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Chromosomal Instability Syndromes Sequencing Panel

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

Sequencing

Test Code TestCPT Code Copy CPT Codes
1214 ATM 81408 Add to Order
BLM 81479
ERCC6 81479
ERCC8 81479
MRE11 81479
NBN 81479
RECQL4 81479
WRN 81479
Full Panel Price* $1590.00
Test Code Test Total Price CPT Codes Copy CPT Codes
1214 Genes x (8) $1590.00 81408, 81479(x7) Add to Order
Pricing Comment

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

Approximately 90% of individuals with Ataxia-telangiectasia have detectable mutations via sequencing of the ATM gene. Approximately 1-2% of patients have large genomic deletions involving ATM. These will not be detected by sequencing, please see our deletion/duplication test using aCGH for the latter (Gatti. GeneReviews. 2010).

Clinical sensitivity is unknown for Ataxia-telangiectasia-like Disorder because it is a rare disease and relatively few patients have been reported.

In a retrospective study (German et al. Hum Mutat 28(8): 743-53, 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.

Mutations in ERCC6 cause Cockayne syndrome complementation group type B, which account for 65% of cases. Approximately 95% of mutations will be detected by sequencing, and approximately 5% are due to gross deletions that will not be detected by sequencing (see aCGH). Mutations in ERCC8 cause Cockayne syndrome complementation group type A, which accounts for 35% of cases. Approximately 80% of mutations will be detected by sequencing, and approximately 20% are due to gross deletions that will not be detected by sequencing (see aCGH). (Laugel. GeneReviews. 2012)

Most NBN mutations reported are based on individuals who are homozygous for the single most common Eastern European mutation, 657_661del, which can be readily detected using sequencing. In the United States, approximately 70% of individuals with NBS tested to date are homozygous for the common allele (657_661del), 15% are heterozygous for c.657_661del5 and a second unique mutation, and 15% are homozygous for a unique mutation (Concannon and Gatti. GeneReviews. 2011). Individuals homozygous for the NBN c.1089C>A mutation have features of Fanconi anemia (Gennery et al. Clin Immunol 113(2):214-9, 2004).

Sequencing of RECQL4 will detect approximately 66% of Rothmund Thomson Syndrome cases. The other 34% may be due to genetic heterogeneity although no other genes have been found. The rate of deletions/duplications is unknown (Wang and Plon. GeneReviews. 2009).

Mutations in WRN are identified by sequencing in approximately 90% of individuals with Werner syndrome (Oshima et al. GeneReviews. 2012).

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Deletion/Duplication Testing via aCGH

Test Code TestIndividual Gene PriceCPT Code Copy CPT Codes
600 ATM$690.00 81479 Add to Order
BLM$690.00 81479
ERCC6$690.00 81479
ERCC8$690.00 81479
MRE11$690.00 81479
NBN$690.00 81479
RECQL4$690.00 81479
WRN$690.00 81479
Full Panel Price* $840.00
Test Code Test Total Price CPT Codes Copy CPT Codes
600 Genes x (8) $840.00 81479(x8) Add to Order
Pricing Comment

# of Genes Ordered

Total Price

1

$690

2

$730

3

$770

4-10

$840

11-30

$1,290

31-100

$1,670

Over 100

Call for quote
Turnaround Time

The great majority of tests are completed within 28 days.

Clinical Features

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, but other solid tumors do occur. Cancer risk is increased by 60-180 fold over population risk (Thoms et al. Experimental Dermatology 16:532–544, 2007). Other features can include premature aging (e.g. grey hair) and insulin-resistant diabetes mellitus. Individuals with A-T are usually sensitive to ionizing radiation (e.g. radiotherapy). Non-classic forms can occur with adult-onset A-T and A-T with early-onset dystonia. Unlike A-T, which is caused by homozygous mutations in the ATM gene, heterozygous carriers of an ATM mutation are at an increased risk of breast cancer (Concannon et al. Cancer Res 68:6486–91, 2008) and heart disease (Swift et al. N Engl J Med 325:1831–6, 1991). The time of onset and progression is variably between and within families of affected individuals. The prevalence of A-T has been reported to be 1:40,000-100,000 in the United States (Gatti. GeneReviews. 2010).

Ataxia-telangiectasia-like disorder (ATLD) is similar to the A-T in that patients show progressive cerebellar ataxia, however they do not show telangiectasias, and show normal immunoglobulin levels, later onset disease and a milder clinical course compared to A-T. At the cellular level, there is chromosomal instability, increased sensitivity to ionizing radiation, defective induction of stress-activated signal transduction pathways, and radioresistant DNA synthesis (Taylor et al. DNA Repair 3(8-9):1219-25, 2004).

