Epigenetic profiling reveals key genes and cis-regulatory networks specific to human parathyroids.

In all terrestrial vertebrates, the parathyroid glands are critical regulators of calcium homeostasis and the sole source of parathyroid hormone (PTH). Hyperparathyroidism and hypoparathyroidism are clinically important disorders affecting multiple organs. However, our knowledge regarding regulatory mechanisms governing the parathyroids has remained limited. Here, we present the comprehensive maps of the chromatin landscape of the human parathyroid glands, identifying active regulatory elements and chromatin interactions. These data allow us to define regulatory circuits and previously unidentified genes that play crucial roles in parathyroid biology. We experimentally validate candidate parathyroid-specific enhancers and demonstrate their integration with GWAS SNPs for parathyroid-related diseases and traits. For instance, we observe reduced activity of a parathyroid-specific enhancer of the Calcium Sensing Receptor gene, which contains a risk allele associated with higher PTH levels compared to the wildtype allele. Our datasets provide a valuable resource for unraveling the mechanisms governing parathyroid gland regulation in health and disease.

Evaluation of diagnostic potential of CD38 in rickets.

BACKGROUND: Rickets occurs in infants and children (aged 2 months to 3 years), compromising their skeletal development and damaging nervous, hematopoietic, immune, and other system functions. This study aimed to explore the significance of CD38 in rickets. METHODS: The microarray dataset GSE22523 was analyzed to obtain differentially expressed genes in rickets patients. A total of 36 rickets patients and healthy controls were recruited for the study, and their blood samples were collected, followed by detecting mRNA levels of CD38 using quantitative real-time polymerase chain reaction (qRT-PCR). Moreover, the significance of CD38 in rickets patients was analyzed by receiver operating characteristic (ROC) analysis, while the correlation between CD38 and 25-hydroxy-vitamin D (25OHD)/parathyroid hormone (PTH) was analyzed with Pearson's correlation. RESULTS: Results showed that CD38 mRNA levels and PTH contents were significantly increased in the rickets patients while 25OHD contents were decreased. Correlation analysis indicated that CD38 was positively correlated with PTH and negatively correlated with 25OHD in both serum and plasma samples of rickets patients. Moreover, ROC analysis showed that serum CD38 was 0.9005 (95 % CI: 0.8313-0.9696), and the AUCs of plasma CD38 was 0.7215 (95 % CI: 0.6031-0.8398) in differentiating rickets patients from healthy persons, advocating serum CD38 had better diagnostic value. CONCLUSION: CD38 mRNA levels were upregulated in rickets patients and closely correlated with PTH and 25OHD contents, indicating CD38 might be a diagnostic marker of rickets patients. Further research on the diagnostic utility of CD38 is necessary for the diagnosis and treatment of ricketsin rickets in the future.

Molecular Genetic Aspects of Sporadic Multiglandular Primary Hyperparathyroidism.

Multiglandular primary hyperparathyroidism (MGD) represents a rare form of primary hyperparathyroidism (PHPT). MGD is associated with hereditary PHPT, but the sporadic MGD is more common and affects a similar patient profile as single gland parathyroid disease (SGD). The distinction between SGD and MGD is of great clinical importance, especially for the strategy of parathyroidectomy. Based on the limited knowledge available, MGD is likely to be a genetically heterogeneous disease resulting from the interaction of germline and somatic DNA mutations together with epigenetic alterations. Furthermore, these events may combine and occur independently in parathyroid tumors within the same individual with MGD. Gene expression profiling has shown that SGD and MGD may represent distinct entities in parathyroid tumorigenesis. We are waiting for studies to analyze exactly which genes are different in SGD and MGD in order to identify potential biomarkers that can distinguish between the two forms of the disease.

GCM2 p.Tyr394Ser variant in Ashkenazi Israeli patients with suspected familial isolated hyperparathyroidism.

