The molecular basis of human 3-methylcrotonyl-CoA carboxylase deficiency Matthias R. Baumgartner, … , E. Regula Baumgartner, David Valle J Clin Invest. 2001;107(4):495-504. https://doi.org/10.1172/JCI11948. Isolated biotin-resistant 3-methylcrotonyl-CoA carboxylase (MCC) deficiency is an autosomal recessive disorder of leucine catabolism that appears to be the most frequent organic aciduria detected in tandem mass spectrometry–based neonatal screening programs. The phenotype is variable, ranging from neonatal onset with severe neurological involvement to asymptomatic adults. MCC is a heteromeric mitochondrial enzyme composed of biotin-containing α subunits and smaller β subunits. Here, we report cloning of MCCA and MCCB cDNAs and the organization of their structural genes. We show that a series of 14 MCC-deficient probands defines two complementation groups, CG1 and 2, resulting from mutations in MCCB and MCCA, respectively. We identify five MCCA and nine MCCB mutant alleles and show that missense mutations in each result in loss of function. Article Find the latest version: https://jci.me/11948/pdf http://www.jci.org http://www.jci.org/107/4?utm_campaign=cover-page&utm_medium=pdf&utm_source=content https://doi.org/10.1172/JCI11948 http://www.jci.org/tags/73?utm_campaign=cover-page&utm_medium=pdf&utm_source=content https://jci.me/11948/pdf https://jci.me/11948/pdf?utm_content=qrcode Introduction MCC (EC 6.4.1.4) is a biotin-dependent carboxylase that catalyzes the fourth step in the leucine catabolic path- way. Isolated, biotin-resistant MCC deficiency (also known as methylcrotonylglycinuria [MIM 210200]) is inherited as an autosomal recessive trait (1). The clinical phenotype is highly variable: some patients present in the neonatal period with seizures and muscular hypoto- nia (2, 3); others are asymptomatic women identified only by detection of abnormal metabolites in the neona- tal screening samples of their healthy babies (4). There is a characteristic organic aciduria with massive excretion of 3-hydroxyisovaleric acid and 3-methylcrotonylglycine, usually in combination with a severe secondary carnitine deficiency. MCC activity in extracts of cultured fibrob- lasts of patients is usually less than 2% of control. No correlation between the level of residual enzyme activity and clinical presentation has been observed. Tandem mass spectrometry (MS/MS), recently intro- duced for newborn screening, provides for the first time to our knowledge a way to detect a large variety of organ- ic acidurias including MCC deficiency (5). Surprisingly, MCC deficiency appears to be the most frequent organ- ic aciduria detected in MS/MS screening programs in North America (4, 6, 7), Europe (8), and Australia (9), with an overall frequency of approximately 1 in 50,000. MCC is a member of the family of biotin-dependent carboxylases, a group of enzymes with diverse meta- bolic functions but common structural features (10, 11). Members of this family have three structurally con- served functional domains: the biotin carboxyl carrier domain, which carries the biotin prosthetic group; the biotin carboxylation domain, which catalyzes the car- boxylation of biotin; and the carboxyltransferase domain, which catalyzes the transfer of a carboxyl group from carboxybiotin to the organic substrate spe- cific for each carboxylase (10, 12). In addition to MCC, there are three other biotin-dependent carboxylases in humans: propionyl-CoA carboxylase (PCC), pyruvate carboxylase (PC), and acetyl-CoA carboxylase (ACC) (10, 11). The genes for all human carboxylases except MCC have been cloned and characterized (13–15). MCC carboxylates 3-methylcrotonyl-CoA at the 4- carbon to form 3-methylglutaconyl-CoA (Figure 1) (1). The reaction uses ATP and bicarbonate and is reversible. Bovine MCC has an approximate size of 835 kDa and appears to comprise six heterodimers (αβ)6 (16). Like PCC, MCC has a larger α subunit, which covalently binds biotin, and a smaller β subunit (13). MCC is predominantly localized to the inner mem- brane of mitochondria and is known to be highly expressed in kidney and liver (1). cDNAs encoding both subunits of MCC recently have been cloned in Ara- bidopsis thaliana and other plants (17, 18). Here we report cloning of human MCCA and MCCB cDNAs, confirmation of their identity by biochemical and molecular studies, and identification of mutations in MCC-deficient patients. The Journal of Clinical Investigation | February 2001 | Volume 107 | Number 4 495 The molecular basis of human 3-methylcrotonyl-CoA carboxylase deficiency Matthias R. Baumgartner,1,2 Shlomo Almashanu,1,3 Terttu Suormala,2 Cassandra Obie,1,3 Robert N. Cole,4 Seymour Packman,5 E. Regula Baumgartner,2 and David Valle1,3 1McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University, Baltimore, Maryland, USA 2Metabolic Unit, Children’s