IL-37 requires the receptors IL-18Rα and IL-1R8 (SIGIRR) to carry out its multifaceted anti-inflammatory program upon innate signal transduction.

Interleukin 37 (IL-37) and IL-1R8 (SIGIRR or TIR8) are anti-inflammatory orphan members of the IL-1 ligand family and IL-1 receptor family, respectively. Here we demonstrate formation and function of the endogenous ligand-receptor complex IL-37-IL-1R8-IL-18Rα. The tripartite complex assembled rapidly on the surface of peripheral blood mononuclear cells upon stimulation with lipopolysaccharide. Silencing of IL-1R8 or IL-18Rα impaired the anti-inflammatory activity of IL-37. Whereas mice with transgenic expression of IL-37 (IL-37tg mice) with intact IL-1R8 were protected from endotoxemia, IL-1R8-deficient IL-37tg mice were not. Proteomic and transcriptomic investigations revealed that IL-37 used IL-1R8 to harness the anti-inflammatory properties of the signaling molecules Mer, PTEN, STAT3 and p62(dok) and to inhibit the kinases Fyn and TAK1 and the transcription factor NF-κB, as well as mitogen-activated protein kinases. Furthermore, IL-37-IL-1R8 exerted a pseudo-starvational effect on the metabolic checkpoint kinase mTOR. IL-37 thus bound to IL-18Rα and exploited IL-1R8 to activate a multifaceted intracellular anti-inflammatory program.

Rad50-CARD9 interactions link cytosolic DNA sensing to IL-1ß production.

Double-stranded DNA (dsDNA) in the cytoplasm triggers the production of interleukin 1ß (IL-1ß) as an antiviral host response, and deregulation of the pathways involved can promote inflammatory disease. Here we report a direct cytosolic interaction between the DNA-damage sensor Rad50 and the innate immune system adaptor CARD9. Transfection of dendritic cells with dsDNA or infection of dendritic cells with a DNA virus induced the formation of dsDNA-Rad50-CARD9 signaling complexes for activation of the transcription factor NF-κB and the generation of pro-IL-1ß. Primary cells conditionally deficient in Rad50 or lacking CARD9 consequently exhibited defective DNA-induced production of IL-1ß, and Card9(-/-) mice had impaired inflammatory responses after infection with a DNA virus in vivo. Our results define a cytosolic DNA-recognition pathway for inflammation and a physical and functional connection between a conserved DNA-damage sensor and the innate immune response to pathogens.

Collaborative evaluation study on 18 candidate diseases for newborn screening in 1.77 million samples.

Analytical and therapeutic innovations led to a continuous but variable extension of newborn screening (NBS) programmes worldwide. Every extension requires a careful evaluation of feasibility, diagnostic (process) quality and possible health benefits to balance benefits and limitations. The aim of this study was to evaluate the suitability of 18 candidate diseases for inclusion in NBS programmes. Utilising tandem mass spectrometry as well as establishing specific diagnostic pathways with second-tier analyses, three German NBS centres designed and conducted an evaluation study for 18 candidate diseases, all of them inherited metabolic diseases. In total, 1 777 264 NBS samples were analysed. Overall, 441 positive NBS results were reported resulting in 68 confirmed diagnoses, 373 false-positive cases and an estimated cumulative prevalence of approximately 1 in 26 000 newborns. The positive predictive value ranged from 0.07 (carnitine transporter defect) to 0.67 (HMG-CoA lyase deficiency). Three individuals were missed and 14 individuals (21%) developed symptoms before the positive NBS results were reported. The majority of tested candidate diseases were found to be suitable for inclusion in NBS programmes, while multiple acyl-CoA dehydrogenase deficiency, isolated methylmalonic acidurias, propionic acidemia and malonyl-CoA decarboxylase deficiency showed some and carnitine transporter defect significant limitations. Evaluation studies are an important tool to assess the potential benefits and limitations of expanding NBS programmes to new diseases.

iBRET Screen of the ABCD1 Peroxisomal Network and Mutation-Induced Network Perturbations.

