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Noonan Spectrum Disorders/Rasopathies Sequencing Panel

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

NGS Sequencing

Test Code Test Copy GenesCPT Code Copy CPT Codes
1309 BRAF 81406 Add to Order
CBL 81479
HRAS 81404
KAT6B 81479
KRAS 81405
LZTR1 81479
MAP2K1 81406
MAP2K2 81406
NRAS 81479
PTPN11 81406
RAF1 81406
RIT1 81479
SHOC2 81405
SOS1 81406
SOS2 81479
Full Panel Price* $680.00
Test Code Test Copy Genes Total Price CPT Codes Copy CPT Codes
1309 Genes x (15) $680.00 81404, 81405(x2), 81406(x6), 81479(x6) Add to Order

New York State Approved Test

Pricing Comment

CPT code 81442 can be used if analysis includes BRAF, CBL, HRAS, KRAS, MAP2K1, MAP2K2, NRAS, PTPN11, RAF1, RIT1, SHOC2, and SOS1. We are happy to accommodate requests for single genes or a subset of these genes. The price will remain the list price. If desired, free reflex testing to remaining genes on panel is available. Alternatively, a single gene or subset of genes can also be ordered on our PGxome Custom Panel.

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 20 days.

Clinical Sensitivity

Clinical Sensitivity for Noonan Syndrome (Aoki et al. 2016)

Gene Sensitivity (%)
PTPN11 50
SOS1 11
RAF1 5
KRAS 1.5
SHOC2 2
BRAF 0.8
NRAS 0.2
RIT1 5
CBL Rare
KATB6 Rare
LZTR1 2.5
SOS2 1

Clinical Sensitivity for CFC Syndrome (Rauen 2016)

Gene Sensitivity (%)
BRAF 75
MAP2K1 12.5
MAP2K2 12.5
KRAS < 2%
SOS1 Rare

Clinical Sensitivity for NSML/LEOPARD Syndrome: ~90% (PTPN11, RAF1) (Gelb and Tartaglia 2010)

Clinical Sensitivity Costello Syndrome: ~ 80-90% (HRAS) (Gripp and Lin 2012)

Sensitivity corresponds to the percentage of all genotyped patients with a clinical diagnosis of the listed phenotype, except for RIT1, where sensitivity corresponds to the percentage of patients with no detectable pathogenic variants in the remaining Noonan-associated genes.

Only two large genomic duplications that span the PTPN11, TBX3 and TBX5 genes have been reported in patients with NS clinical features (Shchelochkov et al. 2008; Graham et al. 2009). The frequency of these duplications is currently unknown, but appears to be quite low.

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Deletion/Duplication Testing via aCGH

Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
600 BRAF$990.00 81479 Add to Order
CBL$990.00 81479
HRAS$990.00 81479
KAT6B$990.00 81479
KRAS$990.00 81479
MAP2K1$990.00 81479
MAP2K2$990.00 81479
NRAS$990.00 81479
PTPN11$990.00 81479
RAF1$990.00 81479
SHOC2$990.00 81479
SOS1$990.00 81479
Full Panel Price* $1490.00
Test Code Test Copy Genes Total Price CPT Codes Copy CPT Codes
600 Genes x (12) $1490.00 81479(x12) Add to Order
Pricing Comment

# of Genes Ordered

Total Price

1

$990

2-5

$1190

6-10

$1290

11-100

$1490

Over 100

Call for quote

Turnaround Time

The great majority of tests are completed within 20 days.

Clinical Sensitivity

Only two large genomic duplications that span the PTPN11, TBX3 and TBX5 genes have been reported in patients with NS clinical features (Shchelochkov et al. 2008; Graham et al. 2009). The frequency of these duplications is currently unknown, but appears to be quite low.

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Clinical Features

The Noonan Spectrum Disorders, also known as RASopathies, are a group of developmental syndromes characterized by extensive clinical and genetic heterogeneity. They include:

1- Noonan syndrome

2- Cardiofaciocutaneous syndrome

3- Noonan syndrome with multiple lentigines, previously known as LEOPARD syndrome

4- Costello syndrome

Although there is a considerable phenotypic overlap among the various syndromes, each syndrome is characterized by distinct clinical features.

