Download Identification of a novel duplication in the APC gene using multiple

Cancer Genetics and Cytogenetics 182 (2008) 130e135
Short communication
Identification of a novel duplication in the APC gene using
multiple ligation probe amplification in a patient with
familial adenomatous polyposis
Lucia Pedacea, Silvia Majorea, Francesca Megiornib, Francesco Binnia,
Carmelilia De Bernardoa, Ivana Antigonia, Nicoletta Preziosia,
Maria Cristina Mazzillib, Paola Grammaticoa,*
a
Medical Genetics, Experimental Medicine Department, University of Rome ‘‘La Sapienza,’’ S. Camillo-Forlanini Hospital,
Circ. ne Gianicolense n. 87, 00152 Rome, Italy
b
Medical Genetics, Experimental Medicine Department, Sapienza University, Rome, Italy
Received 10 October 2007; received in revised form 11 January 2008; accepted 24 January 2008
Abstract
Germline mutations in the adenomatous polyposis coli (APC ) gene cause familial adenomatous
polyposis (FAP), an autosomal dominant disease characterized by hundreds to thousands of adenomatous polyps in the colon and rectum, with progression to colorectal cancer. The majority of APC
mutations are nucleotide substitutions and frameshift mutations that result in truncated proteins.
Recently, large genomic alterations of the APC gene have been reported in FAP. DNA from 15
FAP patients, in whom no APC germline mutations were detected with denaturing high performance liquid chromatography, was analyzed with multiplex ligation-dependent probe amplification
(MLPA) to evaluate gross genomic alterations in the APC gene. In one case, MLPA identified
a novel duplication of exons 2e6 in one copy of the APC gene. Reverse transcriptaseepolymerase
chain reaction revealed that the mutant allele contained an in-frame multiexon duplication including
18 nucleotides located in exon 2, upstream of the ATG initiation codon. The presence of a premature
stop codon in the duplicated sequence leads to the synthesis of a truncated APC polypeptide. These
findings highlight the utility of evaluating infrequent APC mutation events in FAP patients using
MLPA. Ó 2008 Elsevier Inc. All rights reserved.
1. Introduction
Familial adenomatous polyposis (FAP; MIM#175100) is
an autosomal dominant disease that predisposes patients to
colorectal cancer. Patients with FAP typically develop hundreds to thousands of adenomatous polyps in the colon and
rectum, beginning at a mean age of 16 years (range, 7e36
years). Without colectomy, FAP patients inevitably develop
colorectal cancer, usually by the age of 35e40 years [1].
They may have extracolic manifestations, such as upper
gastrointestinal tract tumors, osteomas, desmoid tumors,
epidermal cysts, and congenital hypertrophy of the retinal
pigment epithelium [2]. The phenotypic features of FAP
are classified according to the number of polyps that are
detected during colonoscopy. FAP is deemed classic type
when 100 to O1,000 adenomas are found and attenuated
* Corresponding author. Tel.: and fax: þ39-06-58704646.
E-mail address: [email protected] (P. Grammatico).
0165-4608/08/$ e see front matter Ó 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.cancergencyto.2008.01.009
type (AFAP) when the number of polyps ranges from 10
to 100 [3].
Familial adenomatous polyposis is caused by a germline
mutation in APC gene on chromosome 5q21 (Ensembl
Gene ID ENSG00000134982; http://www.ensembl.org)
[4,5]. APC is composed of 16 exons and encodes a major
10.7-kb transcript. The open reading frame (exons 2e16)
is translated into a 312-kDa, 2843-amino-acid protein [6].
The APC protein is an integral part of the Wnt-signaling
pathway [7], and it also plays a role in cellecell adhesion,
stability of the microtubular cytoskeleton, cell cycle
regulation, and apoptosis [8].
APC mutations have been detected in up to <95% of patients with classic FAP and in ~10% of AFAP patients [9].
More than 800 disease-causing APC mutations have been
identified [10]. Nearly 94% of APC germline mutations
involve the introduction of a premature stop codon, either
by nonsense mutations (33%) or frameshift mutations
(small insertions and deletions; 61%), leading to truncation
L. Pedace et al. / Cancer Genetics and Cytogenetics 182 (2008) 130e135
of the protein product in its C-terminal region [11]. Germline mutations are generally spread over the entire coding
region, but are more frequent in the 50 half [12,13]. Several
recent studies have demonstrated the presence of less common APC mutations in FAP patients, including large genomic rearrangements. Approximately 10% of individuals
with an APC-associated polyposis condition have large
APC deletions [14e18]. A recent study reported an exon
duplication event that led to a truncating germline mutation
in the APC gene [19].
