ORIGINAL ARTICLE
Normophosphatemic Familial Tumoral Calcinosis
Is Caused by Deleterious Mutations in SAMD9,
Encoding a TNF-a Responsive Protein
Ilana Chefetz1,2,3, Danny Ben Amitai4, Sarah Browning5, Karl Skorecki2,3, Noam Adir6, Mark G. Thomas7,
Larissa Kogleck8, Orit Topaz1, Margarita Indelman1, Jouni Uitto9, Gabriele Richard9,10, Neil Bradman5 and
Eli Sprecher1,2,3
Normophosphatemic familial tumoral calcinosis (NFTC) is an autosomal recessive disorder characterized by
calcium deposition in skin and mucosae and associated with unremitting pain and life-threatening skin
infections. A homozygous missense mutation (p.K1495E), resulting in SAMD9 protein degradation, was recently
shown to cause NFTC in five families of Jewish-Yemenite origin. In this study, we evaluated another JewishYemenite NFTC kindred. All patients were compound heterozygous for two mutations in SAMD9: K1495E and a
previously unreported nonsense mutation, R344X, predicted to result in a markedly truncated molecule.
Screening of unaffected population-matched controls revealed heterozygosity for K1495E and R344X only in
individuals of Jewish-Yemenite ancestry, but not in more than 700 control samples of other origins, including 93
non-Jewish Yemenite. These data may be suggestive of positive selection, considering the rarity of NFTC and the
small size of the Jewish-Yemenite population; alternatively, they may reflect genetic drift or the effect of a
population-specific modifier trait. Calcifications in NFTC generally develop over areas subjected to repeated
trauma and are associated with marked inflammatory manifestations, indicating that SAMD9 may play a role in
the inflammatory response to tissue injury. We therefore assessed the effect of cellular stress and tumor
necrosis factor-a (TNF-a), a potent pro-inflammatory cytokine, on SAMD9 gene expression. Whereas exogenous
hydrogen peroxide and heat shock did not affect SAMD9 transcription, osmotic shock was found to markedly
upregulate SAMD9 expression. In addition, incubation of endothelial cells with TNF-a caused a dose-related,
p38-dependant increase in SAMD9 expression. These data link NFTC and SAMD9 to the TNF-a signaling
pathway, suggesting a role for this system in the regulation of extra-osseous calcification.
Journal of Investigative Dermatology (2008) 128, 1423–1429; doi:10.1038/sj.jid.5701203; published online 20 December 2007
INTRODUCTION
Abnormal deposition of calcium salts within skin tissues is
known as calcinosis cutis (Touart and Sau, 1998). Two major
types of acquired calcinosis cutis have been recognized.
1
Laboratory of Molecular Dermatology and Department of Dermatology,
Rambam Health Care Campus, Haifa, Israel; 2Bruce Rappaport Faculty of
Medicine, Haifa, Israel; 3Center for Translational Genetics, Rappaport
Institute for Research in the Medical Sciences, Haifa, Israel; 4Pediatric
Dermatology Unit, Shneider Children’s Medical Center, Petach-Tikvah, Israel;
5
The Centre for Genetic Anthropology, University College London, London,
UK; 6Faculty of Chemistry, Technion—Israel Institute of Technology, Haifa,
Israel; 7Department of Biology, University College London, London, UK;
8
Department of Anthropology, University College London, London, UK;
9
Department of Dermatology and Cutaneous Biology, Thomas Jefferson
University, Philadelphia, Pennsylvania, USA and 10GeneDx Inc.,
Gaithersburg, Maryland, USA
Correspondence: Dr Eli Sprecher, Laboratory of Molecular Dermatology,
Department of Dermatology, Rambam Medical Center, POB 9602, Haifa
31096, Israel. E-mail: e_sprecher@rambam.health.gov.il
Abbreviations: NFTC, normophosphatemic familial tumoral calcinosis;
TNF-a, tumor necrosis factor-a
Received 26 July 2007; revised 18 September 2007; accepted 27 September
2007; published online 20 December 2007
& 2007 The Society for Investigative Dermatology
Metastatic calcinosis cutis refers to calcium deposition in
association with elevated circulating levels of phosphate and/
or calcium, as seen in chronic renal failure or hyperparathyroidism. In contrast, dystrophic calcinosis cutis is usually
secondary to some form of physical insult to the skin, as
seen in autoimmune diseases and skin cancer (Touart and
Sau, 1998).
