Reduced Calcium Sensing Receptor (CaSR) Expression Is Epigenetically Deregulated in Parathyroid Adenomas

Abstract

<jats:title>Abstract</jats:title> <jats:sec> <jats:title>Aim</jats:title> <jats:p>Reduced CaSR expression has been implicated in parathyroid tumorigenesis but the underlying mechanism remains elusive. Accordingly, we aimed to explore the epigenetic changes (DNA methylation and histone modifications) involved in CaSR regulation in sporadic parathyroid adenomas and correlate epigenetic state with disease indices.</jats:p> </jats:sec> <jats:sec> <jats:title>Experimental Design</jats:title> <jats:p>Forty sporadic parathyroid adenomas and 10 normal parathyroid tissues were studied. Real time quantitative polymerase chain reaction (qRT-PCR) for mRNA and immunohistochemistry for protein expression of CaSR were performed. The methylation status of the CaSR promoter 2 was determined by bisulphite sequencing analysis of sodium bisulphite-converted DNA. To determine the role of histone modifications in the CaSR regulation, chromatin immunoprecipitation-qPCR assay was performed.</jats:p> </jats:sec> <jats:sec> <jats:title>Results</jats:title> <jats:p>qRT-PCR revealed reduced CaSR mRNA expression with a fold reduction of 0.12 (P&amp;lt;0.0001) in parathyroid adenomas. Immunohistochemistry revealed reduced protein expression of CaSR in 85% (34/40) of adenomas. The promoter 2 region of CaSR displayed significant hypermethylation in 45% (18/40) of the adenomas compared to the controls (6.7%;1of 10)(P&amp;lt;0.0001). Bisulphite sequencing analysis revealed maximum methylated CpG at GCM2 binding sites on the CaSR promoter compared to other CpG sites. The methylation status of CaSR correlated directly with plasma iPTH levels in patients with parathyroid adenoma. With ChIP-qPCR analysis, H3K9me3 levels showed increased enrichment by 10-fold in adenomas and correlated with CaSR-mRNA expression (r=0.61; P&amp;lt;0.003). Treatment with 5-aza-2´deoxycytidine restored the expression of CaSR in a parathyroid cell line.</jats:p> </jats:sec> <jats:sec> <jats:title>Conclusion</jats:title> <jats:p>Our data suggest that hypermethylation and increased H3K9me3 of the CaSR promoter 2 are involved in silencing CaSR expression in sporadic parathyroid adenoma.</jats:p> </jats:sec>

Preoperative prediction of parathyroid carcinoma in an Asian Indian cohort

“…However, the younger age at presentation for Indian patients with PHPT has been described previously 2,3 . Prevalent vitamin D deficiency and dysregulation of calcium homeostasis molecules have been cited as contributory factors 28‐30 . Accordingly, our study showed younger age at presentation in benign sPHPT when compared to PC.…”

“…2,3 Prevalent vitamin D deficiency and dysregulation of calcium homeostasis molecules have been cited as contributory factors. [28][29][30] Accordingly, our study showed younger age at presentation in benign sPHPT when compared to PC. Still, the difference between the age of PC cases and the larger sPHPT cohort of the Indian PHPT registry (n = 441) was nonsignificant; hence, blurring the impact of age as a predictor of PC.…”

CaSR expression in normal parathyroid and PHPT: new insights into pathogenesis from an autopsy-based study

“…Significantly diminished membranous expression of CaSR was found in all PHPT cases than in normal using both the scoring systems. Although initial studies showed a lack of role of allelic loss or methylation of the CaSR gene promoter [6,11,12,24], a recent study documented hypermethylation and increased H3K9me3 of promoter 2 of CaSR in 45% of their sporadic parathyroid adenomas [25]. It will hence be interesting to analyze normal parathyroid rims for this alteration.…”

Yes-Associated Protein 1 Is a Novel Calcium Sensing Receptor Target in Human Parathyroid Tumors

“…By contrast, in primary PAds cultures, the loss of MEN1 slightly increased YAP1 expression. Therefore, YAP1 and MEN1 mutually and differently regulated their expression levels, suggesting that genetic and epigenetic [ 36 ] aberrations occurring in parathyroid tumors may determine the degree of activity of the two genes and their target genes. Certainly, our data did not provide final insights into the mechanisms underlying such interaction.…”