Active site variants in STT3A cause a dominant type I congenital disorder of glycosylation with neuromusculoskeletal findings.

Congenital disorders of glycosylation (CDGs) form a group of rare diseases characterized by hypoglycosylation. We here report the identification of 16 individuals from nine families who have either inherited or de novo heterozygous missense variants in STT3A, leading to an autosomal-dominant CDG. STT3A encodes the catalytic subunit of the STT3A-containing oligosaccharyltransferase (OST) complex, essential for protein N-glycosylation. Affected individuals presented with variable skeletal anomalies, short stature, macrocephaly, and dysmorphic features; half had intellectual disability. Additional features included increased muscle tone and muscle cramps. Modeling of the variants in the 3D structure of the OST complex indicated that all variants are located in the catalytic site of STT3A, suggesting a direct mechanistic link to the transfer of oligosaccharides onto nascent glycoproteins. Indeed, expression of STT3A at mRNA and steady-state protein level in fibroblasts was normal, while glycosylation was abnormal. In S. cerevisiae, expression of STT3 containing variants homologous to those in affected individuals induced defective glycosylation of carboxypeptidase Y in a wild-type yeast strain and expression of the same mutants in the STT3 hypomorphic stt3-7 yeast strain worsened the already observed glycosylation defect. These data support a dominant pathomechanism underlying the glycosylation defect. Recessive mutations in STT3A have previously been described to lead to a CDG. We present here a dominant form of STT3A-CDG that, because of the presence of abnormal transferrin glycoforms, is unusual among dominant type I CDGs.

Genome and RNA sequencing were essential to reveal cryptic intronic variants associated to defective ATP6AP1 mRNA processing.

The diagnosis of Mendelian disorders has notably advanced with integration of whole exome and genome sequencing (WES and WGS) in clinical practice. However, challenges in variant interpretation and uncovered variants by WES still leave a substantial percentage of patients undiagnosed. In this context, integrating RNA sequencing (RNA-seq) improves diagnostic workflows, particularly for WES inconclusive cases. Additionally, functional studies are often necessary to elucidate the impact of prioritized variants on gene expression and protein function. Our study focused on three unrelated male patients (P1-P3) with ATP6AP1-CDG (congenital disorder of glycosylation), presenting with intellectual disability and varying degrees of hepatopathy, glycosylation defects, and an initially inconclusive diagnosis through WES. Subsequent RNA-seq was pivotal in identifying the underlying genetic causes in P1 and P2, detecting ATP6AP1 underexpression and aberrant splicing. Molecular studies in fibroblasts confirmed these findings and identified the rare intronic variants c.289-233C > T and c.289-289G > A in P1 and P2, respectively. Trio-WGS also revealed the variant c.289-289G > A in P3, which was a de novo change in both patients. Functional assays expressing the mutant alleles in HAP1 cells demonstrated the pathogenic impact of these variants by reproducing the splicing alterations observed in patients. Our study underscores the role of RNA-seq and WGS in enhancing diagnostic rates for genetic diseases such as CDG, providing new insights into ATP6AP1-CDG molecular bases by identifying the first two deep intronic variants in this X-linked gene. Additionally, our study highlights the need to integrate RNA-seq and WGS, followed by functional validation, in routine diagnostics for a comprehensive evaluation of patients with an unidentified molecular etiology.

Exploring genotype-phenotype correlations in glutaric aciduria type 1.

Glutaric aciduria type 1 (GA1) is a rare neurometabolic disease caused by pathogenic variants in the gene encoding the enzyme glutaryl-CoA dehydrogenase (GCDH). We performed an extensive literature search to collect data on GA1 patients, together with unpublished cases, to provide an up-to-date genetic landscape of GCDH pathogenic variants and to investigate potential genotype-phenotype correlation, as this is still poorly understood. From this search, 421 different GCDH pathogenic variants have been identified, including four novel variants; c.179T>C (p.Leu60Pro), c.214C>T (p.Arg72Cys), c.309G>C (p.Leu103Phe), and c.665T>C (p.Phe222Ser).The variants are mostly distributed across the entire gene; although variant frequency in GA1 patients is relatively high in the regions encoding for active domains of GCDH. To investigate potential genotype-phenotype correlations, phenotypic descriptions of 532 patients have been combined and evaluated using novel combinatorial analyses. To do so, various clinical phenotypes were determined for each pathogenic variant by combining the information of all GA1 patients reported with this pathogenic variant, and subsequently mapped onto the 2D and 3D GCDH protein structure. In addition, the predicted pathogenicity of missense variants was analyzed using different in silico prediction score models. Both analyses showed an almost similar distribution of the highly pathogenic variants across the GCDH protein, although some hotspots, including the active domain, were observed. Moreover, it was demonstrated that highly pathogenic variants are significantly correlated with lower residual enzyme activity and the most accurate estimation was achieved by the REVEL score. A clear correlation of the genotype and the clinical phenotype however is still lacking.

