Ion Torrent™ Class-Based Variants
Ion Torrent™ Class-Based Variants are a set of logic-based rules that the Oncomine™ Variant Annotator plugin uses to computationally identify and annotate novel mutations in important genomic variant classes. The annotation logic is based on scientific literature evidence. Details about the logic follow.
Background: Epidermal growth factor receptor (EGFR) is a transmembrane receptor protein made up of 1210 amino acids and located on chromosome 7p11.2. EGFR has 31 exons— exons 2-4 and 8-12 encode the ligand binding domains, exons 5-7 and 13-16 encode the cysteine rich domain, exons 18–24 encode the tyrosine kinase domain (TKD), and exons 25-28 encode the autophosphorylation region [PMID: 17318210]. Mutations in EGFR often target the TKD which make up the ATP-binding pocket of the enzyme [PMID: 17318210]. Exon 19 deletions are indels that range between 15-18 bp in length and usually occur within codons 746-756 [PMID: 23768755, PMID: 22190593, PMID: 26933124, PMID: 24163741]. In non-small cell lung cancer (NSCLC), EGFR exon 19 deletions, located within codons 746-750, represent 45-50% of somatic mutations [PMID: 26933124, PMID: 24163741]. Exon 19 deletions result in constitutive activation of the receptor tyrosine kinase and hyperactivation of downstream signaling pathways [PMID: 29455648].
CBV Criteria: The boundaries defined to capture EGFR exon 19 deletions as a class-based variant start at codon 744 and end at codon 761, which encompass common and rare indels [PMID: 30473385].
Background: Epidermal growth factor receptor (EGFR) is a transmembrane receptor protein made up of 1210 amino acids located on chromosome 7p11.2. EGFR has 31 exons— exons 2-4 and 8-12 encode the ligand binding domains, exons 5-7 and 13-16 encode the cysteine rich domain, exons 18–24 encode the tyrosine kinase domain (TKD), and exons 25-28 encode the autophosphorylation region [PMID: 17318210]. Mutations in EGFR often target the TKD which make up the ATP-binding pocket of the enzyme [PMID: 17318210]. In-frame insertions within exon 20 of EGFR are the third most common type of mutation found in NSCLC, representing 4-12% of all EGFR mutations in NSCLC [PMID: 31208370]. EGFR exon 20 encompasses codons 762 to 823. Exon 20 insertions commonly involve codons 762 to 774 wherein codons 762-766 make up the c-helix and codons 767-774 make up the activation loop [PMID: 31208370, PMID: 30854234]. EGFR exon 20 insertions do not alter the receptor binding affinity; however, it is suggested that the location of exon 20 insertions stabilizes its active confirmation [PMID: 30854234, PMID: 27843613].
CBV Criteria: The boundaries defined to capture EGFR exon 20 insertions as a class-based variant start at codon 762 and end at codon 775. Additionally, known EGFR exon 20 insertions p.A763_Y764insFQEA and p.A763_Y764insLQEA, represented as c.2284–6_2284–5insTCCAGGAAGCCT and c.2284–9_2284–8insCCCTCCAGGAAG due to left alignment into the intronic region, are included in this rule.
Background: Erb-b2 receptor tyrosine kinase 2 (ERBB2) is a transmembrane glycoprotein located on chromosome 17q12 [PMID: 25276427]. ERBB2 (also known as HER2) has 27 exons— exons 2-4 and 9-12 encode the extracellular receptor L domains, exons 5-8 encode the furin like domain, exons 13-16 encode the growth factor receptor domain IV, exon 17 encodes the transmembrane domain, and exons 18–24 encode the tyrosine kinase domain (TKD) [PMID: 22761469, PMID: 29420467]. In lung cancer, the most recurrent ERBB2 activating mutations include in-frame exon 20 insertions [PMID: 30425522]. ERBB2 exon 20 involves codons 770 to 831 and majority of exon 20 insertions occur between codons 775 and 781 within the kinase domain [PMID: 29686424, PMID:22761469]. Insertions at the C-terminal end of ERBB2 exon 20 induce a change in conformation of the α-C helix leading to a constitutively active formation which affects the drug-binding pocket [PMID: 29686424].
CBV Criteria: The boundaries defined to capture ERBB2 exon 20 insertions as a class-based variant start at codon 770 and end at codon 783.
Background: Janus kinase 2 (JAK2) is a non-receptor membrane associated protein tyrosine kinase (PTK) made up of 1132 amino acids and located on chromosome 9p24. JAK2 belongs to the Janus kinase (JAK) family and has 25 exons—exons 3–7 encode the FERM domain, exons 9–11 encode the SH2-pseudokinase domain, exons 12–19 encode the kinase-like or pseudokinase domain (JH2), and exons 19–25 encode the C-terminal tyrosine kinase domain (JH1) [PMID: 23592968; PMID: 17639043; PMID: 29379470]. In addition, codons 523–544 in exon 12 encode the SH2-pseudokinase linker region and codons 817–839 in exon 19 encode the linker between the JH1 and JH2 protein kinase regions [PMID: 23592968, PMID: 17639043]. Mutations in JAK2 often target exon 12 including the region adjacent to the start of the pseudo‐kinase domain [PMID: 21674578]. JAK2 exon 12 mutations (missense and in-frame deletions) are prevalent in myeloproliferative neoplasms including polycythemia vera [PMID: 21674578].
