A multiomics approach to understanding pathology of Combined D,L-2- Hydroxyglutaric Aciduria and phenylbutyrate as potential treatment

Combined D, L-2-Hydroxyglutaric Aciduria (D,L-2HGA) is a rare genetic disorder caused by recessive mutations in the SLC25A1 gene that encodes the mitochondrial citrate carrier protein (CIC). SLC25A1 deficiency leads to a secondary increase in mitochondrial 2-ketoglutarate that, in turn, is reduced to neurotoxic 2-hydroxyglutarate. Clinical symptoms of Combined D,L-2HGA include neonatal encephalopathy, respiratory insufficiency and often with death in infancy. No current therapies exist, although replenishing cytosolic stores by citrate supplementation to replenish cytosolic stores has been proposed. In this study, we demonstrated that patient derived fibroblasts exhibited impaired cellular bioenergetics that were worsened with citrate supplementation. We hypothesized treating patient cells with phenylbutyrate, an FDA approved pharmaceutical drug, would reduce mitochondrial 2-ketoglutarate, leading to improved cellular bioenergetics including oxygen consumption and fatty acid oxidation. Metabolomic and RNA-seq analyses demonstrated a significant decrease in intracellular 2-ketoglutarate, 2-hydroxyglutarate, and in levels of mRNA coding for citrate synthase and isocitrate dehydrogenase. Consistent with the known action of phenylbutyrate, detected levels of phenylacetylglutamine was consistent with the drug acting as 2-ketoglutarate sink in patient cells. Our pre-clinical studies suggest citrate supplementation is unlikely to be an effective treatment of the disorder. However, cellular bioenergetics suggests phenylbutyrate may have interventional utility for this rare disease.


Assessment of whole cell mitochondrial respiration through oximetry
Fibroblasts were seeded in 96-well Seahorse tissue culture microplates (Agilent #101085-004) at a density of 40,000 cells per well and treated with phenylbutyrate at a final concentration of 8 mM in 100 μL complete DMEM media for 72 hrs in a humidified 37 °C, 5% CO2 incubator. For each cell line, 8 replicate wells were used per experimental condition. The oxygen consumption rate (OCR) was measured with the use of a Seahorse XF Cell Mito Stress Test Kit (Agilent #103015-100) according to the manufacturer's protocol. In brief, one day prior to the assessment, the XFe96 FluxPak (Agilent #102416-100) was hydrated overnight using the Seahorse XF Calibrant Solution (Agilent #100840-000) in a humidified 37 °C non-CO2 incubator. On the day of the assessment, the seeded microplate was washed twice with Seahorse XF DMEM media (Agilent #103575-100), followed by a 1 hr incubation in in the same media supplemented with 10 mM glucose (Agilent #103577-100) and 2 mM glutamine (Agilent #103579-100) in a humidified 37 °C non-CO2 incubator. Oligomycin, FCCP and Rot/AA compounds supplied in the Mito Stress Test Kit were prepared using Seahorse XF DMEM media and used at a final well concentration of 1.5 μM, 1.0 μM and 0.5 μM respectively. Fibroblast OCR was measured on a Seahorse XFe96 Analyzer (Agilent Technologies Inc) using the default XF Cell Mito Stress protocol on the Seahorse Wave Controller Software v2.6.1. Following completion of the Seahorse assessment, 20 μL of RIPA buffer (ThermoFisher Scientific #89900) was added to the cells and 5 μL of lysate was used for protein quantification using a DC Protein Assay Kit II (Bio-Rad #5000112). The OCR reading of each well was normalized to its protein concentration.

RNA-sequencing and quantitative PCR
Three replicates of each cell line (experimental, control) per experimental condition were grown to 80% confluence in T175 flasks. Following 72 hrs of phenylbutyrate treatment in complete DMEM media, the cells were washed once with phosphate buffered saline (PBS; ThermoFisher Scientific #10010023) and trypsinized using 0.25% trypsin (Corning Life Sciences #25-053-CI). The collected cell pellets were washed once with PBS and processed for total RNA extraction using a Qiagen RNeasy Mini Kit (Qiagen #74104) with on-column DNase digestion (Qiagen #79254). RNA was eluted using nuclease-free water, followed by integrity assessment using a Fragment Analyzer System (Agilent Technologies Inc) prior to storage at -80 °C. Total RNA was submitted to GENEWIZ (South Plainfield, NJ), an Illumina CSPro certified laboratory, for Illumina HiSeq 2X150 bp sequencing using a PolyA selection approach with ~50 million reads per sample. The resulting FASTQ files were first aligned and mapped to the hg38 genome using Rsubread, followed by gene annotation and counting using featureCounts Bioconductor packages in R 1,2 . Statistical analysis was performed using limma-voom package, and genes with a positive B-statistics value were considered statistically significant 3,4 . Pathway analysis was performed using the WebGestalt tool 5,6 .
The VCF file from the RNA-seq was generated in accordance to GATK RNAseq short variant discovery (SNPs + Indels) best practices workflow 7 , followed by Exomiser analysis to prioritize genes and variants for novel disease-gene discovery or differential diagnosis of Mendelian disease 8 . Aberrant splicing event analysis was performed using the DROP-FRASER tool 9,10 , and the RNA-seq data was mapped to the GENCODE hg19 Release 34 11 using the STAR aligner twoPassMode Basic option 12 Table   6.

