NewNew DevelopmentsDevelopments inin DDisordersisorders ofof IntracellularIntracellular

VitaminVitamin BB12 (Cobalamin)Cobalamin) MetabolismMetabolism

B. Fowler University Children's Hospital Basel, Switzerland StructureStructure ofof VitaminVitamin BB1212 (Cobalamin,(Cobalamin, CblCbl))

Essential cofactor for 2 Enzymes

Methionine Synthase AdenosylMethyl Co Methyl-Cbl Co

Methylmalonyl CoA-Mutase Adenosyl-Cbl FoodsFoods richrich inin VitaminVitamin BB1212 (Cobalamin)(Cobalamin)

Daily requirement 3µg

Vitamin B12 requirement is lowest of all vitamins Ist metabolism is the most complicated Haptocorrin def. GastrointestinalGastrointestinal AbsorptionAbsorption ofof

dietarydietary VitVit--BB12

Cbl = Cobalamin Intrinsic Factor def. HC = Haptocorrin- bound Cbl deg.HC = degraded HC IF = Intrinsic Factor IF-Cbl = IF-bound Cbl z-IF-Cbl = IF-Cbl attached to Cubilin Cubilin / Amnionless def. (Ileal receptor) Transcobalamin def. TCII-Cbl = Transcobalamin bound Cbl. VitaminVitamin BB12 andand HomocysteineHomocysteine MetabolismMetabolism

Methionine Tetrahydrofolate S-adenosyl methionine MS MeCbl S-adenosyl homocysteine

5-Methyl-THF Adenosine Homocysteine Serine Cobalamin

Cystathionine

AdoCbl Cysteine

Sulphate Valine Isoleucine MethylmalonicMethylmalonic acidacid metabolismmetabolism Methionine Threonine Odd-chain fatty acids Cholesterol Cobalamin

Propionyl-CoA Succinate-CoA Ligase AdoCbl ADP ATP D-Methylmalonyl-CoA L-Methylmalony-CoA Succinyl-CoA Succinate GDP GTP Epimerase Mutase α-Ketoglutarate Fumarate

free Methylmalonic Acid Isocitrate Malate

Citrate Oxaloacetate

Acetyl-CoA

Pyruvate Lactate IntracellularIntracellular CobalaminCobalamin Metabolism:Metabolism: 20022002

Cytoplasm Methionine Synthase Methionine Reductase Cbl MeCbl Methionine Synthase OHCbl OHCbl OHCbl Homocysteine TC TC Cbl Lysosome

Cbl

AdoCbl Methylmalonyl-CoA Succinyl-CoA Methylmalonyl CoA-Mutase Mitochondrion TC, Transcobalamin IntracellularIntracellular CobalaminCobalamin Metabolism:Metabolism: 20022002 complementationcomplementation groupsgroups Cytoplasm

Methionine cblE Cbl MeCbl cblG cblF cblC cblD OHCbl OHCbl OHCbl Homocysteine TC TC Cbl Lysosome

cblA Cbl cblB AdoCbl Methylmalonyl-CoA Succinyl-CoA

Mitochondrion CblCCblC defect:defect: earlyearly clinicalclinical presentationpresentation

3.5 w. Feeding problems temperature dysregulation Pale, irritable, unconscious, dystrophic poor growth neurological abnormalities, tachycardia anaemia

Plasma Hcy ÇÇÇ Methionine È Urine Methylmalonic acid ÇÇÇ Treated OH-Cbl i.m 1mg/d. betaine, carnitine

4 months re-admitted to hospital died one day later - multi-organ failure/hyperthermia

Prof. Sengers, Nijmegen CblCCblC defect,defect, latelate ClinicalClinical presentationpresentation

Clinical 12y- 21y. Unsteady gait, urinary incontinence Spinal cord involvement, neuropathy inability to walk respiratory insufficiency (respirator) Thought to have multiple sclerosis: Steroid treatment

