2008 Barcelona - European Society of Human Genetics

Cancer genetics
Mutation screening was performed by SSCP and CSCE in genomic
DNA from 256 HNPCC index cases and 239 controls . Haplotyping
was performed analysing 4 MSH2 extragenic microsatellite markers
(D2S288, D2S2227, D2S1247 and D2S1248) in 50 controls and mutation
carriers by using the PHASE program . We analysed the effect of
the mutation in mRNA processing by RT-PCR and in MSH2 expression
by qRT-PCR using RNA from 5 mutation carriers and 18 controls .
None of the remaining HNPCC cases or controls analysed harboured
the mutation. We identified a common telomeric haplotype and two
centromeric haplotypes, both rare in our population . Although we were
not able to identify any abnormal transcript by RT-PCR with the design
used, we observed a 50% reduction of mRNA MSH2 expression in
carriers when compared with controls .
Haplotype analyses suggest a founder effect of the c .[2635-3T>C;
2635-5C>T] MSH2 mutation and expression studies support a pathogenic
role of this mutation .
P04.025
Biallelic mUtYH germline mutations and endometrial cancer
R. Tricarico 1 , B. Ciambotti 1 , P. Bet 2 , C. Di Gregorio 3 , L. Varesco 2 , M. Genuardi 1 ;
1 Department of Clinical Pathophysiology, Medical Genetics Unit, University of
Florence, Florence, Italy, 2 Center for Hereditary Tumours, National Institute
for Cancer Research, Genoa, Italy, 3 Division of Pathology, Hospital of Carpi,
Modena, Italy.
MAP (MUTYH-Associated Polyposis) is an autosomal recessive condition
predisposing to colorectal cancer, due to inherited defects of the
MUTYH gene causing an excess of somatic G>T mutations in the APC
and KRAS genes . To date, few extracolonic manifestations have been
observed in MAP patients, and the clinical spectrum of this condition
is not yet fully established . Recently, one patient with a diagnosis of
endometrial cancer and biallelic MUTYH germline mutations has been
described (Barnetson et al ., Clin Genet 2007: 72: 551-555) . However,
it is not yet clear if biallelic germline mutations in MUTYH increase
the risk of endometrial tumours . We report on two MAP patients with
biallelic MUTYH germline mutations who developed endometrial carcinoma
. In one of the patients, a diagnosis of Lynch syndrome was originally
considered, and subsequently ruled out by immunohistochemical
analysis of mismatch repair proteins and microsatellite instability
analysis . The endometrial tumours were evaluated for PTEN, PIK3CA,
KRAS, BRAF and CTNNB1 mutations . A G>T transversion at codon 12
of the KRAS gene was observed in one tumor . A single 1-bp frameshift
deletion of PTEN was observed in the same sample . Overall, these results
suggest that endometrial carcinoma may represent a component
manifestation of MAP .
P04.026
Gene conversion rates between Pms2, a mismatch repair gene
involved in Lynch syndrome, and its pseudogene Pms2cL;
impact on the reliability of current mutation scanning
H. M. van der Klift 1 , C. M. Tops 2 , E. C. Bik 2 , M. W. Boogaard 2 , K. Pavlikova 3 , H.
Morreau 4 , P. Devilee 1,4 , J. T. Wijnen 1,2 ;
1 Department of Human Genetics, LUMC, Leiden, The Netherlands, 2 Department
of Clinical Genetics, LUMC, Leiden, The Netherlands, 3 Institute of biology
and medical genetics, University Hospital Motol, Praha, Czech Republic, 4 Department
of Pathology, LUMC, Leiden, The Netherlands.
Mutation detection in PMS2, a Mismatch Repair gene involved in
Lynch syndrome (formerly Hereditary Non Polyposis Colorectal Cancer
or HNPCC), is complicated by the presence of the pseudogene
PMS2CL . It has been shown that gene conversion can occur between
the 3’ part of PMS2 and this 700 kb proximally located pseudogene .
We investigated 83 familial colorectal cancer cases and 25 healthy
controls by semi-quantification of paralogous sequence variants (nucleotides
that differ between PMS2 and PMS2CL) in sequence chromatograms
of non-specific PCR products (amplification of PMS2 and
PMS2CL sequences simultaneously) and show that, from exon 12 onwards,
all PMS2 specific nucleotides on which commonly used MLPA
and PMS2 specific PCR primers are based, can be subject to gene
conversion with an increasing frequency of up to 50 % at the 3’ end of
the gene . Current mutation scanning designs are thus extremely prone
to generate false-negatives or even worse false-positives in this part
of the gene .