Hospital, University of Basel, Basel, Switzerland 3Howard Hughes Medical Institute, and 4Department of Biological Chemistry, Johns Hopkins University, Baltimore, Maryland, USA 5Department of Pediatrics, University of California, San Francisco, California, USA Address correspondence to: David Valle, 802 PCTB, Johns Hopkins University, 725 N. Wolfe Street, Baltimore, Maryland 21205, USA. Phone: (410) 955-4260; Fax: (410) 955-7397; E-mail: dvalle@jhmi.edu. Received for publication December 6, 2000, and accepted in revised form January 7, 2001. Isolated biotin-resistant 3-methylcrotonyl-CoA carboxylase (MCC) deficiency is an autosomal recessive disorder of leucine catabolism that appears to be the most frequent organic aciduria detected in tandem mass spectrometry–based neonatal screening programs. The phenotype is variable, ranging from neonatal onset with severe neurological involvement to asymptomatic adults. MCC is a heteromeric mitochondrial enzyme composed of biotin-containing α subunits and smaller β subunits. Here, we report cloning of MCCA and MCCB cDNAs and the organiza- tion of their structural genes. We show that a series of 14 MCC-deficient probands defines two complementation groups, CG1 and 2, resulting from mutations in MCCB and MCCA, respective- ly. We identify five MCCA and nine MCCB mutant alleles and show that missense mutations in each result in loss of function. J. Clin. Invest. 107:495–504 (2001). 496 The Journal of Clinical Investigation | February 2001 | Volume 107 | Number 4 Methods Patients, complementation analysis, and MCC assay This study includes 16 MCC-deficient probands, all of whom had the diagnostic pattern of organic acid excre- tion and less than 10% MCC activity in extracts of cul- tured skin fibroblasts. Clinical and biochemical data of ten patients have been reported in the literature: 001 (3), 002 (2), 003 (19), 004 (20), 006 (21), 007 (22), 010 (23), 011 (24), 012 (25), and 016 (4). Fibroblasts or DNA of the remaining patients were referred by B.T. Poll-The (patient 005), H.G. Koch (patient 008), J. Smeitink (patient 009), U. von Döbeln (patient 013), R.D. De Kre- mer Dodelson (patient 014), and D.H. Morton (patient 015). Skin fibroblasts were cultured in Eagle’s minimal essential medium (Life Technologies Inc., Rockville, Maryland, USA) supplemented with 2 mmol/l L-gluta- mine and 10% FCS. Complementation analysis was modified from that described by Wolf et al. (26). Briefly, we produced heterokaryons by a 90- or 120-second treatment with 40% PEG harvested the cells 3 days later and measured MCC activity as described previously (3, 27). MCC activity in cultures of mixed but unfused cells was subtracted as blank and self fusions were included as a negative control. Expression of biotin-containing proteins in fibroblasts We harvested cultured fibroblasts by trypsinization, disrupted the washed cells by homogenization, har- vested the mitochondria by differential centrifugation (28), suspended them in SDS-buffer (50 mM Tris- acetate, 1% SDS [wt/vol], 10 mM DTT, 0.02% [wt/vol] bromphenol blue), and dissolved the proteins by boil- ing for 5 minutes. The cellular proteins were separated by SDS-PAGE, and the biotin-containing proteins were detected with an avidin alkaline phosphatase conjugate (avidin-AP, 1:8,000; Bio-Rad Laboratories AG, Glat- tbrugg, Switzerland). Fibroblasts of an unaffected control, a patient with isolated PC deficiency, and a patient with isolated PCCα deficiency were used to confirm the identity of the protein bands. cDNA cloning, sequence analysis, and chromosome mapping We obtained two mouse (AI317324 and AI117072) and two human (AA134548 and AA101775) MCCA EST clones, and one human MCCB (AI564487) EST clone from the IMAGE consortium (Genome Systems Inc., St. Louis, Missouri, USA) and sequenced both strands with an ABI 377 automated sequencer. To design primers corresponding to the 5′ UTR of our putative MCCA, we used the sequence of the MCCA EST clone AA337013 (not available commercially). We used primers DV4418 (sense, 5′-GACGCAGCTGCCTCTG TAC) and DV4417 (sense, 5′-TGGCCGGGCTCCAGGGACATG), complementary to the 5′ UTR, and DV4446 (antisense, 5′-AACTGCTCTTTATGAGACCCC), complementary to the 3′ UTR, to amplify full-length human MCCA from a human retina cDNA library (29); and primers DV4496 (sense, 5′-AGGACCTGAGCTCAGCTTCC) and DV4497 (sense, 5′-TCGGTGCCCGCCGCCATG), complementary to the 5′ UTR, and DV 4495 (antisense, 5′-ACTG- TAACAGCCTCATGTTCG), complementary to the 3′ UTR, to amplify full-length human MCCB from the same human retina cDNA library (29). We gel-purified the PCR products and sequenced them directly. The sequence alignments were prepared with MegAlign (DNASTAR Inc., Madison, Wisconsin, USA). We mapped HsMCCA using the Genebridge 4 radia- tion hybrid panel (Research Genetics Inc., Huntsville, Alabama, USA) using primers DV4621 (sense, 5′- TTTGTCGTCTCAGACTCGATG) and DV4740 (antisense, 5′-AGTCAGAAAAATAAGGCCAACC) corresponding to 5′ flanking intronic sequence of exon 6 and 3′ flanking intronic sequence of exon 7. Isolation and mass spectrometry analysis of biotin- containing proteins Enrichment for biotin-containing proteins. We homogenized 0.32 g of flash frozen male mouse kidney in 1.5 ml buffer A (100 mM Tris-HCl [pH 7.4], 20 mM DTT, 1 mM EDTA, 0.1% (vol/vol) Triton X-100, 20% (vol/vol) glycerol, 1 µM DMSF, 1 tablet cocktail protease inhibitor (Boehringer-Mannheim) and centrifuged the homogenate at 20,000 g for 20 minutes at 4°C. PEG was added to the supernatant to a final concentration of 16% (wt/vol), and the mixture was centrifuged at 20,000 g for 30 minutes at 4°C. The pellet was resus- pended in 1 ml of buffer A, approximately 3 × 108 pre- washed M280 streptavidin Dynabeads (Dynal Inc., Lake Success, New York, USA) were added, and the slur- ry mixed by rotating for 1 hour at 4°C. We washed the beads five times with buffer B (0.25 M KCl in buffer A), resuspended them in 200 µl of XI protein loading buffer, boiled for 5 minutes, and placed the solution on ice. We loaded 25 µl in each lane of a 10% polyacry- lamide gel and stained the separated proteins with Coomassie Brilliant blue R250. S-carboxymethylation and proteolytic digestion. We per- formed in gel digestion of the proteins using the Coomassie blue–stained SDS-polyacrylamide gels according to Williams et al. (30) with the following modifications. Cystines were modified by car- boxymethylation as described elsewhere (31). After rehydrating gel pieces with 5 ng/µl TPCK-treated trypsin in 1% acetic acid, excess trypsin solution was removed and the gel piece was covered with 2 gel vol- umes of 200 mM NH4HCO3 (pH 8), at 37°C for 48 hours. The resulting tryptic peptides were extracted from the gel piece with 60% acetonitrile in 0.1% TFA and concentrated by drying. Mass spectrometry analysis. Tryptic peptides were resus- pended in 1% acetic acid and loaded into a fused silica capillary column (75 µm inner diameter) packed with 10 cm of 5 µm C18 reverse-phase resin (YMC Inc., Atlanta, Georgia, USA) as described elsewhere (32). A 30-minute, 1.5–70% methanol gradient in 1% acetic acid was applied to the column at f low rates of 250–300 nl/min. Eluting peptides were electrosprayed directly into a Finnigan LCQ atmospheric pressure ionization quadrupole ion trap mass spectrometer (ThermoQuest Corp., San Jose, California, USA). Pos- itive-ion mass spectra were obtained at using XCal- ibur software (ThermoQuest Corp.). Peptides were fragmented by a 35% collision energy using a two- atomic-mass-unit isolation width. Fragmentation data were screened against the protein.nrdb.Z data- base from the Frederick Biomedical Supercomputing Center (ftp://ftp.ncifcrf.gov/pub/nonredun/) using the SEQUEST Browser (33) (ThermoQuest Corp.). Mutation analysis by RT-PCR and genomic PCR We extracted RNA and genomic DNA from cultured skin fibroblasts and/or blood using the Puregene RNA and DNA isolation kits (Gentra Systems, Minneapolis, Minnesota, USA) and performed RT-PCR using 5–10 µg fibroblast RNA and a cDNA cycle kit (Invitrogen Corp., Carlsbad, California, USA) following the manu- facturers’ recommendations. We generated first-strand cDNA with primers DV4403 (antisense, 5′-GACC- CAAATGCATGATTCTCC), complementary to sequence in the MCCA 3′ UTR region, and DV 4506 (antisense, 5′-GGTAGAAAAGTACAA TGCACAG), complementary to sequence in the MCCB 3′ UTR region. We then ampli- fied first-strand MCCA cDNA with primers DV4418 and DV4446 to generate a 2,326-bp fragment (–51 to +2275, where +1 is the A of the initiation methionine codon); and we amplified first-strand MCCB cDNA with primers DV4496 and DV 4495 to generate a 1,923- bp fragment (–99 to +1824). In some instances, when the amount of amplified product was inadequate, we went through a second round of PCR with nested primers DV4417 and DV4497. We gel-purified the PCR products and sequenced them directly. To confirm mutations identified in RT-PCR products, we amplified a genomic fragment containing the corre- sponding exon using flanking intronic primers and sequenced the PCR product directly. In the compound heterozygous patients in whom we identified only one of two alleles in RT-PCR products, we amplified and sequenced all exons and flanking intronic sequences. All PCR reactions (50 µl) contained primers (100 ng each), 1× standard PCR buffer (Life Technologies Inc.), dNTPs (200 µM), and Taq polymerase (2.5 U; Life Tech- nologies Inc.). The sequences of all primers are available upon request. To survey a control population for the identified mis- sense mutations, we amplified the relevant exon from genomic DNA and performed allele-specific oligonu- cleotide analysis as described previously (34). Construction of wild-type and mutant