FGFR3 c.742C>T, p.Arg248Cys

Reasoning

PS3 is met at moderate strength. Del Piccolo et al. directly engineered FGFR3 c.742C>T (p.Arg248Cys) and measured ligand-independent receptor dimerization in mammalian plasma-membrane vesicles using a quantitative FRET assay. Wild-type FGFR3 was tested in the same assay as the control. The study analyzed approximately 500 or more vesicles per construct across more than 10 independent experiments. Arg248Cys increased the dimerization constant to 1110 +/- 130 versus 290 +/- 60 for wild type, changed intrinsic FRET to 0.74 +/- 0.03 versus 0.51 +/- 0.04, and produced a dimer-stability difference of -0.8 +/- 0.1 kcal/mol; the Arg248Cys effect was highly statistically significant by the reported analyses, including p < 0.001 for intrinsic FRET and p = 0.001 in the t-test of dimeric-fraction effects. These results provide direct, biologically relevant evidence of altered FGFR3 receptor behavior consistent with a damaging gain-of-function effect. Moderate rather than strong strength is assigned because this is one primary study, the measured dimer-stabilization effect was described as modest, and the assay did not directly measure downstream signaling or use a validated clinical diagnostic-laboratory assay.

Rule

PS3: Well-established in vitro or in vivo functional studies supportive of a damaging effect on the gene or gene product.

Evidence considered

• Del Piccolo et al. 2015, Effect of thanatophoric dysplasia type I mutations on FGFR3 dimerization: FRET-based quantitative dimerization assay of full-length FGFR3 extracellular and transmembrane domains in CHO-cell plasma-membrane vesicles without ligand; wild-type and mutant constructs were compared.
• Del Piccolo et al. 2015, Results/Table 1: approximately 500 or more vesicles per construct across more than 10 independent experiments; Arg248Cys K=1110 +/- 130 versus wild type K=290 +/- 60, intrinsic FRET 0.74 +/- 0.03 versus 0.51 +/- 0.04, and dimer-stability difference -0.8 +/- 0.1 kcal/mol; the Arg248Cys effect on intrinsic FRET had p < 0.001.
• The assay directly tests a biologically relevant FGFR3 property and includes a same-assay wild-type control, but the study is a single primary assay system and describes the stabilization effect as modest; therefore PS3 is calibrated to moderate strength rather than strong.

Still missing

• No independent orthogonal functional assay of downstream FGFR3 signaling or cellular phenotype was identified in the reviewed evidence.

Sources

Reasoning

Generic ACMG/AMP PS4 is met. The exact FGFR3 p.Arg248Cys variant (NM_000142.5:c.742C>T) was reported in 22 of 39 individuals with thanatophoric dysplasia type II and in 0 of 50 controls (PMID:7773297); this is a case-control comparison even though the paper did not print an odds ratio. The uncorrected odds ratio is infinite because the control-cell count is zero. Using the Haldane-Anscombe 0.5-cell correction, OR = (22.5 x 50.5)/(16.5 x 0.5) = 129.86, 95% CI 7.48-2255.60, which excludes 1.0. The exact variant was independently verified in the literature packet and in cohort reports identifying p.Arg248Cys in 66.5% of TD I cases (PMID:25614871) and 16 of 28 Brazilian TDI patients (PMID:29593476).

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

• PMID:7773297 abstract as recorded in literature_pass.json: exact p.Arg248Cys was present in 22 of 39 individuals with thanatophoric dysplasia type II and in none of 50 controls; the case-control OR was computed here rather than taken from the paper.
• For the zero control cell, the uncorrected OR is infinite; Haldane-Anscombe correction gives OR = 129.86 with 95% CI 7.48-2255.60, so the CI excludes 1.0.
• PMID:25614871 full-text extraction: p.Arg248Cys accounted for 66.5% of TD I cases in the International Skeletal Dysplasia Registry cohort.
• PMID:29593476 full-text extraction: c.742C>T (p.R248C) was identified in 16 of 28 Brazilian TDI patients (57%).

