GNAS c.601C>T, p.Arg201Cys

Reasoning

PS3 is met at supporting strength. PMID:20531296 directly studied GNAS p.Arg201Cys (R201C) in an intestine-specific human-GNAS knock-in mouse model. The study measured biologically relevant downstream effects, including increased cAMP, phospho-ERK1/2 and Wnt-pathway target expression, and crypt-cell proliferation; in ApcMin/+ mice, R201C increased mean intestinal adenomas from 30 ± 2.9 to 62 ± 5.2 (n = 11), a two-fold increase with P=0.0001. These findings are concordant with a gain-of-function effect and include quantitative controls and an in vivo disease-relevant model. The evidence does not reach a stronger calibrated level because only one identified study is available and it is not a validated, reproducible clinical diagnostic assay series.

Rule

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

Evidence considered

• The activating mutation R201C in GNAS promotes intestinal tumourigenesis in Apc(Min/+) mice through activation of Wnt and ERK1/2 MAPK pathways: the exact p.Arg201Cys variant was tested in an intestine-specific human-GNAS R201C mouse model.
• Results reported increased cAMP, phospho-ERK1/2, Wnt-pathway target expression, and crypt-cell proliferation; in ApcMin/+ mice the mean adenoma number was 62 ± 5.2 (n = 11), a 2-fold increase over 30 ± 2.9 controls (P=0.0001).
• The in vivo model and quantitative control comparison support a damaging gain-of-function effect, but one non-diagnostic study is insufficient for stronger functional calibration.

Still missing

• No independent replication or validated clinical diagnostic laboratory assay series was identified for this exact variant.

Sources

Reasoning

GNAS p.Arg201Cys lies at codon 201, a recurrent mutational hotspot. The Cancer Hotspots result reports a statistically significant GNAS R201 hotspot, Fecteau et al. directly assayed the R201C/R201H hotspot and found GNAS mutations in 10 of 428 colon tumors, and Lumbroso et al. found Arg201 mutations in 49 of 113 patients with McCune-Albright syndrome. The variant is absent from gnomAD v2.1 and extremely rare in gnomAD v4.1 (8/1,613,500 alleles; AF 4.95817e-06), with no benign variation evidence presented for the hotspot.

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

• Cancer Hotspots result for GNAS R201: the queried variant lies in a statistically significant hotspot; the R201 hotspot row was partially parsed.
• Fecteau et al. 2014, Abstract and Introduction: pyrosequencing targeted the GNAS R201C and R201H hotspots; 10 of 428 colon tumors (2.3%) carried GNAS mutations, and codon 201 was described as a recurrent hotspot.
• Lumbroso et al. 2004, Results: an Arg201 mutation was found in 49 of 113 patients with signs of McCune-Albright syndrome, including 34 Arg201His and 15 Arg201Cys substitutions.
• gnomAD v2.1: 0/251,470 alleles observed for NM_000516.7:c.601C>T.
• gnomAD v4.1: 8/1,613,500 alleles observed, AF 4.95817e-06, with no homozygotes.

Sources

Reasoning

PM2 is met at supporting strength under the supplied generic SVI threshold. No VCEP or disease-specific population-source rule governs GNAS, so the default all-comers gnomAD data are used. gnomAD v2.1 exomes show 0/251,470 alleles, and gnomAD v4.1 shows only 8/1,613,500 alleles with no homozygotes; the filtering grpmax FAF is 1.24e-06, below the generic PM2 supporting cutoff of <=0.0001. The variant is therefore absent or extremely rare in population controls, although it is not literally absent from gnomAD v4.1.

Rule

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

Evidence considered

• gnomAD v4.1: total AF 4.95817e-06 (8/1,613,500 alleles), homozygotes 0, and filtering grpmax FAF 1.24e-06.
• gnomAD v2.1 exomes: AF 0 (0/251,470 alleles), homozygotes 0.
• No governing VCEP or non-cancer/exome-only source rule is present for GNAS; the generic all-comers gnomAD v2.1 and v4.1 entries are therefore used.
• Generic published calibration threshold supplied for this case: PM2 supporting <=0.0001; ClinGen SVI recommendation cited in the case materials with ACMG/AMP 2015 (PMID:25741868).

