Identification of 5 novel germline APC mutations and characterization of clinical phenotypes in Japanese patients with classical and attenuated familial adenomatous polyposis.

Abstract

Familial adenomatous polyposis (FAP) is an autosomal dominant hereditary disease characterized by multiple colorectal adenomatous polyps and frequent extracolonic manifestations. An attenuated form of FAP (AFAP) is diagnosed based on a milder colorectal phenotype, and the colorectal phenotype of (A)FAP has been linked to germline APC mutations. The relationships between the spectrum of mutations and extracolonic manifestations are quite well known, but they need to be further defined.
Nine germline APC mutations, but no large deletions, were identified in the APC locus of 8 (A)FAP patients, and 5 of the mutations, c.446A > T (p.Asp149Val), c.448A > T (p.Lys150X), c.454_457insAGAA (p.Glu152ArgfsX17), c.497insA (p.Thr166AsnfsX2), and c.1958G > C (p.Arg653Ser), were novel mutations. In one patient the p.Asp149Val mutation and p.Lys150X mutation were detected in the same APC allele. The c.1958G > C mutation was located in the last nucleotide of exon 14, and RT-PCR analysis revealed that the mutation resulted in abnormal splicing. The above findings meant that a nonsense mutation, a frameshift mutation, or an exonic mutation leading to abnormal splicing was found in every patient. The following phenotypes, especially extracolonic manifestations, were observed in our (A)FAP patients: (1) multiple gastroduodenal adenomas and early-onset gastric carcinoma in AFAP patients with an exon 4 mutation; (2) a desmoid tumor in two FAP patients with a germline APC mutation outside the region between codons 1403 and 1578, which was previously reported to be associated with the development of desmoid tumors in FAP patients; (3) multiple myeloma in an AFAP patient with an exon 4 mutation.
Nine germline APC mutations, 5 of them were novel, were identified in 8 Japanese (A)FAP patients, and some associations between germline APC mutations and extracolonic manifestations were demonstrated. These findings should contribute to establishing relationships between germline APC mutations and the extracolonic manifestations of (A)FAP patients in the future.

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SHORT REPORTOpen Access
Identification of 5 novel germline APC mutations
and characterization of clinical phenotypes in
Japanese patients with classical and attenuated
familial adenomatous polyposis
Hong Tao1, Kazuya Shinmura1, Hidetaka Yamada1, Masato Maekawa2, Satoshi Osawa3, Yasuhiro Takayanagi3,
Kazuya Okamoto4, Tomohiro Terai3, Hiroki Mori1, Toshio Nakamura5, Haruhiko Sugimura1*
Abstract
Background: Familial adenomatous polyposis (FAP) is an autosomal dominant hereditary disease characterized by
multiple colorectal adenomatous polyps and frequent extracolonic manifestations. An attenuated form of FAP
(AFAP) is diagnosed based on a milder colorectal phenotype, and the colorectal phenotype of (A)FAP has been
linked to germline APC mutations. The relationships between the spectrum of mutations and extracolonic
manifestations are quite well known, but they need to be further defined.
Findings: Nine germline APC mutations, but no large deletions, were identified in the APC locus of 8 (A)FAP
patients, and 5 of the mutations, c.446A > T (p.Asp149Val), c.448A > T (p.Lys150X), c.454_457insAGAA (p.
Glu152ArgfsX17), c.497insA (p.Thr166AsnfsX2), and c.1958G > C (p.Arg653Ser), were novel mutations. In one patient
the p.Asp149Val mutation and p.Lys150X mutation were detected in the same APC allele. The c.1958G > C
mutation was located in the last nucleotide of exon 14, and RT-PCR analysis revealed that the mutation resulted in
abnormal splicing. The above findings meant that a nonsense mutation, a frameshift mutation, or an exonic
mutation leading to abnormal splicing was found in every patient. The following phenotypes, especially
extracolonic manifestations, were observed in our (A)FAP patients: (1) multiple gastroduodenal adenomas and
early-onset gastric carcinoma in AFAP patients with an exon 4 mutation; (2) a desmoid tumor in two FAP patients
with a germline APC mutation outside the region between codons 1403 and 1578, which was previously reported
to be associated with the development of desmoid tumors in FAP patients; (3) multiple myeloma in an AFAP
patient with an exon 4 mutation.
