Loss-of-function mutations in LEMD3 result in osteopoikilosis, Buschke-Ollendorff syndrome and melorheostosis

Article (PDF Available)inNature Genetics 36(11):1213-8 · December 2004with38 Reads
DOI: 10.1038/ng1453 · Source: PubMed
Osteopoikilosis, Buschke-Ollendorff syndrome (BOS) and melorheostosis are disorders characterized by increased bone density. The occurrence of one or more of these phenotypes in the same individual or family suggests that these entities might be allelic. We collected data from three families in which affected individuals had osteopoikilosis with or without manifestations of BOS or melorheostosis. A genome-wide linkage analysis in these families, followed by the identification of a microdeletion in an unrelated individual with these diseases, allowed us to map the gene that is mutated in osteopoikilosis. All the affected individuals that we investigated were heterozygous with respect to a loss-of-function mutation in LEMD3 (also called MAN1), which encodes an inner nuclear membrane protein. A somatic mutation in the second allele of LEMD3 could not be identified in fibroblasts from affected skin of an individual with BOS and an individual with melorheostosis. XMAN1, the Xenopus laevis ortholog, antagonizes BMP signaling during embryogenesis. In this study, LEMD3 interacted with BMP and activin-TGFbeta receptor-activated Smads and antagonized both signaling pathways in human cells.
Loss-of-function mutations in LEMD3 result
in osteopoikilosis, Buschke-Ollendorff syndrome
and melorheostosis
Jan Hellemans
, Olena Preobrazhenska
, Andy Willaert
, Philippe Debeer
, Peter C M Verdonk
, Teresa Costa
Katrien Janssens
, Bjorn Menten
, Nadine Van Roy
, Stefan J T Vermeulen
, Ravi Savarirayan
, Wim Van Hul
Filip Vanhoenacker
, Danny Huylebroeck
, Anne De Paepe
, Jean-Marie Naeyaert
, Jo Vandesompele
Frank Speleman
, Kristin Verschueren
, Paul J Coucke
& Geert R Mortier
Osteopoikilosis, Buschke-Ollendorff syndrome (BOS) and
melorheostosis are disorders characterized by increased bone
. The occurrence of one or more of these phenotypes in
the same individual or family suggests that these entities might
be allelic
. We collected data from three families in which
affected individuals had osteopoikilosis with or without
manifestations of BOS or melorheostosis. A genome-wide
linkage analysis in these families, followed by the identification
of a microdeletion in an unrelated individual with these
diseases, allowed us to map the gene that is mutated in
osteopoikilosis. All the affected individuals that we investigated
were heterozygous with respect to a loss-of-function mutation in
LEMD3 (also called MAN1), which encodes an inner nuclear
membrane protein. A somatic mutation in the second allele of
LEMD3 could not be identified in fibroblasts from affected skin
of an individual with BOS and an individual with
melorheostosis. XMAN1, the Xenopus laevis ortholog,
antagonizes BMP signaling during embryogenesis
. In this study,
LEMD3 interacted with BMP and activin-TGFb receptor–
activated Smads and antagonized both signaling pathways
in human cells.
Osteopoikilosis (OMIM 166700) is an autosomal dominant skeletal
dysplasia characterized by a symmetric but unequal distribution of
multiple hyperostotic areas in different parts of the skeleton (Fig. 1)
These lesions, usually detected incidentally, represent foci of old
remodeled bone with lamellar structure, either connected to adjacent
trabeculae of spongy bone or attached to the subchondral cortex
Osteopoikilosis can occur either as an isolated anomaly or in associa-
tion with other abnormalities of skin and bone. BOS (OMIM 166700),
an autosomal dominant disorder, refers to the association of osteo-
poikilosis with disseminated connective-tissue nevi. Both elastic-type
nevi (juvenile elastoma) and collagen-type nevi (dermatofibrosis
lenticularis disseminata) have been described in BOS
lesions can be absent in some family members, whereas other relatives
may have both
. The co-occurrence of osteopoikilosis and melorheos-
tosis in the same family has been reported in a few instances
Melorheostosis (OMIM 155950) is characterized by a ‘flowing’ (rheos)
hyperostosis of the cortex of tubular bones. These lesions are usually
asymmetric: they may involve only one limb or correspond to a
particular sclerotome. They are often accompanied by abnormalities
of adjacent soft tissues, such as joint contractures, sclerodermatous
skin lesions, muscle atrophy, hemangiomas and lymphoedema
a b
Figure 1 Osteopoikilosis lesions and elastic-type nevus in individual III-3 of
family A. (a) Anteroposterior radiograph of the left shoulder showing multiple
osteopoikilosis lesions, best visible in the left humerus. (b) Light micrograph
of the elastic-type nevus stained with Van Gieson. Original magnification,
100. Thick and coarse collagen bundles with numerous broad and
irregular elastic fibers are present in the mid-dermis.
