Aggeliki Dimopoulos

Somatic USP8 Gene Mutations Are a Common Cause of Pediatric Cushing Disease

Context: Somatic mutations in the ubiquitin-specific protease 8 (USP8) gene have been recently identified as the most common genetic alteration in patients with Cushing disease (CD). However, the frequency of these mutations in the pediatric population has not been extensively assessed. Objective: We investigated the status of the USP8 gene at the somatic level in a cohort of pediatric patients with corticotroph adenomas. Design and Methods: The USP8 gene was fully sequenced in both germline and tumor DNA samples from 42 pediatric patients with CD. Clinical, biochemical, and imaging data were compared between patients with and without somatic USP8 mutations. Results: Five different USP8 mutations (three missense, one frameshift, and one in-frame deletion) were identified in 13 patients (31%), all of them located in exon 14 at the previously described mutational hotspot, affecting the 14-3-3 binding motif of the protein. Patients with somatic mutations were older at disease presentation [mean 5.1 ± 2.1 standard deviation (SD) vs 13.1 ± 3.6 years, P = 0.03]. Levels of urinary free cortisol, midnight serum cortisol, and adrenocorticotropic hormone, as well as tumor size and frequency of invasion of the cavernous sinus, were not significantly different between the two groups. However, patients harboring somatic USP8 mutations had a higher likelihood of recurrence compared with patients without mutations (46.2% vs 10.3%, P = 0.009). Conclusion: Somatic USP8 gene mutations are a common cause of pediatric CD. Patients harboring a somatic mutation had a higher likelihood of tumor recurrence, highlighting the potential importance of this molecular defect for the disease prognosis and the development of targeted therapeutic options.

Folic acid fortification for Europe

Europe should consider mandatory fortification; the current strategies are not solving the problem

Corticotropinoma as a Component of Carney Complex

Known germline gene abnormalities cause one-fifth of the pituitary adenomas in children and adolescents, but, in contrast with other pituitary tumor types, the genetic causes of corticotropinomas are largely unknown. In this study, we report a case of Cushing disease (CD) due to a loss-of-function mutation in PRKAR1A, providing evidence for association of this gene with a corticotropinoma. A 15-year-old male presenting with hypercortisolemia was diagnosed with CD. Remission was achieved after surgical resection of a corticotropin (ACTH)-producing pituitary microadenoma, but recurrence 3 years later prompted reoperation and radiotherapy. Five years after the original diagnosis, the patient developed ACTH-independent Cushing syndrome, and a diagnosis of primary pigmented nodular adrenocortical disease was confirmed. A PRKAR1A mutation (c.671delG, p.G225Afs*16) was detected in a germline DNA sample from the patient, which displayed loss of heterozygosity in the corticotropinoma. No other germline or somatic mutations of interest were found. As corticotropinomas are not a known component of Carney complex (CNC), we performed loss of heterozygosity and messenger RNA stability studies in the patient’s tissues, and analyzed the effect of Prkar1a silencing on AtT-20/D16v-F2 mouse corticotropinoma cells. No PRKAR1A defects were found among 97 other pediatric CD patients studied. Our clinical case and experimental data support a role for PRKAR1A in the pathogenesis of a corticotroph cell tumor. This is a molecularly confirmed report of a corticotropinoma presenting in association with CNC. We conclude that germline PRKAR1A mutations are a novel, albeit apparently infrequent, cause of CD.

Loss-of-function mutations in the CABLES1 gene are a novel cause of Cushing’s disease

The CABLES1 cell cycle regulator participates in the adrenal–pituitary negative feedback, and its expression is reduced in corticotropinomas, pituitary tumors with a largely unexplained genetic basis. We investigated the presence of CABLES1 mutations/copy number variations (CNVs) and their associated clinical, histopathological and molecular features in patients with Cushing’s disease (CD). Samples from 146 pediatric (118 germline DNA only/28 germline and tumor DNA) and 35 adult (tumor DNA) CD patients were screened for CABLES1 mutations. CNVs were assessed in 116 pediatric CD patients (87 germline DNA only/29 germline and tumor DNA). Four potentially pathogenic missense variants in CABLES1 were identified, two in young adults (c.532G > A, p.E178K and c.718C > T, p.L240F) and two in children (c.935G > A, p.G312D and c.1388A > G, and p.D463G) with CD; no CNVs were found. The four variants affected residues within or close to the predicted cyclin-dependent kinase-3 (CDK3)-binding region of the CABLES1 protein and impaired its ability to block cell growth in a mouse corticotropinoma cell line (AtT20/D16v-F2). The four patients had macroadenomas. We provide evidence for a role of CABLES1 as a novel pituitary tumor-predisposing gene. Its function might link two of the main molecular mechanisms altered in corticotropinomas: the cyclin-dependent kinase/cyclin group of cell cycle regulators and the epidermal growth factor receptor signaling pathway. Further studies are needed to assess the prevalence of CABLES1 mutations among patients with other types of pituitary adenomas and to elucidate the pituitary-specific functions of this gene.

