Thanh T. Huynh

A novel EPAS1/HIF2A germline mutation in a congenital polycythemia with paraganglioma

Congenital polycythemias have diverse etiologies, including mutations in the hypoxia sensing pathway. These include HIF2A at exon 12, VHL gene (Chuvash polycythemia), and PHD2 mutations, which in one family was also associated with recurrent pheochromocytoma/paraganglioma (PHEO/PGL). Over the past two decades, we have studied seven unrelated patients with sporadic congenital polycythemia who subsequently developed PHEO/PGL with, until now, no discernible molecular basis. We now report a polycythemic patient with a novel germline HIF2AF374Y (exon 9) mutation, inherited from his mother, who developed PHEO/PGL. We show that this is a gain-of-function mutation and demonstrate no loss-of-heterozygosity or additional somatic mutation of HIF2A in the tumor, indicating HIF2AF374Y may be predisposing rather than causative of PHEO/PGL. This report, in view of two other concomitantly reported PHEO/PGL patients with somatic mutations of HIF2A and polycythemia, underscores the PHEO/PGL-promoting potential of mutations of HIF2A that alone are not sufficient for PHEO/PGL development.

Biochemically silent abdominal paragangliomas in patients with mutations in the succinate dehydrogenase subunit B gene.

CONTEXT Patients with adrenal and extra-adrenal abdominal paraganglioma (PGL) almost invariably have increased plasma and urine concentrations of metanephrines, the O-methylated metabolites of catecholamines. We report four cases of biochemically silent abdominal PGL, in which metanephrines were normal despite extensive disease. OBJECTIVE Our objective was to identify the mechanism underlying the lack of catecholamine hypersecretion and metabolism to metanephrines in biochemically silent PGL. DESIGN This is a descriptive study. SETTING The study was performed at a referral center. PATIENTS One index case and three additional patients with large abdominal PGL and metastases but with the lack of evidence of catecholamine production, six patients with metastatic catecholamine-producing PGL and a mutation of the succinate dehydrogenase subunit B (SDHB) gene, and 136 random patients with catecholamine-producing PGL were included in the study. MAIN OUTCOME MEASURES Plasma, urine, and tumor tissue concentrations of catecholamines and metabolites were calculated with electron microscopy and tyrosine hydroxylase immunohistochemistry. RESULTS All four patients with biochemically silent PGL had an underlying SDHB mutation. In the index case, the tumor tissue concentration of catecholamines (1.8 nmol/g) was less than 0.01% that of the median (20,410 nmol/g) for the 136 patients with catecholamine-producing tumors. Electron microscopy showed the presence of normal secretory granules in all four biochemically silent PGLs. Tyrosine hydroxylase immunoreactivity was negligible in the four biochemically silent PGLs but abundant in catecholamine-producing PGLs. CONCLUSIONS Patients with SDHB mutations may present with biochemically silent abdominal PGLs due to defective catecholamine synthesis resulting from the absence of tyrosine hydroxylase. Screening for tumors in patients with SDHB mutations should not be limited to biochemical tests of catecholamine excess.

Novel HIF2A mutations disrupt oxygen sensing, leading to polycythemia, paragangliomas, and somatostatinomas

Hypoxia-inducible factors (HIFs) control the cellular response to hypoxia and, when dysregulated, contribute to tumorigenesis. Previously, we identified 2 gain-of-function somatic mutations in patients presenting with multiple paragangliomas or somatostatinomas, and polycythemia. Here, we report 2 additional unique HIF2A mutations, which disrupt the hydroxylation domain recognized by prolyl hydroxylase domain-2 containing protein, leading to stabilization of HIF-2α and increased expression of hypoxia-related genes.

Missense mutations in the human SDHB gene increase protein degradation without altering intrinsic enzymatic function