Published online 17 October 2004; doi:10.1038/ng1453
Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium.
Department of Developmental Biology, Flanders Interuniversity Institute for Biotechnology
and Laboratory of Molecular Biology; and
Center for Human Genetics, University of Leuven, Leuven, Belgium.
Department of Orthopedic Surgery, Ghent University
Hospital, Ghent, Belgium.
Medical Genetics Service, Sainte-Justine Hospital and University of Montre
al, Montre
al, Canada.
Department of Medical Genetics,
University Hospital and University of Antwerp, Belgium.
Genetic Health Services Victoria, Murdoch Childrens Research Institute, and University of Melbourne,
Department of Radiology, University Hospital and University of Antwerp, Belgium.
Department of Dermatology, Ghent University Hospital, Ghent, Belgium.
Correspondence should be addressed to G.R.M. (geert.mortier@ugent.be).
NOVEMBER 2004 1213
© 2004 Nature Publishing Group http://www.nature.com/naturegenetics
Affected individuals may be asymptomatic or may complain of
chronic pain in the affected limb
To unravel the genetic defect of osteopoikilosis, we started a
genome-wide linkage analysis in family A (Fig. 2a). Screening of 400
markers with an average spacing of 10 cM resulted in a significant
positive lod score (3.744) for two consecutive markers on chromosome
12q13: D12S368 and D12S83. The results of the linkage analysis in the
two other families, families B and C (Fig. 2b,c), were consistent with
the linkage data obtained in family A. The centromeric boundary of the
genetic interval was determined by a recombination event between
D12S1048 and D12S1663 in individual III-1 (of family A). The
telomeric boundary was defined by a recombination event between
D12S1686 and D12S313 in individual II-5 (of family C), resulting in a
candidate region of 23.55 cM on chromosome 12q1212q14.3. We
calculated a combined maximum two-point lod score of 6.691 at y¼0
for markers D12S1661 and D12S1691 (Supplementary Table 1 online).
The next step in the genetic analysis was the identification
of individual G03-1858, who is affected with proportionate
short stature, microcephaly, learning disabilities, ectopic kidneys and
osteopoikilosis. We hypothesized that this
individual might have a microdeletion,
resulting in the loss of several contiguous
genes, including the gene mutated in osteo-
poikilosis. We investigated this individual for
loss of heterozygosity in the candidate region
on 12q12–12q14.3 and found loss of hetero-
zygosity for marker D12S1686, located in the
telomeric part of the interval. The testing of
additional markers confirmed the presence of
a microdeletion with a centromeric boundary
at marker D12S329 (Fig. 3). The telomeric
boundary of the microdeletion was defined
at the single-nucleotide polymorphism
tsc0527430 using the GeneChip Mapping
10K Array (results for whole genome in
Supplementary Fig. 1 online; results for
chromosome 12 in Fig. 4c). We then tested
more markers in the region of overlap
between the microdeletion and the linkage
interval in family C, which allowed us to
narrow the linkage interval and define a
3.07-Mb critical region for association with
osteopoikilosis between marker D12S329 and
mSAT12.10. This region contains 23 known
genes (National Center for Biotechnology
Information genome viewer; Fig. 3).
Two of these genes, WIF1 (Wnt inhibitory
factor 1) and LEMD3 (LEM domain–
containing 3), are good candidates for invol-
vement in osteopoikilosis. WIF1 is involved
in Wnt signaling, and LEMD3 functions in
BMP signaling, two pathways important in
bone development
. Mutation analysis of
WIF1 did not identify any abnormalities in
the affected individuals. Sequencing of
LEMD3 identified loss-of-function mutations
in all affected individuals of the three families
and in three unrelated individuals with osteo-
poikilosis (Ta ble 1 and Fig. 4e). The splice-
site mutation in individual G03-2881 caused
skipping of exon 6, resulting in a frameshift
and premature stop codon in exon 7 at position 2,021 (Fig. 4d). The
deletion of one of the LEMD3 alleles in individual G03-1858 was
corroborated by fluorescence in situ hybridization (FISH) analysis
with locus-specific probes (Fig. 4b). Re-evaluation of the karyotype
(550-band level) was suggestive of, but not conclusive for, the presence
of a deletion in the 12q14–15 region (Fig. 4a).
