A study investigated paraganglioma/pheochromocytoma tumors in a retrospective cohort and a prospective cohort. Tumour tissue samples were sequenced by next-generation sequencing using a custom gene panel. In some cases, Sanger sequencing was used to detect germline mutations. Some of these tumors were found with germline VHL mutations. Patient ID 100, from the prospective cohort, presented with multiple paragangliomas and a germline mutation in the VHL gene c.191G>C. Other phenotypic and demographic information was not available. The paper performed carbonic anhydrase IX immunohistochemistry and immunostaining with CA9 was observed in patient 100.

PatientID: 94

KindredID: 94

Case: Sex Unknown, 27Y0M, Ethnicity Unknown

DiseaseAssertion: Assumed VHL

FamilyInfo: N/A

CasePresentingHPOs: HP:0002666 (Pheochromocytoma)

CaseHPOFreeText: Multiple benign PCC.

CaseNotHPOs: N/A

CaseNotHPOFreeText: N/A

CasePreviousTesting: N/A

PreviouslyPublished: N/A

SupplementalData: Patient information pulled from Supplemental Table 1a.

Variant: p.V166F (c.496G>T)

LegacyVariant: N/A

CaseProblemVariantFreeText: N/A

ClinVarID: 2224, https://www.ncbi.nlm.nih.gov/clinvar/variation/2224/

CAID: N/A

gnomAD: N/A

VariantEvidence: Carbonic anhydrase 9 (CA9) mRNA level of 26.95 was detected. Membrane CA9 immunostaining was observed.

MutationType: missense_variant;transversion

CivicName: V166F (c.496G>T)

MultipleGeneVariants: N/A

PatientID: 85

KindredID: 85

Case: Sex Unknown, Age Unknown, Ethnicity Unknown

DiseaseAssertion: VHL

FamilyInfo: Family history of VHL but no details provided.

CasePresentingHPOs: HP:0002666 (Pheochromocytoma)

CaseHPOFreeText: N/A

CaseNotHPOs: N/A

CaseNotHPOFreeText: N/A

CasePreviousTesting: N/A

PreviouslyPublished: N/A

SupplementalData: Patient information pulled from Supplemental Table 1. There is additional tumour specific information in Supplemental Table 2.

Variant: p.P138P (c.414A>G)

LegacyVariant: N/A

CaseProblemVariantFreeText: N/A

ClinVarID: 223206, https://www.ncbi.nlm.nih.gov/clinvar/variation/223206/

CAID: N/A

gnomAD: N/A

VariantEvidence: Functionally validated by mRNA analysis, which showed a splicing alteration (data not shown). Membrane CA9 immunostaining was observed (supplemental table 1).

MutationType: missense_variant;transition; synonymous_variant

CivicName: P138= (c.414A>G)

MultipleGeneVariants: N/A

PatientID: 57

KindredID: 57

Case: Sex Unknown, 16Y0M, Ethnicity Unknown

DiseaseAssertion: Pheochromocytoma

FamilyInfo: N/A

CasePresentingHPOs: HP:0002666 (pheochromocytoma)

CaseHPOFreeText: The patient had a single pheochromocytoma.

CaseNotHPOs: N/A

CaseNotHPOFreeText: N/A

CasePreviousTesting: N/A

PreviouslyPublished: N/A

SupplementalData: Patient information pulled from Supplemental Table 1. There is additional tumour specific information in Supplemental Table 2.

Variant: p.R167Q (c.500G>A)

LegacyVariant: N/A

CaseProblemVariantFreeText: N/A

ClinVarID: 2216 https://www.ncbi.nlm.nih.gov/clinvar/variation/2216/

CAID: N/A

gnomAD: 0.000003977 https://gnomad.broadinstitute.org/variant/3-10191507-G-A

VariantEvidence: Carbonic anhydrase 9 (CA9) mRNA level of 25.67 was detected. Membrane CA9 immunostaining was observed (supplemental table 1).

MutationType: missense_variant;transition

CivicName: R167Q (c.500G>A)

MultipleGeneVariants: N/A

PatientID: 74

KindredID: 74

Case: Sex Unknown, 31Y0M, Ethnicity Unknown

DiseaseAssertion: Assumed VHL

FamilyInfo: N/A

CasePresentingHPOs: HP:0002666 (Pheochromocytoma)

CaseHPOFreeText: Single benign PCC.

CaseNotHPOs: N/A

CaseNotHPOFreeText: N/A

CasePreviousTesting: N/A

PreviouslyPublished: N/A

SupplementalData: Patient information pulled from Supplemental Table 1a.

Variant: Complete Deletion (c.1-?_642+?del)

LegacyVariant: N/A

CaseProblemVariantFreeText: N/A

ClinVarID: N/A

CAID: N/A

gnomAD: N/A

VariantEvidence: Carbonic anhydrase 9 (CA9) mRNA level of 34.31 was detected. Membrane CA9 immunostaining was observed.