Rare copy number variants in a population-based investigation of hypoplastic right heart syndrome

BACKGROUND Hypoplastic right heart syndrome (HRHS) is a rare congenital defect characterized by underdevelopment of the right heart structures commonly accompanied by an atrial septal defect. Familial HRHS reports suggest genetic factor involvement. We examined the role of copy number variants (CNVs) in HRHS. METHODS We genotyped 32 HRHS cases identified from all New York State live births (1998-2005) using Illumina HumanOmni2.5 microarrays. CNVs were called with PennCNV and prioritized if they were ≥20 Kb, contained ≥10 SNPs and had minimal overlap with CNVs from in-house controls, the Database of Genomic Variants, HapMap3, and Childrens Hospital of Philadelphia database. RESULTS We identified 28 CNVs in 17 cases; several encompassed genes important for right heart development. One case had a 2p16-2p23 duplication spanning LBH, a limb and heart development transcription factor. Lbh mis-expression results in right ventricular hypoplasia and pulmonary valve defects. This duplication also encompassed SOS1, a factor associated with pulmonary valve stenosis in Noonan syndrome. Sos1-/- mice display thin and poorly trabeculated ventricles. In another case, we identified a 1.5 Mb deletion associated with Williams-Beuren syndrome, a disorder that includes valvular malformations. A third case had a 24 Kb deletion upstream of the TGFβ ligand ITGB8. Embryos genetically null for Itgb8, and its intracellular interactant Band 4.1B, display lethal cardiac phenotypes. CONCLUSION To our knowledge, this is the first study of CNVs in HRHS. We identified several rare CNVs that overlap genes related to right ventricular wall and valve development, suggesting that genetics plays a role in HRHS and providing clues for further investigation. Birth Defects Research 109:16-26, 2017.

Copy number variants in a population-based investigation of Klippel-Trenaunay syndrome.

Klippel–Trenaunay syndrome (KTS) is a rare congenital vascular disorder that is thought to occur sporadically; however, reports of familial occurrence suggest a genetic component. We examined KTS cases to identify novel, potentially causal copy number variants (CNVs). We identified 17 KTS cases from all live‐births occurring in New York (1998–2010). Extracted DNA was genotyped using Illumina microarrays and CNVs were called using PennCNV software. CNVs selected for follow‐up had ≥10 single nucleotide polymorphisms (SNPs) and minimal overlap with in‐house controls or controls from the Database of Genomic Variants. We identified 15 candidate CNVs in seven cases; among them a deletion in two cases within transcripts of HDAC9, a histone deacetylase essential for angiogenic sprouting of endothelial cells. One of them also had a duplication upstream of SALL3, a transcription factor essential for embryonic development that inhibits DNMT3A, a DNA methyltransferase responsible for embryonic de novo DNA methylation. Another case had a duplication spanning ING5, a histone acetylation regulator active during embryogenesis. We identified rare genetic variants related to chromatin modification which may have a key role in regulating vascular development during embryogenesis. Further investigation of their implications in the pathogenesis of KTS is warranted.

Authors’ reply to Smith and colleagues

Some of Smith and colleagues’ statements about our editorial require clarification.1 2 They interpret our statement that: “No important adverse effects have been identified to date, probably because a modest level of fortification has proved very effective in preventing these devastating birth defects” as a claim that fortification is safe because it is effective for one condition. This interpretation is incorrect. Food fortification in the US has resulted in an average exposure to about 200 µg of folic acid per day.3 This is half the recommended dietary allowance (RDA). We were indicating that it is not surprising that no adverse effects have been seen from such a modest dose. As Smith and colleagues point out, WHO states that even high folic acid exposure, in contrast to the small amount fortification delivers, “has not reliably been shown to be associated with negative effects.”4 Thus, the postulated adverse effects, if any, have not been proved. The reduction in neural tube defect rates produced by fortification is proportional to the underlying rate,5 and reported reductions vary depending on the completeness of ascertainment.6 Many …