Targeted Next-Generation Sequencing of MEN 1, RET, CDC 73, and CDKNIB Genes in Familial Primary Hyperparathyroidism: A Study from Northern India.

Primary hyperparathyroidism (PHPT) is characterized by hypercalcemia due to excessive parathyroid hormone (PTH) secretion. About 90–95% of cases of PHPT are sporadic, mostly due to a single adenoma[ 1 ]; 5%–10% are familial, caused by pathogenic germline variants,[ 2 3 ] a condition known as familial primary hyperparathyroidism (FPHPT).[ 4 ] The prevalence increases to 15%–26% in patients with high-risk features suggestive of FPHPT.[ 5 ] FPHPT may occur either as an isolated clinical disorder or as part of a syndrome, such as multiple endocrine neoplasia (MEN) types 1, 2A, and 4, and hyperparathyroidism-jaw tumour syndrome (HPT-jaw tumour) [CDC73] syndrome. Other forms of FHPT include familial hypocalciuric hypercalcemia syndrome (FHH), neonatal severe hyperparathyroidism (NSHPT), and familial isolated hyperparathyroidism (FIHP).[ 6 ] FIHP is diagnosed by exclusion because the genetic cause is often unknown.[ 7 ] Current guidelines[ 8 9 ] recommend genetic analysis in individuals younger than 40 years of age, those with multiglandular disease, those with a family history of PHPT, parathyroid carcinoma (PC), cystic parathyroid adenoma (PA), patients with a strong suspicious of MEN1/MEN2A/MEN4 syndrome and CDC73 related disorder. Genetic testing in familial PHPT helps identify patients at risk for associated neoplasms (e.g., MEN, HPT-jaw tumour syndrome), guides personalised management, enables early detection through surveillance of asymptomatic relatives, reduces the burden in mutation-negative individuals, and facilitates genetic counselling and prenatal diagnosis when necessary.[ 10 ] Although FPHPT is associated with various genes, based on existing evidence, four genes ( MEN1, RET, CDKN1B , and CDC73 ) are commonly implicated in FPHPT.[ 9 ] To determine the prevalence of targeted gene mutations in high-risk patients with PHPT. Compare the prevalence of germline variants in our population with those in other cohorts. Compare biochemical parameters and end-organ complications (renal and/or skeletal disease) in those with a positive and negative genetic test.

The clinical and laboratory characteristics of the 39 patients are shown in Table 1 . Most patients were females, 79.5%. The mean age of our patients was 31.68 ± 10.35 (12–60) years. Renal and skeletal disease were present in 66.7% and 38.4% of patients. Genetic sequencing of the targeted gene panel identified pathogenic germline variants in 11 of the 39 patients (28.2%). Seven (17.9%) patients tested positive for germline variants in MEN1 , while 4 (10.2%) patients tested positive for germline variants in CDC73 . No patient tested positive for germline variants in MEN2A and MEN4 . The clinical and laboratory characteristics of patients with pathogenic germline variants are shown in Table 2 , and the genetic profile is shown in Table 3 . Four out of seven patients with germline variants in MEN1 carried splice variant c. 1351-2A>G. These four patients were unrelated. One patient carried missense variant c. 1763C>T (p.S588L), one patient carried frameshift variant c. 249_252del (p.Ile85fs), and one patient carried missense variant c. 415C>T (p.H139Y). All these germline variants have been previously reported as pathogenic. Two of the seven patients with MEN1 germline variants had microprolactinomas, and one had pancreatic neuroendocrine endocrine. Five patients had multi-glandular disease at surgery. Five patients were cured, while one patient had a persistent disease, and one had a recurrence. Out of four patients with germline variants in CDC73 , one carried c.664C>T(p.R222X), one carried c. 415C>T(p.R139X), one carried c. 687_688dellAG(p.Arg229Serfs37) and one carried c.76delA(p.Ile26SerfsX11). Two patients had a frameshift mutation, and two had a nonsense mutation. All these variants have been previously reported as pathogenic variants. Two participants with germline variants in CDC73 had features of HPT- JT (one had multiple uterine leiomyomas, and one had bilateral renal cysts); none had ossifying fibromas of the jaw. Two patients had a family history of PHPT. All four patients had uniglandular presentation surgery and were subjected to single gland excision and achieved cure. Table 4 shows the difference between the characteristics of patients with positive and negative genetic tests. Those with germline variants were younger than those without germline variants (median 25 vs 30.5 years, P = 0.029) and had significantly higher serum calcium and ALP (median serum calcium 12.9 vs 12 mg/dl, P = 0.045; median ALP 332 vs 134 IU/l, P = 0.004). Patients with germline variants had significantly higher 24-hour urinary calcium levels than those without (median, 600 vs 414; P = 0.029). The median 25 (OH) vitamin D levels were 21.5 ng/mL (range, 13–30) in mutation-positive patients and 22.8 ng/mL (range, 12-32) in mutation-negative patients, P = 0.910. Patients with germline variants had higher PTH levels, [median 250 