Medical records of 26 patients diagnosed with VHL disease in two Korean hospitals between 2003-2012 and harboring germline VHL mutations were retrospectively reviewed. One male patient diagnosed with Type 1 VHL disease at 23Y (Patient 14) presented with renal cell carcinoma, multiple CNS hemangioblastomas and other manifestations. He had a germline inframe deletion in the VHL gene (c.227delTCT) and a negative family history of VHL disease.

Medical records of 26 patients with germline mutations of the VHL gene who had been diagnosed with VHL disease in Severance Hospital (Seoul, Republic of Korea) and Samsung Medical Center (Seoul, Republic of Korea) between 2003 and 2012 were reviewed, in addition to several cases published elsewhere. Peripheral blood leukocytes were isolated and directly sequenced. This revealed a germline nonsense mutation in the VHL gene (c.217C>T; p.Gln73*) in 3 patients. Patient 12, 41Y female, presenting with RCC (no data available for hemangioblastoma) and her daughter, presenting with CNS hemangioblastoma. Data on the mother was originally published by Seo et al. in 2006, J Korean Soc Endocrinol and was translated into English by the current study. Patient 11, 37Y female previously reported in PMID 19270817, presenting with multiple CNS hemangioblastomas, renal cell carcinoma and a family history positive for VHL. All patients were diagnosed with VHL Type 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.N78S (c.233A>G) germline variant of the VHL gene was detected in two individuals (ID 1347/Family 363 and ID 9579/Family 404). Both patients presented with renal cell carcinoma. No family information is provided. Both individuals were previously published in PMID 12202531.

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.F76* (c.227_228delinsAA) germline variant of the VHL gene was detected in a family (ID 512). Member 11876 presented with moderate renal disease (RCC) and member 13998 had unknown renal involvement. Variants c.227T>A and c.228C>A occurred in cis, resulting in an amino acid change of p.Phe76Ter.

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.R167W (c.499C>T) germline variant of the VHL gene was detected in multiple families. Family 1: Members 296, 419, 295 and 414 had no renal involvement. Members 427, 318, 403 and 406 had unknown renal involvement. Members 402, 322, 403 and 1 had mild renal disease (RCC). Members 401 and 404 had moderate renal disease (RCC). Family 23: Member 1037 had no renal involvement. Members 1035 and 1036 had moderate renal disease (RCC). Family 50: Member 6338 had no renal involvement. Members 6340 and 6339 had mild renal disease. The paper states that these families are previously reported in PMID 12202531 and/or 9829912.

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.F76_C77del (c.226_231del) germline variant of the VHL gene was detected in an individual (ID 13773, Family 599). This patient presented with renal cell carcinoma. No family information is provided.

A 49 year old female (Patient 3) from Europe presented with a unilateral adrenal pheochromocytoma. PCR-SSCP genetic testing on paraffin-embedded tissues and peripheral blood confirmed germline mutation in the VHL gene (p.214Gext14 (c.640T>G), referred to in the paper by p.X>Gly+14AA (c.852T>G) due to a difference in transcript sequence). Family history and patient demographic is unknown. This patient underwent surgical resection.

Mutational analysis for 16 patients with clinically diagnosed VHL disease, revealed 12 germline mutations. This mutation was found in a 42-year-old VHL patient with hemangioblastomas of the central nervous system, renal cell carcinoma, and pancreatic cysts (proband no. 12).

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 S168Tfs*2 (c.503_513del) germline mutation (reported as “c.716del11bp” according to legacy reference sequence) was found in a VHL type 1 family of 3. All three family members had renal cell carcinoma, two had hemangioblastomas of the central nervous system, and one had pancreatic cysts or tumors (unspecified) (family ID 63).

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 C162Y (c.485G>A) germline missense mutation of the VHL gene was found in a VHL patient with retinal angiomas, pancreatic cysts or tumor (unspecified), renal cell carcinoma, and pheochromocytoma (family ID 104). The variant was also found in a family of 4 individuals all presenting with CNS hemangioblastoma (family ID 11). This family was previously reported in PMID 8634692.

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. Renal cell carcinoma occurred predominantly in non-missense mutations predicted to lead to gross alteration or complete loss of the VHL protein. This splice site mutation, c.464-1G>A (stated as 677-1G>A in the paper which used the Latif sequence as reference) was found in a VHL type 1 patient with retinal angiomas, pancreatic cysts, and renal cell carcinoma (family ID 68). This study contains very strong evidence for pathogenicity in the form of a -1 splice-site variant in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

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. Renal cell carcinoma occurred predominantly in non-missense mutations predicted to lead to gross alteration or complete loss of the VHL protein. This stop-gain mutation, c.337C>T (stated as c.550C>T in the paper which used the Latif sequence as reference), was found in VHL type 2 patient, who was previously diagnosed with VHL type 1. The patient had a hemangioblastomas of the central nervous system, pancreatic cysts, renal cell carcinoma, and pheochromocytoma (family ID 47).

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 L178_D179insRVKPEL (c.531_532insCTGAGAGTAAAGCCTGAA) mutation was found in 3 related individuals with VHL type 1 (no pheochromocytoma). Patient/proband no. V31. Three unaffected family members were not found to have the mutation.

Screening of 92 unrelated patients with VHL disease revealed 61 DNA variants. Blood samples were isolated and sequenced by direct sequencing using prism ready reaction dye primer cycle sequencing kits (Applied Biosystem Roissy CDG France) and an ABI 373A automated sequencer (AppliedBiosystem Roissy CDG France). No variants were found in 96 control cases. This missense mutation, c.500G>A, was found in a VHL type 1 patient and two VHL type 2B patients (patient no. V322, V36, V262). Patient V322 was asymptomatic but patient V36 and V262 presented with pheochromocytomas. No additional family members were screened in all 3 cases.

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 V170G (c.509T>G) missense mutation was found in a VHL type 1 (no pheochromocytoma) patient (patient no. V81). No unaffected family members were tested. This family was stated as being previously published in PMID 15300849 (no phenotypes specified).

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 L158dup (c.473_474insTTT) mutation was found in 4 related individuals with VHL type 1 (no pheochromocytoma). Patient/proband no. V43. This family was stated as being previously published in PMID 15300849. Three unaffected family members were negative for this mutation.

53 patients with a molecularly confirmed VHL mutation (by PCR through EDTA-anticoagulated blood) and a pancreatic neuroendocrine tumor (ICT) were collected from the German NET-Registry and the German VHL-registry. An additional 296 patients were stated in this paper with a VHL associated germline mutation, but without pancreatic neuroendocrine tumors. 5 patients were found to have the above mutation (that causes the following protein change: E160Afs*12). 2 were VHL patients with ICTs (pancreatic neuroendocrine tumors) taken from the VHL-registry. The other 3 patients were stated to be in this study with the given mutation, however no ICT. Other clinical manifestations outside of a pancreatic neuroendocrine tumor are possible, but were not discussed. ACMG evidence codes: 'PVS1' because they observe a frameshift variant in a gene where loss of function is a known mechanism of disease.

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. 2 patients were found to have the above mutation (c.364_365delinsAT)). 1 was a VHL patient with ICT (pancreatic neuroendocrine tumor) taken from the VHL-registry. The other 1 patient was 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.

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. 1 patients was found to have the above mutation (c.593T>A). They had a VHL patient with an ICT (pancreatic neuroendocrine tumor) taken from the VHL-registry. 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.

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. 1 patient was found to have the above mutation (c.467A>G). They were a VHL patient with an ICT (pancreatic neuroendocrine tumor) taken from the VHL-registry. 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.

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 (L101R (c.302T>G)) was taken from the Bristol Myers Squibb database, with an unknown number of members belonging to Family ID 23BMS. It was reported in the paper as c.515T>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.

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 (F76I (c.226T>A)) was taken from the National Cancer Institute database, with an unknown number of members belonging to Family ID 3682NCI. It was reported in the paper as c.439T>A due to a difference in transcript reference. Additionally, this family ID is stated above in the same table, Table 3, under the mutation c.376delG. Unclear if this is a typo, or if multiple mutations belong to this one family. As reported in Table 3, this mutation is found in VHL family without pheochromocytoma, and with renal cell carcinoma. Later publications reference this cohort as being reported in PMID: 7728151.

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 (E55fs (c.163delG)) was taken from the National Cancer Institute database, with an unknown number of members belonging to Family ID 3682NCI. It was reported in the paper as c.376delG due to a difference in transcript reference. Additionally, this family ID is stated again in Table 3 under the mutation c.439T>A. Unclear if this is a typo, or if multiple mutations belong to this one family. As reported in Table 3, this mutation is found in VHL family without pheochromocytoma, and with renal cell carcinoma. Later publications reference this cohort as being reported in PMID: 7728151.

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 (D121G (c.362A>G)) was taken from the Bristol Myers Squibb database, with an unknown number of members belonging to Family ID 21BMS. It was reported in the paper as c.575A>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.

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 (R113* (c.337C>T)) was taken from the Munich, university of Freiburg and Institute of Pathology database and the Cambridge, Department of Pathology database, with an unknown number of members belonging to Family IDs 55CE, 136CA and 30CA. It was reported in the paper as c.550C>T due to a difference in transcript reference. As reported in Table 3, this mutation is found in VHL families (55CE, 136CA) without pheochromocytoma, and with renal cell carcinoma. Additionally, table 5 lists all ID and mutations associated with individuals without detailed clinical information (including family 30CA). Other phenotypes are possible, however not detailed here.

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 (N78T (c.233A>C)) was taken from the National Cancer Institute database, with an unknown number of members belonging to Family ID 3101NCI. It was reported in the paper as c.446A>C 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.

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 (P192fs (c.575delC)) was taken from the Neuro Olcologie database in Paris, with an unknown number of members belonging to Family ID 51F. It was reported in the paper as c.786delC due to a difference in transcript reference. As reported in Table 4, this mutation is found in VHL families with pheochromocytoma. Other phenotypes are likely, including renal cell carcinoma, however not detailed here.

