CBL-Related Disorders via CBL Gene Sequencing with CNV Detection
- Summary and Pricing
- Clinical Features and Genetics
Sequencing and CNV
|Test Code||Test Copy Genes||Price||CPT Code Copy CPT Codes|
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.
For ordering sequencing of targeted known variants, please proceed to our Targeted Variants landing page.
The great majority of tests are completed within 20 days.
Defects in the CBL gene were found in about 1% of patients with clinical features within the Rasopathy phenotypic spectrum, and without defects in the remaining genes that have been associated with this group of disorders (Martinelli et al. 2015). Heterozygous germline pathogenic variants in CBL were found in 9% patients with a clincal diagnosis of JMML (Strullu et al. 2013).
Noonan syndrome (NS) is characterized by dysmorphic facial features, growth retardation, 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. 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). Pathogenic variants in the CBL gene have been reported both in patients who fulfilled the clinical criteria for the diagnosis of Noonan syndrome and patients with clinical features suggestive of Noonan syndrome, but did not fulfill the clinical criteria for a definitive diagnosis (Martinelli et al. 2010; Lepri et al. 2014). Clinical features identified in these patients include developmental delay; short stature; facial dysmorphism, hypertelorism, ptosis and strabismus; low-set and posteriorly rotated ears, widely spaced nipples; chest abnormalities; dark skin and café-au-lait spots; and cardiologic findings in the form of enlarged left atrium, dysrhythmias and dysplastic leaflets. MRI findings include delayed myelinization without structural abnormalities; abnormal corpus callosum and hypoplasia of the cerebellar vermis. Hypotonia, severe feeding problems, pleural effusions, hydrops and chylothorax were reported in newborn and infant patients (Bulow et al. 2015, Martinelli et al. 2015).
Noonan Syndrome is caused by defects in various genes within the RAS/MAPK pathway. At least 70% of patients with a diagnosis of Noonan syndrome have heterozygous germline pathogenic variants in thirteen genes (Romano et al. 2010; Tartaglia et al. 2010; Aoki et al. 2013). 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 (CBL, KAT6B, and RIT1) encode proteins that are involved in its regulation (Rauen 2013; Martinelli et al. 2010; Niemeyer et al. 2010; Kraft et al. 2011, Aoki et al. 2013). Although most causative Noonan syndrome variants occur de novo, familial cases have been reported. In these families, Noonan syndrome is inherited in an autosomal dominant manner with complete penetrance and variable expressivity (Romano et al. 2010). Defects in the CBL gene appear to be a rare cause of Noonan syndrome. Pathogenic variants are found in about 1% of patients with clinical features within the Rasopathy phenotypic spectrum, and without defects in the remaining genes that have been associated with this group of disorders. Eleven variants have been reported to date. The majority are missense; although splicing, nonsense and small deletions are reported (Martinelli et al. 2015, Human Gene Mutation Database). Germline pathogenic variants in CBL have also been reported in children with juvenile myelomonocytic leukemia (JMML) with or without congenital anomalies that overlap with those of Noonan syndrome (Nieemeyer et al. 2010; Martinelli et al. 2015). The CBL gene encodes an E3 ubiquitin ligase that negatively regulates intracellular signaling through degradation of activated tyrosine kinase receptors. It also positively regulates signal transduction through its role as an adaptor protein (Kales et al. 2010).
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 test provides full coverage of all coding exons of the CBL gene, plus ~10 bases of flanking noncoding DNA. We define full coverage as >20X NGS reads or Sanger sequencing.
Indications for Test
Patients with clinical features suggestive of Noonan syndrome and no pathogenic variants in the PTPN11, SOS1, RAF1, KRAS, HRAS, SHOC2, BRAF, NRAS, MAP2K1, MAP2K1, KAT6B, or RIT1 genes (Martinelli et al. 2015). Children with juvenile myelomonocytic leukemia and congenital anomalies that overlap with those of Noonan syndrome (Nieemeyer et al. 2010).
