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Given the rapidly decreasing cost and increasing speed and accessibility of massively parallel technologies, the integration of comprehensive genomic, transcriptomic, and proteomic data into a "multi-omics" diagnostic pipeline is within reach. Even though genomic analysis has the capability to reveal all possible perturbations in our genetic code, analysis typically reaches a diagnosis in just 35% of cases, with a diagnostic gap arising due to limitations in prioritization and interpretation of detected variants. Here we review the utility of complementing genetic data with transcriptomic data and give a perspective for the introduction of proteomics into the diagnostic pipeline. Together these methodologies enable comprehensive capture of the functional consequence of variants, unobtainable by the analysis of each methodology in isolation. This facilitates functional annotation and reprioritization of candidate genes and variants-a promising approach to shed light on the underlying molecular cause of a patient's disease, increasing diagnostic rate, and allowing actionability in clinical practice.

Flatworms of the species Schmidtea mediterranea are immortal-adult animals contain a large pool of pluripotent stem cells that continuously differentiate into all adult cell types. Therefore, single-cell transcriptome profiling of adult animals should reveal mature and progenitor cells. By combining perturbation experiments, gene expression analysis, a computational method that predicts future cell states from transcriptional changes, and a lineage reconstruction method, we placed all major cell types onto a single lineage tree that connects all cells to a single stem cell compartment. We characterized gene expression changes during differentiation and discovered cell types important for regeneration. Our results demonstrate the importance of single-cell transcriptome analysis for mapping and reconstructing fundamental processes of developmental and regenerative biology at high resolution.

Hydroxyprolinemia is an inborn error of amino acid degradation that is considered a non-disease. Known for more than 50 years, its genetic cause and prevalence have remained unclear. In MS/MS newborn screening, the mass spectrum of hydroxyproline cannot be differentiated from isoleucine and leucine causing false positive newborn screening test results for maple syrup urine disease (MSUD). We studied two siblings with hydroxyprolinemia via exome sequencing and confirmed the candidate gene in five further individuals with hydroxyprolinemia, who were all characterized biochemically and clinically. The prevalence was calculated based on the number of individuals with hydroxyprolinemia detected via MS/MS newborn screening at our centre from 2003 to 2014. In six cases, we identified homozygous or compound heterozygous mutations in PRODH2 as the underlying genetic cause of hydroxyprolinemia. One individual was heterozygous for a deletion in PRODH2 and had an intermittent biochemical phenotype with partial normalization of hydroxyproline concentrations. In one further individual with persistent hydroxyprolinemia no mutation in PRODH2 was found, raising the possibility of another defect of hydroxyproline degradation yet to be identified as the underlying cause of hydroxyprolinemia. Plasma hydroxyproline concentrations were clearly elevated in all individuals with biallelic mutations in PRODH2. All studied individuals remained asymptomatic, giving further evidence that hydroxyprolinemia is a benign condition. The estimated prevalence of hydroxyprolinemia in Germany is about one in 47,300 newborns. Our results establish mutations in PRODH2 as a cause of human hydroxyprolinemia via impaired dehydrogenation of hydroxyproline to delta1-pyroline-3-hydroxy-5-carboxylic acid, and we suggest PRODH2 be renamed HYPDH. Hydroxyprolinemia is an autosomal-recessively inherited benign condition. It is a frequent cause of false positive screening results for MSUD, the prevalence being about 2.5 times higher than that of MSUD.

Acute liver failure (ALF) in infancy and childhood is a life-threatening emergency and in about 50 % the etiology remains unknown. Recently biallelic mutations in NBAS were identified as a new molecular cause of ALF with onset in infancy, leading to recurrent acute liver failure (RALF). The phenotype and medical history of 14 individuals with NBAS deficiency was studied in detail and functional studies were performed on patients’ fibroblasts. The phenotypic spectrum of NBAS deficiency ranges from isolated RALF to a multisystemic disease with short stature, skeletal dysplasia, immunological abnormalities, optic atrophy, and normal motor and cognitive development resembling SOPH syndrome. Liver crises are triggered by febrile infections; they become less frequent with age but are not restricted to childhood. Complete recovery is typical, but ALF crises can be fatal. Antipyretic therapy and induction of anabolism including glucose and parenteral lipids effectively ameliorates the course of liver crises. Patients’ fibroblasts showed an increased sensitivity to high temperature at protein and functional level and a disturbed tethering of vesicles, pointing at a defect of intracellular transport between the endoplasmic reticulum and Golgi. Mutations in NBAS cause a complex disease with a wide clinical spectrum ranging from isolated RALF to a multisystemic phenotype. Thermal susceptibility of the syntaxin 18 complex is the basis of fever dependency of ALF episodes. NBAS deficiency is the first disease related to a primary defect of retrograde transport. Identification of NBAS deficiency allows optimized therapy of liver crises and even prevention of further episodes.

