doi:10.1086/323677 Am. J. Hum. Genet. 69:863–868, 2001 863 Report Maple Syrup Urine Disease: Identification and Carrier-Frequency Determination of a Novel Founder Mutation in the Ashkenazi Jewish Population Lisa Edelmann,1 Melissa P. Wasserstein,1,2 Ruth Kornreich,1 Claude Sansaricq,1,2 Selma E. Snyderman,1,2 and George A. Diaz1,2 Departments of 1Human Genetics and 2Pediatrics, Mount Sinai School of Medicine, New York Maple syrup urine disease (MSUD) is a rare, autosomal recessive disorder of branched-chain amino acid metabolism. We noted that a large proportion (10 of 34) of families with MSUD that were followed in our clinic were of Ashkenazi Jewish (AJ) descent, leading us to search for a common mutation within this group. On the basis of genotyping data suggestive of a conserved haplotype at tightly linked markers on chromosome 6q14, the BCKDHB gene encoding the E1b subunit was sequenced. Three novel mutations were identified in seven unrelated AJ patients with MSUD. The locations of the affected residues in the crystal structure of the E1b subunit suggested possible mechanisms for the deleterious effects of these mutations. Large-scale population screening of AJ individuals for R183P, the mutation present in six of seven patients, revealed that the carrier frequency of the mutant allele was ∼1/113; the patient not carrying R183P had a previously described homozygous mutation in the gene encoding the E2 subunit. These findings suggested that a limited number of mutations might underlie MSUD in the AJ population, potentially facilitating prenatal diagnosis and carrier detection of MSUD in this group. Originally described by Menkes in 1954, as a rapidly fatal neurodegenerative disease (Menkes et al. 1954), maple syrup urine disease (MSUD) (types Ia [MIM 248600], Ib [MIM 248611]), and II [MIM 248610]), is an inborn error of metabolism, resulting from the de- fective activity of branched-chain a-ketoacid dehydrog- enase (BCKAD) (Chuang and Shih 2000). The enzymatic defect, transmitted in an autosomal recessive manner, results in an inability to catabolize leucine, isoleucine, and valine. BCKAD exists as a multienzyme complex composed of a multimeric core of dihydrolipoyl acyl- transferase (E2), to which are bound multiple BCKAD decarboxylase (E1) and dihydrolipoamide dehydroge- nase (E3) subunits (Pettit et al. 1978). Two regulatory subunits, BCKAD kinase and BCKAD phosphatase, are also associated with the E2 core (Damuni et al. 1984; Reed et al. 1985). The E1 subunit exists as a heterotetra- Received July 26, 2001; accepted for publication August 7, 2001; electronically published August 16, 2001. Address for correspondence and reprints: Dr. George A. Diaz, De- partment of Human Genetics, Mount Sinai School of Medicine, New York, NY 10029. E-mail: george.diaz@mssm.edu � 2001 by The American Society of Human Genetics. All rights reserved. 0002-9297/2001/6904-0019$02.00 mer composed of two E1a and two E1b subunits. Be- cause the E3 subunit is shared with the pyruvate and a- ketoglutarate dehydrogenase complexes, decreased activity of this component results in deficiencies of all three enzymes. Five clinical MSUD phenotypes have been described, which differ in their presentation and severity. Classic MSUD, the most common clinical presentation, mani- fests during the newborn period and can result in severe neurological complications and death. The less-common forms are the later-onset intermediate type, an episodic intermittent type, an intermediate-like thiamin-respon- sive type, and E3 deficiency with lactic acidosis. Mu- tations in any of the E1 or E2 components can result in classic, intermediate, or intermittent MSUD, although they are more frequent in E1a and E1b than in E2 (Nellis and Danner 2001). Patients with MSUD can be effec- tively managed with dietary restriction of the branched- chain amino acids and maintenance of strict biochemical control (Snyderman et al. 1964). However, a significant proportion of treated individuals have impaired intel- lectual development or neurological complications, par- ticularly as a result of delayed diagnosis (Kaplan et al. 1991), emphasizing the importance of newborn-screen- 864 Am. J. Hum. Genet. 