Hereditary Breast and Ovarian Cancer Syndrome - HBOC EXPANDED Sequencing and Deletion/Duplication Panel

  • Summary and Pricing
  • Clinical Features and Genetics
  • Citations
  • Methods
  • Ordering/Specimens
Order Kits

Sequencing and Deletion/Duplication Testing via aCGH

Test Code TestCPT Code Copy CPT Codes
1307 ATM 81408, 81479 Add to Order
BARD1 81479, 81479
BRCA1 81214, 81479
BRCA2 81216, 81479
BRIP1 81479, 81479
CDH1 81479, 81406
CHEK2 81479, 81479
MRE11 81479, 81479
MUTYH 81406, 81479
NBN 81479, 81479
PALB2 81406, 81479
PMS2 81317, 81319
PTEN 81321, 81323
RAD50 81479, 81479
RAD51C 81479, 81479
RAD51D 81479, 81479
SMARCA4 81479, 81479
STK11 81405, 81404
TP53 81405, 81479
XRCC2 81479, 81479
Full Panel Price* $1190.00
Test Code Test Total Price CPT Codes Copy CPT Codes
1307 Genes x (20) $1190.00 81214, 81216, 81317, 81319, 81321, 81323, 81404, 81405(x2), 81406(x3), 81408, 81479(x27) Add to Order
Pricing Comment

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. 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

The overall prevalence of germline BRCA1 or BRCA2 pathogenic variants in the general population is 1:400 to 1:800, with higher rates depending on the specific ethnicity, such as 1:40 in the Ashkenazi Jewish population. Pathogenic variants will be detected by sequencing in ~90% of individuals with an identifiable mutation. Previously, BRCA1 variants were observed in 63% of these cases and BRCA2 variants in 37% of these cases (Petrucelli 2013). BRCA1 mutation carriers tend to have breast tumors that are estrogen receptor (ER) negative, progesterone receptor (PR) negative and basal type tumors, whereas BRCA2 mutation carriers have breast tumors that are ER positive, PR positive, and have a luminal phenotype (Pruthi et al. 2010). Individuals with HBOC with a more severe personal or family history tend to have mutations in BRCA1 vs. BRCA2 due to higher penetrance of mutations in the BRCA1 gene (Antoniou et al. 2000). Mutations in genes other than BRCA1 and BRCA2 are often tested selectively, so that the proportion of breast and ovarian cancers with mutations in other genes is not known. Genes tested in this panel have been implicated in hereditary breast and ovarian cancer, and although individually these genes may be involved in a minority of inherited breast cancer genes, the combination of these  variable risk genes may be responsible for a significant portion of these hereditary cancers (Turnbull and Rahman.  2008). For instance, a study by Walsh et al (2011) found approximately 6% of patients with hereditary ovarian cancers who do not have mutations in BRCA1 or BRCA2 have mutations in genes such as BARD1BRIP1, CHEK2, MRE11, NBN, PALB2RAD50RAD51C and TP53 (Walsh et al. 2011). Pathogenic variants in BRCA1 and BRCA2 will be detected by copy number analysis in 10% of individuals with an identifiable BRCA1/2 germline mutation. BRCA1 variants were observed in 90% of these cases and BRCA2 variants in 10% of these cases (Petrucelli 2013). Large rearrangements (e.g. deletions, duplications, tripications), including the five most commonly reported BRCA1 alterations (Hendrickson et al. 2005), can be detected using this test. High-risk patients, defined as individuals with early onset ( Clinical sensitivity for gross deletions/duplications for the other genes in Hereditary Breast and Ovarian Cancer is unknown. HBOC associated gross deletions have been reported for the RAD51C (Vuorela et al.  2011), PALB2 (Antoniou et al. 2014) and TP53 (Melhem-Bertandt et al. 2012) genes. Due to known segmental duplications in CHEK2, gross deletions and duplications are only analyzed for exons 8-10.

