Colorectal Cancer Sequencing And Deletion/Duplication Panel

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  • Clinical Features and Genetics
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Sequencing and Deletion/Duplication Testing via aCGH

Test Code TestCPT Code Copy CPT Codes
1975 APC 81201, 81203 Add to Order
ATM 81408, 81479
BLM 81479, 81479
BMPR1A 81479, 81479
BUB1B 81479, 81479
CDH1 81479, 81406
CHEK2 81479, 81479
EPCAM 81479, 81403
MLH1 81292, 81294
MSH2 81295, 81297
MSH6 81298, 81300
MUTYH 81406, 81479
PMS2 81317, 81319
PTEN 81321, 81323
SMAD4 81406, 81405
STK11 81405, 81404
TP53 81405, 81479
Full Panel Price* $1190.00
Test Code Test Total Price CPT Codes Copy CPT Codes
1975 Genes x (17) $1190.00 81201, 81203, 81292, 81294, 81295, 81297, 81298, 81300, 81317, 81319, 81321, 81323, 81403, 81404, 81405(x3), 81406(x3), 81408, 81479(x13) Add to Order
Pricing Comment

STK11 is analyzed by Multiplex Ligation-dependent Probe Amplification. Targeted testing for gross deletion/duplications discovered by aCGH will be available for family members of the proband only if we are able to test the del/dup by PCR or qPCR. CPT codes 81435 and 81436 can be used if the whole sequencing and deletion/duplication panel is ordered. If you would like to order a subset of these genes contact us to discuss pricing.

Targeted Testing

For ordering targeted known variants, please proceed to our Targeted Variants landing page.

Turnaround Time

The great majority of tests are completed within 28 days.

Clinical Sensitivity

NextGen Sequencing Sensitivity Clinical sensitivity of the tested genes for Hereditary Colorectal Cancers is given for each syndrome. The sensitivity of pathogenic variants that are associated with autosomal recessive disorders and CRC cancers is often unknown, with the exception being MUTYH associated polyposis. Lynch Syndrome

Depending on the clinical criteria used to make a diagnosis (i.e. Amsterdam or “Revised Bethesda”), 30-50% and 15-20% of Lynch patients have a detectable MLH1 and MSH2 mutation, respectively (Syngal et al. 2000). A mutation in MSH6 is detected in < 2% of patients that meet the stringent Amsterdam I criteria, but is detected in ~12% of atypical Lynch/HNPCC families (Peltomäki and Vasen 2004). A mutation in PMS2 is detected in 1-2% of Lynch patients (Peltomäki and Vasen 2004) and ~50% of constitutional mismatch repair-deficiency patients (Wimmer and Etzler 2008). The clinical sensitivity of EPCAM sequence variants in Lynch syndrome is unknown as no sequence variants have been reported for this disease; however sequence variants in the EPCAM gene are known to be causative for congenital tufting enteropathy. The clinical sensitivity of EPCAM deletions is 1-3% of individuals with Lynch Syndrome (Kohlmann and Gruber 2012). Please see deletion/duplication section.

Hereditary Diffuse Gastric Cancer The clinical sensitivity of CDH1 germline mutations is 30% for HDGC families (Carneiro et al. 2007). Juvenile Polyposis Syndrome This test is predicted to identify a BMPR1A mutation in 11-22% and a SMAD4 mutation in 20-26% of patients diagnosed with JPS (Haidle and Howe 2011). PTEN Hamartoma Syndrome This test is predicted to detect causative PTEN mutations in ~80% of patients with CS, ~65% of patients with BRRS and ~20% of patients with PS (Eng 2003). Familial Adenomatous Polyposis This test is predicted to detect >90% of causative APC mutations (Laken et al. 1999). Peutz-Jeghers Syndrome Approximately 55% of patients with a positive family history or 70% of patients with no family history of Peutz-Jeghers syndrome will be detected by STK11 sequencing (McGarrity et al. 1993). Li-Fraumeni Syndrome Sequencing the TP53 gene can detect approximately 95% of patients with Li-Fraumeni syndrome (Schneider et al. 2013). CHEK2-related Cancers The clinical sensitivity of CHEK2 mutations in GI cancers is unknown. MUTYH-Associated Polyposis By definition, nearly all (~99%) MAP patients have biallelic germline mutations in MUTYH. However, mutations in MUTYH are also found in ~25% of patients initially diagnosed with familial adenomatous polyposis (FAP) (Sampson et al. 2003). Ataxia-Telangiectasia Approximately 90% of individuals with Ataxia-telangiectasia have detectable mutations via sequencing of the ATM gene (Gatti. GeneReviews. 2010). Bloom Syndrome In a retrospective study (German et al. 2007), 87% of patients diagnosed with Bloom’s Syndrome were reported to have two BLM mutations. In 6% of the patients, only one mutation was found for this recessive disease, indicating the second mutation was not detectable by DNA sequencing methods. Mosaic Variegated Aneuploidy Syndrome Clinical sensitivity is unknown at this time since MVA syndrome is a rare disease and relatively few patients have been reported.