69:863–868, 2001 Table 1 Primers for Exon-Specific PCR amplifications. GENE AND EXON PRIMER (5′r3′) LENGTH (bp)Forward Reverse E1b: 1 TCCAGTTCCGATTGGTCTGT AGCTGGGATGCAAGGATTG 434 2 CCACAATTCAGGCACATATCAG GCATAATATCTTTCTTTGTACCTTGA 222 3 TCCATACAATGGGGATATGACA TGATCGAGATCTATGGTCCATTT 304 4 TCTCATTTGCCACATTAACCTTT GGGTAGCGGCAATACTTGAA 239 5 CCCCGTCTTTCTTTCTGACC TGAATCAAAATGGAAACATCAAA 237 6 AGCCCTTCTTAGCAGCGAGT TTCTACAAGGAAAATATAAATCAACCA 295 7 TGCACAAGTGTCACCTCAGA GCTTCCAAGCACAACGTAGG 205 8 TTTTTGAGGCTAGAACCTTTGT GCCAAAGGTTTCAGGGAAAT 214 9 CATCAGGTTTTTCTTTTCTCTTTCA TCTTCTGGAATTGGCATGTG 175 10 TTGAAGTTAAGCATCCTGACTCTG CGTTAATGTCAGGGGCACAT 400 E2: 2 TTGTGTTCGCTATTTTC CAGCAGTTGTTTTCAGGA 122 ing programs. Despite early diagnosis and adequate care, episodes of life-threatening metabolic decompensation occur during periods of stress or infection and are as- sociated with significant morbidity and mortality (Ri- viello et al. 1991; Levin et al. 1993; Chuang and Shih 2000). MSUD has been described in all ethnic groups and has an estimated worldwide frequency of 1/185,000 (Naylor 1980). However, a founder effect in the Penn- sylvania Mennonite population has led to an increased incidence (1/176) in this population (Marshall and DiGeorge 1981). In our clinic, we noted that a dispro- portionate percentage (∼30%) of families with MSUD were of Ashkenazi Jewish (AJ) heritage. Here we report the carrier frequency of a common AJ mutation, R183P, and the identification of two novel mutations. Six unrelated AJ patients with classic MSUD (individ- uals 2–6 and 9), one patient with intermediate MSUD (individual 1), and the parents of one classic patient (in- dividuals 7 and 8) participated in this study. The protocol was approved by the Mount Sinai School of Medicine Institutional Review Board, and voluntary informed con- sent was obtained from all participants. DNA was ex- tracted either from peripheral blood (PureGene; Gentra) or from buccal-swab samples (MasterAmp Buccal Kit; Epicentre Technologies). Our initial focus was on E2DAT, a 2-bp deletion in the second exon of the E2 gene, described by Fisher et al. (1993), in individual 9. This mutation destroys an AflIII site in the exon, allowing rapid screening of our AJ patients by amplification of the exon from genomic DNA (table 1), followed by restriction-enzyme digestion. The results of the assay demonstrated that the mutation was found only in individual 9 (not shown). We there- fore identified polymorphic short tandem-repeat (STR) markers flanking the genetic loci encoding E1a, E1b, and E2, to determine whether a conserved haplotype consistent with a common mutation was present in our other patients. It is noteworthy that, in order to assess the BCKDHB locus, we first identified candidate STR markers that were linkage-mapped to 6p21-p22, the published cytogenetic localization given in the OMIM Gene Map (Zneimer et al. 1991). Alignments of gene (see the GenBank Overview web site ) and marker se- quences with the genomic sequence data, by use of the Human Genome Project Working Draft at UCSC, in- dicated that BCKDHB maps to cytogenetic location 6q14. Radiation-hybrid mapping data from Gene- Map’99 supported the localization of BCKDHB to 6q14, so markers from this region were used in the analysis. STRs tightly linked to the relevant genes were ob- tained from ResGen. Radiolabeling, amplification, and electrophoresis were performed by standard methods. Allele assignments were standardized across gels by use of CEPH reference individual 1331-2. The genotyping results were consistent with a disproportionately rep- resented allele at each of the markers flanking the E1b locus (fig. 1). In particular, the 7 allele of marker D6S251 was highly overrepresented, being present in all individ- uals except for individual 9 and accounting for 10 of 12 total alleles. Typing of 50 control AJ chromosomes gave an estimated 7 allele frequency of ∼8%, confirming the striking allele-frequency distortion in the patients