1. Last 7 days
    1. 3050Mnt 712 C to T, Arg to TrpR, 13407

      PatientID: 30

      KindredID: 3407

      Case: M, 50Y0M, Ethnicity Unknown

      DiseaseAssertion: VHL

      FamilyInfo: Another member of his family (patient 31) presented with one radiologic pancreatic neuroendocrine tumor and had the same VHL gene mutation.

      CasePresentingHPOs: HP:0030405 (pancreatic endocrine tumour)

      CaseHPOFreeText: He presented with one radiologic pancreatic endocrine tumour.

      CaseNotHPOs: N/A

      CaseNotHPOFreeText: N/A

      CasePreviousTesting: N/A

      PreviouslyPublished: N/A

      SupplementalData: N/A

      Variant: p.R167W (c.499C>T)

      LegacyVariant: p.R167W (c.712C>T)

      CaseProblemVariantFreeText: N/A

      ClinVar: 2218 https://www.ncbi.nlm.nih.gov/clinvar/variation/2218/

      CAID: CA020450 http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/allele?hgvsOrDescriptor=NM_000551.3%3Ac.499C%3ET

      gnomAD: 0.000007954 https://gnomad.broadinstitute.org/variant/3-10191506-C-T

      VariantEvidence: N/A

      MutationType: missense_variant;transition

      CivicName: R167W (c.499C>T)

      MultipleGeneVariants: N/A

    2. nt 454 C to T, Pro to Ser

      PatientID: 43

      KindredID: 5959

      Case: F, 35Y0M, Ethnicity Unknown

      DiseaseAssertion: VHL

      FamilyInfo: N/A

      CasePresentingHPOs: HP:0030405 (pancreatic endocrine tumour)

      CaseHPOFreeText: She presented with four pathologic pancreatic neuroendocrine tumors.

      CaseNotHPOs: N/A

      CaseNotHPOFreeText: N/A

      CasePreviousTesting: N/A

      PreviouslyPublished: N/A

      SupplementalData: N/A

      Variant: p.P81S (c.241C>T)

      LegacyVariant: p.P81S (c.454C>T)

      CaseProblemVariantFreeText: N/A

      ClinVar: 2233 https://www.ncbi.nlm.nih.gov/clinvar/variation/2233/

      CAID: CA020148 http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/allele?hgvsOrDescriptor=NM_000551.3%3Ac.241C%3ET

      gnomAD: 0.0002179 https://gnomad.broadinstitute.org/variant/3-10183772-C-T

      VariantEvidence: N/A

      MutationType: missense_variant;transition

      CivicName: P81S (c.241C>T)

      MultipleGeneVariants: Germline, VHL, c.562C>G (p.Leu88Val)

    1. PatientID: 50

      KindredID: 17

      Case: F, 30Y0M, Chinese

      DiseaseAssertion: VHL

      FamilyInfo: N/A

      CasePresentingHPOs: HP:0001737; HP:0001732; HP:0005584 (Pancreatic cysts, Abnormality of the pancreas, Renal cell carcinoma)

      CaseHPOFreeText: The proband presented with multiple pancreatic cysts/tumors and renal cell carcinoma at 30.

      CaseNotHPOs: HP:0002666; HP:0009711; HP:0010797 (Pheochromocytoma, Retinal capillary hemangioma, Hemangioblastoma)

      CaseNotHPOFreeText: N/A

      CasePreviousTesting: N/A

      PreviouslyPublished: This patient was also published in a later paper by Wang et al., 2018 (PMID: 29330336).

      SupplementalData: Patient and family genotype-phenotype information is listed in Supplementary Table 1.

      Variant: p.L85P (c.254T>C)

      LegacyVariant: N/A

      CaseProblemVariantFreeText: N/A

      ClinVarID: 376222; https://www.ncbi.nlm.nih.gov/clinvar/variation/376222/

      CAID: N/A

      gnomAD: N/A

      VariantEvidence: N/A

      MutationType: missense_variant;transition

      CivicName: L85P (c.254T>C)

      MultipleGeneVariants: N/A

    2. PatientID: 131

      KindredID: 49

      Case: F, Age 31Y0M, Chinese

      DiseaseAssertion: VHL

      FamilyInfo: N/A

      CasePresentingHPOs: HP:0002666; HP:0005584; HP:0001737; HP:0001732; HP:0010797 (Pheochromocytoma, Renal cell carcinoma, Pancreatic cysts, Abnormality of the pancreas, Hemangioblastoma)

      CaseHPOFreeText: The proband presented with a pheochromocytoma at 18, retinal angioma at 29, and renal cell carcinoma and multiple pancreatic cysts/tumors at 31.

      CaseNotHPOs: HP:0010797 (Hemangioblastoma)

      CaseNotHPOFreeText: N/A

      CasePreviousTesting: N/A

      PreviouslyPublished: This patient was also published in a later paper by Wang et al., 2018 (PMID: 29330336).

      SupplementalData: Patient and family genotype-phenotype information is listed in Supplementary Table 1.

      Variant: p.F136C (c.407T>G)

      LegacyVariant: N/A

      CaseProblemVariantFreeText: N/A

      ClinVarID: 496065; https://www.ncbi.nlm.nih.gov/clinvar/variation/496065/

      CAID: N/A

      gnomAD: N/A

      VariantEvidence: N/A

      MutationType: missense_variant;transversion

      CivicName: F136C (c.407T>G)

      MultipleGeneVariants: N/A

    3. PatientID: 54

      KindredID: 19

      Case: F, 33Y0M, Chinese

      DiseaseAssertion: VHL

      FamilyInfo: The proband (patient 54) has a daughter who was genetically confirmed with the same mutation. The daughter was asymptomatic at the time of analysis.

      CasePresentingHPOs: HP:0009711; HP:0001737; HP:0001732; HP:0005584 (Retinal capillary hemangioma, Pancreatic cysts, Abnormality of the pancreas, Renal cell carcinoma)

      CaseHPOFreeText: The proband presented with a retinal angioma at 12, renal cell carcinoma and multiple pancreatic cysts/tumors at 33.

      CaseNotHPOs: HP:0010797; HP:0002666 (Hemangioblastoma, Pheochromocytoma)

      CaseNotHPOFreeText: N/A

      CasePreviousTesting: N/A

      PreviouslyPublished: This patient was also published in a later paper by Wang et al., 2018 (PMID: 29330336).

      SupplementalData: Patient and family genotype-phenotype information is listed in Supplementary Table 1.

      Variant: p.P86S (c.256C>T)

      LegacyVariant: N/A

      CaseProblemVariantFreeText: N/A

      ClinVarID: 178692; https://www.ncbi.nlm.nih.gov/clinvar/variation/178692/

      CAID: N/A

      gnomAD: N/A

      VariantEvidence: N/A

      MutationType: missense_variant;transition

      CivicName: P86S (c.256C>T)

      MultipleGeneVariants: N/A

    4. PatientID: 231

      KindredID: 86

      Case: M, 54Y0M, Chinese

      DiseaseAssertion: VHL

      FamilyInfo: The proband (patient 229) had a sister (patient 230) who was genetically confirmed with the mutation, and presented with a CNS hemangioblastoma at age 40. The proband's brother 1 (patient 231) was also confirmed with the mutation and had a retinal angioma at 25, and renal cell carcinoma at 54. The proband's brother 2 passed away before they could be diagnosed/ confirmed with the mutation, but they had a CNS hemangioblastoma at 35. Finally, the proband's nephew (son of brother 2) was genetically confirmed with the mutation and presented with renal cell carcinoma at age 30.

      CasePresentingHPOs: HP:0009711; HP:0005584 (Retinal capillary hemangioma, Renal cell carcinoma)

      CaseHPOFreeText: The proband's brother (patient 231) had a retinal angioma at 25 and renal cell carcinoma at 54.

      CaseNotHPOs: HP:0010797; HP:0002666; HP:0001737; HP:0001732 (Hemangioblastoma, Pheochromocytoma, Pancreatic cysts, Abnormality of the pancreas)

      CaseNotHPOFreeText: N/A

      CasePreviousTesting: N/A

      PreviouslyPublished: This patient was also published in a later paper by Wang et al., 2018 (PMID: 29330336).

      SupplementalData: Patient and family genotype-phenotype information is listed in Supplementary Table 1.

      Variant: p.L178R (c.533T>G)

      LegacyVariant: N/A

      CaseProblemVariantFreeText: N/A

      ClinVarID: 625261, https://www.ncbi.nlm.nih.gov/clinvar/variation/625261/

      CAID: N/A

      gnomAD: N/A

      VariantEvidence: N/A

      MutationType: missense_variant;transversion

      CivicName: L178R (c.533T>G)

      MultipleGeneVariants: N/A

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    1. X214L one case (female)

      PatientID: None

      KindredID: 2

      Case: F, 38Y0M, Ethnicity Unknown

      DiseaseAssertion: Retinal hemangioblastoma

      FamilyInfo: N/A

      CasePresentingHPOs: HP:0009711 (Retinal hemangioblastoma)

      CaseHPOFreeText: The patient was diagnosed with bilateral retinal hemangioblastoma at an unknown age.

      CaseNotHPOs: N/A

      CaseNotHPOFreeText: N/A

      CasePreviousTesting: N/A

      PreviouslyPublished: N/A

      SupplementalData: N/A

      Variant: p.*214Lext*14(c.641G>T)

      LegacyVariant: N/A

      ClinVarID: 223236, http://www.ncbi.nlm.nih.gov/clinvar/variation/223236

      gnomAD: N/A

      VariantEvidence: N/A

      MutationType: stop_loss

      CivicName: *214Lext*14(c.641G>T)

      MultipleGeneVariants: N/A

    1. c.473T>A [TRANSCRIPT ID- ENST00000256474, {"type":"entrez-nucleotide","attrs":{"text":"NM_000551","term_id":"1779521775","term_text":"NM_000551"}}NM_000551] (Figure 3), with genomic coordinate chr3: 10,149,796 [hg38 build] leading to substitution of amino acid leucine to glutamine at position 158 [p.L158Q] in the Elongin C binding domain of pVHL. After a thorough search of databases and literature, this variation was previously unreported as a causative variant of VHL. However, according to the dbSNP build 142, this variant has been flagged as clinically associated, the details of which will be scrutinized further under Section 4. Deletion analysis did not reveal any major loss of genetic material in all the three samples. This variant was also not observed in 50 age and ethnicity matched controls. Further in the two affected individuals a variation in intron 1, c.340+5G>C,

      A 42 year old Chinese male presented with cerebral hemangioblastoma, clear cell renal cell carcinoma, and an epididymal tumour. PCR testing of peripheral blood confirmed two familial mutations, a missense (c.256C > T) and a splice site mutation (c.340+5G>C) of the VHL gene. The patient has a 34 year old sister with the same mutations who presented with cerebral hemangioblastoma, pancreatic cysts, and liver cysts.

    1. We describe here a case of von Hippel-Lindau (VHL) disease with a serous cystic neoplasm of the whole pancreas. The patient was a 35-year-old woman suffering from a palpable abdominal tumor. She had a history of hemangioblastomas of the cerebellum. CT revealed large solid tumors in the pancreatic head and body, and multiple cystic lesions in the whole pancreas as well as a right renal tumor. When endoscopic retrograde cholangiopancreatography (ERCP) was performed, bleeding from the duodenal papilla was detected. Since she had some distinguishing clinical features, the diagnosis of VHL disease was made. The preoperative diagnosis of the pancreatic lesion was serous cystic neoplasms with hemosuccus pancreaticus and total pancreatectomy was performed. Histological examination of the specimen revealed serous cystic neoplasms which occupied the entire pancreas. VHL cases operated on for serous cystic neoplasms of the entire pancreas are very rare.

      PatientID: 1

      KindredID: 1

      Case: F, 35Y0M, Japanese

      DiseaseAssertion: VHL

      FamilyInfo: No relevant family history - speculated de novo but did not perform genetic testing of family members

      CasePresentingHPOs: HP:0002894; HP:0005584; HP:0006880 (pancreatic neoplasm; renal cell carcinoma; hemangioblastomas of the cerebellum)

      CaseHPOFreeText: A 35Yo woman became aware of a tumour - giant masses in the whole pancreas and a tumor of the right kidney - after giving birth to a healthy boy. History of cerebal hemangiomas treated by surgery 14Y previously (at 21 years old). (serous cystic neoplasms pancreas; Right renal cell carcinoma; hemangioblastomas of the cerebellum)

      CaseNotHPOs: N/A

      CaseNotHPOFreeText: N/A

      CasePreviousTesting: N/A

      PreviouslyPublished: N/A

      SupplementalData: N/A

      Variant: p.N78I (c.233A>T)

      LegacyVariant: N/A

      CaseProblemVariantFreeText: N/A

      ClinVarID: 223169 https://www.ncbi.nlm.nih.gov/clinvar/variation/223169/

      CAID: N/A

      gnomAD: N/A

      VariantEvidence: N/A

      MutationType: missense_variant;transversion

      CivicName: N78I (c.233A>T)

      MultipleGeneVariants: N/A

    2. We describe here a case of von Hippel-Lindau (VHL) disease with a serous cystic neoplasm of the whole pancreas. The patient was a 35-year-old woman suffering from a palpable abdominal tumor. She had a history of hemangioblastomas of the cerebellum. CT revealed large solid tumors in the pancreatic head and body, and multiple cystic lesions in the whole pancreas as well as a right renal tumor. When endoscopic retrograde cholangiopancreatography (ERCP) was performed, bleeding from the duodenal papilla was detected. Since she had some distinguishing clinical features, the diagnosis of VHL disease was made. The preoperative diagnosis of the pancreatic lesion was serous cystic neoplasms with hemosuccus pancreaticus and total pancreatectomy was performed. Histological examination of the specimen revealed serous cystic neoplasms which occupied the entire pancreas. VHL cases operated on for serous cystic neoplasms of the entire pancreas are very rare.

      ArticleReferenceSequence: Not specified – Assumption: variants assumed to have been documented by the author under reference sequence NM_000551.3

      GenotypingMethod: Not specified

      SamplingMethod: Not specified

    1. Germline mutations were identified in 300/469 (63%) of the families tested; 137 distinct intragenic germline mutations were detected.

      Germline mutational data on VHL was gathered from various countries to assess variation in germline mutations that produce disease within different populations. Families were grouped according to three possible outcomes of disease: renal cell carcinoma without pheochromocytoma, renal cell carcinoma with pheochromocytoma, or pheochromocytoma alone. SSCP and denaturing gradient gel electrophoresis identified mutations in 300/469 (63%) of families affected with VHL. This germline familial VHL mutation (Y175* (c.525C>G)) was taken from the Neuro Olcologie database in Paris, with an unknown number of members belonging to Family ID 63F. It was reported in the paper as c.738C>G due to a difference in transcript reference. As reported in Table 3, this mutation is found in VHL families without pheochromocytoma, and with renal cell carcinoma.

    2. Germline mutations were identified in 300/469 (63%) of the families tested; 137 distinct intragenic germline mutations were detected.

      GroupID/ KindredID: 133CA

      DiseaseAssertion: VHL

      GroupSize: N/A

      GroupNumberMales: N/A

      GroupNumberFemales: N/A

      GroupEthnicity: N/A

      GroupAgeRange: N/A

      GroupInfo: This germline familial VHL mutation was taken from the Cambridge, Department of Pathology database, with an unknown number of members belonging to Family ID 133CA. Table 3 lists all ID and mutations associated with individuals possessing renal carcinoma, without pheochromocytoma.

      GroupPresentingHPOs: HP:0005548 (renal cell carcinoma)

      GroupHPOFreeText: Described as a VHL family with RCC, without pheochromocytoma. Other phenotypes are likely, however not detailed in this publication

      GroupNotHPOs: HP:0002666 (pheochromocytoma)

      GroupNotHPOFreeText: N/A

      GroupPreviousTesting: N/A

      PreviouslyPublished: N/A

      SupplementalData: N/A

      Variant: p.G114C (c.340G>T)

      LegacyVariant: p.G114C (c.553G>T)

      CaseProblemVariantFreeText: N/A

      ClinVarID: N/A

      CAID: CA351751376 https://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/allele?hgvsOrDescriptor=NM_000551.3%3Ac.340G>T

      gnomAD: N/A

      VariantEvidence: N/A

      MutationType: missense_variant; transversion

      CivicName: G114C (c.340G>T)

      MultipleGeneVariants: N/A

    3. Germline mutations were identified in 300/469 (63%) of the families tested; 137 distinct intragenic germline mutations were detected.

      GroupID/ KindredID: 4479NCI, 50CE

      DiseaseAssertion: VHL

      GroupSize: N/A

      GroupNumberMales: N/A

      GroupNumberFemales: N/A

      GroupEthnicity: N/A

      GroupAgeRange: N/A

      GroupInfo: This germline familial VHL mutation was taken from the National Cancer Institute database as well as the Munich, university of Freiburg and Institude of Pathology database, with an unknown number of members belonging to Family ID 4479NCI and 50CE. Table 3 lists all ID and mutations associated with individuals possessing renal carcinoma, without phechromocytoma.

      GroupPresentingHPOs: HP:0005548 (renal cell carcinoma)

      GroupHPOFreeText: Described as a VHL families with RCC, without pheochromocytoma. Other phenotypes are likely, however not detailed in this publication

      GroupNotHPOs: HP:0002666 (pheochromocytoma)

      GroupNotHPOFreeText: N/A

      GroupPreviousTesting: N/A

      PreviouslyPublished: 8707293

      SupplementalData: N/A

      Variant: p.E70* (c.208G>T)

      LegacyVariant: p.E70* (c.421G>T)

      CaseProblemVariantFreeText: N/A

      ClinVarID: 428806 https://www.ncbi.nlm.nih.gov/clinvar/variation/428806/

      CAID: N/A

      gnomAD: N/A

      VariantEvidence: This paper declares this variant is predicted to compromise the Elongin-binding site by truncating the protein.

      MutationType: stop_gained

      CivicName: E70* (c.208G>T)

      MultipleGeneVariants: N/A

    1. PatientID: 10 (Uncle)

      KindredID: 7

      Case: M, 36Y0M, Ethnicity Unknown

      DiseaseAssertion: VHL

      FamilyInfo: 6 members of this family were found to harbor the c.319C>G germline variant of the VHL gene. ID 11 presented with renal and pancreatic abnormalities, pheochromocytoma and other undefined phenotypes. ID 1 presented with pheochromocytoma. ID 2 presented with pancreatic abnormalities. ID 24 presented with renal abnormalities, and pheochromocytoma, and died at age 62. ID 4 presented with pheochromocytoma.

      CasePresentingHPOs: HP:0002666; HP:0001732; HP:0010797 (Pheochromocytoma, Abnormality of the pancreas, Hemangioblastoma)

      CaseHPOFreeText: The patient presented with CNS hemangioblastoma (31Y, 35Y), pancreatic abnormalities (31Y) and pheochromocytoma (13Y).

      CaseNotHPOs: HP:0009711; HP:0005584; HP:0000077 (Retinal capillary hemangioma, Renal cell carcinoma, Abnormality of the kidney)

      CaseNotHPOFreeText: N/A

      CasePreviousTesting: N/A

      PreviouslyPublished: N/A

      SupplementalData: Supplementary Table 1.

      Variant: p.R107G (c.319C>G)

      LegacyVariant: N/A

      CaseProblemVariantFreeText: N/A

      ClinVarID: 43599, https://www.ncbi.nlm.nih.gov/clinvar/variation/43599/

      CAID: N/A

      gnomAD: N/A

      VariantEvidence: N/A

      MutationType: missense_variant;transversion

      CivicName: R107G (c.319C>G)

      MultipleGeneVariants: N/A

    2. PatientID: 1 (Proband)

      KindredID: 1

      Case: M, 20Y0M, Ethnicity Unknown

      DiseaseAssertion: VHL

      FamilyInfo: 9 members of this family were found to harbor the c.193T>G germline variant of the VHL gene. ID 35 was asymptomatic. IDs 28, 2, 6, 17 and 19 all presented with pheochromocytoma. ID 5 presented with CNS hemangioblastoma, retinal angioma and renal cyst OR tumor (not specified). ID 21 presented with retinal angioma and pheochromocytoma.

      CasePresentingHPOs: HP:0002666 (Pheochromocytoma)

      CaseHPOFreeText: The patient presented with a Pheochromocytoma at age 11.

      CaseNotHPOs: HP:0009711; HP:0005584; HP:0001732; HP:0000077; HP:0010797 (Retinal capillary hemangioma, Renal cell carcinoma, Abnormality of the pancreas, Abnormality of the kidney, Hemangioblastoma)

      CaseNotHPOFreeText: N/A

      CasePreviousTesting: N/A

      PreviouslyPublished: N/A

      SupplementalData: Supplementary Table 1.

