d-Glyceric aciduria does not cause nonketotic hyperglycinemia: A historic co-occurrence.

Historically, d-glyceric aciduria was thought to cause an uncharacterized blockage to the glycine cleavage enzyme system (GCS) causing nonketotic hyperglycinemia (NKH) as a secondary phenomenon. This inference was reached based on the clinical and biochemical results from the first d-glyceric aciduria patient reported in 1974. Along with elevated glyceric acid excretion, this patient exhibited severe neurological symptoms of myoclonic epilepsy and absent development, and had elevated glycine levels and decreased glycine cleavage system enzyme activity. Mutations in the GLYCTK gene (encoding d-glycerate kinase) causing glyceric aciduria were previously noted. Since glycine changes were not observed in almost all of the subsequently reported cases of d-glyceric aciduria, this theory of NKH as a secondary syndrome of d-glyceric aciduria was revisited in this work. We showed that this historic patient harbored a homozygous missense mutation in AMT c.350C>T, p.Ser117Leu, and enzymatic assay of the expressed mutation confirmed the pathogeneity of the p.Ser117Leu mutation. We conclude that the original d-glyceric aciduria patient also had classic NKH and that this co-occurrence of two inborn errors of metabolism explains the original presentation. We conclude that no evidence remains that d-glyceric aciduria would cause NKH.

Thirteen new patients with guanidinoacetate methyltransferase deficiency and functional characterization of nineteen novel missense variants in the GAMT gene.

Guanidinoacetate methyltransferase deficiency (GAMT-D) is an autosomal recessively inherited disorder of creatine biosynthesis. Creatine deficiency on cranial proton magnetic resonance spectroscopy, and elevated guanidinoacetate levels in body fluids are the biomarkers of GAMT-D. In 74 patients, 50 different mutations in the GAMT gene have been identified with missense variants being the most common. Clinical and biochemical features of the patients with missense variants were obtained from their physicians using a questionnaire. In 20 patients, 17 missense variants, 25% had a severe, 55% a moderate, and 20% a mild phenotype. The effect of these variants on GAMT enzyme activity was overexpressed using primary GAMT-D fibroblasts: 17 variants retained no significant activity and are therefore considered pathogenic. Two additional variants, c.22C>A (p.Pro8Thr) and c.79T>C (p.Tyr27His) (the latter detected in control cohorts) are in fact not pathogenic as these alleles restored GAMT enzyme activity, although both were predicted to be possibly damaging by in silico analysis. We report 13 new patients with GAMT-D, six novel mutations and functional analysis of 19 missense variants, all being included in our public LOVD database. Our functional assay is important for the confirmation of the pathogenicity of identified missense variants in the GAMT gene.

Novel cases of D-2-hydroxyglutaric aciduria with IDH1 or IDH2 mosaic mutations identified by amplicon deep sequencing.

BACKGROUND: Mosaic IDH1 mutations are described as the cause of metaphyseal chondromatosis with increased urinary excretion of D-2-hydroxyglutarate (MC-HGA), and mutations in IDH2 as the cause of D-2-hydroxyglutaric aciduria (D-2HGA) type II. Mosaicism for IDH2 mutations has not previously been reported as a cause of D-2HGA. Here we describe three cases: one MC-HGA case with IDH1 mosaic mutations, and two D-2HGA type II cases. In one D-2HGA case we identified mosaicism for an IDH2 mutation as the genetic cause of this disorder; the other D-2HGA case was caused by a heterozygous IDH2 mutation, while the unaffected mother was a mosaic carrier. METHODS: We performed amplicon deep sequencing using the 454 GS Junior platform, next to Sanger sequencing, to identify and confirm mosaicism of IDH1 or IDH2 mutations in MC-HGA or D-2HGA, respectively. RESULTS AND CONCLUSIONS: We identified different mutant allele percentages in DNA samples derived from different tissues (blood vs fibroblasts). Furthermore, we found that mutant allele percentages of IDH1 decreased after more passages had occurred in fibroblast cell cultures. We describe a method for the detection and validation of mosaic mutations in IDH1 and IDH2, making quantification with laborious cloning techniques obsolete.

A structural mapping of mutations causing succinyl-CoA:3-ketoacid CoA transferase (SCOT) deficiency.

