Mitochondrial response to the BCKDK-deficiency: Some clues to understand the positive dietary response in this form of autism.

Mutations on the mitochondrial-expressed Branched Chain α-Keto acid Dehydrogenase Kinase (BCKDK) gene have been recently associated with a novel dietary-treatable form of autism. But, being a mitochondrial metabolism disease, little is known about the impact on mitochondrial performance. Here, we analyze the mitochondrial response to the BCKDK-deficiency in patient's primary fibroblasts by measuring bioenergetics, ultra-structural and dynamic parameters. A two-fold increase in superoxide anion production, together with a reduction in ATP-linked respiration and intracellular ATP levels (down to 60%) detected in mutants fibroblasts point to a general bioenergetics depletion that could affect the mitochondrial dynamics and cell fate. Ultrastructure analysis of BCKDK-deficient fibroblasts shows an increased number of elongated mitochondria, apparently associated with changes in the mediator of inner mitochondria membrane fusion, GTPase OPA1 forms, and in the outer mitochondrial membrane, mitofusin 2/MFN2. Our data support a possible hyperfusion response of BCKDK-deficient mitochondria to stress. Cellular fate also seems to be affected as these fibroblasts show an altered proportion of the cells on G0/G1 and G2/M phases. Knockdown of BCKDK gene in control fibroblasts recapitulates most of these features. Same BCKDK-knockdown in a MSUD patient fibroblasts unmasks the direct involvement of the accelerated BCAAs catabolism in the mitochondrial dysfunction. All these data give us a clue to understand the positive dietary response to an overload of branched-chain amino acids. We hypothesize that a combination of the current therapeutic option with a protocol that considers the oxidative damage and energy expenditure, addressing the patients' individuality, might be useful for the physicians.

Cerebrospinal fluid synaptic proteins as useful biomarkers in tyrosine hydroxylase deficiency.

Tyrosine hydroxylase (TH) deficiency is an inborn error of dopamine biosynthesis and a cause of early parkinsonism. Two clinical phenotypes have been described. Type "B": early onset severe encephalopathy; type "A": later onset, less severe and better response to L-dopa. We aimed to study the expression of several key dopaminergic and gabaergic synaptic proteins in the cerebrospinal fluid (CSF) of a series of patients with TH deficiency and their possible relation with the clinical phenotype and response to L-DOPA. Dopamine transporter (DAT), D2-receptor and vesicular monoamine transporter (VMAT2) were measured in the CSF of 10 subjects with TH deficiency by Western blot analysis. In 3 patients, data of pre- and post-treatment with L-DOPA were available, and in one of them, GABA vesicular transporter was determined. Results were compared to an age-matched control population. The concentration of D2-receptors in CSF was significantly higher in patients with TH deficiency than in controls. Similarly, DAT and vesicular monoamine transporter type 2 were up-regulated. Studies performed before L-DOPA, and on L-DOPA therapy showed a paradoxical response with D2 receptor expression increase as L-Dopa doses and homovanillic concentration gradually raised in a B phenotype patient. The opposite results were found in two patients with A phenotype. However, this is a very small sample, and further studies are needed to conclude robust differences between phenotypes. Synaptic proteins are detectable in the CSF and their quantification can be useful for understanding the pathophysiology of neurotransmitter defects and potentially to adjust and personalize treatments in the future.

Clinical and biochemical outcome after hydroxocobalamin dose escalation in a series of patients with cobalamin C deficiency.

