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A Mutation in the Gene for the Neurotransmitter Receptor–Clustering Protein Gephyrin Causes a Novel Form of Molybdenum Cofactor Deficiency

2001; Elsevier BV; Volume: 68; Issue: 1 Linguagem: Inglês

10.1086/316941

1537-6605

Jochen Reiss, Sigrid Gross-Hardt, Ernst Christensen, Peter J. Schmidt, Ralf R. Mendel, Günter Schwarz,

Mitochondrial Function and Pathology

Gephyrin was originally identified as a membrane-associated protein that is essential for the postsynaptic localization of receptors for the neurotransmitters glycine and GABAA. A sequence comparison revealed homologies between gephyrin and proteins necessary for the biosynthesis of the universal molybdenum cofactor (MoCo). Because gephyrin expression can rescue a MoCo-deficient mutation in bacteria, plants, and a murine cell line, it became clear that gephyrin also plays a role in MoCo biosynthesis. Human MoCo deficiency is a fatal disease resulting in severe neurological damage and death in early childhood. Most patients harbor MOCS1 mutations, which prohibit formation of a precursor, or carry MOCS2 mutations, which abrogate precursor conversion to molybdopterin. The present report describes the identification of a gephyrin gene (GEPH) deletion in a patient with symptoms typical of MoCo deficiency. Biochemical studies of the patient's fibroblasts demonstrate that gephyrin catalyzes the insertion of molybdenum into molybdopterin and suggest that this novel form of MoCo deficiency might be curable by molybdate supplementation. Gephyrin was originally identified as a membrane-associated protein that is essential for the postsynaptic localization of receptors for the neurotransmitters glycine and GABAA. A sequence comparison revealed homologies between gephyrin and proteins necessary for the biosynthesis of the universal molybdenum cofactor (MoCo). Because gephyrin expression can rescue a MoCo-deficient mutation in bacteria, plants, and a murine cell line, it became clear that gephyrin also plays a role in MoCo biosynthesis. Human MoCo deficiency is a fatal disease resulting in severe neurological damage and death in early childhood. Most patients harbor MOCS1 mutations, which prohibit formation of a precursor, or carry MOCS2 mutations, which abrogate precursor conversion to molybdopterin. The present report describes the identification of a gephyrin gene (GEPH) deletion in a patient with symptoms typical of MoCo deficiency. Biochemical studies of the patient's fibroblasts demonstrate that gephyrin catalyzes the insertion of molybdenum into molybdopterin and suggest that this novel form of MoCo deficiency might be curable by molybdate supplementation. In mammals, molybdenum cofactor (MoCo) is essential for the activity of sulfite oxidase, xanthine dehydrogenase, and aldehyde oxidase (Johnson and Wadman Johnson and Wadman, 1995Johnson JL Wadman SK Molybdenum cofactor deficiency and isolated sulfite oxidase deficiency.in: Scriver CR Beaudet AL Sly WS Valle D The metabolic and molecular bases of inherited disease. McGraw-Hill, New York1995: 2271-2283Google Scholar). Mutations affecting the MoCo biosynthetic pathway result in the simultaneous loss of all molybdoenzyme activities. The cerebral atrophy associated with MoCo deficiency (MIM 252150) is also observed in isolated sulfite oxidase deficiency (MIM 272300) and can be attributed exclusively to the loss of this enzyme. Both the isolated and the combined