Discovery of the BMPR1A promoter and germline
mutations that cause juvenile polyposis
Daniel Calva-Cerqueira1, Fadi S. Dahdaleh1, George Woodfield1, Sathivel Chinnathambi1,
Peter L. Nagy2, Joy Larsen-Haidle3, Ronald J. Weigel1and James R. Howe1,∗
1Department of Surgery, Carver College of Medicine, University of Iowa Hospitals and Clinics, 200 Hawkins Drive,
Iowa City, IA 52242-1086, USA2Department of Pathology, Columbia University, New York, NY 10032, USA and
3Department of Genetics, Hubert Humphrey Cancer Center, Robbinsdale, MN 55422, USA
Received June 25, 2010; Revised September 2, 2010; Accepted September 8, 2010
Juvenile polyposis (JP) is an autosomal dominant hamartomatous polyposis syndrome where affected indi-
viduals are predisposed to colorectal and upper gastrointestinal cancer. Forty-five percent of JP patients
have mutations or deletions involving the coding regions of SMAD4 and BMPR1A, but the genetic basis of
other cases is unknown. We set out to identify the JP gene in a large kindred having 10 affected members
without SMAD4 or BMPR1A coding sequence mutations or deletions. We found a germline deletion segregat-
ing in all affected members, mapping 119 kb upstream of the coding region of BMPR1A by multiplex ligation-
dependent probe amplification and comparative genomic hybridization. To further understand the genomic
structure of BMPR1A, we performed 5′RACE from lymphoblastoid cell lines and normal colon tissue,
which revealed four non-coding (NC) exons and two putative promoters. Further analysis of this deletion
showed that it encompassed 12 433 bp, including one promoter and NC exon. The activities of each promoter
and deletion constructs were evaluated by luciferase assays, and the stronger promoter sequence analyzed
for changes in JP patients without SMAD4 or BMPR1A alterations. A total of 6 of 65 JP probands were found
to have mutations affecting this promoter. All probands examined had diminished BMPR1A protein by ELISA,
and all promoter mutations but one led to significantly reduced luciferase activity relative to the wild-type
promoter reporter. We conclude that we have identified the promoter for BMPR1A, in which mutations may
be responsible for as many as 10% of JP cases with unknown mutations.
Juvenile polyposis (JP) is a hamartomatous polyposis syndrome
?1 in 100 000 individuals (1). JP predisposes patients to devel-
tinal (GI) tract (2), and a .50% cumulative lifetime risk
of developing colorectal cancer (3), as well as gastric cancer
(3–6). Germline mutations in coding regions of BMPR1A and
SMAD4 have been found in 45% of patients with JP, by sequen-
cing (7,8) and mixed ligation-dependent probe amplification
(MLPA) (9–11). Therefore, the genetic basis of JP is unknown
in 55% of patients, raising the question of whether additional
predisposing genes or other genetic mechanisms affecting non-
coding (NC) regions of BMPR1A or SMAD4 are the cause.
Germline mutations in regulatory regions of genes are more
difficult to detect than in coding regions, but have been shown
to be important in several heritable cancer syndromes. In her-
editary non-polyposis colorectal cancer (HNPCC), mutations
in the promoter regions of hMLH1 and hMSH2 have been
found in affected individuals, with one segregating in a three
generation family (12–14). In Cowdens syndrome, up to
10% of patients have substitutions within the PTEN promoter
region, with corresponding decreases in PTEN protein levels;
all of these cases were sporadic, however (15). In breast
cancer, a large deletion spanning the BRCA1 promoter
region, exons 1a, 1b and 2, was found in eight affected
members of a breast cancer family, and a subset of familial
breast cancer cases are due to deletions involving the
BRCA1 promoter (16).