Context: A germline mutation can be identified in up to 10% of patients with primary hyperparathyroidism (PHPT). In 2017, a high frequency of the GCM2 [(NM_ 004752.4) c.1181A> C; p.Tyr394Ser; rs142287570] variant was reported in PHPT Ashkenazi Jews (AJ). Objective: To evaluate the presence of the GCM2 p.Tyr394Ser variant in Israeli patients addressed for genetic evaluation to characterize their phenotype and clinical management. Method: Patients with PHPT who underwent addressed for genetic screening for suspected familial hypocalciuric hypercalcemia (FHH), a family history of isolated hyperparathyroidism (FIHP), or failed parathyroidectomy with persistent PHPT were recruited. Those with normal initial selected gene sequencing or hyperparathyroid genetic panel completed the GCM2 p.Tyr394Ser variant sequencing. The prevalence of this variant was evaluated using our local genomic database. Results: A total of 42 single individuals from unrelated kindreds were evaluated. A disease-causing mutation was found in 11 (26.1%) patients: 10 were diagnosed with FHH (eight CASR and two AP2S1 mutations), and one patient had a CKN2B mutation. In 28 of the remaining patients, the GCM2 p.Tyr394Ser variant was positive in three (10.7%), and all were AJ. Within AJ (15/28, 53.5%), the rate of the p.Tyr394Ser variant was 3/15 (20%), and of those, two had a history of familial isolated hyperparathyroidism. Multi-glandular parathyroid adenoma/hyperplasia was also observed in two of these patients. No clinical or laboratory findings could discriminate patients with the GCM2 p.Tyr394Ser variant from those with FHH. Cinacalcet normalized the calcium levels in one patient. The prevalence of the GCM2 p.Tyr394Ser variant in 15,407 tests in our local genomic database was 0.98%. Conclusion: In contrast to previous observations, the GCM2 p.Tyr394Ser variant-associated phenotype may be mild in AJ with FIHP, sometimes mimicking FHH. Because surgery may be curative, surgeons should be aware of the possibility of multiple gland diseases in these patients. The clinical spectrum and clinical utility of screening for this variant warrant further investigation.

Biochemical markers and FokI and TaqI vitamin D receptor genes polymorphism in rheumatoid arthritis.

BACKGROUND: Previous studies have reported the role of genes in different metabolic processes in the human body, and any variation in gene polymorphisms could lead to disturbances in these processes and different diseases. OBJECTIVE: This study aimed to compare vitamin D receptor (VDR) FokI and TaqI genotypes in terms of parathyroid hormone (PTH) and some biomarkers of inflammation and susceptibility to rheumatoid arthritis (RA) disease. METHODS: This study included 100 patients with rheumatoid arthritis (RA). Genotyping was performed by polymerase chain reaction (PCR) and examined by specific restriction enzymes using restriction fragment length polymorphism (RFLP). Serum intact PTH, C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), rheumatoid factor (RF), and anti-cyclic citrullinated peptide antibodies (ACCPs) levels were measured. RESULTS: An increased PTH level (> 65 pg/ml) was found in 8% of patients. No significant differences among FokI and TaqI vitamin D receptor genes polymorphism regarding positive and negative RF or ACCPs were found. A significant difference was found among FokI (p = 0.009) and none in TaqI genotypes regarding intact parathyroid hormone level categories. No significant correlation was found between the serum intact PTH level and ESR or CRP levels (P = 0.13 and 0.28, respectively). The parathyroid hormone level was not a good predictor for RF or ACCPs (P = 0.5 and 0.06, respectively). CONCLUSION: The FokI gene may play a role in controlling PTH levels in patients with RA. There was no significant correlation found between the serum intact PTH level and RA severity according to ESR and CRP inflammatory biomarkers. There are no differences between VDR genes FokI and TaqI polymorphism in terms of RA susceptibility (for RF and ACCPs).

Rare Variant of PTH1R Mutation in an Indian Family.

