FGFR2 c.755C>G, p.Ser252Trp

Reasoning

PS3 is met at strong strength. The exact FGFR2 p.Ser252Trp variant (NM_000141.5:c.755C>G) was evaluated against wild-type receptor in surface-plasmon-resonance ligand-binding assays and structural studies. S252W increased affinity for nearly every tested FGF, with 8.6- to 36-fold increases for FGF9/16/20, and enabled binding to FGF10 and FGF19 when wild-type FGFR2c showed negligible or no binding. The study also demonstrated additional receptor-ligand contacts involving Trp252. These concordant biochemical and structural results directly demonstrate an altered, gain-of-function receptor effect relevant to the known FGFR2 disease mechanism.

Rule

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

Evidence considered

• Ibrahimi et al. 2004, Biochemical analysis of pathogenic ligand-dependent FGFR2 mutations: the exact S252W variant was compared with wild-type FGFR2c/FGFR2b using surface plasmon resonance; affinity increased 8.6- to 36-fold for FGF9/16/20 and binding was enabled for FGF10 and FGF19.
• Ibrahimi et al. 2004, Results on effect of Apert syndrome mutations on FGFR2c ligand-binding affinity and specificity, Table 1, and structural analysis: additional receptor-ligand contacts involving Trp252 were observed; the authors concluded that S252W violates FGFR2c ligand-binding specificity.

Sources

Reasoning

The exact FGFR2 p.Ser252Trp variant was reported in 45 of 70 unrelated patients with Apert syndrome. Using the affected-case frequency (45/70) and the gnomAD v2.1 population frequency (1/249,864 alleles, approximated as 1 carrier among 124,932 diploid controls), I computed an approximate case-control OR of 224,875.8 with a Wald 95% CI of 29,829.1-1,695,297.5. The governing PS4 operator is >5.0, and the explicit checks are: 224,875.8 > 5.0? yes; 95% CI 29,829.1-1,695,297.5 includes 1.0? no. This supports PS4. Human review is retained because the available population comparator is not documented as ancestry-matched to the Apert syndrome cases and the paper is a case series rather than a prospectively matched control study.

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:8651276, Differential effects of FGFR2 mutations on syndactyly and cleft palate in Apert syndrome: the abstract states, 'Analysis of DNA from 70 unrelated patients with Apert syndrome showed that 45 had the Ser252Trp' variant.
• gnomAD v2.1 reports 1/249,864 alleles for NM_000141.5:c.755C>G; treating this as approximately 1 carrier among 124,932 diploid controls, the computed 2x2-table OR is (45*124,931)/(25*1) = 224,875.8, with Wald 95% CI 29,829.1-1,695,297.5.
• The literal governing comparison is OR >5.0 and a confidence interval that does not include 1.0: 224,875.8 > 5.0? yes; 95% CI 29,829.1-1,695,297.5 includes 1.0? no.

Still missing

• The case series does not document an ancestry-matched unaffected control cohort; the gnomAD comparator is a population proxy.

Sources

Reasoning

p.Ser252Trp lies in the highly conserved linker between FGFR2 immunoglobulin-like domains II and III, a recurrent Apert syndrome mutational hotspot containing the adjacent pathogenic Pro253Arg substitution. The generic PM1 hotspot/critical-domain definition is satisfied, and no VCEP restriction applies.

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

• The FGFR2 S252W and P253R substitutions are reported in the linker between the second and third extracellular immunoglobulin domains, establishing the location as a recurrent disease-associated hotspot.
• The structural and biochemical study directly evaluates S252W in the FGFR2 ligand-binding region and reports altered ligand interactions involving Trp252.

Sources

Reasoning

The default all-comers population source applies because no governing VCEP specifies a non-cancer or exome-only source. The variant is absent from gnomAD v4.1 and present only once in gnomAD v2.1 exomes (AF 4.00218e-06; 1/249864 alleles; 0 homozygotes). The observed AF is below the generic PM2 supporting threshold of <=0.0001, so PM2 is met at supporting strength. The gnomAD v2.1 non-cancer and gnomAD v3.1 non-cancer query results are also reported as absent, but they are not needed because the generic default all-comers sources already satisfy PM2.

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=4.002177184388308e-06 (1/249864 alleles), homozygotes=0.
• gnomAD v4.1: variant absent.
• The supplied gnomAD non-cancer query results also report absence for GNOMAD_V2_1_NON_CANCER and GNOMAD_V3_1_NON_CANCER; no governing VCEP requires those sources here.
• Generic PM2 calibration supplied for this case: <=0.0001, ClinGen SVI recommendation to downgrade PM2 to Supporting (PMID:25741868).

