methylmalonyl-CoA Mutase Gene Research Articles

Genetic optimization of the human gut bacterium Phocaeicola vulgatus for enhanced succinate production

The demand for sustainably produced bulk chemicals is constantly rising. Succinate serves as a fundamental component in various food, chemical, and pharmaceutical products. Succinate can be produced from sustainable raw materials using microbial fermentation and enzyme-based technologies. Bacteroides and Phocaeicola species, widely distributed and prevalent gut commensals, possess enzyme sets for the metabolization of complex plant polysaccharides and synthesize succinate as a fermentative end product. This study employed novel molecular techniques to enhance succinate yields in the natural succinate producer Phocaeicola vulgatus by directing the metabolic carbon flow toward succinate formation. The deletion of the gene encoding the methylmalonyl-CoA mutase (Δmcm, bvu_0309-0310) resulted in a 95% increase in succinate production, as metabolization to propionate was effectively blocked. Furthermore, deletion of genes encoding the lactate dehydrogenase (Δldh, bvu_2499) and the pyruvate:formate lyase (Δpfl, bvu_2880) eliminated the formation of fermentative end products lactate and formate. By overproducing the transketolase (TKT, BVU_2318) in the triple deletion mutant, succinate production increased from 3.9 mmol/g dry weight in the wild type to 10.9 mmol/g dry weight. Overall, succinate yield increased by 180% in the new mutant strain P. vulgatus Δmcm Δldh Δpfl pG106_tkt relative to the parent strain. This approach is a proof of concept, verifying the genetic accessibility of P. vulgatus, and forms the basis for targeted genetic optimization. The increase of efficiency highlights the huge potential of P. vulgatus as a succinate producer with applications in sustainable bioproduction processes.Key points• Deleting methylmalonyl-CoA mutase gene in P. vulgatus doubled succinate production• Triple deletion mutant with transketolase overexpression increased succinate yield by 180%• P. vulgatus shows high potential for sustainable bulk chemical production via genetic optimization

Clinical outcomes of patients with mut-type methylmalonic acidemia identified through expanded newborn screening in China

BackgroundIsolated methylmalonic acidemia, an autosomal recessive disorder of propionate metabolism, is usually caused by mutations in the methylmalonyl-CoA mutase gene (mut-type). Because no universal consensus was made on whether mut-type methylmalonic acidemia should be included in newborn screening (NBS), we aimed to compare the outcome of this disorder detected by NBS with that detected clinically and investigate the influence of NBS on the disease course.Design & methodsIn this study, 168 patients with mut-type methylmalonic acidemia diagnosed by NBS were compared to 210 patients diagnosed after disease onset while NBS was not performed. Clinical data of these patients from 7 metabolic centers in China were analyzed retrospectively, including initial manifestations, biochemical metabolites, the responsiveness of vitamin B12 therapy, and gene variation, to explore different factors on the long-term outcome.ResultsBy comparison of the clinically-diagnosed patients, NBS-detected patients showed younger age at diagnosis, less incidence of disease onset, better responsiveness of vitamin B12, younger age at start of treatment, lower levels of biochemical features before and after treatment, and better long-term prognosis (P < 0.01). Onset of disease, blood C3/C2 ratio and unresponsiveness of vitamin B12 were more positively associated with poor outcomes of patients whether identified by NBS. Moreover, the factors above as well as older age at start of treatment were positively associated with mortality.ConclusionsThis research highly demonstrated NBS could prevent major disease-related events and allow an earlier treatment initiation. As a key prognostic factor, NBS is beneficial for improving the overall survival of infants with mut-type methylmalonic acidemia.

