European Human Genetics Conference 2007 June 16 – 19, 2007 ...

Concurrent Sessions
C48. CHROMSCAN: Genome-wide association mapping
A. R. Collins;
University of Southampton, Southampton, United Kingdom.
Many genome-wide association mapping studies using high density
arrays of single nucleotide polymorphisms (SNPs) are underway. Typically
these involve 100s to 1000s of DNA samples collected as casecontrol
studies screened for several hundred thousand SNPs across
the genome. Efficient analysis and interpretation of these vast data
sets raises considerable difficulties. If significance is tested at individual
SNPs, large numbers of false positives make interpretation difficult.
Models which utilise information from multiple SNPs simultaneously
offer advantages by reducing the number of tests and providing increased
power. However, the reliable computation of significance levels
remains an issue, given the high density of non-independent SNPs
and consequent auto-correlation. Other concerns include the difficulty
of incorporating information on the underlying linkage disequilibrium
(LD) structure when testing association with disease. Including such
information has been shown to substantially increase power and precision
of mapping.
CHROMSCAN models association with disease in non-overlapping
sliding windows and rapidly and robustly determines candidate regions
and maximum likelihood locations and standard errors for putatively
causal polymorphisms. The program employs a composite-likelihood
based model, estimating a small number of parameters in each region,
thereby reducing the number of tests made. A permutation test
ensures the P-value distribution is not distorted due to autocorrelation
caused by extensive LD. Association with disease is modelled on an
underlying map in linkage disequilibrium units (LDUs). CHROMSCAN,
which runs on UNIX and Linux platforms, is also implemented for parallel
computing on either a Linux-based cluster.
C49. Screening for splice defects: application to BRCA1 and
BRCA2 unknown variants (UV)
V. Caux-Moncoutier1 , S. Pagès-Berhouet1 , D. Michaux1 , A. De Pauw1 , M. Gauthier-Villars1
, D. Stoppa-Lyonnet1,2 , C. Houdayer1,3 ;
1 2 3 Institut Curie, Paris, France, INSERM U509, Paris, France, UMR INSERM
745, Paris, France.
Nearly half of BRCA1 and BRCA2 sequence variations remain of uncertain
clinical significance (UV) and are candidates for splice alterations
e.g. by creating cryptic splice sites. Since an out-of-frame splicing
defect leads to severe reduction in the level of the mutant mRNA, we
developped a cDNA-based test to evidence such allelic imbalance.
Seventy patients without BRCA1/2 mutation and bearing consecutively
identified UVs were included in the study. Following RNA extraction,
DNAse treatment and RT-PCR, 3 exonic SNPs on both BRCA1 and
BRCA2 were asked using a semiquantitative single-nucleotide primer
extension approach.
Data were collected with an ABI3130XL then analysed using Gene-
Mapper (Applied Biosystems). cDNA allelic ratios were corrected using
genomic DNA ratios from the same sample. This approach was
combined with in silico predictions using Splice Site Finder, Splice Site
Prediction, MaxEntScan, ESE Finder and Rescue ESE.
Allelic imbalances were found in 4/21 and 11/49 cases for BRCA1 and
BRCA2, respectively. However, the corresponding UVs did not show
in silico pathogenic effects.
In order to determine UVs’ impact on mRNA instability, sequencing of
their cDNA flanking regions has started, using cDNA from cells treated
with puromycin.
Analysis of the first 5 patients excluded UVs’ involvement therefore
complete cDNA sequencing was performed, evidencing in one case an
out-of-frame deletion of BRCA2 exons 12 and 13. These preliminary
results showed that allelic imbalance screening is a simple way to detect
splicing defects but they also demonstrated that changes in allelic
expression may be due to other cis- or trans-acting factors.
C50. HNPCC-associated missense mutations in MSH2 may lead
to failure of the protein to bind mismatched DNA
S. E. Ollila 1 , D. Dermadi 1 , J. Jiricny 2 , M. Nyström 1 ;
1 Department of Biological and Environmental Sciences, Division of Genetics,
University of Helsinki, Helsinki, Finland, 2 Institute of Molecular Cancer Research,
University of Zürich, Zürich, Switzerland.