Bloom’s Syndrome (BS) was first described in 1954 as a “congenital” skin disorder in “dwarfs” (Bloom. Am J Dis Child 88:754-758, 1954). While patients were found to have additional clinical features, such as immune deficiencies and a propensity for cancer (German et al. Science 148:506-507, 1965; German Am J Hum Genet 21-196-227, 1969), sun-sensitive facial lesions, low birth weight and stunted growth remain the most consistent features of Bloom’s Syndrome today. Symptoms of BS are related to increased mutability of proliferating somatic cells, particularly epithelial cells and lymphocytes. When grown in culture and viewed microscopically, cells of BS patients exhibit extensive “chromosome breakage”, including gaps and breaks, structural rearrangements, and telomeric associations (German and Crippa Ann Genet 9143-154, 1966). Chromosome breakage ultimately leads to excessive somatic recombination and high mutation rates (Groden and German Hum Genet 90:360-367, 1992).

Cockayne Syndrome (CS) presents with sun-sensitivity and postnatal growth failure, but does not have increased skin cancer rates. Other minor features include cachexia, neurological, psychomotoric, mental developmental delays, cataracts, retinopathy, deafness, dental caries, and characteristic facies (Thoms et al. Experimental Dermatology 16:532–544, 2007). Patients also show growth retardation, microcephaly and calcifications of the basal ganglia or elsewhere in the central nervous system. Pathologically, neurological impairment correlates with a primary demyelinization of neurons. This is in contrast with the primary neuronal degeneration found in XP patients. There are three CS forms: (1) CS Type I is the classic/moderate form usually noticed by 1-2 years of age; (2) CS Type II is the severe form noticed in the neonatal period; and (3) CS Type III is the least severe and later-onset (Laugel. GeneReviews. 2012). The prevalence of CS is 2.3 per 1,000,000 livebirths, but this is considered a conservative estimate (Kleijer et al. DNA Repair (Amst) 7:744–50, 2008).

Nijmegen breakage syndrome (NBS) is a rare autosomal recessive disease that causes microcephaly, short stature, immunodeficiency, and predisposition to cancer. Approximately half the individuals with NBS develop non-Hodgkin lymphoma or leukemia. Other cancers observed include medulloblastomas, gliomas, and rhabdomyosarcomas. Individuals with NBS are 50 times more likely to develop cancer than people without. Intellectual development is normal in 40% of patients, borderline-to-mild retardation is found in 50% of patients, and 10% of patients are moderately retarded (Kondratenko et al. Adv Exp Med Biol 601:61-7, 2007). Women with NBS often have premature ovarian insufficiency and are infertile. It is estimated that the incidence of NBS is 1 in 100,000, with a higher prevalence in specific European ancestries (i.e. Slavic population) (Concannon and Gatti. GeneReviews. 2011).

Rothmund Thomson Syndrome (RTS) presents skeletal (e.g. small stature, dysplasias) and cutaneous abnormalities, with a high incidence of osteosarcomas and non-melanoma skin cancer. Individuals with RTS are photosensitive and have facial erythema and swelling in the first months of life. In addition, they present with erythema in their buttocks and extremities, while sparing the trunk, and chronic poikiloderma with hyper/hypopigmentation. Individuals with RTS also have telangiectasias, spot-like skin atrophy, and cataracts. (Thoms et al. Experimental Dermatology 16:532–544, 2007). RTS is considered a rare disorder and its prevalence is unknown (Wang and Plon. GeneReviews. 2009).

Werner Syndrome (WS) presents premature aging (e.g. greying and loss of hair) and various cancers and has been called the "adult form of progeria". Most clinical signs can be observed after 10 years of age. In addition, to premature aging there are clinical features of osteoporosis, hoarseness of voice, short stature, atherosclerosis, cataracts, hypogonadism, and diabetes. A high risk of UV-independent melanomas of the mucosae and acrolentiginous melanomas is noted. Other common cancers include soft tissue sarcomas, thyroid cancers, meningiomas, and osteosarcomas (Thoms et al. Experimental Dermatology 16:532–544, 2007). The prevalence of Werner syndrome is 1:20,000-40,000 in Japanese populations (Satoh et al. Lancet 353:1766, 1999), 1:50,000 in the Sardian population (Masala et al. Eur J Dermatol 17:213–216, 2007), and has been estimated to be 1:200,000 in the United States (Martin et al. J Am Geriatr Soc 47:1136–44, 1999).

