Evgenia Gourgari

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

BACKGROUND Carney 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. SETTING A tertiary care referral center was the setting for analysis of an international cohort of patients with CNC. METHODS Methods 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. RESULTS We 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. CONCLUSIONS A 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.

Large-cell calcifying Sertoli cell tumors of the testes in pediatrics.

Purpose of review The aim of this review is to describe the clinical, biochemical, radiographic, histological, and functional characteristics of large-cell calcifying Sertoli cell tumors of the testes (LCCSCTs). We describe the two main syndromes associated with these tumors: Peutz–Jeghers syndrome (PJS) caused mainly by mutations in the STK11 (aka LKB1) gene, which encodes a serine–threonine kinase, and Carney complex (CNC), which is most often caused by PRKAR1A mutations, the gene encoding regulatory subunit type 1 of protein kinase A. Recent findings Relatively few patients have been reported in the literature with LCCSCTs. In children they often present as prepubertal and/or peripubertal gynecomastia. Although these tumors are very rare, they occur with higher frequency among patients with PJS and CNC. Orchiectomy was often performed in the past; however, these tumors are overwhelmingly benign and, unless there are significant hormonal changes or pain and/or mass effects, there is no need for surgery. Tumors that lead to hyperestrogenemia may be treated efficiently with aromatase inhibitors; any change in appearance should prompt evaluation for malignancy. Summary The detection of LCCSCTs may point to an underlying genetic multiple neoplasia syndrome such as PJS or CNC. Surgery is rarely indicated and aromatase inhibitors constitute an effective treatment for those cases that are associated with gynecomastia and/or advanced skeletal age.

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

CONTEXT The 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. OBJECTIVE The 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. DESIGN AND SETTING Samples 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. METHODS Body 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. RESULTS BMI 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. CONCLUSIONS Increased 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.

Cyclic AMP and c-KIT Signaling in Familial Testicular Germ Cell Tumor Predisposition

BACKGROUND Familial testicular germ cell tumors (FTGCTs) are hypothesized to result from the combined interaction of multiple low-penetrance genes. We reported inactivating germline mutations of the cAMP-binding phosphodiesterase 11A (PDE11A) as modifiers of FTGCT risk. Recent genome-wide association studies have identified single-nucleotide polymorphisms in the KITLG gene, the ligand for the cKIT tyrosine kinase receptor, as strong modifiers of susceptibility to both familial and sporadic testicular germ cell tumors. DESIGN We studied 94 patients with FTGCTs and 50 at-risk male relatives from 63 unrelated kindreds, in whom the PDE11A gene had been sequenced by investigating the association between KITLG genome-wide association study single-nucleotide polymorphisms rs3782179 and rs4474514 and FTGCT risk in these patients and in 692 controls. We also examined cAMP and c-KIT signaling in testicular tissues and cell lines and extended the studies to 2 sporadic cases, one with a PDE11A defect and one without, as a comparison. RESULTS We found a higher frequency of the KITLG risk alleles in FTGCT patients who also had a PDE11A sequence variant, compared with those with a wild-type PDE11A sequence. In NTERA-2 and Tcam-2 cells transfected with the mutated forms of PDE11A (R52T, F258Y, Y727C, R804H, V820M, R867G, and M878V), cAMP levels were significantly higher, and the relative phosphodiesterase activity was lower than in the wild-type cells. KITLG expression was consistently increased in the presence of PDE11A-inactivating defects, both at the RNA and protein levels, in familial testicular germ cell tumors. The 2 sporadic cases that were studied, one with a PDE11A defect and another without, agreed with the data in FTGTCT and in the cell lines. CONCLUSIONS Patients with FTGCT and PDE11A defects also carry KITLG risk alleles more frequently. There may be an interaction between cAMP and c-KIT signaling in predisposition to testicular germ cell tumors.

