RET c.2753T>C, p.Met918Thr

Reasoning

PS3 is met at strong strength. The exact RET p.Met918Thr variant was evaluated in multiple independent cellular functional systems with appropriate comparator constructs and assay controls. In NIH 3T3 transformation experiments, RET(M918T) showed high transforming activity and was assessed across three independent experiments; the variant also produced increased focus formation, soft-agar colony formation, and proliferation relative to controls. In PC12 cells, RET(M918T) caused constitutive phosphorylation, altered neuronal-associated gene expression and promoter activity, and impaired normal NGF-mediated growth inhibition. These concordant results across transformation, kinase-signaling, proliferation, and differentiation-related assays are biologically relevant to the established RET gain-of-function mechanism and directly support a damaging functional effect. The studies are not described as validated clinical diagnostic-laboratory assays, but the replicated exact-variant results across several orthogonal assays support strong rather than moderate evidence under the generic PS3 definition.

Rule

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

Evidence considered

• Iwashita et al. 2000 (PMID:10679286), Results p. 806: RET(M918T) showed approximately two-thirds the transforming activity of RET(C634R), RET(A883F), or RET(E768D/A919P); RET mutant cDNAs were transfected into NIH 3T3 cells and transforming foci were scored after 12 days in three independent experiments.
• Germline RET allelic-variant study (PMID:21810974), Results and Table 3: exact M918T produced 98.5 ± 9.2 focus-forming units per microgram of DNA, formed 20 ± 5 soft-agar colonies per plate, had a 0.6 ± 0.1 day doubling time, and proliferated significantly faster than controls (P<0.05); known RET mutations were used as controls.
• Borrello et al. 1995 (PMID:8755580), Results: PC12 cells transfected with retMEN2B (Met918Thr) showed constitutive RET phosphorylation, neuronal-associated transcript and promoter activation, impaired terminal differentiation/growth arrest, and reduced sensitivity to NGF-mediated growth inhibition.
• The convergent exact-variant findings across NIH 3T3 transformation, soft agar, growth, phosphorylation, and PC12 differentiation-related assays support constitutive RET activation and a damaging gain-of-function effect.

Still missing

• The available studies are research-cell assays rather than explicitly validated and reproducible clinical diagnostic-laboratory assays.

Sources

Reasoning

RET p.Met918Thr lies at residue 918 in the RET tyrosine-kinase domain and specifically in the substrate-binding pocket. The literature describes codon 918 as part of the critical kinase domain and reports that M918T increases RET-ATP binding affinity and stabilizes the active ATP-bound form. This is a well-established functional domain/hotspot context, and the supplied population evidence shows only 1/251,478 alleles in gnomAD v2.1 and absence from gnomAD v4.1.

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

• Molecular mechanisms of RET receptor-mediated oncogenesis in multiple endocrine neoplasia 2, section 'MEN 2B Mutations': M918T is in the RET kinase domain and the residue lies in the substrate-binding pocket; the mutation increases RET-ATP binding affinity and stabilizes the active ATP-bound form.
• The variant is present at 1/251,478 alleles in gnomAD v2.1 (AF 3.97649e-06) and is absent from gnomAD v4.1.

Sources

Reasoning

PM2 is met at supporting strength under the supplied generic SVI threshold. The variant is present only once in gnomAD v2.1 exomes (AF 3.976490985294937e-06; 1/251478 alleles; 0 homozygotes), is absent from gnomAD v4.1, and is absent from the queried gnomAD v2.1 and v3.1 non-cancer datasets. The overall AF is below the supplied PM2 supporting cutoff of <=0.0001. Because the variant is a dominant missense allele, the result is described as extremely rare rather than strictly absent from all controls.

Rule

Absent from controls (or at extremely low frequency if recessive) in Exome Sequencing Project, 1000 Genomes or Exome Aggregation Consortium.