Searching for homozygous haplotype deficiency in Manech Tête Rousse dairy sheep revealed a nonsense variant in the MMUT gene affecting newborn lamb viability

BackgroundRecessive deleterious variants are known to segregate in livestock populations, as in humans, and some may be lethal in the homozygous state.ResultsWe used phased 50 k single nucleotide polymorphism (SNP) genotypes and pedigree data to scan the genome of 6845 Manech Tête Rousse dairy sheep to search for deficiency in homozygous haplotypes (DHH). Five Manech Tête Rousse deficient homozygous haplotypes (MTRDHH1 to 5) were identified, with a homozygous deficiency ranging from 84 to 100%. These haplotypes are located on Ovis aries chromosome (OAR)1 (MTRDHH2 and 3), OAR10 (MTRDHH4), OAR13 (MTRDHH5), and OAR20 (MTRDHH1), and have carrier frequencies ranging from 7.8 to 16.6%. When comparing at-risk matings between DHH carriers to safe matings between non-carriers, two DHH (MTRDHH1 and 2) were linked with decreased insemination success and/or increased stillbirth incidence. We investigated the MTRDHH1 haplotype, which substantially increased stillbirth rate, and identified a single nucleotide variant (SNV) inducing a premature stop codon (p.Gln409*) in the methylmalonyl-CoA mutase (MMUT) gene by using a whole-genome sequencing approach. We generated homozygous lambs for the MMUT mutation by at-risk mating between heterozygous carriers, and most of them died within the first 24 h after birth without any obvious clinical symptoms. Reverse transcriptase-qPCR and western blotting on post-mortem liver and kidney biological samples showed a decreased expression of MMUT mRNA in the liver and absence of a full-length MMUT protein in the mutant homozygous lambs.ConclusionsWe identified five homozygous deficient haplotypes that are likely to harbor five independent deleterious recessive variants in sheep. One of these was detected in the MMUT gene, which is associated with lamb lethality in the homozygous state. A specific management of these haplotypes/variants in the MTR dairy sheep selection program would help enhance the overall fertility and lamb survival.

Novel AAV-mediated genome editing therapy improves health and survival in a mouse model of methylmalonic acidemia.

Methylmalonic acidemia (MMA) is an inborn error of metabolism mostly caused by mutations in the mitochondrial methylmalonyl-CoA mutase gene (MMUT). MMA patients suffer from frequent episodes of metabolic decompensation, which can be life threatening. To mimic both the dietary restrictions and metabolic decompensation seen in MMA patients, we developed a novel protein-controlled diet regimen in a Mmut deficient mouse model of MMA and demonstrated the therapeutic benefit of mLB-001, a nuclease-free, promoterless recombinant AAV GeneRideTM vector designed to insert the mouse Mmut into the endogenous albumin locus via homologous recombination. A single intravenous administration of mLB-001 to neonatal or adult MMA mice prevented body weight loss and mortality when challenged with a high protein diet. The edited hepatocytes expressed functional MMUT protein and expanded over time in the Mmut deficient mice, suggesting a selective growth advantage over the diseased cells. In mice with a humanized liver, treatment with a human homolog of mLB-001 resulted in site-specific genome editing and transgene expression in the transplanted human hepatocytes. Taken together, these findings support the development of hLB-001 that is currently in clinical trials in pediatric patients with severe forms of MMA.

Systemic gene therapy for methylmalonic acidemia using the novel adeno-associated viral vector 44.9

Methylmalonic acidemia (MMA) is a severe and potentially lethal autosomal recessive inborn error of metabolism most frequently caused by mutations in the methylmalonyl-CoA mutase (MMUT) gene. Proof-of-concept adeno-associated virus (AAV) gene therapy studies using mouse models of MMA have demonstrated promise for this therapeutic approach but translation to the clinic could be limited by preexisting capsid immunity and vector potency. Here we explore the efficacy of a novel clade E capsid, 44.9, as a serotype for systemic AAV gene therapy for MMA. An anti-AAV44.9 neutralizing antibody (NAb) survey in adult volunteers (n = 19) and a large cohort of MMA patients (n = 48) revealed a seroprevalence rate of ∼26% and 13%, respectively. The efficacy of AAV44.9 gene delivery was examined in two murine models of MMA, representing neonatal lethal and juvenile phenotypes of MMA. Systemic delivery of the AAV44.9-Mmut vector prevented lethality and lowered disease-related metabolites in MMA mice. Tissue biodistribution and transgene expression studies in treated MMA mice showed that AAV44.9 was efficient at transducing the liver and heart. In summary, we establish that AAV44.9 exhibits a low prevalence of preexisting NAb in humans, is highly efficacious in the treatment of clinically severe MMA mouse models and is therefore a promising vector for clinical translation.