Mutations of succinate dehydrogenase subunit B (SDHB) play a crucial role in the pathogenesis of the most aggressive and metastatic pheochromocytomas (PHEOs) and paragangliomas (PGLs). Although a variety of missense mutations in the coding sequence of the SDHB gene have been found in PHEOs and PGLs, it has been unclear whether these mutations impair mRNA expression, protein stability, subcellular localization, or intrinsic protein function. RT‐PCR and Western blot analysis of SDHB mRNA and protein expression from SDHB‐related PHEOs and PGLs demonstrated intact mRNA expression but significantly reduced protein expression compared to non‐SDHB PHEOs and PGLs. A pulse‐chase assay of common SDHB missense mutations in transfected HeLa cell lines demonstrated that the loss of SDHB function was due to a reduction in mutant protein half‐life, whereas colocalization of SDHB with mitochondria and immunoprecipitation with SDHA demonstrated intact subcellular localization and complex formation. The half‐life of the SDHB protein increased after treatment with histone deacetylase inhibitors (HDACis), implicating the protein quality control machinery in the degradation of mutant SDHB protein. These findings provide the first direct mechanism of functional loss resulting from SDHB mutations and suggest that reducing protein degradation with HDACis may serve as a novel therapeutic paradigm for preventing the development of SDHB‐related tumors.—Yang, C., Matro, J. C., Huntoon, K. M., Ye, D. Y., Huynh, T. T., Fliedner, S. M. J., Breza, J., Zhuang, Z., Pacak, K. Missense mutations in the human SDHB gene increase protein degradation without altering intrinsic enzymatic function. FASEB J. 26, 4506–4516 (2012). www.fasebj.org

Cerebrospinal Fluid Catecholamine Levels as Predictors of Outcome in Subarachnoid Hemorrhage

Objective: Subarachnoid hemorrhage (SAH) is associated with marked sympathetic activation at the time of ictus. The purpose of this study is to determine whether early central catecholamine levels measured from cerebrospinal fluid (CSF) relate to outcome in patients with SAH. Methods: Observational study of consecutive SAH grade 3–5 patients who underwent ventriculostomy placement, but did not undergo open craniotomy for aneurysm obliteration. CSF samples were obtained during the first 48 h following symptom onset and assayed for catecholamine levels. Statistical analyses were performed to determine whether the levels predicted mortality by day 15 or mortality/disability by day 30. Results: For the 102 patients included, mean age was 58, and 73% were female – 21% experienced day-15 mortality, and 32% experienced mortality/disability by day 30. Early mortality was related to Hunt-Hess (H/H) grade (p < 0.001), neurogenic cardiomyopathy (NC) (p = 0.003), cerebral infarction (p = 0.001), elevated intracranial pressure (ICP) (p = 0.029), epinephrine (EPI) level (p = 0.002) and norepinephrine/3,4-dihydroxyphenylglycol (NE/DHPG) ratio (p = 0.003). Mortality/disability was related to H/H grade (p < 0.001), NC (p = 0.018), infarction (p < 0.001), elevated ICP (p = 0.002), EPI (p = 0.004) and NE/DHPG (p = 0.014). Logistic regression identified age [OR 1.09 (95% CI 1.01–1.17)], H/H grade [9.52 (1.19–77)], infarction [10.87 (1.22–100)], ICP elevation [32.26 (2–500)], EPI [1.06 (1.01–1.10)], and (inversely) DHPG [0.99 (0.99–1.00)] as independent predictors of early mortality. For mortality/disability, H/H grade [OR 21.74 (95% CI 5.62–83)], ICP elevation [18.52 (1.93–166)], and EPI [1.05 (1.02–1.09)] emerged as independent predictors. Proportional-hazards analysis revealed age [HR 1.041 (95% CI 1.003–1.08)], H/H grade [6.9 (1.54–31.25)], NC [4.31 (1.5–12.35)], and EPI [1.032 (1.009–1.054)] independently predicted early mortality. Conclusions: CSF catecholamine levels are elevated in SAH patients who experience early mortality or disability. EPI may potentially serve as useful index of outcome in this population of patients with SAH.

Succinate Dehydrogenase Gene Mutations are Strongly Associated with Paraganglioma of the Organ of Zuckerkandl

Organ of Zuckerkandl paragangliomas (PGLs) are rare neuroendocrine tumors that are derived from chromaffin cells located around the origin of the inferior mesenteric artery extending to the level of the aortic bifurcation. Mutations in the genes encoding succinate dehydrogenase subunits (SDH) B, C, and D (SDHx) have been associated with PGLs, but their contribution to PGLs of the organ of Zuckerkandl PGLs is not known. We aimed to describe the clinical presentation of patients with PGLs of the organ of Zuckerkandl and investigate the prevalence of SDHx mutations and other genetic defects among them. The clinical characteristics of 14 patients with PGL of the organ of Zuckerkandl were analyzed retrospectively; their DNA was tested for SDHx mutations and deletions. Eleven out of 14 (79%) patients with PGLs of the organ of Zuckerkandl were found to have mutations in the SDHB (9) or SDHD (2) genes; one patient was found to have the Carney-Stratakis syndrome (CSS), and his PGL was discovered during surgery for gastrointestinal stromal tumor. Our results show that SDHx mutations are prevalent in pediatric and adult PGLs of the organ of Zuckerkandl. Patients with PGLs of the organ of Zuckerkandl should be screened for SDHx mutations and the CSS; in addition, asymptomatic carriers of an SDHx mutation among the relatives of affected patients may benefit from tumor screening for early PGL detection.