form are inherited as autosomal recessive traits, without any symptoms in heterozygous carriers, and they come to clinical attention because of untreatable neonatal seizures, with opisthotonos and facial dysmorphism. The combined form is found more often, with ∼100 cases known worldwide. We had examined 42 of these patients and had found mutations in MOCS1(MIM 603707) (Reiss et al. Reiss et al., 1998bReiss J Cohen N Dorche C Mandel H Mendel RR Stallmeyer B Zabot MT Dierks T Mutations in a polycistronic nuclear gene associated with molybdenum cofactor deficiency.Nat Genet. 1998b; 20: 51-53Crossref PubMed Scopus (98) Google Scholar) or MOCS2 (MIM 603708) (Stallmeyer Stallmeyer et al., 1999aStallmeyer B Drugeon G Reiss J Haenni AL Mendel RR The human molybdopterin synthase gene: identification of a bicistronic transcript with overlapping reading frames.Am J Hum Genet. 1999a; 64: 698-705Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar) in 40 cases (Reiss et al. Reiss et al., 1998aReiss J Christensen E Kurlemann G Zabot MT Dorche C Genomic structure and mutational spectrum of the bicistronic MOCS1 gene defective in molybdenum cofactor deficiency type A.Hum Genet. 1998a; 103: 639-644Crossref PubMed Scopus (53) Google ScholarReiss et al., 1999Reiss J Cohen N Stallmeyer B Mendel RR Dorche C The human molybdopterin synthase gene: genomic structure and mutations in molybdenum cofactor deficiency type B.Am J Hum Genet. 1999; 64: 706-711Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar; Reiss Reiss, 2000Reiss J Genetics of molybdenum cofactor deficiency.Hum Genet. 2000; 106: 157-163Crossref PubMed Scopus (86) Google Scholar). We studied the last of three affected infants born to a Danish mother and father who were cousins. All three died in the neonatal period (day 12, 29, and 3, respectively), with symptoms identical to MoCo deficiency. Three other pregnancies of the mother resulted in two healthy sibs and one spontaneous abortion. There was no family history of genetic disease, and all three affected infants had a normal karyotype. The first infant was a boy, and the other two were girls. All showed hypotonia combined with hyperreflexia, as well as tonic-clonic convulsions. Fibroblasts of the third infant, the patient, were used to verify a MoCo deficiency by biochemical and in vitro complementation assays and to isolate DNA for genetic analysis. The study was approved by the ethics committee of the Medical Faculty of Göttingen. Complete sequencing of all MOCS1 and MOCS2 exons, plus adjacent splice-junction sequences, did not reveal a disease-causing mutation in the patient. The human MOCS3 cDNA and genomic sequence, encoding the molybdopterin synthase sulfurylase (Appleyard et al. Appleyard et al., 1998Appleyard MVCL Sloan J Kana'n JM Heck IS Kinghorn JR Unkles SE The Aspergillus nidulans cnxF gene and its involvement in molybdopterin biosynthesis.J Biol Chem. 1998; 273: 14869-14876Crossref PubMed Scopus (36) Google Scholar), were also found to be devoid of mutations in the patient's DNA (data not shown). We analyzed the GEPH gene (MIM 603930) for the receptor-clustering neuroprotein gephyrin (Kirsch et al Kirsch et al., 1993Kirsch J Wolters I Triller A Betz H Gephyrin antisense oligonucleotides prevent glycine receptor clustering in spinal neurons.Nature. 1993; 366: 745-748Crossref PubMed Scopus (366) Google Scholar; Essrich et al. Essrich et al., 1998Essrich C Lorez M Benson JA Fritschy JM Luscher B Postsynaptic clustering of major GABAA receptor subtypes requires the γ2 subunit and gephyrin.Nat Neurosci. 