Facial Plethora: Modern Technology for Quantifying an Ancient Clinical Sign and Its Use in Cushing Syndrome

CONTEXT Facial plethora is a clinical sign described since ancient times for a variety of diseases. In the 19th century, it was linked to increased blood volume or flow, but this has never been proven. Facial plethora is also one of the earliest described clinical features of Cushings syndrome (CS). OBJECTIVE This study aimed to quantify facial plethora changes in CS as an early assessment of cure after surgery using noninvasive near-infrared multispectral imaging (MSI). DESIGN The longitudinal cohort study was initiated in August 2012 and completed in August 2014. SETTING Clinical research hospital, National Institutes of Health. PATIENTS Thirty-four of the 38 patients who received surgical treatment for CS under protocol 97CH0076 during this period were included. INTERVENTION(S) MSI was performed on the right cheek of patients before surgery and 4.9 ± 3.1 days afterward. MAIN OUTCOME MEASURE(S) Average blood volume fraction as measured by MSI and serum cortisol. RESULTS All but four of the 28 patients (86%) who were assessed as cured by postoperative plasma cortisol measurements of < 3 μg/dL showed a decrease in blood volume fraction (17.7 ± 0.03 vs 15.8 ± 0.03%; P = .0019), whereas an increase was seen in patients with persistent CS (18.5 ± 0.03 vs 21.4 ± 0.04%; P = .0017). Change in blood volume fraction before and after surgery was correlated with postoperative cortisol (rs = 0.58; P = .0003). CONCLUSIONS Clinical data obtained from 34 patients indicate that a decrease in facial plethora after surgery, as evidenced by a decrease in blood volume fraction, is correlated with CS outcome. This novel technology for the first time identified a physiological mechanism associated with an ancient clinical sign. Furthermore, as a proof of principle, MSI is a promising early marker of cure in patients with CS that complements biochemical and clinical data.

Is IGSF1 involved in human pituitary tumor formation

IGSF1 is a membrane glycoprotein highly expressed in the anterior pituitary. Pathogenic mutations in the IGSF1 gene (on Xq26.2) are associated with X-linked central hypothyroidism and testicular enlargement in males. In this study, we tested the hypothesis that IGSF1 is involved in the development of pituitary tumors, especially those that produce growth hormone (GH). IGSF1 was sequenced in 21 patients with gigantism or acromegaly and 92 healthy individuals. Expression studies with a candidate pathogenic IGSF1 variant were carried out in transfected cells and immunohistochemistry for IGSF1 was performed in the sections of GH-producing adenomas, familial somatomammotroph hyperplasia, and in normal pituitary. We identified the sequence variant p.N604T, which in silico analysis suggested could affect IGSF1 function, in two male patients and one female with somatomammotroph hyperplasia from the same family. Of 60 female controls, two carried the same variant and seven were heterozygous for other variants. Immunohistochemistry showed increased IGSF1 staining in the GH-producing tumor from the patient with the IGSF1 p.N604T variant compared with a GH-producing adenoma from a patient negative for any IGSF1 variants and with normal control pituitary tissue. The IGSF1 gene appears polymorphic in the general population. A potentially pathogenic variant identified in the germline of three patients with gigantism from the same family (segregating with the disease) was also detected in two healthy female controls. Variations in IGSF1 expression in pituitary tissue in patients with or without IGSF1 germline mutations point to the need for further studies of IGSF1 action in pituitary adenoma formation.

Comprehensive Analysis of LC/MS Data Using Pseudocolor Plots

AbstractWe have developed new applications of the pseudocolor plot for the analysis of LC/MS data. These applications include spectral averaging, analysis of variance, differential comparison of spectra, and qualitative filtering by compound class. These applications have been motivated by the need to better understand LC/MS data generated from analysis of human biofluids. The examples presented use data generated to profile steroid hormones in urine extracts from a Cushing’s disease patient relative to a healthy control, but are general to any discovery-based scanning mass spectrometry technique. In addition to new visualization techniques, we introduce a new metric of variance: the relative maximum difference from the mean. We also introduce the concept of substructure-dependent analysis of steroid hormones using precursor ion scans. These new analytical techniques provide an alternative approach to traditional untargeted metabolomics workflow. We present an approach to discovery using MS that essentially eliminates alignment or preprocessing of spectra. Moreover, we demonstrate the concept that untargeted metabolomics can be achieved using low mass resolution instrumentation.