Conclusions: Nine germline APC mutations, 5 of them were novel, were identified in 8 Japanese (A)FAP patients,
and some associations between germline APC mutations and extracolonic manifestations were demonstrated.
These findings should contribute to establishing relationships between germline APC mutations and the
extracolonic manifestations of (A)FAP patients in the future.
Background
Familial adenomatous polyposis (FAP) is an autosomal
dominant familial cancer syndrome characterized by the
early onset of large numbers of adenomatous polyps
throughout the entire colon and a nearly 100% lifetime
risk of colorectal cancer (CRC) if the colon is not
removed [1]. A small proportion of familial colorectal
polyposis cases were recently found to be associated
with biallelic germline mutations of the MutYH gene
[2]. However most FAP cases are caused by germline
mutations of the tumor suppressor gene adenomatous
polyposis coli (APC), which encodes a 2843-amino-acid
protein that contains a variety of functional domains
involved in cell cycle control, differentiation, transcrip-
tion, migration, and apoptosis [3]. More than 1000
pathogenetic mutations have been detected throughout
* Correspondence: hsugimur@hama-med.ac.jp
1First Department of Pathology, Hamamatsu University School of Medicine,
1-20-1 Handayama, Higashi Ward, Hamamatsu, Shizuoka 431-3192, Japan
Full list of author information is available at the end of the article
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the APC gene, and the lifetime penetrance of the disease
is close to 100% [3-5]. Some FAP cases have been classi-
fied as ‘attenuated FAP (AFAP)’ because of their attenu-
ated phenotypes. Although there is still no consensus as
to the precise definition of AFAP, some papers have
summarized the characteristics of AFAP as follows:
development of far fewer colorectal adenomatous polyps
in AFAP patients than in classical FAP and the onset of
adenomatous polyps and colorectal cancer 10~15 years
later in AFAP patients than in classical FAP [6-8]. The
germline APC mutations in AFAP patients have been
found to occur at the 5’ end and 3’ end and in a specific
region of exon 9 of the APC gene, in contrast to the
germline APC mutations in classical FAP patients,
which are found in other locations [3,8-10]. Thus, the
analysis of the sites and spectrum of germline APC
mutations in patients with multiple colorectal polyps is
very important to the proper management of (A)FAP.
A number of extracolonic phenotypic manifestations
are associated with FAP: upper gastrointestinal tract
polyps and cancer, desmoid tumors, thyroid cancer,
hepatoblastoma, congenital hypertrophy of the retinal
pigment epithelium (CHRPE), and other extracolonic
malignancies [1,3]. One of them, CHRPE, only occurs in
patients with germline APC mutations between codons
457 and 1444, and desmoid tumors develop only in
patients having mutations between codons 1403 and
1578 [1,3]. Although the correlations between the germ-
line APC genotypes and FAP phenotypes are well
known, they need to be further defined. In this study we
investigated the genes of 8 Japanese (A)FAP patients for
germline APC mutations, and we identified 9 germline
APC mutations, including 5 novel ones. We also discuss
possible relationships between the germline APC muta-
tions and extracolonic manifestations in our (A)FAP
patients.
Methods
Subjects
Blood samples were obtained from 5 patients with clas-
sical FAP and 3 patients with attenuated FAP, all of
whom appeared to be unrelated, in the hospital of
Hamamatsu University School of Medicine. Written
informed consent was obtained from every patient. Lym-
phocyte genomic DNAs were extracted from the blood
samples with a QIAamp DNA Blood Maxi kit (QIA-
GEN, Hilden, Germany). This study was approved by
the Institutional Review Board of the Hamamatsu Uni-
versity School of Medicine (18-4).
