All men and most women with X-linked adrenoleukodystrophy (ALD) develop myelopathy in adulthood. As clinical trials with new potential disease-modifying therapies are emerging, sensitive outcome measures for quantifying myelopathy are needed. This prospective cohort study evaluated spinal cord size (cross-sectional area - CSA) and shape (eccentricity) as potential new quantitative outcome measures for myelopathy in ALD. Seventy-four baseline magnetic resonance imaging (MRI) scans, acquired in 42 male ALD patients and 32 age-matched healthy controls, and 26 follow-up scans of ALD patients were included in the study. We used routine T1 -weighted MRI sequences to measure mean CSA, eccentricity, right-left and anteroposterior diameters in the cervical spinal cord. We compared MRI measurements between groups and correlated CSA with clinical outcome measures of disease severity. Longitudinally, we compared MRI measurements between baseline and 1-year follow-up. CSA was significantly smaller in patients compared to controls on all measured spinal cord levels (P < .001). The difference was completely explained by the effect of the symptomatic subgroup. Furthermore, the spinal cord showed flattening (higher eccentricity and smaller anteroposterior diameters) in patients. CSA correlated strongly with all clinical measures of severity of myelopathy. There was no detectable change in CSA after 1-year follow-up. The cervical spinal cord in symptomatic ALD patients is smaller and flattened compared to controls, possibly due to atrophy of the dorsal columns. CSA is a reliable marker of disease severity and can be a valuable outcome measure in long-term follow-up studies in ALD. SYNOPSIS: A prospective cohort study in 42 adrenoleukodystrophy (ALD) patients and 32 controls demonstrated that the spinal cord cross-sectional area of patients is smaller compared to healthy controls and correlates with severity of myelopathy in patients, hence it could be valuable as a much needed surrogate outcome measure.

Genetic variants in the SLC6A1 gene can cause a broad phenotypic disease spectrum by altering the protein function. Thus, systematically curated clinically relevant genotype-phenotype associations are needed to understand the disease mechanism and improve therapeutic decision-making. We aggregated genetic and clinical data from 172 individuals with likely pathogenic/pathogenic (lp/p) SLC6A1 variants and functional data for 184 variants (14.1% lp/p). Clinical and functional data were available for a subset of 126 individuals. We explored the potential associations of variant positions on the GAT1 3D structure with variant pathogenicity, altered molecular function and phenotype severity using bioinformatic approaches. The GAT1 transmembrane domains 1, 6 and extracellular loop 4 (EL4) were enriched for patient over population variants. Across functionally tested missense variants (n = 156), the spatial proximity from the ligand was associated with loss-of-function in the GAT1 transporter activity. For variants with complete loss of in vitro GABA uptake, we found a 4.6-fold enrichment in patients having severe disease versus non-severe disease (P = 2.9 × 10-3, 95% confidence interval: 1.5-15.3). In summary, we delineated associations between the 3D structure and variant pathogenicity, variant function and phenotype in SLC6A1-related disorders. This knowledge supports biology-informed variant interpretation and research on GAT1 function. All our data can be interactively explored in the SLC6A1 portal (https://slc6a1-portal.broadinstitute.org/).

X-linked adrenoleukodystrophy (ALD), a progressive neurodegenerative disease, is caused by mutations in ABCD1 and characterized by very-long-chain fatty acids (VLCFA) accumulation. Virtually all males develop progressive myelopathy (AMN). A subset of patients, however, develops a fatal cerebral demyelinating disease (cerebral ALD). Hematopoietic stem cell transplantation is curative for cerebral ALD provided the procedure is performed in an early stage of the disease. Unfortunately, this narrow therapeutic window is often missed. Therefore, an increasing number of newborn screening programs are including ALD. To identify new biomarkers for ALD, we developed an Abcd1 knockout mouse with enhanced VLCFA synthesis either ubiquitous or restricted to oligodendrocytes. Biochemical analysis revealed VLCFA accumulation in different lipid classes and acylcarnitines. Both C26:0-lysoPC and C26:0-carnitine were highly elevated in brain, spinal cord, but also in bloodspots. We extended the analysis to patients and confirmed that C26:0-carnitine is also elevated in bloodspots from ALD patients. We anticipate that validation of C26:0-carnitine for the diagnosis of ALD in newborn bloodspots may lead to a faster inclusion of ALD in newborn screening programs in countries that already screen for other inborn errors of metabolism.