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 (R167G (c.499C>G)) was taken from the Cambridge, Department of Pathology database, with an unknown number of members belonging to Family ID 137CA. It was reported in the paper as c.712C>G due to a difference in transcript reference. As reported in Table 4, this mutation is found in VHL families with pheochromocytoma. Other phenotypes are likely, including renal cell carcinoma, however not detailed here.

14 Chinese patients from 10 families were diagnosed with Von Hippel-Lindau disease based on clinical criteria. Genetic testing using single-strand conformation polymorphism (SSCP) was conducted and aberrant bands were purified and sequenced. Patient 9 (58 Y) from family V (no other individuals in this family included in the paper) was found to harbour a 500G>A mutation in the VHL gene (stated as 713G>A in the paper which used the Latif sequence as reference). The patient presented with two hemangioblastomas.

In a study of 426 unrelated VHL patients, 111 were discovered to have alterations in the VHL gene. 18 novel variants were identified in VHL patients, but none were present in 200 unaffected control individuals. DNA samples from peripheral blood leukocytes underwent polymerase chain reaction (PCR) amplification, denaturing high-performance liquid chromatography (DHPLC), and direct sequence analysis of the entire coding sequence and intron-exon boundaries. The L184R (c.551T>G) germline missense mutation of the VHL gene was found in a 44 yr old VHL patient with clear cell renal cell carcinoma as well as central nervous system and retinal hemangioblastomas (index case 363).

In a study of 426 unrelated VHL patients, 111 were discovered to have alterations in the VHL gene. 18 novel variants were identified in VHL patients, but none were present in 200 unaffected control individuals. DNA samples from peripheral blood leukocytes underwent polymerase chain reaction (PCR) amplification, denaturing high-performance liquid chromatography (DHPLC), and direct sequence analysis of the entire coding sequence and intron-exon boundaries. The V84L (c.250G>C) germline mutation of the VHL gene was found in a 24 yr old VHL patient with pheochromocytoma (index case 141). Parents and siblings were negative for the mutation. No phenotype details are noted for these family members.

In a study of 426 unrelated VHL patients, 111 were discovered to have alterations in the VHL gene. 18 novel variants were identified in VHL patients, but none were present in 200 unaffected control individuals. DNA samples from peripheral blood leukocytes underwent polymerase chain reaction (PCR) amplification, denaturing high-performance liquid chromatography (DHPLC), and direct sequence analysis of the entire coding sequence and intron-exon boundaries. The L178P (c.533T>C) germline missense mutation of the VHL gene was found in a 14 yr old VHL patient with retinal hemangioblastomas, multiple pancreatic cysts or tumors, pheochromocytoma, and multiple renal cysts (index case 160).

In a study of 426 unrelated VHL patients, 111 were discovered to have alterations in the VHL gene. 18 novel variants were identified in VHL patients, but none were present in 200 unaffected control individuals. DNA samples from peripheral blood leukocytes underwent polymerase chain reaction (PCR) amplification, denaturing high-performance liquid chromatography (DHPLC), and direct sequence analysis of the entire coding sequence and intron-exon boundaries. The V66G (c.197T>G) germline mutation of the VHL gene was found in a 38 yr old VHL patient with CNS hemangioblastoma, pheochromocytoma, renal cystic lesion and pancreatic cyst or tumor (index case 379). Transmission of the variant could not be determined (n/d) since both parents and family history were not available.

Seven VHL mutations were identified in 10 unrelated, Swedish families. Direct genome sequencing through Southern blot identified this missense mutation (c.485G>T) in family #5 with 2 affected members. One was found with renal cell carcinoma, retinal capillary hemangioma and CNS hemangioblastoma, whereas the other was found with renal cell carcinoma and retinal capillary. Other information on the affected member and relatives was not available.

200 patients with intracranial aneurysms were molecularly analyzed for VHL to determine if it is a candidate gene for aneurysm formation using peripheral blood. Two index patients were analyzed in detail and determined to have the above mutation (292T>C). One patient only had pheochromocytoma and an innocent aneurysm, while the other had pheochromocytoma, spinal hemangioblastoma, cerebellar hemangioblastoma, retinal hemangioblastoma and an innocent saccular aneurysm (dilatation of the cerebral artery) of the left internal carotid artery. ACMG evidence codes: 'PP2' because they observe a missense variant in a gene where missense variants are a common mechanism of disease, and 'PP4' because the patient's phenotypes or family history are highly specific for a disease with single genetic etiology.

200 patients with intracranial aneurysms were molecularly analyzed for VHL to determine if it is a candidate gene for aneurysm formation using peripheral blood. One patient was analyzed in detail and determined to have the above mutation (74C>T). The patient was only recorded to have an intracranial aneurysm (or dilatation of the cerebral artery).

A case report revealed a 34-year-old, Caucasian male diagnosed with bilateral pheochromocytoma and Type 2c VHL disease. Referral for genetic testing and sequencing revealed a c.500G>A (p.Arg167Gln) mutation in the VHL gene. There was no family history of endocrine neoplasms or early cerebral or cardiovascular disease. Genetic testing of family members was not described. ACMG: PM1

8 of 19 patients (42%) presenting with clear cell renal cell carcinoma were found to be heterozygous for the germline variant rs1642742 in VHL (c.*294G>A) whereas controls were heterozygous at a frequency of 22% (134/616) (chi-sq χ2 = 4.397, p = 0.036). Normal tissue was used for sequencing of the VHL gene and analysis was completed at least twice per sample for precision. QIAquick PCR Purification kits, ABI Prism kit and the ABI Model 377 automated DNA sequencer were used. With ages ranging from 50-83Y, and 3:5, M:F ratios. 7 of these 8 members also presented with the germline mutation c.-195G>A. No family data was provided for the patients.

Of 246 patients affected with von Hippel-Lindau disease, 64 patients (26%) from 38 families had pheochromocytoma, including 33 newly diagnosed during screening studies and 31 treated before screening at NIH. Germline mutations in the VHL gene were determined from sequencing peripheral blood using PCR, SSCP (single-strand conformation polymorphism), and Southern Blotting. Family 4885 and family 1112 were found with p.Arg161*/c.481C>T mutations in the VHL gene (legacy notation c.694C>T converted to NM_000551.3). Family 4885 had one member with pheochromocytoma, renal cell carcinoma, CNS hemangioblastoma and cysts. Family 1112 had one member with pheochromocytoma, CNS hemangioblastoma, and retinal angioma.

Of 246 patients affected with von Hippel-Lindau disease, 64 patients (26%) from 38 families had pheochromocytoma, including 33 newly diagnosed during screening studies and 31 treated before screening at NIH. Germline mutations in the VHL gene was determined from peripheral blood. Family 3618 were found with a p.Cys162Trp mutation (legacy notation c.699C>G converted to NM_000551.3) in the VHL gene. Family 3618: 1/1 had pheochromocytoma, 1/1 had hemangioblastoma. 1/1 had cyst and pancreatic neuroendocrine tumor was also found. Family 3618 has been reported previously in whole or in part in the following reports, PMIDs: 8956040; 7728151.

Of 246 patients affected with von Hippel-Lindau disease, 64 patients (26%) from 38 families had pheochromocytoma, including 33 newly diagnosed during screening studies and 31 treated before screening at NIH. Germline mutations in the VHL gene was determined from peripheral blood. Family 4942 was found with p.Arg161Gly mutation in the VHL gene (legacy notation c.695G>A converted to NM_000551.3): 7/8 had pheochromocytoma (one patient had metastatic pheo), 3/8 had hemangioblastoma, 2/6 had retinal angioma.

The medical records of 78 Korean patients with pheochromocytoma (PCC) and paragangliomas (PGL) were examined, 61 patients were diagnosed with PCC and the other 17 patients with PGL. 57 patients were included in the study. After excluding patients, 57 patients underwent sequencing. 14 patients received Sanger sequencing in pre-NGS period, while the other 43 patients underwent genetic testing by either direct sequencing (n=2) or targeted NGS (n=41) in the post-NGS period. 28 patients were found to have germline mutations in 7 PPGL-related genes (SDHB, RET, VHL, NF1, MAX, SDHA, and SDHD). Out of the 28 patients, 6 patients had germline mutations in VHL (5 presented with Pheochromocytoma and 1 with Paraganglioma). This patient had a PGL located in the pancreas and was found to have a E70K(c.208G>A) missense variant in the VHL gene, which was detected by targeted NGS.

A cohort of 141 patients with hemangioblastomas of the central nervous system was analyzed. 81 patients had germline mutations in the VHL gene, analyzed through peripheral blood by Southern blotting and SSCP analysis. This nonsense mutation (c.481C>T) was recorded in the paper as c.694C>T due to a difference in transcript sequence. It was found in 4 patients with hemangioblastomas of the central nervous system. Any family information or relations is unknown. No other phenotypes described.

A cohort of 141 patients with hemangioblastomas of the central nervous system was analyzed. 81 patients had germline mutations in the VHL gene, analyzed through peripheral blood by Southern blotting and SSCP analysis. This nonsense mutation (c.433C>T) was recorded in the paper as c.646C>T due to a difference in transcript sequence. It was found in 3 patients with hemangioblastomas of the central nervous system. Any family information or relations is unknown. No other phenotypes described.

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 splice site mutation (c.464-2A>G) was recorded in the paper as c.677-2A>G due to a difference in transcript sequence. It was found in one patient with a hemangioblastoma of the central nervous system. Any family information is unknown. No other phenotypes described.

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.194C>T) was recorded in the paper as c.407C>T due to a difference in transcript sequence. It was found in one patient with hemangioblastomas of the central nervous system. No other phenotype described.

