Thatjana Gardeitchik

Mutations in the phospholipid remodeling gene SERAC1 impair mitochondrial function and intracellular cholesterol trafficking and cause dystonia and deafness.

Using exome sequencing, we identify SERAC1 mutations as the cause of MEGDEL syndrome, a recessive disorder of dystonia and deafness with Leigh-like syndrome, impaired oxidative phosphorylation and 3-methylglutaconic aciduria. We localized SERAC1 at the interface between the mitochondria and the endoplasmic reticulum in the mitochondria-associated membrane fraction that is essential for phospholipid exchange. A phospholipid analysis in patient fibroblasts showed elevated concentrations of phosphatidylglycerol-34:1 (where the species nomenclature denotes the number of carbon atoms in the two acyl chains:number of double bonds in the two acyl groups) and decreased concentrations of phosphatidylglycerol-36:1 species, resulting in an altered cardiolipin subspecies composition. We also detected low concentrations of bis(monoacyl-glycerol)-phosphate, leading to the accumulation of free cholesterol, as shown by abnormal filipin staining. Complementation of patient fibroblasts with wild-type human SERAC1 by lentiviral infection led to a decrease and partial normalization of the mean ratio of phosphatidylglycerol-34:1 to phosphatidylglycerol-36:1. Our data identify SERAC1 as a key player in the phosphatidylglycerol remodeling that is essential for both mitochondrial function and intracellular cholesterol trafficking.

Metabolic cutis laxa syndromes

Cutis laxa is a rare skin disorder characterized by wrinkled, redundant, inelastic and sagging skin due to defective synthesis of elastic fibers and other proteins of the extracellular matrix. Wrinkled, inelastic skin occurs in many cases as an acquired condition. Syndromic forms of cutis laxa, however, are caused by diverse genetic defects, mostly coding for structural extracellular matrix proteins. Surprisingly a number of metabolic disorders have been also found to be associated with inherited cutis laxa. Menkes disease was the first metabolic disease reported with old-looking, wrinkled skin. Cutis laxa has recently been found in patients with abnormal glycosylation. The discovery of the COG7 defect in patients with wrinkled, inelastic skin was the first genetic link with the Congenital Disorders of Glycosylation (CDG). Since then several inborn errors of metabolism with cutis laxa have been described with variable severity. These include P5CS, ATP6V0A2-CDG and PYCR1 defects. In spite of the evolving number of cutis laxa-related diseases a large part of the cases remain genetically unsolved. In metabolic cutis laxa syndromes the clinical and laboratory features might partially overlap, however there are some distinct, discriminative features. In this review on metabolic diseases causing cutis laxa we offer a practical approach for the differential diagnosis of metabolic cutis laxa syndromes.

Further characterization of ATP6V0A2-related autosomal recessive cutis laxa.

Autosomal recessive cutis laxa (ARCL) syndromes are phenotypically overlapping, but genetically heterogeneous disorders. Mutations in the ATP6V0A2 gene were found to underlie both, autosomal recessive cutis laxa type 2 (ARCL2), Debré type, and wrinkly skin syndrome (WSS). The ATP6V0A2 gene encodes the a2 subunit of the V-type H+-ATPase, playing a role in proton translocation, and possibly also in membrane fusion. Here, we describe a highly variable phenotype in 13 patients with ARCL2, including the oldest affected individual described so far, who showed strikingly progressive dysmorphic features and heterotopic calcifications. In these individuals we identified 17 ATP6V0A2 mutations, 14 of which are novel. Furthermore, we demonstrate a localization of ATP6V0A2 at the Golgi-apparatus and a loss of the mutated ATP6V0A2 protein in patients’ dermal fibroblasts. Investigation of brefeldin A-induced Golgi collapse in dermal fibroblasts as well as in HeLa cells deficient for ATP6V0A2 revealed a delay, which was absent in cells deficient for the ARCL-associated proteins GORAB or PYCR1. Furthermore, fibroblasts from patients with ATP6V0A2 mutations displayed elevated TGF-β signalling and increased TGF-β1 levels in the supernatant. Our current findings expand the genetic and phenotypic spectrum and suggest that, besides the known glycosylation defect, alterations in trafficking and signalling processes are potential key events in the pathogenesis of ATP6V0A2-related ARCL.

Depressive behaviour in children diagnosed with a mitochondrial disorder.