with MSUD. Markers flanking the genes encoding E1a and E2 did not display a pattern consistent with seg- regation of common alleles (not shown). On the basis of these suggestive results, mutation analysis of the E1b exons was performed in individual 4, a homozygote for the putative three-marker haplotype 1-4-7 at markers D6S286, D6S460, and D6S251, respectively. PCR primers were designed to amplify all exons and Reports 865 Figure 1 Genomic organization of polymorphic markers tightly linked to the genes encoding E1a, E1b, and E2 and genotype data for markers flanking E1b. A, Organization of STR markers used in anal- ysis of the BCKDHB locus, and their relative spacing, in both physical and genetic distance. BCKDHB spans ∼240 kb, and the 3′ end lies ∼1 Mb upstream of D6S251. B, Results of genotyping analysis of patients with intermediate (boxed) or classic (underlined) MSUD. The parents of individual 5 are denoted by circles. Individual 9 is a patient with a known mutation (i.e., E2DAT). proximal intronic sequences (table 1). Amplicons were column purified by use of the Qiagen PCR Purification kit and were sequenced bidirectionally on an ABI PRISM 3700 automated sequencer (Applied Biosystems). Traces were processed by the Factura and Autoassembler soft- ware packages (Applied Biosystems). A homozygous missense mutation corresponding to 538GrC in the cDNA sequence was detected in exon 5 (fig. 2A). Over- lapping MspI and NlaIV sites in the amplicon were de- stroyed by the base change, allowing the remaining pa- tients to be screened with the same primer set as was used for sequencing (fig. 2B). The mutation was detected in 10 of 12 chromosomes screened and segregated with the 7 allele of marker D6S251, unambiguously in eight instances and presumptively in two instances (fig. 1B). The 538GrC mutation causes an ArgrPro substi- tution in E1b, at residue 183 in the mature peptide (R183P). R183 is conserved between E1b and the ho- mologous Pseudomonas putida bacterial enzyme and is located in a b-sheet that contributes to a K� ion-binding pocket close to the interface of the E1a and E1b subunits (Ævarsson et al. 2000). Hydrogen-bond interactions made by this residue in the E1 crystal structure were visualized by use of a SwissPdb-Viewer (fig. 3A) (Guex and Peitsch 1997). Three parallel b-sheets (d, e, and g) form extensive interactions in this region of the protein. Sheet e is central to sheets d and g, and R183 makes main-chain and side-chain hydrogen-bond interactions with residues on both adjacent sheets, notably E96 and E189, residues that were also conserved between mam- malian and bacterial subunits (fig. 3A). Structural changes introduced by a Pro substitution would thus be expected to interfere with a conserved structural motif, by altering the conformation of the central b-strand and by disrupting the hydrogen-bond interactions apparently essential for proper folding of the ion-binding pocket and/or for subunit interaction. These predictions are supported by functional data recently described by Wynn et al. (2001), which demonstrate that the R183P mutation renders the E1b subunit thermolabile at phys- iological temperatures. Their studies indicate that the mutation confers a temperature-dependent folding de- fect on the E1b subunit, disrupting its ability to complex with the E1a subunit. Mutation analysis of compound-heterozygous indi- viduals 1 and 2 revealed that they contained two ad- ditional novel mutations, 833GrA and 1114GrT, re- spectively (fig. 2C and D). The latter mutation, found in exon 10, introduced a nonsense codon (E422X) trun- cating the terminal 20 amino acids. The importance that the C-terminal has for E1-E2–subunit association was pointed out by Ævarsson et al. (2000), who reported that addition of a His6 tail onto E1b did not affect E1 heterotetramer assembly but did interfere with the sub- sequent E2-association step. This observation raises the possibility that the truncation mutant might interfere with subunit association, although deletion of such a large region might interfere more generally with protein