Sources

Reasoning

GNAS p.Arg201Cys is a missense change at residue 201, where the different missense change p.Arg201His has been repeatedly observed in affected patients. Lumbroso et al. reported 34 Arg201His cases among 113 patients with signs of McCune-Albright syndrome and found no difference in severity or manifestations between Arg201His and Arg201Cys. Fecteau et al. independently identified both R201H and R201C in colorectal tumors and described both as constitutively activating. These reports establish the alternate same-residue missense change as disease-associated, supporting 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

• Lumbroso et al. 2004, Results: Arg201 mutations occurred in 49/113 patients, including 34 Arg201His and 15 Arg201Cys; no difference in severity or manifestations was noted between the two mutations.
• Fecteau et al. 2014, Introduction: R201C and R201H both result in constitutive activation of Gsα and autonomous cAMP production.
• Zhou et al. 2007, Abstract: McCune-Albright syndrome is caused by embryonic somatic substitutions of His or Cys for Arg at amino acid 201, and the patient carried Arg201Cys.

Sources

Reasoning

The variant is a missense substitution, p.Arg201Cys, so the governing generic rule selects the missense REVEL path only; SpliceAI and BayesDel are not used for PP3 in this situation. REVEL is 0.943, which meets the generic PP3 strong threshold of >= 0.932 from the ClinGen SVI calibration (Pejaver et al. 2022, PMID:36413997).

Rule

Missense variant -> REVEL only. ... PP3 (strong): >= 0.932 — ClinGen SVI REVEL calibration (Pejaver et al. 2022, Am J Hum Genet) (PMID:36413997)

Evidence considered

• NM_000516.7:c.601C>T is a missense variant, NP_000507.1:p.(Arg201Cys).
• Local REVEL predictor lookup: score 0.943 for NM_000516.7:c.601C>T.
• Generic calibration: REVEL PP3 strong threshold >= 0.932, ClinGen SVI REVEL calibration (Pejaver et al. 2022, Am J Hum Genet; PMID:36413997).
• The missense-specific rule requires REVEL only; the available SpliceAI max delta 0.009 and BayesDel score 0.542879 are not counted for PP3.

Sources

Reasoning

The variant is GNAS p.Arg201Cys (NM_000516.7:c.601C>T). The reviewed evidence documents this exact amino-acid substitution, but does not document a previously established pathogenic variant with the same Arg-to-Cys amino-acid change caused by a different nucleotide change. The reports of GNAS Arg201His are a different amino-acid change and therefore support PM5, not PS1.

Rule

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

Evidence considered

• Zhou et al. 2007, abstract and Results: GNAS Arg201Cys was identified as the reported mutation in the patient, corresponding to the queried substitution.
• Lumbroso et al. 2004, Results: codon 201 mutations were Arg-to-His or Arg-to-Cys; the study does not provide a second nucleotide change producing the same Arg-to-Cys protein change.

Sources

Reasoning

PS2 is not met. The directly reviewed case report describes GNAS p.Arg201Cys in peripheral blood and bone tissue as an embryonic/post-zygotic somatic mosaic mutation, with no parental testing or constitutional de novo assessment. The governing PS2 definition requires de novo occurrence with both maternity and paternity confirmed, which is not demonstrated here. A ClinVar submission audit contains the unverified statement that the variant was observed de novo in at least one individual, but it does not provide parental identity testing or a primary pedigree record and cannot override the directly reviewed somatic evidence.