Deletion/Duplication Sensitivity Clinical sensitivity of the tested genes for Hereditary Colorectal Cancers is given for each syndrome. The sensitivity of pathogenic variants that are associated with autosomal recessive disorders and CRC cancers is often unknown, with the exception being MUTYH associated polyposis. Deletion and duplication analysis is not currently available for BUB1B. For CHEK2, only exons 8-10 will be analyzed, which includes all known deletions. The clinical sensitivity of these two genes in CRC is not known. Lynch Syndrome Lynch syndrome is attributed to deletions in the MLH1, MSH2, MSH6, and PMS2 genes in approximately 5%, 20%, 7% and 20% of cases, respectively (Kohlmann and Gruber 2012). EPCAM deletions account for 1-3% of Lynch syndrome cases (Kohlmann and Gruber 2012). Hereditary Diffuse Gastric Cancer Large deletions that usually cannot be detected via sequencing have been detected in the CDH1 gene in up to 4% of patients (Kaurah and Huntsman 2011). Juvenile Polyposis Syndrome This test is predicted to identify a BMPR1A mutation in 1-2% and a SMAD4 mutation in 2-9% of patients diagnosed with JPS (Haidle and Howe 2011). PTEN Hamartoma Syndrome This test is predicted to detect causative PTEN mutations in ~11% of patients with BRRS but not known for other PTEN related disorders (Eng 2003). Familial Adenomatous Polyposis Gross deletions/duplications have been reported in up to 12% of APC patient samples (Jasperson and Burt 2011). Peutz-Jeghers Syndrome Approximately 45% of patients with a positive family history or 21% of patients with no family history of Peutz-Jeghers syndrome will have a pathogenic variant in STK11 by deletion analysis (McGarrity et al. 2013). Li-Fraumeni Syndrome Deletions in the TP53 gene have been detected in 1% of Li-Fraumeni cases (Schneider and Garber 2013). MUTYH-Associated Polyposis Clinical sensitivity for MUTYH deletions/duplications is not currently known. Ataxia-Telangiectasia Approximately 1-2% of Ataxia Telangiectasia patients have large genomic deletions involving the ATM gene that can be detected using aCGH (Gatti 2010). Bloom Syndrome Gross deletions of multiple exons in the BLM gene account for approximately 5% of Bloom Syndrome (German et al. 2007).

