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Noonan Spectrum Disorders/Rasopathies Sequencing Panel with CNV Detection

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

Sequencing and CNV

Test Code Test Copy GenesCPT Code Copy CPT Codes
1309 A2ML1 81479,81479 Add to Order
BRAF 81406,81479
CBL 81479,81479
HRAS 81404,81479
KAT6B 81479,81479
KRAS 81405,81479
LZTR1 81479,81479
MAP2K1 81406,81479
MAP2K2 81406,81479
MAP3K8 81479,81479
NF1 81408,81479
NRAS 81479,81479
PTPN11 81406,81479
RAF1 81406,81479
RASA2 81479,81479
RIT1 81479,81479
RRAS 81479,81479
SHOC2 81405,81479
SOS1 81406,81479
SOS2 81479,81479
SPRY1 81479,81479
Full Panel Price* $680
Test Code Test Copy Genes Total Price CPT Codes Copy CPT Codes
1309 Genes x (21) $680 81404, 81405(x2), 81406(x6), 81408, 81479(x32) Add to Order

New York State Approved Test

Pricing Comments

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 testing single genes in this panel 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.

This test is also offered via our exome backbone with CNV detection (click here). The exome-based test may be higher priced, but permits reflex to the entire exome or to any other set of clinically relevant genes.

Deletion and duplication testing for NF1 is performed using NGS, but CNVs detected in this gene are usually confirmed via multiplex ligation-dependent probe amplification (MLPA). Please see limitations for CNV detection via NGS.

Targeted Testing

For ordering sequencing of 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. PubMed ID: 26446362)

Gene Sensitivity (%)
PTPN11 50
SOS1 11
RAF1 5
KRAS 1.5
SHOC2 2
BRAF 0.8
NRAS 0.2
RIT1 5
CBL Rare
KAT6B Rare
LZTR1 2.5
SOS2 1
NF1 Rare
A2ML1 3 cases
RASA2 3 cases
RRAS 2 cases
MAP3K8 One case
SPRY1 One case

Clinical Sensitivity for CFC Syndrome (Rauen et al. PubMed ID: 20301365)

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 2015. PubMed ID: 20301557)

Clinical Sensitivity Costello Syndrome: ~ 80-90% (HRAS) (Gripp and Lin 2012. PubMed ID: 20301680)

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. PubMed ID: 18348260; Graham et al. 2009. PubMed ID: 19760651). 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 1,000 to 1 in 2,500 births worldwide (Allanson. 1987. PubMed ID: 3543368; Romano et al. 2010. PubMed ID: 20876176; Smpokou et al. 2012. PubMed ID: 23165751; Cao et al. 2017. PubMed ID: 28643916).

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. PubMed ID: 3789005).

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. PubMed ID: 12161596; Sarkozy et al. 2004. PubMed ID: 15121796).

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. PubMed ID: 9934987; Lin et al. 2002. PubMed ID: 12210337). 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. PubMed ID: 16969868).

Genetics

Noonan spectrum disorders are caused by dysregulation of the RAS/mitogen-activated protein kinase (Ras/MAPK) signaling pathway (Tidyman and Rauen. 2009. PubMed ID: 19467855; Wright and Kerr. 2010. PubMed ID: 20371595; Allanson and Roberts. 2016. PubMedID: 20301303).

Heterozygous germline pathogenic variants in the following genes have been reported in patients with Noonan spectrum disorders: PTPN11, SOS1, SOS2, RAF1, KRAS, HRAS, SHOC2, BRAF, NRAS, MAP2K1, MAP2K1, MAP3K8, A2ML1, RASA2, RRAS, SPRY1, NF1, CBL, KAT6B, RIT1, and LZTR1 (Rauen. 2013. PubMed ID: 23875798; Martinelli et al. 2010. PubMed ID: 20619386; Niemeyer et al. 2010. PubMed ID: 20694012; Kraft et al. 2011. PubMed ID: 21804188; Aoki et al. 2013. PubMed ID: 23791108; Vissers et al. 2015. PubMed ID: 24939586).

