VHL c.500G>A, p.Arg167Gln

Reasoning

The queried missense variant is NP_000542.1:p.(Arg167Gln) (R167Q), and ClinVar records it as Pathogenic with a ClinGen VHL Variant Curation Expert Panel review. This satisfies the VHL VCEP's PS1 restriction to variants interpreted by the VHL VCEP or variants with pathogenicity established using VHL VCEP specifications.

VCEP rule

Applied only to variants with interpretation by the VHL VCEP or by a variant with pathogenicity established using VHL VCEP specifications.

Evidence considered

• ClinVar variation 2216, NM_000551.4(VHL):c.500G>A (p.Arg167Gln): Pathogenic, reviewed by expert panel; expert panel is ClinGen VHL Variant Curation Expert Panel.
• The case normalization identifies the queried protein change as NP_000542.1:p.(Arg167Gln).

Sources

Reasoning

PS3 is met at supporting strength. The VHL VCEP accepts damaging functional evidence from HIF1/2a degradation assays, VBC-complex stability assays, or abnormal ECM formation and impaired fibronectin binding. PMID:15574766 directly studied VHL R167Q, corresponding to c.500G>A/p.Arg167Gln, and found reduced Elongin C interaction, failure to restore HIF2a regulation, and defective fibronectin deposition in variant-derived teratomas. These findings match VCEP-relevant damaging functional systems. The governing functional-assay spreadsheet was searched for all three variant notations but contains no variant-specific entry or pre-assigned code; no escalation above supporting is justified from the available control information.

VCEP rule

In-vitro assays should display total loss of HIF1/2a degradation (i.e. HIF1/2a presence) for VHL Type 1, and 2A/B.

Evidence considered

• PMID:15574766: VHL R167Q showed reduced interaction with Elongin C and failed to restore HIF2a regulation in Vhl−/− murine embryonic stem cells; variant-derived teratomas showed defective fibronectin deposition.
• PMID:15574766, abstract and Results: This mutation also failed to restore HIF2a regulation.
• VHL_Functional_Assay_SVI_documentation.xlsx: approved assay categories include VBC-complex stability, HIF1/2a degradation, and fibronectin/ECM assays with PS3_Supporting proposed strength; no row was keyed to this variant.

Still missing

• VHL_Functional_Assay_SVI_documentation.xlsx was searched for c.500G>A, p.Arg167Gln, and p.R167Q; no variant-specific entry or pre-assigned PS3 code was found.
• The available extraction does not establish the complete control count needed to justify a strength above supporting under the VCEP calibration.

Sources

Reasoning

The VHL VCEP permits PS4 scoring from unrelated probands across specific, consistent, and nonspecific phenotypes, assigning 0.25 points per proband when phenotype details are unavailable. Variant-specific reports identify 1 carrier in PMID:10567493, 3 independent patients in PMID:12000816, 1 familial report in PMID:12624160, and 10 individuals from unrelated French families in PMID:15300849, for a conservative aggregate of 15 reported carriers and 3.75 points when counted as phenotype-unavailable probands; this falls within the VCEP's 2–4 point Moderate band. PMID:12624160 provides a familial type 2 context and PMID:12000816 a pheochromocytoma cohort, but the available reports do not provide a case-control comparison, control counts, or a variant-specific odds ratio and possible overlap among historical cohorts must be resolved before final use.

VCEP rule

**See phenotype and points tables.** ... When the phenotype is not available (e.g. from a published article on a VHL cohort that does not detail the phenotype), count 0.25 points per proband. ... **2 – 4 points the PS4 cut-off and Proband Scoring Tables from a mix of any of the following phenotypes: specific, consistent and nonspecific.**

