OR24-6 Non-syndromic Cushing's Disease Due To CDKN1B Mutations: Novel Mutations And Phenotypic Features In A Large Pediatric Cohort

Introduction: Germline loss-of-function mutations in the CDKN1B gene underlie multiple endocrine neoplasia type 4 (MEN4), an infrequent and clinically heterogeneous autosomal dominant syndrome with incomplete penetrance. MEN4 encompasses various neuroendocrine tumors and other neoplasms, including pituitary adenomas. Despite the central role of the CDKN1B protein in corticotroph cell function and the finding that Cdkn1b KO mice develop Pomc-positive pituitary adenomas, only two cases of Cushing’s disease (CD) have so far been described in the setting of MEN4. We aimed to determine the frequency of CDKN1B gene defects in a cohort of pediatric CD patients, and to functionally validate our findings. Methods: We studied 203 pediatric CD patients referred to our center between 1997-2018; 60.6% were female. The median age was 10.8 (interquartile range: 8-13.8) years at disease onset, and 12.8 (10.3-15.5) years at diagnosis. Ten cases had familial or simplex presentation and 103 cases were sporadic. Ninety-eight patients underwent whole-exome sequencing of germline DNA only (n=70) or germline and tumor DNA (n=28); 105 patients were screened by Sanger sequencing. Germline copy number variations (CNVs) were searched for in 197 cases by droplet digital PCR. Loss of heterozygosity (LOH) and CDKN1B immunohistochemistry (IHC) were analyzed in the tumors and inheritance of the variants was investigated. Variants of interest were cloned into expression plasmids and overexpressed in AtT-20/D16v-F2 (mouse corticotropinoma) cells. The expression of CDKN1B protein variants and their impact on POMC quantification were analyzed by Western blot. Results: Four heterozygous germline CDKN1B (NM_004064.4) rare variants were identified in one patient each: c.-32_-29delGAGA, c.320delA (p.Q107Rfs*12), c.356T>C (p.I119T), and c.407A>G (p.D136G). No CNVs or somatic variants were found. Variants c.356T>C and c.-32_-29delGAGA have been reported in MEN4 kindreds. Three c.320delA carriers related to our patient were found; one of them died of a colon adenocarcinoma, which displayed LOH. This tumor represents a novel MEN4 component. In the rest of the cases, variant inheritance could not be determined, and there was no LOH in the corticotropinomas. IHC showed partial (p.D136G) or almost complete (c.-32_-29delGAGA and c.356T>C) loss of CDKN1B nuclear staining in corticotropinomas. All variants were detected at the protein level, but c.320delA resulted in a protein ~10 kDa smaller than the wild type, and c.356T>C caused a slight delay in migration. POMC expression was 1.6±0.1 times higher when overexpressing c.320delA, compared with empty vector (P=0.049). Conclusion: MEN4 explains 2% of the CD cases in our cohort. CDKN1B mutations may lead to apparently isolated CD in young patients, a phenotypic variant that should be noted, given its implications for clinical screening and genetic counseling.