∗To whom correspondence should be addressed. Tel: +1 3193561727; Fax: +1 3193538940; Email: firstname.lastname@example.org
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Human Molecular Genetics, 2010, Vol. 19, No. 23
Advance Access published on September 14, 2010
Understanding the regulatory regions of SMAD4 and
BMPR1A is not only important for us to be able to perform
more comprehensive screening of patients at risk for JP, but
also because these genes have been implicated in other dis-
eases and in the pathogenesis of human cancer. In this
study, we set out to characterize the NC elements of
BMPR1A because of the finding of a germline deletion in a
JP family affecting a region .100 kb upstream from the
Genetic screening studies
In order to search for a new JP gene, we collected DNA from a
large Iowa JP family without germline mutations of BMPR1A
or SMAD4 by sequencing, which consisted of 10 affected, 10
unaffected or unknown individuals and 7 spouses. We began
performing a genome screen using simple tandem repeat poly-
morphisms, but MLPA analysis revealed a possible deletion in
two probes from a NC exon of BMPR1A (Fig. 1A). We tested
for this in the rest of the family, and all 10 affected members
were found to have this deletion. No affected family members
had upper GI polyps documented, or extraintestinal manifes-
tations of JP, including macrocephaly, hereditary hemorrhagic
telangiectasia or cardiac defects (Table 1).
In order to further define the precise deletion, we performed
CGH using DNA from an affected and unaffected member of
this family. This revealed a heterozygous loss of the 10 probes
between 88 515 308 and 88 529 316 on chromosome 10
(Fig. 1B). To determine the precise site of the deletion, PCR
primers were chosen from just inside of the two flanking
CGH probes with normal copy number. Amplification of a
515 bp product was possible in all affected members of the
family (Fig. 2), despite the fact that this region spans
12 433 bp in genomic DNA. Sequencing of this product
revealed that the 5′breakpoint occurred within the second
and third units of three 5 bp repeats (cgggg), and there were
two 4 bp repeats (gctt) beginning 11 bp before and 2 bp after
the 3′end of the deletion (Fig. 3). Genomatix software pre-
dicted that the sequence spanning from 370 bp upstream to
150 bp downstream of the 5′deletion breakpoint was the
likely promoter region for BMPR1A. This region is located
119 379 bp upstream from the 5′UTR and first coding exon
To better define putative promoter regions for BMPR1A, we
performed 5′RACE from both normal and JP patient lympho-
blastoid cell lines (LCLs), and normal colon tissue. This
revealed several different splicing isoforms and four different
NC exons (Fig. 4). These results were compared with cDNAs
and ESTs from the UCSC browser (http://genome.ucsc.edu/),
and suggested two possible promoter regions for these differ-
ent splice variants. Promoter B was the one with 150 bp
deleted in members of JP family 19 and was also the most
likely promoter for the majority of isoforms seen by
5′RACE. Another potential promoter (promoter A) was ident-
ified just upstream of NC exon 1.
Figure 1. MLPA and CGH result in JP family 19. (A) MLPA of the proband showing 50% decreased amplification of the probes within the second NC exon of
BMPR1A. (B) aCGH array probe locations within the genomic DNA sequence upstream from BMPR1A on Chromosome 10 (vertical black lines). The red rec-
tangle represents the deleted region and the green vertical line represents the two MLPA probes that had decreased amplification. The 5′UTR of BMPR1A begins
at 88 635 624.
Human Molecular Genetics, 2010, Vol. 19, No. 234655
Luciferase expression vectors and luminometry
The candidate promoter sequences were cloned into luciferase
vectors and transfected into human embryonic kidney cells
(HEK-293) and colonic fibroblast cells (CRL-1459) to test
their activity. Progressively shorter deletion constructs were
also made to determine the regions of greatest promoter
activity. Promoter A was found to have minimal activity,
with a mean of eight normalized light units (lu) in HEK-293
and 2 lu in CRL-1459 (Fig. 5A). In contrast, promoter B
was much stronger, with 291 lu in HEK-293 and 36 lu in
CRL-1459. Deletion constructs showed a sharp drop in lucifer-
ase activity with loss of the 5′80 bp, and gradually decreasing
to negligible activity in the terminal 120 bp construct
Molecular screening for genetic alterations within
the promoter region
affected and one unaffected member of family 19 with partial
Table 1. Clinical characteristics of JP Family 19
ProbandAge Age at DxColon polyps Number of polypsUGI polyps Extraintestinal manifestationsColon cancer Colectomy
Cyst on wrist
aCarrier by virtue of affected offspring.