A 65Y male patient of Japanese ethnicity presented with hypertension, bilateral adrenal pheochromocytoma, and hepatic hemangioma. A mutation NM_000551.3:c.460C>T (p.Pro154Ser) was found in the VHL gene. Genetic sequencing occurred through PCR testing and RFLP analysis. Please note that the paper does not state a reference sequence, and instead refers to the mutation as the first base of codon 154. His 35Y son had the same mutation and presented with right adrenal pheochromocytoma. His wife, another son, and daughter were healthy and had normal genetic testing results. ACMG: PP2, PP1, PP4

All hemangioblastoma patients treated at The University of Tokyo Hospital from 1954 to 1998 were retrospectively analyzed. Genetic analysis was done on one 31 year-old male patient (patient #15) using isolated peripheral blood and directly sequenced using BigDye Sequencing Kit and Genetic Analyzer 310 (Applied Biosystems, Foster City, CA). Genetic testing revealed a p.Arg161* nonsense germline mutation in the VHL gene. In this 31-year-old man, CT detected 5 cerebellar hemangioblastomas. Fourteen years later, the patient was found to have another cerebellar lesion. The man had no other lesions detected by scans. The patient had no family history of VHL disease.

During the period from 2009 to 2012, 39 families with VHL disease were diagnosed with hereditary VHL disease at Peking University First Hospital Department of Urology were described as part of a broader study examining telomere length and anticipation. In these families, 19 families had two or more than two patients in two or more than two generations, including one family with obscure onset age. Therefore, a total of 57 patients with VHL disease from 18 families were enrolled in this study. Using peripheral blood and southern blotting, DNA sequencing was completed on the following patients. Family 11 involved two individuals with a c.263G>A mutation leading to a p.Trp88Stop change. The first individual, a female, mother, presented with hemangioblastoma at 36. The second individual, a male, son/proband, presented with renal cell carcinoma at 34 and pancreatic cysts at 38. ACMG Evidence Code: 'PP4' because the patients' phenotypes and family histories are highly specific for VHL disease. 'PM4' because of the protein length change caused by the heavily premature stop codon.

Data was collected from 82 VHL mutation carriers in the Dutch VHL surveillance program. Two patients were found with this missense mutation in the VHL gene. One patient was 35Y and the other was 60Y at last follow-up. Their phenotypes were grouped together: retinal hemangioblastoma, spinal cord hemangioblastoma, pheochromocytoma, renal cysts, and pancreatic cysts. Family history was not described. This cohort was published later in PMID 31087189.

Data was collected from 82 VHL mutation carriers in the Dutch VHL surveillance program. One patient was found with this insertion in the VHL gene. The patient was 42Y at the last follow-up and presented with: retinal hemangioblastoma, cerebellar hemangioblastoma, spinal cord hemangioblastoma, renal cell carcinoma, renal cysts and pancreatic cysts. Family history was not described. This cohort was published later in PMID 31087189.

A 9-year-and-5-month old male patient was diagnosed with polyglobulia after an accident at 8 years of age. Proband's mother reported that the patient displayed fatigue and peripheral cyanosis during exercise. The physical exam showed a tumor in the right testicle. This was confirmed to be a cyst in the right epididymis was identified by a testicular ultrasound. Due to family history of VHL disease (4 generations, 12 patients with VHLd), genetic testing was performed on the mother, where an exon 3 deletion in the VHL gene was identified. The mother died due to complications of a CNS hemangioblastoma at 30Y. A peripheral blood sample was then obtained from the patient, where the same exon 3 deletion was identified by MLPA. Complete VHL gene sequencing was then performed on the proband's sample which identified a new S139C(c.416C>G) missense mutation, revealing that the patient was a compound heterozygote. This missense mutation was not previously reported. There was also no information on the proband's father; thus it is not confirmed whether the missense mutation was paternally inherited or de novo. Prediction tools such as PolyPhen2, Mutationassesor, Provean, and SIFT suggests it is deleterious. Due to the clinical presentation and molecular biology studies, the patient was diagnosed with both familial erythrocytosis type 2 and VHL disease.

Screening of 3 patients affected with familial PC for mutations in the RET proto-oncogene (mutations related to MEN 2 syndrome) and the VHL tumor suppressor gene (mutations related to VHL disease) with the use of PCR, SSCP, heteroduplex analysis, and direct sequencing, revealed missense VHL gene mutations. Please note that there was no clinical diagnosis of VHL disease. This missense mutation c.250G>T was found in a Welsh family with 2 siblings (one male and one female) with no evidence of RET; the brother presented with bilateral pheochromocytoma at 11Y and the sister presented with bilateral pheochromocytoma at 17Y. An asymptomatic parent was also documented. Additional phenotypic information was not provided. Please note that the paper states the mutation as c.463G>T because it uses a legacy reference sequence.

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.D143Tfs*29 (c.426_430del) mutation, stated in the paper as c.639_642del4 due to an older transcript. It was seen in cell line A-498. Any family information or other manifestations apart from RCC were unclear.

Tissue analysis from 61 VHL patients, 30 sporadic renal cell carcinoma (RCC) tumor samples, and 6 sporadic RCC cell lines revealed 22 variants within VHL patients, 10 variants in tumor samples and variants in all cell lines. All mutations cluster to the 3’ end of the VHL gene open reading frame, implicating this region as important for the tumor-suppressor function of the VHL protein. Northern blot, SSCP and RT-PCR analysis from normal tissue found a germline p.S65* (c.189_192del) mutation, stated in the paper as c.402_405del4 due to an older transcript. It was seen in family [pedigree No] 19, with an unknown number of members described as ‘VHL-affected’ with RCC. Other manifestations are possible but not clear.

41 non-related patients with retinal capillary hemangioma presenting in the Benjamin Franklin University Eye Clinic between 1988 to 1999 Germany were evaluated for the presence of other VHL lesions. VHL disease was diagnosed in 33 patients based on the presence of other severe VHL lesions (spinal hemangioblastoma, cerebellar hemangioblastoma, renal cysts, pancreatic cyst, renal carcinoma, and pheochromocytoma), molecular genetic testing, and/or family history. VHL germline mutations were identified in 27 out of 29 VHL families screened with molecular genetics. Blood samples were genetically tested using PCR-SSCP sequencing. One patient with VHL-related retinal capillary hemangioma was found to have germline missense c.395A>C VHL variant. 3 other affected relatives were also evaluated and found to have the same mutation but no phenotypes were described. It's unclear which other VHL-related lesions were identified in this patient. This variant was reported as c.608A>C (AF010238.1) and mapped to p.Q132P (c.395A>C) (NM_000551.3).

41 non-related patients with retinal capillary hemangioma presenting in the Benjamin Franklin University Eye Clinic between 1988 to 1999 Germany were evaluated for the presence of other VHL lesions. VHL disease was diagnosed in 33 patients based on the presence of other severe VHL lesions (spinal hemangioblastoma, cerebellar hemangioblastoma, renal cysts, pancreatic cyst, renal carcinoma, and pheochromocytoma), molecular genetic testing, and/or family history. VHL germline mutations were identified in 27 out of 29 VHL families screened with molecular genetics. Blood samples were genetically tested using PCR-SSCP sequencing. One patient with VHL-related retinal angiomatosis was found to have germline missense (c.388G>T) mutation in the VHL gene. 2 other relatives were also evaluated and found to have the same mutation but no phenotypes were described. This variant was reported as c.601G>T (AF010238.1) and mapped to p.V130F (c.388G>T) (NM_000551.3).

A retrospective medical chart review was performed on patients referred to the genetics service. Four related and one unrelated probands were genetically confirmed with a familial mutation at (presumed) c.481C>T of the VHL gene. The first was a 41 year old female who presented with central nervous system hemangioblastoma, renal cell carcinoma, and a pancreatic cyst. The second was a 39 year old male who presented with central nervous system hemangioblastoma, renal cell carcinoma, and a pancreatic cyst. The third was a 67 year old male who presented with central nervous system hemangioblastoma, renal cell carcinoma, and a pancreatic cyst. The fourth was a 32 year old male who presented with central nervous system hemangioblastoma, renal cell carcinoma, and a pancreatic cyst. The fifth was a 29 year old female who presented with central nervous system hemangioblastoma, renal cell carcinoma, and a pancreatic cyst. Family histories are all positive.

This report studied the prevalence of germline variants in Japanese PPGL patients. Of the 370 PPGL original Japanese probands, this cohort consisted of 15 patients with pheochromocytoma and/or paragangliomas with germline VHL varaints. Blood samples were used to examine 7 genes: MAX, SDHB, SDHC, SDHD, TMEM127, VHL, and RET, using Sanger sequencing. Of these 15 VHL patients: 10 had bilateral PCC, 4 had PCC with abdominal/thoracic PGL, and one had metastatic PPGL. The variant F136C (c.407T>G) was one of the 15 found in a patient with PPGL. Their family history or further manifestations were not detailed.

150 index patients with pheochromocytoma/paraganglioma were evaluated. Phenotypic data were collected and germline mutations in five susceptibility genes (RET, VHL, SDHB, SDHD and SDHC) were tested. Out of 30 patients with familial presentation, 13 were clinically diagnosed as VHL syndrome. A 28 year old Asian Indian male presented with unilateral pheochromocytoma. Using PCR and Sanger sequencing through peripheral blood, genetic testing confirmed a non-familial missense mutation at c.509T>C of the VHL gene. Family history is unknown.

150 index patients with pheochromocytoma/paraganglioma were evaluated. Phenotypic data were collected and germline mutations in five susceptibility genes (RET, VHL, SDHB, SDHD and SDHC) were tested. Out of 30 patients with familial presentation, 13 were clinically diagnosed as VHL syndrome. Using PCR and Sanger sequencing through peripheral blood, genetic testing confirmed a mutation at c.482G>A of the VHL gene in three unrelated patients. The first was a 20 year old Asian Indian female who presented with bilateral pheochromocytoma. Family history is positive for her. The second was a 6 year old Asian Indian female who presented with unilateral pheochromocytoma. She has a positive family history. The third was a 34 year old Asian Indian male who presented with bilateral pheochromocytoma. Family history is unknown.

