Summary:

Parathyroid adenoma, is a part of the spectrum of parathyroid disease including parathyroid hyperplasia and parathyroid carcinoma. The etiology of a parathyroid adenoma remains unknown for most patients. Thus, the aim of this study is to investigate the single nucleotide polymorphism in patients attending a tertiary care hospital for parathyroid adenoma with osteoporosis. The study will focus on the genetic polymorphism of genes known to be implicated in the development of parathyroid adenoma, such as cyclin D1.

Keywords: cyclin D1, Parathyroid Adenoma, Osteoporosis

Abbreviations: NIL

Introduction and Background:

Traditionally, the diagnosis of primary hyperparathyroidism (HPT) has been based on the presence of high total and/or ionized serum calcium levels, which are associated with either increased or unsuppressed levels of serum parathyroid hormone (PTH). In addition, HPT has classically been associated with kidney and bone complications, mainly loss of bone mineral density (BMD) [1]. However, in recent years, our understanding of HPT has improved and several distinctive mild clinical forms of HPT have been described, including normocalcemic hyperparathyroidism (NPHPT) and asymptomatic hyperparathyroidism, both of which are increasingly common diagnoses in daily clinical practice [2]. However, these new entities remain only partially understood, particularly concerning their pathophysiology and gene interactions [3,4]. As of now, there is no proper reports for the identification of PTH with osteoporosis. Thus, the present study is to identify the Clinical Correlation of Single Nucleotide Polymorphism in Parathyroid Adenoma with Osteoporosis of Patients Attending a Tertiary Care Hospital in Western UP of India.

Study Objectives:

Objective 1: Isolation of DNA from the selected group of study subjects.

Objective 2: Determination of the SNP on cyclin D1 gene.

Methodology:  

Study Design: Cross-sectional

Sample Size: 30 or less as per availability in six months from the date of start.

Project Implementation Plan:

Participant Selection:

Inclusion Criteria:

·       Patients diagnosed with parathyroid adenoma and osteoporosis.

·       Age: 45-55 Years

Exclusion Criteria:

·       Patients with secondary causes of osteoporosis or parathyroid adenoma, comorbid conditions affecting bone health, and those receiving specific medications affecting bone metabolism.

Informed Consent:

Written informed consent will be obtained from all patients.

Sample Collection:

Patients meeting the inclusion criteria will be recruited from the hospital.

Collection of Blood:

Consumables Requirements:

·       Sterile collection tubes with anticoagulants (e.g., EDTA)

·       Sterile collection tubes without anticoagulants

·       Centrifuge tubes

Procedure:

·       A clean and sterilized needle will be used to collect blood from a vein (commonly from the arm).

·       10 ml of peripheral blood sample will be collected into the anticoagulant-containing tubes (5ml) and without anticoagulant tubes (5ml).

Blood Clotting:

·       The blood will be allowed to clot by leaving the collection tubes undisturbed at room temperature for 30 minutes to 1 hour.

·       The tubes remain upright during clotting.

Centrifuge Setup:

·       Load the collected blood tubes into the centrifuge rotor.

·       Ensure a balanced configuration to prevent imbalance during centrifugation.

Centrifugation parameters:

·       After clotting, centrifuge the blood tubes at an appropriate speed (typically around 1500-2000 rpm) for about 10-15 minutes.

·       This will separate the blood into its components, with the heavier components forming a pellet at the bottom.

Pellet Collection:

·       After centrifugation, carefully remove the tubes from the centrifuge.

·       A sterile pipette will be used to collect the supernatant (plasma or serum) without disturbing the formed pellet.

·       Transfer the plasma or serum to a clean, sterile container for further analysis.

Sample Storage:

·       If immediate analysis is needed, the serum will be keep at 4°°C to maintain stability for a short duration.

·       The serum will be stored at an appropriate temperature, often at -20°C or - 80°C for long-term storage.

Washing (Optional):

·       The cells may have washed with a buffer solution to remove any remaining plasma or serum.

Labelling:

·       Clearly label each sample with patient identifiers, date, and any other relevant information.

Documentation:

·       The serum container will be labelled with patient identifiers, date, and any other relevant information.

·       The entire process will be documented including the time of blood collection, clotting, and centrifugation.

Exams and Tests (Serum):

Blood tests (Soto et al., 2016) may be done to check levels of:

Ø  PTH

Ø  Calcium

Ø  Kidney function (Creatinine, BUN)

Ø  Magnesium

Ø  Phosphorus

Ø  Vitamin D

Plasma - DNA, RNA isolation by kit

 Tissue sample collection:

Tissue Biopsy: Parathyroid adenoma tissue sample will be obtaining through a biopsy or surgical excision - Immunohistochemistry and for cell line development study

Parathyroid Tissue Sample Collection:

Requirements:

Cold Storage: Maintain a cold storage environment (4°C on ice) during and after the collection process to preserve the tissue. To be collected by Central Research Facility, Santosh Deemed to be University.

Procedures:

·       Parathyroid Tissue Sample Storage:

Short-Term Storage: (Temperature) If the analysis will be conducted shortly after collection, store the tissue at a temperature between 2-8°C (refrigerator).

Long-Term Storage:

Flash Freezing: Immediately freeze the tissue using liquid nitrogen or dry ice and store it at - 80°C for extended periods.

·       Cold Storage:

Keep the collection tube with the tissue sample on ice or in a cold storage container to maintain its viability during transportation.

·       Transportation to the Laboratory:

Transport the tissue sample to the laboratory as quickly as possible to minimize degradation.

·       Storage in the Laboratory:

Upon arrival at the laboratory, store the tissue sample at an appropriate temperature for short-term analysis or consider long-term storage at -80°C if needed.

·       Documentation:

Document the entire collection process, including patient information, date, time, and any observations made during the procedure.

Sample Transportation:

·       Packaging: Place samples in certified, leak-proof primary containers, Use gel packs, dry ice, or other cooling elements to maintain the required temperature.

·       Labeling: Clearly label the outer container with handling instructions and Include contact information and delivery address.

·       Documentation: Include all necessary shipping documentation.

·       Transportation Conditions: Ensure the transport method maintains the integrity of the samples (e.g., refrigerated vehicles, priority overnight shipping).

·       Tracking and Monitoring: Track the shipment throughout its journey to ensure timely delivery. Monitor temperature conditions if applicable, using data loggers or temperature indicators.

·       Notification: Inform the receiving party of the shipment details, including tracking number and estimated arrival time.

Additional Considerations:

·       Pathological Assessment of the disease (confirmatory test)

MAX Hospital will do it for the sample at their level

·       For diagnostic purposes, the collected parathyroid tissue may undergo pathological assessment to confirm the presence of adenoma or other conditions.

Expected outcomes:

Polymorphism in cyclin D1 in the study population in the state of Uttar Pradesh is expected in people under high risk of developing parathyroid adenoma.

Future plans based on expected outcomes:

In the future, the study will be carried out on the large population/multicentric.

Timelines:

One year.