BACKGROUND: CblC deficiency produces a combination of methylmalonic aciduria (MMA) and homocystinuria (HCU), and is the most common error of cobalamin metabolism. Patients present a wide spectrum of symptoms, ranging from early severe multisystemic forms, to milder late-onset phenotypes. Cognitive and visual impairment are nearly constant. Hydroxocobalamin (OHCbl), betaine, folinic acid, levocarnitine and eventually dietary protein restriction are the main therapeutic approaches. Although early introduction of OHCbl is crucial, no standardized protocols regarding dose adaptation exist. No reports on long-term outcomes after high doses of this vitamin have been published. METHODS: In this study five patients with CblC deficiency (early severe forms) were treated with high doses of OHCbl for 18 to 30months. Clinical examinations, neurological assessment, and biochemical studies (plasma total homocysteine (tHcy), amino acids, hydroxocobalamin, and methylmalonic acid in urine) were periodically performed. RESULTS: Variable clinical and biochemical outcomes were observed in patients treated with high doses of OHCbl. The best biochemical response was observed in those children with the worse metabolic control. By contrast, those patients with a concentration of tHcy around 50µmol/l or less showed only minor changes. Clinically, a considerable improvement was observed in those patients with severe problems in communication, expressive language and behavior. CONCLUSIONS: According to our study, high OHCbl doses in CblC deficiency could have a greater benefit in those children with a prior history of suboptimal metabolic control, and also in those with severe neurological phenotypes. More specifically, we observed improvements in communication skills and behavior. These results should encourage further prospective trials to determine the optimal OHCbl regimen and to generate protocols and guidelines in this rare disorder.

Mild clinical and biochemical phenotype in two patients with PMM2-CDG (congenital disorder of glycosylation Ia).

Phosphomannomutase 2 deficiency (PMM2-CDG) patients may present as mild phenotypes, with the cerebellum frequently involved. In those cases, false-negative results in screening may occur when applying conventional biochemical procedures. Our aim was to report two patients with a diagnosis of PMM2-CDG presenting with mild clinical phenotype. Patient 1-at 9 months of age, she presented with just psychomotor delay, tremor, hypotonia, and slight lipodystrophy. Patient 2-she presented at 8 months of age with psychomotor delay, hand stereotypes, hypotonia, convergent bilateral strabismus, and tremor but no lipodystrophy. Routine biochemical parameters including blood count, clotting factors, proteins, and thyroid hormone were normal in both cases. Cranial MRI evidenced mild cerebellar atrophy with moderate vermis hypoplasia. In case 1, sialotransferrin pattern showed very slightly increased disialotransferrin with no asialotransferrin, and in case 2, the transferrin pattern was impaired in the first study but nearly normal in the second. Nevertheless, in all the samples, quantification of the patterns obtained by capillary zone electrophoresis analysis gave results out of the control range. High residual PMM2 activity was observed in both cases and the genetic analysis showed that patient 1 was heterozygous for c.722G>C (p.C241S) and c.368G>A (p.R123Q) mutations, and patient 2 showed the c.722G>C and the c.470T>C (p.F157S) mutations in the PMM2 gene. We would like to stress the importance of the use of sensitive semiquantitative methods of screening for CDG in order to achieve early identification of patients with mild phenotypes. Intentional tremor was an atypical but remarkable clinical feature in both cases, and the global cerebellar atrophy with vermis hypoplasia reinforced the early clinical suspicion of a PMM2-CDG disease.

Response to creatine analogs in fibroblasts and patients with creatine transporter deficiency.

Creatine transporter (CRTR) deficiency is one of the most frequent causes of X-linked mental retardation. The lack of an effective treatment for this disease, in contrast to creatine (Cr) biosynthesis disorders that respond to Cr monohydrate (CM), led us to analyze the efficacy of a lipophilic molecule derived from Cr, creatine ethyl ester (CEE), in fibroblasts and patients with CRTR deficiency. CM and CEE uptake studies were performed in six controls and four fibroblast cell lines from patients. We found a significant increase in Cr uptake after 72 h of incubation with CEE (500 micromol/L) in patients and control fibroblasts compared to incubation with CM. Subsequently, we assayed the clinical effect of CEE administration in four patients with CRTR deficiency. After 1 year of treatment, a lack of significant improvement in neuropsychological assessment or changes in Cr level in brain (1)H MRS was observed, and CEE was discontinued. In conclusion, this 12-month trial with CEE did not increase the brain concentration of Cr. Our in vitro data lend support to the idea of a certain passive transport of CEE in both pathological and control cells, although more lipophilic molecules or other cell systems that mimic the BBB should be used for a better approach to the in vivo system.

