Biomedical subjects

Brian H Robinson

Publications and source records attributed to Brian H Robinson.

At least 19 recordsLinked to original sources

PubMed2006

Identification of a canine model of pyruvate dehydrogenase phosphatase 1 deficiency.

Exercise intolerance syndromes are well known to be associated with inborn errors of metabolism affecting glycolysis (phosphorylase and phosphofructokinase deficiency) and fatty acid oxidation (palmitoyl carnitine transferase deficiency). We have identified a canine model for profound exercise intolerance caused by a deficit in PDP1 (EC 3.1.3.43), the phosphatase enzyme that activates the pyruvate dehydrogenase complex (PDHc). The Clumber spaniel breed was originated in 1760 by the Duc de Noailles, as a hunting dog with a gentle temperament suitable for the 'elderly gentleman'. Here we report that 20% of the current Clumber and Sussex spaniel population are carriers for a null mutation in PDP1, and that homozygosity produces severe exercise intolerance. Human pyruvate dehydrogenase phosphatase deficiency was recently characterized at the molecular level. However, the nature of the human mutation (loss of a single amino acid altering PDP1 activity) made it impossible to discern the role of the second phosphatase isoform, PDP2, in the deficient phenotype. Here we show that the null mutation in dogs provides a valuable animal model with which to study the effects of dysregulation of the PDHc. Knowledge of the molecular defect has allowed for the institution of a rapid restriction enzyme test for the canine mutation that will allow for selective breeding and has led to a suggested dietary therapy for affected dogs that has proven to be beneficial. Pharmacological and genetic therapies for PDP1 deficiency can now be investigated and the role of PDP2 can be fully characterized.

Animals↗

PubMed2006

Novel mitochondrial DNA mutations associated with myopathy, cardiomyopathy, renal failure, and deafness.

Patients with mitochondrial disease usually manifest multisystemic dysfunction with a broad clinical spectrum. When the tests for common mitochondrial DNA (mtDNA) point mutations are negative and the mtDNA defects are still hypothesized, it is necessary to screen the entire mitochondrial genome for unknown mutations in order to confirm the diagnosis. We report an 8-year-old girl who had a long history of ragged-red fiber myopathy, short stature, and deafness, who ultimately developed renal failure and fatal cardiac dysfunction. Respiratory chain enzyme analysis on muscle biopsy revealed deficiency in complexes I, II/III, and IV. Whole mitochondrial genome sequencing analysis was performed. Three novel changes: homoplasmic 15458T > C and 15519T > C in cytochrome b, and a near homoplasmic 5783G > A in tRNA(cys), were found in the proband in various tissues. Her mother and asymptomatic sibling also carry the two homoplasmic mutations and the heteroplasmic 5783G > A mutation in blood, hair follicles, and buccal cells, at lower percentage. The 5783G > A mutation occurs at the T arm of tRNA(cys), resulting in the disruption of the stem structure, which may reduce the stability of the tRNA. 15458T > C changes an amino acid serine to proline at a conserved alpha-helix, which may force the helix to bend. These two mutations may have pathogenic significance. This case emphasizes the importance of pursuing more extensive mutational analysis of mtDNA in the absence of common mtDNA point mutations or large deletions, when there is a high suspicion of a mitochondrial disorder.

Abnormalities, Multiple↗

PubMed2006

Lactic acidemia and mitochondrial disease.

Lactic acidemia is present in the majority of patients with mitochondrial oxidative defects as well as in disorders of gluconeogenesis. An understanding of the dynamics of lactic acid metabolism in the human body and the influences on lactate/pyruvate ratios exerted by changes in cellular redox state allows for the development of diagnostic algorithms based on clinical and biochemical phenotypes. Mitochondrial disorders can be due to defects in nuclear genes directly affecting the respiratory chain assembly or function, mtDNA genes affecting the respiratory chain or nuclear genes influencing mtDNA structure and viability. In this review, we look at the classification of mitochondrial disease from the perspective of not just the genetic and biochemical etiology but also from the perspective of the clinical phenotypic expression.

DNA, Mitochondrial↗

PubMed2006

Novel mutations in dihydrolipoamide dehydrogenase deficiency in two cousins with borderline-normal PDH complex activity.