Figure 2. Segregation of the deletion within affected family members. (A) Pedigree of JP family 19; (B) PCR product representing the deleted segment, as
demonstrated by the presence of the 515 bp product; (C) amplification products using control primers located within the deleted segment, representing the wild-
type chromosome 10. LML, low-molecular weight ladder.
4656Human Molecular Genetics, 2010, Vol. 19, No. 23
Figure 3. Sequence surrounding the 12 433 bp deletion in JP family 19 upstream from BMPR1A. (A) The sequence in the box starts at base 88 515 880 and ends
at 88 528 955. Bold letters in blue show the deleted region with...representing most of the nucleotides deleted. The sequence which is underlined is the second
NC exon; the yellow area is the putative promoter sequence as predicted by Genomatix software; the blue highlighted areas show the primers used to sequence
across the deletion; the green highlighted areas represent the two deleted MLPA probes (from Fig. 1B); the red box surrounds the six bases before the deletion,
and the green open box the six bases after the end of the deletion; (B) chromatogram from the 515 bp PCR product sequence flanking the deleted segment.
Figure 4. Clones derived from 5′RACE experiments. The exact positions of the NC exons, the 5′UTR and first coding exon, and the two promoters are located
above the corresponding rectangles [from February 2009 Genome Reference Consortium (GRCh37), UCSC hg19 assembly]. The deletion found in family 19 is
shown in the figure. The numbers in black at right represent the number of 5′RACE clones identified and the numbers in red are the ESTs described in the UCSC
genome browser (44 containing sequence 5′to the coding exons and 57 with coding exons only).
Human Molecular Genetics, 2010, Vol. 19, No. 23 4657
deletion of promoter B revealed that affected members had
ter B was then sequenced in 100 normal controls, and 64
additional JP probands who had previously not been found to
etions by MLPA. Thirty-one of these probands had previously
been sequenced for ENG and PTEN and no disease-causing
mutations were identified (17). Two common polymorphisms
and 62 of 100 controls. Four different substitutions were found
BMPR1A protein levels were reduced in LCLs from these
patients (LCLs were available for three of the five probands
Site-directed mutagenesis of transcription factor
To confirm that the 150 bp deletion seen in our JP family or
the substitutions found in other JP probands would have func-
tional significance, the luciferase activity of constructs with
these changes were compared with the full-length promoter
sequence. Deletion of the terminal 150 bp led to complete
loss of promoter activity, while the proband 86 (2482
A.T) construct had only 24% the activity of the wild-type
promoter, the proband 100 (2320 G.C) 52%, and the pro-
bands 42 and 117 (2328 G.T) construct had 46% activity
(Fig. 6B). In contrast, the proband 13 (2386 G.A) construct
had similar luciferase activity to the wild-type promoter, while
BMPR1A protein levels were reduced to 31% of controls.
In silico promoter analysis
Gene2Promoter software predicted the 520 bp region just
upstream from NC exon 2 to be a promoter for BMPR1A.
Figure 5. Luciferase expression from BMPR1A promoter deletion constructs.
Results of luciferase assays (with standard deviations) from the various del-
etion constructs of promoters A (A) and B (B) from the two cell lines
HEK-293 and CRL-1459; the size of each is the number of bases 5′to the
TSS. The 520 bp construct of promoter B did not have significant increase
in the luciferase activity when NC exon 3 was added to the construct (data
Figure 6. BMPR1A protein levels in family 19 and luciferase activity for
different alterations in the BMPR1A promoter. (A) Results of ELISA normal-
ized to controls for BMPR1A with the corresponding standard deviations from
four members of kindred 19; #350, 202 and 302 have the deletion and are
affected (black), whereas 201 does not and is unaffected (white). (B) Lucifer-
ase activity in HEK-293 cells of genetic alterations in the BMPR1A promoter
found in JP probands, created by SDM of the wild-type promoter construct.