Ancient origin of the CTH alelle carrying the c.200C>T (p.T67I) variant in patients with cystathioninuria.

Hereditary cystathioninuria is due to mutations in the CTH gene that encodes for cystathionase, a pyridoxal-5'-phosphate (PLP) dependent enzyme. To date, mutations in this gene have been described in 10 unrelated cystathioninuric patients. Enzyme assays have showed that mutated cystathionase exhibits lower activity than controls. As cystathioninuria is usually accompanied by a wide variety of symptoms, it has been questioned whether it is a disease or just a biochemical finding not associated with the clinical picture of these patients. This is the first report of Spanish patients with cystathioninuria and mild to severe neurological symptoms in childhood. After oral pyridoxine therapy biochemical parameters have normalized but clinical amelioration was not evident. All patients were homozygotes for the c.200C>T (p.T67I) variant which is the most prevalent inactivating mutation in the CTH gene. To further investigate the history of the alleles carrying the c.200C>T transition in Europe, we also constructed the haplotypes on the CTH locus in our Spanish patients as well as in a clinical series of cystathioninuric patients from the Czech Republic harboring the same nucleotide change. We suggest that the CTH p.T67I substitution could have an ancient common origin, which probably occurred in the Neolithic Era and spread throughout Europe.

Molecular characterization of five patients with homocystinuria due to severe methylenetetrahydrofolate reductase deficiency.

Methylenetetrahydrofolate reductase (MTHFR) plays a major role in folate metabolism. Disturbed function of the enzyme results in hyperhomocysteinemia and causes severe vascular and neurological disorders and developmental delay. Five patients suspected of having non-classical homocystinuria due to MTHFR deficiency were examined with respect to their symptoms, MTHFR enzyme activity and genotypes of the MTHFR gene. All patients presented symptoms of severe central nervous system disease. Two patients died, at the ages of 15 months and 14 years. One patient is currently 32 years old, and is being treated with betaine and folinic acid. The other two patients, with an early diagnosis and a severe course of the disease, are currently improving under treatment. MTHFR enzyme activity in the fibroblasts of four of the patients was practically undetectable. We found four novel mutations, three of which were missense changes c.664G> T (p.V218L), c.1316T> C (p.F435S) and c.1733T> G (p.V574G), and the fourth was the 1-bp deletion c.1780delC (p.L590CfsX72). We also found the previously reported nonsense mutation c.1420G> T (p.E470X). All the patients were homozygous. Molecular modelling of the double mutant allele (p.V218L; p.A222V) revealed that affinity for FAD was not affected in this mutant. For the p.E470X mutation, the evidence pointed to nonsense-mediated mRNA decay. In general, genotype-phenotype analysis predicts milder outcomes for patients with missense changes than for those in which mutations led to severe alterations of the MTHFR protein.

Mutations in the urocanase gene UROC1 are associated with urocanic aciduria.

Urocanase is an enzyme in the histidine pathway encoded by the UROC1 gene. This report describes the first putative mutations, p.L70P and p.R450C, in the coding region of the UROC1 gene in a girl with urocanic aciduria presenting with mental retardation and intermittent ataxia. Computed (in silico) predictions, protein expression studies and enzyme activity assays suggest that none of the mutations can produce a fully functional enzyme. The p.L70P substitution, which probably implies the disruption of an alpha-helix in the N-terminus, would alter its properties and therefore, its function. The p.R450C change would render impossible any interaction between urocanase and its substrate and would loss its enzyme activity. Consequently, these studies suggest that both mutations could alter the correct activity of urocanase, which would explain the clinical and biochemical findings described in this patient.

Quality of dietary control in phenylketonuric patients and its relationship with general intelligence.