Evidence considered

• gnomAD v2.1 exomes: AF 3.976490985294937e-06, 1/251478 alleles, 0 homozygotes; highest subpopulation AF 8.790899660671274e-06 in European (non-Finnish) individuals.
• gnomAD v4.1: absent.
• gnomAD v2.1 non-cancer and gnomAD v3.1 non-cancer: absent.
• Generic SVI PM2 supporting threshold supplied for this case: <=0.0001 (ClinGen SVI 2020 recommendation as supplied in the case calibration block; ACMG/AMP 2015 source PMID:25741868).

Sources

Reasoning

PM6 is supported by the ClinVar exact-variant submission audit, which records de novo origin for two submissions, while the available records do not confirm maternity and paternity. The evidence is therefore consistent with assumed de novo occurrence rather than PS2-level confirmed de novo occurrence.

Rule

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

Evidence considered

• ClinVar exact-variant submission audit lists origin 'de novo' for SCV003918924 and SCV001481264.
• The available ClinVar records do not document confirmation of both maternity and paternity, so the evidence fits PM6 rather than PS2.

Still missing

• Primary case reports and parental testing details for the reported de novo individuals
• Explicit documentation that each reported individual had the relevant disease and no family history

Sources

Reasoning

The variant is a missense substitution, RET NM_020975.6:c.2753T>C, p.(Met918Thr), so the governing generic rule selects the missense REVEL path only; SpliceAI and BayesDel are not used for PP3. REVEL is 0.91, meeting the supplied moderate PP3 threshold of >= 0.773 (ClinGen SVI REVEL calibration, Pejaver et al. 2022, PMID:36413997) but not the strong threshold of >= 0.932. Therefore PP3 is met at moderate strength.

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 lookup for RET NM_020975.6:c.2753T>C (p.Met918Thr): REVEL score 0.91.
• Generic calibration supplied for this case: REVEL PP3 moderate threshold >= 0.773 and strong threshold >= 0.932; ClinGen SVI REVEL calibration, Pejaver et al. 2022, PMID:36413997.
• Because this is a missense variant, the governing one-path rule permits REVEL only; the available SpliceAI max delta 0.014 and BayesDel score 0.417334 were not used for PP3.

Sources

Reasoning

RET p.Met918Thr is an established pathogenic change, but the evidence identifies the same c.2753T>C nucleotide change rather than a different nucleotide substitution producing the same amino-acid change. At the Met918 codon, the observed T>C substitution is the relevant nucleotide change documented in the cited literature; no alternate nucleotide change yielding Met918Thr was established.

Rule

Same amino acid change as a previously established pathogenic variant regardless of nucleotide change.

Evidence considered

• RET p.M918T is reported as the canonical MEN2B change at codon 918 in the RET kinase domain, but the cited records describe the same c.2753T>C substitution rather than an alternate nucleotide change producing M918T.
• The reference codon is Met (ATG); the available evidence does not establish another nucleotide substitution at codon 918 that also produces Thr.

Sources

Reasoning

ClinVar contains exact-variant Pathogenic and Likely pathogenic submissions, but the record reports zero expert-panel submissions and the available assertions are ordinary laboratory, aggregate, literature-only, or other non-expert assertions. Under the requested generic PP5 rule, these do not qualify as a reputable exact-variant source for PP5.

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-variant record NM_020975.6(RET):c.2753T>C (p.Met918Thr) reports Pathogenic/Likely pathogenic classifications but expert_panels is empty and expert_panel_submissions is 0.
• Ordinary laboratory submissions, aggregate ClinVar labels, and non-expert assertions were excluded from PP5.

Sources

Reasoning

BA1 is not met. The highest all-comers population frequency reported is gnomAD v2.1 AF 3.976490985294937e-06 (1/251478 alleles), with the highest subpopulation frequency in European non-Finnish individuals at 8.790899660671274e-06 (1/113754 alleles); gnomAD v4.1 reports the variant as absent. These values are far below the generic BA1 threshold of >=0.05, and the non-cancer gnomAD v2.1 and v3.1 queries also report absence. No indel-calling caveat applies because this is a single-nucleotide substitution.

Rule

Allele frequency is above 5% in Exome Sequencing Project, 1000 Genomes or Exome Aggregation Consortium.

