HUMAN MUTATION 27(6),558^567, 2006
The Spectrum of WRN Mutations in Werner
Shurong Huang,1Lin Lee,1Nancy B. Hanson,1Catherine Lenaerts,2Holger Hoehn,3Martin Poot,4
Craig D. Rubin,5Da-Fu Chen,6Chih-Chao Yang,6Heike Juch,7Thomas Dorn,7Roland Spiegel,8
Elif Arioglu Oral,9Mohammed Abid,10Carla Battisti,11Emanuela Lucci-Cordisco,12Giovanni Neri,12
Erin H. Steed,13Alexa Kidd,14William Isley,15David Showalter,16Janet L. Vittone,17
Alexander Konstantinow,18Johannes Ring,18Peter Meyer,19Sharon L. Wenger,20Axel von Herbay,21
Uwe Wollina,22Markus Schuelke,23Carin R. Huizenga,1Dru F. Leistritz,1George M. Martin,1
I. Saira Mian,24and Junko Oshima1?
1Department of Pathology, University of Washington, Seattle, Washington;2Service de Pediatrie, University of D’Amiens, Amiens Cedex, France;
3Institut fur Humangenetik, der Universitat Wurzburg, Wurzburg, Germany;4Laboratory for Genome Diagnostics, University Medical Center
Utrecht, Utrecht, The Netherlands;5University of Texas Southwestern Medical Center, Dallas, Texas;6Department of Neurology, National
Taiwan University Hospital, Taipei, Taiwan;7Swiss Epilepsy Center, Zurich, Switzerland;8Human Genetics Lab Genetica, Zurich, Switzerland;
9Division of Endocrinology and Metabolism, University of Michigan, Ann Arbor, Michigan;10Pasteur Institute of Morocco in Tangier, Plateau
Marchane, Tangier, Morocco;11O.U. of Neurometabolic Disease, University of Siena, Siena, Italy;12Instituto di Genetica Medica, Facolta di
Medicina ‘‘A Gemelli’’, Universita Cattolica del S. Cuore, Roma, Italy;13North Carolina Department of Health and Human Services, Genetics
and Newborn Screening Unit, Columbus, North Carolina;14Central Regional Genetic Services, Wellington Hospital, Wellington South, New
Zealand;15Departments of Endocrinology, Diabetes, Nutrition, and Metabolism, Mayo Clinic, Rochester, Minnesota;16Medical Genetics, Mayo
Clinic, Rochester, Minnesota;17General Internal Medicine, Mayo Clinic, Rochester, Minnesota;18Division of Environmental Dermatology and
Allergology GSF/TUM, Department of Dermatology and Allergology, Technical University Munich, Munich, Germany;19Genefinder
Technologies Ltd., Munich, Germany;20Division of Medical Genetics, Children’s Hospital of Pittsburgh, Pittsburgh, Pennsylvania;21Institute of
Pathology, University of Heidelberg, Germany;22Department of Dermatology, Hospital Dresden-Friedrichstadt, Dresden, Germany;23Department
of Neuropediatrics, Charite University Hospital, Berlin, Germany;24Lawrence Berkeley National Laboratory, Berkeley, California
Communicated by Mark Paalman
The International Registry of Werner syndrome (www.wernersyndrome.org) has been providing molecular
diagnosis of the Werner syndrome (WS) for the past decade. The present communication summarizes, from
among 99 WS subjects, the spectrum of 50 distinct mutations discovered by our group and by others since the
WRN gene (also called RECQL2 or REQ3) was first cloned in 1996; 25 of these have not previously been
published. All WRN mutations reported thus far have resulted in the elimination of the nuclear localization
signal at the C-terminus of the protein, precluding functional interactions in the nucleus; thus, all could be
classified as null mutations. We now report two new mutations in the N-terminus that result in instability of the
WRN protein. Clinical data confirm that the most penetrant phenotype is bilateral ocular cataracts. Other
cardinal signs were seen in more than 95% of the cases. The median age of death, previously reported to be in
the range of 46–48 years, is 54 years. Lymphoblastoid cell lines (LCLs) have been cryopreserved from the
majority of our index cases, including material from nuclear pedigrees. These, as well as inducible and
complemented hTERT (catalytic subunit of human telomerase) immortalized skin fibroblast cell lines are
available to qualified investigators. Hum Mutat 27(6), 558–567, 2006.
Published 2006 Wiley-Liss, Inc.y
KEY WORDS: Werner syndrome; WRN; RECQL2; RECQ3; Werner helicase; RecQ helicases; progeroid syndromes;
aging; international registries; penetrance; aging
Published online 3 May 2006 in Wiley InterScience (www.
yThis article is a US government work, and as such, is in the public
domain in theUnited States of America.
