Treatment recommendations for glycogen storage disease type IB- associated neutropenia and neutrophil dysfunction with empagliflozin: Consensus from an international workshop.

Glycogen storage disease type Ib (GSD Ib, biallelic variants in SLC37A4) is a rare disorder of glycogen metabolism complicated by neutropenia/neutrophil dysfunction. Since 2019, the SGLT2-inhibitor empagliflozin has provided a mechanism-based treatment option for the symptoms caused by neutropenia/neutrophil dysfunction (e.g. mucosal lesions, inflammatory bowel disease). Because of the rarity of GSD Ib, the published evidence on safety and efficacy of empagliflozin is still limited and does not allow to develop evidence-based guidelines. Here, an international group of experts provides 14 best practice consensus treatment recommendations based on expert practice and review of the published evidence. We recommend to start empagliflozin in all GSD Ib individuals with clinical or laboratory signs related to neutropenia/neutrophil dysfunction with a dose of 0.3-0.4 mg/kg/d given as a single dose in the morning. Treatment can be started in an outpatient setting. The dose should be adapted to the weight and in case of inadequate clinical treatment response or side effects. We strongly recommend to pause empagliflozin immediately in case of threatening dehydration and before planned longer surgeries. Discontinuation of G-CSF therapy should be attempted in all individuals. If available, 1,5-AG should be monitored. Individuals who have previously not tolerated starches should be encouraged to make a new attempt to introduce starch in their diet after initiation of empagliflozin treatment. We advise to monitor certain safety and efficacy parameters and recommend continuous, alternatively frequent glucose measurements during the introduction of empagliflozin. We provide specific recommendations for special circumstances like pregnancy and liver transplantation.

Empagliflozin for treating neutropenia and neutrophil dysfunction in 21 infants with glycogen storage disease 1b.

Empagliflozin has been successfully repurposed for treating neutropenia and neutrophil dysfunction in patients with glycogen storage disease type 1b (GSD 1b), however, data in infants are missing. We report on efficacy and safety of empagliflozin in infants with GSD 1b. This is an international retrospective case series on 21 GSD 1b infants treated with empagliflozin (total treatment time 20.6 years). Before starting empagliflozin (at a median age of 8.1 months with a median dose of 0.3 mg/kg/day) 12 patients had clinical signs and symptoms of neutrophil dysfunction. Six of these previously symptomatic patients had no further neutropenia/neutrophil dysfunction-associated findings on empagliflozin. Eight patients had no signs and symptoms of neutropenia/neutrophil dysfunction before start and during empagliflozin treatment. One previously asymptomatic individual with a horseshoe kidney developed a central line infection with pyelonephritis and urosepsis during empagliflozin treatment. Of the 10 patients who were treated with G-CSF before starting empagliflozin, this was stopped in four and decreased in another four. Eleven individuals were never treated with G-CSF. While in 17 patients glucose homeostasis remained stable on empagliflozin, four showed glucose homeostasis instability in the introductory phase. In 17 patients, no other side effects were reported, while genital (n = 2) or oral (n = 1) candidiasis and skin infection (n = 1) were reported in the remaining four. Empagliflozin had a good effect on neutropenia/neutrophil dysfunction-related signs and symptoms and a favourable safety profile in infants with GSD 1b and therefore qualifies for further exploration as first line treatment.

Endocrine involvement in hepatic glycogen storage diseases: pathophysiology and implications for care.

Hepatic glycogen storage diseases constitute a group of disorders due to defects in the enzymes and transporters involved in glycogen breakdown and synthesis in the liver. Although hypoglycemia and hepatomegaly are the primary manifestations of (most of) hepatic GSDs, involvement of the endocrine system has been reported at multiple levels in individuals with hepatic GSDs. While some endocrine abnormalities (e.g., hypothalamic­pituitary axis dysfunction in GSD I) can be direct consequence of the genetic defect itself, others (e.g., osteopenia in GSD Ib, insulin-resistance in GSD I and GSD III) may be triggered by the (dietary/medical) treatment. Being aware of the endocrine abnormalities occurring in hepatic GSDs is essential (1) to provide optimized medical care to this group of individuals and (2) to drive research aiming at understanding the disease pathophysiology. In this review, a thorough description of the endocrine manifestations in individuals with hepatic GSDs is presented, including pathophysiological and clinical implications.