Evidence considered

• gnomAD v2.1: AF 3.976490985294937e-06, 1/251478 alleles, 0 homozygotes; highest subpopulation AF 8.790899660671274e-06 in European (non-Finnish) individuals, 1/113754 alleles.
• gnomAD v4.1: absent.
• gnomAD v2.1 non-cancer and gnomAD v3.1 non-cancer queries: absent.
• Generic ACMG/AMP 2015 BA1 calibration supplied for this case: >=0.05 (ACMG/AMP 2015 stand-alone benign allele frequency, PMID:25741868).

Sources

Reasoning

BS1 is not met. The observed gnomAD v2.1 AF is 3.976490985294937e-06 overall and 8.790899660671274e-06 in the highest subpopulation, both far below the generic 0.01 default. The variant is associated with a dominantly inherited MEN2B cancer syndrome with early, aggressive disease; there is no population-frequency signal suggesting an allele frequency greater than expected for this disorder. The available data are therefore sufficiently below the default that a maximum-credible-frequency calculation is not needed to exclude BS1 here.

Rule

Allele frequency is greater than expected for the disorder.

Evidence considered

• gnomAD v2.1: overall AF 3.976490985294937e-06 (1/251478 alleles); highest subpopulation AF 8.790899660671274e-06 (1/113754 alleles) in European (non-Finnish) individuals; 0 homozygotes.
• gnomAD v4.1: absent.
• The case summary describes no VCEP coverage and generic ACMG/AMP governance; the disease context is a dominantly inherited MEN2B cancer syndrome with early, aggressive disease.
• Generic SVI BS1 default supplied for this case: >=0.01; per-gene maximum-credible-frequency derivation is preferred when needed (Whiffin et al. 2017, PMID:28518168).

Sources

Reasoning

BS2 is not met. gnomAD v2.1 contains one heterozygous allele among 251478 alleles and zero homozygotes; gnomAD v4.1 and the non-cancer datasets report the variant as absent. These aggregate population observations do not document an identified healthy adult individual carrying the variant, and no healthy adult homozygote, hemizygote, or confirmed unaffected carrier observation is provided. The criterion therefore cannot be applied.

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: 1 allele in 251478, 0 homozygotes; European (non-Finnish) subgroup 1/113754, 0 homozygotes.
• gnomAD v4.1, gnomAD v2.1 non-cancer, and gnomAD v3.1 non-cancer: absent.
• No case-specific report identifies a healthy adult individual carrying RET p.Met918Thr without the relevant early-onset dominant disease phenotype.

Sources

Reasoning

BS3 is not met. The reviewed exact-variant functional studies do not show a lack of damaging effect; instead, RET p.Met918Thr produced increased transforming activity, focus formation, soft-agar colony formation, proliferation, constitutive phosphorylation, and abnormal cellular differentiation-related signaling. No well-established assay result demonstrating preserved protein function or normal splicing was identified for this variant. The observed functional direction is therefore incompatible with applying BS3.

Rule

Well-established in vitro or in vivo functional studies show no damaging effect on protein function or splicing.

Evidence considered

• The exact-variant NIH 3T3 and PC12 studies cited for PS3 report activating or damaging effects, not normal protein function or normal splicing.
• PMID:21810974 reports 98.5 ± 9.2 focus-forming units per microgram of DNA, 20 ± 5 soft-agar colonies per plate, and significantly increased proliferation for M918T relative to controls.
• PMID:10679286 and PMID:8755580 report RET(M918T)-associated transformation, constitutive signaling, and abnormal cellular phenotypes.

Still missing

• No well-established benign functional or splicing assay demonstrating preserved function was identified.

Sources

Reasoning

The available RET literature describes germline activating missense mutations as causative for MEN2 and identifies M918T as a principal disease-associated mutation. This is the opposite of a gene in which disease is known to be caused primarily by truncating variants, so BP1 is not met.

