Spier et al. – Novel candidate genes in adenomatous polyposis
Online supplementary information
Spier et al.
Exome sequencing identifies potential novel candidate genes in patients with
unexplained colorectal adenomatous polyposis
Extracolonic
phenotype / tumors
Duodenal
phenotype
Colectomy (age)
Colorectal cancer
Distribution of
adenomas
No. of colorectal
adenomas
Age at diagnosis
(years) - adenomas
Patient ID
Supplementary Table S1. Phenotype of the seven index patients in whom exome sequencing
was performed (all had an attenuated polyposis and appeared to be sporadic cases)
F710
61
51100
whole colon
no
no
unknown
osteoma?,
lipoma, prostate
cancer (66 y),
bladder cancer
(70 y)
F995
69
51100
whole colon
no
right-sided
hemicolectomy
(69 y)
unknown
unremarkable
F1066
51
100500
whole colon,
proximal
pronounced
no
normal
gastric fundic
gland polyps
F1360
57
100500
proximal
pronounced
no
ileosigmoidostomy
(59 y)
normal
unremarkable
F1526
67
51100
whole colon
no
no
unknown
unremarkable
F1596
41
51100
whole colon
no
no
normal
unremarkable
F1675
35
51100
whole colon
no
ileosigmoidostomy
(39 y)
normal
unremarkable
right-sided
hemicolectomy
(52 y)
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Spier et al. – Novel candidate genes in adenomatous polyposis
Supplementary Table S2. Clinical characteristics of the validation cohort (n=191)
FAP
No. of patients
191
Gender (female / male)
83 / 108
Mean age at diagnosis (years)
Range (years)
45
12-78
No. of colorectal adenomas
< 100 adenomas
118 (62%)
> 100 adenomas
31 (16%)
numerous
15 (8%)
multiple
27 (14%)
Colorectal cancer
64 (34%)
Extracolonic benign lesions *
22 (12%)
Extracolonic malignancies
11 (6%)
Family history **
Familial
26 (14%)
Sporadic
88 (46%)
Unclear (sporadic/familial)
73 (38%)
Unknown
4 (2%)
* gastric fundic gland polyps, congenital hypertrophy of the retinal pigment epithelium (CHRPE),
desmoids, osteomas
** family history refers to the presence of colorectal polyposis in the relatives of colorectal
adenomatous polyposis patients (FAP)
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Spier et al. – Novel candidate genes in adenomatous polyposis
Supplementary Table S3. Overall performance of the enrichment and Next
Generation Sequencing process
Exome sequencing of
leukocyte DNA (n=7)
Target size
Exome sequencing of
polyp DNA (n=12)
51.5 Mb (51542852 bp)
Number of target regions
471155
Targeted sequencing of
leukocyte DNA (validation
cohort, n=191)*
0.6 Mb (622657 bp)
2163
Total reads**
80.13 million (+/- 53%)
60.09 million (+/- 35%)
3.98 million (+/- 14%)
Unique mapped reads**
62.11 million (+/- 58%)
45.44 million (+/- 34%)
3.67 million (+/- 14%)
GC drop out***
16.5% (+/- 5.1%)
9% (+/- 7.4%)
2.2% (+/- 0.1%)
AT drop out***
0.2% (+/- 0.2%)
2.8% (+/- 4%)
11.6% (+/- 1.5%)
Median library insert size***
207 (+/- 58)
157 (+/- 13)
244 (+/- 6)
Read length
101 (paired end)
Mean on-target coverage***
57 (+/- 31)
51 (+/- 27)
304 (+/- 44)
Targets covered at depths
of 10x
Targets covered at depths
of 20x
Targets covered at depths
of 30x
83.0%
80.0%
97.7%
70.0%
64.0%
96.6%
59.0%
51.0%
95.6%
* the enrichment assay included a total of 140 genes, 3 of which are of relevance to the present
study
** mean of each sample +/- relative standard deviation
*** mean of each sample +/- absolute standard deviation
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Spier et al. – Novel candidate genes in adenomatous polyposis
Supplementary Table S4. Screening for the germline mutations identified in the
present study in the corresponding polyp samples (“T”).
