Charalampos Lyssikatos

Succinate Dehydrogenase (SDH) D Subunit (SDHD) Inactivation in a Growth-Hormone-Producing Pituitary Tumor: A New Association for SDH?

BACKGROUNDnMutations in the subunits B, C, and D of succinate dehydrogenase (SDH) mitochondrial complex II have been associated with the development of paragangliomas (PGL), gastrointestinal stromal tumors, papillary thyroid and renal carcinoma (SDHB), and testicular seminoma (SDHD).nnnAIMnOur aim was to examine the possible causative link between SDHD inactivation and somatotropinoma.nnnPATIENTS AND METHODSnA 37-yr-old male presented with acromegaly and hypertension. Other family members were found with PGL. Elevated plasma and urinary levels of catecholamines led to the identification of multiple PGL in the proband in the neck, thorax, and abdomen. Adrenalectomy was performed for bilateral pheochromocytomas (PHEO). A GH-secreting macroadenoma was also found and partially removed via transsphenoidal surgery (TTS). Genetic analysis revealed a novel SDHD mutation (c.298_301delACTC), leading to a frame shift and a premature stop codon at position 133 of the protein. Loss of heterozygosity for the SDHD genetic locus was shown in the GH-secreting adenoma. Down-regulation of SDHD protein in the GH-secreting adenoma by immunoblotting and immunohistochemistry was found. A literature search identified other cases of multiple PGL and/or PHEO in association with pituitary tumors.nnnCONCLUSIONnWe describe the first kindred with a germline SDHD pathogenic mutation, inherited PGL, and acromegaly due to a GH-producing pituitary adenoma. SDHD loss of heterozygosity, down-regulation of protein in the GH-secreting adenoma, and decreased SDH enzymatic activity supports SDHDs involvement in the pituitary tumor formation in this patient. Older cases of multiple PGL and PHEO and pituitary tumors in the literature support a possible association between SDH defects and pituitary tumorigenesis.

Pituitary Adenoma With Paraganglioma/Pheochromocytoma (3PAs) and Succinate Dehydrogenase Defects in Humans and Mice

CONTEXTnGermline mutations in genes coding succinate dehydrogenase (SDH) subunits A, B, C, and D have been identified in familial paragangliomas (PGLs)/pheochromocytomas (PHEOs) and other tumors. We described a GH-secreting pituitary adenoma (PA) caused by SDHD mutation in a patient with familial PGLs. Additional patients with PAs and SDHx defects have since been reported.nnnDESIGNnWe studied 168 patients with unselected sporadic PA and with the association of PAs, PGLs, and/or pheochromocytomas, a condition we named the 3P association (3PAs) for SDHx germline mutations. We also studied the pituitary gland and hormonal profile of Sdhb(+/-) mice and their wild-type littermates at different ages.nnnRESULTSnNo SDHx mutations were detected among sporadic PA, whereas three of four familial cases were positive for a mutation (75%). Most of the SDHx-deficient PAs were either prolactinomas or somatotropinomas. Pituitaries of Sdhb(+/-) mice older than 12 months had an increased number mainly of prolactin-secreting cells and several ultrastructural abnormalities such as intranuclear inclusions, altered chromatin nuclear pattern, and abnormal mitochondria. Igf-1 levels of mutant mice tended to be higher across age groups, whereas Prl and Gh levels varied according to age and sex.nnnCONCLUSIONnThe present study confirms the existence of a new association that we termed 3PAs. It is due mostly to germline SDHx defects, although sporadic cases of 3PAs without SDHx defects also exist. Using Sdhb(+/-) mice, we provide evidence that pituitary hyperplasia in SDHx-deficient cells may be the initial abnormality in the cascade of events leading to PA formation.

Deletions of the PRKAR1A Locus at 17q24.2-q24.3 in Carney Complex: Genotype-Phenotype Correlations and Implications for Genetic Testing

BACKGROUNDnCarney complex (CNC) is a multiple neoplasia syndrome caused by PRKAR1A-inactivating mutations. One-third of the patients, however, have no detectable PRKAR1A coding sequence defects. Small deletions of the gene were previously reported in few patients, but large deletions of the chromosomal PRKAR1A locus have not been studied systematically in a large cohort of patients with CNC.nnnSETTINGnA tertiary care referral center was the setting for analysis of an international cohort of patients with CNC.nnnMETHODSnMethods included genome-wide array analysis followed by fluorescent in situ hybridization, mRNA, and other studies as well as a retrospective analysis of clinical information and phenotype-genotype correlation.nnnRESULTSnWe detected 17q24.2-q24.3 deletions of varying size that included the PRKAR1A gene in 11 CNC patients (of 51 tested). Quantitative PCR showed that these patients had significantly lower PRKAR1A mRNA levels. Phenotype varied but was generally severe and included manifestations that are not commonly associated with CNC, presumably due to haploinsufficiency of other genes in addition to PRKAR1A.nnnCONCLUSIONSnA significant number (21.6%) of patients with CNC that are negative in currently available testing may have PRKAR1A haploinsufficiency due to genomic defects that are not detected by Sanger sequencing. Array-based studies are necessary for diagnostic confirmation of these defects and should be done in patients with unusual and severe phenotypes who are PRKAR1A mutation-negative.

