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Mitochondrial disorders are characterized by a broad clinical spectrum. Identical clinical signs and symptoms can be caused by mutations in different mitochondrial or nuclear genes. Vice versa, the same mutation can lead to different phenotypes. Genetic syndromes and neuromuscular disorders mimicking mitochondrial disorders further complicate the diagnostic process. Whole exome sequencing (WES) is the state of the art next generation sequencing technique to identify genetic defects in mitochondrial disorders. Until recently it has mainly been used as a research tool. In this study, the use of WES in routine diagnostics is described. The WES data of 109 patients, referred under the suspicion of a mitochondrial disorder, were examined in two steps. First, the data were filtered using a virtual gene panel of genes known to be associated with mitochondrial disease. If negative, the entire exome was examined. A molecular diagnosis was achieved in 39 % of the heterogeneous cohort, and in 57 % of the subgroup of 42 patients with the highest suspicion for a mitochondrial disease. In addition to mutations in genes known to be associated with mitochondrial disorders (e.g. TUFM , MTFMT , FBXL4 ), in the subgroup of patients with the lowest suspicion for a mitochondrial disorder we found mutations in several genes associated with neuromuscular disorders ( e.g. SEPN1, ACTA1 ) and genetic syndrome ( e.g. SETBP1, ARID1B ). Our results show that WES technology has been successfully implemented as a state-of-the-art, molecular diagnostic test for mitochondrial disorders as well as for the mimicking disorders in daily clinical practice. It also illustrates that clinical and biochemical phenotyping is essential for successful application of WES to diagnose individual patients.

Background The Treatable ID App was created in 2012 as digital tool to improve early recognition and intervention for treatable inherited metabolic disorders (IMDs) presenting with global developmental delay and intellectual disability (collectively ‘treatable IDs’). Our aim is to update the 2012 review on treatable IDs and App to capture the advances made in the identification of new IMDs along with increased pathophysiological insights catalyzing therapeutic development and implementation. Methods Two independent reviewers queried PubMed, OMIM and Orphanet databases to reassess all previously included disorders and therapies and to identify all reports on Treatable IDs published between 2012 and 2021. These were included if listed in the International Classification of IMDs (ICIMD) and presenting with ID as a major feature, and if published evidence for a therapeutic intervention improving ID primary and/or secondary outcomes is available. Data on clinical symptoms, diagnostic testing, treatment strategies, effects on outcomes, and evidence levels were extracted and evaluated by the reviewers and external experts. The generated knowledge was translated into a diagnostic algorithm and updated version of the App with novel features. Results Our review identified 116 treatable IDs (139 genes), of which 44 newly identified, belonging to 17 ICIMD categories. The most frequent therapeutic interventions were nutritional, pharmacological and vitamin and trace element supplementation. Evidence level varied from 1 to 3 (trials, cohort studies, case–control studies) for 19% and 4–5 (case-report, expert opinion) for 81% of treatments. Reported effects included improvement of clinical deterioration in 62%, neurological manifestations in 47% and development in 37%. Conclusion The number of treatable IDs identified by our literature review increased by more than one-third in eight years. Although there has been much attention to gene-based and enzyme replacement therapy, the majority of effective treatments are nutritional, which are relatively affordable, widely available and (often) surprisingly effective. We present a diagnostic algorithm (adjustable to local resources and expertise) and the updated App to facilitate a swift and accurate workup, prioritizing treatable IDs. Our digital tool is freely available as Native and Web App (www.treatable-id.org) with several novel features. Our Treatable ID endeavor contributes to the Treatabolome and International Rare Diseases Research Consortium goals, enabling clinicians to deliver rapid evidence-based interventions to our rare disease patients.