Here, we describe a novel APC duplication involving
exons 2e6 that was identified by multiplex ligationdependent probe amplification (MLPA).
2. Materials and methods
2.1. Patients
The DNA used in this study was from a cohort of individuals with multiple polyposis coli, referred to the Medical Genetics Unit of the University of Rome ‘‘La
Sapienza,’’ S. Camillo Hospital, for genetic counseling.
Disease status was verified by clinical data examination
(presence of O100 adenomatous polyps or !100 polyps
and a relative with FAP). All patients provided informed
consent.
Genomic DNA was isolated from affected individual
peripheral blood using QIAamp DNA blood mini kits
(Qiagen, Chatsworth, CA). For molecular study of APC,
published primers for the amplification of all exons were
used [4]. Amplicons were treated at 95 C for 10 minutes
and 60 C for 30 minutes to facilitate heteroduplex development and were analyzed by denaturing high performance
liquid chromatography (dHPLC) (Transgenomic, San Jose,
CA) [20]. The chromatogram from each tested patient was
overlaid with the wild-type profile, and samples with an
extra peak was sequenced bidirectionally on an ABI
PRISM 310 DNA sequencer (PE Applied Biosystems,
Foster City, CA).
Fifteen FAP patients for whom APC exon analysis had
failed to identify any pathogenetic germline mutations were
analyzed for large deletions or duplications using MLPA.
2.2. Multiplex ligation-dependent probe amplification
The MLPA test kit (APC kit PO43; MRC-Holland,
Amsterdam, Netherlands) contains 20 paired probes from
the APC region, to examine two fragments of the promoter
region, 50 untranslated mRNA region and coding exons of
the APC gene; the last exon is divided into three fragments
(start, middle, end). Two probes for the APC wild-type
sequence at mutation hotspots (codons 161 and 1309) are
present. The probe mix includes, as controls, 13 probes
for other human genes located on different chromosomes.
Screening was performed according to the manufacturer’s
instructions.
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Briefly, 100 ng of patients genomic DNA in 5 mL Trise
EDTA buffer was heated to 95 C for 5 minutes, cooled, and
then incubated with probe hybridization set for 16 hours at
60 C. Next, hybridized products were mixed with probe ligation at 54 C for 15 minutes and amplified by polymerase
chain reaction (PCR) using 6-FAM-labeled universal
primers. PCR products were separated by capillary electrophoresis on the ABI PRISM 310 analyzer (PE Applied
Biosystems). DNA samples from healthy individuals were
used as controls.
Data were collected and analyzed with GeneScan
(v.3.1.2; PE Applied Biosystems) software. Evaluation of
electropherograms was performed with visual evaluation
of peak height of the APC fragments in relation to the
adjacent control fragments. For each sample, relative peak
areas were calculated and compared with three controls,
using Coffalyser 5.4 software (MRC-Holland).
2.3. RNA analysis
EpsteineBarr viruseimmortalized lymphoblastoid cell
lines were established from peripheral blood lymphocytes
from a patient with APC duplication, for RNA analysis.
Total RNA was isolated using Trizol reagent (Invitrogen,
Carlsbad, CA). cDNA was synthesized with a reverse transcriptaseePCR kit (RT-PCR) (PE Applied Biosystems) and
amplified using forward (F) primers 50 -AGCTTCATA
TGATCAGTTG-30 (exon 2) and 50 -CGATGGAAGAACA
ACTAG-30 (exon 6) and reverse (R) primers 50 -GCTTG
ATACAGATCCTTC-30 (exon 4) and 50 -ATGTGAGCCG
GTTTCATG-30 (exon 8).
We performed amplifications with 2F-4R, 6F-8R, 2F-8R
and 6F-4R primer combinations under the following conditions: 94 C denaturation for 15 seconds; 35 cycles of 94 C
denaturation for 30 seconds, 52 C annealing for 30
seconds, and 72 C extension for 90 seconds; and a final,
10-minute extension at 72 C. RT-PCR of human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as
a control for the quantity of mRNA in each sample. Amplification products were resolved on a 2% agarose gel with
DNA molecular length marker. Novel fragments that were
not detected in normal controls were extracted from the
gel using a QIAquick gel extraction kit (Qiagen) and
analyzed on an ABI PRISM 310 DNA sequencer.