The Supplementary Material referred to in this article can be
accessed at http://www.interscience.wiley.com/jpages/1059-7794/
Received 30 September 2005; accepted revised manuscript 3
?Correspondence to: Junko Oshima, M.D., Ph.D., Department of
Pathology, Box 357470, University of Washington, 1959 NE Paci¢c
Ave, Seattle,WA 98195-7470. E-mail: firstname.lastname@example.org
Grant sponsor: National Cancer Institute; California Breast Cancer
Research Program; Grant sponsor: National Institute of Aging; Grant
Current address of Sharon L. Wenger: Department of Pathology,
WestVirginiaUniversity, Morgantown,WV 26506.
Current address of Axel von Herbay: Academic Department of
Pathology, St. Mark’s Hospital, London,UnitedKingdom.
PUBLISHED 2006 WILEY-LISS, INC.
Werner syndrome (WS; MIM] 277700) is a rare autosomal
recessive disorder characterized by many features suggestive of
accelerated aging [Epstein et al., 1966; Martin, 1978; Goto, 1997].
These include premature graying and loss of hair, bilateral ocular
cataracts, type 2 diabetes mellitus, osteoporosis, various forms
of arteriosclerosis including atherosclerosis, and hypogonadism.
There are scleroderma-like skin changes and regional atrophy of
subcutaneous fat. Deep ulcerations around the Achilles tendon
and malleoli are common and virtually pathognomonic. WS
subjects have an elevated risk of various cancers, particularly
sarcomas [Goto et al., 1996]. As many as five different neoplasms
have been found in a single individual (soft tissue sarcomas,
thyroid carcinoma, osteosarcoma, acral lentigenous melanoma,
and meningioma) [Goto et al., 1996].
WS subjects appear to develop normally until adolescence,
when there is lack of a pubertal growth spurt. Subsequent signs
and symptoms begin to emerge in the early 20s. The median age
of death has been reported to range from 46 years [Epstein et al.,
1966] to 48 years [Goto, 1997], typically as a result of cancer or
atherosclerotic cardiovascular disease. Because the WS phenotype
overlaps with many but not all of the symptoms manifested during
aging as it usually occurs (‘‘normative’’ aging), it has been classified
as a ‘‘segmental progeroid syndrome’’ [Martin, 1978; Martin
et al., 1999].
Several cellular defects have been identified in cultured somatic
cells derived from individuals with WS, including a much shorter
replicative life span when compared to age-matched controls
[Martin et al., 1970]. The genome of WS cells is highly unstable,
with increased chromosomal rearrangements (‘‘variegated trans-
mosaicism’’) [Salk et al., 1985; Melaragno et al., 1995], and large
spontaneous DNA deletions [Fukuchi et al., 1989]. The WS cells
are unable to optimally repair exogenous DNA with double strand
breaks [Chen et al., 2003a]. Telomere length dynamics are
abnormal in WS cells; telomeres shorten rapidly as cell population
doublings increase [Schulz et al., 1996; Tahara et al., 1997]. WS
cells also appear to be deficient in DNA replication and certain
DNA repair pathways. DNA replication initiation sites are
reduced and there is an extended S-phase of DNA replication
[Poot et al., 1992]. WS cells are hypersensitive to a number
of agents that damage DNA, including 4-nitroquinoline-1-oxide
(4-NQO) [Ogburn et al., 1997; Poot et al., 2002], topoisomerase I
inhibitors (camptothecin) [Okada et al., 1998; Poot et al., 1999],
and DNA cross-linking agents [Poot et al., 2001]. In addition, WS
cells are impaired in RNA polymerase II transcription [Balajee
et al., 1999] and p53-mediated apoptosis [Spillare et al., 1999;
Sommers et al., 2005]. The unstable genome of WS cells, and
the associated limited replicative potential and cell loss may be of
central significance to the pathogenesis of WS.
The gene underlying WS (WRN; MIM] 604611) was identified
in 1996 [Yu et al., 1996]. It was shown to be a member of the
RecQ family of DNA helicases. Structural analysis suggests that
the WRN protein contains a RecQ-type helicase domain within
the central region of the polypeptide [Yu et al., 1996] and a
nuclease domain in the N-terminal region [Moser et al., 1997;
Mushegian et al., 1997]. Subsequent biochemical studies con-
firmed that the WRN protein displays both 30-450; helicase [Gray
et al., 1997] and exonuclease [Huang et al., 1998] activities.
