patients.

Finally, we demonstrated that aminoglycoside- and PTC-124-induced read-through of PTC

an

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

M

common mutation in Caucasians) mutation.

AG or PTC-124 -induced read-through of human PTC mutations has therapeutic potential due to

ed

its unique ability to suppress translation termination by nonsense mutations. Xeroderma Pigmentosum, Rett Syndrome, p53 mutations, or adenomatous polyposis coli are several

pt

examples in which this approach has shown enormous efficiency54,55,56,57. The paradigmatic

ce

example is Duchene dystrophy, for which great success has been achieved using this type of therapy in both preclinical and clinical pilot studies58, 59. AGs have the ability to suppress translation termination induced by nonsense mutations and

Ac


mechanisms, namely NMD and increased methylation, might contribute to the low mRNA levels

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 codon60. According to some reports, the presence of a cytosine in the +4 position promotes higher basal and gentamicin-induced read-through than 13

other nucleotides60. Importantly, a U in the -1 position is a key determinant of gentamicin readthrough60 (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,

ip t

but with two As in +4 and -1 positions.

cr

may be preferable in the clinical setting as its use is not associated with the toxicity observed

us

with the prolonged administration of AGs, such as gentamicin. Indeed, PTC-124 has been used with great success to suppress PTC in different pathologies 61,62,63,64. We observed similar results

an

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

M

influences the response of cystic fibrosis patients to gentamicin65. However, as previously mentioned, many WS patients have decreased mRNA either by promoter downregulation, 32, 66

ed

unstable mRNA or both31,

. We found that increased promoter methylation and NMD

pt

correlate to reduced levels of WRN mRNA in the cells of these patients. Importantly, the concomitant contribution of these two mechanisms in regulating the mRNA expression has been

ce

observed for other genes67. Accordingly, considering the case of WS, increasing mRNA concentrations by 5-aza might have therapeutic value in compensating for the effect of nonsensemediated decay if combined with PTC read-through therapy68,69.70,71,72.

Ac


We also observed PTC-124-induced read-through in cells harboring both mutations. This drug

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 14

Cells and cell lines from normal individuals and patients and DNA extraction Blood samples from two 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

ip t

lines were obtained from Coriell Cell Repositories. The samples consisted of 1 WRN-/-

cr

Institute of General Medical Science (NIGMS). All subjects provided written consent for

us

experimental use of their samples.

PBLs from patients and normal individuals were extracted using a lymphocyte Ficoll separation

an

medium and were transformed by EBV propagated in the B95-8 marmoset cell line73,74. To separate CD19-positive cells, microbeads (Miltenyi Biotec) were applied following the

M

manufacturer’s instructions. The cell lines were cultured as described73,74.

ed

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.

pt

For the ribosomal read-through analysis of premature termination codons, cells were left untreated or treated for 24 h with 400 µg/ml streptomycin sulphate (Life Technologies), 400

ce

µg/ml gentamicin sulphate (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

Ac


(AG14426) and 1 control WRN+/+ (AG19614) collected and anonymized by the National

(Santa Cruz), which blocks protein synthesis, for 10 h, as previously described75. DNA was extracted using thephenol/chloroform/isoamylalcohol procedure (Sigma).

Cloning and mutational analysis of WRN 15

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 RTPCR sequencing, the PCR products of the mutated exon were sequenced in the genomic DNA to

ip t

confirm the mutation. The GenBank reference sequences used for the analysis are NG_008870.1,

cr us

Methylation analysis of the WRN gene

an

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

M

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

ed

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

pt

methylation-specific PCR (MSP), we used primers specific for either the methylated or modified unmethylated DNA. The primers used have been previously described33. The primer sequences

ce

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

Ac


NM_000553.4, and ENSG_00000165392.

