Determining the pathogenicity of MUT gene variant by mini-gene splicing assay

ObjectiveTo perform gene mutation splicing analysis on 12 suspected pathogenic MMUT/MUT gene mutation sites to verify the pathogenicity of the mutation.MethodsWild-type and mutant minigenes were inserted into pcMINI vector, and total 5 wild-type recombinant vectors and 12 mutant recombinant vectors were constructed. The total RNA in 293T cells was extracted after the recombinant vectors were transfected into 293T cell line. Then PCR products were detected by agarose gel electrophoresis and analyzed by sequencing.ResultsRT-PCR and sequencing results showed that among 12 mutations, 9 mutations (c.419T>C, 469G>T, c.470T>A, c.626dupC, c.693C>G, c.976A>G, c.1009T>C, c.1777G>T and c.1874A>C) did not affect the gene splicing, and the other 3 mutations (c.454C>T, c.421G>A and c.2125-3C>G) all affected mRNA splicing.ConclusionIn recent case reports of MMUT/MUT gene mutation sites, variant of uncertain significance (VUS) variation is very common. In this study, the pathogenicity of three mutation sites is confirmed by the mini-gene method.

Proteomics Reveals that Methylmalonyl-CoA Mutase Modulates Cell Architecture and Increases Susceptibility to Stress

Methylmalonic acidemia (MMA) is a rare inborn error of metabolism caused by deficiency of the methylmalonyl-CoA mutase (MUT) enzyme. Downstream MUT deficiency, methylmalonic acid accumulates together with toxic metabolites from propionyl-CoA and other compounds upstream of the block in the enzyme pathway. The presentation is with life-threatening acidosis, respiratory distress, brain disturbance, hyperammonemia, and ketosis. Survivors develop poorly understood multi-organ damage, notably to the brain and kidneys. The HEK 293 cell line was engineered by CRISPR/Cas9 technology to knock out the MUT gene (MUT-KO). Shotgun label-free quantitative proteomics and bioinformatics analyses revealed potential damaging biological processes in MUT-deficient cells. MUT-KO induced alteration of cellular architecture and morphology, and ROS overproduction. We found the alteration of proteins involved in cytoskeleton and cell adhesion organization, cell trafficking, mitochondrial, and oxidative processes, as validated by the regulation of VIM, EXT2, SDC2, FN1, GLUL, and CHD1. Additionally, a cell model of MUT-rescuing was developed in order to control the specificity of MUT-KO effects. Globally, the proteomic landscape of MUT-KO suggests the cell model to have an increased susceptibility to propionate- and H2O2-induced stress through an impairment of the mitochondrial functionality and unbalances in the oxidation-reduction processes.

Integration of proteomics and metabolomics data in a novel cellular knock out model of methylmalonic acidemia

Background. Methylmalonic acidemia is a rare inborn error of metabolism caused by mutations in methylmalonyl−CoA mutase (MUT) gene. As intermediate of propionate metabolism, MUT converts methylmalonyl−CoA into succinyl−CoA, which enters the Krebs cycle. Downstream MUT deficiency, methylmalonic acid accumulates in body fluids as biomarker of disease. The long-term complications of the disease can include cognitive and neurological impairment, chronic kidney disease, liver failure, and death. Methods. In order to create a valid cellular model to study the disease, MUT gene was knocked out (KO) in HEK293 cell line by using CRISPR-CAS9 technology. Methylmalonic acid was measured in MUT-KO and wild type (WT) cells by multiple reaction monitoring. A quantitative proteomics analysis was carried out using a label-free mass spectrometry-based approach. Data were processed using MaxQuant software. Moreover, a targeted metabolomics analysis was performed in order to measure an entire panel of amino acids and acylcarnitines. Results. Methylmalonic acid resulted increased in KO cells if compared with WT ones. The proteomic dataset showed a number of 69 differentially expressed proteins, of which 39 down-regulated and 30 up-regulated in the MUT-KO condition. Gene Ontology analysis revealed an enrichment in energy and lipid metabolism categories. The variations in the metabolomic profile are indicative of alterations in fatty acid oxidation processes and lipid metabolism.

Integration of proteomics and metabolomics data in a novel cellular knock out model of methylmalonic acidemia

Background. Methylmalonic acidemia is a rare inborn error of metabolism caused by mutations in methylmalonyl−CoA mutase (MUT) gene. As intermediate of propionate metabolism, MUT converts methylmalonyl−CoA into succinyl−CoA, which enters the Krebs cycle. Downstream MUT deficiency, methylmalonic acid accumulates in body fluids as biomarker of disease. The long-term complications of the disease can include cognitive and neurological impairment, chronic kidney disease, liver failure, and death. Methods. In order to create a valid cellular model to study the disease, MUT gene was knocked out (KO) in HEK293 cell line by using CRISPR-CAS9 technology. Methylmalonic acid was measured in MUT-KO and wild type (WT) cells by multiple reaction monitoring. A quantitative proteomics analysis was carried out using a label-free mass spectrometry-based approach. Data were processed using MaxQuant software. Moreover, a targeted metabolomics analysis was performed in order to measure an entire panel of amino acids and acylcarnitines. Results. Methylmalonic acid resulted increased in KO cells if compared with WT ones. The proteomic dataset showed a number of 69 differentially expressed proteins, of which 39 down-regulated and 30 up-regulated in the MUT-KO condition. Gene Ontology analysis revealed an enrichment in energy and lipid metabolism categories. The variations in the metabolomic profile are indicative of alterations in fatty acid oxidation processes and lipid metabolism.

Methylmalonic Acidemia with Novel MUT Gene Mutations

A 5-year-old boy presented with recurrent episodes of fever, feeding problems, lethargy, from the age of 11 months, and poor weight gain. He was admitted and evaluated for metabolic causes and diagnosed as having methylmalonic acidemia (MMA). He was treated with vit B12 and carnitine supplements and has been on follow-up for the last 3 years. Mutation analysis by next generation sequencing (NGS), supplemented with Sanger sequencing, revealed two novel variants in the MUT gene responsible for MMA in exon 5 and exon 3, respectively. Recently he developed dystonic movements including orofacial dyskinesia. With advent of NGS, judicious use of NGS with Sanger sequencing can help identify causative possibly pathogenic mutations.