Growth advantage of corrected hepatocytes in a juvenile model of methylmalonic acidemia following liver directed adeno-associated viral mediated nuclease-free genome editing.

Methylmalonic acidemia (MMA) is a rare and severe inherited metabolic disease typically caused by mutations of the methylmalonyl-CoA mutase (MMUT) gene. Despite medical management, patients with MMA experience frequent episodes of metabolic instability, severe morbidity, and early mortality. In several preclinical studies, systemic gene therapy has demonstrated impressive improvement in biochemical and clinical phenotypes of MMA murine models. One approach uses a promoterless adeno-associated viral (AAV) vector that relies upon homologous recombination to achieve site-specific in vivo gene addition of MMUT into the last coding exon of albumin (Alb), generating a fused Alb-MMUT transcript after successful editing. We have previously demonstrated that nuclease-free AAV mediated Alb editing could effectively treat MMA mice in the neonatal period and noted that hepatocytes had a growth advantage after correction. Here, we use a transgenic knock-out mouse model of MMA that recapitulates severe clinical and biochemical symptoms to assess the benefits of Alb editing in juvenile animals. As was first noted in the neonatal gene therapy studies, we observe that gene edited hepatocytes in the MMA mice treated as juveniles exhibit a growth advantage, which allows them to repopulate the liver slowly but dramatically by 8-10 months post treatment, and subsequently manifest a biochemical and enzymatic response. In conclusion, our results suggest that the benefit of AAV mediated nuclease-free gene editing of the Alb locus to treat MMA could potentially be therapeutic for older patients.

Genetic testing is necessary for correct diagnosis and treatment in patients with isolated methylmalonic aciduria: a case report

BackgroundIsolated methylmalonic aciduria can be caused by pathogenic mutations in the gene for methylmalonyl-CoA mutase or in the genes encoding enzymes involved in the intracellular metabolism of cobalamin. Some of these mutations may be cobalamin responsive. The type of methylmalonic aciduria cannot always be assumed from clinical manifestation and the responsiveness to cobalamin has to be assessed for appropriate cobalamin administration, or to avoid unnecessary treatment. The cases presented herein highlight the importance of genetic testing in methylmalonic aciduria cases and the need for standardisation of the in vivo cobalamin-responsiveness assessment.Case presentationWe describe two patients who presented in the first week of life with rapid neurological deterioration caused by metabolic acidosis with severe hyperammonaemia requiring extracorporeal elimination in addition to protein restriction, energy support, carnitine, and vitamin B12 treatment. The severity of the clinical symptoms and high methylmalonic acid concentrations in the urine (>30,000 μmol/mmol of creatinine) without hyperhomocysteinaemia in both of our patients suggested isolated methylmalonic aciduria. Based on the neonatal manifestation and the high methylmalonic acid urine levels, we assumed the cobalamin non-responsive form. The in vivo test of responsiveness to cobalamin was performed in both patients. Patient 1 was evaluated as non-responsive; thus, intensive treatment with vitamin B12 was not used. Patient 2 was responsive to cobalamin, but the dose was decreased to 1 mg i.m. every two weeks with daily oral treatment due to non-compliance. Genetic tests revealed bi-allelic mutations in the genes MMAB and MMAA in Patient 1 and 2, respectively. Based on these results, we were able to start intensive treatment with hydroxocobalamin in both patients. After the treatment intensification, there was no acute crisis requiring hospitalisation in Patient 1, and the urine methylmalonic acid levels further decreased in Patient 2.ConclusionsDespite carrying out the in vivo test of responsiveness to cobalamin in both patients, only the results of molecular genetic tests led us to the correct diagnosis and enabled intensive treatment with hydroxocobalamin. The combination of the standardized in vivo test of cobalamin responsiveness and genetic testing is needed for accurate diagnosis and appropriate treatment of isolated methylmalonic aciduria.