X-linked adrenoleukodystrophy (X-ALD) is caused by mutations in the ABCD1 gene and is characterized by impaired beta-oxidation of very-long-chain fatty acids (VLCFA) and subsequent VLCFA accumulation in tissues. In adulthood X-ALD most commonly manifests as a gradually progressive myelopathy, (adrenomyeloneuropathy; AMN) without any curative or disease modifying treatments. We recently showed that bezafibrate (BF), a drug used for the treatment of hyperlipidaemia, reduces VLCFA accumulation in X-ALD fibroblasts by inhibiting ELOVL1, an enzyme involved in the VLCFA synthesis. We therefore designed a proof-of-principal clinical trial to determine whether BF reduces VLCFA levels in plasma and lymphocytes of X-ALD patients. Ten males with AMN were treated with BF for 12 weeks at a dose of 400 mg daily, followed by 12 weeks of 800 mg daily. Every 4 weeks patients were evaluated for side effects and blood samples were taken for analysis. Adherence was good as indicated by a clear reduction in triglycerides. There was no reduction in VLCFA in either plasma or lymphocytes. Plasma levels of BF did not exceed 25 µmol/L. We concluded that BF, at least in the dose given, is unable to lower VLCFA levels in plasma or lymphocytes in X-ALD patients. It is unclear whether this is due to the low levels of BF reached in plasma. Our future work is aimed at the identification of highly-specific inhibitors of ELOVL1 that act at much lower concentrations than BF and are well tolerated. BF appears to have no therapeutic utility in X-ALD.

CTP:phosphoethanolamine cytidylyltransferase (ET), encoded by PCYT2, is the rate-limiting enzyme for phosphatidylethanolamine synthesis via the CDP-ethanolamine pathway. Phosphatidylethanolamine is one of the most abundant membrane lipids and is particularly enriched in the brain. We identified five individuals with biallelic PCYT2 variants clinically characterized by global developmental delay with regression, spastic para- or tetraparesis, epilepsy and progressive cerebral and cerebellar atrophy. Using patient fibroblasts we demonstrated that these variants are hypomorphic, result in altered but residual ET protein levels and concomitant reduced enzyme activity without affecting mRNA levels. The significantly better survival of hypomorphic CRISPR-Cas9 generated pcyt2 zebrafish knockout compared to a complete knockout, in conjunction with previously described data on the Pcyt2 mouse model, indicates that complete loss of ET function may be incompatible with life in vertebrates. Lipidomic analysis revealed profound lipid abnormalities in patient fibroblasts impacting both neutral etherlipid and etherphospholipid metabolism. Plasma lipidomics studies also identified changes in etherlipids that have the potential to be used as biomarkers for ET deficiency. In conclusion, our data establish PCYT2 as a disease gene for a new complex hereditary spastic paraplegia and confirm that etherlipid homeostasis is important for the development and function of the brain.

X-linked adrenoleukodystrophy (ALD) is a progressive neurodegenerative disease that is caused by mutations in the ABCD1 gene and characterized by elevated levels of very long-chain fatty acids (VLCFA) in plasma and tissues, with the most pronounced increase in the central nervous system. Virtually all male patients develop adrenal insufficiency and myelopathy (adrenomyeloneuropathy), but a subset develops a fatal cerebral demyelinating disease (known as cerebral ALD). Female patients may also develop myelopathy, but adrenal insufficiency or leukodystrophy are very rare. ALD has been associated with mitochondrial dysfunction, oxidative stress and bioenergetic failure, but the mechanism by which VLCFA accumulation triggers these effects has not been resolved thus far. In this study, we used primary human fibroblasts from normal subjects and ALD patients to investigate whether VLCFA can induce endoplasmic reticulum stress. We show that saturated VLCFA (C26:0) induce endoplasmic reticulum stress in fibroblasts from ALD patients, but not in controls. Furthermore, there is a clear correlation between the chain-length of the fatty acid and the induction of endoplasmic reticulum stress. Exposure of ALD fibroblasts to C26:0, resulted in increased expression of additional endoplasmic reticulum stress markers (EDEM1, GADD34 and CHOP) and in lipoapoptosis. This new insight into the underlying mechanism of VLCFA-induced toxicity is of great importance for the development of a disease modifying treatment for ALD aimed at the normalization of VLCFA levels in tissues.