Background No curative therapy for mitochondrial disease (MD) exists, prioritizing supportive treatment for symptom relief. In animal and cell models ketones decrease oxidative stress, increase antioxidants and scavenge free radicals, putting ketogenic diets (KDs) on the list of management options for MD. Furthermore, KDs are well-known, safe and effective treatments for epilepsy, a frequent symptom of MD. This systematic review evaluates efficacy and safety of KD for MD. Methods We searched Pubmed, Cochrane, Embase and Cinahl (November 2020) with search terms linked to MD and KD. From the identified records, we excluded studies on Pyruvate Dehydrogenase Complex deficiency. From these eligible reports, cases without a genetically confirmed diagnosis and cases without sufficient data on KD and clinical course were excluded. The remaining studies were included in the qualitative analysis. Results Only 20 cases (14 pediatric) from the 694 papers identified met the inclusion criteria (one controlled trial (n = 5), 15 case reports). KD led to seizure control in 7 out of 8 cases and improved muscular symptoms in 3 of 10 individuals. In 4 of 20 cases KD reversed the clinical phenotype (e.g. cardiomyopathy, movement disorder). In 5 adults with mitochondrial DNA deletion(s) related myopathy rhabdomyolysis led to cessation of KD. Three individuals with POLG mutations died while being on KD, however, their survival was not different compared to individuals with POLG mutations without KD. Conclusion Data on efficacy and safety of KD for MD is too scarce for general recommendations. KD should be considered in individuals with MD and therapy refractory epilepsy, while KD is contraindicated in mitochondrial DNA deletion(s) related myopathy. When considering KD for MD the high rate of adverse effects should be taken into account, but also spectacular improvements in individual cases. KD is a highly individual management option in this fragile patient group and requires an experienced team. To increase knowledge on this—individually—promising management option more (prospective) studies using adequate outcome measures are crucial.

Background Glycogen storage disease type Ib (GSD Ib) is a rare inborn error of glycogen metabolism due to mutations in SLC37A4 . Besides a severe form of fasting intolerance, the disorder is usually associated with neutropenia and neutrophil dysfunction causing serious infections, inflammatory bowel disease, oral, urogenital and perianal lesions as well as impaired wound healing. Recently, SGLT2 inhibitors such as empagliflozin that reduce the plasma levels of 1,5-anhydroglucitol have been described as a new treatment option for the neutropenia and neutrophil dysfunction in patients with GSD Ib. Results We report on a 35-year-old female patient with GSD Ib who had been treated with G-CSF for neutropenia since the age of 9. She had a large chronic abdominal wound as a consequence of recurrent operations due to complications of her inflammatory bowel disease. Treatment with 20 mg empagliflozin per day resulted in normalisation of the neutrophil count and neutrophil function even after termination of G-CSF. The chronic abdominal wound that had been unchanged for 2 years before the start of empagliflozin nearly closed within 12 weeks. No side effects of empagliflozin were observed. Conclusion SGLT2 inhibitors are a new and probably safe treatment option for GSD Ib-associated neutropenia and neutrophil dysfunction. We hypothesize that restoration of neutrophil function and normalisation of neutrophil apoptosis leads to improvement of wound healing and ameliorates symptoms of inflammatory bowel disease.

Cells utilize protein disaggregases to avoid abnormal protein aggregation that causes many diseases. Among these, caseinolytic peptidase B protein homolog (CLPB) is localized in the mitochondrial intermembrane space and linked to human disease. Upon CLPB loss, MICU1 and MICU2, regulators of the mitochondrial calcium uniporter complex (mtCU), and OPA1, a main mediator of mitochondrial fusion, become insoluble but the functional outcome remains unclear. In this work we demonstrate that CLPB is required to maintain mitochondrial calcium signalling and fusion dynamics. CLPB loss results in altered mtCU composition, interfering with mitochondrial calcium uptake independently of cytosolic calcium and mitochondrial membrane potential. Additionally, OPA1 decreases, and aggregation occurs, accompanied by mitochondrial fragmentation. Disease-associated mutations in the CLPB gene present in skin fibroblasts from patients also display mitochondrial calcium and structural changes. Thus, mtCU and fusion activity are dependent on CLPB, and their impairments might contribute to the disease caused by CLPB variants. CLPB is a protein disaggregase that has been linked to human disease. Here, the authors show that in different models of chronic or acute loss of the mitochondrial disaggregase, mitochondrial Ca 2+ handling and fusion dynamics are impaired, revealing their dependence on CLPB.