Sources

Reasoning

FGFR3 p.Arg248Cys is in a statistically significant FGFR3 R248 hotspot according to the Cancer Hotspots result, and the variant is a recurrent disease-associated mutation: it accounted for 66.5% of thanatophoric dysplasia type I cases in the International Skeletal Dysplasia Registry and occurred in 16 of 28 Brazilian TDI patients. The supplied population evidence reports the variant absent from gnomAD v2.1 and v4.1; no benign variation at this hotspot is reported in the supplied evidence. The Cancer Hotspots extraction is explicitly partial and should receive human confirmation.

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 FGFR3 R248 states: This variant lies in a statistically significant hotspot; the extraction warns that the R248 row was only partially parsed.
• Li et al. 2015, PMID:25614871, reports that p.Arg248Cys caused 66.5% of TD I cases in the registry cohort.
• Silva et al. 2018, PMID:29593476, reports c.742C>T (p.R248C) in 16 of 28 Brazilian TDI patients (57%) and characterizes it as recurrent.
• The variant is reported absent from gnomAD v2.1 and gnomAD v4.1; no benign observation at the R248 hotspot is present in the supplied evidence.

Still missing

• Cancer Hotspots extraction is marked partial; verify the hotspot row against the stored screenshot.

Sources

Reasoning

PM2 is met at supporting strength. The exact FGFR3 NM_000142.5:c.742C>T (p.Arg248Cys) variant is absent from gnomAD v2.1 and gnomAD v4.1; the evidence bundle also reports absence from GNOMAD_V2_1_NON_CANCER exomes and GNOMAD_V3_1_NON_CANCER genomes. Thus the observed allele frequency is 0 in each reported source and is below the supplied PM2 supporting threshold of <=0.0001. No VCEP-specific non-cancer or exome-only rule governs this gene, so the generic evidence is used; the non-cancer subsets are concordant rather than required by a governing specification.

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: exact variant absent; observed allele frequency 0 in the queried dataset.
• gnomAD v4.1: exact variant absent; observed allele frequency 0 in the queried dataset.
• GNOMAD_V2_1_NON_CANCER: exact variant absent from the reported exomes-only non-cancer subset.
• GNOMAD_V3_1_NON_CANCER: exact variant absent from the reported genomes-only non-cancer subset; gnomAD v3 has no exome cohort.
• Generic calibration threshold supplied for PM2 supporting: <=0.0001, identified in the prompt as the ClinGen SVI 2020 recommendation and cited to PMID:25741868.
• No VCEP covers FGFR3 in this case; therefore no governing specification requires replacing the default all-comers gnomAD sources with a non-cancer or exome-only source.

Sources

Reasoning

The variant is a missense substitution, NM_000142.5:c.742C>T (p.Arg248Cys), so the governing generic rule selects the REVEL path only; SpliceAI is not used for PP3/BP4 in this missense situation. REVEL is 0.855, meeting the published generic PP3 moderate threshold of >= 0.773. BayesDel is not used because no generic ACMG-strength calibration is established. The result is therefore PP3 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. Its SpliceAI score says nothing about whether the amino-acid substitution is tolerated, so 'no predicted splice impact' is never BP4 support for a missense variant....PP3 (moderate): >= 0.773 — ClinGen SVI REVEL calibration (Pejaver et al. 2022, Am J Hum Genet) (PMID:36413997)

Evidence considered

• The case normalization identifies NM_000142.5:c.742C>T as the missense variant NP_000133.1:p.(Arg248Cys) in exon 7.
• The REVEL lookup reports a score of 0.855 for this variant; this meets the generic PP3 moderate cutoff of >= 0.773.
• The cutoff is the supplied ClinGen SVI REVEL calibration from Pejaver et al. 2022, Am J Hum Genet (PMID:36413997).
• SpliceAI max delta 0.071 was not counted because the missense rule selects REVEL only; BayesDel 0.320063 was not used because no generic ACMG-strength calibration is established.