Some reports have suggested that the asymmetric distribution of
skin lesions in BOS and the segmental involvement usually observed
in melorheostosis result from a somatic mutation
. To investigate
this possibility, we took skin biopsy samples from two affected
individuals, one from an elastic-type nevus in individual III-3
(of family A) with BOS and a second from a hard sclerodermic-like
lesion in individual III-2 (of family B) with melorheostosis. Sequence
analysis of LEMD3 on genomic DNA extracted from both skin
lesions showed no evidence for an additional somatic mutation in
LEMD3 (or ‘second hit’). Analysis of intragenic polymorphisms
showed no loss of heterozygosity or allelic imbalance and therefore
excluded the possible existence of a partial gene deletion as a somatic
mutation. In cDNA from normal and affected skin of the individual
Figure 2 Pedigree structure and haplotypes of the three families with osteopoikilosis, family A (a),
family B (b) and family C (c). Inferred alleles are shown in brackets. The haplotype cosegregating with
the disorder is indicated with a black bar.
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with BOS, we found only the normal sequence, owing to nonsense-
mediated decay of mRNA from the abnormal allele. Because bone
specimens were not available, we could not investigate the possibility
that a somatic mutation in osteoblasts could explain the spotty
occurrence of bone lesions.
LEMD3 is an integral protein of the inner nuclear membrane
contains a nucleoplasmic N- and C-terminal domain and two trans-
membrane segments
. The N-terminal segment shares a conserved
globular domain of B40 amino acids with other inner nuclear
membrane proteins, such as lamina-associated polypeptide 2 (LAP2)
and emerin
.TheX. laevis ortholog of LEMD3 (XMAN1) antag-
onizes BMP signaling. This antagonizing activity of XMAN1 resides in
the C-terminal region that binds to Smad1, Smad5 and Smad8 (ref. 5).
To investigate whether LEMD3 interacts with BMP receptor–
activated and TGFb receptor–activated Smads, we carried out a
yeast two-hybrid analysis using the C-terminal domain of LEMD3
as prey. This analysis identified interactions between the C-terminal
domain of LEMD3 and the MH2 domains of Smad1 (BMP-specific)
and Smad2 (TGFb-specific), suggesting that LEMD3 is involved in
both BMP and TGFb signaling (Fig. 5a).
We further investigated the role of LEMD3 in both signaling path-
ways by overexpressing the protein in two different cell lines. In
HEK293T cells, we measured the expression of several known target
genes in basal conditions and after BMP4 stimulation by quantitative
PCR (Q-PCR). Overexpression of LEMD3 reduced the capacity of
BMP4 to upregulate Smad6, Smad7, Id2 and Id3 (Fig. 5b). In HepG2
cells,wemeasuredtheresponseofTGFb using the activin-TGFb
responsive 3TP-Lux reporter in basal conditions and in the presence of
a constitutively active receptor ALK4, which activates Smad2
and Smad3. Overexpression of LEMD3 reduced the ALK4-induced
3.07 Mb
23 genes
abc d
Figure 3 Ideogram of chromosome 12 showing the linkage interval,
microdeletion and candidate region. (a) Ideogram of chromosome 12.
(b) The 12q12–q14.3 linkage interval with indication of markers at the
boundaries. (c) The microdeletion in individual G03-1858 in relation to the
linkage interval. The region of interest is shown on the right (d), with
LEMD3 as the candidate gene.
50 bp
Exon 5 Exon 6
Exon 5 Exon 7
Nonsense mutation Frameshift mutation Splice-site mutation
with frameshift
ab c
Figure 4 Overview of the cytogenetic and molecular defects found in
affected individuals. (a) Partial karyotype from individual G03-1858 showing
both chromosomes 12. The normal homolog is depicted on the left, and the
homolog with the deletion (arrow) is shown on the right. (b) Metaphase FISH
analysis with the BAC clone encompassing LEMD3 (RP11-30506; filled
arrow) and the centromeric 12 probe (open arrow), showing a microdeletion
on the right homolog. (c) Results from the GeneChip Mapping 10K Array
analysis for chromosome 12 of individual G03-1858. An ideogram of
chromosome 12 is shown on the left; meta-analysis significance of the
genetic copy-number variation of each SNP against the reference mean is
shown on the right. The region with large negative values (bar to the left)
indicates the presence of a microdeletion. (d)EffectoftheLEMD3 mutation
in individual G03-2881. The electrophoretic analysis of an amplified cDNA
fragment containing exons 4–8 shows the presence of a 146-bp shorter
fragment as compared to the control. Partial nonsense-mediated decay is
probably responsible for the weaker signal of this abnormal fragment.