      Variant: p.S65A (c.193T>G)

      LegacyVariant: N/A

      CaseProblemVariantFreeText: N/A

      ClinVarID: 223160, https://www.ncbi.nlm.nih.gov/clinvar/variation/223160/

      CAID: N/A

      gnomAD: N/A

      VariantEvidence: N/A

      MutationType: missense_variant;transversion

      CivicName: S65A (c.193T>G)

      MultipleGeneVariants: N/A

    1. We performed whole-genome (n = 184) and exome (n = 208) sequencing of matched tumour-normal DNA pairs from 392 patients with sporadic pancreatic cancer (PC) as part of the APGI. We identified pathogenic germline mutations in candidate genes (n = 130)

      ArticleReferenceSequence: Not specified – Assumption: variants assumed to have been documented by the author under reference sequence NM_000551.3

      GenotypingMethod: whole-genome and exome sequencing of matched tumour-normal DNA pairs to identify germline mutations.

      SamplingMethod: N/A

    1. GroupID: 6B

      DiseaseAssertion: VHL

      GroupSize: 7

      GroupNumberMales: 7

      GroupNumberFemales: 0

      GroupEthnicity: N/A

      GroupAgeRange: N/A

      GroupInfo: 12 male patients are listed with this mutation. 7 belong to group 6B (EC absent). Any relations are unknown.

      GroupPresentingHPOs: Unknown

      GroupHPOFreeText: Other manifestations may be present although not matched with individuals so could not deduce.

      GroupNotHPOs: HP:0009715 (Papillary cystadenoma of the epididymis)

      GroupNotHPOFreeText: N/A

      GroupPreviousTesting: N/A

      PreviouslyPublished: N/A

      SupplementalData: N/A

      Variant: p.Y98H (c.292T>C)

      LegacyVariant: p.Y98H (c.505T>C)

      CaseProblemVariantFreeText: N/A

      ClinVarID: 2223 https://www.ncbi.nlm.nih.gov/clinvar/variation/2223/

      CAID: N/A

      gnomAD: 0.00001343 https://gnomad.broadinstitute.org/variant/3-10183823-T-C

      VariantEvidence: N/A

      MutationType: missense_variant;transition

      CivicName: Y98H (c.292T>C)

      MultipleGeneVariants: N/A

    2. GroupID: 6A

      DiseaseAssertion: VHL

      GroupSize: 5

      GroupNumberMales: 5

      GroupNumberFemales: 0

      GroupEthnicity: N/A

      GroupAgeRange: N/A

      GroupInfo: 12 male patients are listed with this mutation. 5 belong to group 6A (EC present). Any relations are unknown.

      GroupPresentingHPOs: HP:0009715 (Papillary cystadenoma of the epididymis)

      GroupHPOFreeText: Other manifestations may be present although not matched with individuals so could not deduce.

      GroupNotHPOs: N/A

      GroupNotHPOFreeText: N/A

      GroupPreviousTesting: N/A

      PreviouslyPublished: N/A

      SupplementalData: N/A

      Variant: p.Y98H (c.292T>C)

      LegacyVariant: p.Y98H (c.505T>C)

      CaseProblemVariantFreeText: N/A

      ClinVarID: 2223 https://www.ncbi.nlm.nih.gov/clinvar/variation/2223/

      CAID: N/A

      gnomAD: 0.00001343 https://gnomad.broadinstitute.org/variant/3-10183823-T-C

      VariantEvidence: N/A

      MutationType: missense_variant;transition

      CivicName: Y98H (c.292T>C)

      MultipleGeneVariants: N/A

    1. WM05 VHL RTK2 0.57 

      Of 38 PPGL (pheochromocytoma and paraganglioma) cases studied, one was associated with an individual carrying the germline mutation c.245G>T (ENSP00000256474.2 p.Arg82Leu) of the VHL gene. This mutation was identified using germline clinical sequencing on formalin-fixed and paraffin-embedded (FFPE) tissue and confirmed with sanger sequencing. The authors identified the reference sequence used as NM_000551.3. The patient (ID: WM05) presented with a pheochromocytoma of RTK2 class (8yo). No family history was clear or presented.

    1. Genomic DNA was extracted from fresh or frozen peripheral blood using QIAamp DNA Mini Kit (Qiagene, USA). Somatic DNA was extracted from frozen tumour’s samples after histological confirmation of PHEO/PGL. DNA was extracted by Puregene Core kit A (Qiagene, USA). Quality of DNA was checked by NanoDrop™ 2000/2000c Spectrophotometers (ThermoScientific)Sanger sequencing.PCR primers for BRAF gene have been designed based on GenBank sequences using the Primer 3 software including intron-exon boundaries, reverse primer 5′- CTGTTCAAACTGATGGGACCC– 3′, forward primer 5′– TGCTTGCTCTGATAGGAAAATG– 3′. The BRAF PCR conditions are as follows 25 μl reaction mixture contained 1× PCR buffer (Fermentas), between 50–300 ng of genomic DNA as template, 1.5 mM MgCl2 (Fermentas), 25 pmol of each primer, 200 μM of each deoxynucleotide triphosphate (Fermentas), and 1.0 unit of TaqDNA polymerase (MBI Fermentas). Amplification conditions were included an initial denaturation at 94C° for 3 min., followed by 35 cycles of 45 sec at 94 C°, 45 sec at 60 C°, 1 min. at 72C° and final extension step running 5 min. at 72 C°. DNA fragments were sequenced in both directions using an automatic fluorescent ABI PrismTM 3130 Genetic Analyzer (PE Applied Biosystems) according to the manufactureŕs instructions.

      ArticleReferenceSequence: Not specified – Assumption: variants assumed to have been documented by the author under reference sequence NM_000551.3

      GenotypingMethod: Sanger sequencing

      SamplingMethod: genomic DNA extracted from fresh or frozen peripheral blood

    1. 28F/32ASUnilateral PCCVHLIVS 2c.341-2 A>GSplice Site ChangeSplice site changeDisease causingNANANRNR

      PatientID: 28

      KindredID: 28

      Case: F, 32Y0M, Asian Indian

      DiseaseAssertion: pheochromocytoma

      FamilyInfo: None - Apparently sporadic

      CasePresentingHPOs: HP:0002666 (pheochromocytoma)

      CaseHPOFreeText: Unilateral pheochromocytoma

      CaseNotHPOs: N/A

      CaseNotHPOFreeText: N/A

      CasePreviousTesting: RET/ SDHB/ SDHD/ NF1

      PreviouslyPublished: N/A

      SupplementalData: N/A

      Variant: c.341-2A>G

      CaseProblemVariantFreeText: N/A

      LegacyVariant: N/A

      ClinVarID: 223194 https://www.ncbi.nlm.nih.gov/clinvar/variation/223194/

      CAID: N/A

      gnomAD: N/A

      VariantEvidence: N/A

      MutationType: splice_site_variant

      CivicName: Splice Site (c.341-2A>G)

      MultipleGeneVariants: N/A

    1. c.500G > A (p.Arg167Gln)

      37 Danish patients diagnosed with VHL before 18 years from a prospective cohort study from Denmark's National vHL Research Database (Binderup et al., 2013) were assessed. Parts of the Danish cohort have been previously assesed in the following papers PMID: 19863552, 25834951, 27966541, 27539272. Peripheral blood samples were isolated and sequenced coding exons and exon-intron boundaries using MLPA (Multiplex Ligation-dependent Probe Amplification). One family with 3 patients (Family ID 1) were found to harbor the germline missense mutation in the VHL gene (c.500G>A; p.Arg167Gln). Patient 1 was diagnosed at 2Y old and had no manifestations. Patient 2 was diagnosed at 8Y and had retinal hemangioblastoma (8Y), pheochromocytoma (8Y), and kidney disease (8Y). Patient 3 was diagnosed at 9Y and had 3 retinal hemangioblastomas at 8Y and 16Y.

    2. II 4 5 No manifestations M c.388G > A (p.Val130Ile)

      PatientID: II

      KindredID: 2

      Case: Sex Unknown, 5Y0M, Danish

      DiseaseAssertion: Asymptomatic

      FamilyInfo: One other family member presented with his VHL mutation and was asymptomatic.

      CasePresentingHPOs: N/A

      CaseHPOFreeText: Diagnosed with VHL at age 4Y. Age at the end of study: 5Y.

      CaseNotHPOs: N/A

      CaseNotHPOFreeText: N/A

      CasePreviousTesting: N/A

      PreviouslyPublished: N/A

      SupplementalData: N/A

      Variant: p.V130I (c.388G>A)

      LegacyVariant: N/A

      CaseProblemVariantFreeText: N/A

      ClinVarID: N/A

      CAID: CA351753916, https://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/allele?hgvsOrDescriptor=NM_000551.3%3Ac.388G%3EA

      gnomAD: N/A

      VariantEvidence: N/A

      MutationType: missense_variant;transition

      CivicName: V130I (c.388G>A)

      MultipleGeneVariants: N/A

    3. 8 I 15 35 6 CNS hb (15 years) 2 retinal hb (15 years) M c.319C > T and c.353 T > C (p.Arg107Cys) and (p.Leu118Pro)

      PatientID: I

      KindredID: 8

      Case: Sex Unknown, 35Y0M, Danish

      DiseaseAssertion: VHL

      FamilyInfo: N/A

      CasePresentingHPOs: HP:0009711; HP:0010797 (Retinal capillary hemangioma, Hemangioblastoma)

      CaseHPOFreeText: Diagnosed with VHL at age 15Y. Presented with 6 CNS hemangioblastomas (15Y) and 2 retinal hemangioblastomas (15Y). Age at the end of study: 15Y.

      CaseNotHPOs: N/A

      CaseNotHPOFreeText: N/A

      CasePreviousTesting: N/A

      PreviouslyPublished: N/A

      SupplementalData: N/A

      Variant: p.L118P (c.353T>C)

      LegacyVariant: N/A

      CaseProblemVariantFreeText: N/A

      ClinVarID: 428807, https://www.ncbi.nlm.nih.gov/clinvar/variation/428807/

      CAID: N/A

      gnomAD: N/A

      VariantEvidence: N/A

      MutationType: missense_variant;transition

      CivicName: L118P (c.353T>C)

      MultipleGeneVariants: Germline, VHL, c.319C>T (p.R107C)

    1. In this study, we analyzed the correlations between frequent mutations in the VHL gene and clinical phenotypes in the largest Chinese VHL cohort to date. In total, we screened 540 patients from 187 unrelated VHL families and identified 126 different VHL mutations. Furthermore, we identified 19 frequent mutations and four mutation hotspots and further investigated the genotype–phenotype correlations. Notably, patients or families with the VHL disease have a range of different phenotypes. A variety of factors may contribute to this diversity of phenotypes, including the type of VHL mutation, the site of the mutation, and ethnic background.

      540 patients from 187 unrelated Chinese VHL families at the Peking University First Hospital were all screened for VHL clinical features and germline mutations. Of these, clinical characteristics were observed in 258 patients from 80 unrelated Chinese families. These were reported in this study, grouped by the 21 mutations found. PCR and MLPA on peripheral blood detected the VHL mutations. Possessing the p.L89P (c.266T>C) mutation, 11 members total from 2 families were described, and phenotypic information was given for this group. With ages ranging from 15-52Y, hemangioblastomas were observed in 6/11 members, retinal capillary hemangioma in 5, renal cell carcinoma in 6, pancreatic cysts in 4, and pheochromocytoma in 1 member. See more information as this cohort has been previously published in PMID 29330336; 28388566.

    2. In our study, group 5 represented the c.262T > C p.Trp88Arg mutation that included 21 patients from 2 families (Figure 4 and Table 3). Variability was observed in the mean age at onset for CHB and RCC, which were 41.1 ± 9.1 (range = 29–62) and 37.4 ± 12.9 (range = 23–65), respectively.

      PatientID: 3

      KindredID: 1 (group ID 5)

      Case: M, 63Y0M (deceased), Chinese

      DiseaseAssertion: assumed VHL

      CohortInfo: Clinical characteristics of 258 patients from 80 unrelated Chinese families were reported in this study. Grouped by the 21 mutations found, 2 families with 21 members total were described, and phenotypic information was given for this group.

      FamilyInfo: In family 1, 17 members were described. The first generation describes 5 members with the VHL mutation, each passing on the gene to further generations. Patient 3’s two sons were passed this familial mutation. See Fig 4 for family pedigree; Table 3 for details.

      CasePresentingHPOs: HP:0010797 (hemangioblastoma)

      CaseHPOFreeText: He had CNS hemangioblastomas at 62Y

      CaseNotHPOs: N/A

      CaseNotHPOFreeText: N/A

      CasePreviousTesting: N/A

      PreviouslyPublished: N/A

      SupplementalData: N/A

      Variant: p.W88R (c.262T>C)

      LegacyVariant: N/A

      CaseProblemVariantFreeText: N/A

      ClinVarID: 496053 https://www.ncbi.nlm.nih.gov/clinvar/variation/496053/

      CAID: N/A

      gnomAD: N/A

      VariantEvidence: N/A

      MutationType: missense_variant;transition

      CivicName: W88R (c.262T>C)

      MultipleGeneVariants: N/A

    1. PatientID: 1

      KindredID: 48

      Case: Sex, Age, Ethnicity Unknown

      DiseaseAssertion: VHL

      FamilyInfo: N/A

      CasePresentingHPOs: HP:0005584 (renal cell carcinoma)

      CaseHPOFreeText: one RCC tumor was detected in this patient

      CaseNotHPOs: N/A

      CaseNotHPOFreeText: N/A

      CasePreviousTesting: N/A

      PreviouslyPublished: N/A

      SupplementalData: N/A

      Variant: p.F76del (c.227delTCT)

      CaseProblemVariantFreeText: N/A

      LegacyVariant: p.F76del (c.440delTCT)

      ClinVarID: N/A

      CAID: CA357012 http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/allele?hgvsOrDescriptor=NM_000551.3%3Ac.226_228delTTC

      gnomAD: N/A

      VariantEvidence: LOH detected.

      MutationType: deletion

      CivicName: F76del (c.227delTCT)

      MultipleGeneVariants: N/A

    2. PatientID: 1

      KindredID: 54

      Case: Sex, Age, Ethnicity Unknown

      DiseaseAssertion: VHL

      FamilyInfo: another member (54/2) of the family was detected with an HB and the same mutation

      CasePresentingHPOs: HP:0006880 (hemangioblastoma)

      CaseHPOFreeText: one RCC tumor was detected in this patient

      CaseNotHPOs: N/A

      CaseNotHPOFreeText: N/A

      CasePreviousTesting: N/A

      PreviouslyPublished: N/A

      SupplementalData: N/A

      Variant: p.R177Dfs*25 (c.528delG)

      CaseProblemVariantFreeText: N/A

      LegacyVariant: p.176 FS (c.741delG)

      ClinVarID: N/A

      CAID: CA432423454 http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/allele?hgvsOrDescriptor=NM_000551.3%3Ac.528delG

      gnomAD: N/A

      VariantEvidence: LOH detected.

      MutationType: minus_1_frameshift_variant

      CivicName: R177fs (c.528delG)

      MultipleGeneVariants: N/A

    1. variants of VHL in 18 probands

      PatientID: 3

      KindredID: 3

      Case: Sex Unknown, Age Unknown, Ethnicity Unknown

      DiseaseAssertion: VHL

      FamilyInfo: No family information provided.

      CasePresentingHPOs: HP:0002666; HP:0002668 (Pheochromocytoma; paraganglioma)

      CaseHPOFreeText: Patient had pheochromocytoma and/or paraganglioma (table 2).

      CaseNotHPOs: N/A

      CaseNotHPOFreeText: N/A

      CasePreviousTesting: N/A

      PreviouslyPublished: N/A

      SupplementalData: N/A

      Variant: p.V84M (c.250G>A)

      LegacyVariant: N/A

      CaseProblemVariantFreeText: N/A

      ClinVarID: 428813, https://www.ncbi.nlm.nih.gov/clinvar/variation/428813/

      CAID: N/A

      gnomAD: N/A

      VariantEvidence: N/A

      MutationType: missense_variant, transition

      CivicName: V84M (c.250G>A)

      MultipleGeneVariants: N/A

    2. variants of VHL in 18 probands

      PatientID: 16

      KindredID: 12

      Case: Sex Unknown, Age Unknown, Ethnicity Unknown

      DiseaseAssertion: VHL

      FamilyInfo: No family information provided.

      CasePresentingHPOs: HP:0002666; HP:0002668 (Pheochromocytoma; paraganglioma)

      CaseHPOFreeText: Patient had pheochromocytoma and/or paraganglioma (table 2).

      CaseNotHPOs: N/A

      CaseNotHPOFreeText: N/A

      CasePreviousTesting: N/A

      PreviouslyPublished: N/A

      SupplementalData: N/A

      Variant: p.R167W (c.499C>T)

      LegacyVariant: N/A

      CaseProblemVariantFreeText: N/A

      ClinVarID: 2218, https://www.ncbi.nlm.nih.gov/clinvar/variation/2218/

      CAID: N/A

      gnomAD: N/A

      VariantEvidence: N/A

      MutationType: missense_variant, transition

      CivicName: R167W (c.499C>T)

      MultipleGeneVariants: N/A

    1. VHL_11Frozenc.500G>Ap.Arg167GlnF47AdrenalPCCB

      PatientID: VHL_11 (tumour ID)

      KindredID: 11

      Case: F, 47Y0M, Ethnicity Unknown

      DiseaseAssertion: Adrenal Pheochromocytoma

      FamilyInfo: N/A

      CasePresentingHPOs: HP:0006748 (Adrenal pheochromocytoma)

      CaseHPOFreeText: The patient had a benign adrenal pheochromocytoma at age 47.

      CaseNotHPOs: N/A

      CaseNotHPOFreeText: N/A

      CasePreviousTesting: N/A

      PreviouslyPublished: N/A

      SupplementalData: N/A

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

      LegacyVariant: N/A

      CaseProblemVariantFreeText: N/A

      ClinVar: 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:N/A

      MutationType: missense_variant;transition

      CivicName: R167Q (c.500 G >A)

      MultipleGeneVariants: N/A

    1. Molecular and clinical analysis of 126 French VHL families tested for renal involvement revealed 92 different mutations. 90 of these families had renal involvement. The p.W88S (c.263G>C) germline variant of the VHL gene was detected in an individual (ID 5654, Family 266). The patient presented with renal cell carcinoma. No family information is provided. The paper states that this patient is previously reported in PMIDs ​​12202531 and/or 9829912.

    2. GroupID: 285

      DiseaseAssertion: VHL

      GroupSize: 4

      GroupNumberMales: N/A

      GroupNumberFemales: N/A

      GroupEthnicity: French

      GroupAgeRange: N/A

      GroupInfo: 4 members of this family are reported as carrying the E52K (c.154G>A) germline variant of the VHL gene. Member 6493 had no renal involvement, member 1422 had mild renal involvement and member 6495 had moderate renal involvement (RCC) and member 6497 phenotypes were unknown. Families were recruited from the French VHL Registry. Enrolment in the study required identification of the germline VHL mutation (currently 85% of the families included in the French VHL Registry) and available data regarding renal phenotype. A total of 126 families fulfilled these criteria.

      GroupPresentingHPOs: HP:0009726; HP:0005584 (Renal neoplasm, Renal cell carcinoma)

      GroupHPOFreeText: Mild renal disease (simple renal cyst, multicystic kidney[s], small lesion [less than 10 mm in size] of unknown significance, or small solid tumor without indication for surgical removal [less than 30 mm in size]). Moderate renal disease (less than five solid lesions requiring surgical treatment, either unilateral or bilateral).

      GroupNotHPOs: N/A

      GroupNotHPOFreeText: N/A

      GroupPreviousTesting: N/A

      PreviouslyPublished: N/A

      SupplementalData: Phenotype and genotype information can be found in Supplementary Table S1.

      Variant: p.E52K (c.154G>A)

      LegacyVariant: N/A

      CaseProblemVariantFreeText: N/A

      ClinVarID: 161402, https://www.ncbi.nlm.nih.gov/clinvar/variation/161402/

      CAID: N/A

      gnomAD: 0.00008525, https://gnomad.broadinstitute.org/variant/3-10183685-G-A?dataset=gnomad_r2_1

      VariantEvidence: N/A

      MutationType: missense_variant;transition

      CivicName: E52K (c.154G>A)

      MultipleGeneVariants: N/A

    3. Molecular and clinical analysis of 126 French VHL families tested for renal involvement revealed 92 different mutations. 90 of these families had renal involvement. The p.H191Tfs*11 (c.571del) germline variant of the VHL gene was detected in a family (ID 51). Members 2127, 1184 and 1182 had unknown renal involvement. Member 1183 presented with severe renal disease (RCC). The paper states that this family is previously reported in PMID 12202531 and/or 9829912.

    1. c.292 T>Cap.Y98H

      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. 160 patients were found to have the above mutation (c.292T>C). 2 were VHL patients with ICTs (pancreatic neuroendocrine tumors) taken from the VHL-registry. The other 158 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: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease.