In vivo genome editing at the albumin locus to treat methylmalonic acidemia

Methylmalonic acidemia (MMA) is a metabolic disorder most commonly caused by mutations in the methylmalonyl-CoA mutase (MMUT) gene. Although adeno-associated viral (AAV) gene therapy has been effective at correcting the disease phenotype in MMA mouse models, clinical translation may be impaired by loss of episomal transgene expression and magnified by the need to treat patients early in life. To achieve permanent correction, we developed a dual AAV strategy to express a codon-optimized MMUT transgene from Alb and tested various CRISPR-Cas9 genome-editing vectors in newly developed knockin mouse models of MMA. For one target site in intron 1 of Alb, we designed rescue cassettes expressing MMUT behind a 2A-peptide or an internal ribosomal entry site sequence. A second guide RNA targeted the initiator codon, and the donor cassette encompassed the proximal albumin promoter in the 5′ homology arm. Although all editing approaches were therapeutic, targeting the start codon of albumin allowed the use of a donor cassette that also functioned as an episome and after homologous recombination, even without the expression of Cas9, as an integrant. Targeting the albumin locus using these strategies would be effective for other metabolic disorders where early treatment and permanent long-term correction are needed.

Central nervous system-targeted adeno-associated virus gene therapy in methylmalonic acidemia

Methylmalonic acidemia (MMA) is a severe metabolic disorder most commonly caused by a mutation in the methylmalonyl-CoA mutase (MMUT) gene. Patients with MMA experience multisystemic disease manifestations and remain at risk for neurological disease progression, even after liver transplantation. Therefore, delivery of MMUT to the central nervous system (CNS) may provide patients with neuroprotection and, perhaps, therapeutic benefits. To specifically target the brain, we developed a neurotropic PHP.eB vector that used a CaMKII neuro-specific promoter to restrict the expression of the MMUT transgene in the neuraxis and delivered the adeno-associated virus (AAV) to mice with MMA. The PHP.eB vector transduced cells in multiple brain regions, including the striatum, and enabled high levels of expression of MMUT in the basal ganglia. Following the CNS-specific correction of MMUT expression, disease-related metabolites methylmalonic acid and 2-methylcitrate were significantly (p < 0.02) decreased in serum of treated MMA mice. Our results show that targeting MMUT expression to the CNS using a neurotropic capsid can decrease the circulating metabolite load in MMA and further highlight the benefit of extrahepatic correction for disorders of organic acid metabolism.

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.

Improvement of sabinene tolerance of Escherichia coli using adaptive laboratory evolution and omics technologies

BackgroundBiosynthesis of sabinene, a bicyclic monoterpene, has been accomplished in engineered microorganisms by introducing heterologous pathways and using renewable sugar as a carbon source. However, the efficiency and titers of this method are limited by the low host tolerance to sabinene (in both eukaryotes and prokaryotes).ResultsIn this study, Escherichia coli BL21(DE3) was selected as the strain for adaptive laboratory evolution. The strain was evolved by serial passaging in the medium supplemented with gradually increasing concentration of sabinene, and the evolved strain XYF(DE3), which exhibited significant tolerance to sabinene, was obtained. Then, XYF(DE3) was used as the host for sabinene production and an 8.43-fold higher sabinene production was achieved compared with the parental BL21(DE3), reaching 191.76 mg/L. Whole genomes resequencing suggested the XYF(DE3) strain is a hypermutator. A comparative analysis of transcriptomes of XYF(DE3) and BL21(DE3) was carried out to reveal the mechanism underlying the improvement of sabinene tolerance, and 734 up-regulated genes and 857 down-regulated genes were identified. We further tested the roles of the identified genes in sabinene tolerance via reverse engineering. The results demonstrated that overexpressions of ybcK gene of the DLP12 family, the inner membrane protein gene ygiZ, and the methylmalonyl-CoA mutase gene scpA could increase sabinene tolerance of BL21(DE3) by 127.7%, 71.1%, and 75.4%, respectively. Furthermore, scanning electron microscopy was applied to monitor cell morphology. Under sabinene stress, the parental BL21(DE3) showed increased cell length, whereas XYF(DE3) showed normal cell morphology. In addition, overexpression of ybcK, ygiZ or scpA could partially rescue cell morphology under sabinene stress and overexpression of ygiZ or scpA could increase sabinene production in BL21(DE3).ConclusionsThis study not only obtained a sabinene-tolerant strain for microbial production of sabinene but also revealed potential regulatory mechanisms that are important for sabinene tolerance. In addition, for the first time, ybcK, ygiZ, and scpA were identified to be important for terpene tolerance in E. coli BL21(DE3).