INTRODUCTION: Murk Jansen metaphyseal chondrodysplasia is an extremely rare form of skeletal dysplasia. It is caused by the mutation in PTH1R gene (1). MATERIALS: A 13 year old boy presented with history of progressive bowing of both legs since 5 years of age. He had no history of development delay, seizures, renal stones or abdominal distension. On examination, he was having prominent upper face, prominent tip of nose, long philtrum, small mandible and severe bowing of legs with deformed knee joint. His bone mineral profile came out to be normal. His skeletal survey showed severe metaphyseal dysplasia of long bones of lower limb. His genetic testing revealed heterozygous mutation in PTH1R gene, c.1562G>A variant in exon 16. On extended evaluation, his father and paternal grandmother were also having similar phenotype, however not as severely affected as the index case. RESULT: Murk Jansen metaphyseal chondrodysplasia is characterized by severe short stature, short bowed limbs, clinodactyly and dysmorphic facies with metabolic derangement of hypercalcemia and hypophosphatemia (2). The variant present in our patient has not been reported anywhere yet, hence revealing a new molecular mechanism to an already known rare disease. CONCLUSION: Molecular diagnosis of skeletal dysplasia is of paramount importance as they are a clinically heterogenous group with varied presentation with non-specific radiological findings, however with different treatment and prognostic implications. References Nampoothiri S, Fernández-Rebollo E, Yesodharan D, et al. Jansen metaphyseal chondrodysplasia due to heterozygous H223R PTH1R mutations with or without overt hypercalcemia. J Clin Endocrinol Metab 2016;101(11):4283-4289. Schipani E, Langman CB, Parfitt AM, et al. Constitutively activated receptors for parathyroid hormone and parathyroid hormone- related peptide in Jansen's metaphyseal chondrodysplasia. N Engl J Med 1996;335(10):708-714.

Single-cell RNA sequencing reveals the role of cell heterogeneity in the sex difference in primary hyperparathyroidism.

Objective: To explore the difference in parathyroid tissue-derived cells between male and female PHPT patients. Methods: Resected parathyroid tissues were collected from PHPT patients of both sexes. Single cells were isolated and sequenced for RNA expression profiles. The cell sequencing data were annotated by cell type, followed by population analysis, functional analysis, pathway analysis, cell communication analysis, differential gene expression analysis, and pseudotime trajectory analysis. The subcluster analyses were also performed in the parathyroid cells. Results: No substantial difference in the cell population, function, or communication is found between the two sexes. The interferon-a response, oxidative phosphorylation, and reactive oxygen species pathways are up-regulated in females than in male patients, mainly contributed by fibroblast cells, endothelial cells, parathyroid cells, and myeloid cells, which also have significantly more up-regulated pathways and cellular interactions than the other three cell types. The subcluster analysis of parathyroid cells identified five subpopulations: SPARCL1-OC and ISG15-OC are predominant in females, while more S100A13-PCC and PTHLH-OC are found in males. The cellular functions are also elevated in females compared with males. Cells from female patients show a higher expression level of parathyroid hormone (PTH) but a lower expression level of parathyroid hormone-like hormone (PTHLH). The cell pseudotime trajectory and pathway analyses show that the oxyphil cells may be more mature and functionally active than the chief cells in both sexes. Conclusion: These findings suggest that the sex difference in PHPT may be caused by the differentially expressed genes and activated pathways in different cell types in the parathyroid tissue. The heterogeneity of parathyroid cell subpopulations, especially in oxyphil cells, may be associated with the sex differences in PHPT pathogenesis.

A parathyroid hormone/salt-inducible kinase signaling axis controls renal vitamin D activation and organismal calcium homeostasis.

The renal actions of parathyroid hormone (PTH) promote 1,25-vitamin D generation; however, the signaling mechanisms that control PTH-dependent vitamin D activation remain unknown. Here, we demonstrated that salt-inducible kinases (SIKs) orchestrated renal 1,25-vitamin D production downstream of PTH signaling. PTH inhibited SIK cellular activity by cAMP-dependent PKA phosphorylation. Whole-tissue and single-cell transcriptomics demonstrated that both PTH and pharmacologic SIK inhibitors regulated a vitamin D gene module in the proximal tubule. SIK inhibitors increased 1,25-vitamin D production and renal Cyp27b1 mRNA expression in mice and in human embryonic stem cell-derived kidney organoids. Global- and kidney-specific Sik2/Sik3 mutant mice showed Cyp27b1 upregulation, elevated serum 1,25-vitamin D, and PTH-independent hypercalcemia. The SIK substrate CRTC2 showed PTH and SIK inhibitor-inducible binding to key Cyp27b1 regulatory enhancers in the kidney, which were also required for SIK inhibitors to increase Cyp27b1 in vivo. Finally, in a podocyte injury model of chronic kidney disease-mineral bone disorder (CKD-MBD), SIK inhibitor treatment stimulated renal Cyp27b1 expression and 1,25-vitamin D production. Together, these results demonstrated a PTH/SIK/CRTC signaling axis in the kidney that controls Cyp27b1 expression and 1,25-vitamin D synthesis. These findings indicate that SIK inhibitors might be helpful for stimulation of 1,25-vitamin D production in CKD-MBD.