OBJECTIVES: Assessment of the quality of dietary treatment of phenylketonuria (PKU) patients and investigation of its relationship with the general intelligence of the patients. METHODS: Cross-sectional and longitudinal study of 105 PKU treated patients. The index of dietary control (IDC) was calculated as the phenylalanine (Phe) data reduction in half-year medians and the mean of all medians throughout the patient's life. We calculated four different IDCs related to age: IDC-A (< 6 years), IDC-B (6-12 years), IDC-C (13-18 years) and IDC-D (> 18 years). To evaluate the fluctuation of Phe values we calculated the standard error of the estimate of the regression of Phe concentration over age. Development quotient was calculated with the Brunet-Lezine test (< 4 years). Intelligence quotient was evaluated with the Kaufman Bit Intelligence Test (K-Bit), Wechsler Intelligence Scale for Children-Revised (WISC-R) and Wechsler Adult Intelligence Scale Third Edition (WAIS III). RESULTS: Cross-sectional study: The IDC in age groups were significantly different and so were the number of patients with good, acceptable and poor IDC related to age (p < 0.001). Sampling frequency was good in 72, acceptable in 23 and poor in 10 patients. The general intelligence (101 +/- 10) correlated negatively with the IDC (p < 0.0001) and Phe fluctuations (p < 0.004). Longitudinal study: Significant differences were observed between the IDC through the patients' lifetime except in the adolescent/adult period. CONCLUSIONS: 85% of PKU patients showed good/acceptable quality of dietary control. General intelligence correlates with the IDC at all ages, which highlights the importance of good control to achieve good prognosis.

Creatine transporter deficiency in two adult patients with static encephalopathy.

Creatine transporter deficiency is a recently identified X-linked inborn error of metabolism. The natural course of the disease is not well delineated since clinical data from adult patients have scarcely been reported. A progressive course of the disease has been noted in a few described cases. We report the first two Spanish adult patients with creatine transporter deficiency and compare their clinical phenotype and the evolution of the disease with those of other published cases. The two brothers were identified in a study of a cohort of 610 mentally handicapped male patients. The disease was detected by biochemical studies and confirmed by DNA studies. The most significant clinical features were mental retardation, epilepsy and autistic behaviour, and these symptoms did not worsen, in contrast to other reports. They did not present gastrointestinal problems or movement disorders. Creatine transporter deficiency could be an underdiagnosed metabolic disorder and should be considered in adult patients with mental retardation. Clinical presentation of this disorder showed marked differences among adult patients and the course of the disease was static in our cases. Detection of additional adult patients might allow better understanding of the phenotypic outcome at a later age.

Secondary disorders of glycosylation in inborn errors of fructose metabolism.

Adamowicz and colleagues raised the alert in 2007 about patients with atypical hereditary fructose intolerance (HFI) primarily misdiagnosed as CDG Ix. We describe a girl with neonatal hypertonia, facial trismus, absent swallowing and coughing reflexes, gastro-oesophageal reflux and sporadically elevated Krebs cycle metabolites and lactate. At 14 months microcephaly and hepatomegaly were noted, with hypertransaminasaemia but normal blood coagulation, glucose, phosphate, and absent urinary reducing substances. Neurological impairment persisted. Because of hepatic and neurological abnormalities with developmental delay, Tf IEF was performed and showed a severe type 1 pattern, resulting in a wrong diagnosis of CDG. Subsequently, an aversion to fruits suggested HFI, confirmed by the finding of ALDOB mutations (p.A150P/p.N335K). The girl improved with fructose-free diet, but liver cirrhosis led to hepatic transplantation. She is now 7 years old with good evolution; facial trismus and hypertonia reversed, but microcephaly persists. Transferrin MALDI-TOF MS characterization revealed underoccupation of glycosylation sites and glycan abnormalities, which reversed with dietary treatment. High maternal fructose concentrations might have caused neonatal abnormalities. Although in our patient's mother there is no fructose accumulation at present, it is possible that increased ingestion of fruits and vegetables during pregnancy, together with her heterozygosity, caused an accumulation of fructose that finally affected the fetus. We also describe slightly abnormal transferrin isoelectric focusing and MALDI-TOF MS patterns of intact transferrin and N-glycans in a fructose-1,6-bisphosphatase (FBP1)-deficient patient. While HFI is a well-known cause of secondary CDG, we found no reports of abnormal transferrin isoelectric focusing patterns in FBP1 deficiency and we introduce this condition as a possible secondary cause for altered transferrin isoelectric focusing.