Noonan Syndrome (NS) is characterized by dysmorphic facial features, growth and congenital heart defects, and musculoskeletal abnormalities. Cardiac abnormalities are found in up to 80% of patients and include pulmonary valve stenosis, atrial septal defect, atrioventricular canal defect, and hypertrophic cardiomyopathy. Musculoskeletal abnormalities include short stature, chest deformity with sunken or raised sternum, and short webbed neck. Several additional abnormalities have been described and include renal, genital, hematological, neurologic, cognitive, behavioral, gastrointestinal, dental, and lymphatic findings. Intelligence is usually normal; however, learning disabilities may be present. NS is characterized by extensive clinical heterogeneity, even among members of the same family. Diagnosis is often made in infancy or early childhood. Symptoms often change and lessen with advancing age. Infants with NS are at risk of developing juvenile myelomonocytic leukemia (JMML). The prevalence of NS is estimated at 1 in 1000 to 1 in 2,500 births worldwide (Allanson et al. 1985; Romano et al. 2010; Smpokou et al. 2012).

Cardio-Facio-Cutaneous Syndrome (CFCS) is characterized by distinctive facial appearance, congenital cardiac and ectodermal abnormalities, postnatal growth failure, feeding difficulties with failure to thrive and neurological findings. Facial features include high forehead, short, upturned nose with a low nasal bridge, prominent external ears that are posteriorly angulated and ocular hypertelorism. The most common cardiac abnormalities include pulmonic stenosis and atrial septal defects. Ectodermal abnormalities are heterogeneous in features and severity. They include café au lait spots, erythema, keratosis, ichthyosis, eczema, sparse and brittle hair, and nail dystrophy. The neurological findings include seizures, hypotonia, macrocephaly and various degrees of mental and cognitive delay (Reynolds et al. 1986).

Noonan syndrome with multiple lentigines (NSML), previously known as LEOPARD syndrome (multiple Lentigines, Electrocardiographic-conduction abnormalities, Ocular hyperterlorism, Pulmonary stenosis, Abnormal genitalia, Retardation of growth, sensorineural Deafenss) is characterized by skin pigmentation anomalies including multiple lentigines and café au lait spots, hypertrophic cardiomyopathy, pulmonary valve stenosis, and deafness. Other less common features include short stature, mild mental retardation, and abnormal genitalia (Legius et al. 2002; Sarkozy et al. 2004).

Costello Syndrome (CS) is characterized by coarse facial features with wide forehead, depressed nasal bridge and full cheeks; thick and loose skin of the hands and feet; papillomata; heart defects, mainly pulmonary valve stenosis, rhythm disturbances and hypertrophic cardiomyopathy; increased growth at the prenatal stage followed by postnatal growth retardation; short stature; relative macrocephaly and mild to moderate mental retardation (Van Eeghen et al. 1999; Lin et al. 2002). Patients with CS are at risk of developing benign and malignant tumors, most commonly rhabdomyosarcoma. Neuroblastoma and bladder carcinoma have also been reported (Gripp et al. 2006).

Genetics

Noonan spectrum disorders are caused by dysregulation of the RAS/mitogen-activated protein kinase (Ras/MAPK) signaling pathway (Tidyman et al. 2009; Wright and Kerr 2010).

Heterozygous germline pathogenic variants in fifteen genes have been reported in patients with Noonan spectrum disorders. Ten of these genes (PTPN11, SOS1, RAF1, KRAS, HRAS, SHOC2, BRAF, NRAS, MAP2K1, MAP2K1) encode components of the main Ras/MAPK signaling pathway; while three genes (CBL, KAT6B, and RIT1) encode proteins that are involved in the regulation of this pathway (Rauen 2013; Martinelli et al. 2010; Niemeyer et al. 2010; Kraft et al. 2011; Aoki et al. 2013). Recently, two additional genes, SOS2 and LZTR1, have been implicated in Noonan syndrome. SOS2 is homologue to SOS1. LZTR1 is a member of the BTB-kelch superfamily that had not been previously associated with the RAS/MAPK pathway (Aoki et al. 2016).

Noonan and Noonan-like syndromes are caused by pathogenic variants in PTPN11, SOS1, RAF1, KRAS, SHOC2, BRAF, NRAS, CBL, KAT6B, RIT1, SOS2 and LZTR1. Over 230 pathogenic variants have been reported in these genes. They account for up to 80% of all cases genotyped (Romano et al. 2010; Tartaglia et al. 2010; Aoki et al. 2013; Chen et al. 2014; Cordeddu et al. 2015; Yamamoto et al. 2015). The vast majority are missense, although a few small deletions or insertions, and indels have been reported, which are all predicted to result in in-frame alterations of the translated protein. A de novo balanced chromosomal translocation in the KAT6B gene was reported in one patient with Noonan syndrome-like clinical features (Kraft et al. 2011). To date, three large genomic duplications have been reported in patients with clinical features suggestive of NS. Two of these duplications include the PTPN11, TBX3 and TBX5 genes (Shchelochkov et al. 2008; Graham et al. 2009), while the third includes PTPN11 and TBX3 only (Chen et al. 2014). These three duplications occurred de novo and represent an uncommon cause of NS (Tartaglia et al. 2010).