Germline PRKACA amplification leads to Cushing syndrome caused by 3 adrenocortical pathologic phenotypes

We describe the pathology of 5 patients with germline PRKACA copy number gain and Cushing syndrome: 4 males and 1 female, aged 2 to 43 years, including a mother and son. Imaging showed normal or slightly enlarged adrenal glands in 4 patients and a unilateral mass in the fifth. Biochemically, the patients had corticotropin-independent hypercortisolism. Four underwent bilateral adrenalectomy; unilateral adrenalectomy was performed in the patient with the adrenal mass. Pathologically, 3 patients, including the 1 with the tumor (adenoma), had primary pigmented nodular adrenocortical disease with extranodular cortical atrophy and mild intracapsular and extracapsular extension of cortical cells. The other 2 patients had cortical hyperplasia and prominent capsular and extracapsular micronodular cortical hyperplasia. Immunoperoxidase staining revealed differences for synaptophysin, inhibin-A, and Ki-67 (nuclei) in the atrophic cortices (patients 1, 2, and 3) and hyperplastic cortices (patients 4 and 5) and for Ki-67 (nuclei) and vimentin in the extracortical nodules in the 2 groups of patients. β-Catenin stained the cell membrane, cytoplasm, and nuclei of the adenoma. The patients were well at follow-up (1-23 years); 24-hour urinary cortisol excretion was elevated in the patient who had unilateral adrenalectomy.

A Large Family with Carney Complex Caused by the S147G PRKAR1A Mutation Shows a Unique Spectrum of Disease Including Adrenocortical Cancer

CONTEXTnMost tumors in Carney complex (CNC) are benign, including primary pigmented nodular adrenocortical disease (PPNAD), the main endocrine tumor in CNC. Adrenocortical cancer (AC) has never been observed in the syndrome. Herein, we describe a large Azorean family with CNC caused by a point mutation in the PRKAR1A gene coding for type 1-α (RIα) regulatory subunit of the cAMP-dependent protein kinase A, in which the index patient presented with AC.nnnOBJECTIVEnWe studied the genotype-phenotype correlation in CNC.nnnDESIGN AND SETTINGnWe reported on case series and in vitro testing of the PRKAR1A mutation in a tertiary care referral center.nnnPATIENTSnTwenty-two members of a family were investigated for Cushing syndrome and other CNC components; their DNA was sequenced for PRKAR1A mutations.nnnRESULTSnCushing syndrome due to PPNAD occurred in four patients, including the proposita who presented with AC and three who had Cushing syndrome and/or PPNAD. Lentigines were found in six additional patients who did not have PPNAD. A base substitution (c.439A>G/p.S147G) in PRKAR1A was identified in the proposita, in the three others with PPNAD, in the propositas twin daughters who had lentigines but no evidence of hypercortisolism, and in five other family members, including one without lentigines or evidence of hypercortisolism. Unlike in other RIα defects, loss of heterozygosity was not observed in AC. The S147G mutation was compared to other expressed PRKAR1A mutations; it led to decreased cAMP and catalytic subunit binding by RIα and increased protein kinase A activity in vitro.nnnCONCLUSIONSnIn a large family with CNC, one amino acid substitution caused a spectrum of adrenal disease that ranged from lack of manifestations to cancer. PPNAD and AC were the only manifestations of CNC in these patients, in addition to lentigines. These data have implications for counseling patients with CNC and are significant in documenting the first case of AC in the context of PPNAD.

