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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:

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.

APC Gene DNA Testing

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

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

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

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.

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.