Novel splice-site mutations as
the cause of FAP-related
cancer in two families
K Sweet, B McIlhatton, V McConnell,
W Logan and C Graham
Regional Molecular Genetics
Laboratory, Belfast
APC-Associated polyposis
conditions

Familial adenomatous
polyposis (FAP)

>100 adenomatous colonic
polyps
Often thousands of colonic
polyps
Polyps present throughout the
colon
Early age of onset
Germline mutations in APC
gene
Autosomal dominant

Attenuated FAP (AFAP)

Fewer colonic polyps (<100)
Later age of onset






APC Gene
AFAP
Classic FAP
436
1596
AFAP
MUTATION CLUSTER REGION
1061
(aa)
Ex 1
500
3
5
1000
9/9a 10 11 12 13 14
1309 5bp mutation Hot Spots
2843
1500
2000
15
ARMADILLO REPEATS
B-CATENIN BINDING DOMAIN
FUNCTONAL DOMAINS
MICROTUBULE BINDING DOMAIN
B-CATENIN DEGRADATION DOMAIN
EB1 BINDING DOMAIN
Grady and Markowitz
APC Mutations
Mutation Type
Small deletions
Missense/nonsense
Small insertions
Gross deletions
Splicing
Small indels
Regulatory
No. Mutations (%)
356
(41%)
235
(27%)
131
(15%)
54
(6%)
49
(5%)
17
(2%)
3
(<1%)
Molecular genetic testing using range of techniques
including MLPA, PTT and DNA sequencing
Mutation detection rate – up to 90%
Case 1
?
II:1
9 sibs
II:2
II:3
II:4
II:5
II:6
II:7
III:1
• Age 47- Adenocarcinoma
• Colonoscopy - > 100 + polyps located
proximal and distal to tumour
• Suspected family history of
colon cancer on maternal side
• APC Ex 15 PTT - Negative
• APC Ex 1-14 screen
c.1409-5A>G in intron 10
APC Gene
AFAP
436
(aa)
Ex 1 1
FAP with CHRPE
1596
1000
1500
500
3
5
9/9a 10 11 12 13 14
ARMADILLO REPEATS
AFAP
2000
2843
15
Exon 11
G
c.1409-5 A>G CCCTTTTTAA TTAG GGGGACTAC
A
-Splice acceptor site
• Fruitfly and SpliceSiteFinder prediction programmes predicted use of alternative
splice site. Score – 80.6
• Previously reported in large European study – Pathogenicity undetermined
Friedl & Aretz, 2005
Molecular analysis
c.1409-5A>G
DNA
cDNA
Splice acceptor site
G
CCCTTTTTAA A TTAG GGGGACTAC
Intron 10
Ex 11
Alternative splice site
CCCTTTTTAAGTTAG GGGGACTAC
RNA
4bp insertion – (p.G470Vfsx15)
AATGAACTAGTTAGGGGGACTACAGGC,,,,TGA
Term
Ex11
Ex10
N N M
254bp
250bp
Case 2
Breast Ca
II:1
II:2
? Colon Ca
II:3
Jejunal
Ca
? Colon Ca
III.2
• Age 55 - Carcinoma of sigmoid colon
• Age 62 - Multiple polyps throughout colon
II:4
II:5
II:6
Melanoma
• MYH gene - Negative
• APC Ex 15 PTT - Negative
• APC Ex 1-14 Screen
c.423 G>T (p.R141S)
Exon 4 APC gene
Case 2
Breast Ca
II:1
II:2
II:3
AFAP
II:4
II:5
II:6
AFAP
III:2
III:3
AFAP
III:4
AFAP
c.423 G>T Splicing mutation PRESENT
III:5
APC Gene
AFAP
(aa)
1
AFAP
500
345
9/9a 10 11 12 13 14
1000
1500
2000
15
c.423G>T
p.R141S
• Conserved amino acid sequence
• Predicted to disrupt splice-site – (Fruitfly and SpliceSiteFinder and
prediction programmes)
• Reported by Aretz et al., 2004. Human Mutation 24(5): 370-80
Molecular analysis
Intron 3
DNA
Exon 4
c.423G>T
AAGAGAG GTCAATTGCTTCTTGCT
T
Ex3
Ex4
Ex5
109bp
RNA
II.2
II.2
II.4 III.3
III.3
N
Ex3
301
192
Ex3
Ex4
Ex5
Ex5
Exon skipping confirmed by
sequence analysis of the cDNA
product across junction.
Summary


Case 1 – Atypical FAP
c.1409-5 A>G mutation –


Splice-site prediction programmes
RNA studies



Selection of an alternative splice site.
Case 2 – AFAP
c.423 G>T (p.R141S) missense mutation –




Amino acid conservation
Segregation with disease
Splice site prediction programmes
RNA studies

Exon skipping possibly due to possible effect on exonic splicing
enhancer motifs (ESEs). (Aretz et al., 2004).
To conclude…

Splicing mutations can cause FAP and
warrant further investigation
 CMGS
Best Practice Guidelines on the
interpretation and reporting of unclassified variants