Structure and biochemical characterization of l-2-hydroxyglutarate dehydrogenase and its role in the pathogenesis of l-2-hydroxyglutaric aciduria.

l-2-hydroxyglutarate dehydrogenase (L2HGDH) is a mitochondrial membrane-associated metabolic enzyme, which catalyzes the oxidation of l-2-hydroxyglutarate (l-2-HG) to 2-oxoglutarate (2-OG). Mutations in human L2HGDH lead to abnormal accumulation of l-2-HG, which causes a neurometabolic disorder named l-2-hydroxyglutaric aciduria (l-2-HGA). Here, we report the crystal structures of Drosophila melanogaster L2HGDH (dmL2HGDH) in FAD-bound form and in complex with FAD and 2-OG and show that dmL2HGDH exhibits high activity and substrate specificity for l-2-HG. dmL2HGDH consists of an FAD-binding domain and a substrate-binding domain, and the active site is located at the interface of the two domains with 2-OG binding to the re-face of the isoalloxazine moiety of FAD. Mutagenesis and activity assay confirmed the functional roles of key residues involved in the substrate binding and catalytic reaction and showed that most of the mutations of dmL2HGDH equivalent to l-2-HGA-associated mutations of human L2HGDH led to complete loss of the activity. The structural and biochemical data together reveal the molecular basis for the substrate specificity and catalytic mechanism of L2HGDH and provide insights into the functional roles of human L2HGDH mutations in the pathogeneses of l-2-HGA.

Pathogenic mutations of the human mitochondrial citrate carrier SLC25A1 lead to impaired citrate export required for lipid, dolichol, ubiquinone and sterol synthesis.

Missense mutations of the human mitochondrial citrate carrier, encoded by the SLC25A1 gene, lead to an autosomal recessive neurometabolic disorder characterised by neonatal-onset encephalopathy with severe muscular weakness, intractable seizures, respiratory distress, and lack of psychomotor development, often resulting in early death. Here, we have measured the effect of all twelve known pathogenic mutations on the transport activity. The results show that nine mutations abolish transport of citrate completely, whereas the other three reduce the transport rate by >70%, indicating that impaired citrate transport is the most likely primary cause of the disease. Some mutations may be detrimental to the structure of the carrier, whereas others may impair key functional elements, such as the substrate binding site and the salt bridge network on the matrix side of the carrier. To understand the consequences of impaired citrate transport on metabolism, the substrate specificity was also determined, showing that the human citrate carrier predominantly transports citrate, isocitrate, cis-aconitate, phosphoenolpyruvate and malate. Although D-2- and L-2 hydroxyglutaric aciduria is a metabolic hallmark of the disease, it is unlikely that the citrate carrier plays a significant role in the removal of hydroxyglutarate from the cytosol for oxidation to oxoglutarate in the mitochondrial matrix. In contrast, computer simulations of central metabolism predict that the export of citrate from the mitochondrion cannot be fully compensated by other pathways, restricting the cytosolic production of acetyl-CoA that is required for the synthesis of lipids, sterols, dolichols and ubiquinone, which in turn explains the severe disease phenotypes.

Carnosinase, diabetes mellitus and the potential relevance of carnosinase deficiency.

Carnosinase (CN1) is a dipeptidase, encoded by the CNDP1 gene, that degrades histidine-containing dipeptides, such as carnosine, anserine and homocarnosine. Loss of CN1 function (also called carnosinase deficiency or aminoacyl-histidine dipeptidase deficiency) has been reported in a small number of patients with highly elevated blood carnosine concentrations, denoted carnosinaemia; it is unclear whether the variety of clinical symptoms in these individuals is causally related to carnosinase deficiency. Reduced CN1 function should increase serum carnosine concentrations but the genetic basis of carnosinaemia has not been formally confirmed to be due to CNDP1 mutations. A CNDP1 polymorphism associated with low CN1 activity correlates with significantly reduced risk for diabetic nephropathy, especially in women with type 2 diabetes, and may slow progression of chronic kidney disease in children with glomerulonephritis. Studies in rodents demonstrate antiproteinuric and vasculoprotective effects of carnosine, the precise molecular mechanisms, however, are still incompletely understood. Thus, carnosinemia due to CN1 deficiency may be a non-disease; in contrast, carnosine may potentially protect against long-term sequelae of reactive metabolites accumulating, e.g. in diabetes and chronic renal failure.