Evidence considered

• PMID:10567493, Table 1: VHL nt 713 G/A, codon 167, Arg→Gln, R167Q, identified in 1 patient with symptomatic CNS haemangioblastoma.
• PMID:12000816, Table 2: the equivalent G713A codon-167 Arg-to-Gln mutation was found in 3 independent patients in a cohort of 271 unrelated patients with nonsyndromic pheochromocytoma.
• PMID:12624160, Results/Table 1, family 16: c.500G>A/p.Arg167Gln was reported in a familial type 2 VHL family; the paper notes two codon-167 families overall and describes the phenotype as a type 2B-associated hotspot context.
• PMID:15300849, Table 2: c.500G>A/R167Q was listed in 10 individuals from a cohort of 126 unrelated French VHL families; the paper did not provide variant-specific individual phenotype details.
• PMID:10761708, Table I: the equivalent nucleotide-713 Arg167Gln variant was reported in Japanese VHL families 5, 77, and 79, with type 1 and type 2 family contexts.
• PMID:17024664, Table 1: p.Arg167Gln was included among surface missense substitutions in a genotype–phenotype cohort of 573 individuals from 200 VHL kindreds, but no variant-specific clinical outcome was reported.
• No reviewed source supplied variant-specific affected/control counts, so no odds ratio or 95% confidence interval could be computed.

Still missing

• No variant-specific case-control counts or odds ratio/95% confidence interval were reported in the reviewed sources.
• Potential overlap among historical cohorts and reports has not been established.
• Several reports lack individual phenotype details, so the 0.25-point nonspecific default was used.

Sources

Reasoning

The variant is p.Arg167Gln, and the VHL VCEP PM1 rule permits moderate PM1 for putative missense variants that are known germline hotspots. PMID:12624160 explicitly identifies codon 167 as a VHL hotspot associated with type 2B disease and reports the queried c.500G>A (Arg167Gln) variant in family 16. The VCEP materials contain no domain_tables entry for VHL; the germline-hotspot route is independently sufficient.

VCEP rule

Putative missense variants that are known germline hotspots AND/OR in key functional domains AND/OR somatic variants that have ≥10 instances for the same AA in cancerhotspots.org. See the table of Germline and Somatic Hotspots.

Evidence considered

• PMID:12624160, Results on genotype-phenotype correlations and Table 1, Family 16: c.500G>A causes Arg167Gln; the paper states, "Two families harboured distinct mutations in codon 167, a VHL hotspot associated with high risk of RC, HB, and PH (type 2B)."
• VHL VCEP materials: domain_tables is empty for VHL, so no authoritative domain-table entry was available; PM1 is met through the VCEP-defined known germline-hotspot route.

Sources

Reasoning

The VHL VCEP permits PM2 Supporting when a variant is absent from gnomAD or has GroupMax Filtering Allele Frequency at most 0.00000156 in gnomAD based on gnomAD v4; it also permits PM2 Supporting when no GroupMax Filtering Allele Frequency is calculated. This variant is absent from gnomAD v4.1, satisfying the absence path.

VCEP rule

PM2\_Supporting can be applied for variants either absent from gnomAD or with \<= 0.00000156 (0.000156%) GroupMax Filtering Allele Frequency in gnomAD (based on gnomAD v4 release). If no GroupMax Filtering Allele Frequency is calculated (ex. due to a single variant present), PM2\_Supporting may also be applied.

Evidence considered

• VHL VCEP specification v1.1, PM2 Supporting rule: apply when the variant is absent from gnomAD, has GroupMax Filtering Allele Frequency <=0.00000156, or has no calculated GroupMax Filtering Allele Frequency.
• gnomAD v4.1: NM_000551.4:c.500G>A / GRCh38 3-10149823-G-A is absent.

Sources

Reasoning

The variant is a missense substitution, so the VHL VCEP PP3 missense path applies and names REVEL as the predictor. The available REVEL score is 0.874, meeting the VCEP threshold of >=0.664. The available SpliceAI result and other predictors are not used for PP3 because the governing rule's missense path does not name them.

VCEP rule

For missense variants, use REVEL score >=0.664.

Evidence considered

• VHL VCEP PP3 missense rule: REVEL score >=0.664.
• Local REVEL predictor lookup for NM_000551.4:c.500G>A / NP_000542.1:p.(R167Q): score 0.874.
• SpliceAI and BayesDel results were not counted for PP3 because they are not named by the VCEP missense path.

Sources

Reasoning

Expert panel ClinGen VHL Variant Curation Expert Panel, ClinGen classified as Pathogenic.

VCEP rule

This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee.