Inherited calcinosis cutis is known as familial tumoral
calcinosis. Two forms of familial tumoral calcinosis have
been reported in the literature (Smack et al., 1996), which
recapitulate many features of the two major types of acquired
calcinosis cutis. Hyperphosphatemic familial tumoral calcinosis (MIM211900) is caused by increased renal reabsorption
of phosphate due to mutations in two genes: FGF23 (Araya
et al., 2005; Benet-Pages et al., 2005; Chefetz et al., 2005;
Larsson et al., 2005a, b), coding for a potent phosphaturic
protein (Fukumoto and Yamashita, 2007), and GALNT3,
coding for a glycosyltransferase (Topaz et al., 2004; Ichikawa
et al., 2005, 2007; Specktor et al., 2006), responsible for
FGF23 O-glycosylation (Kato et al., 2006; Frishberg et al.,
2007). Mutations in either gene result in elevated levels of
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I Chefetz et al.
Normophosphatemic Familial Tumoral Calcinosis
circulating phosphate and formation of periarticular subcutaneous calcified masses, sometimes associated with bone
and tooth defects (Frishberg et al., 2005; Specktor et al.,
2006). In contrast, calcified tumor formation in normophosphatemic familial tumoral calcinosis (NFTC; MIM610455) is
generally preceded by a vasculitis-like rash and is associated
with inflammatory manifestations mostly evident in mucosal
tissues (Metzker et al., 1988). Recently, a mutation in
SAMD9 was shown to underlie NFTC in five families of
Jewish-Yemenite origin (Topaz et al., 2006). SAMD9 encodes
a very large protein, thought to be involved in the regulation
of cancer cell proliferation and apoptosis (Li et al., 2007).
In this report, we present data confirming the
pathogenic role of SAMD9 mutations in NFTC, suggesting
that survival of SAMD9 mutations may have occurred in the
Jewish-Yemenite population as a result of positive selection
and indicating that tumor necrosis factor-a (TNF-a) is a major
regulator of SAMD9 gene expression.
RESULTS
Identification of a novel mutation in SAMD9
We assessed a family comprising two affected
children (Figure 1a). Their maternal grandparents are of
Jewish-Yemenite origin as is their paternal grandmother.
The paternal grandfather is of Jewish-Moroccan extraction.
The two patients were dizygotic twins, born after 26 weeks of
gestation. Psychomotor development was normal. Family
history was unremarkable. The male patient was affected by
retinopathy of prematurity. At age 6, the two patients were
first assessed because of the formation of periarticular
b
a
Healthy controls
c
d
e
R344X/WT
WT/WT
K1495E/WT
WT/WT
115 bp 93 bp -
K1495E
200 bp 170 bp -
R344X
Figure 1. Identification of a new mutation in SAMD9. (a) Pedigree and haplotype analysis of an NFTC family and four healthy unrelated control individuals
(C1–C4) carrying mutations p.K1495E and p.R344X. The p.K1495E- and p.R344X-associated haplotypes are boxed in red and blue, respectively. Filled symbols
represent affected individuals. (b) Subepidermal calcified nodule over the left elbow of the male patient. (c) Severe gingivitis displayed by the female patient.