Rule

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

Evidence considered

• Zhou et al. 2007, case report: the abstract states that MAS is caused by embryonic somatic mutations and that Arg201Cys was detected in peripheral blood and bone tissue; the paper further states that MAS is caused by post-zygotic somatic mutation with mosaic distribution.
• ClinVar submission audit, accession SCV003272248: the submitter-derived signal states only that the variant was observed to be de novo in at least one individual; it gives no parental identity testing, parental genotypes, pedigree, or primary evidence sufficient for PS2.

Still missing

• No primary report documenting negative parental samples and identity-confirmed maternity and paternity was located.
• No pedigree establishing a germline de novo occurrence in a patient with the relevant disease was located.

Sources

Reasoning

Generic ACMG/AMP PS4 requires a significant increase in variant prevalence in affected individuals compared with controls, with OR or RR >5.0 and a 95% confidence interval excluding 1. The reviewed PS4-indexed literature reports affected case series, including 4/4 McCune-Albright patients in PMID:1944469, 2 thyroid-carcinoma cases in PMID:12970318, one MAS case in PMID:16543670, one boy with MAS in PMID:20197676, 14 Korean MAS patients in PMID:27506760, and 4/14 sex-cord stromal tumors in PMID:9626141, but none supplies a valid matched control comparison for this exact variant. PMID:20531296 reports R201C in 3/35 colorectal tumors, but this is a somatic tumor series and does not provide a matched-ancestry control group; comparing it with gnomAD germline allele frequency would not be a valid case-control comparison. The available gnomAD v4.1 frequency is 8/1,613,500 alleles overall and 1/29,596 in the highest-frequency Ashkenazi Jewish subgroup, but no compatible affected-case/control table or matched-ancestry case-control design was provided. Therefore PS4 is not met; no valid OR and 95% CI satisfying the governing definition can be established.

Rule

The prevalence of the variant in affected individuals is significantly increased compared to the prevalence in controls. ... 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

• PMID:1944469 abstract: four patients with McCune-Albright syndrome were analyzed and an activating Gs alpha mutation was detected in affected tissues from all four; no unaffected control group was reported.
• PMID:12970318 abstract: two patients with thyroid carcinoma and McCune-Albright syndrome were reported; no control comparison was reported.
• PMID:16543670 abstract: one 52-year-old man with McCune-Albright syndrome and an Arg201Cys-related Gs alpha mutation was described; no control comparison was reported.
• PMID:20197676 abstract: one boy with McCune-Albright syndrome had an Arg201Cys substitution detected in bone tissue; no control comparison was reported.
• PMID:27506760 abstract: a retrospective cohort of 14 Korean patients with McCune-Albright syndrome was described; the abstract provides no unaffected control prevalence for the exact variant.
• PMID:9626141 abstract: R201C was detected in 4 of 14 sex-cord stromal tumors; the abstract provides no matched unaffected control group.
• PMID:20531296 paper extract: R201C had previously been identified in 3 of 35 colorectal tumors (9%); this is a somatic tumor series without a matched-ancestry germline control comparison.
• gnomAD v4.1: exact variant frequency 8/1,613,500 alleles overall and 1/29,596 alleles in the Ashkenazi Jewish subgroup; these population data are not a matched control set for the reported somatic tumor series.

Still missing

• A case-control study or cohort with exact-variant counts in affected individuals and matched-ancestry controls is needed to calculate a valid OR/RR and 95% confidence interval.
• Possible protein-numbering alias in the literature: PMID:35101336 (Arg156Cys)

Sources

Reasoning

PM6 is not met because the available primary evidence supports a post-zygotic somatic mosaic mutation rather than an assumed germline de novo variant. The only de novo indication is a ClinVar submission signal stating that the variant was observed de novo in at least one individual, without reporting parental genotypes, maternity/paternity assumptions, or a germline pedigree. This is insufficient to establish PM6 for generic germline ACMG/AMP interpretation.

Rule

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

Evidence considered

• Zhou et al. 2007, case report: the paper identifies Arg201Cys in blood and bone tissue and explicitly characterizes MAS as caused by post-zygotic somatic mutation with mosaic distribution.
• ClinVar submission audit, accession SCV003272248: the de novo signal is limited to the statement that the variant was observed de novo in at least one individual and supplies no parental genotypes or pedigree details.