pg/ml, (107–1900)], compared to those without germline variants, [median 173 pg/ml, (89–1751], which was not statistically significant, P = 0.179. Skeletal disease, (osteoporosis and subperiosteal bone resorption) was more common in patients with germline variants (63.63% vs 28.57%, P = 0.043). There was no difference in renal disease between the two groups. Multiglandular disease was also more common in patients with germline variants; however, this difference was not statistically significant. Figure 1 shows a summary of our results. Clinical and laboratory characteristics of patients ( n =39) ALP alkaline phosphatase, i PTH intact parathyroid hormone. *Mean±standard deviation Clinical and laboratory characteristics of patients with pathogenic germline variants ( n =11) † Alkaline phosphatase; ‡ PTH intact parathyroid hormone; ¦ PHPT primary hyperparathyroidism Genetic profile of patients with pathogenic germline variants ( n =11) || American College of Medical Genetics and Genomics, ¶ Those mutations bearing this symbol are reported in ClinVar Characteristics of patients with and without germline variants Data expressed as median (Range) unless specified. ALP alkaline phosphatase, i PTH intact parathyroid hormone. Significant P values are in bold ( P< 0.05) Graphical abstract illustrating the diagnostic yield of targeted next-generation sequencing in FPHPT, focusing on MEN1, RET, CDC73 , and CDKN1B mutations. This summary reflects findings in a resource-limited tertiary care setting

Our cohort found a remarkable prevalence of genetic variants with features suggestive of familial PHT. This is consistent with the hypothesis that familial PHPT is likely an underdiagnosed entity due to the limited number of genetic studies performed in routine clinical practice, non-standardised genetic screening, and an insufficient understanding of the genetics of PHPT. We recommend genetic testing in patients with PHPT who present any risk characteristics considered in our study’s inclusion criteria. Although our custom NGS panel focused on four high-risk genes, this approach was deliberate, targeting genes with the highest clinical relevance in familial PHPT. Future studies incorporating broader sequencing approaches may provide additional insights into the genetic architecture of this condition. However, the strength of our research lies in its targeted approach, which ensures precise, clinically actionable results. AQ prepared the manuscript, RAM supervised the study, AC and AQ helped in data collection, IAB helped in data processing, MIB and AIW finalised the manuscript, and MAW, AAM and JAS helped in statistical analysis and preparation of tables. There are no conflicts of interest. No artificial Intelligence was used while preparing this manuscript. The data are available from the corresponding author.

In this study, we describe a well-defined cohort of 39 patients, who presented with features indicative of FPHPT. This is the largest cohort of patients with suspected FHPT reported from India, where a customised gene panel related to FPHPT was studied. We identified nine germline variants in 11 participants (28.2%), seven in MEN1 , and four in CDC73 . We selected this specific panel of genes (MEN1, RET, CDKN1B , and CDC73) because they account for most pathogenic variants in FPHPT,[ 9 ] making them the highest-yield targets in resource-limited settings. In our cohort, most patients had target organ damage, making Familial Hypocalciuric Hypercalcemia (FHH) unlikely; therefore, sequencing of the Calcium-Sensing Receptor (CASR) , Guanine Nucleotide-Binding Protein Subunit Alpha-11 (GNA11) , and Adaptor-Related Protein Complex 2 Sigma 1 Subunit (AP2S1) was not pursued. Custom panels provided deeper coverage, higher sensitivity, and lower cost than exome/genome sequencing, ensuring clinically relevant and feasible testing in our setting. The frequency of germline variants identified in our study exceeds the rates reported in previous research involving individuals clinically diagnosed with FPHPT. For example, a recent study of a Mediterranean cohort of 40 patients reported germline variants in 22.5% of cases.[ 19 ] Studies from other regions have shown varying prevalence rates: 9.3% in an American cohort,[ 20 ] 15% in South Australia,[ 5 ] and 26% in New Zealand.[ 5 ] One of the largest studies conducted so far identified 19 pathogenic germline variants (including 11 in CASR , 6 in MEN1 , 1 in CDC73 , and 1 in AP2S1 ) among 121 British patients suspected of having FPHPT, corresponding to a prevalence of 16%.[ 21 ] These discrepancies may reflect differences in diagnostic criteria for FPHPT and the specific genes analysed across studies.[ 5 21 22 ] MEN1 pathogenic variants in PHPT cases have been reported in approximately 15%–21% of patients.[ 23 24 ] Our findings align with these results, as 17.9% of participants in our study carried MEN1 pathogenic variants. However, a study of 40 patients[ 24 ] found no MEN1 pathogenic variants, and another involving 29 Finnish patients with suspected FHPT identified only one MEN1 mutation (3%) among all genes analysed.