Laboratory Urine MMA ÇÇÇ Plasma total homocysteine ÇÇ Treated i.m. OH-Cbl 500µg / d. - 10mg /week

Gold et al. 1995 TheThe CblCCblC defectdefect ofof cellularcellular cobalamincobalamin metabolismmetabolism

NATURE GENETICS 38 I NUMBER1 I JANUARY 2006 93-100

• Discovered by homozygosity mapping • Located on chromosome 1p • Codes for ca. 30 kDa protein TheThe cblCcblC genegene

•• In 204 individuals, 42 different mutations •• One mutation, 271dupA, accounted for 40% of all disease alleles. •• In further 118 individuals, 11 additional mutations 42% 271dupA (Lerner-Ellis…Fowler:Hum Mutation 2009)

cblCcblC genotypegenotype // phenotypephenotype correlationscorrelations

Early-onset disease c.271dupA c.331C>T

Late-onset disease c.394C>T (stop codon) FunctionFunction ofof thethe cblCcblC proteinprotein

• Similar motifs to those seen in bacterial genes with cobalamin-related functions e.g. TonB • TonB is involved in transducing energy generated from the proton motive force to the transport of iron and cobalamin across the outer bacterial membrane • Recombinant MMACHC binds cobalamin and functions in vitro as a CNCbl decyanase (Kim et al. 2008) • Exact function of MMACHC remains to be proved Evidence suggests MMACHC is an intracellular cobalamin trafficking chaperone that delivers cobalamin to and from other cobalamin related proteins.

MMACHC homology model (red) and the three-dimensional NMR structure of the monomeric C-terminal domain of TonB (blue). TheThe CblDCblD DefectDefect

TheThe cblDcblD complementationcomplementation groupgroup (Willard et al. 1978, Am. J. Hum. Genet.)

Originally Assigned to two siblings with homocystinuria / MMAuria (Goodman et al. 1970, Biochem. Med.)

Fibroblasts studies È uptake of Cbl È synthesis of AdoCbl and MeCbl È MMCoA-mutase and Methionine synthase activity

BUT less severe than those in cblC defect TheThe CblDCblD defectdefect ofof cellularcellular CblCbl metabolismmetabolism oneone genegene –– threethree phenotypesphenotypes •• Original cblD 2 siblings with combined defect methylmalonic- aciduria (MMA) and homocystinuria (Hcy) • Our study (2004, Suormala, Coelho, Fowler et al. J B Chem:279: 42742) - 2 patients with isolated Hcy - normal MMA - 1 patient with isolated MMA –normal Hcy Complementation studies proved that these patients belong to the cblD complementation group = gene

• Now 13 patients known 4 combined defect (2 described 1980, 2 new ones) 4 isolated homocystinuria, 5 isolated Methylmalonic acidaemia IntracellularIntracellular CobalaminCobalamin MetabolismMetabolism

Cytoplasm

Methionine cblE Cbl MeCbl cblG cblF cblC cblD Var.1 OHCbl OHCbl OHCbl Cbl Homocysteine TC Var.2 TC Cbl Lysosome

cblA Cbl cblB AdoCbl Methylmalonyl-CoA Succinyl-CoA

Mitochondrion ClassificationClassification ofof cblDcblD patientspatients

Urine Plasma Clinical findings

CblD-MMA/HC MMA ÇÇÇ Hcy ÇÇÇ Development delay 4 patients Methionine È Seizures Hypotonia Lethargy Feeding difficulties Megaloblastic anemia

CblD-HC MMA normal Hcy ÇÇÇ Development delay 4 patients Methionine È Ataxia Absent ankle reflex Megaloblastic anemia

CblD-MMA MMA ÇÇÇ Hcy normal Respiratory distress 5 patients Methionine normal Cranial haemorrhage Seizures EEG abnormal SearchSearch forfor thethe cblDcblD genegene

ƒ Identification of the chromosome Microcell Mediated Chromosome Transfer (MMCT)