Succinyl-CoA:3-ketoacid CoA transferase (SCOT) deficiency is a rare inherited metabolic disorder of ketone metabolism, characterized by ketoacidotic episodes and often permanent ketosis. To date there are ~20 disease-associated alleles on the OXCT1 gene that encodes the mitochondrial enzyme SCOT. SCOT catalyzes the first, rate-limiting step of ketone body utilization in peripheral tissues, by transferring a CoA moiety from succinyl-CoA to form acetoacetyl-CoA, for entry into the tricarboxylic acid cycle for energy production. We have determined the crystal structure of human SCOT, providing a molecular understanding of the reported mutations based on their potential structural effects. An interactive version of this manuscript (which may contain additional mutations appended after acceptance of this manuscript) may be found on the web address: http://www.thesgc.org/jimd/SCOT .

The molecular basis of aminoacylase 1 deficiency.

Aminoacylase 1 is a zinc-binding enzyme which hydrolyzes N-acetyl amino acids into the free amino acid and acetic acid. Deficiency of aminoacylase 1 due to mutations in the aminoacylase 1 (ACY1) gene follows an autosomal-recessive trait of inheritance and is characterized by accumulation of N-acetyl amino acids in the urine. In affected individuals neurological findings such as febrile seizures, delay of psychomotor development and moderate mental retardation have been reported. Except for one missense mutation which has been studied in Escherichia coli, mutations underlying aminoacylase 1 deficiency have not been characterized so far. This has prompted us to approach expression studies of all mutations known to occur in aminoacylase 1 deficient individuals in a human cell line (HEK293), thus providing the authentic human machinery for posttranslational modifications. Mutations were inserted using site directed mutagenesis and aminoacylase 1 enzyme activity was assessed in cells overexpressing aminoacylase 1, using mainly the natural high affinity substrate N-acetyl methionine. Overexpression of the wild type enzyme in HEK293 cells resulted in an approximately 50-fold increase of the aminoacylase 1 activity of homogenized cells. Most mutations resulted in a nearly complete loss of enzyme function. Notably, the two newly discovered mutations p.Arg378Trp, p.Arg378Gln and the mutation p.Arg393His yielded considerable residual activity of the enzyme, which is tentatively explained by their intramolecular localization and molecular characteristics. In contrast to aminoacylase 1 variants which showed no detectable aminoacylase 1 activity, aminoacylase 1 proteins with the mutations p.Arg378Trp, p.Arg378Gln and p.Arg393His were also detected in Western blot analysis. Investigations of the molecular bases of additional cases of aminoacylase 1 deficiency contribute to a better understanding of this inborn error of metabolism whose clinical significance and long-term consequences remain to be elucidated.

MCAD deficiency in Denmark.

Medium-chain acyl-CoA dehydrogenase deficiency (MCADD) is the most common defect of fatty acid oxidation. Many countries have introduced newborn screening for MCADD, because characteristic acylcarnitines can easily be identified in filter paper blood spot samples by tandem mass spectrometry (MS/MS), because MCADD is a frequent disease, and because of the success of early treatment initiated before clinical symptoms have emerged. In Denmark we have screened 519,350 newborns for MCADD by MS/MS and identified 58 affected babies. The diagnosis of MCADD was confirmed in all 58 newborns by mutation analysis. This gives an incidence of MCADD detected by newborn screening in Denmark of 1/8954. In sharp contrast to this we found that the incidence of clinically presenting MCADD in Denmark in the 10 year period preceding introduction of MS/MS-based screening was only 1 in 39,691. This means that four times more newborns with MCADD are detected by screening than what is expected based on the number of children presenting clinically in an unscreened population. The mutation spectrum in the newborns detected by screening is different from that observed in clinically presenting patients with a much lower proportion of newborns being homozygous for the prevalent disease-causing c.985A>G mutation. A significant number of the newborns have genotypes with mutations that have not been observed in patients detected clinically. Some of these mutations, like c.199T>C and c.127G>A, are always associated with a milder biochemical phenotype and may cause a milder form of MCADD with a relatively low risk of disease manifestation, thereby explaining part of the discrepancy between the frequency of clinically manifested MCADD and the frequency of MCADD determined by screening. In addition, our data suggest that some of this discrepancy can be explained by a reduced penetrance of the c.985A>G mutation, with perhaps only 50% of c.985A>G homozygotes presenting with disease manifestations. Interestingly, we also report that the observed number of newborns identified by screening who are homozygous for the c.985A>G mutation is twice that predicted from the estimated carrier frequency. We therefore redetermined the carrier frequency in a new sample of 1946 blood spots using a new assay, but this only confirmed that the c.985A>G carrier frequency in Denmark is approximately 1/105. We conclude that MCADD is much more frequent than expected, has a reduced penetrance and that rapid genotyping using the initial blood spot sample is important for correct diagnosis and counseling.

