A fifty-five-year-old woman came to Yokaichi National Hospital
on August 13, 1996, with a chief complaint of pain in the left calcaneus.
At the age of thirty-six years, the patient had undergone bilateral
cataract extraction. At the age of fifty years, she had been admitted
to another private hospital with a complaint of pain in the left
calcaneus and was diagnosed with osteoporosis (Fig. 1, a).
We believe that the pain was related to the onset of the tumor that
was later diagnosed by us. Bone-grafting of the left calcaneus was
performed for the treatment of osteoporosis (Fig. 1, b).
The patient continued to have pain in the left foot until the time
of presentation.
The family history revealed that the patient's younger brother
had undergone bilateral cataract surgery at the age of forty years
and had died of a malignant tumor at the age of fifty-two years.
Her parents were consanguineous; her paternal grandfather was also
her maternal grandfather, and her paternal and maternal grandmothers
were siblings (Fig. 2).
Physical examination revealed that the patient was 139.4 centimeters
tall and weighed 32.9 kilograms. She appeared much older than her
stated age. The trunk was normally developed, but the extremities
were slender. The scalp hair was white, sparse, and receding. The
eyebrows, eyelashes, and axillary and pubic hair were white and sparse.
The mouth was small, and the nose was small and beak-shaped. The
skin of the face was shiny, thin, and taut (Fig. 3). There was
little subcutaneous tissue, but the skin was moveable. There was
atrophy of the skin, subcutaneous tissue, and muscles of the extremities. The
bones of the feet were prominent, and the skin was stretched over
them. Local heat, redness, and swelling were present in the region
of the left calcaneus. The lungs were clear. The blood pressure was
102/54 millimeters of mercury (13.60/7.20 kilopascals) in the right
arm and 126/60 millimeters of mercury (16.80/8.00 kilopascals) in
the left arm. The pulse was eighty beats per minute, with regular
rhythm. The heart was not enlarged, and there was no hepatosplenomegaly.
Urinalysis revealed a specific gravity of 1.015 and a pH of 5.5,
and the test was negative for protein and sugar. Histological examination
revealed occasional white blood cells, occasional red blood cells,
and occasional epithelial cells.
The hematocrit was 42.8 percent, and the red blood-cell count
was 4.36 × 106 per microliter (4.36 ×
1012 per liter). The hemoglobin level
was 13.4 grams per deciliter (8.3 millimoles per liter), with a
mean corpuscular volume of 98.2 cubic micrometers, a mean corpuscular
hemoglobin of 30.7 picograms, and a mean corpuscular hemoglobin
concentration of 31.3 percent. The white blood-cell count was 4.4
× 109 per liter, with a normal differential.
The erythrocyte sedimentation rate was thirty-six millimeters per
hour. The platelet count was 21.1 × 104 per
microliter (211 × 109 per liter). The
serum potassium level was 4.0 milliequivalents per liter (4.0 millimoles
per liter), the serum chloride level was 102 milliequivalents per liter
(102 millimoles per liter), and the serum sodium level was 143 milliequivalents
per liter (143 millimoles per liter). The total protein level was 8.4
grams per deciliter (84.0 grams per liter), and the albumin-to-globulin
ratio was 1.56. The serum alkaline phosphatase level was 397 international units
per liter (normal, 109 to 344 international units per liter), the
serum total cholesterol level was 207 milligrams per deciliter (5.35
millimoles per liter), the triglyceride level was 132 milligrams per
deciliter (1.49 millimoles per liter), the blood urea nitrogen level
was 22.0 milligrams per deciliter (7.9 millimoles per liter), and
the C-reactive protein level was 0.0 milligrams per deciliter (0.0 milligrams
per liter). The twenty-four-hour urinary excretion rate of 17-ketosteroids
was 2.0 milligrams per day (7.0 micromoles per day) (volume, 600
milliliters), and a serological test for syphilis was negative.