(d) Sequence analysis revealed two distinct mutations in the patients: an A4G transition at cDNA position 4483, which is predicted to result in missense
mutation p.K1495E (right upper panel); and a C4T transition at cDNA position 1030, resulting in premature termination codon p.R344X (left upper panel). The
corresponding wild-type sequences are given in lower panels. (e) PCR–restriction fragment length polymorphism analysis confirms segregation of the mutations
in the family. PCR amplification was performed as described in the text. Mutation p.K1495E creates a novel recognition site for MboII. Thus, heterozygous
carriers display a 93 bp fragment and healthy homozygous individuals show only a 115 bp fragment; mutation p.R344X abolishes a recognition site for
endonuclease BSh1236I. Thus healthy individuals display a 170 bp fragment, whereas carriers of the mutation also display a 200 bp fragment.
1424 Journal of Investigative Dermatology (2008), Volume 128
I Chefetz et al.
Normophosphatemic Familial Tumoral Calcinosis
calcified masses. No preceding rash had been observed. On
examination, the patients displayed subcutaneous mobile
calcified masses above the knees and elbows (Figure 1b) and
showed edema, erythema, and minute calcified nodules over
the sides of their feet (not shown). In addition, both patients
displayed marked edema and redness of the gingivae (Figure
1c). Complete blood count and routine chemistry (including
phosphate levels) were normal.
To confirm NFTC at the molecular level, we sequenced
the entire coding region of the SAMD9 gene (including
exon–intron boundaries) (Figure 1d). Both patients were
found to be compound heterozygous for two distinct
mutations: an A4G transition at cDNA position 4483, which
is predicted to result in missense mutation p.K1495E
previously reported in families affected with NFTC (Topaz
et al., 2006); and a C4T transition at cDNA position 1030,
resulting in the substitution of a stop codon for an arginine
residue at amino-acid position 344 (p.R344X). p.K1495E has
previously been shown to result in protein degradation
(Topaz et al., 2006); p.R344X is predicted to result in
significant truncation of the native 1590 amino-acid long
SAMD9 molecule.
Using PCR–restriction fragment length polymorphism
assays, we confirmed segregation of the two mutations in
the family (Figure 1e).
Mutational screening
To assess the carrier rate for both mutations in the general
population, we screened a cohort of healthy unrelated
individuals of Jewish-Yemenite, Jewish-non-Yemenite, and
Yemenite-non-Jewish origins. Both mutations were exclusively found among controls of Jewish-Yemenite extraction
(Table 1). These data were suggestive of two separate founder
events in the Jewish-Yemenite population. To assess this
possibility, we used microsatellite markers spanning the
SAMD9 locus to genotype all family members and four
controls shown to carry a heterozygous mutation in SAMD9.
As shown in Figure 1a, patients and carrier controls were
shown to share the same mutation-associated hemihaplotype,
demonstrating that p.K1495E and p.R344X arose in the
Jewish-Yemenite population from two independent events.
Regulation of SAMD9 gene expression
The physiological function of SAMD9 is currently unknown.
Recent data suggest that SAMD9 may inhibit proliferation
and promote apoptosis in cancer cells (Li et al., 2007). In
addition, SAMD9 mutations are associated with prominent
Table 1. Population screening of unrelated healthy
individuals
Yemenite Jews
Non-Yemenite
Non-Jewish
Yemenite
K1495E
1 (n=154)
0 (n=612)
0 (n=93)
R344X
5 (n=154)
0 (n=183)
0 (n=93)
Mutation
inflammatory manifestations in NFTC (Metzker et al.,
1988; Topaz et al, 2006). We therefore hypothesized
that SAMD9 may be under the regulation of TNF-a, a major
pro-inflammatory cytokine and inducer of apoptosis, which
has been suggested to be involved in the pathogenesis of
extra-osseous calcification (Dellegrottaglie et al., 2006).