[ 25 ] Similarly, detection of germline MEN1 variants was observed in over half of Indian sporadic PHPT patients, with specific variants (for example, c.-35A>T, c. 1525C>A) linked to recurrence and multiglandular disease.[ 26 ] Although guidelines recommend genetic screening for PHPT in individuals aged 30–45 years, we adopted a more stringent cutoff of < 35 years. This choice reflects the natural history of MEN- related and syndromic PHPT, which often manifests in the second and third decades of life. Lowering the age threshold enhances the likelihood of detecting hereditary syndromes, enables timely surveillance for associated tumours, and facilitates appropriate surgical planning. This approach aligns with MEN-focused screening protocols, supporting earlier diagnosis and intervention in FPHPT. Negative genetic results in clinically high-risk individuals in our study could be attributed to: (1) variants in genes not covered in our panel (e.g., CASR, GNA11, AP2S1, GCM2 ), (2) presence of variants of uncertain significance or deep intronic mutations not detectable on targeted panels, (3) possible mosaicism or epigenetic modifications, (4) and finally, phenocopies, where the clinical features mimic familial disease but are in fact sporadic. The most frequent MEN1 pathogenic variant in our cohort was the splice-site mutation c. 1351-2A>G, observed in 4 of 7 unrelated participants. This mutation involves the substitution of adenine with guanine at codon 1351. Of these individuals, two were diagnosed with microprolactinoma. Data regarding the prevalence of pathogenic germline CDC73 variants in PHPT patients are scarce. We observed pathogenic germline CDC73 variants in four (10.2%) patients. This is almost like a study reported by Van Der et al. ,[ 17 ] in which pathogenic germline CDC73 variants were identified in 11 of the 89 PHPT patients (12.4%). In this study, 10 of the 11 patients were male (91%). In a case series from Western India[ 27 ] involving seven patients with germline CDC73 variants, there were four males and three females. In our study, there were two males and two females. In our study, all four patients had uniglandular disease. This is consistent with the case series from Western India, which reported uniglandular disease in all seven patients.[ 27 ] In our study, two participants had features of HPT-JT (one had multiple uterine leiomyomas, and one had bilateral renal cysts). In the case series from Western India,[ 27 ] renal cysts were present in three patients, jaw tumours in two patients and uterine endometrial involvement in two patients. Out of 11 patients with pathogenic germline CDC73 variants, Van Der et al .[ 17 ] reported HPT- JT in three patients, renal abnormalities in one patient and uterine abnormalities in none. In our patients with germline CDC73 variants, family history of PHPT was positive in 2/4 (50%). A previous study[ 17 ] reported a family history of PHPT in 73% of CDC73 mutation carriers. Previous studies have associated atypical parathyroid tumours with mutations in CDC73 .[ 28 ] None of our patients had atypical parathyroid tumours. In a recent study by Van Der et al. ,[ 17 ] germline variants in CDC73 were not found in 11 patients with atypical parathyroid tumours. We found several clinical traits that were significantly more frequent in patients with germline variants. Patients with germline variants were younger and had higher serum calcium and ALP. A study[ 29 ] reported higher calcium and PTH concentrations in patients with a positive genetic test compared to the group with a negative test, but this was not statistically significant. Additionally, skeletal disease was more common in patients with germline variants. The earlier onset of disease in genetically driven PHPT leads to more skeletal involvement in the form of osteoporosis and subperiosteal bone resorption. Burgess et al .[ 30 ] have reported that a reduction in bone mass is evident in most women with MEN1 by 35 years of age. Eller-Vainicher C et al .[ 31 ] reported that MEN1 -related PHPT patients show more severe bone involvement than sporadic PHPT. Our study found no statistically significant difference between renal disease in patients with a positive genetic mutation. This is consistent with what was reported by Eller-Vainicher C et al .[ 31 ] A summary of relevant Indian and international studies on genetic testing in familial PHPT is provided in Table 5 . Shows a summary of the latest Indian and International Studies on Genetic Testing in FPHT CES: Clinical Exome Sequencing, MLPA: Multiplex Ligation-dependent Probe Amplification, GCM2: Glial Cells Missing Transcription Factor 2, RET: REarranged during Transfection proto-oncogene We have not tested our participants for FHH forms (caused by variants of CASR , GNA11 , or AP2S1 ), neonatal severe hyperparathyroidism, and other forms caused by several genes classified as FIHP due to a lack of funds and the less frequent occurrence of these mutations. Also, we have not tested family members of patients with pathogenic variants.