Mapping the network of proteins provides a powerful means to investigate the function of disease genes and to unravel the molecular basis of phenotypes. We present an automated informatics-aided and bioluminescence resonance energy transfer-based approach (iBRET) enabling high-confidence detection of protein-protein interactions in living mammalian cells. A screen of the ABCD1 protein, which is affected in X-linked adrenoleukodystrophy (X-ALD), against an organelle library of peroxisomal proteins demonstrated applicability of iBRET for large-scale experiments. We identified novel protein-protein interactions for ABCD1 (with ALDH3A2, DAO, ECI2, FAR1, PEX10, PEX13, PEX5, PXMP2, and PIPOX), mapped its position within the peroxisomal protein-protein interaction network, and determined that pathogenic missense variants in ABCD1 alter the interaction with selected binding partners. These findings provide mechanistic insights into pathophysiology of X-ALD and may foster the identification of new disease modifiers.

Age-dependent favorable visual recovery despite significant retinal atrophy in pediatric MOGAD: how much retina do you really need to see well?

BACKGROUND: To investigate age-related severity, patterns of retinal structural damage, and functional visual recovery in pediatric and adult cohorts of myelin oligodendrocyte glycoprotein antibody-associated disease (MOGAD) optic neuritis (ON). METHODS: All MOGAD patients from the 5 participating centers were included. Patients with initial manifestation <18 years were included in the pediatric (MOGADped) cohort and patients with ≥18 years in the adult (MOGADadult) cohort. For patients with MOGAD ON, examinations at least ≥6 months after ON onset were included in the analyses. Using spectral domain optical coherence tomography (SD-OCT), we acquired peripapillary retinal nerve fiber layer thickness (pRNFL) and volumes of combined ganglion cell and inner plexiform layer (GCIPL). High- and 2.5% low-contrast visual acuity (HCVA, LCVA) and visual-evoked potentials (VEP) were obtained. RESULTS: Twenty MOGADped (10.3±3.7 years, 30 MOGAD ON eyes) and 39 MOGADadult (34.9±11.6 years, 42 MOGAD ON eyes) patients were included. The average number of ON episodes per ON eye was similar in both groups (1.8±1.3 and 2.0±1.7). In both pediatric and adult MOGAD, ON led to pronounced neuroaxonal retinal atrophy (pRNFL: 63.1±18.7 and 64.3±22.9 µm; GCIPL: 0.42±0.09 and 0.44±0.13 mm3, respectively) and moderate delay of the VEP latencies (117.9±10.7 and 118.0±14.5 ms). In contrast, visual acuity was substantially better in children (HCVA: 51.4±9.3 vs. 35.0±20.6 raw letters, p=0.001; LCVA: 22.8±14.6 vs. 13.5±16.4, p=0.028). Complete visual recovery (HCVA-logMAR 0.0) occurred in 73.3% of MOGADped and 31% MOGADadults ON eyes, while 3.3% and 31% demonstrated moderate to severe (logMAR > 0.5) visual impairment. Independent of retinal atrophy, age at ON onset significantly correlated with visual outcome. CONCLUSION: Pediatric MOGAD ON showed better visual recovery than adult MOGAD ON despite profound and almost identical neuroaxonal retinal atrophy. Age-related cortical neuroplasticity may account for the substantial discrepancy between structural changes and functional outcomes.

Diagnostic Challenges Using a 2-Tier Strategy for Methylmalonic Acidurias: Data from 1.2 Million Dried Blood Spots.