We initially assessed the effect of increasing concentrations of TNF-a on SAMD9 expression in endothelial cells,
where SAMD9 expression is known to be the highest (Topaz
et al., 2006). We observed a maximal 4.5-fold increase in
SAMD9 expression upon exposure of the cells to 10 ng ml1
TNF-a (Figure 2a). At this concentration, a maximal increase
in mRNA levels was observed 6 hours post-exposure (Figure
2b). This effect could be blocked by methylprednisolone (Figure 2c) but not by doxycycline (not shown), another
known inhibitor of TNF-a signaling (Festoff et al., 2006; Shen
et al., 2006).
TNF-a has been shown to act through a number of
cellular pathways (Holtmann and Neurath, 2004; Liu,
2005). To identify the molecular mechanisms underlying
TNF-a-mediated increase in SAMD9, we assessed SAMD9
expression in the presence of TNF-a and known inhibitors of
p38, c-Jun N-terminal kinase, and NF-kB. Down-regulation
of c-Jun N-terminal kinase did not significantly affect the
extent of SAMD9 induction (Figure 3a). In contrast, inhibition
of p38 almost completely abrogated TNF-a-induced
SAMD9 transcription (Figure 3a). p38 is known to mostly
mediate TNF-a pro-inflammatory and pro-apoptotic signals
(Saklatvala, 2004; Schieven, 2005), which is in line with
recent data indicating that SAMD9 promotes apoptosis
(Li et al., 2007). In contrast, TNF-a-mediated NF-kB activation is known to counteract pro-apoptotic signals (Papa et al.,
2006). Accordingly, inhibition of NF-kB translocation to the
nucleus was found to markedly upregulate SAMD9 transcription and to further augment the response of SAMD9
expression to TNF-a stimulation (Figure 3b).
Because dystrophic calcinosis is known to occur following
cellular damage and as TNF-a and cellular stresses have been
shown to induce similar cellular responses (Alpert et al.,
1999), we assessed the effect of a number of cellular stresses
on SAMD9 expression (Figure 4). Whereas both hydrogen
peroxide (Figure 4b), inducing oxidative stress, and heat
stress (not shown) did not cause increased SAMD9 expression, hyperosmotic shock led to a concentration-dependant
increase in SAMD9 expression (Figure 4a), with sorbitol
0.5 M inducing SAMD9 expression by more than 100-fold.
DISCUSSION
Calcification, defined as the precipitation of calcium salts, is
a process known to occur mainly in bone tissues, under
physiological conditions. A growing body of evidence points
to extra-osseous calcification as a major cause of morbidity
and mortality in humans (Atzeni et al., 2006; Shao et al.,
2006; Stoll and Bendszus, 2006; Sharma et al., 2007; Uitto
and Jiang, 2007). More importantly, interventions aimed at
attenuating extra-osseous calcification may increase survival
among susceptible individuals (Schmitt et al., 2006). These
data emphasize the need for a better understanding of the
www.jidonline.org 1425
I Chefetz et al.
Normophosphatemic Familial Tumoral Calcinosis
SAMD9 expression (%)
600
500
400
300
200
100
SAMD9 expression (%)
0
0
0.1
1
TNF-α (ng ml–1)
10
800
700
600
500
400
300
200
100
0
0
5
15
10
Time (hours)
20
T
T+M
25
700
SAMD9 expression (%)
600
500
400
300
200
100
0
C
Figure 2. Effect of TNF-a on SAMD9 gene expression. (a) EaHy cells
were cultured in the presence of increasing concentrations of TNF-a, and
SAMD9 gene expression was measured as described in Materials and
Methods; (b) EaHy cells were exposed for increasing periods of time to
10 ng ml1 of recombinant TNF-a, and SAMD9 gene expression was
quantified by quantitative real-time PCR; (c) EaHy cells were cultured for
6 hours in the presence of 0 (C), 10 ng ml1 of recombinant TNF-a (T), or
10 ng ml1 of recombinant TNF-a and 0.5 mM of methylprednisolone (T þ M),
and SAMD9 gene expression was assessed by quantitative real-time PCR.