Sequence analysis shows skipping of exon 6 in the mutated allele (causing
a frameshift with a premature stop codon in exon 7 at position 2,021; data
not shown). (e) The positions of all LEMD3 mutations identified in this study
are shown below the structure of LEMD3 (see also Table 1). Functional
domains are indicated in gray: the LEM-containing N-terminal domain, the
two transmembrane (TM) domains and the C-terminal domain with the
RNA-recognition motif (RRM) motif. The black horizontal bar indicates the
Smad-interacting part and BMP-antagonizing portion (as shown in XMAN1;
ref. 5) of the C-terminal domain.
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activation of the 3TP-Lux reporter (Fig. 5c). These results indicate that
LEMD3 can antagonize both BMP and TGFb signaling in human cells.
Finally, we investigated the effect of the LEMD3 mutations that we
identified in affected individuals. We made three mutant LEMD3
constructs containing the 1185dupT, 1609C-T and 2154dupA muta-
tions, respectively. We carried out a luciferase assay in HEK293T cells
transfected with a TGFb-responsive reporter and found that each
mutant construct was unable to reduce TGFb signaling, unlike
the wild-type construct (Fig. 5d). The loss-of-function effect of these
mutations most probably resulted from the absence of the Smad-
interacting C-terminal domain in the truncated proteins, present on
western blots. We therefore believe that the mutations found in this
study are hypomorphic, either because of nonsense-mediated decay
or because of the production of a truncated protein lacking the
C-terminal domain. We measured by Q-PCR the expression of several
target genes in skin fibroblasts from individual III-3 of family A with
the 2154dupA mutation. This analysis confirmed that fibroblasts from
this affected individual were haploinsufficient with respect to LEMD3.
In addition, we found that expression of the gene Id3 after TGFb
stimulation was significantly higher in these fibroblasts than in
controls (Fig. 5e). This is the first evidence to our knowledge that
haploinsufficiency of LEMD3 in human fibroblasts results in enhanced
TGFb signaling with upregulation of target genes downstream in the
pathway. We observed no significant differences between fibroblasts
from the elastic-type nevus and normal skin of the affected individual
in all conditions tested.
In conclusion, we found that loss-of-function mutations in LEMD3
can result in osteopoikilosis, BOS and melorheostosis. The failure to
detect a somatic mutation in LEMD3 in affected skin fibroblasts from
an individual with BOS and one with melorheostosis may suggest that
Table 1 LEMD3 mutations
Affected family or individual LEMD3 mutation
G03-1858 Total gene deletion
1 G03-2882 457C-T
2 G03-1885 1033-1035delGGGinsC
3 Family C (G03-2457) 1185dupT
4 Family B (G02-1757) 1609C-T
5 G03-2881 1941+5delG
6 Family A (G02-1389) 2154dupA
The numbers refer to the position of each mutation as shown in Figure 4e.
Numbering is
according to cDNA sequence NM-014319.
Bait Smad1
0.5 µg LEMD3
1 µg LEMD3
2 µg LEMD3
Smad6 Smad7 ld2 ld3
Normalized gene expression
Basal ALK4
LEMD3 overexpression
Relative luciferase activity
Relative luciferase activity
Empty WT LEMD3 1185dupT
Basal BMP4
Basal BMP4
ld3 LEMD3
Expression ratios (affected/control)
cd e
Figure 5 Analysis of normal and mutant LEMD3 in BMP and TGFb signaling. (a) Yeast two-hybrid analysis shows that the C-terminal part of LEMD3
(LEMD3-C) specifically interacts with the MH2 domain of Smad1 and Smad2. SIP1–Smad1 interaction is included as a positive control. Interaction was
assessed by activation of the gene MEL1 encoding a-galactosidase, visualized by blue staining of the yeast. (b) Normalized gene expression for four known
target genes in basal () and BMP4-stimulated (+) HEK293T cells. Overexpression of LEMD3 reduces the capacity of BMP4 to upregulate Smad6, Smad7,
Id2 and Id3. (c) Relative luciferase activity from the 3TP-Lux reporter construct in HepG2 cells. Overexpression of LEMD3 reduces activation of the 3TP-Lux
reporter gene in the presence of the constitutively active ALK4 receptor. (d) Relative luciferase activity from the (CAGA)
reporter construct cotransfected
with plasmids expressing different LEMD3 mutants in HEK293T cells. Reduction of TGFb signaling is not observed for the different mutant LEMD3
constructs. (e) Expression ratios between fibroblasts from an affected individual and control fibroblasts for LEMD3 and Id3 as measured by Q-PCR. The
ratios for Id3 in basal conditions and after BMP4 or TGFb stimulation are shown on the left. Significantly (P o 0.05) higher expression of Id3 was observed
after TGFb stimulation. The ratios for LEMD3 are shown on the right and are indicative of haploinsufficiency in fibroblasts from the affected individual
(P o 0.05).