BACKGROUND: The detection of methylmalonic acid (MMA) by second-tier analysis has been shown to reduce the number of false positives in newborn screening (NBS) for genetically determined methylmalonic acidurias (MMAuria). In addition to genetic conditions, MMA is an indicator of vitamin B12 status, thus applicable to detect maternal vitamin B12 deficiency in the newborns screened. METHODS: Biochemical and clinical follow-up data of a 7.5-year pilot study with 1.2 million newborns screened were reviewed. RESULTS: Among 1,195,850 NBS samples, 3,595 (0.3%) fulfilled criteria for second-tier analysis of MMA. In 37 (0.003%; 1/32,000) samples, elevated concentrations of MMA were detected, resulting in diagnostic workup at a metabolic center in 21 newborns. In 6 infants (1/199,000), genetic conditions were established, 1 infant with cobalamin C deficiency (CblC) showed only a moderate elevation of MMA. The remaining 15 newborns (1/79,000) displayed significantly lower concentrations of MMA and were evaluated for maternal vitamin B12 deficiency. In 9 mothers, vitamin B12 deficiency was verified, and 6 showed no indication for vitamin B12 deficiency. Treatment with vitamin B12 normalized biochemical parameters in all 15 infants. CONCLUSIONS: Applying a 2-tier strategy measuring MMA in NBS identified genetic conditions of MMAuria. It was possible to separate severe, early-onset phenotypes from maternal vitamin B12 deficiency. However, the detection of CblC deficiency with mildly elevated MMA interferes with impaired vitamin B12 status of unknown relevance and thus burdens possibly healthy newborns. Regarding maternal vitamin B12 deficiency, testing and supplementing mothers-to-be is preferable. This might decrease straining follow-up of newborns and improve quality and overall perception of NBS.

An interactive network of elastase, secretases, and PAR-2 protein regulates CXCR1 receptor surface expression on neutrophils.

CXCL8 (IL-8) recruits and activates neutrophils through the G protein-coupled chemokine receptor CXCR1. We showed previously that elastase cleaves CXCR1 and thereby impairs antibacterial host defense. However, the molecular intracellular machinery involved in this process remained undefined. Here we demonstrate by using flow cytometry, confocal microscopy, subcellular fractionation, co-immunoprecipitation, and bioluminescence resonance energy transfer that combined α- and γ-secretase activities are functionally involved in elastase-mediated regulation of CXCR1 surface expression on human neutrophils, whereas matrix metalloproteases are dispensable. We further demonstrate that PAR-2 is stored in mobilizable compartments in neutrophils. Bioluminescence resonance energy transfer and co-immunoprecipitation studies showed that secretases, PAR-2, and CXCR1 colocalize and physically interact in a novel protease/secretase-chemokine receptor network. PAR-2 blocking experiments provided evidence that elastase increased intracellular presenilin-1 expression through PAR-2 signaling. When viewed in combination, these studies establish a novel functional network of elastase, secretases, and PAR-2 that regulate CXCR1 expression on neutrophils. Interfering with this network could lead to novel therapeutic approaches in neutrophilic diseases, such as cystic fibrosis or rheumatoid arthritis.

Use of sapropterin dihydrochloride in maternal phenylketonuria. A European experience of eight cases.

Sapropterin dihydrochloride (SD) is the first drug treatment for phenylketonuria (PKU), but due to the lack of data, its use in maternal PKU must be undertaken with caution as noted in the FDA and EMEA labels. We collected data from eight pregnancies in PKU women treated with SD and we analysed the phenotypes of these patients, their tetrahydrobiopterin (BH4) responsiveness, the indications for SD treatment, the efficacy (metabolic control, phenylalanine (Phe) tolerance and offspring outcome) and the safety data. Results showed that in the seven patients known to be responsive to BH4, the use of SD during pregnancy was efficient in terms of metabolic control and Phe tolerance. The indications for giving SD included the failure of the low-Phe diet (n = 3), the fact that some of these women had never experienced the low Phe diet (n = 2), one unexpected pregnancy in a woman currently on SD and one pregnancy where the foetus was known to have PKU. The offspring of these seven pregnancies were all normal babies with normal birth measurements and outcomes. No side effect related to SD was observed in these seven cases. In the eighth case, SD was prescribed as a rescue treatment without previous knowledge of the BH4 responsiveness to a woman who was already 8 weeks pregnant without diet. The birth occurred at 33 weeks of gestational age with Potter syndrome (probably related to the absence of metabolic control during the first trimester) and the baby died in the first hours of life. In conclusion, the data presented here provides the first evidence that treatment with pharmacological doses of SD appears to be efficient and safe in women with PKU during pregnancy. Its use should, however, be restricted to those women previously identified to be clear responders to BH4.