    2. c.499 C>Tap.R167W

      GroupID: 24B

      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: 15

      GroupNumberMales: N/A

      GroupNumberFemales: N/A

      GroupEthnicity: N/A

      GroupAgeRange: N/A

      GroupInfo: 15 patients with the given mutation were grouped as group 24(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.R167W (c.499C>T)

      LegacyVariant: N/A

      CaseProblemVariantFreeText: N/A

      ClinVarID: 2218 https://www.ncbi.nlm.nih.gov/clinvar/variation/2218/

      CAID: N/A

      gnomAD: 0.000007954 https://gnomad.broadinstitute.org/variant/3-10191506-C-T

      VariantEvidence: N/A

      MutationType: missense_variant; transition

      CivicName: R167W (c.499C>T)

      MultipleGeneVariants: N/A

    3. c.583 C>Tap.Q195X

      GroupID: 28B

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

      GroupSize: 3

      GroupNumberMales: N/A

      GroupNumberFemales: N/A

      GroupEthnicity: N/A

      GroupAgeRange: N/A

      GroupInfo: 3 patients with the given mutation were grouped as group 28(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

      GroupPreviousTesting: N/A

      PreviouslyPublished: N/A

      SupplementalData: N/A

      Variant: p.Q195* (c.583C>T)

      CaseProblemVariantFreeText: N/A

      LegacyVariant: N/A

      ClinVarID: 428794 https://www.ncbi.nlm.nih.gov/clinvar/variation/428794/

      CAID: CA70052558 http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/allele?hgvsOrDescriptor=NM_000551.3%3Ac.583C%3ET

      gnomAD: N/A

      VariantEvidence: N/A

      MutationType: stop_gained

      CivicName: Q195* (c.583C>T)

      MultipleGeneVariants: N/A

    1. GroupID: 8

      DiseaseAssertion: VHL

      GroupSize: 2

      GroupNumberMales: N/A

      GroupNumberFemales: N/A

      GroupEthnicity: N/A

      GroupAgeRange: N/A

      GroupInfo: Family of 2 VHL Type 1 affected individuals.

      GroupPresentingHPOs: HP:0010797 (Hemangioblastoma)

      GroupHPOFreeText: CNS hemangioblastoma (2/2 affected members).

      GroupNotHPOs: HP:0009711; HP:0002894; HP:0002666; HP:0005584 (Retinal capillary hemangioma, Neoplasm of the pancreas, Pheochromocytoma, Renal cell carcinoma)

      GroupNotHPOFreeText: N/A

      GroupPreviousTesting: N/A

      PreviouslyPublished: N/A

      SupplementalData: N/A

      Variant: c.340+7G>A

      LegacyVariant: N/A

      CaseProblemVariantFreeText: N/A

      ClinVarID: N/A

      CAID: CA2573131791, https://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA2573131791

      gnomAD: N/A

      VariantEvidence: Variant reported as IVS1+7G>A which is assumed to be equivalent to c.340+7G>A. This variant was not found in 120 alleles from normal subjects in the Brazilian population.

      MutationType: splice_site_variant; missense_variant;transition

      CivicName: c.340+7G>A

      MultipleGeneVariants: N/A

    2. PatientID: None (Mother)

      KindredID: 13

      Case: Sex Unknown, Age Unknown, Ethnicity Unknown

      DiseaseAssertion: Assumed VHL

      FamilyInfo: Child of this patient presented with CNS hemangioblastoma, retinal angioma, renal carcinoma and a renal tumor <3cm in size and the c.436delC germline variant of the VHL gene.

      CasePresentingHPOs: HP:0006880; HP:0009713; HP:0001737 (Cerebellar hemangioblastoma, Spinal hemangioblastoma, Pancreatic cysts)

      CaseHPOFreeText: This patient was originally thought to be an asymptomatic carrier but was later discovered to have two occult hemangioblastomas (spinal and cerebellar) as well as multiple pancreatic cysts.

      CaseNotHPOs: N/A

      CaseNotHPOFreeText: N/A

      CasePreviousTesting: N/A

      PreviouslyPublished: N/A

      SupplementalData: N/A

      Variant: p.P146Lfs*13 (c.436del)

      LegacyVariant: N/A

      CaseProblemVariantFreeText: N/A

      ClinVarID: N/A

      CAID: CA432421892, https://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/allele?hgvsOrDescriptor=NM_000551.3%3Ac.436del

      gnomAD: N/A

      VariantEvidence: This variant was not found in 120 alleles from normal subjects in the Brazilian population.

      MutationType: deletion; minus_1_frameshift_variant

      CivicName: P146fs (c.436del)

      MultipleGeneVariants: N/A

    1. VHL (n = 10)

      PatientID: N/A

      KindredID: 1

      Case: M, 65Y0M, Ethnicity Unknown

      DiseaseAssertion: bladder carcinoma

      FamilyInfo: No family information provided.

      CasePresentingHPOs: HP:0002862 (bladder carcinoma)

      CaseHPOFreeText: Patient was a white male diagnosed with bladder urothelial carcinoma, who passed away at age 65Y0M.

      CaseNotHPOs: N/A

      CaseNotHPOFreeText: N/A

      CasePreviousTesting: N/A

      PreviouslyPublished: N/A

      SupplementalData: See table s3 for patient-related information.

      Variant: p.L188V (c.562C>G)

      LegacyVariant: N/A

      CaseProblemVariantFreeText: N/A

      ClinVarID: 2225, https://www.ncbi.nlm.nih.gov/clinvar/variation/2225/

      CAID: N/A

      gnomAD: 0.00002121, https://gnomad.broadinstitute.org/variant/3-10191569-C-G

      VariantEvidence: N/A

      MutationType: missense variant; transversion

      CivicName: L188V (c.562C>G)

      MultipleGeneVariants: N/A

    2. p.L188V

      Pathogenic variants from 10,389 individuals across 33 cancer types were examined from The Cancer Genome Atlas (TCGA) cohort. 12 of these individuals had a germline mutation in the VHL gene. The L188V (c.562C>G) missense VHL mutation was found in two individuals, a white male diagnosed with bladder urothelial carcinoma who died at age 65Y0M, as well as a white 66Y0M female who was diagnosed with lung adenocarcinoma. No family information was provided.

    1. PatientID: 221

      KindredID: 221

      Case: Sex Unknown, 16Y0M, Ethnicity Unknown

      DiseaseAssertion: VHL

      FamilyInfo: N/A

      CasePresentingHPOs: HP:0009711 (Retinal capillary hemangioma)

      CaseHPOFreeText: N/A

      CaseNotHPOs: HP:0010797; HP:0006770; HP:0002666; HP:0000107; HP:0001732 (Hemangioblastoma, Clear cell renal cell carcinoma, Pheochromocytoma, Renal cyst, Abnormality of the pancreas)

      CaseNotHPOFreeText: N/A

      CasePreviousTesting: N/A

      PreviouslyPublished: 17006605

      SupplementalData: Table S1, S2 and S3

      Variant: c.-61_-51dup

      LegacyVariant: N/A

      CaseProblemVariantFreeText: N/A

      ClinVarID: 517152, https://www.ncbi.nlm.nih.gov/clinvar/variation/517152/

      CAID: N/A

      gnomAD: 0.00009581, https://gnomad.broadinstitute.org/variant/3-10183466-C-CTCCGCCCCGCG?dataset=gnomad_r2_1

      VariantEvidence: Variant was reported as c.-65_-55dup11 which was mapped to c.-61_-51dup.

      MutationType: splice_site_variant; duplication; plus_2_frameshift_variant; 5_prime_UTR_variant

      CivicName: c.-61_-51dup11

      MultipleGeneVariants: N/A

    1. 6418Y18Y44NNNYNc.500G>AExon 3p.Arg167GlnMissense

      PatientID: 6

      KindredID: 4

      Case: Sex Unknown, 44Y0M, Ethnicity Unknown

      DiseaseAssertion: VHL

      FamilyInfo: Four other family members were found to have the same mutation (Patient 7,8,9,10).

      CasePresentingHPOs: HP:0002668, HP:0002666, HP:0030405, HP:0010797 (Paraganglioma, pheochromocytoma, pancreatic endocrine tumor, hemangioblastoma)

      CaseHPOFreeText: The patient was diagnosed with pancreatic endocrine tumor at age 44 and diagnosed with pheochromocytoma and paraganglioma at age 18.

      CaseNotHPOs: HP:0009711, HP:0001737, HP:0000107, HP:0005584 (retinal capillary hemangioma, pancreatic cysts, renal cyst, renal cell carcinoma)

      CaseNotHPOFreeText: N/A

      CasePreviousTesting: N/A

      PreviouslyPublished: N/A

      SupplementalData: N/A

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

      LegacyVariant: N/A

      CaseProblemVariantFreeText: N/A

      ClinVar: 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:N/A

      MutationType: missense_variant;transition

      CivicName: R167Q (c.500 G >A)

      MultipleGeneVariants: N/A

    1. nt 544 A/C* 111 Ser→Arg S111R Missense 1

      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 missense mutation (c.331A>C) was recorded in the paper as c.544A>C due to a difference in transcript sequence. It was found in 1 patient with a hemangioblastoma of the central nervous system. Any family information is unknown. No other phenotypes described.

    2. nt 479 T/C 89 Leu→Pro L89P Missense 6

      GroupID: 13

      DiseaseAssertion: hemangioblastoma

      GroupSize: 6

      GroupNumberMales: N/A

      GroupNumberFemales: N/A

      GroupEthnicity: N/A

      GroupAgeRange: N/A

      GroupInfo: 6 out of the 81 patients listed in this hemangioblastoma cohort were found with this c.266T>C mutation. Any relation is unknown.

      GroupPresentingHPOs: HP:0010797 (hemangioblastoma)

      GroupHPOFreeText: all patients had hemangioblastoma of the CNS; other phenotypes are unknown. 2 patents were said to be found with primary asymptomatic tumors.

      GroupNotHPOs: N/A

      GroupNotHPOFreeText: N/A

      GroupPreviousTesting: N/A

      PreviouslyPublished: N/A

      SupplementalData: N/A

      Variant: p.L89P (c.266T>C)

      LegacyVariant: p.L89P (c.479T>C)

      CaseProblemVariantFreeText: N/A

      ClinVarID: 182979 https://www.ncbi.nlm.nih.gov/clinvar/variation/182979/

      CAID: N/A

      gnomAD: N/A

      VariantEvidence: N/A

      MutationType: missense_variant; transition

      CivicName: L89P (c.266T>C)

      MultipleGeneVariants: N/A

    1. PMID: 7977367

      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. RT-PCR analysis in RCC cell lines found a p.I180N (c.539T>A) mutation, stated in the paper as c.752T>A due to an older transcript. It was seen in 2 cell lines: A-704 and 769-P. Any family information or other manifestations apart from RCC were unclear.

    1. RNA sequencing (RNA-seq) analysis of patients' LCLs and tumors demonstrated that genomic variants in E1′ were associated with an upregulation of transcripts containing the E1′ cryptic exon compared with controls (Figure 3A; supplemental Figure 8). In addition, the mutated allele was preferentially expressed (>70%), suggesting a causal relationship between the genetic variants and E1′ retention
    2. F12c.414A>G/WTVHL p.Pro138Pro/WTNNNNVHL disease—

      PatientID: II (daughter)

      KindredID: F12

      Case: F, Age Unknown, Ethnicity Unknown

      DiseaseAssertion: VHL

      FamilyInfo: 4 members of this family (I.mother, II.daughter, II.son, III.granddaughter) presented with pheochromocytoma and were found to have the P138= (c.414A>G) germline variant of the VHL gene.

      CasePresentingHPOs: HP:0002666 (Pheochromocytoma)