Sources

Reasoning

The variant is NP_000133.1:p.Arg248Cys from NM_000142.5:c.742C>T. The reviewed pathogenic reports establish this same nucleotide and amino-acid change, but no previously established pathogenic variant with the same p.Arg248Cys amino-acid change caused by a different nucleotide change was identified. The splice-impact caveat does not change this conclusion because SpliceAI's maximum delta score is 0.071 and the variant is a coding missense substitution.

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

• ClinVar variation 16332 identifies the exact variant NM_000142.5(FGFR3):c.742C>T (p.Arg248Cys) as Pathogenic; this is the same nucleotide change, not an alternate nucleotide change.
• Li et al. 2015, PMID:25614871, reports p.Arg248Cys as the recurrent FGFR3 mutation, without presenting a different nucleotide change for the same amino-acid substitution.
• Silva et al. 2018, PMID:29593476, reports c.742C>T (p.R248C), again the same nucleotide change.
• SpliceAI reports a maximum delta score of 0.071, providing no significant splice-impact signal for this coding missense variant.

Sources

Reasoning

PP5 is not met under the specified criterion policy. Although PMID:7773297 reports the exact variant as causing thanatophoric dysplasia, ClinVar variation 16332 has no expert-panel submissions; its Pathogenic assertions are from ordinary laboratory or other non-expert submissions. Such assertions cannot trigger 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 variation 16332: Pathogenic overall, but expert_panel_submissions = 0; the record contains laboratory and non-expert assertions rather than an exact-variant expert-panel classification.
• PMID:7773297 abstract reports the exact p.Arg248Cys variant in thanatophoric dysplasia, but a primary publication is not sufficient for PP5 under the specified expert-panel-only policy.

Sources

Reasoning

BA1 is not met. The exact FGFR3 NM_000142.5:c.742C>T (p.Arg248Cys) variant was absent from gnomAD v2.1 and gnomAD v4.1, including the reported non-cancer subsets; therefore no filtering allele frequency (grpmax FAF) is available and the allele-frequency requirement of >=0.05 is not reached. The variant's established association with lethal thanatophoric dysplasia and the concordant pathogenic ClinVar record further preclude treating an unobserved variant as a stand-alone benign high-frequency allele.

Rule

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

Evidence considered

• gnomAD v2.1: exact variant absent; no allele count, allele frequency, or grpmax FAF reported.
• gnomAD v4.1: exact variant absent; no allele count, allele frequency, or grpmax FAF reported.
• The evidence bundle also reports the exact variant absent from GNOMAD_V2_1_NON_CANCER exomes and GNOMAD_V3_1_NON_CANCER genomes; no non-cancer frequency is available.
• ClinVar exact-variant record NM_000142.5(FGFR3):c.742C>T (p.Arg248Cys): Pathogenic, multiple submitters, no conflicts; this is contextual evidence against a stand-alone benign call, not the frequency basis.
• Generic calibration threshold supplied for BA1: >=0.05, attributed to ACMG/AMP 2015 (PMID:25741868).

Sources

Reasoning

BS1 is not met. The exact variant was absent from gnomAD v2.1 and gnomAD v4.1, including the reported non-cancer subsets, so there is no filtering allele frequency (grpmax FAF) demonstrating that the allele is more frequent than expected for FGFR3-associated skeletal dysplasia. The generic 0.01 default is therefore not reached; no maximum-credible-frequency calculation is needed because no elevated frequency was observed.

Rule

Allele frequency is greater than expected for disorder.

