Long-term follow-up with filter paper samples in patients with propionic acidemia.

BACKGROUND: Propionic acidemia (PA) is an inherited disorder caused by deficiency of propionyl CoA carboxylase. Most patients with this disorder are diagnosed during the neonatal period because of severe metabolic acidosis and hyperammonemia. Patients are required to undergo blood and urine analysis at least 3 to 4 times per year, depending on age and metabolic control. METHODS: We designed a prospective study in which we investigated the results from blood and urinary samples collected monthly in filter paper from 10 PA patients followed in a single metabolic reference center from January 2015 to September 2017. The aim of this study was to evaluate the usefulness of filter paper samples in the follow-up of the PA patients. RESULTS: During the follow-up period, 163 dried blood spot (DBS) and 119 urine dried spot samples were analyzed and compared with 160 plasma and 103 liquid urine specimens; 64 specimens of plasma were analyzed for odd-numbered long-chain fatty acids (OLCFAs). A total of 40 metabolic crises, 18 of them with hyperammonemia were documented. We observed a strong correlation between the filter paper and the urine/plasma samples for the main PA parameters both in stable metabolic conditions as well as in acute decompensations. Also, there was a strong correlation between OLCFAs measured in plasma and quantification of odd number acylcarnitines in DBS. CONCLUSIONS: We conclude that filter paper blood and urinary samples can be used for the follow-up of the patients with PA, correctly reflecting their metabolic situation.

Genes and Variants Underlying Human Congenital Lactic Acidosis-From Genetics to Personalized Treatment.

Congenital lactic acidosis (CLA) is a rare condition in most instances due to a range of inborn errors of metabolism that result in defective mitochondrial function. Even though the implementation of next generation sequencing has been rapid, the diagnosis rate for this highly heterogeneous allelic condition remains low. The present work reports our group's experience of using a clinical/biochemical analysis system in conjunction with genetic findings that facilitates the taking of timely clinical decisions with minimum need for invasive procedures. The system's workflow combines different metabolomics datasets and phenotypic information with the results of clinical exome sequencing and/or RNA analysis. The system's use detected genetic variants in 64% of a cohort of 39 CLA-patients; these variants, 14 of which were novel, were found in 19 different nuclear and two mitochondrial genes. For patients with variants of unknown significance, the genetic analysis was combined with functional genetic and/or bioenergetics analyses in an attempt to detect pathogenicity. Our results warranted subsequent testing of antisense therapy to rescue the abnormal splicing in cultures of fibroblasts from a patient with a defective GFM1 gene. The discussed system facilitates the diagnosis of CLA by avoiding the need to use invasive techniques and increase our knowledge of the causes of this condition.

Expanding the genetic and phenotypic spectrum of branched-chain amino acid transferase 2 deficiency.

The first step in branched-chain amino acid (BCAA) catabolism is catalyzed by the two BCAA transferase isoenzymes, cytoplasmic branched-chain amino acid transferase (BCAT) 1, and mitochondrial BCAT2. Defects in the second step of BCAA catabolism cause maple syrup urine disease (MSUD), a condition which has been far more extensively investigated. Here, we studied the consequences of BCAT2 deficiency, an ultra-rare condition in humans. We present genetic, clinical, and functional data in five individuals from four different families with homozygous or compound heterozygous BCAT2 mutations which were all detected following abnormal biochemical profile results or familial mutation segregation studies. We demonstrate that BCAT2 deficiency has a recognizable biochemical profile with raised plasma BCAAs and, in contrast with MSUD, low-normal branched-chain keto acids (BCKAs) with undetectable l-allo-isoleucine. Interestingly, unlike in MSUD, none of the individuals with BCAT2 deficiency developed acute encephalopathy even with exceptionally high BCAA levels. We observed wide-ranging clinical phenotypes in individuals with BCAT2 deficiency. While one adult was apparently asymptomatic, three individuals had presented with developmental delay and autistic features. We show that the biochemical characteristics of BCAT2 deficiency may be amenable to protein-restricted diet and that early treatment may improve outcome in affected individuals. BCAT2 deficiency is an inborn error of BCAA catabolism. At present, it is unclear whether developmental delay and autism are parts of the variable phenotypic spectrum of this condition or coincidental. Further studies will be required to explore this.