We have diagnosed dihydrolipoamide dehydrogenase (DLD) deficiency in two male second cousins, who presented with markedly different clinical phenotypes. Patient 1 had a recurrent encephalopathy, and patient 2 had microcephaly and lactic acidosis. Their presentation is unusual, in that the DLD subunit deficiency had little effect on pyruvate dehydrogenase complex activity, but caused a severe reduction in the activities of other enzymes that utilize this subunit. We have identified two mutations in the DLD gene in each patient. The second cousins have one novel mutation in common resulting in a substitution of isoleucine for threonine (I47T), which has not been previously reported in the literature. Patient 1 has a second mutation that has been reported to be common in the Ashkenazi Jewish population, G229C. Patient 2 has a second mutation, E375K, which has also been previously reported in the literature. Enzyme kinetic measurements on patient fibroblasts show that under certain conditions, one heteroallelic mutation may have a higher K(m). This may account for the differing clinical phenotypes. These findings have important repercussions for other patients with similar clinical phenotypes, as DLD activity is not normally measured in cases with normal PDHc activity.

Acidosis, Lactic↗

PubMed2006

Intermittent peripheral weakness as the presenting feature of pyruvate dehydrogenase deficiency.

Two unrelated children presenting with episodic isolated peripheral weakness were found to have pyruvate dehydrogenase (PDH) deficiency (OMIM 312170) due to previously undescribed mutations (Pro250Thr, Arg88Cys) in the gene for the E1alpha subunit (PDHA1). Taken in context with the literature, these patients suggest that acute weakness initially resembling Guillain-Barré syndrome is a potentially reversible and probably underdiagnosed manifestation of PDH deficiency and that peripheral nerve function should be evaluated in PDH-deficient patients.

Child↗

PubMed2006

Kinetic studies of the interaction of fatty acids with phosphatidylcholine vesicles (liposomes).

The kinetics of addition of fatty acids (as alkaline solutions of the fatty acid anions) to pre-existing unilamellar phospholipid vesicles (mean diameter 100 nm) has been studied. The phospholipid DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) has been mainly used, together with three fatty acids, oleic acid (cis-9-octadecenoic acid), linoleic acid (cis,cis-9,12-octadecadienoic acid) and capric acid (decanoic acid). Experiments were performed above as well as below the main phase transition temperature (Tm) of DMPC vesicles. The pH chosen to study the fatty acid vesicle interaction (after fatty acid and vesicle mixing) was 8.5 in the case of oleic acid and linoleic acid and 7.4 for capric acid. In the absence of any pre-existing phospholipid vesicles, the addition of alkaline solutions of the fatty acid anions to corresponding buffer solutions of pH 8.5 or 7.4 leads to a partial protonation of the fatty acid anions again resulting in the formation of fatty acid vesicles. This process is rather slow, taking place over a period of hours/days, and the vesicles formed are very polydisperse and include a range of vesicle sizes/shapes. However, in the presence of pre-existing phospholipid vesicles the added fatty acids equilibrate readily within a few minutes and the size of the vesicles that form are then closely related to the size of the originally present phospholipid vesicles; the vesicles formed being generally somewhat larger than the pre-existing vesicles. In the case of the phospholipid DMPC, the mixed fatty acid/phospholipid vesicle system is often formed rather rapidly (particularly above Tm), so that stopped-flow methods have been applied to follow the kinetics of the process. It is proposed that most of the fatty acid molecules are initially rapidly incorporated into the bilayers of the pre-exisiting phospholipid vesicles as monomers, rather than that the added fatty acids form separate fatty acid vesicles. The mean vesicle sizes formed in the systems investigated have been analysed by using dynamic light scattering measurements. The behaviour of the DMPC system was found to be slightly different from the POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) system studied before, but the results are consistent with a model that involves growth and subsequent fission of the mixed vesicles. The study provides further support of the "matrix effect" in this type of system [S. Lonchin, P.L. Luisi, P. Walde, B.H. Robinson, J. Phys. Chem. B 103 (1999) 10910-10916]. The pre-existing DMPC vesicles act as a kind of seed to control the behavior of the system in the presence of added fatty acid anions.

Buffers↗

PubMed2005

Automated spectrophotometric analysis of mitochondrial respiratory chain complex enzyme activities in cultured skin fibroblasts.