2.4. Western blot analysis
Total protein was extracted from lymphoblastoid cell
lines with RIPA buffer: 50 mmol/L Tris-HCl pH 8.0, 150
mmol/L NaCl, 1% NP-40, 0.5% deoxycholic sodium salt,
0.1% SDS, 0.2 mmol/L PMSF, and 1 protease inhibitor
mixture (Complete Mini, EDTA free; Roche, Indianapolis,
IN). A 200-mg quantity of the proteins was loaded onto
a 3% low-melting-point agarosee0.1% sodium dodecyl
sulfate gel or onto a 12% polyacrylamide gel and transferred
by capillarity to a polyvinylidene fluoride filter (Millipore,
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L. Pedace et al. / Cancer Genetics and Cytogenetics 182 (2008) 130e135
Billerica, MA). Blots were incubated with mouse monoclonal antibody against APC protein (Ab-1; Calbiochem, San
Diego, CA) for 2 hours at room temperature and then with
the horseradish peroxidase-conjugated anti-mouse antibody
(Jackson Immunoresearch Laboratories, West Grove, PA).
Specific bands were visualized by the enhanced chemiluminescence method (Amersham, Piscataway, NJ).
3. Results
Among a series of unrelated patients clinically diagnosed with FAP, 15 negative results were obtained by
screening for APC germline mutations using dHPLC and
direct sequencing of all samples exhibiting abnormal
dHPLC profiles. We then used MLPA analysis to screen
those samples for exon deletions or duplications. In one
patient, we observed an increase in copy number of exons
2e6, suggesting a duplication event involving one allele
of the APC gene (Fig. 1). The result was confirmed by
MLPA in three affected relatives of the patient (the proband’s father, uncle, and cousin), whereas two unaffected
family members did not have changes in copy number.
To determine if DNA duplication was maintained in
APC transcripts, we performed RT-PCR, using primers
flanking the duplication borders. cDNA amplified by 2F4R, 6F-8R, and 2F-8R primer combinations showed
additional bands in the patient sample, compared with the
lymphoblastoid cell control. In particular, PCR performed
with the 6F-4R primers produced an amplicon only in the
patient’s cDNA, showing the presence of a contiguous duplication (Fig. 2). Direct sequencing of the PCR fragments
from the patient sample confirmed that the mutated mRNA
contained in-tandem duplication of exons 2e6 inserted between exons 6 and 7, without a shift in the open reading
frame (Fig. 3A).
The duplicated segment includes 18 nucleotides of the 50
exon 2 untranslated region, located upstream of the ATG
initiation codon in the wild-type gene (Fig. 3B). The mutant
protein was predicted to have 218 amino acids, with an
expected molecular weight around 22 kDa. Western blot
analysis revealed the wild-type APC protein in the patient
and control cell lines; an additional band corresponding
to the mutant APC was observed only in the patient sample
(Fig. 4).
We investigated the mechanisms underlying the genome
rearrangement using the web-based RepeatMasker facility
(http://www.repeatmasker.org/) and BLAST (http://www.
ncbi.nlm.nih.gov/blast/). We identified 23 interspersed
repeat elements, including 9 Alu repeats in the 50 UTR of
the APC gene, and 19 interspersed elements, including 9
Alu repeats in intron 6. We detected two types of Alu
sequences: AluJ (oldest primate family), which showed
no recombination, and AluS (younger family), in which rearrangements have been recognized as a common source of
local recombination [21]. BLAST analysis of the two
sequences revealed 68 homologous regions (length,
30e750 bp) potentially able to form misalignments and
subsequent nonallelic homologous recombination, which
could explain the detected duplication.
Fig. 1. Results of multiplex ligation-dependent probe amplification (MLPA) analysis representing normalized ratios vs. position of the probes: APC exons
and control regions on other chromosomes (c). Ratio results: normal (ratio ~1), gain (ratio O 1.5), loss (ratio ! 0.5). The error bars indicate standard
deviation. DNA from the patient with two copies of exons 1e5 (exons 2e6, according to Ensembl Gene ID ENSG00000134982 [http://www.ensembl.org])
per cell suggests large APC duplications from one chromosome.