Noonan and Noonan-like syndromes are caused by pathogenic variants in PTPN11, SOS1, RAF1, KRAS, SHOC2, BRAF, NRAS, CBL, KAT6B, RIT1, SOS2, LZTR1, A2ML1, MAP3K8, RASA2, RRAS, SPRY1, and NF1. Over 330 pathogenic variants have been reported in these genes. They account for up to 80% of all cases genotyped (Romano et al. 2010. PubMed ID: 20876176; Tartaglia et al. 2010. PubMed ID: 20648242; Aoki et al. 2013. PubMed ID: 23791108; Chen. 2014. PubMed ID: 25049390; Cordeddu. 2015. PubMed ID: 26173643; Yamamoto et al. 2015. PubMed ID: 25795793). 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. PubMed ID: 21804188). 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. PubMed ID: 18348260; Graham et al. 2009. PubMed ID: 19760651), while the third includes PTPN11 and TBX3 only (Chen et al. 2014. PubMed ID: 24739123). These three duplications occurred de novo and represent an uncommon cause of NS (Tartaglia et al. 2010. PubMed ID: 20648242).

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. PubMed ID: 20876176).

CFCS is caused by defects in the follwoing RAS/MAPK genes: BRAF, HRAS, KRAS, MAP2K1, MAP2K2, and NRAS. To date, over 100 heterozygous pathogenic variants have been detected (Rodriguez-Viciana et al. 2006. PubMed ID: 16439621; Niihori et al. 2006. PubMed ID: 16474404; Schubbert et al. 2006. PubMed ID: 16474405; Tumurkhuu et al. 2010. PubMed ID: 20030748; Narumi et al. 2008. PubMed ID: 18651097). The vast majority of variants have been reported in the HRAS gene. They affect codons Gly12 and Gly13. 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. More recently, four different indels that are predicted to result in substitutions of codon Gly12 have been identified in patients with Costello syndrome. 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. PubMed ID: 20358587). 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. 2006. PubMed ID: 16733669; Pandit et al. 2007. PubMed ID: 17603483). 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. PubMed ID: 18159945). 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. 2006. PubMed ID: 16987887).

CS syndrome is caused by pathogenic variants in the HRAS gene (Aoki et al. 2005. PubMed ID: 16170316). To date, 20 heterozygous activating germline variants have been reported in patients with clinical features of CS. Most of these are missense. A few indels, which are expected to result in amino acid substitutions; and a 21-bp duplication that is predicted to result in an inframe insertion of seven residues was reported in a patient with mild clinical features suggestive of CS (Lorenz et al. 2013. PubMed ID: 23335589). The majority of variants affect codons G12 and G13. However, variants affecting other codons have been reported. 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) involves somatic PTPN11 pathogenic variants in about 34% of all cases genotyped (Tartaglia et al. 2003. PubMed ID: 12717436). Somatic RAF1 pathogenic variants have been implicated in several human cancers (Pandit et al. 2007. PubMed ID: 17603483; Sarkozy et al. 2009. PubMed ID: 19206169). Somatic KRAS pathogenic variants have been implicated in several human cancers, including JMML (Reimann et al. 2006. PubMed ID: 16826224) and colon cancer (Edkins et al. 2006. PubMed ID: 16969076). Somatic NRAS pathogenic variants were detected in patients with JMML (Flotho et al. 1999. PubMed ID: 10049057). Somatic recurrent MAP2K1 pathogenic variants have been implicated in several human cancers including melanoma (Nikolaev et al. 2011. PubMed ID: 22197931). Somatic HRAS pathogenic variants have been reported in patients with Costello syndrome (Gripp et al. 2006. PubMed ID: 16329078; Sol-Church et al. 2009. PubMed ID: 19206176).

Testing Strategy

For this Next Generation Sequencing (NGS) test, 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 regions not captured or with insufficient number of sequence reads.