Contains fulltext : 154784.pdf (Publisher’s version ) (Closed access) FBXL4 deficiency is a recently described disorder of mitochondrial maintenance associated with a loss of mitochondrial DNA in cells. To date, the genetic diagnosis of FBXL4 deficiency has been established in 28 individuals. This paper retrospectively reviews proxy-reported clinical and biochemical findings and evaluates brain imaging, morphological and genetic data in 21 of those patients. Neonatal/early-onset severe lactic acidosis, muscular hypotonia, feeding problems and failure to thrive is the characteristic pattern at first presentation. Facial dysmorphic features are present in 67 % of cases. Seven children died (mean age 37 months); 11 children were alive (mean age at follow-up 46 months), three children were lost to follow-up. All survivors developed severe psychomotor retardation. Brain imaging was non-specific in neonates but a later-onset, rapidly progressive brain atrophy was noted. Elevated blood lactate and metabolic acidosis were observed in all individuals; creatine kinase was elevated in 45 % of measurements. Diagnostic workup in patient tissues and cells revealed a severe combined respiratory chain defect with a general decrease of enzymes associated with mitochondrial energy metabolism and a relative depletion of mitochondrial DNA content. Mutations were detected throughout the FBXL4 gene albeit with no clear delineation of a genotype-phenotype correlation. Treatment with "mitochondrial medications" did not prove effective. In conclusion, a clinical pattern of early-onset encephalopathy, persistent lactic acidosis, profound muscular hypotonia and typical facial dysmorphism should prompt initiation of molecular genetic analysis of FBXL4. Establishment of the diagnosis permits genetic counselling, prevents patients undergoing unhelpful diagnostic procedures and allows for accurate prognosis.

Congenital disorders of glycosylation (CDG) have a broad spectrum of clinical manifestations. They can affect multiple organ systems, including skin and subcutaneous tissue. We report on an infant with severe ichthyosis caused by MPDU1 mutations. The case illustrates that skin manifestations are an important feature of CDG syndromes. Therefore, metabolic investigations should be included in the workup of infantile ichthyosis disorders.

Brown-Vialetto-Van Laere syndrome (BVVLS [MIM 211530]) is a rare neurological disorder characterized by infancy onset sensorineural deafness and ponto-bulbar palsy. Mutations in SLC52A3 (formerly C20orf54), coding for riboflavin transporter 2 (hRFT2), have been identified as the molecular genetic correlate in several individuals with BVVLS. Exome sequencing of just one single case revealed that compound heterozygosity for two pathogenic mutations in the SLC52A2 gene coding for riboflavin transporter 3 (hRFT3), another member of the riboflavin transporter family, is also associated with BVVLS. Overexpression studies confirmed that the gene products of both mutant alleles have reduced riboflavin transport activities. While mutations in SLC52A3 cause decreased plasma riboflavin levels, concordant with a role of SLC52A3 in riboflavin uptake from food, the SLC52A2-mutant individual had normal plasma riboflavin concentrations, a finding in line with a postulated function of SLC52A2 in riboflavin uptake from blood into target cells. Our results contribute to the understanding of human riboflavin metabolism and underscore its role in the pathogenesis of BVVLS, thereby providing a rational basis for a high-dose riboflavin treatment.

Inherited disorders of mitochondrial energy metabolism form a large and heterogeneous group of metabolic diseases. More than 250 gene defects have been reported to date and this number continues to grow. Mitochondrial diseases can be grouped into (1) disorders of oxidative phosphorylation (OXPHOS) subunits and their assembly factors, (2) defects of mitochondrial DNA, RNA and protein synthesis, (3) defects in the substrate-generating upstream reactions of OXPHOS, (4) defects in relevant cofactors and (5) defects in mitochondrial homeostasis. Deficiency of more than one respiratory chain enzyme is a common finding. Combined defects are found in 49 % of the known disease-causing genes of mitochondrial energy metabolism and in 57 % of patients with OXPHOS defects identified in our diagnostic centre. Combined defects of complexes I, III, IV and V are typically due to deficiency of mitochondrial DNA replication, RNA metabolism or translation. Defects in cofactors can result in combined defects of various combinations, and defects of mitochondrial homeostasis can result in a generalised decrease of all OXPHOS enzymes. Noteworthy, identification of combined defects can be complicated by different degrees of severity of each affected enzyme. Furthermore, even defects of single respiratory chain enzymes can result in combined defects due to aberrant formation of respiratory chain supercomplexes. Combined OXPHOS defects have a great variety of clinical manifestations in terms of onset, course severity and tissue involvement. They can present as classical encephalomyopathy but also with hepatopathy, nephropathy, haematologic findings and Perrault syndrome in a subset of disorders.

Pyruvate oxidation defects (PODs) are among the most frequent causes of deficiencies in the mitochondrial energy metabolism and represent a substantial subset of classical mitochondrial diseases. PODs are not only caused by deficiency of subunits of the pyruvate dehydrogenase complex (PDHC) but also by various disorders recently described in the whole pyruvate oxidation route including cofactors, regulation of PDHC and the mitochondrial pyruvate carrier. Our own patients from 2000 to July 2014 and patients identified by a systematic survey of the literature from 1970 to July 2014 with a pyruvate oxidation disorder and a genetically proven defect were included in the study (n=628). Of these defects 74.2% (n=466) belong to PDHC subunits, 24.5% (n=154) to cofactors, 0.5% (n=3) to PDHC regulation and 0.8% (n=5) to mitochondrial pyruvate import. PODs are underestimated in the field of mitochondrial diseases because not all diagnostic centres include biochemical investigations of PDHC in their routine analysis. Cofactor and transport defects can be missed, if pyruvate oxidation is not measured in intact mitochondria routinely. Furthermore deficiency of the X-chromosomal PDHA1 can be biochemically missed depending on the X-inactivation pattern. This is reflected by an increasing number of patients diagnosed recently by genetic high throughput screening approaches. PDHC deficiency including regulation and import affect mainly the glucose dependent central and peripheral nervous system and skeletal muscle. PODs with combined enzyme defects affect also other organs like heart, lung and liver. The spectrum of clinical presentation of PODs is still expanding. PODs are a therapeutically interesting group of mitochondrial diseases since some can be bypassed by ketogenic diet or treated by cofactor supplementation. PDHC kinase inhibition, chaperone therapy and PGC1α stimulation is still a matter of further investigations.