A higher incidence of depression has been described in adults with primary oxidative phosphorylation disease. We evaluated the psychological characteristics of eighteen non-retarded pediatric patients diagnosed with a disorder of the oxidative phosphorylation. We found significantly higher rate of withdrawn, depressive behaviour compared to population norm scores, to children with other types of inborn errors of metabolism and also in comparison to patients with Sotos syndrome. The occurrence of depressive behaviour showed no correlation with the degree of mitochondrial dysfunction. These findings support the hypothesis that mood disorders could be associated to abnormal cerebral energy metabolism.

Genotype-phenotype spectrum of PYCR1-related autosomal recessive cutis laxa

Autosomal recessive cutis laxa type 2B (ARCL2B; OMIM # 612940) is a segmental progeroid disorder caused by mutations in PYCR1 encoding pyrroline-5-carboxylate reductase 1, which is part of the conserved proline de novo synthesis pathway. Here we describe 33 patients with PYCR1-related ARCL from 27 families with initial diagnoses varying between wrinkly skin syndrome, gerodermia osteodysplastica, De Barsy syndrome or more severe progeria syndromes. Given the difficult differential diagnosis of ARCL syndromes we performed a systematic comparison of clinical features of PYCR1-related ARCL. Intrauterine growth retardation, a characteristic triangular facial gestalt, psychomotor retardation, and hypotonia were the most relevant distinctive hallmarks of ARCL due to proline de novo synthesis defects. Corneal clouding or cataracts, athetoid movements, and finger contractures were rather rare features, but had a high predictive value. In our cohort we identified 20 different PYCR1 mutations of which seven were novel. Most of the mutations accumulated in exons 4 to 6. Missense alterations of highly conserved residues were most frequent followed by splice site changes and a single nonsense mutation. Analysis of genotype-phenotype correlation revealed that patients with mutations in the first two exons had lower average clinical scores and absent or only mild intellectual disability. Structural analyses predicted interference with PYCR1 multimerization for a subset of missense mutations. These findings have implications for the clinics as well as the pathomechanism of PYCR1-related ARCL.

Perinatal and early infantile symptoms in congenital disorders of glycosylation.

Congenital disorders of glycosylation (CDG) are a rapidly growing family of inborn errors. Screening for CDG in suspected cases is usually performed in the first year of life by serum transferrin isoelectric focusing or mass spectrometry. Based on the transferrin analysis patients can be biochemically diagnosed with a type 1 or type 2 transferrin pattern, and labeled as CDG‐I, or CDG‐II. The diagnosis of CDG is frequently delayed due to the highly variable phenotype, some cases showing single organ involvement and others mimicking syndromes, like skeletal dysplasia, cutis laxa syndrome, or congenital muscle dystrophy. The aim of our study was to evaluate perinatal abnormalities and early discriminative symptoms in 58 patients consecutively diagnosed with diverse CDG‐subtypes. Neonatal findings and clinical features in the first months of life were studied in 36 children with CDG‐I and 22 with CDG‐II. Maternal complications were found in five, small for gestational age in nine patients. Five children had abnormal neonatal screening results for hypothyroidism. Congenital microcephaly and neonatal seizures were common in CDG‐II. Inverted nipples were uncommon with 5 out of 58 children. Dysmorphic features were mostly nonspecific, except for cutis laxa. Early complications included feeding problems, cardiomyopathy, thrombosis, and bleeding. Cases presenting in the neonatal period had the highest mortality rate. Survival in CDG patients is highly dependent on early intervention therapy. We recommend low threshold screening for glycosylation disorders in infants with neurologic symptoms, even in the absence of abnormal fat distribution. Growth retardation and neonatal bleeding increase suspicion for CDG.

Mutations in ATP6V1E1 or ATP6V1A Cause Autosomal-Recessive Cutis Laxa

Defects of the V-type proton (H+) ATPase (V-ATPase) impair acidification and intracellular trafficking of membrane-enclosed compartments, including secretory granules, endosomes, and lysosomes. Whole-exome sequencing in five families affected by mild to severe cutis laxa, dysmorphic facial features, and cardiopulmonary involvement identified biallelic missense mutations in ATP6V1E1 and ATP6V1A, which encode the E1 and A subunits, respectively, of the V1 domain of the heteromultimeric V-ATPase complex. Structural modeling indicated that all substitutions affect critical residues and inter- or intrasubunit interactions. Furthermore, complexome profiling, a method combining blue-native gel electrophoresis and liquid chromatography tandem mass spectrometry, showed that they disturb either the assembly or the stability of the V-ATPase complex. Protein glycosylation was variably affected. Abnormal vesicular trafficking was evidenced by delayed retrograde transport after brefeldin A treatment and abnormal swelling and fragmentation of the Golgi apparatus. In addition to showing reduced and fragmented elastic fibers, the histopathological hallmark of cutis laxa, transmission electron microscopy of the dermis also showed pronounced changes in the structure and organization of the collagen fibers. Our findings expand the clinical and molecular spectrum of metabolic cutis laxa syndromes and further link defective extracellular matrix assembly to faulty protein processing and cellular trafficking caused by genetic defects in the V-ATPase complex.