Repurposing empagliflozin in individuals with glycogen storage disease Ib: A value-based healthcare approach and systematic benefit-risk assessment.

Off-label repurposing of empagliflozin allows pathomechanism-based treatment of neutropenia/neutrophil-dysfunction in glycogen storage disease type Ib (GSDIb). From a value-based healthcare (VBHC) perspective, we here retrospectively studied patient-reported, clinical and pharmacoeconomic outcomes in 11 GSDIb individuals before and under empagliflozin at two centers (the Netherlands [NL], Austria [AT]), including a budget impact analysis, sensitivity-analysis, and systematic benefit-risk assessment. Under empagliflozin, all GSDIb individuals reported improved quality-of-life-scores. Neutrophil dysfunction related symptoms allowed either granulocyte colony-stimulating factor cessation or tapering. Calculated cost savings per patient per year ranged between € 6482-14 190 (NL) and € 1281-41 231 (AT). The budget impact analysis estimated annual total cost savings ranging between € 75 062-225 716 (NL) and € 37 697-231 790 (AT), based on conservative assumptions. The systematic benefit-risk assessment was favorable. From a VBHC perspective, empagliflozin treatment in GSDIb improved personal and clinical outcomes while saving costs, thereby creating value at multiple pillars. We emphasize the importance to reimburse empagliflozin for GSDIb individuals, further supported by the favorable systematic benefit-risk assessment. These observations in similar directions in two countries/health care systems strongly suggest that our findings can be extrapolated to other geographical areas and health care systems.

Repurposing SGLT2 inhibitors: Treatment of renal proximal tubulopathy in Fanconi-Bickel syndrome with empagliflozin.

Renal proximal tubulopathy in Fanconi-Bickel syndrome is caused by impaired basolateral glucose transport via GLUT2 and consequently, intracellular accumulation of glucose and glycogen. SGLT2 inhibitors act on apical glucose reabsorption of renal proximal tubular cells. The purpose of this study was to retrospectively describe the first experiences with repurposing the SGLT2 inhibitor empagliflozin to treat the generalized tubulopathy in Fanconi-Bickel syndrome. A case series was conducted of seven persons from five families (five males, two females; three children, who were 14y5m, 2y9m, and 1y6m old) with genetically confirmed Fanconi-Bickel syndrome, off-label treated with empagliflozin. Median (range) age at start of empagliflozin was 27 years (1y6m - 61y) and duration of follow-up under empagliflozin treatment was 169 days (57-344). Under empagliflozin (up to 25 mg/d), biochemical parameters of tubular cell integrity (urinary N-acetyl-glucosaminidase) and/or tubular functions (including urinary α1-microglobulin) improved in all persons with Fanconi-Bickel syndrome, albeit to varying degrees. Clinically, supplementations (i.e., phosphate, alkali, carnitine, and alfacalcidol) could be completely discontinued in the three children, whereas results in the four adult patients were more variable and not as significant. Empagliflozin was well-tolerated and no symptomatic hypoglycemia was observed. In conclusion, SGLT2 inhibitors such as empagliflozin shift the metabolic block in Fanconi-Bickel syndrome, that is, they intervene specifically in the underlying pathophysiology and can thus attenuate renal proximal tubulopathy, especially when started in early childhood.

Personalised modelling of clinical heterogeneity between medium-chain acyl-CoA dehydrogenase patients.