MutationType: deletion

CivicName: Deletion

MultipleGeneVariants: N/A

PatientID: 42

KindredID: 42

Case: Sex Unknown, 40Y0M, Ethnicity Unknown

DiseaseAssertion: Assumed VHL

FamilyInfo: N/A

CasePresentingHPOs: HP:0002666 (Pheochromocytoma)

CaseHPOFreeText: Multiple benign PCC.

CaseNotHPOs: N/A

CaseNotHPOFreeText: N/A

CasePreviousTesting: N/A

PreviouslyPublished: N/A

SupplementalData: Patient information pulled from Supplemental Table 1a.

Variant: p.L178P (c.533T>C)

LegacyVariant: N/A

CaseProblemVariantFreeText: N/A

ClinVarID: 428795, https://www.ncbi.nlm.nih.gov/clinvar/variation/428795/

CAID: N/A

gnomAD: N/A

VariantEvidence: Membrane CA9 immunostaining was observed.

MutationType: missense_variant;transition

CivicName: L178P (c.533T>C)

MultipleGeneVariants: N/A

PatientID: 100

KindredID: 100

Case: Sex Unknown, Age Unknown, Ethnicity Unknown

DiseaseAssertion: Paraganglioma

FamilyInfo: N/A

CasePresentingHPOs: HP:0002668 (paraganglioma)

CaseHPOFreeText: The patient had multiple paragangliomas.

CaseNotHPOs: N/A

CaseNotHPOFreeText: N/A

CasePreviousTesting: N/A

PreviouslyPublished: N/A

SupplementalData: Patient information pulled from Supplemental Table 1. There is additional tumour specific information in Supplemental Table 2.

Variant: p.R64P (c.191G>C)

ClinVarID: 2226 https://www.ncbi.nlm.nih.gov/clinvar/variation/2226/

gnomAD: N/A

VariantEvidence: Membrane CA9 immunostaining was observed (supplemental table 1).

MutationType: missense_variant;transversion

CivicName: R64P(c.191G>C)

MultipleGeneVariants: N/A

PatientID: 40/41

KindredID: 40/41

Case: Sex Unknown, 15Y0M, Ethnicity Unknown

DiseaseAssertion: Assumed VHL

FamilyInfo: N/A

CasePresentingHPOs: HP:0002666 (Pheochromocytoma)

CaseHPOFreeText: Multiple benign PCC.

CaseNotHPOs: N/A

CaseNotHPOFreeText: N/A

CasePreviousTesting: N/A

PreviouslyPublished: N/A

SupplementalData: Patient information pulled from Supplemental Table 1a.

Variant: p.L178P (c.533T>C)

LegacyVariant: N/A

CaseProblemVariantFreeText: N/A

ClinVarID: 428795, https://www.ncbi.nlm.nih.gov/clinvar/variation/428795/

CAID: N/A

gnomAD: N/A

VariantEvidence: Patient data is extracted from two rows assumed to be referring to the same patient (i.e. tumor samples 40 and 41 in Supplementary Table 1a). Carbonic anhydrase 9 (CA9) mRNA level of 795.70 (PCC) and 815.38 (PCC) were detected. Membrane CA9 immunostaining was observed in both PCC samples.

MutationType: missense_variant;transition

CivicName: L178P (c.533T>C)

MultipleGeneVariants: N/A

PatientID: 10

KindredID: 10

Case: Sex Unknown, 26Y0M, Ethnicity Unknown

DiseaseAssertion: Pheochromocytoma

FamilyInfo: N/A - not stated

CasePresentingHPOs: HP:0002666 (pheochromocytoma)

CaseHPOFreeText: A pheochromocytoma (PCC) diagnosed first at 11Y was found, with follow up of a 15 year duration.

CaseNotHPOs: N/A

CaseNotHPOFreeText: N/A

CasePreviousTesting: N/A

PreviouslyPublished: N/A

SupplementalData: Yes

Variant: c.340+682T>C

LegacyVariant: N/A

CaseProblemVariantFreeText: N/A

ClinVarID: 964770 https://www.ncbi.nlm.nih.gov/clinvar/variation/964770

CAID: N/A

gnomAD: N/A

VariantEvidence: Tumor analysis was performed since there was a paraffin embedded sample. Patient 10 did not have loss of heterozygosity, but was found to have a somatic variant in the exon 3 of VHL (c.482G>A; p.Arg161Gln). This was the only patient observed to have a cluster of tumor cells with a positive membranous CA9 immunostaining, this can be seen in VHL-related PPGL. CA9 is a protein reported to influence regulation cell proliferation and tumor progression and is known to be expressed at the membrane of tumour cells in the case of VHL inactivation can be seen in VHL-related PPGL. Subsequently as expected, the PPGL of patient 10 had a significantly low expression of VHL E1-E1' mRNA VHL and PNMT mRNA compared to VHL-tumours used as controls. This suggests that the two VHL variants change the VHL mRNA splicing.

MutationType: splice_site_variant

CivicName: Splice Site (c.340+682T>C); intron_variant

MultipleGeneVariants: Somatic, VHL, c.482G>A (p.Arg161Gln)