Genetics

Cellular DNA is constantly being bombarded by endogenous damage, such as oxygen radicals, inappropriate methylation, and exogenous damage, such as chemicals, chemotherapeutics and ionizing radiation. DNA damage can be also induced during cellular processes, such as mobile element transposition, rearrangements of immune genes, and meiotic recombination (Duker. American Journal of Medical Genetics (Semin. Med. Genet.) 115:125–129, 2002). Correction of these damaging insults require a coordinated effort of genes that are involved in DNA damage sensing, checkpoint control and repair. Mutations in these genes can lead to genomic instability within syndromes leading to an elevated risk of cancers.

Ataxia-telangiectasia is an autosomal recessive disorder that is caused by homozygous 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. DNA Repair 3(8-9):1219-25, 2004). Mutations in ATM result in defective checkpoint cycling. Over 500 private mutations are described with no common hot spots for mutations.  In North America most affected individuals are compound heterozygotes for two ATM mutations. Founder mutations for ATM mutations have been observed in several populations (Gatti. GeneReviews. 2010). 

Ataxia-telangiectasia-like disorder is caused by mutations in the MRE11/MRE11A gene, which is involved in homologous recombination, telomere length maintenance, cell cycle checkpoint control and DNA double-strand break repair (Taylor et al. DNA Repair 3(8-9):1219-25, 2004). The MRE11A protein forms a complex with the RAD50 and NBS1 proteins to form the MRN complex, which is required for proper DNA repair.  The serine protein kinase mutated in A-T acts upstream of the MRN complex and is responsible for MRN activation through phosphorylation. The MRN complex also has a role in ATM recruitment to DNA damage foci (Wu et al. Mol Cell  46(3):351-61, 2012) and has been shown to associate with mismatch repair proteins (Wu et al. Mol Cancer Res 9(11):1443-8, 2011). ATLD has an autosomal recessive mode of inheritance and mutations reported to date include missense, splicing, and a small deletion (Human Gene Mutation Database).

Bloom’s syndrome is an autosomal recessive disorder, caused by homozygous or compound heterozygous mutations in the BLM gene (German et al. Hum Mutat 28:743-753, 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 Nat Rev Cancer 3:169-178, 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 Crit Rev Biochem Mol Biol 39:79-97, 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 et al. J Biol Chem 276:30031-30035, 2001; Pedrazzi et al. Biol Chem 384:1155-1164, 2003). Indeed, there is some evidence that heterozygous carriers of a BLM mutation have an increased risk for colorectal cancer (Gruber et al. Science 297:2013, 2002), a disease most commonly associated with heterozygous mutations in the MMR genes: MLH1, MSH2 and MSH6.

Cockayne syndrome is an autosomal recessive disorder. ERCC6 and ERCC8 appear to play an important role in the temporary removal of the stalled polymerase II to allow repair followed by the continuation of the transcription. Interestingly, nucleotide excision repair from cells of CS patients are defective in transcription couple repair (TCR), but exhibit normal global genome repair. This may help to explain the lack of cancer development, whereas both pathways are usually defective in Xeroderma Pigmentation patients who have high rates of skin cancer. Interestingly, CS cells can repair 6-4 photoproducts, unlike cells from XP patients. Finally, CS cells exhibit an increased rate of apoptosis because of TCR failure and blockage of transcription. Enhanced apoptosis of initially damaged cells may also prevent tumor cell development. (Thoms et al. Experimental Dermatology, 16:532–544, 2007). There are no reported genotype-phenotype correlations (Laugel. GeneReviews. 2012).

Nijmegen Breakage Syndrome is and autosomal recessive disease caused by mutations in the NBN gene.  The protein product of NBN, Nibrin, normally associates with the MRE11A and RAD50 proteins to form the MRN complex. The MRN complex, upon DNA damage, is involved in DNA repair and cell cycle arrest via the ATM kinase; pathogenic mutations in NBN lead to faulty DNA repair and improper cell cycle control. NBS is inherited in an autosomal recessive manner, although carriers of NBN mutations may be at a higher risk of malignancies (Ciara et al. Acta Neuropathol 119(3):325-34, 2010; Steffen et al. Int J Cancer 10;111(1):67-71, 2004). Most mutations reported to date in NBN result in truncation mutations of Nibrin. The c.657_661del mutation is the most common mutation found in Eastern Europeans with NBS, accounting for more than 90% of all mutant alleles. Other mutations are private mutations which occur in one or a small number of families (Concannon and Gatti. GeneReviews. 2011).