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

16

Total cell extracts were prepared with radioimmunoprecipitation assay buffer76. Cell extracts and western blots were performed as previously described76. The following antibodies were used: mouse anti-WRN [8H3] amino-terminal antibody (1:200; ab66601, Abcam); and mouse antiWRN [4H12] antibody (1:200; ab66606, Abcam). RNA was isolated using TRIzol (Life

ip t

Technologies). RNA (2 µg) was reverse-transcribed using SuperScript II reverse transcriptase

cr us

Immunofluorescence analysis

Cells were attached to adhesion slides (Marilienfeld) and immunostaining was performed as

an

previously described76. 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-

M

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]

ed

amino-terminal antibody (1:200; ab66601, Abcam); mouse anti-WRN [4H12] antibody (1:200;

pt

ab66606, Abcam); rabbit anti-WRN [H-300] antibody (1:100; sc-5629, Santa Cruz); mouse antinucleolin [C23] antibody (1:300; MS-3; sc-8031, Santa Cruz), and rabbit anti-nucleolin [C23]

ce

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

Ac


(Life Technologies) and amplified using specific primers for WRN described elsewhere 33.

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 (LiteLeica Microsystems), respectively. Apoptosis analysis 17

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 described12.. β-terminal deoxynucleotidyl-transferase dUTP nick-end labeling staining

ip t

was performed using the in situ cell death detection kit (Roche Diagnostics). Nuclei were

cr

experiment (n2 = 9) were performed. Three hundred nuclei were counted per condition in each

an

us

experiment. The P-values were determined by Student’s t-test.

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

and the AGs described above.

M

WRN-deficient and control lymphocytes were left untreated or treated for 24 h with PTC-124

ed

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

pt

previously described12.

ce

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-488conjugated anti-mouse IgG (Molecular Probes). Nuclei were counterstained with DAPI. Three

Ac


counterstained with DAPI. Three independent experiments (n1 = 3) with three replications per

independent experiments (n1 = 3) with three 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

18

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

ip t

fixative. Metaphase spreads were then made by placing the cells onto a glass slide and staining

cr

(Olympus) and then analyzed using Image-Pro Plus (Media Cybernetics, Inc.) software. Three

us

independent experiments were performed (n1 = 3) with three replications per experiment (n2 = 9). One hundred metaphases were analyzed per condition in each experiment. The P-values were

M

an

determined by Student’s t-test.

DNA fiber assays

ed

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

pt

30 min and immediately washed with cold phosphate-buffered saline (PBS). The DNA spreads

ce

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

Ac


with DAPI. Images were captured with an OLYMPUS IX81 epifluorescence microscope

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 19

(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 fibre length (μm). Three different experiments (n1 = 3) with three replications per experiment (n2 = 9) were performed. Two

cr us

Competing Interests: No competing Interests

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

an

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

ce

pt

ed

M

Structural Convergence Fund; COF 03/11).

Ac


ip t

hundred fibers were counted for each condition for all three experiments.

20

Bibliography 1) Epstein CJ, Martin GM, Schultz AL, Motulsky AG. Werner's syndrome a review of its symptomatology, natural history, pathologic features, genetics and relationship to the natural

ip t

aging process. Medicine (Baltimore) 1966 May;45:177-21.

2) Salk D.Werner's syndrome: a review of recent research with an analysis of connective tissue

cr us

62:1-5.

3) Goto M Hierarchical deterioration of body systems in Werner's syndrome: implications for

an

normal ageing. Mech Ageing Dev. 1997;98 :239-54.

M

4) Lauper JM, Krause A, Vaughan TL, Monnat RJ Jr.Spectrum and risk of neoplasia in Werner syndrome: a systematic review.PLoS One. 2013; 8(4):e59709. PLoS One. 2013;8(4):e59709.

ed

doi: 10.1371/journal.pone.0059709

pt

5)Goto M, Miller RW, Ishikawa Y, Sugano H. Excess of rare cancers in Werner syndrome (adult

ce

progeria).Cancer Epidemiol Biomarkers Prev.1996;5:239–46.

6) Yamamoto K, Imakiire A, Miyagawa N, Kasahara T. A report of two cases of Werner's syndrome and review of the literature.JOrthopSurg (Hong Kong) 2003; 11:224–33.