Bilateral Adrenal Medullary Hyperplasia Associated With an SDHB Mutation

The patient is a 61-year-old white man that has a succinate dehydrogenase subunit B (SDHB) mutation who underwent bilateral laparoscopic adrenalectomy and was found to have adrenal medullary hyperplasia in both glands. He first presented with chest pain during exercise that prompted a cardiac work-up. Coronary artery catheterization showed a critical coronary artery stenosis that would require cardiac stent placement. Because of his SDHB mutation and a family history of four deaths secondary to pheochromocytoma or paraganglioma, an investigation was done to be sure the patient did not have those tumors before the cardiac stent placement. The patient’s father died at the age of 27 from cardiac complications caused by a pheochromocytoma that was found at autopsy. Two of his father’s brothers also died in their late 20s secondary to cardiac complications that were assumed to be related to paragangliomas or pheochromocytomas. Finally, the year before this patient was hospitalized, his nephew died at the age of 29 years from complications of metastatic paraganglioma. After the patient’s brother was found to have a pheochromocytoma, the family was tested for gene mutations that cause familial pheochromocytoma or paraganglioma including the von Hippel-Lindau gene, the RET proto-oncogene (for multiple endocrine neoplasia type 2), the neurofibromatosis I gene, and the SDHB and SDHD genes. The patient and his family were found to have an alanine to guanine germline mutation (c.587G A, ex6: p.Cys196Tyr) in the SDHB gene. After the cardiac catheterization, an extensive pheochromocytoma/paraganglioma work-up was done that showed conflicting results. The patient remained in the hospital during the work-up because of continual episodes of paroxysmal hypertension resulting in systolic blood pressures greater than 200 mmHg. A 24-hour urine collection was negative for increased catecholamines, metanephrines, or vanillylmandelic acid. At the same time plasma-free metanephrines were also drawn while the patient was normotensive and were negative. However, a plasma-free metanephrine level of 1.76 nmol/L (normal, 0.9 nmol/L) was obtained when the labs were drawn during an episode of paroxysmal hypertension. A magnetic resonance imaging did not show any abnormal masses suggestive of pheochromocytoma or paraganglioma, but an iodine-123–metaiodobenzylguanidine single photon emission computed tomography did show enlarged adrenal glands and bilateral, abnormally intense focal uptake (Figs 1A and 1B, arrows). The diagnosis was not straightforward. Because of the patient’s personal history of an SDHB mutation, the family’s history

Association between acute sympathetic response, early onset vasospasm, and delayed vasospasm following spontaneous subarachnoid hemorrhage

Subarachnoid hemorrhage (SAH) is accompanied by a marked acute sympathetic response, and evidence exists for sympathetic participation in the development of cerebral vasospasm (VS). The purpose of this observational investigation was to assess the association between acute central catecholaminergic activity, early VS and delayed VS following SAH. SAH grade 3-5 patients who received ventriculostomy, and in whom bilateral temporal transcranial insonation was performed, were enrolled. Cerebrospinal fluid (CSF) was sampled (<48 hours) and assayed for catecholamines, which were correlated to measures of early and delayed sonographic anterior circulation VS. Clinical independent predictors of early VS included age (odds ratio .946 [95% confidence interval .902-.991]), CT scan score (4.27 [1.30-14.0]) and neurogenic cardiomyopathy (6.5 [1.24-34.1]). Age (.925 [.859-.996]) and CT scan score (8.30 [1.33-5.17]) also independently predicted delayed VS. Any early VS independently predicted conventionally defined delayed VS (10.9 [2.64-45.0]), and severe delayed VS was independently predicted by any early VS (9.87 [2.45-39.7]) and by conventionally defined early VS (12.3 [2.80-54.1]). The norepinephrine:3,4-dihydroxyphenylglycol ratio (NE/DHPG) independently predicted severe delayed VS (3.38 [1.01-11.35]), for which DHPG was a negative predictor (.356 [.151-.839]). Epinephrine was a negative predictor of any early VS (.574 [.357-.921]), any delayed VS (.372 [.158-.875]), and delayed conventional VS (.402 [.200-.807]). Early and delayed VS appear to be related processes that are generally unrelated to the acute central sympathetic response following SAH. The one exception may be severe delayed VS which may be associated with noradrenergic activation.