Arginine supplementation in four patients with X-linked creatine transporter defect.

BACKGROUND: Treatment with oral creatine monohydrate has not shown efficacy in patients with creatine transporter deficiency (CRTR-D). Another therapeutic option proposed is L-arginine, the substrate for the enzyme L-arginine:glycine amidinotransferase (AGAT). We evaluate clinical characteristics and cerebral creatine replenishment after L-arginine therapy in four patients with CRTR-D. PATIENTS AND METHODS: Four boys with genetically confirmed diagnosis of CRTR-D (ages 9-16 years) were supplemented with L-arginine (0.4 g/kg per day) for a period of 9 months. Treatment efficacy was evaluated by clinical and neuropsychological assessment and determination of creatine signals by brain proton magnetic resonance spectroscopy ((1)H-MRS). RESULTS: Epileptic seizures remained well controlled with antiepileptic drugs in three cases, both before and after L-arginine supplementation. Vineland Adaptive Behaviour Scale did not show any change in communication, daily living skills, socialization or motor skills, and a lack of improvement in brain (1)H-MRS follow-up was observed. L-Arginine was discontinued at the end of the observation period. CONCLUSIONS: Nine months of L-arginine supplementation did not show effectiveness in the four patients affected with CRTR-D in this protocol.

Hypermanganesemia due to mutations in SLC39A14: further insights into Mn deposition in the central nervous system.

BACKGROUND: The SLC39A14, SLC30A10 and SLC39A8 are considered to be key genes involved in manganese (Mn) homeostasis in humans. Mn levels in plasma and urine are useful tools for early recognition of these disorders. We aimed to explore further biomarkers of Mn deposition in the central nervous system in two siblings presenting with acute dystonia and hypermanganesemia due to mutations in SLC39A14. These biomarkers may help clinicians to establish faster and accurate diagnosis and to monitor disease progression after chelation therapy is administered. RESULTS: A customized gene panel for movement disorders revealed a novel missense variant (c.311G > T; p.Ser104Ile) in SLC39A14 gene in two siblings presenting at the age of 10 months with acute dystonia and motor regression. Mn concentrations were analyzed using inductively coupled mass spectrometry in plasma and cerebrospinal fluid, disclosing elevated Mn levels in the index case compared to control patients. Surprisingly, Mn values were 3-fold higher in CSF than in plasma. We quantified the pallidal index, defined as the ratio between the signal intensity in the globus pallidus and the subcortical frontal white matter in axial T1-weighted MRI, and found significantly higher values in the SLC39A14 patient than in controls. These values increased over a period of 10 years, suggesting the relentless pallidal accumulation of Mn. Following genetic confirmation, a trial with the Mn chelator Na2CaEDTA led to a reduction in plasma Mn, zinc and selenium levels. However, parents reported worsening of cervical dystonia, irritability and sleep difficulties and chelation therapy was discontinued. CONCLUSIONS: Our study expands the very few descriptions of patients with SLC39A14 mutations. We report for the first time the elevation of Mn in CSF of SLC39A14 mutated patients, supporting the hypothesis that brain is an important organ of Mn deposition in SLC39A14-related disease. The pallidal index is an indirect and non-invasive method that can be used to rate disease progression on follow-up MRIs. Finally, we propose that patients with inherited defects of manganese transport should be initially treated with low doses of Na2CaEDTA followed by gradual dose escalation, together with a close monitoring of blood trace elements in order to avoid side effects.

Secondary alteration of the transferrin isoelectric focusing pattern in a case of bacterial meningitis.