Pre-implantation genetic diagnosis in an Iranian family with a novel mutation in MUT gene

BackgroundMethylmalonic acidemia (MMA), which is an autosomal recessive metabolic disorder, is caused by mutations in methylmalonyl-CoA mutase (MUT) gene. As a result, the conversion of methylmalonyl-CoA to succinyl-CoA is impaired in this disorder, leading to a wide range of clinical manifestations varying from no signs or symptoms to severe lethargy and metabolic crisis in newborn infants. Since identification of novel mutations in MUT gene can help discover the exact pathogenesis of MMA and also use these disease-causing mutations in prenatal diagnosis, this study was conducted to uncover the possible mutations in an Iranian couple with a deceased offspring clinically diagnosed as having organic acidemia. Moreover, to prevent the occurrence of the mutation in the next pregnancy, we took the advantage of pre-implantation genetic diagnosis (PGD), which resulted in a successful pregnancy.Case presentationThe affected individual was a 15-month-old boy who passed away due to aspiration pneumonia. The child presented at the age of 3 months with lethargy, protracted vomiting, hypotonia, and decreased level of consciousness. To find the mutated gene, Next Generation Sequencing (NGS) was performed as carrier testing for the parents and the results revealed a novel (private) heterozygous missense mutation in MUT gene (c.1055A > G, p.Q352R). After performing PGD on three blastomeres, one was identified as being homozygous wild-type that was followed by successful pregnancy.ConclusionsOur study identified a novel, deleterious, heterozygous missense mutation in MUT gene in a couple and helps to consider the genetic counselling and prenatal diagnosis more seriously for this family with clinical phenotypes of organic acidemia.

Interplay between mitochondria and diet mediates pathogen and stress resistance in Caenorhabditis elegans.

Diet is a crucial determinant of organismal biology; interactions between the host, its diet, and its microbiota are critical to determining the health of an organism. A variety of genetic and biochemical means were used to assay stress sensitivity in C. elegans reared on two standard laboratory diets: E. coli OP50, the most commonly used food for C. elegans, or E. coli HT115, which is typically used for RNAi-mediated gene knockdown. We demonstrated that the relatively subtle shift to a diet of E. coli HT115 had a dramatic impact on C. elegans’s survival after exposure to pathogenic or abiotic stresses. Interestingly, this was independent of canonical host defense pathways. Instead the change arises from improvements in mitochondrial health, likely due to alleviation of a vitamin B12 deficiency exhibited by worms reared on an E. coli OP50 diet. Increasing B12 availability, by feeding on E. coli HT115, supplementing E. coli OP50 with exogenous vitamin B12, or overexpression of the B12 transporter, improved mitochondrial homeostasis and increased resistance. Loss of the methylmalonyl-CoA mutase gene mmcm-1/MUT, which requires vitamin B12 as a cofactor, abolished these improvements, establishing a genetic basis for the E. coli OP50-incurred sensitivity. Our study forges a mechanistic link between a dietary deficiency (nutrition/microbiota) and a physiological consequence (host sensitivity), using the host-microbiota-diet framework.

BRANCHED CHAIN AMINO ACIDS AT THE EDGE BETWEEN MENDELIAN AND COMPLEX DISORDERS.