pathogenesis of extra-osseous calcification. Rare monogenic
disorders offer a unique setting for the discovery of novel
physiological functions. Indeed, animal models do not
always faithfully replicate human pathologies and, despite
recent technological advances, the study of complex traits
still poses formidable conceptual and technical challenges
(Antonarakis and Beckmann, 2006). Hyperphosphatemic
familial tumoral calcinosis and NFTC adequately model the
two major forms of acquired calcinosis cutis (Sprecher,
2007). Hyperphosphatemic familial tumoral calcinosis very
much resembles metastatic calcinosis, and indeed the
discovery of mutations in GALNT3 in hyperphosphatemic
familial tumoral calcinosis led to the delineation of its role in
the regulation of FGF23, a key protein involved in the
1426 Journal of Investigative Dermatology (2008), Volume 128
pathogenesis of renal failure-associated hyperphosphatemia,
which in turn is causally related to metastatic calcinosis and
calciphylaxis in kidney disease (White et al., 2006).
In contrast, NFTC is thought to replicate many aspects of
dystrophic calcinosis, a common form of extra-osseous
calcification, occurring in various tissues including skin and
often associated with inflammation and tissue injury as
observed in acne, autoimmune diseases, and cancer (Touart
and Sau, 1998).
Although the current report of a second SAMD9 mutation
in NFTC definitely establishes the molecular etiology of this
disorder, the physiological function of SAMD9 remains to be
fully elucidated. Recent data have suggested that SAMD9
may promote cell apoptosis in cancer cells and thereby
function as a tumor suppressor gene (Li et al., 2007). Given
the prominent inflammatory manifestations associated with
NFTC caused by SAMD9 deficiency (Metzker et al., 1988),
our observations in human patients suggest a role for SAMD9
in the regulation of pro-inflammatory signals. TNF-a, a key
cytokine playing an important role in homeostatic maintenance of epithelial tissues, links inflammation to the
apoptotic machinery (Holtmann and Neurath, 2004). Our
observations indicate that SAMD9 may be a downstream
target of TNF-a signaling; whether it mediates pro-apoptotic
signals, activates counter-regulatory anti-inflammatory
activities, or both remains to be determined. Interestingly,
hyperosmotic sorbitol, which, like TNF-a, signals through
p38 (Figure 3a; Cheng et al., 2002), induced SAMD9
expression, suggesting a common regulatory pathway for
SAMD9, involving inducers of cellular stress and inflammatory cell response.
The present data also shed new light on the molecular
genetics of NFTC. The identification of two founder events
causing an exceedingly rare disease in the Jewish Yemenite
population amounting to approximately 174,600 individuals
(www.cbs.gov.il) is surprising. Although recessive diseases
are often thought to persist throughout evolution because of
the fact that heterozygous mutations confer a selective
advantage to carriers, this conjecture has only rarely been
formally proven (Allison, 2004). In the present case, given the
lack of information regarding the physiological function of
SAMD9, we can only speculate that the occurrence of two
rare mutations in a very small population is suggestive of
positive selection, although random drift cannot be excluded.
Owing to various historical circumstances, Yemenite Jews
have been living in closed communities since the 10th
century. Consequently, numerous specific genetic variants
have emerged and have been maintained within this closed
and endogamous population as attested by the existence of
unique mutations (Avigad et al., 1990) and genetic conditions
highly prevalent among Yemenite Jews (Ostrer, 2001). It
could be envisaged that excessive tissue inflammation
associated with deleterious mutations in SAMD9 may have
conferred a survival advantage to Yemenite Jews otherwise
exposed to potentially lethal transmissible diseases, a major
cause of mortality and morbidity in the Jewish-Yemenite
community until its immigration to Israel in the late 1940s
(Borkan, 1993). Furthermore, the state of isolation of the
I Chefetz et al.