Still missing

• No primary germline case report documenting assumed de novo inheritance without confirmed parental identity was located.
• The reported tissue distribution does not establish a constitutional germline event.

Sources

Reasoning

PP5 requires a reputable source to recently report the exact variant as pathogenic when the evidence is not available for independent evaluation. The exact variant has Pathogenic and Likely pathogenic ClinVar submissions, but the ClinVar audit shows zero expert-panel submissions; the available assertions are ordinary laboratory, OMIM/literature-only, or other non-expert submissions. These do not satisfy the required expert-panel-type source restriction for this adjudication, so PP5 is not met.

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_000516.7(GNAS):c.601C>T (p.Arg201Cys) reports Pathogenic and Likely pathogenic submissions, but the submission audit records expert_panel_submissions = 0.
• The available ClinVar assertions include ordinary clinical-laboratory submissions and OMIM/literature-only submissions; no exact-variant expert-panel classification was identified.

Sources

Reasoning

BA1 is not met. Under the generic default, BA1 requires an allele frequency of at least 0.05. The appropriate gnomAD v4.1 filtering measure is grpmax FAF = 1.24e-06, based on 8/1,613,500 total alleles; the highest point estimate is only 1/29,596 in Ashkenazi Jewish individuals, whose observed allele count is below the required 2,000-allele population minimum. gnomAD v2.1 exomes reported 0/251,470 alleles. The robust filtering frequency is therefore far below 5%, and no disease-specific or consensus evidence justifies treating this pathogenic-consensus variant as a benign stand-alone BA1 exception.

Rule

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

Evidence considered

• gnomAD v4.1: total AF 4.95817e-06 (8/1,613,500 alleles), homozygotes 0, and filtering grpmax FAF 1.24e-06.
• gnomAD v4.1 highest point estimate: Ashkenazi Jewish AF 3.37883e-05 (1/29,596 alleles); this population has fewer than 2,000 observed alleles and is not used to establish BA1.
• gnomAD v2.1 exomes: AF 0 (0/251,470 alleles), homozygotes 0.
• Generic published calibration threshold supplied for this case: BA1 >= 0.05, ACMG/AMP 2015 (PMID:25741868).

Sources

Reasoning

BS1 is not met on the available population data. The generic default calibration is >=0.01, while the appropriate gnomAD v4.1 filtering frequency is grpmax FAF = 1.24e-06, with total AF 4.95817e-06 from 8/1,613,500 alleles and no homozygotes. The only higher population point estimate is 1/29,596 in Ashkenazi Jewish individuals, below the 2,000-observed-allele minimum and therefore not a reliable basis for BS1. This frequency is not greater than expected for a disorder under the supplied generic threshold; no ancestry-specific maximum-credible-frequency calculation is needed because the observed filtering frequency is orders of magnitude lower than 1%.

Rule

Allele frequency is greater than expected for the disorder.

Evidence considered

• gnomAD v4.1: total AF 4.95817e-06 (8/1,613,500 alleles), homozygotes 0, and filtering grpmax FAF 1.24e-06.
• gnomAD v4.1 highest point estimate: Ashkenazi Jewish AF 3.37883e-05 (1/29,596 alleles); below the 2,000-observed-allele requirement for frequency-based BS1/BA1 assessment.
• gnomAD v2.1 exomes: AF 0 (0/251,470 alleles), homozygotes 0.
• Generic published calibration threshold supplied for this case: BS1 >= 0.01; disease-specific maximum-credible-frequency considerations are described by Whiffin et al. 2017 (PMID:28518168).

Sources

Reasoning

The reviewed GNAS disease literature documents recurrent activating missense variants, especially at Arg201, as a disease mechanism: Lumbroso et al. found codon-201 missense mutations in 49 of 113 affected patients, and Zhou et al. identified an activating Arg201Cys mutation in McCune-Albright syndrome. This evidence does not support a gene in which disease is known to be caused primarily by truncating variants.

