Ignite Creation Date:
2024-05-05 @ 10:59 PM
Last Modification Date:
2024-10-26 @ 10:26 AM
Study NCT ID:
NCT01228786
Status:
UNKNOWN
Last Update Posted:
2012-07-20
First Post:
2010-10-26
Brief Title:
Regulation of Vitamin D Receptor VDRCalcium Sensing Receptor CaSR Cyclin D1Ki67 and Proliferating Cell Nuclear Antigen PCNA in Primary Hyperparathyroidism
Sponsor:
Post Graduate Institute of Medical Education and Research Chandigarh
Organization:
PIMERIndia
Study Overview
Official Title:
Transcriptional and Translational Regulation of Vitamin D Receptor VDR and Calcium Sensing Receptor CaSR in Patients With Sporadic Primary Hyperparathyroidism
Status:
UNKNOWN
Status Verified Date:
2012-07
Last Known Status:
RECRUITING
Delayed Posting:
No
If Stopped, Why?:
Not Stopped
Has Expanded Access:
False
If Expanded Access, NCT#:
N/A
Has Expanded Access, NCT# Status:
N/A
Acronym:
None
Brief Summary:
The present study is designed to examine the expression of VDR CaSR PTH Cyclin D1 Ki67 and PCNA and to find out its relationship with clinical parameters in parathyroid adenomas Examination of the contribution of genes expression can elucidate the critical link between proliferation and functional abnormalities in parathyroid adenomas Alternative to DNA and RNA protein expression can provide a better understanding of this disease
Detailed Description:
Introduction
Primary hyperparathyroidism PHPT is the third most frequent endocrine disorder after diabetes mellitus and thyroid disorders that predominantly affects postmenopausal women with an incidence of 1 -5 in 1000 people It can occur at any age though young people are rarely affected PHPT is characterized by hypersecretion of parathyroid hormone PTH and resultant hypercalcemia Most cases of PHPT 85 result from a solitary adenoma in one of the parathyroid glands Multi-gland hyperplasia is found in about 10-15 of the patients while carcinoma occurs rarely 1-2
The parathyroid glands regulate calcium homeostasis The major target organs for parathyroid hormone PTH are bone and the kidneys PTH increases bone resorption to mobilize calcium into the circulation In the kidney PTH enhances calcium reabsorption and increases phosphate excretion PTH also stimulates renal production of 1α25-dihydroxyvitamin D 125OH2D which in turn enhances intestinal absorption of calcium Thus the physiological effects of PTH are to increase the concentration of calcium in the circulation Negative feedback from calcium and 125OH2D modulate parathyroid function These effects are mediated via the calcium-sensing receptor CaSR expressed on the parathyroid cell surface Similarly 125OH2D acts through vitamin D receptor VDR and suppresses PTH synthesis and secretion Thus PTH secretion is tightly coupled to the parathyroid cells ambient calcium level
Increased parathyroid cell proliferation and decreased calcium-mediated control of the PTH secretion are characteristic findings in all types of hyperparathyroidism 1-4 Calcium via its receptor the CaSR and the 125OH2D-VDR complex are the most important regulators in this respect Decreased actions of these regulators would stimulate the parathyroid cells to proliferate Molecular analyses have revealed the presence of tumor-specific DNA rearrangements in a subset of adenomas In such rearrangements the 5 PTH gene regulatory region combines upstream of the Cyclin D1 gene which results in over expression of cyclins that could induce proliferation by increasing mitotic rate In fact Cyclin D1 over expression was first demonstrated in a patient with parathyroid adenoma 5
Impaired CaSR gene expression is found in parathyroid lesions of both primary and secondary hyperparathyroidism 6-11 No mutations could be found in CaSR gene in parathyroid neoplasias 1112 Similar present findings shows reduced VDR gene expression 813-15 and concur with studies on the apparent lack of VDR gene mutations in hyperparathyroidism 16-18 Differential expression of VDR and CaSR gene has been reported in a few parathyroid adenomas but exact mechanism is not known These observations raise the possibility that altered expression of these genes could be related to the pathogenesis of parathyroid adenomas