Inherited mutations in mismatch repair (MMR) genes, MLH1 MSH2,
MSH6 and PMS2 predispose to hereditary non-polyposis colorectal
cancer (HNPCC). A significant proportion of all identified MMR gene
mutations are non-truncating, which complicates the interpretation of
their clinical relevance.
We have previously conducted functional studies on non-truncating
MSH2 mutations to clarify their role in the pathogenesis of HNPCC. In
this study we characterized the biochemical defects of mutated MSH2
proteins in more detail, by assessing the ability of the mutated proteins
to bind base-base mismatches, and to dissociate from mismatched
DNA in vitro.
Wild-type and fifteen mutated MSH2 proteins, twelve of which had
showed functional defects in our previous studies, were expressed
in the baculovirus system together with his-tagged wild-type MSH6.
The recombinant heterodimeric MSH2/MSH6 complexes were purified
by fast protein liquid chromatography or partially purified on Ni-NTA
columns. The heterodimers were then used in bandshift assays to examine
their affinity for homo- and heteroduplex DNA. In addition, their
ATP-mediated dissociation from the mismatches was assessed.
None of the proteins showed detectable binding to homoduplex DNA.
Preliminary data showed that some of the mutants, also those defective
in other functional assays, displayed mismatch binding comparable
to the wild-type protein complex. Some failed to bind mismatches, and
some weak binders could also be identified. The ATP-mediated dissociation
from DNA was not impaired in any of the proteins analysed
thus far. This study will provide additional information for interpreting
the pathogenicity of HNPCC-associated missense mutations.
C51. MUTYH-associated polyposis (MAP): spectrum and
frequency of extracolonic lesions
D. Christian, V. Steinke, S. Uhlhaas, P. Propping, W. Friedl, S. Aretz;
Institute of Human Genetics, University Hospital Bonn, Bonn, Germany.
MUTYH-associated polyposis (MAP) is a recently discovered autosomal-recessive
precancerous condition of the colorectum which is
caused by germline mutations in the base excision repair (BER) gene
MUTYH. MAP is associated with a colorectal cancer lifetime risk of
up to 100%, comparable to familial adenomatous polyposis. However,
there are only sporadic descriptions of extraintestinal manifestations.
Here we report on a systematic evaluation of the tumour spectrum in
German MAP patients, based on medical records and anamnestic information.
The study is part of a collaborative European trial performed
together with two centres in the Netherlands and UK. To date, 83 biallelic
German MUTYH mutation carriers were included. The median
age at evaluation was 54 years (range 28-85). In 17% of the patients
duodenal polyps are reported; 49% had extraintestinal lesions (10 patients
had two, 6 had three different tumours), 23% had at least one
extracolonic malignancy. The following tumours were observed more
than once:
Tumour Frequency Age of diagnosis
Breast cancer 10% (4/42 females) 49-60
Endometrial cancer 5% (2/42 females) 32-54
Ovarian cancer 5% (2/42 females) 45-56
Skin cancer 5% (4/83) 32-68
Bladder carcinoma 2,4% (2/83) 45-62
Teratoma 2,4% (2/83) 28-33
Lipomas 8% (7/83) 30-50
Benign skin tumours 15% (12/83) 15-62
Compared to the age-related population-based tumour risk these preliminary data
indicate an increased incidence of gynaecological cancer (endometrium, ovary) and
cutaneous tumours in MAP patients. The risk of breast cancer is close to the agerelated
female population risk. The results may influence future surveillance recommendations
and contribute to the understanding of the underlying pathophysiological
mechanisms in MAP.
The study was supported by the Deutsche Krebshilfe (German Cancer Aid),
project no. 106244
C52. A genome-wide SNP screen for bowel cancer susceptibility
alleles
I. Tomlinson1 , R. Houlston2 , CORGI consortium, NSCCG;
1 2 London Research Institute, London, United Kingdom, Institute of Cancer Research,
Sutton, United Kingdom.
We present the results of a genome-wide screen using the Illumina
Hap550 SNP arrays to search for susceptibility alleles for colorectal
carcinoma. 1,000 familial cases and 1,000 controls from the UK Caucasian
population were screened in stage 1 of this survey and we re-
2