1998; 1: 563-571Crossref PubMed Scopus (695) Google Scholar; Kneussel and Betz Kneussel and Betz, 2000Kneussel M Betz H Receptors, gephyrin and gephyrin-associated proteins: novel insights into the assembly of inhibitory postsynaptic membrane specializations.J Physiol. 2000; 525: 1-9Crossref PubMed Scopus (144) Google Scholar) as a candidate gene for the MoCo deficiency in this family. Gephyrin consists of two distinct protein domains separated by a linker sequence (fig. 1a). The N-terminal domain of gephyrin is homologous to the bacterial protein MogA, and the C-terminal domain is homologous to bacterial MoeA, both proteins being involved in the biosynthesis of MoCo (Stewart Stewart, 1988Stewart V Nitrate respiration in relation to facultative metabolism in Enterobacteria.Microbiol Rev. 1988; 52: 190-232Crossref PubMed Google Scholar). This architecture is shared by the Drosophila protein Cinnamon (Kamdar et al. Kamdar et al., 1994Kamdar KP Shelton ME Finnerty V The Drosophila molybdenum cofactor gene Cinnamon is homologous to three Escherichia coli cofactor proteins and to the rat protein gephyrin.Genetics. 1994; 137: 791-801Crossref PubMed Google Scholar) and, with a reversed domain order, the plant protein Cnx1 (Stallmeyer et al. Stallmeyer et al., 1995Stallmeyer B Nerlich A Schiemann J Brinkmann H Mendel RR Molybdenum cofactor biosynthesis: the Arabidopsis thaliana cDNA cnx1 encodes a multifunctional two-domain protein homologous to a mammalian neuroprotein, the insect protein Cinnamon and three Escherichia coli proteins.Plant J. 1995; 8: 751-762Crossref PubMed Scopus (87) Google Scholar). The bacterial moaB gene is also homologous to the 5′ region of the GEPH cDNA, as well as to the bacterial mogA gene. However, no bacterial mutant and no function is known for moaB. The moeA gene product is believed to activate molybdenum before its incorporation into molybdopterin (Hasona et al. Hasona et al., 1998Hasona A Ramesh MR Shanmugam KT Physiological and genetic analyses leading to identification of a biochemical role for the moeA (molybdate metabolism) gene product of Escherichia coli.J Bacteriol. 1998; 180: 1466-1472Crossref PubMed Google Scholar; Kuper et al. Kuper et al., 2000Kuper J Palmer T Mendel RR Schwarz G Mutations in the molybdenum cofactor biosynthetic protein Cnx1G from Arabidopsis thaliana define functions for molybdopterin binding, molybdenum insertion, and molybdenum cofactor stabilization.Proc Natl Acad Sci USA. 2000; 97: 6475-6480Crossref PubMed Scopus (39) Google Scholar). Bacterial mogA mutants can produce active MoCo in media supplemented with high concentrations of molybdate (Stewart and MacGregor Stewart and MacGregor, 1982Stewart V MacGregor CH Nitrate reductase in Escherichia coli K-12: involvement of chlC, chlE, and chlG loci.J Bacteriol. 1982; 151: 788-799Crossref PubMed Google Scholar). This "molybdenum rescue" is also observed for the corresponding plant mutants (Stallmeyer et al. Stallmeyer et al., 1995Stallmeyer B Nerlich A Schiemann J Brinkmann H Mendel RR Molybdenum cofactor biosynthesis: the Arabidopsis thaliana cDNA cnx1 encodes a multifunctional two-domain protein homologous to a mammalian neuroprotein, the insect protein Cinnamon and three Escherichia coli proteins.Plant J. 1995; 8: 751-762Crossref PubMed Scopus (87) Google Scholar) and the MoCo-deficient murine cell line L929 (Falciani