Conventional sequencing analysis of the APC gene
The 1st-15th exons of APC and their boundary regions
were amplified by polymerase chain reaction (PCR) and
directly sequenced with ABI BigDye Terminator Ready
Reaction Mix (Applied Biosystems, Foster City, CA,
USA) and an ABI 3100 Genetic Analyzer (Applied Bio-
systems) [11]. Information regarding the PCR primers is
available upon request. Subcloning of the PCR frag-
ments was performed by using a pGEM-T Easy TA
Cloning Kit (Promega, Madison, WI) according to the
supplier’s protocol.
Calculation of splicing efficiency and detection of the
exonic splicing enhancer (ESE) sequence
The splicing efficiency of the wild-type allele and the
mutant-type allele of the patient with c.1958G > C
mutation was predicted by using the Berkeley Droso-
phila Genome Project (BDGP) splice prediction program
[12]. The effect of the exonic mutation on putative ESE
sites was predicted by the ESEfinder software program
[13]. ESEfinder is a web-based resource that facilitates
rapid analysis of exon sequences to identify binding
motifs for serine/arginine-rich (SR) proteins.
APC mRNA transcript analysis
RNAs were extracted from blood samples with a PAX-
gene Blood RNA Kit (QIAGEN) and converted to first-
strand cDNAs by using a SuperScript First-Strand
Synthesis System for reverse transcription (RT)-PCR
(Invitrogen, Carlsbad, CA, USA) according to the suppli-
er’s protocol [14]. RT-PCR was performed with a set of
primers, i.e., 5’-aaa gac gtt gcg aga agt tg-3’ for the
sequence of exon 13 and 5’-caa acc tcg ctt tga aga ag-3’
for the sequence of exon 15, and the products were
separated on 2% agarose gel and stained with ethidium
bromide before being examined with an ultraviolet ima-
ging system. The splicing rates were evaluated by com-
paring the intensities of the two main bands detected in
each sample by using ImageJ software (National Insti-
tutes of Health, USA) as reported previously [15].
Multiple ligation-dependent probe amplification (MLPA)
analysis
A MLPA kit (P043 APC) was purchased from MRC-
Holland (Amsterdam, The Netherlands), and reactions
were carried out according to the manufacturer’s
instructions. Probe ratios below 0.7 and above 1.3 are
regarded as indicative of a decrease and increase, respec-
tively, of gene dosage.
Results
Identification of 5 novel germline APC mutations
Genomic DNA sequencing of the entire APC coding
regions and exon-intron boundaries enabled identifica-
tion of a total of 9 germline APC mutations in 8 unre-
lated Japanese (A)FAP patients (Table 1). Five of the 9
germline APC mutations identified in this study, i.e.,
c.446A > T (p.Asp149Val), c.448A > T (p.Lys150X),
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c.454_457insAGAA (p.Glu152ArgfsX17), c.497insA (p.
Thr166AsnfsX2), and c.1958G > C (p.Arg653Ser), had
never been reported in any articles according to the
information in the Human Gene Mutation Database or
in the APC variant databases in the Leiden Open Varia-
tion Database (LOVD) [16-18], and none were found in
a thorough review of the literature, indicating that they
are novel mutations. Sequencing of the subcloned APC
fragments revealed that the c.446A > T mutation and
c.448A > T mutation detected in Patient 1 were located
on the same APC allele (Figure 1a). Since the c.448A >
T mutation is a nonsense mutation, the c.448A > T
mutation is more likely than the c.446A > T missense
mutation to be a disease-causing mutation. The
c.454_457insAGAA mutation detected in Patient 2 and
the c.497insA mutation detected in Patient 3 are frame-
shift type mutations and lead to the formation of pre-
mature stop codons (Figure 1b, c). The c.1958G > C
mutation detected in Patient 4 was associated with an
amino acid substitution (p.Arg653Ser) and was located
in the last nucleotide of exon 14 (Figure 1d). The
remaining 4 APC mutations had been reported pre-
viously [19-21].
