Piebaldism and Familial Gastrointestinal Stromal Tumors (GISTs) via the KIT Gene
- Summary and Pricing
- Clinical Features and Genetics
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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.
For ordering targeted known variants, please proceed to our Targeted Variants landing page.
The great majority of tests are completed within 28 days.
This test is predicted to detect a loss-of-function pathogenic variant in nearly all patients with piebaldism (Syrris et al. 2002). The clinical sensitivity of germline pathogenic variants in KIT from individuals with GIST is considered to be low, but at least 16 variants have been reported in several families with multiple occurrences of GIST (Bachet et al. 2013).
Deletion/Duplication Testing via aCGH
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The great majority of tests are completed within 28 days.
Clinical sensitivity is not known; however gross deletions have been reported for Piebaldism (Ezoe et al. 1995).
Piebaldism is caused by inactivating pathogenic variants in KIT, while neoplasms (GISTs, GCTs, AML, CML and lymphomas) are caused by activating, or gain-of-function, pathogenic variants (Akin and Metcalf 2004). KIT encodes a transmembrane receptor tyrosine kinase that responds to the Stem Cell Factor (SCF) ligand. Kit is expressed on the surface of several different cell types, including melanocytes, gastrointestinal pacemaker cells, hematopoietic progenitor cells, mast cells and germ cells. In melanocytes, Kit signaling is required for migration from the embryonic neural crest; loss of Kit function results in the absence of melanocytes in midline patches of hair and skin. In other cells, signaling through the SCF/c-kit pathway promotes proliferation and survival; constitutive activation of Kit in these cells causes excessive proliferation, malignancy and metastasis. Loss-of-function variants are found throughout all 21 exons of the KIT gene, whereas activating variants appear to be clustered in exons 2, 8, 9, 11, 13 and 17 (Heinrich et al. 2002). Importantly, the selective Kit inhibitor Gleevec (Novartis Pharmaceuticals, East Hanover, NJ) is a very effective treatment for KIT-dependent neoplasms (Tuveson et al. 2001). However, a few activating variants, such as D816V, have been shown to be insensitive to Gleevec (Miettinen et al. 2002). As a result, identifying the causative variant in these neoplasms is critical for determining the most effective mode of treatment.
Pathogenic variants in the c-kit proto-oncogene (KIT) cause a variety of disorders, including piebaldism, gastrointestinal stromal tumors (GISTs), germ cell tumors (GCTs) and hematopoietic neoplasms such as acute myeloid leukemia (AML), chronic myeloid leukemia (CML) and malignant lymphoma (Akin and Metcalfe 2004). Piebaldism is an autosomal dominant disorder characterized by patches of skin and hair that entirely lack pigment (Murakami et al. 2004). These white patches are mainly found on the scalp and forehead, resulting in a distinctive white forelock trait. Gastrointestinal stromal tumors, or GISTs, are rare mesenchymal tumors that specifically express the Kit protein and originate in the gastrointestinal (GI) tract or abdomen (Miettinen et al. 2002). In most cases, GISTs spontaneously arise due to somatic pathogenic variants of the KIT gene. However, families with germline pathogenic variants in the KIT gene have also been described (Isozaki et al. 2000; Maeyama et al. 2001). In these families, inheritance of heterozygous KIT variants leads to cutaneous hyperpigmentation and development of multiple GISTs.
For this NextGen test, the full coding regions plus ~20 bp of non-coding DNA flanking each exon are sequenced for the gene listed below. 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 any regions not captured or with insufficient number of sequence reads. All pathogenic, likely pathogenic, or variants of uncertain significance are confirmed by Sanger sequencing.
Indications for Test
Candidates for this test are patients with piebaldism or suspicion of familial GISTs.
This test is specifically designed for heritable germline mutations and is not appropriate for the detection of somatic mutations in tumor tissue. This test is also not recommended for patients with mastocytosis (Valent et al. 2007).