Table 2. GA of the BMPR1A promoter found in JP probands
Del 2150 to 21 2 MZF-1, TF2B, 2 SP-1 39%
2306 G/G.C/G E2F
2328 G/G.T/G MZF-1
2328 G/G.T/G MZF-1
2386 G/G.A/G None identified
GA, genetic alterations.
aPutative RBSs at the site of each substitution.
bBMPR1Aproteinlevelsrelative to controlsforthe fourJP probands evaluated.
cPercent of luciferase activity of SDM clone relative to the wild-type promoter
4658Human Molecular Genetics, 2010, Vol. 19, No. 23
This region had 78 CpG sites, and MatInspector software pre-
dicted many potential transcription factor regulatory-binding
sites (RBSs) (Fig. 7A). This was evaluated by RegionMiner
for orthologous sequences within 12 different species
(Macaca mulatta, Pan troglodytes, Mus musculus, Rattus nor-
vegicus, Equus caballus, Canis familiaris, Bos taurus, Sus
scrofa, Monodelphis domestica, Ornithorhnuchus anatinus,
Danio rerio and Gallus gallus) and 4 species were found to
have levels of conservation ranging between 47.2 and 66.8%
(Table 3). Sequences from these four species were then ana-
lyzed alongside the human BMPR1A promoter B sequence
using FrameWorker, and two 10-element models were
factors (EGRF, DEAF, AP2F, ZBPF, ZF5F, CTCF, SP1F
and MTEN) across the five species. Both ZBPF and EGRF-
binding sites were seen within the last 150 bp of promoter B
(Fig. 7B), which would be lost in the deletion affecting JP
By current sequencing methods and MLPA screening, ?40–
45% of JP patients have genetic alterations detected in
BMPR1A or SMAD4 (9). The failure to detect mutations in
over half of the JP patients is a serious obstacle for the screen-
ing of patients at risk, and our further understanding of the
molecular basis of JP. JP kindred 19 clearly shows that del-
etion of the promoter and NC exon 2 of BMPR1A causes aber-
rant BMP signaling, with decreased levels of BMPR1A
protein in affected members, and elimination of luciferase
activity of this promoter construct.
Two candidate promoter regions were selected for study due
to their high GC content, proximity to transcription start sites
(TSS), the presence of regulatory sequences (such as a
TATA-box, SP1 and AP2 sites), and gene structure. There
were six different splice variants found, with only one contain-
ing NC exon 1, two with NC exon 2, five with NC exon 3, two
with NC exon 4 and one without NC exons (Fig. 4). Isoforms
with NC exon 1 splicing into NC exon 3 and NC exon 2 into 3
were found, but not 1 into 2, suggesting that there might be sep-
arate promoter regions upstream of NC exons 1 and 2. These
Figure 7. (A) Map of the 520 nucleotides that make up BMPR1A promoter B. The various deletion constructs that were cloned into luciferase vectors are shown,
and the putative transcription factor-binding sites are depicted. JP patient (Pt) substitutions are also shown. (B) Cross-species comparison of the BMPR1A pro-
moter reveals two 10-element models that are conserved (key in inset for both A + B).
Table 3. Percentage of conservation between species for the BMPR1A promo-
ter B region
Species Percent conservation
Human Molecular Genetics, 2010, Vol. 19, No. 23 4659
regions were cloned into luciferase vectors, as were the
sequences immediately upstream from NC exon 3 and the
5′UTR. No luciferase activity was found in the latter two
regions (data not shown), while promoter A (upstream of NC
exon 1) showed some weak activity and promoter B much
greater activity in HEK-293 and CRL-1459 cells (Fig. 5).