human MCCA/B expression vectors We TA cloned the full-length human MCCA (–51 to +2275) and MCCB (–99 to +1824) cDNAs into pCR Blunt II TOPO (Invitrogen Corp.). To introduce the MCCA mutations R385S, A289V, and L437P, we har- vested an 896-bp ACCI fragment from RT-PCR–ampli- fied patient cDNA and subcloned this fragment into the pMCCA-TOPO construct. We then transferred the wild-type and mutant MCCA constructs into a mam- malian expression vector (pTracer-CMV2; Invitrogen- Corp.) at the EcoR I site. This vector contains a green fluorescent protein (GFP) gene fused to the Zeocin resistance gene. Similarly, to introduce the MCCB mis- sense mutation E99Q, we harvested a 193-bp EcoN I/BstE II fragment from RT-PCR amplified patient cDNA, subcloned this fragment into the pMCCB- TOPO construct and then transferred the wild-type and mutant constructs into pTracer-CMV2. To intro- duce the MCCB missense mutations S173L, V339M, R155C, P310R and R193C, we harvested a 965-bp BstE II/Sfi I fragment from RT-PCR–amplified patient cDNA and subcloned this fragment directly into MCCB-pTracer-CMV2. We sequenced all constructs in both directions to validate the sequences. Transfections We transformed primary fibroblasts from proband 010 (homozygous for MCCA Q421fs(+1), from proband 001 (homozygous for MCCB S173L), and from a con- trol as described previously (34). For expression stud- ies, we electroporated the indicated constructs into transformed cells as described elsewhere (34). We har- vested the cells after 72 hours and measured MCC and PCC activity radioisotopically as described previously (27). Our standard MCC assay enables us to reliably detect activity as low as 5–10 pmol/min/mg protein. All The Journal of Clinical Investigation | February 2001 | Volume 107 | Number 4 497 Figure 1 The MCC-catalyzed reaction and its position in the leucine catabol- ic pathway. The dashed arrow indicates the metabolites that accu- mulate due to deficiency of MCC. 498 The Journal of Clinical Investigation | February 2001 | Volume 107 | Number 4 transfections were in duplicates. Transformed fibrob- lasts from an unaffected individual were used as con- trol. Transfection efficiency was assessed by coexpress- ing GFP in the same construct. Results Genetic complementation and assignment to MCCA or MCCB As an initial step in defining the molecular basis of MCC deficiency, we performed biochemical and somatic cell genetic studies with fibroblasts from 14 MCC-deficient probands. Using restoration of MCC activity in PEG-induced heterokaryons of fibroblasts as an end point, we defined two complementation groups (CGs), one comprising eight probands (MCC- CG1, 001-008) and the other, six probands (MCC- CG2, 009-014) (data available upon request). Given that MCC is composed of αβ heteromers, we antici- pated that the two CGs likely corresponded to muta- tions in genes encoding the α and β subunits of MCC (encoded by MCCA and MCCB, respectively). To investigate the complementation phenotype at the protein level, we examined the expression of the MCCα subunit using the covalently bound biotin as a tag in fibroblasts of five of six CG2 and in all of the eight CG1 probands (Figure 2). In CG2, MCCα was not detected in four of five probands, whereas in the remaining CG2 cell line (proband 011), the MCCα band was at least as intense as in controls (Figure 2). In all the CG1 cell extracts, MCCα was reduced but present. These results suggested that MCC-CG2 is caused by mutations in MCCA and, by exclusion, MCC-CG1, by mutations in MCCB. Identification of mammalian candidate MCCA and MCCB cDNAs Database search. We used the amino acid sequences of A. thaliana MCCA and MCCB (17, 18) and the TBLASTN algorithm to probe the public EST data- bases to identify murine and human cDNAs encoding candidate MCCAs and MCCBs. We used the murine candidates to assemble full-length mouse putative MCCA cDNA (GenBank accession number: MmMC- CA: AF310338) and the human candidate ESTs to design primers corresponding to the predicted 5′ and 3′ UTR of the putative human MCCA and MCCB cDNAs. Using these primer pairs, we amplified a sin- gle fragment of the predicted size for both MCCA and MCCB from a human retina cDNA library (29). Gen- Bank accession numbers: HsMCCA: AF310339. HsM- CCB: AF301000. We used additional ESTs to extend the 5′ and 3′ UTR sequences. The sequence of the human MCCA candidate has 57 bp of 5′ UTR, a 2,175-bp ORF, and 222 bp of 3′ UTR extending to a polyadenylation signal (AAUAAA). The murine can- didate MCCA cDNA has 42 bp of 5′ UTR, a 2,151-bp ORF, and 190 bp of 3′ UTR extending to a probable polyadenylation signal (AUAAA). The sequence of the amplified human MCCB candidate has 99 bp of 5′ UTR, a 1,689-bp ORF, and 540 bp of 5′ UTR. The candidate cDNAs predict a human MCCα of 725 amino acids with a calculated molecular mass of 80 kDa, a mouse MCCα