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Novel Inborn Error of Vitamin B12 Metabolism Caused by Mutations in ABCD4

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Novel Inborn Error of Vitamin B12 Metabolism Caused by Mutations in ABCD4 Novel inborn error of vitamin B12 metabolism caused by mutations in ABCD4 Jaeseung Kim Department of Human Genetics McGill University Montréal, Québec, Canada June 2012 A thesis submitted to McGill University in partial fulfillment of the requirements of the degree of Master of Science © Jaeseung Kim 2012 ABSTRACT Vitamin B12 (cobalamin, Cbl) is an essential cofactor for two human enzymes: methylmalonyl-CoA mutase (MUT) and methionine synthase (MTR). MUT utilizes 5’-deoxyadenosylcobalamin (AdoCbl) to convert methylmalonyl- CoA to succinyl-CoA in the mitochondria, whereas MTR utilizes methylcobalamin (MeCbl) to convert homocysteine to methionine in the cytoplasm. To date, eight complementation groups (cblA-G and mut), each the result of mutations at a different gene, have been discovered to be involved in the intracellular metabolism of cobalamin. A patient presented at birth, following an abnormal newborn screen, with hypotonia, lethargy, poor feeding and bone marrow suppression. There were elevated levels of methylmalonic acid and homocysteine, suggestive of a defect in vitamin B12 metabolism. Studies of cultured fibroblast showed decreased function of the cobalamin-dependent enzymes, MTR and MUT. There was increased uptake of labelled cyanocobalamin (CNCbl) but decreased synthesis of the cobalamin cofactors MeCbl and AdoCbl, with accumulation of “free” (i.e. non-protein bound) CNCbl in the cells. The cellular phenotype mimicked that of the cblF disorder caused by mutations in the LMBRD1 gene encoding the lysosomal membrane protein LMBD1 that is thought to play a role in transfer of cobalamin across the lysosomal membrane into the cytoplasm. However, cells from the patient 2 complemented those from all known complementation groups, including cblF, and no mutations in LMBRD1 were found. Whole-exome sequencing led to the identification of two mutations in the ABCD4 gene: c.956A>G (p.Y319C) and c.1746_1747insCT (p.E583LfsX9). Two additional patients with deleterious ABCD4 mutations were later found. Transfection of patient fibroblasts with wild type ABCD4 led to rescue of all abnormal cellular phenotypes. This thesis reports that this novel disorder, named cblJ, is an autosomal recessive disorder caused by mutations in ABCD4. The findings suggest that ABCD4, an ABC half-transporter, is another essential component of intracellular cobalamin metabolism. 3 RÉ SUME La vitamine B12 (cobalamine, Cbl) est un cofacteur essentiel pour deux enzymes de l'homme: la méthylmalonyl-CoA mutase (MUT) et la méthionine synthase (MTR). La MUT utilise 5'-deoxyadenosylcobalamin (AdoCbl) pour convertir la méthylmalonyl-CoA en succinyl-CoA dans la mitochondrie, alors que MTR utilise la méthylcobalamine (MeCbl) pour convertir l'homocystéine en méthionine dans le cytoplasme. À ce jour, huit groupes de complémentation (cblA-G et mut) ont été découverts à être impliqués dans le métabolisme intracellulaire de la cobalamine. Chacun est le résultat de mutations au niveau d'un gène différent. Un patient s’est présenté à la naissance, suite à une anomalie sue le dépistage nouveau-né, avec l’hypotonie, la léthargie, la mauvaise alimentation et la suppression de la moelle osseuse. Le patient avait des niveaux élevés d'acide méthylmalonique et d'homocystéine, suggérant un défaut dans le métabolisme de la vitamine B12. Les études de fibroblastes cultivés ont démontré une diminution de la fonction des enzymes dépendants sur la cobalamine, MTR et MUT. Il avait aussi une augmentation de l’absorption de la cyanocobalamine (CNCbl), mais une diminution de la synthèse de cofacteurs cobalamine la MeCbl et AdoCbl, avec une accumulation de CNCbl “libre” (c'est à dire non liée aux protéines plasmatiques) dans les cellules. Le phénotype cellulaire imitait celle de la maladie cblF, causée par des mutations dans LMBRD1, le gène codant pour la 4 protéine membrane lysosomale LMBRD1, qui semble jouer un rôle dans le transfert de la cobalamine à travers la membrane lysosomale dans le cytoplasme. Cependant, les cellules des deux patients complémentaient celles de tous les groupes de complémentation connus, y compris cblF, et aucune des mutations dans LMBRD1 ont été trouvés. Le séquençage de l’exome a mené à l'identification de deux mutations dans le gène ABCD4: c.956A>G (p.Y319C) et c.1746_1747insCT (p.E583LfsX9). Deux autres patients avec des mutations dans ABCD4 ont été retrouvés. Toutes les mutations ont été prévues d’être nocives. La transfection de fibroblastes de patients avec ABCD4 de type sauvage a conduit à sauver tous les phénotypes cellulaires anormaux. Cette thèse rapporte que ce trouble inédit, nommé cblJ, est une maladie autosomique récessive causée par des mutations dans ABCD4. Les résultats suggèrent qu’ABCD4, un demi-ABC transporteur, est un autre élément essentiel du métabolisme de la cobalamine intracellulaire. 