Germline PRKACA amplification causes variable phenotypes that may depend on the extent of the genomic defect: molecular mechanisms and clinical presentations

OBJECTIVEnWe have recently reported five patients with bilateral adrenocortical hyperplasia (BAH) and Cushings syndrome (CS) caused by constitutive activation of the catalytic subunit of protein kinase A (PRKACA). By doing new in-depth analysis of their cytogenetic abnormality, we attempted a better genotype-phenotype correlation of their PRKACA amplification.nnnDESIGNnThis study is a case series.nnnMETHODSnMolecular cytogenetic, genomic, clinical, and histopathological analyses were performed in five patients with CS.nnnRESULTSnReinvestigation of the defects of previously described patients by state-of-the-art molecular cytogenetics showed complex genomic rearrangements in the chromosome 19p13.2p13.12 locus, resulting in copy number gains encompassing the entire PRKACA gene; three patients (one sporadic case and two related cases) were observed with gains consistent with duplications, while two sporadic patients were observed with gains consistent with triplications. Although all five patients presented with ACTH-independent CS, the three sporadic patients had micronodular BAH and underwent bilateral adrenalectomy in early childhood, whereas the two related patients, a mother and a son, presented with macronodular BAH as adults. In at least one patient, PRKACA triplication was associated with a more severe phenotype.nnnCONCLUSIONSnConstitutional chromosomal PRKACA gene amplification is a recently identified genetic defect associated with CS, a trait that may be inherited in an autosomal dominant manner or occur de novo. Genomic rearrangements can be complex and can result in different copy number states of dosage-sensitive genes, e.g., duplication and triplication. PRKACA amplification can lead to variable phenotypes clinically and pathologically, both micro- and macro-nodular BAH, the latter of which we speculate may depend on the extent of amplification.

Ectopic Adrenocorticotropic Hormone and Corticotropin-Releasing Hormone Co-Secreting Tumors in Children and Adolescents Causing Cushing Syndrome: A Diagnostic Dilemma and How to Solve It

CONTEXTnEctopic ACTH/CRH syndrome is a rare cause of Cushing syndrome (CS), especially in children. The localization, work-up, and management of ACTH/CRH-secreting tumors are discussed.nnnSETTINGnA retrospective study was conducted of patients under 21 years of age evaluated at the National Institutes of Health (NIH) for CS and diagnosed with ectopic ACTH/CRH-secreting tumors during the period 2009-2014.nnnPATIENTSnSeven patients with ectopic ACTH/CRH CS are included in this study with a median age 13.6 years (range 1-21), and 3 are female.nnnMEASUREMENTSnClinical, biochemical, radiological features, treatment, and histological findings are described.nnnRESULTSnSeven patients were found to have ACTH/CRH-secreting tumors, all with neuroendocrine features. The site of the primary lesion varied: pancreas (3), thymus (2), liver (1), right lower pulmonary lobe (1). PATIENTS underwent biochemical evaluation for CS, including diurnal serum cortisol and ACTH levels, urinary free cortisol levels (UFC), and CRH stimulation tests. All patients underwent radiological investigations including MRI, CT, and PET scan; imaging with octreotide and 68 gallium DOTATATE scans were performed in individual cases. Five patients underwent inferior petrosal sinus sampling; 4 patients had sampling for ACTH and CRH levels from additional sites. Three patients underwent trans-sphenoidal surgery (TSS), and 3 patients required bilateral adrenalectomy. Three patients (43%) died due to metastatic disease, demonstrating the high mortality rate. One of the unique findings in these seven patients is that in each case, their neuroendocrine tumors were ultimately proven to be co-secreting ACTH and CRH. This explains the enigmatic presentation, in which 3 patients initially thought to have Cushings disease (CD) with corresponding pituitary hyperplasia underwent TSS prior to the correct localization of the causative tumor.nnnCONCLUSIONSnEctopic ACTH/CRH co-secreting tumors are extremely rare in children and adolescents. The diagnosis of this condition is frequently missed and is sometimes confused with CD due to the effect of CRH on the pituitary.

Familial pituitary apoplexy as the only presentation of a novel AIP mutation

Growth hormone (GH)-producing tumors in AIP mutation-positive patients are typically more aggressive than their sporadic counterparts. They are also frequently associated with onset in childhood and adolescence and appear to be less responsive to medical therapy (Beckers et al. 2013). Amale predominance has been observed (Daly et al. 2010). We report on a unique clinical presentation of a kindred with a novel AIPmutation: only one of the three affected members had all the typical signs and symptoms of patients with an AIP mutation. On the contrary, the carrier father and brother had both partially empty sella turcica and, in fact, the latter ended being treated for anterior pituitary lobe hormonal deficiencies, including GH. This was presumably the result of pituitary apoplexy due to a rapidly growing AIP-induced adenoma. Briefly, a 12 years 2 month-old boy was seen for tall stature and accelerated growth velocity that was first noticed at the age of 4 years. At initial presentation, his height was 186.6 cm (z-score C4.72), over the calculated mid-parental height (180.5G4.5 cm), and his weight was 78.7 kg (z-score C2.59). He was also macrocephalic and he had several facial characteristics typical of gigantism. Hormonal investigation showed increased IGF1: 906 ng/ml (nv: 183–850) and GH: 18.3 ng/ml that did not suppress to oral glucose tolerance test (OGTT). The rest of pituitary hormones were in the lower normal range (Supplementary Table 1, see section on supplementary data given at the end of this article). Pituitary MRI showed the presence of a 1.3!1.2!1.1 cm pituitary adenoma with suprasellar extension (Fig. 1A). He underwent partial resection of the pituitary adenoma. The surgical pathology evaluation confirmed the presence of a GH-positive, sparsely granulated tumor with globules of keratin (fibrous bodies) and lack of normal reticulin framework (Fig. 2A, B, C, and D). Areas of expanded acini clearly distinctive from the adenoma were detected suggestive of hyperplasia (Fig. 2E and F). Both parents had normal heights and no history of acromegaly.