OBJECTIVES To achieve the above aim the following objectives would be under taken
1 Comparison of expression of VDR and CaSR genes in parathyroid adenomas with normal parathyroid tissue obtained during surgery from non-hyperparathyroid patients by Real Time-PCR
2 To confirm expression of VDR CaSRPTH cyclin D1 Ki67 and PCNA in parathyroid cells by immunohistochemistry
3 Comparison of protein expression profile of parathyroid adenomas with normal parathyroid tissue by proteomic analysis
MATERIALS AND METHODS
Subjects
All the PHPT patients attending outdoor patient clinic of Endocrinology and General Surgery department of Nehru Hospital PGIMER Chandigarh will be included for this study with following inclusion and exclusion criteria
Inclusion Criteria Patients with surgically verified sporadic PHPT and controls will be included as follows Group A 20 sporadic PHPT patients Group B 10 normocalcemic euthyroid patients operated for thyroid disorders will provide normal parathyroid tissue to serve as control tissue
Exclusion Criteria Patients with hyperplasia renal failure and multiple endocrine neoplasia will be excluded
Sample collection Parathyroid tissue samples will be collected immediately after surgery snap frozen and stored at -80oC until use
Assay Methods Preoperative levels of total serum calcium reference range 86-102 mgdL will be determined by auto-analyzer Modular P Roche Diagnostics Germany The serum Calcium level will be corrected with the serum albumin level The serum intact PTH reference range 12-55 ngLand 25-hydroxyvitamin D 111-429 ngml will be measured using immunochemiluminiscence ELECSYS-2010 Roche Diagnostics Germany
1 Gene expression study for VDR and CaSR-
1 RNA extraction
Total RNA will be extracted from the cryopreserved normal and adenomatous parathyroid tissue samples by using the TRIZOL reagent Life Technologies Inc according to the vendors instructions The samples will be pretreated with DNase and stored at -80oC until use The cDNA will be generated from the 3-4 mg of RNA by reverse transcriptase with random hexamers as primers
2 Real-Time Polymerase Chain Reaction
The mRNA levels of VDR and CaSR will be estimated by Real-PCR Stratagene Robocycler Gradient 40 system using specific primer sequences and PCR conditions The PCR products will be separated by electrophoresis on 6 agarose gel the products will be then stained with ethidium bromide and located by fluorescence using UV light Bio Max 1D TM 151 Kodak Rochester NY would be used to evaluate the band intensities of the PCR bands
2 Expression study of VDR CaSRPTH Cyclin D1 Ki67 and PCNA protein-
Immunohistochemistry Monoclonal antibodies will be used for immunostaining of VDR CaSR PTH Cyclin D1 Ki67 and PCNA proteins on paraffin sections Source- Pharmingen USA Secondary antibody goat anti-mouse horse radish peroxidase conjugated BD Biosciences Pharmingen USA specific for primary antibodies will be used in each test Indirect immunoperoxidase method will be used for the staining of paraffin sections In this assay the unconjugated primary antibody binds to the antigen in the specimen To localize this attachment a peroxidase conjugated specific secondary antibody is used which binds to the primary antibody A substrate is than added to localize the reaction
The slides will be then examined under the light microscope Staining will be graded as negative 0 mild moderate and marked depending on intensity of immunoreactivity
3 Comparison of protein expression profile-
1 Extraction of Proteins 100mg of tissue will be homogenized with 05 ml of cold 4C lysis buffer Protein will be estimated by Bicin chronic Acid BCA method and store the remaining at -80C
2 First Dimension IEF and Second Dimension SDS-PAGE separation The concentrated sample will be solubilized in rehydration buffer A total 125 - 200 µl will be loaded onto 7 cm IPG strip Amersham IEF run will be according to manufacturers protocol
For second dimension the IPG strips will be equilibrated for 15 min with 6 ml equilibration buffer Each IPG strip will be loaded onto 12 polyacrylamide gel and electrophoresed 100 or 200 V for 6 h After electrophoresis proteins will be fixed and are visualized using Coomassie and Silver stains
