Ketogenic Diet Treatment of Defects in the Mitochondrial Malate Aspartate Shuttle and Pyruvate Carrier.

The mitochondrial malate aspartate shuttle system (MAS) maintains the cytosolic NAD+/NADH redox balance, thereby sustaining cytosolic redox-dependent pathways, such as glycolysis and serine biosynthesis. Human disease has been associated with defects in four MAS-proteins (encoded by MDH1, MDH2, GOT2, SLC25A12) sharing a neurological/epileptic phenotype, as well as citrin deficiency (SLC25A13) with a complex hepatopathic-neuropsychiatric phenotype. Ketogenic diets (KD) are high-fat/low-carbohydrate diets, which decrease glycolysis thus bypassing the mentioned defects. The same holds for mitochondrial pyruvate carrier (MPC) 1 deficiency, which also presents neurological deficits. We here describe 40 (18 previously unreported) subjects with MAS-/MPC1-defects (32 neurological phenotypes, eight citrin deficiency), describe and discuss their phenotypes and genotypes (presenting 12 novel variants), and the efficacy of KD. Of 13 MAS/MPC1-individuals with a neurological phenotype treated with KD, 11 experienced benefits-mainly a striking effect against seizures. Two individuals with citrin deficiency deceased before the correct diagnosis was established, presumably due to high-carbohydrate treatment. Six citrin-deficient individuals received a carbohydrate-restricted/fat-enriched diet and showed normalisation of laboratory values/hepatopathy as well as age-adequate thriving. We conclude that patients with MAS-/MPC1-defects are amenable to dietary intervention and that early (genetic) diagnosis is key for initiation of proper treatment and can even be lifesaving.

Efficacy and safety of empagliflozin in glycogen storage disease type Ib: Data from an international questionnaire.

PURPOSE: This paper aims to report collective information on safety and efficacy of empagliflozin drug repurposing in individuals with glycogen storage disease type Ib (GSD Ib). METHODS: This is an international retrospective questionnaire study on the safety and efficacy of empagliflozin use for management of neutropenia/neutrophil dysfunction in patients with GSD Ib, conducted among the respective health care providers from 24 countries across the globe. RESULTS: Clinical data from 112 individuals with GSD Ib were evaluated, representing a total of 94 treatment years. The median age at start of empagliflozin treatment was 10.5 years (range = 0-38 years). Empagliflozin showed positive effects on all neutrophil dysfunction-related symptoms, including oral and urogenital mucosal lesions, recurrent infections, skin abscesses, inflammatory bowel disease, and anemia. Before initiating empagliflozin, most patients with GSD Ib were on G-CSF (94/112; 84%). At the time of the survey, 49 of 89 (55%) patients previously treated with G-CSF had completely stopped G-CSF, and another 15 (17%) were able to reduce the dose. The most common adverse event during empagliflozin treatment was hypoglycemia, occurring in 18% of individuals. CONCLUSION: Empagliflozin has a favorable effect on neutropenia/neutrophil dysfunction-related symptoms and safety profile in individuals with GSD Ib.

100 years of inherited metabolic disorders in Austria-A national registry of minimal birth prevalence, diagnosis, and clinical outcome of inborn errors of metabolism in Austria between 1921 and 2021.