2,500
12,000
2,000
10,000
1,500
1,000
500
0
Inhibitor
TNF-α
Control
–
SB203580
+
–
SP600126
+
–
SAMD9 expression (%)
SAMD9 expression (%)
Normophosphatemic Familial Tumoral Calcinosis
+
8,000
6,000
4,000
2,000
0
63,000
0
62,000
61,000
0.25
Sorbitol (M)
0.5
140
SAMD9 expression (%)
SAMD9 expression (%)
120
7,000
6,000
5,000
4,000
3,000
100
80
60
40
20
2,000
0
1,000
0
TNF-α
–
+
–
+
NF-κΒ inh.
–
–
+
+
Figure 3. TNF-a-mediated SAMD9 induction. (a) EaHy cells were cultured in
the presence ( þ ) or absence () of 10 ng ml1 TNF-a, p38 inhibitor
(SB203580), or c-Jun N-terminal kinase inhibitor (SP600126), and SAMD9
gene expression was measured after 6 hours; (b) EaHy cells were cultured in
the presence ( þ ) or absence () of 10 ng ml1 TNF-a and NF-kB inhibitor
JSH-23, and SAMD9 gene expression was assessed as described above.
Jewish community in Yemen may have contributed to the
local adaptation, amplification, and confinement of SAMD9
mutations to this small ethnic group. On the other hand,
neighboring populations must have been, at least in part,
exposed to similar conditions, suggesting the possible
existence of modifier traits specific to the Jewish-Yemenite
population.
In summary, we have identified a novel mutation in
SAMD9 underlying NFTC, definitively establishing mutations
in SAMD9 as a cause of NFTC, and suggesting random drift, a
selection effect in the Jewish-Yemenite population, and/or the
effect of population-specific modifier traits. In addition, given
the role of SAMD9 in NFTC and the fact that SAMD9
expression was found to be regulated by TNF-a, the present
data indicate that further studies of the role of TNF-a in the
regulation of extra-osseous calcification in the skin are
warranted.
0
0.25
0.5
Hydrogen peroxide (mM)
1
Figure 4. Effect of cellular stress on SAMD9 gene expression. EaHy cells
were cultured in the presence of increasing concentrations of (a) sorbitol or
(b) hydrogen peroxide, and SAMD9 gene expression was monitored using
quantitative real-time PCR.
Declaration of Helsinki Principles. DNA was extracted according to
standard procedures. Fifteen milliliters of blood were drawn from
each participant, and DNA was extracted using a salt/chloroform
extraction method.
Microsatellite analysis
Polymorphic microsatellite markers spanning the SAMD9 locus were
selected from the GDB database (http://www.gdb.org), and genotypes were established by PCR amplification of genomic DNA using
Supertherm Taq polymerase (Eisenberg Bro Co., Givat Schmuel,
Israel) and fluorescently labeled primer pairs (Research Genetics,
Invitrogen, Carlsbad, CA) according to the manufacturer’s recommendations. PCR conditions were 5 minutes at 951C followed by 35
cycles for 30 seconds at 951C, 30 seconds at 561C, 30 seconds at
721C, and a final extension step at 721C for 5 minutes. PCR products
were separated by PAGE on an ABI 310 sequencer system, and allele
sizes were determined with Genescan 3.1 and Genotyper 2.0
software. Parsimonious haplotypes were subsequently established
for each individual.
DNA sequencing
MATERIALS AND METHODS
Patients
All affected and healthy control participants or their legal guardian
provided written and informed consent according to a protocol
approved by local institutional review boards in adherence to the
SAMD9 was PCR-amplified using oligonucleotide primer pairs listed
in Table S1, Taq polymerase and Q solution (Qiagen, Valencia, CA)
using the following cycling conditions: 941C for 5 minutes followed
by 35 cycles at 941C for 1 minute, 591C (unless otherwise indicated
in Table S1) for 1 minute, 721C for 1 minute 30 seconds. Gel-purified
www.jidonline.org 1427
I Chefetz et al.
Normophosphatemic Familial Tumoral Calcinosis
(QIAquick gel extraction kit, Qiagen) amplicons were subjected to
bi-directional DNA sequencing using the BigDye terminator
system on an ABI Prism 3100 sequencer (PE Applied Biosystems,
Foster City, CA).
