Human adrenocorticotropin-secreting pituitary adenomas show frequent loss of heterozygosity at the glucocorticoid receptor gene locus

Corticotropinomas are characterized by a relative resistance to the negative feedback action of cortisol on ACTH secretion. In this respect there is a similarity with the clinical syndrome of cortisol resistance. As cortisol resistance can be caused by genetic abnormalities in the glucocorticoid receptor (GR) gene, we investigated whether the insensitivity of corticotropinomas to cortisol is also caused by de novo mutations in the GR gene. We screened for the GR gene in leukocyte and tumor DNA from 22 patients with Cushing's disease for mutations using PCR/single strand conformation polymorphism analysis. In a previous study, we identified 5 polymorphisms in the GR gene in a normal population. These polymorphisms were used as markers for the possible occurrence of loss of heterozygosity (LOH) at the GR gene locus. Except for 1 silent point mutation, we did not identify novel mutations in the GR gene in leukocytes or corticotropinomas from these patients. Of the 22 patients, 18 were heterozygous for at least 1 of the polymorphisms. In 6 of these patients, LOH had occurred in the tumor DNA. Of 21 patients examined for LOH on chromosome 11q13, only 1, with a corticotroph carcinoma, showed allelic deletion. As controls we studied 28 pituitary tumors of other subtypes (11 clinically nonfunctioning, 8 prolactinomas, and 9 GH-producing adenomas) and found evidence for LOH in only 1 prolactinoma. In six patients LOH was found at the GR gene locus (chromosome 5) in DNA derived from adenoma cells. Our observations indicate for the first time that LOH at the GR gene locus is a relatively frequent phenomenon in pituitary adenomas of patients with Cushing's disease. This might explain the relative resistance of the adenoma cells to the inhibitory feedback action of cortisol on ACTH secretion. The specificity of the GR LOH to corticotropinomas supports this concept. Somatic mutations of the GR are not a frequent cause of relative cortisol resistance in these cells

A Solve-RD ClinVar-based reanalysis of 1522 index cases from ERN-ITHACA reveals common pitfalls and misinterpretations in exome sequencing

Purpose Within the Solve-RD project (https://solve-rd.eu/), the European Reference Network for Intellectual disability, TeleHealth, Autism and Congenital Anomalies aimed to investigate whether a reanalysis of exomes from unsolved cases based on ClinVar annotations could establish additional diagnoses. We present the results of the “ClinVar low-hanging fruit” reanalysis, reasons for the failure of previous analyses, and lessons learned. Methods Data from the first 3576 exomes (1522 probands and 2054 relatives) collected from European Reference Network for Intellectual disability, TeleHealth, Autism and Congenital Anomalies was reanalyzed by the Solve-RD consortium by evaluating for the presence of single-nucleotide variant, and small insertions and deletions already reported as (likely) pathogenic in ClinVar. Variants were filtered according to frequency, genotype, and mode of inheritance and reinterpreted. Results We identified causal variants in 59 cases (3.9%), 50 of them also raised by other approaches and 9 leading to new diagnoses, highlighting interpretation challenges: variants in genes not known to be involved in human disease at the time of the first analysis, misleading genotypes, or variants undetected by local pipelines (variants in off-target regions, low quality filters, low allelic balance, or high frequency). Conclusion The “ClinVar low-hanging fruit” analysis represents an effective, fast, and easy approach to recover causal variants from exome sequencing data, herewith contributing to the reduction of the diagnostic deadlock

Supplementary Material for: The EFEMP1 Gene: A Frequent Target for Epigenetic Silencing in Multiple Human Pituitary Adenoma Subtypes

<strong><em>Background/Aims:</em></strong> In a genome-wide investigation we recently identified the EGF-containing fibulin-like extracellular matrix protein 1 gene, <i>EFEMP1</i>, as hypermethylated in growth hormone-secreting adenoma. <b><i>Methods:</i></b> In an independent cohort we determined expression of EFEMP1, CpG island methylation and histone tail modification status. The causal consequences of epigenetic modification were determined through epidrug-induced reversal and enforced EFEMP1 expression in GH3 cells. <b><i>Results:</i></b> The majority of adenomas, irrespective of subtype, show reduced EFEMP1 expression. However, epigenetic change, as determined by CpG island methylation, was not invariantly associated with decreased EFEMP1 expression. Conversely, chromatin immunoprecipitation assays revealed enrichment for modifications associated with either active or silenced genes in adenoma that did or did not express EFEMP1 respectively. In AtT-20 and GH3 cells a causal relationship between epigenetic silencing and expression of EFEMP1 was established where co-incubation with the epidrugs zebularine and TSA induced expression of EFEMP1 and concomitant histone tail modifications toward those associated with expressed genes. Enforced expression of EFEMP1 in GH3 cells was without effect on cell proliferation or apoptotic end-points, however inhibition of endogenous matrix metalloproteinase (MMP)-2 expression was apparent. Primary adenomas did not show this relationship, however a positive correlation was apparent with the MMP7 transcript and perhaps reflects cell or species differences. <b><i>Conclusions:</i></b> The protein product of the <i>EFEMP1</i> gene, fibulin-3, is reported to impact on multiple pathways in a cell-specific context. Subtype-independent loss of EFEMP1 expression in the majority of primary adenomas should prompt more detailed investigation in this tumour type