Umasuthan Srirangalingam

Tumor Risks and Genotype-Phenotype-Proteotype Analysis in 358 Patients With Germline Mutations in SDHB and SDHD

Succinate dehydrogenase B (SDHB) and D (SDHD) subunit gene mutations predispose to adrenal and extraadrenal pheochromocytomas, head and neck paragangliomas (HNPGL), and other tumor types. We report tumor risks in 358 patients with SDHB (n=295) and SDHD (n=63) mutations. Risks of HNPGL and pheochromocytoma in SDHB mutation carriers were 29% and 52%, respectively, at age 60 years and 71% and 29%, respectively, in SDHD mutation carriers. Risks of malignant pheochromocytoma and renal tumors (14% at age 70 years) were higher in SDHB mutation carriers; 55 different mutations (including a novel recurrent exon 1 deletion) were identified. No clear genotype–phenotype correlations were detected for SDHB mutations. However, SDHD mutations predicted to result in loss of expression or a truncated or unstable protein were associated with a significantly increased risk of pheochromocytoma compared to missense mutations that were not predicted to impair protein stability (most such cases had the common p.Pro81Leu mutation). Analysis of the largest cohort of SDHB/D mutation carriers has enhanced estimates of penetrance and tumor risk and supports in silicon protein structure prediction analysis for functional assessment of mutations. The differing effect of the SDHD p.Pro81Leu on HNPGL and pheochromocytoma risks suggests differing mechanisms of tumorigenesis in SDH‐associated HNPGL and pheochromocytoma. Hum Mutat 31:41–51, 2010.

Characterization of aryl hydrocarbon receptor interacting protein (AIP) mutations in familial isolated pituitary adenoma families

Familial isolated pituitary adenoma (FIPA) is an autosomal dominant condition with variable genetic background and incomplete penetrance. Germline mutations of the aryl hydrocarbon receptor interacting protein (AIP) gene have been reported in 15–40% of FIPA patients. Limited data are available on the functional consequences of the mutations or regarding the regulation of the AIP gene. We describe a large cohort of FIPA families and characterize missense and silent mutations using minigene constructs, luciferase and β‐galactosidase assays, as well as in silico predictions. Patients with AIP mutations had a lower mean age at diagnosis (23.6±11.2 years) than AIP mutation‐negative patients (40.4±14.5 years). A promoter mutation showed reduced in vitro activity corresponding to lower mRNA expression in patient samples. Stimulation of the protein kinase A‐pathway positively regulates the AIP promoter. Silent mutations led to abnormal splicing resulting in truncated protein or reduced AIP expression. A two‐hybrid assay of protein–protein interaction of all missense variants showed variable disruption of AIP‐phosphodiesterase‐4A5 binding. In summary, exonic, promoter, splice‐site, and large deletion mutations in AIP are implicated in 31% of families in our FIPA cohort. Functional characterization of AIP changes is important to identify the functional impact of gene sequence variants. Hum Mutat 31:1–11, 2010.

Heterogeneous Genetic Background of the Association of Pheochromocytoma/Paraganglioma and Pituitary Adenoma: Results From a Large Patient Cohort

Context: Pituitary adenomas and pheochromocytomas/paragangliomas (pheo/PGL) can occur in the same patient or in the same family. Coexistence of the two diseases could be due to either a common pathogenic mechanism or a coincidence. Objective: The objective of the investigation was to study the possible coexistence of pituitary adenoma and pheo/PGL. Design: Thirty-nine cases of sporadic or familial pheo/PGL and pituitary adenomas were investigated. Known pheo/PGL genes (SDHA-D, SDHAF2, RET, VHL, TMEM127, MAX, FH) and pituitary adenoma genes (MEN1, AIP, CDKN1B) were sequenced using next generation or Sanger sequencing. Loss of heterozygosity study and pathological studies were performed on the available tumor samples. Setting: The study was conducted at university hospitals. Patients: Thirty-nine patients with sporadic of familial pituitary adenoma and pheo/PGL participated in the study. Outcome: Outcomes included genetic screening and clinical characteristics. Results: Eleven germline mutations (five SDHB, one SDHC, one SDHD, two VHL, and two MEN1) and four variants of unknown significance (two SDHA, one SDHB, and one SDHAF2) were identified in the studied genes in our patient cohort. Tumor tissue analysis identified LOH at the SDHB locus in three pituitary adenomas and loss of heterozygosity at the MEN1 locus in two pheochromocytomas. All the pituitary adenomas of patients affected by SDHX alterations have a unique histological feature not previously described in this context. Conclusions: Mutations in the genes known to cause pheo/PGL can rarely be associated with pituitary adenomas, whereas mutation in a gene predisposing to pituitary adenomas (MEN1) can be associated with pheo/PGL. Our findings suggest that genetic testing should be considered in all patients or families with the constellation of pheo/PGL and a pituitary adenoma.

