2008 Barcelona - European Society of Human Genetics

Cancer genetics
Techniques
There is good evidence for the hypothesis that colorectal carcinoma
initiation is progression from benign adenoma to carcinoma via a series
of sequential somatic genetic and epigenetic changes . However
it is not known whether genomic instability is a necessary initiating
factor .
We are currently recruiting patients with a known family history of
colorectal carcinoma or who are undergoing surveillance colonoscopy
for recurrent polyps . These patients attend screening colonoscopy,
where polyps are identified and removed. To date, 137 patients have
been recruited with a total of 195 polyps .
A pilot study, looking at markers (K-ras, BRAF, MSI, LOH) of genomic
instability in 168 polyps in 107 patients demonstrated that this was
technically feasible .
We will collect 50 hyperplastic polyps and 300 adenomatous polyps,
including 30 serrated adenomas, and 10 polyps from patients with
Lynch syndrome .
Isolated crypts will be extracted from polyps and tested for genomic instability
using several parameters: chromosomal loss/gain, copy number
variation, LoH, MSI and CPG-Island Promoter Methylation . We will
also screen for mutations in known target genes .
Results will be compared with normal tissue, to determine when/if
genomic instability occurs, and it’s relationship to other (epi)genetic
changes
Conclusions
We hope to elucidate the earliest drivers of colorectal carcinogenesis:
when each form of genomic instability occurs, whether they initiate
carcinogenesis and how this relates to family history and clinico-pathological
features .
P04.009
microsatellites instability and mismatch repair genes mutations
in hereditary nonpolyposis colorectal cancer
C. IANCU1 , C. Vrabie2 , D. Iancu1 , G. Girbea1 , A. Constantinescu1 , C. Constantinescu1
, E. Neagu1 , L. Barbarii1 ;
1 2 National Institute of Legal Medicine, Bucharest, Romania, National Institute
“Victor Babes”, Bucharest, Romania.
Background: Hereditary nonpolyposis colorectal cancer (HNPCC) is
an autosomal dominant cancer predisposition syndrome caused by
germ-line mutations in DNA mismatch repair genes . Therefore diagnosis
of genomic deletions of one or more exons of the MSH2 and MLH1
genes and the analyse of microsatellite instability (MSI) from a minimal
amount of highly damaged DNA is difficult.
Methods. Formalin-fixed and paraffin-embedded tissue samples from
25 HNPCC patients were analysed for microsatellite instability (MSI)
using a panel of 7 markers (BAT-25, BAT-26, NR-21, NR-24, MONO-
27, Penta C and Penta D) included in a commercial kit . MLPA tests
were also performed to screen for major mutation in the mismatch repair
genes MLH1 and MSH2 .
Results . The study revealed a 58% microsatellite instability pattern for
the 25 unrelated Romanian HNPCC patients . 5 to 10% of major alterations
detected on the MLH1 and MSH2 genes are subject for further
characterization .
Conclusions . So far, family history, MSI and MMR gene mutation analysis
become critical parameters for HNPCC genetic characterization
and genetic counseling .
P04.010
microsatellite instability and promotor hypermethylation of
MLH and MSH in Bulgarian patients with sporadic colorectal
cancer
A. V. Mitkova 1 , T. Vlaykova 2 , G. Stancheva 1 , T. Kadiyska 3 , M. Gulubova 4 , E.
Kostadinov 5 , D. Damyanov 5 , G. Cirovski 6 , Y. Yovchev 6 , I. Kremensky 3 , V. Mitev 7 ,
R. Kaneva 1 ;
1 Molecular Medicine Centre, Medical University-Sofia, Sofia, Bulgaria, 2 Dept.
Chemistry and Biochemistry, Trakia University, Stara Zagora, Bulgaria, 3 National
Genetic Laboratory, University Hospital of Obstetrics and Gynaecology
“Majchin Dom”, Medical University-Sofia, Sofia, Bulgaria, 4 Dept. General and
Clinical Pathology, Trakia University, Stara Zagora, Bulgaria, 5 Dept. Gastroenterology,
University Hospital “Queen Giovana”, Medical University-Sofia, Sofia,
Bulgaria, 6 Dept. General Surgery, Medical Faculty, Trakia University, Stara
Zagora, Bulgaria, 7 Dept. Chemistry and Biochemistry, Medical University-Sofia,
Sofia, Bulgaria.