Alternative DNA structures that can be accidentally generated
during various DNA metabolic pathways are shown to be preferred
substrates of the WRN protein [Huang et al., 2000; Mohaghegh
et al., 2001].
A nuclearlocalizationsignal resides betweenresidues
1369–1402 at the C-terminus [Matsumoto et al., 1997b; Suzuki
et al., 2001]. An additional nucleolar localization signal is
implicated between residue 949–1092 within the RecQ consensus
domain [von Kobbe and Bohr, 2002]. The WRN protein is
localized primarily in the nucleoli in human cells and relocates
to form nucleoplasmic foci at sites of DNA damage [Sakamoto
et al., 2001].
In addition, there are two consensus domains in the C-terminal
region of the WRN protein: the RecQ helicase conserved region
(RQC) and the ‘‘helicase, RNaseD, C-terminal conserved region’’
(HRDC) [Morozov et al., 1997]. Structural analysis predicts
that WRN protein interacts with the substrate DNA and proteins
via its RQC region [Bennett and Keck, 2004] and with DNA
through the HRDC region [Liu et al., 1999]. Subsequent
biochemical studies demonstrated that substrate-specific DNA
binding activity occurs in three domains: N-terminal, RQC, and
HRDC [von Kobbe et al., 2003]. The WRN protein was shown to
accumulate at sites of double strand breaks via its HRDC domain
[Lan et al., 2005].
Following the initial report of four mutations [Yu et al., 1996],
additional mutations were reported in the WRN gene [Oshima
et al., 1996; Goto et al., 1997; Meisslitzer et al., 1997; Yu et al.,
1997; Matsumoto et al., 1997a]. The spectrum of 19 WRN
mutations was reviewed in 1999 [Moser et al., 1999]. All
mutations so far reported result in truncations of the WRN
protein, with the loss of the nuclear localization signal. This
precludes participation of the WRN protein in its nuclear
functions. We now extend the Moser et al.  report to a
total of 50 WRN mutations, 25 of which have not been previously
Samples were collected from individuals with Werner syndrome
participating in our International Registry of Werner Syndrome
(www.wernersyndrome.org), supplemented by materials banked
at the Coriell Institute for Medical Research (Camden, NJ). The
International Registry of Werner Syndrome has ongoing approval
from the University of Washington Institutional Review Board.
Blood or skin samples were processed using the follow protocols:
1) cryopreservation of an aliquot of the primary cells as a resource
for the cell bank; 2) isolation of DNA from primary cells;
3) establishment of lymphoblastoid cell line with Epstein-Barr
virus or of fibroblast lines with hTERT transformation; 4) isolation
of DNA, RNA, and protein from such lines; 5) Western analysis
with positive and negative controls; 6) RT-PCR, with confirmation
of successful amplification by agarose gel electrophoresis and
sequencing of the entire coding region; and 7) verification
of mutations by genomic DNA sequencing (Fig. 1).
Generally, 20–30ml of peripheral blood was received, from
which 1–2ml was used for DNA isolation and the rest was
fractionated to obtain plasma and buffy coat. The plasmas were
stored at –801C for future studies and buffy coats were used to
establish LCLs. Surplus white blood cells were frozen and stored in
liquid nitrogen for future use. Once LCLs were established,
a minimum of 10 frozen vials were stored in two different
locations. DNA, RNA, and protein were isolated from all LCLs.
LCLs were cultured in RPMI-1640 (Invitrogen/GIBCO, Carls-
bad, CA) containing 15% heat-inactivated fetal bovine serum
HUMAN MUTATION 27(6),558^567, 2006559
Human Mutation DOI 10.1002/humu
(FBS; Atlanta Biologicals, Norcross, GA), 100 units/ml penicillin,
and 100mg/ml streptomycin at 371C in an atmosphere of 5% CO2
and ambient oxygen concentrations. Fibroblast cells were similarly
cultured except that the medium was Dulbecco’s modified Eagle’s
medium (DMEM high glucose formulation, Invitrogen/GIBCO).
RT-PCR, PCR, and Sequencing
RT-PCR sequencing of the WRN gene and Western analysis
of the WRN protein were performed in samples, together with
positive and negative controls as previously described [Oshima
et al., 1996]. RT-PCR rather than genomic PCR was used as our
initial screen because it can detect splicing mutations; these were
frequent. Moreover, the WRN gene has a total of 35 exons, making
genomic screening rather cumbersome. When mutations were
identified by RT-PCR, the PCR products of the mutated exons
were sequenced in genomic DNA to confirm the mutation
[Yu et al., 1997]. When exon skipping was identified by RT-PCR
sequencing, the skipped exon and the two adjacent exons were
sequenced with special attention to the exon/intron boundary
mutations [Yu et al., 1997]. When mutations were not identified,
RT-PCR sequencing was repeated to ensure the absence of
mutations. In all cases, Western analyses were performed to
confirm the sequencing results. Typical cases of WS are due to null
mutations, in which case there is an absence of WRN protein.