Clinical manifestations of familial paraganglioma and phaeochromocytomas in succinate dehydrogenase B (SDH-B) gene mutation carriers

Objective  Phaeochromocytomas and paragangliomas are familial in up to 25% of cases and can result from succinate dehydrogenase (SDH) gene mutations. The aim of this study was to describe the clinical manifestations of subjects with SDH‐B gene mutations.

Evaluation of SDHB, SDHD and VHL gene susceptibility testing in the assessment of individuals with non‐syndromic phaeochromocytoma, paraganglioma and head and neck paraganglioma

Research studies have reported that about a third of individuals with phaeochromocytoma/paraganglioma (PPGL) have an inherited predisposition, although the frequency of specific mutations can vary between populations. We evaluated VHL, SDHB and SDHD mutation testing in cohorts of patients with non‐syndromic PPGL and head and neck paraganglioma (HNPGL).

Contrasting clinical manifestations of SDHB and VHL associated chromaffin tumours

Mutations in succinate dehydrogense-B (SDHB) and the von Hippel-Lindau (VHL) genes result in an increased risk of developing chromaffin tumours via a common aetiological pathway. The aim of the present retrospective study was to compare the clinical phenotypes of disease in subjects developing chromaffin tumours as a result of SDHB mutations or VHL disease. Thirty-one subjects with chromaffin tumours were assessed; 16 subjects had SDHB gene mutations and 15 subjects had a diagnosis of VHL. VHL-related tumours were predominantly adrenal phaeochromocytomas (22/26; 84.6%), while SDHB-related tumours were predominantly extra-adrenal paragangliomas (19/25; 76%). Median age at onset of the first chromaffin tumour was similar in the two cohorts. Tumour size was significantly larger in the SDHB cohort in comparison with the VHL cohort (P=0.002). Multifocal disease was present in 9/15 (60%) of the VHL cohort (bilateral phaeochromocytomas) and only 3/16 (19%) of the SDHB cohort, while metastatic disease was found in 5/16 (31%) of the SDHB cohort but not in the VHL cohort to date. The frequency of symptoms, hypertension and the magnitude of catecholamine secretion appeared to be greater in the SDHB cohort. Renal cell carcinomas were a feature in 5/15 (33%) of the VHL cohort and 1/16 (6%) of the SDHB cohort. These data indicate that SDHB-related tumours are predominantly extra-adrenal in location and associated with higher catecholamine secretion and more malignant disease, in subjects who appear more symptomatic. VHL-related tumours tend to be adrenal phaeochromocytomas, frequently bilateral and associated with a milder phenotype.

Outcomes of annual surveillance imaging in an adult and paediatric cohort of succinate dehydrogenase B mutation carriers.

For ‘asymptomatic carriers’ of the succinate dehydrogenase subunit B (SDHB) gene mutations, there is currently no consensus as to the appropriate modality or frequency of surveillance imaging. We present the results of a surveillance programme of SDHB mutation carriers.