[Analysis of L2HGDH gene mutation in a patient with 2-hydroxyglutaric aciduria].

OBJECTIVE: To explore pathogenic mutation in a family affected with 2-hydroxyglutaric aciduria. METHODS: Exons of 3 candidate genes, including L2HGDH, D2HGDH and SLC25A1, were amplified with polymerase chain reaction and subjected to direct sequencing. RESULTS: DNA sequencing has found that the proband and his affected younger brother have both carried a heterozygous mutation c.845G>A (p.R282Q) in the exon 7 of the L2HGDH gene. The same mutation was not detected in the his sister who was healthy. Pedigree analysis has confirmed that the above mutation was inherited from the mother. No mutation was detected in exons and flanking sequences of the D2HGDH and SLC25A1 genes. CONCLUSION: Mutation of the L2HGDH gene probably underlies the 2-hydroxyglutaric aciduria in this family.

Effects of hematopoietic stem cell transplantation on acyl-CoA oxidase deficiency: a sibling comparison study.

OBJECTIVE: Acyl-CoA oxidase (ACOX1) deficiency is a rare disorder of peroxisomal very-long chain fatty acid oxidation. No reports detailing attempted treatment, longitudinal imaging, or neuropathology exist. We describe the natural history of clinical symptoms and brain imaging in two siblings with ACOX1 deficiency, including the younger sibling's response to allogeneic unrelated donor hematopoietic stem cell transplantation (HSCT). METHODS: We conducted retrospective chart review to obtain clinical history, neuro-imaging, and neuropathology data. ACOX1 genotyping were performed to confirm the disease. In vitro fibroblast and neural stem cell fatty acid oxidation assays were also performed. RESULTS: Both patients experienced a fatal neurodegenerative course, with late-stage cerebellar and cerebral gray matter atrophy. Serial brain magnetic resonance imaging in the younger sibling indicated demyelination began in the medulla and progressed rostrally to include the white matter of the cerebellum, pons, midbrain, and eventually subcortical white matter. The successfully engrafted younger sibling had less brain inflammation, cortical atrophy, and neuronal loss on neuro-imaging and neuropathology compared to the untreated older sister. Fibroblasts and stem cells demonstrated deficient very long chain fatty acid oxidation. INTERPRETATION: Although HSCT did not halt the course of ACOX1 deficiency, it reduced the extent of white matter inflammation in the brain. Demyelination continued because of ongoing neuronal loss, which may be due to inability of transplant to prevent progression of gray matter disease, adverse effects of chronic corticosteroid use to control graft-versus-host disease, or intervention occurring beyond a critical point for therapeutic efficacy.

Characterization of patient mutations in human persulfide dioxygenase (ETHE1) involved in H2S catabolism.

Hydrogen sulfide (H(2)S) is a recently described endogenously produced gaseous signaling molecule that influences various cellular processes in the central nervous system, cardiovascular system, and gastrointestinal tract. The biogenesis of H(2)S involves the cytoplasmic transsulfuration enzymes, cystathionine ß-synthase and γ-cystathionase, whereas its catabolism occurs in the mitochondrion and couples to the energy-yielding electron transfer chain. Low steady-state levels of H(2)S appear to be controlled primarily by efficient oxygen-dependent catabolism via sulfide quinone oxidoreductase, persulfide dioxygenase (ETHE1), rhodanese, and sulfite oxidase. Mutations in the persulfide dioxgenase, i.e. ETHE1, result in ethylmalonic encephalopathy, an inborn error of metabolism. In this study, we report the biochemical characterization and kinetic properties of human persulfide dioxygenase and describe the biochemical penalties associated with two patient mutations, T152I and D196N. Steady-state kinetic analysis reveals that the T152I mutation results in a 3-fold lower activity, which is correlated with a 3-fold lower iron content compared with the wild-type enzyme. The D196N mutation results in a 2-fold higher K(m) for the substrate, glutathione persulfide.

Complex II deficiency--a case report and review of the literature.