      CaseHPOFreeText: N/A

      CaseNotHPOs: N/A

      CaseNotHPOFreeText: N/A

      CasePreviousTesting: N/A

      PreviouslyPublished: N/A

      SupplementalData: N/A

      Variant: p.P138= (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: N/A

      MutationType: missense_variant;transition; synonymous_variant

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

    3. Expression study of the new VHL isoforms in patients' cellsRNA sequencing (RNA-seq) analysis of patients' LCLs and tumors demonstrated that genomic variants in E1′ were associated with an upregulation of transcripts containing the E1′ cryptic exon compared with controls (Figure 3A; supplemental Figure 8). In addition, the mutated allele was preferentially expressed (>70%), suggesting a causal relationship between the genetic variants and E1′ retention (supplemental Figure 8C).Figure 3. Expression study of the new VHL transcript isoforms in patient cells. (A) Sashimi plots from RNA-seq data obtained from samples (LCLs or pheochromocytomas [Pheo]) of 3 patients from F8. The positions of the different VHL exons are indicated, with the maximal number of reads indicated at the right. Splice junctions are denoted by the horizontal links, with details provided in supplemental Figure 8. (B) TaqMan quantification of the different VHL isoforms from samples of F8 performed using probes specific to VHL E1-E2, the translated sequence upstream E1′, or E1-E1′ junctions. Relative gene expression was normalized to LCL control (C1) fixed at 1 (mean results of technical duplicates). (C) TaqMan quantification of the different VHL isoforms in LCLs (established from 2 independent controls and from patients of F1, F2, and F8) cultured in the absence or presence of puromycin (puro), an inhibitor of nonsense-mediated mRNA decay. TaqMan probes were specific to the VHL E1-E1′ or E1-E2 junction. The graph resumes experiments performed on LCLs cultured independently 3 times and quantified in duplicate. Data are represented as the mean ± standard error of the mean. (D) RT-PCR using primers specific for E1 and E3 was performed on mRNA extracted from LCLs established from controls and patients of F6 (carrying the mutation c.340+574A>T that targets the SA site of E1′). Patient with erythrocytosis is indicated in red. On the right, the spliced isoforms are schematically represented. **P < .05, ***P < .01 based on Student t test. *Denotes larger fragments that contained E1′ spliced with E1, but with 15 nucleotides deleted (represented in red) by the use of an alternative SA site (sequences of the cloned bands are detailed in supplemental Figure 10).Download : Download high-res image (230KB)Download : Download full-size imageFigure 3. Expression study of the new VHL transcript isoforms in patient cells. (A) Sashimi plots from RNA-seq data obtained from samples (LCLs or pheochromocytomas [Pheo]) of 3 patients from F8. The positions of the different VHL exons are indicated, with the maximal number of reads indicated at the right. Splice junctions are denoted by the horizontal links, with details provided in supplemental Figure 8. (B) TaqMan quantification of the different VHL isoforms from samples of F8 performed using probes specific to VHL E1-E2, the translated sequence upstream E1′, or E1-E1′ junctions. Relative gene expression was normalized to LCL control (C1) fixed at 1 (mean results of technical duplicates). (C) TaqMan quantification of the different VHL isoforms in LCLs (established from 2 independent controls and from patients of F1, F2, and F8) cultured in the absence or presence of puromycin (puro), an inhibitor of nonsense-mediated mRNA decay. TaqMan probes were specific to the VHL E1-E1′ or E1-E2 junction. The graph resumes experiments performed on LCLs cultured independently 3 times and quantified in duplicate. Data are represented as the mean ± standard error of the mean. (D) RT-PCR using primers specific for E1 and E3 was performed on mRNA extracted from LCLs established from controls and patients of F6 (carrying the mutation c.340+574A>T that targets the SA site of E1′). Patient with erythrocytosis is indicated in red. On the right, the spliced isoforms are schematically represented. **P < .05, ***P < .01 based on Student t test. *Denotes larger fragments that contained E1′ spliced with E1, but with 15 nucleotides deleted (represented in red) by the use of an alternative SA site (sequences of the cloned bands are detailed in supplemental Figure 10).We performed quantitative RT-PCR using TaqMan probes specific for the different splicing junctions or for the region upstream of E1′ (location of probes shown in Figure 1C), which showed a strong upregulation of a unique isoform containing E1′ spliced with E1 in LCLs and tumors from patients of F8 compared with controls (Figure 3B). The study therefore focused on the isoform E1-E1′. Transcripts that retained E1′ spliced with E1 contained (in-frame with E1) a premature termination codon (Figure 1C) and could likely be targeted for degradation according to nonsense-mediated mRNA decay (NMD) mechanisms. We therefore investigated the expression of transcripts that contained E1 spliced with E1′ in the absence or presence of puromycin, an inhibitor of NMD. Without puromycin, higher levels of expression were seen in samples with mutated E1′ (Figure 3C) compared with samples containing WT E1′. Exposure to puromycin resulted in a profound induction of isoforms containing E1 spliced with E1′ vs other isoforms (Figure 3C; supplemental Figure 9). These results indicated that isoforms with E1-E1′ junction were degraded by NMD and may have failed to produce proteins. We performed RT-PCR on LCL samples of F6 carrying the mutation in the SA site, using primers in VHL exons E1 and E3. We observed fragments of larger size in samples from patients carrying the mutation, which were highly visible in the presence of puromycin (Figure 3D). Cloning and sequencing of these fragments demonstrated a dysregulated splicing of E1′ with the use of an alternative SA site located 15 nucleotides downstream (supplemental Figure 10).To measure the impact of the splicing dysregulation on pVHL expression, we performed an immunoblot with an antibody able to recognize the amino acids encoded by E1 and therefore able to detect the different pVHL isoforms.8 The antibody detected overexpressed exogenous X1 protein after transfection of the plasmid encoding X1 (Figure 4A). However, it failed to detect the endogenous protein, even in patients' samples that overexpressed the mRNA containing E1-E1′. Instead, the immunoblots showed a lower expression of all the VHL protein isoforms in patients with mutated E1′ (Figures 4A-B).Figure 4. Functional studies of mutations in VHL E1′. (A-B) Immunoblot analysis of patient LCLs. A representative immunoblot (A) and quantification of 3 different immunoblots (B) are presented. Relative gene expression was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression, and results obtained with LCL control (C1) were fixed at 100%. Data are represented as the mean ± standard error of the mean. A VHL antibody that recognized VHL E1 was able to detect the hypothetical X1 protein. In the most left lane, control LCLs were transfected with an expression vector containing the coding sequence for X1; 5 µg of proteins were blotted vs 45 µg for other samples to avoid signal saturation with overexpressed X1. Patients with erythrocytosis are indicated in red. (C) Functional hypoxia response element (HRE)–dependent reporter assays were performed in 786-O cells (ie, VHL− cells that constitutively express HIF-2α). The results are expressed as relative firefly luciferase activity with Renilla luciferase as an internal control; 1.0 unit denotes the basal activity of endogenous HIF-2α using the HRE luciferase reporter plasmid. The ability of WT and mutated X1 to downregulate firefly luciferase activity (related to HIF activity) was compared with pVHL and in competition with pVHL. An immunoblot using an antibody specific to the hemagglutinin tag (HA) was used to detect HA-VHL and HA-X1. X1-L128V+L138P corresponds to a potential impact of the c.340+648T>C and c.340+617C>G mutations on the hypothetical X1 protein. Three independent experiments were performed. (D) Characterization of VHL E1′ retention by the minigene experiment (representative picture of agarose gel; n = 3). RT-PCR was performed on mRNA obtained from cell lines transfected with a minigene construct containing VHL E1′ (WT or mutated) flanked by large intronic sequences cloned between the SERPING1 exons (exons A and B, targeted by the RT-PCR primers). The plasmids were transfected, and the expression of the spliced chimeric transcripts (containing EA and EB ± E1′) was analyzed. Two WT constructs containing E1′ were used; 1 contained the single-nucleotide polymorphism c.340+1150T>C (rs779808). Bands corresponding to EA and EB spliced together or with VHL E1′ are indicated on the right. *P < .05, **P < .01, ***P < .001, ****P < .0001 based on Student t test. †Corresponds to unspecific bands verified by sequencing. The minigene experiment performed with the construction carrying the mutation c.340+574A>T (that targets the SA site of E1′ in F6) confirmed the use of an alternative SA site (right panel) with the deletion of 15 nucleotides (represented in red). ns, not significant.Download : Download high-res image (204KB)Download : Download full-size imageFigure 4. Functional studies of mutations in VHL E1′. (A-B) Immunoblot analysis of patient LCLs. A representative immunoblot (A) and quantification of 3 different immunoblots (B) are presented. Relative gene expression was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression, and results obtained with LCL control (C1) were fixed at 100%. Data are represented as the mean ± standard error of the mean. A VHL antibody that recognized VHL E1 was able to detect the hypothetical X1 protein. In the most left lane, control LCLs were transfected with an expression vector containing the coding sequence for X1; 5 µg of proteins were blotted vs 45 µg for other samples to avoid signal saturation with overexpressed X1. Patients with erythrocytosis are indicated in red. (C) Functional hypoxia response element (HRE)–dependent reporter assays were performed in 786-O cells (ie, VHL− cells that constitutively express HIF-2α). The results are expressed as relative firefly luciferase activity with Renilla luciferase as an internal control; 1.0 unit denotes the basal activity of endogenous HIF-2α using the HRE luciferase reporter plasmid. The ability of WT and mutated X1 to downregulate firefly luciferase activity (related to HIF activity) was compared with pVHL and in competition with pVHL. An immunoblot using an antibody specific to the hemagglutinin tag (HA) was used to detect HA-VHL and HA-X1. X1-L128V+L138P corresponds to a potential impact of the c.340+648T>C and c.340+617C>G mutations on the hypothetical X1 protein. Three independent experiments were performed. (D) Characterization of VHL E1′ retention by the minigene experiment (representative picture of agarose gel; n = 3). RT-PCR was performed on mRNA obtained from cell lines transfected with a minigene construct containing VHL E1′ (WT or mutated) flanked by large intronic sequences cloned between the SERPING1 exons (exons A and B, targeted by the RT-PCR primers). The plasmids were transfected, and the expression of the spliced chimeric transcripts (containing EA and EB ± E1′) was analyzed. Two WT constructs containing E1′ were used; 1 contained the single-nucleotide polymorphism c.340+1150T>C (rs779808). Bands corresponding to EA and EB spliced together or with VHL E1′ are indicated on the right. *P < .05, **P < .01, ***P < .001, ****P < .0001 based on Student t test. †Corresponds to unspecific bands verified by sequencing. The minigene experiment performed with the construction carrying the mutation c.340+574A>T (that targets the SA site of E1′ in F6) confirmed the use of an alternative SA site (right panel) with the deletion of 15 nucleotides (represented in red). ns, not significant.Functional characterization of the mutated VHL E1′Because the hypoxia pathway represents the major pathway involved in the genesis of secondary erythrocytosis and VHL disease,13, 14, 1528 we performed functional studies of the hypothetical WT or mutated X1 proteins using hypoxia response element–dependent reporter assays. These functional tests failed to reveal any substantial effects of X1 on this pathway, either alone or in competition with pVHL (Figure 4C; supplemental Figure 11). We next focused our study on a potential impact of the E1′ variants on splicing by performing splicing reporter minigene assays in various cell lines. The experiments showed that splicing of WT E1′ was barely detected (Figure 4D). Interestingly, higher molecular weight bands corresponding to the expected spliced isoforms containing E1′ appeared in the presence of the mutations. Cloning and sequencing of the isoforms confirmed a retention of E1′ at the expected splicing sites. The level of expression of this upper isoform, specific to E1′ inclusion, was higher for mutations associated with cancers than for those with erythrocytosis, independently of the cell lines. Notably, the combination of both variants associated with cancer had a more pronounced effect than each variant individually (the single-nucleotide polymorphism showing very low effect), suggesting a synergistic effect of the variants on splicing (supplemental Figure 12). In silico analysis of c.340+617C>G consistently predicted a severe alteration of splicing by the creation of an exonic splicing enhancer site (supplemental Figure 13).The minigene experiment performed with mutated E1′ at the SA site (F6) confirmed the inclusion of E1′ during splicing (Figure 4D, right panel), using the same downstream alternative SA site identified in mRNA extracted from patients' LCLs (supplemental Figure 10).Synonymous mutations in VHL E2 induce exon skippingWe then focused our study on the synonymous heterozygous D143D mutation in E2, identified in the proband of F1. This mutation was also identified in the homozygous state in 2 patients with erythrocytosis (F9 and F10; Figure 5A; Table 1). The mRNA extracted from LCLs established from the different members of these 2 families was reverse transcribed and sequenced. Comparison of chromatograms obtained by sequencing of DNA vs cDNA displayed a weaker peak of the mutated allele in cDNA, reflecting a decreased expression of the mRNA transcripts carrying the mutation.Download : Download high-res image (221KB)Download : Download full-size imageFigure 5. Genetic and expression study of synonymous mutations in VHL E2. (A-B) Pedigree and sequence chromatograms of germline DNA, tumor (pheochromocytoma) DNA, or cDNA prepared from 2 families (F9 and F10) with erythrocytosis (A) and 2 families (F11 and F12) with VHL disease (B). (C) Results of RT-PCR using mRNA extracted from LCLs (F9 and F10) and leukocytes and tumor material (pheochromocytoma) (F11 and F12). (D) TaqMan quantification of the different VHL isoforms in LCLs (established from patients of F9, F10, and F11) cultured in the absence or presence of puromycin. TaqMan probes are specific to the VHL E1-E2 or E1-E3 junction. Relative gene expression was normalized to LCL control (C1). (E) Immunoblot analysis of patient LCLs. Patients with erythrocytosis are indicated in red. A representative immunoblot (upper panel) and quantification of 4 different immunoblots are displayed (lower panel). Relative gene expression was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression, and results obtained with C1 were fixed at 100%. Data are represented as the mean ± standard error of the mean. *P < .05, **P < .01, ***P < .001, ****P < .0001 based on Student t test. CNS, central nervous system.A different synonymous mutation in E2, c.414A>G p.Pro138Pro (P138P), was identified in 2 families (F11 and F12) with VHL disease (Figure 5B). This heterozygous mutation segregated with the disease in 3 generations. Sequencing of DNA extracted from the pheochromocytoma of F11-III.1 showed a loss of the WT allele in the tumor, demonstrating loss of heterozygosity as currently described in classical VHL disease.Suspecting an effect of the synonymous mutations on splicing, we next assessed the expression of the VHL transcripts in patients' samples by RT-PCR (Figure 5C). We found a significant change in the ratio of expressed VHL isoforms, with a higher expression of the E1-E3 transcripts in LCLs of patients homozygous for D143D and in the pheochromocytoma with P138P mutation. These results suggest an effect of the mutation on splicing regulatory elements, leading to E2 skipping. In silico analysis of the synonymous mutations indicated a potential effect on exonic splicing enhancer motifs (supplemental Figure 14).To specifically quantify the different VHL isoforms, we performed quantitative RT-PCR on LCLs using TaqMan probes complementary to the VHL E1-E2 or E1-E3 junction (Figure 5D). All mutated samples displayed an increase of the isoform with skipped E2 (E1-E3). The patients carrying a homozygous D143D mutation presented a severe decrease of the WT isoform expression level. These results demonstrated that synonymous mutations in E2 affected mRNA isoform production.To evaluate a potential impact on protein expression, we performed a western blot analysis. We did not observe any overexpression of the pVHL172 isoform as expected from mRNA quantification studies. However, in patients homozygous for D143D, we detected a strong downregulation of all the pVHL isoforms, which was also observed to a lesser extent in LCL samples from heterozygous patients (Figure 5E).Proportion of VHL E2 skipping is correlated with disease severity in minigene experimentsTo study the impact of the synonymous E2 mutations on splicing in different cell lines, we performed minigene assays. We tested the implications of the D143D and P138P mutations on splicing in addition to the nearby mutations described in patients with erythrocytosis (P138L and G144R).4,6,29 Indeed, these mutations may also affect splicing rather than VHL protein function. We first evaluated the loss of function of these mutants by reporter assays. We found that these could downregulate HIF in a manner similar to the WT protein, in contrast to VHL proteins lacking E2 (pVHLΔE2), which are described as nonfunctional in terms of their regulation of HIF activity8, 9, 1012 (Figure 6A). Minigene experiments were performed in a variety of cell lines relevant to the studied diseases, using LCLs as control (Figure 6B). We demonstrated that all mutations caused E2 skipping in LCLs. In other cell lines, these experiments demonstrated a major impact on the VHL E2 splicing of the P138P mutation, which is associated with cancer. The mutations P138L, D143D, and G144R, which are all associated with erythrocytosis, displayed a weaker effect on splicing, with slight variations among cell lines but with a stronger effect in the erythroid cell line (UT7 cultured with erythropoietin).Figure 6. Functional study of synonymous mutations in VHL E2. (A) Functional hypoxia response element (HRE)–dependent reporter assays were performed in 786-O cells to evaluate the impact of VHL mutations in E2 (P138L and G144R) on VHL protein activity. The VHL protein lacking E2 (pVHL172/VHLΔE2) was used as a negative control. The results are expressed as firefly luciferase activity relative to Renilla luciferase as an internal control; 1.0 unit denotes the basal activity of endogenous HIF-2α using the HRE luciferase reporter plasmid. Immunoblots using an antibody specific for the hemagglutinin tag (HA) were used to detect HA-VHL. (B) Characterization of VHL E2 skipping by minigene analyses. Minigene experiments were performed in a variety of cell lines relevant to the studied diseases: renal (293T, 786-O, and HK2), pheochromocytoma (pheo; PC12), erythroid cell line (UT7 cultured with erythropoietin), and LCL. RT-PCR was performed on mRNA obtained from cell lines transfected with a minigene construct containing VHL E2 flanked by intronic sequences cloned between the SERPING1 exons (exons A and B; targeted by the RT-PCR primers). Bands corresponding to EA and EB spliced together or with VHL E2 are indicated on the right. (C) Sashimi plots from RNA-seq data. The positions of the different VHL exons are indicated, with the maximum number of reads for each exon indicated at the right. Splice junctions are denoted by the horizontal links, with details provided in supplemental Figure 15A. (D) Heatmap of pheochromocytoma transcriptome data. A comparison of transcriptome data for the pheochromocytoma from patient F11 III.1 (with P138P mutation) vs Affymetrix data from the largest available cohort of paragangliomas/pheochromocytomas (recruited by the French COMETE network) that identified homogeneous molecular subgroups associated with susceptibility genes (Burnichon et al30).Download : Download high-res image (254KB)Download : Download full-size imageFigure 6. Functional study of synonymous mutations in VHL E2. (A) Functional hypoxia response element (HRE)–dependent reporter assays were performed in 786-O cells to evaluate the impact of VHL mutations in E2 (P138L and G144R) on VHL protein activity. The VHL protein lacking E2 (pVHL172/VHLΔE2) was used as a negative control. The results are expressed as firefly luciferase activity relative to Renilla luciferase as an internal control; 1.0 unit denotes the basal activity of endogenous HIF-2α using the HRE luciferase reporter plasmid. Immunoblots using an antibody specific for the hemagglutinin tag (HA) were used to detect HA-VHL. (B) Characterization of VHL E2 skipping by minigene analyses. Minigene experiments were performed in a variety of cell lines relevant to the studied diseases: renal (293T, 786-O, and HK2), pheochromocytoma (pheo; PC12), erythroid cell line (UT7 cultured with erythropoietin), and LCL. RT-PCR was performed on mRNA obtained from cell lines transfected with a minigene construct containing VHL E2 flanked by intronic sequences cloned between the SERPING1 exons (exons A and B; targeted by the RT-PCR primers). Bands corresponding to EA and EB spliced together or with VHL E2 are indicated on the right. (C) Sashimi plots from RNA-seq data. The positions of the different VHL exons are indicated, with the maximum number of reads for each exon indicated at the right. Splice junctions are denoted by the horizontal links, with details provided in supplemental Figure 15A. (D) Heatmap of pheochromocytoma transcriptome data. A comparison of transcriptome data for the pheochromocytoma from patient F11 III.1 (with P138P mutation) vs Affymetrix data from the largest available cohort of paragangliomas/pheochromocytomas (recruited by the French COMETE network) that identified homogeneous molecular subgroups associated with susceptibility genes (Burnichon et al30).Splicing dysregulation of VHL is causal in the development of diseaseRNA-seq confirmed that the synonymous mutations were not silent but instead induced potent E2 skipping (Figure 6C; supplemental Figure 15A). Additional transcriptome analyses of the pheochromocytoma carrying P138P showed an upregulation of HIF target genes (typically seen in VHL-related pheochromocytomas) compared with the pheochromocytoma carrying a RET mutation (supplemental Figure 15B).30 We then reanalyzed our independent cohort of pheochromocytomas30 using RNA-seq data from both tumors. After unsupervised classification, we observed a segregation of the P138P pheochromocytoma with other VHL-related tumors (C1B cluster), whereas the control pheochromocytoma bearing the RET mutation was grouped with other RET-related tumors (Figure 6D).
    1. 6f557.6n16.6↑407 C/T+9.6

      PatientID: 6

      KindredID: 6

      Case: F, 55Y0M, Ethnicity Unknown

      DiseaseAssertion: Assumed VHL

      FamilyInfo: No details on family information.

      CasePresentingHPOs: HP:0010797 (hemangioblastoma)

      CaseHPOFreeText: N/A

      CaseNotHPOs: N/A

      CaseNotHPOFreeText: N/A

      CasePreviousTesting: N/A

      PreviouslyPublished: N/A

      SupplementalData: N/A

      Variant: p.S65L (c.194C>T)

      LegacyVariant: c.407C>T

      CaseProblemVariantFreeText: N/A

      ClinVarID: 182975, https://www.ncbi.nlm.nih.gov/clinvar/variation/182975/

      CAID: N/A

      gnomAD: 0, https://gnomad.broadinstitute.org/variant/3-10183725-C-T

      VariantEvidence: N/A

      MutationType: missense_variant; transition

      CivicName: S65L (c.194C>T)

      MultipleGeneVariants: N/A

    2. 18m395.6n16.1n407 C/G+0.2

      PatientID: 18

      KindredID: 18

      Case: M, 39Y0M, Ethnicity Unknown

      DiseaseAssertion: Assumed VHL

      FamilyInfo: No details on family information.

      CasePresentingHPOs: HP:0010797 (hemangioblastoma)

      CaseHPOFreeText: N/A

      CaseNotHPOs: N/A

      CaseNotHPOFreeText: N/A

      CasePreviousTesting: N/A

      PreviouslyPublished: N/A

      SupplementalData: N/A

      Variant: p.S65W (c.194C>G)

      LegacyVariant: c.407C>G

      CaseProblemVariantFreeText: N/A

      ClinVarID: 43597, https://www.ncbi.nlm.nih.gov/clinvar/variation/43597/

      CAID: N/A

      gnomAD: N/A

      VariantEvidence: N/A

      MutationType: missense_variant; transversion

      CivicName: S65W (c.194C>G)

      MultipleGeneVariants: N/A

    1. 12PCCF25+VHL30Uni−− VHL 3Germlinec.499C>Tp.Arg167TrpPathogenic

      PatientID: 12

      KindredID: 2

      Case: F, 25Y0M, Ethnicity Unknown

      DiseaseAssertion: VHL

      FamilyInfo: Patient has 2 siblings (patients 10 and 11) with the same mutation and pheochromocytoma.

      CasePresentingHPOs: HP:0002666 (Pheochromocytoma)

      CaseHPOFreeText: Unilateral pheochromocytoma (30mm)

      CaseNotHPOs: N/A

      CaseNotHPOFreeText: N/A

      CasePreviousTesting: SDHB (NM_003000.2), SDHC (NM_003001.3), SDHD (NM_003002.2), SDHAF2 (NM_017841.2), RET (exons 10–11 and 13–16, NM_020975.4), TMEM127 (NM_017849.3), MAX (NM_002382.3).

      PreviouslyPublished: N/A

      SupplementalData: Table 2

      Variant: p.R167W (c.499C>T)

      LegacyVariant: N/A

      CaseProblemVariantFreeText: N/A

      ClinVarID: 2218, https://www.ncbi.nlm.nih.gov/clinvar/variation/2218/

      CAID: N/A

      gnomAD: 0.000007954, https://gnomad.broadinstitute.org/variant/3-10191506-C-T

      VariantEvidence: Loss of heterozygosity could be detected in tumour DNA.

      MutationType: missense_variant;transition

      CivicName: R167W (c.499C>T)

      MultipleGeneVariants: N/A

    1. c.266T > C

      A Mexican family with Von Hippel-Lindau disease was identified. The family consisted of 7 clinically affected members, 2 of which received genetic testing that demonstrated the presence of the L89P (c.266T>C) germline mutation of the VHL gene. Genomic DNA was extracted from peripheral blood leukocytes and the entire VHL coding sequence was amplified using PCR and direct automated sequencing of VHL was performed. Genetic testing was also performed in 11 clinically asymptomatic family members. 4 of these individuals were found to carry the same mutation; it was concluded 3 members were too young to manifest the disease and 1 was a non-penetrant female. Clinical manifestations present in this family include: cerebellar hemangioblastoma, spinal hemangioblastoma, and renal cell carcinoma as well as other unspecified CNS tumors.

    1. The patient was a 24-year-old male who presented with masses in his bilateral adrenals. In February 2013, he underwent an abdominal CT scan that revealed masses in his bilateral adrenals (Fig. 1C, upper image) and a cyst in his left kidney (Fig. 1C, bottom image). The masses were subsequently resected by laparoscopic surgery and pathological analysis confirmed the presence of Pheos. His mother and aunt died from HBs in their brain, while his sister and cousin had Pheos in their bilateral adrenals and right adrenal, respectively. We decide to sequence the relevant susceptible genes in the proband and confirmed that he had a heterozygous missense VHL mutation c.500G>A (p.Arg167Gln)

      Genotype-phenotype correlations were analyzed in five unrelated Chinese families with a family history VHL. The proband (Patient 3) of family 3 was a 24-year-old male who presented with adrenal pheochromocytomas and a cyst in his left kidney. The proband's family history of VHL included a mother and aunt who died from brain hemangiomas, a sister with pheochromocytomas in her bilateral adrenals and a female cousin with pheochromocytomas in her right adrenal. Genetic analysis of the proband was done using DNA isolated from peripheral blood and was screened for potential mutations in a panel of susceptibility genes for pheochromocytoma’s and/or RCC using Target Capture-Based Deep Sequencing. Identification of mutations were further screened by Sanger Sequencing. In the proband, a mutation in VHL was confirmed: c.500G>A (p.Arg167Gln). The same mutation, c.500G>A (p.Arg167Gln), was also found in the proband (Patient 4) from an unrelated family (family 4). The proband was a 21-year-old male who reported blurry vision and metamorphopsia in his left eye for 10 days. Tissue pathology revealed a hemangioblastoma in his left retina. After 8 months, he presented with masses in his bilateral adrenals. Pathological report confirmed the presence of pheochromocytomas. Additionally, an MRI scan showed a mass in his basal ganglia region , a mass in his left frontal lobe, as well as a mass in his cervical cord, which were most likely CNS hemangioblastomas, but were not confirmed with pathological investigation. The proband's family history of VHL included his father who also had pheochromocytomas in his bilateral adrenals.

    1. A previous study of 26 Japanese, VHL families was extended to 41 additional families. Germline mutations were detected in 55 of 77 Japanese families using SSCP, direct sequencing, and Southern Blot analysis of peripheral blood samples. The F76del (c.227_229del) germline mutation was found in 2 VHL type 1 patients, both presenting with hemangioblastomas of the central nervous system, pancreatic cyst or tumor, and renal cell carcinoma (families ID 80 and 2). The variant was reported as c.437_439del and mapped to c.224_226del which is equivalent to c.227_229del. This family was previously reported in PMID 8634692.

    2. PatientID: None

      KindredID: 104

      Case: Sex Unknown, Age Unknown, Japanese

      DiseaseAssertion: VHL

      FamilyInfo: N/A

      CasePresentingHPOs: HP:0005584; HP:0001732; HP:0002666; HP:0009711 (Renal cell carcinoma, Abnormality of the pancreas, Pheochromocytoma, Retinal capillary hemangioma)

      CaseHPOFreeText: Pheochromocytoma, renal cell carcinoma, pancreatic cyst or tumor (unspecified) and retinal angioma.

      CaseNotHPOs: HP:0010797 (Hemangioblastoma)

      CaseNotHPOFreeText: N/A

      CasePreviousTesting: N/A

      PreviouslyPublished: N/A

      SupplementalData: N/A

      Variant: p.C162Y (c.485G>A)

      LegacyVariant: c.698G>A

      CaseProblemVariantFreeText: N/A

      ClinVarID: 223225, https://www.ncbi.nlm.nih.gov/clinvar/variation/223225/

      CAID: N/A

      gnomAD: N/A

      VariantEvidence: N/A

      MutationType: missense_variant;transition

      CivicName: C162Y (c.485G>A)

      MultipleGeneVariants: N/A

    1. 28 y

      PatientID: N/A

      KindredID: 5

      Case: Sex Unknown, 28Y0M, Ethnicity Unknown

      DiseaseAssertion: Asymptomatic

      FamilyInfo: Two first degree relatives were found to have the same mutation in the VHL gene. One relative had no features of VHL disease at 38 years old, and the other first degree relative presented with a cerebellar hemangioblastoma at 39 years old.

      CasePresentingHPOs: N/A

      CaseHPOFreeText: No clinical features of VHL disease

      CaseNotHPOs: N/A

      CaseNotHPOFreeText: N/A

      CasePreviousTesting: N/A

      PreviouslyPublished: N/A

      SupplementalData: N/A

      Variant: p.R200W (c.598C>T)

      LegacyVariant: p.R200W (c.811C>T)

      ClinVarID: 2232 (https://www.ncbi.nlm.nih.gov/clinvar/variation/2232/)

      CAID: N/A

      gnomAD: 0.0002016 (https://gnomad.broadinstitute.org/variant/3-10191605-C-T)

      VariantEvidence: N/A

      MutationType: missense_variant;transition

      CivicName: R200W(c.598C>T)

      MultipleGeneVariants: N/A

    1. proband (III-2

      PatientID: III-2

      KindredID: 1

      Case: M, 62Y0M, Ethnicity Unknown

      DiseaseAssertion: VHL

      FamilyInfo: His mother (II,2) did not carry the mutation. His father (II-1) died age 42 from a cerebellar hemangioblastoma, while his paternal grandparents died at an advanced age and had no known history of VHL manifestations. His brother (III,1) did not harbour the mutation. The patient and his wife (III,3) who did not carry the mutation, had 3 daughters; two with the mutation and one without. Two of his daughters who carried the mutation, aged 28 and 36 (IV-2, IV-3), had a personal history of CNS and retinal hemangioblastomas. Patient IV-2 recently developed a ccRCC and a pheochromocytoma, and patient IV-3 a pancreatic neuroendocrine tumor. Refer to pedigree in Figure 1.