Branched chained amino acids (BCAA) are essential components of the human diet and important nutrient signals, which regain particular interest in recent years with the avenue of metabolomics studies suggesting their potential role as biomarkers. There is now compelling evidence for predictive role of BCAA in progression of diabetes, but causality relationship is still debated concerning insulin resistance and genetic versus non-genetic pathogenesis. Mendelian randomization studies in large cohorts of diabetes indicated pathogenic role of PPM1K (protein phosphatase Mg2+/Mn2+ dependent 1K) on Chr 4q22.1 gene, encoding for a phosphatase that activates BCKDH (branched chain keto acid dehydrogenase) complex. Recent studies indicated that insulin rapidly and dose-dependently regulates gene expression of the same complex, but the relationship with systemic insulin resistance and glucose levels is complex. Rare genetic syndromes due to Mendelian mutations in key genes in BCAA catabolism may be good models to understand potential role of gene of BCAA catabolism. However, in studying complex disorders geneticists are faced to complete new aspects of metabolic regulation complicating understanding genetics of obesity, diabetes or metabolic syndrome. A review of genetic syndromes of BCAA metabolism suggests that insulin resistance is not present, except rare cases of methylmalonic aciduria due to MUT (methylmalonyl-coA mutase) gene on Chr 6p12.3. Another aspect that complicates understanding is the new role of central nervous system (CNS) in insulin resistance. For a long time the hypothalamic hunger/satiety neuronal system was considered a key site of nutrient regulation. Genes may also affect the brain rewarding system (BRS) that would regulate food intake by modulating the motivation to obtain food and considering hedonic properties. Nutrigenomic and nutrigenetic investigations taking into account concurrently BCAA intake, metabolic regulation and gene variation have large perspectives to merge genetic and nutritional understanding in complex disorders.

Molecular Genetic Characterization of 151 Mut-Type Methylmalonic Aciduria Patients and Identification of 41 Novel Mutations in MUT.

Isolated methylmalonic aciduria (MMA) is an autosomal-recessive disorder of propionate metabolism that is most commonly caused by mutations in the methylmalonyl-CoA mutase (MUT) gene (mut-type MMA). We investigated a cohort of 151 patients, classifying 114 patients as mut(0) and 32 as mut(-) (five not defined). As per the definition, mut(-) patients showed a higher propionate incorporation ratio in vitro, which was correlated to a considerably later age of onset compared with mut(0) patients. In all patients, we found a total of 110 different mutations, of which 41 were novel. While the missense alleles p.Asn219Tyr, p.Arg369His, and p.Arg694Trp recurred in >10 alleles, 47 mutations were identified only once, suggesting many patients carry private mutations. Deficient alleles in the mut(-) subclass were almost exclusively caused by missense mutations, found disproportionately in the C-terminal cofactor binding domain. On the contrary, only half of the mut(0) mutations were of the missense type. Western blot analysis revealed reduced MUT protein for all 34 cell lines (27 mut(0) , seven mut(-) ) tested, suggesting protein instability as a major mechanism of deficiency in mut-type MMA. This large-scale evaluation helps to characterize the landscape of MUT mutations and their relationship to dysfunction and disease.

168. Assaying Hepatic Correction Mediated by Varied AAV Vectors in a Knock-Out Transgenic Mouse Model of Methylmalonic Acidemia (MMA)