Inherited metabolic disorders (IMDs) are a heterogeneous group of rare disorders characterized by disruption of metabolic pathways. To date, data on incidence and prevalence of IMDs are limited. Taking advantage of a functioning network within the Austrian metabolic group, our registry research aimed to update the data of the "Registry for Inherited Metabolic Disorders" started between 1985 and 1995 with retrospectively retrieved data on patients with IMDs according to the Society for the Study of Inborn Errors of Metabolism International Classification of Diseases 11 (SSIEM ICD11) catalogue. Included in this retrospective register were 2631 patients with an IMD according to the SSIEM ICD11 Classification, who were treated in Austria. Thus, a prevalence of 1.8/10 000 for 2020 and a median minimal birth prevalence of 16.9/100 000 (range 0.7/100 000-113/100 000) were calculated for the period 1921 to February 2021. We detected a male predominance (m:f = 1.2:1) and a mean age of currently alive patients of 17.6 years (range 5.16 months-100 years). Most common diagnoses were phenylketonuria (17.7%), classical galactosaemia (6.6%), and biotinidase deficiency (4.2%). The most common diagnosis categories were disorders of amino acid and peptide metabolism (819/2631; 31.1%), disorders of energy metabolism (396/2631; 15.1%), and lysosomal disorders (395/2631; 15.0%). In addition to its epidemiological relevance, the "Registry for Inherited Metabolic Disorders" is an important tool for enhancing an exchange between care providers. Moreover, by pooling expertise it prospectively improves patient treatment, similar to pediatric oncology protocols. A substantial requirement for ful filling this goal is to regularly update the registry and provide nationwide coverage with inclusion of all medical specialties.

A retrospective study on disease management in children and adolescents with phenylketonuria during the Covid-19 pandemic lockdown in Austria.

BACKGROUND: In classical phenylketonuria (PKU) phenylalanine (Phe) accumulates due to functional impairment of the enzyme phenylalanine hydroxylase caused by pathogenic variants in the PAH gene. PKU treatment prevents severe cognitive impairment. Blood Phe concentration is the main biochemical monitoring parameter. Between appointments and venous blood sampling, Austrian PKU patients send dried blood spots (DBS) for Phe measurements to their centre. Coronavirus disease-19 (COVID-19), caused by the SARS CoV-2 virus, was classified as a pandemic by the World Health Organization in March 2020. In Austria, two nationwide lockdowns were installed during the first and second pandemic wave with variable regional and national restrictions in between. This retrospective questionnaire study compared the frequency of Phe measurements and Phe concentrations during lockdown with the respective period of the previous year in children and adolescents with PKU and explored potential influencing factors. RESULTS: 77 patients (30 female, 47 male; mean age 12.4 [8-19] years in 2020) from five centres were included. The decline of venous samples taken on appointments in 2020 did not reach significance but the number of patients with none or only one DBS tripled from 4 (5.2%) in 2019 to 12 (15.6%) in 2020. Significantly more patients had a decline than a rise in the number of DBS sent in between 2019 and 2020 (p < 0.001; Chi2 = 14.79). Especially patients ≥ 16 years sent significantly less DBS in 2020 (T = 156, p = 0.02, r = 0.49). In patients who adhered to DBS measurements, Phe concentrations remained stable. Male or female sex and dietary only versus dietary plus sapropterin treatment did not influence frequency of measurements and median Phe. CONCLUSION: During the COVID pandemic, the number of PKU patients who stopped sending DBS to their metabolic centre increased significantly, especially among those older than 16 years. Those who kept up sending DBS maintained stable Phe concentrations. Our follow-up system, which is based on DBS sent in by patients to trigger communication with the metabolic team served adherent patients well. It failed, however, to actively retrieve patients who stopped or reduced Phe measurements.

Project "Backtoclinic I": An overview on the state of care of adult PKU patients in Austria.