STXBP1 syndrome is a rare neurodevelopmental disorder caused by heterozygous variants in the STXBP1 gene and is characterized by psychomotor delay, early-onset developmental delay, and epileptic encephalopathy. Pathogenic STXBP1 variants are thought to alter excitation-inhibition (E/I) balance at the synaptic level, which could impact neuronal network dynamics; however, this has not been investigated yet. Here, we present the first EEG study of patients with STXBP1 syndrome to quantify the impact of the synaptic E/I dysregulation on ongoing brain activity. We used high-frequency-resolution analyses of classical and recently developed methods known to be sensitive to E/I balance. EEG was recorded during eyes-open rest in children with STXBP1 syndrome (n = 14) and age-matched typically developing children (n = 50). Brain-wide abnormalities were observed in each of the four resting-state measures assessed here: (i) slowing of activity and increased low-frequency power in the range 1.75-4.63 Hz, (ii) increased long-range temporal correlations in the 11-18 Hz range, (iii) a decrease of our recently introduced measure of functional E/I ratio in a similar frequency range (12-24 Hz), and (iv) a larger exponent of the 1/f-like aperiodic component of the power spectrum. Overall, these findings indicate that large-scale brain activity in STXBP1 syndrome exhibits inhibition-dominated dynamics, which may be compensatory to counteract local circuitry imbalances expected to shift E/I balance toward excitation, as observed in preclinical models. We argue that quantitative EEG investigations in STXBP1 and other neurodevelopmental disorders are a crucial step to understand large-scale functional consequences of synaptic E/I perturbations.

Classical Galactosemia (CG) is an inherited disorder of galactose metabolism caused by a deficiency of the galactose-1-phosphate uridylyltransferase (GALT) enzyme resulting in neurocognitive complications. As in many Inborn Errors of Metabolism, the metabolic pathway of CG is well-defined, but the pathophysiology and high variability in clinical outcome are poorly understood. The aim of this study was to investigate structural changes of the brain of CG patients on MRI and their association with clinical outcome.

Newborn screening (NBS) for X-linked adrenoleukodystrophy (ALD) can identify affected individuals before the onset of life-threatening manifestations. Some countries have decided to only screen boys (sex-specific screening). This study investigates the attitudes of individuals with ALD towards sex-specific NBS for ALD. A questionnaire was sent to all patients in the Dutch ALD cohort. Invitees were asked who they thought should be screened for ALD: only boys, both boys and girls or neither. The motives and background characteristics of respondents were compared between screening preferences. Out of 108 invitees, 66 participants (61%), 38 men and 28 women, participated in this study. The majority (n = 53, 80%) favored screening both newborn boys and girls for ALD, while 20% preferred boys only. None of the respondents felt that newborns should not be screened for ALD. There were no differences in the background characteristics of the respondents between screening preferences. Our study revealed a diverse range of motivations underlying respondents' screening preferences. This study is one of the first to investigate the attitudes of patients towards sex-specific screening for ALD. The outcomes of this study can offer insights to stakeholders engaged in the implementation of NBS programs. ALD patients are important stakeholders who can provide valuable input in this process.

STXBP1-related disorder (STXBP1-RD) is a neurodevelopmental disorder caused by pathogenic variants in the STXBP1 gene. Its gene product MUNC18-1 organizes synaptic vesicle exocytosis and is essential for synaptic transmission. Patients present with developmental delay, intellectual disability, and/or epileptic seizures, with high clinical heterogeneity. To date, the cellular deficits of neurons of patients with STXBP1-RD are unknown.