Methylmalonic acidemia (MMA) is an inborn error of metabolism most commonly caused by deficient methylmalonyl-CoA mutase (MUT) activity. The disorder can have multiple clinical manifestations, including metabolic instability, stroke of the basal ganglia, pancreatitis, end-stage renal failure, growth impairment, osteoporosis and developmental delay. Unfortunately, current non-invasive therapies fail to chronically manage the disease, and patients still suffer from increased morbidity and early mortality. Solid organ transplantation, including elective liver, combined liver-kidney and isolated kidney transplantation, has been used to provide sustained benefit to patients, but the procedures come with substantial risks as well as the post-operative requirement for life-long immunosuppression. To address the large and unmet need for new therapies for patients with MMA, we have developed an effective adeno-associated viral (AAV) gene therapy that has been previously validated in a neonatal lethal mouse model of Mut deficiency (Mut−/−). The current project compares how two distinct AAV8 vectors that express the human MUT gene under the control of either the liver specific, human alpha 1-antitrypsin (hAAT) promoter, or the ubiquitous CMV-enhanced chicken β-actin (CBA) promoter differentially affect metabolite levels following systemic delivery to adult mice. The animals used in this study (Mut−/−;TgINS-MCK-Mut) express wild-type Mut in a muscle-specific fashion via a stable germline transgene and completely lack transgene expression in the liver. Mut−/−;TgINS-MCK-Mut) mice accurately model the hepatorenal manifestations of MMA, but afford an opportunity to assess gene therapy vectors at or after weaning because mice are rescued from neonatal lethality, yet experience massive elevations of the characteristic metabolites (methylmalonic and 2-methylcitric acid) because of the lack of hepatic Mut activity. Disease related metabolites were measured in plasma samples derived from Mut−/−; TglNS-MCK-Mut mice (n=7) prior to AAV gene delivery. The mice were then injected via the retro-orbital route with 1.5X1011 GC of either AAV8-hAAT-MUT or AAV8-CBA-MUT. At 10 days and 30 days post-treatment, mice treated with either vector showed a significant reduction in methylcitrate and methymalonic acid levels, with AAV8-CBA-MUT (n=2) treated mice manifesting methylcitrate and methylmalonic acid levels that trended lower than those measured in mice injected with AAV8-hAAT-MUT (n=4) at both time points. Further studies will be needed to precisely compare the differences between the hAAT and CBA promoters in MMA mouse models, but our preliminary results demonstrate that the Mut−/−; TgINS-MCK-Mut mice can be used to easily ascertain hepatic correction of Mut deficiency, which should help inform the selection of regulatory elements that will provide maximal therapeutic efficacy to treat patients with MMA.

48. Treatment of Methylmalonic Acidemia by Promoterless Gene-Targeting Using Adeno-Associated Viral (AAV) Mediated Homologous Recombination

Methylmalonic acidemia (MMA) is an autosomal recessive inborn error of metabolism most typically caused by mutations in methylmalonyl-CoA mutase (MUT). While the hallmark of this disease is elevated levels of methylmalonic acid in the plasma, other disease related metabolites such as methylcitrate are elevated in the plasma as well. Patients with MMA suffer from frequent and potential lethal bouts of metabolic instability that can be eliminated by liver transplantation. Adeno-associated viral (AAV) gene therapy has shown great promise as the treatment for MMA in a murine model of the disease. However, a majority of the AAV-treated mice developed hepatocellular cancer, which was determined to arise from AAV-mediated insertional mutagenesis. In an attempt to create a safer gene therapy platform for the treatment of MMA, we created a novel vector for site-specific gene addition of human MUT into the mouse albumin (Alb) locus. This promoterless AAV vector contains a 2A-peptide coding sequence proximal to a codon-optimized human MUT gene. The 2A-MUT sequence is flanked by arms of homology immediately upstream of mouse Alb stop codon. Since albumin is expressed exclusively in hepatocytes, we prepared an AAV serotype 8 vector to take advantage of the murine liver tropism conferred by this capsid. This newly created vector was named AAV8-Alb-A2-MUT. After site-specific integration of the vector into the Alb locus in the liver, ribosomal skipping generates both Alb and MUT as separate proteins derived from the same transcript. We delivered a dose of 2.5e12 GC of AAV8-Alb-2A-MUT to five mice with MMA by intraperitoneal injection at birth. At one month post-injection, we observed increased hepatic expression of the MUT by western blot, improved growth and a significant reduction of disease related metabolites, in the treated MMA mice (Table 1Table 1). This gene delivery approach is anticipated to provide permanent hepatic transgene expression while reducing the risk of off-target integration and vector-mediated insertional mutagenesis.Table 1Markers of Correction in MMA Murine ModelUntreated (n=3-8)AAV8-Alb-A2-MUT (n=5)P ValuePercent of Wild-type Mut ExpressionND11.3+/-9.1NAPlasma Methylmalonic Acid (uM)969+/−396563+/−98<0.05Plasma Methylcitrate (nM)5, 020+/−8732, 904+/−638<0.0001Weight at 1 Month6.9+/−1.610.7+/−0.6<0.003 View Table in HTML

Mutation analysis of methylmalonyl CoA mutase gene exon 2 in Egyptian families: Identification of 25 novel allelic variants