The genetic testing program for VHL disease included 17 families, ten of which had definite VHL disease and seven had possible VHL disease. 17 families (n=109 individuals) were tested for VHL mutations. Mutation analysis was performed by direct sequencing of the VHL gene. Mutations were identified in 36 patients. Mutations-carriers were screened for VHL disease-related tumors and were offered follow-up annual examinations. Tumor status and phenotypes were documented at initial screening and at follow ups. 1 individual from family 19 was tested positive for p.P86S mutation. 1 hemangioblastoma, renal cyst, pancreatic cyst, 1 renal cell carcinoma were diagnosed at initial screening. 1 additional renal cell carcinoma, 1 hemangioblastoma and endolymphatic sac tumor were diagnosed at follow ups. The patients deceased at last follow up. 1 individual from family 43 was also tested positive for p.P86S mutation. 1 hemangioblastoma was diagnosed at initial screening. 3 more hemangioblastomas, 2 renal cell carcinoma, pancreatic cancer and 1 pheochromocytoma were diagnosed at follow-ups. ACMG evidence codes: 'PP4' because the patients' family history are highly specific for a disease with single genetic etiology.

Fresh frozen (nZ69) and formalin-fixed paraffinembedded (FFPE; nZ30) specimens, including PCCs/PGLs and normal adrenal medullas (nAM), were collected by hospitals through the Spanish National Tumor Bank Network (CNIO) and by the Instituto Oncologico Veneto in Italy. Tumor ids VHL_1-12 were frozen samples. Tumor VHL_7 was an adrenal pheochromocytoma found with a c.277G>C germline mutation in a 36Y old male. It should be noted that this mutation is described as c.277G>C with p.G39R protein change. However, the cDNA given does not match the protein position and therefore was assumed to be p.G93R. Other demographics and pheonotype information was not available.

Peripheral blood from unrelated patients with pheochromocytoma was tested for mutations of proto-oncogene RET, tumor suppressor gene VHL, succinate dehydrogenase subunit D (SDHD) gene, and the succinate dehydrogenase subunit B (SDHB) gene. Of 271 patients with nonsydromic pheochromocytoma and no family history of the disease, 30 mutations were found in VHL. Multiple pheochromocytomas were statistically more frequent among patients with mutations than among patients without mutations (21 of 66 vs. 5 of 205, P<0.001). The mutation A122I (c.364_365GC>AT) was detected in the patient, referred to in the paper by c.577_578GC>AT due to a difference in transcript sequence. This missense mutation was found in the VHL gene and one patient with pheochromocytoma.

Peripheral blood from unrelated patients with pheochromocytoma was tested for mutations of proto-oncogene RET, tumor suppressor gene VHL, succinate dehydrogenase subunit D (SDHD) gene, and the succinate dehydrogenase subunit B (SDHB) gene. Of 271 patients with nonsydromic pheochromocytoma and no family history of the disease, 30 mutations were found in VHL. Multiple pheochromocytomas were statistically more frequent among patients with mutations than among patients without mutations (21 of 66 vs. 5 of 205, P<0.001). The S68W (c.203C>G) mutation was detected in the patient, referred to in the paper by c.416C>G due to a difference in transcript sequence. This missense mutation was found in the VHL gene and one patient with pheochromocytoma.

A cohort of 314 patients with pheochromocytoma and/or functional paraganglioma had blood samples collected and all five major pheo-pgl susceptibility genes (RET, VHL, SDHB, SDHD, and SDHC) were screened for mutations. 25 patients had germline VHL mutations, 16 of which had a family history and 9 patients had an apparently sporadic presentation, 11 were male and 14 were female, the median age of diagnosis is 23.9 and range of diagnosis was 4-49. Out of the 25 patients 23 had adrenal tumours, 4 had para-adrenal tumours, and 9 had other VHL lesions (renal tumours and hemangioblastomas). 1 patient with postive family history and/or a syndromic presentation was found with pheochromocytoma or functional paraganglioma and a germline missense VHL mutation (c.292T>C). Other phenotypes unique to the patient are unknown. ACMG: PP4

A cohort of 314 patients with pheochromocytoma and functional paraganglioma had blood samples collected and all five major pheo-pgl susceptibility genes (RET, VHL, SDHB, SDHD, and SDHC) were screened for mutations. 25 patients had germline VHL mutations, 16 of which had a family history and 9 patients had an apparently sporadic presentation, 11 were male and 14 were female, the median age of diagnosis is 23.9 and range of diagnosis was 4-49. Out of the 25 patients 23 had adrenal tumours, 4 had para-adrenal tumours, and 9 had other VHL lesions (renal tumours and hemangioblastomas). 2 patients with postive family history and/or a syndromic presentation were found with pheochromocytoma or functional paraganglioma and a germline missense VHL mutation (c.290C>T). Other phenotypes unique to the patient are unknown. ACMG: PP4

A retrospective cohort study included all the VHL patients diagnosed at Peking University First Hospital (Beijing, China) prior to June 1 2016. A total of 291 patients from 115 different families were included for analysis. A female patient (patient #41) was confirmed for germline mutation in the VHL gene (c.239del; p.Ser80Ilefs*79). She was diagnosed with renal cell carcinoma at 44Y and multiple pancreatic cysts or tumours at 44Y. This cohort was also published in a paper by Wang et al., 2018 (PMID: 29330336).

Forty-four of the 50 patients with pheochromocytoma and paraganglioma treated at the Christian Medical College, Vellore between January 2010 and June 2012 were included in this study. Four patients were confirmed with VHL disease and with mutations in the VHL gene from DNA obtained from peripheral blood. One patient was a 21Y female who presented with right adrenal tumor confirmed with a VHL germline mutation (c.533T>A; p.Leu178Gln). ACMG: PP4

A 17 year old female confirmed with a germline VHL mutation (c.337C>T) presented with cerebellar and spinal hemangioblastomas. Imaging showed obstructive hydrocephalus and the head MRI revealed a large cystic mass with enhancing mural nodule involving the left cerebellar hemisphere, measuring 4 × 3 × 3.1 cm. There were associated small enhancing lesions scattered throughout the cerebellar hemispheres bilaterally involving the medulla and cerebellar vermis. Spinal MRI revealed additional satellite lesions throughout the cervical and thoracic spine with the appearance of drop metastasis. One month later, she underwent another surgery for removal of her large enhancing cervicothoracic tumor found to be a hemangioblastoma. Five months later, the patient was found to have retinal capillary hemangioblastoma and preretinal fibrosis in both eyes. There was a limited serous detachment of the left eye. An external cryopexy was performed at that time. However, after a few months, she developed tractional retinal detachment with vitreous hemorrhage of the left eye then posterior subcapsular cataract of the left eye. Two years later, a brain MRI showed a cystic lesion with enhancing foci in the superior portion of the cerebellum and multiple enhancing lesions in the medulla and inferior portion of the cerebellar hemispheres. Her spinal MRI showed enlarging thoracic and cervical lesions ranging from 3 to 6 mm. Her abdominal MRI showed a 14-mm enhancing nodule in the left lobe of the liver and multiple cysts throughout the pancreas. In addition, she had progressive retinal hemagioblastomas in the right eye. She underwent implantation of nitric oxide into her right eye. The patient's mother died of colon cancer at 36 years of age. Her maternal grandmother died of a brain tumour at 50 years of age and her aunt died of a brain tumour at 18 years of age. Her maternal grandfather died of colon cancer (age unknown). No one in her family was tested for the mutation. The patient also had a germline NF1 mutation c.7987A.

Nine Chinese index patients were referred for the molecular analysis of VHL based on clinical suspicion. PCR and MLPA were performed on whole blood. Proband 9 presented with VHL disease type 2B, and was found to have a CNS hemangioblastoma at age 26. They were found to have a c.239G>T [p.Ser80Ile] mutation in the VHL gene. Proband 9 underwent prenatal testing twice, which revealed the presence of the same mutation in their 2 fetus children.

Nine Chinese index patients were referred for the molecular analysis of VHL based on clinical suspicion. PCR and MLPA were performed on whole blood. One index patient was found to harbor the p.R161* mutation. Clinical manifestations included epididymal cysts, presenting at 40 years of age, and CNS hemangioblastoma and renal cell carcinoma, both presenting at 47 years of age. Familial screening found the mutation present in a sister with renal cell carcinoma (age 45), and absent in 2 asymptomatic daughters.

Germline mutations were found in 20 Brazilian, VHL probands and their families. The L101R (c.302T>G) germline missense mutation was found in 2 affected family members with hemangioblastomas of the central nervous system (VHL family 5).

Germline mutations were found in 20 Brazilian, VHL probands and their families. Peripheral blood samples were taken and genomic DNA was sequenced using PCR and Southern Blotting. The three exons of the VHL gene were amplified by PCR, sequenced in both strands with Big Dye terminators (Applied Biosystems), and collected and analysed on an ABI Prism 377 (Applied Biosystems). Germline rearrangements involving part or the entire VHL gene were detected by quantitative Southern blotting using g7 cDNA11 and a β globin probe. This missense mutation, R167Q (c.500G>A), was found in a VHL type 2 family of four affected individuals (VHL family 16). Two patients had hemangioblastomas of the central nervous system and three had pheochromocytomas. The family was also found to have an intronic mutation, 462-95T>A, that was found to not interfere with the protein structure. ACMG codes as follows: Cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease (PP1).

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 mutation was found in a 1 patient without retinal hemangioblastomas, however other manifestations are not clearly stated. No family information or relation was provided. Recorded in the paper as c.179delG, this automaps to c.180delG (p.V62Cfs*5). This is possibly previously published in PMID: 15300849

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 missense mutation (p.G114R, c.340G>C) was found in a VHL patient with retinal hemangioblastomas. Other manifestations are not clearly stated. No family information was provided. This is possibly previously published in PMID: 15300849

93 families examined at the Clinical Center of the National Institutes of Health (Bethesda, MD) underwent genetic testing for germline mutations of the VHL gene. Whole blood was extracted and genomic DNA was amplified by PCR and products were sequences using a cycle sequencing kit with dye-labeled terminators and automatically sequenced. This nonsense mutation was found in VHL type 1 family (lineage 80). The variant was reported as c.407C>A (AF010238.1) and was mapped to S65* (c.194C>A) (NM_000551.3).