Over 80% of women with X-linked adrenoleukodystrophy (ALD) develop spinal cord disease in adulthood for which treatment is supportive only. For future clinical trials quantitative data on disease progression rates are essential. Moreover, diagnosis can be challenging in ALD women, as the most important diagnostic biomarker is normal in 15-20%. Better biomarkers are needed. The purpose of this single centre cross-sectional follow-up study in women with ALD was to assess whether Expanded Disability Status Scale (EDSS), AMC Linear Disability Scale (ALDS) and Short Form (36) Health Survey (SF-36) can detect disease progression and to model the effect of age and duration of symptoms on the rate of progression. Moreover, we performed a pilot study to assess if a semi-targeted lipidomics approach can identify possible new diagnostic biomarkers.

Flubendazole, originally developed to treat infections with intestinal nematodes, has been shown to be efficacious in animal models of filarial infections. For treatment of filarial nematodes, systemic exposure is needed. For this purpose, an orally bioavailable amorphous solid dispersion (ASD) formulation of flubendazole was developed. As this formulation results in improved systemic absorption, the pharmacokinetic and toxicological profile of the flubendazole ASD formulation have been assessed to ensure human safety before clinical trials could be initiated.

De novo mutations in PURA have recently been described to cause PURA syndrome, a neurodevelopmental disorder characterised by severe intellectual disability (ID), epilepsy, feeding difficulties and neonatal hypotonia.

Males with X-linked adrenoleukodystrophy (ALD) are at high risk for developing adrenal insufficiency and/or progressive leukodystrophy (cerebral ALD) at an early age. Pathogenic variants in ABCD1 result in elevated levels of very long-chain fatty acids (VLCFA), including C26:0-lysophosphatidylcholine (C26:0-LPC). Newborn screening for ALD enables prospective monitoring and timely therapeutic intervention, thereby preventing irreversible damage and saving lives. The Dutch Health Council recommended to screen only male newborns for ALD without identifying untreatable conditions associated with elevated C26:0-LPC, like Zellweger spectrum disorders and single peroxisomal enzyme defects. Here, we present the results of the SCAN (Screening for ALD in the Netherlands) study which is the first sex-specific newborn screening program worldwide. Males with ALD are identified based on elevated C26:0-LPC levels, the presence of one X-chromosome and a variant in ABCD1, in heel prick dried bloodspots. Screening of 71 208 newborns resulted in the identification of four boys with ALD who, following referral to the pediatric neurologist and confirmation of the diagnosis, enrolled in a long-term follow-up program. The results of this pilot show the feasibility of employing a boys-only screening algorithm that identifies males with ALD without identifying untreatable conditions. This approach will be of interest to countries that are considering ALD newborn screening but are reluctant to identify girls with ALD because for girls there is no direct health benefit. We also analyzed whether gestational age, sex, birth weight and age at heel prick blood sampling affect C26:0-LPC concentrations and demonstrate that these covariates have a minimal effect.

Primary coenzyme Q10 deficiency-7 is caused by homozygous or compound heterozygous mutations in the COQ4 gene. Until now 12 patients have been reported, most presenting with a lethal infantile phenotype with encephalopathy, epilepsy and cardiomyopathy. We report on a new phenotype of COQ4 deficiency: a childhood onset spinocerebellar ataxia with stroke-like episodes.

