Novel splice-site mutations as the cause of FAP
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Transcript Novel splice-site mutations as the cause of FAP
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