Any discrepant results would trigger a repeat set of assays.
When only genomic DNA was available, all the coding exons
were sequenced, including 50–100 nucleotide of intron sequences
flanking all exons [Yu et al., 1997]. When mutations were
identified, the genomic PCR and sequencing analysis of the
mutated exons were repeated to confirm the results. When
mutations were not identified, all coding exons were resequenced
before negative results were reported.
The reaction conditions and technical details of the RT-PCR
and PCR sequencing has been described previously [Oshima et al.,
1996; Yu et al., 1997]. All the data are independently examined
and analyzed by two different individuals by comparisons with the
wild type WRN sequence (GenBank accession number L76937.1,
Generation and Detection of Recombinant
Constructs containing WRN cDNA encoding either the wild
type, E84A exonuclease mutant [Huang et al., 1998], K125N
mutant, K135E mutant, or [K125N1K135E] double mutant
N-terminal region (amino acids 1–333) of the WRN protein were
expressed in baculoviruses according to the published protocol
[Huang et al., 2000]. For the protein analysis, total protein was
prepared from insect cells infected with baculoviruses carrying the
indicated cDNAs, and purified by an affinity column [Huang
et al., 2000]. The purified WRN N-terminal fragment proteins
were resolved by a 10% PAGE followed by staining with Coomassie
brilliant blue. For Western analysis, 30mg total proteins were
resolved by a 10% PAGE and then blotted to a nylon membrane
and detected by a rabbit polyclonal anti-WRN antibody followed
by chemiluminescent autography. For Northern blotting, total
RNAs (5mg) were loaded for each of the samples, blotted to a
nylon membrane, and detected with a WRN-specific32P-labelled
cDNA probe followed by autoradiography.
International Registry of Werner Syndrome
The International Registry of Werner Syndrome at the
Department of Pathology, University of Washington, Seattle,
WA, was established in 1988. The original purpose of the Registry
was to collect samples from WS patients for positional cloning
of WRN. Between 1988 and the time of identification of the WRN
gene in 1996, we collected a total of 81 index cases. During this
early period, we focused on cases with a definite diagnosis of WS
based on a set of clinical criteria [Nakura et al., 1994]. In order to
identify unusual WRN mutations and to investigate other genes
responsible for segmental progeroid syndromes whose features
partially overlap with those of the WS, we expanded our Registry
to include probable and possible diagnoses of WS [Nakura
et al., 1994].
Clinicians of patients with WS symptoms have contacted us via
several linkages: 1) review articles published by our group; 2) an
internet search directing them to our website (www.wernersyn
drome.org); 3) the GeneTests/GeneClinics online database
(www.genetests.org); and 4) the Genetic Alliance patient
advocacy listing (www.geneticalliance.org). Clinicians are asked
to complete registry forms. Informed consent for the registry and
repository was done by our genetic counselor or the clinician if
local regulations allow (forms can be downloaded at www.werner
syndrome.org/registry/participate.html). As of November 2005, we
have enrolled a total of 144 pedigrees, or a total of 274 individuals,
including index cases and their family members.
In many cases, peripheral blood samples were available; rarely,
skin biopsies were sent to our laboratory (see LCL or Fibroblast in
Materials Available column in Supplementary Table S1 (available
suppmat). We successfully established LCLs in more than 95%
of the cases including blood samples that arrived at our laboratory
several days after sampling. For samples that arrived 1 week or
more after sampling (mostly from abroad), the success rate was
approximately 30%. In some instances, the molecular genetic
analysis was based upon previously established cell cultures or
previously extracted DNA (DNA in Supplementary Table S1,
Materials Available column).