Inactivation of the genes involved in DNA mismatch repair is associated
with microsatellite instability (MSI) and loss of heterozygosity
(LOH) . Mutations in MLH1 and MSH2 genes are usually the cause of
hereditary colorectal cancers (CRC), such as HNPCC, while microsatellite
instability in sporadic CRC often results from epigenetic inactivation
of hMLH1 due to DNA promotor methylation .
We examined MSI in 98 patients with sporadic CRC and the methylation
status of the MLH1 and MSH2 promotor regions in the cases with
MSI/LOH. MSI was evaluated in a panel of five microsatellite markers
(BAT26, D5S346, D18S35, D2S123 and FGA) and the hypermethylation
assessed by methyl-specific PCR (MSP) of bisulfite converted
DNA . The PCR products were analyzed on agarose gel electrophoresis,
following by direct sequencing .
Our results demonstrated MSI/LOH in 29 patients (29 .5%) with sporadic
colorectal cancer . Among them the promoter hypermethylation
of MLH1 was observed in 14 (48 .3%) of the cases, whereas promotor
hypermethylation of MSH2 was observed only in one case (3 .4%) . The
epigenetic changes of MLH1 were associated with loss of the protein
expression .
Conclusions: Widespread promotor methylation of tumor suppressor
genes is a common mechanism of gene inactivation during tumorigenesis
. Our results suggest that microsatellite instability in Bulgarian
patients with sporadic CRC and the epigenetic inactivation of hMLH1
in association with DNA methylation occurs in similar frequency to relative
studies of sporadic colorectal cancer . The promoter hypermetilation
of MSH2, although a rare event, is also a possible cause of mismatch
repair system deficiency in this type of CRC.
P04.011
implication of MSH mutations in colorectal cancer (cRc)
patients carrying a monoallelic MYH mutation
M. D. Giráldez 1 , F. Balaguer 1 , T. Caldés 2 , N. Gómez-Fernández 3 , C. Ruiz-Ponte
3 , J. Muñoz 1 , V. Gonzalo 1 , T. Ocaña 1 , J. Clofent 4 , A. Carracedo 3 , A. Castells 1 ,
S. Castellvi-Bel 1 , A. E. G. Gastrointestinal Oncology Group 5 ;
1 Gastroenterology, Hospital Clínic, IMDiM, CIBEREHD, IDIBAPS, Barcelona,
Spain, 2 Oncology Molecular Lab, Hospital Clínico Universitario San Carlos, Madrid,
Spain, 3 Grupo de Medicina Xenomica-USC-FPGMX-CIBERER, Santiago
de Compostela, Spain, 4 Gastroenterology Department, Hospital Meixoeiro-
CHUVI, Vigo, Spain, 5 Asociación Española de Gastroenterología, Barcelona,
Spain.
Background . CCR risk associated with germline monoallelic MYH mutations
remains controversial . Nevertheless, a slight increased risk has
been suggested . MYH and MSH6 proteins act together during the DNA
reparation process . It has been shown that individuals harboring mutations
in both genes could have an increased CRC risk .
Aim . To evaluate the prevalence of MSH6 mutations in CRC patients
carrying a monoallelic mutation in MYH .
Patients and methods . We analyzed the prevalence of MSH6 mutations
in a CRC patients cohort with and without a monoallelic MYH mutation
(group I, n=26 and group II, n=50, respectively), and in another
cohort including healthy controls with and without a monoallelic MYH
mutation (group III, n=21, group IV, n=21, respectively) . Patients and
controls were recruited from EPICOLON and Hospital Clinic, respectively
. Mutational screening was performed by TaqMan, SSCP, DHPLC
and sequencing .
Results . We detected three germline MSH6 mutations in group I
(11 .5%), a missense variant (R635G), a change in 3’UTR region
(4098A>C) and a nonsense mutation (Q982X) . We did not detect any
MSH6 mutation in groups II and IV, whereas only one was found in
group III (4 .8%) (M1326G) . MSH6 mutations were more frequently
found in CRC patients with a monoallelic MYH mutation than in noncarriers
(11 .5% vs 0%, p=0 .037) .
Conclusions . CRC patients harboring a monoallelic MYH mutation may
carry more frequently a MSH6 mutation than patients without them .
We could hypothesize that the CRC predisposition associated with a
mutation in each gene could add up and confer an increased risk .