CRISPR/Cas9-based functional genomics strategy to decipher the pathogenicity of genetic variants in inherited metabolic disorders.

The determination of the functional impact of variants of uncertain significance (VUS) is one of the major bottlenecks in the diagnostic workflow of inherited genetic diseases. To face this problem, we set up a CRISPR/Cas9-based strategy for knock-in cellular model generation, focusing on inherited metabolic disorders (IMDs). We selected variants in seven IMD-associated genes, including seven reported disease-causing variants and four benign/likely benign variants. Overall, 11 knock-in cell models were generated via homology-directed repair in HAP1 haploid cells using CRISPR/Cas9. The functional impact of the variants was determined by analyzing the characteristic biochemical alterations of each disorder. Functional studies performed in knock-in cell models showed that our approach accurately distinguished the functional effect of pathogenic from non-pathogenic variants in a reliable manner in a wide range of IMDs. Our study provides a generic approach to assess the functional impact of genetic variants to improve IMD diagnosis and this tool could emerge as a promising alternative to invasive tests, such as muscular or skin biopsies. Although the study has been performed only in IMDs, this strategy is generic and could be applied to other genetic disorders.

Functional Evidence of CCDC186 as a New Disease-Associated Gene with Endocrine and Central Nervous System Alterations.

CCDC186 protein is involved in the maturation of dense-core vesicles (DCVs) in the trans-Golgi network in neurons and endocrine cells. Mutations in genes involved in DCV regulation, other than CCDC186, have been described in patients with neurodevelopmental disorders. To date, only one patient, within a large sequencing study of 1000 cases, and a single case report with variants in CCDC186, had previously been described. However, no functional studies in any of these two cases had been performed. We identified three patients from two gypsy families, unrelated to each other, with mutations in the CCDC186 gene. Clinically, all patients presented with seizures, frontotemporal atrophy, hypomyelination, recurrent infections, and endocrine disturbances such as severe non-ketotic hypoglycemia. Low levels of cortisol, insulin, or growth hormone could only be verified in one patient. All of them had a neonatal onset and died between 7 months and 4 years of age. Whole exome sequencing identified a homozygous variant in the CCDC186 gene (c.2215C>T, p.Arg739Ter) in the index patients of both families. Protein expression studies demonstrated that CCDC186 was almost undetectable in fibroblasts and muscle tissue. These observations correlated with the transcriptomic analysis performed in fibroblasts in one of the patients, which showed a significant reduction of CCDC186 mRNA levels. Our study provides functional evidence that mutations in this gene have a pathogenic effect on the protein and reinforces CCDC186 as a new disease-associated gene. In addition, mutations in CCDC186 could explain the combined endocrine and neurologic alterations detected in our patients.

Clinico-radiological features, molecular spectrum, and identification of prognostic factors in developmental and epileptic encephalopathy due to inosine triphosphate pyrophosphatase (ITPase) deficiency.

Developmental and epileptic encephalopathy 35 (DEE 35) is a severe neurological condition caused by biallelic variants in ITPA, encoding inosine triphosphate pyrophosphatase, an essential enzyme in purine metabolism. We delineate the genotypic and phenotypic spectrum of DEE 35, analyzing possible predictors for adverse clinical outcomes. We investigated a cohort of 28 new patients and reviewed previously described cases, providing a comprehensive characterization of 40 subjects. Exome sequencing was performed to identify underlying ITPA pathogenic variants. Brain MRI (magnetic resonance imaging) scans were systematically analyzed to delineate the neuroradiological spectrum. Survival curves according to the Kaplan-Meier method and log-rank test were used to investigate outcome predictors in different subgroups of patients. We identified 18 distinct ITPA pathogenic variants, including 14 novel variants, and two deletions. All subjects showed profound developmental delay, microcephaly, and refractory epilepsy followed by neurodevelopmental regression. Brain MRI revision revealed a recurrent pattern of delayed myelination and restricted diffusion of early myelinating structures. Congenital microcephaly and cardiac involvement were statistically significant novel clinical predictors of adverse outcomes. We refined the molecular, clinical, and neuroradiological characterization of ITPase deficiency, and identified new clinical predictors which may have a potentially important impact on diagnosis, counseling, and follow-up of affected individuals.