Value of genetic analysis for confirming inborn errors of metabolism detected through the Spanish neonatal screening program.

The present work describes the value of genetic analysis as a confirmatory measure following the detection of suspected inborn errors of metabolism in the Spanish newborn mass spectrometry screening program. One hundred and forty-one consecutive DNA samples were analyzed by next-generation sequencing using a customized exome sequencing panel. When required, the Illumina extended clinical exome panel was used, as was Sanger sequencing or transcriptional profiling. Biochemical tests were used to confirm the results of the genetic analysis. Using the customized panel, the metabolic disease suspected in 83 newborns (59%) was confirmed. In three further cases, two monoallelic variants were detected for two genes involved in the same biochemical pathway. In the remainder, either a single variant or no variant was identified. Given the persistent absence of biochemical alterations, carrier status was assigned in 39 cases. False positives were recorded for 11. In five cases in which the biochemical pattern was persistently altered, further genetic analysis allowed the detection of two variants affecting the function of BCAT2, ACSF3, and DNAJC12, as well as a second, deep intronic variant in ETFDH or PTS. The present results suggest that genetic analysis using extended next-generation sequencing panels can be used as a confirmatory test for suspected inborn errors of metabolism detected in newborn screening programs. Biochemical tests can be very helpful when a diagnosis is unclear. In summary, simultaneous genomic and metabolomic analyses can increase the number of inborn errors of metabolism that can be confirmed following suggestive newborn screening results.

Improving the diagnosis of cobalamin and related defects by genomic analysis, plus functional and structural assessment of novel variants.

BACKGROUND: Cellular cobalamin defects are a locus and allelic heterogeneous disorder. The gold standard for coming to genetic diagnoses of cobalamin defects has for some time been gene-by-gene Sanger sequencing of individual DNA fragments. Enzymatic and cellular methods are employed before such sequencing to help in the selection of the gene defects to be sought, but this is time-consuming and laborious. Furthermore some cases remain undiagnosed because no biochemical methods have been available to test for cobalamin absorption and transport defects. RESULTS: This paper reports the use of massive parallel sequencing of DNA (exome analysis) for the accurate and rapid genetic diagnosis of cobalamin-related defects in a cohort of affected patients. The method was first validated in an initial cohort with different cobalamin defects. Mendelian segregation, the frequency of mutations, and the comprehensive structural and functional analysis of gene variants, identified disease-causing mutations in 12 genes involved in the absorption and synthesis of active cofactors of vitamin B12 (22 cases), and in the non-cobalamin metabolism-related genes ACSF3 (in four biochemically misdiagnosed patients) and SUCLA2 (in one patient with an unusual presentation). We have identified thirteen new variants all classified as pathogenic according to the ACGM recommendation but four were classified as variant likely pathogenic in MUT and SUCLA2. Functional and structural analysis provided evidences to classify them as pathogenic variants. CONCLUSIONS: The present findings suggest that the technology used is sufficiently sensitive and specific, and the results it provides sufficiently reproducible, to recommend its use as a second-tier test after the biochemical detection of cobalamin disorder markers in the first days of life. However, for accurate diagnoses to be made, biochemical and functional tests that allow comprehensive clinical phenotyping are also needed.

Isolated and Combined Remethylation Disorders: Biochemical and Genetic Diagnosis and Pathophysiology

Abstract Genetic defects affecting the remethylation pathway cause hyperhomocysteinemia. Isolated remethylation defects are caused by mutations of the 5, 10-methylenetetrahydrofolate reductase (MTHFR), methionine synthase reductase(MTRR), methionine synthase(MTR), and MMADHC genes, and combined remethylation defects are the result of mutations in genes involved in the synthesis of either methylcobalamin or adenosylcobalamin, that is, the active cofactors of MTRR and methylmalonyl-CoA mutase. Diagnosis is based on the biochemical analysis of amino acids, homocysteine, propionylcarnitine, methylmalonic acid, S-adenosylmethionine, and 5-methylentetrahydrofolate in physiological fluids. Gene-by-gene Sanger sequencing has long been the gold standard genetic analysis for confirming the disorder and identifying the gene involved, but massive parallel sequencing is now being used to examine all those potentially involved in one go. Early treatment to rescue metabolic homeostasis is based on the following of an appropriate diet, betaine administration, and, in some cases, oral or intramuscular administration of vitamin B12 or folate. Elevated ROS levels, apoptosis, endoplasmic reticulum (ER) stress, the activation of autophagy, and alterations in Ca2+ homeostasis may all contribute toward the pathogenesis of the disease. Pharmacological agents to restore the function of the ER and mitochondria and/or to reduce oxidative stress-induced apoptosis might provide novel ways of treating patients with remethylation disorders.