Background Congenital myasthenic syndromes (CMS) are a heterogeneous group of disorders caused by genetic defects resulting in impaired neuromuscular transmission. Although effective treatments are available, CMS is probably underdiagnosed, and systematic clinico-genetic investigations are warranted. Methods We used a nationwide approach to collect Austrian patients with genetically confirmed CMS. We provide a clinical and molecular characterization of this cohort and aimed to ascertain the current frequency of CMS in Austria. Results Twenty-eight cases with genetically confirmed CMS were identified, corresponding to an overall prevalence of 3.1 per million (95% CI 2.0–4.3) in Austria. The most frequent genetic etiology was CHRNE ( n  = 13), accounting for 46.4% of the cohort. Within this subgroup, the variant c.1327del, p.(Glu443Lysfs*64) was detected in nine individuals. Moreover, causative variants were found in DOK7 ( n  = 4), RAPSN ( n  = 3), COLQ ( n  = 2), GMPPB ( n  = 2), CHAT ( n  = 1), COL13A1 ( n  = 1), MUSK ( n  = 1) and AGRN ( n  = 1). Clinical onset within the first year of life was reported in one half of the patients. Across all subtypes, the most common symptoms were ptosis (85.7%), lower limb (67.9%), upper limb (60.7%) and facial weakness (60.7%). The majority of patients (96.4%) received specific treatment, including acetylcholinesterase inhibitors in 20, adrenergic agonists in 11 and 3,4-diaminopyridine in nine patients. Conclusions Our study presents the first systematic characterization of individuals with CMS in Austria, providing prevalence estimates and genotype–phenotype correlations that may help to improve the diagnostic approach and patient management.

Congenital disorders of glycosylation are a genetically and phenotypically heterogeneous family of diseases affecting the co‐ and posttranslational modification of proteins. Using exome sequencing, we detected biallelic variants in GFUS (NM_003313.4) c.[632G>A];[659C>T] (p.[Gly211Glu];[Ser220Leu]) in a patient presenting with global developmental delay, mild coarse facial features and faltering growth. GFUS encodes GDP‐L‐fucose synthase, the terminal enzyme in de novo synthesis of GDP‐L‐fucose, required for fucosylation of N‐ and O‐glycans. We found reduced GFUS protein and decreased GDP‐L‐fucose levels leading to a general hypofucosylation determined in patient's glycoproteins in serum, leukocytes, thrombocytes and fibroblasts. Complementation of patient fibroblasts with wild‐type GFUS cDNA restored fucosylation. Making use of the GDP‐L‐fucose salvage pathway, oral fucose supplementation normalized fucosylation of proteins within 4 weeks as measured in serum and leukocytes. During the follow‐up of 19 months, a moderate improvement of growth was seen, as well as a clear improvement of cognitive skills as measured by the Kaufmann ABC and the Nijmegen Pediatric CDG Rating Scale. In conclusion, GFUS‐CDG is a new glycosylation disorder for which oral L‐fucose supplementation is promising. Synopsis A novel type of congenital disorder of glycosylation (CDG) was identified in a child carrying biallelic variants in GDP‐L‐fucose synthase (GFUS). Oral L‐fucose supplementation resulted in clinical improvements. The index patient presented with stunted growth and no speech at the age of 3 6/12 years, coarse facial features, aversion to feeding, and recurrent vomiting on tube feeding. Biallelic variants in GFUS lead to a reduced level of GDP‐L‐fucose and subsequently to a general hypofucosylation of proteins. GFUS deficiency can be bypassed by oral L‐fucose supplementation triggering the salvage pathway for the generation of GDP‐L‐fucose. After the fucose therapy, the growth parameters have stabilized, and the patient started to speak and showed improved attention span and cognitive skills. Graphical Abstract A novel type of congenital disorder of glycosylation (CDG) was identified in a child carrying biallelic variants in GDP‐L‐fucose synthase (GFUS). Oral L‐fucose supplementation resulted in clinical improvements.

