Somatic activating mutations in MAP2K1 cause melorheostosis

Abstract

Melorheostosis is a sporadic disease of uncertain etiology characterized by asymmetric bone overgrowth and functional impairment. Using whole exome sequencing, we identify somatic mosaic MAP2K1 mutations in affected, but not unaffected, bone of eight unrelated patients with melorheostosis. The activating mutations (Q56P, K57E and K57N) cluster tightly in the MEK1 negative regulatory domain. Affected bone displays a mosaic pattern of increased p-ERK1/2 in osteoblast immunohistochemistry. Osteoblasts cultured from affected bone comprise two populations with distinct p-ERK1/2 levels by flow cytometry, enhanced ERK1/2 activation, and increased cell proliferation. However, these MAP2K1 mutations inhibit BMP2-mediated osteoblast mineralization and differentiation in vitro, underlying the markedly increased osteoid detected in affected bone histology. Mosaicism is also detected in the skin overlying bone lesions in four of five patients tested. Our data show that the MAP2K1 oncogene is important in human bone formation and implicate MEK1 inhibition as a potential treatment avenue for melorheostosis.

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ARTICLE
Somatic activating mutations in MAP2K1 cause
melorheostosis
Heeseog Kang1, Smita Jha2,3, Zuoming Deng4, Nadja Fratzl-Zelman5, Wayne A. Cabral1,10, Aleksandra Ivovic6,
Françoise Meylan 6, Eric P. Hanson7, Eileen Lange8, James Katz8, Paul Roschger5, Klaus Klaushofer5,
Edward W. Cowen9, Richard M. Siegel6, Joan C. Marini1& Timothy Bhattacharyya2
Melorheostosis is a sporadic disease of uncertain etiology characterized by asymmetric bone
overgrowth and functional impairment. Using whole exome sequencing, we identify somatic
mosaic MAP2K1 mutations in affected, but not unaffected, bone of eight unrelated patients
with melorheostosis. The activating mutations (Q56P, K57E and K57N) cluster tightly in the
MEK1 negative regulatory domain. Affected bone displays a mosaic pattern of increased
p-ERK1/2 in osteoblast immunohistochemistry. Osteoblasts cultured from affected bone
comprise two populations with distinct p-ERK1/2 levels by flow cytometry, enhanced ERK1/2
activation, and increased cell proliferation. However, these MAP2K1 mutations inhibit BMP2-
mediated osteoblast mineralization and differentiation in vitro, underlying the markedly
increased osteoid detected in affected bone histology. Mosaicism is also detected in the skin
overlying bone lesions in four of five patients tested. Our data show that the MAP2K1
oncogene is important in human bone formation and implicate MEK1 inhibition as a potential
treatment avenue for melorheostosis.
DOI: 10.1038/s41467-018-03720-z OPEN
1Section on Heritable Disorders of Bone and Extracellular Matrix, National Institute of Child Health and Human Development, National Institutes of Health,
Bethesda, MD 20892, USA. 2Clinical and Investigative Orthopedics Surgery Unit, National Institute of Arthritis and Musculoskeletal and Skin Diseases,
National Institutes of Health, Bethesda, MD 20892, USA. 3Program in Reproductive and Adult Endocrinology, Eunice Kennedy Shriver National Institute of
Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA. 4Biodata Mining and Discovery Section, Office of Science
and Technology, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892, USA. 5Ludwig
Boltzmann Institute of Osteology at the Hanusch Hospital of WGKK and AUVA Trauma Center Meidling, 1st Medical Department Hanusch Hospital, UKH
Meidling, Kundratstr. 37, Vienna 1120, Austria. 6Immunoregulation Section, Autoimmunity Branch, National Institute of Arthritis and Musculoskeletal and
Skin Diseases, National Institutes of Health, Bethesda, MD 20892, USA. 7Immunodeficiency and Inflammation Unit, Autoimmunity Branch, National Institute
of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda 20892, USA. 8Office of the Clinical Director, National Institute of
Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892, USA. 9Dermatology Branch, National Institute of
Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
10
Present address: Molecular Genetics Section,
Medical Genomics and Metabolic Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA.
These authors contributed equally: Heeseog Kang, Smita Jha, Zuoming Deng. These authors jointly supervised this work: Richard M. Siegel, Joan C. Marini,
Timothy Bhattacharyya. Correspondence and requests for materials should be addressed to T.B. (email: timothy.bhattacharyya@nih.gov)
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Melorheostosis (OMIM%155950) is a rare dysostosis
characterized by excess bone formation in the classic
“dripping candle wax”pattern on the surface of bone,
revealed radiographically (Fig. 1a, b and Supplementary Fig. 1)1.
The disease occurs in males and females equally, without familial
clustering, and often affects multiple contiguous bones uni-
laterally in an asymmetric distribution2. Symptoms of melor-
heostosis may begin in childhood; the diagnosis is usually
apparent by the age of 20 years3. The bone overgrowth lesions are
associated with pain, functional impairment, joint contracture,
and deformity. There is a wide spectrum of clinical appearance2
with skin lesions overlying the affected bone noted in up to 30%
of patients. Bone lesions do not metastasize, but conversion to
osteosarcoma has rarely been reported4. Increased uptake on
bone scintigraphy suggests that melorheostotic bone is
metabolically active (Fig. 1c–e). Patients are diagnosed by a
combination of clinical findings, radiographs and bone scan.
There is no definitive diagnostic test or specific treatment for
melorheostosis2.
At present, two patterns of occurrence of melorheostosis have
been differentiated by genetics. Patients with the autosomal
dominant “spotted bone disease”osteopoikilosis and the related
condition Buschke–Ollendorff syndrome (OMIM #166700) may
display features of melorheostosis, and have been shown to have
germline loss-of-function mutations in LEMD3, encoding the
inner nuclear membrane protein MAN15.LEMD3 mutations
have not been found in patients with the more common sporadic
melorheostosis without osteopoikilosis6; the genetic cause of their
condition is undefined7. Recently, a patient with melorheostosis
and familial osteopoikilosis was found to have a germline LEMD3
mutation and a somatic KRAS mutation in an overlying area of
scleroderma-like skin8. Whether the KRAS mutation was causa-
tive for the bone findings in this one patient remains unde-
termined. The lack of vertical genetic transmission and
asymmetric involvement suggests that sporadic melorheostosis
may be caused by somatic mutations in bone-forming cells9, but
bone tissue has not been previously investigated for somatic
mutations in bone forming cells.
We biopsied affected and unaffected bone of 15 patients with
melorheostosis and compared the tissue whole exome sequences
for each patient. We identified mutations in the MAP2K1 gene in
affected, but not unaffected, bone of eight patients. MAP2K1
encodes the protein MEK1, a kinase whose activity is modulated
by its negative regulatory domain. The MAP2K1 mutations
identified in melorheostosis patients cause substitutions in two
residues of the MEK1 negative regulatory domain. We present
genetic, functional and histological data supporting the
enhancement of MEK1 activity predicted by the location of the
mutations and the causative role of the MAP2K1 mutations in
melorheostosis. This study provides evidence of a bone disease
caused by mutations in MAP2K1.