Rule

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

Evidence considered

• Lumbroso et al. 2004, Abstract and Results: GNAS activating Arg201 mutations were found in 49 of 113 patients with signs of McCune-Albright syndrome.
• Zhou et al. 2007, Abstract and Results: an activating GNAS Arg201Cys missense mutation was identified in a patient with McCune-Albright syndrome.
• Fecteau et al. 2014, Introduction: GNAS is a known oncogene with recurrent activating codon-201 mutations in neoplasms.

Sources

Reasoning

The variant is a missense substitution, p.Arg201Cys, so the governing generic rule selects the missense REVEL path only; SpliceAI and BayesDel are not used for BP4 in this situation. REVEL is 0.943, which does not meet any generic BP4 threshold, including the supporting cutoff of <= 0.29 from the ClinGen SVI calibration (Pejaver et al. 2022, PMID:36413997).

Rule

Missense variant -> REVEL only. ... BP4 (supporting): <= 0.29 — ClinGen SVI REVEL calibration (Pejaver et al. 2022, Am J Hum Genet) (PMID:36413997)

Evidence considered

• NM_000516.7:c.601C>T is a missense variant, NP_000507.1:p.(Arg201Cys).
• Local REVEL predictor lookup: score 0.943 for NM_000516.7:c.601C>T.
• Generic calibration: REVEL BP4 supporting threshold <= 0.29, ClinGen SVI REVEL calibration (Pejaver et al. 2022, Am J Hum Genet; PMID:36413997).
• The missense-specific rule requires REVEL only; the available SpliceAI max delta 0.009 and BayesDel score 0.542879 are not substituted for the applicable REVEL result.

Sources

Reasoning

BP6 requires a reputable source to recently report the exact variant as benign when the evidence is not available for independent evaluation. The exact-variant ClinVar record contains Pathogenic and Likely pathogenic submissions and zero expert-panel submissions; no exact-variant expert-panel benign or likely benign classification is present. Ordinary submissions, aggregate labels, and non-expert assertions cannot trigger BP6, 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 NM_000516.7(GNAS):c.601C>T (p.Arg201Cys) reports Pathogenic and Likely pathogenic submissions, with expert_panel_submissions = 0.
• No exact-variant expert-panel benign or likely benign classification is present in the ClinVar submission audit.

Sources

Reasoning

PP1 cannot be assessed. No affected family members, informative meioses, cosegregating genotypes, or pedigree-based phenotype data were reported for this variant. The reviewed report is a single sporadic mosaic case and therefore does not provide segregation evidence.

Rule

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

Evidence considered

• Zhou et al. 2007 reports one 32-year-old man with MAS and tissue-specific mosaic testing; it does not report affected relatives, familial testing, or segregation analysis.

Still missing

• No pedigree with affected relatives and variant testing was located.
• No segregation counts or informative meioses were reported.

Sources

Reasoning

The available evidence supports an important GNAS activating-missense mechanism at Arg201, but it does not establish the gene-wide requirement that GNAS has a low rate of benign missense variation. No validated gene-level benign-missense constraint metric or sufficiently comprehensive benign missense dataset was supplied, so PP2 cannot be assessed under the stated rule.

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

• Lumbroso et al. 2004: recurrent activating Arg201 missense mutations are a major mechanism in McCune-Albright syndrome.
• Zhou et al. 2007: Arg201Cys is an activating GNAS missense mutation associated with McCune-Albright syndrome.
• The case materials provide no validated gene-wide benign-missense constraint metric or comprehensive benign missense rate for GNAS.

Still missing

• A validated gene-wide assessment showing a low rate of benign GNAS missense variation is required.