Identification of a new fatty acid synthesis-transport machinery at the peroxisomal membrane.

The neurodegenerative disease X-linked adrenoleukodystrophy (X-ALD) is characterized by the abnormal accumulation of very long chain fatty acids. Mutations in the gene encoding the peroxisomal ATP-binding cassette half-transporter, adrenoleukodystrophy protein (ALDP), are the primary cause of X-ALD. To gain a better understanding of ALDP dysfunction, we searched for interaction partners of ALDP and identified binary interactions to proteins with functions in fatty acid synthesis (ACLY, FASN, and ACC) and activation (FATP4), constituting a thus far unknown fatty acid synthesis-transport machinery at the cytoplasmic side of the peroxisomal membrane. This machinery adds to the knowledge of the complex mechanisms of peroxisomal fatty acid metabolism at a molecular level and elucidates potential epigenetic mechanisms as regulatory processes in the pathogenesis and thus the clinical course of X-ALD.

Optical Coherence Tomography: Retinal Imaging Contributes to the Understanding of Brain Pathology in Classical Galactosemia.

It remains unresolved whether central nervous system involvement in treated classical galactosemia (CG) is a progressive neurodegenerative process. This study aimed to investigate retinal neuroaxonal degeneration in CG as a surrogate of brain pathology. Global peripapillary retinal nerve fibre layer (GpRNFL) and combined ganglion cell and inner plexiform layer (GCIPL) were analysed in 11 CG patients and 60 controls (HC) using spectral-domain optical coherence tomography. Visual acuity (VA) and low-contrast VA (LCVA) were acquired to test visual function. GpRNFL and GCIPL did not differ between CG and HC (p > 0.05). However, in CG, there was an effect of intellectual outcome on GCIPL (p = 0.036), and GpRNFL and GCIPL correlated with neurological rating scale scores (p < 0.05). A single-case follow-up analysis showed GpRNFL (0.53-0.83%) and GCIPL (0.52-0.85%) annual decrease beyond the normal aging effect. VA and LCVA were reduced in CG with intellectual disability (p = 0.009/0.006), likely due to impaired visual perception. These findings support that CG is not a neurodegenerative disease, but that brain damage is more likely to occur early in brain development. To clarify a minor neurodegenerative component in the brain pathology of CG, we propose multicenter cross-sectional and longitudinal studies using retinal imaging.

Serum glial fibrillary acidic protein and neurofilament light chain in patients with early treated phenylketonuria.

To pave the way for healthy aging in early treated phenylketonuria (ETPKU) patients, a better understanding of the neurological course in this population is needed, requiring easy accessible biomarkers to monitor neurological disease progression in large cohorts. The objective of this pilot study was to investigate the potential of glial fibrillary acidic protein (GFAP) and neurofilament light chain (NfL) as blood biomarkers to indicate changes of the central nervous system in ETPKU. In this single-center cross-sectional study, GFAP and NfL concentrations in serum were quantified using the Simoa® multiplex technology in 56 ETPKU patients aged 6-36 years and 16 age matched healthy controls. Correlation analysis and hierarchical linear regression analysis were performed to investigate an association with disease-related biochemical parameters and retinal layers assessed by optical coherence tomography. ETPKU patients did not show significantly higher GFAP concentrations (mean 73 pg/ml) compared to healthy controls (mean 60 pg/ml, p = 0.140). However, individual pediatric and adult ETPKU patients had GFAP concentrations above the healthy control range. In addition, there was a significant association of GFAP concentrations with current plasma tyrosine concentrations (r = -0.482, p = 0.036), a biochemical marker in phenylketonuria, and the retinal inner nuclear layer volume (r = 0.451, p = 0.04). There was no evidence of NfL alterations in our ETPKU cohort. These pilot results encourage multicenter longitudinal studies to further investigate serum GFAP as a complementary tool to better understand and monitor neurological disease progression in ETPKU. Follow-up investigations on aging ETPKU patients are required to elucidate the potential of serum NfL as biomarker.