Biochemical screening of 504,049 newborns in Denmark, the Faroe Islands and Greenland--experience and development of a routine program for expanded newborn screening.

Expanded newborn screening for selected inborn errors of metabolism (IEM) in Denmark, the Faroe Islands and Greenland was introduced in 2002. We now present clinical, biochemical, and statistical results of expanded screening (excluding PKU) of 504,049 newborns during nine years as well as diagnoses and clinical findings in 82,930 unscreened newborns born in the same period. The frequencies of diagnoses made within the panel of disorders screened for are compared with the frequencies of the disorders in the decade preceding expanded newborn screening. The expanded screening was performed as a pilot study during the first seven years, and the experience obtained during these years was used in the development of the routine neonatal screening program introduced in 2009. Methods for screening included tandem mass spectrometry and an assay for determination of biotinidase activity. A total of 310 samples from 504,049 newborns gave positive screening results. Of the 310 results, 114 were true positive, including results from 12 newborns in which the disease in question was subsequently diagnosed in their mothers. Thus, the overall frequency of an IEM in the screening panel was 1:4942 (mothers excluded) or 1:4421 (mothers included). The false positive rate was 0.038% and positive predictive value 37%. Overall specificity was 99.99%. All patients with true positive results were followed in The Center for Inherited Metabolic Disorders in Copenhagen, and the mean follow-up period was 45 months (range 2109 months). There were no deaths among the 102 children, and 94% had no clinically significant sequelae at last follow-up. Our study confirms the higher frequency of selected IEM after implementation of expanded newborn screening and suggests an improved outcome for several disorders. We argue that newborn screening for these disorders should be standard of care, though unresolved issues remain, e.g. about newborns with a potential for remaining asymptomatic throughout life. Well organized logistics of the screening program from screening laboratory to centralized, clinical management is important.

Heterozygosity for an in-frame deletion causes glutaryl-CoA dehydrogenase deficiency in a patient detected by newborn screening: investigation of the effect of the mutant allele.

A patient with suspected glutaric aciduria type 1 (GA-1) was detected by newborn screening. GA-1 is known as an autosomal recessively inherited disease due to defects in the gene coding for glutaryl-CoA dehydrogenase (GCDH), a mitochondrial enzyme involved in the catabolism of the amino acids hydroxylysine, lysine and tryptophan. DNA and cDNA sequencing revealed a 18 bp deletion (c.553_570del18) resulting in deletion of six amino acids (p.Gly185_Ser190del) in one allele and no sequence changes in the other allele. Confirmatory biochemical analysis of blood, urine and cultured fibroblasts from the proband were consistent with a mild biochemical GA-1 phenotype. Recombinant expression of the mutant variant in E. coli showed that the GCDH-(p.Gly185_Ser190del) protein displayed severely decreased assembly into tetramers and enzyme activity. To discover a potential dominant negative effect of the mutant protein, we engineered a prokaryotic expression system in which expression of a wild type and a mutant GCDH allele is controlled by separately inducible promoters. These cells displayed decreased levels of GCDH tetramer and enzyme activity when expressing both the wild type and the mutant GCDH variant protein compared to the situation when only the wild type allele was expressed. Further experiments suggest that the major impact of the GCDH-(p.Gly185_Ser190del) protein in heterozygous cells consists of hampering the assembly of wild type GCDH into tetramers. Our experimental data are consistent with the hypothesis that heterozygosity for this mutation confers a dominant negative effect resulting in a GCDH enzyme activity that is significantly lower than the expected 50%.

Diagnosis of glutaric aciduria type 1 by measuring 3-hydroxyglutaric acid in dried urine spots by liquid chromatography tandem mass spectrometry.

