The clinical characterization of patients with methylmalonic acidemia (MMA) and related disorders, via a dedicated natural history study, NHGRI protocol Clinical and Basic Investigations of Methylmalonic Acidemia and Related Disorders (ClinicalTrials.gov Identifier: NCT00078078) has continued. Through our clinical protocol, we have continued to accrue patients with MMA and cobalamin metabolic disorders and have evaluated >160 affected individuals;this is the largest single center cohort of such patients in the world. In the past year, we have continued to focus on the clinical characterization of patients with previously undefined forms of MMA as well as studies that expand our understanding of the natural history of well-recognized forms of MMA. One collaborative effort has led to the identification of a new inborn error of metabolism caused by mutations in a transcriptional co-regulator, HCFC1. This gene product participates in the regulation of transcription at many cellular promoters and had been previously identified and studied by others for more than a decade for its role in transcriptional co-regulation. As part of a multicenter approach to characterize a subset of severely affected males who were previously diagnosed with cblC or cblD deficiency by cellular biochemistry but lacked mutations in the respective genes, we determined the genetic basis of a novel, X-linked form of combined methylmalonic acidemia and hyperhomocysteinemia called cobalamin X deficiency (cblX). These severely affected males were previously diagnosed with cblC or cblD deficiency by cellular biochemistry but lacked mutations in the respective genes. Reassessment of these boys, first using exome sequencing and then traditional Sanger sequencing, revealed pathogenic mutations in HCFC1, an X-linked transcriptional co-regulator, in 14 patients, establishing the first human metabolic disorder caused by aberrant transcription Reference 1. These studies demonstrate the novel insights into intermediary metabolism that can be achieved through the study of rare metabolic disorders and highlights the rapid and comprehensive advances that can be achieved in the diverse and highly collaborative NHGRI IRP environment. Our group also participated in the further delineation of the pathology underlying MMA associated kidney disease. As part of a collaboration with a group at Harvard Medical School, we helped establish that megamitochondrion formation in the proximal tubule, associated with a functional electron transport chain defect, characterizes ultrastructure of affected nephrons Reference 2. We also contributed to a large, retrospective, international study that described the natural history of transcobalamin 2 deficiency Reference 3 and to a manscript detailing the response of a patient with MMA to hemodialysis and combined liver-kidney transplantation Reference 4. Lastly, a comprehensive book chapter on the topic of branched chain amino acid disorders was contributed Reference 5. A manuscript describing the neuroradiographic manifestations in patients with MMA who have suffered from metabolic strokes of the basal ganglia has recently been accepted for publication (Baker EH, Sloan JL, Hauser N, Gropman AL, et al. MRI Characteristics of Globus Pallidus Infarcts in Isolated Methylmalonic Acidemia, Am J of Neuroradiology, accepted 6/2014, in press). Active efforts include clinical and CNS neuroradiographic characterization of our cohort using MRI, MRS and DTI analyses, , analyses of the spectroscopic properties of MMA, a description of the state of MMA post solid organ transplantation, delineation of the natural history and ophthalmological phenotypes in patients with MUT MMA and cblC deficiency, renal phenotyping, and a critical reappraisal of the formula composition and dietary management of patients with MMA. A collaborative intra- and extramural Bench to Bedside grant with Dr Mendel Tuchman (CNMC) to fund our studies using stable isotopes in MMA patients ended this year. A publication describing the results from stable isotopic metabolic studies in MMA patients is in preparation. Laboratory investigations have continued to focus on generating and characterizing mouse models of methylmalonic acidemia and gene therapy studies. We propose to use transgenesis to enable the examination of the effects of organ and/or cell specific correction on the biochemical and pathological phenotype of Mut -/- mice, a strategy that is supported by our natural history observations of MMA patients with various forms of solid organ transplantation. In the next year, we will continue to characterize murine models that have an physiologically apparent intermediate or inducible phenotype yet are robust to allow the assessment of gene therapy approaches and the exploration of pathophysiological mechanisms. We will then characterize the disease state using genomic, proteomic and metabolomic approaches to define mechanisms and identify biomarkers that might be translated to patient care. We have studied viral gene therapy as treatment for MMA, using preclinical cellular and animal models to gather efficacy and safety data. We had previously developed and tested adeno-associated viral (AAV) vectors to transfer the Mut gene into mice and used vectors of serotypes 2, 8 and 9 that express the Mut or MUT gene under the control of a the enhanced, chicken beta-actin promoter (CBA) or the liver-specific, thyroid-binding globulin promoter (TBG) and had delivered them to Mut-/- mice in the neonatal period. The results in the all the prior studies were striking: while the untreated Mut-/- mice uniformly perish in early life, the treated Mut-/- mice had near normal long-term survival, behaved normally, displayed an ameliorated metabolic phenotype and demonstrated enzymatic activity as long as two years after treatment with an AAV8 or AAV9 vector. During the last cycle, we noted that many AAV treated mice, in both mutant and control groups, developed hepatocellular carcinoma (HCC). Over the past year, in collaboration with Dr Shawn Burgess of the NHGRI IRP, we have defined the genotoxicity of AAV in mice. These efforts have defined new safety considerations for AAV and helped direct the construction of new vectors with improved safety. We are preparing a manuscript describing our results as we develop AAV vectors with ameliorated genotoxicity. Such vectors will lead to the creation of an optimal vector for use in humans, affording an opportunity to pursue an IND submission to the FDA as a step toward translating gene therapy to the clinic. We have also served as a resource for other groups in the NHGRI who strive to develop gene therapy vectors, including a study members of the section led on AAV gene therapy for citrullinemia Reference 6.
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