Results
Identification of mosaicism for MAP2K1. Fifteen patients with
melorheostosis underwent biopsies of affected and unaffected
bone (Supplementary Table 1and 2). A consistent intraoperative
finding was extremely dense, rigid affected bone that often dulled
the osteotomes and drill bits. Genomic DNA was extracted from
both tissues and subjected to high-depth whole exome sequen-
cing. We identified 8365 sequence variants that were present in a
subject’s affected bone but not unaffected bone. After restricting
these variants to those that were found in more than 1% of
sequences (but less than germline frequency), rare in the general
population, and predicted to alter protein coding or mRNA
splicing, we were left with 284 variants (Fig. 2a and Table 1). Only
five genes contained putative somatic mutations in more than one
affected bone sample (MAP2K1,USH2A, CCDC13, SIRT5 and
RAB44). Among these five genes, it was striking that sequences
from five affected bone samples contained one of three tightly
clustered missense mutations in the coding region of MAP2K1,
c.167 A > C (p.Q56P), c.169 A > G (p.K57E), and c.171 G > T
(p.K57N), encoding substitutions in two adjacent amino acids of
the protein MEK1 (Fig. 2b and Supplementary Data 1). These
coding variants are not present in the exomes of 123,136 healthy
individuals searchable with the ExAC10. Both substituted residues
are located in a MEK1 α-helix that negatively regulates kinase
function (Fig. 2b and Supplementary Fig. 2)11. All three of these
mutations have been previously shown to lead to gain-of-function
through loss of the inhibitory role of the negative regulatory
domain and have been identified in malignancies, including
lung12, melanoma13 and hairy cell leukemia14 (Supplementary
Table 3). All other 281 variants were unique to the affected bone
of 1 of the 15 subjects (Supplementary Data 1). No germline or
somatic LEMD3,MAP2K2 or MAP2K3 variants were identified in
automated or manual re-examination of exome sequencing data
Fig. 1 Clinical findings in melorheostosis. aClinical appearance of Melo-10 with melorheostosis of right lower extremity. Note irregular thickening of the
right leg. The affected bone was found to harbor a MAP2K1 mutation. bRadiograph of the right tibia/fibula of Melo-10 with classic candle-wax appearance.
cMaximal intensity projection (MIP) 18F-NaF PET image of the lower extremities showing intensely increased 18F-NaF activity in the bones of the right leg,
primarily along the medial and distal femur, and along the tibia extending into the foot. d,eAxial CT and fused 18F-NaF PET/CT images at the level of
mid-calf demonstrating dense cortical bone formation in the anterior and posterior aspect of the right tibia (red arrows in dwith corresponding abnormally
increased 18F-NaF uptake (white arrows, in e)
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123
P loop
Negative
regulatory
ERK
binding
4 5 6 7 8 910 11
Catalytic core
ATP binding
Catalytic loop
Mg
activation
segment
K57E
K57N
Q56P
Proline rich Catalytic core
10,000
8000
6000
4000
2000
0
HEX channel (mutant) [RFU]HEX channel (mutant) [RFU]
10,000
8000
6000
4000
2000
FAM channel (wildtype) [RFU] FAM channel (wildtype) [RFU]
39.2%
6.95%0%
0%
Unaffected
Bone biopsies from 15 subjects with
melorheostosis
Affected bone Contralateral
unaffected bone
Exome sequencing >100X depth
BWA genome alignment (hg19)
↓
GATK/Picard:
duplicate removal, base quality score recalibration,
InDel realignment
↓
MuTect and Strelka: somatic
variant calling in affected bone
↓
Annovar: variant annotation
8365 sequence variants
Variant filtering
Called by both MuTect and Strelka (1073)
Population frequency <1% in EXAC and 1KGP
Alters protein coding sequence or splicing
Not in duplicate genomic region
Variants in same gene found in
multiple patients (frequency)
Mutations cluster in same domain
Shared mutations between patients
Mutation confirmed by ddPCR
Q56P (3)
K57E (1)
K57N (4)
MAP2K1 (8)
MAP2K1 (5)
CCDC13 (2)
USH2A (2)
SIRT5 (2)
RAB44 (2)
284 variants
Bone
Cultured
osteoblasts
Affected
1000 60002000 3000 4000 50000 1000 2000 3000 4000 5000 6000
a
c
b
Fig. 2 MAP2K1 somatic mutations in melorheostotic bone. aFlowchart of genetic analysis. bSchematic of the MAP2K1 exon structure encoding MEK1
protein domains. The three mutations from the eight patients are clustered in the negative regulatory domain. cQuantification of mutant allele abundance
by ddPCR in unaffected and affected bone biopsies (top row), as well as in cultured osteoblasts from the respective biopsies (bottom row), from patient
Melo-2 identified by WES to have a p.K57N mutation in affected bone. Each dot represents a droplet, with blue being mutant positive, green being wildtype
positive, and orange being positive for both. Color-matched numbers correspond to count of droplets per quadrant. The boxed number is the fractional
abundance of mutant allele in each sample
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from any patient. A KRAS p.Q61R mutation was found in both
the affected and unaffected bone of one MAP2K1 mutation-
negative patient with skin lesions consistent with RASopathy,
who likely has melorheostosis as part of a more complex early
embryonic mosaic RASopathy, which will be reported separately.
Thus 5 of 15 patients were identified to have MAP2K1 mutations
by whole exome sequencing.
The allele frequency of these somatic heterozygous
MAP2K1 mutations identified by automated somatic mutation
calling was relatively high (10–25%). We performed amplicon-
based targeted sequencing to validate the exome sequencing
findings and screen the remaining patients for MAP2K1
mutations at higher sensitivity. This approach revealed three
additional patients with MAP2K1 p.K57N mutations at lower
mutant allele frequencies (Table 1). Amplicon-seq polymerase
errors were less than 1% at the base positions in question
(Supplementary Tables 4,5and 6). Their mutations were missed
by WES due to low allele frequencies in patients Melo-16 and
Melo-6 (2.7% and 4.8%, respectively) or low coverage in patient
Melo-2 (58×). All eight MAP2K1 mutations were then confirmed
by droplet digital PCR (ddPCR) of DNA from affected bone
(Fig. 2c). The MAP2K1 mutant allele frequency in affected bone
by ddPCR ranged from 9 to 28% for p.Q56P, 3–34% for p.K57N,
and 18% for p.K57E and correlates well with amplicon and
WES data.
The ddPCR mutant allele frequency in osteoblasts cultured
from melorheostotic bone of seven subjects, varied from 0.05 to
47% and was similar in different samples from the same cell
culture, but often did not match the frequency found in bone
tissue, consistent with tissue mosaicism. For example, there was
a markedly higher frequency of the p.K57N mutation in
cultured osteoblasts than bone tissue from patient Melo-2
(40–47% in cell passages 1–5 vs. 6.9% in bone) and a lower
frequency of p.Q56P in Melo-4 (0.05–0.2% in cell passages 1–5
vs. 10% in bone tissue).
Mosaicism for their respective MAP2K1 mutations
was also detected in skin tissue overlying affected bone
of patients Melo-2, Melo-4, Melo-6 and Melo-18, but not
detected in skin from patient Melo-16, who has a lower
disease burden. To look for germline mutations, we tested
blood samples available from seven patients. MAP2K1 mutations
were not identified in blood samples including those with
mutation-positive skin tissue, further confirming mosaicism
(Table 1).
Melorheostosis histology reveals intense remodeling. To gain
insights into the effects of MAP2K1 mutations on bone structure,
remodeling and mineralization, we compared histomorphometry
of tissue sections from affected and unaffected bone in six
patients. Melorheostotic bone was characterized in the outer
regions by distinctive parallel layers of primary lamellar bone, an
organization that underlies the surgical hardness of the bone
(Fig. 3a). This newly formed compact tissue was intensely
remodeled into a highly porous osteonal-like bone at greater
depth from the surface (Fig. 3b). Compared to unaffected bone,
affected bone showed increased active bone-resorbing osteoclasts,
elevated eroded surfaces and a higher number of bone-forming
osteoblasts (Fig. 3c). There was an approximately six-fold increase
in the thickness of osteoid (unmineralized bone matrix) and a
greater than 50-fold increase in osteoid surface/bone surface in
the affected bone samples compared to their respective unaffected
counterparts (Fig. 3c, d).