et al. Falciani et al., 1994Falciani F Terao M Goldwurm S Ronchi A Gatto A Minoia C Calzi ML Salmona M Cazzaniga G Garattini E Molybdenum (VI) salts convert the xanthine oxidoreductase apoprotein into the active enzyme in mouse L929 fibroblastic cells.Biochem J. 1994; 298: 69-77Crossref PubMed Scopus (27) Google Scholar). Geph cDNA derived from rats could restore molybdenum-repairable plant mutants and bacterial mogA mutants, as well as L929 cells (Stallmeyer et al. Stallmeyer et al., 1999bStallmeyer B Schulze J Schwarz G Nerlich A Reiss J Kirsch J Mendel RR The neurotransmitter receptor-anchoring protein gephyrin reconstitutes molybdenum cofactor biosynthesis in bacteria, plants, and mammalian cells.Proc Natl Acad Sci USA. 1999b; 96: 1333-1338Crossref PubMed Scopus (131) Google Scholar). Likewise, Geph knockout mice showed the expected absence of synaptic glycine-receptor clustering and developed symptoms identical to those of MoCo deficiency (Feng et al. Feng et al., 1998Feng G Tintrup H Kirsch J Nichol MC Kuhse J Beth H Sanes JR Dual requirement for gephyrin in glycine receptor clustering and molybdoenzyme activity.Science. 1998; 282: 1321-1324Crossref PubMed Scopus (324) Google Scholar). Therefore, gephyrin combines an evolutionarily novel function (neuroreceptor clustering) with a conserved old function (MoCo biosynthesis), the latter being essential for activation (E domain) and insertion (G domain) of molybdenum into molybdopterin, thereby forming the biologically active cofactor (fig. 1b). The rat Geph cDNA sequence (Prior et al. Prior et al., 1992Prior P Schmitt B Grenningloh G Priballa I Multhaup G Beyreuther K Maulet Y Werner P Langosch D Kirsch J Betz H Primary structure and alternative splice variants of gephyrin, a putative glycine receptor-tubulin linker protein.Neuron. 1992; 8: 1161-1170Abstract Full Text PDF PubMed Scopus (273) Google Scholar) was used to screen the nonredundant GenBank database for human cDNA sequences with the homology search program Advanced BLAST. A 4,193-bp cDNA clone derived from brain tissue contains the complete coding sequence for human gephyrin, with the start codon at position 1122 and the stop codon at position 3330. The nucleotide and amino acid sequence homologies are 93% and 100%, respectively. The human cDNA sequence was used to identify the genomic sequences in the HTGS (high throughout genomic sequences) library. A homology search revealed that the gene is located on chromosome 14 and consists of 22 exons covering a genomic region of ⩾375 kbp. These genomic sequences were used as primer targets to amplify all exons individually, plus each of their adjacent splice-signal sequences (table 1). Amplification of patient and control DNA revealed a deletion encompassing exons 2 and 3 in the patient (fig. 2a). This deletion was verified by reverse transcriptase PCR, in which exclusively truncated transcripts were detected (fig. 2b), followed by sequencing of the amplification products (fig. 3a). On the mRNA level, this deletion results in a frameshift after only 21 codons of the normal coding sequence. Therefore, neither of the two gephyrin domains (fig. 1) is expressed. Western blot analysis confirmed the complete absence of gephyrin protein in crude extracts of the patient's fibroblasts, whereas the expression of at least two splice variants (Prior et al. Prior et al., 1992Prior P Schmitt B Grenningloh G Priballa I Multhaup G Beyreuther K Maulet Y Werner P Langosch D Kirsch J Betz H Primary structure and alternative splice variants of gephyrin, a putative glycine receptor-tubulin linker protein.Neuron. 