Accumulation of glutaric acid (GA) and 3-hydroxyglutaric acid (3HGA) in body fluids is the biochemical hallmark of type 1 glutaric aciduria (GA1), a disorder characterized by acute striatal degeneration and a subsequent dystonia. To date, methods for quantification of 3HGA are mainly based on stable isotope dilution gas chromatography mass spectrometry (GC-MS) and require extensive sample preparation. Here we describe a simple liquid chromatography tandem MS (LC-MS/MS) method to quantify this important metabolite in dried urine spots (DUS). This method is based on derivatization with 4-[2-(N,N-dimethylamino)ethylaminosulfonyl]-7-(2-aminoethylamino)-2,1,3-benzoxadiazole (DAABD-AE). Derivatization was adopted to improve the chromatographic and mass spectrometric properties of the studied analytes. Derivatization was performed directly on a 3.2-mm disc of DUS as a sample without extraction. Sample mixture was heated at 60°C for 45 min, and 5 µl of the reaction solution was analyzed by LC-MS/MS. Reference ranges obtained were in excellent agreement with the literature. The method was applied retrospectively for the analysis of DUS samples from established low- and high-excreter GA1 patients as well as controls (n = 100). Comparison of results obtained versus those obtained by GC-MS was satisfactory (n = 14). In populations with a high risk of GA1, this approach will be useful as a primary screening method for high- or low-excreter variants. In these populations, however, DUS analysis should not be implemented before completing a parallel comparative study with the standard screening method (i.e., molecular testing). In addition, follow-up DUS GA and 3HGA testing of babies with elevated dried blood spot C5DC acylcarnitines will be useful as a first-tier diagnostic test, thus reducing the number of cases requiring enzymatic and molecular analyses to establish or refute the diagnosis of GA1.

Diagnosis and management of glutaric aciduria type I--revised recommendations.

Glutaric aciduria type I (synonym, glutaric acidemia type I) is a rare organic aciduria. Untreated patients characteristically develop dystonia during infancy resulting in a high morbidity and mortality. The neuropathological correlate is striatal injury which results from encephalopathic crises precipitated by infectious diseases, immunizations and surgery during a finite period of brain development, or develops insidiously without clinically apparent crises. Glutaric aciduria type I is caused by inherited deficiency of glutaryl-CoA dehydrogenase which is involved in the catabolic pathways of L-lysine, L-hydroxylysine and L-tryptophan. This defect gives rise to elevated glutaric acid, 3-hydroxyglutaric acid, glutaconic acid, and glutarylcarnitine which can be detected by gas chromatography/mass spectrometry (organic acids) or tandem mass spectrometry (acylcarnitines). Glutaric aciduria type I is included in the panel of diseases that are identified by expanded newborn screening in some countries. It has been shown that in the majority of neonatally diagnosed patients striatal injury can be prevented by combined metabolic treatment. Metabolic treatment that includes a low lysine diet, carnitine supplementation and intensified emergency treatment during acute episodes of intercurrent illness should be introduced and monitored by an experienced interdisciplinary team. However, initiation of treatment after the onset of symptoms is generally not effective in preventing permanent damage. Secondary dystonia is often difficult to treat, and the efficacy of available drugs cannot be predicted precisely in individual patients. The major aim of this revision is to re-evaluate the previous diagnostic and therapeutic recommendations for patients with this disease and incorporate new research findings into the guideline.

Genotypic and phenotypic spectrum of pyridoxine-dependent epilepsy (ALDH7A1 deficiency).