      CasePresentingHPOs: HP:0006770, HP:0010797, HP:0009711 (clear cell renal cell carcinoma, hemangioblastoma, retinal capillary hemangioma)

      CaseHPOFreeText: The patient died at age 62 of CNS complications.

      CaseNotHPOs: HP:0002666, HP:0030405 (pheochromocytoma, pancreatic endocrine tumour)

      CaseNotHPOFreeText: No erythrocytosis; the hematocrit, red blood cells count and Epo levels were all normal.

      CasePreviousTesting: N/A

      PreviouslyPublished: N/A

      SupplementalData: Yes supplemental data and figures. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5555745/bin/NIHMS881280-supplement-Supplemental_Data.pdf

      https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5555745/bin/NIHMS881280-supplement-Supplemental_Figures.pdf

      Variant: p.R161Q (c.482G>A)

      LegacyVariant: N/A

      CaseProblemVariantFreeText: N/A

      ClinVarID: 182983 https://www.ncbi.nlm.nih.gov/clinvar/variation/182983/

      CAID: CA020413 http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/allele?hgvsOrDescriptor=NM_000551.3%3Ac.482G%3EA

      gnomAD: N/A

      VariantEvidence: The patient was a heterozygous carrier of a single R200W and R161Q mutation (both on the same VHL allele).

      MutationType: missense_variant;transition

      CivicName: R200W (c.598C>T) and R161Q (c.482G>A)

      MultipleGeneVariants: Germline, VHL, c.598C>T (p.Arg200Trp)

    1. GroupID: 20

      Case: 19Y0M, Sex Unknown, Ethnicity Unknown

      DiseaseAssertion: retinal capillary hemangioma

      FamilyInfo: 1 of the 573 patients discussed in this paper were found to have this variant. This mutation was found in 1 VHL family with a single patient.

      CasePresentingHPOs: HP:0009711 (retinal capillary hemangioma)

      CaseHPOFreeText: patient had retinal angiomas diagnosed at 19Y

      CaseNotHPOs: HP:0005584; HP:0002666; HP:0006880 (renal cell carcinoma; pheochromocytoma; cerebellar hemangioblastoma)

      CaseNotHPOFreeText: N/A

      CasePreviousTesting: N/A

      PreviouslyPublished: N/A

      SupplementalData: Supplementary Table S1.

      Variant: p.T124Hfs*35 (c.369delG)

      LegacyVariant: p.Thr124Hisfs*35 (c.367delG)

      CaseProblemVariantFreeText: c.367delG automaps to c.369delG - most up to date transcript

      ClinVarID: N/A

      CAID: CA432421810 http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/allele?hgvsOrDescriptor=NM_000551.3%3Ac.369delG

      gnomAD: N/A

      VariantEvidence: N/A

      MutationType: minus_1_frameshift_variant

      CivicName: T124fs (c.369del)

      MultipleGeneVariants: N/A

    1. Patients presenting with juxtapapillary capillary retinal angioma in Germany were queried for the presence of VHL mutations. Peripheral blood samples were obtained, exons of the VHL gene were amplified with polymerase chain reaction (PCR), and products were separated by single-stranded conformation polymorphism (SSCP) for the detection of intragenic mutations. Aberrant bands were sequenced to identify mutations. Southern blotting was performed to detect large deletions if the SSCP failed to show a band shift. This patient was diagnosed at age 12 with a c.194C>G VHL mutation (stated as 407C>G in the paper which used the Latif sequence as reference) and a juxtapapillary capillary retinal angioma, with an, indicated but not described family history of VHL disease. The patient was followed up 3.9 years later.

    1. Figure 1.

      PatientID: III:8

      KindredID: 1

      Case: F, Age Unknown, Ethnicity Unknown

      DiseaseAssertion: Asymptomatic

      FamilyInfo: Look to proband (III:3) for extensive family info.

      CohortInfo: Patients with the diagnosis of familial or sporadic PHEO and their relatives.

      CasePresentingHPOs: N/A

      CaseHPOFreeText: N/A

      CaseNotHPOs: HP:0005584 (Renal cell carcinoma)

      CaseNotHPOFreeText: N/A

      CasePreviousTesting: N/A

      PreviouslyPublished: N/A

      SupplementalData: Figure 1

      Variant: p.P138= (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: N/A

      MutationType: missense_variant;transition

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

      MultipleGeneVariants: N/A

    1. PatientID: V-21

      KindredID: 1

      Case: M, Age Unknown, Ethnicity Unknown

      DiseaseAssertion: VHL

      FamilyInfo: Family’s roots were traced to the German town of Leipzig in Saxony, from whence the original 11 siblings had immigrated to America around 1670. The following report is a 2011 updated pedigree on the previously reported Tisherman kindred. At the time of this study, 43/49 patients have undergone mutational analysis revealing the VHL familial mutation (Y112H) and 35/49 patients have clinical VHL manifestations. Phenotypic cumulative lifetime risk was 71% for Pheo/PGL, 15% for hemangioblastoma, 33% for retinal angioma, 3% for renal cell carcinoma, and 3% for pancreatic cysts. 6 members of the family (all deceased; 4 males, 2 females) had suspected VHL and 2 (males) of these were suspected to have had Pheo/PGL.

      CasePresentingHPOs: HP:0002666; HP:0002668; HP:0009711 (Pheochromocytoma, Paraganglioma, Retinal capillary hemangioma)

      CaseHPOFreeText: Phenotype noted as: Pheo/PGL (unclear which of the two or if both apply to this patient). Deceased.

      CaseNotHPOs: HP:0005584 (Renal cell carcinoma)

      CaseNotHPOFreeText: N/A

      CasePreviousTesting: N/A

      PreviouslyPublished: 9201016; 8239848 (no genetic information; only phenotypes)

      SupplementalData: N/A

      Variant: p.Y112H (c.334T>C)

      LegacyVariant: N/A

      CaseProblemVariantFreeText: N/A

      ClinVarID: 2222, https://www.ncbi.nlm.nih.gov/clinvar/variation/2222/

      CAID: N/A

      gnomAD: N/A

      VariantEvidence: N/A

      MutationType: missense_variant;transition

      CivicName: Y112H (c.334T>C)

      MultipleGeneVariants: N/A

    2. PatientID: VI-17

      KindredID: 1

      Case: F, Age Unknown, Ethnicity Unknown

      DiseaseAssertion: VHL (Asymptomatic)

      FamilyInfo: Family’s roots were traced to the German town of Leipzig in Saxony, from whence the original 11 siblings had immigrated to America around 1670. The following report is a 2011 updated pedigree on the previously reported Tisherman kindred. At the time of this study, 43/49 patients have undergone mutational analysis revealing the VHL familial mutation (Y112H) and 35/49 patients have clinical VHL manifestations. Phenotypic cumulative lifetime risk was 71% for Pheo/PGL, 15% for hemangioblastoma, 33% for retinal angioma, 3% for renal cell carcinoma, and 3% for pancreatic cysts. 6 members of the family (all deceased; 4 males, 2 females) had suspected VHL and 2 (males) of these were suspected to have had Pheo/PGL.

      CasePresentingHPOs: N/A

      CaseHPOFreeText: N/A

      CaseNotHPOs: HP:0005584; HP:0009711; HP:0002666; HP:0002668 (Renal cell carcinoma, Retinal capillary hemangioma, Pheochromocytoma, Paraganglioma)

      CaseNotHPOFreeText: N/A

      CasePreviousTesting: N/A

      PreviouslyPublished: 9201016; 8239848 (no genetic information; only phenotypes)

      SupplementalData: N/A

      Variant: p.Y112H (c.334T>C)

      LegacyVariant: N/A

      CaseProblemVariantFreeText: N/A

      ClinVarID: 2222, https://www.ncbi.nlm.nih.gov/clinvar/variation/2222/

      CAID: N/A

      gnomAD: N/A

      VariantEvidence: N/A

      MutationType: missense_variant;transition

      CivicName: Y112H (c.334T>C)

      MultipleGeneVariants: N/A

    1. 1 (100)

      GroupID: 23

      Case: Sex Unknown, Age Unknown, Ethnicity Unknown

      DiseaseAssertion: VHL

      CohortInfo: a retrospective cohort study from an eye centre in Germany involved 148 VHL patients and their genetic data involved in retinal capillary hemangiomas.

      CasePresentingHPOs: HP:0009711 (retinal capillary hemangioma)

      CaseHPOFreeText: This individual patient was the only one discussed with this mutation. They had retinal hemangiomas; Other VHL manifestations were discussed but not per- variant.

      CaseNotHPOs: N/A

      CaseNotHPOFreeText: N/A

      CasePreviousTesting: N/A

      PreviouslyPublished: N/A

      SupplementalData: Yes - Supplementary Table S1

      Variant: p.P86A (c.256C>G)

      LegacyVariant: N/A

      GroupProblemVariantFreeText: N/A

      ClinVarID: 93327 https://www.ncbi.nlm.nih.gov/clinvar/variation/93327/

      CAID: N/A

      gnomAD: N/A

      VariantEvidence: N/A

      MutationType: missense_variant; transversion

      CivicName: P86A (c.256C>G)

      MultipleGeneVariants: N/A

    1. Our series comprised 125 eligible subjects with germlineVHL c.505 T/C mutation, including 64 previously known and 61 newly diagnosed mutation carriers.

      GroupID: None

      DiseaseAssertion: VHL

      GroupSize: 125

      GroupNumberMales: 58

      GroupNumberFemales: 67

      GroupEthnicity: German

      GroupAgeRange: 4-86 Y

      GroupInfo: The subjects included 64 patients belonging to 15 families with a known VHL c.505 T/C mutation. From their test programme, another 10 c.505 T/C mutation carriers were identified. As well, all consenting first degree relatives (at risk subjects) of mutation carriers, irrespective of disease status, were subjected to mutation analysis, resulting in another 51 mutation carriers detected.

      GroupPresentingHPOs: HP:0002666, HP:0009713, HP:0009711, HP:0010797, HP:0006748, HP:0006737, HP:0006770, HP:0002893, HP:0100616, HP:0030357, HP:0002894 (pheochromocytoma, spinal hemangioblastoma, retinal capillary hemangioma, hemangioblastoma, adrenal pheochromocytoma, extraadrenal pheochromocytoma, clear cell renal cell carcinoma, pituitary adenoma, testicular teratoma, small cell lung carcinoma, neoplasm of the pancreas)

      GroupHPOFreeText: 96 patients had clinical manifestations of VHL. 55 subjects (31 males, 24 females) developed pheochromocytoma during their lifetime. Overall penetrance for pheochromocytomas was estimated to be 31% by age 35 and 60% by age 70. Other frequent lesions were retinal angioma (36%), hemangioblastoma of the spine (36%), and, to a lesser extent, hemangioblastoma of the brain (16%). One patient with angiomatosis retinae developed at least 25 retinal angiomas, whereas the remaining patients had an average of two angiomas with no more than five lesions in one patient. 15 patients had bilateral and 25 unilateral involvement of the retinae. Pheochromocytoma was observed as a single tumour with adrenal or extra-adrenal localization in 32 patients, but 23 mutation carriers developed multiple pheochromocytomas. Four patients developed renal clear cell carcinoma (estimated overall penetrance 13% by the age of 70). Two of these patients died of metastases. All renal clear cell carcinomas were unilateral, single tumours, and no kidney showed renal cysts. Three of the patients with renal cell carcinoma were female. The kidney tumours were detected at the ages of 46 (male patient), 48, 63, and 74 years. Two patients with renal cell carcinoma also had multiple spinal hemangioblastomas. Other rare tumours were one pancreatic islet cell tumour and one pituitary adenoma; both neoplasms had been incidentally found in a dead 34 year old woman at necropsy. One 68 year old male patient was noted to have small cell lung cancer, and two other males developed testicular teratocarcinoma both at 39 years of age.

      GroupNotHPOs: N/A

      GroupNotHPOFreeText: N/A

      GroupPreviousTesting: N/A

      PreviouslyPublished: N/A

      SupplementalData: N/A

      Variant: p.Y98H (c.292T>C)

      LegacyVariant: p.Y98H (c.505T>C)

      CaseProblemVariantFreeText: N/A

      ClinVarID: 2223 https://www.ncbi.nlm.nih.gov/clinvar/variation/2223/

      CAID: N/A

      gnomAD: 0.00001343 https://gnomad.broadinstitute.org/variant/3-10183823-T-C

      VariantEvidence: N/A

      MutationType: missense_variant;transition

      CivicName: Y98H (c.292T>C)

      MultipleGeneVariants: N/A

    1. Case report: We report a young girl who first presented with a unilateral total exudative retinal detachment diagnosed as Coats' disease. Eight years later, when she presented with classical retinal hemangioblastomas with reduced vision in the fellow eye, the diagnosis of von Hippel-Lindau (VHL) disease was confirmed.

      PatientID: 1 (case)

      KindredID: 1

      Case: F, 16Y0M, Ethnicity Unknown

      DiseaseAssertion: VHL

      FamilyInfo: Family history revealed that a paternal aunt had kidney surgery at age 12 for unknown reason, whose daughter was reported to have poor vision in her left eye due to retinal problems. These individuals were not available so no genetic testing was done.

      CasePresentingHPOs: HP:0009711 (retinal capillary hemangioma)

      CaseHPOFreeText: first presented with a unilateral total exudative retinal detachment diagnosed as Coats’ disease (no vision in her right eye since age 2 years). 8Y later (age 15Y), when she presented with classical retinal hemangioblastomas with reduced vision in the fellow eye, the diagnosis of VHL disease was confirmed. By age 16, she developed 3 consecutive total bullous retinal detachments with additional complications of macular dragging, chronic cystoid macular edema with a macular hole, subretinal fibrosis, and anterior and posterior vitreoretinal proliferation that required leaving silicone oil in the eye to maintain the retina in an anatomic position.

      CaseNotHPOs: N/A

      CaseNotHPOFreeText: N/A

      CasePreviousTesting: N/A

      PreviouslyPublished: N/A

      SupplementalData: N/A

      Variant: p.L128Ffs*31 (c.381delG)

      LegacyVariant: p.128 (c.595delG)

      CaseProblemVariantFreeText: Paper describes, a deletion of a single base (delG, guanine) at nucleotide 594 of the VHL gene, resulting in a shift in the translational frame of the mRNA after amino acid 128 and causing a premature stop codon at position 158. Therefore, c.381delG (LRG_322p1:p.(Leu128PhefsTer31)) confirms this.

      ClinVarID: 625241 https://www.ncbi.nlm.nih.gov/clinvar/variation/625241/

      CAID: N/A

      gnomAD: N/A

      VariantEvidence: N/A

      MutationType: minus_1_frameshift_variant

      CivicName: L128fs (c.381delG)

      MultipleGeneVariants: N/A

    1. In this paper we report the presence of a single nucleotide mutation in exon 3 of VHL gene c499 C>T causing substitution of Arginine by Tryptophan at position 167 (R 167 W).

      PatientID: 3 (III:3)

      KindredID: 1

      Case: F, 21Y0M (deceased), Persian

      DiseaseAssertion: pheochromocytoma

      FamilyInfo: 4 sisters were treated with bilateral adrenal mass resection. Patient 2 died at 23Y and patient 3 died at 21Y due to complications associated with VHL. 2 years later, 3 of their other sisters were found to have the mutation. It is assumed that all 5 of them had the VHL mutation. Their mother and the siblings examined normal, and the father had passed earlier and had a family history of hypertension.

      CasePresentingHPOs: HP:0002666 (pheochromocytoma)

      CaseHPOFreeText: died at the age of 21 years because of cerebral hemorrhage and malignant hypertension.

      CaseNotHPOs: N/A

      CaseNotHPOFreeText: N/A

      CasePreviousTesting: N/A

      PreviouslyPublished: N/A

      SupplementalData: N/A

      Variant: p.R167W (c.499C>T) [assumed]

      LegacyVariant: N/A

      CaseProblemVariantFreeText: N/A

      ClinVarID: 2218, https://www.ncbi.nlm.nih.gov/clinvar/variation/2218/

      CAID: N/A

      gnomAD: 0.000007954, https://gnomad.broadinstitute.org/variant/3-10191506-C-T?dataset=gnomad_r2_1

      VariantEvidence: N/A

      MutationType: missense_variant;transition

      CivicName: R167W (c.499C>T)

      MultipleGeneVariants: N/A

    1. PatientID: III-24

      KindredID: 1

      Case: F, Age unknown, Ethnicity Unknown

      DiseaseAssertion: assumed VHL

      FamilyInfo: Large multigenerational family with a history of both VHL and spinocerebellar ataxia type 2 (SCA2). Screening for clinical and genetic diagnostics were gathered as well as thorough information on non-living members. 1/2 of her sons also appeared to have the VHL mutation. See family pedigree (Fig. 1).

      CasePresentingHPOs: HP:0002011; HP:0009711; HP:0000077 (Morphological central nervous system abnormality, retinal capillary hemangioma; Abnormality of the kidney)

      CaseHPOFreeText: Patient mentioned to have CNS (including retinal angiomas) and renal involvement.

      CaseNotHPOs: N/A

      CaseNotHPOFreeText: N/A

      CasePreviousTesting: N/A

      PreviouslyPublished: N/A

      SupplementalData: N/A

      Variant: p.E160Sfs*10 (c.477delA)

      LegacyVariant: c.688delA

      CaseProblemVariantFreeText: c.477del, p.Glu160Serfs10 should be used. Details: 477del same as 475delA bc that codon is AAA so doesn't matter which A gets deleted: there is an A at 475 476 477 so for HGVS we say that 477 (the latter) is deleted since that follows HGVS nomenclature. Thus use c.477del: p.Glu160Serfs10

      ClinVarID: 182959 https://www.ncbi.nlm.nih.gov/clinvar/variation/182959/

      CAID: N/A

      gnomAD: N/A

      VariantEvidence: N/A

      MutationType: minus_1_frameshift_variant

      CivicName: E160fs (c.477del)

      MultipleGeneVariants: N/A

    1. A study investigated paraganglioma/pheochromocytoma tumors in a retrospective cohort and a prospective cohort. Some of these tumors were found with germline VHL mutations. Tumor ID 11 (paraganglioma) and 12 (pheochromocytoma) belonged to one 26Y old patient with a germline p.Pro154Ser VHL mutation. Patient data is extracted from two rows assumed to be referring to the same patient (i.e. tumor samples 11 and 12 in Supplementary Table 1a). Other phenotypic and demographic information was not available.

    1. PatientID: 19 (tumour 33)

      KindredID: 4

      Case: Sex Unknown, Age Unknown, Ethnicity Unknown

      DiseaseAssertion: VHL

      FamilyInfo: Two other members of this patient's family (patient 20 and patient 21) were found to harbour the same VHL gene mutation.

      CasePresentingHPOs: HP:0002666 (pheochromocytoma)

      CaseHPOFreeText: Benign pheochromocytoma

      CaseNotHPOs: N/A

      CaseNotHPOFreeText: N/A

      CasePreviousTesting: RET

      PreviouslyPublished: 7759077, 9681856

      SupplementalData: N/A

      Variant: p.L188V (c.562C>G)

      LegacyVariant: p.L188V (c.775C>G)

      CaseProblemVariantFreeText: N/A

      ClinVar: 2225 https://www.ncbi.nlm.nih.gov/clinvar/variation/2225/

      CAID: CA020488 http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/allele?hgvsOrDescriptor=NM_000551.3%3Ac.562C%3EG

      gnomAD: 0.00002121 https://gnomad.broadinstitute.org/variant/3-10191569-C-G

      VariantEvidence: Refer to Table 1 for LOH information.

      MutationType: missense_variant;transversion

      CivicName: L188V (c.562C>G)

      MultipleGeneVariants: N/A

    2. PatientID: 1 (tumour 1)

      KindredID: 1

      Case: Sex Unknown, Age Unknown, Ethnicity Unknown

      DiseaseAssertion: VHL

      FamilyInfo: N/A

      CasePresentingHPOs: HP:0002666 (pheochromocytoma)

      CaseHPOFreeText: Patient had one benign pheochromocytoma.

      CaseNotHPOs: N/A

      CaseNotHPOFreeText: N/A

      CasePreviousTesting: RET

      PreviouslyPublished: 7759077, 9681856

      SupplementalData: N/A

      Variant: p.Y98H (c.292T>C)

      LegacyVariant: p.Y98H (c.505T>C)

      CaseProblemVariantFreeText: N/A

      ClinVar: 2223 https://www.ncbi.nlm.nih.gov/clinvar/variation/2223/

      CAID: CA020246 http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/allele?hgvsOrDescriptor=NM_000551.3%3Ac.292T%3EC

      gnomAD: 0.00001343 https://gnomad.broadinstitute.org/variant/3-10183823-T-C

      VariantEvidence: Refer to Table 1 for LOH information.