Sources

Reasoning

ClinVar clinical-testing records report the variant as de novo and describe negative parental samples, while the available record does not confirm maternity and paternity; this supports assumed de novo status under PM6, pending primary-record review.

Rule

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

Evidence considered

• ClinVar submission audit: SCV004046053 states that analysis of parental samples was negative for the variant, indicating that it likely occurred de novo.
• ClinVar submission audit: SCV000659618 records that the variant was observed to be de novo in at least one individual, but does not document confirmation of maternity and paternity.

Still missing

• Primary clinical documentation should verify the parental testing methods, sample identity, and whether maternity and paternity were formally confirmed.

Sources

Reasoning

ClinVar clinical-testing records report that the variant segregates with disease in related individuals and in several families with multiple affected individuals, supporting PP1; the available record does not provide family-level genotypes, phenotype details, or meiosis counts for independent verification.

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

• ClinVar submission audit: SCV000659618 reports that the variant has been observed to segregate with disease in related individuals.
• ClinVar submission audit: SCV005088771 reports that the variant segregates with the disease in several families with multiple affected individuals.

Still missing

• Primary reports with family pedigrees, affected-member phenotypes, genotypes, and informative meioses are needed to verify and potentially upgrade segregation strength.

Sources

Reasoning

NM_000141.5:c.755C>G causes the missense change NP_000132.3:p.(Ser252Trp), so the governing generic rule selects the REVEL path only; SpliceAI is not used for PP3/BP4 in this missense situation. The REVEL score is 0.796, which meets the supplied generic PP3 moderate threshold of >= 0.773. BayesDel is not used because no generic ACMG-strength calibration is established.

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

• Variant consequence is missense: NM_000141.5:c.755C>G (NP_000132.3:p.(Ser252Trp)); the applicable computational path is REVEL only.
• Local REVEL lookup reports a score of 0.796.
• The supplied ClinGen SVI REVEL calibration sets generic PP3 moderate at >= 0.773 (Pejaver et al. 2022, Am J Hum Genet, PMID:36413997); 0.796 meets this threshold.
• SpliceAI max delta 0.001 was not counted because SpliceAI is not the governing path for a missense variant. BayesDel 0.408986 was not used because no generic ACMG-strength calibration is established.

Sources

Reasoning

ClinVar submission records report the variant as de novo in at least one individual, but the available evidence does not confirm both maternity and paternity; therefore the PS2 requirement is not satisfied.

Rule

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

Evidence considered

• ClinVar submission audit: SCV000659618 reports that in at least one individual the variant was observed to be de novo, but the record does not provide confirmation of both maternity and paternity.
• ClinVar submission audit: SCV004046053 reports that parental samples were negative for the variant, indicating a likely de novo event, without documented maternity and paternity confirmation.

Still missing

• Documented confirmation of both maternity and paternity is required for PS2.

Sources

Reasoning

ClinVar contains an exact-variant Pathogenic record for NM_000141.5:c.755C>G, but the record reports zero expert-panel submissions and instead consists of ordinary laboratory, literature-only, or other non-expert assertions. Under the required exact-variant expert-panel restriction, this does not 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 13272 exactly matches NM_000141.5(FGFR2):c.755C>G (p.Ser252Trp) and reports Pathogenic, but expert_panels is empty and expert_panel_submissions is 0; the listed submissions are not expert-panel assertions.

Sources

Reasoning

The default all-comers population source applies because no governing VCEP specifies a non-cancer or exome-only source. gnomAD v2.1 exomes reports 1/249864 alleles (AF 4.00218e-06; 0.000400%), with no homozygotes; gnomAD v4.1 reports the variant as absent. This is far below the generic BA1 stand-alone threshold of >=0.05 (5%), so BA1 is not met. The gnomAD v2.1 grpmax FAF is unavailable, but this does not affect the conclusion because the observed overall AF is already orders of magnitude below 5%.

Rule

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

Evidence considered

• gnomAD v2.1 exomes: 1/249864 alleles, AF=4.002177184388308e-06 (0.000400%), homozygotes=0; the highest observed subpopulation AF was 1/16152=6.19118e-05 in African/African American participants.
• gnomAD v4.1: variant absent.
• Generic BA1 calibration supplied for this case: >=0.05 (5%), ACMG/AMP 2015 stand-alone benign allele-frequency threshold (PMID:25741868).

