Lab Dept:

Anatomic Pathology

Test Name:

COLARIS AP®COMPREHENSIVE SEQUENCING

General Information

Lab Order Codes:

COLAP

Synonyms:

Full sequence APC Gene; FAP/AFAP; MYH

CPT Codes:

83891 - Isolation or extraction of highly purified nucleic acid
83898 x42 - Amplification of patient nucleic acid, single primer pair, each pair
83904 x42 - Mutation identification by sequencing, single segment, each segment
83894 x3 - Separation by gel electrophoresis

Test Includes:

This test includes a Full sequence and large rearrangement analysis of the APC gene for FAP/AFAP & MYH mutation panel analysis for MYH-associated polyposis. NOTE: Patients heterozygous for a single MYH panel mutation will automatically receive full sequence analysis of the MYH gene. The MYH mutation panel is appropriate only for individuals of European ancestry. Full sequence analysis of MYH may be more useful in other individuals.

Logistics

Test Indications:

Comprehensive Colaris AP® assesses an individual’s risk of developing colorectal polyps and cancer based on detection of mutations in the APC and MYH genes.

Adenomatous polyposis syndromes are hereditary colorectal cancer syndromes that are associated with multiple adenomatous polyps in the colon. There are three adenomatous polyposis syndromes: Familial adenomatous polyposis (FAP) and attenuated FAP (AFAP), which are caused by a mutation in the adenomatous polyposis coli (APC) gene, and MYH–associated polyposis (MAP), which is associated with mutations in the mutY homolog (MYH) gene.

Lab Testing Sections:

Anatomic Pathology - Send-Outs

Referred to:

Myriad Genetic Laboratories, Inc.

Phone Numbers:

Minneapolis:

Saint Paul:

612-813-6280

651-220-6555

Test Availability:

Sunday - Thursday

Turnaround Time:

3 to 4 weeks

Special Instructions:

Obtain special kit from Myriad Genetic Laboratories. Complete forms in kit and send to the lab with the patient or specimen.

Must specify variant(s), and relationship of known mutation carrier to patient (e.g. sister). An Informed consent signed by an individual legally authorized to do so (e.g. The Health Care Provider) is required. (Note: Test requests without a signature will not be processed). Ancestry and patient history are requested.

Specimen

Specimen Type:

Whole blood

Container:

Lavender top (EDTA) tube
(2-10 mL EDTA tubes are provided in the Myriad kit)

Draw Volume:

20 mL blood

Processed Volume:

Same as Draw Volume

Collection:

Routine venipuncture

Collect 20 mL of whole blood into 2-10 mL lavender top ( EDTA) tubes provided in Myriad Kit. Invert specimens gently to mix. Label tubes appropriately. Send collected specimens and collection kit to the laboratory as soon as possible. Specimens are viable for 72 hours. Avoid collections on Friday, if possible. It is recommended that specimens not be shipped on Friday. Contact the laboratory prior to collections on Friday or Saturday.

Special Processing:

Lab Staff: Do not centrifuge specimens. Specimens should remain in original collection containers. Ship at ambient temperature Monday through Thursday. Specimens must be received by reference lab within 72 hours of collection.

Specify relationship of known mutation carrier to patient. Specify Mutations. Ship specimen in kit and mailer provided by Myriad.

Specimens should be kept at room temperature and shipped according to the test kit instructions.

Patient Preparation:

If patient is receiving chemotherapy, collected specimen before administration of chemo.

Sample Rejection:

Wrong tube type, insufficient DNA extracted, specimen too old, mislabeled or unlabeled specimens

Interpretive

Reference Range:

The test will be resulted with the following as appropriate:

Positive for a deleterious mutation: Includes all nonsense and frameshift mutations in APC that occur at or before amino acid 2644 (based on documentation of deleterious mutations in APC).

In addition, specific missense mutations and non-coding intervening sequence (IVS) mutations are recognized as deleterious on the basis of data derived from linkage analysis of high risk families, functional assays, biochemical evidence and/or demonstration of abnormal mRNA transcript processing.

Deletions and duplications of an entire exon(s) identified by Southern blot analysis may also be interpreted to be deleterious. Deleterious large genomic rearrangements include single exon and multi exonic deletions and duplications that are out of frame. In frame deletions/duplications are interpreted on an individual basis and the specific evidence supporting the classification of these mutations is included in the individual patient report.

Genetic variant, suspected deleterious: Includes genetic variants for which the available evidence indicates a likelihood, but not proof, that the mutation is deleterious. The specific evidence supporting such an interpretation will be summarized for individual variants on each such report.

Genetic variant, favor polymorphism: Includes genetic variants for which available evidence indicates that the variant is highly unlikely to contribute substantially to cancer risk. The specific evidence supporting such an interpretation will be summarized for individual variants on each such report.

Genetic variant of uncertain significance: Includes missense mutations and mutations that occur in analyzed intronic regions whose clinical significance has not yet been determined, as well as nonsense and frameshift mutations that occur distal to amino acid position 2644 in APC.

