Exome sequencing and metabolomic analysis of a chronic kidney disease and hearing loss patient family revealed RMND1 mutation induced sphingolipid metabolism defects.

Mitochondrial disorders (MIDs) shows overlapping clinical presentations owing to the genetic and metabolic defects of mitochondria. However, specific relationship between inherited mutations in nuclear encoded mitochondrial proteins and their functional impacts in terms of metabolic defects in patients is not yet well explored. Therefore, using high throughput whole exome sequencing (WES), we screened a chronic kidney disease (CKD) and sensorineural hearing loss (SNHL) patient, and her family members to ascertain the mode of inheritance of the mutation, and healthy population controls to establish its rare frequency. The impact of mutation on biophysical characteristics of the protein was further studied by mapping it in 3D structure. Furthermore, LC-MS tandem mass spectrophotometry based untargeted metabolomic profiling was done to study the fluctuations in plasma metabolites relevant to disease causative mutations and kidney damage. We identified a very rare homozygous c.631G > A (p.Val211Met) pathogenic mutation in RMND1 gene in the proband, which is inherited in an autosomal recessive fashion. This gene is involved in the mitochondrial translational pathways and contribute in mitochondrial energy metabolism. The p.Val211Met mutation is found to disturb the structural orientation (RMSD is -2.95 Å) and stability (ΔΔG is -0.552 Kcal/mol) of the RMND1 protein. Plasma metabolomics analysis revealed the aberrant accumulation of metabolites connected to lipid and amino acid metabolism pathways. Of these metabolites, pathway networking has discovered ceramide, a metabolite of sphingolipids, which plays a role in different signaling cascades including mitochondrial membrane biosynthesis, is highly elevated in this patient. This study suggests that genetic defects in RMND1 gene alters the mitochondrial energy metabolism leading to the accumulation of ceramide, and subsequently promote dysregulated apoptosis and tissue necrosis in kidneys.

LC-MS/MS-based metabolic profiling of Escherichia coli under heterologous gene expression stress.

Escherichia coli is frequently exploited for genetic manipulations and heterologous gene expression studies. We have evaluated the metabolic profile of E. coli strain BL21 (DE3) RIL CodonPlus after genetic modifications and subjecting to the production of recombinant protein. Three genetically variable E. coli cell types were studied, normal cells (susceptible to antibiotics) cultured in simple LB medium, cells harboring ampicillin-resistant plasmid pET21a (+), grown under antibiotic stress, and cells having recombinant plasmid pET21a (+) ligated with bacterial lactate dehydrogenase gene grown under ampicillin and standard isopropyl thiogalactoside (IPTG)-induced gene expression conditions. A total of 592 metabolites were identified through liquid chromatography-mass spectrometry/mass spectrometry analysis, feature and peak detection using XCMS and CAMERA followed by precursor identification by METLIN-based procedures. Overall, 107 metabolites were found differentially regulated among genetically modified cells. Quantitative analysis has shown a significant modulation in DHNA-CoA, p-aminobenzoic acid, and citrulline levels, indicating an alteration in vitamin K, folic acid biosynthesis, and urea cycle of E. coli cells during heterologous gene expression. Modulations in energy metabolites including NADH, AMP, ADP, ATP, carbohydrate, terpenoids, fatty acid metabolites, diadenosine tetraphosphate (Ap4A), and l-carnitine advocate major metabolic rearrangements. Our study provides a broader insight into the metabolic adaptations of bacterial cells during gene manipulation experiments that can be prolonged to improve the yield of heterologous gene products and concomitant production of valuable biomolecules.

Hypermethylation of CpG islands and shores around specific microRNAs and mirtrons is associated with the phenotype and presence of bladder cancer.

PURPOSE: To analyze the role and translational potential for hypermethylation of CpG islands and shores in the regulation of small RNAs within urothelial cell carcinoma (UCC). To examine microRNAs (miR) and mirtrons, a new class of RNA located within gene introns and processed in a Drosha-independent manner. EXPERIMENTAL DESIGN: The methylation status of 865 small RNAs was evaluated in normal and malignant cell lines by using 5-azacytidine and microarrays. Bisulfite sequencing was used for CpG regions around selected RNAs. Prognostic and diagnostic associations for epigenetically regulated RNAs were examined by using material from 359 patients, including 216 tumors and 121 urinary samples (68 cases and 53 controls). Functional analyses examined the effect of silencing susceptible RNAs in normal urothelial cells. RESULTS: Exonic/UTR-located miRs and mirtons are most susceptible to epigenetic regulation. We identified 4 mirtrons and 16 miRs with CpG hypermethylation across 35 regions in normal and malignant urothelium. For several miRs, hypermethylation was more frequent and dense in CpG shores than islands (e.g., miRs-9/149/210/212/328/503/1224/1227/1229), and was associated with tumor grade, stage, and prognosis (e.g., miR-1224 multivariate analysis OR = 2.5; 95% CI, 1.3-5.0; P = 0.006). The urinary expression of epigenetically silenced RNAs (miRs-152/328/1224) was associated with the presence of UCC (concordance index, 0.86; 95% CI, 0.80-0.93; ANOVA P < 0.016). CONCLUSIONS: Hypermethylation of mirtrons and miRs is common in UCC. Mirtrons appear particularly susceptible to epigenetic regulation. Aberrant hypermethylation of small RNAs is associated with the presence and behavior of UCC, suggesting potential roles as diagnostic and prognostic biomarkers.