Pyridoxine-dependent epilepsy was recently shown to be due to mutations in the ALDH7A1 gene, which encodes antiquitin, an enzyme that catalyses the nicotinamide adenine dinucleotide-dependent dehydrogenation of l-alpha-aminoadipic semialdehyde/L-Delta1-piperideine 6-carboxylate. However, whilst this is a highly treatable disorder, there is general uncertainty about when to consider this diagnosis and how to test for it. This study aimed to evaluate the use of measurement of urine L-alpha-aminoadipic semialdehyde/creatinine ratio and mutation analysis of ALDH7A1 (antiquitin) in investigation of patients with suspected or clinically proven pyridoxine-dependent epilepsy and to characterize further the phenotypic spectrum of antiquitin deficiency. Urinary L-alpha-aminoadipic semialdehyde concentration was determined by liquid chromatography tandem mass spectrometry. When this was above the normal range, DNA sequencing of the ALDH7A1 gene was performed. Clinicians were asked to complete questionnaires on clinical, biochemical, magnetic resonance imaging and electroencephalography features of patients. The clinical spectrum of antiquitin deficiency extended from ventriculomegaly detected on foetal ultrasound, through abnormal foetal movements and a multisystem neonatal disorder, to the onset of seizures and autistic features after the first year of life. Our relatively large series suggested that clinical diagnosis of pyridoxine dependent epilepsy can be challenging because: (i) there may be some response to antiepileptic drugs; (ii) in infants with multisystem pathology, the response to pyridoxine may not be instant and obvious; and (iii) structural brain abnormalities may co-exist and be considered sufficient cause of epilepsy, whereas the fits may be a consequence of antiquitin deficiency and are then responsive to pyridoxine. These findings support the use of biochemical and DNA tests for antiquitin deficiency and a clinical trial of pyridoxine in infants and children with epilepsy across a broad range of clinical scenarios.

D-glyceric aciduria is caused by genetic deficiency of D-glycerate kinase (GLYCTK).

D-glyceric aciduria is a rare inborn error of serine and fructose metabolism that was first described in 1974. Most affected individuals have presented with neurological symptoms. The molecular basis of D-glyceric aciduria is largely unknown; possible causes that have been discussed are deficiencies of D-glycerate dehydrogenase, triokinase, and D-glycerate kinase. In 1989, van Schaftingen has reported decreased D-glycerate kinase activity in the liver of a single patient with D-glyceric aciduria. However, this analysis has not been performed in other affected individuals, and the underlying defect has remained unknown on the gene level until now. We report three patients with deficiency of D-glycerate kinase. They are of Serbian, Mexican, and Turkish origin and include the patient initially reported in 1974. All had homozygous mutations in exon 5 of the GLYCTK gene encoding D-glycerate kinase: c.1448delT (p.Phe483SerfsX2), c.1478T>G (p.Phe493Cys), or c.1558delC (p.Leu520CysfsX108). Transient overexpression of the variant GLYCTK genes in HEK293 cells clearly showed loss of enzyme activity and immunoreactivity when compared to the reference enzyme. Our work has revealed mutations in the GLYCTK gene as the cause of D-glycerate kinase deficiency and D-glyceric aciduria and provides a noninvasive approach for further diagnostic workup and research.

Evidence for genetic heterogeneity in D-2-hydroxyglutaric aciduria.

We performed molecular, enzyme, and metabolic studies in 50 patients with D-2-hydroxyglutaric aciduria (D-2-HGA) who accumulated D-2-hydroxyglutarate (D-2-HG) in physiological fluids. Presumed pathogenic mutations were detected in 24 of 50 patients in the D-2-hydroxyglutarate dehydrogenase (D2HGDH) gene, which encodes D-2-hydroxyglutarate dehydrogenase (D-2-HGDH). Enzyme assay of D-2-HGDH confirmed that all patients with mutations had impaired enzyme activity, whereas patients with D-2-HGA whose enzyme activity was normal did not have mutations. Significantly lower D-2-HG concentrations in body fluids were observed in mutation-positive D-2-HGA patients than in mutation-negative patients. These results imply that multiple genetic loci may be associated with hyperexcretion of D-2-HG. Accordingly, we suggest a new classification: D-2-HGA Type I associates with D-2-HGDH deficiency, whereas idiopathic D-2-HGA manifests with normal D-2-HGDH activity and higher D-2-HG levels in body fluids compared with Type I patients. It remains possible that several classifications for idiopathic D-2-HGA patients with diverse genetic loci will be revealed in future studies.

Disease-causing missense mutations affect enzymatic activity, stability and oligomerization of glutaryl-CoA dehydrogenase (GCDH).