Sources

Reasoning

The governing PP4 definition requires the patient's phenotype or family history to be highly specific for a disease with a single genetic etiology. The available case materials identify the variant and literature disease contexts but do not provide the current patient's phenotype or family history, so PP4 cannot be assessed.

Rule

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

Still missing

• Current-patient phenotype and family-history details are required.

Reasoning

BS2 cannot be established from the available evidence. gnomAD reports zero homozygotes and eight total heterozygous alleles in v4.1, but population-database carrier observations do not document that a named healthy adult individual carried the variant, nor do they establish age, phenotype, penetrance, or the relevant inheritance model. The available literature concerns somatic GNAS R201C in tumors and does not provide a qualifying healthy-adult observation for this germline criterion. A clinical review of individual healthy adult carriers with adequate age and phenotype information is required.

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 v4.1: 8/1,613,500 total alleles and 0 homozygotes; this is an aggregate population count without individual phenotype or age data.
• gnomAD v2.1 exomes: 0/251,470 alleles and 0 homozygotes.
• The available variant-specific literature describes somatic GNAS R201C tumor biology, not a qualifying healthy adult observation for BS2.

Still missing

• No documented named healthy adult individual with verified NM_000516.7:c.601C>T and adequate age, phenotype, and inheritance context.
• No established early-onset full-penetrance disease model for applying BS2 to this GNAS variant.
• gnomAD aggregate carrier counts cannot by themselves establish healthy-adult status.

Sources

Reasoning

BS3 is not assessed because no well-established functional study demonstrating no damaging effect on GNAS protein function or splicing was identified. The available exact-variant in vivo study instead reports increased cAMP signaling, pathway activation, proliferation, and adenoma formation, so it cannot provide benign functional evidence.

Rule

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

Evidence considered

• The exact-variant functional study evaluated GNAS R201C in vivo but reported activating rather than normal functional results; therefore it does not support BS3.
• No independent benign-control assay or validated clinical diagnostic laboratory study showing no damaging effect on protein function or splicing was identified.

Still missing

• No validated assay showing normal GNAS protein function or splicing for p.Arg201Cys was identified.

Sources

Reasoning

BS4 cannot be assessed. No affected family members with negative variant testing were reported, and no family segregation study was available. The absence of a reported pedigree is not evidence of lack of segregation.

Rule

Lack of segregation in affected members of a family.

Evidence considered

• The reviewed case report describes a single sporadic mosaic patient and provides no affected-relative testing or family-based non-segregation analysis.

Still missing

• No affected family members were described or tested.
• No family-based non-segregation result was located.

Sources

Reasoning

BP2 requires an observation in trans with a pathogenic variant for a fully penetrant dominant gene/disorder, or an observation in cis with a pathogenic variant in any inheritance pattern. The available case evidence provides no affected-proband observation with a second pathogenic variant, no parental or offspring testing, and no cis/trans phase result for NM_000516.7:c.601C>T (p.Arg201Cys). Therefore the required allelic observation 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

• ClinVar's exact-variant record provides classifications and literature citations but no documented cis/trans phase, parental testing, offspring testing, or second pathogenic variant observed with NM_000516.7:c.601C>T (p.Arg201Cys).
• The reviewed variant-specific paper extraction for PMID:20531296 concerns an intestine-specific mouse model of GNAS R201C and reports functional tumorigenesis data, not a human cis/trans allelic observation; it therefore does not support BP2.

Still missing

• Parental or offspring testing to establish phase with a second pathogenic variant
• A documented affected-proband observation containing the queried variant and a second pathogenic variant

Sources

Reasoning

BP5 requires that the variant be found in a case with an alternate molecular basis for disease. The available materials contain reports of GNAS R201C in somatic tumors and McCune-Albright syndrome tissues, but do not document a case in which this exact variant co-occurs with a demonstrated alternate molecular cause of the relevant disease. BP5 therefore cannot be assessed from the available evidence.

Rule

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

Still missing

• A case report or cohort record showing the exact variant plus an independently established alternate molecular basis is required.