53 patients with a molecularly confirmed VHL mutation (by PCR through EDTA-anticoagulated blood) and a pancreatic neuroendocrine tumor (ICT) were collected from the German NET-Registry and the German VHL-registry. An additional 296 patients were stated in this paper with a VHL associated germline mutation, but without pancreatic neuroendocrine tumors. 5 patients were found to have the above mutation (c.548C>A). 1 was a VHL patient with an ICT (pancreatic neuroendocrine tumor) taken from the VHL-registry. The other 4 patients were stated to be in this study with the given mutation, however no ICT. Other clinical manifestations outside of a pancreatic neuroendocrine tumor are possible, but were not discussed. ACMG evidence codes: 'PVS1' because they observe a nonsense variant in a gene where loss of function is a known mechanism of disease .

PatientID: 2

KindredID: 2

Case: M, 53Y0M, Ethnicity Unknown

DiseaseAssertion: VHL

FamilyInfo: N/A

CasePresentingHPOs: HP:0006880; HP:0000107 (Cerebellar hemangioblastoma, Renal cyst)

CaseHPOFreeText: Cerebellum hemangioblastoma.

CaseNotHPOs: N/A

CaseNotHPOFreeText: N/A

CasePreviousTesting: N/A

PreviouslyPublished: N/A

SupplementalData: N/A

Variant: p.S183* (c.548C>A)

LegacyVariant: c.761C>A

CaseProblemVariantFreeText: N/A

ClinVarID: 2215, https://www.ncbi.nlm.nih.gov/clinvar/variation/2215/

CAID: N/A

gnomAD: N/A

VariantEvidence: Preoperative hemoglobin: 17.9. Postoperative hemoglobin: 15.1.

MutationType: stop_gained; missense_variant;transversion

CivicName: S183* (c.548C>A)

MultipleGeneVariants: N/A

Tissue analysis from 61 VHL patients, 30 sporadic renal cell carcinoma (RCC) tumor samples, and 6 sporadic RCC cell lines revealed 22 variants within VHL patients, 10 variants in tumor samples and variants in all cell lines. All mutations cluster to the 3’ end of the VHL gene open reading frame, implicating this region as important for the tumor-suppressor function of the VHL protein. Northern blot, SSCP and RT-PCR analysis from normal tissue found a germline p.S183* (c.548C>A) mutation, stated in the paper as c.761C>A due to an older transcript. It was seen in family [pedigree No] 22, with an unknown number of members described as ‘VHL-affected’ with RCC. Other manifestations are possible but not clear.

GroupID/ KindredID: VHL 6

DiseaseAssertion: assumed VHL

GroupSize: 3

GroupNumberMales: N/A

GroupNumberFemales: N/A

GroupEthnicity: Italian

GroupAgeRange: N/A

GroupInfo: 3 patients with the given mutation belonging to VHL family 6

GroupPresentingHPOs: HP:0009711; HP:0010797; HP:0000107; HP:0005584; HP:0001737 (retinal capillary hemangioma, hemangioblastoma, renal cyst, renal cell carcinoma, pancreatic cysts)

GroupHPOFreeText: all 3 patients have pancreatic cysts, 2 had retinal capillary hemangiomas, hemangioblastoma, and renal cysts, and 1 had RCC

GroupNotHPOs: N/A

GroupNotHPOFreeText: N/A

GroupPreviousTesting: N/A

PreviouslyPublished: N/A

SupplementalData: N/A

Variant: p.S183* (c.548C>A)

CaseProblemVariantFreeText: N/A

LegacyVariant: p.S183* (c.761C>A)

ClinVarID: 2215 https://www.ncbi.nlm.nih.gov/clinvar/variation/2215/

CAID: N/A

gnomAD: N/A

VariantEvidence: N/A

MutationType: stop_gained

CivicName: S183* (c.548C>A)

MultipleGeneVariants: N/A

Between 1994 and 2001, the Dutch VHL working group conducted DNA analysis on 146 probands with VHL-associated manifestations. DNA of probands was extracted from peripheral blood samples. Exons 1, 2 and 3 of the VHL gene and their flanking untranslated and intronic coding sequences were amplified by PCR and subjected to semi-automated sequence analysis. Screening for structural rearrangements, including deletions, involving the VHL gene was performed by Southern blot analysis and the multiplex ligation-dependent probe amplification (MLPA) assay. Deletions encompassing the entire VHL gene were confirmed by FISH analysis on lymphocytes from affected individuals. VHL mutations were identified in 43 families. This mutation (c.548C>A) was detected in 1 member of family 22. The patient presented with CNS hemangioblastoma and retinal capillary hemangioma. The patient does not have a family history of VHL disease. The genetic information of the patient's parents are not available.