Molecular diagnosis of glycogen storage disease and disorders with overlapping clinical symptoms by massive parallel sequencing.

PURPOSE: Glycogen storage disease (GSD) is an umbrella term for a group of genetic disorders that involve the abnormal metabolism of glycogen; to date, 23 types of GSD have been identified. The nonspecific clinical presentation of GSD and the lack of specific biomarkers mean that Sanger sequencing is now widely relied on for making a diagnosis. However, this gene-by-gene sequencing technique is both laborious and costly, which is a consequence of the number of genes to be sequenced and the large size of some genes. METHODS: This work reports the use of massive parallel sequencing to diagnose patients at our laboratory in Spain using either a customized gene panel (targeted exome sequencing) or the Illumina Clinical-Exome TruSight One Gene Panel (clinical exome sequencing (CES)). Sequence variants were matched against biochemical and clinical hallmarks. RESULTS: Pathogenic mutations were detected in 23 patients. Twenty-two mutations were recognized (mostly loss-of-function mutations), including 11 that were novel in GSD-associated genes. In addition, CES detected five patients with mutations in ALDOB, LIPA, NKX2-5, CPT2, or ANO5. Although these genes are not involved in GSD, they are associated with overlapping phenotypic characteristics such as hepatic, muscular, and cardiac dysfunction. CONCLUSIONS: These results show that next-generation sequencing, in combination with the detection of biochemical and clinical hallmarks, provides an accurate, high-throughput means of making genetic diagnoses of GSD and related diseases.Genet Med 18 10, 1037-1043.

Newborn screening for homocystinurias and methylation disorders: systematic review and proposed guidelines.

Newborn screening (NBS) is justified if early intervention is effective in a disorder generally not detected early in life on a clinical basis, and if sensitive and specific biochemical markers exist. Experience with NBS for homocystinurias and methylation disorders is limited. However, there is robust evidence for the success of early treatment with diet, betaine and/or pyridoxine for CBS deficiency and good evidence for the success of early betaine treatment in severe MTHFR deficiency. These conditions can be screened in dried blood spots by determining methionine (Met), methionine-to-phenylanine (Met/Phe) ratio, and total homocysteine (tHcy) as a second tier marker. Therefore, we recommend NBS for cystathionine beta-synthase and severe MTHFR deficiency. Weaker evidence is available for the disorders of intracellular cobalamin metabolism. Early treatment is clearly of advantage for patients with the late-onset cblC defect. In the early-onset type, survival and non-neurological symptoms improve but the effect on neurocognitive development is uncertain. The cblC defect can be screened by measuring propionylcarnitine, propionylcarnitine-to-acetylcarnitine ratio combined with the second tier markers methylmalonic acid and tHcy. For the cblE and cblG defects, evidence for the benefit of early treatment is weaker; and data on performance of Met, Met/Phe and tHcy even more limited. Individuals homozygous or compound heterozygous for MAT1A mutations may benefit from detection by NBS using Met, which on the other hand also detects asymptomatic heterozygotes. Clinical and laboratory data is insufficient to develop any recommendation on NBS for the cblD, cblF, cblJ defects, glycineN-methyltransferase-, S-adenosylhomocysteinehydrolase- and adenosine kinase deficiency.

Carnitine-acylcarnitine translocase deficiency: experience with four cases in Spain and review of the literature.