Mutation screening
Mutations were screened in the family using PCR–restriction
fragment length polymorphism assays. To screen for p.R344X, a
200-bp fragment was PCR-amplified, using allele-specific reverse
primer 50 -CCTGTCAATAATTTAACCAACTTTGGTCCC-30 and forward primer 50 -GAACAAAGTAAAAAATTCTCACTATTTGCG-30 ; the
former creates a recognition site for BSh1236I, which is abolished in
the presence of p.R344X. After incubation at 371C for 4 hours,
digested PCR products were electrophoresed in a 2.5% agarose gel
and visualized under UV light. p.K1495E was identified using a
PCR–restriction fragment length polymorphism assay described
previously (Topaz et al., 2006).
Mutations were screened in ethnically and geographically related
healthy individuals using TaqMan technology (Applied Biosciences,
Warrington, UK). Reactions were performed on 384-well microplates in accordance with the manufacturer’s recommended protocol and analyzed using ABI TaqMan 7900HT software. Primers and
MGB probes are detailed in Table S1.
Cell cultures and reagents
EaHy cells, a human umbilical vein endothelial cells-derived
transformed endothelial cell line, were maintained in high-glucose
DMEM medium (Beit-Ha-Emek, Beit-Ha-Emek, Israel) supplemented
with 10% (v/v) fetal calf serum (Beit-Ha-Emek) and 2 mM L-glutamine
(Beit-Ha-Emek).
Cells were treated with a number of molecular reagents: TNF-a
(PeproTech, Rocky Hill, NJ), sorbitol (Sigma, St Louis, MO),
hydrogen peroxide (Sigma), SB203580 (3 mM; Calbiochem),
SP600125 (25 mM; Calbiochem), and NF-kB activation inhibitor-II
JSH-23 (10 mg ml1; Calbiochem).
Quantitative real-time PCR
For quantitative real-time PCR, cDNA was synthesized from 1 mg of
total RNA using the Reverse-iT first strand synthesis kit (Abgene,
Epson, UK) and random hexamers. cDNA PCR amplification was
carried out using the SYBR Green JumpStart Taq ReadyMix (Sigma)
on a Mx3000p/5p multifilter system (Stratagene, Cedar Creek, TX)
with gene-specific intron-crossing oligonucleotide pairs listed in
Table S1. To ensure the specificity of the reaction conditions, at the
end of the individual runs, the melting temperature (Tm) of the
amplified products was measured to confirm its homogeneity.
Cycling conditions were as follows: 951C for 10 minutes, 951C for
10 seconds, 621C for 25 seconds, and 721C for 15 seconds for a total
of 40 cycles. Each sample was analyzed in triplicate. For
quantification, standard curves were obtained using serially diluted
cDNA amplified in the same real-time PCR run. Results were
normalized to ACTB mRNA levels. After the quantification
procedure, the products were resolved by 2.5% agarose gel
electrophoresis to confirm that the reaction had amplified the
correct DNA fragments of known size.
CONFLICT OF INTEREST
The authors state no conflict of interest.
1428 Journal of Investigative Dermatology (2008), Volume 128
ACKNOWLEDGMENTS
We are grateful to the family members for their enthusiastic and generous
participation in our study. We wish to thank Vered Friedman and Rita FuhrerMor for their help with nucleic acid analysis, Uri Seligshon for providing DNA
samples, and Ariel Miller and Tamar Paperna for the gift of the EaHy cells.
This study was supported in part by grants provided by Israel Science
Foundation, the Rappaport Institute for Research in the Medical Sciences,
NIH/NIAMS Grant R01 AR052627 and the Veronique Simone and Barry and
Carole Rose Funds of the Canadian Technion Society.
SUPPLEMENTARY MATERIAL
Table S1. Oligonucleotide primer sequences.
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