We report clinical and molecular data compiled from a total
of 99 WS subjects with confirmed mutations (50 were female,
FIGURE 1. Diagram of standard protocols at the International
Registry of Werner Syndrome. Typical processing steps of the
560 HUMAN MUTATION 27(6),558^567, 2006
Human Mutation DOI 10.1002/humu
44 male and 5 unknown). This is consistent with the expected
gender ratios observed in autosomal recessive disorders. They
represent 21 ethnic/geographic groups: German, French, Japanese,
Italian, Swiss, Austrian, United States-Caucasian, Brazil, New
Zealand, Sardinia, African, Indian, Ashkenazi Jewish, Korean,
Taiwanese, Puerto Rican, Turk, Celtic, Dutch, Syrian, and
WRN Mutations inWS Patients
Fifty individually distinct WRN mutations were observed, 25 of
which have not been previously published. Of these 50 mutations,
41 occur in the coding sequence and nine are in introns. These are
summarized in Supplementary Table S1 and their locations are
mapped on the WRN gene in Figure 2. The mutations can be
grouped into four categories: 1) nonsense mutations which change
an amino acid codon to a stop codon and lead to the termination
of protein translation; 2) insertions and/or deletions which lead
to a reading frameshift and subsequent termination of protein
translation; 3) substitutions at the splice junctions that cause the
skipping of exons and a subsequent frameshift; and 4) missense
mutations which lead to amino acid changes in the protein.
The mutations were located throughout the coding region
of the WRN gene. There was one mutation at the 50region, six
mutations in the 30-450exonuclease domain, 10 mutations
between the exonuclease and the helicase domains, 11 mutations
within the helicase domain, 10 mutations in the RQC domain, five
mutations between the RQC and HRDC domains, four mutations
in the HRDC domain, and three mutations between HRDC and
the nuclear localization signal region. The most frequent mutation
among non-Japanese WS cases was a C to T transition
substitution at nucleotide 1105, changing amino acid codon 369
from arginine (CGA) to stop codon (TGA) [Oshima et al., 1996;
Yu et al., 1997; Matsumoto et al., 1997a]. This mutation occurred
in 24 of the 99 WS subjects (24%) compiled in the study either as
homozygotes or compound heterozygotes. The most common
mutation among Japanese WS was a transversion substitution from
G to C at the consensus splicing sequence at the junction of intron
25 and exon 26 that abolishes the splicing site and leads to
deletion of exon 26 [Yu et al., 1997]. This c.3139–1G4C
mutation was seen in 67% of our Japanese cases, and has not been
seen in cases from other ethnic origins. The skipping of exon
26 was found in one French pedigree, due to the substitution from
G to C at the splicing consensus sequence in intron 26 adjacent
to exon 26 (c.323311G4C) [Moser et al., 2000].
Novel Mutations That AlterWRN Protein Stability
Two new missense mutations in the exonuclease domain,
c.375A4T and c.403A4G, were identified as homozygous
FIGURE 2. Locations of WRN mutations. Exons are designated as boxes 1^35. Introns are indicated by thin lines. The numbers
above the exons indicate the nucleotide positions in theWRN cDNA (GenBank accession no. L76937.1) with the ¢rst ATG being 1.
The localizations of the functional domains are indicated. Purple boxes,exonuclease domain;yellow boxes, helicase domain; green
boxes, RQC, the RecQ helicase conserved region; pink boxes, HRDC, the Helicase, RnaseD, C-terminal conserved region; and gray
box, NLS, the nuclear localization signal; CDS, coding sequence; FS/Ter, frameshift and premature translation termination; IVS,
interveningsequence. I-VI indicate themotifs inexonuclease and helicasedomains.
HUMAN MUTATION 27(6),558^567, 2006 561
Human Mutation DOI 10.1002/humu
mutations in one Caucasian German subject (STUTT1010)
([c.375A4T 1 c.403A4G]1 [c.375A4T 1 c.403A4G]). A
transversion substitution from A to T at nucleotide 375 changed
amino acid codon 125 from lysine (AAA) to asparagine (AAT)
(K125N). The other missense mutation in this subject was the
result of a transversion (A to G at nucleotide 403, changing amino
acid codon 135 from lysine (AAG) to glutamate (GAG) (K135E)
Sequence and structural analysis predicted that the K125N and
K135E mutations are unlikely to affect exonuclease activity, but
are more likely to affect protein stability (Fig. 3). The three-
dimensional structure of the Klenow fragment of E. coli DNA
polymerase I complex with single-stranded DNA was used to
interpret the potential impact of the two missense mutations
[Mian, 1997]. The WRN mutations are predicted to occur towards
the end of an a helix and the beginning of a b strand (Fig. 3A).
