Transcript Variable gene expression and reduced penetrance in familial

Variable gene expression and reduced penetrance in
familial adenomatous polyposis
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
In our material, 96 unrelated FAP patients from the Swedish
polyposis register were screened for mutations in the APC and
MUTYH genes. 61 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 .
The one patient with classical FAP without any identified
mutation belongs to a large kindred including 150 individuals of
whom 57 are affected. Two individuals from this family were
analysed with TaqMan quantitative RT-PCR analysis. A lower
APC expression was detected, but no deletion or methylation of
the promoter region has been found so far.
The Exon- and SNP arrays from Affymetrix were used in order to
reveal the genetic cause of the AFAP cases without identified
mutations in the APC or MUTYH genes. Further we used the
exon-arrays to investigate the lower APC gene expression in
cases with classical FAP and with the SNP arrays we verified
the extension of larger deletions of the APC region previous
found with mlpa.
Results
The exonarrays were analyzed as follows; subsequent to ANOVA
analysis a threshold cutoff was set to p-values less than 0.001 and at
least a 2-fold geometric change in gene-level expression between
controls and patients. For the AFAP cases two examples of two
different spliced gene are shown i fig1a and fig1b. In the classical
FAP cases the APC gene showed a difference in expression and
splicepattern.
1A
1B
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.
Material and Methods
Five patients with AFAP from two families and two patients with
classical FAP from one family were analysed with the 1.0HuEx
arrays from affymetrix.
The 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 patients having a deletions including at least the entire APC
region previously found with mlpa were analyzed using the 250K
and the 6.0 SNP arrays to reveal the extension of the deletions.
A microarray (Affymetrix 250K) analysis of a patient
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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