Sources

Reasoning

The default all-comers population source applies because no governing VCEP specifies a non-cancer or exome-only source. The filtering grpmax FAF is unavailable in the supplied gnomAD record, so a FAF-based BS1 threshold cannot be positively established. The available gnomAD v2.1 total AF is only 4.00218e-06 (1/249864), and the highest observed subpopulation point estimate is 6.19118e-05 (1/16152), both far below the generic 0.01 default. The disorder context is early-onset autosomal dominant Apert syndrome/FGFR2-related craniosynostosis, and the variant has a strong pathogenic consensus; no prevalence, penetrance, or allelic-heterogeneity calculation supports a frequency greater than expected for the disorder. BS1 is therefore not met.

Rule

Allele frequency is greater than expected for disorder.

Evidence considered

• gnomAD v2.1 exomes: total AF=4.002177184388308e-06 (1/249864 alleles), homozygotes=0; highest observed African/African American point estimate=6.191183754333828e-05 (1/16152 alleles), homozygotes=0; grpmax FAF=null.
• gnomAD v4.1: variant absent.
• ClinVar: Pathogenic, criteria provided, multiple submitters, no conflicts; this pathogenic consensus argues against treating the very rare population observation as excess frequency.
• Generic BS1 calibration supplied for this case: >=0.01 is a default only; the governing criterion remains allele frequency greater than expected for the disorder.

Still missing

• A filtering grpmax FAF is not reported for the available gnomAD record.
• No disorder-specific maximum credible allele-frequency calculation with explicit prevalence, penetrance, and allelic-heterogeneity inputs is available.

Sources

Reasoning

The available FGFR2 disease literature identifies activating missense variants, including S252W, as a disease mechanism for craniosynostosis; it does not support a gene-level pattern in which disease is caused primarily by truncating variants. Therefore BP1 is not met.

Rule

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

Evidence considered

• The literature describes gain-of-function missense mutations in FGFR2 as responsible for craniosynostosis syndromes and specifically studies S252W as an activating mutation.
• Structural and biochemical experiments show altered ligand binding for S252W, supporting the established activating-missense mechanism.

Sources

Reasoning

NM_000141.5:c.755C>G causes the missense change NP_000132.3:p.(Ser252Trp), so the governing generic rule selects the REVEL path only; the SpliceAI max delta of 0.001 cannot be used as BP4 evidence for this missense variant. REVEL is 0.796, above the supplied generic BP4 supporting cutoff of <= 0.29, so BP4 is not met. BayesDel is not used because no generic ACMG-strength calibration is established.

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

• Variant consequence is missense: NM_000141.5:c.755C>G (NP_000132.3:p.(Ser252Trp)); the applicable computational path is REVEL only.
• Local REVEL lookup reports a score of 0.796.
• The supplied ClinGen SVI REVEL calibration sets generic BP4 supporting at <= 0.29 (Pejaver et al. 2022, Am J Hum Genet, PMID:36413997); 0.796 does not meet this threshold.
• SpliceAI max delta 0.001 was not substituted for the missense REVEL result. BayesDel 0.408986 was not used because no generic ACMG-strength calibration is established.

Sources

Reasoning

ClinVar contains an exact-variant Pathogenic record for NM_000141.5:c.755C>G, not a benign or likely benign expert-panel classification. The record reports zero expert-panel submissions, and ordinary laboratory or aggregate/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 13272 exactly matches NM_000141.5(FGFR2):c.755C>G (p.Ser252Trp), is classified Pathogenic, and has no expert-panel submissions; no exact-variant benign or likely benign expert-panel assertion is present.

Sources

Reasoning

The variant is p.Ser252Trp, and this amino-acid change is well documented in Apert syndrome, but the available evidence does not establish a previously pathogenic p.Ser252Trp change caused by a different nucleotide substitution. The PS1 same-amino-acid/different-nucleotide requirement therefore cannot be confirmed.

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

• The literature identifies FGFR2 Ser252Trp as a recurrent Apert syndrome mutation and directly studies the protein-level substitution, but does not provide a distinct nucleotide change producing the same p.Ser252Trp alteration.
• The exact variant is normalized as NP_000132.3:p.(Ser252Trp).

Still missing

• A validated pathogenic FGFR2 nucleotide substitution different from c.755C>G that also produces p.Ser252Trp is not documented.

Sources

Reasoning

The available evidence establishes pathogenic p.Ser252Trp and identifies the adjacent p.Pro253Arg substitution, but it does not document a different pathogenic missense substitution at residue 252. A complete same-residue comparator set is not available.