93 families examined at the Clinical Center of the National Institutes of Health (Bethesda, MD) underwent genetic testing for germline mutations of the VHL gene. Whole blood was extracted and genomic DNA was amplified by PCR and products were sequences using a cycle sequencing kit with dye-labeled terminators and automatically sequenced. This frameshift mutation was found in a VHL type 1 family (lineage 175). The variant was reported as c.393delG (AF010238.1) and was mapped to V62Cfs*5 (c.180del) (NM_000551.3). This family is stated as being previously reported in PMID 7728151 and/or 8956040. Both these reports include a family presenting with this variant and it is unclear if one or both coincide with the current family.

Patients with ELSTs were identified and included in the International Endolymphatic Sac Tumor Registry. The registry contained 93 patients as of July 2014, and of those, 25 were sporadic, and 68 associated with VHL. Patients with ELSTs and clinically confirmed diagnoses of VHL were included in this study. Statistics of this VHL subgroup are as follows: mean age 30, 62% female, 91% symptomatic, 54% with retinal angioma, 72% with CNS hemangioblastoma, 46% with renal cell carcinoma, 19% with pheochromocytoma, and 12% with pancreatic neuroendocrine tumour. In the data from this study, only endolymphatic sac tumours can be confirmed as a phenotype for this mutation (c.499C>T), which was found in 3 individuals each from France, UK, or Germany.

Patients with ELSTs were identified and included in the International Endolymphatic Sac Tumor Registry. The registry contained 93 patients as of July 2014, and of those, 25 were sporadic, and 68 associated with VHL. Patients with ELSTs and clinically confirmed diagnoses of VHL were included in this study. Statistics of this VHL subgroup are as follows: mean age 30, 62% female, 91% symptomatic, 54% with retinal angioma, 72% with CNS hemangioblastoma, 46% with renal cell carcinoma, 19% with pheochromocytoma, and 12% with pancreatic neuroendocrine tumour. In the data from this study, only endolymphatic sac tumours can be confirmed as a phenotype for this mutation (c.256C>T), which was found in 1 individual from France.


Patients with ELSTs were identified and included in the International Endolymphatic Sac Tumor Registry. The registry contained 93 patients as of July 2014, and of those, 25 were sporadic, and 68 associated with VHL. Patients with ELSTs and clinically confirmed diagnoses of VHL were included in this study. Statistics of this VHL subgroup are as follows: mean age 30, 62% female, 91% symptomatic, 54% with retinal angioma, 72% with CNS hemangioblastoma, 46% with renal cell carcinoma, 19% with pheochromocytoma, and 12% with pancreatic neuroendocrine tumour. In the data from this study, only endolymphatic sac tumours can be confirmed as a phenotype for this mutation (c.500G>A), which was found in 4 individuals from France or Italy. 


Patients with ELSTs were identified and included in the International Endolymphatic Sac Tumor Registry. The registry contained 93 patients as of July 2014, and of those, 25 were sporadic, and 68 associated with VHL. Patients with ELSTs and clinically confirmed diagnoses of VHL were included in this study. Statistics of this VHL subgroup are as follows: mean age 30, 62% female, 91% symptomatic, 54% with retinal angioma, 72% with CNS hemangioblastoma, 46% with renal cell carcinoma, 19% with pheochromocytoma, and 12% with pancreatic neuroendocrine tumour. In the data from this study, only endolymphatic sac tumours can be confirmed as a phenotype for this mutation (c.236_241delGCAGTC), which was found in 1 individual from Germany.


Of 65 VHL families from central Europe, 53 were identified with germline mutations using SSCP analysis of PCR products by peripheral blood. This missense mutation was found in 2 German, VHL type 2 family, members with retinal angiomas, hemangioblastomas of the central nervous system, and pheochromocytomas (family VHL 29). The L178Q (c.533T>A) mutation was detected in the patient, referred to in the paper by c.746T>A due to a difference in transcript sequence. Each patient's phenotype is highly specific for a disease with a single genetic etiology (ACMG code: PP4).

Of 65 VHL families from central Europe, 53 were identified with germline mutations using SSCP analysis of PCR products by peripheral blood. The R161 (c.481C>T) mutation was detected in the patient, referred to in the paper by R161 (c.694C>T) due to a difference in transcript sequence. This nonsense mutation was found in 2 unrelated, German families. One VHL type 1 family of 2 affected individuals had one patient with retinal angiomas, 2 with hemangioblastomas of the central nervous system, one with renal cell carcinoma and renal cysts, one with pancreatic cysts, and one with cystadenomas of the epididymis (family VHL 32). Another VHL type 1 family of 3 affected individuals had 3 patients with retinal angiomas, one with hemangioblastomas of the central nervous system, 2 with renal cysts and renal cell carcinoma, one with pancreatic cysts, and one with cystadenomas of the epididymis (family VHL 49). ACMG codes as follows: A nonsense mutation in a gene where loss-of-function is a known mechanism of disease (PVS1). Cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease (PP1). HPO terms: papillary cystadenomas of the broad ligament.

Of 65 VHL families from central Europe, 53 were identified with germline mutations. The mutation of c.464-1G>A was referred to in the paper by c.677-1G>A due to a difference in transcript sequence. This splice mutation was found in a German, VHL type 1 patient with hemangioblastomas of the central nervous system (family VHL 48). This study contains very strong evidence of pathogenicity in the form of a canonical -1 splice site variant in a gene where loss-of-function is a known mechanism of disease (ACMG code: PVS1).

Genotype-phenotype correlations of 573 VHL patients from 200 kindreds were analyzed and confirmed that higher risk of pheochromocytoma is associated with missense mutations that result in substitution of a surface amino acid or that disrupt the structural integrity of the VHL gene product (pVHL). Age-related risks of retinal hemangoblastomas and renal cell carcinoma were higher in patients with nonsense or frameshift mutations. Southern blot and/or MLPA analysis by whole blood detected this mutation. It was found in a VHL type 1 family of 5 individuals. Four family members had retinal angiomas and 3 had hemangioblastomas of the cerebellum.

Genotype-phenotype correlations of 573 VHL patients from 200 kindreds were analyzed and confirmed that higher risk of pheochromocytoma is associated with missense mutations that result in substitution of a surface amino acid or that disrupt the structural integrity of the VHL gene product (pVHL). Age-related risks of retinal hemangoblastomas and renal cell carcinoma were higher in patients with nonsense or frameshift mutations. Southern blot and/or MLPA analysis by whole blood detected this mutation D197FS (c.589del) was found in a VHL type 1 family with 3 individuals. 1 had retinal angiomas diagnosed at 38Y and 1 with renal cell carcinoma diagnosed at 25Y.

216 patients with clinically expected VHL disease due to family history or presence of VHL typical tumours were routinely examined in an eye centre in Germany between January 2019 and January 2020, majority of which were of European descent (~95%). Since data on the genotype–phenotype correlation of the onset of Retinal Hemangioblastoma (RH) is scarce in relation to VHL disease, this study wanted to inversitage the relationship between VHL gene variants (truncating vs single amino acid substitutions) and the ocular phenotype of the disease. 42 different rare VHL gene variants in 148 patients were detected - some of whom had RH and some who did not. Although date of onset of RH, as well as data on central nervous system haemangioblastomas (CNSH), pheochromocytoma/paraganglioma (PPGL), clear cell renal cell carcinoma (ccRCC) and pancreatic neuroendocrine neoplasm (PNEN) were collected, it was not reported on a per patient basis and therefore could not be discussed per variant. There was 1 patient reported with the c.233A>G (p.N78S) mutation. This member presented with retinal hemangiomas. No family information was provided.

216 patients with clinically expected VHL disease due to family history or presence of VHL typical tumours were routinely examined in an eye centre in Germany between January 2019 and January 2020, majority of which were of European descent (~95%). Since data on the genotype–phenotype correlation of the onset of Retinal Hemangioblastoma (RH) is scarce in relation to VHL disease, this study wanted to inversitage the relationship between VHL gene variants (truncating vs single amino acid substitutions) and the ocular phenotype of the disease. 42 different rare VHL gene variants in 148 patients were detected - some of whom had RH and some who did not. Although date of onset of RH, as well as data on central nervous system haemangioblastomas (CNSH), pheochromocytoma/paraganglioma (PPGL), clear cell renal cell carcinoma (ccRCC) and pancreatic neuroendocrine neoplasm (PNEN) were collected, it was not reported on a per patient basis and therefore could not be discussed per variant. There were 2 patients reported with the p.R79G (c.235C>G) mutation. One member presented with retinal hemangiomas, while the other did not - however no other clinical manifestations were discussed. Additionally, no family information was provided.

74 patients (38 males, 36 females; 24 with prior sequencing data) with pheochromoctyoma were first analyzed by denaturing high performance liquid chromatography (dHPLC). PCR of all exons of VHL was performed on leukocyte DNA extracted from stored blood samples, and those showing variance were sequenced. In addition, 35 positive controls were used to confirm this method. This patient was a positive control who has been previously sequenced (not referenced). One patient was found with the R167W (c.499C>T) VHL mutation. No family history was mentioned or clearly stated of their phenotypes.

Of 16 patients presenting with clinical VHL features that underwent genetic testing, one presented with the c.484T>C mutation of the VHL gene (stated as c.697T>C in the paper which used the Latif sequence as reference). Peripheral blood lymphocytes were collected and products of the polymerase chain reaction performed on this sample were analyzed with single-stranded conformational polymorphism analysis (SSCP), PCR sequencing and Southern analysis. This missense mutation was confirmed using restriction fragment length polymorphism or allele-specific oligonucleotide analysis. This patient has no family history of clinical VHL features. The patient presented with renal cell carcinoma.