Out of 103 patients with PHPT evaluated in the Department of Endocrinology at our University Hospital over two years (February 2021 to February 2023), 39 patients met the criteria for genetic analysis based on one or more of the following characteristics: HPT- JT (Hyperparathyroidism jaw tumour) features include ossifying fibromas of the mandible and/or maxilla, uterine tumours (e.g., adenofibromas, leiomyomas, adenomyosis, hyperplasia, adenosarcomas) and malignant and non-malignant renal lesions (Wilms tumour, clear cell renal carcinoma, papillary renal cell tumour, and renal cysts). Family history of PHPT Parathyroid cancer (PC) Patient age less than 35 years. Cystic Parathyroid adenoma (PA) Multiglandular disease. Patients with a strong suspicion of MEN 1/MEN 2A,4 syndromes. HPT- JT (Hyperparathyroidism jaw tumour) features include ossifying fibromas of the mandible and/or maxilla, uterine tumours (e.g., adenofibromas, leiomyomas, adenomyosis, hyperplasia, adenosarcomas) and malignant and non-malignant renal lesions (Wilms tumour, clear cell renal carcinoma, papillary renal cell tumour, and renal cysts). Family history of PHPT Parathyroid cancer (PC) Patient age less than 35 years. Cystic Parathyroid adenoma (PA) Multiglandular disease. Patients with a strong suspicion of MEN 1/MEN 2A,4 syndromes. Exclusion criteria: Chronic kidney disease, estimated glomerular filtration rate, <60 ml/min/1.73 m 2 Malabsorption syndrome Secondary hyperparathyroidism Chronic kidney disease, estimated glomerular filtration rate, <60 ml/min/1.73 m 2 Malabsorption syndrome Secondary hyperparathyroidism An automated chemistry analyser analysed fasting serum samples for calcium, phosphate, creatinine, and alkaline phosphatase (ALP). A 24-hour urine collection was analysed for calcium and creatinine. The normal laboratory range is 8.5–10.5 mg/dl for serum calcium and 2.5–4.5 mg/dl for serum phosphate. Serum iPTH and 25-hydroxy vitamin D (25-OHD) were measured by DXI 800, Beckman Coulter Chemiluminescence random access analyser (Brea, CA), following the manufacturer’s protocol. The reference range for PTH levels is 12–88 pg/ml. The intra-assay and inter-assay coefficients of variation of the iPTH assay were 2.1% and 3.9%, respectively, and that of the 25-OHD assay were 3.6% and 6.5%, respectively. A radiological survey of the hands, skull, lumbar spine, pelvis, and any other suspected or known fracture site was performed. Three-site bone mineral density (BMD), including the lumbar spine, dual femur, and distal radius, was measured with the help of GE lunar Dual Energy X-ray Absorptiometry (DXA). Patients were reported as having osteopenia or osteoporosis or normal, according to WHO.[ 11 ] Renal ultrasonography (USG) was performed to diagnose renal stones, nephrocalcinosis, or renal cysts. Parathyroid adenoma was localised by USG neck, technetium-99m ( 99m Tc) sestamibi scan, and, in cases with negative or discordant USG and 99m Tc, 4D-computerised tomography (CT) neck. Female patients underwent pelvic USG for any uterine tumours. From the patients chosen for genetic analysis, 5 ml of peripheral blood from a peripheral vein was collected in EDTA vials and stored at − 80°C. Genomic DNA was isolated from blood samples by phenol-chloroform method/DNA extraction Kits. DNA quality and the content were checked by Agarose gel electrophoresis and spectrophotometry, respectively. Polymerase chain reaction (PCR) was performed using a Thermal Cycler to amplify the desired targets. A customized NGS panel was used for target sequencing of MEN1, RET, CDKNIB and CDC73 genes. The nucleic acid (DNA) received was quality-checked. Briefly, 10 ng of DNA was amplified using a custom thermos assay designed by the client (covering four genes and having 14,626 bp in the target region) as per the instruction manual, and sequencing was performed using the Ion S5 platform as per the user manual. The sequencing reads QC, mapping on hg19 human reference genome, variant calling (SNVs, small InDels, CNVs) and annotation was carried out with IonReporter™ (IR) Software 