Table 1 Table of individual patients showing the mutation location, cDNA and protein consequences, and variant allele
frequencies (VAF)
Subject AA Changes VAF WES
affected
bone
VAF WES
unaffected
bone
VAF amplicon
affected bone
VAF ddPCR
affected bone
VAF ddPCR
unaffected
bone
VAF
ddPCR
skin
VAF ddPCR
unaffected
skin
VAF
ddPCR
blood
melo4 NM_002755:
exon2:c.A167C:
p.Q56P
10.1%a0.00% N.D. 9.4% 0.0% 12.5% 0.0% 0.0%
melo9 NM_002755:
exon2:c.A167C:
p.Q56P
17.19%a0.00% N.D. 20.0% 0.0% N.D. N.D. 0.0%
melo19 NM_002755:
exon2:c.A167C:
p.Q56P
30.86%a0.99% 27.9% 27.8% 0.0% N.D. N.D. N.D.
melo10 NM_002755:
exon2:c.
A169G:
p.K57E
12.07%a0.00% 16.7% 18.3% 0.0% N.D. N.D. 0.0%
melo2 NM_002755:
exon2:c.G171T:
p.K57N
6.09% 0.00% 7.2% 7.0% 0.0% 4.1% 0.0% 0.0%
melo6 NM_002755:
exon2:c.G171T:
p.K57N
3.12% 0.00% 4.8% 4.4% 0.0% 16.2% 0.0% 0.0%
melo16 NM_002755:
exon2:c.G171T:
p.K57N
1.04% 0.00% 2.7% 2.8% 0.0% 0.0% 0.0% 0.0%
melo18 NM_002755:
exon2:c.G171T:
p.K57N
25.21%a0.0% 34.4% 33.6% 0.0% 6.5% N.D. 0.0%
aCases where MAP2K1 mutations were identified through automated filtering of WES for somatic mutations. Others were found by manual inspection. Mutations were not identified in normal control
bone. Melo-19 had one mutant read in unaffected bone that was not seen in ddPCR, designating it as a sequencing error. The VAF results from ddPCR, amplicon and whole exome sequencing are highly
correlated (R=0.96 or greater for all comparisons, p< 0.003). MAP2K1 mutations are present in overlying skin in four of five patients tested while contralateral skin is negative. N.D. not done
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MEK1 signaling increased in melorheostosis. To determine how
the MAP2K1 mutations identified in our cohort affected the
MEK1 target kinases ERK1 and ERK2, we examined MEK1
activity by immunohistochemical analysis of phosphorylated
ERK1/2 (p-ERK1/2) in sections of affected bone tissue from two
patients with MAP2K1 mutations. Osteocytes and osteoblasts in
affected bone displayed stronger p-ERK1/2 signals than control
bone, but no p-ERK1/2 signal was detected in osteoclasts
(Fig. 4a). Consistent with somatic mosaicism, the p-ERK1/2
positive cells occurred in patches in affected bone tissue.
Furthermore, osteoblasts cultured from affected bone and having a
high frequency of mutant MAP2K1 cells (approximately 80%)
exhibited a bimodal ERK1/2 activation after serum-stimulation when
examined by flow cytometry for intracellular p-ERK1/2. Bimodal
ERK1/2 activation was not observed in cells from unaffected bone.
Both peaks of ERK1/2 activation in osteoblasts from affected bone,
Unaffected Affected Affected
MAP2K1 p.K57N (Melo-18)MAP2K1 p.K57N (Melo-2)
MAP2K1 p.Q56P (Melo-9)
Osteoid
thickness
Unaff Aff Unaff
Unaff
Unaffacted Affacted
Unaff
****
Aff
Aff Aff
Unaff Aff
20
15
10
5
0
4
3
2
1
0
10
8
6
4
2
0
80
60
40
20
0
Osteoid
surface
Eroded
surface
Osteoclast
surface
% Bone surface
% Bone surface
% Bone surface
% Bone surface
(μm)
Osteoblast
surface
*********
12
10
8
6
4
2
0
*
*
**
100 μm 100 μm
*
100 μm 100 μm
1000 μm
ab
c
d
Fig. 3 Intense bone remodeling and abnormal mineralization in melorheostosis. aLight microscopy images of cortical bone from patient Melo-2 showing
the orientation of the collagenous matrix. Goldner’s trichrome staining is viewed under polarized light. Affected bone tissue shows in the periosteal regions
appositional growth through deposition of layers of compact primary lamellar bone oriented parallel to the periosteal surface (open arrows). Subsequently
these regions become highly remodeled. The solid arrows point towards large canals traversing the parallel layers of primary bone. Note the thick osteoid
seams (red, unmineralized matrix) in affected bone tissue, whereas no osteoid is seen in unaffected bone (asterisks). bA backscattered electron image
(BEI) of affected bone tissue from patient Melo-18. Gray represents the mineralized bone tissue, black is non-mineralized tissue. Note the extreme porosity
of the bone tissue as a consequence of an intense bone remodeling activity (solid arrows). The open arrow is the same area labeled by the open arrow in a.
cBone histomorphometry. Comparison of indices of bone formation and bone resorption of affected vs. unaffected bone tissue from six patients with
MAP2K1 mutations. All indices of bone formation and bone resorption are significantly increased in affected bone tissue compared to unaffected bone of
the same patient. *p< 0.05; **p< 0.01; ***p< 0.001; ****p< 0.0001 vs. values of unaffected bone tissue (paired t-test). Unaff: unaffected bone tissue, Aff:
affected bone tissue. dOverviews of unaffected and affected bone tissue obtained from patient Melo-9. Affected bone can be easily recognized by its
porosity and by the substantial portions of unmineralized matrix (solid arrows), while osteoid formation is not seen around osteonal canals in the
unaffected bone sample (asterisks). Goldner’s trichrome staining followed by light microscopy in the bright-field mode. Green indicates mineralized bone
matrix and red indicates unmineralized matrix
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likely representing wild-type and mutant cells, were reduced by the
MEK-specific inhibitor U0126. Baseline p-ERK1/2 was also higher in
a proportion of affected cells than in unaffected cells (Fig. 4b). These
findingswouldbeexpectedfromamixedcellpopulationand
confirm MAP2K1 mosaicism at the cellular level.
We also investigated the MEK1-ERK1/2 pathway in cultured
osteoblasts harboring MAP2K1 mutations by western blot analysis
in seven patients (Fig. 4c). Osteoblasts from the same patient and
passage as those analyzed by flow cytometry showed increased p-
ERK1/2 compared to cells from unaffected bone, indicating MEK1
gain-of-function (Supplementary Fig. 4). FBS-induced ERK1/2
activation could more readily be seen in unaffected osteoblasts by
western blotting, likely due to the increased sensitivity in
comparison to flow cytometry. Pre-treatment of cells with
U0126 significantly diminished serum-stimulated p-ERK1/2, sup-
porting enhanced MEK1 activity as mediating the increased ERK1/
2 activation. The level of ERK1/2 activation by MEK1 generally
correlated with mutant allele frequency. Activation was clearly seen
in Melo-2 and Melo-18, with the highest percentage of mutant cells.
In cells cultured from affected bone of Melo-10 with lower mutant
IgG control
p-ERK1/2
Colon cancer Normal bone Melorheostosis bone
MAP2K1 p.K57N (Melo-2)
p-ERK1/2
MAP2K1 p.K57N (Melo-2)
Melo-2
MFI
low peak
p-ERK1/2
MFI
high peak
p-ERK1/2
261
2301 0
345
Baseline
Baseline
Baseline
DMSO
U0126
U0126
Starv.
Starv.