Varicella zoster virus ORF25 gene product: an essential hub protein linking encapsidation proteins and the nuclear egress complex.

Varicella zoster virus (VZV) ORF25 is a 156 amino acid protein belonging to the approximately 40 core proteins that are conserved throughout the Herpesviridae. By analogy to its functional orthologue UL33 in Herpes simplex virus 1 (HSV-1), ORF25 is thought to be a component of the terminase complex. To investigate how cleavage and encapsidation of viral DNA links to the nuclear egress of mature capsids in VZV, we tested 10 VZV proteins that are predicted to be involved in either of the two processes for protein interactions against each other using three independent protein-protein interaction (PPI) detection systems: the yeast-two-hybrid (Y2H) system, a luminescence based MBP pull-down interaction screening assay (LuMPIS), and a bioluminescence resonance energy transfer (BRET) assay. A set of 20 interactions was consistently detected by at least 2 methods and resulted in a dense interaction network between proteins associated in encapsidation and nuclear egress. The results indicate that the terminase complex in VZV consists of ORF25, ORF30, and ORF45/42 and support a model in which both processes are closely linked to each other. Consistent with its role as a central hub for protein interactions, ORF25 is shown to be essential for VZV replication.

Subcutaneous vitamin B12 administration using a portable infusion pump in cobalamin-related remethylation disorders: a gentle and easy to use alternative to intramuscular injections.

BACKGROUND: Cobalamin (cbl)-related remethylation disorders are a heterogeneous group of inherited disorders comprising the remethylation of homocysteine to methionine and affecting multiple organ systems, most prominently the nervous system and the bone marrow. To date, the parenteral, generally intramuscular, lifelong administration of hydroxycobalamin (OHCbl) is the mainstay of therapy in these disorders. The dosage and frequency of OHCbl is titrated in each patient to the minimum effective dose in order to account for the painful injections. This may result in undertreatment, a possible risk factor for disease progression and disease-related complications. RESULTS: We describe parenteral administration of OHCbl using a subcutaneous catheter together with a portable infusion pump in a home therapy setting in four pediatric patients with remethylation disorders, two patients with cblC, one patient with cblG, and one patient with cblE deficiency, in whom intramuscular injections were not or no longer feasible. The placement of the subcutaneous catheters and handling of the infusion pump were readily accomplished and well accepted by the patients and their families. No adverse events occurred. The use of a small, portable syringe driver pump allowed for a most flexible administration of OHCbl in everyday life. The concentrations of total homocysteine levels were determined at regular patient visits and remained within the therapeutic target range. This approach allowed for the continuation of OHCbl therapy or the adjustment of therapy required to improve metabolic control in our patients. CONCLUSIONS: Subcutaneous infusion using a subcutaneous catheter system and a portable pump for OHCbl administration in combined and isolated remethylation disorders is safe, acceptable, and effective. It decreases disease burden in preventing frequent single injections and providing patient independence. Thus, it may promote long-term adherence to therapy in patients and parents.

Edgetic Perturbations Contribute to Phenotypic Variability in PEX26 Deficiency.