ƒ Chromosomal region Polymorphic markers analysis

ƒ Candidate gene Homology search with bacterial proteins MappingMapping analysisanalysis ofof chromosomechromosome 22

ƒ 10.2 Mb chromosomal interval (2006 29 Mb) ƒ containing 28 genes (2006 168, 2 candidates studied) ƒ 8 uncharacterized genes

cM Markers Size HET (%) 154,4 150 218-242 83 154,4 2286 218-264 70 154,4 2334 259-273 77 154,6 349 129-135 47 154,8 129 162-180 77 155,9 132 189-213 76 156,4 381 298-312 60 156,4 2270 209-221 56 156,4 151 211-229 80 156,9 2335 153-173 76 157 2301 108-135 73 158,6 2277 245-259 58 158,6 2324 130-156 71 ChromosomalChromosomal locationlocation ofof thethe candidatecandidate genegene M151 M132 M2184 M2236 M2275 M2324 M2313 M2301 M2335 M151 M132 M2184 M2236 M2275 M2324 M2313 M2301 M2335 Centromere Telomere

144 Mb 145 Mb 146 Mb 147 Mb 148 Mb 149 Mb 150 Mb 151 Mb 152 Mb 153 Mb

MMADHC

ƒ Significant homology with bacterial genes related to ABC transporters ƒ Encodes a polypeptide of 296 aminoacids (32.8 kDa) ƒ Mutations were found in all cblD patients → MethylMalonic Aciduria and HomoCystinuria cblD type (MMADHC) ƒ Maps to chromosome 2q23.2 TransfectionTransfection ofof combinedcombined defectdefect fibroblastsfibroblasts withwith wildwild typetype andand mutantmutant MMADHCMMADHC

50 45 40 35 30 25 20 15 Methylcobalamin (% of total) Methylcobalamin (% of total) 10 5 0 vector wild type 545C>A 746A>G 776T>C 57-64del

cblD -Hcy cblD-MMA MMADHCMMADHC mutationsmutations

cblD-MMA/HC S228X S228X Y140X R250X Y140X R250X F204_A232del F204_A232del

2 3 4 5 6 7 8 R54X R54X L259P L259P T182N T182N D246G D246G Y249W Y249W L20fsX22 L20fsX22 C19fsX20 C19fsX20 T152fsX162 T152fsX162 L103_S108dup L103_S108dup

cblD-MMA cblD-HC

Frameshift/Stop codon Nonsense Splice site deletion Missense Inframe duplication MMADHCMMADHC mutationsmutations inin cblDcblD--MMAMMA

Start Start codon codon

2 3 4 5 6 7 8 R54X R54X L20fsX21 L20fsX21 C19fsX20 C19fsX20

cblD-MMA PossiblePossible functionfunction ofof thethe cblDcblD ProteinProtein

ƒ Protein sequence highly conserved in mammalian species

ƒ MMADHC not member of previously identified gene family

ƒ Shares similarity with putative ATPase component of bacterial ABC transporter

ƒ Lacks critical domains of most ABC transporters

ƒ Possible mitochondrial leader sequence

ƒ Vitamin B12 binding motif DxHxxG

MMADHC

Mitochondrial B12 sequence binding IdentificationIdentification ofof AdoCblAdoCbl andand MeCblMeCbl functionalfunctional domainsdomains

methyl-Cbl synthesis

mitochondrial adenosyl-cbl synthesis targeting

2 3 4 5 6 7 8 Met Met Gln Start 62 116 118 292 codon cblFcblF defectdefect Clinical Presentation (Fernandes book, 2006) • Seven of eight presented in the first year of life. (now 14 patients known) • Megaloblastic anemia, neutropenia and thrombocytopenia. • Failure to thrive, recurrent infections, developmental delay, lethargy, hypotonia, aspiration pneumonia, hepatomegaly and encephalopathy, pancytopenia, and heart anomalies. • Original infant girl had glossitis and stomatitis in first week of life. - severe feeding difficulties requiring tube feeding. - Tooth abnormalities and dextrocardia were present. • One infant died suddenly at home in the first year of life. • One boy developed juvenile rheumatoid arthritis at the age of 4 yearsyears and a pigmentedpigmented skinskin abnormalityabnormality atat 1010 years.years. cblFcblF defectdefect