Complex II deficiency is a rare cause of mitochondrial respiratory chain defects with a prevalence of 2-23%. It is exclusively nuclear encoded and functions in the citric acid cycle by oxidizing succinate to fumarate and in the mitochondrial electron transport chain (ETC) by transferring electrons to ubiquinone. Of the four subunits, SDHA and SDHB are catalytic and SDHC and SDHD are anchoring. Mutations in SDHA and SDHAF1 (assembly factor) have been found in patients with CII deficiency and a mitochondrial phenotype. We present a patient with CII deficiency with a previously undescribed phenotype of dilated cardiomyopathy, left ventricular noncompaction, failure to thrive, hypotonia, and developmental delay. Also, a comprehensive review of 36 cases published in the literature was undertaken. The results show that CII deficiency has a variable phenotype with no correlation with residual complex activity in muscle although the phenotype and enzyme activities are comparable within a family. For some, the condition was fatal in infancy, others had multisystem involvement and some had onset in adulthood with mild symptoms and normal cognition. Neurological involvement is most commonly observed and brain imaging commonly shows leukoencephalopathy, Leigh syndrome, or cerebellar atrophy. Mutations in SDHAF1 are associated with leukoencephalopathy. Other organ systems like heart, muscle, and eyes are only involved in about 50% of the cases but cardiomyopathy is associated with high mortality and morbidity. In some patients, riboflavin has provided clinical improvement.

A lymphoblast model for IDH2 gain-of-function activity in d-2-hydroxyglutaric aciduria type II: novel avenues for biochemical and therapeutic studies.

The recent discovery of heterozygous isocitrate dehydrogenase 2 (IDH2) mutations of residue Arg(140) to Gln(140) or Gly(140) (IDH2(wt/R140Q), IDH2(wt/R140G)) in d-2-hydroxyglutaric aciduria (D-2-HGA) has defined the primary genetic lesion in 50% of D-2-HGA patients, denoted type II. Overexpression studies with IDH1(R132H) and IDH2(R172K) mutations demonstrated that the enzymes acquired a new function, converting 2-ketoglutarate (2-KG) to d-2-hydroxyglutarate (D-2-HG), in lieu of the normal IDH reaction which reversibly converts isocitrate to 2-KG. To confirm the IDH2(wt/R140Q) gain-of-function in D-2-HGA type II, and to evaluate potential therapeutic strategies, we developed a specific and sensitive IDH2(wt/R140Q) enzyme assay in lymphoblasts. This assay determines gain-of-function activity which converts 2-KG to D-2-HG in homogenates of D-2-HGA type II lymphoblasts, and uses stable-isotope-labeled 2-keto[3,3,4,4-(2)H(4)]glutarate. The specificity and sensitivity of the assay are enhanced with chiral separation and detection of stable-isotope-labeled D-2-HG by ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Eleven potential inhibitors of IDH2(wt/R140Q) enzyme activity were evaluated with this procedure. The mean reaction rate in D-2-HGA type II lymphoblasts was 8-fold higher than that of controls and D-2-HGA type I cells (14.4nmolh(-1)mgprotein(-1) vs. 1.9), with a corresponding 140-fold increase in intracellular D-2-HG level. Optimal inhibition of IDH2(wt/R140Q) activity was obtained with oxaloacetate, which competitively inhibited IDH2(wt/R140Q) activity. Lymphoblast IDH2(wt/R140Q) showed long-term cell culture stability without loss of the heterozygous IDH2(wt/R140Q) mutation, underscoring the utility of the lymphoblast model for future biochemical and therapeutic studies.

Severe infantile encephalomyopathy caused by a mutation in COX6B1, a nucleus-encoded subunit of cytochrome c oxidase.

Cytochrome c oxidase (COX) deficiency, one of the most common respiratory-chain defects in humans, has been associated with mutations in either mitochondrial DNA genes or nucleus-encoded proteins that are not part in but promote the biogenesis of COX. Mutations of nucleus-encoded structural subunits were sought for but never found in COX-defective patients, leading to the conjecture that they may be incompatible with extra-uterine survival. We report a disease-associated mutation in one such subunit, COX6B1. Nuclear-encoded COX genes should be reconsidered and included in the diagnostic mutational screening of human disorders related to COX deficiency.

Whole-exome sequencing detects somatic mutations of IDH1 in metaphyseal chondromatosis with D-2-hydroxyglutaric aciduria (MC-HGA).