Ac


metabolism, growth control of cultured cells, and chromosomal aberrations. Hum Genet. 1982;

7) Salk D.Werner's syndrome: a review of recent research with an analysis of connective tissue metabolism, growth control of cultured cells, and chromosomal aberrations. Hum Genet. 1982; 62:1-5

21

8)Salk D, Au K, Hoehn H, et al. Cytogenetics of Werner’s syndrome cultured skin fibroblasts: variegated translocation mosaicismCytogenet Cell Genet 1981;30:92-107.

9) Gebhart E, Bauer R, Raub U, Schinzel M, Ruprecht KW, Jonas JB. Spontaneous and induced

ip t

chromosomal instability in Werner syndrome. Hum Genet. 1988;80:135-9.

cr

T, Goto M, Hayashi M. Chromosomal instability in B-lymphoblasotoid cell lines from Werner

us

and Bloom syndrome patients. Mutat Res. 2002; 26;15-24.

an

11) Poot M, Gollahon KA, Emond MJ, Silber JR, Rabinovitch PS. Werner syndrome diploid fibroblasts are sensitive to 4-nitroquinoline-N-oxide and 8-methoxypsoralen: implications for the

M

disease phenotype. FASEB J. 2002; 16:757-8.

ed

12) Ogburn CE, Oshima J, Poot M, Chen R, Hunt KE, Gollahon KA, Rabinovitch PS, Martin GM.An apoptosis-inducing genotoxin differentiates heterozygotic carriers for Werner helicase

pt

mutations from wild-type and homozygous mutants. Hum Genet. 1997; 101:121-5

ce

13)Poot M, Gollahon KA, Rabinovitch PS. Werner syndrome lymphoblastoid cells are sensitive to camptothecin-induced apoptosis in S-phase. Hum Genet. 1999; 104 :10-14.

14) Poot M, Yom JS, Whang SH, Kato JT, Gollahon KA, RabinovitchPS.Werner syndrome cells

Ac


10)Honma M1, Tadokoro S, Sakamoto H, Tanabe H, Sugimoto M, Furuichi Y, Satoh T, Sofuni

are sensitive to DNA cross-linking drugs.FASEB J. 2001 ;15:1224-6.

15) Yu CE, Oshima J, Fu YH, Wijsman EM, Hisama F, Alisch R, Matthews S, Nakura J, Miki T, Ouais S, Martin GM, Mulligan J, Schellenberg GD. Positional cloning of the Werner's syndrome gene Science. 1996;272:258-62. 22

16)

Larsen NB, Hickson ID RecQ Helicases: Conserved Guardians of Genomic Integrity.

AdvExp Med Biol. 2013;767 :161-84.

17). Rossi ML, Ghosh AK, Bohr VA. Roles of Werner syndrome protein in protection of

ip t

genome integrity. DNA Repair 2010;9:331-44

cr

J, Martin GM, SchellenbergGD.Mutations in the consensus helicase domains of the Werner

us

syndrome gene. Werner's Syndrome Collaborative Group. Am J Hum Genet. 1997;60 :330-41.

an

19) Gray MD, Shen JC, Kamath-Loeb AS, Blank A, Sopher BL, Martin GM, Oshima J, Loeb

M

LA. The Werner syndrome protein is a DNA helicase. Nat Genet. 1997 Sep; 17(1):100-3.

20) Suzuki N, Shimamoto A, Imamura O, Kuromitsu J, Kitao S, Goto M, Furuichi Y.DNA

ed

helicase activity in Werner's syndrome gene product synthesized in a baculovirus system. Nucleic Acids Res. 1997; 25(15):2973-8.

pt

21) The premature ageing syndrome protein, WRN, is a 3'-->5' exonuclease. Huang S, Li B,

ce

Gray MD, Oshima J, Mian IS, Campisi J. Nat Genet. 1998; 20 (2):114-6.

22) Spillare EA1, Robles AI, Wang XW, Shen JC, Yu CE, Schellenberg GD, Harris CC p53mediated apoptosis is attenuated in Werner syndrome cells. Genes Dev. 1999; 13:1355-60.