Although most causative NS pathogenic variants occur de novo, familial cases have been reported. In these families, NS is inherited in an autosomal dominant manner with complete penetrance and variable expressivity (Romano et al. 2010).

CFCS is caused by defects in five RAS/MAPK genes (BRAF, MAP2K1, MAP2K2, KRAS and SOS1). Over 90 heterozygous pathogenic variants have been detected (Rodriguez-Viciana et al. 2006; Niihori et al. 2006; Schubbert et al. 2006; Tumurkhuu et al. 2010; Narumi et al. 2008). Although most CFCS causative variants are missense, small deletions and duplications have been documented. These are all predicted to result in in-frame alterations of the translated protein. No complex rearrangements have been reported. Most patients with CFCS do not reproduce. With one exception, all causative variants reported to date occurred de novo. The exception consists of a familial c.383C>A (p.Pro128Gln) variant in the MAP2K2 gene that was shown to be transmitted through four generations (Rauen et al. 2010). In this family, CFCS is inherited in an autosomal dominant manner.

NSML, previously known as LEOPARD syndrome is caused by defects in PTPN11 and RAF1 (Digilio et al. 2002; Pandit et al. 2007). Unlike NS, NSML-causative variants in the PTPN11 gene act through a dominant negative effect, which appears to disrupt the function of the wild-type gene product (SHP2 protein) (Jopling et al. 2007). PTPN11 pathogenic variants are the most common cause of NSML and account for over 90% of all cases genotyped. Ten different PTPN11 pathogenic variants, all missense, have been reported in patients with NSML (Human Gene Mutation Database). RAF1 pathogenic variants appear to be a rare cause of NSML. Parents of NSML patients are often asymptomatic, and de novo pathogenic variants are common. However, familial cases have been reported. In these families, affected relatives are diagnosed only after the birth of a visibly affected child, and the disease is transmitted in an autosomal dominant manner with variable penetrance and expressivity (Gelb and Tartaglia 2010).

CS syndrome is caused by pathogenic variants in the HRAS gene (Aoki et al. 2005). To date, 16 heterozygous activating germline missense variants and three indels, which are expected to result in amino acid substitutions, have been reported in patients with clinical features of CS. The majority of variants affect codons G12 and G13. However, variants affecting other codons have been reported. Recently, a 21-bp duplication that is predicted to result in an inframe insertion of seven amino acids p.Glu63-Asp69dup was reported in a patient with mild clinical features suggestive of CS (Lorenz et al. 2013). Most CS cases are sporadic resulting from de novo HRAS pathogenic variants.

Somatic mosaicism has been reported for several RAS/MAPK genes. Non-syndromic Juvenile Myelomonocytic Leukemia (JMML) is caused by somatic PTPN11 pathogenic variants in about 34% of all cases genotyped (Tartaglia et al. 2003). Somatic RAF1 pathogenic variants have been implicated in several human cancers (Pandit et al. 2007; Sarkozy et al. 2009). Somatic KRAS pathogenic variants have been implicated in several human cancers, including JMML (Reimann et al. 2006) and colon cancer (Edkins et al. 2006). Somatic NRAS pathogenic variants were detected in patients with JMML (Flotho et al. 1999). Somatic recurrent MAP2K1 pathogenic variants have been implicated in several human cancers including melanoma (Nikolaev et al. 2011). Somatic HRAS pathogenic variants have been reported in patients with Costello syndrome (Gripp et al. 2006; Sol-Church et al. 2009).

Testing Strategy

For this NextGen test, the full coding regions plus ~10 bp 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 kit, followed by massively parallel sequencing of the captured DNA fragments. Additional Sanger sequencing is performed for any regions not captured or with insufficient number of sequence reads. All pathogenic, likely pathogenic, or variants of uncertain significance are confirmed by Sanger sequencing.

Indications for Test

Candidates for this NGS RASopathies panel are: patients with clinical features suggestive of Noonan syndrome, Cardio-Facio-Cutaneous Syndrome, or Noonan syndrome with multiple lentigines, also known as LEOPARD syndrome, patients with a clinical diagnosis of these syndromes that previously tested negative in a subset of genes included in this panel, patients with a clinical diagnosis of Costello syndrome and no mutations in HRAS, and patients with Noonan syndrome-like clinical features.