SPP1 and UMOD gene variants are synergistically associated with risk of renal stone disease.

OBJECTIVE: Calcium and oxalate are the most abundant metabolites present in the stone matrix. The SPP1 and UMOD gene has specific expression in kidneys and are involved in various stages of stone formation. Therefore, genetic variants in the SPP1 and UMOD genes may enhance the development of renal stone disease. This study has been designed to understand the association of genetic variants of SPP1 and UMOD genes with renal stone disease. MATERIALS AND METHOD: A prospective study has been carried out, including 150 renal stone disease patients and 150 healthy individuals. Biochemical parameters were performed, including serum calcium levels, creatinine, parathyroid hormone, and 24-Hour urine metabolites. The genotyping of SPP1 (rs1126616) and UMOD (rs4293393) gene variants were performed using a customized TaqMan probe. T-test was used for continuous biochemical data analysis. The Chi-square test has been applied to assess the risk of a particular genotype associated with renal stone disease. In addition, correlation analysis for biochemical parameters and genetic variants with the renal stone disease has been performed using Shapley additive explanations (SHAP) values calculated with the help of the pycaret library. RESULT: Renal stone patients had significantly higher levels of parathyroid hormone (93.37 ± 52.78 pg/ml vs 64.67 ± 31.50 pg/ml, P=<0.0001), serum creatinine (0.94 ± 0.38 mg/dl vs 0.77 ± 0.17 mg/dl, P=<0.0001) and 24hr urine metabolites in comparison to the healthy controls. Heterozygous (CT) variant of SPP1 and homozygous (GG) variant of UMOD genes were significantly associated with an increased risk of developing the renal stone disease (p = 0.0100, OR = 2.06, 95 %CI = 1.13-3.75; p=<0.0001, OR = 5.773, 95 % CI = 2.03-16.38, respectively). Individuals with hyperparathyroidism and CC (SPP1) and GG (UMOD) genotypes have a high risk (P = 0.0055, OR = 2.75, 95 %CI = 1.35-5.67; P = 0.0129, OR = 10.03, 95 %CI = 1.60-110.40, respectively) of developing a renal stone. In addition, individuals with hypercalciuria and TT genotype of SPP1 (P = 0.0112, OR = 2.92, 95 % CI = 1.33-6.35), AG genotype of UMOD (P=<0.0001, OR = 5.45, 95 %CI = 2.24-13.96) and GG genotype of UMOD (P=<0.0001, OR = 10.02, 95 %CI = 3.53-24.63) have high risk of developing renal stones. Moreover, Individuals with hyperoxaluria and AG + GG (UMOD) genotype have a greater risk (P=<0.0001, OR = 7.35, 95 % CI = 3.83-13.68) of developing a renal stone. The renal stone risk was persistent (P=<0.0002, OR = 2.44, 95 % CI = 1.52-3.86) when analyzed for the synergistic effect of risk genotypes of SPP1 (CT) and UMOD (GG) gene. Further, correlation analysis also confirmed the strong association between genetic variants and renal stone development. CONCLUSION: Genetic variants of the SPP1 and UMOD genes were associated with renal stone disease. In the presence of risk genotype and hyperparathyroidism, hypercalciuria, and hyperoxaluria, the susceptibility to develop the renal stone disease risk gets modulated.

Homozygous Ser-1 to Pro-1 mutation in parathyroid hormone identified in hypocalcemic patients results in secretion of a biologically inactive pro-hormone.