of 717 amino acids with a calcu- lated molecular mass of 79 kDa, and a human MCCβ of 563 amino acids with a calculated molecular mass of 61 kDa. Human MCCα has 84% and 45% identity to MCCα of mouse and A. thaliana, respectively (Figure 3a). Human MCCβ has 60% identity to MCCβ of A. thaliana (Figure 3b). Similar to PCC, the MCCα subunit contains an NH2-terminal biotin carboxylation domain and a COOH-terminal biotin carrier domain (10). The biotin carrier domain is centered on the motif AMKM, which is found in most biotinylated proteins (Figure 3a) (10). Biotin is covalently attached to the ε-amino group of lysine 681; the ε-biotinyl lysine amide is termed biocytin (Figure 3a). As with other biotin- dependent carboxylases, there is a conserved (A)PM motif 29 residues NH2-terminal and a hydrophobic residue (F714) 33-31 residues COOH-terminal of bio- cytin (35). The biotin carboxylation domain is located in the NH2-terminal two-thirds of MCCα and is linked to the biotin carrier domain by a less-conserved (only 25% identity to A. thaliana residues 427-664) “hinge” region of residues 441-650. Additionally, there is per- fect conservation of 11 residues (Figure 3a, asterisks) that are highly conserved among all biotin-dependent Figure 2 Expression of the biotin-containing MCCα subunit in fibroblasts. Proteins in mitochondrial enriched fractions from cultured fibrob- lasts were separated by SDS-PAGE, and the biotin-containing sub- units of MCC, PCC, and PC were detected with an avidin alkaline phosphatase conjugate. Most, but not all, CG2-probands lack the MCCα subunit, while it is detectable in all CG1 probands. The Journal of Clinical Investigation | February 2001 | Volume 107 | Number 4 499 Figure 3 Sequence alignment of human MCCα and MCCβ with orthologs from mouse and A. thaliana. Amino acids identical to the human sequence are highlighted. Missense mutations identified in MCC-defi- cient patients are indicated above with the substituted amino acid. Potential cleavage sites for the NH2-terminal mitochondrial leader sequences are indicated by vertical arrowheads. (a) Sequence align- ment of human, mouse, and A. thaliana MCCα. The predicted ATP- binding site in the NH2-terminal biotin carboxylation domain and the predicted COOH-terminal biotin carboxyl carrier domain are indicated by solid and dashed over- lines, respectively. The arrow indi- cates the lysine residue that links covalently to biotin (biocytin). The residues marked with an asterisk within the biotin carboxylation domain are thought to play an important role in catalysis (36, 37). (b) Sequence alignment of human and A. thaliana MCCβ. The putative 3-methylcrotonyl-CoA binding domain is indicated by a solid over-line. 500 The Journal of Clinical Investigation | February 2001 | Volume 107 | Number 4 Figure 4 Coomassie-stained SDS-PAGE gel of biotin-containing proteins puri- fied from a mouse kidney extract with streptavidin Dynabeads. We excised the proteins from the gel, digested with trypsin, and analyzed the tryptic peptides using MALDI-TOF and liquid chromatography coupled to ESI-MS/MS. We analyzed all the indicated bands includ- ing those that we identified as MCCα and β. carboxylases and thought to play a role in catalysis (12, 36, 37). Consistent with this prediction, the putative carboxylation domain contains the conserved sequence GGGGKGMRIV at positions 209-218 (Figure 3a), which is part of the ATP binding pocket in the biotin carboxylation domain of Escherichia coli ACC (37) and is similar to a consensus P-loop ATP-binding site [G′XGK(TS)] (38). The β subunit has a putative CoA binding motif (Figure 3b) (10, 39). MCCα and β have candidate NH2-terminal mito- chondrial targeting sequences with multiple arginine residues and a paucity of acidic residues (40). Possible cleavage sites are indicated by vertical arrowheads in Figure 3. Because the sequence of mitochondrial lead- ers is not highly conserved, we favor the more COOH- terminal cleavage site in MCCα just before a highly conserved region (Figure 3a). Isolation and mass spectrometry analysis of biotin-contain- ing proteins. We also used a biochemical strategy to identify MCCα and β. We enriched biotin-containing proteins in a mouse kidney extract using streptavidin Dynabeads and separated these by SDS-PAGE (Figure 4). Using MALDI-TOF and electrospray ionization mass spectrometry (ESI-MS/MS), we analyzed tryptic fragments of these proteins to confirm the identity expected on the basis of the size of ACC, PC, and PCCβ. The analysis of the expected PCCα identified, in addition to fragments derived from PCCα, unique fragments corresponding to the conceptual transla- tion of the putative murine MCCA cDNA (Figure 4). We identified 35 peptides with 100% identity to the putative murine MCCα subunit covering 571 amino acids or 80% of the putative full-length murine MCCα. The