Of 16 patients presenting with clinical VHL features that underwent genetic testing, one presented with the c.250G>T mutation of the VHL gene (stated as c.463G>T in the paper which used the Latif sequence as reference). Peripheral blood lymphocytes were collected and products of the polymerase chain reaction performed on this sample were analyzed with single-stranded conformational polymorphism analysis (SSCP), PCR sequencing and Southern analysis. This missense mutation was confirmed using restriction fragment length polymorphism or allele-specific oligonucleotide analysis. This patient has a family history of multiple tumor types indicative of VHL disease. The patient presented with a retinal angiomata, hemangioblastoma and pheochromocytoma.

A case report details a 27 year old male presenting with spinal hemangioblastoma, pancreatic cysts, and kidney cysts. Genetic testing of unclear nature revealed the presence of an NM_000551.3:c.313A>C mutation, with a predicted p.Thr105Pro change. No family information was provided. ACMG Evidence Codes: 'PP4' because the pattern of presentations observed is highly specific for VHL disease.

A retrospective study described polyglobulia in 19 patients precise germline VHL mutations and with hemangioblastomas. 10 other patients followed with VHL mutations without an exact location. Patient 26 was a 41Y male. They had a hemangioblastoma tumor 6.7cm3 in size. The mutation p.G114S (c.340G>A) was described in the paper as c.553G>A due to a difference in transcript reference. No family information was provided.

27 index patients suspected of having VHL disease from unrelated Chinese families and their relatives were studied. The nucleotide were numbered according to the sequence numbering by Latif et al, 1993 and this was converted to NM_000551.3. The paper did not differentiate between a pancreatic cysts and tumor and presented these together as a 'pancreatic lesion'. The paper summariezed the phenotypes of the families together. Family 18 was found with p.Cys162Tyr mutation in the VHL gene: 2/2 members had CNS hemangioblastoma, 1/2 had RCC and 1/2 had a pancreatic lesion. ACMG PP4

27 index patients suspected of having VHL disease from unrelated Chinese families and their relatives were studied. The nucleotide were numbered according to the sequence numbering by Latif et al, 1993 and this was converted to NM_000551.3. The paper did not differentiate between a pancreatic cysts and tumor and presented these together as a 'pancreatic lesion'. The paper summarized the phenotypes of the families together. Family 6 was found with p.Asn78Ser mutation in the VHL gene: 22 family members were recorded with the given mutation. 18 had VHL manifestations while 4 were asymptomatic carriers. 10/18 members had CNS hemangioblastoma, 5/18 had retinal angioma, 12/18 had RCC, 7/18 members had a pancreatic lesion, and 1/18 had pheochromocytoma. ACMG PP4

VHL germline mutation carriers who had an ophthalmologic examination at the Erasmus Medical Centre or the Rotterdam Eye Hospital between 1984 and 2017, and were diagnosed with retinal hemangioblastoma (RH) were included in this study. A diagnosis of RH was based on the clinical appearance of a vasoproliferative retinal tumour. Peripheral blood samples from every member that had clinically suspected VHL disease of each family were collected for VHL mutation analysis. A total of 21 patients from 10 families were included on the basis of both a VHL germline mutation and ocular manifestations. Copy number analyses were carried out by multiplex ligation dependent probe amplification (MLPA) and fluorescence in situ hybridization (FISH). Mutations were described as one of the following two groups: truncating (complete deletion, partial deletion, nonsense mutation, splice site mutation, duplication and insertion) or missense. Ocular manifestations were not reported on an individual level, but rather were broken down based on the two groups. From Pedigree (family) 7, six individuals were suspected of VHL disease and mutational analysis revealed a missense V130P(c.388G>T) mutation. 5/6 individuals developed RH, while 1/6 did not.

42 CNS hemangioblastoma samples were obtained from 31 patients, who were consecutively treated between 1993 and 1997 at the Department of Neurosurgery of the Freiburg University Medical Centre were analyzed for VHL mutations. One patient of 17 presenting with VHL germline mutations presented with the C162W (c.486C>G) mutation (stated as c.699C>G in the paper which used the Latif sequence as reference). Single strand conformation polymorphism and southern blotting for variant identification in the DNA found in blood samples. The patient was established as having VHL disease by standard of Neumann at al., 1987. The patient presented with two cerebellum tumors (a,b).

53-year-old male presented to the hospital with recurrent chest pain, sweating, palpitation and hypertension. Transthoracic echocardiography revealed a left ventricular enlargement. An abdominal CT scan revealed a tumor in the right adrenal gland, 5.3cm in size. He was hospitalized again due deteriorating symptoms involving a left anterior descending stenosis. He was admitted to the intensive care unit and died of cardiogenic shock. A diagnosis of VHL syndrome was confirmed once genetic analysis revealed a heterozygous V84L(c.250G>C) missense mutation in the patient and his daughter, however his elder son did not possess the mutation. A diagnosis of VHL type 2C was made due to the pheochromocytoma complicated with myocardial damage, myocardial infarction and heart failure.

A 39-year-old man presented with bilateral clear cell renal cell carcinoma. Bilateral nephron sparing surgery was performed. After 2 years multiple pancreatic cysts were noted. Two years later 6 bilateral renal cell carcinoma lesions developed. Repeat bilateral nephron sparing surgery was performed. Five years later at age 48 years, he presented with mental confusion and brain MRI revealed a cerebellar lesion; histology confirmed this to be a hemangioblastoma. Deoxyribonucleic acid analysis of the peripheral blood demonstrated a missense germline mutation of the von Hippel-Lindau gene c.699C>G (mapped to c.486C>G (p.Cys162Trp)). Family history was unremarkable and the patient was childless.

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 C162F (c.485G>T) missense VHL mutation was found in two African American females diagnosed with clear cell renal cell carcinoma, one of whom was 40Y0M and the other 55Y0M. No family information was provided on either patient.

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 R167W (c.499C>T) missense VHL mutation was found in two individuals, a white 67Y0M female diagnosed with clear cell renal cell carcinoma, as well as a white 19Y0M female diagnosed with pheochromocytoma. No family information was provided.

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.C162W (c.486C>G) mutation, 15 members total from 4 families were described, and phenotypic information was given for this group. With ages of onset ranging from 12-56Y, hemangioblastomas were observed in 12/15 members, retinal capillary hemangioma in 4, renal cell carcinoma in 8, pancreatic cysts in 6, pheochromocytoma in 1 member and and a papillary cystadenoma of the epididymis genital system tumor (epididymis or broad ligament) in 1 member. This cohort has been previously published in PMID 29330336 (2 families with 7 members altogether), and 28388566 (as family #67 and #69).

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 c.464-1G>C mutation, 11 members total from 2 families were described, and phenotypic information was given for this group. With ages of onset ranging from 10-66Y, hemangioblastomas were observed in 6/11 members, renal cell carcinoma in 4, pancreatic cysts in 4, and a papillary cystadenoma of the epididymis genital system tumor (epididymis or broad ligament) in 1 member. These families have been previously published: see more information in PMID 29330336 (2 families of 11 people total), and PMID 28388566 (as family #52 (9 members), and #53 (2 members)).

This study focused on observing SDHx function on the development of PPGLs. DNA was extracted from peripheral blood samples and next generation sequencing of a clinical gene panel included; SDHA, SDHB, SDHC, SDHD, KIT, PDGFRA and NF1 (for GIST) and SDHA, SDHB, SDHC, SDHD, SDHAF2, MAX, TMEM127, VHL, RET, FH for (PPGL). The lowest SDHC expression level was seen in case #0026. This case was a pheochromocytoma with a known pathogenic germline VHL variant (c.499C>G, p.Arg167Gly). She was a 25Y patient who showed an equivocal result on SDHB immunohistochemistry testing, but no evidence of a somatic SDHx mutation. No family history was noted.

In a study from the National Cancer Institute, 6 unrelated VHL patients with known germline VHL mutations underwent surgery for uni- or bilateral renal lesions. The paper used legacy notation for VHL point mutations, so mutations were converted and reported in VHL NM_ 000551.3. DNA was prepared from either cells in culture (grown from renal lesions) or primary formalin- or ethanol-fixed tumor lysates under salt-free conditions.Patient #4 was 27Y female (current age at time of study) with a germline VHL missense mutation (c.233A>G, originally written in the paper as legacy notation c.446A>G) and presented with CNS hemangioblastoma, renal cell carcinoma, renal cysts, pancreas lesion, and adrenal lesion. Family history was not described.

A 64 year old female was found to have a germline VHL missense mutation (Arg167Trp). The paper does not provide any information about nucleotide change. The patient presented with a large suprasellar hemangioblastoma, and had a documented past history of severe VHL disease, with bilateral ccRCC, bilateral pheochromocytomas, pancreatic cysts, retinal hemangioblastomas, and multiple CNS hemangioblastomas. No information about patient family history was provided. Neurological exam was significant for Romberg sign and mild dysmetria of bilateral upper extremities. A visual field test of the left eye revealed new onset inferior temporal quadrantanopsia. MRI confirmed a suprasellar hemangioblastoma within the circle of Willis with increased optic tract edema and compression of the optic chiasm. Left eye extraocular movements and pupillary reaction intact. The patient was treated with octoreotide for 9 months, leading to resultant clinical stability and decreased tumor volume.

Using polymerase chain reaction (PCR)-direct sequencing and universal primer quantitative fluorescent multiplex–PCR, mutations in VHL gene in 19 VHL disease families recruited from the Department of Urology, Peking University First Hospital in the period from 2009 to 2012 were examined. Two probands were found to carry the mutation c.481C>T in exon 3 of the VHL gene leading to an early stop codon. The first proband was from family 14 and presented with hemangioblastoma and renal cell carcinoma at age 29. At age 33, the patient presented with pancreatic cysts or tumours. The patient had positive VHL family history and his father had a hemangioblastoma of the central nervous system at age 42. He was deceased before diagnosis could occur. The other proband was from family 15 and had no VHL family history. The patient presented with hemangioblastoma, renal cell carcinoma, and pancreatic cysts or tumours at age 39. Both of these families were previously published in 29330336.