Genes

Official Gene Symbol OMIM ID
BRAF 164757
CBL 165360
HRAS 190020
KAT6B 605880
KRAS 190070
LZTR1 600574
MAP2K1 176872
MAP2K2 601263
NRAS 164790
PTPN11 176876
RAF1 164760
RIT1 609591
SHOC2 602775
SOS1 182530
SOS2 601247
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT

Related Tests

Name
BRAF-Related Disorders via BRAF Gene Sequencing with CNV Detection
CBL-Related Disorders via the CBL Gene
KAT6B-Related Disorders via the KAT6B Gene
KRAS-Related Disorders via the KRAS Gene
PTPN11-Related Disorders via PTPN11 Gene Sequencing with CNV Detection
RAF1-Related Disorders via RAF1 Gene Sequencing with CNV Detection
Autism Spectrum Disorders and Intellectual Disability (ASD-ID) Comprehensive Sequencing Panel with CNV Detection
Autism Spectrum Disorders Sequencing Panel with CNV Detection
Autoimmune Lymphoproliferative Syndrome/ALPS Sequencing Panel with CNV Detection
Cardio-Facio-Cutaneous Syndrome via MAP2K1 Gene Sequencing with CNV Detection
Cardio-Facio-Cutaneous Syndrome via MAP2K2 Gene Sequencing with CNV Detection
Comprehensive Cardiology Sequencing Panel with CNV Detection
Costello Syndrome via the HRAS Gene
Dilated Cardiomyopathy Sequencing Panel with CNV Detection
Fetal Concerns Sequencing Panel with CNV Detection
Hypertrophic Cardiomyopathy Sequencing Panel with CNV Detection
Neonatal Crisis Sequencing Panel with CNV Detection
Noonan Syndrome via RIT1 Gene Sequencing with CNV Detection
Noonan Syndrome via the LZTR1 Gene
Noonan Syndrome via the NRAS Gene
Noonan Syndrome via the SOS1 Gene
Noonan Syndrome via the SOS2 Gene
Noonan-Like Syndrome with Loose Anagen Hair via the SHOC2 Gene
Pan Cardiomyopathy Sequencing Panel with CNV Detection
Schwannomatosis Sequencing Panel with CNV Detection
Sudden Cardiac Arrest Sequencing Panel with CNV Detection