BACKGROUND: High rates of lost to follow-up (LTFU) adult patients are a major concern in the long-term management of phenylketonuria (PKU). To address this issue, we designed the project "Backtoclinic" with the purpose of identifying LTFU adult PKU patients in Austria as a first step to reestablish appropriate treatment. SUBJECTS AND METHODS: Individuals born between 1966 and 1999 and diagnosed with PKU through the National Austrian Newborn Screening Program (NANSP) were identified using the NANSP's database. Follow-up data were collected in the Austrian metabolic centers (Medical University of Vienna, Graz, Innsbruck and Salzburg). Patients with no contact to any of these centers within the previous two years were classified as LTFU. Epidemiological characteristics of the whole study population as well as of LTFU- and currently in follow-up patients were analyzed. RESULTS: Between 1966 and 1999, 281 individuals were diagnosed with PKU through the NANSP. Two patients died in their first year of life and were excluded from the analysis. Of the remaining 279 patients (mean age ± SD: 36.7 ± 9.1 y, 42.7% females), 177 (63.4%) are currently LTFU. The rate of LTFU patients is higher in men than in women (68.1% vs 57.5%), and markedly increases with age in both sexes. The gender gap is greatest in young adults (52.6% vs. 25.0% in the age range 20.0-24.9 y) and declines with age (94.4% vs. 80.0% in the age range > 45.0 y). CONCLUSIONS: We found an alarming rate of 63.4% of LTFU adult PKU patients in Austria, and observed a gender gap in the PKU state of care. Our findings illustrate the urgent need for the metabolic community to identify LTFU adult PKU patients and to develop strategies to reestablish appropriate treatment for men and women with PKU.

Severe Deoxyguanosine Kinase Deficiency in Austria: A 6-Patient Series.

Mutations in the nuclear gene DGUOK, encoding deoxyguanosine kinase, cause an infantile hepatocerebral type of mitochondrial depletion syndrome (MDS). We report 6 MDS patients harboring bi-allelic DGUOK mutations, of which 3 are novel, including a large intragenic Austrian founder deletion. One patient was diagnosed with hepatocellular carcinoma aged 6 months, supporting a link between mitochondrial DNA depletion and tumorigenesis; liver transplantation proved beneficial with regard to both tumor treatment and psychomotor development.

The genotypic and phenotypic spectrum of MTO1 deficiency.

BACKGROUND: Mitochondrial diseases, a group of multi-systemic disorders often characterized by tissue-specific phenotypes, are usually progressive and fatal disorders resulting from defects in oxidative phosphorylation. MTO1 (Mitochondrial tRNA Translation Optimization 1), an evolutionarily conserved protein expressed in high-energy demand tissues has been linked to human early-onset combined oxidative phosphorylation deficiency associated with hypertrophic cardiomyopathy, often referred to as combined oxidative phosphorylation deficiency-10 (COXPD10). MATERIAL AND METHODS: Thirty five cases of MTO1 deficiency were identified and reviewed through international collaboration. The cases of two female siblings, who presented at 1 and 2years of life with seizures, global developmental delay, hypotonia, elevated lactate and complex I and IV deficiency on muscle biopsy but without cardiomyopathy, are presented in detail. RESULTS: For the description of phenotypic features, the denominator varies as the literature was insufficient to allow for complete ascertainment of all data for the 35 cases. An extensive review of all known MTO1 deficiency cases revealed the most common features at presentation to be lactic acidosis (LA) (21/34; 62% cases) and hypertrophic cardiomyopathy (15/34; 44% cases). Eventually lactic acidosis and hypertrophic cardiomyopathy are described in 35/35 (100%) and 27/34 (79%) of patients with MTO1 deficiency, respectively; with global developmental delay/intellectual disability present in 28/29 (97%), feeding difficulties in 17/35 (49%), failure to thrive in 12/35 (34%), seizures in 12/35 (34%), optic atrophy in 11/21 (52%) and ataxia in 7/34 (21%). There are 19 different pathogenic MTO1 variants identified in these 35 cases: one splice-site, 3 frameshift and 15 missense variants. None have bi-allelic variants that completely inactivate MTO1; however, patients where one variant is truncating (i.e. frameshift) while the second one is a missense appear to have a more severe, even fatal, phenotype. These data suggest that complete loss of MTO1 is not viable. A ketogenic diet may have exerted a favourable effect on seizures in 2/5 patients. CONCLUSION: MTO1 deficiency is lethal in some but not all cases, and a genotype-phenotype relation is suggested. Aside from lactic acidosis and cardiomyopathy, developmental delay and other phenotypic features affecting multiple organ systems are often present in these patients, suggesting a broader spectrum than hitherto reported. The diagnosis should be suspected on clinical features and the presence of markers of mitochondrial dysfunction in body fluids, especially low residual complex I, III and IV activity in muscle. Molecular confirmation is required and targeted genomic testing may be the most efficient approach. Although subjective clinical improvement was observed in a small number of patients on therapies such as ketogenic diet and dichloroacetate, no evidence-based effective therapy exists.