      MutationType: missense_variant;transition

      CivicName: Y98H (c.292T>C)

      MultipleGeneVariants: N/A

    1. c.407T > C

      All patients included in the study were VHL mutation carriers of 18 years or older examined between 1972 and 2012 in the VHL expertise centers University Medical Center Groningen (UMCG) and University Medical Center Utrecht (UMCU). Clinical data of 75 VHL mutation carriers were retrospectively retrieved from electronic and written patient record. Of these patients, one was found to harbour the c.407T>C germline mutation of the VHL gene. They presented with a cerebellar hemangioblastoma, spinal hemangioblastoma, renal cell carcinoma, renal cyst and pancreatic cysts. Their age at the last follow-up was 41. Previously published in: PMID 24132471.

    1. c.340G>C (p.Gly114Arg)

      Thirty-nine cases of sporadic or familial pheo/PGL and pituitary adenomas (PA) were investigated. Known pheo/PGL genes (SDHA-D, SDHAF2, RET, VHL, TMEM127, MAX, FH) and pituitary adenoma genes (MEN1, AIP, CDKN1B) were sequenced using next generation or Sanger sequencing. One patient (id: 20) presented at age 15 with a pituitary adenoma. He has a family history of clear-cell renal tumor and multiple hemangioblastomas. Genetic testing showed a germline mutation c.340G>C (p.Gly114Arg) of the VHL gene. Mother, maternal aunt, and grandmother are noted as having VHL. There was no LOH at the VHL locus in the pituitary adenoma. This patient’s information was previously published (PMID: 222652326). CIViC entry is only published for this patient in association with the alternative PMID.

    1. For instance, a patient identified with an SDHD gene mutation actually had von Hippel-Lindau (VHL) syndrome, as determined by the presence of a pathogenic VHL gene variant

      PatientID: 4

      KindredID: 4

      Case: F, 51Y0M, Ethnicity Unknown

      DiseaseAssertion: VHL

      FamilyInfo: Patient’s mother had duodenal carcinoid and her second son had pheochromocytoma.

      CasePresentingHPOs: (HP:0002666; HP:0030405; HP:0009713 (Pheochromocytoma; pancreatic endocrine tumor; spinal hemangioblastoma)

      CaseHPOFreeText: Patient presented with right pheochromocytoma at 23Y0M and left pheocromoctytoma at 32Y0M, pancreatic endocrine tumor at 32Y0M, and spinal cord hemangioblastoma was suspected.

      CaseNotHPOs: N/A

      CaseNotHPOFreeText: N/A

      CasePreviousTesting: BRCA1, BRCA2, TP53, STK11, MLH1, MLH2, MLH6, PMS2, APC, MEN1, RET, NTRK1, PTEN, RB1, SDHD, SDHAF2, SDHC, SDHB, TSC1, TSC2 and WT1 (table 2)

      PreviouslyPublished: N/A

      SupplementalData: N/A

      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: N/A

      MutationType: missense_variant; transversion

      CivicName: V166F (c.496G>T)

      MultipleGeneVariants: Germline, DHD, c.331G>A (p.Val111Ile); Germline, WT1, c.1249+7G>C (p.?)

    1. 2 Germany c.461C>T p.Pro154Leu

      GroupID: 19

      DiseaseAssertion: VHL

      GroupSize: 2

      GroupNumberMales: N/A

      GroupNumberFemales: N/A

      GroupEthnicity: German

      GroupAgeRange: N/A

      GroupInfo: 2 registrants from Germany were grouped in this study via the mutation of c.461C>T

      GroupPresentingHPOs: HP:0030393 (Endolymphatic sac tumor)

      GroupHPOFreeText: N/A

      GroupNotHPOs: N/A

      GroupNotHPOFreeText: N/A

      GroupPreviousTesting: N/A

      PreviouslyPublished: N/A

      SupplementalData: N/A

      Variant: p.P154L (c.461C>T)

      CaseProblemVariantFreeText: N/A

      LegacyVariant: N/A

      ClinVarID: 649525 https://www.ncbi.nlm.nih.gov/clinvar/variation/649525/

      CAID: N/A

      gnomAD: N/A

      VariantEvidence: N/A

      MutationType: missense_variant; transition

      CivicName: P154L (c.461C>T)

      MultipleGeneVariants: N/A

    2. 1 France c.197_209del p.Val66Glyfs*89

      GroupID: 1

      Case: Sex unknown, Age unknown, French

      DiseaseAssertion: VHL

      FamilyInfo: Unknown. 1 registrant was found with this variant; no family info was mentioned

      CasePresentingHPOs: HP:0030393 (Endolymphatic sac tumor)

      CaseHPOFreeText: N/A

      CaseNotHPOs: N/A

      CaseNotHPOFreeText: N/A

      CasePreviousTesting: N/A

      PreviouslyPublished: N/A

      SupplementalData: N/A

      Variant: p.V66Gfs*89 (c.197_209del)

      CaseProblemVariantFreeText: N/A

      LegacyVariant: N/A

      ClinVarID: N/A

      CAID: CA432536400 http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/allele?hgvsOrDescriptor=NM_000551.3%3Ac.197del

      gnomAD: N/A

      VariantEvidence: N/A

      MutationType: minus_1_frameshift_variant

      CivicName: V66Gfs*89 (c.197_209del)

      MultipleGeneVariants: N/A

    1. Patient P5 A 32-year-old man accessed the ophthalmic emergency service in 2010. BCVA was 20/32 in OD, and the fundoscopic examination revealed three peripheral RCHs with retinal detachment and a juxtapapillary fibrovascular membrane (Figure 4). Moreover, two small retinal lesions were detected in OS, and BCVA of OS was 20/20. The patient underwent retinal surgery in OS (scleral buckle and transscleral cryotherapy); then, the lesions of both eyes were treated with multiple laser sessions. The CNS imaging revealed the presence of multiple cerebellar and spinal hemangioblastomas. In 2012, cataract surgery was performed in OD. In August 2019, BCVA was 20/20 OU, and regression of all the RCHs was observed.

      PatientID: 5

      KindredID: 3

      Case: M, 41Y0M, Ethnicity Unknown

      DiseaseAssertion: VHL

      FamilyInfo: A family pedigree is provided in figure 1. Both the son and daughter of patient 5 were found to have the same mutation. Neither the 10-year-old daughter, nor the 7-year-old son had RCHs or any other tumors.

      CasePresentingHPOs: HP:0006880; HP:0009711; HP:0009713; HP:0000541 (Cerebellar hemangioblastoma; Retinal capillary hemangioma; Spinal hemangioblastoma; Retinal detachment)

      CaseHPOFreeText: Patient had an ophthalmic emergency service in 2010 at age 32Y0M. His visual acuity was 20/32 in the right eye, but fundoscopic examination revealed three peripheral RCHs with retinal detachment. Moreover, two small retinal lesions were detected in the left eye, yet the visual acuity of this eye was 20/20. The patient underwent retinal surgery in the left eye and lesions of both eyes were treated with multiple laser sessions. CNS imaging revealed multiple cerebellar and spinal hemangioblastomas. In 2012, cataract surgery was performed in the right eye. In 2019, visual acuity was 20/20 in both eyes and all the RCHs regressed.

      CaseNotHPOs: N/A

      CaseNotHPOFreeText: N/A

      CasePreviousTesting: N/A

      PreviouslyPublished: N/A

      SupplementalData: N/A

      Variant: p.R161* (c.481C>T)

      LegacyVariant: N/A

      CaseProblemVariantFreeText: N/A

      ClinVarID: 2217, https://www.ncbi.nlm.nih.gov/clinvar/variation/2217/

      CAID: N/A

      gnomAD: N/A

      VariantEvidence: N/A

      MutationType: stop_gained

      CivicName: R161* (c.481C>T)

      MultipleGeneVariants: N/A

    1. (c.482 G > A) VHL gene

      A 15-year-old Indian male presented in 2017 with a history of acute-onset painless vision loss of both eyes. He also had intermittent history of headache, which was not severe before the onset of vision loss for about 3 months and was treated as a case of optic neuritis. 2 months later he presented with episodes of headache, palpitation, chest pain, and persisting low visual acuity and high blood pressure. He was evaluated for endocrine causes of hypertension and a CT scan of abdomen revealed bilateral pheochromocytoma, which was more likely considering his young age and presenting symptoms of episodic headache and hypertension. He was given antihypertensive medications to control blood pressure and was taken up for adrenalectomy, following which his blood pressure stabilized. At his 1 year follow up, his visual acuity was more improved in both eyes. Due to his bilateral pheochromocytoma, genetic testing was performed and was found to have the p.R161Q (c.482G>A) VHL mutation consistent with VHL disease. Further follow up showed no novel tumors, no family history and his relatives have been advised to get screened for the mutation.

    1. Case 2A 13-year-old male was noted to be intermittently tachycardic and hypertensive during hospitalization for a pneumonia episode, and found to have a 6 cm right adrenal mass on MRI (Fig. 2). Patient’s father has von Hippel-Lindau disease and had undergone bilateral adrenalectomy for pheochromocytoma and bilateral nephrectomy for renal cell carcinoma. A pathogenic VHL mutation (c.583C > T, p.Q195X) was found in the patient,

      PatientID: 1

      KindredID: 2

      Case: M, 14Y7M, Ethnicity Unknown

      DiseaseAssertion: VHL

      FamilyInfo: Patient’s father has VHL disease and undergone bilateral adrenalectomy for pheochromocytoma and bilateral nephrectomy for renal cell carcinoma. His father has the same nonsense VHL mutation.

      CasePresentingHPOs: HP:0006748 (adrenal pheochromocytoma)

      CaseHPOFreeText: At 13Y0M, patient 2 presented to the hospital with pneumonia and was noted as intermittently tachycardic and hypertensive. An MRI revealed an adrenal mass. Three weeks after detection a right adrenalectomy was performed. Patient remains disease-free at the 19 month post-operative follow-up.

      CaseNotHPOs: N/A

      CaseNotHPOFreeText: N/A

      CasePreviousTesting: N/A

      PreviouslyPublished: N/A

      SupplementalData: N/A

      Variant: p.Q195* (c.583C>T)

      LegacyVariant: N/A

      CaseProblemVariantFreeText: N/A

      ClinVarID: 428794, https://www.ncbi.nlm.nih.gov/clinvar/variation/428794/

      CAID: N/A

      gnomAD: N/A

      VariantEvidence: N/A

      MutationType: stop_gained

      CivicName: Q195* (c.583C>T)

      MultipleGeneVariants: N/A

    1. PatientID: None (Brother)

      KindredID: 24

      Case: M, 27Y0M, EthnicityUnknown

      DiseaseAssertion: Asymptomatic

      FamilyInfo: 2/3 genetically tested individuals from a family with no family history of VHL disease were found to harbor the c.492G>T mutation causing a Gln164His missense variant (noted in the paper as c.705G>T due to Latif reference transcript being used). The proband presented with retinal capillary hemangioma and the other had unremarkable ophthalmoscopic findings (27Y, brother). The proband’s sister was negative for the variant.

      CasePresentingHPOs: N/A

      CaseHPOFreeText: N/A

      CaseNotHPOs: N/A

      CaseNotHPOFreeText: Paper states that ophthalmoscopic findings were unremarkable.

      CasePreviousTesting: N/A

      PreviouslyPublished: N/A

      SupplementalData: N/A

      Variant: p.Q164H (c.492G>T)

      LegacyVariant: c.705G>T

      CaseProblemVariantFreeText: N/A

      ClinVarID: 456566, https://www.ncbi.nlm.nih.gov/clinvar/variation/456566/

      CAID: N/A

      gnomAD: N/A

      VariantEvidence: N/A

      MutationType: missense_variant;transversion

      CivicName: Q164H (c.492G>T)

      MultipleGeneVariants: N/A

    1. In a study of 114 unrelated VHL families, 85 germline mutations were found. VHL mutations were detected in affected family members, but not in unaffected family members or 96 normal individuals.  This missense mutation was found in 2 family members with VHL type 1 phenotype (family ID 4480), using PCR and SSCP sequencing. The mutation of c.497T>A was referred to in the paper by c.710T>A due to a difference in transcript sequence. Both individuals were identified with CNS hemangioblastomas and one with retinal hemangioblastoma.

    1. c695G→A, R161Q mutation was identified in two unrelated kindreds

      PatientID: None

      KindredID: 3

      Case: Sex Unknown, Age Unknown, Ethnicity Unknown

      DiseaseAssertion: pheochromocytoma

      FamilyInfo: Not specified past “familial pheochromocytoma”

      CasePresentingHPOs: HP:0002666 (pheochromocytoma)

      CaseHPOFreeText: N/A

      CaseNotHPOs: N/A

      CaseNotHPOFreeText: N/A

      CasePreviousTesting: RET

      PreviouslyPublished: N/A

      SupplementalData: N/A

      Variant: p.R161Q (c.482G>A)

      LegacyVariant: p.R161Q (c.695G>A)

      CaseProblemVariantFreeText: N/A

      ClinVarID: 182983 https://www.ncbi.nlm.nih.gov/clinvar/variation/182983/

      CAID: N/A

      gnomAD: N/A

      VariantEvidence: N/A

      MutationType: missense_variant;transition

      CivicName: R161Q (c.482G>A)

      MultipleGeneVariants: N/A

    1. Genomic DNA was purified from whole blood by using the PureGene Blood Core Kit C (Qiagen, Hilden, Germany, cat. no. 158389) according to the manufacturer’s instructions. The VHL mutation analysis was performed using direct Sanger sequencing of exons and exon-intron boundaries (Supplementary Table ​Table1).1). PCR was carried out in a 15 μl reaction mixture containing 1.5 μl 10x Key Buffer, 0.3μl dNTPs (10μM), 1 μl forward and reverse primer (10μM), 0.1 μl Taq DNA polymerase, and 1μl DNA sample template (50 ng/μl). The thermal cycling setting for the primer sets include a 5 min initial denaturation at 95°C followed by 40 amplification cycles (20 sec denaturation at 95°C, 20 sec annealing at 55-65°C depending on the primer sets, and 30 sec extension at 72°, finished with a final extension at 72°C for 5 min). PCR products were bi-directionally sequenced with an ABI PRISM 3130XL Genetic Analyzer (Applied Biosystems, California, USA) using the BigDye Terminator Cycle Sequencing-Kit version 1.1 (Applied Biosystems, California, USA) according to the manufactures’ manual. The sequence was visualized and evaluated using ChromasPro, version 1.7.6 (Technelysium Pty Ltd, South Brisbane, Australia).

      ArticleReferenceSequence: NM_000551.3

      GenotypingMethod: Sanger sequencing of exons and exon-intron boundaries.

      SamplingMethod: whole blood

    1. Von Hippel-Lindau (VHL) is a tumor suppressor that negatively regulates the production of angiogenic factors. Mutations in the VHL gene cause VHL syndrome, which is characterized by highly vascularized tumors. Here we report a c.464T>A mutation of the VHL gene in three patients with hemangioblastoma from a Chinese family. This mutation was not reported previously and was absent in the unaffected family members. The mutation is predicted to cause Val to Glu substitution at VHL protein residue 155 in a conserved region. Previous biochemical studies demonstrated that residue Val-155 was critical for VHL protein binding to chaperonin TRiC/CCT, an essential step for proper VHL protein folding. Our finding of naturally occurring VHL V155E mutation in patients with VHL syndrome supports the functional importance of Val-155 residue in VHL protein and illustrates the diversity of VHL gene defects underlying VHL syndrome.
    1. 445delG

      GroupID/ KindredID: 13

      DiseaseAssertion: VHL

      GroupSize: 3

      GroupNumberMales: N/A

      GroupNumberFemales: N/A

      GroupEthnicity: Polish

      GroupAgeRange: N/A

      GroupInfo: Familial VHL stated; 3 family members recorded with this mutation.

      GroupPresentingHPOs: HP:0009711; HP:0010797; HP:0005584 (retinal capillary hemangioma, hemangioblastoma; renal cell carcinoma)

      GroupHPOFreeText: All 3 patients were tallied with a retinal capillary hemangiomas, 2 with cHemangioblastomas, and 1 with RCC. These phenotypes couldn’t be paired as it was not clear with each member.

      GroupNotHPOs: N/A

      GroupNotHPOFreeText: N/A

      GroupPreviousTesting: N/A

      PreviouslyPublished: N/A

      SupplementalData: N/A

      Variant: p.A149Pfs*10 (c.445delG)

      CaseProblemVariantFreeText: N/A

      LegacyVariant: N/A

      ClinVarID: N/A

      CAID: CA432421902 http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/allele?hgvsOrDescriptor=NM_000551.3%3Ac.445delG

      gnomAD: N/A

      VariantEvidence: N/A

      MutationType: minus_1_frameshift_variant

      CivicName: A149fs (c.445delG)

      MultipleGeneVariants: N/A

    1. Eight patients with VHL disease type 2 were selected from the group of familial VHL patients with known VHL germline mutations followed on a clinical protocol at the Urologic Oncology Branch, National Cancer Institute, National Institutes of Health (NIH). Patient 4 was a female diagnosed with VHL type 2A, a left adrenal pheochromocytoma (44Y), neuroendocrine tumor of the pancreas and retinal angioma and no metastasis. Her last follow-up was 31 years after initial diagnosis. She had a c.381_382delinsTT/p.Leu128Phe germline mutation (legacy notation c.594GC>TT converted to NM_000551.3). Suspected to be previously published, since the papers states that reported VHL patients were selected from the group of familial VHL patients with known VHL germline mutations followed on a clinical protocol at the Urologic Oncology Branch, National Cancer Institute, National Institutes of Health (NIH).

    1. Screening of 92 unrelated patients with VHL disease revealed 61 DNA variants. No variants were found in 96 control cases. Direct sequencing on peripheral blood was systematically performed on exon 1 PCR products. Southern blotting was used to search for larger alterations to the gene. The P71Lfs*61 (c.211_212insT) mutation was found in a VHL type 1 (no pheochromocytoma) patient (patient no. V96).

    2. GroupID: V13

      DiseaseAssertion: VHL

      GroupSize: 4

      GroupNumberMales: N/A

      GroupNumberFemales: N/A

      GroupEthnicity: French

      GroupAgeRange: N/A

      GroupInfo: Patients are grouped by family.

      FamilyInfo: 4/8 members of this family that underwent genetic testing had the same mutation.

      GroupPresentingHPOs: N/A

      GroupHPOFreeText: Diagnosed with VHL Type 1.

      GroupNotHPOs: HP:0002666 (Pheochromocytoma)

      GroupNotHPOFreeText: N/A

      GroupPreviousTesting: N/A

      PreviouslyPublished: N/A

      SupplementalData: N/A

      Variant: p.W117C (c.351G>T)

      LegacyVariant: N/A

      CaseProblemVariantFreeText: N/A

      ClinVarID: 167827, https://www.ncbi.nlm.nih.gov/clinvar/variation/167827/

      CAID: N/A

      gnomAD: N/A

      VariantEvidence: N/A

      MutationType: missense_variant;transversion

      CivicName: W117C (c.351G>T)

      MultipleGeneVariants: N/A

    1. GroupID/ KindredID: 4

      DiseaseAssertion: VHL

      GroupSize: 3

      GroupNumberMales: N/A

      GroupNumberFemales: N/A

      GroupEthnicity: Swedish

      GroupAgeRange: N/A

      GroupInfo: 3 patients grouped under family 4 from this Swedish VHL family

      GroupPresentingHPOs: HP:0005584; HP:0009711; HP:0010797 (renal cell carcinoma; retinal capillary hemangioma; hemangioblastoma)

      GroupHPOFreeText: 2/3 had renal cell carcinoma, 2/3 had retinal capillary hemangioma, 1/3 hemangioblastoma. Could not distinguish each patient from the other.

      GroupNotHPOs: HP:0002666 (pheochromocytoma)

      GroupNotHPOFreeText: none of the 3 patients had pheochromocytoma

      GroupPreviousTesting: N/A

      PreviouslyPublished: N/A

      SupplementalData: N/A

      Variant: p.R161* (c.481C>T)

      CaseProblemVariantFreeText: N/A

      LegacyVariant: N/A

      ClinVarID: 2217 https://www.ncbi.nlm.nih.gov/clinvar/variation/2217/

      CAID: N/A

      gnomAD: N/A

      VariantEvidence: N/A

      MutationType: stop_gained

      CivicName: R161* (c.481C>T)

      MultipleGeneVariants: N/A

    1. KindredID:71

      GroupID: 10

      GroupSize: Unknown

      GroupNumberMales: Unknown

      GroupNumberFemales: Unknown

      GroupEthnicity: Unknown

      GroupAgeRange: Unknown

      DiseaseAssertion: VHL

      GroupInfo: Kindred no.71 describes a family where at least one member (unspecified how many) had this mutation in the VHL gene. Family history indicates that pheochromocytoma is present in this kindred.