BACKGROUND: Monogenetic inborn errors of metabolism cause a wide phenotypic heterogeneity that may even differ between family members carrying the same genetic variant. Computational modelling of metabolic networks may identify putative sources of this inter-patient heterogeneity. Here, we mainly focus on medium-chain acyl-CoA dehydrogenase deficiency (MCADD), the most common inborn error of the mitochondrial fatty acid oxidation (mFAO). It is an enigma why some MCADD patients-if untreated-are at risk to develop severe metabolic decompensations, whereas others remain asymptomatic throughout life. We hypothesised that an ability to maintain an increased free mitochondrial CoA (CoASH) and pathway flux might distinguish asymptomatic from symptomatic patients. RESULTS: We built and experimentally validated, for the first time, a kinetic model of the human liver mFAO. Metabolites were partitioned according to their water solubility between the bulk aqueous matrix and the inner membrane. Enzymes are also either membrane-bound or in the matrix. This metabolite partitioning is a novel model attribute and improved predictions. MCADD substantially reduced pathway flux and CoASH, the latter due to the sequestration of CoA as medium-chain acyl-CoA esters. Analysis of urine from MCADD patients obtained during a metabolic decompensation showed an accumulation of medium- and short-chain acylcarnitines, just like the acyl-CoA pool in the MCADD model. The model suggested some rescues that increased flux and CoASH, notably increasing short-chain acyl-CoA dehydrogenase (SCAD) levels. Proteome analysis of MCADD patient-derived fibroblasts indeed revealed elevated levels of SCAD in a patient with a clinically asymptomatic state. This is a rescue for MCADD that has not been explored before. Personalised models based on these proteomics data confirmed an increased pathway flux and CoASH in the model of an asymptomatic patient compared to those of symptomatic MCADD patients. CONCLUSIONS: We present a detailed, validated kinetic model of mFAO in human liver, with solubility-dependent metabolite partitioning. Personalised modelling of individual patients provides a novel explanation for phenotypic heterogeneity among MCADD patients. Further development of personalised metabolic models is a promising direction to improve individualised risk assessment, management and monitoring for inborn errors of metabolism.

Duocarmycin SA Reduces Proliferation and Increases Apoptosis in Acute Myeloid Leukemia Cells In Vitro.

Acute myeloid leukemia (AML) is a hematological malignancy that is characterized by an expansion of immature myeloid precursors. Despite therapeutic advances, the prognosis of AML patients remains poor and there is a need for the evaluation of promising therapeutic candidates to treat the disease. The objective of this study was to evaluate the efficacy of duocarmycin Stable A (DSA) in AML cells in vitro. We hypothesized that DSA would induce DNA damage in the form of DNA double-strand breaks (DSBs) and exert cytotoxic effects on AML cells within the picomolar range. Human AML cell lines Molm-14 and HL-60 were used to perform 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT), DNA DSBs, cell cycle, 5-ethynyl-2-deoxyuridine (EdU), colony formation unit (CFU), Annexin V, RNA sequencing and other assays described in this study. Our results showed that DSA induced DNA DSBs, induced cell cycle arrest at the G2M phase, reduced proliferation and increased apoptosis in AML cells. Additionally, RNA sequencing results showed that DSA regulates genes that are associated with cellular processes such as DNA repair, G2M checkpoint and apoptosis. These results suggest that DSA is efficacious in AML cells and is therefore a promising potential therapeutic candidate that can be further evaluated for the treatment of AML.

Studies on the binding of CO to low-spin [Fe(II)(Por)L2] complexes: an aid to understanding the binding of CO to haemoglobin and myoglobin.

The visible and Mössbauer spectra of [Fe(II)(Por)L2] and [Fe(II)(Por)L(CO)] complexes (where Por = protoporphyrin IX (PPIX) or tetra(p-sulfophenyl)porphyrin (TPPS) and L = an aliphatic or aromatic nitrogenous base) are reported and discussed. The results are compared to those of previously reported [Fe(II)(Por)L(CO)] complexes (where Por = PPIX, TPPS, PMXPP, TPP, OMTBP and OEP; L = a nitrogenous aromatic ligand) and HbCO (where Hb = haemoglobin) and MyCO (where My = myoglobin). A new approach, to extracting information from the Mössbauer parameters has been developed by plotting those of the [Fe(II)(Por)L2] complexes against those of [Fe(II)(Por)L(CO)] complexes for the same ligands, has yielded a series of trend lines that show a significant dependence on both the nature of the porphyrin and also of the nitrogenous ligand. Different trend lines were found for aromatic nitrogenous ligands to aliphatic nitrogenous ligands showing that the porphyrins could donate different amounts of charge to the Fe(II) cations as the L ligand changed, and hence, they display electron sink properties. From the plots, it was shown that haemoglobin and myoglobin both bind CO very strongly compared to the model complexes studied herein. Using the reported structural and Mössbauer data for the [Fe(II)(Por)L2] and [Fe(II)(Por)L(CO)] complexes, it proved possible and instructive to plot the Mössbauer parameters against a number of the bond lengths around the Fe(II) cations. The interpretation of the resulting trend lines both supported and facilitated the extension of our findings enabling further understanding of the geometry of the bonding in CO haemoglobin and CO myoglobin.