216 patients with clinically expected VHL disease due to family history or presence of VHL typical tumours were routinely examined in an eye centre in Germany between January 2019 and January 2020, majority of which were of European descent (~95%). Since data on the genotype–phenotype correlation of the onset of Retinal Hemangioblastoma (RH) is scarce in relation to VHL disease, this study wanted to inversitage the relationship between VHL gene variants (truncating vs single amino acid substitutions) and the ocular phenotype of the disease. 42 different rare VHL gene variants in 148 patients were detected - some of whom had RH and some who did not. Although date of onset of RH, as well as data on central nervous system haemangioblastomas (CNSH), pheochromocytoma/paraganglioma (PPGL), clear cell renal cell carcinoma (ccRCC) and pancreatic neuroendocrine neoplasm (PNEN) were collected, it was not reported on a per patient basis and therefore could not be discussed per variant. There were 3 patients reported with the p.S183* (c.548C>A) mutation. All 3 members presented with retinal hemangiomas, while no other clinical manifestations were discussed. Additionally, no family information was provided.

The authors described the growth kinetics of 96 renal tumours in 64 VHL patients (32 males, 32 females). All patients had VHL disease with renal involvement. 54/64 had undergone renal surgery or a radiofrequency thermal ablation procedure for RCC. In 10/64 patients, at least one or more RCC was diagnosed. However, at the time of diagnosis, an indication for treatment was not yet present. For all patients, several CT/MRI scans existed for retrospective description of tumour growth kinetics. 10-ml EDTA anticoagulated blood samples were available from each patient for mutation screening of all exons of the VHL gene. Genomic DNA from blood samples was extracted using standard methods. Small deletions/insertions or point mutations were identified by DNA sequencing followed by complete mutation screening using PCR/single-strand conformation polymorphism and denaturing high-performance liquid chromatography. A germline nonsense variant p.S183*(c.548C>A) was identified in patient #57. This mutation was noted as c.761C>A in the paper due to a difference in reference transcript used (see Table S2).

A cohort of 141 patients with hemangioblastomas of the central nervous system was analyzed. 81 patients had germline mutations in the VHL gene, analyzed through peripheral blood by Southern blotting and SSCP analysis. This nonsense mutation (c.548C>A) was recorded in the paper as c.761C>A due to a difference in transcript sequence. It was found in 4 patients with hemangioblastomas of the central nervous system. Any family information or relations is unknown. No other phenotypes described.

GroupID: 27B

DiseaseAssertion: Unknown. Patients were pulled from either the VHL-Registry or NET-Registry and recorded with the given mutation. Symptoms or manifestations are not clear.

GroupSize: 4

GroupNumberMales: N/A

GroupNumberFemales: N/A

GroupEthnicity: N/A

GroupAgeRange: N/A

GroupInfo: 4 patients with the given mutation were grouped as group 27(B) in this study. These patients were asymptomatic or unclear of symptoms.

GroupPresentingHPOs: N/A

GroupHPOFreeText: N/A

GroupNotHPOs: HP:0030405 (pancreatic endocrine tumor)

GroupNotHPOFreeText: N/A

PreviouslyPublished: N/A

SupplementalData: N/A

Variant: p.S183* (c.548C>A)

LegacyVariant: N/A

CaseProblemVariantFreeText: N/A

ClinVarID: 2215 https://www.ncbi.nlm.nih.gov/clinvar/variation/2215/

CAID: N/A

gnomAD: N/A

VariantEvidence: N/A

MutationType: stop_gained

CivicName: S183* (c.548C>A)

MultipleGeneVariants: N/A

GroupID: 27A

Case: Sex, Age, Ethnicity unknown

DiseaseAssertion: VHL

FamilyInfo: None. The patient with the given mutation in this study was a VHL patient with pancreatic ICTs.

CasePresentingHPOs: HP:0030405 (pancreatic endocrine tumor)

CaseHPOFreeText: Patient has islet cell tumors (ICTs), or listed as pancreatic endocrine tumor.

CaseNotHPOs: N/A

CaseNotHPOFreeText: N/A

CasePreviousTesting: N/A

PreviouslyPublished: N/A

SupplementalData: N/A

Variant: p.S183* (c.548C>A)

LegacyVariant: N/A

CaseProblemVariantFreeText: N/A

ClinVarID: 2215 https://www.ncbi.nlm.nih.gov/clinvar/variation/2215/

CAID: N/A

gnomAD: N/A

VariantEvidence: N/A

MutationType: stop_gained

CivicName: S183* (c.548C>A)

MultipleGeneVariants: N/A