With a newly developed protocol including non-specific sequencing,
cDNA analysis and Southern Blot analysis that excludes false-nega-
tive and false-positive results due to gene conversion events, we
scanned colorectal cancer patients including strong candidates for a
PMS2 germline mutation based on immunohistochemistry analysis
of their tumours . We show that several mutations detected in the 3’
part of PMS2 using the current mutation scanning designs are actually
false-positives and that also mutations can be missed .
P04.027
screening for genomic rearrangements of the MMR and APC
genes in Galician families with hereditary colorectal cancer
C. Fernández-Rozadilla 1 , N. Gómez-Fernández 1 , I. Madrigal 2 , O. Lortes 1 , M.
Mila 2 , M. Magdalena 1 , S. Castellví-Bel 3 , A. Vega 1 , A. Castells 3 , A. Carracedo 1 ,
C. Ruiz-Ponte 1 ;
1 Fundación Pública Galega de Medicina Xenomica-SERGAS, Grupo de Medicina
Xenomica-USC, CIBER-ER, Santiago de Compostela, Spain, 2 Biochemistry
and Molecular Genetics Department, Hospital Clínic, CIBER-ER, IDIBAPS, Barcelona,
Spain, 3 Department of Gastroenterology, Institut de Malalties Digestives
i Metabòliques, Hospital Clínic, CIBEREHD, IDIBAPS, Barcelona, Spain.
Lynch syndrome and Familial Adenomatous Polyposis (FAP) are two
well-known inherited colorectal cancer syndromes caused by germline
mutations in susceptibility genes (MMR, APC, MYH) . Molecular diagnosis
has until very recently relied only on detection of germline point
mutations in these genes . Nevertheless, it is known that large rearrangements
also play an important role in pathogenesis and should
therefore be considered .
Thirty-nine FAP and 16 Lynch syndrome unrelated Galician families
that were negative for germlime mutations were analysed by MLPA
to determine the frequency of large rearrangements . Positive results
were further confirmed by other methods (FISH, cDNA analysis).
Three different deletions in APC were detected in 4 families (8% of the
total APC families): an exon 4 deletion, an exon 1-15 deletion, and two
whole gene deletions . Carriers of whole allelic deletions presented a
severe polyposis phenotype with an early onset of symptoms .
In Lynch syndrome families, 3 deletions where found in MSH2 (exon 7-
16, exon 2-3 and one whole gene deletion), whereas only one deletion
was found in MLH1 (exon 4-6), making up to 14% (10% MSH2 and 4%
MLH1) of the Lynch syndrome germline mutations . Large rearrangements
were mainly detected in families fulfilling Amsterdam criteria.
The overall results resemble those previously published in other populations
and confirm that large rearrangements represent an important
percentage of FAP and Lynch syndrome germline mutations . They
should therefore be taken into account in molecular genetic testing of
Galician families .
P04.028
New germline mutations of the MLH mismatch repair gene in
brazilian hereditary non-polyposis colorectal cancer
M. V. Dominguez 1 , E. S. Monteiro 2 , D. M. Carraro 1 , B. M. Rossi 2 ;
1 Laboratory of Genomic and Molecular Biology of Treatment and Investigation
Center of Hospital A.C.Camargo, São Paulo, Brazil, 2 Department of Pelvic
Surgery of Hospital A.C. Camargo, São Paulo, Brazil.
Background: Hereditary non-polyposis colorectal cancer (HNPCC) is
an autosomal dominant syndrome predisposing to the early development
of various cancers including those of colon, rectum, endometrium,
ovarium, small bowel, stomach and urinary tract . HNPCC is caused by
germline mutations in the DNA mismatch repair genes, mostly MLH1
and MSH2 . The mutation spectrum in the Brazilian population is still
poorly documented .
Aim: To report our experience on the mutation screening and investigation
of the pathogenicity of the mutations . in MLH1 and MSH2 genes
in 20 unrelated Brazilian kindreds with suspected HNPCC .
Methods: Twenty brazilian patients with familial clustering of CRC
were genetically tested. The families were identified by applying the
Amsterdam and Bethesda Criteria . Point mutations in the MLH1 and
MSH2 genes were screened by denaturing high performance liquid
chromatography (DHPLC) followed by direct sequencing .
Results: Two novel nonpathogenic mutations (2 of 20 [10%]) in MLH1exon
15 (intron) nt . 1707 a->g and MLH1-exon 13 (intron) nt . 1632
g->a were identified in brazilian CRC patients.
Conclusion: According to the Human Gene Mutation Database (HGMD)
and the International Society of Gastrointestinal Hereditary Tumors
(InSIGHT) Database, this is the first report of this mutation.