Methylmalonic aciduria (MMA) is an autosomal recessive disorder of methylmalonate and cobalamin (cbl; vitamin B12) metabolism. It is an inborn error of organic acid metabolism which commonly results from a defect in the gene encoding the methylmalonyl-CoA mutase (MCM) apoenzyme. Here we report the results of mutation study of exon 2 of the methylmalonyl CoA mutase (MUT) gene, coding MCM residues from 1 to 128, in ten unrelated Egyptian families affected with methylmalonic aciduria. Patients were presented with a wide-anion gap metabolic acidosis. The diagnosis has established by the measurement of C3 (propionylcarnitine) and C3:C2 (propionylcarnitine/acetylcarnitine) in blood by using liquid chromatography–tandem mass spectrometry (LC/MS–MS) and was confirmed by the detection of an abnormally elevated level of methylmalonic acid in urine by using gas chromatography–mass spectrometry (GC/MS) and isocratic cation exchange high-performance liquid-chromatography (HPLC). Direct sequencing of gDNA of the MUT gene exon 2 has revealed a total of 26 allelic variants: ten of which were intronic, eight were located upstream to the exon 2 coding region, four were novel modifications predicted to affect the splicing region, three were novel mutations within the coding region: c.15G>A (p.K5K), c.165C>A (p.N55K) and c.7del (p.R3EfsX14), as well as the previously reported mutation c.323G>A (p.R108H).

Functional characterization and categorization of missense mutations that cause methylmalonyl-CoA mutase (MUT) deficiency.

ABSTRACTMethylmalonyl‐CoA mutase (MUT) is an essential enzyme in propionate catabolism that requires adenosylcobalamin as a cofactor. Almost 250 inherited mutations in the MUT gene are known to cause the devastating disorder methylmalonic aciduria; however, the mechanism of dysfunction of these mutations, more than half of which are missense changes, has not been thoroughly investigated. Here, we examined 23 patient missense mutations covering a spectrum of exonic/structural regions, clinical phenotypes, and ethnic populations in order to determine their influence on protein stability, using two recombinant expression systems and a thermostability assay, and enzymatic function by measuring MUT activity and affinity for its cofactor and substrate. Our data stratify MUT missense mutations into categories of biochemical defects, including (1) reduced protein level due to misfolding, (2) increased thermolability, (3) impaired enzyme activity, and (4) reduced cofactor response in substrate turnover. We further demonstrate the stabilization of wild‐type and thermolabile mutants by chemical chaperones in vitro and in bacterial cells. This in‐depth mutation study illustrates the tools available for MUT enzyme characterization, guides future categorization of further missense mutations, and supports the development of alternative, chaperone‐based therapy for patients not responding to current treatment.

Mutation Analysis of Methylmalonyl CoA Mutase Gene Exon 2 in Egyptian Families: Identification of 25 Novel Allelic Variants

Methylmalonic aciduria (MMA) is an autosomal recessive disorder of methylmalonate and cobalamin (cbl; vitamin B12) metabolism. It is an inborn error of organic acid metabolism results commonly from a defect in the gene encoding the methylmalonyl-CoA mutase apoenzyme (MCM). Here we report the results of mutation study of Exon 2 of MUT gene (coding MCM residues from 1 to 128) in ten unrelated Egyptian families affected with methylmalonic aciduria. Patients were presented with a wide-anion gap metabolic acidosis. The diagnosis has established by measurement of C3 (propionylcarnitine) and C3:C2 (propionylcarnitine/acetylcarnitine) in blood by tandem mass spectrometry, and confirmed by detection of abnormally elevated methylmalonic acid level in urine by gas chromatography-mass spectrometry GC/MS and by isocratic cation exchange “high-performance liquid-chromatography” (HPLC). Direct sequencing of gDNA of the MUT gene exon 2 has revealed a total of 26 allelic variants, ten of which were intronic, four were novel modifications predicted to affect splicing region, eight were located upstream to exon 2 coding region, three were novel mutations within coding region (c.15G>A (p.K5K), c.165C>A (p.N55K) and c.7del (p.R3EfsX14) and the last one was a previously reported mutation c.323G>A.