protein comigrating with the 66-kDa marker (Fig- ure 4) contained tryptic fragments with sequences cor- responding to the conceptual translation of the puta- tive human MCCB cDNA. We identified 25 peptides with 100% identity to the putative human MCCβ sub- unit covering 414 amino acids or 74% of the putative full-length MCCβ. These sequence results strongly supported the identity of the mammalian MCCA and MCCB cDNAs. Organization of human MCCA and MCCB structural genes During the course of determining the structural organization of these genes by long-range PCR and direct sequencing, we identified sequences correspon- ding to MCCA and MCCB in the human high through- out genome sequence database (GenBank accession numbers AC026920 and AC026775, respectively). These clones contain exons encoding the complete MCCA and MCCB cDNAs and provide all exon/intron boundaries including the flanking intronic sequences with one exception (5′ flanking intronic sequence of MCCA exon 8). MCCA has 19 exons and MCCB 17 exons. Given that the draft sequence has gaps, our information on the size of some introns is incomplete. Because the MCCA gene was not mapped in the Uni- Gene database, we searched the flanking sequence in the BAC clone containing MCCA for other mapped genes. We identified the UniGene cluster Hs.10887 represented by several ESTs present in the MCCA genomic clone and localized to 3q25-q27 (D3S1553- D3S1580). To confirm this localization, we used the Genebridge 4 radiation hybrid panel to regionally localize MCCA 3.56 cR from the WI-6365 marker cor- responding to the same region on chromosome 3. Similarly, we identified UniGene cluster Hs.167531 representing several EST clones covering the 3′ end of the MCCB cDNA. This cluster maps to chromosome 5q12-q13.1 (D5S637-D5S1977). Patients with isolated MCC deficiency have mutations in MCCA or MCCB To confirm the identity of MCCA and MCCB, we sur- veyed these genes for mutations in our collection of 14 unrelated MCC-deficient probands. We grouped the probands according to their CGs and used RT-PCR to amplify the corresponding mRNA from their cultured skin fibroblasts. We sequenced the entire ORF in each proband and confirmed all mutations by direct sequencing of PCR-amplified genomic DNA. In two additional probands (015 and 016) (ref. 4) from the Amish/Mennonite population in Lancaster County, Pennsylvania, we searched for mutations by direct sequencing of PCR-amplified genomic DNA. We iden- tified five MCCA mutant alleles in CG2 cell lines accounting for ten of 12 possible mutant MCCA genes (Table 1). The mutations include three uncomplicated missense mutations (Figure 3a), one missense mutation that alters splicing (Figure 5a), and one 1-bp insertion. In CG1 cell lines, we identified nine MCCB mutant alle- les accounting for 12 of 16 possible mutant genes (Table 2). The mutations include 6 uncomplicated missense mutations (Figure 3b), one missense mutation that alters splicing (Figure 5b), one splice site mutation, and one 1-bp insertion. In spite of sequencing all exons and flanking intronic sequences (with the exception of the alleles has a low frequency in this population. We did not screen for alleles for which we did not have the appropriate control population (Vietnamese, MCCB- R155Q, -P310R; Turkish, MCCB-S173L, -V339M). Discussion Using a combination of homology probing and mass spectrometry we cloned human MCCA and MCCB full length cDNAs. The conceptual translation of MCCA shows the expected NH2-terminal biotin carboxylation domain and the COOH-terminal biotin carboxyl carrier domain (Figure 3a), separated by a less conserved “hinge” region (10, 12). Presumably because of differ- ences in substrate specificities, carboxyltransferase domains are less conserved among biotin-dependent carboxylases. MCC catalyzes the carboxylation of methylcrotonyl-CoA (1). The acceptor binding site is thought to be on MCCβ (10, 39). Consistent with this, human MCCβ shares high identity with A. thaliana MCCβ (60%) and only 28% identity with human PCCβ (13) that supports the role of the β subunit in determin- ing substrate specificity of these enzymes (Figure 3b). Our series of 6 MCCA- and 10 MCCB-deficient 5′ flanking intronic sequence of MCCA exon 8), we were not able to identify a second allele in two CG2 and four CG1 probands. In five of these probands, the one allele identi- fied appeared to be homozygous in the RT-PCR product, but was clearly heterozygous in genomic DNA, sug- gesting that the steady level of mRNA from the second allele was not detectable as would be the case for a promoter mutation or an intra- genic deletion or insertion missed by genomic PCR. These results strong- ly support the identification of the MCCA and MCCB genes and their assignment to MCC deficiency in CG2 and CG1, respectively. Expression of MCCA and MCCB alleles As a final test