BACKGROUND: Mitochondrial respiratory chain complex (RCC) disorders may occur as commonly as 1 in 8500 individuals. Because of the great variability of phenotypic presentations, measurement of individual RCC enzyme activities is a crucial diagnostic process. Current assay methods are time-consuming and labor-intensive and thus constitute a major impediment to clinical practice. A method with a faster turnaround time would therefore be beneficial. METHOD: We developed an automated spectrophotometric method for measuring the respiratory chain enzyme activities of complex I, complex II + III, and complex IV with the Hitachi 912, an automated spectrophotometer. Mitochondrial citrate synthase was also determined for normalization of the RCC activities. RESULTS: A blinded method comparison with samples from an external testing center yielded a 91% concordance of interpretations. Mean intraassay imprecision (as CV; n = 20) in a single batch analysis of each RCC was 5.9%. Interassay imprecision, evaluated on 2 samples harvested and analyzed 3 times each, gave mean CVs of 10%-18%. CONCLUSIONS: With this automated method, a panel of RCC enzyme activities can be determined in <2 h. In addition, an immunoblot assay using monoclonal antibodies against specific subunits of RCC enzyme complexes can be informative in cases of borderline enzyme activity. Our results suggest that in vitro diagnosis of RCC enzyme deficiencies in skin fibroblasts is an effective alternative to invasive muscle biopsy.

Autoanalysis↗

PubMed2005

Pyruvate dehydrogenase phosphatase deficiency: identification of the first mutation in two brothers and restoration of activity by protein complementation.

CONTEXT: Pyruvate dehydrogenase phosphatase (PDP) deficiency has been previously reported as an enzymopathy, but the genetic basis for such a defect has never been established. OBJECTIVE: The aim of this study was to identify the cause of the defect in two patients who presented with PDP deficiency. PATIENTS: We studied two brothers of consanguineous parents who presented with neonatal hypotonia, elevated lactate, and less than 25% native pyruvate dehydrogenase complex (PDHc) activity in skin fibroblasts compared with controls. The activity of the complex could be restored to normal values by preincubation of the cells with dichloroacetate or by treating cell extracts with calcium. RESULTS: These two individuals were found to be homozygous for a 3-bp deletion in the coding sequence of the PDP isoform 1 (PDP1), which removes the amino acid residue leucine from position 213 of the protein. A recombinant version of this protein was synthesized and found to have a very reduced (<5%) ability to activate purified PDHc. Reduced steady-state levels of PDP1 in the patient's fibroblasts coupled with the low catalytic activity of the mutant PDP1 resulted in native PDHc activity being reduced, but this could be corrected by the addition of recombinant PDP1 (wild type). CONCLUSION: We have identified mutations in PDP1 in two brothers with PDP deficiency and have proven that the mutation is disease-causing. This is the first demonstration of human disease due to a mutation in PDP1.

Amino Acid Sequence↗

PubMed2005

A novel mutation in the dihydrolipoamide dehydrogenase E3 subunit gene (DLD) resulting in an atypical form of alpha-ketoglutarate dehydrogenase deficiency.

The alpha-ketoglutarate dehydrogenase complex (KGDC) catalyses the decarboxylation of alpha-ketoglutarate into succinyl-coenzyme A in the Krebs cycle. This enzymatic complex is made up of three subunits (E1, encoded by PDHA1; E2, encoded by DLST; and E3, encoded by DLD). The E3 subunit is common to two other enzymatic complexes, namely pyruvate dehydrogenase complex (PDC) and branched-chain ketoacid dehydrogenase complex (BCKDC). KGDC deficiency is a rare autosomal recessive disorder, most often presenting with severe encephalopathy and hyperlactatemia with neonatal onset. We found a KGDC deficiency in cultured skin fibroblasts from three siblings born to consanguinous parents. E3 subunit activity was shown to be deficient (20% of control values), despite the absence of usual clinical clues to E3 deficiency, i.e. accumulation of pyruvate and branched-chain amino acids in plasma and branched-chain alpha-ketoacids in urine. RT-PCR of E3 mRNA from the three patients, followed by sequencing, revealed an homozygous c.1444A>G substitution located in E3 exon 13, predictive of a p.R482G (or R447G in the processed gene product) substitution in a highly conserved domain of the protein. Only eleven E3 mutations have been reported so far. The only other case of E3 deficiency without clinical or biochemical evidences of PDC and BCKDC deficiencies has been ascribed to a c.1436A>T (p.D479V; or D444V in the processed gene product) mutation, very close to the mutation reported herein. Since c.1444A>G (p.R482G; or R447G in the processed gene product) and c.1436A>T (p.D479V; or D444V in the processed gene product) lie within the interface domain of E3 with E2 (KGDC and BCKDC) or the E3-binding protein (PDC), our data suggest that interaction of E3 with these other subunits differs in some extent among KGDC, PDC, and BCKDC.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗

PubMed2005

Gene-expression analysis after alcohol exposure in the developing mouse.