SDHA mutated paragangliomas may be at high risk of metastasis

We report the clinical outcomes of eleven patients with succinate dehydrogenase subunit A (SDHA) germline mutations from three UK tertiary referral centres to highlight a more diverse and expanding clinical spectrum of associated phenotypes. We suggest that SDHA paraganglioma-related disease is not a low-risk condition as first described. Of our six index cases, two developed metastatic disease and a further one had local vascular invasion. One patient developed multiple metachronous disease. Therefore, we believe these patients, like those with SDHB and SDHD mutations, should be part of a surveillance programme. Paraganglioma (PGL)-associated mutations in SDHA have only been reported in a small number of patients worldwide. There is controversy over the necessity for surveillance screening in these patients, compared to SDHB and SDHD, as penetrance is thought to be lower (Benn et al. 2015) and variants exist with uncertain pathogenicity. Initial reports associated SDHA with autosomal recessive causes of juvenile encephalopathy (Leigh syndrome) (Bourgeron et al. 1995) and homozygous mutations in SDHA cause severe neurological dysfunction and cardiomyopathy (Renkema et al. 2015). SDHA mutations have now been associated with phaeochromocytoma and paraganglioma (PPGL) formation in an autosomal dominant manner. SDHA mutations account for only 3% of cases of familial PGL cases, with presumed low penetrance (Korpershoek et al. 2011) and therefore very little data on clinical features of SDHA-related PPGL exist. Six index cases were originally diagnosed between 1973 and 2011 and had histologically proven PPGL, who subsequently underwent genetic testing during the course of their follow-up and were confirmed to have an underlying SDHA germline mutation. We performed a retrospective analysis of their notes and describe their clinical outcomes. From these six index cases, cascade genetic testing occurred and identified five further asymptomatic carriers of SDHA mutations. All patients are now being followed up in specialised endocrine clinics and are undergoing annual screening, including annual clinical and biochemical assessment and cross-sectional imaging, although the frequency and modality of imaging differs between centres. To predict the pathogenicity of the DNA variants, the missense variants were investigated in silico using PloyPhen2 and SIFT. Table 1 provides a detailed summary of the patients described. The six index patients originally presented were aged 18, 34, 36, 46, 47 and 68 years. Five patients presented with a single lesion at diagnosis: intrathyroidal PGL, mediastinal PGL, phaeochromocytoma and two extraadrenal PGLs. One patient (patient 3) presented with two synchronous lesions: she had a 3-methoxytyramine (3MT)-secreting carotid body tumour and a noradrenalinesecreting thoracic PGL. All patients underwent surgical resection of the primary tumours. Two patients developed recurrence in the surgical bed and both patients went on to develop metastatic disease 16 and 37 years later (patients 8 and 9). One of these two patients (patient 8) also developed an additional five metachronous lesions 7–10 years after original diagnosis. These two cases are described in more detail. Patient 8 presented aged 46 years with headaches and malignant hypertension (210/130 mmHg). Urinary noradrenaline was very raised (Table 1) and imaging confirmed a 5 cm para-adrenal PGL, which was subsequently resected. He developed a symptomatic recurrence one year later, which was surgically resected. Eight years after his original diagnosis, he presented with symptoms of catecholamine excess and four new lesions were identified and resected. On surveillance imaging three years later, a new non-secretory lesion was identified. Surgical resection was undertaken one year subsequently due to increasing PGL size and plasma catecholamine levels. He remained well with no evidence of further disease on imaging until five years later when rising noradrenaline levels were noted and uptake in the left adrenal bed and in the vertebral body of L4 was

Modeling Congenital Adrenal Hyperplasia and Testing Interventions for Adrenal Insufficiency Using Donor-Specific Reprogrammed Cells

Summary Adrenal insufficiency is managed by hormone replacement therapy, which is far from optimal; the ability to generate functional steroidogenic cells would offer a unique opportunity for a curative approach to restoring the complex feedback regulation of the hypothalamic-pituitary-adrenal axis. Here, we generated human induced steroidogenic cells (hiSCs) from fibroblasts, blood-, and urine-derived cells through forced expression of steroidogenic factor-1 and activation of the PKA and LHRH pathways. hiSCs had ultrastructural features resembling steroid-secreting cells, expressed steroidogenic enzymes, and secreted steroid hormones in response to stimuli. hiSCs were viable when transplanted into the mouse kidney capsule and intra-adrenal. Importantly, the hypocortisolism of hiSCs derived from patients with adrenal insufficiency due to congenital adrenal hyperplasia was rescued by expressing the wild-type version of the defective disease-causing enzymes. Our study provides an effective tool with many potential applications for studying adrenal pathobiology in a personalized manner and opens venues for the development of precision therapies.

Four generations of SDHB-related disease: complexities in management

SDHB mutations are linked to the familial paraganglioma syndrome type 4 (PGL4), which is associated with predominantly extra-adrenal disease and has high metastatic rates. Despite the lower penetrance rates in carriers of SDHB mutations compared to mutations in other paraganglioma susceptibility genes, the aggressive behavior of SDHB-linked disease warrants intensive surveillance to identify and resect tumors early. Patients with similar SDHB genotypes in whom the PGL syndrome manifests often exhibit very heterogeneous phenotypes. Tumors can arise in various locations, and management can be considerably different, depending on tumor site and pathology. We present a case series of five SDHB mutation carriers over four generations from the same family to illustrate the complexities in management.