Germline engineering of the chicken genome using CRISPR/Cas9 by in vivo transfection of PGCs.

Development of simple and readily adoptable methods to mediate germline engineering of the chicken genome will have many applications in research, agriculture and industrial biotechnology. We report germline targeting of the endogenous chicken Interferon Alpha and Beta Receptor Subunit 1 (IFNAR1) gene by in vivo transgenic expression of the high-fidelity Cas9 (Cas9-HF1) and guide RNAs (gRNAs) in chickens. First, we developed a Tol2 transposon vector carrying Cas9-HF1, IFNAR1-gRNAs (IF-gRNAs) and green fluorescent protein (GFP) transgenes (pTgRCG) and validated in chicken fibroblast DF1 cells. Next, the pTgRCG plasmid was directly injected into the dorsal aorta of embryonic day (ED) 2.5 chicken embryos targeting the circulating primordial germ cells (PGCs). The resulting chimera roosters generated a fully transgenic generation 1 (G1) hen with constitutive expression of Cas9-HF1 and IF-gRNAs (G1_Tol2-Cas9/IF-gRNA). We detected a spectrum of indels at gRNA-targeted loci in the G1_Tol2-Cas9/IF-gRNA hen and the indels were stably inherited by the G2 progeny. Breeding of the G1_Tol2-Cas9/IF-gRNA hen resulted in up to 10% transgene-free heterozygote IFNAR1 mutants, following null-segregation of the Tol2 insert. The method described here will provide new opportunities for genome editing in chicken and other avian species that lack PGC culture.

Glycogen storage disease type 1a is associated with disturbed vitamin A metabolism and elevated serum retinol levels.

Glycogen storage disease type 1a (GSD Ia) is an inborn error of metabolism caused by mutations in the G6PC gene, encoding the catalytic subunit of glucose-6-phosphatase. Early symptoms include severe fasting intolerance, failure to thrive and hepatomegaly, biochemically associated with nonketotic hypoglycemia, fasting hyperlactidemia, hyperuricemia and hyperlipidemia. Dietary management is the cornerstone of treatment aiming at maintaining euglycemia, prevention of secondary metabolic perturbations and long-term complications, including liver (hepatocellular adenomas and carcinomas), kidney and bone disease (hypovitaminosis D and osteoporosis). As impaired vitamin A homeostasis also associates with similar symptoms and is coordinated by the liver, we here analysed whether vitamin A metabolism is affected in GSD Ia patients and liver-specific G6pc-/- knock-out mice. Serum levels of retinol and retinol binding protein 4 (RBP4) were significantly increased in both GSD Ia patients and L-G6pc-/- mice. In contrast, hepatic retinol levels were significantly reduced in L-G6pc-/- mice, while hepatic retinyl palmitate (vitamin A storage form) and RBP4 levels were not altered. Transcript and protein analyses indicate an enhanced production of retinol and reduced conversion the retinoic acids (unchanged LRAT, Pnpla2/ATGL and Pnpla3 up, Cyp26a1 down) in L-G6pc-/- mice. Aberrant expression of genes involved in vitamin A metabolism was associated with reduced basal messenger RNA levels of markers of inflammation (Cd68, Tnfα, Nos2, Il-6) and fibrosis (Col1a1, Acta2, Tgfß, Timp1) in livers of L-G6pc-/- mice. In conclusion, GSD Ia is associated with elevated serum retinol and RBP4 levels, which may contribute to disease symptoms, including osteoporosis and hepatic steatosis.

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.

Modeling Phenotypic Heterogeneity of Glycogen Storage Disease Type 1a Liver Disease in Mice by Somatic CRISPR/CRISPR-associated protein 9-Mediated Gene Editing.