C",1,0,1,0,0pc,0pc,0pc,0pc>Detection of abnormal
splicing caused by the novel APC mutation c.1958G > C
The novel mutation c.1958G > C detected in Patient 4
was localized in the last nucleotide of exon 14, and
three germline APC mutations, i.e., c.1956C > T,
c.1957A > C, and c.1957A > G very close to c.1958G >
C, have previously been reported to induce abnormal
splicing of exon 14 [22]. Moreover, a severe reduction
in splicing efficiency was predicted for the c.1958G > C
mutation by the BDGP splice prediction program, and
disruption of the binding site for one of the SR proteins,
SC35, by the mutation was predicted by the ESEfinder
program for splicing enhancer’s motif prediction
(Table 2). These predictions prompted us to examine
the effect of the c.1958G > C mutation on splicing by
mRNA transcript analysis. An RNA sample from the
patient with the c.1958G/C genotype and two RNA
samples from two control subjects with the c.1958G/G
Table 1 Germline APC mutations and clinical phenotypes identified in 8 Japanese (A)FAP patients
Patient
IDmutation1(Exon2)
Germline APC
ConsequenceReference3FAP type
(age4)
Number5of
colorectal polyps
Colorectal
cancer (age)
Absent (386)
Extracolonic
manifestation (age4)
1c.446A > T (4) p.Asp149Val This study AFAP (34)40-100
Gastric adenocarcinoma
(34)
Multiple gastroduodenal
adenomas(34)
Gastric hyperplastic
polyps (29)
Multiple gastroduodenal
adenomas (69)
Multiple myeloma (69)
Multiple gastroduodenal
adenomas (32)
Duodenal
adenocarcinoma (32)
Desmoid tumor (31)
Multiple gastroduodenal
adenomas (41)
Small intestinal
adenocarcinoma (41)
Multiple gastroduodenal
polyps (19)
Multiple gastroduodenal
adenomas (22)
Desmoid tumor (25)
Multiple gastroduodenal
polyps (31)
Papillary thyroid cancer
(31)
c.448A > T (4)p.Lys150X This study
2 c.454_457insAGAA (4)p.Glu152ArgfsX17 This studyAFAP (29)40-100Present (516)
3 c.497insA (4)p.Thr166AsnfsX2 This study AFAP (69)> 100Present (694)
4c.1958G > C (14)p.Arg653Ser This studyFAP (32) > 300Present (324)
Aberrant splicing
5c.1993_1994delTT (15)p.Leu664IlefsX8[19]FAP (28)> 300NA7
6c.3505_3509delGAGAA
(15)
c.3747C > A (15)
p.Glu1169ThrfsX8[20]FAP (19)> 300Absent (196)
7 p.Cys1249X[21]FAP (22) > 1000Present (224)
8c.3927_3931delAAAGA
(15)
p.Glu1309AspfsX4[21]FAP (31) > 1000Present (314)
1The reference sequence of the APC gene [GenBank:NM_000038] was used. Nucleotide +1 is the A of the ATG-translation initiation codon.
2The exon that contained the ATG-translation initiation codon was regarded as the first exon.
3When a germline APC mutation had been reported previously, the paper reporting it is cited.
4Age at diagnosis is shown.
5The number of colorectal polyps refers to the cumulative number.
6Age at last observation is shown.
7NA: not available.
