RET c.2428G>C, p.Gly810Arg

Reasoning

PM2 is met at supporting strength. NM_020975.6:c.2428G>C (RET p.Gly810Arg) is absent from gnomAD v2.1 and gnomAD v4.1, and is also reported absent from gnomAD v2.1 non-cancer and gnomAD v3.1 non-cancer. The generic ClinGen SVI threshold supplied for PM2 supporting is <=0.0001; an absent variant has an observed allele frequency of 0 in these queried datasets and is below that threshold. No VCEP-specific population source or threshold governs this case.

Rule

Absent from controls (or at extremely low frequency if recessive) in Exome Sequencing Project, 1000 Genomes or Exome Aggregation Consortium. ... PM2 (supporting): <= 0.0001 — SVI recommendation to downgrade PM2 to Supporting (ClinGen SVI 2020) (PMID:25741868)

Evidence considered

• gnomAD v2.1 reports the variant as absent.
• gnomAD v4.1 reports the variant as absent.
• The case population evidence also reports absence from gnomAD v2.1 non-cancer and gnomAD v3.1 non-cancer.
• Generic calibration threshold supplied for PM2 supporting: <=0.0001 (ClinGen SVI 2020 recommendation; ACMG/AMP 2015 source PMID:25741868).

Sources

Reasoning

NM_020975.6:c.2428G>C is a missense variant, p.(Gly810Arg), so the governing generic rule selects the missense REVEL path only; SpliceAI and BayesDel are not used for PP3/BP4 in this situation. The REVEL score is 0.809, which meets the published generic PP3 moderate threshold of >= 0.773 from the ClinGen SVI REVEL calibration (Pejaver et al. 2022, PMID:36413997).

Rule

PP3: Multiple lines of computational evidence support a deleterious effect on the gene or gene product (conservation, evolutionary, splicing impact, etc.). ... Missense variant -> REVEL only. ... PP3 (moderate): >= 0.773 — ClinGen SVI REVEL calibration (Pejaver et al. 2022, Am J Hum Genet) (PMID:36413997)

Evidence considered

• Local REVEL predictor lookup for NM_020975.6:c.2428G>C (p.Gly810Arg): REVEL = 0.809.
• ClinGen SVI REVEL calibration, Pejaver et al. 2022 (PMID:36413997): generic PP3 moderate threshold >= 0.773.
• Variant consequence is missense; under the governing one-path rule, REVEL is the applicable predictor and the SpliceAI max delta 0.001 and BayesDel score 0.456153 are not counted for PP3.

Sources

Reasoning

PMID:31988000 directly evaluated RET G810R (the protein consequence of c.2428G>C) in RET kinase enzyme assays and HEK-293 cells expressing KIF5B-RET variants. The study found reduced inhibition by selpercatinib and other RET inhibitors, while ATP affinity was only modestly changed (Km 49.8 μM for G810R versus 15.3 μM for wild-type RET), and interpreted the result as impaired drug binding rather than increased kinase activity. These experiments demonstrate altered pharmacologic response in an oncogenic, drug-resistance context, but they do not establish a damaging effect on RET function relevant to the generic ACMG/AMP disease mechanism for this germline variant. The paper also does not provide clinical-diagnostic laboratory validation, reproducibility data across independent laboratories, or a disease-relevant normal-function control sufficient to support PS3.

Rule

Well-established in vitro or in vivo functional studies supportive of a damaging effect on the gene or gene product.

Evidence considered

• PMID:31988000, Results: RET G810R was identified in post-treatment metastatic lesions and was absent from pretreatment material, with convergent G810 substitutions across metastatic sites.
• PMID:31988000, Table 1 and Figure 4: enzyme and cell-based assays showed reduced RET inhibitor activity against G810R; the study reported Km = 49.8 μM for G810R versus 15.3 μM for wild-type RET.
• The functional readout was acquired resistance to RET inhibitors, not a validated demonstration of damaging RET function in the germline MEN2 disease mechanism.

Still missing

• No validated clinical-diagnostic assay demonstrates that RET G810R disrupts normal RET function or activates the RET disease mechanism.
• No independent replication or disease-relevant functional calibration supports a PS3 strength assignment.

Sources

Reasoning

PP5 is not met. The exact-variant ClinVar record for NM_020975.6:c.2428G>C has one single-submitter clinical-laboratory submission classified as uncertain significance and zero expert-panel submissions. The literature-pass papers do not report this exact variant as pathogenic: PMID:15604628, PMID:11739416, and PMID:25810047 do not mention it, while PMID:31988000 reports a somatic acquired-resistance RET G810R observation rather than a reputable germline pathogenic classification. The generic PP5 condition therefore is not satisfied.