Like other secreted peptides, nascent parathyroid hormone (PTH) is synthesized with a pre- and a pro-sequence (25 and 6 amino acids, respectively). These precursor segments are sequentially removed in parathyroid cells before packaging into secretory granules. Three patients from two unrelated families who presented during infancy with symptomatic hypocalcemia were found to have a homozygous serine (S) to proline (P) change affecting the first amino acid of the mature PTH. Unexpectedly, biological activity of synthetic [P1]PTH(1-34) was indistinguishable from that of unmodified [S1]PTH(1-34). However, in contrast to conditioned medium from COS-7 cells expressing prepro[S1]PTH(1-84), medium from cells expressing prepro[P1]PTH(1-84) failed to stimulate cAMP production despite similar PTH levels when measured by an intact assay that detects PTH(1-84) and large amino-terminally truncated fragments thereof. Analysis of the secreted, but inactive PTH variant led to the identification of pro[P1]PTH(-6 to +84). Synthetic pro[P1]PTH(-6 to +34) and pro[S1]PTH(-6 to +34) had much less bioactivity than the corresponding PTH(1-34) analogs. Unlike pro[S1]PTH(-6 to +34), pro[P1]PTH(-6 to +34) was resistant to cleavage by furin suggesting that the amino acid variant impairs preproPTH processing. Consistent with this conclusion, plasma of patients with the homozygous P1 mutation had elevated proPTH levels, as determined with an in-house assay specific for pro[P1]PTH(-6 to +84). In fact, a large fraction of PTH detected by the commercial intact assay represented the secreted pro[P1]PTH. In contrast, two commercial biointact assays that use antibodies directed against the first few amino acid residues of PTH(1-84) for capture or detection failed to detect pro[P1]PTH.

Circ_CUX1/miR-130b-5p/p300 axis for parathyroid hormone-stimulation of Runx2 activity in rat osteoblasts: A combined bioinformatic and experimental approach.

Parathyroid hormone (PTH) regulates the expression of bone remodeling genes by enhancing the activity of Runx2 in osteoblasts. p300, a histone acetyltransferase, acetylated Runx2 to activate the expression of its target genes. PTH stimulated the expression of p300 in rat osteoblastic cells. Increasing studies suggested the potential of non-coding RNAs (ncRNAs), such as microRNAs (miRNAs) and circular RNAs (circRNAs), in regulating gene expression under both physiological and pathological conditions. In this study, we hypothesized that PTH regulates Runx2 activity via ncRNAs-mediated p300 expression in rat osteoblastic cells. Bioinformatics and experimental approaches identified PTH-upregulation of miR-130b-5p and circ_CUX1 that putatively target p300 and miR-130b-5p, respectively. An antisense-mediated knockdown of circ_CUX1 was performed to determine the sponging activity of circ_CUX1. Knockdown of circ_CUX1 promoted miR-130b-5p activity and reduced p300 expression, resulting in decreased Runx2 acetylation in rat osteoblastic cells. Further, bioinformatics analysis identified the possible signaling pathways that regulate Runx2 activity and osteoblast differentiation via circ_CUX1/miR-130b-5p/p300 axis. The predicted circ_CUX1/miR-130b-5p/p300 axis might pave the way for better diagnostic and therapeutic approaches for bone-related diseases.

Resistance to Thyroid Hormones: A Case-Series Study.

The aim of the study is to describe the clinical features of two unrelated patients with resistance to thyroid hormones (RTH), the first, a total thyroidectomized patient, and the second, a pregnant woman. We report the features found in her newborn who also showed RTH. Patient 1 is a 38-year-old man with total thyroidectomy managed for excessive thyroid stimulating hormone (TSH) production, which poorly responded to the replacement therapy. He was found with a THRß c.1378G>A p.(Glu460Lys) heterozygous mutation, which was also present in other members of his family (son, brother, and father). Interestingly, Patient 1 had hypertension, dyslipidemia, and hepatic steatosis, which have been recently suggested as RTH-related comorbidities. Patient 2 is a 32-year-old pregnant woman with multinodular goiter, and the THRß heterozygous variant c.959G>C, that, to the best of our knowledge, has been reported in literature only once. Her newborn had tachycardia and increased thyroid hormone levels, and showed the same mutation. After delivery, high parathyroid hormone (PTH) and calcium serum levels were found in Patient 2 and the scintigraphy showed the presence of adenoma of a parathyroid gland. This case-series study provides a practical example of the management of RTH in a thyroidectomized patient, a pregnant woman, and a newborn. A novel RTH pathogenic mutation is described for the second time in literature. Furthermore, the importance of metabolic assessment in patients with RTHß has been highlighted and the possible correlation between RTH and primary hyperparathyroidism is discussed.