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other genetic factors contribute to the presence and distribution of
skin and bone lesions in these disorders. In addition, we found
evidence that LEMD3 can antagonize both the BMP and TGFb
signaling pathway. These results are in accordance with previous
studies showing that genetic defects in both signaling pathways can
result in hyperostotic bone disorders. Loss-of-function mutations in
SOST, encoding the extracellular BMP antagonist sclerostin, lead to
(OMIM 269500), whereas activating missense muta-
tions in TGFB1 can result in Camurati-Engelmann disease
(OMIM 131300). Finally, increased signaling in the TGFb pathway
has been observed in other fibrotic skin disorders, such as sclero-
derma, and may therefore explain the skin lesions in individuals with
BOS and melorheostosis
Material from affected individuals. We obtained appropriate informed con-
sent from all subjects involved in the study. Family A is a three-generation
family of Belgian origin. All affected individuals had osteopoikilosis. Two
affected individuals (II-5 and III-3) also had skin manifestations of BOS. In
individual III-3, skin biopsy samples were taken from a connective-tissue nevus
on the left thigh and from normal skin on the right thigh. Light microscopy
showed more numerous elastin fibers in the middle and deep dermis on the
affected side. These fibers had a slightly granular and thickened appearance
(Fig. 1b). Ultrastructurally, the elastin fibers were much thicker and more
numerous in the affected skin than in the normal skin. They had a well-
developed amorphous matrix with delicate peripheral microfibrils. Results of
light microscopy and electron microscopy analysis were consistent with the
diagnosis of connective-tissue nevus of the elastic-tissue type. Fibroblasts from
both biopsies were cultured for molecular analysis.
Family B is another three-generation family of Belgian origin, previously
, illustrating the co-occurrence of osteopoikilosis and melorheostosis
in one family. Family C is a three-generation British family with autosomal
dominant osteopoikilosis and skin lesions reminiscent of BOS. Individual G03-
1885 is an Australian with BOS. Individuals G03-2881 and G03-2882 have
osteopoikilosis and are both of Belgian origin but are unrelated. Individual
G03-1858 had a history of prenatal and postnatal growth retardation and
learning disability that was diagnosed as Russell-Silver syndrome in early
childhood. Investigations for hypertension in infancy revealed ectopic kidneys
and aberrant renal arteries. Evaluation at the age of 16 years showed propor-
tionate short stature with height of 131.5 cm (50th centile for a 9-y-old girl),
weight of 31.8 kg (50th centile for a 10-y-old girl) and head circumference of
49 cm (50th centile for a 3-y-old girl). Physical examination revealed a subtly
dysmorphic face with synophrys, mild hypertelorism, broad and high nasal
bridge, micrognathia and maxillary overbite. In addition, a diffuse hyper-
pigmentation spot was noted on the left thigh. Osteopoikilosis lesions were
found on skeletal survey.
Linkage analysis. We used all autosomal markers from the Applied Biosystems
Linkage mapping set version 2 for a genome-wide linkage analysis. We used an
improved protocol from the Centre National de Ge
notypage for pooling an
average of four markers per PCR to carry out all reactions. Additional markers
were taken from the Marshfield map or designed based on the simple-tandem-
repeat finder in the University of California Santa Cruz genome browser. We
carried out genotyping on an Applied Biosystems Prism 3100 Genetic Analyzer
running Genemapper v2.0 software. We used the MLINK program of the
LINKAGE software package
to calculate two-point lod scores between the
disease phenotype and each of the markers, assuming a dominant mode of
inheritance with a penetrance of 0.95 and a disease allele frequency of 0.0001.
The frequency of most marker alleles was set to 1/8. For markers with more
than eight alleles in the pedigrees, the allele frequency was set to 1/n,where
n is the number of alleles.