Combined Respiratory Chain Deficiency and UQCC2 Mutations in Neonatal Encephalomyopathy: Defective Supercomplex Assembly in Complex III Deficiencies.

Vertebrate respiratory chain complex III consists of eleven subunits. Mutations in five subunits either mitochondrial (MT-CYB) or nuclear (CYC1, UQCRC2, UQCRB, and UQCRQ) encoded have been reported. Defects in five further factors for assembly (TTC19, UQCC2, and UQCC3) or iron-sulphur cluster loading (BCS1L and LYRM7) cause complex III deficiency. Here, we report a second patient with UQCC2 deficiency. This girl was born prematurely; pregnancy was complicated by intrauterine growth retardation and oligohydramnios. She presented with respiratory distress syndrome, developed epileptic seizures progressing to status epilepticus, and died at day 33. She had profound lactic acidosis and elevated urinary pyruvate. Exome sequencing revealed two homozygous missense variants in UQCC2, leading to a severe reduction of UQCC2 protein. Deficiency of complexes I and III was found enzymatically and on the protein level. A review of the literature on genetically distinct complex III defects revealed that, except TTC19 deficiency, the biochemical pattern was very often a combined respiratory chain deficiency. Besides complex III, typically, complex I was decreased, in some cases complex IV. In accordance with previous observations, the presence of assembled complex III is required for the stability or assembly of complexes I and IV, which might be related to respirasome/supercomplex formation.

Mutation screen reveals novel variants and expands the phenotypes associated with DYNC1H1.

Dynein, cytoplasmic 1, heavy chain 1 (DYNC1H1) encodes a necessary subunit of the cytoplasmic dynein complex, which traffics cargo along microtubules. Dominant DYNC1H1 mutations are implicated in neural diseases, including spinal muscular atrophy with lower extremity dominance (SMA-LED), intellectual disability with neuronal migration defects, malformations of cortical development, and Charcot-Marie-Tooth disease, type 2O. We hypothesized that additional variants could be found in these and novel motoneuron and related diseases. Therefore, we analyzed our database of 1024 whole exome sequencing samples of motoneuron and related diseases for novel single nucleotide variations. We filtered these results for significant variants, which were further screened using segregation analysis in available family members. Analysis revealed six novel, rare, and highly conserved variants. Three of these are likely pathogenic and encompass a broad phenotypic spectrum with distinct disease clusters. Our findings suggest that DYNC1H1 variants can cause not only lower, but also upper motor neuron disease. It thus adds DYNC1H1 to the growing list of spastic paraplegia related genes in microtubule-dependent motor protein pathways.

Clinical outcome, biochemical and therapeutic follow-up in 14 Austrian patients with Long-Chain 3-Hydroxy Acyl CoA Dehydrogenase Deficiency (LCHADD).