BACKGROUND: Carnitine-acylcarnitine translocase (CACT) deficiency is a rare autosomal recessive disease in the mitochondrial transport of long-chain fatty acids. Despite early diagnosis and treatment, the disease still has a high mortality rate. METHODS: Clinical symptoms, long-term follow-up, and biochemical and molecular results of four cases are described and compared with the reviewed literature data of 55 cases. RESULTS: Two cases with neonatal onset, carrying in homozygosity the novel variant sequences p.Gly20Asp (c.59G>A) and p.Arg179Gly (c.536A>G), died during an intercurrent infectious process in the first year of life despite adequate dietetic treatment (frequent feeding, high-carbohydrate/low-fat diet, MCT, carnitine). The other two cases, one with infantile onset and the other diagnosed in the newborn period after a previous affected sibling, show excellent development at 4 and 16 years of age under treatment. The review shows that the most frequent presenting symptoms of CACT deficiency are hypoketotic hypoglycemia, hyperammonemia, hepatomegaly, cardiomyopathy and/or arrhythmia, and respiratory distress. The onset of symptoms is predominantly neonatal in 82% and infantile in 18%. The mortality rate is high (65%), most in the first year of life due to myocardiopathy or sudden death. Outcomes seem to correlate better with the absence of cardiac disease and with a higher long-chain fatty acid oxidation rate in cultured fibroblasts than with residual enzyme activity. CONCLUSION: Diagnosis before the occurrence of clinical symptoms by tandem MS-MS and very early therapeutic intervention together with good dietary compliance could lead to a better prognosis, especially in milder clinical cases.

Selected reaction monitoring as an effective method for reliable quantification of disease-associated proteins in maple syrup urine disease.

Selected reaction monitoring (SRM) mass spectrometry can quantitatively measure proteins by specific targeting of peptide sequences, and allows the determination of multiple proteins in one single analysis. Here, we show the feasibility of simultaneous measurements of multiple proteins in mitochondria-enriched samples from cultured fibroblasts from healthy individuals and patients with mutations in branched-chain α-ketoacid dehydrogenase (BCKDH) complex. BCKDH is a mitochondrial multienzyme complex and its defective activity causes maple syrup urine disease (MSUD), a rare but severe inherited metabolic disorder. Four different genes encode the catalytic subunits of BCKDH: E1α (BCKDHA), E1ß (BCKDHB), E2 (DBT), and E3 (DLD). All four proteins were successfully quantified in healthy individuals. However, the E1α and E1ß proteins were not detected in patients carrying mutations in one of those genes, whereas mRNA levels were almost unaltered, indicating instability of E1α and E1ß monomers. Using SRM we elucidated the protein effects of mutations generating premature termination codons or misfolded proteins. SRM is a complement to transcript level measurements and a valuable tool to shed light on molecular mechanisms and on effects of pharmacological therapies at protein level. SRM is particularly effective for inherited disorders caused by multiple proteins such as defects in multienzyme complexes.

Proposed guidelines for the diagnosis and management of methylmalonic and propionic acidemia.

Methylmalonic and propionic acidemia (MMA/PA) are inborn errors of metabolism characterized by accumulation of propionic acid and/or methylmalonic acid due to deficiency of methylmalonyl-CoA mutase (MUT) or propionyl-CoA carboxylase (PCC). MMA has an estimated incidence of ~ 1: 50,000 and PA of ~ 1:100'000 -150,000. Patients present either shortly after birth with acute deterioration, metabolic acidosis and hyperammonemia or later at any age with a more heterogeneous clinical picture, leading to early death or to severe neurological handicap in many survivors. Mental outcome tends to be worse in PA and late complications include chronic kidney disease almost exclusively in MMA and cardiomyopathy mainly in PA. Except for vitamin B12 responsive forms of MMA the outcome remains poor despite the existence of apparently effective therapy with a low protein diet and carnitine. This may be related to under recognition and delayed diagnosis due to nonspecific clinical presentation and insufficient awareness of health care professionals because of disease rarity.

Two novel mutations in the BCKDK (branched-chain keto-acid dehydrogenase kinase) gene are responsible for a neurobehavioral deficit in two pediatric unrelated patients.

