Melanoma Predisposition via CDK4 Gene Sequencing with CNV Detection
- Summary and Pricing
- Clinical Features and Genetics
Sequencing and Del/Dup via NGS
|Test Code||Test Copy Genes||Individual Gene Price||CPT Code Copy CPT Codes|
Our most cost-effective testing approach is NextGen sequencing with Sanger sequencing supplemented as needed to ensure sufficient coverage and to confirm NextGen calls that are pathogenic, likely pathogenic or of uncertain significance. If, however, full gene Sanger sequencing only is desired (for purposes of insurance billing or STAT turnaround time for example), please see link below for Test Code, pricing, and turnaround time information.
The great majority of tests are completed within 28 days.
Only 2% of the families in the Geno-MEL study carried CDK4 mutations (Goldstein et al. Cancer Res 66:9818–9828, 2006), and less than 15 families have been reported worldwide (Meyle at al. Hum Genet 126:499–510, 2009). To our knowledge, no causative copy number variants have been reported in the CDK4 gene.
Melanoma is a malignant tumor that originates in melanocytes, a specialized cell type that produces melanin pigments that determine skin, hair and eye color (Lin and Fisher. Nature 445:843–850, 2007). Over the past few decades there has been a rise in the incidence of melanoma due to improved awareness leading to additional diagnoses and to lifestyle changes that have resulted in an increase in sun exposure (Mehnert and Kluger. Curr Oncol Rep 14:449–457, 2012). Most melanomas occur as sporadic cases with no recognized familial component; however, melanoma appears to be twice as common in people with an affected parent, three times as common if a sibling is affected, and nine times as common if both a parent and a sibling are affected (Hemminki et al. J Invest Dermatol 120:217–223, 2003). Familial clustering is likely the result of genetic and environmental factors. Heritable alleles for melanoma susceptibility range from high-risk, high-penetrance alleles that are rare, to low-risk, low-penetrance alleles that are rather ubiquitous (Nelson et al. Clinics in Dermatology 27, 46–52, 2009). Mutations in the highly penetrant CDKN2A, and less commonly the CDK4 gene, are responsible for the majority of predisposition to melanoma cases. CDK4 mutations appear to be associated with a similar median age at melanoma diagnosis as CDKN2A mutations (i.e. 36 years) (Meyle at al. Hum Genet 126:499–510, 2009; Goldstein et al. Cancer Res 66:9818–9828, 2006).
Melanoma predisposition is inherited in an autosomal dominant manner. The strongest genetic risk for the development of melanoma results from heritable alterations in the cyclin-dependent kinase inhibitor 2A (CDKN2A) gene, however mutations in the CDK4 gene have been reported. CDK4 is an oncogene that encodes a protein kinase. The two most common CDK4 mutations leading to melanoma are both at codon 24 (ie, Arg24Cys and Arg24His). Mutations in CDK4 are therefore oncogenic alleles, with only a single mutation necessary to result in tumorigenesis (Nelson et al. Clinics in Dermatology 27, 46–52, 2009). The function of the upstream p16/INK4a protein is to inhibit CDK4/6 protein-mediated phosphorylation of the Rb (Retinoblastoma) protein; p16/INK4a therefore keeps the Rb protein dephosphorylated, which is the active state of Rb. Mutated CDK4 protein is resistant to the inhibition of p16/INK4a (Ibrahim et al. Annu. Rev. Pathol. Mech. Dis. 4:551-579, 2009), thus causing the phosphorylation of the Rb protein, which leads to release of the bound transcription factor, E2F, which then allows the cell to undergo unregulated cell division leading to the development of melanoma.
For this Next Generation Sequencing (NGS) test, sequencing is accomplished by capturing specific regions with an optimized solution-based hybridization kit, followed by massively parallel sequencing of the captured DNA fragments. Additional Sanger sequencing is performed for regions not captured or with insufficient number of sequence reads. All reported pathogenic, likely pathogenic, and variants of uncertain significance are confirmed by Sanger sequencing.
For Sanger sequencing, polymerase chain reaction (PCR) is used to amplify targeted regions. After purification of the PCR products, cycle sequencing is carried out using the ABI Big Dye Terminator v.3.0 kit. PCR products are resolved by electrophoresis on an ABI 3730xl capillary sequencer. In nearly all cases, cycle sequencing is performed separately in both the forward and reverse directions.
Copy number variants (CNVs) are also detected from NGS data. We utilize a CNV calling algorithm that compares mean read depth and distribution for each target in the test sample against multiple matched controls. Neighboring target read depth and distribution and zygosity of any variants within each target region are used to reinforce CNV calls. All CNVs are confirmed using another technology such as aCGH, MLPA, or PCR before they are reported.