1992; 8: 1161-1170Abstract Full Text PDF PubMed Scopus (273) Google Scholar) can be seen in control cells (fig. 3b).Table 1Oligonucleotides Used in Study of Patient with Gephyrin Gene DeletionMethodaA = amplification; E = exon.PrimerSequenceEA: E1G1F5′-CCTAGCTGTCGCGCTCTCCT-3′G1R5′-TCCCGAGGCCGCAGAGAAGG-3′ E2G2F5′-GGTCAGCAATAGCTTAAATG-3′G2R5′-CTCTTTTGAGAAAAGGAACAC-3′ E3G3F5′-GCATTCTGATGGTAATGGCA-3′G3R5′-ACCCCTCACCAAGATGCTAA-3′ E4G4F5′-GGGATGTTTTGAGCAAGCAG-3′G4R5′-CCATGATTAGTTTAATCCTTG-3′ E5G5F5′-GTGGGTTTTACTAGTCTGAC-3′G5R5′-ACAGTTCACCTAGCAAATGG-3′ E6G6F5′-CTTAATGTATTTAAACCGGGC-3′G6R5′-TTTGGCTCCCTAACTTTCAC-3′ E7G7F5′-CAGTTTGATTGCCACCATCTG7R5′-TTACCTGTGGGTCCTTTAGG-3′ E8G8F5′-AAGGGGGTCTTGATTCTACA-3′G8R5′-CCCAGATTACTATAGAAGAGC-3′ E9G9F5′-ACCTCAGGAGCTTGCCCATT-3′G9R5′-AAGCTCTAGTTCAGCAGCCC-3′ E10G10F5′-GTCATTGCCACTTTTTAATCA-3′G10R5′-CAGGAAAACTGTGCATTAATG-3′ E11G11F5′-CAAGCACTCATGCCCATCTT-3′G11R5′-CAGTGCCTGATTATGTTTAAG-3′ E12G12F5′-CTTGTTCCATGCTGTAGGTC-3′G12R5′-TTCCACTAAACTGATAGGAGA-3′ E13G13F5′-CTTTTCCTTTGCAGCAGCAA-3′G13R5′-ACTGCCATAGGAACAACAGC-3′ E14G14F5′-TATCCTGGGCCTATCTGATG-3′G14R5′-GCCAGGGTTTCCTGAGTAAA-3′ E15G15F5′-CACTAAAGTTTCCCTCTGAG-3′G15R5′-CAACACAGAACATATGTCAG-3′ E16G16F5′-TATGCAACATTAACCTAATAG-3′G16R5′-ATGAGTATTCCAAAAACTCG-3′ E17G17F5′-GCCTATTAGTGAATAAGGCG-3′G17R5′-AGATGCCTACCAGACCACAG-3′ E18G18F5′-TCATTTAAAGTGTTGAAAGTC-3′G18R5′-CCCATATATGAGATAACAAGA-3′ E19G19F5′-AAAACACTGGAGTACTTAATG-3′G19R5′-CCAGAAAAAGGAAAGGAAAC-3′ E20G20F5′-TAGACAGACATAATTATTTGGC-3′G20R5′-TTAGGAAATCATATCCCTAAC-3′ E21G21F5′-GTCCACTGTATTCTTTGCAC-3′G21R5′-CAGGATAGGTGTCTAGGAA-3′ E22G22F5′-AGGGCCCAACTGTATACGCC-3′G22R5′-GCTTTCTCCTGCTGGTGACC-3′RT-PCR: RTRT15′-CAGGGCCATCCCTGGTGCTT-3′ PCR1G1F5′-CCTAGCTGTCGCGCTCTCCT-3′RT15′-CAGGGCCATCCCTGGTGCTT-3′ PCR2RT25′-TTCTCCCGGCTCCTGTCAGT-3′RT35′-TCGTGGTGCAAATCCTGTTC-3′a A = amplification; E = exon. Open table in a new tab Figure 3a, Sequence analysis of the RT-PCR product of control (top) and patient (bottom). Sequencing was done commercially (SeqLab) after purification of the amplification products with the QIA quick PCR purification kit (Qiagen) with the PCR primers and dideoxy fluorescent dye terminators (ABI). The transition from exon 1 to exon 2 or 4, respectively, is indicated by the arrow. b, Western blot analysis of gephyrin expression in crude protein extracts using a polyclonal anti-gephyrin antibody. Crude protein extracts were prepared from fibroblasts, separated by SDS-PAGE using a 7.5 % polyacrylamide gel, and blotted onto a polyvinyldiene fluoride membrane. Positive controls were recombinant rat Geph P1 and P2 splice forms expressed in Escherichia coli (G.S., unpublished data). A primary polyclonal antibody generated against recombinant rat gephyrin P1 protein was used (diluted in serum at 1:2000), followed by chemiluminescent detection (secondary antibody, Promega; ECL system, Amersham/Pharmacia). Lane 1, recombinant rat gephyrin P2 (20 ng); lane 2, recombinant rat gephyrin P1 (20 ng); lane 3, size marker (labeled in kDa); lane 4, positive control (18 μg); lane 5, patient with gephyrin gene deletion (20 μg).