Molecular diagnostic is an important
tool in risk assessment and early detection of many cancers and in
particular of colorectal cancer. It helps in the daily management
of many families attending Genetic Counseling Services. The Hereditary
Colorectal Cancer Molecular Diagnostics Group currently offers DNA
testing for the following cancer susceptibility syndromes:
Familial Adenomatous Polyposis (FAP),
Hereditary Nonpolyposis Colorectal Cancer (HNPCC) and
Li-Fraumeni Syndrome (LFS).
DNA testing of these syndromes are directed
to complement the clinical diagnosis and to perform carrier testing
in families and individuals. The Molecular Diagnostic Group is part
of a multidisciplinary team involved in familial cancer, which includes
the Genetic Counselling Unit and the Psyconcology
Unit. The Molecular
Diagnostic Group has participated in the External Quality Assessment
schemes organised by the EMQN for HNPCC since 2003.
Indications for testing
Cancer patients with familial adenomatous polyposis (FAP)
may consider APC gene (NM_00038) sequence analysis. This test should
be offered in the context of genetic counselling before and after testing.
Methodology
All coding exons and associated intron junctions of the APC gene (15
exons) are analysed by direct DNA sequence analysis using an automated
fluorescent sequencer. Sequence analysis is performed in both directions.
Limitations
The method will not detect mutations located in regions of the APC gene not analysed
such as intron sequences other than the splice junctions or upstream and downstream
sequences. The method also will not detect gross alterations in the APC gene
including most large deletions, duplications, and inversions. We are currently
setting up the methodology to assess APC gene gross deletions.
Specimen Requirements
Testing on peripheral blood Link
MYH Gene DNA Testing
Indications for testing
Cancer patients with attenuated familial adenomatous polyposis (AFAP,
harbouring less than 100 adenomas) or multiple colorectal adenomas
(MCA) may consider MYH gene (NM_012222) sequence analysis. This test
should be offered in the context of genetic counselling before and
after testing.
Methodology
The sequence variants Y165C and G382D are analysed by allele discrimination
using the PCR-light cycler approach. These variants represent >80%
of MYH mutations in Caucasian population. Depending on the
result, all exons and associated intron junctions of the MYH gene (16
exons) are analysed by direct DNA sequence analysis using an
automated fluorescent sequencer. Sequence analysis is performed
in both directions.
Limitations
The method will not detect mutations located in regions of the
MYH gene not analysed (intron sequences other than the splice
junctions, and upstream and downstream sequences). The method
also will not detect gross alterations in the MYH gene including
most large deletions, duplications, and inversions.
Specimen Requirements
Testing on peripheral blood
Microsatellite Instability Analysis (MSI)
Indications for testing
Individuals with a suspected diagnosis of Hereditary Nonpolyposis Colorectal
Cancer Syndrome (HNPCC) are candidates for MSI testing. This test
should be offered in the context of genetic counselling before and
after testing.
Methodology
A panel of two microsatellite markers (BAT26, D12S95), plus a panel
of four markers if necessary (D4S2948, D21S1235, D21S415, BAT25)
are tested (González I, et al JNCI, Vol. 92, Nº.
7, April 5, 2000). Fluorescent labelled primers are used in the
PCR amplification.
Limitations MSI analysis is dependent upon specimen quality and purity. For
MSI, the determination of instability requires comparison of
matched normal and tumour samples. As an average 80% of all specimens
are amenable for DNA analysis
Specimen Requirements
1) Paraffin-embedded tissue with areas of normal tissue
and tumor
Testing on paraffin-embedded tumour tissues or
2) Paraffin-embedded tissue tumor and peripheral blood.
Testing on paraffin-embedded tumour tissues Link
Testing on Peripheral Blood
Mismatch repair genes: MLH1 and MSH2 Genes
DNA Testing
Indications for testing
Individuals with a suspected diagnosis of Hereditary Nonpolyposis Colorectal
Cancer Syndrome (HNPCC) and MSI+ tumours, may consider mismatch repair
genes sequence analysis. This test should be offered in the context
of genetic counselling before and after testing.
Methodology
All coding exons and associated intron junctions of the MLH1 gene
(NM_ 000249; 19 exons) and the MSH2 gene (NM_000251; 16 exons)
are analyzed by direct DNA sequence analysis using an automated
fluorescent sequencer. Sequence analysis is performed in both
directions.
Limitations
This method will not detect mutations located in regions of the
genes that are not analyzed such as intron sequences other than
the splice junctions or upstream and downstream sequences. Direct
sequencing will not detect certain genetic alterations including
most large deletions, duplications, and inversions. As HNPCC is
genetically heterogeneous, mutations in genes other than MLH1 and
MSH2 are possible; these other genes are not analysed in our department.
We are currently setting up the methodology to assess APC gene
gross deletions.
Specimen Requirements
Testing on peripheral blood.
p53 Gene DNA Testing
Indications for testing
Cancer patients with family history indicative of Li-Fraumeni syndrome
(LFS) or Li-Fraumeni syndrome-like, may consider p53 gene sequence
analysis. This test should be offered in the context of genetic counselling
before and after testing.
Methodology
The coding region of exons 4, 5, 6, 7, 8 and 9 of the p53 gene
and associated intron junctions (where >90% of mutations have
been reported) are analysed by direct DNA sequence analysis using
an automated fluorescent sequencer. Sequence analysis is performed
in both directions.
Limitations
The method will not detect mutations located in regions of the
p53 gene not analysed (exons 1,2, 3, 10 and 11, intron sequences
other than the splice junctions, and upstream and downstream
sequences). The method also will not detect gross alterations
in the p53 gene including most large deletions, duplications,
and inversions.
Specimen Requirements
Testing on peripheral blood.
Specimen Requirements - Testing on Peripheral Blood
Peripheral blood samples collected in purple-topped tubes (sodium EDTA
anticoagulant) should be shipped to the laboratory promptly at room
temperature. In general, 20 ml of peripheral blood is needed. For certain
tests, it is also possible to submit RNA directly for testing. These
should be shipped on dry ice for optimal preservation. Delayed shipment
of the specimen may compromise the quality of the RNA required for
testing.
Do not freeze whole blood before sending.
- Testing on Paraffin-Embedded Tumour Tissues
For DNA testing, we can utilize paraffin-embedded tissue or sections
prepared on glass slides with the areas to be analysed indicated.