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Figure 1 5 novel germline APC mutations identified by sequencing analysis. Results of sequencing the APC gene locus in DNA derived
from the blood of Japanese (A)FAP patients. An asterisk indicates the location of the mutation. a. Results of sequencing analysis of the PCR
product covering the c.446A > T and c.448A > T mutations. An amino acid sequence based on the mutated nucleotide sequence is shown
below the electropherograms. b. Results of sequencing analysis of the subcloned PCR product covering the c.454_457insAGAA mutation. An
amino acid sequence based on the mutated nucleotide sequence is shown below the electropherograms. c. Results of sequencing analysis of
the subcloned PCR product covering the c.497insA mutation. An amino acid sequence based on the mutated nucleotide sequence is shown
below the electropherograms. d. Results of sequencing analysis of the PCR product covering the c.1958G > C mutation. The boundary between
exon 14 and intron 14 is indicated by a vertical line.
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genotype were prepared and used for RT-PCR analysis
with a forward primer for the sequence on exon 13 and
a reverse primer for the sequence on exon 15. Two
main bands were detected in all samples (Figure 2), and
direct sequencing of the two bands revealed that the
upper band (band A) represented the wild-type fragment
and the lower band (band B) represented the whole
exon 14-skipped product (data not shown), findings that
are consistent with those reported in a previous paper
[22]. Calculation of the splicing rate of the mutant-type
transcript by dividing the intensity of band B by the
intensity of band A with the ImageJ program showed
that the splicing rate in samples with the G/C genotype
was higher than in samples with the G/G genotype (1.24
vs 0.23 and 0.31) (Figure 2), suggesting that the c.1958G
> C mutation caused abnormal splicing.
Confirmation of the c.454_457insAGAA mutation and the
c.3927_3931delAAAGA mutation by MLPA analysis
To better evaluate the state of the genomic DNA of (A)
FAP patients, MLPA analysis, which is useful for detect-
ing large deletions and duplications, was also performed
on all 8 samples. No increased signals were detected,
but decreased signals were detected in two patients.
One decreased signal was detected in APC exon 4 of the
DNA derived from Patient 2 (Figure 3a). However, no
large deletions were detected in mRNA transcripts from
Patient 2 by RT-PCR analysis with a forward primer for
the sequence of exon 2 and a reverse primer for the
sequence of exon 6 (data not shown). Examination of
the sequence of the DNA probe for APC exon 4
revealed that the probe for exon 4 overlapped with the
c.454_457insAGAA mutation (p.Glu152ArgfsX17) in
Patient 2. Thus, it is likely that the overlapping caused
the disruption of the MLPA reactions in the DNA. The
other decreased signal was detected with the p.1309
mutation-specific probe in Patient 8 (Figure 3b). These
results were consistent with the results of the sequen-
cing analysis (p.Glu1309AspfsX4 mutation) in the
patient. The results of the MLPA analysis in conjunction
with the sequencing analysis indicated that base substi-
tutions or microdeletions, not large deletions or duplica-
tions, in the APC gene locus caused the production of
truncated APC proteins in our (A)FAP patients.
Characterization of clinical phenotypes of patients with
different germline APC mutations
The clinical phenotypes of all 8 patients are summarized
in Table 1. All 3 patients (Patients 1-3) with a mutation
in APC exon 4 had been diagnosed with AFAP because
of their attenuated colorectal phenotypes. From 40 to
100 colorectal polyps were detected in Patient 1. Patient
2 was found to have a relatively small number of color-
ectal polyps at 29 years of age, and clinical follow up
was performed instead of surgical treatment. At 51 years
of age, however, an early-stage CRC and approximately
100 colorectal adenomatous polyps were detected, and
total colectomy was performed. A sister of Patient 2 was
also diagnosed with AFAP and found to have the same
Table 2 Prediction of the effects of the c.1958G > C
mutation on splicing
AlleleSplice prediction1
ESE score for SC352
Wild type (G)
Mutant type (C)
0.91
<0.01
4.595
-3
1Predicted by the Berkeley Drosophila Genome Project (BDGP) splice
prediction program.