BACKGROUND: LCHADD is a long-fatty acid oxidation disorder with immediate symptoms and long-term complications. We evaluated data on clinical status, biochemical parameters, therapeutic regimens and outcome of Austrian LCHADD patients. STUDY DESIGN: Clinical and outcome data including history, diagnosis, short- and long-term manifestations, growth, psychomotor development, hospitalizations, therapy of 14 Austrian patients with LCHADD were evaluated. Biochemically, we evaluated creatine kinase (CK) and acyl carnitine profiles. RESULTS: All LCHADD patients are homozygous for the common mutation. Three are siblings. Diagnosis was first established biochemically. Nine/14 (64%) were prematures, with IRDS occurring in six. In nine (64%), diagnosis was established through newborn screening, the remaining five (36%) were diagnosed clinically. Four pregnancies were complicated by HELLP syndrome, one by preeclampsia. In two, intrauterine growth retardation and placental insufficiency were reported. Five were diagnosed with hepatopathy at some point, seven with cardiomyopathy and eight with retinopathy, clinically relevant only in one patient. Polyneuropathy is only present in one. Three patients have a PEG, one is regularly fed via NG-tube. Growth is normal in all, as well as psychomotor development, except for two extremely premature girls. In 11 patients, 165 episodes with elevated creatine kinase concentrations were observed with 6-31 (median 14) per patient; three have shown no elevated CK concentrations. Median total carnitine on therapy was 19 µmol/l (range 11-61). For 14 patients, there have been 181 hospitalizations (median 9 per patient), comprising 1337 in-patient-days. All centres adhere to treatment with a fat-defined diet; patients have between 15% and 40% of their energy intake from fat (median 29%), out of which between 20% and 80% are medium-chain triglycerides (MCT) (median 62%). Four patients have been treated with heptanoate (C7). CONCLUSION: Our data show LCHADD outcome can be favourable. Growth and psychomotor development is normal, except in two prematures. Frequency of CK measurements decreases with age, correlating with a decreasing number of hospitalizations. About 50% develop complications affecting different organ systems. There is no relevant difference between the patients treated in the respective centers. Concluding from single case reports, anaplerotic therapy with heptanoate should be further evaluated.

Cross-sectional study of 168 patients with hepatorenal tyrosinaemia and implications for clinical practice.

BACKGROUND: Hepatorenal tyrosinaemia (Tyr 1) is a rare inborn error of tyrosine metabolism. Without treatment, patients are at high risk of developing acute liver failure, renal dysfunction and in the long run hepatocellular carcinoma. The aim of our study was to collect cross-sectional data. METHODS: Via questionnaires we collected retrospective data of 168 patients with Tyr 1 from 21 centres (Europe, Turkey and Israel) about diagnosis, treatment, monitoring and outcome. In a subsequent consensus workshop, we discussed data and clinical implications. RESULTS: Early treatment by NTBC accompanied by diet is essential to prevent serious complications such as liver failure, hepatocellular carcinoma and renal disease. As patients may remain initially asymptomatic or develop uncharacteristic clinical symptoms in the first months of life newborn mass screening using succinylacetone (SA) as a screening parameter in dried blood is mandatory for early diagnosis. NTBC-treatment has to be combined with natural protein restriction supplemented with essential amino acids. NTBC dosage should be reduced to the minimal dose allowing metabolic control, once daily dosing may be an option in older children and adults in order to increase compliance. Metabolic control is judged by SA (below detection limit) in dried blood or urine, plasma tyrosine (<400 µM) and NTBC-levels in the therapeutic range (20-40 µM). Side effects of NTBC are mild and often transient. Indications for liver transplantation are hepatocellular carcinoma or failure to respond to NTBC. Follow-up procedures should include liver and kidney function tests, tumor markers and imaging, ophthalmological examination, blood count, psychomotor and intelligence testing as well as therapeutic monitoring (SA, tyrosine, NTBC in blood). CONCLUSION: Based on the data from 21 centres treating 168 patients we were able to characterize current practice and clinical experience in Tyr 1. This information could form the basis for clinical practice recommendations, however further prospective data are required to underpin some of the recommendations.

Unrelated CD3/CD19-depleted peripheral stem cell transplantation for Hurler syndrome.