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.

Copy number variants (CNVs) are also detected from NGS data. We utilize a CNV calling algorithm that compares mean read depth and distribution for each target in the test sample against multiple matched controls. Neighboring target read depth and distribution and zygosity of any variants within each target region are used to reinforce CNV calls. All CNVs are confirmed using another technology such as aCGH, MLPA, or PCR before they are reported.

This panel provides 100% coverage of all coding exons of the genes listed, plus ~10 bases of flanking noncoding DNA. We define coverage as ≥20X NGS reads or Sanger sequencing.

Deletion and duplication testing for NF1 is performed using NGS, but CNVs detected in this gene are usually confirmed via multiplex ligation-dependent probe amplification (MLPA). Please see limitations for CNV detection via NGS.

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
A2ML1 610627
BRAF 164757
CBL 165360
HRAS 190020
KAT6B 605880
KRAS 190070
LZTR1 600574
MAP2K1 176872
MAP2K2 601263
MAP3K8 191195
NF1 613113
NRAS 164790
PTPN11 176876
RAF1 164760
RASA2 601589
RIT1 609591
RRAS 165090
SHOC2 602775
SOS1 182530
SOS2 601247
SPRY1 602465
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT

Diseases

Name Inheritance OMIM ID
Cardio-Facio-Cutaneous Syndrome AD 115150
Cardiofaciocutaneous syndrome 2 AD 615278
Cardiofaciocutaneous syndrome 3 AD 615279
Cardiofaciocutaneous syndrome 4 AD 615280
Costello Syndrome AD 218040
Genitopatellar Syndrome AD 606170
LEOPARD Syndrome AD 151100
LEOPARD Syndrome 2 AD 611554
LEOPARD Syndrome 3 AD 613707
Neurofibromatosis, Type 1 AD 162200
Noonan Syndrome 1 AD 163950
Noonan Syndrome 10 AD 616564
Noonan Syndrome 3 AD 609942
Noonan Syndrome 4 AD 610733
Noonan Syndrome 5 AD 611553
Noonan Syndrome 6 AD 613224
Noonan Syndrome 7 AD 613706
Noonan Syndrome 8 AD 615355
Noonan Syndrome 9 AD 616559
Noonan Syndrome-Like Disorder With Or Without Juvenile Myelomonocytic Leukemia AD 613563
Noonan-Like Syndrome With Loose Anagen Hair AD 607721
Young Simpson Syndrome AD 603736

