6.13 von Hippel–Lindau disease and succinate dehydrogenase subunit (SDHB, SDHC, and SDHD) genes

This chapter considers the clinical and molecular features of von Hippel–Lindau (VHL) disease (OMIM 193300) and mutations in succinate dehydrogenase subunit genes (SDHB (OMIM 115310), SDHC (OMIM 605373), and SDHD (OMIM 168000)). Both disorders are important causes of phaeochromocytoma and, in addition to having overlapping clinical phenotypes, also share some similarities in mechanisms of tumourigenesis.

VHL is a dominantly inherited familial cancer syndrome with multisystem involvement. The most frequent features are retinal and central nervous system haemangioblastomas, renal cell carcinoma (RCC), and renal, pancreatic, and epididymal cysts (1). The most important endocrine complications are phaeochromocytoma and pancreatic islet cell tumours.

The earliest features of VHL disease are usually retinal or central nervous system haemangioblastomas (CHB) (Table 6.13.1 and Fig. 6.13.1) (4). However there is marked phenotypic variability. Thus phaeochromocytoma or RCC can be the presenting feature (5). In such cases the detection of subclinical haemangioblastomas (e.g. retinal by ophthalmological screening, or cerebellar by brain MRI) or the detection of visceral cysts and tumours by abdominal imaging can aid diagnosis. If there is a positive family history, a clinical diagnosis of VHL disease can be made in an at risk individual by the identification of a single retinal or cerebellar haemangioblastoma, RCC, or phaeochromocytoma (6). In isolated cases conventional diagnostic criteria require the presence two or more retinal or cerebellar haemangioblastomas or a single haemangioblastoma and a visceral tumour. However, in many cases molecular genetic testing can allow a diagnosis of VHL disease to be made in patients who do not satisfy clinical diagnostic criteria (7). When a mutation has been identified in a family, other relatives can be tested to determine their mutation status and hence their need for surveillance.

There are marked interfamilial differences in phaeochromocytoma frequency in VHL disease. Thus in some families phaeochromocytoma is the most common manifestation, but in others it is rare. These differences reflect genotype–phenotype correlations and the high risk of phaeochromocytoma associated with certain VHL missense mutations. Large deletions, protein truncating mutations, and missense mutations that disrupt protein stability are associated with a high risk of retinal angioma, CHB, and RCC but a low risk of phaeochromocytoma (type 1 VHL phenotype) whereas missense mutations affecting amino acids on the VHL protein (pVHL) surface predominate in VHL patients with phaeochromocytoma (8, 9, 11). However not all phaeochromocytoma-associated missense mutations are equivalent. Most cause a high risk of retinal angioma, CHB, RCC, and phaeochromocytoma (type 2B VHL disease), but rare missense mutations may cause type 2A (haemangioblastomas and phaeochromocytoma but rarely RCC) or type 2C (phaeochromocytoma only) phenotypes (3, 5, 10, 11).

The clinical presentation of phaeochromocytoma in VHL disease is similar to that in sporadic cases except that there is a higher frequency of bilateral or multiple tumours and, on average, an earlier onset (mean approximately 30 years) in VHL disease. As with sporadic tumours, phaeochromocytomas in VHL disease may be extra-adrenal and, in about 5% of cases, malignant. Early detection of phaeochromocytoma in VHL disease facilitates management and so screening for phaeochromocytoma should be offered to all VHL patients and at-risk individuals irrespective of whether there is a family history of phaeochromocytoma. However, the presence of a positive family history or a missense mutation known to be associated with a high risk of phaeochromocytoma indicate a need for enhanced phaeochromocytoma surveillance. Patients with apparently nonsyndromic familial or bilateral phaeochromocytoma, or phaeochromocytoma at a young age may have a germline VHL gene mutation (4, 12) and should be offered VHL mutation analysis. In such cases the nature of the VHL mutation identified will indicate the risk of other types of VHL related tumours (e.g. whether a type 2A, 2B, or 2C associated mutation).

The most frequent pancreatic feature of VHL disease is multiple cystadenomas, which rarely cause clinical disease. However, pancreatic tumours, most commonly nonsecretary islet cell tumours, occur in a minority (5–10%) of cases. These tumours are often asymptomatic and are detected by routine abdominal imaging. Initial experience of pancreatic tumours in VHL disease suggested a high frequency of malignancy, but more recent studies have suggested that surgery may be delayed for small tumours (13). Although there is a clinical impression that there are interfamilial differences in pancreatic tumour incidence and that the risk of pancreatic islet cell tumours and phaeochromocytomas may be correlated, the genotype–phenotype correlations reported for pancreatic tumours are less clear than for phaeochromocytoma.