Post-operative growth is different in various forms of pediatric Cushing's syndrome

Cushing’s syndrome (CS) is a rare disease in children it is associated with weight gain and stunting of their linear growth (McArthur et al. 1980, Magiakou et al. 1994a, Stratakis 2012). In this study, we assessed growth in pediatric patients with CS after cure, caused by either adrenocorticotropic hormone (ACTH)-dependent Cushing’s disease (CD) or a form of ACTH-independent CS, and patients with micronodular adrenal hyperplasia (MAH). We reviewed medical records of patients who had successful transsphenoidal surgery or adrenalectomy at the NIH between the years of 2002 and 2012. A total of 18 children with CD (9 F, mean age 12.2G3.0 years) and 19 children with MAH (15 F, mean age 9.8G4.4 years) were included. All patients were evaluated under the clinical protocol 97CH0076 and 95CH0059 that were approved by the NICHD Institutional Review Board. Informed consent was signed from the patients’ parents. The diagnosis of CS was established as previously reported (Batista et al. 2007). Patients that were at Tanner stage 5 at the time of surgery were excluded from the analysis of annual growth velocity and insulin-like growth factor 1 (IGF1) z scores. The mean follow-up after surgery was 402G27 days for the CD and 365G87 days for the MAH patients (PZ0.09). No significant difference was found in terms of mean age (although CD patients were 2.4 years older), gender distribution, duration of symptoms, midnight cortisol levels, IGF1 z score, and mean urinary free cortisol at the time of surgery. The demographics are presented in Table 1. The baseline height (Ht) z scores and BMI z scores were not significantly different between the two groups (PZ0.85 and PZ0.66 respectively; Fig. 1A). The mean Ht z score in CD before and after surgery was K1.24G1.14 and K0.91G1.30 respectively (PZ0.03). The mean Ht z score in MAH before and after surgery was K1.33G1.64 and K0.62G1.37 respectively (PZ0.0002; Fig. 1B). The mean BMI z score in CD before and after

Growth hormone and risk for cardiac tumors in Carney complex

Carney complex (CNC) is a multiple neoplasia syndrome that is caused mostly by PRKAR1A mutations. Cardiac myxomas are the leading cause of mortality in CNC patients who, in addition, often develop growth hormone (GH) excess. We studied patients with CNC, who were observed for over a period of 20 years (1995-2015) for the development of both GH excess and cardiac myxomas. GH secretion was evaluated by standard testing; dedicated cardiovascular imaging was used to detect cardiac abnormalities. Four excised cardiac myxomas were tested for the expression of insulin-like growth factor-1 (IGF-1). A total of 99 CNC patients (97 with a PRKAR1A mutation) were included in the study with a mean age of 25.8 ± 16.6 years at presentation. Over an observed mean follow-up of 25.8 years, 60% of patients with GH excess (n = 46) developed a cardiac myxoma compared with only 36% of those without GH excess (n = 54) (P = 0.016). Overall, patients with GH excess were also more likely to have a tumor vs those with normal GH secretion (OR: 2.78, 95% CI: 1.23-6.29; P = 0.014). IGF-1 mRNA and protein were higher in CNC myxomas than in normal heart tissue. We conclude that the development of cardiac myxomas in CNC may be associated with increased GH secretion, in a manner analogous to the association between fibrous dysplasia and GH excess in McCune-Albright syndrome, a condition similar to CNC. We speculate that treatment of GH excess in patients with CNC may reduce the likelihood of cardiac myxoma formation and/or recurrence of this tumor.