Baseline
Affected
Affected
Affected
Unaffected
Unaffected
UnaffectedMelo-2
p-ERK1/2
Baseline Baseline
1.00 1.06 3.52 1.50 1.02 1.09 5.23 3.18
U0126U0126 DMSODMSO No. stim.No. stim.
FBS stim. FBS stim.
FBS stim.
DMSO
DMSO
DMSOU0126 U0126
409
2057 2725
447 713
5709 3784
637
FBS stimulation
Baseline DMSO U0126
LHLHLH
IgG control
Unaffected
Affected
–42/44 kDa
–42/44 kDa
–17 kDa
p-ERK1/2
Tot ERK1/2
COX IV
a
b
c
FBS stim.
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allele frequency of 10%, the activation of ERK1/2 by MEK1 was
equivocal (Supplementary Fig. 4).
MEK1 activation increases cell proliferation.MAP2K1 muta-
tions did not affect MAP2K1 transcripts (Fig. 5a) or MEK1
protein levels (Fig. 5b), supporting a gain-of-function mechanism
intrinsic to the protein itself. ERK1/2 activation promotes G1 to
S-phase cell cycle progression and accelerates cell proliferation.
The enhanced MEK1-ERK1/2 activation in affected osteoblasts
increased cell proliferation in vitro (Fig. 5c). For example, the
doubling time of osteoblasts from affected bone of patient Melo-2
(mutant allele frequency 45%) was 18 h, while osteoblasts from
this patient’s unaffected bone had a doubling time of 54 h.
Osteoblasts from affected bone did not lose contact inhibition.
Furthermore, affected osteoblasts showed increased levels of
cyclin D3 expression compared to unaffected cells, consistent
with accelerated G1-S cell cycle transition (Fig. 5d).
MEK1 activation decreases osteoblast differentiation in vitro.
To further investigate the functional consequences of the
MAP2K1 mutations causing melorheostosis, osteoblast differ-
entiation and mineralization were assessed in vitro. The presence
of the MAP2K1 mutation strikingly inhibited BMP2-stimulated
mineralization in affected compared to unaffected osteoblasts
(Fig. 5e). Expression of marker genes for osteoblast differentiation
was also reduced in cells with the MAP2K1 mutation during
BMP2-stimulated differentiation (Fig. 5f). Reduced expression of
RUNX2 (runt-related transcription factor 2), ALPL(alkaline
phosphatase) and COL1A1 (the α1 chain of type I collagen)
indicates the prevalence of immature bone forming cells. Osteo-
blasts early in differentiation secrete proteins to stimulate osteo-
clastogenesis15. In fact, we observed a markedly increased ratio of
RANKL/OPG transcripts in osteoblasts from affected bone, which
would generate a strongly positive context for osteoclastogenic
stimulus (Fig. 5f)16.
Together, these data support key histological findings in
affected bone. Inhibition of mineralization by overactive MEK1
accounts for the massive accumulation of unmineralized osteoid
in affected bone tissue (Fig. 3d). Furthermore, the elevated
RANKL/OPG ratio stimulates the increased osteoclast number
and intense remodeling that occurs in melorheostotic bone
(Fig. 3c).
Discussion
Genetic osteosclerotic disorders are a rare but interesting subset
of bone diseases which can provide important insights into
bone biology. Identification of a germline mutation causing
sclerosteosis led to the development of a potential treatment for
osteoporosis17–19. Investigations into fibrodysplasia ossificans
progressiva elucidated the role of inflammation in heterotopic
ossification and exposed targets for treatment20. Here, our
investigation has revealed a role for MEK1 in a disorder char-
acterized by excessive bone formation.
We identified mutations in MAP2K1 in the affected, but not
unaffected, bone of 8 of 15 patients diagnosed clinically and
radiologically with melorheostosis. The mutations cluster at two
residues in the MEK1 negative regulatory domain, p.Q56 and p.
K57, where identical substitutions have been identified in multi-
ple malignancies, but not primary bone tumors. The occurrence
of the mutations in a MEK1 “hot spot,”and the evidence of
enhanced MEK1-ERK1/2 signaling in melorheostotic bone and
osteoblasts, strongly support MAP2K1 somatic mutations as
causing about half of cases of sporadic melorheostosis. In our
cohort, we found no occurrences of somatic or germline muta-
tions in LEMD3, which have previously been reported in patients
with familial osteopoikilosis and features of melorheostosis6,8.
Mosaicism for cells with increased ERK1/2 activation was
confirmed directly in melorheostotic bone by immunohis-
tochemistry. Furthermore, two populations of cells with distinct
levels of ERK1/2 activation were detected by flow cytometry.
Increased ERK1/2 activation was also detected in cultured
osteoblasts from affected, but not unaffected bone. In individual
patients, the proportion of cultured mutant osteoblasts often
differed from the proportion of mutant allele in genomic DNA
extracted from bone chips from the same lesion, consistent with
clustering of affected cells in mosaic conditions.
The three MAP2K1 mutations we identified in melorheostosis
have been shown to increase MEK1 activity in transfection stu-
dies21, and likely act by destabilizing the α-helix comprising the
MEK1 negative regulatory region, which normally keeps the ATP-
bindingsiteinaninactiveconformation
11.MEK1isadownstream
activator in the RAS pathway. Mutations in RAS pathway com-
ponents are associated with developmental defects falling under the
umbrella designation RASopathies22 and involve variable cardiac,
facial and neurodevelopmental defects. Interestingly, germline
mutations identified in leukocytes from patients with the RASo-
pathy cardio-facio-cutaneous syndrome23,24 include two MAP2K1
mutations (p.G128V/p.Y130C) in the catalytic core, and putatively
activating mutations in the negative regulatory region of MAP2K1
(p.F53S) or MAP2K2 (p.F57V, analogous to MAP2K1 p.F53). Bone
overgrowth is not reported in these RAS-MAPK syndromes,
implying that the somatic mutations we report in osteoblasts pro-
duce a different phenotype than the same mutation in the germline.
Mosaicism for mutations at the same residues (p.Q56P and
p.K57E) we identified in the MEK1 negative regulatory domain in
Fig. 4 Activation of the MEK1-ERK1/2 pathway by MAP2K1 mutations. aImmunohistochemical analysis of ERK1/2 activation in bone tissues from a
melorheostosis patient. Left column shows sections of colon cancer stained with p-ERK1/2-specific antibodies. Marked brown staining is visible. Middle
column shows a section of normal bone stained in a similar fashion. Right column shows representative section from melorheostotic bone (Melo-18,
MAP2K1 p.K57N, VAF 46%) stained with p-ERK1/2-specific antibodies. Osteocytes stain brown surrounded by woven bone. Cells positive for p-ERK1/2 are
also seen in the periosteum. Inset shows high-power view of positively staining cells. A multinucleated osteoclast which does not stain for p-ERK1/2 is
marked by the solid arrow. Cells negative for p-ERK1/2 are noted by the open arrows. See Supplementary Figure 3for staining of SW48 colon cancer cells,
which harbor the MAP2K1 p.K56P mutation. bp-ERK1/2-specificflow cytometry analysis. Affected and unaffected osteoblasts from Melo-2 (MAP2K1 p.
K57N, VAF 45%) were stimulated with serum with or without MEK inhibitor U0126. Two peaks in the histogram indicates cell subpopulations of distinct
level of p-ERK1/2 upon serum-stimulation in osteoblasts from affected bone (red), cells from unaffected bone (blue) only showed a single peak. U0126
reduced p-ERK1/2 in cells from affected bone. Cells stained with matching rabbit IgG isotype control are also shown (gray). The geometrical mean channel
fluorescence is shown below for the high and low peaks of p-ERK1/2 marked on the histograms. cWestern blot analysis of osteoblasts from affected and
unaffected bone of patient Melo-2 (MAP2K1 p.K57N, VAF 45%) shows increased ERK1/2 activation (p-ERK1/2) by MEK1 mutation in affected osteoblasts
(lane 7 of p-ERK1/2 blot), as compared to unaffected osteoblasts (lane 3), by serum-stimulation. Inhibition of MEK1 with U0126 significantly diminished p-
ERK1/2 in both affected and unaffected, but the level of p-ERK1/2 was still higher in affected osteoblasts compared to unaffected (lanes 4 and 8).