Diagnostic Odyssey in an Adult Patient with Ophthalmologic Abnormalities and Hearing Loss: Contribution of RNA-Seq to the Diagnosis of a PEX1 Deficiency.

Peroxisomal biogenesis disorders (PBDs) are a heterogeneous group of genetic diseases. Multiple peroxisomal pathways are impaired, and very long chain fatty acids (VLCFA) are the first line biomarkers for the diagnosis. The clinical presentation of PBDs may range from severe, lethal multisystemic disorders to milder, late-onset disease. The vast majority of PBDs belong to Zellweger Spectrum Disordes (ZSDs) and represents a continuum of overlapping clinical symptoms, with Zellweger syndrome being the most severe and Heimler syndrome the less severe disease. Mild clinical conditions frequently present normal or slight biochemical alterations, making the diagnosis of these patients challenging. In the present study we used a combined WES and RNA-seq strategy to diagnose a patient presenting with retinal dystrophy as the main clinical symptom. Results showed the patient was compound heterozygous for mutations in PEX1. VLCFA were normal, but retrospective analysis of lysosphosphatidylcholines (LPC) containing C22:0-C26:0 species was altered. This simple test could avoid the diagnostic odyssey of patients with mild phenotype, such as the individual described here, who was diagnosed very late in adult life. We provide functional data in cell line models that may explain the mild phenotype of the patient by demonstrating the hypomorphic nature of a deep intronic variant altering PEX1 mRNA processing.

Variants in the ethylmalonyl-CoA decarboxylase (ECHDC1) gene: a novel player in ethylmalonic aciduria?

Ethylmalonic acid (EMA) is a major and potentially cytotoxic metabolite associated with short-chain acyl-CoA dehydrogenase (SCAD) deficiency, a condition whose status as a disease is uncertain. Unexplained high EMA is observed in some individuals with complex neurological symptoms, who carry the SCAD gene (ACADS) variants, c.625G>A and c.511C>T. The variants have a high allele frequency in the general population, but are significantly overrepresented in individuals with elevated EMA. This has led to the idea that these variants need to be associated with variants in other genes to cause hyperexcretion of ethylmalonic acid and possibly a diseased state. Ethylmalonyl-CoA decarboxylase (ECHDC1) has been described and characterized as an EMA metabolite repair enzyme, however, its clinical relevance has never been investigated. In this study, we sequenced the ECHDC1 gene (ECHDC1) in 82 individuals, who were reported with unexplained high EMA levels due to the presence of the common ACADS variants only. Three individuals with ACADS c.625G>A variants were found to be heterozygous for ECHDC1 loss-of-function variants. Knockdown experiments of ECHDC1, in healthy human cells with different ACADS c.625G>A genotypes, showed that ECHDC1 haploinsufficiency and homozygosity for the ACADS c.625G>A variant had a synergistic effect on cellular EMA excretion. This study reports the first cases of ECHDC1 gene defects in humans and suggests that ECHDC1 may be involved in elevated EMA excretion in only a small group of individuals with the common ACADS variants. However, a direct link between ECHDC1/ACADS deficiency, EMA and disease could not be proven.

Delineating the neurological phenotype in children with defects in the ECHS1 or HIBCH gene.