Rule

Reputable source recently reports variant as pathogenic, but the evidence is not available to the laboratory to perform an independent evaluation.

Evidence considered

• ClinVar exact-match audit: one clinical-laboratory submission, uncertain significance, criteria provided/single submitter, and zero expert-panel submissions.
• PMID:15604628, PMID:11739416, and PMID:25810047 do not mention the exact RET c.2428G>C/p.Gly810Arg variant.
• PMID:31988000 reports somatic RET G810R acquired resistance and does not provide a germline pathogenic classification for this variant.

Sources

Reasoning

BA1 is not met. The variant NM_020975.6:c.2428G>C (RET p.Gly810Arg) is absent from gnomAD v2.1, gnomAD v4.1, gnomAD v2.1 non-cancer, and gnomAD v3.1 non-cancer. Therefore, no filtering allele frequency (grpmax FAF) is available, and the generic BA1 threshold of >=0.05 is not reached. No population-frequency evidence supports a stand-alone benign call.

Rule

Allele frequency is above 5% in Exome Sequencing Project, 1000 Genomes or Exome Aggregation Consortium. ... BA1 (stand-alone): >= 0.05 — ACMG/AMP 2015 stand-alone benign allele frequency (PMID:25741868)

Evidence considered

• gnomAD v2.1 reports the variant as absent; no allele count, allele frequency, or grpmax FAF is available.
• gnomAD v4.1 reports the variant as absent; no allele count, allele frequency, or grpmax FAF is available.
• The case population evidence also reports absence from gnomAD v2.1 non-cancer and gnomAD v3.1 non-cancer; no non-cancer frequency is available.
• Generic calibration threshold supplied for BA1: >=0.05 (ACMG/AMP 2015 stand-alone benign allele frequency, PMID:25741868).

Sources

Reasoning

BS1 is not met. The variant is absent from gnomAD v2.1, gnomAD v4.1, gnomAD v2.1 non-cancer, and gnomAD v3.1 non-cancer. Thus, there is no filtering allele frequency (grpmax FAF) demonstrating a frequency greater than expected for the disorder, and the generic default threshold of >=0.01 is not reached. Because no elevated population frequency is observed, ancestry, penetrance, prevalence, and allelic-heterogeneity considerations do not create a BS1 path.

Rule

Allele frequency is greater than expected for the disorder. ... BS1 (strong): >= 0.01 — Generic SVI default; per-gene derivation preferred (Whiffin et al. 2017) (PMID:28518168)

Evidence considered

• gnomAD v2.1 reports the variant as absent; no filtering allele frequency (grpmax FAF) is available.
• gnomAD v4.1 reports the variant as absent; no filtering allele frequency (grpmax FAF) is available.
• The case population evidence also reports absence from gnomAD v2.1 non-cancer and gnomAD v3.1 non-cancer; no ancestry-specific or non-cancer frequency is available.
• Generic calibration threshold supplied for BS1: >=0.01; BS1 is otherwise defined by frequency being greater than expected for the disorder (Whiffin et al. 2017, PMID:28518168).

Sources

Reasoning

BP1 is not supported because RET-associated MEN2 literature describes activating germline RET mutations as the disease mechanism, rather than a disorder caused primarily by truncating variants. PMID:11739416 states that each MEN2 index case probably has an activating germline RET mutation and that the specific RET codon mutation correlates with disease phenotype. This is inconsistent with the BP1 premise.

Rule

Missense variant in a gene for which primarily truncating variants are known to cause disease.

Evidence considered

• PMID:11739416 states that MEN2 index cases probably have an activating germline RET mutation and that the specific RET codon mutation correlates with phenotype and disease aggressiveness.

Sources

Reasoning

NM_020975.6:c.2428G>C is a missense variant, p.(Gly810Arg), so BP4 must be assessed using REVEL only. The REVEL score is 0.809, which is above the strongest generic BP4 threshold of <= 0.016 and the supporting threshold of <= 0.29; it is therefore not evidence for no impact. The SpliceAI max delta 0.001 cannot substitute for the applicable missense REVEL path, and BayesDel has no established generic ACMG-strength calibration.