|Official Gene Symbol||OMIM ID|
|Cancer Sequencing and Deletion/Duplication Panel|
|Familial Gastrointestinal Stromal Tumors (GISTs) via the PDGFRA Gene|
- Genetic Counselor Team - firstname.lastname@example.org
- Jerry Machado, PhD, DABMG, FCCMG - email@example.com
- Akin C., Metcalfe D.D. 2004. The Journal of Allergy and Clinical Immunology. 114: 13-9; quiz 20. PubMed ID: 15241338
- Bachet J.B. et al. 2013. European Journal of Cancer (oxford, England : 1990). 49: 2531-41. PubMed ID: 23648119
- Ezoe K. et al. 1995. American Journal of Human Genetics. 56: 58-66. PubMed ID: 7529964
- Heinrich M.C. et al. 2002. Journal of Clinical Oncology : Official Journal of the American Society of Clinical Oncology. 20: 1692-703. PubMed ID: 11896121
- Isozaki K. et al. 2000. The American Journal of Pathology. 157: 1581-5. PubMed ID: 11073817
- Maeyama H. et al. 2001. Gastroenterology. 120: 210-5. PubMed ID: 11208730
- Miettinen M. et al. 2002. European Journal of Cancer (oxford, England : 1990). 38 Suppl 5: S39-51. PubMed ID: 12528772
- Murakami T. et al. 2004. Journal of Dermatological Science. 35: 29-33. PubMed ID: 15194144
- Syrris P. et al. 2002. Human Mutation. 20: 234. PubMed ID: 12204004
- Tuveson D.A. et al. 2001. Oncogene. 20: 5054-8. PubMed ID: 11526490
- Valent P. et al. 2007. European Journal of Clinical Investigation. 37: 435–453. PubMed ID: 17537151
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.
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.
Deletion/Duplication Testing Via Array Comparative Genomic Hybridization
Equal amounts of genomic DNA from the patient and a gender matched reference sample are amplified and labeled with Cy3 and Cy5 dyes, respectively. To prevent any sample cross contamination, a unique sample tracking control is added into each patient sample. Each labeled patient product is then purified, quantified, and combined with the same amount of reference product. The combined sample is loaded onto the designed array and hybridized for at least 22-42 hours at 65°C. Arrays are then washed and scanned immediately with 2.5 µM resolution. Only data for the gene(s) of interest for each patient are extracted and analyzed.
PreventionGenetics' high density gene-centric custom designed aCGH enables the detection of relatively small deletions and duplications within a single exon of a given gene or deletions and duplications encompassing the entire gene. PreventionGenetics has established and verified this test's accuracy and precision.
Our dense probe coverage may allow detection of deletions/duplications down to 100 bp; however due to limitations and probe spacing this cannot be guaranteed across all exons of all genes. Therefore, some copy number changes smaller than 100-300 bp within a targeted large exon may not be detected by our array.
This array may not detect deletions and duplications present at low levels of mosaicism or those present in genes that have pseudogene copies or repeats elsewhere in the genome.
aCGH will not detect balanced translocations, inversions, or point mutations that may be responsible for the clinical phenotype.
Breakpoints, if occurring outside the targeted gene, may be hard to define.
The sensitivity of this assay may be reduced when DNA is extracted by an outside laboratory.
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.
- The first four pages of the requisition form must accompany all specimens.
- Billing information is on the third and fourth pages.
- Specimen and shipping instructions are listed on the fifth and sixth pages.
- All testing must be ordered by a qualified healthcare provider.
(Delivery accepted Monday - Saturday)
- Collect 3-5 ml (5 ml preferred) of whole blood in EDTA (purple top tube) or ACD (yellow top tube). For Test #500-DNA Banking only, collect 10-20 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 good for up to 48 hours.
- If refrigerated, blood specimen is good for up to one week.
- Label the tube with the patient name, date of birth and/or ID number.
(Delivery accepted Monday - Saturday)
- NextGen Sequencing Tests: Send in screw cap tube at least 10 µg of purified DNA at a concentration of at least 50 µg/ml
- Sanger Sequencing Tests: Send in a screw cap tube at least 15 µg of purified DNA at a concentration of at least 20 µg/ml. For tests involving the sequencing of more than three genes, send an additional 5 µg DNA per gene. DNA may be shipped at room temperature.
- Deletion/Duplication via aCGH: Send in screw cap tube at least 1 µg of purified DNA at a concentration of at least 100 µg/ml.
- Whole-Genome Chromosomal Microarray: Collect at least 5 µg of DNA in TE (10 mM Tris-cl pH 8.0, 1mM EDTA), dissolved in 200 µl at a concentration of at least 100 ng/ul (indicate concentration on tube label). DNA extracted using a column-based method (Qiagen) or bead-based technology is preferred.
(Delivery accepted Monday - Thursday)
- PreventionGenetics should be notified in advance of arrival of a cell culture.
- Ship at least two T25 flasks of confluent cells.
- Label the flasks with the patient name, date of birth, and/or ID number.
- We do not culture cells.