abnormality results from double point mutations of the
emopamil binding protein gene (Ebp)
Kwang Won Seo,
Richard I. Kelley,
Department of Experimental Animal Science, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan
Kennedy Krieger Institute and Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA
Received: 9 January 2001 / Accepted: 1 April 2001
Abstract. Mouse Td
(Tattered-Hokkaido) was described as be-
ing allelic with Td in our previous study. Both allelic genes, which
are located at the same position on the centromere of the X Chro-
mosome (Chr), generate similar phenotypes such as male embry-
onic lethality, and in heterozygous females, hyperkeratotic skin,
skeletal abnormalities, and growth retardation. The emopamil
binding protein gene (Ebp) emerged as a candidate for mouse Td
mutation, since the Td gene was recently determined to result from
a point mutation of Ebp. In this study, Ebp cDNA of Td
demonstrated to possess double point mutations that cause two
amino acid changes from Leu to Pro at position 132 and from Ser
to Cys at 133 in EBP protein. EBP participates in cholesterol
biosynthesis, and cholest-8(9)-en-3␤-ol was found to be increased
in the plasma of Td
adult females but not in that of normal mice.
From these results, a loss of function was expected for the EBP
protein encoded by Td
. Both the phenotypes and genes respon-
sible for Td
as well as Td are quite similar to those of human
X-linked chondrodysplasia punctata (CDPX2).
(Tattered-Hokkaido) was described as being allelic
with Td in our previous study (Seo et al. 1997). Both genes, which
are located near the centromere of the X Chr, generate similar
phenotypes such as male embryonic lethality, and in heterozygous
females, hyperkeratotic skin, skeletal abnormalities, and growth
retardation. In this Td critical area, there are several genes and
many kinds of ESTs (Blair et al. 1995; Schindelhauer et al. 1996).
As one of these genes, Ebp (emopamil binding protein gene) was
recently identified to be the gene responsible for Td, which pos-
sesses a point mutation at the Ebp gene (Derry et al. 1999) and
was, therefore, evaluated as a candidate gene for Td
Ebp has been identified as a gene that encodes a nonglyco-
sylated type I integral membrane protein of endoplasmic reticulum
and shows high level expression in epithelial tissues such as liver,
intestine, adrenal gland, testis, ovary, and uterus of guinea pigs
(Hanner et al. 1995; Moebius et al. 1993). The EBP protein has
emopamil binding domains, including the sterol acceptor site and
the catalytic center (Moebius et al. 1996), which show ⌬
isomerase activity (Silve et al. 1996). Human sterol isomerase, a
homolog of mouse EBP, is suggested not only to play a role in
cholesterol biosynthesis, but also to affect lipoprotein internaliza-
tion (Dussossoy et al. 1999). In humans, mutations of EBP are
known to cause the genetic disorder of X-linked dominant chon-
drodysplasia punctata (CDPX2) (Braverman et al. 1999; Derry et
al. 1999). This syndrome of humans is lethal in most males, and
affected females display asymmetric hyperkeratotic skin and skel-
etal abnormalities (Bruch et al. 1995; Happle 1995; Heymans et al.
1985), which are similar to the phenotypes of Td and Td
this study, we examined whether or not the Td
sults from mutation of the Ebp gene.
Materials and methods
Cloning and sequencing of Ebp gene.
gene has been kept in
the congenic CKH strain in our laboratory. Total RNAs of Td
normal congenic males were extracted from each three independent em-
bryos at 12.5 days post-coitum (dpc) using Isogen (Nippon Gene, Tokyo,
Japan). The full coding region of the Ebp cDNA was synthesized with
reverse transcription-polymerase chain reaction (RT-PCR) from 3 gof
total RNA using Superscript™ II (Gibco-BRL, Gaithersburg, Md.). PCR
was carried out with primer 1 (TAT GTA CGA AGC TGC CAG CG;
position 104–123) and primer 2 (CCT GTG CGA GGA GGA AGA AG;
position 914–895) with a Takara PCR thermal cycler (Takara, Kyoto,
Japan), starting at 94°C for 5 min (one cycle), followed by 30 cycles of
94°C for 1 min, 65°C for 1 min, and 72°C for 1 min, and at 4°C at the end.
The PCR mixture and enzymes were purchased from Toyobo (Osaka,
Japan). For ligation of the PCR product, pGEM-T Easy vector (Promega,
Madison, WI) was used. DNA sequencing was performed with an ABI
Prism 377 genetic analyzer (Perkin Elmer Cetus, Norwalk, Conn.) with an
ABI Prism™ dye terminator cycle sequencing reaction kit (Perkin Elmer
Sterol content of plasma.
Plasma (100 l) was obtained from 8-week-
/+ females and age-matched controls from CKH normal
females. Sterol content of plasma samples was determined by gas chro-
matography and selected-ion mass spectrometry (GC/MS) in comparison
with purified sterol standards as described (Kelley 1995).
Northern blot analysis.
Total RNAs from the embryos at 12.5 dpc and
the livers of 8-week-old females were isolated with Isogen, and 20 gof
each RNA was loaded in a lane of electrophoresed agarose gel for Northern
blot analysis. Northern blotting was performed in hybridization buffer
containing 5× SSC (1× SSC consisted of 150 m
NaCl and 15 m
citrate, pH 7.0), 5× Denhart’s solution (1× Denhart’s solution consisted of
20 mg of polyvinyl pyrrolidone, 20 mg of Ficoll 400, and 20 mg of bovine
serum albumin per 100 ml), 0.5% SDS, 0.2 mg/ml salmon sperm DNA, and
50% formamide for 16 h at 42°C with the [␣-
P]dCTP labeled probe of
Ebp cDNA, which was made from the PCR product using primers 1 and 2.
After hybridization, the filter was washed twice with 2× SSC and 2% SDS,
each time for 5 min at 42°C, and 0.1× SSC and 0.1% SDS for 5 min at
65°C, and then exposed to X-ray film (Fuji Photofilm Co., Tokyo, Japan)
at −80°C. The filters were reused for the internal standard probe of the
glyceraldehyde-3-phosphate dehydrogenase gene after dehybridization.
The sequence of Ebp cDNA. The full coding region of Ebp cDNA
from the Td
male embryo was isolated and sequenced. Double
Correspondence to: T. Watanabe; E-mail: firstname.lastname@example.org.
Mammalian Genome 12, 602–605 (2001).
© Springer-Verlag New York Inc. 2001