Evidence considered

• gnomAD v2.1: exact variant absent; no allele count, allele frequency, or grpmax FAF reported.
• gnomAD v4.1: exact variant absent; no allele count, allele frequency, or grpmax FAF reported.
• The evidence bundle also reports the exact variant absent from GNOMAD_V2_1_NON_CANCER exomes and GNOMAD_V3_1_NON_CANCER genomes; no ancestry-specific elevated frequency is available.
• Generic calibration threshold supplied for BS1: >=0.01; the prompt identifies the default as a generic SVI threshold and cites Whiffin et al. 2017 (PMID:28518168) for preferred per-gene derivation, but no elevated frequency requiring that calculation is present.
• The variant is associated with thanatophoric dysplasia, a severe early-onset skeletal dysplasia; this disease context does not create a BS1 observation in the absence of population frequency evidence.

Sources

Reasoning

BS2 is not met. No qualifying healthy adult individual carrying FGFR3 p.Arg248Cys as a heterozygote, homozygote, or hemizygote was identified in the supplied evidence. The variant is absent from gnomAD v2.1 and v4.1, and the available literature describes affected individuals with thanatophoric dysplasia rather than healthy adult carriers; the generic BS2 requirement is therefore not satisfied.

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 exact variant as absent, providing no healthy-carrier observation or homozygote count.
• The variant-specific literature reviewed in the evidence bundle reports affected individuals with thanatophoric dysplasia and does not provide a qualifying healthy adult observation for this variant.
• Generic BS2 assessment uses the supplied ACMG/AMP definition and requires an observation in a healthy adult; absence from a database alone is not treated as an additional BS2 observation.

Sources

Reasoning

BS3 is not met. The directly relevant FRET study did not show that FGFR3 p.Arg248Cys has no damaging effect; instead, compared with wild type, it demonstrated altered ligand-independent dimerization, a higher dimerization constant (1110 +/- 130 versus 290 +/- 60), altered intrinsic FRET (0.74 +/- 0.03 versus 0.51 +/- 0.04), and statistically significant structural effects. Thus, the available functional evidence is directionally inconsistent with the BS3 definition. No separate well-established assay demonstrating preserved protein function or splicing was identified.

Rule

BS3: Well-established in vitro or in vivo functional studies show no damaging effect on protein function or splicing.

Evidence considered

• Del Piccolo et al. 2015 directly compared Arg248Cys with wild-type FGFR3 in a quantitative FRET dimerization assay and found altered dimerization and structure rather than an unchanged functional result.
• The available study therefore provides evidence against applying BS3; it is not evidence that the variant has no damaging effect.

Still missing

• No independent well-established functional study demonstrating preserved FGFR3 protein function or normal splicing was identified.

Sources

Reasoning

The available FGFR3 disease evidence describes skeletal-dysplasia variants as activating missense changes and identifies p.Arg248Cys as a recurrent, frequent disease-associated missense variant. This supports a missense disease mechanism rather than a gene in which disease is primarily caused 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

• Li et al. 2015, PMID:25614871, states that FGFR3 skeletal-dysplasia mutations are gain-of-function and reports p.Arg248Cys in 66.5% of TD I cases.
• Gandhi et al. 2015, PMID:25606676, experimentally studies the FGFR3 missense mutations Arg248Cys, Ser249Cys, and Tyr373Cys in a dimerization assay.
• Silva et al. 2018, PMID:29593476, reports recurrent c.742C>T (p.R248C) in Brazilian TDI patients.

Sources

Reasoning

The variant is a missense substitution, so the governing generic rule selects the REVEL path only and does not permit SpliceAI to substitute for a non-supportive REVEL result. REVEL is 0.855, above the supplied BP4 supporting cutoff of <= 0.29 and therefore does not support a benign computational effect. BayesDel is not used because no generic ACMG-strength calibration is established. BP4 is not met.