3 Image Analysis In-gel trypsin digestion and MALDI mass spectrometry analysis Stained gels will be scanned with GS-800 densitometer Bio-Rad using PDQuest software Bio-Rad and the proteins of interest will be analyzed by the MALDI-TOFMS Voyager DE-PRO Applied Biosystems at Institute of microbial technology IMTech
Proteins will be identified by peptide mass fingerprinting using Mascot web based Search engine with the help of peptide mass spectra
Primary hyperparathyroidism PHPT is the third most frequent endocrine disorder after diabetes mellitus and thyroid disorders that predominantly affects postmenopausal women with an incidence of 1 -5 in 1000 people It can occur at any age though young people are rarely affected PHPT is characterized by hypersecretion of parathyroid hormone PTH and resultant hypercalcemia Most cases of PHPT 85 result from a solitary adenoma in one of the parathyroid glands Multi-gland hyperplasia is found in about 10-15 of the patients while carcinoma occurs rarely 1-2
The parathyroid glands regulate calcium homeostasis The major target organs for parathyroid hormone PTH are bone and the kidneys PTH increases bone resorption to mobilize calcium into the circulation In the kidney PTH enhances calcium reabsorption and increases phosphate excretion PTH also stimulates renal production of 1α25-dihydroxyvitamin D 125OH2D which in turn enhances intestinal absorption of calcium Thus the physiological effects of PTH are to increase the concentration of calcium in the circulation Negative feedback from calcium and 125OH2D modulate parathyroid function These effects are mediated via the calcium-sensing receptor CaSR expressed on the parathyroid cell surface Similarly 125OH2D acts through vitamin D receptor VDR and suppresses PTH synthesis and secretion Thus PTH secretion is tightly coupled to the parathyroid cells ambient calcium level
Increased parathyroid cell proliferation and decreased calcium-mediated control of the PTH secretion are characteristic findings in all types of hyperparathyroidism 1-4 Calcium via its receptor the CaSR and the 125OH2D-VDR complex are the most important regulators in this respect Decreased actions of these regulators would stimulate the parathyroid cells to proliferate Molecular analyses have revealed the presence of tumor-specific DNA rearrangements in a subset of adenomas In such rearrangements the 5 PTH gene regulatory region combines upstream of the Cyclin D1 gene which results in over expression of cyclins that could induce proliferation by increasing mitotic rate In fact Cyclin D1 over expression was first demonstrated in a patient with parathyroid adenoma 5
Impaired CaSR gene expression is found in parathyroid lesions of both primary and secondary hyperparathyroidism 6-11 No mutations could be found in CaSR gene in parathyroid neoplasias 1112 Similar present findings shows reduced VDR gene expression 813-15 and concur with studies on the apparent lack of VDR gene mutations in hyperparathyroidism 16-18 Differential expression of VDR and CaSR gene has been reported in a few parathyroid adenomas but exact mechanism is not known These observations raise the possibility that altered expression of these genes could be related to the pathogenesis of parathyroid adenomas
OBJECTIVES To achieve the above aim the following objectives would be under taken
1 Comparison of expression of VDR and CaSR genes in parathyroid adenomas with normal parathyroid tissue obtained during surgery from non-hyperparathyroid patients by Real Time-PCR
2 To confirm expression of VDR CaSRPTH cyclin D1 Ki67 and PCNA in parathyroid cells by immunohistochemistry
3 Comparison of protein expression profile of parathyroid adenomas with normal parathyroid tissue by proteomic analysis
MATERIALS AND METHODS
Subjects
All the PHPT patients attending outdoor patient clinic of Endocrinology and General Surgery department of Nehru Hospital PGIMER Chandigarh will be included for this study with following inclusion and exclusion criteria