Evidence considered

• VHL VCEP CSPEC PP5 entry: applicability is Not Applicable for this VCEP and the criterion is not for use.
• ClinVar expert panel classification

Sources

Reasoning

The VHL PM5 rule requires a prior pathogenic variant at the same residue and comparison using Grantham distance, with splice metapredictors excluding a splice effect. The collected PM5 review found zero same-residue comparator variants at Arg167, so no qualifying prior pathogenic comparator and no Grantham comparison are available.

VCEP rule

Pathogenicity of prior variant is established by interpretation of the VHL VCEP or variants with pathogenicity established using VHL VCEP specifications. The Grantham distance should be used to compare variants. The variant under consideration must be equal or a larger distance than the classified pathogenic variant (Grantham, 1974, Table 2 [¹⁶](#pmid_4843792) ). Splice metapredictors should be used to ensure the variant is not predicted to have an effect on splicing.

Evidence considered

• PM5 comparator collection for NP_000542.1:p.(R167Q): 6 candidates seen, 0 same-residue candidates, 0 expert-panel pathogenic/likely pathogenic candidates, and 0 non-expert-majority pathogenic/likely pathogenic candidates.

Still missing

• A qualifying prior pathogenic missense variant at residue 167 with an available Grantham comparison.

Sources

Reasoning

The governing VHL VCEP BA1 threshold is a GroupMax Filtering Allele Frequency of at least 0.000156 in gnomAD based on the gnomAD v4 release. The variant is absent from gnomAD v4.1, so no GroupMax Filtering Allele Frequency reaches or exceeds the BA1 threshold.

VCEP rule

Use a BA1 cut off of >=0.000156 (0.0156%) GroupMax Filtering Allele Frequency in gnomAD (based on gnomAD v4 release).

Evidence considered

• VHL VCEP specification v1.1, BA1 rule: GroupMax Filtering Allele Frequency >=0.000156 (0.0156%) in gnomAD based on gnomAD v4.
• gnomAD v4.1: NM_000551.4:c.500G>A / GRCh38 3-10149823-G-A is absent; therefore no gnomAD v4.1 GroupMax Filtering Allele Frequency is available or above threshold.

Sources

Reasoning

The governing VHL VCEP BS1 threshold is a GroupMax Filtering Allele Frequency of at least 0.0000156 in gnomAD based on gnomAD v4. The variant is absent from gnomAD v4.1, so no GroupMax Filtering Allele Frequency reaches or exceeds the BS1 threshold.

VCEP rule

Use BS1 cut off of  >=0.0000156 (0.00156%) GroupMax Filtering Allele Frequency in gnomAD (based on gnomAD v4).

Evidence considered

• VHL VCEP specification v1.1, BS1 rule: GroupMax Filtering Allele Frequency >=0.0000156 (0.00156%) in gnomAD based on gnomAD v4.
• gnomAD v4.1: NM_000551.4:c.500G>A / GRCh38 3-10149823-G-A is absent; therefore no gnomAD v4.1 GroupMax Filtering Allele Frequency is available or above threshold.

Sources

Reasoning

BS3 is not met. The VHL VCEP requires benign functional evidence showing that HIF1/2a degradation replicates WT function, VBC-complex stability is unaffected, or ECM formation/fibronectin binding is unaffected. The variant-specific study instead reports reduced Elongin C interaction, failure to restore HIF2a regulation, and defective fibronectin deposition for VHL R167Q, which is inconsistent with the benign functional paths specified for BS3.

VCEP rule

Evidence of benign effect for VHL Type 1 and 2A/B can be seen when HIF1/2a displays degradation (i.e. replicates WT function), and/or the VHL Elongin C, Elongin B, Cullin2 RBX1 (VCB-CR) E3 ubiquitin ligase complex stability is not affected and/or ECM formation/fibronectin binding is unaffected.

Evidence considered

• PMID:15574766: VHL R167Q showed reduced Elongin C interaction, failure to restore HIF2a regulation, and defective fibronectin deposition.
• PMID:15574766, abstract and Results: This mutation also failed to restore HIF2a regulation.
• VHL_Functional_Assay_SVI_documentation.xlsx was searched as the governing functional-assay file; it contains general assay specifications but no variant-specific row.