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Because of the presumed function of gephyrin in MoCo formation (fig. 1b), loss of this protein should result in the accumulation of molybdenum-free molybdopterin. We demonstrated the presence of the fluorescent oxidation product of molybdopterin ("form A") (Johnson and Wadman Johnson and Wadman, 1995Johnson JL Wadman SK Molybdenum cofactor deficiency and isolated sulfite oxidase deficiency.in: Scriver CR Beaudet AL Sly WS Valle D The metabolic and molecular bases of inherited disease. McGraw-Hill, New York1995: 2271-2283Google Scholar) by high-performance liquid chromatography analysis (HPLC) (fig. 4a). Biologically active molybdopterin and MoCo can be quantified by the in vitro nit-1 reconstitution assay, which is based on the transfer of MoCo to the nitrate reductase apoprotein of the Neurospora crassa nit-1 mutant (Nason et al. Nason et al., 1971Nason A Lee KY Pan SS Ketchum PA Lamberti A De Vries J In vitro formation of assimilatory reduced nicotinamide adenine dinucleotide phosphate: nitrate reductase from a Neurospora mutant and a component of molybdenum-enzymes.Proc Natl Acad Sci USA. 1971; 68: 3242-3246Crossref PubMed Scopus (151) Google Scholar). Depending on presence or absence of molybdate in the assay, it can be used for the detection of total molybdopterin (+Mo), including all forms of the pterin and MoCo (−Mo). HPLC analysis of the patient's cells shows an increased level of molybdopterin (fig. 4b), compared with control cells. Active MoCo could be detected only in control cells and in those patient cells that were supplemented with 1 mM molybdate (fig. 4b). This molybdate repair of the cofactor could also be demonstrated by the regained sulfite oxidase activity (fig. 4c). After the primary sequence of the receptor-associated gephyrin revealed homologies to MoCo biosynthetic enzymes, it appeared counterintuitive that a eukaryotic protein might have two such different functions as (i) a structural role in receptor clustering and (ii) a biosynthetic activity in MoCo formation. It has been suggested that two bacterial biosynthetic genes (mogA and moeA) have been fused during evolution to form a multidomain protein with a novel function (Kamdar et al. Kamdar et al., 1994Kamdar KP Shelton ME Finnerty V The Drosophila molybdenum cofactor gene Cinnamon is homologous to three Escherichia coli cofactor proteins and to the rat protein gephyrin.Genetics. 1994; 137: 791-801Crossref PubMed Google Scholar; Stallmeyer et al. Stallmeyer et al., 1995Stallmeyer B Nerlich A Schiemann J Brinkmann H Mendel RR Molybdenum cofactor biosynthesis: the Arabidopsis thaliana cDNA cnx1 encodes a multifunctional two-domain protein homologous to a mammalian neuroprotein, the insect protein Cinnamon and three Escherichia coli proteins.Plant J. 1995; 8: 751-762Crossref PubMed Scopus (87) Google Scholar; Feng et al. Feng et al., 1998Feng G Tintrup H Kirsch J Nichol MC Kuhse J Beth H Sanes JR Dual requirement for gephyrin in glycine receptor clustering and molybdoenzyme activity.Science. 1998; 282: 1321-1324Crossref PubMed Scopus (324) Google Scholar). This has happened under persistence of the biosynthetic activities, whose abrogation, as shown here, results in a lethal malfunction. Moreover, the fusion of the same prokaryotic genes, in reversed orientation, in higher plants (Stallmeyer et al. Stallmeyer et al., 1995Stallmeyer B Nerlich A Schiemann J Brinkmann H Mendel RR Molybdenum cofactor biosynthesis: the Arabidopsis thaliana cDNA cnx1 encodes a multifunctional two-domain protein homologous to a mammalian neuroprotein, the insect protein Cinnamon and three Escherichia coli proteins.Plant J. 1995; 8: 751-762Crossref PubMed Scopus (87) Google Scholar) (fig. 1a) demonstrates that there is a strong selective pressure for this convergent development in eukaryotes. The gravity of the disease in the family described here is very similar to the serious phenotype seen in the Geph knockout mice (Feng et al. Feng et al., 1998Feng G Tintrup H Kirsch J Nichol MC Kuhse J Beth H Sanes JR Dual requirement for gephyrin in glycine receptor clustering and molybdoenzyme activity.Science. 