Metabolomics
Five replicates of fibroblasts per cell line (experimental, control) per experimental condition were grown to 80% confluence in T175 flasks with or without phenylbutyrate treatment in complete DMEM media.
Following 72 hrs of phenylbutyrate treatment, the cells were harvested as described earlier and stored at -80 °C prior to submission to Metabolon (Morrisville, NC) for global metabolomics analysis as previously described 15,16 . Per Metabolon, samples were prepared using the automated MicroLab STAR® system from Hamilton Company. Several recovery standards were added prior to extraction for QC purposes. To remove protein and dissociate small molecules bound to protein or trapped in the precipitated protein matrix, proteins were precipitated with methanol under vigorous shaking for 2 min (Glen Mills GenoGrinder 2000) followed by centrifugation. The resulting extract was divided into five fractions: two for analysis by two separate reverse phase (RP)/UPLC-MS/MS methods with positive ion mode electrospray ionization (ESI), one for analysis by RP/UPLC-MS/MS with negative ion mode ESI, one for analysis by HILIC/UPLC-MS/MS with negative ion mode ESI, and one sample was reserved for backup. Samples were placed briefly on a TurboVap® (Zymark) to remove the organic solvent. The sample extracts were stored overnight under nitrogen before preparation for analysis.
All analytical methods utilized a Waters ACQUITY ultra-performance liquid chromatography (UPLC) and a Thermo Scientific Q-Exactive high resolution/accurate mass spectrometer interfaced with a heated electrospray ionization (HESI-II) source and Orbitrap mass analyzer operated at 35,000 mass resolution. The sample extract was dried then reconstituted in solvents compatible to each of the four methods. Each reconstitution solvent contained a series of standards at fixed concentrations to ensure injection and chromatographic consistency. One aliquot was analyzed using acidic positive ion conditions, chromatographically optimized for more hydrophilic compounds. In this method, the extract was gradient eluted from a C18 column (Waters UPLC BEH C18-2.1x100 mm, 1.7 µm) using water and methanol, containing 0.05% perfluoropentanoic acid (PFPA) and 0.1% formic acid (FA). Another aliquot was also analyzed using acidic positive ion conditions; however it was chromatographically optimized for more hydrophobic compounds. In this method, the extract was gradient eluted from the same afore mentioned C18 column using methanol, acetonitrile, water, 0.05% PFPA and 0.01% FA and was operated at an overall higher organic content. Another aliquot was analyzed using basic negative ion optimized conditions using a separate dedicated C18 column. The basic extracts were gradient eluted from the column using methanol and water, however with 6.5 mM Ammonium Bicarbonate at pH 8. The fourth aliquot was analyzed via negative ionization following elution from a HILIC column (Waters UPLC BEH Amide 2.1x150 mm, 1.7 µm) using a gradient consisting of water and acetonitrile with 10mM Ammonium Formate, pH 10.8. The MS analysis alternated between MS and data-dependent MSn scans using dynamic exclusion. The scan range varied slighted between methods but covered 70-1000 m/z. Raw data was extracted, peak-identified and QC processed using Metabolon's hardware and software.
Compounds were identified by comparison to library entries of purified standards or recurrent unknown entities. Metabolon maintains a library based on authenticated standards that contains the retention time/index (RI), mass to charge ratio (m/z), and chromatographic data (including MS/MS spectral data) on all molecules present in the library. Furthermore, biochemical identifications are based on three criteria: retention index within a narrow RI window of the proposed identification, accurate mass match to the library +/-10 ppm, and the MS/MS forward and reverse scores between the experimental data and authentic standards. The MS/MS scores are based on a comparison of the ions present in the experimental spectrum to the ions present in the library spectrum. The resulting data were further analyzed using the limma-voom Bioconductor package in R to identify differential metabolites with an adjusted pvalue ≤ 0.05 3,4 .