We used exome sequencing of blood DNA in four unrelated patients to identify the genetic basis of metaphyseal chondromatosis with urinary excretion of D-2-hydroxy-glutaric acid (MC-HGA), a rare entity comprising severe chondrodysplasia, organic aciduria, and variable cerebral involvement. No evidence for recessive mutations was found; instead, two patients showed mutations in IDH1 predicting p.R132H and p.R132S as apparent somatic mosaicism. Sanger sequencing confirmed the presence of the mutation in blood DNA in one patient, and in blood and saliva (but not in fibroblast) DNA in the other patient. Mutations at codon 132 of IDH1 change the enzymatic specificity of the cytoplasmic isocitrate dehydrogenase enzyme. They result in increased D-2-hydroxy-glutarate production, α-ketoglutarate depletion, activation of HIF-1α (a key regulator of chondrocyte proliferation at the growth plate), and reduction of N-acetyl-aspartyl-glutamate level in glial cells. Thus, somatic mutations in IDH1 may explain all features of MC-HGA, including sporadic occurrence, metaphyseal disorganization, and chondromatosis, urinary excretion of D-2-hydroxy-glutaric acid, and reduced cerebral myelinization.

Succinyl-CoA:3-oxoacid coenzyme A transferase (SCOT) deficiency: A rare and potentially fatal metabolic disease.

Succinyl-CoA:3-oxoacid coenzyme A transferase deficiency (SCOTD) is a rare autosomal recessive disorder of ketone body utilization caused by mutations in OXCT1. We performed a systematic literature search and evaluated clinical, biochemical and genetic data on 34 previously published and 10 novel patients with SCOTD. Structural mapping and in silico analysis of protein variants is also presented. All patients presented with severe ketoacidotic episodes. Age at first symptoms ranged from 36 h to 3 years (median 7 months). About 70% of patients manifested in the first year of life, approximately one quarter already within the neonatal period. Two patients died, while the remainder (95%) were alive at the time of the report. Almost all the surviving patients (92%) showed normal psychomotor development and no neurologic abnormalities. A total of 29 missense mutations are reported. Analysis of the published crystal structure of the human SCOT enzyme, paired with both sequence-based and structure-based methods to predict variant pathogenicity, provides insight into the biochemical consequences of the reported variants. Pathogenic variants cluster in SCOT protein regions that affect certain structures of the protein. The described pathogenic variants can be viewed in an interactive map of the SCOT protein at https://michelanglo.sgc.ox.ac.uk/r/oxct. This comprehensive data analysis provides a systematic overview of all cases of SCOTD published to date. Although SCOTD is a rather benign disorder with often favourable outcome, metabolic crises can be life-threatening or even fatal. As the diagnosis can only be made by enzyme studies or mutation analyses, SCOTD may be underdiagnosed.

An overview of L-2-hydroxyglutarate dehydrogenase gene (L2HGDH) variants: a genotype-phenotype study.

L-2-Hydroxyglutaric aciduria (L2HGA) is a rare, neurometabolic disorder with an autosomal recessive mode of inheritance. Affected individuals only have neurological manifestations, including psychomotor retardation, cerebellar ataxia, and more variably macrocephaly, or epilepsy. The diagnosis of L2HGA can be made based on magnetic resonance imaging (MRI), biochemical analysis, and mutational analysis of L2HGDH. About 200 patients with elevated concentrations of 2-hydroxyglutarate (2HG) in the urine were referred for chiral determination of 2HG and L2HGDH mutational analysis. All patients with increased L2HG (n=106; 83 families) were included. Clinical information on 61 patients was obtained via questionnaires. In 82 families the mutations were detected by direct sequence analysis and/or multiplex ligation dependent probe amplification (MLPA), including one case where MLPA was essential to detect the second allele. In another case RT-PCR followed by deep intronic sequencing was needed to detect the mutation. Thirty-five novel mutations as well as 35 reported mutations and 14 nondisease-related variants are reviewed and included in a novel Leiden Open source Variation Database (LOVD) for L2HGDH variants (http://www.LOVD.nl/L2HGDH). Every user can access the database and submit variants/patients. Furthermore, we report on the phenotype, including neurological manifestations and urinary levels of L2HG, and we evaluate the phenotype-genotype relationship.

Myopathy and parkinsonism in phosphoglycerate kinase deficiency.

A 25-year-old man with exertional myoglobinuria had no evidence of hemolytic anemia, but he had severe parkinsonism that was responsive to levodopa. Phosphoglycerate kinase (PGK) activity was markedly decreased in muscle, and molecular analysis of the PGK1 gene identified the p.T378P mutation that was recently reported in a patient with isolated myopathy. This case reinforces the concept that PGK deficiency is a clinically heterogeneous disorder and raises the question of a relationship between PGK deficiency and idiopathic juvenile Parkinson disease.