This test provides full coverage of all coding exons of the CDK4 gene, plus ~10 bases of flanking noncoding DNA. We define full coverage as >20X NGS reads or Sanger sequencing.
Indications for Test
Individuals who have multiple family members with melanoma, multiple primary melanomas within individual members, and diagnosis of additional tumors within a family. Also individuals who want to know their carrier status of CDK4 mutations. This test is specifically designed for heritable germline mutations and is not appropriate for the detection of somatic mutations in tumor tissue.
|Official Gene Symbol||OMIM ID|
|Melanoma Sequencing Panel with CNV Detection|
- Genetic Counselor Team - email@example.com
- Jerry Machado, PhD, DABMG, FCCMG - firstname.lastname@example.org
- Goldstein et al. (2006). "High-risk melanoma susceptibility genes and pancreatic cancer, neural system tumors, and uveal melanoma across GenoMEL." Cancer Res 66:9818–9828. PubMed ID: 17047042
- Hemminki et al. (2003). "Familial and attributable risks in cutaneous melanoma: effects of proband and age." J Invest Dermatol 120:217–223. PubMed ID: 12542525
- Ibrahim et al. (2009). "Molecular pathogenesis of cutaneous melanocytic neoplasms." Annu. Rev. Pathol. Mech. Dis. 4:551-579. PubMed ID: 19400696
- Lin JY, Fisher DE. 2007. Melanocyte biology and skin pigmentation. Nature 445: 843–850. PubMed ID: 17314970
- Mehnert and Kluger. (2012). "Driver mutations in melanoma: lessons learned from bench-to-bedside studies." Curr Oncol Rep 14:449–457. PubMed ID: 22723080
- Meyle at al. (2009). "Genetic risk factors for melanoma." Hum Genet 126:499–510. PubMed ID: 19585149
- Nelson AA, Tsao H. 2009. Melanoma and genetics. Clinics in Dermatology 27: 46–52. PubMed ID: 19095153
Sequencing and Deletion/Duplication Testing via NextGen Sequencing using PG-Select Capture Probes
We use a combination of Next Generation Sequencing (NGS) and Sanger sequencing technologies to cover the full coding regions of the listed genes plus ~20 bases of non-coding DNA flanking each exon. As required, genomic DNA is extracted from the patient specimen. For NGS, patient DNA corresponding to these regions is captured using an optimized set of DNA hybridization probes. Captured DNA is sequenced using Illumina’s Reversible Dye Terminator (RDT) platform (Illumina, San Diego, CA, USA). Regions with insufficient coverage by NGS are covered by Sanger sequencing. All pathogenic, likely pathogenic, or variants of uncertain significance are confirmed by Sanger sequencing.
For Sanger sequencing, Polymerase Chain Reaction (PCR) is used to amplify targeted regions. After purification of the PCR products, cycle sequencing is carried out using the ABI Big Dye Terminator v.3.0 kit. PCR products are resolved by electrophoresis on an ABI 3730xl capillary sequencer. In nearly all cases, cycle sequencing is performed separately in both the forward and reverse directions.
Patient DNA sequence is aligned to the genomic reference sequence for the indicated gene region(s). All differences from the reference sequences (sequence variants) are assigned to one of five interpretation categories, listed below, per ACMG Guidelines (Richards et al. 2015).
(1) Pathogenic Variants
(2) Likely Pathogenic Variants
(3) Variants of Uncertain Significance
(4) Likely Benign Variants
(5) Benign, Common Variants
Human Genome Variation Society (HGVS) recommendations are used to describe sequence variants (http://www.hgvs.org). Rare variants and undocumented variants are nearly always classified as likely benign if there is no indication that they alter protein sequence or disrupt splicing.
Deletion and Duplication Testing via NGS
As of March 2016, 6.36 Mb of sequence (83 genes, 1557 exons) generated in our lab was compared between Sanger and NextGen methodologies. We detected no differences between the two methods. The comparison involved 6400 total sequence variants (differences from the reference sequences). Of these, 6144 were nucleotide substitutions and 256 were insertions or deletions. About 65% of the variants were heterozygous and 35% homozygous. The insertions and deletions ranged in length from 1 to over 100 nucleotides.
In silico validation of insertions and deletions in 20 replicates of 5 genes was also performed. The validation included insertions and deletions of lengths between 1 and 100 nucleotides. Insertions tested in silico: 2200 between 1 and 5 nucleotides, 625 between 6 and 10 nucleotides, 29 between 11 and 20 nucleotides, 25 between 21 and 49 nucleotides, and 23 at or greater than 50 nucleotides, with the largest at 98 nucleotides. All insertions were detected. Deletions tested in silico: 1813 between 1 and 5 nucleotides, 97 between 6 and 10 nucleotides, 32 between 11 and 20 nucleotides, 20 between 21 and 49 nucleotides, and 39 at or greater than 50 nucleotides, with the largest at 96 nucleotides. All deletions less than 50 nucleotides in length were detected, 13 greater than 50 nucleotides in length were missed. Our standard NextGen sequence variant calling algorithms are generally not capable of detecting insertions (duplications) or heterozygous deletions greater than 100 nucleotides. Large homozygous deletions appear to be detectable.