The neurological phenotype of 3-hydroxyisobutyryl-CoA hydrolase (HIBCH) and short-chain enoyl-CoA hydratase (SCEH) defects is expanding and natural history studies are necessary to improve clinical management. From 42 patients with Leigh syndrome studied by massive parallel sequencing, we identified five patients with SCEH and HIBCH deficiency. Fourteen additional patients were recruited through collaborations with other centres. In total, we analysed the neurological features and mutation spectrum in 19 new SCEH/HIBCH patients. For natural history studies and phenotype to genotype associations we also included 70 previously reported patients. The 19 newly identified cases presented with Leigh syndrome (SCEH, n = 11; HIBCH, n = 6) and paroxysmal dystonia (SCEH, n = 2). Basal ganglia lesions (18 patients) were associated with small cysts in the putamen/pallidum in half of the cases, a characteristic hallmark for diagnosis. Eighteen pathogenic variants were identified, 11 were novel. Among all 89 cases, we observed a longer survival in HIBCH compared to SCEH patients, and in HIBCH patients carrying homozygous mutations on the protein surface compared to those with variants inside/near the catalytic region. The SCEH p.(Ala173Val) change was associated with a milder form of paroxysmal dystonia triggered by increased energy demands. In a child harbouring SCEH p.(Ala173Val) and the novel p.(Leu123Phe) change, an 83.6% reduction of the protein was observed in fibroblasts. The SCEH and HIBCH defects in the catabolic valine pathway were a frequent cause of Leigh syndrome in our cohort. We identified phenotype and genotype associations that may help predict outcome and improve clinical management.

Biallelic mutations in NDUFA8 cause complex I deficiency in two siblings with favorable clinical evolution.

Isolated complex I (CI) deficiency is the most common cause of oxidative phosphorylation (OXPHOS) dysfunction. Whole-exome sequencing identified biallelic mutations in NDUFA8 (c.[293G > T]; [293G > T], encoding for an accessory subunit of CI, in two siblings with a favorable clinical evolution. The individuals reported here are practically asymptomatic, with the exception of slight failure to thrive and some language difficulties at the age of 6 and 9 years, respectively. These observations are remarkable since the vast majority of patients with CI deficiency, including the only NDUFA8 patient reported so far, showed an extremely poor clinical outcome. Western blot studies demonstrated that NDUFA8 protein was strongly reduced in the patients' fibroblasts and muscle extracts. In addition, there was a marked and specific decrease in the steady-state levels of CI subunits. BN-PAGE demonstrated an isolated defect in the assembly and the activity of CI with impaired supercomplexes formation and abnormal accumulation of CI subassemblies. Confocal microscopy analysis in fibroblasts showed rounder mitochondria and diminished branching degree of the mitochondrial network. Functional complementation studies demonstrated disease-causality for the identified mutation as lentiviral transduction with wild-type NDUFA8 cDNA restored the steady-state levels of CI subunits and completely recovered the deficient enzymatic activity in immortalized mutant fibroblasts. In summary, we provide additional evidence of the involvement of NDUFA8 as a mitochondrial disease-causing gene associated with altered mitochondrial morphology, CI deficiency, impaired supercomplexes formation, and very mild progression of the disease.

Clinical presentation and proteomic signature of patients with TANGO2 mutations.

Transport And Golgi Organization protein 2 (TANGO2) deficiency has recently been identified as a rare metabolic disorder with a distinct clinical and biochemical phenotype of recurrent metabolic crises, hypoglycemia, lactic acidosis, rhabdomyolysis, arrhythmias, and encephalopathy with cognitive decline. We report nine subjects from seven independent families, and we studied muscle histology, respiratory chain enzyme activities in skeletal muscle and proteomic signature of fibroblasts. All nine subjects carried autosomal recessive TANGO2 mutations. Two carried the reported deletion of exons 3 to 9, one homozygous, one heterozygous with a 22q11.21 microdeletion inherited in trans. The other subjects carried three novel homozygous (c.262C>T/p.Arg88*; c.220A>C/p.Thr74Pro; c.380+1G>A), and two further novel heterozygous (c.6_9del/p.Phe6del); c.11-13delTCT/p.Phe5del mutations. Immunoblot analysis detected a significant decrease of TANGO2 protein. Muscle histology showed mild variation of fiber diameter, no ragged-red/cytochrome c oxidase-negative fibers and a defect of multiple respiratory chain enzymes and coenzyme Q10 (CoQ10 ) in two cases, suggesting a possible secondary defect of oxidative phosphorylation. Proteomic analysis in fibroblasts revealed significant changes in components of the mitochondrial fatty acid oxidation, plasma membrane, endoplasmic reticulum-Golgi network and secretory pathways. Clinical presentation of TANGO2 mutations is homogeneous and clinically recognizable. The hemizygous mutations in two patients suggest that some mutations leading to allele loss are difficult to detect. A combined defect of the respiratory chain enzymes and CoQ10 with altered levels of several membrane proteins provides molecular insights into the underlying pathophysiology and may guide rational new therapeutic interventions.

Mutations in TIMM50 cause severe mitochondrial dysfunction by targeting key aspects of mitochondrial physiology.