Differences in Adiposity in Cushing Syndrome Caused by PRKAR1A Mutations: Clues for the Role of Cyclic AMP Signaling in Obesity and Diagnostic Implications

CONTEXTnThe cAMP signaling pathway is implicated in bilateral adrenocortical hyperplasias. Bilateral adrenocortical hyperplasia is often associated with ACTH-independent Cushing syndrome (CS) and may be caused by mutations in genes such as PRKAR1A, which is responsible for primary pigmented nodular adrenocortical disease (PPNAD). PRKAR1A regulates cAMP-dependent protein kinase (PKA), an essential enzyme in the regulation of adiposity. Although CS is invariably associated with obesity, its different forms, including those associated with PKA defects, have not been compared.nnnOBJECTIVEnThe purpose of this study was to characterize the phenotypic and molecular differences in periadrenal adipose tissue (PAT) between patients with CS with and without PRKAR1A mutations.nnnDESIGN AND SETTINGnSamples from adrenalectomies of 51 patients were studied: patients with CS with (n = 13) and without (n = 32) PRKAR1A mutations and a comparison group with aldosterone-producing adenomas (APAs) (n = 6). In addition, clinical data from a larger group of patients with Cushing disease (n = 89) and hyperaldosteronism (n = 26) were used for comparison.nnnMETHODSnBody mass index (BMI), abdominal computed tomography scans, and cortisol data were collected preoperatively. PAT was assayed for PKA activity, cAMP levels, and PKA subunit expression.nnnRESULTSnBMI was lower in adult patients with CS with PRKAR1A mutations. cAMP and active PKA levels in PAT were elevated in patients with CS with PRKAR1A mutations.nnnCONCLUSIONSnIncreased PKA signaling in PAT was associated with lower BMI in CS. Differences in fat distribution may contribute to phenotypic differences between patients with CS with and without PRKAR1A mutations. The observed differences are in agreement with the known roles of cAMP signaling in regulating adiposity, but this is the first time that germline defects of PKA are linked to variable obesity phenotypes in humans.

Death in pediatric Cushing syndrome is uncommon but still occurs

AbstractCushing syndrome (CS) in children is rare. Delayed diagnosis and treatment of CS may be associated with increased morbidity and, unfortunately, mortality. We performed a retrospective review of all patients with CS under the age of 18xa0years referred to the National Institutes of Health (NIH) from 1998 to 2013 in order to describe deceased patients among cases of pediatric CS referred to the National Institutes of Health (NIH). The deaths of four children (three females and one male), aged 7.5–15.5xa0years (mean age 11.2xa0years) with length of disease 2–4xa0years, were recorded among 160 (2.5xa0%) children seen at or referred to the NIH over the last 15xa0years. All died at different institutions, prior to coming to the NIH (two) or after leaving NIH (two). Presenting symptoms included increasing weight and decreasing height gain, facial plethora, dorsocervical fat pad (webbed neck), striae, headache, vision disturbances, and depression and other mood or behavior changes; there were no differences between how these patients presented and the others in our cohort. The causes of CS in the deceased patients were also not different, in fact, they spanned the entire spectrum of CS: pituitary disease (one), ectopic corticotropin production (one), and primary adrenal hyperplasia (one). In one patient, the cause of CS could not be verified. Three died of sepsis and one due to residual disease and complications of the primary tumor.n Conclusions: Despite the advances in early diagnosis and treatment of pediatric CS, a 2.5xa0% mortality rate was identified in a large cohort of patients with this condition referred to an experienced, tertiary care referral center (although these deaths occurred elsewhere). Pediatricians need to recognize the possibility of death, primarily due to sepsis, in a patient with pediatric CS and treat accordingly.