Glutaric aciduria type 1 (GA1) is an autosomal recessive neurometabolic disorder caused by mutations in the glutaryl-CoA dehydrogenase gene (GCDH), leading to an accumulation and high excretion of glutaric acid and 3-hydroxyglutaric acid. Considerable variation in severity of the clinical phenotype is observed with no correlation to the genotype. We report here for the first time on expression studies of four missense mutations c.412A > G (p.Arg138Gly), c.787A > G (p.Met263Val), c.1204C > T (p.Arg402Trp) and c.1240G > A (p.Glu414Lys) identified in GA1 patients in mammalian cells. Biochemical analyses revealed that all mutants were enzymatically inactive with the exception of p.Met263Val which showed 10% activity of the expressed wild-type enzyme. Western blot and pulse-chase analyses demonstrated that the amount of expressed p.Arg402Trp protein was significantly reduced compared with cells expressing wild-type protein which was due to rapid intramitochondrial degradation. Upon cross-linkage the formation of homotetrameric GCDH was strongly impaired in p.Met263Val and p.Arg402Trp mutants. In addition, GCDH appears to interact with distinct heterologous polypeptides to form novel 97, 130 and 200 kDa GCDH complexes. Molecular modeling of mutant GCDH suggests that Met263 at the surface of the GCDH protein might be part of the contact interface to interacting proteins. These results indicate that reduced intramitochondrial stability as well as the impaired formation of homo- and heteromeric GCDH complexes can underlie GA1.

Clinical and biochemical monitoring of patients with fatty acid oxidation disorders.

Evidence-based guidelines for monitoring patients with disorders in fatty acid oxidation (FAO) are lacking, and most protocols are based on expert statements. Here, we describe our protocol for Danish patients. Clinical monitoring is the most important measure and has the main aims of checking growth, development and diet and of bringing families to the clinic regularly to remind them of their child's risk and review how they cope and adjust, e.g. to an acute intercurrent illness. Most of these measures are simple and can be carried out during a routine out-patient visit; we seldom do more complicated assessments by a neuropsychologist, speech therapist, or physical and occupational therapists. Paraclinical measurements are not used for short-chain and medium-chain disorders; electrocardiography (including 24 h monitoring) and echocardiography are done for most patients with long-chain and carnitine transporter deficiencies. Eye examination is done in all, and liver ultrasonography in some patients with long-chain 3-hydroxyacyl-coenzyme A dehydrogenase/tri-functional protein (LCHAD/TFP) deficiencies. Biochemical follow-up includes determination of free carnitine and acylcarnitines. Free carnitine is measured to monitor carnitine supplementation in patients with multiple acyl-coenzyme A dehydrogenase deficiency (MADD) and carnitine transporter deficiency (CTD) and to follow metabolic control and disclose deficiency states in other FAO disorders. We are evaluating long-chain acylcarnitines in patients with long-chain disorders; so far there does not seem to be any clear-cut benefit in following these levels. An erythrocyte fatty acid profile is done in patients with long-chain disorders to test for essential fatty acid and docosahexanoic acid (DHA) deficiencies. The measurement of creatine kinase is helpful in long-chain disorders. Ongoing follow-up and education of the patient is important throughout life to prevent disease morbidity or death from metabolic crises.

A novel missense mutation in SUCLG1 associated with mitochondrial DNA depletion, encephalomyopathic form, with methylmalonic aciduria.

Mitochondrial DNA depletion, encephalomyopathic form, with methylmalonic aciduria is associated with mutations in SUCLA2, the gene encoding a beta subunit of succinate-CoA ligase, where 17 patients have been reported. Mutations in SUCLG1, encoding the alpha subunit of the enzyme, have been reported in only one family, where a homozygous 2 bp deletion was associated with fatal infantile lactic acidosis. We here report a patient with a novel homozygous missense mutation in SUCLG1, whose phenotype is similar to that of patients with SUCLA2 mutations.

Prolidase deficiency: it looks like systemic lupus erythematosus but it is not.

Three siblings with recalcitrant leg ulceration, splenomegaly, photosensitive rash, and autoantibodies were suspected of having prolidase deficiency. Urine was checked for iminodipeptiduria, fibroblasts were cultured and analyzed for prolidase activity, and DNA was extracted for identifying the causative mutation. Glycyl proline was found as the dominant dipeptide in the urine. The activity of proline dipeptidase in fibroblasts was 2.5% of control fibroblasts. Sequence analysis of the PEPD gene revealed a homozygous nonsense C-->G transition at nucleotide 768. In conclusion, prolidase deficiency was diagnosed in siblings with skin ulceration autoantibodies and a lupus-like disease. A novel nonsense mutation was found, associated with the severe outcome of our patients.