The region of WRN protein containing these mutations (Fig. 3B,
green side chains) would be distal from the substrate binding and
catalytic sites and close to the surface, especially K125N; this face
of the exonuclease domain would be available for interaction with
another molecule. Inspection of the Klenow fragment structure
indicates that the surface exposed residues K406 (K125 in WRN),
E410 (E129 in WRN), and R436 (K156 in WRN) are aligned such
that the positively and negatively side charged chains can form
stabilizing charge-charge interactions (Fig. 3C). The NZ atom of
Klenow residue K416 (K135 in WRN) can form stabilizing
interactions with the peptide carbonyl oxygen atoms of the loop
containing E410 and R436. Thus, the WRN mutations, K125N
and K135E, are likely to perturb and/or eliminate this intricate
network of stabilizing interactions thereby disrupting the local
structure of the exonuclease domain without influencing exonu-
In order to verify the structural analysis, we expressed the
N-terminal regions of the mutant WRN proteins, including the
exonuclease region, in eukaryotic cells to examine protein stability
(Fig. 4). Following the baculovirus-mediated transfection of wild
type, K125N mutant, K135E mutant, or [K125N1K135E] double
mutant, mRNA and proteins were isolated from sf9 insect cells
every 24hr. As controls, wild type and E84A exonuclease mutant
were employed. The latter is the mutant that had been shown
to abolish exonuclease activity without affecting protein stability
[Huang et al., 1998]. The mRNAs transcribed from the cDNAs
containing the missense mutations (K125N, K135E, [K125N1
K135E]) were at levels similar to those from the wild type and the
E84A mutant (Fig. 4A). On the other hand, the protein levels
produced from the single mutant cDNAs, either K125N or K135E,
were much lower compared to the wild type and exonuclease
mutant controls. [K125N1K135E] double mutant proteins were
virtually undetectable with this method (Fig. 4B and C). This is
not due to the overall reduction of protein production (Fig. 4B).
We tested protein stability using full-length WRN cDNA. Because
of its relatively large size, however, protein levels were overall low
and the differences between the wild type and the mutants were
not clearly seen (data not shown). We were unable to obtain blood
or fibroblast samples from this patient to further examine these
mutations. In conclusion, these results are consistent with the
sequence and structural observations described above.
Clinical Features of PatientsWithWRN Mutations
Previous clinical reviews of WS cases have based the diagnosis
on clinical signs and symptoms and did not include molecular data
[Epstein et al., 1966; Tollefsbol and Cohen, 1984; Goto, 1997].
We therefore have evaluated the known signs and symptoms
of WS in genetically diagnosed WS patients. The patients were
referred to us by collaborating physicians who suspected a
diagnosis of WS. Most of the clinical information was provided
at the time of referral, along with biological samples. We have
received periodic medical updates on some patients. The median
age of referral was 46.4 years, the youngest being 15.9 years
(Fig. 5). Table 1 summarizes the frequency of well-documented
abnormalities of WS. Most notably, bilateral ocular cataracts were
reported in all of the cases in which information was available.
Skin alterations (scleroderma-like skin, tight skin, thin skin,
hyperkeratosis, etc.), graying and/or loss of hair and short stature
were reported in 98.6, 96.3, and 94.7% of cases, respectively;
90.9% of the cases presented all four cardinal signs of WS [Nakura
et al., 1994]. Other signs that are known to develop at later stages
of WS or ones that require additional laboratory tests to determine
were reported in slightly lower percentages. Atherosclerosis and
neoplasia, two major causes of death, were reported in 39.5 and
43.6% of the molecularly confirmed cases, respectively. Diabetes
and osteoporosis were reported in 70.8 and 90.6% of the cases,
respectively. The median age of death of WS patients in our
registry of molecularly confirmed WS was 54.3 years (Fig. 5),
7 years older than the median age of death reported by Epstein
et al. . The mean age of onset of cataracts was
approximately 31 years, comparable to that reported by Epstein
et al. .
The WRN gene consists of 35 exons that encode a multi-
functional protein of 1,432 amino acids (Fig. 2; Supplementary
Table S1). The WRN mutations so far identified are located
all across the entire coding region, including six mutations within
the exonuclease domain, eleven mutations within the helicase
domain, 10 mutations within the RQC domain, and four
mutations within the HRDC domain. All the mutations so far
identified in WS patients, except for the two missense mutations
in the exonuclease domain, are nonsense mutations or insertion/
deletion/substitution mutations, which result in truncation of
protein translation before the nuclear localization signal. These
truncated proteins cannot be transported into the nucleus
to perform their functions [Matsumoto et al., 1997b; Suzuki
et al., 2001]. Mutated mRNAs are known to be degraded via an
alternative pathway [Jacobson and Peltz, 1996]. Mutant WRN
mRNA has been shown to have a shorter half-life than that of the
wild type [Yamabe et al., 1997]. These appear to be satisfactory
explanations as to how the mutations in WRN abolish enzymatic
activities and why all the mutations are functionally null.