The Gene2Promoter prediction software program identified
to be the most important promoter for BMPR1A. However, we
cannot rule out the possibility that promoter A (or others) plays
a role in different tissues, during various stages of development,
or in different contexts influenced by the abundance and combi-
nations of specific transcription factors. Further evaluation of
promoter B using MatInspector (http://www.genomatix.de/p
://www-bimas.cit.nih.gov/molbio/proscan/) identified several
potential binding sites that may regulate transcription of
BMPR1A (Fig. 7). One important transcription factor may be
the myeloid zinc finger 1 factor (MZF1), for when this site was
lost in the 440 bp deletion construct, there was a 59–81% drop
in luciferase activity (in CRL-1459 and HEK-293, respectively)
the 520 bp construct) there are four SP-1 sites,one E2F site, one
AP-2 site and one additional MZF1-binding site. The 120 bp
construct had .95% reduction in promoter activity relative to
the 520 bp construct, and there is one SP-1 site, one AP-2, an
RNA PolII transcription 2B-binding site (TF2B) and a core pro-
moter motif 10 (MTEN) element located in the sequence
est decrease in luciferase activity was seen between the 520 and
225 bp constructs, and this region contains two MZF1-binding
sites, two AP-2 sites, one E2F site, one ZF5F site and four
latory elements for BMPR1A transcription. Also of interest was
the fact that JP patient substitutions were found within MZF1,
E2F and ZF5F sites. Finally, since in silico analysis determines
potential binding sites for transcription factors based upon
degrees of homology to various consensus sequences, further
species comparison. This 520 bp region was conserved across
several species, with 47% orthology to the brown rat and 68%
to the wild boar. The 10 element transcription factor models
were remarkably similar across these species, thereby confirm-
ing their potential relevance (Fig. 7B).
Deletions of BMPR1A have previously been reported in JP
patients, but these have been very large, usually involving the
entire gene. Delnatte et al. (18) described four patients with JP
having early age of onset (1–18 months), both upper and
lower GI polyps, and macrocephaly who all had contiguous
deletions of both PTEN and BMPR1A. Salviati et al. (19)
reported one patient with a 12 Mb deletion on chromosome
BMPR1A), with lower GI polyps (but no upper GI), onset at
age 5, and no macrocephaly. Menko et al. presented four
additional patients with deletions encompassing PTEN,
BMPR1A and at least eight other genes. All these patients
had macrocephaly, facial dysmorphism and psychomotor
retardation, three had juvenile polyps (presenting at ages 2–
4 years) and the other had metastatic rectal cancer diagnosed
at age 24. They concluded that the clinical phenotype seen
with contiguous deletion of these two genes was variable,
did not always fit the criteria for JP of infancy, and was not
necessarily related to the size of the deletion (20).
Others have found deletions of BMPR1A by MLPA. Aretz
et al. (11) found one JP patient with deletions of all probes
of PTEN and BMPR1A (but did not describe the phenotype),
one with loss of two BMPR1A NC exon probes and 2 exon
1 probes (age of onset 2 years, 11 total colonic polyps), and
the final with loss of exon 1 probes (onset at age 8, total of
.30 colonic polyps). van Hattem et al. (10) found one
patient with deletion of BMPR1A exons 10 and 11, and two
patients had deletions of all probes for both PTEN and
BMPR1A, but no clinical information was provided. We pre-
viously described a patient with deletions of all MLPA
probes for PTEN and BMPR1A (9). This individual was diag-
nosed with rectal bleeding at age 20 and had a partial colect-
omy at age 21, later had a gastrectomy for gastric polyps at
age 54, and then developed rectal cancer at age 55 and had
a total proctocolectomy for a T1N0M0 lesion. He had head
circumference in the 95th percentile (55.7 cm), asymmetric
mouth opening, inguinal hernia repairs in infancy and child-
hood, sensorineural hearing loss and had had trichoepithelio-
mas and dermatofibromas removed in the past. His parents
and two siblings were phenotypically normal, without
known polyps or rectal bleeding, and one of his sons had
polyps and a colectomy performed at age 24.
Members of our JP family with a 12.4 kb deletion segregat-
ing in 10 affected individuals from two generations did not
have upper GI polyps, macrocephaly or an early age of
onset (Table 1). This deletion does not involve any other
genes on 10q23, besides the NC region of BMPR1A. More
specifically, this deletion results in loss of the last 150 bases
of promoter B, which by luciferase experiments caused a
.95% drop in activity relative to the 520 bp construct
(Fig. 6B). This loss of activity is perhaps due to the loss of
an SP-1 site from positions 2113 to 2120 or another from
positions 2150 to 2161, which has the last base at position
2150 within the large deletion, or the TF2B-binding site
(from 2119 to 2124), or 2 MZF1 sites (Fig. 7A).