Congenital disorders of glycosylation (CDG) are a group of inherited defects in the synthesis and processing of the linked glycans of glycoproteins and other glycoconjugates. The phenotypic spectrum presents wide variability, and clinical diagnosis is not reliable in most cases. Isoelectric focusing (IEF) of serum transferrin is widely used as a tool to detect CDG. We describe a paediatric patient presenting an altered serum transferrin pattern due to a secondary disorder of glycosylation caused by pneumococcal meningitis (Streptococcus pneumoniae, serotype 19A). During admission, brain CT scan and MRI showed acute ischaemic lesions in brain frontotemporal parenchyma, and enlarged subarachnoidal spaces in the frontal area resembling a chronic injury. This led us to screen for inborn errors of metabolism potentially associated with these findings (homocystinuria, glutaric aciduria, CDG syndromes). Biochemical studies for the screening of these inborn errors of metabolism were normal except for sialotransferrin isoelectric focusing, which showed a type 2 pattern. However, 16 days later, together with the remission of the meningitis process, the sialotransferrin pattern had normalized. The apolipoprotein C-III (an O-glycoprotein) profile was normal in all samples analysed. In conclusion, infectious events should be ruled out in the differential diagnosis of CDG syndromes. Furthermore, our findings highlight the possibility that the type 2 IEF pattern of serum sialotransferrin detected in some patients with neonatal death due to organ failure and septic events might be secondary to the infectious process.

[Cerebral creatine transporter deficiency: an infradiagnosed neurometabolic disease]. / Deficiencia del transportador de creatina cerebral: una enfermedad neurometabolica infradiagnosticada.

INTRODUCTION: Brain creatine deficiencies are a group of inborn errors of metabolism recently recognized which are caused by arginine: glycine amidinotransferase (AGAT) deficiency, guanidinoacetate metiltransferase (GAMT) deficiency and defects in creatine transporter (CRTR). Although all of them are characterized by a brain creatine deficiency, clinical and biochemical features are different. CASE REPORTS: We present a retrospective study about four patients of masculine sex affected of creatine transporter defects who were recently diagnosed in our centre. We describe the clinical presentation features, the different tests that we used in the diagnosis process (brain magnetic resonance spectroscopy, biochemical analysis of guanidinoacetate and creatine/creatinine ratio in urine), evolution aspects and the response to treatment. The most significative clinical feature was developmental delay mainly in expressive speech, they also presented epilepsy (three cases), autism (three cases), hypotonia (one case) and microcephalia (one case). Brain magnetic resonance spectroscopy showed a low (three cases) or an absence (one case) of creatine level. To confirm the defect we studied the creatine uptake in fibroblasts and molecular analysis of the SLC6A8/creatine transporter gene. Patients with creatine transporter deficiency are being treated with arginine, because a lack of response to creatine. CONCLUSION: Cerebral creatine transporter deficiency can present with different neurological symptoms but developmental and language delay and epilepsy are the most significative; diagnosis is easy and there are some therapeutical options.

[Diagnosis and treatment of brain creatine deficiency syndromes]. / Diagnostico y tratamiento de los sindromes de deficiencia de creatina cerebral.

AIM: To review the clinical, biochemical and genetic aspects of brain creatine deficiency syndromes, as well as the therapeutic options available. DEVELOPMENT: Brain creatine deficiency syndrome has recently been described as a series of inborn errors of metabolism that affect the synthesis and transport of creatine. Three metabolic defects are known: two affect synthesis -guanidinoacetate methyltransferase (GAMT) and arginine:glycine amidinotransferase (AGAT)- and one affects the transport of creatine. Clinically, these patients can display mental retardation, language disorders, epilepsy, autistic behaviour, neurological impairment and movement disorders. After the clinical selection, the different defects can be identified by a biochemical study involving the analysis of metabolites in biological fluids (guanidinoacetate and creatine/ creatinine ratio). Before continuing with the molecular studies, it is important to confirm the deficiency of brain creatine by means of magnetic resonance imaging with spectroscopy. Diagnostic confirmation of AGAT and GAMT deficits is carried out by determining the enzymatic activity in fibroblasts or lymphoblasts, or the incorporation of creatine in the case of studies of transport defects. The study of mutations in AGAT, GAMT (autosomal recessive inheritance) and SLC6A8 (X-linked) genes completes the diagnosis. CONCLUSIONS: Brain creatine deficiency syndromes are mainly associated to mental retardation and autism. GAMT and AGAT deficiencies respond to treatment with creatine, whereas patients with transport defects do not respond to this therapy; new therapeutic approaches are therefore being evaluated for this disease.