Ac


18) Yu CE, Oshima J, Wijsman EM, Nakura J, Miki T, Piussan C, Matthews S, Fu YH, Mulligan

23) Crabbe L, Jauch A, Naeger CM, Holtgreve-Grez H, KarlsederJ.Telomere dysfunction as a cause of genomic instability in Werner syndrome. ProcNatlAcadSci U S A. 2007 13; 104 :220510.

23

24) Matsumoto T, Shimamoto A, Goto M, Furuichi Y. Impaired nuclear localization of defective DNA helicases in Werner's syndrome. Nat Genet. 1997; 16:335-6.

25)vonKobbe C, Bohr VA. A nucleolar targeting sequence in the Werner syndrome protein

ip t

resides within residues 949-1092. J Cell Sci. 2002 ;115:3901-7.

cr

us

Werner syndrome protein in human cells. ProcNatlAcadSci U S A. 1998 9;95:6887-92

27)Huang S, Lee L, Hanson NB, Lenaerts C, Hoehn H, Poot M, Rubin CD, Chen DF, Yang CC,

an

Juch H, Dorn T, Spiegel R, Oral EA, Abid M, Battisti C, Lucci-Cordisco E, Neri G, Steed EH, Kidd A, Isley W, Showalter D, Vittone JL, Konstantinow A, Ring J, Meyer P, Wenger SL, von

M

Herbay A, Wollina U, Schuelke M, Huizenga CR, Leistritz DF, Martin GM, Mian IS, Oshima J.

ed

The spectrum of WRN mutations in Werner syndrome patients. Hum Mutat. 2006;27:558–67.

28)Friedrich K, Lee L, Leistritz DF, Nürnberg G, Saha B, Hisama FM, Eyman DK, Lessel D,

pt

Nürnberg P, Li C, Garcia-F-Villalta MJ, Kets CM, Schmidtke J, Cruz VT, Van den Akker PC, Boak J, Peter D, Compoginis G, Cefle K, Ozturk S, López N, Wessel T, Poot M, Ippel PF,

ce

Groff-Kellermann B, Hoehn H, Martin GM, Kubisch C, Oshima J.WRN mutations in Werner syndrome patients: genomic rearrangements, unusual intronic mutations and ethnic-specific alterations. Hum Genet. 2010; 128:103-11

Ac


26) Marciniak RA1, Lombard DB, Johnson FB, Guarente L. Nucleolar localization of the

29) Huang S, Lee L, Hanson NB, et al. The spectrum of WRN mutations in Werner syndrome patients. Hum Mutat 2006;27: 558-67.

24

30) Goto M, Imamura O, Kuromitsu J, Matsumoto T, Yamabe Y, Tokutake Y, Suzuki N, Analysis of helicase gene mutations in Japanese Werner's syndrome patients.Mason B, Drayna D, Sugawara M, Sugimoto M, Furuichi Y. Hum Genet. 1997; 99 (2):191-3.

ip t

31) Yamabe Y, Sugimoto M, Satoh M, Suzuki N, Sugawara M, Goto M, Furuichi Y. Downregulation of the defective transcripts of the Werner's syndrome gene in the cells of patients.

cr

us

32)Wang L1, Hunt KE, Martin GM, Oshima J. Structure and function of the human Werner syndrome gene promoter: evidence for transcriptional modulation.Nucleic Acids Res. 1998 Aug

an

1;26:3480-5.

M

33)Agrelo R1, Cheng WH, Setien F, Ropero S, Espada J, Fraga MF, Herranz M, Paz MF, Sanchez-Cespedes M, Artiga MJ, Guerrero D, Castells A, von Kobbe C, Bohr VA, Esteller M.

ed

Epigenetic inactivation of the premature aging Werner syndrome gene in human cancer.

pt

ProcNatlAcadSci U S A. 2006;103:8822-7.

34) Bidou L, Allamand V, Rousset JP, Namy O. Sense from nonsense: therapies for premature

ce

stop codon diseases. Trends Mol Med. 2012;18:679-88.

35) Palombo, M; Monroy, S, Achenbach, RE. Síndrome de Werner: Dos nuevos casos. Rev.

Ac


BiochemBiophys Res Commun.1997; 236:151–54.

argent. dermatol. 2010 ; 91: 1-9.

