Targeted Sequencing to Screen Germline and Somatic Variants, its Validation and Expression at Gene and Protein Levels in Indian Primary Hyperparathyroidism Patients
Recommended Citation
Garg S, Arya A, Kumari P, Ritambhara N, Sachdeva N, Dahiya D, Rao SD, Mithal A, Bhadada SK. Targeted Sequencing to Screen Germline and Somatic Variants, its Validation and Expression at Gene and Protein Levels in Indian Primary Hyperparathyroidism Patients. J Bone Miner Res 2024; 39:128.
Document Type
Conference Proceeding
Publication Date
9-27-2024
Publication Title
J Bone Miner Res
Abstract
Background: Primary hyperparathyroidism (PHPT) has been characterized by hypercalcemia with elevated parathyroid hormone. In India, the genetic causes of familial forms of PHPT have been known, however, there has been no comprehensive study of genetic analysis of sporadic and familial PHPT cases till now. Aim: To establish an Indian database of germline and somatic variants using 18 genes panel clinically relevant to parathyroid tumors Methods: Thirty histopathologically proven, PHPT patients and five controls were recruited between 2019-2023. A panel of 18 genes (MEN1, CDC73, CASR, CDKN1B, RET, GCM2, AP2S1, GNA11, PTH, CDKN1A, CDKN2B, CDKN2C, EZH2, ZFX, GATA3, CTNNB1, LRP5, and CCND1) clinically relevant to PHPT was designed for next generation sequencing. DNA extracted from both blood and tissue samples from patients and controls were prepared in two different pools of libraries and run on NovaSeq 6000 Illumina platform. Variant calling was performed to obtain germline and somatic variants as per ACMG guidelines which were validated through sanger sequencing. Besides, gene and protein expression of genes as well as in-silico functional characterization (Mutalyzer 3 and CharmGUI) was done. Results: The mean age of the patients recruited was 48.33 ± 15.70 years (28-70) with a sporadic: familial ratio of 25:5. Clinical manifestations were bone pain (n=20;66.6%), abdominal pain (n=15;50%), weakness and fatigue (n=21;70%), gall stone disease (n=6; 20%), renal stone disease (n=18;60%), fractures (n=10;33.3%), and osteoporosis (n=18;60%). Biochemically, the corrected serum calcium (11.68 ± 1.41 mg/dl), vitamin D (24.52 ± 13.53 ng/ml), serum PTH (811.3 ± 904.5 pg/ml), serum phosphorus (2.36 ± 0.76 mg/dL), serum alkaline phosphatase (267.7±237) and serum creatinine (0.96 ± 0.47 mg/dl) levels were fluctuated in PHPT patients in comparison to control. Out of total 197 germline and 108 somatic variations; the benign mutations reported were 190 (germline) and 103 (somatic) respectively. In germline, the pathogenic missense (c.T818C, exon 5) and frameshift insertion mutations (c.252dupT, exon 2; c.566dupA, exon3) were screened in MEN1 gene and VUS (possibly pathogenic) in GCM2 (c.T1102A, exon 5), LRP5 (c.C3919T, exon 18; c.G229A, exon 2) and RET (c.C1423T, exon 7) genes. In somatic, the pathogenic stopgain (c.C981G, exon7; c.C590T, exon 3); and missense (c.C496T, exon 3; c.T1673G, exon 10; c.G467A, exon 3) mutations were screened in MEN1 gene and further validated through sanger sequencing. Loss of function was observed in the MEN1 gene at gene and protein expression level. In-silico analysis showed truncated MEN1 protein in stopgain and frameshift insertions affecting structure and function of the protein. Conclusion: Two novel mutations have been reported in the blood and four in tissue samples of Indian PHPT patients. Approx. 17% of MEN1 mutations were screened in sporadic PHPT patients.
Volume
39
First Page
128