Retinal and central nervous system haemangioblastomas are benign vascular tumours consisting of endothelial lined vascular channels and surrounding stromal cells and pericytes. Although benign, they are frequently cystic and neurological symptoms result from compression of the adjacent structures and/or raised intracranial pressure. Cerebellar involvement is most frequent and these usually respond well to surgery. However, both retinal and CHB are frequently multiple. Surgery for brainstem and spinal haemangioblastomas can be hazardous and CNS lesions remain an important cause of morbidity and mortality. Although the natural history of retinal lesions is to enlarge and cause retinal detachment and haemorrhage resulting in blindness, most small haemangioblastomas respond to laser- or cryotherapy so early detection is important (see below).

The lifetime risk of RCC in most cases of VHL disease (types 1 and 2B) is high (>70%) (4, 11). VHL disease is characterized not only by a high risk of RCC but also by an earlier age at onset (mean age 44 years for symptomatic lesions but as early as 16 years for early tumours detected by renal imaging) and a high risk of bilateral and multicentric tumours. Microscopically, VHL kidneys may contain numerous, small tumours and the risk of recurrence (from new primary tumours) after local excision for RCC is very high. However, a nephron-sparing approach is considered the optimal management for RCC in VHL disease in most centres. Thus, renal tumours detected at an early presymptomatic stage by routine surveillance are followed until 3 cm in size when nephron sparing resection is performed and other small lesions are also excised. The aim of this conservative approach to surgery is to delay dialysis for as long as possible. Although there is a high rate of reoperation for new primary tumours with this approach, the risk of metastatic spread appears small.

Endolymphatic sac tumours have also been recognized as a complication of VHL disease (3). These papillary adenocarcinomas may be asymptomatic or cause patients to present with symptoms such as tinnitus or deafness.

The VHL tumour suppressor gene was isolated in 1993 and encodes a 213-amino acid protein (pVHL) which is widely expressed in human tissues (7). A wide variety of germline VHL gene mutations have been identified, including large deletions, protein truncating mutations, and missense amino acid substitutions (11). Tumours from VHL patients show inactivation (by loss, mutation, or methylation) of the wild-type allele so that the mechanism of tumourigenesis appears similar to that of a classical tumour suppressor gene such as the retinoblastoma gene (14). However, an added complexity in VHL disease is the existence of intricate genotype–phenotype correlations, which suggested that the VHL gene product (pVHL) had multiple and tissue-specific functions (see above).

Although pVHL has been implicated in multiple signalling pathways, the signature pVHL function is the ability to regulate expression of the hypoxia-inducible transcription factors HIF-1 and HIF-2 (15, 16). Thus pVHL is the recognition component of an E3 ubiquitin ligase complex that, in normoxic cells, binds to hydroxylated prolines on the HIF-1 and HIF-2 α subunits, resulting in ubiquitylation and proteosomal degradation of the subunits (Fig. 6.13.2). Oxygen is an essential cofactor of the prolyl hydroxylation enzymes that regulate the ability of pVHL to bind to HIF-α subunits (16). In hypoxic conditions, pVHL is unable to bind to HIF-α subunits and HIF-1 and HIF-2 transcription factors are stabilized and cause activation of hypoxic-response genes, promoting angiogenesis, alterations in cell metabolism, and proliferation. Targets of HIF-2 are thought to be particularly implicated in the pathogenesis of RCC (17). Although many VHL mutations that are associated with phaeochromocytoma lead to dysregulation of HIF pathways, a second VHL-regulated pathway has been implicated in the pathogenesis of phaeochromocytoma in VHL disease. Thus pVHL has been implicated in a developmental apoptotic pathway that is normally activated when nerve growth factor becomes limiting for neuronal progenitor cells and results in developmental culling of the sympathetic neuronal (thought to be the phaeochromocytoma precursor cells) in late fetal life. Germline VHL− mutations associated with phaeochromocytoma are thought to impair this developmental apoptosis pathway and so predispose to phaeochromocytoma (17).