|Official Gene Symbol||OMIM ID|
|Noonan Syndrome-Like Disorder With Or Without Juvenile Myelomonocytic Leukemia||AD||613563|
- Genetic Counselor Team - email@example.com
- Brett Deml, PhD - firstname.lastname@example.org
- Allanson JE, Hall JG, Hughes HE, Preus M, Witt RD. 1985. Noonan syndrome: the changing phenotype. Am. J. Med. Genet. 21: 507-514. PubMed ID: 4025385
- Aoki Y, Niihori T, Banjo T, Okamoto N, Mizuno S, Kurosawa K, Ogata T, Takada F, Yano M, Ando T, Hoshika T, Barnett C, Ohashi H, Kawame H, Hasegawa T, Okutani T, Nagashima T, Hasegawa S, Funayama R, Nagashima T, Nakayama K, Inoue S, Watanabe Y, Ogura T, Matsubara Y. 2013. Gain-of-function mutations in RIT1 cause Noonan syndrome, a RAS/MAPK pathway syndrome. Am. J. Hum. Genet. 93:173-180.
PubMed ID: 23791108
- Bülow L, Lissewski C, Bressel R, Rauch A, Stark Z, Zenker M, Bartsch O. 2015. Hydrops, fetal pleural effusions and chylothorax in three patients with CBL mutations. Am. J. Med. Genet. A 167A: 394–399. PubMed ID: 25358541
- Human Gene Mutation Database (Bio-base).
- Kales SC, Ryan PE, Nau MM, Lipkowitz S. 2010. Cbl and human myeloid neoplasms: the Cbl oncogene comes of age. Cancer Res. 70: 4789–4794. PubMed ID: 20501843
- Kraft M, Cirstea IC, Voss AK, Thomas T, Goehring I, Sheikh BN, Gordon L, Scott H, Smyth GK, Ahmadian MR, Trautmann U, Zenker M, Tartaglia M, Ekici A, Reis A, Dörr HG, Rauch A, Thiel CT. 2011. Disruption of the histone acetyltransferase MYST4 leads to a Noonan syndrome–like phenotype and hyperactivated MAPK signaling in humans and mice. Journal of Clinical Investigation 121: 3479–3491. PubMed ID: 21804188
- Lepri FR, Scavelli R, Digilio MC, Gnazzo M, Grotta S, Dentici ML, Pisaneschi E, Sirleto P, Capolino R, Baban A, Russo S, Franchin T, Angioni A, Dallapiccola B. 2014. Diagnosis of Noonan syndrome and related disorders using target next generation sequencing. BMC Med. Genet. 15: 14. PubMed ID: 24451042
- Martinelli S, Luca A De, Stellacci E, Rossi C, Checquolo S, Lepri F, Caputo V, Silvano M, Buscherini F, Consoli F, Ferrara G, Digilio MC, Cavaliere ML, van Hagen JM, Zampino G, van der Burgt I, Ferrero GB, Mazzanti L, Screpanti I, Yntema HG, Nillesen WM, Savarirayan R, Zenker M, Dallapiccola B, Gelb BD, Tartaglia M. 2010. Heterozygous Germline Mutations in the CBL Tumor-Suppressor Gene Cause a Noonan Syndrome-like Phenotype. Am J Hum Genet 87: 250–257. PubMed ID: 20619386
- Martinelli S, Stellacci E, Pannone L, D’Agostino D, Consoli F, Lissewski C, Silvano M, Cencelli G, Lepri F, Maitz S, Pauli S, Rauch A, Zampino G, Selicorni A, Melançon S, Digilio MC, Gelb BD, De Luca A, Dallapiccola B, Zenker M, Tartaglia M. 2015. Molecular Diversity and Associated Phenotypic Spectrum of Germline CBL Mutations. Human Mutation n/a–n/a. PubMed ID: 25952305
- Niemeyer CM, Kang MW, Shin DH, Furlan I, Erlacher M, Bunin NJ, Bunda S, Finklestein JZ, Sakamoto KM, Gorr TA, Mehta P, Schmid I, Kropshofer G, Corbacioglu S, Lang PJ, Klein C, Schlegel PG, Heinzmann A, Schneider M, Starý J, van den Heuvel-Eibrink MM, Hasle H, Locatelli F, Sakai D, Archambeault S, Chen L, Russell RC, Sybingco SS, Ohh M, Braun BS, Flotho C, Loh ML. 2010. Germline CBL mutations cause developmental abnormalities and predispose to juvenile myelomonocytic leukemia. Nature Genetics 42: 794–800. PubMed ID: 20694012
- Rauen KA. 2013. The RASopathies. Annu Rev Genomics Hum Genet 14: 355–369. PubMed ID: 23875798
- Romano AA, Allanson JE, Dahlgren J, Gelb BD, Hall B, Pierpont ME, Roberts AE, Robinson W, Takemoto CM, Noonan JA. 2010. Noonan syndrome: clinical features, diagnosis, and management guidelines. Pediatrics 126: 746-759. PubMed ID: 20876176