5 TABLE OF CONTENTS ABSTRACT .......................................................................................................... 2 RÉ SUME ............................................................................................................... 4 TABLE OF CONTENTS ..................................................................................... 6 LIST OF ABBREVIATIONS ............................................................................ 11 LIST OF FIGURES ........................................................................................... 13 LIST OF TABLES .............................................................................................. 14 ACKNOWLEDGEMENTS ............................................................................... 15 CHAPTER 1: INTRODUCTION ..................................................................... 16 1.1 Vitamin B Group ........................................................................................... 16 1.2 Vitamin B12 .................................................................................................... 16 1.2.1 Historical Aspects ......................................................................... 16 1.2.2 Structure ........................................................................................ 18 1.2.3 Biosynthesis .................................................................................. 20 1.2.4 Function ........................................................................................ 20 1.2.4.1 Cofactor for Three Enzyme Classes ............................. 20 1.2.4.2 Two Human Enzymes ................................................... 23 1.3 Absorption and Transport of Vitamin B12 ..................................................... 23 1.3.1 Carrier Proteins ............................................................................. 23 1.3.2 Absorption and Transport Pathway ............................................... 24 1.3.3 Inborn Errors of Vitamin B12 Absorption and Transport .............. 25 1.3.3.1 Haptocorrin Deficiency ................................................ 26 1.3.3.2 Inherited Intrinsic Factor Deficiency ........................... 26 1.3.3.3 Imerslund-Gräsbeck Syndrome .................................... 28 1.3.3.4 Transcobalamin Deficiency .......................................... 29 6 1.3.3.5 Transcobalamin Receptor Deficiency .......................... 29 1.3.4 Related Proteins ............................................................................ 30 1.3.4.1 Megalin/LRP2 .............................................................. 30 1.3.4.2 ABCC1/MRP1 .............................................................. 31 1.4 Intracellular Metabolism of Vitamin B12 ....................................................... 32 1.4.1 The Patients ................................................................................... 32 1.4.2 Somatic Cell Complementation Analysis ..................................... 33 1.4.3 Discoveries of Eight Complementation Groups ........................... 34 1.4.3.1 Four Complementation Groups .................................... 35 1.4.3.2 A Complementation Group with Three Phenotypes ..... 36 1.4.3.3 Isolated HC with Megaloblastic Anemia ...................... 37 1.4.3.4 Failure of Lysosomal Release of Vitamin B12 .............. 38 1.4.3.5 Heterogeneity Among Patients with HC ...................... 38 1.4.4 Inborn Errors of Vitamin B12 Metabolism .................................... 39 1.4.4.1 cblF, cblC, and cblD ..................................................... 40 1.4.4.2 cblB, cblA, and mut ....................................................... 44 1.4.4.3 cblE and cblG ............................................................... 47 1.4.5 Other Causes of MMA .................................................................. 49 1.4.6 Other Causes of HC ...................................................................... 49 1.5 cblF Disease .................................................................................................. 50 1.5.1 Background on Lysosome ............................................................. 50 1.5.1.1 Role of Lysosome ......................................................... 50 1.5.1.2 Lysosomal Soluble Proteins ......................................... 51 1.5.1.3 Lysosomal Membrane Proteins .................................... 51 1.5.1.4 Disorders of Lysosomal Export .................................... 52 1.5.2 LMBRD1 Gene .............................................................................. 53 1.5.3 Pathophysiology and Treatment ................................................... 54 1.6
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