Quantification data of band intensities are shown in a table. COX IV was used as control for equal amount protein loading
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melorheostosis patients has recently been reported in endothelial,
but not skin or blood, cells of patients with extracranial arter-
iovenous malformations (AVM)25. Similar to melorheostotic bone
lesions, AVM lesions caused by MAP2K1 mutations expand but do
not metastasize.
While the MAPK pathway is known to be involved in skele-
togenesis and MEK1 is essential in embryogenesis, our study
documents the importance of the MEK1-ERK1/2 pathway in
human bone cell biology. While somatic mutations in MAP2K1
have also been reported in Langerhans cell histiocytosis and
Relative expression
1.5
1.0
0.5
0.0
Unaff Aff Unaff Aff Unaff Aff Unaff Aff Unaff Aff Unaff Aff Unaff Aff
Melo-18
Melo-06Melo-02
0 8 16 24 32 40 48 56 64 72 80 88 96 104 112 120
Time (h)
100
80
60
40
20
0
% confluence
Melo-10
K57E K57N
Q56P
MAP2K1
MAP2K1 p.K57N (Melo-18)
RANKL/OPGALPLRUNX2 COL1A1
Unaffected
Affected
Melo-04 Melo-09 Melo-19 Melo-10 Melo-02 Melo-06 Melo-18
Unaff
8
6
4
2
0
1.5
1.0
0.5
0.0
4
3
2
1
0
Relative expression
Relative expression
Relative expression
Relative expression
Aff
Unaff
Aff
Unaff
Aff
Unaff
Aff
50
40
30
20
10
0
–45 kDa
–17 kDa
MEK1
COXIV
Aff
Unaff
Aff
Unaff
Aff
Unaff
Aff
Unaff
Aff
Unaff
Aff
Unaff
Aff
Unaff
Melo-19Melo-09Melo-04
Affected
Unaffected D1 D3 D6
p-ERK1/2
Cyclin D3
GAPDH
–42/44 kDa
–31 kDa
–37 kDa
Aff
Unaff
Aff
Unaff
Aff
Unaff
MAP2K1 p.K57N (Melo-18)
K57NK57E
MEK1
Q56P
a
c
b
d
e
fg
Fig. 5 Increased cell proliferation and delayed osteoblast differentiation and mineralization by MAP2K1 mutations. aMAP2K1 variants found in seven
melorheostosis patients do not affect the levels of MAP2K1 transcripts. There was no statistically significant difference in MAP2K1 transcript levels between
affected and unaffected osteoblasts (*p<0.05, paired t-test). Real-time qPCR was carried out in triplicate for each patient sample. bWestern blot analysis
displays comparable levels of MEK1 protein in affected and unaffected osteoblasts. cCell proliferation assay using live-cell imaging. Affected and
unaffected osteoblasts from melorheostosis patient, Melo-2 (MAP2K1 p.K57N, VAF 45%) were plated at various densities (1000~5000 cells/well) (n=
30). Percent cell confluence is shown at 2-h intervals with symbols indicating mean of replicates (error bars: SEM). Doubling time calculated from the
linear phase growth yielded a doubling time of 18 h for affected osteoblasts compared to 54 h for unaffected. Results are representative of two independent
experiments. (p< 0.0001). dWestern blot analysis shows that affected osteoblasts (lanes 2, 4, and 6) expressed higher level of cyclin D3 compared to
unaffected (lanes 1, 3, and 5), correlating with the increased p-ERK1/2 level in affected cells shown in Fig. 4c. Note that levels of cyclin D3 and p-ERK1/2
decreased on day 3 (D3) and day 6 (D6) compared to day 1 (D1), because culture media was not refreshed after day 1, similar to conditions used during the
live-cell imaging shown in Fig. 5c. eAlizarin Red S staining of mineralization in osteoblast cultures from patient Melo-18 (MAP2K1 p.K57N, VAF 46%). After
7 weeks of BMP2-stimulated mineralization in vitro, significantly inhibited mineralization was observed in affected cells compared to unaffected. fReal-
time qPCR analysis of expression of osteogenic marker genes, RUNX2,COL1A1, and ALPL in osteoblasts from melorheostosis patient Melo-2 (MAP2K1 p.
K57N, VAF 45%) after two weeks of osteogenic stimulation. Expression level of RUNX2, COL1A1, and ALPL was significantly lower in affected osteoblasts
compared to unaffected. gThe RANKL/OPG transcript ratio, an index of osteoclastogenic stimulus, was assessed by real-time qPCR with Melo-2 patient
osteoblasts as in Fig. 5f. Significantly higher ratio of RANKL/OPG in affected osteoblasts indicates increased osteoclastogenesis compared to unaffected
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Erdheim–Chester disease (ECD)26, the mutated cells in ECD are
derived from monocytes rather than osteoblasts. Bone lesions in
ECD are typically bilateral and symmetrical and occur in trabe-
cular bone of the epiphysis27. In contrast, activation of ERK1/2
MAP kinases in melorheostosis occurs in osteoblast lineage and
the lesions are primarily cortical.
Transgenic murine models, in which the entire MEK1 negative
regulatory domain was deleted, share the elevated ERK1/2 sig-
naling seen in melorheostosis28. However, the mice displayed
accelerated bone remodeling rather than bone overgrowth. The
mouse osteoblasts also showed increased differentiation and
mineralization, but unchanged proliferation in vitro.
In melorheostosis, activation of the MEK1-ERK1/2 pathway in
mature bone results in an increase in osteoblast surface and
increased production of unmineralized bone matrix (osteoid).
This is consistent with the faster proliferation of osteoblasts
cultured from melorheostotic bone and with the less differ-
entiated osteoblast phenotype revealed in vitro in BMP2-
stimulated cells, in which transcripts of osteogenic genes
RUNX2,COL1A1, and ALPL were decreased. Histologically, we
observed a significant increase in osteoid formation and osteoclast
numbers in affected bone from the MAP2K1 variant cohort.
Interestingly, these features were previously noted in a single case
report29 with unknown genetics. The increased osteoid seen
in vivo is consistent with the decreased mineralization deposited
by affected osteoblasts in vitro with BMP2 stimulation. Increased
osteoclast numbers and bone turnover in affected bone is con-
sistent with the increased RANKL secretion by cultured cells,
which would stimulate osteoclast development and intense bone
remodeling. Furthermore, it is known that chronic activation of
ERK1/2 strongly inhibits BMP2-mediated mineralization, as
previously shown in a murine model for neurofibromatosis30, and
provides an underlying mechanism for the increased unminer-
alized osteoid found in melorheostotic bone. The phenotypic
distinction between murine models and melorheostosis patients
may be ascribed to overexpression of the MEK1 transgene in all
murine osteoblasts as opposed to the mosaic mutations in
melorheostosis, or greater disruption of function by deletion of
the full negative regulatory domain28,31. Furthermore, studies in
fruit flies and zebrafish indicate that activating MAP2K1 muta-
tions affect ERK1/2 activation differently depending on pathways
intrinsic to the target tissue and developmental state22.