CONTACTS

Genetic Counselors
Geneticist
Citations
  • Allanson J.E. et al. 1985. American Journal of Medical Genetics. 21: 507-14. PubMed ID: 4025385
  • Aoki Y. et al. 2005. Nature Genetics. 37: 1038-40. PubMed ID: 16170316
  • Aoki Y. et al. 2013. American Journal of Human Genetics. 93: 173-80. PubMed ID: 23791108
  • Aoki Y. et al. 2016. Journal of Human Genetics. 61: 33-9. PubMed ID: 26446362
  • Chen J.L. et al. 2014. Molecular Cytogenetics. 7: 28. PubMed ID: 24739123
  • Chen P.C. et al. 2014. Proceedings of the National Academy of Sciences of the United States of America. 111: 11473-8. PubMed ID: 25049390
  • Cordeddu V. et al. 2015. Human Mutation. 36: 1080-7. PubMed ID: 26173643
  • Digilio M.C. et al. 2002. American Journal of Human Genetics. 71: 389-94. PubMed ID: 12058348
  • Edkins S. et al. 2006. Cancer Biology & Therapy. 5: 928-32. PubMed ID: 16969076
  • Flotho C. et al. 1999. Leukemia. 13: 32-7. PubMed ID: 10049057
  • Gelb B.D, Tartaglia M. 2010. LEOPARD Syndrome. In: Pagon RA, Adam MP, Ardinger HH, Bird TD, Dolan CR, Fong C-T, Smith RJ, and Stephens K, editors. GeneReviews(®), Seattle (WA): University of Washington, Seattle. PubMed ID: 20301557
  • Graham J.M. Jr. et al. 2009. American Journal of Medical Genetics. Part A. 149A: 2122-8. PubMed ID: 19760651
  • Gripp K.W. et al. 2006. American Journal of Medical Genetics. Part A. 140: 1-7. PubMed ID: 16329078
  • Gripp K.W. et al. 2006. American Journal of Medical Genetics. Part A. 140: 2163-9. PubMed ID: 16969868
  • Gripp K.W., Lin A.E. 2012. Costello Syndrome. In: Pagon RA, Adam MP, Ardinger HH, Bird TD, Dolan CR, Fong C-T, Smith RJ, and Stephens K, editors. GeneReviews(®), Seattle (WA): University of Washington, Seattle. PubMed ID: 20301680
  • Human Gene Mutation Database (Bio-base).
  • Jopling C. et al. 2007. Plos Genetics. 3: e225. PubMed ID: 18159945
  • Kraft M. et al. 2011. The Journal of Clinical Investigation. 121: 3479-91. PubMed ID: 21804188
  • Legius E. et al. 2002. Journal of Medical Genetics. 39: 571-4. PubMed ID: 12161596
  • Lin A.E. et al. 2002. American Journal of Medical Genetics. 111: 115-29. PubMed ID: 12210337
  • Lorenz S. et al. 2013. Human Molecular Genetics. 22: 1643-53. PubMed ID: 23335589
  • Martinelli S. et al. 2010. American Journal of Human Genetics. 87: 250-7. PubMed ID: 20619386
  • Narumi Y. et al. 2008. Journal of Human Genetics. 53: 834-41. PubMed ID: 18651097
  • Niemeyer C.M. et al. 2010. Nature Genetics. 42: 794-800. PubMed ID: 20694012
  • Niihori T. et al. 2006. Nature Genetics. 38: 294-6. PubMed ID: 16474404
  • Nikolaev S.I. et al. 2011. Nature Genetics. 44: 133-9. PubMed ID: 22197931
  • Pandit B. et al. 2007. Nature Genetics. 39: 1007-12. PubMed ID: 17603483
  • Rauen K.A. 2013. Annual Review of Genomics and Human Genetics. 14: 355-69. PubMed ID: 23875798
  • Rauen K.A. 2016. Cardiofaciocutaneous Syndrome. In: Pagon RA, Adam MP, Bird TD, Dolan CR, Fong C-T, Smith RJ, and Stephens K, editors. GeneReviews™, Seattle (WA): University of Washington, Seattle. PubMed ID: 20301365
  • Rauen K.A. et al. 2010. American Journal of Medical Genetics. Part A. 152A: 807-14. PubMed ID: 20358587
  • Reimann C. et al. 2006. Leukemia. 20: 1637-8. PubMed ID: 16826224
  • Reynolds J.F. et al. 1986. American Journal of Medical Genetics. 25: 413-27. PubMed ID: 3789005
  • Rodriguez-Viciana P. et al. 2006. Science. 311: 1287-90. PubMed ID: 16439621
  • Romano A.A. et al. 2010. Pediatrics. 126: 746-59. PubMed ID: 20876176
  • Sarkozy A. 2004. Journal of Medical Genetics. 41: e68-e68. PubMed ID: 15121796
  • Sarkozy A. et al. 2009. Human Mutation. 30: 695-702. PubMed ID: 19206169
  • Schubbert S. et al. 2006. Nature Genetics. 38: 331-6. PubMed ID: 16474405
  • Shchelochkov O.A. et al. 2008. American Journal of Medical Genetics. Part A. 146A: 1042-8. PubMed ID: 18348260
  • Smpokou P. et al. 2012. American Journal of Medical Genetics. Part A. 158A: 3106-11. PubMed ID: 23165751
  • Sol-Church K. et al. 2009. American Journal of Medical Genetics. Part A. 149A: 315-21. PubMed ID: 19206176
  • Tartaglia M. et al. 2003. Nature Genetics. 34: 148-50. PubMed ID: 12717436
  • Tartaglia M. et al. 2010. Molecular Syndromology. 1: 2-26. PubMed ID: 20648242
  • Tidyman W.E., Rauen K.A. 2009. Current Opinion in Genetics & Development. 19: 230-6. PubMed ID: 19467855
  • Tumurkhuu M. et al. 2010. Pediatrics International. 52: 557-62. PubMed ID: 20030748
  • van Eeghen A.M. et al. 1999. American Journal of Medical Genetics. 82: 187-93. PubMed ID: 9934987
  • Wright E.M., Kerr B. 2010. Archives of Disease in Childhood. 95: 724-30. PubMed ID: 20371595
  • Yamamoto G.L. et al. 2015. Journal of Medical Genetics. 52: 413-21. PubMed ID: 25795793
Order Kits
TEST METHODS

NextGen Sequencing using PG-Select Capture Probes

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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