      GroupPresentingHPOs: HP:0002666 (Pheochromocytoma)

      GroupHPOFreeText: This study investigated the frequency of different mutation classes between kindreds with and without pheochromocytoma. No other information about disease presentation within members of a kindred could be inferred.

      GroupNotHPOs: N/A

      GroupNotHPOFreeText: N/A

      GroupPreviousTesting: N/A

      PreviouslyPublished: N/A

      SupplementalData: N/A

      Variant: p.C77Lfs*55 (c.228dup)

      LegacyVariant: c.441insC (p.147fs)

      GroupProblemVariantFreeText: N/A

      ClinVarID: N/A

      CAID: CA658795177, http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/allele?hgvsOrDescriptor=NM_000551.3%3Ac.228_229insC

      gnomAD: N/A

      VariantEvidence:

      MutationType: plus_1_frameshift_variant; duplication

      CivicName: C77Lfs*55(c.228dup)

      MultipleGeneVariants: N/A

    1. We identified deletions ranging from 0.5 kb to 250 kb affecting part of or the entire VHL and flanking genes in 54 families.

      GroupID: 21

      DiseaseAssertion: VHL

      GroupSize: N/A GroupNumberMales: N/A GroupNumberFemales: N/A GroupEthnicity: N/A GroupAgeRange: N/A

      GroupInfo: 54 VHL deletion families from the Freiburg registry were selected in order to assist in the correlation of VHL deletions to clinical phenotypes.

      GroupPresentingHPOs: HP:0005584; HP:0002666; HP:0009711; HP:0010797; HP:0009713; HP:0001737; HP:0030424 (Renal cell carcinoma, Pheochromocytoma, Retinal capillary hemangioma, Hemangioblastoma, Spinal hemangioblastoma, Pancreatic cysts, Epididymal cyst)

      GroupHPOFreeText: # of affected individuals: Renal cell carcinoma (1), Pheochromocytoma (1), Retinal capillary hemangioma (1), Hemangioblastoma/brain/cerebellum (1), Spinal hemangioblastoma (1), Pancreatic cysts (1), Epididymal cyst (1)

      GroupNotHPOs: N/A

      GroupNotHPOFreeText: N/A

      GroupPreviousTesting: N/A

      PreviouslyPublished: N/A

      SupplementalData: Supplemental Table 2, Table 1, Table 3

      Variant: Exon 2-3 Deletion (c.341-?_642+?del)

      LegacyVariant: N/A

      CaseProblemVariantFreeText: N/A

      ClinVarID: N/A

      CAID: N/A

      gnomAD: N/A

      VariantEvidence: N/A

      MutationType: exon_loss_variant

      CivicName: Exon 2-3 Deletion

      MultipleGeneVariants: N/A

    1. III/6 (44)-----Pro25Leu

      PatientID: IIII-6

      KindredID: 1

      Case: F, 44Y0M, Hungarian

      DiseaseAssertion: Asymptomatic

      FamilyInfo: The patient's mother (II-5) carried the Ser80Asn mutation and presented with renal cell carcinoma. The patient's siblings (III-7 and III-8) both carried the Ser80Asn mutation. Patient III-7 presented with hemangioblastoma of the brain and retina hemangioblastoma and her children carried the Ser80Asn mutation with clinical presentation. Patient III-8 presented with brain hemangioblastoma, pheochromocytoma, and retina hemangioma. The patient's maternal aunt (II-1) carried both Ser80Asn and Pro25Leu mutation but was asymptomatic. She passed on the Ser80Asn mutation to her daughter (III-1), the proband and this patient's cousin. Refer to figure 1 for pedigree.

      CasePresentingHPOs: N/A

      CaseHPOFreeText: N/A

      CaseNotHPOs: HP:0005584, HP:0002666, HP:0010797, HP:0009711 (renal cell carcinoma, pheochromocytoma, hemangioblastoma, retinal capillary hemangioma)

      CaseNotHPOFreeText: N/A

      CasePreviousTesting: N/A

      PreviouslyPublished: N/A

      SupplementalData: N/A

      Variant: p.P25L (c.74C>T)

      LegacyVariant: N/A

      CaseProblemVariantFreeText: N/A

      ClinVarID: 93330 https://www.ncbi.nlm.nih.gov/clinvar/variation/93330/

      CAID: CA020538 http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/allele?hgvsOrDescriptor=NM_000551.3%3Ac.74C%3ET

      gnomAD: 0.002988 https://gnomad.broadinstitute.org/variant/3-10183605-C-T

      VariantEvidence: N/A

      MutationType: missense_variant;transition

      CivicName:P25L (c.74C>T)

      MultipleGeneVariants: N/A

    1. An asymptomatic 47-year-old woman was diagnosed as an obligate carrier of a mutant von Hippel-Lindau gene. The diagnosis of von Hippel-Lindau disease in her daughter had led to her retrospective diagnosis and that of her son. The patient was screened and found with metastatic renal cell carcinoma and extra-adrenal pheochromocytoma. The VHL mutation in the family is c.257C>T (p.Pro86Leu). The patient died 31 months after being diagnosed and the phenotype of her children is unknown.

    1. CASE REPORTA young lady (18 years of age) presented with recurrent episodes of headache for 3 years, associated with episodic sweating and palpitations. She was recently documented to have blood pressure (200/160 mmHg) and was referred to us for further management.A detailed family history revealed the occurrence of central nervous system (CNS) tumors in her father and two of his siblings [Figure 1]. Her father had undergone C7–D3 decompression laminectomy for D2, D3 hemangioblastomas. He recovered, but subsequently developed recurrence of the spinal tumor. At this stage, he was also detected to have a right adrenal mass, and both tumors were left untreated. He developed paraplegia and eventually died following a cerebrovascular accident. His two brothers underwent surgery for cerebellar hemangioblastomas at age 46 and 50 years, and they were reportedly well following surgery. Our subject has two siblings aged 17 and 20 years, both of whom had no symptoms suggestive of VHL.Open in a separate windowFigure 1Pedigree analysis of patientClinical examination revealed no neurocutaneous markers. Her blood pressure was 170/140 mmHg (supine) and 150/120 mmHg (standing). Fundus examination revealed bilateral papilledema. Her neurological examination was normal.Her 24-hour urine metanephrine level was 100 mcg and normetanephrine level was >5700 μg [normal range up to 600 mcg (metanephrine) and 900 mcg (normetanephrine)]. She also had impaired glucose tolerance. Computed tomography (CT) scan revealed a large heterogeneous right adrenal mass with patchy peripheral enhancement, a smaller left adrenal mass and an enhancing lesion in the distal body of the pancreas [Figure 2]. Metaiodobenzylguanidine (MIBG) scan showed significantly increased uptake over both adrenal glands [Figure 3]. At surgery, she was found to have an 8-cm tumor in the right adrenal, a 2-cm tumor in the left adrenal and a 2-cm hard pancreatic neoplasm in the distal pancreas [Figure 4]. She underwent bilateral adrenalectomy (right total adrenalectomy, left cortical sparing adrenalectomy) and distal pancreatectomy with splenectomy. Histology of the tumor showed bilateral pheochromocytoma and well-differentiated neuroendocrine tumor of the pancreas. The postoperative period was uneventful, and she was advised regular follow-up. We also advised genetic screening to all her first-degree relatives. After surgery, she was asked to continue Tab. Prednislone 5 mg once daily (in the morning). This patient is on telephonic follow-up and came last month back for follow-up. We planned to taper off steroids and assess response but she herself stopped the medicines and her serum cortisols were checked which were in normal range off all medicines. She is not on any antihypertensive medication. Following surgery, she had no visual complaints and her papilledema had resolved.

      PatientID: 1 (case)

      KindredID: 1

      Case: F, 18Y, Indian

      DiseaseAssertion: assumed VHL

      FamilyInfo: A detailed family history revealed the occurrence of central nervous system (CNS) tumors in her father and two of his siblings. Her father had recurrence of spinal tumors, adrenal masses, paraplegia, and eventually died following a cerebrovascular accident. His two brothers underwent surgery for cerebellar hemangioblastomas at age 46 and 50 years. The subject has two siblings aged 17 and 20 years, both of whom had no symptoms suggestive of VHL. No other members have been tested.

      CasePresentingHPOs: HP:0006748; HP:0030405; HP:0001085 (adrenal pheochromocytoma, pancreatic endocrine tumour, Papilledema)

      CaseHPOFreeText: presented with recurrent episodes of headache for 3 years, associated with episodic sweating and palpitations. Fundus examination revealed bilateral papilledema (right adrenal mass with patchy peripheral enhancement, a smaller left adrenal mass (bilateral pheochromocytoma) and an enhancing lesion in the distal body of the pancreas)

      CaseNotHPOs: N/A

      CaseNotHPOFreeText: Her neurological examination was normal.

      CasePreviousTesting: N/A

      PreviouslyPublished: N/A

      SupplementalData: N/A

      Variant: c.463+3A>G

      LegacyVariant: c.IVS2+3A>G

      CaseProblemVariantFreeText: N/A

      ClinVarID: 428796 https://www.ncbi.nlm.nih.gov/clinvar/variation/428796/

      CAID: N/A

      gnomAD: N/A

      VariantEvidence: N/A

      MutationType: splice_site_variant

      CivicName: Splice Region (c.463+3A>G)

      MultipleGeneVariants: N/A

    1. Mutations

      45 Lebanese patients with a reported family history of breast cancer were observed in this study. Using Whole Exome Sequencing (WES) with Sanger sequencing, various germline mutations were found. One BRCA2 mutation was found in a patient with an accompanying VHL mutation. Patient code B22 (belonging to family 3) had a M221L (c.631A>C) VHL mutation. This patient also had mutations in the following genes: ARL11, TP53, ERCC3, MRE11A, and PTCH1. Other than a family history of breast/ovarian cancer, this patient’s specific manifestations are unclear.

    1. 612–613delGC Fs Exon 3 204 GAG E No stop

      A study of 103 patients with VHL retinal manifestations and 108 patients without VHL retinal manifestations extracted from the French VHL database revealed that the number of hemangioblastomas appeared higher in patients who had a substitution when compared with patients with a truncated protein. Detected by PCR and SSCP, this frameshift mutation p.R205Hfs*50 (c.612_613delGC) was found in a VHL patient without retinal hemangioblastomas. Other manifestations are not clearly stated. No family information was provided.

    1. PatientID: 10

      KindredID: Unknown

      Case: M, 40Y0M, Dutch

      DiseaseAssertion: VHL

      FamilyInfo: Unknown - study sampled 15 patients from 5 families

      CasePresentingHPOs: HP:0001737; HP:0000107; HP:0010797; HP:0006880; HP:0005584 (pancreatic cysts, renal cyst, cerebellar hemangioblastoma, spinal hemangioblastoma, renal cell carcinoma)

      CaseHPOFreeText: N/A

      CaseNotHPOs: HP:0009711 (retinal capillary hemangioma)

      CaseNotHPOFreeText: No evidence of left or right eye angiomas

      CasePreviousTesting: N/A

      PreviouslyPublished: N/A

      SupplementalData: N/A

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

      LegacyVariant: p.R238Q

      CaseProblemVariantFreeText: Variant determined through the assumption of the authors using Latif et al 1993 (AF010238.1) transcript and adding 71bp to the protein sequence.

      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:N/A

      MutationType: missense_variant;transition

      CivicName: R167Q (c.500G>A)

      MultipleGeneVariants: N/A

  2. www.researchsquare.com www.researchsquare.com
    1. was not concerned that freedom fighters were referred to as terrorists, did not demand proof of God’s existence nor did I think that the missionaries, along with all the other Whites in Rhodesia, ought to have stayed at home.

      This sentence is easy to miss, but it shows what Tambu’s “good girl” behavior costs her. She is praised as a model daughter because of what she does not question: the labeling of freedom fighters as terrorists (colonial politics), God’s existence (religion), and the missionaries’ place in Rhodesia (race). The book’s central tension is that being “well-behaved” depends on not noticing injustice. Babamukuru rewards that silence, while Nyasha is punished for asking exactly these questions. The older narrator is criticizing her younger self here, since the passage reads as a list of everything she failed to see. This suggests the book is about political awareness as much as gender.

    2. The victimisation, I saw, was universal. It didn’t depend on poverty, on lack of education or on tradition. It didn’t depend on any of the

      Women in society face many challenges simply because of their gender. Regardless of their education, wealth or social status, they can still experience unfair treatment from men. Some women are forced to sacrifice their personal ambitions to satisfy family and cultural expectations. However, women deserve equal opportunities, respect and the freedom to make their own decisions.

    3. hido went into Standard Six in the year that Babamukuru returned from England. This meant that he was going to secondary school the following year. Babamukuru had intended him to stay on at the mission to counteract the unAfrican exposure he had been subjected to in England, but Mr Baker, as Nyaradzo’s father is called, arranged for Chido to take the entrance examination to his son’s school. So determined was this good missionary that Chido should have the best in life that he personally drove my cousin to Salisbury, where the examination was taking place. Not surprisingly, since Whites were indulgent towards promising young black boys in those days, provided that the promise was a peaceful promise, a grateful promise to accept whatever was handed out to them and not to expect more, Chido was offered a place at the school and a scholarship to go with it. Nyasha was sure that Mr Baker had had a hand in that scholarship. To ease his conscience, she said. ‘A word with the headmasters’ she told me when Nyaradzo was not present. ‘You know how it is, bwana to bwana: “The boy needs the cash, old man!”; “He’s a good boy, what. Pity to waste him. We'll see what we can do.” So Chido gets his scholarship and Mr Baker feels better about sending his sons there in the first place. Really! The things they get up to to pull the wool over our eyes. Really!’ Nyasha’s analysis made sense because Babamukuru did not approve of European habits, easy options or unnecessary expenditure. Without the scholarship Chido certainly would not have gone to that school and Mr Baker would have had his sons’ superior education nibbling away at his conscience. The upshot of all this was that Chido did go to this boarding school and by the time I went to the mission he had adapted remarkably well to private-school life. He had acquired the usual courteous, self- indulgent, private —- school habits and was firm friends with the Baker boys. As a result I did not see much of him or of the Bakerboys, except for a day or two at the beginning or the end of the holidays, because their school ran according toa calendar that was different from the one we used at the mission. Since Nyasha was in Form Two in that year, my first year at the mission, she was writing her first set of public examinations. Actually, they were her second set, because she had sat her Standard Six examinations, but Nyasha, who had benefited froma head-start programme for gifted children in England, pooh- poohed those Standard Six examinations. Characteristicall

      In this passage, the characters experience different forms of struggle. Nyasha struggles with educational pressure and psychological exhaustion as she faces demanding examinations and high expectations. Babamukuru struggles with cultural identity as he tries to balance African traditions with Western education. Tambu also experiences social and educational inequality by observing the advantages enjoyed by Chido through his father's connections. These struggles reveal the effects of education, cultural differences and social inequality on the characters' lives.

    4. the way she had begun to call me ‘Sisi’, because she was older than I and so did not need to cali me that; nor had she done so when she came home for Nhamo’s funeral or when I arrived, a matter of a few hours ago. Not that it was important in i

      Anna struggles with the changes in social relationships and hierarchy .After Tambu arrives at Babamukuru's home ,Anna begins to treat her differently by "sisi" and kneeling while speaking to her .This shows how social status influences the way people interact with one another .Anna also struggle with communication because she becomes quiet and reverved toward Tambu .Her behaviour reveals how change in social position can create distance and discomfort between people

    5. urther than I had even thought of reaching, was beginning to indicate that there were other directions to be taken, other struggles to engage in besides the consuming desire to emancipate myself and my family. Nyasha gave me the impression of moving, always moving and striving towards some state that she had seen and accepted a long time ago. Apprehensive as I was, vague as I was about the nature of her destination, I wanted to go with her. I did not want to be left behind. And being so young, time measured in hours and half hours, was important, so I did not want to spend three whole weeks away from my cousin. Knowing I would be at a loss without her, I was tempted to ask Babamukuru whether I could go with them to the mission, but in the end I decided not to because I knew he would refuse and be justified in doing so. There was much work to be done in the fields, in the garden, in the home. And, of course, I could not leave my mother, who was not well. We all gathered in the yard, my father, my mother, Takesure, Lucia, my little sisters and myself, to wave them off, with everybody laughing and talking, saying my uncle must be sure to come back soon, bringing Maiguru, Nyasha and Chido as well with him. And then my relatives were in the car, rolling out of the yard and out of sight. A sigh escaped from us and we all felt oddly relieved. “Whew! It was good to have Mukoma here, it was good,’ observed my father, ‘but it puts a weight on your shoulders, a great weight on your shoulders!’ ‘It’s true,” nodded Takesure, leering lasciviously at Lucia. ‘Dhiya! There’s nothing to be afraid of any more. Won’t youcome back to the Aozi tonight?’ ‘Maybe if you cut my hands off,’ retorted Lucia. ‘Then you might be useful.’ “But you have the hands and you are still here. So you are waiting, aren’t you? Just waiting to come back into the hozi.’ Lucia was careful not to be provoked. ‘You know why I’m waiting? For my sister, isn’t it? As soon as my sister makes up her mind what she wants, you won’t see me here any more.’ My father and Takesure found this amusing. They had a good laugh at Lucia’s expense. ‘Now what is this lam hearing!’ gurgled Takesure. ‘The woman

      In this passage, the characters experience different forms of struggle. Nyasha struggles with educational pressure and psychological exhaustion as she faces demanding examinations and high expectations. Babamukuru struggles with cultural identity as he tries to balance African traditions with Western education. Tambu also experiences social and educational inequality by observing the advantages enjoyed by Chido through his father's connections. These struggles reveal the effects of education, cultural differences and social inequality on the characters' lives.

    6. blessing but one that

      In this passage, the characters experience different struggles. Tambu struggles with family expectations and maintaining her identity while trying to succeed in education. Babamukuru struggles with his responsibilities as a guardian and his desire to guide Tambu towards a successful future. Nyasha struggles with personal discomfort and adapting to life with others. These struggles reveal the challenges of family relationships, social expectations and personal differences in the novel.

    7. ‘Even if you do ignore me,” Lucia continued, ‘it doesn’t mean I’m not here. And anyway, Mwaramu, maybe you can tell me plainly: where do you want me to go? We both know I can’t go home. Their sending me here in the first place, it was because there was no food, and no work either at that place, isn’t it? It’s true, you know it. So where do you want me to go? As for going with Takesure, ha-a-a! I know it’s the way you joke, Babamukuru. What would I go to do at Takesure’s home?’ I wished Lucia would be quiet. Her case was a serious one and it did not help to be rude to Babamukuru. Lucia was my mother’s sister, several years younger than my mother and a wild woman in spite of - or maybe because of — her beauty. She was dark like my mother, but unlike my mother her complexion always had a light shining from underneath the skin, so she could afford to scoff at the skin-lightening creams tha@other girls used. ‘Fanta and Coca- Cola!’ she laughed. ‘Aiwa! Not me. I prefer to be the same colour all over.’ Asa result, her skin was not deteriorating and breaking; it remained glowing and healthy. My mother’s family was very poor, poorer even than my own. At the time that my father took my mother, there were no cattle at all in my grandfather’s kraal. Because of this, some people had believed it was a blessing that the first two of my maternal grandparents’ children were girls. ‘Otherwise,’ they reasoned optimistically, ‘if he had had sons, how would those sons have taken wives? See now, the daughters will bring cattle, the cattle will enable the old man to work his fields, the family will prosper, and when the sons are of an age to marry, by then they will have acculuated their roora.’ The wise and the cynical disagreed. ‘Who knows?’ they dissented. ‘If there had been sons earlier, they would have helped the old man on the land. The family would have been better off than they are now. Besides,” they added significantly, ‘a man can’t be sure about daughters!” Thus the debate continued in my mother’s village, way up in the north-west of the country, until my father, visiting a distant relative, saw my mother, impregnated her and was obliged to take her home with him. It was unfortunate that it happened this way because, under these circumstances, my grandfather could not

      In this passage, the characters experience different forms of struggle. Nyasha struggles with educational pressure and psychological exhaustion as she faces demanding examinations and high expectations. Babamukuru struggles with cultural identity as he tries to balance African traditions with Western education. Tambu also experiences social and educational inequality by observing the advantages enjoyed by Chido through his father's connections. These struggles reveal the effects of education, cultural differences and social inequality on the characters' lives.

    8. I’m glad,’ said Nyasha when we began to talk seriously, ‘that we have to share this room. It means we’ll be friends. But you curled your lip at us when we came back from England, so I didn’t know what was going to happen when you came. I just said to myself, P'll try to be nice, I'll try to be friendly and we'll see what happens.’ ‘But I did not sneer at you,’ I protested, speaking in Shona. Our conversation was laboured and clumsy because when Nyasha spoke seriously her thoughts came in English, whereas with me, the little English I had disappeared wh

      This passage shows the challenges Nyasha faces in building relationships and expressing herself .its highlight how societal and cultural expectations can affect girl's experience and their ability to feel understood .This suggests that girls may face emotional struggles while trying to find their place in society

    9. onally, I thought it was a great shame that Maiguru had been deprived of the opportunity to make the most of herself, even if she had accepted that deprivation. I was all for people being given opportunities.