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Clinical Features

Colorectal cancer (CRC) is the development of tumors in the colon and rectum that occur in approximately 8% of individuals with cancer (Siegel et al. 2015). Similar to other cancers, CRC is caused by genetic and environmental factors (e.g. smoking, diet). Approximately 30% of CRCs are considered familial and 5% of CRCs are caused by a Mendelian disorder (Esteban-Jurado 2014). Colorectal cancer is generally broken down into the presence or absence of polyposis (numerous internal polyps). Identification of pathogenic variants in the germline of CRC patients is important for cancer surveillance (i.e. colonoscopy) for the affected individual and family members, since early surveillance and treatment has been shown to decrease morbidity and mortality (Kohlmann and Gruber 2014). CRC inherited diseases include: Lynch Syndrome Lynch syndrome, also known as Hereditary Nonpolyposis Colorectal Cancer (HNPCC), is an inherited cancer syndrome mainly caused by germline mutations in DNA mismatch repair (MMR) genes. MMR genes are responsible for repairing small sequence errors, or mismatches, during DNA replication. Mutations in mismatch repair genes can cause widespread genomic instability characterized by the expansion and contraction of short tandem repeat sequences (microsatellites) (Grady and Carethers 2008). This phenomenon of microsatellite instability (MSI) leads to somatic mutations in oncogenes and/or tumor suppressor genes, including TGFBR2, NF1, and others (Wang et al. 2003). As a result, Lynch syndrome is marked by early onset and a high lifetime risk of cancers, particularly in the right colon but also in the endometrium, ovary, stomach, bile duct, kidney, bladder, ureter, and brain (Jang and Chung 2010). Hereditary Diffuse Gastric Cancer Hereditary diffuse gastric cancer is a highly penetrant diffuse-type of gastric cancer. Patients with HDGC typically present at 40 years of age and have a cumulative cancer risk of 67% for men and 83% for women by 80 years of age (Pharoah et al. 2001). HDGC is caused by CDH1 mutations, which are found in all ethnic groups, but are rare in countries with high rates of sporadic gastric cancer, such as Japan and Korea (Guilford et al. 2010). Approximately 10% of gastric cancers show familial clustering and about 1–3% of cases are known to be hereditary (e.g. CDH1 mutations). Mutations in CDH1 can also cause lobular breast cancer in women (Carneiro et al. 2007). Juvenile Polyposis Syndrome Juvenile polyposis syndrome is a rare, inherited hamartomatous polyposis syndrome with increased susceptibility to colorectal cancer. Clinical diagnosis of JPS is typically made when one of the following criteria is met: more than five juvenile polyps in the colorectum; multiple juvenile polyps throughout the GI tract; or any number of juvenile polyps and a family history of gastrointestinal polyps (Chow and Macrae 2005). Juvenile refers to the developmentally immature nature of the polyp, not the age of disease onset. In addition to polyposis, 10-20% of JPS patients also have extracolonic abnormalities such as congenital heart defects, cleft lip or palate, microcephaly and malrotations (Eng et al. 2001). Although a solitary juvenile polyp in the general population has very little malignant potential (Nugent et al. 1993), patients with JPS have a 68% chance of developing gastrointestinal cancer by the age of 60 (Chow and Macrae, 2005). Thus, confirming a diagnosis of JPS is important for the appropriate surveillance and management of cancer in individuals with juvenile polyps. PTEN Hamartoma Syndrome PTEN hamartoma tumor syndrome (PHTS) is a cluster of related clinical conditions, all caused by germline mutations in the PTEN tumor suppressor gene. Included in PHTS are Cowden syndrome, Bannayan-Riley-Ruvalcaba syndrome, Proteus syndrome and Proteus-like syndromes, and VACTERL Association with Hydrocephalus. While each PHTS condition has its own unique pathognomonic features (Blumenthal and Dennis 2008), hamartomatous overgrowth, macrocephaly and vascular malformations appear to be common to all conditions (Zhou et al. 2001). A presumptive diagnosis of PHTS is typically made based on clinical symptoms, but a definitive diagnosis requires the identification of a heterozygous PTEN mutation. Patients with a germline mutation in PTEN have a 5-10 fold higher chance of developing cancer at a much earlier age (< 30 y/o) than the general population (Eng 2003). In addition to confirming the diagnosis of PHTS, testing patients for a germline PTEN mutation is essential to accurately assess their risk for cancer and to make appropriate recommendations regarding prevention and treatment of malignancy. Familial Adenomatous Polyposis Familial adenomatous polyposis is an inherited cancer syndrome characterized clinically by the development of hundreds to thousands of adenomatous