Cytogenetic and FISH analysis. We carried out karyotyping in accordance
with standard procedures. We analyzed metaphases at the 550-band level. We
carried out FISH analysis as described
using BAC clone RP11-30506 from the
12q14.3 region.
GeneChip Mapping 10K Array analysis. We analyzed DNA from individual
G03-1858 using the GeneChip Mapping 10K Array (Affymetrix) as described
We analyzed data with both the Affymetrix GCOS and GDAS software and the
Affymetrix GeneChip Chromosome Copy Number Tool. We used copy number
estimation (meta P value) to identify the microdeletion, the boundaries of
which were defined by loss of heterozygosity. Results were visualized with
arrayCGHbase (B. Menten et al., unpublished data).
Sequence analysis. We amplified all exons by PCR using intronic primers and
additional exonic primers for larger exons (primer sequences are available on
request). We used a touchdown PCR program with an annealing temperature
decreasing from 60 1Cto481C over 12 cycles, followed by 20 cycles with an
annealing temperature of 48 1C. We sequenced PCR products using the BigDye
v3.1 ET terminator cycle sequencing kit from Applied Biosystems. Sequencing
reactions were loaded onto an Applied Biosystems Prism 3100 Genetic Analyzer
and analyzed with Sequencing Analysis v3.7 and SeqScape v1.1 software
(Applied Biosystems).
Yeast two-hybrid analysis. We cloned the C-terminal domain of human
LEMD3, encoding amino acids 520–853, as a PvuII-XhoI fragment into the
SmaIandXhoI restriction sites of the prey vector pAct2 (Clontech). We
cotransformed yeast strain AH109 with this vector or a positive control
(SIP1) prey vector with different Smad bait vectors
, using the Yeastmaker
yeast transformation system 2 (Clontech). We assayed activation of the gene
MEL1 by interacting hybrid proteins in yeast on Trp-Leu drop-out medium
containing the chromogenic substrate X-a-Gal (Clontech).
Luciferase assay. We carried out transfections of HepG2 cells in triplicate in 24-
well plates using Fugene (Roche). Each well was transfected with a total amount
of 500 ng of plasmid DNA, including 50 ng of 3TP-Lux reporter, 50 ng of RSV-
promoter-based lacZ reporter and combinations of 50 ng of the constitutively
active ALK4 receptor with 100 ng of LEMD3 or empty expression vector. We
maintained a constant amount of DNA by adding pBluescript vector. Forty-
eight hours after transfection, we assayed cell extracts for luciferase and
b-galactosidase activities in accordance with the manufacturers’ protocols
(Promega and Clontech, respectively). We normalized the data by calculating
the ratio of luciferase activity to b-galactosidase activity.
Q-PCR. We isolated RNA using RNeasy Mini Kit (Qiagen) and synthesized
cDNA using SuperScript II Reverse Transcriptase Kit with random hexamer
primers (Invitrogen) in a total volume of 20 ml. We used 5 mlofcDNA(1:10
dilution) in combination with the Q-PCR Core Kit for SYBR Green I
(Eurogentec) and 250 nM gene-specific primers to carry out Q-PCR on a
GeneAmp 5700 Sequence Detector (Applied Biosystems). The Q-PCR program
consists of 40 cycles with 15 s at 95 1C and 1 min at 60 1C, followed by a
dissociation run to determine melting curves. We carried out all reactions in
duplicate and normalized them to the geometric mean of three stable reference
genes (GAPD, HPRT1 and YWHAZ)
To investigate the effect of LEMD3 on Smad signaling, we seeded HEK293T
cells in 9-cm dishes and transfected them with a total of 2 mgofLEMD3
plasmid DNA (provided by H. Worman; Department of Medicine, College of
Physicians and Surgeons, Columbia University, New York, New York, USA)
using Fugene (Roche). Twenty-four hours after transfection, cells were serum-
starved for 4 h and then stimulated with 5 ng ml
of human recombinant
BMP4 (R&D Systems) for 1 h followed by RNA extraction.
In the screening for differentially expressed genes, we used fibroblasts from
a control individual and from normal and affected skin of affected individual
III-3 (of family A). We grew the cells in 13.5-cm dishes to 80% confluency
and then serum-starved them for 2 h. We collected cells after 1 h of treatment
with 1 ng ml
human recombinant TGFb1 (R&D Systems) or with 5 ng ml
of BMP4. Expression levels for all genes were determined in four independent
experiments. Differential gene expression was considered significant when the
difference was at least 50% and the 95% confidence interval of the mean
expression ratio did not include 1 (equivalent with P o 0.05).