Multiple sources of metabolic disturbance in ETHE1-related ethylmalonic encephalopathy.

Ethylmalonic encephalopathy (EE) is a rare metabolic disorder caused by dysfunction of ETHE1, a mitochondrial dioxygenase involved in hydrogen sulfide (H2S) detoxification. Patients present in infancy with psychomotor retardation, chronic diarrhea, orthostatic acrocyanosis and relapsing petechiae. High levels of lactic acid, ethymalonic acid (EMA) and methylsuccinic acid (MSA) are detected in body fluids. Several pathways may contribute to the pathophysiology, including isoleucine, methionine and fatty acid metabolism. We report on a 15-month-old male presenting with typical EE associated with a homozygous ETHE1 mutation. We investigated oral isoleucine (150 mg/kg), methionine (100 mg/kg), fatty acid loading tests and isoleucine-restricted diet (200 mg/day) for any effects on several metabolic parameters. Before loading tests or specific dietary interventions, EMA, C4-C5 acylcarnitines and most acylglycines were elevated, indicating functional deficiency of short chain acyl-CoA (SCAD) as well as all branched acyl-CoA dehydrogenases. Excretion of EMA and n-butyrylglycine increased following each of the loads, and isoleucine led to increased levels of derivative metabolites. An isoleucine-restricted diet for 8 days corrected some of the abnormalities but led to no obvious clinical improvement and only partial effects on EMA. A principal component analysis supports the inference that these dietary conditions have consistent effects on the global metabolic profile. Our results suggest that multiple pathways modulate EMA levels in EE. They might all interact with H2S toxicity. Prolonged dietary interventions involving the restriction for branched aminoacids, fatty acids and methionine could be discussed as auxiliary therapeutical strategies in EE.

RNaseH2 mutants that cause Aicardi-Goutieres syndrome are active nucleases.

Mutations in the genes encoding the RNaseH2 and TREX1 nucleases have been identified in patients with Aicardi-Goutieres syndrome (AGS). To determine if the AGS RNaseH2 mutations result in the loss of nuclease activity, the human wild-type RNaseH2 and four mutant complexes that constitute the majority of mutations identified in AGS patients have been prepared and tested for ribonuclease H activity. The heterotrimeric structures of the mutant RNaseH2 complexes are intact. Furthermore, the ribonuclease H activities of the mutant complexes are indistinguishable from the wild-type enzyme with the exception of the RNaseH2 subunit A (Gly37Ser) mutant, which exhibits some evidence of altered nuclease specificity. These data indicate that the mechanism of RNaseH2 dysfunction in AGS cannot be simply explained by loss of ribonuclease H activity and points to a more complex mechanism perhaps mediated through altered interactions with as yet identified nucleic acids or protein partners.

Peripheral neuropathy in a patient with D-2-hydroxyglutaric aciduria.

D-2-hydroxyglutaric aciduria (D-2-HGA; OMIM 600721) is a rare autosomal recessive neurometabolic disorder with a wide clinical spectrum. The severe phenotype is homogeneous and is characterized by early infantile-onset epileptic encephalopathy with hypotonia, delayed cerebral visual development, cardiomyopathy and facial dysmorphic features. The mild phenotype has a more variable clinical expression with hypotonia and developmental delay. We present peripheral neuropathy as an additional clinical and electrophysiological feature in a 16-year-old boy with a homozygous missense mutation in exon 3 of the D-2-hydroxyglutarate dehydrogenase gene (D2HGDH) at position c.458T>C. This mutation results in replacement of a methionine residue, which was highly conserved during evolution, by threonine (p.Met153Thr).

Impairment of CDKL5 nuclear localisation as a cause for severe infantile encephalopathy.