Still missing

• VHL_Functional_Assay_SVI_documentation.xlsx was searched for c.500G>A, p.Arg167Gln, and p.R167Q; no variant-specific entry or pre-assigned BS3 code was found.

Sources

Reasoning

The VCEP requires de novo scoring based on probands and phenotype specificity, but the available variant reports do not document confirmed or assumed de novo status, parental testing, maternity/paternity, or a VCEP-defined Danish-criteria phenotype for this variant. The variant is reported in several families and cohorts, but those reports do not establish de novo occurrence for any proband; therefore PS2 cannot be assessed without substituting a non-VCEP rule.

VCEP rule

A single proband cannot be very strong evidence, but multiple probands can be combined to reach very strong (4+ points). ... Phenotype highly specific for the gene (Danish Criteria) (≥2 but less than 4 _de novo_ points). ... Phenotype consistent but not highly specific. Ex. VHL spectrum cancer without family history or strong indication of VHL phenotype. (≥1 but less than 2 _de novo_ points) ... Phenotype consistent but not highly specific (≥0.5 but less than 1 _de novo_ points) Ex. subject included in a VHL cohort, but specific information on tumor types is not provided.

Evidence considered

• The VCEP PS2 rule assigns strength by de novo points and phenotype specificity, including Danish Criteria for highly specific VHL phenotypes.
• Gläscker et al. reported R167Q in one patient but did not report variant-specific parental testing, de novo status, or phenotype details sufficient for PS2.
• Ong et al. listed Arg167Gln in VHL families, but the extracted report does not establish de novo status or parental testing for the variant.
• Neumann et al. reported the corresponding codon-167 substitution in three independent patients without variant-specific de novo or parental data.
• Rocha et al. reported c.500G>A in family 16 but did not provide the parental/de novo evidence or phenotype scoring required by the VCEP PS2 rule.
• Brauch et al. listed c.500G>A/p.R167Q in multiple families but the extracted report does not provide the required de novo evidence.
• Ong et al. analyzed genotype-phenotype correlations but did not report a variant-specific de novo observation or usable PS2 phenotype score.

Still missing

• Variant-specific de novo observations in probands are not documented.
• Maternity and paternity confirmation or exclusion are not documented.
• Phenotype details needed to apply the VCEP's Danish Criteria are not documented for de novo probands.

Sources

Reasoning

The VCEP PM6 rule requires PS2-style de novo scoring and distinguishes assumed de novo observations from cases with confirmed maternity and paternity. The available reports identify R167Q in patients or families but do not document assumed or confirmed de novo status, parental testing, or maternity/paternity for any observation; PM6 therefore cannot be assessed.

VCEP rule

See PS2 evidence code for scoring and phenotypes. Assumed _de novo_ receives half the points as compared to maternity and paternity confirmed _de novo_. If paternity and maternity are not confirmed, score as the PM6 code. PM6 can receive “VeryStrong” strength. For example, if there are >4 de novo probands with Danish Criteria and none have paternity confirmed, this can receive PM6_VeryStrong. Note: the VCI as of Nov 2022 does not allow PM6_VeryStrong. Instead apply the PS2 evidence code and increase the strength to “VeryStrong” with a note that paternity and/or maternity is not confirmed.

Evidence considered

• The VCEP PM6 rule directs scoring to PS2 evidence, assigns half the points to assumed de novo observations compared with maternity- and paternity-confirmed de novo observations, and specifies how unconfirmed parentage is handled.
• The reviewed R167Q reports identify affected patients or families but do not state that the variant was assumed de novo or document maternity/paternity testing for any carrier.
• Rocha et al. reported c.500G>A in family 16, but familial occurrence alone does not establish the PM6 de novo evidence required by the VCEP.

Still missing

• No variant-specific assumed de novo observations are documented.
• No maternity or paternity confirmation status is documented.
• No PS2-style phenotype scoring is available for a de novo observation.

Sources

Reasoning

The VCEP PP1 rule requires a count of meioses across families. Although R167Q is reported in multiple families and cohorts, the available extracts do not provide variant-specific affected/unaffected segregation, phase, or a countable number of informative meioses. Multiple reported families cannot be converted into meioses under the VCEP rule, so PP1 is not assessed.