Identification of a somatic mosaic mutation in osteoblasts
accounts for the unique clinical features of melorheostosis. The
“dripping candle wax”pattern observed on radiographs is due to
marked appositional bone growth followed by aggressive remo-
deling. The radiographic pattern of multiple contiguous bones
affected while others are spared may be due to longitudinal
expansion of mutant osteoblasts precursors along the axis of
development (Supplementary Fig. 5)32. The marked variability in
the pattern of bone lesions between patients is likely related to
mutations arising during different points during development,
with earlier mutations resulting in an increased burden of disease.
Further studies will delineate the roles of specificMAP2K1
mutations in different tissues at various developmental stages.
Melorheostosis can now be viewed as a genetically hetero-
geneous group of dysostoses. The melorheostosis seen in patients
who have “spotted bone”osteopoikilosis is associated with a
mutation in germline LEMD36. Whyte et al. have shown that
additional somatic mutations in KRAS (and possibly other genes)
may play a role in the development of “dripping candle wax”
bone lesions on radiographs in some patients in an osteopoiki-
losis kindred8. Patients who have sporadic melorheostosis are a
distinct group and do not have germline6,33, or as we now show,
somatic mutations in LEMD3. It remains to be seen if the
radiographic similarities in melorheostosis caused by LEMD3
mutations and MAP2K1 mutations are due to involvement of a
common pathway with activation of SMADs, or are simply a
similar clinical outcome.
Our finding that three of four patients demonstrated mutations
in the overlying skin raises the possibility that testing of skin for
MAP2K1 mutations may be a diagnostic test for melorheostosis.
However, if the skin sample is negative, bone biopsy may still be
necessary to detect the mutation, especially in patients with a low
mutant allele frequency. Although the causative gene is an
oncogene, the clinical data from many years is reassuring to
patients that malignant degeneration is rare2,4.
Development of a diagnostic test is especially valuable because
identification of somatic MAP2K1 mutations in melorheostosis
raises the possibility of inhibiting MEK1 to treat melorheostotic
bone lesions. Several MEK1 inhibitors have already been devel-
oped for malignancies with MAP2K1 mutations identical to those
we identified in melorheostosis34. Because melorheostosis is one
of few osteosclerotic bone-forming diseases, study of the MEK1-
ERK1/2 pathway may lead to insights relevant to diseases where
bone formation is impaired, such as delayed fracture healing and
osteoporosis.
Methods
Patient cohort and bone sampling. We recruited 20 unrelated adults with a
diagnosis of melorheostosis to a NIAMS Institutional Review Board-approved
protocol (NCT02504879). Diagnoses were confirmed by radiographs and increased
uptake on 18F-NaF bone scan (Fig. 1). Patients were excluded if pregnant or
lactating, if under the age of 18, if actively infected or if unable to provide informed
consent. Study subjects were not compensated for their participation. Fifteen
patients consented to bone biopsies of affected and contralateral unaffected bone
(see Supplementary Table 1for clinical characteristics). Patients consented to
publication of photographs.
Patients underwent open surgical biopsy of the affected bone under fluoroscopic
guidance. The site of the biopsy of affected bone was chosen to minimize risk. Thus
the most superficial area of melorheostotic bone was chosen. Under general
anesthesia, the limb was exsanguinated and a tourniquet elevated (in all but one
patient with proximal femur lesion). The bone was exposed and any thickened
periosteum divided. Fluoroscopy was used to confirm the site of affected bone.
Four drill holes were used to create a rectangle, and an osteotome used to connect
the drill holes. The rectangular piece of cortical bone was then removed and
sectioned in the operating room. The sections were placed immediately in cell
culture media (for DNA extraction and osteoblast culture) or 70% ethanol for
histology. Hemostasis was obtained with electrocautery and the wound closed in
layers. A contralateral control sample of bone with no radiographic evidence of
melorheostosis was also obtained.
For some patients, such as Melo-2 and Melo-18, abundant melorheostotic bone
was available for evaluation. For other patients, only a small amount of affected
bone could be safely sampled; priority was given to bone for DNA extraction
followed by osteoblast culture over other studies. In all patients, the smallest
informative sample of normal bone was taken to minimize morbidity. Thus,
normal bone was often a limiting reagent. See Supplementary Table 2for details on
which patient samples underwent particular experiments.
Additional tissue sampling. In our initial study design, we were concerned that
surgical biopsy of melorheostosis (which had never been done before) could lead to
worsening of the disease, infection, or other complication. Therefore, only the
smallest amount of usable bone was collected. After a shared mutation was iden-
tified and the absence of surgical complications assured, four patients returning for
a follow-up visit underwent 6 mm punch biopsy of skin overlying an area of
affected bone, and a control biopsy of unaffected skin from the contralateral side.
Seven of the eight patients with MAP2K1 mutations agreed to have blood tested for
mutations. DNA from peripheral blood leukocytes was tested by ddPCR for pre-
sence of the mutation.
Genomic DNA extraction and mutation detection in bone tissue. Soft tissues
and cartilage were manually dissected from affected and unaffected bone of each
patient. Following repeated washes in phosphate-buffered saline (PBS) containing
no Ca2+or Mg2+to remove marrow, mineralized tissues were minced using a
rongeur and spring scissors in a sterile conical bottom glass tube (Roboz, Gai-
thersburg, MD).
Genomic DNA was isolated from minced bone chips following the
manufacturer’s instructions (Gentra Puregene, Qiagen). In brief, minced bone
samples were resuspended in cell lysis solution and digested with 0.2 mg/ml
proteinase K at 55 °C for 12 h. Digested proteins were precipitated and removed by
centrifugation at 14,000×gfor 3 min. The cleared supernatant was transferred to a
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new tube and genomic DNA was precipitated with isopropanol and collected by
centrifugation at 14,000×gfor 5 min. The DNA pellet was washed once with 70%
ethanol and air-dried at room temperature for 5 min. DNA was resuspended in
DNA hydration buffer (10 mM Tris, 1 mM EDTA, pH 8.0) and its concentration
was measured using a NanoDrop spectrophotometer (ThermoFischer Scientific).
Genomic DNA samples underwent deep Whole Exome Sequencing (Otogenetics),
with 107–270× coverage for each sample. Most exome targets (82%) had at least
50× coverage in each sample. Agilent SureSelect Human 51 Mbp All Exon kit was
used for exome capture. Paired-end sequencing was performed on Illumina HiSeq
sequencers. Sequencing reads were returned in FASTQ format and were aligned to
Human Reference Genome Build 37 using Burrows–Wheeler alignment Tool. The
standard PICARD-GATK pipeline was used to remove duplicate reads, refine
alignment around indels, and recalibrate base quality scores. The resulting BAM
files (one per sample) served as inputs to somatic mutation callers (Fig. 2a).
Two somatic mutation callers (muTect v1.1.7 and Strelka v1.0.1435) were used,
with default parameters for WES data. Somatic variants called by either method
were annotated with functional impact and population frequency using
ANNOVAR. We filtered somatic variants for those that were not present in the
unaffected bone, caused protein or ncRNA sequence changes or splicing changes,
had less than 1% frequency in ExAC and 1000 Genomes Project databases, were
not in a duplicated genomic region, and were called by both muTect and Strelka.
Using this method, 284 putative somatic variants were identified in 15 affected
bone samples (Supplementary Data 1and Supplementary Fig. 2).
Exome sequencing was complemented by high-coverage amplicon sequencing
with an average sequencing coverage of more than 10,000 × per sample. A targeted
NGS amplicon library prep kit from Swift Biosciences (56 G Oncology) was used to
screen patients for MAP2K1 somatic mutations. The panel targets cancer-
associated mutations in 56 genes including 5 mutation hotspot regions in MAP2K1
(exons 2, 3, 6, 7, 11), as well as the RAS codon 12, 13 and 61 hotspot mutations.
The amplicon libraries were sequenced on Illumina MiSeq and variants were called
with LoFreq 2.1.2.
