Clinical Profile and Mutations Associated with Multiple Endocrine Neoplasia-Type1 (MEN1) and Their First-Degree Relatives at Risk of Developing MEN1: A Prospective Study

Multiple Endocrine Neoplasia type-1 (MEN1) is an autosomal dominant disorder with a combined occurrence of tumours of parathyroid glands, pancreatic islets, and anterior pituitary. About 90% of these patients carry mutations in the MEN1 gene, though the spectrum is not well defined in India. Forty clinically suspected cases of MEN1 were enrolled prospectively over six years; 32 patients (23 index-cases and nine affected relatives) with≥2 classical endocrine tumours of MEN1 were considered definite, and eight were categorised as ‘MEN1-like’. Details of their clinical presentation, treatment and mutational analysis including MEN1 gene, 3′ and 5′ untranslated regions (UTR) of MEN1, CDKN1B, and CaSR genes were collated. Asymptomatic first-degree relatives were also screened. Among the 32 definite MEN1 patients, all had primary hyperparathyroidism, 22 (68.7%) had gastroentero-pancreatic neuroendocrine tumours, and 21 (66%) had pituitary adenoma. Of the 23 definite index-cases, 13 (56.5%) carried mutations in the MEN1 gene. Five of nine affected first-degree relatives (55.5%), and four of 10 asymptomatic relatives (40%) also had MEN1 mutations. Seven of 10 MEN1 mutation-negative definite index-cases harboured p.V109G polymorphism in the CDKN1B gene. All eight MEN1-like cases were negative for mutations and large deletions in MEN1, mutations in 3′ and 5′ UTR of MEN1, CaSR and CDKN1B genes. The study has helped to clearly document the pattern of mutations among Indian MEN1 patients. However, the absence of MEN1 mutation in ~44% of cases and the presence of p.V109G polymorphism in CDKN1B gene raise the question whether such polymorphisms could independently contribute to pathogenesis.

Germline CDKN1B Loss-of-Function Variants Cause Pediatric Cushing’s Disease With or Without an MEN4 Phenotype

Context Germline loss-of-function CDKN1B gene variants cause the autosomal dominant syndrome of multiple endocrine neoplasia type 4 (MEN4). Even though pituitary neuroendocrine tumors are a well-known component of the syndrome, only 2 cases of Cushing’s disease (CD) have so far been described in this setting. Aim To screen a large cohort of CD patients for CDKN1B gene defects and to determine their functional effects. Patients We screened 211 CD patients (94.3% pediatric) by germline whole-exome sequencing (WES) only (n = 157), germline and tumor WES (n = 27), Sanger sequencing (n = 6), and/or germline copy number variant (CNV) analysis (n = 194). Sixty cases were previously unpublished. Variant segregation was investigated in the patients’ families, and putative pathogenic variants were functionally characterized. Results Five variants of interest were found in 1 patient each: 1 truncating (p.Q107Rfs*12) and 4 nontruncating variants, including 3 missense changes affecting the CDKN1B protein scatter domain (p.I119T, p.E126Q, and p.D136G) and one 5’ untranslated region (UTR) deletion (c.-29_-26delAGAG). No CNVs were found. All cases presented early (10.5 ± 1.3 years) and apparently sporadically. Aside from colon adenocarcinoma in 1 carrier, no additional neoplasms were detected in the probands or their families. In vitro assays demonstrated protein instability and disruption of the scatter domain of CDKN1B for all variants tested. Conclusions Five patients with CD and germline CDKN1B variants of uncertain significance (n = 2) or pathogenic/likely pathogenic (n = 3) were identified, accounting for 2.6% of the patients screened. Our finding that germline CDKN1B loss-of-function may present as apparently sporadic, isolated pediatric CD has important implications for clinical screening and genetic counselling.