36)Muftuoglu M, Oshima J, von Kobbe C, Cheng WH, Leistritz DF, Bohr VA. The clinical characteristics of Werner syndrome: molecular and biochemical diagnosis. Hum Genet. 2008; 124:369–77.

25

37)Yeong EK, Yang CC. Chronic leg ulcers in Werner's syndrome. Br J Plast Surg. 2004; 57:86– 8.

38)Goto M, Yamabe Y, Shiratori M, Okada M, Kawabe T, Matsumoto T, Sugimoto M,

ip t

FuruichiY.Immunological diagnosis of Werner syndrome by down-regulated and truncated gene

cr

39) Agrelo R. Epigenetic Silencing of Progeroid Syndromes. In Epigenetics ofAging.Tollefsbol,

Keeling KM, Xue X, Gunn G, Bedwell DM. .Therapeutics based on stop codon

an

40)

us

TO.ed.1sted. Springer Verlag, New York, 2010

M

readthroughAnnu Rev Genomics Hum Genet. 2014;15:371-94.

41)Rodríguez-López AM1, Jackson DA, Iborra F, Cox LS. Asymmetry of DNA replication fork

ed

progression in Werner's syndrome.Aging Cell.2002 ;1:30-9.

42) Fraga MF, Agrelo R, Esteller M. Cross-talk between aging and cancer: the epigenetic

pt

language. Ann N Y Acad Sci. 2007; 1100:60-74

ce

43)Kim J, Kim Y-J, Issa JP. Aging and DNAmethylation.CurrChemBiol 2009;3:321-9.

44)Bachrati CZ, Hickson ID. RecQhelicases:suppressors of tumorigenesis and premature aging.

Ac


products. HumGenet. 1999; 105:301-7.

journalBiochem J 2003;15:577-606

45)EstellerM.Epigenetic gene silencing in cancer: the DNA hypermethylome. Hum Mol Genet. 2007 15;16 Spec No 1:R50-9.

26

46)Christensen BC, Houseman EA, MarsitCJ, Aging and Environmental ExposuresAlter TissueSpecific DNA MethylationDependent upon CpG Island Context. PlosGenet 2009:5:e1000602

47)Thompson RF, Atzmon G, Gheorghe C, et al. Tissue-specific dysregulation of

ip t

DNAmethylation in aging. Aging Cell 2010;9: 506-18

cr

us

methylation changes in mice.Genome Res 2010; 20:332-40.

49)Addo BK, Chung W, Shen L, et al. Age-Related DNA Methylation Changes in Normal

an

Human Prostate Tissues. Clin Cancer Res 2007;13:3796-802.

M

50)Koch CM, Suschek CV, Lin Q, et al. Specific age-associated DNA methylation changes in human dermal fibroblasts. PLoS One 2011;6:e16679

ed

51)Heyn H1, Li N, Ferreira HJ, Moran S, Pisano DG, Gomez A, Diez J, Sanchez-Mut JV, Setien F, Carmona FJ, Puca AA, Sayols S, Pujana MA, Serra-Musach J, Iglesias-Platas I, Formiga F,

pt

Fernandez AF, Fraga MF, Heath SC, Valencia A, Gut IG, Wang J, Esteller M. Distinct DNA

ce

methylomes of newborns and centenarians. ProcNatlAcadSci U S A. 2012 Jun 26;109:10522-7

52)Fraga MF, Ballestar E, Paz MF, et al.Epigenetic differences arise during the lifetime of monozygotic twins. ProcNatlAcadSci USA 2005;102:10604-9.

Ac


48)Maegawa S, Hinkal G, Kim HS, et al.Widespread and tissue specific age-related DNA

53)Goto M1, Ishikawa Y, Sugimoto M, Furuichi Y. BiosciTrends. Werner syndrome: a changing pattern of clinical manifestations in Japan (1917~2008). 2013;7:13-22

27

54)Brendel C, Belakhov V, Werner H, Wegener E, Gärtner J, Nudelman I, Baasov T, Huppke P. Readthrough of nonsense mutations in Rett syndrome: evaluation of novel aminoglycosides and generation of a new mouse model. J Mol Med (Berl). 2011 Apr;89:389-98

ip t

55)Floquet C, Deforges J, Rousset JP, BidouL.Rescue of non-sense mutated p53 tumor

cr

56)Floquet C, Rousset JP, Bidou L. Readthrough of premature termination codons in the

us

adenomatous polyposis coli gene restores its biological activity in human cancer cells.PLoS One.

an

2011;6:e24125.