5.18.2.0. Later, the RefSeq database was used to identify and characterise genes-associated variants. The annotation for variants was derived using various disease databases like OMIM and ClinVar. The population frequency information from 1,000 genomes, ExAC, GnomAD, dbSNP and ESP , was used to eliminate common variants/polymorphism. For the prediction of the possible impact of coding variants on the structure and function of a protein, PolyPhen-2 and SIFT scores were used. Also, all variants were separately analysed by multiple other prediction tools for in-silicon variant effect prediction. All variants were then interpreted based on ACMG guidelines[ 12 ] and reported. 1) PHPT: PHPT was defined as persistent hypercalcemia and concomitantly raised or inappropriately normal serum intact PTH (iPTH). 2) FPHPT: It was defined as PHPT with one or more characteristics listed in the inclusion criteria. 3) Renal disease was defined as ultrasonographic evidence of renal stones and/or nephrocalcinosis. 4) Skeletal disease: Skeletal disease was defined as X-ray evidence of subperiosteal resorption, fractures, a salt-and-pepper appearance of the skull or brown tumour, and/or DXA evidence of osteopenia or osteoporosis. 5) Multiglandular disease: Multiglandular disease was defined as a biopsy documented involvement of 2 or more glands affected by either adenomas or hyperplasia. 6) Persistent PHPT: Persistent PHPT was defined as hypercalcemia and raised iPTH at any time during the six-month period after parathyroid surgery. 7) Recurrent PHPT: Recurrent PHPT was defined as hypercalcemia and raised iPTH following a period of normokalaemia for 6 months or longer after parathyroid surgery. 1) PHPT: PHPT was defined as persistent hypercalcemia and concomitantly raised or inappropriately normal serum intact PTH (iPTH). 2) FPHPT: It was defined as PHPT with one or more characteristics listed in the inclusion criteria. 3) Renal disease was defined as ultrasonographic evidence of renal stones and/or nephrocalcinosis. 4) Skeletal disease: Skeletal disease was defined as X-ray evidence of subperiosteal resorption, fractures, a salt-and-pepper appearance of the skull or brown tumour, and/or DXA evidence of osteopenia or osteoporosis. 5) Multiglandular disease: Multiglandular disease was defined as a biopsy documented involvement of 2 or more glands affected by either adenomas or hyperplasia. 6) Persistent PHPT: Persistent PHPT was defined as hypercalcemia and raised iPTH at any time during the six-month period after parathyroid surgery. 7) Recurrent PHPT: Recurrent PHPT was defined as hypercalcemia and raised iPTH following a period of normokalaemia for 6 months or longer after parathyroid surgery. All participants with germline variants in MEN1 underwent subtotal parathyroidectomy, while patients who tested positive for germline variants in CDC73 underwent single-gland excision. Patients with either vitamin D deficiency or insufficiency were given the appropriate correction before undergoing surgery. The recorded data was compiled and entered in a spreadsheet (Microsoft Excel) and then exported to the data editor of SPSS Version 20.0 (SPSS Inc., Chicago, Illinois, USA). Continuous variables were expressed as Mean ± SD and categorical variables were summarised as frequencies and percentages. Students’ independent t -test or Mann-Whitney U-test, whichever is feasible, was employed to compare continuous variables. The chi-square test or Fisher’s exact test, whichever is appropriate, was applied to compare categorical variables. A P -value of less than 0.05 was considered statistically significant. The study was approved by the Institutional Ethics Committee (IEC) of Sher-i-Kashmir Institute of Medical Sciences (SKIMS), Srinagar, under protocol number IEC/SKIMS #RP 208/2021, which was issued on October 15, 2021. Informed consent was obtained for participation in the study and the use of patient data for research and educational purposes. All procedures followed the guidelines laid down in the Declaration of Helsinki 1964 and as revised later.