of the identity of our candidate human MCCA and MCCB cDNAs, we subcloned them into a mammalian expression vector (pTracer-CMV2), electroporated the recombinant constructs into a SV40T transformed reference CG2 or CG1 cell line, and measured MCC activity 72 hours later (27). As a ref- erence, we also measured PCC activ- ity (27). Wild-type MCCA and MCCB alleles restored MCC activity to 43% and 53% of untransfected control fibroblasts, respectively (Table 3). Transfection efficiency, assessed by scoring a subset of cells in each transfection for the presence of the coexpressed GFP, ranged from 10 to 20% in these experiments. Similarly, to test the functional consequences of the missense mutations, we expressed three MCCA and six MCCB missense alleles. MCCB-R193C and -V339M had activity of 6 and 7 pmol/min/mg protein, respectively, or about 4% of the activity produced by the wild-type allele, whereas the remaining four MCCB alleles and the three MCCA alleles produced no detectable activity (Table 3). These results confirm the deleterious func- tional consequences of the tested missense mutations. Population frequency of selected MCCA and MCCB alleles Additionally, we used allele-specific oligonucleotide analysis (34) to survey a North American control popu- lation of 66 individuals for three MCCA alleles (R385S, A289V, and L437P) and two MCCB alleles (E99Q and R193C). Aside from one individual heterozygous for R385S, we did not identify any of these alleles in this collection of 132 control chromosomes (data not shown). These results indicate that each of these mutant The Journal of Clinical Investigation | February 2001 | Volume 107 | Number 4 501 Figure 5 MCCA and B missense mutations that alter splicing. (a) MCCA D532H. The 1594G→C trans- version of the last bp of exon 13 results in the missense mutation D532H. The 3′ base of an exon also contributes to donor splice site recognition and, as shown in the lower panel, RT-PCR of MCCA cDNA in this patient with primers corresponding to the 5′ and 3′ UTR resulted in a product smaller than in wild-type. Sequence analysis of this product showed that exon 13 (217 bp) is skipped, which shifts the reading frame. Thus, the deleterious consequences of this mis- sense mutation appear to be entirely due to the splicing defect. (b) MCCB I437V. As shown in the upper panel, the 1309A→G transition in exon 14 results in the replacement of isoleucine by valine, a conservative change. However, the mutation also activates a cryptic splice donor. Use of this new donor splice site deletes the last 64 bp of exon 14 from the mature transcript. As shown in the lower panel, direct sequencing of the RT-PCR product shows that virtually all the transcript present uses the new, more 5′ splice donor. The second allele of this compound heterozygous patient does not produce detectable RNA. wt, wild-type; mut, mutant. 502 The Journal of Clinical Investigation | February 2001 | Volume 107 | Number 4 probands is characterized by the fact that almost every proband had a unique genotype with no prevalent mutant allele for either gene. In agreement with this observation, using allele-specific oligonucleotide analy- sis to screen 66 North American controls (132 chro- mosomes), we found only a single heterozygote from one allele (MCCA-R385S) and no carriers for the others tested (MCCA-A289V, -L437P; MCCB-E99Q, -R193C). For MCCA, we assume functional significance for the frameshift mutation Q421fs(+1) and the missense mutation D532H, which alters splicing (Figure 5a), because both result in truncated proteins lacking func- tionally important domains. The MCCA mutations R385S, A289V, and L437P all change conserved residues (Figure 3a), and the corresponding alleles con- fer no detectable MCC activity when expressed in the CG2 reference cell line (Table 3). In contrast to the other four CG2 probands tested, who had no detectable MCCα, we detected normal amounts of MCCα protein in the proband 011 homozygous for R385S (Figure 2). This result is consistent with the pre- diction based on the structure of the biotin carboxyla- tion domain of E. coli ACC that the residue correspon- ding to MCCα R385 is part of a positively charged pocket for bicarbonate binding (36, 37). For MCCB, we assume functional significance for the frameshift mutation S173fs(+1), the splice site muta- tion In5ac-1G→A and the missense mutation I437V, which alters splicing (Figure 5b). The remaining six MCCB missense mutations all change conserved residues and were the only coding alterations we found in sequencing the full-length ORF (Figure 3b). In expression studies, we showed that the MCCB-R155Q, -P310R, -S173L, and -E99Q alleles had no detectable MCC activity, whereas MCCB-V339M and -R193C had some residual activity, about 4% of the experimental control value (Table 3), when expressed in CG1-deficient reference cell