1998; 282: 1321-1324Crossref PubMed Scopus (324) Google Scholar). Such mice could be used to test whether MoCo-deficient patients with GEPH mutations could be treated successfully by administering high concentrations of molybdate. In attempts reported elsewhere, administration of an oral (Duran et al. Duran et al., 1978Duran M Beemer FA Heiden CVD Korteland J Bree PKD Brink M Wadman SK Combined deficiency of xanthine oxidase and sulphite oxidase: a defect of molybdenum metabolism or transport?.J Inherit Metab Dis. 1978; 1: 175-178Crossref PubMed Scopus (91) Google Scholar; Munnich et al. Munnich et al., 1983Munnich A Saudubray JM Charpentier C Ogier H Coude FX Frezal J Multiple molybdoenzyme deficiencies due to an inborn error of molybdenum cofactor metabolism: two additional cases in a new family.J Inherit Metab Dis Suppl. 1983; 6: 95-96Crossref PubMed Scopus (12) Google Scholar; Endres et al. Endres et al., 1988Endres W Shin YS Günther R Ibel H Duran M Wadman SK Report on a new patient with combined deficiencies of sulphite oxidase and xanthine dehydrogenase due to molybdenum cofactor deficiency.Eur J Pediatr. 1988; 148: 246-249Crossref PubMed Scopus (21) Google Scholar) or an intravenous (Bamforth et al. Bamforth et al., 1990Bamforth FJ Johnson JL Davidson AGF Wong LTK Lockitch G Applegarth DA Biochemical investigation of a child with molybdenum cofactor deficiency.Clin Biochem. 1990; 23: 537-542Crossref PubMed Scopus (16) Google Scholar) molybdate supplementation to MoCo-deficient patients did not lead to any clinical or biochemical improvement. These patients, however, have not been examined genetically, and, on the basis of the observed mutation frequencies, it seems likely that they carried mutations in the "nonrepairable" genes MOCS1 or MOCS2 (Reiss Reiss, 2000Reiss J Genetics of molybdenum cofactor deficiency.Hum Genet. 2000; 106: 157-163Crossref PubMed Scopus (86) Google Scholar). The dysfunction of gephyrin as a receptor-clustering molecule in the postsynaptic membrane cannot be restored by these means. Hyperekplexia (MIM 149400), as a consequence of isolated receptor dysfunction, is a relatively mild neurological disorder, which can be ameliorated by pharmaceutical agents (Andrew and Owen Andrew and Owen, 1997Andrew M Owen MJ Hyperekplexia: abnormal startle response due to glycine receptor mutations.Br J Psychiatry. 1997; 170: 106-108Crossref PubMed Scopus (37) Google Scholar). These drugs could be combined with a molybdate therapy in gephyrin-deficient patients to treat the synaptic receptor dysfunction, as well as the biosynthetic deficiency. As in the other forms of MoCo deficiency, the most successful approaches may include prenatal treatment to prevent neurological damage. Technical assistance by T. Otte is gratefully acknowledged. We thank U. Lenz and D. Schmalz for helpful advice and W. Engel for continued support. This study has been supported by the Deutsche Forschungsgemeinschaft (support to J.R., R.R.M., and G.S.) and the Fritz Thyssen Stiftung (support to R.R.M.).