3-Methylglutaconic aciduria (3-MGA-uria) syndromes comprise a heterogeneous group of diseases associated with mitochondrial membrane defects. Whole-exome sequencing identified compound heterozygous mutations in TIMM50 (c.[341 G>A];[805 G>A]) in a boy with West syndrome, optic atrophy, neutropenia, cardiomyopathy, Leigh syndrome, and persistent 3-MGA-uria. A comprehensive analysis of the mitochondrial function was performed in fibroblasts of the patient to elucidate the molecular basis of the disease. TIMM50 protein was severely reduced in the patient fibroblasts, regardless of the normal mRNA levels, suggesting that the mutated residues might be important for TIMM50 protein stability. Severe morphological defects and ultrastructural abnormalities with aberrant mitochondrial cristae organization in muscle and fibroblasts were found. The levels of fully assembled OXPHOS complexes and supercomplexes were strongly reduced in fibroblasts from this patient. High-resolution respirometry demonstrated a significant reduction of the maximum respiratory capacity. A TIMM50-deficient HEK293T cell line that we generated using CRISPR/Cas9 mimicked the respiratory defect observed in the patient fibroblasts; notably, this defect was rescued by transfection with a plasmid encoding the TIMM50 wild-type protein. In summary, we demonstrated that TIMM50 deficiency causes a severe mitochondrial dysfunction by targeting key aspects of mitochondrial physiology, such as the maintenance of proper mitochondrial morphology, OXPHOS assembly, and mitochondrial respiratory capacity.

Newborn screening for homocystinurias: Recent recommendations versus current practice.

PURPOSE: To assess how the current practice of newborn screening (NBS) for homocystinurias compares with published recommendations. METHODS: Twenty-two of 32 NBS programmes from 18 countries screened for at least one form of homocystinuria. Centres provided pseudonymised NBS data from patients with cystathionine beta-synthase deficiency (CBSD, n = 19), methionine adenosyltransferase I/III deficiency (MATI/IIID, n = 28), combined remethylation disorder (cRMD, n = 56) and isolated remethylation disorder (iRMD), including methylenetetrahydrofolate reductase deficiency (MTHFRD) (n = 8). Markers and decision limits were converted to multiples of the median (MoM) to allow comparison between centres. RESULTS: NBS programmes, algorithms and decision limits varied considerably. Only nine centres used the recommended second-tier marker total homocysteine (tHcy). The median decision limits of all centres were ≥ 2.35 for high and ≤ 0.44 MoM for low methionine, ≥ 1.95 for high and ≤ 0.47 MoM for low methionine/phenylalanine, ≥ 2.54 for high propionylcarnitine and ≥ 2.78 MoM for propionylcarnitine/acetylcarnitine. These decision limits alone had a 100%, 100%, 86% and 84% sensitivity for the detection of CBSD, MATI/IIID, iRMD and cRMD, respectively, but failed to detect six individuals with cRMD. To enhance sensitivity and decrease second-tier testing costs, we further adapted these decision limits using the data of 15 000 healthy newborns. CONCLUSIONS: Due to the favorable outcome of early treated patients, NBS for homocystinurias is recommended. To improve NBS, decision limits should be revised considering the population median. Relevant markers should be combined; use of the postanalytical tools offered by the CLIR project (Collaborative Laboratory Integrated Reports, which considers, for example, birth weight and gestational age) is recommended. tHcy and methylmalonic acid should be implemented as second-tier markers.

Cerebrospinal fluid monoamines, pterins, and folate in patients with mitochondrial diseases: systematic review and hospital experience.