The ACADS gene variation spectrum in 114 patients with short-chain acyl-CoA dehydrogenase (SCAD) deficiency is dominated by missense variations leading to protein misfolding at the cellular level.

Short-chain acyl-CoA dehydrogenase (SCAD) deficiency is an inherited disorder of mitochondrial fatty acid oxidation associated with variations in the ACADS gene and variable clinical symptoms. In addition to rare ACADS inactivating variations, two common variations, c.511C > T (p.Arg171Trp) and c.625G > A (p.Gly209Ser), have been identified in patients, but these are also present in up to 14% of normal populations leading to questions of their clinical relevance. The common variant alleles encode proteins with nearly normal enzymatic activity at physiological conditions in vitro. SCAD enzyme function, however, is impaired at increased temperature and the tendency to misfold increases under conditions of cellular stress. The present study examines misfolding of variant SCAD proteins identified in patients with SCAD deficiency. Analysis of the ACADS gene in 114 patients revealed 29 variations, 26 missense, one start codon, and two stop codon variations. In vitro import studies of variant SCAD proteins in isolated mitochondria from SCAD deficient (SCAD-/-) mice demonstrated an increased tendency of the abnormal proteins to misfold and aggregate compared to the wild-type, a phenomenon that often leads to gain-of-function cellular phenotypes. However, no correlation was found between the clinical phenotype and the degree of SCAD dysfunction. We propose that SCAD deficiency should be considered as a disorder of protein folding that can lead to clinical disease in combination with other genetic and environmental factors.

Mitochondrial encephalomyopathy with elevated methylmalonic acid is caused by SUCLA2 mutations.

We have identified 12 patients with autosomal recessive mitochondrial encephalomyopathy with elevated methylmalonic acid. The disorder has a high incidence of 1 in 1700 in the Faroe Islands due to a founder effect, and a carrier frequency of 1 in 33. The symptoms comprise hypotonia, muscle atrophy, hyperkinesia, severe hearing impairment and postnatal growth retardation. Neuroimaging showed demyelination and central and cortical atrophy, including atrophy of the basal ganglia, and some of the patients fulfilled the criteria for Leigh syndrome. Urine and plasma methylmalonic acid were elevated. Homozygosity mapping with the Affymetrix 10 K array revealed a homozygous region on chromosome 13q14 harbouring the SUCLA2 gene. Mutations in SUCLA2 were recently shown to cause a similar disorder in a small Israeli family. Mutation analysis identified a novel splice site mutation in SUCLA2, IVS4 + 1G --> A, leading to skipping of exon 4. The SUCLA2 gene encodes the ATP-forming beta subunit of the Krebs cycle enzyme succinyl-CoA ligase. The hallmark of the condition, elevated methylmalonic acid, can be explained by an accumulation of the substrate of the enzyme, succinyl-CoA, which in turn leads to elevated methylmalonic acid, because the conversion of methylmalonyl-CoA to succinyl-CoA is inhibited.

Treatment of mitochondrial neurogastrointestinal encephalomyopathy with dialysis.

OBJECTIVE: To study the effect of continuous ambulatory peritoneal dialysis on nucleoside levels and clinical course in a patient with mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). Patient We studied a patient with genetically verified MNGIE, who prior to treatment had lost weight progressively, developed amenorrhea, vomited multiple times daily, and had abdominal pain. Intervention The patient was treated with peritoneal dialysis for 3 years, and the effect on symptoms and plasma concentrations of thymidine and deoxyuridine were monitored. RESULTS: Dialysis stopped vomiting and reduced abdominal pain, and the patient gained 5 kg in weight and started to menstruate again. Symptoms returned if dialysis was paused. Dialysis did not affect plasma nucleoside levels. CONCLUSIONS: This study shows an unambiguous clinical benefit of peritoneal dialysis on gastrointestinal symptoms in MNGIE. Dialysis did not affect nucleoside levels, indicating elevated thymidine and deoxyuridine levels are not solely responsible for the pathogenesis of MNGIE.