Exposure to alcohol in the embryonic mouse can lead to structural and neurophysiologic changes. The cause of these changes is poorly understood, but they are likely the result of numerous mechanisms. Here we investigate ethanol-induced alterations in gene expression in the fetal brain. Using complementary-DNA microarrays, we identified 25 genes that were down-regulated by prenatal ethanol exposure on days 7 and 9 of gestation. None were found to be up-regulated. Of those that were repressed, 6 (Timp4, Bmp15, Rnf25, Akt1, Tulp4, Dexras1) have been identified, and they are discussed here in the context of the developing fetus. The identified genes have been shown to be involved in cell proliferation, differentiation, and apoptosis, and they contribute to tissue growth and remodeling, as well as neuronal growth and survival. Microarray studies may be useful in the identification of a genetic marker for fetal alcohol syndrome, the discovery of novel pathways that may be involved in its origin, or both.

Animals↗

PubMed2005

Enzyme hyperactivity in AOT water-in-oil microemulsions is induced by 'lone' sodium counterions in the water-pool.

Water-in-oil microemulsions are thermodynamically stable single-phase dispersions of water and surfactant within a continuous oil phase. The classical ternary system, based on the surfactant sodium bis(2-ethylhexyl)sulfosuccinate ('AOT'), water and an alkane such as n-heptane, is an optically transparent monodispersion of spherical water-droplets coated with a close-packed surfactant monolayer and the droplet radius is, to a good first approximation, directly proportional to the molar water: surfactant ratio, R. Enzymes dissolved in the water droplets retain activity and stability. These systems have attracted interest as media for biotransformations. Principally based upon studies in AOT-stabilized w/o microemulsions, a peculiar feature of the kinetics of enzyme-catalyzed reactions has long been apparent: the reaction rate characteristically increases from around zero at R=3, through a maximum, in the range R= 10-20, and thereafter decreases again, so that plots of rate vs. R are characteristically 'bell-shaped'. Furthermore, at optimal R, enzymes seem to be 'hyperactive', i.e., they are more active, by a modest but significant factor of 2-3-fold, than in aqueous solution. In this paper we propose the hypothesis that this kind of R-dependence arises because of the presence of freely mobile lone surfactant counterions (Na+) within the water-pool. These ions have no charge partners within the water pool and consequently have a high electrochemical potential. According to our model, lone counterions facilitate the hydrolysis of ester or amide substrates, for example, by stabilizing the tetrahedral intermediate formed during the reaction through ion-pairing with the carbonyl oxygen of the substrate, thus facilitating transfer of negative charge from the carbonyl carbon as it is attacked by the incoming nucleophile. An expression for the relationship between the concentration of free counterions in the water-pool and the compositional parameter R leads directly, through Debye-Hückel theory, to an expression for the relationship between the reaction rate and R, log k(R)= log k(o) + C(1/R)1/2 where k(R) is the rate constant at some finite R, k(o) is the rate constant extrapolated to R = infinity and C is an R-independent coefficient. For enzymes that display bell-shaped kinetics, such as bovine alpha-chymotrypsin and Chromobacterium viscosum lipase, the descending part of the plot (i.e. from optimal R to high R) obeys this equation very well. Inspection of the above equation shows that the rate constant, k(R) is greater than k(o). Furthermore it is reasonable to equate k(o) with k(aq), the aqueous solution value of k since the condition R = infinity may be equated with the condition of infinite dilution with respect to counterions, so eliminating their specific effect on the kinetics. It follows from the inequality, k(R) > k(o) approximately equal to k(aq), that the enzyme is 'hyperactive' in the microemulsion compared with aqueous solution. We show that this is indeed the case for the chymotrypsin-catalyzed hydrolysis of N-trans-cinnamoylimidazole and the lipase catalyzed hydrolysis of 4-nitrophenyl acetate. The tailing off of enzyme activity at low R (< 10) is most likely due to conformational immobilization, probably due to partial dehydration in these low-water preparations (water activity, a(w), drops off rapidly below R = 15). We show that the reaction of glycylglycine with 4-nitrophenyl acetate, a 'hyperactive' non-enzymic reaction, does not suffer from this effect and obeys the above equation across the whole range of R.

Arylsulfonates↗

PubMed2004

Deficiency of pyruvate dehydrogenase caused by novel and known mutations in the E1alpha subunit.