Inactivating mutations in the BCKDK gene, which codes for the kinase responsible for the negative regulation of the branched-chain α-keto acid dehydrogenase complex (BCKD), have recently been associated with a form of autism in three families. In this work, two novel exonic BCKDK mutations, c.520C>G/p.R174G and c.1166T>C/p.L389P, were identified at the homozygous state in two unrelated children with persistently reduced body fluid levels of branched-chain amino acids (BCAAs), developmental delay, microcephaly, and neurobehavioral abnormalities. Functional analysis of the mutations confirmed the missense character of the c.1166T>C change and showed a splicing defect r.[520c>g;521_543del]/p.R174Gfs1*, for c.520C>G due to the presence of a new donor splice site. Mutation p.L389P showed total loss of kinase activity. Moreover, patient-derived fibroblasts showed undetectable (p.R174Gfs1*) or barely detectable (p.L389P) levels of BCKDK protein and its phosphorylated substrate (phospho-E1α), resulting in increased BCKD activity and the very rapid BCAA catabolism manifested by the patients' clinical phenotype. Based on these results, a protein-rich diet plus oral BCAA supplementation was implemented in the patient homozygous for p.R174Gfs1*. This treatment normalized plasma BCAA levels and improved growth, developmental and behavioral variables. Our results demonstrate that BCKDK mutations can result in neurobehavioral deficits in humans and support the rationale for dietary intervention.

Determination of urinary alpha-aminoadipic semialdehyde by LC-MS/MS in patients with congenital metabolic diseases.

This paper describes a full detailed high performance liquid chromatography/tandem mass spectrometry method for the identification and quantification of human urine alpha-aminoadipic semialdehyde, biomarker of pyridoxine-dependent epilepsy. The ionization mode of the electrospray interface was negative and the metabolite was detected in the multiple reaction monitoring mode. Intra-day and inter-day laboratory precision were 4.64% and 7.30%, respectively, total run time was 3.5min. The calibration curve was linear between 0.25 and 10nmol with a correlation coefficient of the calibration line (R(2)≥0.9984); the limit of quantification was 0.25nmol within the control group. This simple, fast, high reproducible and robust procedure facilitates a rapid diagnosis of patients with pyridoxine-dependent epilepsy and can also be used to confirm the elevated urinary alpha-aminoadipic semialdehyde excretion in patients with other metabolic diseases as molybdenum cofactor and isolated sulphite oxidase deficiencies.

Pharmacological chaperones as a potential therapeutic option in methylmalonic aciduria cblB type.

Methylmalonic aciduria (MMA) cblB type is caused by mutations in the MMAB gene. This encodes the enzyme ATP:cob(I)alamin adenosyltransferase (ATR), which converts reduced cob(I)alamin to an active adenosylcobalamin cofactor. We recently reported the presence of destabilizing pathogenic mutations that retain some residual ATR activity. The aim of the present study was to seek pharmacological chaperones as a tailored therapy for stabilizing the ATR protein. High-throughput ligand screening of over 2000 compounds was performed; six were found to enhance the thermal stability of purified recombinant ATR. Further studies using a well-established bacterial system in which the recombinant ATR protein was expressed in the presence of these six compounds, showed them all to increase the stability of the wild-type ATR and the p.Ile96Thr mutant proteins. Compound V (N-{[(4-chlorophenyl)carbamothioyl]amino}-2-phenylacetamide) significantly increased this stability and did not act as an inhibitor of the purified protein. Importantly, compound V increased the activity of ATR in patient-derived fibroblasts harboring the destabilizing p.Ile96Thr mutation in a hemizygous state to within control range. When cobalamin was coadministrated with compound V, mutant ATR activity further improved. Oral administration of low doses of compound V to C57BL/6J mice for 12 days, led to increase in steady-state levels of ATR protein in liver and brain (disease-relevant organs). These results hold promise for the clinical use of pharmacological chaperones in MMA cblB type patients harboring chaperone-responsive mutations.

Clinical, biochemical, and molecular studies in pyridoxine-dependent epilepsy. Antisense therapy as possible new therapeutic option.