Glycogen storage disease type Ib (GSD1b) is due to a defect in the glucose-6-phosphate transporter (G6PT) of the endoplasmic reticulum, which is encoded by the SLC37A4 gene. This transporter allows the glucose-6-phosphate that is made in the cytosol to cross the endoplasmic reticulum (ER) membrane and be hydrolyzed by glucose-6-phosphatase (G6PC1), a membrane enzyme whose catalytic site faces the lumen of the ER. Logically, G6PT deficiency causes the same metabolic symptoms (hepatorenal glycogenosis, lactic acidosis, hypoglycemia) as deficiency in G6PC1 (GSD1a). Unlike GSD1a, GSD1b is accompanied by low neutrophil counts and impaired neutrophil function, which is also observed, independently of any metabolic problem, in G6PC3 deficiency. Neutrophil dysfunction is, in both diseases, due to the accumulation of 1,5-anhydroglucitol-6-phosphate (1,5-AG6P), a potent inhibitor of hexokinases, which is slowly formed in the cells from 1,5-anhydroglucitol (1,5-AG), a glucose analog that is normally present in blood. Healthy neutrophils prevent the accumulation of 1,5-AG6P due to its hydrolysis by G6PC3 following transport into the ER by G6PT. An understanding of this mechanism has led to a treatment aimed at lowering the concentration of 1,5-AG in blood by treating patients with inhibitors of SGLT2, which inhibits renal glucose reabsorption. The enhanced urinary excretion of glucose inhibits the 1,5-AG transporter, SGLT5, causing a substantial decrease in the concentration of this polyol in blood, an increase in neutrophil counts and function and a remarkable improvement in neutropenia-associated clinical signs and symptoms.

Purpose To investigate monoallelic CLPB variants. Pathogenic variants in many genes cause congenital neutropenia. While most patients exhibit isolated hematological involvement, biallelic CLPB variants underlie a neurological phenotype ranging from nonprogressive intellectual disability to prenatal encephalopathy with progressive brain atrophy, movement disorder, cataracts, 3-methylglutaconic aciduria, and neutropenia. CLPB was recently shown to be a mitochondrial refoldase; however, the exact function remains elusive. Methods We investigated six unrelated probands from four countries in three continents, with neutropenia and a phenotype dominated by epilepsy, developmental issues, and 3-methylglutaconic aciduria with next-generation sequencing. Results In each individual, we identified one of four different de novo monoallelic missense variants in CLPB . We show that these variants disturb refoldase and to a lesser extent ATPase activity of CLPB in a dominant-negative manner. Complexome profiling in fibroblasts showed CLPB at very high molecular mass comigrating with the prohibitins. In control fibroblasts, HAX1 migrated predominantly as monomer while in patient samples multiple HAX1 peaks were observed at higher molecular masses comigrating with CLPB thus suggesting a longer-lasting interaction between CLPB and HAX1. Conclusion Both biallelic as well as specific monoallelic CLPB variants result in a phenotypic spectrum centered around neurodevelopmental delay, seizures, and neutropenia presumably mediated via HAX1.

Background ATPase family AAA-domain containing protein 3A (ATAD3A) is a nuclear-encoded mitochondrial membrane-anchored protein involved in diverse processes including mitochondrial dynamics, mitochondrial DNA organization, and cholesterol metabolism. Biallelic deletions (null), recessive missense variants (hypomorph), and heterozygous missense variants or duplications (antimorph) in ATAD3A lead to neurological syndromes in humans. Methods To expand the mutational spectrum of ATAD3A variants and to provide functional interpretation of missense alleles in trans to deletion alleles, we performed exome sequencing for identification of single nucleotide variants (SNVs) and copy number variants (CNVs) in ATAD3A in individuals with neurological and mitochondrial phenotypes. A Drosophila Atad3a Gal4 knockin-null allele was generated using CRISPR-Cas9 genome editing technology to aid the interpretation of variants. Results We report 13 individuals from 8 unrelated families with biallelic ATAD3A variants. The variants included four missense variants inherited in trans to loss-of-function alleles (p.(Leu77Val), p.(Phe50Leu), p.(Arg170Trp), p.(Gly236Val)), a homozygous missense variant p.(Arg327Pro), and a heterozygous non-frameshift indel p.(Lys568del). Affected individuals exhibited findings previously associated with ATAD3A pathogenic variation, including developmental delay, hypotonia, congenital cataracts, hypertrophic cardiomyopathy, and cerebellar atrophy. Drosophila studies indicated that Phe50Leu, Gly236Val, Arg327Pro, and Lys568del are severe loss-of-function alleles leading to early developmental lethality. Further, we showed that Phe50Leu, Gly236Val, and Arg327Pro cause neurogenesis defects. On the contrary, Leu77Val and Arg170Trp are partial loss-of-function alleles that cause progressive locomotion defects and whose expression leads to an increase in autophagy and mitophagy in adult muscles. Conclusion Our findings expand the allelic spectrum of ATAD3A variants and exemplify the use of a functional assay in Drosophila to aid variant interpretation.