polyps in the colon and rectum. If untreated, nearly all FAP patients will develop colorectal cancer (CRC) by age 40 (Fearnhead et al. 2001). In addition to CRC, FAP patients are also predisposed to desmoid tumors, small bowel cancer, thyroid cancer, hepatoblastoma and medulloblastoma (Galiatsatos and Foulkes 2006). About 60% of families with FAP also display congenital hypertrophy of the retinal pigment epithelium (CHRPE), a condition that does not affect sight or have malignant potential but can be easily detected by ophthalmoscopy at any age. CHRPE is highly diagnostic of FAP and can be useful for identifying FAP patients and at-risk family members, well before the appearance of polyps (Díaz-Llopis and Menezo 1988). Peutz-Jeghers Syndrome Peutz-Jeghers syndrome is characterized by hamartomatous polyps in the gastrointestinal tract and melanin pigmentation around the mouth, eyes, nostrils, buccal mucosa, fingers, toes and other sites. PJS patients typically present in early childhood with pigmentation or with complications of polyposis, such as intussusception, bowel obstruction and/or bleeding. Compared to the general population, patients with PJS have an increased risk of intestinal and various extra-intestinal malignancies, including breast, pancreatic, ovarian, testicular and cervical cancer; their lifetime risk is ~4 fold higher for gastrointestinal cancer and ~6 fold higher for breast cancer compared to individuals without PJS (Hearle 2006). Approximately 75% of PJS cases are known to be familial; while the remainder appears to be sporadic (Lim et al. 2003). Li-Fraumeni Syndrome Li-Fraumeni Syndrome is a hereditary cancer syndrome that predisposes individuals to multiple neoplasms at an early age. The most common neoplasms associated with LFS are bone and soft-tissue sarcomas, pre-menopausal breast carcinomas, adrenocortical carcinomas and brain tumors. Although much less common, melanomas, germ cell tumors, gastric carcinomas and Wilms tumors have also been described in LFS patients (Varley et al. 1997). The average age of malignancy for individuals with LFS is typically between 20 and 45, which is at least 2-3 decades sooner than reported for the general population (Nichols et al. 2001). CHEK2-related Cancers Mutations in the CHEK2 gene have been reported to cause a Li-Fraumeni-like syndrome (Bell 1999), although subsequent studies have indicated that CHEK2 mutations are only very rarely found in patients with classic symptoms of LFS (Lee et al. 2001). However, mutations in CHEK2 have been found more frequently found in patients who have hereditary breast cancer (Vahteristo et al. 2002; Meijers-Heijboer et al. 2003). CHEK2 mutations have also been reported in a breast and colon cancer family, and specific variants may predispose individuals to colorectal cancer (Abud and Prolla 2012; Narod 2010). MUTYH-Associated Polyposis MUTYH-associated polyposis is an autosomal recessive condition of FAP caused exclusively by mutations in the MUTYH gene (Al-Tassan et al. 2002; Sieber et al. 2003). Individuals with MAP typically present by age 55 with multiple (between 10 and 1000) colorectal adenomas, some of which have or will become colorectal tumors (Poulsen and Bisgaard 2008). Ataxia-Telangiectasia Ataxia telangiectasia (A-T) is characterized by early onset (1-4 years) progressive cerebellar ataxia, telangiectasias of the conjunctivae, oculomotor apraxia, choreoathetosis, immunodeficiency, slurred speech, frequent infections, and an increased risk of cancers, especially leukemia and lymphoma. Unlike A-T, which is caused by homozygous or compound heterozygous mutations in the ATM gene, heterozygous carriers of an ATM causative mutation are at an increased risk of breast cancer (Concannon et al. 2008) and heart disease (Swift et al. 1991). In addition, one mutated copy of ATM has been associated with an increased risk of stomach and colorectal cancer (Thompson et al. 2005). Bloom Syndrome Bloom’s Syndrome was first described in 1954 as a “congenital” skin disorder in “dwarfs” (Bloom 1954). Additional clinical features, such as immune deficiencies and a propensity for cancer (German et al. 1965; German 1969), sun-sensitive facial lesions, low birth weight and stunted growth remain the most consistent features of Bloom Syndrome today. There is some evidence that heterozygous carriers of a BLM mutation have an increased risk for colorectal cancer (Gruber et al. 2002). Mosaic Variegated Aneuploidy Syndrome Mosaic Variegated Aneuploidy syndrome is a rare disease generally characterized by prenatal growth retardation, microcephaly, mental retardation, central nervous system abnormalities, and cancer predisposition (García-Castillo et al., 2008; Callier et al., 2005). An individual with gastric neoplasia and without the characteristic features of MVA syndrome was found to have an intronic homozygous intronic BUB1B mutation (Rio Frio et al. 2010).