The ascertainment, diagnosis and surveillance of patients and relatives at risk of VHL disease is essential to prevent morbidity and mortality. The multisystem nature of the disease can lead to inconsistent and uncoordinated follow-up and it is important that a process is established to coordinate the multidisciplinary surveillance required. Following the diagnosis of VHL disease in an individual, all at-risk relatives should be contacted and informed of the need for investigation. Surveillance should commence in childhood (Box 6.13.1) and continue until there is no evidence of VHL disease at an advanced age (penetrance is almost complete by age 65 years). However, in most families it is possible to determine the need for surveillance by molecular genetic testing. Lifelong surveillance is indicated in affected individuals and asymptomatic gene carriers, whilst noncarriers can be reassured and discharged. The introduction of systematic surveillance protocols for following up affected and at-risk members of VHL kindreds has led to the early diagnosis of VHL tumours with a reduction in morbidity.

Succinate dehydrogenase is a heterotetrameric protein consisting of A, B, C, and D subunits located on the inner mitochondrial membrane (18). Succinate dehydrogenase has a critical role in cellular energy metabolism through its dual role in the Krebs citric acid cycle and as part of the respiratory chain (mitochondrial complex 2). The SDH-B subunit (also known as iron-sulphur protein), contains three iron-sulphur clusters ([2Fe-2S], [4Fe-4S], and [3Fe-4S]), is part of the hydrophilic catalytic domain, and binds to the A subunit, which contains a covalently attached flavin adenine dinucleotide cofactor and the substrate binding site. The B subunit also binds to the two hydrophobic membrane anchor subunits, C and D. The SDH-C and -D subunits attach the complex to the mitochondrial inner membrane and also contain the ubiquinone binding site to which the electrons are transferred from the SDH-B subunit iron-sulphur clusters within the B subunit.

Germline mutations in the gene encoding the D subunit of succinate hydrogenase were first found to be associated with familial head and neck paragangliomas (HNPGL) and then phaeochromocytoma (19, 20). Thereafter, germline mutations in the B subunit gene (SDHB) were also demonstrated to cause susceptibility to HNPGL and adrenal and extra-adrenal phaeochromocytoma (21). Germline SDHB and SDHD mutations are now recognized as a major cause of phaeochromocytoma susceptibility. Mutations in SDHA have been associated rarely with neoplasia (but can cause an autosomal recessive juvenile encephalopathy (22)) and SDHC mutations are an infrequent cause of HNPGL and a rare cause of phaeochromocytoma (23). With increasing availability and application of molecular testing for SDHB and SDHD mutations the phenotype has been expanded to include renal and thyroid tumours and gastrointestinal stromal cell tumours. Furthermore, SDHB mutations have been associated with a high risk of malignant phaeochromocytoma (24).

There is considerable overlap between the clinical features associated with mutations in the three genes that encode the B, C, and D subunits of succinate dehydrogenase, but there are also some important differences with respect to inheritance pattern and risks of individual tumours.

SDHB mutations Although germline mutations in SDHB, SDHC, and SDHD mutations can each be associated with the development of phaeochromocytoma and HNPGL, SDHB mutations are particularly associated with phaeochromocytoma and SDHD and SDHC with HNPGL. Thus in molecular genetic studies of population-based cohorts of phaeochromocytoma and HNPGL patients, the frequency of SDHB mutations is higher in the former group and SDHD in the latter (24–26). Mean age of phaeochromocytoma in SDHB mutation carriers is younger than in sporadic cases and similar to that in VHL disease. In contrast to sporadic and VHL-associated phaeochromocytomas, many phaeochromocytomas in SDHB mutation carriers occur at extra-adrenal sites (such tumours are also known as ‘paragangliomas’). Furthermore, there is a high frequency of malignancy in SDHB-associated phaeochromocytomas such that germline SDHB mutations may be detected in 30–50% of patients with malignant phaeochromocytoma. Patients with germline SDHB mutations are at risk for RCC. although the lifetime of approximately 15% is much less than in VHL disease. Familial or bilateral RCC without a personal or family history of phaeochromocytoma or HNPGL can be the presenting feature of germline SDHB mutations (27). Germline mutations in SDHB (and SDHC and SDHD) may also present with phaeochromocytoma and gastrointestinal stromal tumours (Carney−Stratakis syndrome) (28).