To validate and quantitate the allele frequency of somatic mutations in other
tissues and cells cultured ex vivo, we utilized ddPCR (Fig. 2c). The approach
determines the abundance of each allele using allele-specific hydrolysis probes
(Supplementary Table 7) complementary to mutant allele (FAM-labeled) and wild-
type allele (HEX-labeled) and is based on partitioning of each sample into >10,000
nanoliter-sized droplets, in which PCR is carried out. gBlock oligos of 200 bp
bearing the mutation of interest were designed by Integrated DNA Technologies,
Inc. (Coralville, IA) and used as mutation-positive controls. Reactions were
performed on the QX200 Droplet Digital PCR System (Bio-Rad), using standard
PCR cycling conditions based on manufacturer’s instructions (10-min enzyme
activation at 95 °C, followed by 40 cycles of 30-s 94 °C denaturation and 1-min 55 °
C annealing/extension, finishing with a 10-min enzyme deactivation at 98 °C).
QuantaSoft software was used to quantitate the concentration of each allele
as the number of copies per microliter, using a Poisson distribution model to
account for the number of target copies per individual droplet. Fractional
abundance of mutant allele was calculated as the concentration (copies per
microliter) obtained in FAM channel divided by sum of concentrations of FAM
and HEX channels.
Bone histology and histomorphometry. Bone samples from a subset of 6 patients
with MAP2K1 mutations were embedded in polymethylmethacrylate and
prepared for histomorphometric analyses using standard procedures36. For
histological examinations, thin sections (4 µm) were cut from the tissue blocks
with a hard tissue microtome (Leica SM2500, Leica Microsystems Nußloch
GmbH), deplasticized with 2-methoxyethyl-acetate and stained with
modified Goldner’s Trichrome36 (Fig. 3d). A light microscope equipped with a
video camera (Zeiss Axiophot, Zeiss AxioCam, Oberkochen, Germany) was used to
obtain digital images of the sections that were analyzed using NIH ImageJ software
(version 1.63)37. Bone histomorphometric analyses were performed on 4 randomly
chosen areas throughout each bone section (Fig. 3c). Because bone samples
from the various skeletal sites contained predominantly compact/osteonal bone,
only indices of bone formation and resorption were evaluated: osteoid thickness
(O.Th), osteoid surface per bone surface (OS/BS), osteoblast surface per bone
surface (Ob.S/BS), osteoclast surface per bone surface (Oc.S/BS) and eroded
surface per bone surface (ES/BS). Bone lamellar organization was observed
under polarized light.
Subsequently, the residual blocks were prepared for backscattered electron
imaging by grinding and polishing to obtain plane parallel surfaces, then carbon
coated (Fig. 3a, b). The entire cross-sectioned bone sample area was imaged with a
spatial resolution of 1.8 µm per pixel using a field emission scanning electron
microscope (FESEM) (Zeiss Supra 40, Oberkochen, Germany) equipped with a
four-quadrant semiconductor backscatter electron detector. The FESEM was
operated with electron energy of 20 keV. The gray levels reflecting the mineral/
calcium content were calibrated by the material contrast of pure carbon and
aluminum.
Statistical evaluation was performed with GraphPad Prism 6.0 (GraphPad
Software, Inc., La Jolla, CA, USA). Comparison of histomorphometric indices were
based on paired t-test comparing affected vs. unaffected tissue in each patient.
Statistical significance was considered as p< 0.05.
Cell culture. Primary osteoblasts were grown from freshly minced bone chips
according to the method of Robey and Termine38. Bone chips (~ 1 mm) were
incubated in alpha-minimal essential medium (α-MEM, Life Technologies) con-
taining type II collagenase (200 U/ml, Worthington Biochemical Corp., Lakewood,
NJ) for 2 h at 37 °C with gentle rocking. Bone chips were allowed to settle briefly,
then collagenase solution was removed and washed twice with PBS. Bone chips
were placed in T75 tissue culture flasks with α-MEM supplemented with 10% fetal
bovine serum (Gemini Bio-Products, West Sacramento, CA) and antibiotics
(penicillin (100 U/ml) and streptomy cin (100 µg/ml), Thermo Fisher Scientific
Inc.). Bone chips were incubated for 2–3 weeks at 37 °C/8% CO
2
, refreshing culture
media every 3 days. Osteoblasts of passages 1–4 were used in this study.
Genomic DNA was extracted from cultured osteoblasts using the Gentra
Puregene kit (Qiagen), as described above. The proportion of mutant MAP2K1
allele in DNA from multiple cell passages and revivals was determined by ddPCR
as above.
Cell proliferation assay by live-cell imaging. Osteoblasts were plated at the
indicated densities in 96-well tissue culture dish and imaged at 2 h intervals for 120
h using the IncuCyte ZOOM Kinetic Imaging System (Essen Bioscience) at 10×
magnification (Fig. 5c). During the live-cell imaging, cells were incubated in 37 °C/
8% CO
2
. Percent confluence was calculated from the percentage of the well area
occupied by cells. Doubling time was calculated using GraphPad Prism 6.0 using
non-linear regression. To analyze comparable populations of cells between groups,
only wells with confluency at the first time-point of image acquisition within one
half standard deviation of the mean were included in the analysis. Statistical
comparison was done by two-way ANOVA.
Gene expression in osteoblasts by real-time quantitative PCR. Total RNA was
extracted from osteoblasts using the RNeasy mini kit (Qiagen) following the
manufacturer’s instruction. RNA concentration was measured by the NanoDrop
spectrophotometer. Synthesis of cDNA was performed using 1 µg of total RNA and
the High‐Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific),
following the manufacturer’s instructions. Comparative real-time PCR was per-
formed in triplicate with an Applied Biosystems Prism 7500 Fast Sequence
Detection System using TaqMan universal PCR master mix, according to the
manufacture’s protocol (Applied Biosystems Inc., Foster City, CA) (Fig. 5a, f, g).
The TaqMan probes and primers were purchased from Applied Biosystems:
Human RUNX2 (Hs01047973_m1), COL1A1 (Hs00164004_m1), ALPL
(Hs01029144_m1), RANKL (TNFSF11, Hs00243522_m1), OPG (TNFRSF11B,
Hs00900358_m1). Human GAPDH (Hs02786624_g1) and TBP (Hs00427620_m1)
were used as endogenous controls for normalization. Levels of MAP2K1,RUNX2,
ALPL and COL1A1, RANKL (TNFSF11), and OPG (TNFRSF11B) transcripts
were determined using the 7500 Fast System SDS software version 1.3.1 (Applied
Biosystems). Relative expression was calculated using the comparative ΔΔCt
method.
Flow cytometric analysis for intracellular p-ERK1/2. Osteoblasts cultured from
affected and unaffected bone of melorheostosis patients were serum-starved for 1 h
and stimulated with 20% FBS with or without MEK inhibitor U0126 (10 μM) for
30 min. Osteoblasts were then harvested for intracellular staining with antibodies
for p-ERK1/2. After fixation with 4% paraformaldehyde and permeabilization with
ice cold methanol, cells were washed in PBS containing 1% BSA. Cells were stained
for 60 min in the dark at room temperature with the following antibodies (Cell
Signaling Technology, Cambridge, MA): Phospho-p44/42 MAPK (ERK1/2)
(Thr202/Tyr204), (197G2), (clone E10) rabbit monoclonal antibody (Alexa Fluor
647®Conjugate) (#13148) (1:100 dilution) or rabbit IgG isotype control (Alexa
Fluor®647 Conjugate) #3452). Cells were acquired on a FACSVerse™flow cyt-
ometer (Becton Dickinson) and analyses for p-ERK1/2 levels were performed by
using FlowJo®software (Tree Star) on live gated cells (Fig. 4b). The gating strategy
is shown in Supplementary Figure 7.