MLH1, MSH2, MSH6, PMS2, and EPCAM Lynch syndrome is an autosomal dominant disease mainly caused by germline mutations in one of four described MMR genes: MLH1, MSH2, MSH6, and PMS2 (Peltomäki and Vasen 2004; Kohlmann and Gruber 2012). Mutations in the MLH1 and MSH2 genes account for 80-90% of all Lynch syndrome patients and most frequently occur in families meeting the stringent Amsterdam I criteria. Mutations in the MSH6, and PMS2 genes account for most of the remaining Lynch patients and are often found in families with atypical symptoms, such as extracolonic carcinomas; and have been found to have a low rate of MSI. Mutations in another gene, EPCAM, which encodes a calcium-independent cell adhesion molecule and not a mismatch repair protein, are involved in Lynch syndrome. Germline mutations in the EPCAM gene cause inactivation of the nearby MSH2 gene via hypermethylation in approximately 1-3% of individuals with Lynch syndrome (Kohlmann and Gruber 2012). The only reported mutations of the EPCAM gene that are causative for Lynch Syndrome are large deletions. Missense, nonsense and splicing mutations are involved in congenital tufting enteropathy (Human Gene Mutation Database; The cumulative incidence of colon cancer risk from EPCAM deletions has been estimated to be 75% by 70 years of age, and 12% for endometrial cancer in women (Kempers et al. 2011). CDH1 Hereditary diffuse gastric cancer is an autosomal dominant disease that is caused by mutations in the CDH1 gene. CDH1 encodes epithelial cadherin (E-cadherin), which is a transmembrane membrane protein that is responsible for cell-to-cell adhesion and cellular invasion suppression. It also plays important roles in signal transduction, differentiation, gene expression, cell motility, and inflammation (Kaurah and Huntsman. GeneReviews. 2011). The activity of E-cadherin in coordination with the actin cytoskeleton through catenins (e.g. α-, β-, and γ-) is responsible for cellular adhesion. Many human cancers show low levels of E-cadherin compared to normal tissue, which causes defects in cellular adhesion and ultimately leads to metastasis. HDGC is mostly inherited from an affected family member. The majority of germline mutations are truncating mutations in E-cadherin, the second hit of the normal allele in normal gastric tissue is usually due to CDH1 promoter hypermethylation and secondly due to CDH1 deletions (Schrader and Huntsman 2010). BMPR1A and SMAD4 Juvenile polyposis syndrome is caused by heterozygous germline mutations in one of two genes: BMPR1A and SMAD4 (Howe 1998; Howe et al. 2001). Both genes mediate the biological effects of the Transforming Growth Factor-β (TGF-β) superfamily of cytokines (Miyazono et al. 2009). In epithelial cells, the TGF-β pathway normally inhibits growth and proliferation; mutations in BMPR1A and SMAD4 decrease TGF-β signaling and lead to neoplasia. BMPR1A encodes a transmembrane serine/threonine kinase receptor that binds the Bone Morphogenetic Protein (BMP) subfamily of TGF-β ligands (Heldin et al. 1997). Approximately 70 pathogenic variations have been identified throughout the BMPR1A gene and most (~90%) are detectable by DNA sequencing (Human Gene Mutation Database). In addition to causing JPS, one BMPR1A mutation (p.Ala338Asp) has also been identified in a family with Cowden Syndrome, indicating BMPR1A mutations might also define a small subset of CS cases (Zhou et al. 2001). SMAD4 mediates the biological effects of the Transforming Growth Factor-β (TGF-β) superfamily of cytokines (Miyazono et al. 2009). In epithelial cells, the TGF-β pathway normally inhibits growth and proliferation; mutations in SMAD4 decrease TGF-β signaling and can lead to carcinoma. PTEN PTEN hamartoma tumor syndrome inherited in an autosomal dominant manner, and PTEN is the only known gene to be associated with the disease. In addition to PHTS, germline mutations in PTEN have been identified in 16% of patients with Autism Spectrum Disorders (ASD) and macrocephaly, 12.5% of patients with adenomatous and hyperplastic polyps, and 5% of women with at least two different types of cancer (Zbuk and Eng 2006; Lintas and Persico 2009). To date, >200 mutations have been reported for the PTEN gene, and most (~95%) are of the