Pyruvate dehydrogenase (PDH)-complex deficiency (OMIM 312170) is a clinically heterogeneous disorder, with phenotypes ranging from fatal lactic acidosis (LA) in the newborn to chronic neurological dysfunction. To date, over 80 different mutations have been identified in the PDHA1 gene in patients with PDH complex deficiency, which are thus thought to contribute to the PDH deficient phenotype. We have identified 14 additional patients with total PDH complex deficiency, all of whom were found to contain mutations within the PDHA1 gene (E(1)alpha subunit). The mutations include both missense mutations and duplications. Eight of these patients had novel mutations, and the remaining had mutations that have been identified previously in PDH complex deficient patients, with residual fibroblast activity ranging from 2.4 to 69% of control values. The nature of these mutations illustrates the variability in phenotype for a given gene defect, with intermittent ataxia being the mildest presentation, Leigh syndrome being the most common and severe neonatal LA the most severe.

Adult↗

PubMed2004

Respiratory chain analysis of skin fibroblasts in mitochondrial disease.

NADH:ubiquinone dehydrogenase (complex I) deficiency can be diagnosed from cultured skin fibroblasts using a number of methods, the most commonly used is a linked assay of rotenone-sensitive complex I + III activity (NADH:cytochrome c reductase). Because of interference from diaphorases, this method requires either the isolation of mitochondria (or at least partial purification). For a suitable mitochondrial preparation from skin fibroblasts, this requires the culturing of 4-20 individual 100mm tissue culture plates, depending on the purity of preparation required. These assays are therefore time-consuming, and do not assist in a rapid diagnosis. There is also no clear demarkation between the normal range of activity and the deficient range since mild mutations can produce only partial decreases in complex I activity. Equally, assaying patient cells that do not have a specific deficiency may prove to be time-wasting in the process of providing a quick, definitive clinical diagnosis. The lactate/pyruvate ratio of fibroblasts has been used to indicate the extent of respiratory chain involvement, as cells with a metabolic defect usually produce more lactate with an increased ratio from 25:1 to much higher values [Methods Enzymol. 264 (1996) 454]. This measurement may not always be conclusive, as the values can fluctuate as a result of culture medium, cell passage number, cell number and viability. In this report, we evaluate the use of pyruvate oxidation measurements from whole cells prepared from a single plate of cultured fibroblasts as an alternative to lactate/pyruvate ratios, or other methods both direct and indirect as indicators of the extent of respiratory chain involvement and the possibility of a defect within complex I. Whole cell 2-14C pyruvate oxidation appears to indicate the presence of a complex I defect in patients compared to normal controls more reliably than L/P ratios, but this has some puzzling exceptions.

Journal Article↗

PubMed2004

The role of the LRPPRC (leucine-rich pentatricopeptide repeat cassette) gene in cytochrome oxidase assembly: mutation causes lowered levels of COX (cytochrome c oxidase) I and COX III mRNA.

Leigh syndrome French Canadian (LSFC) is a variant of cytochrome oxidase deficiency found in Québec and caused by mutations in the LRPPRC (leucine-rich pentatricopeptide repeat cassette) gene. Northern blots showed that the LRPPRC mRNA levels seen in skeletal muscle>heart>placenta>kidney>liver>lung=brain were proportionally almost opposite in strength to the severity of the enzymic cytochrome oxidase defect. The levels of COX (cytochrome c oxidase) I and COX III mRNA visible on Northern blots were reduced in LSFC patients due to the common (A354V, Ala354-->Val) founder mutation. The amount of LRPPRC protein found in both fibroblast and liver mitochondria from LSFC patients was consistently reduced to <30% of control levels. Import of [(35)S]methionine LRPPRC into rat liver mitochondria was slower for the mutant (A354V) protein. A titre of LRPPRC protein was also found in nuclear fractions that could not be easily accounted for by mitochondrial contamination. [35S]Methionine labelling of mitochondrial translation products showed that the translation of COX I, and perhaps COX III, was specifically reduced in the presence of the mutation. These results suggest that the gene product of LRPPRC, like PET 309p, has a role in the translation or stability of the mRNA for mitochondrially encoded COX subunits. A more diffuse distribution of LRPPRC in LSFC cells compared with controls was evident when viewed by immunofluorescence microscopy, with less LRPPRC present in peripheral mitochondria.

Autopsy↗

PubMed2004

Clinical variability in maternally inherited leber hereditary optic neuropathy with the G14459A mutation.