Related Test

Name
PGxome®

CONTACTS

Genetic Counselors
Geneticist
Citations
  • Allanson and Roberts. 2016. PubMed ID: 20301303
  • Allanson. 1987. PubMed ID: 3543368
  • Aoki et al. 2005. PubMed ID: 16170316
  • Aoki et al. 2013. PubMed ID: 23791108
  • Aoki et al. 2016. PubMed ID: 26446362
  • Cao et al. 2017. PubMed ID: 28643916
  • Chen et al. 2014. PubMed ID: 24739123
  • Chen et al. 2014. PubMed ID: 25049390
  • Cordeddu et al. 2015. PubMed ID: 26173643
  • Digilio et al. 2006. PubMed ID: 16733669
  • Edkins et al. 2006. PubMed ID: 16969076
  • Flotho et al. 1999. PubMed ID: 10049057
  • Gelb and Tartaglia M. 2015. PubMed ID: 20301557
  • Gelb and Tartaglia. 2006. PubMed ID: 16987887
  • Graham et al. 2009. PubMed ID: 19760651
  • Gripp and Lin. 2012. PubMed ID: 20301680
  • Gripp et al. 2006. PubMed ID: 16329078
  • Gripp et al. 2006. PubMed ID: 16969868
  • Human Gene Mutation Database (Bio-base).
  • Jopling et al. 2007. PubMed ID: 18159945
  • Kraft et al. 2011. PubMed ID: 21804188
  • Legius et al. 2002. PubMed ID: 12161596
  • Lin et al. 2002. PubMed ID: 12210337
  • Lorenz et al. 2013. PubMed ID: 23335589
  • Martinelli et al. 2010. PubMed ID: 20619386
  • Narumi et al. 2008. PubMed ID: 18651097
  • Niemeyer et al. 2010. PubMed ID: 20694012
  • Niihori et al. 2006. PubMed ID: 16474404
  • Nikolaev et al. 2011. PubMed ID: 22197931
  • Pandit et al. 2007. PubMed ID: 17603483
  • Rauen et al. 2010. PubMed ID: 20358587
  • Rauen. 2013. PubMed ID: 23875798
  • Rauen. 2016. PubMed ID: 20301365
  • Reimann et al. 2006. PubMed ID: 16826224
  • Reynolds et al. 1986. PubMed ID: 3789005
  • Rodriguez-Viciana et al. 2006. PubMed ID: 16439621
  • Romano et al. 2010. PubMed ID: 20876176
  • Sarkozy et al. 2004. PubMed ID: 15121796
  • Sarkozy et al. 2009. PubMed ID: 19206169
  • Schubbert et al. 2006. PubMed ID: 16474405
  • Shchelochkov et al. 2008. PubMed ID: 18348260
  • Smpokou et al. 2012. PubMed ID: 23165751
  • Sol-Church et al. 2009. PubMed ID: 19206176
  • Tartaglia et al. 2003. PubMed ID: 12717436
  • Tartaglia et al. 2010. PubMed ID: 20648242
  • Tidyman and Rauen. 2009. PubMed ID: 19467855
  • Tumurkhuu et al. 2010. PubMed ID: 20030748
  • van Eeghen et al. 1999. PubMed ID: 9934987
  • Vissers et al. 2015. PubMed ID: 24939586
  • Wright and Kerr. 2010. PubMed ID: 20371595
  • Yamamoto et al. 2015. PubMed ID: 25795793
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TEST METHODS

Sequencing and CNV Detection via NextGen Sequencing using PG-Select Capture Probes

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 ~10 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.

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 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.

Deletion and Duplication Testing via NGS

Copy number variants (CNVs) such as deletions and duplications are detected from next generation sequencing data. We utilize a CNV calling algorithm that compares mean read depth and distribution for each target in the test sample against multiple matched controls. Neighboring target read depth and distribution, and zygosity of any variants within each target region are used to reinforce CNV calls. All CNVs are confirmed using another technology such as PCR, aCGH or MLPA before they are reported.
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 and Duplication Testing via NGS
 
In general, sensitivity for single, double, or triple exon CNVs is ~80% and for CNVs of four exon size or larger is close to 100%, but may vary from gene-to-gene based on exon size, depth of coverage, and characteristics of the region.
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 ~10 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 and Duplication Testing via NGS
 
This CNV calling algorithm used in this test detects most deletions and duplications; however aberrations in a small percentage of regions may not be accurately detected due to sequence paralogy (e.g. pseudogenes, segmental duplications), sequence properties, deletion/duplication size (e.g. single vs. two or more exons), and inadequate coverage. 
 
Balanced translocations or inversions within a targeted gene, or large unbalanced translocations or inversions that extend beyond the genomic location of a targeted gene are not detected.
 
In nearly all cases, our ability to determine the exact copy number change within a targeted gene is limited. In particular, when we find copy excess within a targeted gene, we cannot be certain that the region is duplicated, triplicated etc. In many duplication cases, we are unable to determine the genomic location or the orientation of the duplicated segment with respect to the gene. In particular, we often cannot determine if the duplicated segment is inserted in tandem within the gene or inserted elsewhere in the genome. Similarly, we may not be able to determine the orientation of the duplicated segment (direct or inverted), and whether it will disrupt the open reading frame of the given gene.
 
Our ability to detect CNVs due to somatic mosaicism is limited.
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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