type that can be detected by DNA sequencing (Human Gene Mutation Database). The PTEN gene consists of 9 exons and encodes a dual lipid and protein phosphatase. Mutations have been reported throughout the coding region, and sequencing of all 9 exons is recommended (Eng 2003). Five mutations have also been reported within the minimal promoter about 800 bp upstream of the start codon and sequencing of this region is also recommended (Teresi et al. 2007). APC Familial adenomatous polyposis is an autosomal dominant disorder caused by germline mutations in the adenomatous polyposis coli (APC) gene. More than 1200 mutations have been reported in APC (Human Gene Mutation Database), and >90% are nonsense or frameshift mutations that result in a dysfunctional truncated protein product (Nagase and Nakamura 1993). Germline mutations are spread throughout the coding region (Béroud and Soussi 1996). Several pathogenic mutations have also been documented in the promoter, 3’ untranslated region (UTR) and deep within intron 14 (Heinimann et al. 2001; Rosa De et al. 2009). Severe FAP (i.e. more than 1000 polyps) typically occurs in patients with mutations between codons 1250 and 1464 (Caspari et al. 1994). In contrast, patients with attenuated FAP (i.e. fewer than 100 colorectal polyps) usually have mutations at the very 5’ and 3’ ends of the gene, or in an alternatively spliced region of exon 9 (Young et al. 1998; Soravia et al. 1998). Congenital hypertrophy of retinal pigment epithelium (CHRPE) is limited to patients with mutations between codons 457 and 1444 (Caspari et al. 1995). Two missense variants, p.Ile1307Lys and p.Glu1317Lys, commonly found in Ashkenazi Jewish populations predispose carriers to multiple colorectal adenomas (generally less than 100) and carcinoma, but with low and variable penetrance (Frayling et al. 1998). Pathogenic variants in the exon 1B promoter of APC have also been associated with gastric adenocarcinoma and proximal polyposis of the stomach (Li et al. 2016). STK11 Peutz-Jeghers syndrome is caused by heterozygous germline mutations in the tumor suppressor gene STK11. STK11, also called LKB1, consists of 9 exons and encodes a serine/threonine kinase that inhibits cellular proliferation by promoting cell-cycle arrest (Tiainen et al. 1999). Second hit mutations in STK11 ultimately lead to unfettered growth and tumorigenesis. To date, ~100 unique mutations have been described throughout the STK11 gene (Human Gene Mutation Database). Most (80%) are truncating mutations (i.e. frameshift, nonsense, splice-site, or exonic deletions) that result in early protein termination (Hearle 2006). The remaining mutations are mostly missense or in-frame deletions. Large genomic deletions in STK11 have also been described. TP53 Li-Fraumeni Syndrome is inherited in an autosomal dominant manner and caused by heterozygous germline mutations in the TP53 gene (Malkin et al. 1990; Srivastava et al. 1990). TP53 encodes the well described cellular tumor p53 antigen (Soussi 2010). p53 is a ubiquitously expressed DNA-binding protein that plays a major role in the regulation of cell division, DNA repair, programmed cell death, and metabolism. More than 200 pathogenic variations have been reported throughout the TP53 gene, and nearly all are detectable by DNA sequencing (Human Gene Mutation Database). Three gross deletions encompassing one or more exons of the TP53 gene have been described but these account for less than 1% of all LFS patients. The risk of developing cancer for carriers of TP53 mutations has been estimated to be ~73% for men and nearly 100% for women (Chompret et al. 2000). CHEK2 CHEK2 encodes a protein kinase that protects the genome from ionizing radiation and genotoxic insults. To date, approximately 40 mutations have been reported throughout the CHEK2 gene, and >95% are detectable by this DNA sequencing test (Human Gene Mutation Database). MUTYH The MUTYH gene encodes a vital component of the base excision repair (BER) system, which protects DNA from oxidative damage and the misincorporation of adenines opposite guanines during DNA replication (Lu et al. 2006). As such, the molecular profile of colorectal adenomas and carcinomas taken from MAP patients includes G:C → T:A transversions