Rothmund Thomson Syndrome is an autosomal recessive disease that is caused by mutations in the RECQL4 gene. The RECQL4 gene encodes the protein ATP-dependent DNA helicase Q4, which functions in unwinding DNA for many biological processes (e.g. replication initiation) (Sangrithi et al. Cell 121:887–98, 2005). The majority of mutations result in absent protein from truncating mutations (Siitonen et al. Eur J Hum Genet 17:151–8, 2009; Wang and Plon. GeneReviews. 2009)

Werner syndrome is an autosomal recessive disease that is caused by mutations in the WRN gene. WRN encodes an ATP-dependent helicase, belonging to the RECQ family of helicases. It is involved in unwinding DNA for processes such as DNA replication, repair, recombination, and transcription. It also has is an exonuclease and functions in the maintenance of telomeres (Rossi et al. DNA Repair (Amst) 9:331–44, 2010). The most common mutation is c.1105C>T, which is prevalent in European and Japanese populations (Matsumoto et al. Hum Genet 100:123–30, 1997; .Friedrich et al. Hum Genet 128:103–11, 2010). Founder mutations exist in other populations (Oshima et al. GeneReviews. 2012). Most mutations result in loss of protein expression via frameshift.

Testing Strategy

This Chromosomal Instability Syndromes Next Generation Sequencing (NGS) Panel analyzes 8 genes. For this NGS panel, the full coding regions, plus ~20bp of non-coding DNA flanking each exon, are sequenced for each of the Chromosomal Instability Syndrome 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

Each gene can also be tested individually using our Sanger sequencing assays. Please see our test menu.

Indications for Test

This test especially aids in a differential diagnosis of similar phenotypes, rules out particular syndromes, and provides the analysis of multiple genes simultaneously. Individuals who are suspected of any of these disorders, especially if clinical diagnosis is unclear, and individuals who have been found to be negative by mutation analysis for a single gene test are candidates. Laboratory findings that support the diagnosis include: protein levels, chromosome translocations, immunodeficiency, and radiosensitivity demonstrated by in vitro assay. This test is specifically designed for heritable germline mutations and is not appropriate for the detection of somatic mutations in tumor tissue.

Individuals presenting with a Cockayne syndrome phenotype may have a mutation in the Xeroderma Pigmentosum genes ERCC2, ERCC3, or ERCC5 (Rapin et al Neurology 28;55(10):1442-9, 2000). Please see Xeroderma Pigmentosum gene testing in our test menu.

Genes

Official Gene Symbol OMIM ID
ATM 607585
BLM 604610
ERCC6 609413
ERCC8 609412
MRE11 600814
NBN 602667
RECQL4 603780
WRN 604611
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT

Related Tests

Name
Ataxia telangiectasia Syndrome via the ATM Gene
Ataxia-telangiectasia-like disorder via the MRE11/MRE11A Gene
Bloom's Syndrome via the BLM Gene
Cancer Sequencing and Deletion/Duplication Panel
Cockayne Syndrome via the ERCC6 Gene
Cockayne Syndrome via the ERCC8 Gene
Colorectal Cancer Sequencing And Deletion/Duplication Panel
Hereditary Breast and Ovarian Cancer Syndrome - HBOC EXPANDED Sequencing and Deletion/Duplication Panel
Nijmegen Breakage Syndrome via the NBN Gene
Pancreatic Cancer Sequencing Panel
Rothmund Thomson Syndrome via the RECQL4 Gene
Werner Syndrome via the WRN Gene