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 reviewed FGFR2 literature reports p.Ser252Trp and the adjacent p.Pro253Arg substitution; p.Pro253Arg does not satisfy the same-residue requirement for PM5.
• The available comparator collection contains no confirmed same-residue pathogenic alternative.

Still missing

• A validated pathogenic missense variant at FGFR2 residue 252 other than p.Ser252Trp is not documented in the available evidence.

Sources

Reasoning

FGFR2 disease-associated missense gain-of-function is documented, but the available case evidence does not establish the required gene-level low rate of benign missense variation. The single observed gnomAD allele and absence in gnomAD v4 cannot define that gene-level property.

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

• FGFR2-associated craniosynostosis is described as being caused by activating missense mutations.
• The queried variant has one allele in gnomAD v2.1 (AF 4.00218e-06) and is absent from gnomAD v4.1, which is insufficient by itself to establish the gene-wide benign-missense rate required for PP2.

Still missing

• A gene-level analysis demonstrating a low rate of benign FGFR2 missense variation is unavailable.

Sources

Reasoning

The available evidence describes the Apert syndrome phenotype in published cohorts, but this case bundle does not provide the patient's phenotype or family history. Therefore, the required assessment of whether this patient's presentation is highly specific for a single-genetic-etiology disease cannot be completed.

Rule

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

Still missing

• Patient-level phenotype and family-history details are needed to assess PP4.

Reasoning

The gnomAD v2.1 record contains one observed allele and no homozygotes, while gnomAD v4.1 reports absence. However, the available population record does not document that the carrier was a phenotypically healthy adult or provide age and phenotype ascertainment sufficient to apply BS2. Because this is an early-onset autosomal dominant FGFR2 craniosynostosis-associated variant with a strong pathogenic consensus, the single gnomAD allele cannot be treated as a confirmed healthy-adult observation without individual-level phenotype and quality review. BS2 is not assessed.

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 exomes: 1/249864 alleles and 0 homozygotes; the record does not provide carrier age or phenotypic ascertainment.
• gnomAD v4.1: variant absent.
• ClinVar: Pathogenic, criteria provided, multiple submitters, no conflicts; this creates a material reason not to infer a benign healthy-adult observation from an unphenotyped population carrier.

Still missing

• Individual-level age, phenotype, and variant-quality information for the single gnomAD carrier is unavailable.

Sources

Reasoning

BS3 was not assessed because the available exact-variant functional evidence evaluates increased ligand binding and altered receptor specificity, not a well-established benign effect or absence of damage. No validated assay evidence showing normal FGFR2 protein function or splicing was identified for p.Ser252Trp.

Rule

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

Evidence considered

• Ibrahimi et al. 2004, Biochemical analysis of pathogenic ligand-dependent FGFR2 mutations: the exact S252W assays measured increased ligand affinity and altered ligand specificity, so this source does not support BS3.

Still missing

• No well-established benign functional study demonstrating normal FGFR2 protein function or splicing for p.Ser252Trp was identified.

Sources

Reasoning

The available evidence reports de novo occurrence and positive segregation but does not identify an affected family member who lacks the variant; lack-of-segregation evidence is therefore insufficient to assess BS4.

Rule

Lack of segregation in affected members of a family.

Still missing

• A family pedigree with phenotype assessment and variant testing in affected relatives is required to evaluate non-segregation.

Reasoning

FGFR2-related Apert syndrome is a dominant disorder, so the trans branch of BP2 would require observation of this variant in trans with a pathogenic variant in a fully penetrant dominant disorder; the cis branch would require a pathogenic variant in cis. The available evidence documents the evaluated variant and reports de novo/segregation claims but does not provide a second pathogenic variant, a cis/trans relationship, or parent-tested phase for this case. Therefore 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

• The available case evidence identifies NM_000141.5:c.755C>G (p.Ser252Trp) as an FGFR2 variant associated with the autosomal dominant Apert syndrome context.
• No case-level observation of a second pathogenic variant in cis or trans, and no direct phase result, was found in the supplied evidence.

Still missing

• A second pathogenic variant and validated cis/trans phase in the same affected individual are not documented.
• Parent testing or another direct phase assay is not provided.

Reasoning

No patient-level report in the supplied evidence establishes that this variant occurred in a case with an alternate molecular basis for disease. The published Apert syndrome case-series and functional literature do not provide the required alternate molecular diagnosis for a case.

Rule

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

Still missing

• A patient-level case with an independently established alternate molecular diagnosis is required to assess BP5.