PURPOSE: Pyridoxine-dependent epilepsy seizure (PDE; OMIM 266100) is a disorder associated with severe seizures that can be controlled pharmacologically with pyridoxine. In the majority of patients with PDE, the disorder is caused by the deficient activity of the enzyme α-aminoadipic semialdehyde dehydrogenase (antiquitin protein), which is encoded by the ALDH7A1 gene. The aim of this work was the clinical, biochemical, and genetic analysis of 12 unrelated patients, mostly from Spain, in an attempt to provide further valuable data regarding the wide clinical, biochemical, and genetic spectrum of the disease. METHODS: The disease was confirmed based on the presence of α-aminoadipic semialdehyde (α-AASA) in urine measured by liquid chromatography tandem mass spectrometry (LC-MS/MS) and pipecolic acid (PA) in plasma and/or cerebrospinal fluid (CSF) measured by high performance liquid chromatography (HPLC)/MS/MS and by sequencing analysis of messenger RNA (mRNA) and genomic DNA of ALDH7A1. KEY FINDINGS: Most of the patients had seizures in the neonatal period, but they responded to vitamin B6 administration. Three patients developed late-onset seizures, and most patients showed mild-to-moderate postnatal developmental delay. All patients had elevated PA and α-AASA levels, even those who had undergone pyridoxine treatment for several years. The clinical spectrum of our patients is not limited to seizures but many of them show associated neurologic dysfunctions such as muscle tone alterations, irritability, and psychomotor retardation. The mutational spectrum of the present patients included 12 mutations, five already reported (c.500A>G, c.919C>T, c.1429G>C c.1217_1218delAT, and c.1482-1G>T) and seven novel sequence changes (c.75C>T, c.319G>T, c.554_555delAA, c.757C>T, c.787 + 1G>T, c.1474T>C, c.1093-?_1620+?). Only one mutation, p.G477R (c.1429G>C), was recurrent; this was detected in four different alleles. Transcriptional profile analysis of one patient's lymphoblasts and ex vivo splicing analysis showed the silent nucleotide change c.75C>T to be a novel splicing mutation creating a new donor splice site inside exon 1. Antisense therapy of the aberrant mRNA splicing in a lymphoblast cell line harboring mutation c.75C>T was successful. SIGNIFICANCE: The present results broaden our knowledge of PDE, provide information regarding the genetic background of PDE in Spain, afford data of use when making molecular-based prenatal diagnosis, and provide a cellular proof-of concept for antisense therapy application.

A novel regulatory defect in the branched-chain α-keto acid dehydrogenase complex due to a mutation in the PPM1K gene causes a mild variant phenotype of maple syrup urine disease.

This article describes a hitherto unreported involvement of the phosphatase PP2Cm, a recently described member of the branched-chain α-keto acid dehydrogenase (BCKDH) complex, in maple syrup urine disease (MSUD). The disease-causing mutation was identified in a patient with a mild variant phenotype, involving a gene not previously associated with MSUD. SNP array-based genotyping showed a copy-neutral homozygous pattern for chromosome 4 compatible with uniparental isodisomy. Mutation analysis of the candidate gene, PPM1K, revealed a homozygous c.417_418delTA change predicted to result in a truncated, unstable protein. No PP2Cm mutant protein was detected in immunocytochemical or Western blot expression analyses. The transient expression of wild-type PPM1K in PP2Cm-deficient fibroblasts recovered 35% of normal BCKDH activity. As PP2Cm has been described essential for cell survival, apoptosis and metabolism, the impact of its deficiency on specific metabolic stress variables was evaluated in PP2Cm-deficient fibroblasts. Increases were seen in ROS levels along with the activation of specific stress-signaling MAP kinases. Similar to that described for the pyruvate dehydrogenase complex, a defect in the regulation of BCKDH caused the aberrant metabolism of its substrate, contributing to the patient's MSUD phenotype--and perhaps others.

Novel features in the evolution of adenylosuccinate lyase deficiency.