We analyzed the following genes (their ID numbers in the RTPrimerDB
are given in parentheses): GAPD (3), HPRT1 (5), YWHAZ (9),
MMP2 (113), CTGF (596), COL1A1 (1089), COL3A1 (1090), COL5A1
(1091), FN1 (1092), ELN (1093), ID1 (1094), ID2 (1095), ID3 (1096),
NOVEMBER 2004 1217
© 2004 Nature Publishing Group http://www.nature.com/naturegenetics
ID4 (1097), LEMD3 (1098), RUNX2 (1099), SMAD6 (1100), SMAD7 (1101)
and SERPINE1 (1102).
Analysis of mutant LEMD3 constructs. We modified the wild-type LEMD3
construct (provided by H. Worman; Department of Medicine, College of
Physicians and Surgeons, Columbia University, New York, New York, USA)
to contain the 1185dupT, 1609C-T or 2154dupA mutation using the
QuickChange Site-Directed Mutagenesis kit (Stratagene) in accordance with
the manufacturers instructions. We plated HEK293T cells at a density of
4 10
cells per well in Dulbeccos modified Eagle medium plus (Invitrogen)
and transferred them to Opti-MEM I (Invitrogen) after overnight adherence.
We transiently transfected cells with 2 mgofeachplasmidand0.5mgofthe
TGFb-responsive reporter construct
in duplicate using Lipofecta-
mine (Invitrogen). We also transfected cells with 20 ng of pRL-TK (Promega)
to correct for transfection efficiency. Cells were serum-starved for 7 h before
stimulation with 7 ng ml
recombinant human TGFb (R&D systems). We
lysed cells 24 h after transfection. We quantified activities of firefly and Renilla
luciferase using the Dual-Luciferase Reporter Assay System (Promega).
URLs. Improved linkage analysis protocols from the Centre National de
notypage are available at http://www.cng.fr/. The arrayCGHbase visualiza-
tion tool and RTPrimerDB database are available at http://medgen.ugent.be/
arraycghbase/ and http://medgen.ugent.be/rtprimerdb/, respectively.
Note: Supplementary information is available on the Nature Genetics website.
We thank the affected individuals and families for their interest and cooperation,
M. Godfrey for critical review of the manuscript, P. Tylzanowski for suggestions
and plasmid stocks and H. Worman for the expression vector encoding human
LEMD3. This study was supported, in part, by the Fund for Scientific Research,
Flanders, with a mandate fundamental clinical research to P.D. and G.R.M.;
a research assistantship to P.C.M.V.; and research projects to F.S., S.J.T.V., W.V.H.
and G.R.M. This study was also supported by an Interuniversity Attraction Pole
grant to W.V.H. and by the Fifth Framework of the specific research and
technological development program Quality of Life and Management of Living
Resources of the European Commission to J.H., A.D.P. and G.R.M. J.H. is funded
by, and J.V. is a postdoctoral researcher with, the Institute for the Promotion
of Innovation by Science and Technology in Flanders. N.V.R. and K.J. are
postdoctoral researchers of the Fund for Scientific Research, Flanders. The research
at Flanders Interuniversity Institute for Biotechnology was supported by the Fund
for Scientific Research, Flanders and the University of Leuven. O.P. is holder of a
DWTC (Federal Services for Scientific, Technical and Cultural affairs) postdoctoral
fellowship and is supported by the Interuniversity Attraction Pole Network. This
text presents research results of the Belgian program of Interuniversity Poles of
attraction initiated by the Belgian State, Prime Ministers Office, Science Policy
Programming. The scientific responsibility is assumed by the authors.
The authors declare that they have no competing financial interests.