Rule

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

Evidence considered

• The RET review states that germline mutations causing MEN2 are activating and that more than 95% of MEN2B cases involve M918T.
• The original RET study describes germline missense mutations as causative events and M918T as the MEN2B kinase-domain substitution.

Sources

Reasoning

The variant is a missense substitution, so the governing generic rule selects the missense REVEL path only; SpliceAI cannot be substituted for a gray-zone or non-benign REVEL result, and BayesDel has no generic calibration. REVEL is 0.91, which exceeds all supplied BP4 thresholds, including the supporting threshold of <= 0.29. Thus BP4 is not met; the SpliceAI max delta of 0.014 is not evidence for BP4 in this missense situation.

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 lookup for RET NM_020975.6:c.2753T>C (p.Met918Thr): REVEL score 0.91.
• Generic calibration supplied for this case: REVEL BP4 supporting threshold <= 0.29, moderate threshold <= 0.183, and strong threshold <= 0.016; ClinGen SVI REVEL calibration, Pejaver et al. 2022, PMID:36413997.
• The available SpliceAI max delta is 0.014, but the governing one-path rule excludes SpliceAI for this missense variant; BayesDel 0.417334 is not usable because no generic ACMG-strength calibration is established.

Sources

Reasoning

The exact-variant ClinVar record contains Pathogenic and Likely pathogenic submissions and no benign or likely benign expert-panel assertion. No qualifying reputable exact-variant benign source is available, so BP6 is not met.

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-variant record reports Pathogenic/Likely pathogenic classifications, with no expert-panel submission and no benign or likely benign assertion.
• Ordinary laboratory submissions, aggregate ClinVar labels, and non-expert assertions were not used to trigger BP6.

Sources

Reasoning

PS2 cannot be applied because the available evidence does not document confirmation of both maternity and paternity for an affected individual. ClinVar's exact-variant submission audit contains de novo origin labels, but those labels do not establish confirmed maternity and paternity or provide a documented family-history assessment.

Rule

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

Evidence considered

• ClinVar exact-variant submission audit lists de novo origin for SCV003918924 and SCV001481264, but provides no parental identity-confirmation details.
• No case-level pedigree or explicit confirmed-maternity/confirmed-paternity report was available in the reviewed evidence.

Still missing

• Documented maternity and paternity confirmation for an affected individual
• Documented absence of family history in that individual

Sources

Reasoning

The literature packet identifies an exact-variant case series reporting p.Met918Thr in 34 unrelated individuals with MEN2B and absence in 93 unaffected individuals. This is a case-control comparison in direction, but the available report does not provide the total number of affected individuals tested, so the affected-allele prevalence and a valid OR cannot be calculated. The generic ACMG/AMP rule requires OR or RR >5.0 with a confidence interval excluding 1.0; neither a calculable OR/RR nor its 95% CI is available. The variant is also present at 1/251478 gnomAD v2.1 exome alleles and absent from gnomAD v4.1, but no matched case frequency is available for a case-versus-population calculation.

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

• The literature_pass packet reports the exact p.Met918Thr variant in 34 unrelated MEN2B individuals and absence in 93 unaffected individuals; this is a case-control comparison, but no total affected cohort denominator is supplied.
• Because the affected denominator is missing, an OR and 95% CI could not be computed from the 34 affected carriers and 0/93 control carriers.
• gnomAD v2.1 reports 1/251478 alleles (AF 3.97649e-06); gnomAD v4.1 reports the variant as absent, but no matched case frequency is available.

Still missing

• Total number of affected individuals tested in the exact-variant case series is needed to calculate affected prevalence.
• A valid OR or RR and 95% CI excluding 1.0 are required under the generic PS4 rule.

Sources

Reasoning

A different missense change at residue 918, M918V, is reported in a germline medullary thyroid carcinoma study and was experimentally evaluated. However, that study describes M918V as low or non-transforming and does not establish it as pathogenic; the available evidence therefore does not confirm the required pathogenic comparator for PM5.

Rule

Novel missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before.