57)Kuschal C, DiGiovanna JJ, Khan SG, Gatti RA, Kraemer KH.Repair of UV photolesions in

M

xerodermapigmentosum group C cells induced by translational readthrough of premature

ed

termination codons.ProcNatlAcadSci U S A. 2013;110(48):19483-8.

58)Finkel RS, Flanigan KM, Wong B, Bönnemann C, Sampson J, Sweeney HL, Reha A,

pt

Northcutt VJ, Elfring G, Barth J, Peltz SW. .Phase 2a study of ataluren-mediated dystrophin production in patients with nonsense mutation Duchenne muscular dystrophy. PLoS One. 2013

ce

Dec 11;8:e81302.

59)Foster H, Popplewell L, Dickson G. Genetic therapeutic approaches for Duchenne muscular

Ac


suppressor gene by aminoglycosides. Nucleic Acids Res. 2011 ;39:3350-62.

dystrophy.Hum Gene Ther. 2012 Jul;23:676-87.

60)Floquet C, Hatin I, Rousset JP, Bidou L. Statistical analysis of readthrough levels for nonsense mutations in mammalian cells reveals a major determinant of response to gentamicin. PLoS Genet. 2012;8:e1002608. 28

61)Tan L, Narayan SB, Chen J, Meyers GD, Bennett MJ. PTC124 improves readthrough and increases enzymatic activity of the CPT1A R160X nonsense mutation J Inherit Metab Dis. 2011 Apr;34:443-7.

ip t

62)Goldmann T, Overlack N, Wolfrum U, Nagel-Wolfrum K. PTC124-mediated translational readthrough of a nonsense mutation causing Usher syndrome type 1C. Hum Gene Ther. 2011

cr

us

63)Wilschanski M, Miller LL, Shoseyov D, Blau H, Rivlin J, Aviram M, Cohen M, Armoni S, Yaakov Y, Pugatsch T, Cohen-Cymberknoh M, Miller NL, Reha A, Northcutt VJ, Hirawat S,

an

Donnelly K, Elfring GL, Ajayi T, Kerem E Chronic ataluren (PTC124) treatment of nonsense

M

mutation cystic fibrosis. EurRespir J. 2011;38:59-69.

64Welch EM, Barton ER, Zhuo J, Tomizawa Y, Friesen WJ, Trifillis P, Paushkin S, Patel M,

ed

Trotta CR, Hwang S, Wilde RG, Karp G, Takasugi J, Chen G, Jones S, Ren H, Moon YC, Corson D, Turpoff AA, Campbell JA, Conn MM, Khan A, Almstead NG, Hedrick J, Mollin A,

pt

Risher N, Weetall M, Yeh S, Branstrom AA, Colacino JM, Babiak J, Ju WD, Hirawat S,

ce

Northcutt VJ, Miller LL, Spatrick P, He F, Kawana M, Feng H, Jacobson A, Peltz SW, Sweeney HL. PTC124 targets genetic disorders caused by nonsensemutations.Nature. 2007 May 3;447:8791.

Ac


May;22:537-47

65)Linde L, Boelz S, Nissim-Rafinia M, Oren YS, Wilschanski M, Yaacov Y, Virgilis D, NeuYilik G, Kulozik AE, Kerem E, Kerem B. .Nonsense-mediated mRNA decay affects nonsense transcript levels and governs response of cystic fibrosis patients to gentamicin. J Clin Invest. 2007;117:683-92. 29

66)Müller FB1, Tsianakas A, Kuwert C, Korge BP, Hunzelmann N A novel compound heterozygous mutation in Werner syndrome results in WRN transcript decay. Br J Dermatol. 2005; 152:1030-2

ip t

67) Kaneko R, Kawaguchi M, Toyama T, Taguchi Y, Yagi T. Expression levels of Protocadherin-alpha transcripts are decreased by nonsense-mediated mRNA decay with

cr us

430:86-94

68)Characterization of the biochemical properties of the human Upf1 gene product that is

an

involved in nonsense-mediated mRNA decay.Bhattacharya A, Czaplinski K, Trifillis P, He F, Jacobson A, Peltz SW.RNA. 2000;6:1226-35.