Peroxisomes share metabolic pathways with other organelles and peroxisomes are embedded into key cellular processes. However, the specific function of many peroxisomal proteins remains unclear and restricted knowledge of the peroxisomal protein interaction network limits a precise mapping of this network into the cellular metabolism. Inborn peroxisomal disorders are autosomal or X-linked recessive diseases that affect peroxisomal biogenesis (PBD) and/or peroxisomal metabolism. Pathogenic variants in the PEX26 gene lead to peroxisomal disorders of the full Zellweger spectrum continuum. To investigate the phenotypic complexity of PEX26 deficiency, we performed a combined organelle protein interaction screen and network medicine approach and 1) analyzed whether PEX26 establishes interactions with other peroxisomal proteins, 2) deciphered the PEX26 interaction network, 3) determined how PEX26 is involved in further processes of peroxisomal biogenesis and metabolism, and 4) showed how variant-specific disruption of protein-protein interactions (edgetic perturbations) may contribute to phenotypic variability in PEX26 deficient patients. The discovery of 14 novel protein-protein interactions for PEX26 revealed a hub position of PEX26 inside the peroxisomal interactome. Analysis of edgetic perturbations of PEX26 variants revealed a strong correlation between the number of affected protein-protein interactions and the molecular phenotype of matrix protein import. The role of PEX26 in peroxisomal biogenesis was expanded encompassing matrix protein import, division and proliferation, and membrane assembly. Moreover, the PEX26 interaction network intersects with cellular lipid metabolism at different steps. The results of this study expand the knowledge about the function of PEX26 and refine genotype-phenotype correlations, which may contribute to our understanding of the underlying disease mechanism of PEX26 deficiency.

Newborn screening for carnitine transporter defect in Bavaria and the long-term follow-up of the identified newborns and mothers: Assessing the benefit and possible harm based on 19 ½ years of experience.

Carnitine transporter defect (CTD) is a potentially life-threatening disorder causing acute metabolic decompensation, cardiac arrhythmia, and cardiac and skeletal myopathies. CTD is included in many newborn screening (NBS) programs. The screening parameter free carnitine, however, is influenced by maternal conditions due to placental transfer. This study reviewed the NBS results for CTD as part of a pilot study in Bavaria, Germany, and the long-term follow-up of the identified patients treated in our center between January 1999 and June 2018. Among 1,816,000 Bavarian NBS samples, six newborns were diagnosed with CTD (incidence of 1:302,667; positive predictive value (PPV) of 1.63% from 2008 to 2018). In the 24 newborns presented to our center for confirmatory testing, we detected four newborns and six mothers with CTD, one newborn and three mothers in whom CTD was presumed but not genetically confirmed, and one mother with glutaric aciduria type I. In 11 newborns, no indication for an inborn error of metabolism was found. The newborns and mothers with CTD had no serious cardiac adverse events or relevant muscular symptoms at diagnosis and during treatment for up to 14 years. Three mothers were lost to follow-up. Revealing a lower incidence than expected, our data confirm that NBS for CTD most likely misses newborns with CTD. It rather produces high numbers of false-positives and a low PPV picking up asymptomatic mothers with a diagnosis of uncertain clinical significance. Our data add to the growing evidence that argues against an implementation of CTD in NBS programs.

Optical Coherence Tomography to Assess Neurodegeneration in Phenylalanine Hydroxylase Deficiency.

In phenylalanine hydroxylase (PAH) deficiency, an easily feasible method to access the progression of neurodegeneration is warranted to contribute to current discussions on treatment indications and targets. The objective of the present study was to investigate whether optical coherence tomography (OCT) measures as markers of neurodegeneration differ between patients with PAH deficiency and healthy controls (HCs) according to phenotype and metabolic control. In this single-center cross-sectional study, 92 patients with different phenotypes of PAH deficiency [PAH deficiency not requiring treatment, early treated phenylketonuria (ETPKU), and late-diagnosed phenylketonuria (PKU)] compared with 76 HCs were examined using spectral-domain OCT. Indices of phenylalanine elevation and variability were correlated with OCT parameters. Late-diagnosed PKU patients showed reduced peripapillary retinal nerve fiber layer (pRNFL) thickness and combined ganglion cell and inner plexiform layer (GCIPL) volume. Adult ETPKU patients were found to have lower GCIPL volume (p = 0.016), which correlated with the indices of phenylalanine control. In pediatric ETPKU patients with poor metabolic control, pRNFL was significantly reduced (p = 0.004). Patients with PAH deficiency not requiring treatment did not exhibit retinal degeneration. Inner nuclear layer (INL) was significantly increased in the pediatric ETPKU patients, driven by those with current poor metabolic control (p = 0.006). Our data provide evidence of retinal neuroaxonal degeneration and INL swelling, depending on the phenotype, current age, and metabolic control. These findings suggest that OCT is suitable to investigate neurodegeneration in PKU and we propose OCT as a sensitive, reliable, safe, low-burden, and low-cost examination for future multicenter studies.