Diseases of the Synaptic Vesicle: A Potential New Group of Neurometabolic Disorders Affecting Neurotransmission.

The general concept of inborn error of metabolism is currently evolving into the interface between classical biochemistry and cellular biology. Basic neuroscience is providing increasing knowledge about the mechanisms of neurotransmission and novel related disorders are being described. There is a necessity of updating the classic concept of "inborn error of neurotransmitters (NT)" that considers mainly defects of synthesis and catabolism and transport of low weight NT molecules. Monogenic defects of the synaptic vesicle (SV), and especially those affecting the SV cycle are a potential new group of NT disorders since they end up in abnormal NT turnover and release. The most common clinical manifestations include epilepsy, intellectual disability, autism and movement disorders, and are in the continuum symptoms of synaptopathies. Interestingly, brain malformations and neurodegenerative conditions are also present within SV diseases. Metabolomics, proteomics, and other -omic techniques probably will provide biomarkers and contribute to therapeutic targets in the future.

Dataset reporting BCKDK interference in a BCAA-catabolism restricted environment.

This data article contains complementary figures to the research article "Mitochondrial response to the BCKDK-deficiency: some clues to understand the positive dietary response in this form of autism" [1]. Herein we present data relative to the effect of knocking down BCKDK gene on the real time oxygen consumption rate of fibroblasts obtained from a Maple Syrup Urine Disease (MSUD) patient. Interference of BCKDK expression on such cells showing a reduced branched-chain α-ketoacid dehydrogenase (BCKDHc) activity; let us generate a scenario to study the direct effect of BCKDK absence in an environment of high branched-chain amino acids (BCAAs) concentrations. Data relative to the effectiveness of the knockdown together with the potentiality of the BCKDK-knockdown to increase the deficient branched-chain α-ketoacid dehydrogenase activity detected in MSUD patients are also shown.

A statistical algorithm showing coenzyme Q10 and citrate synthase as biomarkers for mitochondrial respiratory chain enzyme activities.

Laboratory data interpretation for the assessment of complex biological systems remains a great challenge, as occurs in mitochondrial function research studies. The classical biochemical data interpretation of patients versus reference values may be insufficient, and in fact the current classifications of mitochondrial patients are still done on basis of probability criteria. We have developed and applied a mathematic agglomerative algorithm to search for correlations among the different biochemical variables of the mitochondrial respiratory chain in order to identify populations displaying correlation coefficients >0.95. We demonstrated that coenzyme Q10 may be a better biomarker of mitochondrial respiratory chain enzyme activities than the citrate synthase activity. Furthermore, the application of this algorithm may be useful to re-classify mitochondrial patients or to explore associations among other biochemical variables from different biological systems.

Muscle coenzyme Q10 concentrations in patients with probable and definite diagnosis of respiratory chain disorders.

Coenzyme Q(10) (CoQ) deficiency syndrome is a disorder of unknown ethiology that may cause different forms of mitochondrial encephalomyopathy. In the present study our aim was to analyse CoQ concentration and mitochondrial respiratory chain (MRC) enzyme activities in muscle biopsies of patients with clinical suspicion and/or biochemical-molecular diagnosis of a mitochondrial disorder. We studied 36 patients classified into 3 groups: 1) 14 patients without a definitive diagnosis of mitochondrial disease, 2) 13 patients with decreased CI + III and II + III activities of the MRC, and 3) 9 patients with definitive diagnosis of mitochondrial disease. Only 1 of the 14 patients of group 1 showed slightly reduced CoQ values in muscle. Six of the 13 patients from group 2 showed partial CoQ deficiency in muscle and 1 of the 9 cases from group 3 presented a slight CoQ deficiency. Significantly positive correlation was observed between CI + III and CII + III activities with CoQ concentrations in the 36 muscle homogenates from patients (r = 0.555; p = 0.001; and r = 0.460; p = 0.005, respectively). In conclusion, measurement of MRC enzyme activities is a useful tool for the detection of CoQ deficiency, which should be confirmed by CoQ quantification.