A 35-year old woman was diagnosed with VHL due to clinical presentation of pancreatic serous cystic neoplasm and renal cell carcinoma at age 35, and cerebellar hemangioblastomas that were surgically removed at age 21. She had no family history. Genetic testing performed for her previous case report (PMID: 21673464) revealed a N78I (c.233A>T) missense mutation in the VHL gene. The patient had previously underwent total pancreatectomy and right nephrectomy for pancreatic serous cystic neoplasm (SCN) and renal cell carcinoma (RCC), respectively. Since her surgery, she had follow-up examinations performed every 6 months. At the 4th year follow up, tumors in the liver were detected by CT scan and radiographically diagnosed as metastases of the pancreatic SCN or RCC. 53 months after the initial surgery, partial resections of the liver were performed. Immunohistochemical findings of the resected liver tumour were similar to the primary pancreatic SCN, and not the RCC. Thus, the liver tumors were pathologically diagnosed as liver metastases of the pancreatic SCN. After the second surgery, follow up examinations continued to be performed every 6 months. At the first follow-up, 3 tumors in the liver were detected by CT and MRI. After 6 months, no increase in the number of tumours were detected. Anterior segmentectomy and partial resection of segment III of the liver were done 66 months after 1st surgery and 13 months after second surgery, respectively. Again, morphology and immunohistochemical findings of the resected tumors again were similar to the primary pancreatic SCN, thus diagnosed as multiple metachronous liver metastases of pancreatic SCN. Since then the patient has remained disease-free without any recurrence after the third operation for 6.5 years until the last follow-up.

Seventeen patients (age range 3–73) with VHL, including 12 probands and 5 relatives, that were observed between 2005 and 2020 were recruited for a retrospective analysis in relation to pancreatic neuroendocrine tumors (PanNETs). A total of 13/17 patients had PanNETs. In 4 patients, PanNETs were found at VHL diagnosis and in 9 patients they were found during follow-up after a median time of 12 years. Six of these patients underwent surgery and seven were conservatively treated. Four of these patients had lymph node metastases and a tumordiameter significantly larger than the nonmetastatic PanNETs. Five patients who underwent surgery had stable disease after a 3 year follow up, while one patient showed liver metastases. Similarly, six non-resected PanNETs were stable after a 2 year, while one patient developed a novel small PanNET and slight growth of the pre-existing PanNET. Genomic DNA from blood was extracted and high-coverage sequencing for VHL gene was performed using an AmpliSeq custom panel that targets all exons, as well as intron–exon junctions of genes that are frequently mutated in PanNETs (VHL; RET; MEN1). A total of nine different VHL mutations were identified. Three patients were found to be de novo, but authors did not specify who. This proband (Patient 2a) with Renal Cell Carcinoma, CNS/Retinal Hemangioblastoma, Pancreatic cysts, and PanNET was found to have a germline missense variant N78S(c.233A>G) in exon 1 of the VHL gene. A relative (Patient 2b) was found to carry the same mutation with the same clinical presentation, except with additional Paraganglioma.

Fourty-four of 389 patients with VHL (between 1988-1999) screened positive for PNETs either by pathologic analysis of tissue specimens or by characteristic radiographic appearance on CT and MRI. VHL gene germline mutations were determined by quantitative Southern blotting to detect deletions of the gene and gene rearrangements as well as complete gene sequencing. One patient (21), a 23 year old female, was found to harbour the c.293A>C mutation in the VHL gene (stated as 506 A to C in the paper which used the Latif sequence as reference) leading to an amino acid change of Tyr to Ser at protein position 98. She presented with two pathologic pancreatic neuroendocrine tumors. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

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.89_297del germline mutation of the VHL gene. They presented with a retinal hemangioblastoma, cerebellar hemangioblastoma, spinal hemangioblastoma, renal cell carcinoma, pancreatic endocrine tumor, renal cyst and pancreatic cysts. Their age at the last follow-up was 70. They were previously published in PMID 24132471.

Of 31 Brazilian patients, one was found to harbour the c.233A>G (p.Asn78Ser) mutation of the VHL gene. Genomic DNA was extracted and all VHL coding regions were analyzed by Sanger sequencing. MLPA was performed to investigate large deletions using SALSA® MLPA® P016 VHL probe mix. Data analysis was performed with Genescan 3.7. This patient, age 33, presented with a paraganglioma, pheochromocytoma, pancreatic endocrine tumor, renal cell carcinoma, renal cyst, pancreatic cyst and a CNS hemangioblastoma. It is not clear if this mutation is familial. ACMG evidence codes: 'PP4' since the patients present phenotypes highly specific for a disease with a single genetic etiology.

A retrospective study reviewed cases of 23 patients with advanced and complicated VHL eye disease who underwent pars plana vitrectomy in the years 1999 to 2012 that were a part of 223 VHL patients consulted at the International Hereditary Cancer Center and Polish VHL Registry. Genetic molecular analysis was performed in 22/23 patients through PCR and MLPA, and 24 eyes from 23 patients were studied. A 23 year old (patient 21) with a germline intronic mutation c.464-1G>T, stated in the paper as c.IVS2-1G>T, with previous history of HbCNS. Patient was examined in a study of advanced VHL eye disease and had multiple retinal capillary hemangioblastomas requiring surgery.

A 20-year-old male was admitted to the General Hospital of Tianjin Medical University in 1998 and was found with a cerebral hemangioblastoma. He went on to develop another hemangioblastoma 3 years later. In 2012, the 34Y old patient was found with RCC, pancreatic cysts, and an assumed pancreatic neuroendocrine tumour. Genomic DNA was extracted from the peripheral blood leukocytes and PCR determined a germline p.Leu158Pro mutation in the VHL gene. There was a recorded family history of VHL disease, with the patient’s mother, grandmother, two uncles and three aunts also suffering from the disease. Additionally, one uncle had succumbed to RCC and five other individuals in the family had succumbed to cerebral hemangioblastomas. Whether these family members had other VHL phenotypes is unclear.

17 patients from 10 families affected with VHL syndrome, particularly with retinal capillary hemangioblastomas (RCHs) were examined. 24-year-old woman (patient 1) from family 1 had her first retinal examination due to the detection of a cerebellar hemangioblastoma. Her vision was 20/20 in both eyes, but a peripheral retinal capillary hemangioblastoma (RCH) was present in the left eye. Laser was performed on the retina surrounding the lesion. During the follow-up, the tumor was stable in size and there was no development of RCHs in the right eye. Genomic DNA was extracted from whole blood for sanger sequencing of the VHL gene and multiplex ligation-dependent probe amplification was performed to identify genomic rearrangements. A novel frameshift P154Qfs4*(c.461delC) mutation was identified in exon 2 of the VHL gene. The parents of patient 1 were molecularly tested and neither possessed the mutation; thus, the mutation was identified as de novo. The parents nor the sister of patient 1 had VHL syndrome characterized by RCHs.

Between 1994 and 2001, the Dutch VHL working group conducted DNA analysis on 146 probands with VHL-associated manifestations. DNA of probands was extracted from peripheral blood samples. Exons 1, 2 and 3 of the VHL gene and their flanking untranslated and intronic coding sequences were amplified by PCR and subjected to semi-automated sequence analysis. Screening for structural rearrangements, including deletions, involving the VHL gene was performed by Southern blot analysis and the multiplex ligation-dependent probe amplification (MLPA) assay. Deletions encompassing the entire VHL gene were confirmed by FISH analysis on lymphocytes from affected individuals. VHL mutations were identified in 43 families. Family 30 has 1 affected member. Genetic analysis reveals the mutation C162Wfs*12(c.485dupG) in the VHL gene, which the paper reports in less up-to-date as c.485dupG (p.L163fs). The patient was said to be sporadic in their history of VHL disease. They have CNS and retinal hemangioblastomas.

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 detected in 2 VHL type 1 family members (Family ID 3682), using PCR and SSCP sequencing. The mutation of c.226T>A was referred to in the paper by c.439T>A due to a difference in transcript sequence. Both patients carried CNS hemangioblastomas. Supporting evidence of pathogenicity because cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease (ACMG code: PP1).

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 5 VHL type 2 family members (family ID 2547), using PCR and SSCP sequencing. The mutation of c.278G>A was referred to in the paper by c.491G>A due to a difference in transcript sequence. Three of 5 individuals were identified with retinal hemangioblastomas, 4 with CNS hemangioblastomas, and 2 with pheochromocytoma.

33 patients diagnosed with VHL disease, according to (Maher et al. 1990), were tested for germline VHL mutations. Genetic testing was performed on high-molecular-weight DNA from peripheral blood using Southern analysis and PCR. One patient was found to harbour the 500G>A (Arg167Gln) VHL gene mutation, documented in the paper as 713G>A due to a difference in transcript sequence. They presented with a pancreatic tumor. ACMG evidence codes: 'PP4' since the patient presents phenotypes highly specific for a disease with a single genetic etiology.

36 VHL-related pheochromocytomas (from 21 patients) and 10 VHL-related CNS hemangioblastomas (from 6 patients) were studied using comparative genomic hybridization (CGH) to detect genetic imbalances. Patient III in VHL-Associated hemangioblastoma was found with germline insertion mutation (c.230insTCT) in the VHL gene (stated as c.443TCT in the paper which used the Latif sequence as reference) in the VHL gene. All patients have familial VHL.

Analysis of peripheral blood samples from 5 Japanese families with PCR and SSCP revealed 4 germline mutations. This inframe deletion NM_000551.3:c.227_229delTCT was found in a family of 3 individuals (Family B). Please note that the paper states the mutation as c.440_442delTCT because it uses a legacy reference sequence. Only one patient in this family developed symptoms, which included a brain stem hemangioblastoma and retinal angioma discovered at age 30. Parents of this individual showed no manifestations of VHL disease and were negative for bands via single-strand conformational polymorphism (SSCP) analysis where the proband showed extra bands.