Mutations in the human X-linked cyclin-dependent kinase-like 5 (CDKL5) gene have been shown to cause infantile spasms as well as Rett syndrome-like phenotype. To date, fewer than 20 different mutations have been reported. So far, no clear genotype-phenotype correlation has been established. We screened the entire coding region of CDKL5 in 151 affected girls with a clinically heterogeneous phenotype ranging from encephalopathy with epilepsy to atypical Rett syndrome by denaturing high liquid performance chromatography and direct sequencing, and we identified three novel missense mutations located in catalytic domain (p.Ala40Val, p.Arg65Gln, p.Leu220Pro). Segregation analysis showed that p.Arg65Gln was inherited from the healthy father, which rules out the involvement of CDKL5 in the aetiology of the phenotype in this patient. However, the de novo occurrence was shown for p.Ala40Val and p.Leu220Pro. The p.Ala40Val mutation was observed in two unrelated patients and represented the first recurrent mutation in the CDKL5 gene. For the two de novo mutations, we analysed the cellular localisation of the wild-type and CDKL5 mutants by transfection experiments. We showed that the two CDKL5 mutations cause mislocalisation of the mutant CDKL5 proteins in the cytoplasm. Interestingly these missense mutations that result in a mislocalisation of the CDKL5 protein are associated with severe developmental delay which was apparent within the first months of life characterised by early and generalised hypotonia, and autistic features, and as well as early infantile spasms.

Carbonic anhydrase II deficiency a novel mutation.

Carbonic anhydrase II (CA II) deficiency is an extremely rare autosomal recessive disorder, characterised by a triad of osteopetrosis, renal tubular acidosis and cerebral calcifications. A 12 year old boy with classical features of CA II deficiency is reported who was found to be homozygous for the mutation in CA II gene and parents were heterozygous for the same mutation .To the best of our knowledge this is the first case report of mutation proven CA II deficiency from India.

Biochemical characterization of human D-2-hydroxyglutarate dehydrogenase and two disease related variants reveals the molecular cause of D-2-hydroxyglutaric aciduria.

D-2-hydroxyglutaric aciduria is a neurometabolic disorder, characterized by the accumulation of D-2-hydroxyglutarate (D-2HG) in human mitochondria. Increased levels of D-2HG are detected in humans exhibiting point mutations in the genes encoding isocitrate dehydrogenase, citrate carrier, the electron transferring flavoprotein (ETF) and its downstream electron acceptor ETF-ubiquinone oxidoreductase or D-2-hydroxyglutarate dehydrogenase (hD2HGDH). However, while the pathogenicity of several amino acid replacements in the former four proteins has been studied extensively, not much is known about the effect of certain point mutations on the biochemical properties of hD2HGDH. Therefore, we recombinantly produced wild type hD2HGDH as well as two recently identified disease-related variants (hD2HGDH-I147S and -V444A) and performed their detailed biochemical characterization. We could show that hD2HGDH is a FAD dependent protein, which is able to catalyze the oxidation of D-2HG and D-lactate to α-ketoglutarate and pyruvate, respectively. The two variants were obtained as apo-proteins and were thus catalytically inactive. The addition of FAD failed to restore enzymatic activity of the variants, indicating that the cofactor binding site is compromised by the single amino acid replacements. Further analyses revealed that both variants form aggregates that are apparently unable to bind the FAD cofactor. Since, D-2-hydroxyglutaric aciduria may also result from a loss of function of either the ETF or its downstream electron acceptor ETF-ubiquinone oxidoreductase, ETF may serve as the cognate electron acceptor of reduced hD2HGDH. Here, we show that hD2HGDH directly reduces recombinant human ETF, thus establishing a metabolic link between the oxidation of D-2-hydroxyglutarate and the mitochondrial electron transport chain.

A molecular chaperone for mitochondrial complex I assembly is mutated in a progressive encephalopathy.

NADH:ubiquinone oxidoreductase (complex I) deficiency is a common cause of mitochondrial oxidative phosphorylation disease. It is associated with a wide range of clinical phenotypes in infants, including Leigh syndrome, cardiomyopathy, and encephalomyopathy. In at least half of patients, enzyme deficiency results from a failure to assemble the holoenzyme complex; however, the molecular chaperones required for assembly of the mammalian enzyme remain unknown. Using whole genome subtraction of yeasts with and without a complex I to generate candidate assembly factors, we identified a paralogue (B17.2L) of the B17.2 structural subunit. We found a null mutation in B17.2L in a patient with a progressive encephalopathy and showed that the associated complex I assembly defect could be completely rescued by retroviral expression of B17.2L in patient fibroblasts. An anti-B17.2L antibody did not associate with the holoenzyme complex but specifically recognized an 830-kDa subassembly in several patients with complex I assembly defects and coimmunoprecipitated a subset of complex I structural subunits from normal human heart mitochondria. These results demonstrate that B17.2L is a bona fide molecular chaperone that is essential for the assembly of complex I and for the normal function of the nervous system.