Download and MUTYH mutation negative FAP and AFAP patients

Variable gene expression in APC- and MUTYH mutation
negative FAP and AFAP patients
Rohlin A*1, Wernersson J1, Björk J2, and Nordling M1 Dept of Clinical Genetics, Sahlgrenska University Hospital, Gothenburg, 2) The Swedish
Polyposis Registry, Department of Gastroenterology and Hepatology, Karolinska University Hospital, Solna .
Introduction
Familial adenomatous polyposis (FAP) has been linked to
germline mutations in the APC tumor suppressor gene and in
the case of MAP (MUTYH associated polyposis), the baseexcision repair gene MUTYH. 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 and
MUTYH genes. Sixty-one different mutations in the APC gene
were found in 81 of the families and 6 additional families were
found to have biallelic MUTYH mutations. A disease-causing
mutation was found in all except one of the patients with a
classical phenotype (Kanter-Smoler et al. 2008). In AFAP the
genetic cause remains undetected in up to 70–80% of the
patients .
Results
Subsequent to ANOVA analysis a threshold cutoff was set to pvalues less than 0.001 and at least a 2-fold geometric change in
gene-level expression between controls and patients. This yielded 6
downregulated genes and 2 upregulated genes in total. The
alternative splice analysis showed a significant difference in
splicepattern for 5 genes (cutoff p-value 0.001). Two different spliced
genes are shown i fig1 a and b.
1A
1B
We used the Exon- and SNP arrays from Affymetrix in an
attempt to reveal the genetic cause of the AFAP cases without
identified mutations in the APC or MUTYH genes and to
investigate larger deletions of the APC region previous found
with mlpa. The exon-arrays reveal the expression levels and the
differences in isoforms generated by alternative splicing events.
Additionally, we used this platform to investigate if expression of
different isoforms might in part explain the variable penetrance
of FAP observed within families and between families with the
same mutation.
Methods
Two families with AFAP with two and three patients respectively
were analyzed with the 1.0 HuEx arrays from Affymetrix. The
exon-arrays include over 40 probes for each gene and four
probes (one probeset) for every exon for all well annotated
genes. The robust multi-array analysis (RMA) algorithm was
used for probeset (gene-level) and (exon-level) intensity
analysis. This generates a core gene list with 17800 transcripts
for analysis regarding expression and alternative splicing events.
Five deletions including at least the entire APC region have
been found with mlpa. The 250K SNP array was used to reveal
the extension of the deletions.
A microarray (Affymetrix 250K) analysis of APC entire gene deletion
previously identified with mlpa, shows that the deletion extends at
least 5 genes 5´ of the APC gene and includes a region of 7.7Mb
(fig2a and fig2b).
2A
2B
Conclusions
A combination of exon-arrays and SNP-arrays can be used in
order to get a more detailed picture of copy number changes and
its correlation with differentially expressed/alternative spliced
transcripts. This will give valuable information about regulation of
genes and add information regarding new mutational
mechanisms.
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