Structural pharmacology of PTH and PTHrP.

Parathyroid hormone (PTH) and PTH-related peptide (PTHrP) regulate extracellular phosphate and calcium homeostasis as well as bone remodeling. PTH is a classic endocrine peptide hormone whose synthesis and negative feedback by multiple factors control release from the parathyroid glands. PTHrP is ubiquitously expressed (pre- and postnatally) and acts in an autocrine/paracrine manner. This review considers the structural pharmacology and actions of PTH and PTHrP, biological consequences of inherited mutations, engineered analogs that illuminate similarities and differences in physiologic actions, and targeted therapeutic opportunities.

Parathyroid hormone-related protein (PTHrP)-dependent modulation of gene expression signatures in cancer cells.

PTHrP is encoded by PTHLH gene which can generate by alternative promoter usage and splicing mechanisms at least three mature peptides of 139, 141 and 173 amino acids with distinct carboxy terminus. PTHrP may undergo proteolytic processing into smaller bioactive forms, comprising an amino terminus peptide, which is the mediator of the "classical" PTH-like effect, as well as midregion and carboxy terminus peptides that act as multifaceted critical regulator of proliferation, differentiation and apoptosis via the reprogramming of gene expression in normal and neoplastic cells. Moreover, a nuclear/nucleolar localization signal sequence is present in the [87-107] domain allowing PTHrP nuclear import and "intracrine" effect additional to the autocrine/paracrine one. Within the large number of data available in the literature on PTHrP bioactivities, the goal of this chapter is to pick up selected studies that report the detection of molecular signatures of cancer cell exposure to PTHrP, either as full-length protein or discrete peptides, demonstrated by individual gene or whole genome expression profiling, briefly recapitulating the biological implications associated with the specific gene activation or silencing.

Gene expression profiles in parathyroid adenoma and normal parathyroid tissue.

A parathyroid adenoma comprises 80-85% as a cause of primary hyperparathyroidism. The clonal origin of most parathyroid adenomas suggests a defect at the level of the gene controlling growth of the parathyroid cell or the expression of parathyroid hormone (PTH). Two genes, MEN1 and CCND1, a tumor suppressor and a proto-oncogene respectively, have been solidly established as primary tumorigenic drivers in parathyroid adenomas. As well, germline and somatic mutation of other genes involved in cell cycle regulation or PTH regulation have been discovered in parathyroid adenomas. Moreover, comparative genomic studies between parathyroid adenomas and normal parathyroid tissues have suggested more complex genetic landscape. Microarray analysis have revealed differential expression profiles of genes involved in cell cycle regulation, growth factors, apoptotic pathway, or PTH synthesis or regulation pathway such as CASR, GCM2 and KL (Klotho). Furthermore, recent next-generation sequencing analysis reconfirmed previous finding or revealed novel finding, suggesting signal peptidase complex subunit (SPCS2), ribosomal proteins (RPL23, RPL26, RPN1, RPS25), the endoplasmic reticulum membrane (SEC11C, SEC11A, SEC61G), Klotho, cyclin D1, ß-catenin, VDR, CaSR and GCM2 may be important factors contributing to the parathyroid adenoma.

The parathyroid glands and parathyroid hormone: Insights from PTH gene mutations.