Interpretation of the test results is limited by the information that is currently available. Better interpretation should be possible in the future as more data and knowledge about human genetics and this specific disorder are accumulated.
When Sanger sequencing does not reveal any difference from the reference sequence, or when a sequence variant is homozygous, we cannot be certain that we were able to detect both patient alleles. Occasionally, a patient may carry an allele which does not amplify, due to a large deletion or insertion. In these cases, the report will contain no information about the second allele. Our Sanger and NGS Sequencing tests are generally not capable of detecting Copy Number Variants (CNVs).
We sequence all coding exons for each given transcript, plus ~20 bp of flanking non-coding DNA for each exon. Test reports contain no information about other portions of the gene, such as regulatory domains, deep intronic regions or any currently uncharacterized alternative exons.
In most cases, we are unable to determine the phase of sequence variants. In particular, when we find two likely causative mutations for recessive disorders, we cannot be certain that the mutations are on different alleles.
Our ability to detect minor sequence variants due to somatic mosaicism is limited. Sequence variants that are present in less than 50% of the patient’s nucleated cells may not be detected.
Runs of mononucleotide repeats (eg (A)n or (T)n) with n >8 in the reference sequence are generally not analyzed because of strand slippage during PCR.
Unless otherwise indicated, DNA sequence data is obtained from a specific cell-type (usually leukocytes from whole blood). Test reports contain no information about the DNA sequence in other cell-types.
We cannot be certain that the reference sequences are correct.
Rare, low probability interpretations of sequencing results, such as for example the occurrence of de novo mutations in recessive disorders, are generally not included in the reports.
We have confidence in our ability to track a specimen once it has been received by PreventionGenetics. However, we take no responsibility for any specimen labeling errors that occur before the sample arrives at PreventionGenetics.
myPrevent - Online Ordering
- The test can be added to your online orders in the Summary and Pricing section.
- Once the test has been added log in to myPrevent to fill out an online requisition form.
- A completed requisition form must accompany all specimens.
- Billing information along with specimen and shipping instructions are within the requisition form.
- All testing must be ordered by a qualified healthcare provider.
(Delivery accepted Monday - Saturday)
- Collect 3 ml -5 ml (5 ml preferred) of whole blood in EDTA (purple top tube) or ACD (yellow top tube). For Test #500-DNA Banking only, collect 10 ml -20 ml of whole blood.
- For small babies, we require a minimum of 1 ml of blood.
- Only one blood tube is required for multiple tests.
- Ship blood tubes at room temperature in an insulated container. Do not freeze blood.
- During hot weather, include a frozen ice pack in the shipping container. Place a paper towel or other thin material between the ice pack and the blood tube.
- In cold weather, include an unfrozen ice pack in the shipping container as insulation.
- At room temperature, blood specimen is stable for up to 48 hours.
- If refrigerated, blood specimen is stable for up to one week.
- Label the tube with the patient name, date of birth and/or ID number.
(Delivery accepted Monday - Saturday)
- Send in screw cap tube at least 5 µg -10 µg of purified DNA at a concentration of at least 20 µg/ml for NGS and Sanger tests and at least 5 µg of purified DNA at a concentration of at least 100 µg/ml for gene-centric aCGH, MLPA, and CMA tests, minimum 2 µg for limited specimens.
- For requests requiring more than one test, send an additional 5 µg DNA per test ordered when possible.
- DNA may be shipped at room temperature.
- Label the tube with the composition of the solute, DNA concentration as well as the patient’s name, date of birth, and/or ID number.
- We only accept genomic DNA for testing. We do NOT accept products of whole genome amplification reactions or other amplification reactions.
(Delivery preferred Monday - Thursday)
- PreventionGenetics should be notified in advance of arrival of a cell culture.
- Culture and send at least two T25 flasks of confluent cells.
- Some panels may require additional flasks (dependent on size of genes, amount of Sanger sequencing required, etc.). Multiple test requests may also require additional flasks. Please contact us for details.
- Send specimens in insulated, shatterproof container overnight.
- Cell cultures may be shipped at room temperature or refrigerated.
- Label the flasks with the patient name, date of birth, and/or ID number.
- We strongly recommend maintaining a local back-up culture. We do not culture cells.