folding. The missense 833GrA mutation in exon 7 re- sulted in the substitution G278S. This residue was also conserved between mammalian and bacterial E1 homo- logues, and it is situated at a hairpin turn between b- sheet j and a-helix 9 (fig. 3B). The adjacent residue on helix 9, W277, forms a hydrogen-bond interaction with E231 in a hydrophobic pocket important in E1b-E1b′ interactions (Ævarsson et al. 2000). The G278S mutant might therefore destabilize E1b-subunit interactions dur- ing heterotetramer assembly. The severity of these mu- tations corresponds well with the observed clinical phe- notypes, since G278S is associated with the intermediate phenotype and E372X is associated with the classic phenotype. On the basis of the mutation-analysis results, we screened for the R183P mutation in DNA samples from 1,014 AJ individuals from the New York metropolitan area that were obtained with informed consent. All per- sonal identifiers were removed, and the samples were tested anonymously. PCR amplification was performed with genomic DNA in a volume of 50 ml containing 10 pmol of exon 5–specific primers (table 1), with 100 mM 866 Am. J. Hum. Genet. 69:863–868, 2001 Figure 2 Results of mutation analysis of AJ patients with MSUD. A, C, and D, Changes (arrows) from expected wild-type sequence, which is shown below the traces. The affected codon is boxed, and the predicted amino acid change is indicated below the codon. The different sequence traces correspond to exon 5 in individual 4 (A), to exon 10 in individual 2 (C), and to exon 7 in individual 1 (D). B, Representative digestion of exon 5 amplimers with NlaIV (New England Biolabs). The sizes of 100-bp-ladder marker fragments (lane M) are indicated to the left of the gel. The samples shown were amplified from the genomic DNA of individuals 1 (lane 1), 5 (lane 2), 6 (lane 3), 7 (lane 4), and 9 (lane 5). Undigested amplimers are 237 bp in length, whereas digested products are predicted to be 99 bp and 128 bp in length. of each dNTP (Roche Molecular Biochemicals), 5 U Taq DNA Polymerase (Roche), 10 mM Tris-HCl, 50 mM KCl, 0.1% Triton X-100, and 1.5 mM MgCl2. Aliquots (4 ml) of the PCR products were blotted onto replicate 8#12-cm Hybond-N� membranes (Amersham Phar- macia Biotech), by a Biomek 2000 automated pipetting workstation (Beckman Coulter), for hybridization with allele-specific oligonucleotides (ASOs) for the R183P mutation (5′-TCACTATCCCGTCCCCTT-3′) and the wild-type sequence (5′-TCACTATCCGGTCCCCTT-3′). Mutation detection was performed by radioactive meth- ods as described elsewhere (Fodor et al. 1998). For ra- dioactive detection, the membranes were hybridized for 2 h at 42�C, with ∼106 cpm [32P]-ATP end-labeled probe/ ml and 10-fold molar excess of the mutant or wild-type competitor oligonucleotide. The membranes were then washed sequentially in 5 # SSC for 5 min at room temperature and in 5 # SSC for 5 min at 45�C, followed by a wash in 0.1 # SSC/0.1% SDS for 5 min at 45�C, and then were exposed to autoradiography. Nine carriers were identified, which corresponded to a carrier fre- quency of ∼1/113. Samples giving a positive signal with the mutant ASO were reamplified, and carrier status confirmed by the NlaIV PCR-RFLP assay. The AJ R183P allele, which accounts for 10 of 12 mutant E1b alleles in our limited series, was present at a frequency significantly higher than the estimated MSUD-carrier frequency (∼1/215) in the general pop- ulation (Peinemann et al. 1993; Danner and Doering 1998; Chuang and Shih 2000) This finding is consistent with our observation of a disproportionate number of AJ cases of MSUD. The expected disease incidence for R183P homozygotes is 1/51,000. Additional compound- heterozygous cases are expected, but the degree of allelic heterogeneity in the population remains to be elucidated. A rough estimate of the number of AJ individuals born after the initiation (in 1968) of New York State newborn screening for MSUD was derived on the basis of infor- mation from the most recent (i.e., 1991) available United Jewish Appeal Federation Population Estimates and the 2000 