Primary Hyperparathyroidism and Gut Microbiota Unravelling an Unexpected Relationship: the HYPOGEUM Study

Methods. The project will be organized in three working packages (WP). WP1. Determine if the propensity of patients with PHPT to develop bone loss can be predicted by the composition of stool microbiome (month 0-11). We will screen men and women age 30-80 years diagnosed with PHPT and evaluated by 3-site DXA (lumbar spine, hip, and distal radius- both 1/3 and ultra-distal), and bone turnover markers. Two groups with 45 patients each will be enrolled: 1) Subjects with normal BMD or mild osteopenia (distal radius T-score > -1.5), no fragility fractures and CTX levels that are associated with normal bone turnover (100-250 pg/ml) (not bone looser). 2) Subjects with osteoporosis (distal radius T-score < -2.5) with or without one or more fragility fractures) and CTX levels that are associated with active disease (>500 pg/ml) (bone looser). Blood samples and a faecal sample will be collected by each participant. Stool samples will be stored immediately in a cooler with -20°C freezer packs and stored at -80°C until shipped to COSMOSID for Metagenomic Shotgun sequencing and analysis of the microbiome community structure.

Metagenomics Shotgun sequencing and analysis. The microbiome diversity and composition of each stool sample will be determined by CosmosID Inc., Rockville, MD. Briefly, DNA from human faecal samples will be isolated using the QIAGEN DNeasy PowerSoil Pro Kit. DNA libraries will be prepared using the Illumina Nextera XT library preparation kit and Library quantity assessed with Qubit (ThermoFisher). Libraries will be then sequenced on an Illumina HiSeq platform 2x150bp. Bioinformatic analysis of sequencing reads will be done by CosmosID bioinformatics platform that uses high performance data mining algorithms and the highly curated dynamic comparator database GenBook®.

End points and interpretation: We will determine if PHPT patients loosing bone have a higher frequency of one or more Th17 cell-inducing-SFB equivalent bacteria as compared to control PHPT patients. This analysis will therefore yield evidence pertaining to species and strains that may exacerbate bone loss in PHPT. The DXA endpoints will be BMD, T-scores and Z-scores of the distal and 1/3 radius, lumbar spine and hip.

Power Calculation: Our analysis of the preliminary data set revealed that BL was abundant at mean relative abundance of ~5.8% (SD 0.055). From these data we calculated that enrolling 45 per group will provide 80% statistical power of detecting a difference for BL if the mean relative abundance of BL is > 1.5-fold (8.7%) higher in PHPT patients with low bone density as compared to control PHPT patients.

WP2. Evaluate how GM composition modulates immune system in humans (month 3-10). In order to evaluate Th subsets, T cells will be analysed by real time RT-PCR measuring FOXP3, IL-17A, TNF-alpha and IL-4 gene expression. Briefly, red cells will be lysed in peripheral blood samples and total nucleated cells collected and frozen at -80°C until RNA extraction. RNA will be isolated using TRIzol reagent, chloroform extraction, and subsequent isopropanol precipitation according to standard procedure. Relative cytokine expression will be determined using the 2-ΔΔCT method with normalization to β-Actin. In order to evaluate inflammation we will measure IL-17A (the main species of IL-17 produced by Th17 cells), free RANKL, OPG, TNF by ELISA technique. To assess bone turnover P1NP (a marker of bone formation), CTX (a marker of bone resorption), 25OHvitamin D (25OHD) will be measured by ELISA technique.