2Exonic splicing enhancer (ESE) score as predicted by ESEfinder. Its threshold
score for SC35 protein is 2.383.
3Below the threshold score.
Figure 2 RT-PCR analysis for evaluation of the effect of the c.1958G > C mutation on splicing. RT-PCR with a forward primer for the
sequence on exon 13 and a reverse primer for the sequence on exon 15 and subsequent agarose gel electrophoresis were performed on blood
samples derived from two control subjects with the c.1958G/G genotype (lanes 1 and 2) and a patient with the c.1958G/C genotype (lane 3).
The two main bands identified are indicated as band A (upper) and band B (lower). The intensity ratio (intensity of band B/intensity of band A)
calculated with ImageJ software is shown under each lane of the panel. dw: distilled water. M: 100-bp ladder.
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APC mutation, but none of the 6 unaffected members of
this family was found to have the family-specific muta-
tion. Colon fiberscopy was performed for the first time
in Patient 3 when he was 69 years old because of rectal
bleeding, and more than 100 colorectal adenomatous
polyps and multiple advanced-stage CRCs were
observed. Total colectomy was performed, but the
patient died of liver and lung metastases 7 years later.
By contrast, more than 300 colorectal adenomatous
polyps were found in each of the other 5 patients, who
were diagnosed with classical FAP. The mother of
Patient 4 and the daughter of Patient 5 were found to
have their family-specific APC mutation, consistent with
their FAP phenotype. Among the extracolonic manifes-
tations, gastroduodenal polyps, which are also common
in FAP patients, were observed in all of the patients
Figure 3 MLPA analysis for evaluation of the blood DNA of (A)FAP patients. The results of the MLPA analysis for Patient 2 with a
c.454_457insAGAA (p.Glu152ArgfsX17) mutation (a) and Patient 8 with a c.3927_3931delAAAGA (p.Glu1309AspfsX4) mutation (b). Names of
MLPA probes are shown below the panels. A decreased signal is indicated by an asterisk. Results are shown as means + standard deviation.
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with classical and attenuated FAP in this study. Most of
the polyps in the stomach were histologically diagnosed
as fundic gland polyps. However, multiple adenomas
were observed in the stomach and duodenum of
Patients 1, 3, 4, 5, and 7. Desmoid tumors are common
in FAP patients and were observed in Patients 5 and 7.
Malignant neoplasms in the form of gastric carcinoma,
multiple myeloma, duodenal carcinoma, small intestinal
carcinoma, and thyroid carcinoma developed in Patient
1, Patient 3, Patient 4, Patient 5, and Patient 8, respec-
tively. No extracolonic manifestations were detected in
the other (A)FAP affected members of any of the
families, or such information was unavailable.
Discussion
Sequencing analysis, RT-PCR analysis, and MLPA analy-
sis of the APC genes of 8 Japanese (A)FAP patients
from 8 unrelated families revealed a nonsense mutation,
a frameshift mutation, or an exonic mutation leading to
abnormal splicing, all of which resulted in the produc-
tion of a truncated APC protein, in every patient. No
large deletions or duplications in the APC locus were
detected in any of the patients. Five of the 9 germline
APC mutations detected in this study had never been
reported before, meaning that they are novel mutations.
Since two mutations, c.446A > T (p.Asp149Val) and
c.448A > T (p.Lys150X), are on the same allele and
located close to each other, it is possible that these two
mutations are in complete linkage disequilibrium but
are not two independent mutations. Consistent with the
previous finding that mutations in the 5’ and 3’ ends
and in exon 9 of the APC gene are associated with hav-
ing the attenuated type of FAP [6-10], the mutations in
exon 4 were found in the 3 AFAP patients in this study,
and the mutations in exons 14, and 15 were found in
the 5 classical FAP patients in this study.