T1
ZSWIM7:c.231_232del;
p.Cys78Phefs*21
T2
(homozygous)
T3
yes
% mutant
reads
(number of
mutant
reads /
coverage)
100 (27/27)
yes
94 (16/17)
yes
61 (28/46)
DSC2:
c.2686_2687dupGA;
p.Ala897Lysfs*4
T1
yes
40 (18/45)
T4
yes
41 (24/58)
T1
PIEZO1:
c.5289C>G;p.Tyr1763* T2
yes
yes
50 (1/2)
71 (5/7)
Patient
Mutation
ID
F710
F995
F1526
Polyp
Mutation confirmed
sample
in polyp sample?
ID
4
Spier et al. – Novel candidate genes in adenomatous polyposis
Supplementary Table S5. Additional information for potential pathogenic mutations identified in exome sequencing
patients (n=7, shown in bold) and the validation sample (n=191). With the exception of the mutation in ZSWIM7, all
mutations were heterozygous.
Gene
DSC2
Patient
Mutation
ID
Type
Remarks
Variation
Phenotype
Family history
Segregation
F386;
F929
COSMIC: 1x in large
intestine, 1x in
hematopoietic and lymphoid
tissue); same mutation in
missense
patients with
cardiomyopathy [1, 2];
Cadherin tandem repeat
domain
F386: typical (+
duodenal adenomas,
osteomas, fibromas);
F929: atypical
F386: familial (son,
daughter, sister
and her son with
polyps, father
gastric cancer);
F929: Unclear
F386: no (affected
sister and daughter
don´t carry the
mutation)
F995+F1360:
attenuated; F807:
typical (+duodenal
adenomas,
retroperitoneal
fibrosis)
F995+F1360:
unclear; F807:
familial (father
CRC, grandfather
polyps, son
polyps)
F807: ? (both sons
carry the mutation,
one is affected, the
other was not
affected at the age
of 24 y)
c.907G>A; p.Val303Met
same mutation in patients
with arrhythmogenic right
ventricular cardiomyopathy
[3-5]; only 7 codons in front
of regular stop codon
DSC2
F995;
c.2686_2687dupGA;
F1360;
p.Ala897Lysfs*4
F807
ins
DSC2
F998
c.2701A>G;p.Arg901Gly
missense penultimate codon
attenuated
familial? (mother
CRC (61 y),
deceased)
not possible
PIEZO1
F906
c.2104C>T;p.His702Tyr
missense
attenuated
unclear
not possible
PIEZO1
F1899
c.3021C>G;p.Ile1007Met
missense
attenuated
sporadic
not possible
F1909
COSMIC: mutation in
adjacent codon
c.5134G>A;p.Val1712Met missense
(c.5131C>T;p.P1711S) in
large intestine
attenuated
sporadic
not possible
PIEZO1
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Spier et al. – Novel candidate genes in adenomatous polyposis
PIEZO1
F1526
c.5289C>G;p.Tyr1763*
PIEZO1
F1445
ZSWIM7 F710
stop
759 codons before regular
stop codon
attenuated
sporadic
not possible
c.5863C>T;p.Arg1955Cys missense
attenuated
sporadic
not possible
c.231_232del;
p.Cys78Phefs*21
(homozygous)
attenuated
unclear
not possible
del
6
Spier et al. – Novel candidate genes in adenomatous polyposis
Supplementary Table S6. List of genes, which were screened for somatic
mutations in adenoma samples
Gene
NCBI transcript ID
APC
NM_000038.5
BMPR1A
NM_004329.2
BRAF
NM_004333.4
CTNNB1
NM_001098210.1
EGFR
NM_005228.3
FBXW7
NM_033632.3
KRAS
NM_033360.2
MAP2K4
NM_003010.2
MLH1
NM_000249.3
MSH2
NM_000251.2
MSH6
NM_000179.2
NRAS
NM_002524.4
PDGFRA
NM_006206.4
PIK3CA
NM_006218.2
PIK3R1
NM_181523.2
PMS2
NM_000535.5
POLD1
NM_002691.3
POLE
NM_006231.2
PTEN
NM_000314.4
SMAD4
NM_005359.3
STK11
NM_000455.4
TP53
NM_000546.5
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Spier et al. – Novel candidate genes in adenomatous polyposis
Supplementary Table S7. Histological findings of all polyp samples (“T”) and
results of screening for somatic mutations in known cancer genes (cf.