in the adenomatous polyposis coli (APC) and K-ras tumor suppressor (KRAS) genes, among others (Lipton et al. 2003; Jones et al. 2004). To date, about 100 pathogenic mutations have been reported in the MUTYH gene, nearly all (~99%) of which are single nucleotide variations, small insertions or deletions, or splice-site mutations (; Human Gene Mutation Database). While MAP occurs in patients from various ethnic groups, specific MUTYH mutations are found in different populations. In European and North American MAP populations, two missense mutations, p.Tyr179Cys and p.Gly396Asp, are most common; both homozygous and compound heterozygous mutations contribute to the disease (Jones et al. 2002). In Asian MAP populations, common mutations include the missense mutation p.Arg245Cys, splice-site mutation c.934-2A>G, and p.Glu480* nonsense mutation; in these cases only homozygous mutations have been reported to contribute to disease (Tao 2004, 2004; Miyaki et al. 2005). The penetrance of colorectal cancer (CRC) for biallelic carriers of MUTYH mutations is nearly 100% by the age of 60 (Farrington et al. 2005). ATM Ataxia-telangiectasia is an autosomal recessive disorder that is caused by mutations in the ATM gene. ATM encodes a serine protein kinase (ATM) that is involved in DNA repair via phosphorylation of downstream proteins. It senses double-stranded DNA breaks, coordinates cell-cycle checkpoints prior to repair, and recruits repair proteins to damaged DNA sites (Taylor et al. 2004). Mutations in ATM result in defective checkpoint cycling. Over 500 private causative mutations are described with no common hot spots for mutations. In North America most affected individuals are compound heterozygotes for two ATM mutations. However, founder ATM mutations have been observed in several populations (Gatti 1993). BLM Bloom syndrome is an autosomal recessive disorder, caused by homozygous or compound heterozygous mutations in the BLM gene (German et al. 2007); more than 60 unique mutations have been identified. Most (60%) are single nucleotide changes leading to nonsense, missense or splicing mutations, while the remaining are small insertions/deletions (35%) or large deletions of multiple exons (5%). The BLM gene encodes a DNA helicase of the RecQ family. RecQ proteins are critical for maintaining the efficiency and integrity of DNA replication (Hickson 2003); they resolve secondary structures ahead of replication forks, limit recombination to identical sequences, and assist in the replication and maintenance of telomeres (Bennett and Keck 2004). In addition to these cellular functions, the BLM protein may also be important for the Mismatch Repair (MMR) pathway through its interaction with the MLH1 and MSH6 proteins (Langland 2001; Pedrazzi et al. 2003). BUB1B MVA syndrome is generally considered a rare autosomal recessive disorder that can be caused by pathogenic variants in the BUB1B gene; however individuals can also be affected with only one pathogenic mutation present. At the cellular level, >10% of cells exhibit mosaic aneuploidies. In addition, approximately 2/3 of metaphases from patients show PCS (García-Castillo et al. 2008). The BUB1B gene's product (BUBR1) is thought to be involved in the mitotic spindle checkpoint, which includes several proteins that sense microtubule attachment to kinetochores. BUB1B mutations can cause defects in the mitotic spindle checkpoint, spindle attachment or cause reduction of BUBR1 protein levels (Suijkerbuijk et al., 2010). There does not appear to be preferential specific chromosome gains and losses, and thus trisomies and monosomies appear to be random (Hanks and Rahman, 2005). Interestingly, while BUB1B mutations can occur in individuals who are predisposed to cancer (MVA syndrome), BUB1B mutations are rarely observed in somatic cancers (Hanks et al., 2006). Most pathogenic variants are missense or nonsense, but other mutations such as splice site changes, small insertions and duplications have also been reported (Human Gene Mutation Database). Missense mutations tend to occur in or near the BUBR1 kinase domain (Suijkerbuijk et al. 2010), and biallelic mutations appear to usually consist of one truncating and one missense variant (Hanks et al. 2006).