For patients with mucopolysaccharidosis type IH (MPS1-H; Hurler syndrome), early allogeneic hematopoietic stem cell transplantation (HSCT) is the treatment of choice. One boy and one girl aged 20.5 and 22 months, respectively, with MPS1-H received a conditioning regimen consisting of thiotepa, fludarabine, treosulfan, and ATG. Grafts were peripheral blood stem cells from unrelated donors (10/12 and 11/11 matched), that were manipulated by CD3/CD19 depletion and contained 20.3 and 28.2 × 10(6) CD34+ cells/kg body weight, respectively. Both patients achieved stable hematopoietic engraftment and stable donor chimerism. Neither acute or chronic graft-versus-host disease (GVHD) nor other severe transplant-related complications occurred. At a follow-up of 48 and 37 months, both patients are alive and well with normal levels of α-L-iduronidase and have made major neurodevelopmental progress. Treosulfan-based conditioning offers the advantage of reduced toxicity; the use of unrelated CD3/CD19-depleted peripheral stem cell grafts allows transfusion of high CD34+ cell numbers together with a "tailored" number of CD3+ cells as well as engraftment facilitating cells in order to achieve rapid hematopoietic engraftment while reducing the risk of graft rejection and GVHD. This regimen might be an additional option when unrelated donor HSCT is considered for a patient with MPS1-H.

Propionic acidemia: clinical course and outcome in 55 pediatric and adolescent patients.

BACKGROUND: Propionic acidemia is an inherited disorder caused by deficiency of propionyl-CoA carboxylase. Although it is one of the most frequent organic acidurias, information on the outcome of affected individuals is still limited. STUDY DESIGN/METHODS: Clinical and outcome data of 55 patients with propionic acidemia from 16 European metabolic centers were evaluated retrospectively. 35 patients were diagnosed by selective metabolic screening while 20 patients were identified by newborn screening. Endocrine parameters and bone age were evaluated. In addition, IQ testing was performed and the patients' and their families' quality of life was assessed. RESULTS: The vast majority of patients (>85%) presented with metabolic decompensation in the neonatal period. Asymptomatic individuals were the exception. About three quarters of the study population was mentally retarded, median IQ was 55. Apart from neurologic symptoms, complications comprised hematologic abnormalities, cardiac diseases, feeding problems and impaired growth. Most patients considered their quality of life high. However, according to the parents' point of view psychic problems were four times more common in propionic acidemia patients than in healthy controls. CONCLUSION: Our data show that the outcome of propionic acidemia is still unfavourable, in spite of improved clinical management. Many patients develop long-term complications affecting different organ systems. Impairment of neurocognitive development is of special concern. Nevertheless, self-assessment of quality of life of the patients and their parents yielded rather positive results.

Prevalence of tetrahydrobiopterine (BH4)-responsive alleles among Austrian patients with PAH deficiency: comprehensive results from molecular analysis in 147 patients.

Phenylketonuria (PKU, MIM 261600) is an autosomal recessive disorder caused by mutations of the phenylalanine hydroxylase gene (PAH, GenBank U49897.1, RefSeq NM_000277). To date more than 560 variants of the PAH gene have been identified. In Europe there is regional distribution of specific mutations. Due to recent progress in chaperone therapy, the prevalence of BH4-responsive alleles gained therapeutic importance. Here we report the mutational spectrum of PAH deficiency in 147 unrelated Austrian families. Overall mutation detection rate was 98.6 %. There was a total of 62 disease-causing mutations, including five novel mutations IVS4 + 6T>A, p.H290Y, IVS8-2A>G, p.A322V and p.I421S. The five most prevalent mutations found in patients were p.R408W, IVS12 + 1G>A, p.R261Q, p.R158Q and IVS2 + 5G>C. Neonatal phenylalanine levels before treatment were available in 114/147 patients. Prediction of BH4-responsiveness in patients with full genotypes was exclusively made according to published data. Among the 133 patients needing dietary treatment, 28.4 % are expected to be BH4 "non-responsive", 4.5 % are highly likely BH4-responsive, 35.8 % are probably BH4-responsive while no interpretation was possible for 31.3 %. The mutation data reflect the population history of Austria and provide information on the likely proportion of Austrian PKU patients that may benefit from BH4-therapy.