M

69)Tools for turnover: methods for analysis of mRNA stability in eukaryotic cells. Jacobson A, PeltzSW.Methods. 1999 Jan;17:1-2.

ed

70)Interrelationships of the pathways of mRNA decay and translation in eukaryotic cells.Jacobson A, PeltzSW.Annu Rev Biochem. 1996;65:693-739.

pt

71).Peltz SW, Welch EM, Trotta CR, Davis T, Jacobson A. Targeting post-transcriptional

ce

control for drug discovery RNA Biol. 2009 Jul-Aug;6:329-34. Epub 2009 Jul 7. Review.

72)Hinzpeter A1, Aissat A, de Becdelièvre A, Bieth E, Sondo E, Martin N, Costes B, Costa C,

Ac


frameshift mutations and by high DNA methylation in their promoter regions. Gene; 2009

Goossens M, Galietta LJ, Girodon E, FanenP.Alternative splicing of in-frame exon associated with premature termination codons: implications for readthrough therapies. Hum Mutat. 2013 ;34:287-91.

73)Tahara H, Tokutake Y, Maeda S, Kataoka H, Watanabe T, Satoh M, Matsumoto T, Sugawara M, Ide T, Goto M, Furuichi Y, Sugimoto M. Abnormal telomere dynamics of B-lymphoblastoid 30

cell strains from Werner's syndrome patients transformed by Epstein-Barr virus. Oncogene. 1997;15:1911-20. 74) Miller G, Lipman M. Release of infectious Epstein-Barr virus by transformed marmoset

ip t

leukocytes. ProcNatlAcadSci U S A 1973; 70:190 – 4 75) Ionov Y, Nowak N, Perucho M, Markowitz S, Cowell JK.Manipulation of nonsense

cr

us

Oncogene; 2004;23:639-45.

76)Leeb M, Wutz A. Ring1B is crucial for the regulation of developmental control genes and

ce

pt

ed

M

an

PRC1 proteins but not X inactivation in embryonic cells. J Cell Biol 2007; 178: 219–229.

Ac


mediated decay identifies gene mutations in colon cancer Cells with microsatellite instability.

31

ip t cr us an

M

sequence with codons in alternate colors as well as the protein sequence. B) Sequencing results

ed

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

pt

the normal and mutated allele. No mutation was found in the wild type control (CTRL). C)

ce

Photographs of the two patients showing the typical WS premature appearance. Their mother, who is heterozygous for the mutation, did not show the WS phenotype.

Ac


Fig. 1 Novel WS mutation A) The representation of the mutation localization shows the DNA

32

ip t cr us an M ed pt


ce Ac

Fig.2 WRN is not expressed in WS cells harboring the p.S1256X mutation: Methylation analysis of the WRN promoter in patients and controls. 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 33

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-WS+/+), heterozygous (HTWRN+/-), p.S1256X(WRN-/-), and p.R369X (WRN-/-) cell lines. Actin was used as a loading

ip t

control. C) Immunofluorescence using the 4H12 antibody (which only detects full-length WRN)

cr

(WRN-/-) showing no WRN expression. Blue indicates DAPI staining (first panel), and green

us

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

an

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

M

PCR primers are indicated as white and grey arrows, respectively. The bisulphite genomic

ed

sequencing results are shown for five 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

pt

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

ce

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.