A genetic variant of uncertain significance in APC is considered less likely to be deleterious if it has been observed in one or more individuals with a known deleterious mutation in the same gene.

No deleterious mutation detected: Includes non-truncating genetic variants observed at an allele frequency of approximately 1% of a suitable control population (providing that no data suggest clinical significance), as well as all genetic variants for which published data demonstrate absence of substantial clinical significance. Also includes mutations in the protein-coding region that neither alter the amino acid sequence nor are predicted to significantly affect exon splicing, and base pair alterations in non-coding portions of the gene that have been demonstrated to have no deleterious effect on the length or stability of the mRNA transcript. Data on polymorphic variants are available upon request.

There may be uncommon genetic abnormalities in APC that will not be detected by Colaris AP (see Limitations of Method). This analysis, however, is believed to rule out the majority of abnormalities in this gene, which is believed to be responsible for most Familial Adenomatous Polyposis (FAP) and attenuated FAP (AFAP).

Y165C and G382D in MYH were not detected: There may be other mutations in MYH that were not detected because this test was designed to detect Y165C and G382D.

Specific variant/mutation not identified: Indicates that specific and designated mutations or variants are not present in the individual being tested. If a specific deleterious mutation has been identified in a family member, a negative analysis for the specific mutation indicates that the tested individual is at the general population risk of developing those cancers and benign findings associated with FAP and AFAP.

Positive for two MYH mutations: Includes observations of Y165C and G382D together, or observations of two alleles of Y165C or G382D. The presence of these two MYH mutations has been documented in recent literature to be associated with colorectal polyposis and cancer.

One MYH mutation detected, colorectal polyposis and cancer risk unknown: Includes observations of one allele of Y165C or G382D. It is currently unknown whether individuals who carry a single MYH mutation are at some measure of increased risk for colorectal polyposis and cancer. Patients with one MYH mutation will automatically receive full sequence analysis of the MYH gene (see MYH Technical Specifications).

Critical Values:

N/A

Limitations:

Analytical Sensitivity: The incidence of a false report of a genetic variant or mutation resulting from technical error is considered negligible because of independent confirmation of all genetic variants (see above). No false-positive results were seen in a sample set consisting of thirty-three DNA samples obtained from individuals that were analyzed by the sequencing method described above. In addition, no false positive results were seen in Southern blot analysis of a set of twenty-two samples that were previously examined for deletions and duplications in APC.

Failure to detect a genetic variant or mutation in the analyzed DNA regions may result from errors in specimen handling and tracking, amplification and sequencing reactions, or computer-assisted analysis and data review. The sequencing method described above accurately identified each of twenty-four mutations in APC samples that had been analyzed previously by independent laboratories. In addition, seven samples that were previously examined by alternative methods for deletions and duplications in APC were correctly identified by the Southern blot method.

Limitations of Method: There may be limited portions of APC for which sequence determination can be performed only in the forward or reverse direction. Unequal allele amplification may result from rare polymorphisms under primer sites. This assay will not detect some types of errors in RNA transcript processing.

Analysis of MYH includes analysis for Y165C and G382D, and does not rule out the possibility of other mutations. Unequal allele amplification may result from rare polymorphisms under primer sites.

Additional information about performance characteristics of the MYH analysis is available in the MYH Technical Specifications.

Methodology:

DNA analysis, by direct sequence or Southern blot methods, for a specified mutation in APC. Southern blot analysis of all exons of APC is performed for all requests for single site mutation analysis of a large rearrangement.

Blood samples are assigned a unique bar code for robotic specimen tracking. DNA is extracted and purified from white cells isolated from each sample.

Sequence Analysis: Aliquots of patient DNA are each subjected to polymerase chain reaction (PCR) amplification reactions. The amplified products are each directly sequenced in forward and reverse directions using fluorescent dye-labeled sequencing primers. Chromatographic tracings of each amplicon are analyzed by a proprietary computer-base review followed by visual inspection and confirmation. Genetic variants are detected by comparison with a consensus wild-type sequence constructed for each gene. All potential genetic variants are independently confirmed by repeated PCR amplification of the indicated gene region(s) and sequence determination as above.

Southern blot analysis: Aliquots of genomic DNA are digested individually with three restriction enzymes or combinations of enzymes for APC analysis. Digested DNA is electrophoresed in an agarose gel, transferred to a membrane, and hybridized with a gene-specific probe labeled with 32P. The probe binds to all fragments containing coding; sequences of that gene Autoradiographs and phosphorimages are produced and analyzed for the presence of novel bands and for fragment dosage, from which it is determined which, if any, exons have been deleted or duplicated, Positive and negative controls are run with deleted or duplicated. Positive and negative controls are run with each batch. All potential mutations are independently confirmed.

Contraindications:

No family history

References:

Colaris AP Technical Specifications, Myraid Genetic Laboratories, Inc. 29 Aug 2005

Myriad Genetic Laboratories Website (June 2006)
(801) 584-3600 Fax (801) 584-3640