Western blot analysis of MEK1 and ERK1/2 activation. Osteoblast lysates were
analyzed by western blotting using standard methods and indicated antibodies
(Fig. 4c, b, d). Briefly, osteoblasts from affected and unaffected bone of melor-
heostosis patients were grown in α-MEM supplemented with 10% FBS (Gemini
Bio-Products, West Sacramento, CA) and antibiotics (penicillin (100 U/ml) and
streptomycin (100 µg/ml), Thermo Fi sher Scientific Inc.). Cells were seeded at
75,000 cells/well in 12-well tissue culture vessel containing 1 ml/well of culture
media. On the next day, cells were pre-treated with U0126 (1, 4-diamino-2, 3-
dicyano-1,4-bis[2-aminophenylthio] butadiene, a MEK1/2 inhib itor, for 1 h.
Inhibitor concentration in culture was 10 µM (#9903, Cell Signaling Technology)
or DMSO (vehicle control, Sigma) in serum-free α-MEM. Cells were then stimu-
lated with FBS (20% in media) for 30 min before being lysed in cell lysis buffer (50
mM Tris (pH 8.0), 150 mM NaCl, 1% IGEPAL, 0.5% sodium deoxycholate, 0.1%
sodium disodium sulfate, 1× protease inhibitor cocktail, and 1× phosphatase
inhibitor cocktail). Cell lysates were cleared by centrifugation at 20,000×gfor 20
min at 4 °C. Soluble cell lysates were mixed with SDS-PAGE sample buffer (50 mM
Tris, pH 6.8, 2% SDS, 10% glycerol, 0.1% bromophenol blue, and 100 mM
dithiothreitol) and denatured at 80 °C for 10 min. Proteins were resolved in 4–15%
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mini-PROTEAN precast gels (Bio-Rad, Hercules, CA) at 100 V for 90 min. The
resolved proteins were transferred to nitrocellulose membranes (Bio-Rad) at 100 V
for 90 min at 4 °C. The membranes were immersed in 5% bovine serum albumin in
Tris-buffered saline containing 0.5% Tween-20 (TBS-T) for 1 h at room tem-
perature to block non-specific binding of primary antibodies. Subsequently, the
membranes were probed with the following antibodies (Cell Signaling Technology,
Cambridge, MA): phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (clone
#D13.14.4E) rabbit monoclonal antibody (#4370, 1:2000), p44/42 MAPK (Erk1/2)
(clone # L34F12) mouse monoclonal antibody (#4696, 1:2000), MEK1/2 rabbit
antibody (#9122, 1:1000), cyclin D3 (clone #DCS22) mouse monoclonal antibody
(#2936, 1:2000), GAPDH (clone #D16H11) rabbit monoclonal antibody (#5174,
1:1000), and COX IV (clone # 3E11) rabbit monoclonal antibody (#4850, 1:1000).
After incubating membranes with primary antibodies for 16 h at 4 °C, membranes
were washed in TBS-T three times for 5 min each at room temperature. Protein
bands were visualized by incubating membranes for 1 h at room temperature with
the following secondary antibodies (LI-COR Biosciences, Lincoln, NE): IRDye®
680RD anti-rabbit IgG (#926-68071, 0.1 ug/ml) or IRDye®800CW anti-mouse IgG
(#926-32210, 0.1 ug/ml). The membranes were washed in TBS-T three times for 5
min each at room temperature before scanning with Odyssey®Infrared Imaging
Systems (LI-COR Biosciences, Lincoln, NE). Uncropped western blots are available
in Supplementary Figure 6.
Immunohistochemistry for p-ERK1/2. Fresh bone tissue was fixed in 10%
neutral buffered formalin, decalcified by formic acid, and embedded in paraffin.
Thin sections (5 µm) were mounted onto tape adhesive slides. Following depar-
affinization and rehydration with ethanol, endogenous peroxidase was blocked
using 3% hydrogen peroxide/distilled water. Antigen retrieval was performed
using a citrate buffer (BioGenex) for 10 s at 100 °C, then slid es were cooled and
rinsed in distilled water and Tris-buffered saline/Tween-20 (TBS-T). After blocking
in 5% normal goat serum (Vector Labs), sections were incubated with anti-phos-
pho-p44/42 MAPK ERK1/2 rabbit monoclonal antibody (Thr202/Tyr204; diluted
1:200, Cell Signaling Technology, USA #4376) overnight at 4 °C or monoclonal
rabbit IgG isotype control. Subsequently, biotinylated goat anti-rabbit IgG (1:100,
Vector Labs) was applied for 30 min, before slides were incubated with ABC
Elite Standard (Vector Labs) for 30 min. p-ERK1/2 was visualized by a 5-min
incubation in DAB Reagent (Sigma) and subsequent hematoxylin counterstain.
Slides were dehydrated, cleared in xylene and mounted with coverslips with Per-
mount. Paraffin embedded colon cancer cells (SW48, carrying a p.Q56P mutation
in MAP2K1) was used as positive control13. Normal bone from patients under-
going surgical resection for unrelated indications served as negative control
(Fig. 4a).
Osteoblast mineralization and differentiation in vitro. Osteoblasts from affected
and unaffected bone of patient Melo-18 were plated at passage 2 with 50,000 cells/
well in 12-well plates and cultured to confluency in α-MEM supplemented with
10% FBS and antibiotics at 37 °C/8% CO
2
. Following confluence, osteoblasts were
grown in osteoblast differentiation medium (50 µg/mL L-ascorbic acid, 10 nM
dexamethasone, and 2.5 mM β-glycerop hosphate), with or without 100 ng/mL
recombinant BMP2 (#355-BM, R&D Systems, Minneapolis, MN), refreshing
osteogenic media every 3 days for 7 weeks.
To visualize mineralization in osteoblast culture, cells were washed once with
PBS and fixed with 4% paraformaldehyde for 30 min at room temperature. Fixed
cells were washed three times with PBS and stained with 2% Alizarin Red S
solution (pH 4.2) for 30 min at room temperature. Excess Alizarin Red S stain was
removed with three distilled water washes. Cells were air-dried in the dark before
imaging (Fig. 5e). During osteoblast mineralization in osteogenic medium, total
RNA was collected at 2 weeks’post-induction and used to examine gene expression
by real-time quantitative PCR analysis.
Data availabilty. The data that support the findings of this study are available from
the corresponding author upon reasonable request.
Received: 7 November 2017 Accepted: 7 March 2018
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Acknowledgements
We thank Dr. William Gahl for early support of the study, Donna Butcher for assistance
with immunohistochemistry, Daniela Gabriel, Petra Keplinger, Sonja Lueger and Phae-
dra Messmer for careful preparation of bone samples for histomorphometry. We are
especially grateful to Kathleen Harper, the Melorheostosis Association, and the patients
for their support of this study.
Author contributions
T.B. conceived the overall project. S.J. and T.B. designed and wrote the clinical protocol,
provided clinical care for patients and analyzed data. J.K., E.W.C. and E.L. supported
clinical care of patients. With assistance from E.P.H., W.A.C. and F.M. H.K.
designed, conducted experiments with cultured osteoblasts and analyzed data under
supervision of J.C.M. Z.D., A.I., and R.S. analyzed the data from whole exome sequen-
cing. N.F.Z., P.R. and K.K. performed histological evaluation of bone specimens. E.W.C.
and S.J. obtained and analyzed skin samples. J.C.M., T.B., R.S., H.K., Z.D., and S.J. wrote
the manuscript.
Additional information
Supplementary Information accompanies this paper at https://doi.org/10.1038/s41467-
018-03720-z.
Competing interests: The authors declare no competing interests.
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ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-03720-z
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