Received 22 July; accepted 17 September 2004
Published online at http://www.nature.com/naturegenetics/
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© 2004 Nature Publishing Group http://www.nature.com/naturegenetics
    • "Of note, increased TGF-β signaling has also been associated with other fibrotic skin disorders (Hellemans et al., 2004; Saito et al., 2001; Mori et al., 2003) and hypertrophic scarring (Korekawa et al., 2012). The skeletal findings in BOS may be directly attributed to loss of LEMD3 function, as increased activity of both bone morphogenic protein and TGF-β have been implicated in increased bone formation in a variety of sclerosing bone disorders (de Vernejoul and Kornak, 2010; Hellemans et al., 2004). To date, 125 pathogenic mutations of the LEMD3 gene have been detected (Kratzsch et al., 2016), which cause the skeletal and dermatologic findings often encountered in BOS through a multitude of different mechanisms, including nonsense-mediated decay of LEMD3 mRNA (Burger et al., 2010 ) and nonsense-mediated obliteration of the functional domains of the protein (Yuste-Chaves et al., 2011). "
    Full-text · Article · Jul 2016
    • "The LEMD3 gene [29], detected as a selective sweep in the Gir breed, is a specific repressor of the transforming growth factor beta (TGF-beta) receptor, activin, and BMP signaling, and is involved in negative regulation of skeletal muscle cell differentiation, which might have been selected for in Gir, a breed developed for milk production [35]. In humans, mutations leading to loss of function of this gene are associated with diseases causing sclerosing bone lesions and increased bone density, such as osteopoikilosis [39,40]. This selection signature was reported by Ramey et al. [41], between 48.67 and 48.9 Mb on BTA5, using an approach based on sliding windows estimations of minor allele frequency (MAF). "
    [Show abstract] [Hide abstract] ABSTRACT: Signatures of selection are regions in the genome that have been preferentially increased in frequency and fixed in a population because of their functional importance in specific processes. These regions can be detected because of their lower genetic variability and specific regional linkage disequilibrium (LD) patterns. By comparing the differences in regional LD variation between dairy and beef cattle types, and between indicine and taurine subspecies, we aim at finding signatures of selection for production and adaptation in cattle breeds. The VarLD method was applied to compare the LD variation in the autosomal genome between breeds, including Angus and Brown Swiss, representing taurine breeds, and Nelore and Gir, representing indicine breeds. Genomic regions containing the top 0.01 and 0.1 percentile of signals were characterized using the UMD3.1 Bos taurus genome assembly to identify genes in those regions and compared with previously reported selection signatures and regions with copy number variation. For all comparisons, the top 0.01 and 0.1 percentile included 26 and 165 signals and 17 and 125 genes, respectively, including TECRL, BT.23182 or FPPS, CAST, MYOM1, UVRAG and DNAJA1. The VarLD method is a powerful tool to identify differences in linkage disequilibrium between cattle populations and putative signatures of selection with potential adaptive and productive importance.
    Full-text · Article · Mar 2014
    • "Although this reduction could at least partially be due to the role of NE components in heterochromatin formation, it has also been proposed that INM proteins can sequester transcription factors to the nuclear periphery and impede their binding to target genes. For example, in humans and mice, emerin physically interacts with lmo7 and β-catenin, two transcription factors involved in muscle differentiation [23,24], whereas in humans LEMD3/MAN1 tethers Smads to the NE, thereby affecting connective tissue differentiation [25-27]. "
    [Show abstract] [Hide abstract] ABSTRACT: Laminopathies are diseases characterized by defects in nuclear envelope structure. A well-known example is Emery-Dreifuss muscular dystrophy, which is caused by mutations in the human lamin A/C and emerin genes. While most nuclear envelope proteins are ubiquitously expressed, laminopathies often affect only a subset of tissues. The molecular mechanisms underlying these tissue-specific manifestations remain elusive. We hypothesize that different functional subclasses of genes might be differentially affected by defects in specific nuclear envelope components. Here we determine genome-wide DNA association profiles of two nuclear envelope components, lamin/LMN-1 and emerin/EMR-1 in adult Caenorhabditis elegans. Although both proteins bind to transcriptionally inactive regions of the genome, EMR-1 is enriched at genes involved in muscle and neuronal function. Deletion of either EMR-1 or LEM-2, another integral envelope protein, causes local changes in nuclear architecture as evidenced by altered association between DNA and LMN-1. Transcriptome analyses reveal that EMR-1 and LEM-2 are associated with gene repression, particularly of genes implicated in muscle and nervous system function. We demonstrate that emr-1, but not lem-2, mutants are sensitive to the cholinesterase inhibitor aldicarb, indicating altered activity at neuromuscular junctions. We identify a class of elements that bind EMR-1 but do not associate with LMN-1, and these are enriched for muscle and neuronal genes. Our data support a redundant function of EMR-1 and LEM-2 in chromatin anchoring to the nuclear envelope and gene repression. We demonstrate a specific role of EMR-1 in neuromuscular junction activity that may contribute to Emery-Dreifuss muscular dystrophy in humans.
    Full-text · Article · Feb 2014
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