We are currently analyzing whole exome sequencing (WES) with appropriate follow up to identify important genetic factors associated with Cushing's disease (CD) and related abnormal physical features. The ultimate goal is to identify a genetic variant or variants that cause CD. CD is a condition in which the pituitary gland produces inappropriately high levels of adrenocorticotropic hormone (ACTH). The ACTH stimulates the adrenal gland to produce excess cortisol, leading to clinical disease. CD is caused by ACTH secreting pituitary tumors. It is noteworthy that patients who have CD also have abnormal features: abnormal facial features including abnormal facial height and nasal length. CD is a serious condition. It requires surgery to remove the tumor. The tumors sometimes recur in which case radiation or medical therapy is required which is not always successful. CD can cause a wide range of problems due to the high cortisol levels. These include diabetes, fractures, poor growth, and hypertension. CD can be fatal. Whole exome sequencing (WES) is a powerful tool for identifying important genetic variants associated with medical conditions. It is an efficient method of determining the genetic code (sequence) of all the regions in the genome that are translated into protein, the exons. The exons constitute about 1% of DNA, thus sequencing exons provides a large amount of information at a lower cost than sequencing the entire genome. Another advantage of the exome sequencing approach is that the areas of the genome sequenced are those for which data interpretation is the most robust. WES is now one of the most important methods for genetic investigation because it provides data on the coding regions of the genome and because it is a cost effective method of searching for important genetic variants. Pediatric aged patients seen at NICHD with a confirmed diagnosis of CD are evaluated for this study. Those who have histopathologically confirmed disease in conjunction with DNA, hormonal documentation of the disease and complete clinical data are potential cases. Analysis comparing variants found in the cases with large control populations has been done. In addition, the data are being examined for copy number variants in the exons to determine if they play a role in Cushing's disease. Whole exome sequencing has now been performed on tumor tissue in patients where tumor tissue is available to determine if there are specific tumor-associated variants in the exome. These data have been analyzed in the same manner as the blood samples. In addition, samples from patients who have ectopic posterior pituitaries have been obtained and whole exome sequenced. The data have been screened using standard data sets to identify rare variants. The most important variants are being selected for additional follow up. Since the last report, the group has published on corticotropinoma as a component of Carney complex. A case of Cushing disease (CD) due to a loss-of-function mutation in PRKAR1A was discovered, 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 cause of CD.

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U.S. National Inst/Child Hlth/Human Dev
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Hernández-Ramírez, Laura C; Tatsi, Christina; Lodish, Maya B et al. (2017) Corticotropinoma as a Component of Carney Complex. J Endocr Soc 1:918-925
Hernández-Ramírez, Laura C; Gam, Ryhem; Valdés, Nuria et al. (2017) Loss-of-function mutations in the CABLES1 gene are a novel cause of Cushing's disease. Endocr Relat Cancer 24:379-392
Faucz, Fabio R; Tirosh, Amit; Tatsi, Christina et al. (2017) Somatic USP8 Gene Mutations Are a Common Cause of Pediatric Cushing Disease. J Clin Endocrinol Metab 102:2836-2843