See More

See Less

Clinical Features

Hereditary breast and ovarian cancer (HBOC) syndrome is an inherited disorder that is mainly associated with tumors of the breasts and ovaries. Other malignancies in HBOC families can include melanoma, pancreatic and prostate cancer. In comparison to sporadic breast and ovarian cancers, HBOC syndrome tends to occur at an earlier age (i.e. < 50 years), tumors often occur bilaterally, consist of multiple affected family members, including males with breast cancer, and occur with a higher predisposition in specific ethnicities, such as the Ashkenazi Jewish population (Petrucelli et al. 2011; Pruthi et al. 2010). Identifying individuals with a high risk for developing HBOC may allow for early detection of tumor formation and allow for prophylactic mastectomy and/or oophorectomy or other treatments (Smith 2012).

Breast and ovarian cancers can show a familial inheritance due to shared environment or inherited genes of low penetrance, which confer a moderate risk (Berliner et al. 2013). In addition, approximately 5-10% of breast, and 10-15% of ovarian cancer cases are the result of genetic predisposition due to gene specific mutations that significantly increase an individual's risk of developing these cancers (Marchina et al. 2010). HBOC syndrome is mainly due to pathogenic variants in the BRCA1 and BRCA2 genes, however pathogenic variants have also been found in other genes. This was noted because higher incidences of breast and/or ovarian cancer has been observed in several syndromes, albeit with different cancer spectrums, including but not limited to Li-Fraumeni syndrome, Cowden syndrome, Peutz-Jeghers syndrome, Fanconi Anemia, Ataxia telangiectasia, Ataxia-telangiectasia-like disorder and Nijmegen breakage syndrome caused by mutations in TP53, PTEN, STK11, PALB2, ATM, MRE11/MRE11A and NBN genes, respectively.


Hereditary breast and ovarian cancer is inherited in an autosomal dominant manner and presents with high, although incomplete penetrance. Pathogenic variants in a number of genes have been reported to significantly increase an individual’s likelihood for developing breast cancer (Tan et al. 2008). Among those, germline mutations in the most common highly penetrant mutated breast cancer genes, BRCA1 and BRCA2 (Miki et al. 1994; Wooster et al. 1995), appear to provide the highest relative risk, ~10- to 20-fold. Hereditary BRCA1 and BRCA2 mutations account for approximately 25-60% of inherited breast cancer (Pruthi et al. 2010; Meindl et al. 2011) and 11-39% of inherited ovarian cancer (Berliner et al. 2013). Other genes have also been implicated in hereditary breast and ovarian cancer, and although individual mutations in these genes may cause only a small fraction of inherited breast and ovarian cancer, the combination of moderately and mildly penetrant gene variants may be responsible for a significant portion of these hereditary cancers (Turnbull and Rahman. 2008).

Genes related to other syndromes (e.g. Cowden syndrome, and Li-Fraumeni syndrome) that are mutated and inherited in a dominant manner may predispose individuals to breast cancer and/or ovarian cancer with moderate penetrance. Early-onset breast cancer is a major component of Li-Fraumeni Syndrome (LFS), and mutations in the LFS-associated gene TP53 provide a 10- to 20-fold increased risk for developing bilateral mammary carcinomas, in addition to other cancers (i.e. ovarian). Individuals with Cowden syndrome caused by mutations in PTEN have a lifetime risk of 50% for breast cancer and 5-10% for endometrial cancer (Hearle et al. 2006; Eng 2000). Peutz-Jeghers syndrome, caused by mutations in STK11, can reach a breast cancer incidence of 32% by 60 years of age (Lim et al. 2004). RAD51C is a tumor suppressor that has a role in DNA repair. It is mutated in approximately 1-5% of individuals with a family history of breast and ovarian cancer (Meindl et al. 2011). CDH1 pathogenic variants predispose individuals to higher rates of breast cancer, but not ovarian cancer (Pennington and Swisher 2012). The relative risk of breast cancer due to PALB2 pathogenic variants has been estimated at 2.3, with a higher risk for women under 50 years of age (3.0 relative-risk) versus a lower risk in women older than 50 years of age (1.9 relative-risk) (Walsh and King 2007). A larger recent study has found a higher relative risk (<9) depending on age for PALB2 mutation carriers, and the mean cumulative risk of breast cancer by 70 was 35% (Antoniou et al. 2014).