Clinical and biochemical features guiding the diagnostics in neurometabolic cutis laxa

Patients with cutis laxa (CL) have wrinkled, sagging skin with decreased elasticity. Skin symptoms are associated with variable systemic involvement. The most common, genetically highly heterogeneous form of autosomal recessive CL, ARCL2, is frequently associated with variable metabolic and neurological symptoms. Progeroid symptoms, dysmorphic features, hypotonia and psychomotor retardation are highly overlapping in the early phase of these disorders. This makes the genetic diagnosis often challenging. In search for discriminatory symptoms, we prospectively evaluated clinical, neurologic, metabolic and genetic features in our patient cohort referred for suspected ARCL. From a cohort of 26 children, we confirmed mutations in genes associated with ARCL in 16 children (14 probands), including 12 novel mutations. Abnormal glycosylation and gyration abnormalities were mostly, but not always associated with ATP6V0A2 mutations. Epilepsy was most common in ATP6V0A2 defects. Corpus callosum dysgenesis was associated with PYCR1 and ALDH18A1 mutations. Dystonic posturing was discriminatory for PYCR1 and ALDH18A1 defects. Metabolic markers of mitochondrial dysfunction were found in one patient with PYCR1 mutations. So far unreported white matter abnormalities were found associated with GORAB and RIN2 mutations. We describe a large cohort of CL patients with neurologic involvement. Migration defects and corpus callosum hypoplasia were not always diagnostic for a specific genetic defect in CL. All patients with ATP6V0A2 defects had abnormal glycosylation. To conclude, central nervous system and metabolic abnormalities were discriminatory in this genetically heterogeneous group, although not always diagnostic for a certain genetic defect in CL.

Recognizable phenotype with common occurrence of microcephaly, psychomotor retardation, but no spontaneous bone fractures in autosomal recessive cutis laxa type IIB due to PYCR1 mutations

Recognizable Phenotype With Common Occurrence of Microcephaly, Psychomotor Retardation, but no Spontaneous Bone Fractures in Autosomal Recessive Cutis Laxa Type IIB Due to PYCR1 Mutations Dorus Kouwenberg, Thatjana Gardeitchik, Ron A Wevers, Johannes H€aberle, and Eva Morava* Department of Pediatrics, Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands Laboratory of Genetic, Endocrine and Metabolic Diseases, IGMD, Nijmegen, The Netherlands University Hospital for Children, Zurich, Switzerland

Long-term clinical outcome, therapy and mild mitochondrial dysfunction in hyperprolinemia

Although hyperprolinemia type-II has a discriminative metabolic phenotype and is frequently associated with neurological system involvement, the casual relation between the metabolic abnormalities and the clinical features, except for those of the secondary B6 deficiency, has been frequently debated. In order to evaluate disease frequency and the neuro-metabolic outcome we searched our laboratory database between 1992 and 2010, including 20,991 urinary organic acid profiles. From these individuals 16,720 parallel blood samples were available, and were investigated by serum amino acid analysis. We also evaluated the clinical, neurological, psychological features, laboratory data and vitamin levels and therapeutic effect in metabolically confirmed hyperprolinemia. Due to the mitochondrial localization of both ALDH4A1 and PRODH mitochondrial enzyme complex activity was evaluated and oxygen consumption was measured to assess ATP production in patient-fibroblasts. The Mitochondrial Disease Score was used to evaluate clinical mitochondrial dysfunction. The child behavior checklist was used to screen for psychopathology. We found four patients with increased urinary P5C diagnosed with hyperprolinemia type II, and only one patient had hyperprolinemia type I. All children with hyperprolinemia type II had low normal B6 concentration, and three of the patients had biochemical markers suggesting mitochondrial dysfunction. Mitochondrial dysfunction was confirmed in a muscle biopsy in one case. Intellectual disability was found in two adolescent patients. All patients showed seizures and significant behavioral problems, including anxiety and hallucinations. The clinical course was non-progressive and independent from the B6 concentration and B6 therapy. Hyperprolinemia is a rare inborn error. Individuals with hyperprolinemia should be monitored closely due to their frequent behavioral problems.