Mitochondrial diseases are a group of genetic disorders leading to the dysfunction of mitochondrial energy metabolism pathways. We aimed to assess the clinical phenotype and the biochemical cerebrospinal fluid (CSF) biogenic amine profiles of patients with different diagnoses of genetic mitochondrial diseases. We recruited 29 patients with genetically confirmed mitochondrial diseases harboring mutations in either nuclear or mitochondrial DNA (mtDNA) genes. Signs and symptoms of impaired neurotransmission and neuroradiological data were recorded. CSF monoamines, pterins, and 5-methyltetrahydrofolate (5MTHF) concentrations were analyzed using high-performance liquid chromatography with electrochemical and fluorescence detection procedures. The mtDNA mutations were studied by Sanger sequencing, Southern blot, and real-time PCR, and nuclear DNA was assessed either by Sanger or next-generation sequencing. Five out of 29 cases showed predominant dopaminergic signs not attributable to basal ganglia involvement, harboring mutations in different nuclear genes. A chi-square test showed a statistically significant association between high homovanillic acid (HVA) values and low CSF 5-MTHF values (chi-square = 10.916; p = 0.001). Seven out of the eight patients with high CSF HVA values showed cerebral folate deficiency. Five of them harbored mtDNA deletions associated with Kearns-Sayre syndrome (KSS), one had a mitochondrial point mutation at the mtDNA ATPase6 gene, and one had a POLG mutation. In conclusion, dopamine deficiency clinical signs were present in some patients with mitochondrial diseases with different genetic backgrounds. High CSF HVA values, together with a severe cerebral folate deficiency, were observed in KSS patients and in other mtDNA mutation syndromes.

Mutations in TRAPPC11 are associated with a congenital disorder of glycosylation.

Congenital disorders of glycosylation (CDG) are a heterogeneous and rapidly growing group of diseases caused by abnormal glycosylation of proteins and/or lipids. Mutations in genes involved in the homeostasis of the endoplasmic reticulum (ER), the Golgi apparatus (GA), and the vesicular trafficking from the ER to the ER-Golgi intermediate compartment (ERGIC) have been found to be associated with CDG. Here, we report a patient with defects in both N- and O-glycosylation combined with a delayed vesicular transport in the GA due to mutations in TRAPPC11, a subunit of the TRAPPIII complex. TRAPPIII is implicated in the anterograde transport from the ER to the ERGIC as well as in the vesicle export from the GA. This report expands the spectrum of genetic alterations associated with CDG, providing new insights for the diagnosis and the understanding of the physiopathological mechanisms underlying glycosylation disorders.

Small molecules as therapeutic agents for inborn errors of metabolism.

Most inborn errors of metabolism (IEM) remain without effective treatment mainly due to the incapacity of conventional therapeutic approaches to target the neurological symptomatology and to ameliorate the multisystemic involvement frequently observed in these patients. However, in recent years, the therapeutic use of small molecules has emerged as a promising approach for treating this heterogeneous group of disorders. In this review, we focus on the use of therapeutically active small molecules to treat IEM, including readthrough agents, pharmacological chaperones, proteostasis regulators, substrate inhibitors, and autophagy inducers. The small molecules reviewed herein act at different cellular levels, and this knowledge provides new tools to set up innovative treatment approaches for particular IEM. We review the molecular mechanism underlying therapeutic properties of small molecules, methodologies used to screen for these compounds, and their applicability in preclinical and clinical practice.

Free-thiamine is a potential biomarker of thiamine transporter-2 deficiency: a treatable cause of Leigh syndrome.

Thiamine transporter-2 deficiency is caused by mutations in the SLC19A3 gene. As opposed to other causes of Leigh syndrome, early administration of thiamine and biotin has a dramatic and immediate clinical effect. New biochemical markers are needed to aid in early diagnosis and timely therapeutic intervention. Thiamine derivatives were analysed by high performance liquid chromatography in 106 whole blood and 38 cerebrospinal fluid samples from paediatric controls, 16 cerebrospinal fluid samples from patients with Leigh syndrome, six of whom harboured mutations in the SLC19A3 gene, and 49 patients with other neurological disorders. Free-thiamine was remarkably reduced in the cerebrospinal fluid of five SLC19A3 patients before treatment. In contrast, free-thiamine was slightly decreased in 15.2% of patients with other neurological conditions, and above the reference range in one SLC19A3 patient on thiamine supplementation. We also observed a severe deficiency of free-thiamine and low levels of thiamine diphosphate in fibroblasts from SLC19A3 patients. Surprisingly, pyruvate dehydrogenase activity and mitochondrial substrate oxidation rates were within the control range. Thiamine derivatives normalized after the addition of thiamine to the culture medium. In conclusion, we found a profound deficiency of free-thiamine in the CSF and fibroblasts of patients with thiamine transporter-2 deficiency. Thiamine supplementation led to clinical improvement in patients early treated and restored thiamine values in fibroblasts and cerebrospinal fluid.

Mutations in the lipoyltransferase LIPT1 gene cause a fatal disease associated with a specific lipoylation defect of the 2-ketoacid dehydrogenase complexes.