VCEP rule

>7 meioses across >=2 families ... 5 – 6 meioses across ≥1 family. ... 3 – 4 meioses across ≥1 family.

Evidence considered

• The VCEP PP1 rule assigns Supporting for 3–4 meioses, Moderate for 5–6 meioses, and Strong for more than 7 meioses across at least 2 families.
• Brauch et al. and Rocha et al. report R167Q in families, but the extracted findings do not provide variant-specific segregation or informative-meiosis counts.
• The reviewed cohort reports identify R167Q in multiple individuals or families without enough affected-relative genotype data to calculate PP1 meioses.

Still missing

• Informative meioses are not reported for the target variant.
• Variant-specific affected and unaffected relative genotypes are not reported.
• Segregation phase and family-level counts needed to distinguish 3–4, 5–6, or >7 meioses are unavailable.

Sources

Reasoning

The VHL VCEP requires at least three unaffected individuals aged 65 years or older harboring the same variant, with either full phenotyping and screening for strong BS2 or incomplete phenotyping and screening with no noted VHL-related cancers for BS2 Supporting. The available case evidence contains no qualifying individual-level age, phenotype, screening, and carrier data.

VCEP rule

VHL is not highly penetrant _at an early age._ BS2 can be applied if: There are at least 3 individuals, all >=65yo, unaffected, harboring the same variant, with full phenotyping and screening for the absence of VHL-related cancers. BS2_Supporting can be applied if: At least 3 individuals, all >=65yo, unaffected, harboring the same variant, lacking full phenotyping and screening, with no noted VHL-related cancers.

Evidence considered

• VHL VCEP specification v1.1, BS2 requires at least three unaffected carriers aged >=65 years; strong BS2 additionally requires full phenotyping and screening, while BS2 Supporting permits incomplete phenotyping and screening only when no VHL-related cancers are noted.
• No qualifying individual-level unaffected-carrier cohort or homozygote evidence is present for this variant.

Still missing

• Individual-level evidence for at least three carriers aged >=65 years, including carrier confirmation, VHL cancer screening, and completeness of phenotyping is unavailable.

Sources

Reasoning

The VCEP BS4 rule requires lack of segregation in affected members of at least one family, with Supporting for one family and Strong for two or more families. The available variant-specific extracts do not document affected relatives who were tested and lacked the variant; therefore non-segregation cannot be established and BS4 is not assessed.

VCEP rule

Lack of segregation is seen in affected members of ≥2 families ... Lack of segregation is seen in 1 family.

Evidence considered

• The VCEP BS4 rule requires lack of segregation in affected members of at least two families for Strong or one family for Supporting.
• The reviewed family reports mention R167Q but do not document affected relatives who tested negative for the variant, so lack of segregation cannot be counted.

Still missing

• No affected family member with a documented negative test for R167Q is reported.
• The number of families with demonstrated lack of segregation is unavailable.

Sources

Reasoning

BP2 is applicable under the VHL VCEP, but the available evidence does not document a phase-confirmed trans observation with a known pathogenic variant, a homozygous unaffected individual, or an observation in cis or with unknown phase with three or more different pathogenic VHL variants. The reviewed variant reports document occurrence of p.Arg167Gln in affected cohorts and families, but do not provide the phase or qualifying co-occurrence information required by the VCEP. No qualifying absence of congenital polycythemia is documented either, so the rule cannot be evaluated.

VCEP rule

BP2 should not be used in the presence of any clinical or molecular findings of congenital polycythemia ... i) -variant observed in trans with a known pathogenic variant (phase confirmed), in the absence of congenital polycythemia (clinical manifestations or molecular) ii) -OR observed in the homozygous state in an individual without personal &/or family history of Von Hippel-Lindau disease or congenital polycythemia iii) -OR observed _in cis_ or with unknown phase with three or more different pathogenic _VHL_ variants ... \-variant is observed _in cis_ (or phase is unknown) w/ a pathogenic _VHL_ variant