Rule

BP4: Multiple lines of computational evidence suggest no impact on gene or gene product (conservation, evolutionary, splicing impact, etc). ... Missense variant -> REVEL only. ... BP4 (supporting): <= 0.29 — ClinGen SVI REVEL calibration (Pejaver et al. 2022, Am J Hum Genet) (PMID:36413997)

Evidence considered

• Local REVEL predictor lookup for NM_020975.6:c.2428G>C (p.Gly810Arg): REVEL = 0.809.
• ClinGen SVI REVEL calibration, Pejaver et al. 2022 (PMID:36413997): generic BP4 thresholds are <= 0.016 strong, <= 0.183 moderate, and <= 0.29 supporting.
• Variant consequence is missense; under the governing one-path rule, the SpliceAI max delta 0.001 is not used for BP4, and BayesDel is not usable because no generic ACMG-strength calibration is established.

Sources

Reasoning

BP6 is not met. The exact-variant ClinVar audit shows only one single-submitter uncertain-significance clinical-laboratory submission and zero expert-panel submissions; there is no exact-variant expert-panel benign or likely-benign classification. The cited literature does not report NM_020975.6:c.2428G>C as benign. Ordinary laboratory submissions, the aggregate uncertain-significance label, and unrelated general RET/MEN2 references cannot trigger BP6.

Rule

Reputable source recently reports variant as benign, but the evidence is not available to the laboratory to perform an independent evaluation.

Evidence considered

• ClinVar exact-match audit: one clinical-laboratory submission classified as uncertain significance, single submitter, with zero expert-panel submissions.
• PMID:15604628, PMID:11739416, and PMID:25810047 do not report the exact variant as benign or likely benign.

Sources

Reasoning

The variant is RET p.Gly810Arg. PMID:31988000 reports the same amino-acid substitution as an acquired somatic resistance mutation, but the available evidence does not establish it as a pathogenic germline variant. No qualifying previously established pathogenic variant with the same amino-acid change and a different nucleotide change was identified. The rule's splicing caveat is not material because SpliceAI's maximum delta score is 0.001.

Rule

Same amino acid change as a previously established pathogenic variant regardless of nucleotide change. ... Caveat: Beware of changes that impact splicing rather than at the amino acid/protein level.

Evidence considered

• PMID:31988000 explicitly reports RET G810R in post-treatment metastatic lesions as an acquired resistance mutation and does not establish it as a pathogenic germline variant.
• SpliceAI reports a maximum delta score of 0.001, so there is no substantial splice-impact concern for applying the protein-level rule.

Still missing

• A previously established pathogenic variant producing the same RET amino-acid change through a different nucleotide change is not documented.

Sources

Reasoning

The available case evidence does not document a disease-affected proband with this variant, testing of both biological parents confirming maternity and paternity, a de novo result, or absence of family history. Therefore PS2 cannot be assessed.

Rule

De novo (both maternity and paternity confirmed) in a patient with the disease and no family history.

Evidence considered

• The case evidence identifies RET c.2428G>C (p.Gly810Arg) but provides no proband-level phenotype, parental testing, maternity/paternity confirmation, de novo statement, or family-history record.
• PMID:31988000 describes acquired somatic RET G810R resistance in metastatic cancer and does not establish a germline de novo observation in an affected proband.

Still missing

• Affected-proband phenotype and diagnosis
• Variant testing results for both biological parents
• Confirmed maternity and paternity
• Documented family history

Sources

Reasoning

No PS4 case-control comparison could be established for NM_020975.6:c.2428G>C (p.Gly810Arg). The literature-pass PS4 candidates were checked against the available paper extracts: PMID:15604628, PMID:25810047, and PMID:11739416 do not mention the exact variant, and PMID:20301434 and PMID:26389271 have no available abstract or full text in this case. PMID:31988000 reports RET G810R as an acquired resistance mutation in a somatic RET-fusion cancer study, not a germline affected-versus-unaffected case-control analysis. The variant is absent from gnomAD v2.1 and v4.1, but no affected-case count, control count, case frequency, odds ratio, or confidence interval is available. The governing numeric operator is >5.0, and the required comparison would be OR > 5.0 with a 95% confidence interval excluding 1.0; because no OR or confidence interval is available, the inequality cannot be instantiated and PS4 is not assessed rather than inferred from rarity or a paper's unrelated classification.

Rule

The prevalence of the variant in affected individuals is significantly increased compared to the prevalence in controls. ... Note 1: Relative risk (RR) or odds ratio (OR), as obtained from case-control studies, is >5.0 and the confidence interval around the estimate of RR or OR does not include 1.0.

Evidence considered

• gnomAD v2.1 and v4.1 report the exact variant absent, which supplies no affected-versus-control prevalence or effect estimate.
• PMID:15604628 lists RET-associated MEN2 phenotypes but does not mention c.2428G>C or p.Gly810Arg and provides no case-control data.
• PMID:25810047 states that RET mutations other than codons M918T and C634 are in the ATA-MOD category but does not mention G810R or provide variant-specific cohort data.
• PMID:11739416 discusses genotype-phenotype correlations for MEN2 generally but does not mention the exact variant.
• PMID:20301434 and PMID:26389271 were PS4 literature-pass candidates, but no abstract or full text was available to verify exact-variant case-control evidence.
• PMID:31988000 reports acquired somatic RET G810R resistance in a RET-fusion-positive cancer patient and does not provide a germline affected-versus-control comparison.