Knowledge of genotype-phenotype correlations in (A)
FAP has been accumulating, and it is useful in the clini-
cal management of (A)FAP families, but the relation-
ships between the locations of the APC mutations and
extracolonic manifestations are still not fully under-
stood. Furthermore, as the number of patients diagnosed
with (A)FAP has increased, a broad range of variable
extracolonic manifestations has gradually come to be
recognized in (A)FAP patients. Based on the results of
APC mutations and extracolonic manifestations
observed in our study, we focused our attention on the
following three issues. First, fundic gland polyps, not
gastric adenoma, have been reported to be the main gas-
tric lesions in AFAP patients [1,8]. Interestingly, multi-
ple adenomas in the stomach, in addition to the
commonly detected fundic gland polyps, as well as in
the duodenum were observed in 2 of our 3 AFAP
patients with germline mutations in APC exon 4, and a
gastric adenocarcinoma was also found in one of them
at 34 years of age. As far as we know [7,8], this is the
first report of such an early-onset gastric cancer in
AFAP patients. Gastric adenocarcinomas have been
reported in (A)FAP patients [23-26], but the mechanism
of involvement of the APC mutation in the gastric carci-
nogenesis remains largely unknown. There have been
reports of gastric adenocarcinomas in (A)FAP patients
having arisen from a fundic gland polyp or adenoma,
and the reports suggested the existence of an adenoma-
adenocarcinoma sequence in their carcinogenesis
[23-25]. However, there have been other reports claim-
ing to have found no relation between a gastric adeno-
carcinoma in an AFAP patient and existing fundic gland
polyps or adenomas [26]. Analysis of the APC inactiva-
tion status in the adenomas and adenocarcinomas
detected in our patients may help to better understand
the mechanism of the involvement of APC mutations in
tumorigenesis. In addition to genetic factors, various
environmental factors have been reported to affect the
risk of gastric cancer [27-29], environmental factors and
genes that modify the development of gastric lesions
may be involved in the clinical phenotype. The second
issue is that it has previously been reported that there is
a close association between the presence of a germline
APC mutation between codons 1403 and 1578 in FAP
patients and the occurrence of desmoid tumors [1,3],
however, both of the FAP patients with a desmoid
tumor in our series had a germline APC mutation out-
side the region, i.e., in codon 664 and codon 1249.
Some other recent papers have also reported the occur-
rence of desmoid tumors in FAP patients with APC
mutations outside the region between codons 1403 and
1578 [30,31]. Thus, the results of this study and others
together with the findings described in previous reviews
[1,3] have suggested that although FAP-associated des-
moids predominantly occur in patients carrying APC
mutations between codons 1403 and 1578, some of
them occur outside the region. The third issue that we
focused our attention on is that a multiple myeloma was
detected in one of our 3 AFAP patients with a germline
exon 4 mutation. This is the first report of complication
of AFAP by multiple myeloma. However, evidence of
APC involvement, such as a second somatic mutation of
APC in multiple myeloma, needs to be found in order
to rule out the possibility that the occurrence of the
multiple myeloma was a coincidental unrelated event.
We think that although the number of cases analyzed
was relatively small, the above three findings will contri-
bute to establishing relationships between germline APC
abnormalities and clinical phenotypes in (A)FAP
patients and to better characterizing the differences
between APC-related polyposis and MutYH-associated
polyposis in the future. However, since many examples
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of deviations from observed APC genotype-FAP pheno-
type correlations and highly variable phenotypic traits
have been reported [16,32-34], it has been pointed out
that the family history is important in (A)FAP genotype-
phenotype analyses and the factors other than the APC
genotype may be involved in producing the (A)FAP
phenotype.
Although a nearly 100% risk of CRC has been
reported in patients with classical FAP, the lifetime risk
of CRC in AFAP patients is unclear. At the time this
study was performed CRC had been diagnosed in
Patient 2 at 51 years of age and Patient 3 at 69 years of
age, but not in Patient 1, who was 38 years old. Lifetime
risk of CRC is an important factor in the clinical man-
agement and genetic counselling of AFAP patients,
especially because it affects the decision as to whether
to proceed with prophylactic colectomy. The results of
this study should contribute to determining the lifetime
risk of CRC in AFAP patients in the future.