lines. We identified V339M in two com- pound heterozygous Turkish probands (Table 2), the only patients in our collection with residual MCC activity in fibroblasts. Although 4% residual activity is at the detec- tion limit of the standard MCC assay we used for the expression studies (27), these results are in accordance with the residual activity detect- ed in fibroblasts of these patients with a modi- fied MCC assay of increased sensitivity (3, 22). MCCα was reduced, but clearly present, in all CG1 cell lines in our biochemical detection of the biotin-containing α subunit (Figure 2). This suggests that the MCCα subunit is less stable when the β subunit is absent or defective. Interestingly, we detected the MCCβ S173fs(+1), a T insertion, as one allele in a mild- ly affected Swiss compound heterozygote (20) and in an asymptomatic Mennonite homozy- gote from Lancaster County, Pennsylvania (Table 2). The ancestors of the Lancaster County Amish/Men- nonite population originated from Switzerland (41) and may have brought this allele with them. Haplotype analysis will be necessary to confirm a founder muta- tion. Moreover, the Amish proband is homozygous for a different MCCB allele, E99Q. Thus, despite the small size and common origins of the Amish/Mennonite population in this region, there is allelic heterogeneity for MCC deficiency. Combining our results and the published clinical reports (2–4, 19–25), we were not able to discern a phenotype-genotype correlation. Probands 010 and 015, homozygous for truncating mutations in MCCA or MCCB (Tables 1 and 2), have, in one case, no symp- toms and, in the other, a mild phenotype with late onset and no residual damage (23). By contrast, probands 011, 001, and 002, homozygous for mis- sense mutations MCCA-R385S, MCCB-S173L, and - E99Q, have a severe phenotype with early-onset, major neurological involvement and, in one case, a fatal outcome (Table 1) (2, 3, 24). Proband 016, an adult Amish patient homozygous for the same MCCB-E99Q, has only mild symptoms (4). Further- more, the two Turkish patients with MCCB-V339M that have some residual MCC activity both have a severe phenotype. Taken together, these results sug- gest that factors other than the genotype at the Table 2 MCCB mutant alleles No. Allele Exon Nucleotide Clinical Ethnic Proband change phenotypeA origin 1 R155Q 5 464G→A Mild Vietnamese 006 2 P310R 10 929C→G Mild Vietnamese 006 3 S173fs(+1) 6 518insT Mild Swiss/MennoniteB 004C, 015D 4 I437V 14 1309A→GE Mild Dutch 003C 5 R193C 6 577C→T Mild Dutch 005 6 In5ac-1G→A In5ac-1G→A Mild Dutch 005 7 S173L 6 518C→T Severe Turkish 001D 8 E99Q 4 295G→C Severe/mild Turkish/Amish 002D, 016D 9 V339M 11 1015G→A Severe Turkish 007C, 008C AMild: late onset, good recovery after acute attack, no or mild developmental delay; severe: onset in infancy, severe neurological involvement with severe developmental delay. BDetect- ed by newborn screening. COnly one allele identified. DHomozygous. EActivates cryptic splice donor (Figure 5b). Table 1 MCCA mutant alleles No. Allele Exon Nucleotide Clinical Ethnic Proband change phenotypeA origin 1 R385S 11 1155A→C Severe German 011B 2 Q421fs(+1) 11 1264insG Mild Swedish/Amer. 013C, 010B 3 A289V 8 866C→T Mild American 012C 4 D532H 13 1594G→CD Severe Turkish 009B 5 L437P 12 1310T→C Severe Argentine 014B AMild: late onset, good recovery after acute attack, no or mild developmental delay; severe: onset in infancy, severe neurological involvement with severe developmental delay. BHomozygous. COnly one allele identified. DAlters splicing (Figure 5a). The Journal of Clinical Investigation | February 2001 | Volume 107 | Number 4 503 MCCA and MCCB loci (modifying genes, environ- mental variables) must have a major influence on the phenotype of MCC deficiency. Since the widespread introduction of MS/MS to new- born screening, many new patients with MCC defi- ciency have been detected. Surprisingly, MCC deficien- cy appears to be the most frequent organic aciduria in these screening programs (4, 6–9) with an overall fre- quency of approximately 1 in 50,000. Studies of these prospectively identified individuals should provide insight into the factors that determine the phenotypic severity of MCC deficiency. Note added in proof. We have recently learned that the molecular basis of MCC deficiency has also been identi- fied by Gallardo et al. (2001, Am. J. Hum. Genet. 68:334). Acknowledgments We thank A. Kohlschütter, U. von Döbeln, S. Berry, J. Smeitink, R.D. De Kremer Dodelson, W. Lehnert, U. Wiesmann, U. Wendel, W.J. Kleijer, B. Steinmann, B.T. Poll-The, D.H. Morton, and H.G Koch for referring fibroblasts of their patients, and J. Nathans for provid- ing the human retina cDNA library and S. Muscelli for assistance in preparing the manuscript. M.R. Baum- gartner, T. Suormala, and E.R. 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