Spasticity and dystonia have been associated with mitochondrial (mt) DNA mutations at A11696G, G14459A, and T14596A. We describe the clinical features and molecular analysis of two Caucasian pedigrees with the 14,459 guanosine (G) --> adenine (A) transition. The maternally inherited Leber hereditary optic neuropathy (LHON) phenotypes showed extreme clinical variability and the only screening test that was abnormal in the patient with spasticity/dystonia was a high T2 signal in the putamen bilaterally. The male patient in the second pedigree showed features of optic neuropathy without spasticity/dystonia. These results further support that the 14,459 G --> A transition mutation is causally related to LHON and spasticity/dystonia.

Adolescent↗

PubMed2004

Altered mitochondrial structure and motion dynamics in living cells with energy metabolism defects revealed by real time microscope imaging.

Using the real time microscope (RTM), a system applying new developments in light microscopy, we documented the spatial and temporal dynamics of mitochondrial behavior in human cultured skin fibroblasts. Without the use of stains or probes, we resolved fibroblast mitochondria as dark slender filaments of approximately 0.2 m wide and up to 10 m long, as well as a few smaller ovoid forms. In the living cell, the three most common mitochondrial movements were: (1) small oscillatory movements; (2) larger movements including filament extension, retraction, and branching as well as combinations of these actions; and (3) whole transit movements of single mitochondrial filaments. Skin fibroblasts from patients with mitochondrial complex I deficiency and normal fibroblasts during incubation with rotenone, or antimycin A, contained higher proportions of mitochondria in the swollen filamentous forms, nodal filaments, and ovoid forms rather than the slender filamentous forms in normal cells. Interestingly, decreased motility was observed with the more ovoid mitochondrial forms compared to the filamentous forms. We conclude that mitochondrial morphology and dynamic motion are strongly associated with changes in mitochondrial energy metabolism. Images documenting our observations are presented both at single time points and as QuickTime videos.

Cell Line↗

PubMed2004

Regulation of NADH/CoQ oxidoreductase: do phosphorylation events affect activity?

We had previously suggested that phosphorylation of proteins by mitochondrial kinases regulate the activity of NADH/CoQ oxidoreductase. Initial data showed that pyruvate dehydrogenase kinase (PDK) and cAMP-dependent protein kinase A (PKA) phosphorylate mitochondrial membrane proteins. Upon phosphorylation with crude PDK, mitochondria appeared to be deficient in NADH/cytochrome c reductase activity associated with increased superoxide production. Conversely, phosphorylation by PKA resulted in increased NADH/cytochrome c reductase activity and decreased superoxide formation. Current data confirms PKA involvement in regulating Complex I activity through phosphorylation of an 18 kDa subunit. Beef heart NADH/ cytochrome c reductase activity increases to 150% of control upon incubation with PKA and ATP-gamma-S. We have cloned the four human isoforms of PDK and purified beef heart Complex I. Incubation of mitochondria with PDK isoforms and ATP did not alter Complex I activity or superoxide production. Radiolabeling of mitochondria and purified Complex I with PDK failed to reveal phosphorylated proteins.

Adenosine Triphosphate↗

PubMed2004

Slc25a13-knockout mice harbor metabolic deficits but fail to display hallmarks of adult-onset type II citrullinemia.

Adult-onset type II citrullinemia (CTLN2) is an autosomal recessive disease caused by mutations in SLC25A13, the gene encoding the mitochondrial aspartate/glutamate carrier citrin. The absence of citrin leads to a liver-specific, quantitative decrease of argininosuccinate synthetase (ASS), causing hyperammonemia and citrullinemia. To investigate the physiological role of citrin and the development of CTLN2, an Slc25a13-knockout (also known as Ctrn-deficient) mouse model was created. The resulting Ctrn-/- mice were devoid of Slc25a13 mRNA and citrin protein. Liver mitochondrial assays revealed markedly decreased activities in aspartate transport and the malate-aspartate shuttle. Liver perfusion also demonstrated deficits in ureogenesis from ammonia, gluconeogenesis from lactate, and an increase in the lactate-to-pyruvate ratio within hepatocytes. Surprisingly, Ctrn-/- mice up to 1 year of age failed to show CTLN2-like symptoms due to normal hepatic ASS activity. Serological measures of glucose, amino acid, and ammonia metabolism also showed no significant alterations. Nitrogen-loading treatments produced only minor changes in the hepatic ammonia and amino acid levels. These results suggest that citrin deficiency alone may not be sufficient to produce a CTLN2-like phenotype in mice. These observations are compatible, however, with the variable age of onset, incomplete penetrance, and strong ethnic bias seen in CTLN2 where additional environmental and/or genetic triggers are now suspected.

Amino Acids↗