- Smpokou P, Tworog-Dube E, Kucherlapati RS, Roberts AE. 2012. Medical complications, clinical findings, and educational outcomes in adults with Noonan syndrome. American Journal of Medical Genetics Part A 158A: 3106–3111. PubMed ID: 23165751
- Strullu M, Caye A, Cassinat B, Fenneteau O, Touzot F, Blauwblomme T, Rodriguez R, Latour S, Petit A, Barlogis V, Galambrun C, Leblanc T, Baruchel A, Chomienne C, Cavé H. 2013. In hematopoietic cells with a germline mutation of CBL, loss of heterozygosity is not a signature of juvenile myelo-monocytic leukemia. Leukemia 27: 2404–2407. PubMed ID: 23823657
- Tartaglia M, Zampino G, Gelb BD. 2010. Noonan Syndrome: Clinical Aspects and Molecular Pathogenesis. Mol Syndromol 1: 2–26. PubMed ID: 20648242
Sequencing and CNV Detection via NextGen Sequencing using PG-Select Capture Probes
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
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.
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.
myPrevent - Online Ordering
- The test can be added to your online orders in the Summary and Pricing section.
- Once the test has been added log in to myPrevent to fill out an online requisition form.
- A completed requisition form must accompany all specimens.
- Billing information along with specimen and shipping instructions are within the requisition form.
- All testing must be ordered by a qualified healthcare provider.
(Delivery accepted Monday - Saturday)
- Collect 3 ml -5 ml (5 ml preferred) of whole blood in EDTA (purple top tube) or ACD (yellow top tube). For Test #500-DNA Banking only, collect 10 ml -20 ml of whole blood.
- For small babies, we require a minimum of 1 ml of blood.
- Only one blood tube is required for multiple tests.
- Ship blood tubes at room temperature in an insulated container. Do not freeze blood.
- During hot weather, include a frozen ice pack in the shipping container. Place a paper towel or other thin material between the ice pack and the blood tube.
- In cold weather, include an unfrozen ice pack in the shipping container as insulation.
- At room temperature, blood specimen is stable for up to 48 hours.
- If refrigerated, blood specimen is stable for up to one week.
- Label the tube with the patient name, date of birth and/or ID number.
(Delivery accepted Monday - Saturday)
- Send in screw cap tube at least 5 µg -10 µg of purified DNA at a concentration of at least 20 µg/ml for NGS and Sanger tests and at least 5 µg of purified DNA at a concentration of at least 100 µg/ml for gene-centric aCGH, MLPA, and CMA tests, minimum 2 µg for limited specimens.
- For requests requiring more than one test, send an additional 5 µg DNA per test ordered when possible.
- DNA may be shipped at room temperature.
- Label the tube with the composition of the solute, DNA concentration as well as the patient’s name, date of birth, and/or ID number.
- We only accept genomic DNA for testing. We do NOT accept products of whole genome amplification reactions or other amplification reactions.
(Delivery preferred Monday - Thursday)
- PreventionGenetics should be notified in advance of arrival of a cell culture.
- Culture and send at least two T25 flasks of confluent cells.
- Some panels may require additional flasks (dependent on size of genes, amount of Sanger sequencing required, etc.). Multiple test requests may also require additional flasks. Please contact us for details.
- Send specimens in insulated, shatterproof container overnight.
- Cell cultures may be shipped at room temperature or refrigerated.
- Label the flasks with the patient name, date of birth, and/or ID number.
- We strongly recommend maintaining a local back-up culture. We do not culture cells.