Individuals with heterozygous mutations in low to moderately penetrant genes BRIP1, MRE11 and ATM increase their breast cancer risk from 1.5-5 fold (Meindl et al. 2011; Pennington and Swisher 2012). Others have reported that lifetime risks of BRIP1 and PALB2 mutations are associated with a 20-50% lifetime risk of breast cancer (Seal et al. 2006; Rahman et al. 2007). Female relatives of individuals with ataxia-telangiectasia and who are carriers of an ATM mutation have a 2-5 fold increased risk of breast cancer (Swift et al. 1987; Thompson et al. 2005). This is an important increase considering approximately 0.2-1.0% of the general population are ATM mutation heterozygotes (Lindor et al. 2008). Mutations in CHEK2 have been found in patients who have hereditary breast cancer, but do not have detectable BRCA1 or BRCA2 mutations (Vahteristo et al. 2002; Meijers-Heijboer et al. 2003). CHEK2 mutations increase the risk of breast cancer 2-3 fold, and up to 4-5 fold if there is a family history (Weischer et al. 2008). NBN mutations are associated with a 2-fold increased risk of breast cancer (Walsh and King 2007). Pathogenic variants in the MUTYH, PMS2, RAD51D, SMARCA4 and XRCC2 genes have also been associated with hereditary breast and/or ovarian cancer (Maxwell et al. 2014; Gutiérrez-Enríquez et al. 2013; Jelinic et al. 2014; Park et al. 2012), although there risk is not well defined. It has been estimated that approximately 6% of patients with hereditary ovarian cancers who do not have pathogenic variants in BRCA1 or BRCA2 may have pathogenic variants in genes such as BARD1BRIP1, CHEK2, MRE11, NBN, PALB2,  RAD50RAD51C and TP53 (Walsh et al. 2011). 

See individual gene test descriptions for information on molecular biology of gene products.

Testing Strategy

This HBOC High Risk NGS panel analyzes 20 genes, in which pathogenic variants have been associated with low to moderate to high risks of developing hereditary breast and/or ovarian cancer. For this NGS panel, the full coding regions, plus ~20bp of non-coding DNA flanking each exon, are sequenced for each of the HBOC 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. All pathogenic, undocumented and questionable variant calls are confirmed by Sanger sequencing. Deletion/Duplication testing is performed by aCGH.

Each gene can also be tested individually using our Sanger sequencing assays. Deletion and duplication testing is available for all genes in the panel, except SMARCA4 and XRCC2. Please see our test menu.

Indications for Test

Individuals with a clinical presentation of Hereditary Breast and Ovarian Cancer syndrome and a family history of HBOC. Clinical presentation or family history includes early-onset of breast cancer (i.e.

 This is a predictive test and it only provides information regarding the likelihood of breast and/or ovarian cancer.  A positive test does not mean that a person will have breast and/or ovarian cancer and a negative test does not mean that a person will not get breast and/or ovarian cancer.


Official Gene Symbol OMIM ID
ATM 607585
BARD1 601593
BRCA1 113705
BRCA2 600185
BRIP1 605882
CDH1 192090
CHEK2 604373
MRE11 600814
MUTYH 604933
NBN 602667
PALB2 610355
PMS2 600259
PTEN 601728
RAD50 604040
RAD51C 602774
RAD51D 602954
SMARCA4 603254
STK11 602216
TP53 191170
XRCC2 600375
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT

Related Tests

Ataxia telangiectasia Syndrome via the ATM Gene
Ataxia-telangiectasia-like disorder via the MRE11/MRE11A Gene
Cancer Sequencing and Deletion/Duplication Panel
Chromosomal Instability Syndromes Sequencing Panel
Coffin-Siris Syndrome Sequencing Panel
Colorectal Cancer Sequencing And Deletion/Duplication Panel
Fanconi Anemia Sequencing Panel
Fanconi Anemia via the BRCA2/FANCD1 Gene
Fanconi Anemia via the BRIP1/FANCJ Gene
Fanconi Anemia via the PALB2/FANCN Gene
Fanconi Anemia via the RAD51C/FANCO Gene
Hereditary Breast and Ovarian Cancer Syndrome - HBOC HIGH RISK Sequencing and Deletion/Duplication Panel
Hereditary Breast and Ovarian Cancer via the BARD1 Gene
Hereditary Breast and Ovarian Cancer via the RAD50 Gene
Hereditary Breast and Ovarian Cancer via the RAD51D Gene
Hereditary Breast Cancer via the CHEK2 Gene
Hereditary Diffuse Gastric Cancer via the CDH1 Gene
Hereditary Myelodysplastic Syndrome (MDS) / Acute Myeloid Leukemia (AML) Sequencing Panel
Li-Fraumeni Syndrome via the TP53 Gene
Lynch Syndrome Sequencing and Deletion/Duplication Panel
Lynch Syndrome via the PMS2 Gene
MUTYH Associated Polyposis (MAP) Syndrome via the MUTYH Gene
Nijmegen Breakage Syndrome via the NBN Gene
Ovarian Cancer and Rhabdoid Tumor Predisposition Syndrome via the SMARCA4 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
  • Antoniou AC, Casadei S, Heikkinen T, Barrowdale D, Pylkäs K, Roberts J, Lee A, Subramanian D, Leeneer K De, Fostira F, Tomiak E, Neuhausen SL, et al. 2014. Breast-Cancer Risk in Families with Mutations in PALB2. New England Journal of Medicine 371: 497–506. PubMed ID: 25099575
  • Berliner JL, Fay AM, Cummings SA, Burnett B, Tillmanns T. 2012. NSGC Practice Guideline: Risk Assessment and Genetic Counseling for Hereditary Breast and Ovarian Cancer. Journal of Genetic Counseling 22: 155–163. PubMed ID: 23188549
  • Eng. 2000. Will the real Cowden syndrome please stand up: revised diagnostic criteria. J Med Genet 37:828-830.
    PubMed ID: 11073535
  • Gutiérrez-Enríquez S, Bonache S, Ruíz de Garibay G, Osorio A, Santamariña M, Ramón y Cajal T, Esteban-Cardeñosa E, Tenés A, Yanowsky K, Barroso A, Montalban G, Blanco A, et al. 2013. About 1% of the breast and ovarian Spanish families testing negative for BRCA1 and BRCA2 are carriers of RAD51D pathogenic variants: RAD51D germline mutations in breast and ovarian Spanish families. International Journal of Cancer n/a–n/a. PubMed ID: 24130102
  • Hearle N. 2006. Frequency and Spectrum of Cancers in the Peutz-Jeghers Syndrome. Clinical Cancer Research 12: 3209–3215. PubMed ID: 16707622
  • Hendrickson BC, Judkins T, Ward BD, Eliason K, Deffenbaugh AE, Burbidge LA, Pyne K, Leclair B, Ward BE, Scholl T. 2005. Prevalence of five previously reported and recurrentBRCA1 genetic rearrangement mutations in 20,000 patients from hereditary breast/ovarian cancer families. Genes, Chromosomes and Cancer 43: 309–313. PubMed ID: 15846789
  • Jelinic P, Mueller JJ, Olvera N, Dao F, Scott SN, Shah R, Gao J, Schultz N, Gonen M, Soslow RA, Berger MF, Levine DA. 2014. Recurrent SMARCA4 mutations in small cell carcinoma of the ovary. Nature Genetics 46: 424–426. PubMed ID: 24658004