BACKGROUND AND AIMS: Patients with glycogen storage disease type 1a (GSD-1a) primarily present with life-threatening hypoglycemia and display severe liver disease characterized by hepatomegaly. Despite strict dietary management, long-term complications still occur, such as liver tumor development. Variations in residual glucose-6-phosphatase (G6PC1) activity likely contribute to phenotypic heterogeneity in biochemical symptoms and complications between patients. However, lack of insight into the relationship between G6PC1 activity and symptoms/complications and poor understanding of the underlying disease mechanisms pose major challenges to provide optimal health care and quality of life for GSD-1a patients. Currently available GSD-1a animal models are not suitable to systematically investigate the relationship between hepatic G6PC activity and phenotypic heterogeneity or the contribution of gene-gene interactions (GGIs) in the liver. APPROACH AND RESULTS: To meet these needs, we generated and characterized a hepatocyte-specific GSD-1a mouse model using somatic CRISPR/CRISPR-associated protein 9 (Cas9)-mediated gene editing. Hepatic G6pc editing reduced hepatic G6PC activity up to 98% and resulted in failure to thrive, fasting hypoglycemia, hypertriglyceridemia, hepatomegaly, hepatic steatosis (HS), and increased liver tumor incidence. This approach was furthermore successful in simultaneously modulating hepatic G6PC and carbohydrate response element-binding protein, a transcription factor that is activated in GSD-1a and protects against HS under these conditions. Importantly, it also allowed for the modeling of a spectrum of GSD-1a phenotypes in terms of hepatic G6PC activity, fasting hypoglycemia, hypertriglyceridemia, hepatomegaly and HS. CONCLUSIONS: In conclusion, we show that somatic CRISPR/Cas9-mediated gene editing allows for the modeling of a spectrum of hepatocyte-borne GSD-1a disease symptoms in mice and to efficiently study GGIs in the liver. This approach opens perspectives for translational research and will likely contribute to personalized treatments for GSD-1a and other genetic liver diseases.

Treating neutropenia and neutrophil dysfunction in glycogen storage disease type Ib with an SGLT2 inhibitor.

Neutropenia and neutrophil dysfunction cause serious infections and inflammatory bowel disease in glycogen storage disease type Ib (GSD-Ib). Our discovery that accumulating 1,5-anhydroglucitol-6-phosphate (1,5AG6P) caused neutropenia in a glucose-6-phosphatase 3 (G6PC3)-deficient mouse model and in 2 rare diseases (GSD-Ib and G6PC3 deficiency) led us to repurpose the widely used antidiabetic drug empagliflozin, an inhibitor of the renal glucose cotransporter sodium glucose cotransporter 2 (SGLT2). Off-label use of empagliflozin in 4 GSD-Ib patients with incomplete response to granulocyte colony-stimulating factor (GCSF) treatment decreased serum 1,5AG and neutrophil 1,5AG6P levels within 1 month. Clinically, symptoms of frequent infections, mucosal lesions, and inflammatory bowel disease resolved, and no symptomatic hypoglycemia was observed. GCSF could be discontinued in 2 patients and tapered by 57% and 81%, respectively, in the other 2. The fluctuating neutrophil numbers in all patients were increased and stabilized. We further demonstrated improved neutrophil function: normal oxidative burst (in 3 of 3 patients tested), corrected protein glycosylation (2 of 2), and normal neutrophil chemotaxis (1 of 1), and bactericidal activity (1 of 1) under treatment. In summary, the glucose-lowering SGLT2 inhibitor empagliflozin, used for type 2 diabetes, was successfully repurposed for treating neutropenia and neutrophil dysfunction in the rare inherited metabolic disorder GSD-Ib without causing symptomatic hypoglycemia. We ascribe this to an improvement in neutrophil function resulting from the reduction of the intracellular concentration of 1,5AG6P.

Studies on the binding of nitrogenous bases to protoporphyrin IX iron(II) in aqueous solution at high pH values.

Studies are reported on the formation of low-spin six-coordinate [Fe(PPIX)L2] complexes from iron(II) protoporphyrin where L is one of a series of nitrogenous ligands (aliphatic, aromatic or heterocyclic). The bonding constants have been determined by titration of the metal complex with these ligands and are compared in relation to previous studies. The adduct formation was monitored utilising optical spectroscopy. In addition, MÓ§ssbauer spectroscopic experiments were conducted to monitor the electronic environment around the central iron atom in these complexes. The two complementary spectroscopic methods indicated that all nitrogen ligands formed low-spin octahedral complexes. The magnitude of the overall binding constants (ß2 values) are discussed and related to (a) the pKa values of the free ligands and (b) the Mössbauer parameter ΔEQ, which represents the quadrupole splitting of the haem iron. The ß2 and ΔEQ values are also discussed in terms of the structure of the ligand. Cooperative binding was observed for nearly all the ligands with Hill coefficients close to 2 for iron(II) protoporphyrin; one of these ligands displayed a much greater affinity than any we previously studied, and this was a direct consequence of the structure of the ligand. Overall conclusions on these and previous studies are drawn in terms of aliphatic ligands versus aromatic ring structures and the absence or presence of sterically hindered nitrogen atoms. The implications of the work for the greater understanding of haem proteins in general and in particular how the nitrogenous ligand binding results are relevant to and aid the understanding of the binding of inhibitor molecules to the cytochrome P450 mono-oxygenases (for therapeutic purposes) are also discussed. Changes in the electronic absorption spectra of five-coordinate [Fe(II)(PPIX)(2-MeIm)] that occurred as the temperature was lowered from room temperature to 78° K.