CBV Criteria: The boundaries defined to capture JAK2 exon 12 in-frame insertions and deletions (indels) as a class-based variant start at codon 536 and end at codon 547. These boundaries capture the most common JAK2 exon 12 indels, including N542_E543del, E543_D544del, F537_K539delinsL and R541_E543delinsK mutations.
KIT exon 11 insertion and KIT exon 11 deletion
Background: KIT, also known as CD117, is a proto-oncogene receptor tyrosine kinase made up of 976 amino acids located on chromosome 4q12 [PMID: 16689459]. KIT is a member of the PDGFR type III receptor tyrosine kinase family [PMID: 26579483; PMID: 26309392]. KIT has 21 exons— exons 1–9 encode the extracellular ligand-binding region containing five Ig-like domains, exon 10 encodes the transmembrane (TM) domain, and exon 11 encodes a cytoplasmic juxtamembrane region. The intracellular region of KIT is encoded by exons 12–21, wherein, exons 12–14 encode the tyrosine kinase domain 1, exons 15–16 encode the kinase insert (KI) region, and exons 17–19 encode the tyrosine kinase domain 2 [PMID: 24449920, PMID: 27536065, PMID: 31378162, PMID: 15342366, PMID: 17555444]. Recurrent somatic KIT alterations are observed in both solid and hematological cancers and include activating mutations such as single nucleotide variants (SNVs), small duplications, and complex in-frame insertions or deletions (indels). Mutations in KIT exons 8, 9, 11, and 17 disrupt the auto-inhibitory mechanisms and lead to constitutive activity [PMID: 27536065]. While mutations in exons 8 and 17 are common in myeloid cancers, 60–70% of somatic mutations (including SNVs and indels) in gastrointestinal stromal tumor (GIST) occur in exon 11, which encodes the juxtamembrane domain [PMID: 27536065, PMID: 22588877, PMID: 23678293; NCCN-GIST].
CBV Criteria: The boundaries defined to capture KIT exon 11 insertions and deletions as a class-based variant start at codon 550 and end at codon 580.
Background: The MET proto-oncogene is a receptor tyrosine kinase made up of 1390 amino acids and is located on chromosome 7q31 [PMID: 15735036]. MET has 21 exons containing three main structural domains—an extracellular Sema domain in exon 2, a juxtamembrane domain in exon 14, and a tyrosine kinase domain in exons 15-21 [PMID: 28376232, PMID: 9380410]. Splice site mutations flanking exon 14 are observed in 4% of non-small cell lung cancer (NSCLC). These mutations include canonical splice site mutations affecting exon 14 and deletions that extend into the splicing motifs within intron 13 [PMID: 25971938, PMID: 27343443]. Such mutations disrupt splicing leading to the formation of an alternative transcript that joins exon 13 directly to exon 15 and skips exon 14 entirely. The MET exon 14 skipping transcript lacks the juxtamembrane domain that contains the recognition motif for ubiquitin-dependent proteolysis and thus leads to a marked increase in steady-state level of the MET protein [PMID: 28164087].
CBV Criteria: The boundaries defined to capture MET exon 14 skipping as a class-based variant can be summarized into four individual rules: (a) has transcript = NM_001127500.3, exon = 14, and location = splicesite_3 (at c.2942) or splicesite_5 (at c.3082), (b) is an intronic deletion ≥ 4 bp impacting 30 nucleotides preceding exon 14 (c) is a missense variant at c.3082 [PMID: 27343443, PMID: 26729443, PMID: 25971938, PMID: 25898962, PMID: 25898965, PMID: 31472177], (d) is one of the following variants with confirmed skipping defined as c.3082delG [PMID: 25971938, PMID: 27343443], c.3066_3081delAGCTACTTTTCCAGAA, c.3075_3082del [PMID: 31472177], or c.3080_3081delAA [PMID: 26729443].
Background: Platelet derived growth factor receptor alpha (PDGFRA) is a cell surface tyrosine kinase receptor made up of 1089 amino acids and located on chromosome 4q12. PDGFRA contains 23 exons –exons 1-2 encode the signaling peptide, exons 3-10 encode the extracellular domain, exon 11 encodes the transmembrane domain, exon 12 encodes the juxtamembrane domain, and exons 13-23 encode the kinase domain [PMID: 33449152]. Mutations in PDGFRA often target the activation loop located within exon 18 and, to a lesser extent, target the juxtamembrane domain and the N-lobe of the kinase domain found within exons 12 and 14 [PMID: 30778083, PMID: 33449152, PMID: 15146165]. Activating PDGFRA exon 18 mutations such as V561D, N659K, D842V, and in-frame deletions are observed in 30-40% of KIT-negative gastrointestinal stromal tumors (GISTs) [PMID: 17193822, PMID: 15928335, PMID: 12522257, PMID: 23970477]. Specifically, PDGFRA in-frame deletions in codons 841-847 make up 23% of all exon 18 mutations [PMID: 15146165].
CBV Criteria: The boundaries defined to capture PDGFRA exon 18 deletions as a class-based variant start at codon 841 and end at codon 847.