  • Judkins T, Rosenthal E, Arnell C, Burbidge LA, Geary W, Barrus T, Schoenberger J, Trost J, Wenstrup RJ, Roa BB. 2012. Clinical significance of large rearrangements in BRCA1 and BRCA2. Cancer 118: 5210–5216. PubMed ID: 22544547
  • Lim W, Olschwang S, Keller JJ, Westerman AM, Menko FH, Boardman LA, Scott RJ, Trimbath J, Giardiello FM, Gruber SB. 2004. Relative frequency and morphology of cancers in STK11 mutation carriers. Gastroenterology 126: 1788-1794. PubMed ID: 15188174
  • Lindor NM, McMaster ML, Lindor CJ, Greene MH. 2008. Concise Handbook of Familial Cancer Susceptibility Syndromes - Second Edition. JNCI Monographs 2008: 3–93. PubMed ID: 18559331
  • Marchina E, Fontana MG, Speziani M, Salvi A, Ricca G, Di Lorenzo D, Gervasi M, Caimi L, Barlati S. 2010. BRCA1 and BRCA2 genetic test in high risk patients and families: counselling and management. Oncology Reports 24: 1661-1667. PubMed ID: 21042765
  • Maxwell KN, Wubbenhorst B, D’Andrea K, Garman B, Long JM, Powers J, Rathbun K, Stopfer JE, Zhu J, Bradbury AR, Simon MS, DeMichele A, et al. 2014. Prevalence of mutations in a panel of breast cancer susceptibility genes in BRCA1/2-negative patients with early-onset breast cancer. Genetics in Medicine. PubMed ID: 25503501
  • Meijers-Heijboer H, Wijnen J, Vasen H, Wasielewski M, Wagner A, Hollestelle A, Elstrodt F, Bos R van den, Snoo A de, Fat GTA, Brekelmans C, Jagmohan S, et al. 2003. The CHEK2 1100delC mutation identifies families with a hereditary breast and colorectal cancer phenotype. Am. J. Hum. Genet. 72: 1308–1314. PubMed ID: 12690581
  • Meindl A, Ditsch N, Kast K, Rhiem K, Schmutzler RK. 2011. Hereditary breast and ovarian cancer: new genes, new treatments, new concepts. Dtsch Arztebl Int 108: 323–330. PubMed ID: 21637635
  • Melhem-Bertrandt A, Bojadzieva J, Ready KJ, Obeid E, Liu DD, Gutierrez-Barrera AM, Litton JK, Olopade OI, Hortobagyi GN, Strong LC, Arun BK. 2012. Early onset HER2-positive breast cancer is associated with germline TP53 mutations. Cancer 118: 908–913. PubMed ID: 21761402
  • Miki Y, Swensen J, Shattuck-Eidens D, Futreal PA, Harshman K, Tavtigian S, Liu Q, Cochran C, Bennett LM, Ding W. 1994. A strong candidate for the breast and ovarian cancer susceptibility gene BRCA1. Science 266: 66–71. PubMed ID: 7545954
  • Park DJ, Lesueur F, Nguyen-Dumont T, Pertesi M, Odefrey F, Hammet F, Neuhausen SL, John EM, Andrulis IL, Terry MB, Daly M, Buys S, et al. 2012. Rare Mutations in XRCC2 Increase the Risk of Breast Cancer. The American Journal of Human Genetics 90: 734–739. PubMed ID: 22464251
  • Pennington KP, Swisher EM. 2012. Hereditary ovarian cancer: Beyond the usual suspects. Gynecologic Oncology 124: 347-353. PubMed ID: 22264603
  • Petrucelli N, Daly MB, Feldman GL. 2013. BRCA1 and BRCA2 Hereditary Breast and Ovarian Cancer. In: Pagon RA, Adam MP, Bird TD, Dolan CR, Fong C-T, and Stephens K, editors. GeneReviews™, Seattle (WA): University of Washington, Seattle. PubMed ID: 20301425
  • Pruthi S, Gostout BS, Lindor NM. 2010. Identification and Management of Women With BRCA Mutations or Hereditary Predisposition for Breast and Ovarian Cancer. Mayo Clinic Proceedings 85: 1111–1120. PubMed ID: 21123638
  • Rahman N, Seal S, Thompson D, Kelly P, Renwick A, Elliott A, Reid S, Spanova K, Barfoot R, Chagtai T, Jayatilake H, McGuffog L, Hanks S, Evans DG, Eccles D; Breast Cancer Susceptibility Collaboration (UK), Easton DF, Stratton MR. 2007. PALB2, which encodes a BRCA2-interacting protein, is a breast cancer susceptibility gene. Nature Genetics 39: 165-167. PubMed ID: 17200668