      I’d cut the sentence ‘I was all for people being given opportunities’ and replace the explanation with a short exchange between Tambu and Maiguru so we see Tambu’s reaction instead of being told it.

    10. What was needed in that kitchen was a combination of Maiguru’s detachment and Lucia’s direction. Everybody needed to broaden out a little, to stop and consider the alternatives, but the matter was too intimate. It stung too saltily, too sharply and agonisingly the sensitive images that the women had of themselves,images that were really no more than reflections. But the women had been taught to recognise these reflections as self and it was frightening now to even begin to think that, the very facts which set them apart as a group, as women, as a certain kind of person, were only myths; frightening to acknowledge that generations of threat and assault and neglect had battered these myths into the extreme, dividing reality they faced, of the Maigurus or the Lucias.So instead of a broadening from both positions, instead of an encompassing expansion and a growth, the fear made it necessary to tighten up. Each retreated more resolutely into their roles, pretending while they did that actually they were advancing, had in fact initiated an offensive, when really, for each one of them, it was a last solitary, hopeless defence of the security of their illusions.

      COMMENT: This paragraph emphasizes how women in this society are confined to inflexible, defensive roles. Rather than coming together or breaking away, the pressure and long-standing neglect compel them to protect their own illusions of security, revealing the profound psychological cost of the burdens society places on them.

      REWRITE: Within the kitchen, the thick atmosphere was filled with a quiet, desperate strain. Instead of uniting or breaking down the systemic barriers erected around them, each woman withdrew further into her own familiar defenses. They carried out their designated roles with bleak exactness acting as though survival required obedience transforming every ordinary chore into a silent, isolated guard against a world that gave them no way out.

    11. Nhamo laughed when I related my story. ‘So what! I don’t care what he says,’ he shrugged, shocking me with this disrespectful language that I had not heard before. ‘I’m at school, aren’t I? It doesn’t worry me what he says about me. So what’s your problem? It doesn’t even affect you.” ‘But you can’t study.” ‘Who says? I should know. I go to schooi. You go nowhere.’ *But I want to go to school.’ ‘Wanting won’t help.” ‘Why not?’ He hesitated, then shrugged. ‘It’s the same everywhere. Because you are a girl.’ It was out. ‘That’s what Baba said, remember?’ I was no longer listening. My concern for my brother died an unobtrusive death.

      This passage highlights one of the novel's central tensions: the struggle between traditional expectations and individual freedom. Through the characters, Dangarembga questions a society that gives men greater authority while limiting women's opportunities.

    12. Today there are fewer white people on the mission. They are called expatriates, not missionaries, and can be seen living in unpainted brick houses. But they are deified in the same way as the missionaries were because they are white so that their coming is still an honour. J am told that whether you are called an expatriate ora missionary depends on how and by whom you were recruited. Although the distinction was told to me by a reliable source, it does not stick in my mind since I have not observed it myself in my dealings with these people. I often ask myself why they come, giving up the comforts and security of their more advanced homes. Which brings us back to matters of brotherly love, contribution and lightening of diverse darknesses.

      The ongoing "deification" of white figures on the mission reinforces an implicit social lesson that authority, wisdom, and value remain rooted in Western, colonial systems rather than local traditions. For girls like Tambu and Nyasha, growing up in this environment means their personal development and education are bound to a system that demands cultural self-effacement to gain approval. Ultimately, this racial dynamic adds an inescapable layer of displacement to their struggle as young women, forcing them to negotiate their identity under both colonial condescension and patriarchal rule.

    13. nversation I had with my mother about the toilet happened four or five days after we arrived. On the day we arrived, when the car rolled to a stop under the shade of the mango trees, we were greeted by depressing silence. When people did come to welcome us it was only Netsai and Rambanai, more naked than dressed in their tattered frocks, their legs and their arms and even their hair and faces grey with shena. Dirty and dusty, they embraced us, wrapping their arms around Babamukuru first and then Maiguru, Nyasha, myself and lastly Anna: ‘You have come, Babamukuru. You have come, Maiguru. You have come, Sisi Nyasha,’ and so on until we had all been ritually embraced.

      Netsai and Rambanai's primary role during the arrival is immediately framed through subservience and ritualistic duty as they dutifully welcome and embrace the higher-status family members. Ultimately, their neglected physical state stands in stark contrast to the privileged, educated life Tambu and Nyasha experience at the mission, exposing how poverty severely limits a girl's dignity and development within the homestead.

    14. Anna came and knelt down in the doorway to tell us that supper was nearly ready. ‘For heaven’s sake, Anna, stand up!’ commanded Nyasha irritably. ‘Every time you come in here I tell you not to kneel down, but you keep on doing it. What’s the matter with you?’ And then she was ashamed of herself. ‘Now, honestly, just listen to me. Getting all cross and crotchety!

      Class and gender: Anna is a poor woman serving the household, and her kneeling shows the hierarchy.

    15. t was embarrassing the way Lucia wouldn't give up on Maiguru. Maiguru, who thought that Lucia ought to suffer the consequences of her fecund appetites, perhaps felt sorry for her too, but preferred all the same not to become involved in matters of the flesh or the earth, although she did not like this preference to be too obvious since it meant she was setting herself above the rest of us. Now, with Lucia insisting that Maiguru take sides, come out in the open, we were in a very delicate situation.

      Lucia’s relentless pressure on Maiguru was painful to watch. Maiguru believed Lucia should lie in the bed her runaway passions had made, yet a sliver of sympathy lingered beneath her cool exterior. Still, she preferred to keep her hands clean of dirty, earthbound affairs—though she hid that superiority carefully to avoid looking like she was too good for the rest of us. But now, with Lucia forcing her into a corner and demanding she take a stand, we were all walking on razor-thin ice.

    16. I felt sorry for Maiguru because she could not use the money she earned for her own purposes and had been prevented by marriage from doing the things she wanted to do. But it was not so simple, because she had been married by my Babamukuru, which defined her situation as good. I

      It’s wild that Maiguru has a Master’s degree and works full-time, but still has to ask her husband for basic spending money. Even with her own education and paycheck, the family dynamics still treat her like she has no say

    17. Personally, I thought it was a great shame that Maiguru had been deprived of the opportunity to make the most of herself, even if she had accepted that deprivation.

      It shows that even an educated woman like Maiguru can experience gender inequality within marriage. Despite having her own qualifications and earning money, she has sacrificed some of her personal ambitions and freedom for her husband and family. It suggests that education alone does not guarantee women independence in a patriarchal society. I find this important because Maiguru's situation challenges the assumption that a successful marriage automatically means a woman is fulfilled.

    18. Coming to the mission, continuing my education and doing well at it, these had been the things that mattered. And since these things had been progressing according to plan for nearly two years, I had thought that ambiguities no longer existed. I had thought that issues would continue to be clearly delimited, with Babamu- kuru, who was as nearly divine as any human being could hope to be, imposing the limits. Through him, because of him, black would remain definitely sombre and white permanently clear, even in , Spite of Nyasha, whose strange disposition hinted at shades and * textures within the same colour. My vagueness and my reverence for my uncle, what he was, what he had achieved, what he represented and therefore what he wanted, had stunted the growth of my faculty of criticism, sapped the energy that in childhood I had used to define my own position. It had happened insidiously,

      I would rewrite this paragraph by giving Tambu more opportunity to express her personal feelings and thoughts. I would add more detail about her internal conflict between following society's expectations and pursuing her educational ambitions. I would keep the original message but make her determination and emotional struggle more visible.

    19. Maiguru grew very distant. “This matter is not my concern,’ she shrugged, carelessly turning down the corners of her mouth. ‘Am I of their totem? I am not. I was taken. Let them sort out their own problems, and as for those who want to get involved, well, that’s up to them. I don’t want to intrude into the affairs of my husband’s family. I shall just keep quiet and go to bed.’

      Although Maiguru is an educated, highly accomplished woman holding a Master's degree, her societal role is reduced to a passive dependent within Babamukuru's patriarchal family structure. Her cynical detachment ("I was taken") and refusal to get involved reflect her coping mechanism for surviving an environment where her intellect and voice carry no institutional weight. Ultimately, her deliberate retreat into silence ("I shall just keep quiet and go to bed") underscores the tragic purpose of the novel: illustrating how patriarchal dominance and colonial structures force even the most educated women into self-suppression and emotional disengagement.

    20. hen Nyasha came back with the gravy, Anna following with the rest of the meal. Maiguru said that Babamukuru’s old meal was no longer fresh. She said she would eat it herself, that Babamukuru should serve himself another portion of food. Babamukuru thought his wife was making a fuss about nothing, but she insisted, so the ritual dishing out of my uncle’s food was performed again. This time Nyasha did not wait for her father to finish. By the time he was on to the third dish, she was helping herself to rice.

      This passage demonstrates how women are expected to follow traditional gender roles rather than pursue their own ambitions. The author shows that being a woman in this society means facing restrictions on education, independence and personal freedom.

    21. did understand why I could not go back to school, but I loved going to school and I was good at it. Therefore, my circumstances affected me badly. My father thought I should not mind. ‘Is that anything to worry about? Ha-a-a, it’s nothing,’ he reassured me,

      for the second question Tambu is denied education even though she is a girl

    22. As far as I was concerned, people only made clay pots when they were very young and playing at being grown up, or when they were grown up because they had to have pots for storing water and mahewu and the like. But we used two- and five-gallon drums for water these days and I had never seen anybody make a proper dari although we had several in the house. So those pots were definitely Nyasha’s craze, not mine, and while she was very concerned about making sure that they did not crack, while she was meticulous in scratching her designs, they did not matter much one way or the other to me, serving only to pass the tim

      Tambu’s dismissal of the activity as "Nyasha’s craze" underscores how Nyasha's Westernized desire for creative autonomy alienates her from other girls who view womanhood strictly through the lens of utilitarian labor. Ultimately, obsessing over the clay pots provides Nyasha a rare, private space where she can exert agency and control in a world where her thoughts, behavior, and body are otherwise heavily policed.

    23. Although she had been brought up in abject poverty, she had not, like my mother, been married to it at fifteen. Her spirit, unfettered in this respect, had experimented with living and drawn its own conclusions. Consequently, she was a much bolder woman than my mother; and my father, who no longer felt threatened by a woman’s boldness since he had proved his mettle by dispiriting my mother, was excited by the thought of possessing a woman like Lucia, like possessing a thunderstorm to make it crackle and thunder and lightning at your command.

      What I took away: I streamlined the wordy phrasing around her "unfettered" spirit and "proving his mettle" to make the text flow more naturally.

      What I added/emphasized: I made it clearer that Jeremiah’s attraction to Lucia’s independence wasn't real respect—it was just a desire to control a strong woman the same way he had already broken Tambu’s mother. "Because Lucia was not forced into marriage at fifteen like my mother, her spirit remained free to test life and form her own opinions. This made her far bolder than my mother. My father was actually drawn to that boldness—not because he respected her, but because he had already broken my mother's spirit and now wanted the thrill of controlling a woman as wild and powerful as a thunderstorm"

    24. Babamukuru thought his wife was making a fuss about nothing, but she insisted, so the ritual dishing out of my uncle’s food was performed again.

      Maiguru's actions show that women in the family are expected to take responsibility for serving men. Although she is Babamukuru's wife, her work in the kitchen is treated as normal. Nyasha's decision not to wait for her father to finish eating suggests that she does not fully accept these traditional expectations.

    25. ‘This business of :-womanhood is a heavy burden,” she said. ‘How could it not be? ‘Aren’t we the ones who bear children? When itis like that you can’t just decided today I want to do this, tomorrow I want to do that, ‘ the next day I want to be educated! When there are sacrifices to be ‘ made, you are the one who has to make them.

      one of the answers of the first questions

    26. It happens,’ she assured me. ‘You get so comfortable and used to the way things are. Look at me now. I was comfortable in England but now I’m a whore with dirty habits.’ ‘But, Nyasha,’ I began. ‘I know,’ she interrupted. ‘It’s not England any more and I ought to adjust. But when you’ve seen different things you want to be sure you’re adjusting to the right thing. You can’t go on all the time being whatever’s necessary. You’ve got to have some conviction, and I’m convinced I don’t want to be anyone’s underdog. It’s not right for anyone to be that. But once you get used to it, well, it just seems natural and you just carry on. And that’s the end of you.

      This is literally the main point of the whole book. Nyasha points out how dangerous it is to just "adjust" to unfair expectations because eventually it starts feeling normal and you completely lose your identity. She’d rather fight back than silently accept being treated like an underdog.

    27. the decision at least was mine.

      Tambu’s first act of resistance against Babamukuru. It shows the tension between obedience and self-determination. Even after she sees the photos and regrets it, she values that the choice was hers.

    28. But I did not sneer at you,’ I protested, speaking in Shona. Our conversation was laboured and clumsy because when Nyasha spoke seriously her thoughts came in English, whereas with me, the little English I had disappeared when I dropped my vigilance to speak of things that mattered.

      This slightly highlights the main idea of the book, telling us in a way that girls were less educated in the society than men were.

    29. Babamukuru insisted he would kill Nyasha and then hang himself. ‘She has dared,’ he said, sweat pouring off him, his chest heaving with the grossness of the thought, ‘to raise her fist against me. She has dared to challenge me. Me! Her father. I am tellingyou,’ and he began to struggle again, ‘today she will not live. We cannot have two men in this house. Not even Chido, you hear that Nyasha? Not even your brother there dares to challenge my authority. Do you hear what I am saying, do you hear? Your salvation lies in going away from my house. Forever. Otherwise,’ he spat in her face because, still securely held, he could not strike her, ‘otherwise I-will-kill-you.’ He spat again. Nyasha rose from the floor, walked out of the room. ‘She walks! She just walks away. She is proud. That is her problem. She is proud. Pthu! Sis! She is not my daughter.’

      What i added :Enhanced sensory details and physical pacing (e.g., "sweat plastering his shirt," "veins bulging") to heighten the visceral tension and claustrophobia of the domestic explosion.

      Intensified the emotional weight of Babamukuru's dialogue, underscoring how deeply his patriarchal ego and sense of god-like authority have been shattered by a fourteen-year-old girl.

      What was taken away:

      Cleaned up minor transcription artifacts and repetitive interruptions from the raw text (such as raw bullet-like punctuation fragments or awkward line breaks).

      Tightened the prose to make the sequence of physical restraint, the final act of degradation (spitting), and Nyasha’s silent, chilling exit flow with cinematic continuity

      Rewritten Version of the Paragraph Babamukuru shook with rage, sweat plastering his shirt as he hissed that he would end her life and his own. "She has dared," he choked, his chest heaving with the blasphemy of it, "to raise her fist against me. To challenge me! Me, her father!" He lunged forward again, held back only by force, screaming that she would not see another day. "There cannot be two men in this house—not even Chido dares to question my authority! Do you hear me? Your only salvation is to leave this house forever. Otherwise..." Unable to swing his arms, he spat directly into her face. "Otherwise..." He spat a second time. Slowly, Nyasha stood up from the floor and walked past him into the dark hallway. "She walks away! She just walks away!" Babamukuru roared, veins bulging as he condemned her pride. "That is her sickness, she is proud! Pthu! Get out! She is no longer my daughter.!!

    30. Maiguru represents the struggles of educated women in a patriarchal society. Despite having a Master's degree, she cannot fully enjoy her independence because her responsibilities as a wife and mother take priority. This shows that education alone does not guarantee freedom or equality for women.

    31. Maiguru snorted. ‘And what do you expect? Why should a woman go all that way and put up with all those problems if not to look after her husband?’

      This passage highlights the struggle between personal ambitions and family responsibilities. Maiguru is educated and has her own dreams, but she has sacrificed many of them because of her marriage. The author shows that women can lose their independence even when they are educated and financially productive.

    32. Then Nyasha came back with the gravy, Anna following with the rest of the meal. Maiguru said that Babamukuru’s old meal was no longer fresh. She said she would eat it herself, that Babamukuru should serve himself another portion of food. Babamukuru thought his wife was making a fuss about nothing, but she insisted, so the ritual dishing out of my uncle’s food was performed again. This time Nyasha did not wait for her father to finish.

      this passage reveals the gender roles and unequal expectations within Babamukuru's family. Maiguru takes responsibility for ensuring that her husband receives special treatment, while the women are expected to serve him. The repeated ritual of serving his food shows how male authority is deeply rooted in their family traditions. However, Nyasha's decision not to wait for her father to finish eating may suggest her resistance to these traditional expectations. It makes me question why men are given special privileges while women are expected to remain obedient and serve them

    33. What are you doing, Nyasha?’ Babamukuru asked, withoutlooking at her, so that one wondered how he had noticed. ‘I thought you had finished with the rice,’ Nyasha replied, ladling meat and gravy on to her rice. ‘What about your mother, here?’ Babamukuru asked conver- sationally. ‘Do you think she doesn’t know what she’s doing, waiting on me like this?’ ‘I don’t like cold food,’ Nyasha pointed out, as willing as her father to engage in dialogue.

      This shows us that the women in their household didn't have the freedom to even eat food whenever they wanted to.

    34. as more concerned with how early it was, much too early to be eating the last meal of the day. At that rate I would be hungry by the time I went to bed, and I had nightmares when I slept without a comforting mound of sadza warming my stomach.

      Represents struggle of food and sleep.

    35. at I noticed, very early on, was that some of the missionaries were definitely strange, strange in the way that Nyasha and Chido were Strange when they came back from England. These missionaries, the strange ones, liked to speak Shona much more than they liked to speak English. And when you, wanting to practise your English, spoke to them in English, they always answered in Shona. It was disappointing, and confusing too for people like me who were bilingual, since we had developed a kind of reflex which made us speak English when we spoke to whiteskins and reserved our own language for talking to each other.

      Struggle and confusion in communication with bilingual people.

    36. It made me reckless in the way that draining the dregs in my father’s gourd had done when I was little. It made me tell Nyasha frankly what I thought. ‘Even if you have been to England, you should respect your mother,’ I told her. ‘I would not speak to my mother in the way I have heard you speak to Maiguru.

      Shows Tambu's struugle in thegourd field as compared to Nyasha's living in England

    37. But I did not sneer at you,’ I protested, speaking in Shona. Our conversation was laboured and clumsy because when Nyasha spoke seriously her thoughts came in English, whereas with me, the little English I had disappeared when I dropped my vigilance to speak of things that mattered.

      This conversation shows us the gap in expertise of language between the two characters showing us the struggle of learning english language.

    38. Having sat down in that chair I began to wonder whether it would have been more appropriate in this English place to sit closer to my uncle and aunt. I debated seriously, agonisingly, for at least two minutes, whether to change my seat to the sofa or not.

      The Westernized setting instills a sense of insecurity within Tambu. It reveals her psychological burden of upholding unspoken social decorum.

    39. onse- . quently, I was relieved when Anna came and knelt down in the doorway to tell us that supper was nearly ready. ‘For heaven’s sake, Anna, stand up!’ commanded Nyasha irritably. ‘Every time you come in here I tell you not to kneel down, but you keep on doing it. What’s the matter with you?’ And then she was ashamed of herself. ‘Now, honestly, just listen to me. Getting all cross and crotchety! But really, Anna, there’s no need to kneel when you speak to me.

      This paragraph shows Anna on struggling

    40. To our surprise, Maiguru did leave, by bus, early the next morning. She did not slink away in the dark, but quite openly packed a suitcase, put on her travelling clothes, had her breakfast and left. Babamukuru was still feeling injured, which was why, I thought, he let her go, but Nyasha had a different theory. She thought Babamukuru simply did not believe that Maiguru would doit. Would do it, could do it. It made no difference, she said. The point was that he did not believe. Babamukuru, she said, expected his wife to get cold feet before she got to the bus-stop or, at the latest, before the bus pulled away. It would have been useful, Nyasha said, if things had turned out that way, because then Babamukuru would always have been able to remind his wife that she had tried to leave and had failed. Unfortunately, she told me, Babamukuru had to wait until Maiguru had boarded that bus and had gone to discover whether he was right or not, and by that time it was too late to do anything about it.

      This passage shows that Maiguru is not completely powerless. Despite the challenges in her marriage, she finally takes action to express her dissatisfaction. I think this demonstrates the importance of women having the courage to stand up for their rights.

    41. She did tell us, didn’t she, what she thinks, and did anyone say anything? No. Why not? Because Maiguru is educated. That’s why you all kept quiet. Because she’s rich and comes here and flashes her money around, so you listen to her as though you want to eat the words that come out of her mouth. But me, I’m not educated, am I? I’m just poor and ignorant, so you want me to keep quiet, you say I mustn’t talk.

      This shows how economic differences can affect relationships between women. Mainini feels that her opinions are ignored because she is poor. It reminds me that every woman deserves to be heard regardless of her financial position.

  3. drive.google.com drive.google.com
    1. Injustice anywhere is a threat to justice everywhere.

      This statement by him is one of the many that fill me with empathy. His desire to create fairness and equality all over is relatable.