Metabolic Derangement • Failure of Cbl transport across the lysosomal membrane following degradation of TC in the lysosome. • No conversion of Cbl to either AdoCbl or MeCbl. • Abnormal Schilling test suggests IF-Cbl has to pass through a lysosomal stage in the enterocyte before Cbl is released into the portal circulation. Genetics • Autosomal recessive. • The gene responsible for cblF has not been identified Treatment Parenteral OHCbl, 1mg/day, (first daily and then biweekly, or even less frequently) seems to be effective in correcting the metabolic and clinical findings. cblFcblF defect:defect: DiagnosticDiagnostic TestsTests

Plasma Hcy ÇÇÇ Methionine È cobalamin È / Normal Urine Methylmalonic acid ÇÇÇ Schilling test abnormal

Precise diagnosis requires tests in cultured fibroblasts. - Incorporation of [14C] propionate into macromolecules - screen for integrity of methylmalonyl-CoA mutase activity - Conversion of [14C] labeled formate to methionine reliably measures methionine synthase function - Total incorporation of [57Co] CNCbl and conversion to both MeCbl and AdoCbl, can differentiate Cbl disorders.

In cblF patients, total incorporation of labeled CNCbl elevated, but CNCbl not converted to AdoCbl or MeCbl. 2009: 41;234

Homozygosity mapping Multi-point linkage analysis of the genome-wide scan using the Affymetrix GeneChip® Human Mapping 250K Sty Array.

6 Fine polymorphic mapping and haplotype analysis reveals LMBRD1 as candidate gene

LMBRD1 Localisation of transiently expressed LMBD1-EGFP with the lysosomal marker LAMP1 • Mutations in the LMBRD1 gene (encoding LMBD1, a lysosomal membrane protein) found in cblF patients

• Transfection of cblF fibroblasts with wild-type LMBD1 rescued cobalamin coenzyme synthesis

• LMBRD1 identifed as cblF gene and suggests that LMBD1 is a lysosomal membrane exporter for cobalamin

Homozygosity mapping Mutations in the LMBRD1 gene (encoding LMBD1, a lysosomal membrane protein) found in cblF patients No. 5 No. 2 L352fs T172fs G G G G 457 78 88 347 G Intra- N-terminus G 448 lysosomal 170 •6q13 10 66 101 165 202 328 368 432 489 •18 exons 1 2 3 4 5 6 7 8 9 •61.4 kDa cytosolic protein 32 44 123 143 224 306 390 410 511 •540 amino T134fs C-terminus K281fs acids 540

No. 1T237X Site of mutation (fs = frameshift)No. 4 G Putative glycosylation site No. 3 The 1056delG allele (No. 5) leading to a frameshift and premature termination codon in exon 11 was present on 18 of the 24 disease chromosomes (common 1.34 Mb haplotype).

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A Methylcobalamin Coelho D, Suormal T, Stucki M, Lerner-Ellis JP, Rosenblatt DS, Newbold R, Baumgartner M, Fowler B.

TcR

TcR = Transcobalamin AcknowledgementsAcknowledgements

ƒ Terttu Suormala, David Coelho (Basel), ƒ Matthias Baumgartner, Martin Stucki (Zürich) ƒ Jordan Lerner-Ellis, David Rosenblatt (Montreal) ƒ Petra Zavadakova, Viktor Kožich (Prague) ƒ Hans Georg Koch, Martin Berghäuser (Münster) ƒ James E. Wraith (Manchester) , Alberto Burlina (Padua) ƒ Adrian Sewell, Jürgen Herwig (Frankfurt) ƒ Many colleagues who sent us fibroblasts

ƒ Swiss National Science Foundation grant No 3200-066878