Nine mutations have been discovered in the parathyroid hormone (PTH) gene since it was initially sequenced in 1983. An autosomal dominant C18R mutation in the signal peptide was first reported in 1990, followed by an exon skipping mutation, leading to loss of exon 2 in 1992; the latter mutation prevents PTH biosynthesis, as exon 2 contains the initiation codon. The S23P and S23X mutations affecting the same residue were reported in 1999 and 2012, respectively, while in 2008, the somatic mutation, R83X, was detected in a parathyroid adenoma tissue sample from a patient with overt hyperparathyroidism. In 2013, the heterozygous p.Met1_Asp6del mutation was discovered incidentally in a case-control study, while another heterozygous mutation, M14K, was detected in the signal peptide 4 years later. In 2015, a homozygous R56C mutation was reported, and was the first hypoparathyroidism-causing mutation identified that affects the mature bioactive portion of PTH; this mutation has significantly contributed to the understanding of the molecular mechanisms involved in signal transduction through the PTH receptor. Recently, a novel homozygous S32P mutation was identified, which is also situated in the bioactive portion of PTH. The discovery of these nine mutations in the PTH gene and determination of the molecular mechanisms underlying their effects has provided deep insights into the synthesis, processing, and secretion of PTH. Future attempts to discover other such mutations will help elucidate as yet unknown functions of PTH, with potential clinical implications.

Interactome of PTH-Regulated miRNAs and Their Predicted Target Genes for Investigating the Epigenetic Effects of PTH (1-34) in Bone Metabolism.

Osteoporosis is a metabolic bone disease that mostly affects the elderly. A lot of drugs are available, mostly with an antiresorptive effect but just a few with an osteoanabolic effect, meaning they promote bone building. PTH (1-34) or teriparatide is an osteoanabolic drug, but its efficacy varies between individuals. We performed a literature review and extracted a dataset of 62 microRNAs (miRNAs) from 10 different studies; predicted miRNA target interactions (MTIs) were obtained with the help of four software tools: DIANA, miRWalk, miRDB and TargetScan. With the construction of an interactome of PTH-regulated miRNAs and their predicted target genes, we elucidated miR-146a-5p, miR-551b-5p, miR-205-3p, miR-33a-3p, miR-338-5p as miRNAs with the most interactions and miR-410-3p as the miRNA targeting bone-related pathways with the highest significance. These miRNAs could help in further understanding the mechanism of action of PTH on bone metabolism and osteoporosis. They also have the potential for novel network-based biomarkers for osteoporosis treatment efficacy and safety and as new therapeutic targets.

Familial Pseudohypoparathyroidism Type IB Associated with an SVA Retrotransposon Insertion in the GNAS Locus.

Loss of methylation (LOM) at GNAS-A/B:TSS-differentially methylated regions (DMRs) in the GNAS locus is observed in pseudohypoparathyroidism type 1B (PHP1B). Many PHP1B cases are sporadic, but autosomal dominant-PHP1B has a deletion involving NESP55 expressed from the maternal allele or STX16 located upstream of the GNAS locus on the maternal allele. We report the possible first familial PHP1B cases with retrotransposon insertion in the GNAS locus on the maternal allele. To our knowledge, they are the possible first cases with imprinting disorders caused by retrotransposon insertion. The two sibling cases experienced tetany and/or cramps from school age and had hypocalcemia and an increased serum intact parathyroid hormone (PTH) level together with overweight, round face, and normal intellectual levels. Methylation analysis for DMRs in the GNAS locus showed only LOM of the GNAS-A/B:TSS-DMR. Copy number abnormalities at STX16 and the GNAS locus were not detected by array comparative genomic hybridization. Whole-genome sequencing and Sanger sequencing revealed an approximately 1000-bp SVA retrotransposon insertion upstream of the first exon of A/B on the GNAS locus in these siblings. Whole-genome methylome analysis by Enzymatic Methyl-Seq in the siblings showed normal methylation status in the region surrounding the insertion site and mild LOM of the GNAS-A/B:TSS-DMR. We conducted transcriptome analysis using mRNA from skin fibroblasts and induced pluripotent stem cells (iPSCs) derived from the siblings and detected no aberrant NESP55 transcripts. Quantitative reverse-transcriptase PCR (qRT-PCR) analysis in skin fibroblasts showed increased A/B expression in the patients and no NESP55 expression, even in a control. qRT-PCR analysis in iPSCs showed decreased NESP55 expression with normal methylation status of the GNAS-NESP:TSS-DMR in the patients. The retrotransposon insertion in the siblings likely caused decreased NESP55 expression that could lead to increased A/B expression via LOM of the GNAS-A/B:TSS-DMR, subsequent reduced Gsα expression, and finally, PHP1B development. © 2022 American Society for Bone and Mineral Research (ASBMR).