SDHC mutations Patients with germline SDHC mutations are less common than those with SDHB and SDHD mutations. Germline SDHC mutation carriers most commonly present with HNPGL (which tend to be unifocal) and only occasionally with phaeochromocytoma.

SDHD mutations Germline SDHD mutations were first characterized in patients with familial HNPGL and subsequently in familial phaeochromocytoma (19, 20). HNPGL in SDHD mutation carriers are often bilateral and multifocal. On average, the risk of HNPGL is higher in SDHD mutation carriers than in SDHB mutation carriers, whereas the reverse is true for phaeochromocytoma (Fig. 6.13.3). Nevertheless, germline SDHD mutations are an important cause of phaeochromocytoma susceptibility. The risk of malignancy is highest, but not confined to, SDHB mutations. The unusual inheritance pattern of SDHD-associated tumours may often lead to the possibility of familial disease being overlooked. Thus both SDHB and SDHC mutations cause dominantly inherited disease (so the risk of a child inheriting the mutation from an affected parent is 1 in 2) although age-dependent penetrance is apparent and incomplete penetrance is common. However, SDHD mutations display an unusual pattern of inheritance. Thus although the risk of a child inheriting the mutation from an affected parent is 1 in 2, the risk of a child who inherits a mutation becoming clinically affected is dependent on which parent has transmitted the mutation (19). Thus children who inherit a mutation from their father have a high risk of tumours but children who inherit the mutation from their mother are almost always unaffected. This parent of origin effect on disease expression is reminiscent of genomic imprinting, but the SDHD gene has not been demonstrated to be imprinted.

More than 200 different germline SDHB, SDHC, and SDHD mutations have been described (see the LOVD database (http://chromium.liacs.nl/LOVD2/SDH/home.php)). These mutations represent a wide variety of mutation types (e.g. missense, frameshift, splice-site and exonic deletions) and are loss of function mutations. As with any relatively recently described gene, the pathogenic significance of rare variants may be difficult to assess.

Tumours from individuals with SDHB/C/D subunit mutations demonstrate loss of the wild-type allele, as seen in VHL disease and other classic tumour suppressor genes. Several mechanisms have been implicated in the development of SDHB/C/D-related phaeochromocytomas. Thus inactivation of SDHB/D can result in a pseudohypoxic state (similar to that seen in VHL tumours) (29) and activation of HIF pathways with SDH inactivation has been linked to accumulation of succinate and resulting inhibition of prolyl hydroxylase enzymes that are necessary for proteosomal degradation of HIF-α subunits (Fig. 6.13.2) (30). Also, animal models of SDH inactivation suggest that reactive oxygen species may be increased and these might also provoke a pseudohypoxic state (31). As in VHL disease (see above), germline SDHB/D mutations have also been reported to predispose to a failure of normal developmental apoptosis of sympathetic neuronal cells, leading to persistence of ‘phaeochromocytoma precursor cells’ (17).

Unlike VHL disease, there is relatively little experience of the utility of surveillance in SDHB/C/D gene carriers. However, anecdotal evidence suggests that surveillance of asymptomatic gene carriers can lead to early tumour detection. As experience with surveillance programmes increases, a consensus should emerge as to the optimum methodologies and frequency of surveillance. However, currently no such consensus exists and the protocol proposed in Box 6.13.2 is provided as an example of a programme used in one centre.

Up to a third of patients with phaeochromocytoma will have an underlying genetic cause. In some cases this will have been suspected because of a family or personal history of other features of a known phaeochromocytoma susceptibility syndrome (e.g. VHL disease, multiple endocrine neoplasia type 2, neurofibromatosis type 1 (von Recklinghausen’s disease)). However genetic testing of apparently sporadic, nonsyndromic cases can reveal a germline mutation in 12–25% of cases (24, 26). Although this observation led to suggestions that all patients with phaeochromocytoma might be offered mutation analysis of RET, SDHB, SDHD, and VHL the detection rate for mutations in older patients with sporadic, nonsyndromic adrenal phaeochromocytomas is very low. Hence mutation analysis in sporadic patients with a single phaeochromocytoma should be prioritized for those with: (1) features of a known inherited phaeochromocytoma syndrome (e.g. RCC, HNPGL, medullary thyroid cancer, etc.); (2) malignant tumours (SDHB); (c) extra-adrenal phaeochromocytoma (SDHB, SDHD); or (3) age at diagnosis less than 40 years (VHL, SDHB, SDHD).