Ac


in cell lines showing WRN expression and nuclear localization in HT (WRN+/+) and p.S1256X

34


ce

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

Ac


Fig. 3PTC read-through treatment in WS cells harboring the p.S1256X mutation restores

indicating that the protein is stable in the cytosol. B) Western analysis of WRN protein with the 8H3 antibody, which recognises 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 35

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,

ip t

gentamicin, streptomycin, or G418 showing restored nuclear WRN expression. Plots of the

cr

WRN expression after being treated with the three different AGs and PTC-124 by read-through

ce

pt

ed

M

an

us

therapy of PTC mutations are shown.

Ac


percentage (%) of cells harboring the p.S1256X and p.R369X mutations and expressing nuclear

36


ce

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

Ac


Fig. 4 Restoration of nuclear localization and functionality of WRN as measured by

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 three different AGs in cells harboring the p.S1256X or p.R369Xmutation. C) WS cells are sensitive to 37

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

ip t

(WRN-/-). Error bars represent the standard deviation (n = 9); **P < 0.01. P-values were

cr

conditions. E) Statistical representation of γ-H2AX foci in WS cells harboring the p.S1256X

us

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

ce

pt

ed

M

untreated or treated with PTC-124 or AGs.

an

determined by Student’s t-test.(F)Representative images showing γ-H2AX foci in WS cells left

Ac


determined by Student’s t-test. D) The apoptotic cells are shown in green for the different

38


ce

through therapy in WS cells harboring the p.S1256X mutation. Chromosomal breakage measured by cytogenetic analysis of metaphase chromosomes. Untreated WS cells harboring the

Ac


Fig.5 Chromosomal damage and impaired DNA replication are reduced by PTC read-

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 39

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 69 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,

ip t

which was similar to the fiber length in the control. Two plots of one representative experiment

cr

were performed with the control cells and WS cells harboring the p.S1256X mutation left

ce

pt

ed

M

an

us

untreated or treated with PTC-124 or gentamicin. BrdU-labelled fibers are stained in green.

Ac


out of three biological replicas are shown (n = 200 for each condition). The DNA fiber assays

40

Accelerated epigenetic aging in Werner syndrome.

Three novel mutations in the cystic fibrosis gene detected by chemical cleavage: analysis of variant splicing and a nonsense mutation.

[Nonsense readthrough therapy for Duchenne muscular dystrophy].

Epigenetic Regulation of Werner Syndrome Gene in Age-Related Cataract.

Translational readthrough at nonsense mutations in the HSF1 gene of Saccharomyces cerevisiae.

Association of a Novel Nonsense Mutation in KIAA1279 with Goldberg-Shprintzen Syndrome.

A novel nonsense mutation of the KAL1 gene (p.Trp204*) in Kallmann syndrome.

Adenine phosphoribosyltransferase (APRT) deficiency: identification of a novel nonsense mutation.

Detailed assessment of renal function in a proband with Harboyan syndrome caused by a novel homozygous SLC4A11 nonsense mutation.

Review of pharmacological therapies in fibromyalgia syndrome.

A homozygous nonsense CEP250 mutation combined with a heterozygous nonsense C2orf71 mutation is associated with atypical Usher syndrome.

A patient with a novel homozygous missense mutation in FTO and concomitant nonsense mutation in CETP.

Mutation Spectrum and Phenotypic Features in Noonan Syndrome with PTPN11 Mutations: Definition of Two Novel Mutations.

A Nonsense Mutation in Mycobacterium marinum That Is Suppressible by a Novel Mechanism.

Factor V deficiency caused by a novel nonsense mutation (Gln2031stop) in a Chinese patient.

Cardiac conduction defects and Brugada syndrome: A family with overlap syndrome carrying a nonsense SCN5A mutation.

Clinical characteristics and treatment of Herlyn-Werner-Wunderlich syndrome.

A novel missense mutation in POMT1 modulates the severe congenital muscular dystrophy phenotype associated with POMT1 nonsense mutations.

Novel nutraceutic therapies for the treatment of metabolic syndrome.

A system for coordinated analysis of translational readthrough and nonsense-mediated mRNA decay.

Suppressibility of recA, recB, and recC mutations by nonsense suppressors.

atypical Werner syndrome with heterozygous LMNA mutation.

Epigenetic targets for novel therapies of lung diseases.

Novel pharmacological therapies for management of chronic constipation.