Multilevel Annotation of Germline MEN1 Variants of Synonymous, Nonsynonymous, and Uncertain Significance in Indian Patients With Sporadic Primary Hyperparathyroidism.

Primary hyperparathyroidism (PHPT) is third most common endocrine disorder characterized by hypercalcemia with elevated or nonsuppressed parathyroid hormone levels by parathyroid tumors. Familial PHPT, as part of multiple endocrine type-1, occurs due to the germline mutation in the MEN1 gene. The involvement and the role of germline MEN1 variations in sporadic PHPT of Indian PHPT patients are unknown. Precise classifications of different types of MEN1 variations are fundamental for determining clinical relevance and diagnostic role. This prospective cohort study was performed on 82 patients with PHPT (with no clinical or history of MEN1) who underwent screening for MEN1 variations through Sanger sequencing. Multilevel computational analysis was performed to determine the structure-function relationship of synonymous, nonsynonymous, and variants of uncertain significance (VUS). Of the 82 PHPT patients, 42 (51%) had 26 germline MEN1 variants, including eight nonsynonymous, seven synonymous, nine VUS, one splice site, and one regulatory variation. Five most common germline variations (c.1838A>G, c.1817C>T, c.1525C>A, c.-35A>T, and c.250T>C) were observed in this study. c.-35A>T (5' untranslated region [UTR]) was associated with recurrence of PHPT (odds ratio [OR] = 5.4; p = 0.04) and subsequent detection of other endocrine tumors (OR = 13.6, p = 0.035). c.1525C>A was associated with multi glandular parathyroid tumor (OR = 13.6, p = 0.035). Align-Grantham variation and Grantham deviation (Align-GVGD), functional analysis through hidden Markov MODEL (FATHMM), and MutationTaster analysis reported the disease-specific potential of VUS and synonymous variations. Significant linkage disequilibrium was observed in c.1785G>A and c.1817C>T (r2  = 0.3859, p = 0.0001), c.1475C>G and c.1525C>A (r2  = 0.385, p = 0.0004), and c.1569T>C and c.1838A>G (r2  = 0.488, p = 0.0001). The detection of MEN1 variations, especially those with disease-specific potential, can prompt early screening for other MEN1-related tumors and disease recurrence. © 2022 American Society for Bone and Mineral Research (ASBMR).

Bioactive, full-length parathyroid hormone delivered using an adeno-associated viral vector.

Delivering the parathyroid hormone (PTH) gene has been attempted preclinically in a handful of studies, but delivering full-length PTH (1-84) using adeno-associated viral (AAV) vectors has not. Given the difficulty in achieving therapeutic levels of secreted proteins using gene therapy, this study seeks to determine the feasibility of doing so with PTH. An AAV vector was used to deliver human PTH driven by a strong promoter. We demonstrate the ability to secrete full-length PTH from various cell types in vitro. PTH secretion from hepatocytes was measured over time and a fluorescent marker was used to compare the secretion rate of PTH in various cell types. Potency was measured by the ability of PTH to act on the PTH receptors of osteosarcoma cells and induced proliferation. PTH showed potency in vitro by inducing proliferation in two osteosarcoma cell lines. In vivo, AAV was administered systemically in immunocompromised mice which received xenografts of osteosarcoma cells. Animals that received the highest dose of AAV-PTH had higher liver and plasma concentrations of PTH. All dosing groups achieved measurable plasma concentrations of human PTH that were above the normal range. The high-dose group also had significantly larger tumors compared to control groups on the final day of the study. The tumors also showed dose-dependent differences in morphology. When looking at endocrine signaling and endogenous bone turnover, we observed a significant difference in tibial growth plate width in animals that received the high-dose AAV as well as dose-dependent changes in blood biomarkers related to PTH. This proof-of-concept study shows promise for further exploration of an AAV gene therapy to deliver full-length PTH for hypoparathyroidism. Additional investigation will determine efficacy in a disease model, but data shown establish bioactivity in well-established models of osteosarcoma.