The results of the RT-PCR analysis in this study
showed that a G to C transversion at c.1958, which cor-
responds to the last nucleotide of exon 14 of the APC
gene, causes abnormal splicing. Since APC mRNA tran-
script analyses in previous studies have demonstrated
that exonic single-base substitutions of c.423G > T,
c.834G > C, c.1869G > T, c.1918C > G, c.1956C > T,
c.1957A > C, and c.1957A > G affect splicing [22,35-37],
c.1958G > C is the 8th APC exonic mutation that has
been demonstrated to result in abnormal splicing. Inter-
estingly, most (6 out of 8) of the exonic mutations asso-
ciated with abnormal splicing have been located in exon
14, but the reason for this clustering is unclear.
Conclusions
In the present study, 9 germline APC mutations, 5 of
which were novel, were identified in 8 Japanese (A)FAP
patients. The following three findings regarding the rela-
tion between the location of the germline APC muta-
tions and extracolonic manifestations were also obtained
in this study: 1) severe gastric lesions occurred in AFAP
patients with an exon 4 mutation, 2) desmoid tumors
developed in FAP patients with germline APC mutations
outside the region between codons 1403 and 1578, 3) a
multiple myeloma developed in an AFAP patient with
an exon 4 mutation. These findings should contribute to
increasing our knowledge of the associations between
APC genotypes and (A)FAP phenotypes, which are
informative for proper clinical management and genetic
counselling of (A)FAP patients and their families.
Acknowledgements
This work was supported in part by a Grant-in-Aid from the Ministry of
Health, Labour and Welfare for the Comprehensive 10-Year Strategy for
Cancer Control (19-19) and the Third Term Comprehensive Control Research
for Cancer, JSPS KAKENHI (22590356), MEXT KAKENHI (18014009), and a
Grant-in-Aid from the Smoking Research Foundation.
Author details
1First Department of Pathology, Hamamatsu University School of Medicine,
1-20-1 Handayama, Higashi Ward, Hamamatsu, Shizuoka 431-3192, Japan.
2Department of Laboratory Medicine, Hamamatsu University School of
Medicine, 1-20-1 Handayama, Higashi Ward, Hamamatsu, Shizuoka 431-3192,
Japan.3First Department of Medicine, Hamamatsu University School of
Medicine, 1-20-1 Handayama, Higashi Ward, Hamamatsu, Shizuoka 431-3192,
Japan.4Department of Surgery, Fujinomiya City General Hospital, 3-1 Nishiki-
cho, Fujinomiya, Shizuoka 418-0076, Japan.5Second Department of Surgery,
Hamamatsu University School of Medicine, 1-20-1 Handayama, Higashi Ward,
Hamamatsu, Shizuoka 431-3192, Japan.
Authors’ contributions
HT carried out the mutation detection and molecular genetic analyses,
collected up the clinical data, participated in the design of the study, and
drafted the manuscript. KS participated in the design of the study and
drafted the manuscript. HY extracted the total RNAs from blood samples.
MM, SO, YT, KO, TT, HM and TN identified relevant patients for genetic
testing and were responsible for clinicopathological data. HS and MM
conceived of the study, participated in the design of the study, and drafted
the manuscript. All authors read and approved the final manuscript.
Competing interests
The authors declare that they have no competing interests.
Received: 22 June 2010 Accepted: 16 November 2010
Published: 16 November 2010
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doi:10.1186/1756-0500-3-305
Cite this article as: Tao et al.: Identification of 5 novel germline APC
mutations and characterization of clinical phenotypes in Japanese
patients with classical and attenuated familial adenomatous polyposis.
BMC Research Notes 2010 3:305.
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