Evidence considered

• VHL VCEP CSPEC BP2 entry: BP2 should not be used in the presence of any clinical or molecular findings of congenital polycythemia; the VCEP provides strong paths for phase-confirmed trans, homozygous unaffected, or three-or-more pathogenic VHL variants in cis/unknown phase, and a supporting path for one pathogenic VHL variant in cis or with unknown phase.
• Gläs​ker et al. 1999 (PMID:10567493), Results Table 1: VHL R167Q (nt 713 G/A; Arg→Gln) was identified in 1 patient, but the extraction reports no variant-specific phase, segregation, or congenital-polycythemia observation.
• Ong et al. 2007 (PMID:17024664), Table 1: p.Arg167Gln is listed among surface missense substitutions in a cohort of 573 individuals from 200 VHL kindreds; no variant-specific phase or qualifying allelic co-occurrence is reported.
• Neumann et al. 2002 (PMID:12000816), Table 2: the corresponding Arg167Gln mutation was found in 3 independent patients; no variant-specific phase, segregation, or qualifying second pathogenic allele is reported.
• Rocha et al. 2003 (PMID:12624160), Results/Table 1 Family 16: c.500G>A (Arg167Gln) was identified in a familial type 2 VHL family; the extraction reports no phase-resolved co-occurrence with another pathogenic VHL variant or congenital-polycythemia assessment.
• Crossey et al. 1994 (PMID:8270255) was not used because no paper_extracts entry was available for this source and the available case evidence did not establish BP2 phase or allelic observations.
• The case evidence contains no documented phase-confirmed trans/cis result, homozygous unaffected individual, or three pathogenic VHL variants in cis/unknown phase for this variant.

Still missing

• Phase-resolved family or proband data identifying a pathogenic variant in trans or cis with NM_000551.4:c.500G>A.
• Documentation that the relevant individual lacks clinical or molecular congenital polycythemia when required by the applicable BP2 path.
• Alternatively, documentation of a homozygous unaffected individual or three different pathogenic VHL variants in cis or with unknown phase.

Sources

Reasoning

The variant is a missense substitution and is within the VCEP's splice-impact assessment scope. The governing BP4 rule requires concordance of both SpliceAI (<=0.1) and VarSeak (Class 1 or Class 2). SpliceAI is available with a maximum delta score of 0.026, which meets its cutoff, but no VarSeak class result is available; therefore the required concordance cannot be evaluated. The SpliceAI result is not reused for BP7.

VCEP rule

BP4 can be applied to assess lack of splicing impact, with concordance of Splice AI (≤0.1) and VarSeak (Class 1 or Class 2).

Evidence considered

• VHL VCEP BP4 rule requires concordance of SpliceAI (<=0.1) and VarSeak (Class 1 or Class 2).
• SpliceAI Lookup for NM_000551.4:c.500G>A: DS_AG 0.002, DS_AL 0.011, DS_DG 0.026, DS_DL 0.000; maximum delta score 0.026.
• No VarSeak Class 1 or Class 2 result is present for this variant, so BP4 cannot be assigned.

Still missing

• VarSeak classification for NM_000551.4:c.500G>A is unavailable; BP4 requires SpliceAI and VarSeak concordance.

Sources

Reasoning

BP5 is applicable under the VHL VCEP, but the available case evidence does not document two or more qualifying co-occurrences with a highly penetrant pathogenic variant in another gene that fully explains each patient's phenotype and meets the VHL-specific exclusion conditions.

VCEP rule

BP5 can be applied for two or more co-occurrences with pathogenic variants in a different gene that fully explained the patient's phenotype, but specific circumstances would need to be met in order for a case to be considered for inclusion. First, the variant in the other gene must be considered highly penetrant, with both the individual's age, tumour type and gender taken into consideration. Additionally, the patient's personal and family history (including up to 2nd degree relatives) should not overlap with features seen in VHL and VHL tumour histologies.

Evidence considered

• The reviewed variant-specific publications report VHL carriers and phenotypes but do not document two qualifying co-occurrences with pathogenic variants in different genes under the VHL VCEP conditions.

Still missing

• Need documented co-occurrence data for at least two individuals, including the other pathogenic variant, its penetrance, and phenotype fit.
• Need confirmation that each patient's personal and second-degree family history does not overlap VHL manifestations or VHL tumour histologies.

Sources