CONTACTS

Genetic Counselors
Geneticist
Citations
  • Bennett RJ, Keck JL. 2004. Structure and function of RecQ DNA helicases. Crit. Rev. Biochem. Mol. Biol. 39: 79-97. PubMed ID: 15217989
  • Bloom, D. 1954. Congenital telangiectatic erythema resembling lupus erythematosus in dwarfs; probably a syndrome entity. AMA Am J Dis Child 88(6): 754-8. PubMed ID: 13206391
  • Ciara et al. (2010). PubMed ID: 19908051
  • Concannon and Gatti. (2011). PubMed ID: 20301355
  • Concannon P, Haile RW, Borresen-Dale A-L, Rosenstein BS, Gatti RA, Teraoka SN, Diep AT, Jansen L, Atencio DP, Langholz B, Capanu M, Liang X, et al. 2008. Variants in the ATM Gene Associated with a Reduced Risk of Contralateral Breast Cancer. Cancer Research 68: 6486–6491. PubMed ID: 18701470
  • Duker. (2002). PubMed ID: 12407692
  • Friedrich et al. (2010). PubMed ID: 20443122
  • Gatti R. 2010. Ataxia-Telangiectasia. 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: 20301790
  • Gennery et al. (2004). PubMed ID: 15451479
  • German J, Sanz MM, Ciocci S, Ye TZ, Ellis NA. 2007. Syndrome-causing mutations of the BLM gene in persons in the Bloom’s Syndrome Registry. Human Mutation 28: 743–753. PubMed ID: 17407155
  • German, J. 1969. Bloom's syndrome. I. Genetical and clinical observations in the first twenty-seven patients. Am J Hum Genet 21(2): 196-227. PubMed ID: 5770175
  • German, J., et.al. 1965. Chromosomal Breakage in a Rare and Probably Genetically Determined Syndrome of Man. Science 148: 506-7. PubMed ID: 14263770
  • Groden, J., German, J. (1992). PubMed ID: 1483691
  • Gruber SB, Ellis NA, Scott KK, Almog R, Kolachana P, Bonner JD, Kirchhoff T, Tomsho LP, Nafa K, Pierce H, Low M, Satagopan J, et al. 2002. BLM heterozygosity and the risk of colorectal cancer. Science 297: 2013.. PubMed ID: 12242432
  • Hickson ID. 2003. RecQ helicases: caretakers of the genome. Nature Reviews Cancer 3: 169–178. PubMed ID: 12612652
  • Kleijer WJ, Laugel V, Berneburg M, Nardo T, Fawcett H, Gratchev A, Jaspers NGJ, Sarasin A, Stefanini M, Lehmann AR. 2008. Incidence of DNA repair deficiency disorders in western Europe: Xeroderma pigmentosum, Cockayne syndrome and trichothiodystrophy. DNA Repair 7: 744–750. PubMed ID: 18329345
  • Kondratenko et al. (2007). PubMed ID: 17712992
  • Langland G. 2001. The Bloom’s Syndrome Protein (BLM) Interacts with MLH1 but Is Not Required for DNA Mismatch Repair. Journal of Biological Chemistry 276: 30031–30035. PubMed ID: 11325959
  • Laugel. (2012). PubMed ID: 20301516
  • Martin et al. (1999). PubMed ID: 10484259
  • Masala et al. (2007). PubMed ID: 17478382
  • Matsumoto et al. (1997). PubMed ID: 9225981
  • Oshima et al. (2012). PubMed ID: 20301687
  • Pedrazzi G, Bachrati CZ, Selak N, Studer I, Petkovic M, Hickson ID, Jiricny J, Stagljar I. 2003. The Bloom’s syndrome helicase interacts directly with the human DNA mismatch repair protein hMSH6. Biol. Chem. 384: 1155–1164. PubMed ID: 12974384
  • Rapin I, Lindenbaum Y, Dickson DW, Kraemer KH, Robbins JH. 2000. Cockayne syndrome and xeroderma pigmentosum. Neurology 55: 1442–1449. PubMed ID: 11185579
  • Rossi et al. (2010). PubMed ID: 20075015
  • Sangrithi et al. (2005). PubMed ID: 15960976
  • Satoh et al. (1999). PubMed ID: 10347997
  • Siitonen et al. (2009). PubMed ID: 18716613
  • Steffen et al. (2004). PubMed ID: 15185344
  • Swift M, Morrell D, Massey RB, Chase CL. 1991. Incidence of cancer in 161 families affected by ataxia-telangiectasia. N. Engl. J. Med. 325: 1831–1836. PubMed ID: 1961222
  • Taylor et al. (2004). PubMed ID: 15279810
  • Thoms et al. (2007). PubMed ID: 17518994
  • Wang and Plon. (2009). PubMed ID: 20301415
  • Wu et al. (2011). PubMed ID: 21849470
Order Kits
TEST METHODS

NextGen Sequencing

Test Procedure

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.

Analytical Validity

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.   

Analytical Limitations

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

Test Procedure

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.

Analytical Validity

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.

Analytical Limitations

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.

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.
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.
SPECIMEN TYPES
WHOLE BLOOD

(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.
DNA

(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.
CELL CULTURE

(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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