Besides affecting this promoter, the deletion also included
the contiguous NC exon 2 and 12 kb of downstream intron.
The role of BMPR1A NC exons is more difficult to define,
and further study into their activity and molecular structure
will help to unravel the role they play in transcription, trans-
lation, cellular localization, mRNA stability or interaction
with other molecules involved in BMP signaling. Differential
expression of the BMPR1A NC exons may be tissue specific,
and may be an important influence on the variability and
timing of gene and protein expression. Whether the loss of
NC exon 2 in family 19 plays a role in the development of
JP in addition to promoter B remains to be seen.
There are several examples of inherited GI cancer syn-
dromes that result from genetic alterations of promoter
regions of their predisposing genes. Up to 10% of patients
with Cowden syndrome have substitutions within the PTEN
promoter region with a corresponding 50% decrease in
PTEN protein levels. Furthermore, these substitutions lead to
4660 Human Molecular Genetics, 2010, Vol. 19, No. 23
increased phosphorylation of Akt, which is believed to lead to
increased activity of the pro-proliferative PI3K/Akt pathway,
possibly leading to the genesis of cancer in patients with
Cowden’s sydnrome (15). All of these cases were sporadic,
however, and inheritance of these mutations within families
was not demonstrated.
In HNPCC, Shin et al. described hMLH1 and hMSH2 core
promoter mutations in 96 cases or suspected cases, and found
germline changes in the hMSH2 promoter that segregated
within affected members of 2 families. One was an A insertion
at 280 in an affected mother and son but not the unaffected
daughter, and the other was a G-C transversion at 2225 in the
proband of a family with multiple cancers. The former change
reduced the activity of a luciferase reporter vector by 82%,
and was within an E1A-F consensus sequence (12). Yan et al.
screened 37 suspected HNPCC cases for changes in the promo-
ters of hMSH2, hMLH1 and hMSH6, since mutations could not
be found in the coding regions. They found a deletion of GT at
278–9 in the promoter of hMSH2 segregating in three affected
members of an HNPCC family. This mutation led to an 87%
reduction in luciferase activity, and also appeared to affect
E1A-F binding (14). Green et al. found a C to T heterozygous
mutation at position 242 upstream of the translation start site
in the promoter region of hMLH1 in two affected members of
an HNPCC family and another individual with adenomatous
polyps. This change affected a putative Myb-binding site, and
led to reduced luciferase activity relative to the wild-type
Consequently, there is precedent for promoter mutations
causing autosomal dominant diseases, although these are rela-
tively uncommon. However, greater understanding of NC
regions of DNA will likely lead to many more such discov-
eries in the future. The specific example described here of a
12 kb deletion 119 kb upstream from the coding region of
BMPR1A segregating in all 10 affected members of a JP
family is particularly striking. The potential significance of
alterations of the promoter was confirmed by the finding that
5 of 64 additional JP probands without coding region
mutations of BMPR1A or SMAD4 had single base pair substi-
tutions associated with reduced BMPR1A protein levels and
reduced luciferase activity. Therefore, we conclude that
BMPR1A promoter mutations cause JP, and may be respon-
sible for up to 10% of genetically undefined JP cases.
MATERIALS AND METHODS
DNA screening and sequencing
All JP patient blood samples were collected under a protocol
approved by the Institutional Review Board at the University of
Iowa. Genomic DNA was extracted from peripheral blood by
salting out (21) or the Puregene DNA purification kit (Gentra
Systems, Minneapolis, MN, USA), and from LCLs by the
Qiagen AllPrep DNA mini kit (Qiagen, Valencia, CA, USA).
DNA was amplified using primers flanking each intron-exon
boundary of the 11 coding exons of BMPR1A and SMAD4, and
products were purified using Qiagen spin columns (Qiagen).
PCR products were sequenced by dideoxy cycle sequencing fol-
lowed byelectrophoresisthrough an ABImodel 3730automated
sequencer (Applied Biosystems, Foster City, CA, USA).