Testing Strategy

The Colorectal Cancer NextGen Sequencing Panel analyzes 17 genes that have been associated with hereditary colorectal cancers. For this NGS panel, the full coding regions, plus ~20bp of non-coding DNA flanking each exon, are sequenced for each of the genes listed below. Sequencing is accomplished by capturing specific regions with an optimized solution-based hybridization method, 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, including the exon 1B promoter region of APC. All pathogenic, undocumented and questionable variant calls are confirmed by Sanger sequencing. Due to known PMS2 pseudogenes, the PMS2 gene is also analyzed via Sanger sequencing using a long-range PCR strategy. Each gene/group of genes can also be tested individually using our Sanger sequencing assays. Please see our test menu. Deletion and duplication testing of 17 genes is analyzed using array comparative genomic hybridization. For deletion/duplication testing, STK11 is analyzed by Multiplex Ligation-dependent Probe Amplification.

Indications for Test

This test is suitable for individuals with a clinical history of hereditary colorectal cancers. This test especially aids in a differential diagnosis of similar phenotypes, rules out particular syndromes, and provides the analysis of multiple genes simultaneously. Individuals with multiple colorectal tumors, multifocal, recurrent, family history, and early onset (e.g. < 50 years) tumors should be assessed with this panel. Germline mutations in the Lynch syndrome genes have also been shown to be associated with ovarian cancer (Watson et al. 2008). 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
APC 611731
ATM 607585
BLM 604610
BMPR1A 601299
BUB1B 602860
CDH1 192090
CHEK2 604373
EPCAM 185535
MLH1 120436
MSH2 609309
MSH6 600678
MUTYH 604933
PMS2 600259
PTEN 601728
SMAD4 600993
STK11 602216
TP53 191170
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT

Related Tests

Ataxia telangiectasia Syndrome via the ATM Gene
Autism Spectrum Disorders and Intellectual Disability (ASD-ID) Comprehensive Panel
Bloom's Syndrome via the BLM Gene
Cancer Sequencing and Deletion/Duplication Panel
Chromosomal Instability Syndromes Sequencing Panel
Comprehensive Cardiology Sequencing Panel
Familial Adenomatous Polyposis via the APC Gene
Hereditary Breast and Ovarian Cancer Syndrome - HBOC EXPANDED Sequencing and Deletion/Duplication Panel
Hereditary Breast and Ovarian Cancer Syndrome - HBOC HIGH RISK Sequencing and Deletion/Duplication Panel
Hereditary Breast Cancer via the CHEK2 Gene
Hereditary Diffuse Gastric Cancer via the CDH1 Gene
Hereditary Hemorrhagic Telangiectasia (HHT) Sequencing Panel
Hereditary Myelodysplastic Syndrome (MDS) / Acute Myeloid Leukemia (AML) Sequencing Panel
Juvenile Polyposis Syndrome (JPS) and Hereditary Hemorrhagic Telangiectasia (HHT) via the SMAD4 Gene
Juvenile Polyposis Syndrome (JPS) via the BMPR1A Gene
Li-Fraumeni Syndrome via the TP53 Gene
Lynch Syndrome Sequencing and Deletion/Duplication Panel
Lynch Syndrome via the EPCAM Gene
Lynch Syndrome via the MLH1 Gene
Lynch Syndrome via the MSH2 Gene
Lynch Syndrome via the PMS2 Gene
Lynch Syndrome via the MSH6 Gene
Mosaic Variegated Aneuploidy Syndrome via the BUB1B Gene
MUTYH Associated Polyposis (MAP) Syndrome via the MUTYH Gene
Pancreatic Cancer Sequencing Panel
Peutz-Jeghers Syndrome via the STK11 Gene
Peutz-Jeghers Syndrome via the STK11 Gene
PTEN Hamartoma Tumor Syndrome via the PTEN Gene
Renal Cancer Sequencing Panel


Genetic Counselors
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Order Kits

NextGen Sequencing and Deletion/Duplication Testing Via Array Comparative Genomic Hybridization

Test Procedure

NextGen Sequencing

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 (  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/Duplication Testing via aCGH

As required, DNA is extracted from the patient specimen. 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 hours at 65ºC.  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 (HDGC) aCGH is designed to have comprehensive coverage for both coding and non-coding regions for each targeted gene with very high density probe coverage.  The average probe spacing within each exon is 47 bp or a minimum of three probes per exon covering all targeted exons and UTRs.  The average probe spacing is 289 bp covering all intronic, 2kb upstream and downstream regions of each targeted gene.  In addition, the flanking 300-bp intronic sequence on either side of targeted exons has enriched probe coverage.  Therefore, PreventionGenetics’ aCGH enables the detection of relatively small deletion and amplification mutations within a single exon of a given gene or deletion and amplification mutations encompassing the entire gene.

Analytical Validity

NextGen Sequencing

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. 

Deletion/Duplication Testing via aCGH

PreventionGenetics’ high density gene-centric custom designed aCGH enables the detection of relatively small deletion and amplification mutations (down to ~300 bp) within a single exon of a given gene or deletion and amplification mutations encompassing the entire gene. PreventionGenetics has established and verified this test’s accuracy and precision.

Analytical Limitations

NextGen Sequencing

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 aCGH

Any copy number changes smaller than 300bps (within the targeted region) may not be detected by our array.

This array may not detect deletion and amplification mutations 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 happened 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

Order Kits

Ordering Options

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.
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