The two missense mutations in the exonuclease domain, K125N
and K135E, are located close to exonuclease motif II [Mian, 1997]
(Fig. 3A, green triangles). Both of these missense mutations appear
to render the WRN protein unstable (Fig. 4), an experimental
finding that can be rationalized by inspection of the three-
dimensional structure of the Klenow fragment of E. coli DNA
polymerase I (Fig.3B and C). Therefore, these missense mutations
may also act as null mutations. Our preliminary studies showed
that the K125N and K135E mutations do not significantly alter
the activities of WRN protein, as predicted by the structural
analysis. It is possible, however, that these mutations may
affect other properties of the WRN protein, such as its interactions
with other proteins or protein complexes. A recent publication
demonstrated that site directed mutation of lysine at codon 1016
562HUMAN MUTATION 27(6),558^567, 2006
Human Mutation DOI 10.1002/humu
FIGURE 3. Sequenceandstructuralanalysisof theN-terminalregionof theWRN protein. A: A multiplesequencealignment ofa 30-50
exonuclease domain present in the following selected proteins: EcDPOIII, Escherichia coli DNA polymerase III (RCSB code 1J54);
EcExoI, E. coli exonuclease I (RCSB1FXX); EcRNaseT, E. coli RNaseT (RefSeq NP_416169); EcKlenow_1QSL, Klenow fragment of
E.coliDNApolymeraseI (RCSB1QSL); HsWRN: HomosapiensWRN protein (RefSeqNP_000544). Numbersindicatethenumberof
residues not shown explicitly; residues that are conserved in three of the ¢ve sequences are highlighted.The annotations below the
alignment refer toWRN (HsWRN) and indicate the locations and names of mutations discussed in this study.The annotations above
the alignment refer toDNA polymeraseI (EcKlenow_1QSL) andrefer tofeatures observedin thecrystal structureof theKlenow frag-
ment complexed with single-stranded substrate and taken from the 1QSL entry in the PDBsum Resource (www.ebi.ac.uk/thornton
srv/databases/pdbsum).The arrows and cylinders represent beta-strands and alpha-helices, respectively. Red and blue triangles in-
dicate the locations and names of residues that interact with a metal ion in the active site and DNA substrate respectively. Green
triangles mark the residues in the Klenow fragment (K406 and K416) predicted to be equivalent to theWRN mutations K125N and
K135E.Yellow triangles mark residues and the boxed region indicates the region shown in the crystal structure. B: Images of the
Klenow fragment shown in two di¡erent orientations using the same color scheme as in panel A.The thin gray tube is a smoothed
representation of the alpha carbon backbone and the small gray spheres denote the N- and C-terminal residues.The N-termini are
representedby thegrayspheres atthebottoms of themolecules.Thesingle-strandedDNA substrateisshownincyan (all atomrepre-
chains (K406 and K416) are the positions equivalent to theWRN mutations K125N and K135E; the yellow side chains (E410 and
R436) are discussed in the text. C: Images of part of the Klenow fragment shown in two di¡erent orientations and corresponding to
theregionboxedin the alignment.Only atoms in thepeptide backboneofKlenow fragment residues 406 to 438 areshown (C: gray,
N: blue,O: red).Thesidechains of fourspeci¢c residues aredrawnexplicitly, K406 andK416 (green), andE410 andR436 (yellow).
HUMAN MUTATION 27(6),558^567, 2006 563
Human Mutation DOI 10.1002/humu
in the RQC domain significantly reduces helicase activity and the
binding activity for various DNA substrates [Lee et al., 2005].
c.1105C4T, which changes codon 369 CGA to stop codon
TGA. This mutation accounted for approximately 25% of our
cases, depending on whether pedigrees, cases or affected
chromosomes were counted. The ethnicities of this mutation
include French, Austrian, Japanese, German, and US subjects.