Individuals with no identifiable mutations by sequencing
were screened by MLPA for exonic deletions (MRC
Holland, Amsterdam). The large deletion identified in a JP
proband by the MLPA method at the 5′end of BMPR1A
was further characterized using an oligonucleotide array com-
parative genomic hybridization (aCGH) chip (HG18_WG_
CGH_5 of 8 array; NimbleGen Systems, Madison, WI,
USA), which has probes spaced approximately every 700 bp.
Primers were designed using the Primer3 program (http://
frodo.wi.mit.edu/) (22) to make probes flanking the deletion
as determined by the aCGH chip.
Sandwich ELISA was performed in the four members of
family 19 to determine relative protein levels of BMPR1A
Systems, Minneapolis, MN, USA), and for actin as a control
Gilbertsville, PA, USA). The detection antibodies used were
BMPR1A-ab59947-100 (Abcam, Cambride, MA, USA) and
Actin-A5316-Sigma (Sigma). Quantification of protein was
performed by measuring optical density as read at 450 nm in
a Spectra Max Plus 384 (Molecular Devices, Sunnyvale,
CA, USA) after addition of secondary antibody conjugated
with HRP (Anti-Rabbit-HRP-sc-2030 or Anti-Mouse-HRP-
sc-3697; SantaCruz Biotechnology, Santa Cruz, CA, USA).
cDNA was created using gene-specific primers from RNA
extracted from a control LCL and fresh colon tissue, using
the Invitrogen 5′RACE kit (Invitrogen). Clones with inserts
were sequenced to determine different splice variants, poten-
tial TSS and the most abundant mRNA isoforms.
The putative promoter regions were evaluated using the
genomatix.de) and Promoter Scan 1.7 (http://www-bimas.cit.
nih.gov/molbio/proscan/). Deletion constructs from the puta-
tive promoters were then amplified from genomic DNA by
PCR using successively shorter sequences upstream from
each putative TSS. The primers used to amplify the deletion
constructs had a MluI restriction endonuclease site incorpor-
ated into the 5′end and BglII at the 3′end. The PCR products
were then ligated into the pGL3 luciferase basic reporter
vector (Promega, Madison, WI, USA).
Five micrograms of pGL3 constructs was transfected using
Transfast (Promega) in a 1:2 ratio into a normal human
CRL-1459 and the HEK-293 cell lines (American Type
Culture Collection, Manassas, VA, USA), and co-transfected
ter (pRL-CMV; Promega). Cells were incubated with MEM
with 10% serum and penicillin/streptomycin/amphotericin for
72 h. Triplicate reactions were performed for each construct.
Cells were lysed and firefly luciferase activity was measured
at 562 nm for 10 s (using the Dual-Luciferase Reporter Assay
kit, Promega) with the TD 20/20 luminometer (Turner BioSys-
Human Molecular Genetics, 2010, Vol. 19, No. 234661
tems, Sunnyvale, CA, USA). Renilla luciferase activity was Download full-text
then measured at 480 nm for 10 s. Firefly luciferase activity
for each construct was determined by subtracting the back-
ground firefly luciferase activity from the control pGL3
basic vector and normalizing to the Renilla luciferase activity.
MatInspector software (www.genomatix.de/products/MatInsp
ector) was used to identify potential RBSs from each promoter
sequence, RegionMiner to search for orthologous sequences in
different species, and FrameWorker to identify different tran-
(23–26). The promoter B region was screened by sequencing
in 64 JP probands that did not have any coding mutations in
both SMAD4 and BMPR1A or large exonic deletions by
MLPA, and 100 normal controls. Site-directed mutagenesis
(SDM) of potential RBSs was performed using the Quik-
Change II kit to evaluate the luciferase activity of mutations
in the promoter (Stratagene, La Jolla, CA, USA).
We are grateful to all the patients who donated samples for
this study and to the physicians and genetic counselors who
Conflict of Interest statement. None declared.
This work was supported by NIH 1R01CA136884-01, the
Roy J. Carver Charitable Trust and the Susser family.
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