L. Pedace et al. / Cancer Genetics and Cytogenetics 182 (2008) 130e135
133
Fig. 2. Reverse transcriptaseepolymerase chain reaction products of the patient sample (mut) and normal samples (wt) on 2% agarose gel with DNA
molecular length marker (M).
Fig. 3. (A) cDNA sequence chromatograms showing abnormal junction between exon 6 and duplicated exon 2 (panel 1), and splice junction between
duplicated exon 6 and exon 7 without shift on the open reading frame (panel 2). (B) Exoneintron structure of wild-type and duplicated APC genes. Arrows
indicate primer position and amplification direction. Black areas indicate the five in-tandem duplicated exons. White areas indicate regions located upstream
of the ATG codon in exon 1and exon 2, that are not translated in the wild-type protein.
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L. Pedace et al. / Cancer Genetics and Cytogenetics 182 (2008) 130e135
Fig. 4. APC protein analysis in total extracts from lymphoblastoid cells of
the patient with the DNA microduplication (lane 1) and control lymphoblastoid cells (lane 2). Western blot probed with a monoclonal APC antibody (Ab-1; Calbiochem, San Diego, CA) shows expression of full-length
APC (~312 kDa) in both samples. The mutant APC protein with a predicted
22 kDa molecular weight was detected only in the patient’s cells (lane 1).
4. Discussion
Familial adenomatous polyposis is a clinically welldefined hereditary disease caused by germline mutations
in the APC gene. FAP is characterized by diffuse adenomatous polyposis in the colon and rectum, with variable
extracolonic manifestations. In the present study, we used
MLPA to screen 15 unrelated FAP cases that were negative
according to dHPLC. We identified one patient with a duplication event involving exons 2e6 in one copy of the APC
gene (Ensembl Gene ID ENSG00000134982). This patient
was 33 years old, and the age of FAP clinical diagnosis was
21 years; she had thousands of adenomatous colorectal
polyps and jaw-bone osteomas, an extracolonic manifestation that is often found in FAP patients with mutations between codons 767 and 1513 [22]. The family pedigree of
our patient showed a clear dominant inheritance pattern,
and all affected family members showed classic FAP. These
observation justified further investigation to confirm the
effect of duplication on gene expression in the affected
individuals.
The RT-PCR and subsequent sequencing revealed in the
patient the presence of a mutant mRNA copy containing an
in-tandem duplication of exons 2e6 that conserved the
APC open reading frame. The duplication event includes
insertion of the 18 nucleotides (GTCCAAGGGTAGCC
AAGG) located in exon 2 upstream of the APC coding sequence. Therefore, the first six codons of the duplicated
segment in our patient contain an in-frame stop signal that
leads to a truncated APC protein. Results of the Western
blot studies confirmed the presence of a shorter APC
protein.
We then supposed that mRNA presumably escapes
cytoplasmic degradation via nonsense-mediated RNA
decay (NMD) pathway [23]. The mutant allele is thus
predicted to result in a translated product containing APC
amino acid residues 1e215, plus three novel amino acids
(V, Q and G) at the COOH end. The predicted protein
should retain the N-terminus coil-coiled region, containing
the oligomerization domain (amino acids 6e57) and two
out of five nuclear export signals (NESs; amino acids
68e77 and 165e174) to shuttle the b-catenin-APC complex between nucleus and cytoplasm. Therefore, presence
of the oligomerization domain might lead mutant APC to
form dimers of wild-type APC protein.
Our findings allowed us to support the hypothesis that
the predicted mutant protein may display a dominant negative effect by reducing APC tumor-suppressor wild-type
allele function. In favor of this hypothesis, it has been
shown that truncated APC protein influences migration of
colon cancer cells, enhances chromosomal instability in
a dominant manner, and counteracts the degradation of
b-catenin catalyzed by wild-type APC [24e27], unlike
null alleles APC, which support the haploinsufficiency
model in which an unstable truncated protein with a reduced
amount of normal APC induces a less severe phenotype.
Moreover, such proteic complexes might be dysfunctional
or recognized and destroyed. The dominant negative effect
of mutant APC protein could partially explain the classic
phenotype and early age of FAP onset in our patient.
In summary, we here report a novel APC duplication of
exon 2e6 in a patient with FAP. This mutation was identified by MLPA and characterized by RT-PCR and direct
sequencing. Our finding indicates that the combination of
MLPA and dHPLC analysis is useful for genetic testing
of common and uncommon APC mutations in FAP patients.
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