Predator Scent-Induced Sensitization of Hypertension and Anxiety-like Behaviors.

Post-traumatic stress disorder (PTSD), an anxiety-related syndrome, is associated with increased risk for cardiovascular diseases. The present study investigated whether predator scent (PS) stress, a model of PTSD, induces sensitization of hypertension and anxiety-like behaviors and underlying mechanisms related to renin-angiotensin systems (RAS) and inflammation. Coyote urine, as a PS stressor, was used to model PTSD. After PS exposures, separate cohorts of rats were studied for hypertensive response sensitization (HTRS), anxiety-like behaviors, and changes in plasma levels and mRNA expression of several components of the RAS and proinflammatory cytokines (PICs) in the lamina terminalis (LT), paraventricular nucleus (PVN), and amygdala (AMY). Rats exposed to PS as compared to control animals exhibited (1) a significantly greater hypertensive response (i.e., HTRS) when challenged with a slow-pressor dose of angiotensin (ANG) II, (2) significant decrease in locomotor activity and increase in time spent in the closed arms of a plus maze as well as general immobility (i.e., behavioral signs of increased anxiety), (3) upregulated plasma levels of ANG II and interleukin-6, and (4) increased expression of message for components of the RAS and PICs in key brain nuclei. All the PS-induced adverse effects were blocked by pretreatment with either an angiotensin-converting enzyme antagonist or a tumor necrosis factor-α inhibitor. The results suggest that PS, used as an experimental model of PTSD, sensitizes ANG II-induced hypertension and produces behavioral signs of anxiety, probably through upregulation of RAS components and inflammatory markers in plasma and brain areas associated with anxiety and blood pressure control.

Towards values-based healthcare for inherited metabolic disorders: An overview of current practices for persons with liver glycogen storage disease and fatty acid oxidation disorders.

Value-based healthcare (VBHC) intends to achieve better outcomes for patients, to improve quality of patient care, with reduced costs. Four dimensions define a model of intimately related value-pillars: personal value, allocative value, technical value, and societal value. VBHC is mostly applied in common diseases, and there are fundamental challenges in applying VBHC strategies to low volume, high complex healthcare situations, such as rare diseases, including inherited metabolic disorders. This article summarizes current practices at various academical domains (i.e., research, healthcare, education, and training) that (aim to) increase values at various value-pillars for persons with liver glycogen storage diseases or fatty acid oxidation disorders and their families. Future perspectives may include facilitating virtual networks to function as integrated practice units, improving measurement of outcomes, and creating information technology platforms to overcome the ethical, legal, societal, and technical challenges of data sharing for healthcare and research purposes.

Plasma carnitine concentrations in Medium-chain acyl-CoA dehydrogenase deficiency: lessons from an observational cohort study.

Our aim was to study the effect of secondary carnitine deficiency (SCD) and carnitine supplementation on important outcome measures for persons with medium-chain Acyl-CoA dehydrogenase deficiency (MCADD). We performed a large retrospective observational study using all recorded visits of persons with MCADD in the University Medical Center Groningen, the Netherlands, between October 1994 and October 2019. Frequency and duration of acute unscheduled preventive hospital visits, exercise tolerance, fatigue, and muscle pain were considered important clinical outcomes and were studied in relation to (acyl)carnitine profile and carnitine supplementation status. The study encompassed 1228 visits of 93 persons with MCADD. >60% had SCD during follow-up. This included only persons with severe MCADD. Carnitine supplementation and SCD were unrelated to the frequency and duration of the acute unscheduled preventive hospital visits (P > 0.05). The relative risk for fatigue, muscle ache, or exercise intolerance was equal between persons with and without SCD (RR 1.6, 95% CI 0.48-5.10, P = 0.4662). No episodes of metabolic crisis were recorded in non-carnitine-supplemented persons with MCADD and SCD. In some persons with MCADD, SCD resolved without carnitine supplementation. There is absence of real-world evidence in favor of routine carnitine analysis and carnitine supplementation in the follow-up of persons with MCADD.