BACKGROUND: Adenylosuccinate lyase (ADSL) deficiency is an autosomal recessive disorder of the purine synthesis which results in accumulation of succinylpurines (succinyladenosine (S-Ado) and succinylamino-imidazole carboxamide riboside (SAICAr)) in body fluids. Patients present developmental delay, often accompanied by epilepsy and autistic spectrum disorders. OBJECTIVES: To describe atypical neurological features in the evolution of three novel unrelated cases of ADSL deficiency. PATIENTS: A 9-year-old boy with severe cognitive impairment and autistic behaviour received d-ribose therapy for one year. Drug withdrawal was associated with acute neurological deterioration, severe brain atrophy and demyelination on MRI. The second patient is a 5.5-year-old girl with mild developmental delay who presented a benign course with moderate cognitive impairment as the only feature in her evolution. The final patient is a 14-year-old boy with severe cognitive impairment who developed drug-resistant epilepsy and bathing reflex seizures, progressive spasticity in the lower limbs and thoracic deformity. METHODS: SAICAr and S-Ado in urine were analysed by HPLC with diode array detection. Diagnosis was confirmed by molecular analysis of the ADSL gene. RESULTS: An elevation of S-Ado and SAICAr excretion in urine was detected in all three patients. The patients were homozygous for the missence change p.I369L and for the novel change p.M389V. CONCLUSION: Drug-resistant epilepsy and specific therapeutic interventions may modify the neurological outcome in ADSL deficiency. d-ribose must be considered with caution as, in our experience, it returns no clinical benefit and drug withdrawal can precipitate status epilepticus and acute neurological deterioration.

A fatal mitochondrial disease is associated with defective NFU1 function in the maturation of a subset of mitochondrial Fe-S proteins.

We report on ten individuals with a fatal infantile encephalopathy and/or pulmonary hypertension, leading to death before the age of 15 months. Hyperglycinemia and lactic acidosis were common findings. Glycine cleavage system and pyruvate dehydrogenase complex (PDHC) activities were low. Homozygosity mapping revealed a perfectly overlapping homozygous region of 1.24 Mb corresponding to chromosome 2 and led to the identification of a homozygous missense mutation (c.622G > T) in NFU1, which encodes a conserved protein suggested to participate in Fe-S cluster biogenesis. Nine individuals were homozygous for this mutation, whereas one was compound heterozygous for this and a splice-site (c.545 + 5G > A) mutation. The biochemical phenotype suggested an impaired activity of the Fe-S enzyme lipoic acid synthase (LAS). Direct measurement of protein-bound lipoic acid in individual tissues indeed showed marked decreases. Upon depletion of NFU1 by RNA interference in human cell culture, LAS and, in turn, PDHC activities were largely diminished. In addition, the amount of succinate dehydrogenase, but no other Fe-S proteins, was decreased. In contrast, depletion of the general Fe-S scaffold protein ISCU severely affected assembly of all tested Fe-S proteins, suggesting that NFU1 performs a specific function in mitochondrial Fe-S cluster maturation. Similar biochemical effects were observed in Saccharomyces cerevisiae upon deletion of NFU1, resulting in lower lipoylation and SDH activity. Importantly, yeast Nfu1 protein carrying the individuals' missense mutation was functionally impaired. We conclude that NFU1 functions as a late-acting maturation factor for a subset of mitochondrial Fe-S proteins.

Genotype-phenotype correlations in sepiapterin reductase deficiency. A splicing defect accounts for a new phenotypic variant.

Sepiapterin reductase (SR) catalyzes the final step in the de novo synthesis of tetrahydrobiopterin, essential cofactor for phenylalanine, tyrosine, and tryptophan hydroxylases. SR deficiency is a very rare disease resulting in monoamine neurotransmitter depletion. Most patients present with clinical symptoms before the first year of age corresponding to a dopa-responsive dystonia phenotype with diurnal fluctuations, although some patients exhibit more complex motor and neurological phenotypes. Herein, we describe four new cases from Spain, their clinical phenotype and the biochemical and genetic analyses. Two mutations in the SPR gene were functionally expressed to provide a basis to establish genotype-phenotype correlations. Mutation c.751A>T is functionally null, correlating with the severe phenotype observed. The novel mutation c.304G>T was identified in three siblings with a strikingly mild phenotype without cognitive delay and close to asymptomatic in the eldest sister. Minigene analysis demonstrated that this mutation located in the last nucleotide of exon 1 affects splicing although some normal transcripts can be produced, resulting in the missense mutant p.G102C that retains partial activity. These results may account for the mild phenotype and the variable clinical presentations observed, which could depend on interindividual differences in relative abundance of correctly spliced and aberrant transcripts.