Supplementary Table S6). ND = not detectable.
Polyp
Patient
sample Histology
ID
ID
T1
F710
T2
T3
T1
tubular
adenoma
hyperplastic
polyp? /
tubular
adenoma?
tubular
adenoma
Grade of
Gene
dysplasia
Somatic mutation
% mutant
reads
(number of
mutant
reads /
coverage)
low grade CTNNB1
c.490G>A;
p.Asp164Asn
29 (6/21)
Deleterious /
benign
c.3469G>T;
p.Glu1157*
16 (5/31)
-
42 (11/26)
-
32 (18/57)
-
42 (26/62)
-
41 (37/91)
-
APC
low grade
ND
mainly low APC
tubulovillous grade,
adenoma
partly high
APC
grade
APC
F995
T4
tubulovillous
low grade
adenoma
APC
FBXW7
T1
F1066
T2
KRAS
tubular
adenoma
low grade
tubular
adenoma
low grade KRAS
TP53
APC
T1
tubular
adenoma
low grade
T2
T1
F1675
APC
MSH2
F1526
T2
T3
tubular
adenoma
tubular
adenoma
tubular
adenoma
tubular
adenoma
In silico
prediction
tools (SIFT /
PolyPhen2)
APC
low grade
MSH2
c.2626C>T;
p.Arg876*
c.4612delG;
p.Glu1538Asnfs*27
c.3049delA;
p.Asn1017Metfs*5
c.4385_4386delAG;
p.Ser1465Trpfs*3
c.1393C>T;
p.Arg465Cys
c.38G>A;
p.Gly13Asp
c.804C>G;
p.Asn268Lys
c.35G>A;
p.Gly12Asp
c.694C>T;p.Arg232*
c.4221delT;
p.Ser1407Argfs*8
c.589A>G;
p.Lys197Glu
c.4233delT;
p.Ser1411Argfs*4
c.589A>G;
p.Lys197Glu
42 (28/67)
Deleterious /
prob. dam.
Deleterious /
poss. dam.
Deleterious /
prob. dam.
Deleterious /
benign
-
37 (30/82)
-
48 (23/48)
Deleterious /
prob. dam.
30 (27/90)
-
35 (49/139)
17 (32/184)
11 (6/55)
22 (46/211)
39 (29/74)
low grade CTNNB1 c.97T>C;p.Ser33Pro 20 (6/30)
low grade
Deleterious /
prob. dam.
Deleterious /
prob. dam.
ND
low grade CTNNB1 c.104T>G;p.Ile35Ser 44 (17/39)
8
Deleterious /
prob. dam.
Spier et al. – Novel candidate genes in adenomatous polyposis
Suppl. Fig. S1 (a) Heterozygous DSC2 germline mutation c.2686_2687dupGA;p.Ala897Lysfs*4
in patient F995 (exome sequencing data, reverse sequence). (b) Sanger sequencing of the
corresponding region (reverse sequence).
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Spier et al. – Novel candidate genes in adenomatous polyposis
Suppl. Fig. S2 (a) Homozygous ZSWIM7 mutation c.231_232del;p.Cys78Phefs*21 in patient
F710 (exome sequencing data, reverse sequence). (b) Sanger sequencing of corresponding
region (reverse sequence).
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Spier et al. – Novel candidate genes in adenomatous polyposis
Suppl. Fig. S3 (a) PIEZO1 mutation c.5289C>G;p.Tyr1763* in patient F1526. The variant
seemed to be homozygous in exome sequencing data with low read depth of 7x (reverse
sequence). (b) Sanger sequencing of corresponding region revealing the mutation as
heterozygous (reverse sequence).
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Spier et al. – Novel candidate genes in adenomatous polyposis
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