Inclusion Criteria Patients with surgically verified sporadic PHPT and controls will be included as follows Group A 20 sporadic PHPT patients Group B 10 normocalcemic euthyroid patients operated for thyroid disorders will provide normal parathyroid tissue to serve as control tissue
Exclusion Criteria Patients with hyperplasia renal failure and multiple endocrine neoplasia will be excluded
Sample collection Parathyroid tissue samples will be collected immediately after surgery snap frozen and stored at -80oC until use
Assay Methods Preoperative levels of total serum calcium reference range 86-102 mgdL will be determined by auto-analyzer Modular P Roche Diagnostics Germany The serum Calcium level will be corrected with the serum albumin level The serum intact PTH reference range 12-55 ngLand 25-hydroxyvitamin D 111-429 ngml will be measured using immunochemiluminiscence ELECSYS-2010 Roche Diagnostics Germany
1 Gene expression study for VDR and CaSR-
1 RNA extraction
Total RNA will be extracted from the cryopreserved normal and adenomatous parathyroid tissue samples by using the TRIZOL reagent Life Technologies Inc according to the vendors instructions The samples will be pretreated with DNase and stored at -80oC until use The cDNA will be generated from the 3-4 mg of RNA by reverse transcriptase with random hexamers as primers
2 Real-Time Polymerase Chain Reaction
The mRNA levels of VDR and CaSR will be estimated by Real-PCR Stratagene Robocycler Gradient 40 system using specific primer sequences and PCR conditions The PCR products will be separated by electrophoresis on 6 agarose gel the products will be then stained with ethidium bromide and located by fluorescence using UV light Bio Max 1D TM 151 Kodak Rochester NY would be used to evaluate the band intensities of the PCR bands
2 Expression study of VDR CaSRPTH Cyclin D1 Ki67 and PCNA protein-
Immunohistochemistry Monoclonal antibodies will be used for immunostaining of VDR CaSR PTH Cyclin D1 Ki67 and PCNA proteins on paraffin sections Source- Pharmingen USA Secondary antibody goat anti-mouse horse radish peroxidase conjugated BD Biosciences Pharmingen USA specific for primary antibodies will be used in each test Indirect immunoperoxidase method will be used for the staining of paraffin sections In this assay the unconjugated primary antibody binds to the antigen in the specimen To localize this attachment a peroxidase conjugated specific secondary antibody is used which binds to the primary antibody A substrate is than added to localize the reaction
The slides will be then examined under the light microscope Staining will be graded as negative 0 mild moderate and marked depending on intensity of immunoreactivity
3 Comparison of protein expression profile-
1 Extraction of Proteins 100mg of tissue will be homogenized with 05 ml of cold 4C lysis buffer Protein will be estimated by Bicin chronic Acid BCA method and store the remaining at -80C
2 First Dimension IEF and Second Dimension SDS-PAGE separation The concentrated sample will be solubilized in rehydration buffer A total 125 - 200 µl will be loaded onto 7 cm IPG strip Amersham IEF run will be according to manufacturers protocol
For second dimension the IPG strips will be equilibrated for 15 min with 6 ml equilibration buffer Each IPG strip will be loaded onto 12 polyacrylamide gel and electrophoresed 100 or 200 V for 6 h After electrophoresis proteins will be fixed and are visualized using Coomassie and Silver stains
3 Image Analysis In-gel trypsin digestion and MALDI mass spectrometry analysis Stained gels will be scanned with GS-800 densitometer Bio-Rad using PDQuest software Bio-Rad and the proteins of interest will be analyzed by the MALDI-TOFMS Voyager DE-PRO Applied Biosystems at Institute of microbial technology IMTech
Proteins will be identified by peptide mass fingerprinting using Mascot web based Search engine with the help of peptide mass spectra
Study Oversight
Has Oversight DMC:
None
Is a FDA Regulated Drug?:
None
Is a FDA Regulated Device?:
None
Is an Unapproved Device?:
None
Is a PPSD?:
None
Is a US Export?:
None
Is an FDA AA801 Violation?:
None
Secondary IDs
| Secondary ID | Type | Domain | Link |
|---|---|---|---|
| BHADADA-SK | OTHER | ICMR India | None |