U.S. census (U.S. Census Bureau, American Fact Finder). Assuming that the proportion of AJ individuals !30 years of age is the same as that in the general pop- ulation and that the percentage of AJ families has re- mained constant, we estimated that ∼480,000 AJ indi- viduals were born after the initiation of screening. The expected total of approximately nine homozygous cases is somewhat greater than our clinical observations but within reason, considering the crudeness of the estimate. The high prevalence of R183P in our patients sug- Reports 867 Figure 3 Ribbon representations of portions of E1b crystal struc- ture, showing positions of mutated residues. The E1 crystal-structure data were obtained from the NCBI Structure (Molecular Modeling Database). Swissprot-Pdb Viewer was used to generate models and image files. A, Ribbon diagram of b-sheets d, e, and g, with the side chains of E146, R183, and E239, respectively, which are shown in white. The mutated residue, R183, is indicated by the arrow, side- chain hydrogen-bond interactions are indicated by dashed lines, and the coordinated K� ion is labeled directly. For clarity of presentation, main-chain hydrogen interactions have been omitted. B, Overview of entire molecule, except for structures overlying the sheet containing R183. The residues altered by missense mutations, R183 and G228, are indicated by bent and dashed arrows, respectively, and their side chains are shown in white. The a-carbon backbone of residues trun- cated by the nonsense mutation E372X are shown in white. gests that the identification of this mutation will facilitate prenatal diagnosis of affected families and may permit carrier screening after the identification of additional common mutations in the AJ population. Population screening is currently being offered for other diseases with similar allele frequencies as R183P, including Bloom syndrome (1/107) (Li et al. 1998), mucolipidosis type IV (1/100) (Bargal et al. 2001), Niemann-Pick type A (∼1/90) (Schuchman and Miranda 1997), and Fanconi anemia group C (1/89) (Verlander et al. 1995; Auerbach 1997). Effective carrier detection may decrease the in- cidence and/or the morbidity associated with delayed diagnosis of this disorder in the AJ population. Acknowledgments The authors would like to express their appreciation to all of the families who agreed to participate in the study and to Shihong Zhang for excellent technical assistance. G.A.D. was supported in part by Mount Sinai Child Health Research Cen- ter grant 5 P30 HD 28822. Electronic-Database Information Accession numbers and URLs for data in this article are as follows: GenBank Overview, http://www.ncbi.nlm.nih.gov/Genbank/ GenbankOverview.html (for human branched-chain a-keto dehydrogenase E1b-subunit mRNA [accession U50708]) GeneMap’99, http://www.ncbi.nlm.nih.gov/genemap99/ Human Genome Project Working Draft at UCSC, http:// genome.ucsc.edu NCBI Structure (Molecular Modeling Database), http:// www.ncbi.nlm.nih.gov/Structure/MMDB/mmdb.shtml (for human BCKAD [accession number 1DTW]) Online Mendelian Inheritance in Man (OMIM), http://www .ncbi.nlm.nih.gov/Omim (for MSUD types Ia [MIM 248600], Ib [MIM 248611]), and II [MIM 248610]) U.S. Census Bureau, American Fact Finder, http://factfinder .census.gov/servlet/BasicFactsServlet References Ævarsson A, Chuang JL, Wynn RM, Turley S, Chuang DT, Hol WG (2000) Crystal structure of human branched-chain a-ketoacid dehydrogenase and the molecular basis of mul- tienzyme complex deficiency in maple syrup urine disease. Structure Fold Des 8:277–291 Auerbach AD (1997) Fanconi anemia: genetic testing in Ash- kenazi Jews. Genet Test 1:27–33 Bargal R, Avidan N, Olender T, Ben Asher E, Zeigler M, Raas- Rothschild A, Frumkin A, Ben-Yoseph O, Friedlender Y, Lancet D, Bach G (2001) Mucolipidosis type IV: novel MCOLN1 mutations in Jewish and non-Jewish patients and the frequency of the disease in the Ashkenazi Jewish pop- ulation. Hum Mutat 17:397–402 Chuang DT, Shih VE (2000) Disorders of branched chain amino acid and keto acid metabolism. In: Scriver CR, Beau- det A, Sly WL, Valle D (eds) The metabolic and molecular bases of inherited disease, 8th ed. McGraw-Hill, New York, pp 1971–2006 Damuni Z, Merryfield ML, Humphreys JS, Reed LJ (1984) Purification and properties of branched-chain alpha-keto 868 Am. J. Hum. Genet. 