Rule

BP4: Multiple lines of computational evidence suggest no impact on gene or gene product (conservation, evolutionary, splicing impact, etc.)...Missense variant -> REVEL only. Its SpliceAI score says nothing about whether the amino-acid substitution is tolerated, so 'no predicted splice impact' is never BP4 support for a missense variant....BP4 (supporting): <= 0.29 — ClinGen SVI REVEL calibration (Pejaver et al. 2022, Am J Hum Genet) (PMID:36413997)

Evidence considered

• The case normalization identifies NM_000142.5:c.742C>T as the missense variant NP_000133.1:p.(Arg248Cys) in exon 7.
• The REVEL lookup reports a score of 0.855 for this variant; this exceeds the generic BP4 supporting cutoff of <= 0.29, so BP4 is not met.
• The cutoff is the supplied ClinGen SVI REVEL calibration from Pejaver et al. 2022, Am J Hum Genet (PMID:36413997).
• SpliceAI max delta 0.071 was not counted because a missense variant uses the REVEL path only; its presence cannot establish BP4 when REVEL is not in the benign range.

Sources

Reasoning

BP6 is not met under the specified criterion policy. ClinVar variation 16332 has no exact-variant expert-panel classification as Benign or Likely benign; the available record is Pathogenic with zero expert-panel submissions. Ordinary laboratory submissions, aggregate labels, and non-expert assertions 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 variation 16332: overall classification Pathogenic and expert_panel_submissions = 0; no exact-variant Benign or Likely benign expert-panel assertion is present.

Sources

Reasoning

PS2 cannot be assessed because the available evidence does not document a specific affected proband with confirmed maternity and paternity, nor does it establish absence of family history. Reports describing sporadic cases or de novo origin in ClinVar submissions do not provide the required case-level parental testing evidence.

Rule

PS2: De novo (both maternity and paternity confirmed) in a patient with the disease and no family history.

Evidence considered

• The available case-level literature does not document parental testing for the exact variant.
• ClinVar submission origin labels such as de novo, maternal, or unknown are submission metadata without an auditable affected-proband parental-testing record and were not used to satisfy PS2.

Still missing

• Affected-proband-level evidence linked to NM_000142.5:c.742C>T is not documented.
• Confirmed maternity and paternity testing are not documented.
• Family-history status is not documented.

Reasoning

The available same-residue comparator collection contains zero candidates and does not establish whether a different missense substitution at FGFR3 residue 248 has been determined to be pathogenic. The reviewed papers report p.Arg248Cys itself, but no qualifying alternate missense change at residue 248. A complete residue-level pathogenic comparator search is therefore still required.

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

• The same-residue comparator collection reports zero candidates and zero expert-panel or non-expert majority pathogenic/likely pathogenic candidates.
• Li et al. 2015, PMID:25614871, and Silva et al. 2018, PMID:29593476, report the observed p.Arg248Cys/c.742C>T variant rather than a different missense substitution at residue 248.

Still missing

• Confirm through a complete ClinVar and literature review whether any different pathogenic missense substitution occurs at residue 248.

Sources

Reasoning

PM6 cannot be assessed from the available evidence. A Brazilian cohort reported 16 of 28 thanatophoric dysplasia type I patients with c.742C>T and described all 16 as sporadic, but it did not document case-level parental testing or otherwise provide sufficient evidence that the variant was assumed de novo in those patients.

Rule

PM6: Assumed de novo, but without confirmation of paternity and maternity.

Evidence considered

• Novel and Recurrent Mutations in the FGFR3 Gene and Double Heterozygosity Cases in a Cohort of Brazilian Patients with Skeletal Dysplasia, Table 1 and Results: c.742C>T (p.R248C) was identified in 16 of 28 TDI patients (57%), and the table classified all 16 as sporadic cases; parental testing was not documented.
• The report that cases were sporadic is insufficient by itself to establish an assumed de novo event for PM6 without case-level parental or family evidence.

Still missing

• Case-level parental testing is not reported for the 16 variant-positive patients.
• The publication does not establish which specific patients were assumed de novo under PM6.