Retinal axonal degeneration in Niemann-Pick type C disease.

OBJECTIVE: Niemann-Pick disease type C1 (NPC1) is a rare autosomal-recessive lysosomal storage disorder presenting with a broad clinical spectrum ranging from a severe infantile-onset neurovisceral disorder to late-onset neurodegenerative disease. Optical coherence tomography (OCT) is established to detect retinal degeneration in vivo. We examined NPC1-patients (NPC1-P), clinically asymptomatic NPC1-mutation carriers (NPC1-MC), and healthy controls (HC) to (1) identify retinal degeneration in NPC1-disease and (2) to investigate possible subclinical retinal degeneration in NPC1-MC. METHODS: Fourteen NPC1-P, 17 NPC1-MC, and 31 age-matched HC were examined using spectral-domain OCT. Neurological examinations, clinical scales [modified Disability Rating Scale (mDRS); Scale for the Rating and Assessment of Ataxia (SARA); Spinocerebellar Ataxia Functional Index (SCAFI)], and video-oculography (VOG) were correlated with OCT data. RESULTS: Macular retinal nerve fiber layer and volumes of combined ganglion cell and inner plexiform layer were significantly lower in NPC1-P compared to HC [mRNFL (µm):0.13 ± 0.01 vs. 0.14 ± 0.02; p = 0.01; GCIPL (mm3):0.60 ± 0.05 vs. 0.62 ± 0.04; p = 0.04]. No significant differences were found in NPC1-MC in comparison to HC. In NPC1-P, the amplitude of upward vertical saccades showed positive associations with peripapillary RNFL (ρ = 0.645; p < 0.05), and thinned GCIP (ρ = 0.609; p < 0.05), but not in NPC1-MC. In NPC1-P correlations between combined outer plexiform layer and outer nuclear layer (OPONL) with mDRS (r = - 0.617; p < 0.05) and GCIP with SARA (r = - 0.622; p < 0.05) were observed. Furthermore, in NPC1-MC, motor scores were negatively associated with pRNFL (ρ = - 0.677; p < 0.01). CONCLUSIONS: Using OCT, we showed retinal degeneration in NPC1-P and significant correlation between retinal neuroaxonal degeneration with clinical measurements. We observed a non-significant trend of retinal degeneration in NPC1-MC correlating with subclinical motor abnormalities. Based on these preliminary data, OCT may be an important marker of neurodegeneration in NPC1-disease after onset of clinical symptoms.

Isoform-specific domain organization determines conformation and function of the peroxisomal biogenesis factor PEX26.

Peroxisomal biogenesis factor PEX26 is a membrane anchor for the multi-subunit PEX1-PEX6 protein complex that controls ubiquitination and dislocation of PEX5 cargo receptors for peroxisomal matrix protein import. PEX26 associates with the peroxisomal translocation pore via PEX14 and a splice variant (PEX26Δex5) of unknown function has been reported. Here, we demonstrate PEX26 homooligomerization mediated by two heptad repeat domains adjacent to the transmembrane domain. We show that isoform-specific domain organization determines PEX26 oligomerization and impacts peroxisomal ß-oxidation and proliferation. PEX26 and PEX26Δex5 displayed different patterns of interaction with PEX2-PEX10 or PEX13-PEX14 complexes, which relate to distinct pre-peroxisomes in the de novo synthesis pathway. Our data support an alternative PEX14-dependent mechanism of peroxisomal membrane association for the splice variant, which lacks a transmembrane domain. Structure-function relationships of PEX26 isoforms explain an extended function in peroxisomal homeostasis and these findings may improve our understanding of the broad phenotype of PEX26-associated human disorders.