A glucose tolerance test revealed that the blood sugar level was
101 milligrams per deciliter (5.6 millimoles per liter) (normal,
seventy to 110 milligrams per deciliter [3.9 to 6.1 millimoles per
liter]) at the beginning of fasting, 139 milligrams per deciliter
(7.7 millimoles per liter) at one-half hour, 146 milligrams per
deciliter (8.1 millimoles per liter) at one hour, 141 milligrams per
deciliter (7.8 millimoles per liter) at one and one-half hours,
and 134 milligrams per deciliter (7.4 millimoles per liter) at two
hours. All laboratory data, with the exception of the erythrocyte sedimentation
rate, the alkaline phosphatase level, and the results of the glucose
tolerance test, were normal. The patient was diagnosed as having
diabetes mellitus.
An electrocardiogram revealed normal findings. Radiographs of
the chest revealed normal findings except for moderate calcification
of the aorta.
Radiographs of the calcanei revealed marked calcification of
the Achilles tendon bilaterally. The left calcaneus had a solitary
osteolytic lesion in the marrow cavity, with cortical destruction.
There was no sclerotic rim around the lesion, and no cortical expansion
was observed. Microcalcification was present in the bone marrow
(Fig. 1, c and d).
Bone scintigraphy with technetium-99m methylene diphosphonate showed
uptake of tracer in the left calcaneus. T1-weighted coronal magnetic resonance
images revealed an interosseous area of low signal intensity, consistent
with an expansile lesion in the calcaneus and the overlying cortex
(Fig. 4, a).
T2-weighted coronal and sagittal magnetic resonance images showed
a high-signal-intensity lesion with regions of high contrast that
represented necrosis and hemorrhage (Fig. 4, b and c).
Computerized tomography scans revealed a pathological fracture of
the calcaneus.
We diagnosed this lesion as a bone tumor. A biopsy was performed
on December 4, 1996, to determine whether the tumor was benign or
malignant. Histological evaluation of the biopsy specimen revealed
irregularly shaped regions of partially calcified osteoid formation
associated with solid growth of tumor cells and occasional tumorous cartilage.
The tumor cells had ovoid nuclei with conspicuous nucleoli and exhibited
some degree of pleomorphism (Fig. 5). The diagnosis of osteoblastic
osteosarcoma was made on January 11, 1997. A left below-the-knee amputation
was performed on February 5, 1997.
Mutation analysis according to the mutant allele-specific amplification
method with use of peripheral blood leukocytes was carried out as
previously described (Fig. 6)10,11.
With this method, specimens of DNA are obtained from skin fibroblasts,
B-lymphoblastoid cells cultured in the laboratory, or heparinized whole
blood. In the present study, we used whole blood cells. The blood
samples were first diluted eightfold with sucrose-Triton solution
containing 0.32-molar sucrose, ten-millimolar Tris-hydrochloric
acid (pH 7.5), five-millimolar magnesium chloride, and 1 percent
Triton X-100. Samples were placed on ice for twenty minutes and
then centrifuged at 1000 times gravity for ten minutes to pellet
the cell nuclei. The pellet was resuspended and incubated at 37
degrees Celsius overnight in 2.5 milliliters of lysis solution containing
67.5-millimolar sodium chloride, 22.5-millimolar EDTA, 0.5 percent
sodium dodecyl sulfate, and proteinase K (0.2 milligram per milliliter).
The mixture was treated with a combination of phenol, chloroform,
and isoamyl alcohol (in a ratio of 50:48:2 by volume), and the aqueous
layer was extracted from the lysate. Finally, genomic DNA was precipitated
with ethanol and resuspended in a solution of ten-millimolar Tris-hydrochloric
acid (pH 8.0) and 0.2-millimolar EDTA. Polymerase chain reaction
primers (Sawaday Technology, Tokyo, Japan) were purchased, oligonucleotide
primers specific to wild-type alleles or mutant alleles were designed,
and the optimal temperature of annealing for each set of primers
was determined with use of the OLIGO primer analysis software program
(Lifescience Software Resource, Long Lake, Minnesota). Polymerase
chain reaction was performed in twenty-five microliters of 4 percent glycerin
solution containing Taq polymerase (Perkin-Elmer Biosystems, Foster
City, California), ten picomoles of mutant allele-specific primer
(or wild-type allele-specific primer), ten picomoles of a common
primer, and twenty-five to fifty nanograms of genomic DNA as a template.