A Novel Regulatory Region of the p27 Locus Is Required for Normal Erythroid p27 Expression, and Produces a Long Noncoding RNA with No Detectable Function

Mammalian cells express thousands of lineage-restricted long noncoding RNAs (lncRNAs), although the functions of most are unknown. "Knockout" studies to investigate lncRNA function by gene deletion in cells or whole animals have been informative. However, since most lncRNAs show open chromatin and transcription factor (TF) binding at their promoters as well as elsewhere in their genomic loci, it is essential to distinguish the roles of the RNA transcript from those of potential cis-regulatory elements, both of which are eliminated in deletion studies. We identified a novel mouse lncRNA named Drop27 (Downstream of p27) that is transcribed 4 kb downstream of the Cdkn1b gene encoding the cell-cycle inhibitor p27, which is strongly upregulated during erythroid maturation. Drop27 is conserved between mice and humans, has two exons separated by a 21 kb intron, and produces a 400 bp spliced, polyadenylated RNA that is abundantly transcribed and highly enriched in mouse erythroblasts compared to other tissues. We used the CRISPR/Cas system to delete the entire Drop27 locus in the G1E erythroid cell line. Microarray analysis showed reduced Cdkn1b mRNA in Drop27-deleted cells with very few other changes in the transcriptome. Quantitative PCR verified this finding by demonstrating that heterozygous deletion of Drop27 caused reduced Cdkn1b mRNA by 35%, while homozygous deletion led to a 70% reduction. This effect also occurred at the primary transcript level, demonstrating that deletion of the Drop27 reduced Cdkn1b transcription. To distinguish the effects of loss of the lncRNA transcribed from Drop27 from the potential loss of a cis-acting regulatory module in the Drop27 genomic locus, we used homologous recombination to insert a bovine growth hormone (BGH) polyadenylation (polyA) cassette into the first exon of Drop27, 80 bp downstream of the transcription start site. This caused premature termination of the lncRNA transcript while maintaining all genomic sequences, including potential cis-regulatory elements. Analysis of multiple clones showed that homozygous polyA cassette integration reduced full-length Drop27 transcript levels by more than 99%, although Cdkn1b mRNA levels were unaffected. These findings demonstrate that Drop27 lncRNA is dispensable for Cdkn1b transcription, while its genomic sequences are required, indicating that the Drop27 gene locus contains an erythroid cis-regulatory element (enhancer) for the Cdkn1b gene. Several strong candidates for the proposed enhancer are found in the Drop27 gene. Multiple epigenetic features strongly associated with enhancers map in several distinct locations in the Drop27 locus in erythroid and other hematopoietic cells, including DNase hypersensitivity, p300 binding, and multiple transcription factor sites. A functional role for the Drop27 lncRNA is not identified by this experiment, and it is possible that it arose as a byproduct of enhancer activity. Our findings provide new insight into mechanisms of hematopoietic gene expression and are of more broad relevance to the lncRNA field in general. In particular, we demonstrate that the genomic loci of some lncRNA genes may function as cis elements, irrespective of the transcripts arising from them. Disclosures No relevant conflicts of interest to declare.

Abstract 498: A Single Nucleotide Polymorphism in the CDKN1B Gene is Associated With Carotid Plaque Progression

Objective: Studies suggest that a SNP in the promoter region of the C DKN1B gene (-838 C>A; rs36228499), a cell cycle inhibitor, may be associated with decreased vascular SMC proliferation and increased risk of MI. Our objective was to test the hypothesis that the -838A allele is associated with decreased carotid plaque progression. Methods: 214 AIM-HIGH participants underwent annual carotid MRI over 2 years using a standardized protocol. Images were analyzed by Core Lab reviewers blinded to the genotyping results. Plaques with ulceration or hemorrhage were excluded as these local features may contribute excess variability or override genotype. Changes in wall thickness (WT) and lumen, wall, fibrous tissue and total vessel volumes were measured. Associations between number of A alleles and remodeling patterns were tested using linear regression. Results: Of the 214 subjects, 161 had suitable MRI and genotype data available for analysis. There was a significant trend toward less growth (more regression) in mean WT for each A allele (p=0.043) (Figure 1). Other plaque variables were not significantly associated with genotype but directionally showed weak trends toward more expansive remodeling for each C allele. Conclusions: These data suggest that -838 C>A SNP of the C DKN1B gene is significantly associated with carotid mean WT progression among subjects with established vascular disease well treated for LDL-C and blood pressure.