Cofactor disorders of mitochondrial energy metabolism are a heterogeneous group of diseases with a wide variety of clinical symptoms, particular metabolic profiles and variable enzymatic defects. Mutations in NFU1, BOLA3, LIAS and IBA57 have been identified in patients with deficient lipoic acid-dependent enzymatic activities and defects in the assembly and activity of the mitochondrial respiratory chain complexes. Here, we report a patient with an early onset fatal lactic acidosis presenting a biochemical phenotype compatible with a combined defect of pyruvate dehydrogenase (PDHC) and 2-ketoglutarate dehydrogenase (2-KGDH) activities, which suggested a deficiency in lipoic acid metabolism. Immunostaining analysis showed that lipoylated E2-PDH and E2-KGDH were extremely reduced in this patient. However, the absence of glycine elevation, the normal activity of the glycine cleavage system and the normal lipoylation of the H protein suggested a defect of lipoic acid transfer to particular proteins rather than a general impairment of lipoic acid biosynthesis as the potential cause of the disease. By analogy with yeast metabolism, we postulated LIPT1 as the altered candidate gene causing the disease. Sequence analysis of the human LIPT1 identified two heterozygous missense mutations (c.212C>T and c.292C>G), segregating in different alleles. Functional complementation experiments in patient's fibroblasts demonstrated that these mutations are disease-causing and that LIPT1 protein is required for lipoylation and activation of 2-ketoacid dehydrogenases in humans. These findings expand the spectrum of genetic defects associated with lipoic acid metabolism and provide the first evidence of a lipoic acid transfer defect in humans.

Differential diagnosis of lipoic acid synthesis defects.

Lipoic acid (LA) is an essential cofactor required for the activity of five multienzymatic complexes that play a central role in the mitochondrial energy metabolism: four 2-oxoacid dehydrogenase complexes [pyruvate dehydrogenase (PDH), branched-chain ketoacid dehydrogenase (BCKDH), 2-ketoglutarate dehydrogenase (2-KGDH), and 2-oxoadipate dehydrogenase (2-OADH)] and the glycine cleavage system (GCS). LA is synthesized in a complex multistep process that requires appropriate function of the mitochondrial fatty acid synthesis (mtFASII) and the biogenesis of iron-sulphur (Fe-S) clusters. Defects in the biosynthesis of LA have been reported to be associated with multiple and severe defects of the mitochondrial energy metabolism. In recent years, disease-causing mutations in genes encoding for proteins involved in LA metabolism have been reported: NFU1, BOLA3, IBA57, LIAS, GLRX5, LIPT1, ISCA2, and LIPT2. These studies represented important progress in understanding the pathophysiology and molecular bases underlying these disorders. Here we review current knowledge regarding involvement of LA synthesis defects in human diseases with special emphasis on the diagnostic strategies for these disorders. The clinical and biochemical characteristics of patients with LA synthesis defects are discussed and a workup for the differential diagnosis proposed.

Cholestane-3ß,5α,6ß-triol: high levels in Niemann-Pick type C, cerebrotendinous xanthomatosis, and lysosomal acid lipase deficiency.

Niemann-Pick type C (NPC) is a progressive neurodegenerative disease characterized by lysosomal/endosomal accumulation of unesterified cholesterol and glycolipids. Recent studies have shown that plasma cholestane-3ß,5α,6ß-triol (CT) and 7-ketocholesterol (7-KC) could be potential biomarkers for the diagnosis of NPC patients. We aimed to know the sensitivity and specificity of these biomarkers for the diagnosis of NPC compared with other diseases that can potentially lead to oxysterol alterations. We studied 107 controls and 122 patients including 16 with NPC, 3 with lysosomal acid lipase (LAL) deficiency, 8 with other lysosomal diseases, 5 with galactosemia, 11 with cerebrotendinous xanthomatosis (CTX), 3 with Smith-Lemli-Opitz, 14 with peroxisomal biogenesis disorders, 19 with unspecific hepatic diseases, 13 with familial hypercholesterolemia, and 30 with neurological involvement and no evidence of an inherited metabolic disease. CT and 7-KC were analyzed by HPLC-ESI-MS/MS as mono-dimethylglycine derivatives. Levels of 7-KC were high in most of the studied diseases, whereas those of CT were only high in NPC, LAL, and CTX patients. Consequently, although CT is a sensitive biomarker of NPC disease, including those cases with doubtful filipin staining, it is not specific. 7-KC is a very unspecific biomarker.