The other is c.3139–1G4C, which leads to the deletion of exon
26 (r.3139_3233del95) and truncation of the protein immediately
after the RQC domain. This mutation occurred in 22 Japanese WS
were noted.One is
subjects all as homozygotes, and accounts for 67% of Japanese WS
cases among our Registry cases and 62% in another study [Satoh
et al., 1999]. This mutation has been seen exclusively in Japanese
WS patients and a founder effect has been postulated [Matsumoto
et al., 1997a]. This can be also partly attributed to an increased
awareness of the syndrome among Japanese physicians as well
as to higher consanguinity among the Japanese population as
compared to the U.S. population. Other mutations only occurred
in one or a few WS subjects and appear to be due to sporadic
Out of 99 cases with clinical presentation of WS and/or absent
WRN protein by Western blot, indicating two mutant alleles,
six have only one mutation identified (indicated as 1mut in
Supplementary Table 1, Mutation Status column). In four cases,
FIGURE 4. K125NandK135EmutationsrenderWRNproteinsun-
stable. A: Northern blotting of total RNA isolated from insect
cells expressing segments of theWRN proteinwith the indicated
mutations. B: Coomassie blue staining of the proteins isolated
from the culture. Left panel shows the patterns of 1mg of total
proteins. Rightpanelshowsthepatterns of100mgof thepuri¢ed
proteins. C:Western analysis of thepuri¢edproteins.
TABLE 1. ClinicalFeatures of Werner SyndromePatientsWith
Signorsymptom Percent frequency
Type 2 diabetes mellitus
100 (87 /87)
?Clinical information was obtained at the time of referrals. Actual
numbers of the cases where information was available is given in the
parentheses. Diagnosis of atherosclerosis is based on the primary
aCardinal signs (bilateral ocular cataracts, characteristic dermatological
pathology, short stature, premature graying and/or thinning of scalp
FIGURE 5. Distribution of the ages of onset of ocular cataracts
in molecularly con¢rmed WS (A), distribution of the ages of
the patients at the time of referral to our registry (B), and
the distribution of the ages of death (C). X-axes indicate the
ageindecades, andY-axes show the numberofcases.
564HUMAN MUTATION 27(6),558^567, 2006
Human Mutation DOI 10.1002/humu
this was due to insufficient DNA or low quality of the DNA.
Based upon this, we estimated that approximately 1% (2/198) of
mutations might have been missed in our screening.
The WRN protein is thought to be a ‘‘caretaker of the genome’’
[Hickson, 2003]. The studies on interacting proteins suggest that
WRN protein is able to participate in multiple DNA metabolic
pathways when recruited to the site of gene action by binding
proteins. At least 16 proteins interacting with WRN protein have
been identified to date, many of which bind at the overlapping
RQC and HRDC regions. Most noticeably, the WRN protein is
involved in DNA double strand break repair by nonhomologous
end-joining in concert with the DNA-PK complex (DNA-PKcs,
Ku70 and 80) [Cooper et al., 2000; Li and Comai, 2001; Yannone
et al., 2001]. Physical and functional interactions with FEN-1
[Brosh et al., 2001; Sharma et al., 2005], DNA polymerase b
[Harrigan et al., 2003; Ahn et al., 2004], PARP-1 [Li et al., 2004;
von Kobbe et al., 2004], and APE1 [Ahn et al., 2004] suggest a
role of WRN in base excision repair. WRN protein may modulate
DNA replication via interaction with Topo I [Laine et al., 2003]
and DNA polymerases d [Szekely et al., 2000]. Physical
association of TRF-2 and WRN protein [Johnson et al., 2001],
in conjunction with biochemical data demonstrating the displace-
ment of D-loops by WRN protein [Orren et al., 2002], suggests
that WRN may participate in the maintenance of telomeres.
WRN protein has been shown to be involved in the synthesis of
lagging strand of telomeres [Crabbe et al., 2004]. The tumor
suppressor, p53, binds to and inhibits WRN helicase activity,
while signaling downstream transcriptional activity that regulates
the initiation of apoptosis following DNA damage [Sommers et al.,
2005]. Taken together, WRN exonuclease and helicase activities
may be used for various DNA transactions, particularly during
various modalities of DNA repair. The specifics of the DNA
transaction may depend on the protein complexes which direct
WRN protein to the site of DNA damage and coordinate its
We have reported four pedigrees among our Werner Registry
families with wild-type WRN but with heterozygous mutations
at the LMNA locus [Chen et al., 2003b]. It is possible that other
‘‘atypical WS’’ cases with wild-type WRN and LMNA are due
to mutations in these various WRN interacting proteins.
WS cases followed up by our registry presented as either
probands or nuclear families. Figure 6 shows the WS pedigrees in
which samples from more than one individual have been examined
and biological samples were obtained. These materials can be
made available to investigators upon request.
We thank Mark Steele, MD, for his efforts in identifying
the PCH family, Johannes Ring, MD, for his contributions with the
MUNCH family, and Dr Hassani Benyouness, an endocrinologist
in Oujda, Morocco, for facilitating enrollment of MOROC-
CO1010. This work was jointly supported by the National Cancer
Institute and the National Institute on Aging (CA78088 to
G.S.M.), and the California Breast Cancer Research Program
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