Download Detection of complex mutations in Swedish FAP familes

Detection of complex mutations in Swedish FAP familes
Anna Rohlin,1 Yvonne Engwall,1Josephine Wernersson 1,Jan Björk,2 and Margareta Nordling1
1Department of Molecular and Clinical Genetics, Institute of Biomedicine, Sahlgrenska Academy at University of Gothenburg, Gothenburg,
Sweden.2The Swedish Polyposis Registry, Department of Medicine, Karolinska Institute, Stockholm, Sweden.
Introduction
Familial adenomatous polyposis (FAP) has been linked to germline
mutations in the APC tumor suppressor gene. Classical FAP and AFAP
(attenuated familial polyposis) show a phenotypic heterogeneity, which is
partially related to the mutation site or type.
In our material, 96 unrelated FAP patients from the Swedish polyposis
register were screened for mutations in the APC gene.
By using a combination of several different mutation detection
techniques, the mutations were revealed in >95% in families with a
classical FAP phenotype (Kanter-Smoler et al. 2008).
Around 12%-15% of our families with a classical phenotype are shown to
have larger deletions including parts of the APC gene and or the whole
APC gene(identified with mlpa). In several studies larger
deletion/rearragements of the APC gene seem to be more common than
previous expected.
In this study we used the Exon- and SNP arrays from Affymetrix to
investigate more complex larger deletions including the whole gene and
the promotor regions 1A and 1B of the APC gene.
Material and Methods
Four families with larger deletions (previously found with mlpa) were
analyzed with the SNP arrays 6.0 and the 1.0 HuEx arrays both from
Affymetrix.
The GeneChip 6.0 platform consists of about 906 600 SNP sequences and
about 900 000 nonpolymorphic pobes, which cover the whole genome
with an average spacing of 0.7Kb.
The exon-arrays include over 40 probes for each gene and four probes
(one probeset) for every exon for all well annotated genes. The exonarrays reveal the expression levels and the differences in isoforms
generated by alternative splicing events. The expression level were also
invesigated with TAGman analysis using three different probes for the
APC transcripts.
A
C1466
B
C1929
C
C2348
D
C2581
Fig1
Fig2
Results
The results of the SNP array and exon array analysis for the four large
deletions are shown in fig1A-D and table1. Table 1 gives the extensions
of the abbreviations, the deleted regions nand the results of the exon
expression analysis. The exon expression arrays results include the
three different transcripts generated from the promotor 1A and 1B
(expression fold change). In fig 2 the analysis (Partek GS) shows the -1.8
times (fold change) reduced APC expression for patient C2348 having a
deletion including both the promotor 1A and 1B.
The TAGman results are showing the reduced expression levels with
three different probes were probe 3 only includes the transcript
generated from promotor 1B and probe1 and 2 all the transcripts
generated from both the promotors (Fig3). In table 2 the TAGman
results for two deletions including promotor 1A and promotor 1A and
1B are shown.
Fig3
Table2
Patient
Deletions in the APC
region
C2348
del APC and promotor 1A
and B
-51%
-65%
-75%
C2581
del APC and promotor 1A
-75%
-82%
-60%
probe1
probe2
probe3
Table1
Patient
Abbreviation
Expression
Fold change
C1929
114Kb deletion
n.a
Deleted Region
coding region of APC
Conclusions
promotor 1A and 1B and coding region of APC 1
gene upstream and 6 genes downstream of APC
C2348
203 Kb deletion
-1.8
promotor 1A and coding region of APC and 12
genes upstream and 2
C1466
6008Kb deletion
C2581
116Kb deletion
n.a
genes downstream of APC
A combination of exon-arrays and SNP-arrays can be used in order to get a
more detailed picture of copy number changes and correlations with
differentially expressed/alternative spliced transcripts. In this study the
reduced expression reveals the importance of both the promotor1A and 1B,
although a higher overall reduced expression was seen for promotor 1A only.
This will give valuable information about regulation of the APC gene and add
information regarding new mutational mechanisms.
-2.2promotor 1A and coding region of APC
[email protected]
Reference: "Clinical characterization and the mutation spectrum in Swedish adenomatous polyposis families"
Kanter-Smoler, G., Fritzell, F., Rohlin, A,. Engwall, Y., Hallberg, B., Bergman, A., Meuller, J., Grönberg, H.,
Karlsson, P., Björk, J. and Nordling, M.
BMC Med. (2008) Apr 24;6:10.