Genetic, biochemical, and clinical spectrum of patients with mitochondrial trifunctional protein deficiency identified after the introduction of newborn screening in the Netherlands.

Long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD) is included in many newborn screening (NBS) programs. Acylcarnitine-based NBS for LCHADD not only identifies LCHADD, but also the other deficiencies of the mitochondrial trifunctional protein (MTP), a multi-enzyme complex involved in long-chain fatty acid ß-oxidation. Besides LCHAD, MTP harbors two additional enzyme activities: long-chain enoyl-CoA hydratase (LCEH) and long-chain ketoacyl-CoA thiolase (LCKAT). Deficiency of one or more MTP activities causes generalized MTP deficiency (MTPD), LCHADD, LCEH deficiency (not yet reported), or LCKAT deficiency (LCKATD). To gain insight in the outcomes of MTP-deficient patients diagnosed after the introduction of NBS for LCHADD in the Netherlands, a retrospective evaluation of genetic, biochemical, and clinical characteristics of MTP-deficient patients, identified since 2007, was carried out. Thirteen patients were identified: seven with LCHADD, five with MTPD, and one with LCKATD. All LCHADD patients (one missed by NBS, clinical diagnosis) and one MTPD patient (clinical diagnosis) were alive. Four MTPD patients and one LCKATD patient developed cardiomyopathy and died within 1 month and 13 months of life, respectively. Surviving patients did not develop symptomatic hypoglycemia, but experienced reversible cardiomyopathy and rhabdomyolysis. Five LCHADD patients developed subclinical neuropathy and/or retinopathy. In conclusion, patient outcomes were highly variable, stressing the need for accurate classification of and discrimination between the MTP deficiencies to improve insight in the yield of NBS for LCHADD. NBS allowed the prevention of symptomatic hypoglycemia, but current treatment options failed to treat cardiomyopathy and prevent long-term complications. Moreover, milder patients, who might benefit from NBS, were missed due to normal acylcarnitine profiles.

Marker counter-selection via CRISPR/Cas9 co-targeting for efficient generation of genome edited avian cell lines and germ cells.

Efficient isolation of genetically modified cells that are phenotypically indistinguishable from the unmodified cells remains a major technical barrier for the broader utilization of CRISPR/Cas9. Here, we report a novel enrichment approach to select the genome engineered cells by co-targeting a genomically integrated GFP gene along with the endogenous gene of interest (GOI). Using this co-targeting approach, multiple genomic loci were successfully targeted in chicken (DF1) and quail (CEC-32) fibroblast cell lines by transient transfection of Cas9 and guide RNAs (gRNAs). Clonal isolation of co-targeted DF1 cells showed 75% of cell clones had deletion of GFP and biallelic deletion of the GOI. To assess the utility of this approach to generate genome modified animals, we tested it on chicken primordial germ cells (PGCs) expressing GFP by co-targeting with gRNAs against GFP and endogenous ovomucoid (OVM) gene. PGCs enriched for loss of GFP and confirmed for OVM deletion, derived by co-targeting, were injected into Hamburger and Hamilton stage 14-15 chicken embryos, and their ability to migrate to the genital ridge was confirmed. This simple, efficient enrichment approach could easily be applied to the creation of knock-out or edited cell lines or animals.

Double Rayleigh scattering in a digitally enhanced, all-fiber optical frequency reference.

We demonstrate a passive, all-optical fiber frequency reference using a digitally enhanced homodyne interferometric phase readout. We model the noise contributions from fiber thermal noise and the coupling of double Rayleigh scattering in a digitally enhanced homodyne interferometer. A system frequency stability of 0.1 Hz/Hz is achieved above 100 Hz, which coincides with the double Rayleigh scattering estimate and is approximately a factor of five above the thermo-dynamic noise limit.