  • Seal S, Thompson D, Renwick A, Elliott A, Kelly P, Barfoot R, Chagtai T, Jayatilake H, Ahmed M, Spanova K, North B, McGuffog L, Evans DG, Eccles D; Breast Cancer Susceptibility Collaboration (UK), Easton DF, Stratton MR, Rahman N. 2006. Truncating mutations in the Fanconi anemia J gene BRIP1 are low-penetrance breast cancer susceptibility alleles. Nature Genetics 38: 1239-1241. PubMed ID: 17033622
  • Smith EC. 2012. An Overview of Hereditary Breast and Ovarian Cancer Syndrome. Journal of Midwifery & Women’s Health 57: 577–584. PubMed ID: 23050669
  • Swift M, Reitnauer PJ, Morrell D, Chase CL. 1987. Breast and other cancers in families with ataxia-telangiectasia. N. Engl. J. Med. 316: 1289-1294. PubMed ID: 3574400
  • Tan DSP, Marchio C, Reis-Filho JS. 2008. Hereditary breast cancer: from molecular pathology to tailored therapies. Journal of Clinical Pathology 61: 1073–1082. PubMed ID: 18682420
  • Thompson D, Duedal S, Kirner J, McGuffog L, Last J, Reiman A, Byrd P, Taylor M, Easton DF. 2005. Cancer Risks and Mortality in Heterozygous ATM Mutation Carriers. JNCI Journal of the National Cancer Institute 97: 813–822. PubMed ID: 15928302
  • Turnbull C, Rahman N. 2008. Genetic Predisposition to Breast Cancer: Past, Present, and Future. Annual Review of Genomics and Human Genetics 9: 321-345. PubMed ID: 18544032
  • Vahteristo P, Bartkova J, Eerola H, Syrjäkoski K, Ojala S, Kilpivaara O, Tamminen A, Kononen J, Aittomäki K, Heikkilä P, Holli K, Blomqvist C, et al. 2002. A CHEK2 Genetic Variant Contributing to a Substantial Fraction of Familial Breast Cancer. American Journal of Human Genetics 71: 432. PubMed ID: 12094328
  • Vuorela M, Pylkäs K, Hartikainen JM, Sundfeldt K, Lindblom A, Wachenfeldt Wäppling A von, Haanpää M, Puistola U, Rosengren A, Anttila M, Kosma V-M, Mannermaa A, Winqvist R. 2011. Further evidence for the contribution of the RAD51C gene in hereditary breast and ovarian cancer susceptibility. Breast Cancer Res. Treat. 130: 1003-1010. PubMed ID: 21750962
  • Walsh T, Casadei S, Lee MK, Pennil CC, Nord AS, Thornton AM, Roeb W, Agnew KJ, Stray SM, Wickramanayake A, Norquist B, Pennington KP, et al. 2011. Mutations in 12 genes for inherited ovarian, fallopian tube, and peritoneal carcinoma identified by massively parallel sequencing. Proceedings of the National Academy of Sciences 108: 18032–18037. PubMed ID: 22006311
  • Walsh T, King M-C. 2007. Ten Genes for Inherited Breast Cancer. Cancer Cell 11: 103-105. PubMed ID: 17292821
  • Weischer M, Bojesen SE, Ellervik C, Tybjaerg-Hansen A, Nordestgaard BG. 2008. CHEK2*1100delC Genotyping for Clinical Assessment of Breast Cancer Risk: Meta-Analyses of 26,000 Patient Cases and 27,000 Controls. Journal of Clinical Oncology 26: 542–548. PubMed ID: 18172190
  • Wooster R, Bignell G, Lancaster J, Swift S, Seal S, Mangion J, Collins N, Gregory S, Gumbs C, Micklem G. 1995. Identification of the breast cancer susceptibility gene BRCA2. Nature 378: 789–792. PubMed ID: 8524414
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.
  • 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.