The polymerase chain reaction products were separated by electrophoresis
in a 3 percent gel made of Agarose L 03 TAKARA (Takara, Tokyo, Japan).
In the present study, three sets of primers (one each for mutations
1, 4, and 6) were applied and the amplification of mutation 6 was
detected (Fig. 7),
implying that the patient was homozygous for this type of mutation
(designated as mutation 6/6 according to Matsumoto et al.11,12).
In 1904, Otto Werner described four siblings who had premature
aging, cataracts, and skin disorders15.
In 1934, Oppenheimer and Kugel established the eponym of Werner
syndrome13. Since then, more than
1000 cases have been reported. Werner syndrome has been frequently
associated with malignancy2,4,7.
The patient in the present report had an unusual body habitus (short
stature and a stocky trunk with spindly limbs), signs of premature
senescence (gray hair, cataracts, osteoporosis, and arteriosclerosis),
and scleroderma-like skin changes, all of which are consistent with
Werner syndrome6.
The locus of Werner syndrome was recently mapped to chromosome
8 (8p11-12)5, and the gene responsible
(known as WRN) was identified by positional cloning16. WRN encodes a protein, 1432 amino
acids long, that is homologous to the RecQ-type DNA helicase. Nineteen
different mutations of Werner syndrome have been found9; mutations 4 and 6 are major mutations
in Japan. To our knowledge, we are the first to report the case
of a patient with Werner syndrome and osteosarcoma whose diagnosis
was confirmed by mutation analysis. As 75 percent of alleles of
Japanese patients with Werner syndrome are explained by only three
types of mutations, determining the diagnosis on the basis of genetic mutations
is relatively easy. Our patient exhibited mutation 6/6. Mutation
6 is predicted to produce a truncated protein that lacks enzymatic
activity, culminating in the loss of Werner syndrome helicase function.
A possible genotype-phenotype relationship regarding the cell lines
of thyroid carcinomas has been noted (one mutation seems to be associated
with follicular thyroid carcinoma and another, with papillary thyroid
carcinoma)9, but a thorough clinical
evaluation has failed thus far to reveal any difference in symptoms
among patients with different mutations11.
The tumor in our patient was different from conventional osteosarcoma
in that it affected the calcaneus and was diagnosed when the patient
was fifty-five years old (and not younger). However, this lesion
demonstrated the typical characteristics of osteosarcoma arising
in patients with Werner syndrome; as reported previously8, the average age of patients who
have osteosarcoma in association with Werner syndrome is forty years
and the lesion affects sites that are atypical for osteosarcoma.
The cause of the malignancy complicating Werner syndrome is not
well known. Boyd and Grant proposed that premature aging of all
organ systems might be the cause of the high prevalence of malignant
tumors in elderly individuals1.
This hypothesis was supported by the finding that skin fibroblasts
from a patient with Werner syndrome lived only one-third or one-eighth
as long as normal skin fibroblasts10,
and that, in vitro, the DNA replication rate was
slower in fibroblasts from a patient with Werner syndrome3. However, Goto et al. showed that
Werner syndrome was not only a premature aging disease but also
a cancer syndrome, as the malignant tumors seen in patients who
had Werner syndrome were different from those seen in patients who
did not have Werner syndrome with respect to site, histological
type, and age at onset6. Specifically,
Goto et al. reported that (1) the rate of occurrence of nonepithelial
tumors was ten times the usual rate, (2) nonepithelial tumors included
bone and soft-tissue tumors, melanoma, leukemia, and myelodysplastic
syndrome, (3) gastrointestinal cancer, lung cancer, and prostate
cancer were rare, whereas thyroid cancer was frequent9, and (4) meningioma was frequent6. Thus, additional genetic analyses
should be done to determine why Werner syndrome is associated with
malignant tumors.
Previously, Werner syndrome was diagnosed on the basis of clinical
findings only. To our knowledge, we are the first to confirm the
diagnosis of Werner syndrome associated with calcaneal osteosarcoma
with use of mutant allele-specific amplification.