Study of a German family of 16 family members with cosegregation of the pheochromocytoma-only phenotype with two concurrent germline mutations in the VHL protein alpha and beta domains. These mutations were c.562 C>T (stated as 775C>G in the paper which used the Latif sequence as reference) and c.241C>T (stated as 454C>T in the paper). Patient 1 was unaffected and had the wild-type allele. His spouse, patient 2, presented with pheochromocytoma and had both mutations. They had four children; one had a pheochromocytoma and died, two (patients 5 and 7) had both mutations and presented with pheochromocytomas, and one child (patient 6) was unaffected and carried neither mutation. Patient 7 had a child (patient 9) who also carried both mutations and presented with pheochromocytoma. In the other branch of the family, patient 3 who carried both mutations and presented with pheochromocytoma had a child with patient 4 who was unaffected. Their child (patient 8) carried both mutations and presented with pheochromocytoma. Patient 8 had two children; one who did not carry the mutation and was unaffected (patient 10) and another that carried both mutations but was disease-free (patient 11). DNA was isolated with whole blood and denaturing high-performance liquid chromatography (DHPLC) and dye terminator method on an ABI Prism 310 genetic analyzer (Applied Biosystems, Weiterstadt, Germany) were used for sequencing. Cosegregation with disease in multiple affected family members in a gene definitively known to cause the disease (ACMG code: PP1).

301 were enrolled in a prospective clinical protocol to evaluate the natural history of vHL-associated pancreatic lesions (NCT00062166). Of these, 69 patients with pancreatic manifestations of vHL disease and missense VHL mutations were included in the current study. Individual patient phenotype was not described, but overall of the 60 patients, 57 had RCC, 41 had CNS hemangioblastomas, 28 had retinal hemangioblastoma, 23 had pheochromocytoma. 36/69 patients were female and the mean age was 50.5 years old. All patients underwent genetic testing using Sanger sequencing and Southern Blotting. One patient was listed with this missense mutation, c.278G>T.

Tissues embedded in paraffin blocks were collected from 203 cHAB patients operated in different Medical Centres in Poland. Eight of these harboured germline mutations in unrelated patients from distinct undiagnosed VHL families. Various variants were unverified as somatic or germline as they were detetced in tumour but blood samples were not tested. Other phenotypes and demographic information is not known. Patient 51 was found with a germline p.Arg161* mutation in the VHL gene with hemangioblastomas.

Thirteen patients with supratentorial hemangioblastoma were studied at Le Kremlin-Bicêtre University. Other phenotypes included: 6 patients with pancreatic cysts, 1 patient with an endocrine pancreatic tumor, 5 patients with renal cell carcinoma, 3 patients with pheochromocytoma, and 6 patients with retina hemangioblastomas, although specific phenotypes for each patient was not described. Blood samples were isolated from patients and the coding sequence and flanking intronic sequences were directly sequenced. Quantitative Southern blotting and multiplex ligation-dependent probe amplification studies were performed to detect large genomic rearrangements. Patient 8 (male) had a germline VHL p.Arg161* mutation. The patient had a meckel cave hemangioblastoma at age 28 and a papillary edema and pituitary stalk hemangioblastoma at age 34.

Thirteen patients from four families (A, B, C and D) with known VHL disease and known mutations in the VHL gene were examined.  MLPA using venous blood showed 4 genetic variants. Family C was found with a p.L198P mutation in the VHL gene, only confirmed in patient II-1 (male), and was identified together with the polymorphism c.74C>T, p.P25L. The asymptomatic father was also a carrier of the same polymorphism (c.74C>T, p.P25L). No further familial information was provided although suggestion of a de novo mutation. Patient II-1 presented with adrenal pheochromocytoma. pancreatic neuroendocrine tumour, and retinal hemangioblastoma.

Three patients with mutations in the VHL gene were diagnosed with pancreatic neuroendocrine tumours before the age of 18. Case 2 was a young female who developed bilateral phaeochromocytomas at 12 and underwent staged bilateral adrenalectomies at 14. At the age of 16, she was discovered to have a 2.5 cm tail of pancreas pancreatic neuroendocrine tumour. This enlarged during follow-up and additional smaller pancreatic neuroendocrine tumours in the pancreatic body became visible. A second 1 mm grade 1 pNET was also present and was completely excised. In the 7 years of follow-up since this operation, new pNETs developed within the pancreatic head, body and tail and have continued to enlarge. During a period of follow-up, she was found to have a small asymptomatic spinal haemangioblastoma. Genetic testing was done when the patient was 11-years-old and confirmed an inherited mutation in the VHL gene. She was found to have inherited a mutation in exon 3 of the VHL gene (c.499C>T, p.R167W). This missense mutation is noted in the paper as c.712C>T due to a difference in the VHL reference sequence notation used. The proband's mother died at the age of 45 from a progressive metastatic pancreatic neuroendocrine tumour diagnosed at the age of 35. The mother also had a pheochromocytoma, hemangioblastoma, and renal cell carcinoma. The proband's uncle is also described as having VHL disease and presented with pheochromocytoma, hemangioblastoma, renal cell carcinoma. The proband's maternal cousin, 'Case 3' in the paper, was a 3-year-old male who was diagnosed with VHL disease after cascade genetic screening and was found to have the same mutation as 'Case 2' in the VHL gene (c.499C>T, p.R167W). At the age of 11, he underwent resection of an abdominal paraganglioma, followed by staged bilateral adrenalectomies for phaeochromocytomas at the age of 14. Post-operative surveillance imaging detected a 1 cm body of pancreas pancreatic neuroendocrine tumour at the age of 12. Case 3's brother was a 26 year old male with a pheochromocytoma and hemangioblastoma.

Through PCR purification and Sanger sequencing of peripheral blood (using an alcohol precipitation method), 121 consecutive, unrelated, index PCC/PGL patients underwent genetic testing for five PCC/PGL susceptibility genes (RET, VHL, SDHB, SDHD and SDHC) and were evaluated for clinical diagnosis of neurofibromatosis type1. Two unrelated probands were genetically confirmed with a familial mutation at c.482G>A of the VHL gene. The first was an 20 year old female who presented with bilateral pheochromocytoma. The second was a 6 year old female who presented with bilateral metachronous pheochromocytoma. Family histories are unknown.

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. Two families (Family ID 9 and 13) were found with a germline missense mutation in the VHL gene (S65W; c.194C>G). Family 9 had three patients: Patient I and II were both diagnosed at 0Y and had no manifestations by the end of the study at 5Y and 10Y respectively. Patient III was diagnosed at age 16 and developed two CNS hemangioblastomas and two pancreatic cysts at age 16. Family 13 had one patient who was diagnosed at age 13 and had eight retinal hemangioblastomas at that presented at ages 13, 16, 17, and 18. This patient also had one pancreatic cyst and one CNS hemangioblastoma at age 17. This patient died at age 48.

Whole blood samples and PCR and SSCP was used to investigate a family in Kuwait with Arabian and Persian genetic admixture. The germline VHL mutation p.Trp117Cys was found in a second extended von Hippel-Lindau (VHL) family. The mutation c.351G>T was referred to in the paper by c.564G>T due to a difference in transcript sequence. The index patient, III-9, presented with a history of VHL syndrome: CNS hemangioblastoma, spinal hemangioblastoma, retinal hemangioblastoma, cerebellar hemangioblastoma, and renal cell carcinoma. The clinical history of all other patients revealed multiple CNS hemangioblastomas in various organs and renal cell carcinoma without pheochromocytomas. One affected member of the family (III-8) had a bladder tumor and an unspecified brain tumor in addition. Index patient's Persian grandmother had VHL (hemangioblastoma and renal cell carcinoma), 5/6 of her children (2 females, 3 males), and 7/20 of her grandchildren (4 females, 3 males) including the index patient. All affected members of the family, lacked any evidence of pheochromocytoma, leading to the categorization of this family as VHL type 1. This mutation was found in all patients with clinical diagnoses but in none of the healthy members of the family or in the 55 healthy control subjects. ACMG: PP4, PP1

26 VHL patients from 18 families, who had undergone a partial adrenalectomy for pheochromocytoma were retrospectively analyzed. 1 patient from a single family was confirmed to have the mutation (Y112H). Additional manifestations aside from pheochromocytoma 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.

26 VHL patients from 18 families, who had undergone a partial adrenalectomy for pheochromocytoma were retrospectively analyzed. 1 patient from a single family was confirmed to have the mutation (L118P). Additional manifestations aside from pheochromocytoma 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.

Tissue specimens of apparent sporadic pheochromocytoma were retrieved from the archives of the Department of Pathology of the Erasmus MC (Rotterdam), the Radboud University Nijmegen Medical Center, and the University Hospital Zurich (Switzerland). Mutation analysis was performed for 136 VHL cases and six germline VHL mutations were found.Mutation analysis was performed using SSCP analysis and BigDye® Terminator V3.1 cycle sequencing kit (Applied Biosystems, Foster City, CA). 136 VHL cases and six germline VHL mutations were found. A c.431G>A (stated as c.644G>A in the paper which used the Latif sequence as reference) mutation was found in one 39Y female patient with benign adrenal pheochromocytoma. Note: the protein change was recorded improperly by the author as p.Gly144Gln when in fact it is p.Gly144Glu. No other phenotypes, patient demographics, or family history was described.

An update on the genetic findings pertaining to a previously published (pmids: 8239848, 21713522) large familial pheochromocytoma Pennsylvania kindred. The family is comprised of: 18 living individuals with only pheochromocytoma; 7 living individuals with only retinal angioma; 6 living individuals with both; 51 other first-degree relatives; 5 additional relatives with pheochromocytomas, including a 69Y woman and her 42Y son described previously as non-penetrant individuals (pmid 8239848). Family classified as a VHL Type 2A (pheochromocytoma, retinal angioma, CNS hemangioblastoma, and little to no RCC). All 31 individuals (including 6 archival specimens) with a diagnosis of pheochromocytoma, retinal angioma or both had the identical T547C (legacy nomenclature) germline mutation determined through PCR, single-strand conformational polymorphism analysis and direct sequencing.