Evidence considered

• Cosci et al. 2011 studied RET M918V as a different missense change at the same residue and reported intermediate focus-formation activity, but concluded that M918V was low or non-transforming and did not establish it as pathogenic.
• The available PM5 candidate artifact contains no confirmed same-residue pathogenic comparator.

Still missing

• A source-registry-cited determination that a different missense variant at RET residue 918 is pathogenic or likely pathogenic is still required.

Sources

Reasoning

PP1 cannot be assessed because the evidence identifies unrelated affected individuals and reported de novo cases, but does not provide a pedigree showing the variant and disease status across multiple affected family members or a count of informative meioses.

Rule

Co-segregation with disease in multiple affected family members in a gene definitively known to cause the disease.

Evidence considered

• ClinVar records and the reviewed literature leads describe multiple affected or de novo individuals, not a documented family segregation analysis for this exact variant.
• No qualifying count of affected variant carriers, affected non-carriers, or informative meioses was available.

Still missing

• Family pedigrees with genotype and phenotype for multiple affected relatives
• Informative meioses and segregation counts

Sources

Reasoning

RET is clearly associated with a disease mechanism involving activating missense variants, but the supplied evidence does not provide a gene-level estimate or complete analysis demonstrating a low rate of benign missense variation. The criterion requires both components, so PP2 cannot be assessed from the available evidence.

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

• RET disease-associated biology is described as being driven by activating missense mutations, including M918T, but no complete RET gene-level benign-missense-rate metric is supplied.
• The available gnomAD records provide frequency for this variant, not the gene-wide benign missense rate required by PP2.

Still missing

• A validated gene-level assessment of benign missense variation for RET is required.

Sources

Reasoning

The rule requires the patient's phenotype or family history to be highly specific for a disease with a single genetic etiology. The available case materials establish that RET p.Met918Thr is associated with MEN2B, but they do not provide a patient-specific phenotype or family-history description for this case that can be judged against the PP4 requirement.

Rule

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

Evidence considered

• The literature describes MEN2B as associated with RET p.Met918Thr, but no case-specific phenotype or family history is supplied for this criterion.

Still missing

• Patient-specific phenotype and family-history details are needed to judge whether the presentation is highly specific for a single genetic etiology.

Sources

Reasoning

BS4 cannot be assessed because no affected family members are documented as lacking the variant. The available evidence contains no pedigree-level non-segregation data and therefore cannot distinguish true lack of segregation from phenocopy or another familial pathogenic variant.

Rule

Lack of segregation in affected members of a family.

Evidence considered

• No affected relative is documented as variant-negative in the reviewed case evidence.
• No family-level non-segregation analysis was available.

Still missing

• Pedigree-level genotypes and disease status for affected relatives
• Evaluation for phenocopies or additional pathogenic variants if non-segregation is observed

Reasoning

BP2 is applicable to a fully penetrant dominant disorder when the evaluated variant is observed in trans with a pathogenic variant, or to any inheritance pattern when it is observed in cis with a pathogenic variant. The case materials establish a dominant MEN2B context but do not document a second pathogenic allele, a cis/trans relationship, or parent/offspring phase testing for NM_020975.6:c.2753T>C (p.Met918Thr). The required allelic observation is therefore unavailable, so 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.

Evidence considered

• PMID:7824936 supports the relevant RET MEN2B inheritance context as dominant.
• The reviewed case materials contain no documented pathogenic second allele and no phase result showing the evaluated RET variant in cis or trans with another pathogenic variant.

Still missing

• Identify any additional pathogenic or likely pathogenic allele in the same affected proband.
• Obtain parent/offspring testing or equivalent molecular phasing to establish whether the alleles are in cis or trans.

Sources

Reasoning

No evidence in the case materials shows that this variant was found in a case with an alternate molecular basis for disease. The available MEN2B and RET literature describes the variant and its disease association, but does not establish an alternative molecular cause for the relevant case.

Rule

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

Evidence considered

• No alternate pathogenic molecular diagnosis is documented in the supplied case evidence.

Still missing

• Evidence of a confirmed alternate molecular basis in the same affected case is required.