69:863–868, 2001 acid dehydrogenase phosphatase from bovine kidney. Proc Natl Acad Sci USA 81:4335–4338 Danner DJ, Doering CB (1998) Human mutations affecting branched chain alpha-ketoacid dehydrogenase. Front Biosci 3:d517–d524 Fisher CW, Fisher CR, Chuang JL, Lau KS, Chuang DT, Cox RP (1993) Occurrence of a 2-bp (AT) deletion allele and nonsense (G-to-T) mutant allele at the E2 (DBT) locus of six patients with maple syrup urine disease: multiple-exon skipping as a secondary effect of the mutations. Am J Hum Genet 52:414–424 Fodor FH, Weston A, Bleiweiss IJ, McCurdy LD, Walsh MM, Tartter PI, Brower ST, Eng CM (1998) Frequency and carrier risk associated with common BRCA1 and BRCA2 mutations in Ashkenazi Jewish breast cancer patients. Am J Hum Genet 63:45–51 Guex N, Peitsch MC (1997) SWISS-MODEL and the Swiss- PdbViewer: an environment for comparative protein mod- eling. Electrophoresis 18:2714–2723 Kaplan P, Mazur A, Field M, Berlin JA, Berry GT, Heidenreich R, Yudkoff M, Segal S (1991) Intellectual outcome in chil- dren with maple syrup urine disease. J Pediatr 119:46–50 Levin ML, Scheimann A, Lewis RA, Beaudet AL (1993) Ce- rebral edema in maple syrup urine disease. J Pediatr 122: 167–168 Li L, Eng C, Desnick RJ, German J, Ellis NA (1998) Carrier frequency of the Bloom syndrome blmAsh mutation in the Ashkenazi Jewish population. Mol Genet Metab 64:286–290 Marshall L, DiGeorge A (1981) Maple syrup urine disease in the Old Order Mennonites. Am J Hum Genet Suppl 33:139A Menkes J, Hurst P, Craig JM (1954) A new syndrome: pro- gressive familial infantile cerebral dysfunction associated with an unusual urinary substance. Pediatrics 14:462–466 Naylor EW (1980) Newborn screening for maple syrup urine disease (branched-chain ketoaciduria) In: Bickel H, Guthrie R, Hammersen G (eds) Neonatal screening for inborn errors of metabolism. Springer-Verlag, Berlin, pp 19–28 Nellis MM, Danner DJ (2001) Gene preference in maple syrup urine disease. Am J Hum Genet 68:232–237 Peinemann F, Wendel U, Danner DJ (1993) Molecular genetic characterization of maple syrup urine disease in European families. Biochem Med Metab Biol 50:338–345 Pettit FH, Yeaman SJ, Reed LJ (1978) Purification and char- acterization of branched chain a-keto acid dehydrogenase complex of bovine kidney. Proc Natl Acad Sci USA 75:4881– 4885 Reed LJ, Damuni Z, Merryfield ML (1985) Regulation of mammalian pyruvate and branched-chain a-keto acid de- hydrogenase complexes by phosphorylation-dephosphory- lation. Curr Top Cell Regul 27:41–49 Riviello JJ Jr, Rezvani I, DiGeorge AM, Foley CM (1991) Ce- rebral edema causing death in children with maple syrup urine disease. J Pediatr 119:42–45 Schuchman EH, Miranda SR (1997) Niemann-Pick disease: mutation update, genotype/phenotype correlations and prospects for genetic testing. Genet Test 1:13–19 Snyderman SE, Norton PM, Roitman E, Holt LE Jr (1964) Maple syrup urine disease with particular reference to dieto- therapy. Pediatrics 34:454 Verlander PC, Kaporis A, Liu Q, Zhang Q, Seligsohn U, Auer- bach AD (1995) Carrier frequency of the IVS4 � 4 ArT mutation of the Fanconi anemia gene FAC in the Ashkenazi Jewish population. Blood 86:4034–4038 Wynn RM, Chuang JL, Sansaricq C, Mandel H, Chuang DT (2001) Biochemical basis of type 1B (E1b) mutations in ma- ple syrup urine disease: a prevalent allele in patients from the Druze kindred in Israel. J Biol Chem (in press) Zneimer SM, Lau KS, Eddy RL, Shows TB, Chuang JL, Chuang DT, Cox RP (1991) Regional assignment of two genes of the human branched-chain a-keto acid dehydrog- enase complex: the E1 b gene (BCKDHB) to chromosome 6p21-22 and the E2 gene (DBT) to chromosome 1p31. Ge- nomics 10:740–747 Maple Syrup Urine Disease: Identification and Carrier-Frequency Determination of a Novel Founder Mutation in the Ashkenazi Jewish Population Acknowledgments References