Still missing

• A case-control study or equivalent affected-case and matched-control counts for the exact variant is required.
• An odds ratio or relative risk with a confidence interval is required to test OR > 5.0 and exclusion of 1.0.

Sources

Reasoning

No VCEP domain table applies to RET. PMID:31988000 places G810R in the RET solvent-front region and reports convergent G810 resistance substitutions with functional drug-binding effects, providing a plausible critical-region/hotspot lead. However, the available evidence does not establish the required absence of benign variation across the relevant region, and Cancer Hotspots returned no statistically significant hotspot record. The PM1 threshold therefore cannot be completed from the supplied evidence.

Rule

Located in a mutational hot spot and/or critical and well-established functional domain (e.g. active site of an enzyme) without benign variation.

Evidence considered

• PMID:31988000 reports RET G810R in the solvent-front region, identifies distinct G810 substitutions across metastatic sites, and shows impaired RET-inhibitor binding in enzyme and cell assays.
• Cancer Hotspots returned no statistically significant hotspot record for RET G810.
• No RET entry was present in vcep_materials.json's domain_tables, so no VCEP-authoritative domain list applies.

Still missing

• A documented RET critical-domain or hotspot definition with evidence excluding benign variation across the relevant region is needed.

Sources

Reasoning

The target residue is RET Gly810. The available paper reports other G810 substitutions in the context of acquired drug resistance, but it does not establish a different missense change at residue 810 as pathogenic for a germline disorder. No qualifying pathogenic alternate missense comparator was identified.

Rule

Novel missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before. ... Caveat: Beware of changes that impact splicing rather than at the amino acid/protein level.

Evidence considered

• PMID:31988000 describes distinct RET G810 substitutions as acquired resistance mutations, not as established pathogenic germline variants.

Still missing

• A different missense variant at RET residue 810 with an established pathogenic classification is not documented.

Sources

Reasoning

The available case evidence does not document an assumed de novo occurrence of RET c.2428G>C in an affected patient, and it does not provide the clinical or parental observations needed to distinguish PM6 from no de novo evidence. Therefore PM6 cannot be assessed.

Rule

Assumed de novo, but without confirmation of paternity and maternity.

Evidence considered

• The case evidence contains no report of RET c.2428G>C as de novo or assumed de novo in a patient with the relevant disease.
• PMID:31988000 concerns an acquired somatic mutation in a RET fusion-positive tumor, not a germline de novo observation.

Still missing

• Affected-patient phenotype and diagnosis
• A report that the variant is de novo or assumed de novo
• Available parental testing information

Sources

Reasoning

The available case evidence does not identify multiple affected family members carrying RET c.2428G>C, provide informative meioses, or report cosegregation with disease. Therefore PP1 cannot be assessed.

Rule

Co-segregation with disease in multiple affected family members in a gene definitively known to cause the disease. ... Note: May be used as stronger evidence with increasing segregation data.

Evidence considered

• The case evidence provides no pedigree, affected-relative genotypes, meioses, segregation table, or variant-specific cosegregation result.
• The available literature extracts do not report family segregation for RET c.2428G>C (p.Gly810Arg).

Still missing

• Family pedigree and affected-relative diagnoses
• Genotypes of affected family members
• Informative meioses or segregation counts

Reasoning

The supplied evidence does not provide a gene-level analysis showing both a low rate of benign RET missense variation and missense variants as a common disease mechanism. The absence of this variant from gnomAD is variant-level evidence and cannot establish the two gene-level PP2 conditions.

Rule

Missense variant in a gene that has a low rate of benign missense variation and where missense variants are a common mechanism of disease.

Evidence considered

• The case evidence reports the variant as absent from gnomAD v2.1 and v4.1, but those observations do not establish PP2's required gene-level conditions.

Still missing

• Gene-level benign missense rate and disease-mechanism evidence satisfying both PP2 conditions are unavailable.

Reasoning

The available case materials identify RET and the associated MEN2 condition in ClinVar context, but they do not provide the patient's clinical phenotype, family history, or a disease-specific presentation. General descriptions of MEN2 in PMID:15604628, PMID:11739416, and PMID:25810047 cannot substitute for the patient's phenotype and do not establish that this patient's presentation is highly specific for a single genetic etiology. PP4 therefore cannot be assessed.