Sources

Reasoning

PP1 cannot be assessed because no informative family with multiple affected members carrying the exact variant is documented. The available exact-variant reports describe unrelated affected cases or sporadic cases rather than cosegregation across affected relatives.

Rule

PP1: Co-segregation with disease in multiple affected family members in a gene definitively known to cause the disease.

Evidence considered

• The reviewed exact-variant literature reports cohort-level affected individuals and sporadic cases but does not provide an informative family segregation analysis.

Still missing

• No family pedigree with multiple affected members is documented.
• No variant-positive and phenotype-positive relatives with meioses or segregation data are documented.

Reasoning

The evidence supports that FGFR3 skeletal-dysplasia disease commonly involves activating missense variants, including recurrent p.Arg248Cys, but it does not provide a gene-level assessment demonstrating both a low rate of benign missense variation and a missense-common disease mechanism under the stated rule. The variant's absence from gnomAD is not a gene-level benign-missense-rate assessment.

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

• Li et al. 2015, PMID:25614871, states that FGFR3 skeletal-dysplasia mutations are gain-of-function and reports p.Arg248Cys as the most frequent TD I mutation in the cohort.
• The supplied evidence reports this variant absent from gnomAD v2.1 and v4.1, but does not quantify the gene's benign missense variation rate.

Still missing

• Provide a gene-level benign missense variation analysis and a documented FGFR3 missense-mechanism assessment.

Sources

Reasoning

The generic PP4 definition requires the patient's phenotype or family history to be highly specific for a disease with a single genetic etiology. The supplied case evidence contains no patient phenotype or family-history description for this individual, so PP4 cannot be assessed from the available case-specific evidence.

Rule

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

Still missing

• Patient phenotype and family history are required to assess whether the presentation is highly specific for an FGFR3-related disease.

Reasoning

BS4 cannot be assessed because no family is documented in which affected members were tested and shown not to carry NM_000142.5:c.742C>T. The absence of reported segregation data is not evidence of lack of segregation.

Rule

BS4: Lack of segregation in affected members of a family.

Evidence considered

• The available evidence contains no affected-family-member genotype data demonstrating that the variant fails to segregate with disease.

Still missing

• No affected family members with negative variant testing are documented.
• No pedigree-level evaluation excluding phenocopies or another pathogenic variant is documented.

Reasoning

BP2 is applicable to a phase-specific observation, but the reviewed evidence does not establish either qualifying configuration. The Brazilian cohort reports sporadic cases and confirms heterozygosity for c.742C>T after correcting allele-dropout artifacts, but it does not report a pathogenic variant in trans or a pathogenic variant in cis. The reviewed registry cohort likewise provides affected-case frequency without cis/trans testing. Therefore, the available evidence is insufficient to assess BP2.

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

• Rios et al. 2018, Table 1 and Results: the c.742C>T cases were reported as sporadic; two apparent homozygous results were shown by resequencing to be heterozygous because of a polymorphism affecting amplification of the wild-type allele, with no pathogenic second allele or phase relationship reported.
• Li et al. 2015, International Skeletal Dysplasia Registry cohort: the paper reports affected individuals carrying p.Arg248Cys but does not provide cis/trans phase with another pathogenic variant.
• No reviewed source documents the evaluated variant in trans with a pathogenic variant for a fully penetrant dominant disorder or in cis with a pathogenic variant.

Still missing

• Parental or family-phase testing, or molecular haplotyping, is needed to determine whether c.742C>T is in cis or trans with a pathogenic variant.

Sources

Reasoning

The generic BP5 definition requires the variant to be found in a case with an alternate molecular basis for disease. The supplied evidence documents FGFR3-related disease reports but provides no case-specific alternate molecular diagnosis or competing pathogenic molecular basis, so BP5 cannot be assessed.

Rule

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

Still missing

• A confirmed alternate molecular diagnosis in the relevant case is required to assess BP5.