Fatal pitfalls in newborn screening for mitochondrial trifunctional protein (MTP)/long-chain 3-Hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency.

BACKGROUND: Mitochondrial trifunctional protein (MTP) and long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency are long-chain fatty acid oxidation disorders with particularly high morbidity and mortality. Outcome can be favorable if diagnosed in time, prompting the implementation in newborn screening programs. Sporadic cases missed by the initial screening sample have been reported. However, little is known on pitfalls during confirmatory testing resulting in fatal misconception of the diagnosis. RESULTS: We report a series of three patients with MTP and LCHAD deficiency, in whom diagnosis was missed by newborn screening, resulting in life-threatening metabolic decompensations within the first half year of life. Two of the patients showed elevated concentrations of primary markers C16-OH and C18:1-OH but were missed by confirmatory testing performed by the maternity clinic. A metabolic center was not consulted. Confirmatory testing consisted of analyses of acylcarnitines in blood and organic acids in urine, the finding of normal excretion of organic acids led to rejection and underestimation of the diagnosis, respectively. The third patient, a preterm infant, was not identified in the initial screening sample due to only moderate elevations of C16-OH and C18:1-OH and normal secondary markers and analyte ratios. CONCLUSION: Our observations highlight limitations of newborn screening for MTP/LCHAD deficiency. They confirm that analyses of acylcarnitines in blood and organic acids in urine alone are not suitable for confirmatory testing and molecular or functional analysis is crucial in diagnosing MTP/LCHAD deficiency. Mild elevations of primary biomarkers in premature infants need to trigger confirmatory testing. Our report underscores the essential role of specialized centers in confirming or ruling out diagnoses in suspicious screening results.

Efficacy, safety and population pharmacokinetics of sapropterin in PKU patients <4 years: results from the SPARK open-label, multicentre, randomized phase IIIb trial.

BACKGROUND: Sapropterin dihydrochloride, a synthetic formulation of BH4, the cofactor for phenylalanine hydroxylase (PAH, EC 1.14.16.1), was initially approved in Europe only for patients ≥4 years with BH4-responsive phenylketonuria. The aim of the SPARK (Safety Paediatric efficAcy phaRmacokinetic with Kuvan®) trial was to assess the efficacy (improvement in daily phenylalanine tolerance, neuromotor development and growth parameters), safety and pharmacokinetics of sapropterin dihydrochloride in children <4 years. RESULTS: In total, 109 male or female children <4 years with confirmed BH4-responsive phenylketonuria or mild hyperphenylalaninemia and good adherence to dietary treatment were screened. 56 patients were randomly assigned (1:1) to 10 mg/kg/day oral sapropterin plus a phenylalanine-restricted diet or to only a phenylalanine-restricted diet for 26 weeks (27 to the sapropterin and diet group and 29 to the diet-only group; intention-to-treat population). Of these, 52 patients with ≥1 pharmacokinetic sample were included in the pharmacokinetic analysis, and 54 patients were included in the safety analysis. At week 26 in the sapropterin plus diet group, mean phenylalanine tolerance was 30.5 (95% confidence interval 18.7-42.3) mg/kg/day higher than in the diet-only group (p < 0.001). The safety profile of sapropterin, measured monthly, was acceptable and consistent with that seen in studies of older children. Using non-linear mixed effect modelling, a one-compartment model with flip-flop pharmacokinetic behaviour, in which the effect of weight was substantial, best described the pharmacokinetic profile. Patients in both groups had normal neuromotor development and stable growth parameters. CONCLUSIONS: The addition of sapropterin to a phenylalanine-restricted diet was well tolerated and led to a significant improvement in phenylalanine tolerance in children <4 years with BH4-responsive phenylketonuria or mild hyperphenylalaninemia. The pharmacokinetic model favours once per day dosing with adjustment for weight. Based on the SPARK trial results, sapropterin has received EU approval to treat patients <4 years with BH4-responsive phenylketonuria. TRIAL REGISTRATION: ClinicalTrials.gov, NCT01376908 . Registered June 17, 2011.