Rule

Patient’s phenotype or family history is highly specific for a disease with a single genetic etiology.

Evidence considered

• ClinVar identifies the exact variant as associated with Multiple endocrine neoplasia, type 2, but the available record does not provide this patient's phenotype or family history.
• PMID:15604628, PMID:11739416, and PMID:25810047 describe MEN2 generally; none supplies the patient's phenotype or a variant-specific PP4 assessment.

Still missing

• Patient phenotype and relevant family history are required.
• The phenotype must be shown to be highly specific for a disease with a single genetic etiology.

Sources

Reasoning

BS2 cannot be assessed from the available population evidence. The gnomAD records establish absence of the variant but provide no observed healthy adult carrying the variant in the heterozygous state, no homozygote or hemizygote observation, and no phenotype or age information. The requirement for an observed healthy adult with the relevant genotype and an early-onset, fully penetrant disorder therefore remains untested.

Rule

Observed in a healthy adult individual for a recessive (homozygous), dominant (heterozygous), or X-linked (hemizygous) disorder, with full penetrance expected at an early age.

Evidence considered

• gnomAD v2.1 and v4.1 report the variant as absent and therefore do not provide a healthy-carrier observation.
• The available non-cancer gnomAD entries also report absence and do not provide a genotype-level healthy-adult observation, age, or phenotype assessment.

Still missing

• A documented healthy adult carrying the variant in the relevant genotype, with age and phenotype information, is unavailable.

Sources

Reasoning

The available functional paper, PMID:31988000, did not establish that RET G810R has no damaging effect on protein function or splicing. Its assays measured RET inhibitor sensitivity and ATP affinity in an oncogenic fusion-cell and kinase context; they did not assess normal RET signaling, normal protein function, or splicing with a validated benign control design. Therefore, the evidence cannot support BS3, but it also does not justify treating the criterion as met or not met; the criterion remains not assessed.

Evidence considered

• PMID:31988000, Table 1 and Figure 4: available experiments assessed inhibitor sensitivity and ATP affinity, not absence of damaging effect on normal RET function or splicing.
• No benign-control or clinical-diagnostic validation framework was reported for applying BS3.

Still missing

• No validated assay demonstrates preserved normal RET protein function or signaling for G810R.
• No splicing assay is available for this exonic missense variant.

Sources

Reasoning

The available case evidence does not identify affected family members who lack RET c.2428G>C, nor does it provide a pedigree, genotype data, or clinical information sufficient to evaluate phenocopies or another pathogenic variant. Therefore BS4 cannot be assessed.

Rule

Lack of segregation in affected members of a family.

Evidence considered

• The case evidence contains no affected-relative genotypes or documented non-segregation for RET c.2428G>C.
• No family-level clinical data are available to address the stated BS4 caveat concerning phenocopies or more than one pathogenic variant contributing to an autosomal dominant disorder.

Still missing

• Affected-family-member list and diagnoses
• Genotypes of affected relatives
• Pedigree and inheritance structure
• Assessment for phenocopies or multiple pathogenic variants

Reasoning

BP2 requires either observation in trans with a pathogenic variant for a fully penetrant dominant gene/disorder or observation in cis with a pathogenic variant. The available case evidence documents neither a pathogenic second variant nor parental or offspring testing establishing cis/trans phase, and it does not establish the fully penetrant condition required for the trans pathway. Consequently, BP2 cannot be assessed.

Rule

Observed in trans with a pathogenic variant for a fully penetrant dominant gene/disorder or observed in cis with a pathogenic variant in any inheritance pattern.

Still missing

• Documented pathogenic second variant
• Parental or offspring testing establishing cis or trans phase
• Evidence that the relevant dominant RET disorder is fully penetrant

Reasoning

No evidence establishes that the exact RET c.2428G>C (p.Gly810Arg) variant was found in a case with an alternate molecular basis for disease. The available literature includes general MEN2 material and a separate report of a patient with double germline MSH2 and RET mutations, but that report is not in the case's source registry as an exact-variant source and the provided evidence does not identify this c.2428G>C variant in that case. BP5 therefore remains not assessed rather than being inferred from the existence of another RET-related case.

Rule

Variant found in a case with an alternate molecular basis for disease.

Evidence considered

• The available exact-variant ClinVar record is an uncertain-significance single-submitter record and does not document an alternate molecular basis.
• The literature extracts reviewed for this group do not document RET c.2428G>C in a case with another molecular diagnosis.

Still missing

• An exact-variant case report must document an alternate molecular diagnosis that explains the phenotype.