The Onchocerca ochengi adult implant and Brugia malayi microfilariemic Severe-Combined Immunodeficient (SCID) mouse models are validated screens to measure macrofilaricidal and microfilaricidal activities of candidate onchocerciasis drugs. The purpose of this study was to assess whether 5 daily sub-cutaneous (s.c.) injections of standard flubendazole (FBZ) suspension (10mg/kg), a single s.c. injection (10mg/kg) or 5 daily repeated oral doses of FBZ amorphous solid dispersion (ASD) formulation (0.2, 1.5 or 15mg/kg) mediated macrofilaricidal efficacy against O. ochengi male worms implanted into SCID mice. The direct microfilaricidal activity against circulating B. malayi microfilariae of single dose FBZ ASD formulation (2 or 40 mg/kg) was also evaluated and compared against the standard microfilaricide, ivermectin (IVM). Systemic exposures of FBZ/FBZ metabolites achieved following dosing were measured by pharmacokinetic (PK) bioanalysis. At necropsy, five weeks following start of FBZ SC injections, there were significant reductions in burdens of motile O. ochengi worms following multiple injections (93%) or single injection (82%). Further, significant proportions of mice dosed following multiple injections (5/6; 83%) or single injection (6/10; 60%) were infection negative (drug-cured). In comparison, no significant reduction in recovery of motile adult O. ochengi adult worms was obtained in any multiple-oral dosage group. Single oral-dosed FBZ did not mediate any significant microfilaricidal activity against circulating B. malayi mf at 2 or 7 days compared with >80% efficacy of single dose IVM. In conclusion, multiple oral FBZ formulation doses, whilst achieving substantial bioavailability, do not emulate the efficacy delivered by the parenteral route in vivo against adult O. ochengi. PK analysis determined FBZ efficacy was related to sustained systemic drug levels rather than achievable Cmax. PK modelling predicted that oral FBZ would have to be given at low dose for up to 5 weeks in the mouse model to achieve a matching efficacious exposure profile.

Men with the hereditary peroxisomal disorder X-linked adrenoleukodystrophy (ALD) are at risk of developing inflammatory demyelinating lesions in the brain. In the absence of inflammatory (post-contrast enhancing) lesions on MRI cognitive function is considered spared, but some form of cognitive dysfunction may nevertheless be present. The aim of this cross-sectional study was to characterize cognitive functioning of ALD men with no or minimal MRI abnormalities, which will define cognitive functioning in this category of patients.

X-linked adrenoleukodystrophy (ALD) is a peroxisomal metabolic disorder with a highly complex clinical presentation. ALD is caused by mutations in the ABCD1 gene, and is characterized by the accumulation of very long-chain fatty acids in plasma and tissues. Disease-causing mutations are 'loss of function' mutations, with no prognostic value with respect to the clinical outcome of an individual. All male patients with ALD develop spinal cord disease and a peripheral neuropathy in adulthood, although age of onset is highly variable. However, the lifetime prevalence to develop progressive white matter lesions, termed cerebral ALD (CALD), is only about 60%. Early identification of transition to CALD is critical since it can be halted by allogeneic hematopoietic stem cell therapy only in an early stage. The primary goal of this study is to identify molecular markers which may be prognostic of cerebral demyelination from a simple blood sample, with the hope that blood-based assays can replace the current protocols for diagnosis. We collected six well-characterized brother pairs affected by ALD and discordant for the presence of CALD and performed multi-omic profiling of blood samples including genome, epigenome, transcriptome, metabolome/lipidome, and proteome profiling. In our analysis we identify discordant genomic alleles present across all families as well as differentially abundant molecular features across the omics technologies. The analysis was focused on univariate modeling to discriminate the two phenotypic groups, but was unable to identify statistically significant candidate molecular markers. Our study highlights the issues caused by a large amount of inter-individual variation, and supports the emerging hypothesis that cerebral demyelination is a complex mix of environmental factors and/or heterogeneous genomic alleles. We confirm previous observations about the role of immune response, specifically auto-immunity and the potential role of PFN1 protein overabundance in CALD in a subset of the families. We envision our methodology as well as dataset has utility to the field for reproducing previous or enabling future modifier investigations.

Traumatic brain injury (TBI) in children can be associated with poor outcome in crucial functional domains, including motor, neurocognitive and behavioural functioning. However, outcome varies between patients and is mediated by complex interplay between demographic factors, premorbid functioning and (sub)acute clinical characteristics. At present, methods to understand let alone predict outcome on the basis of these variables are lacking, which contributes to unnecessary follow-up as well as undetected impairments in children. Therefore, this study aims to develop prognostic models for the individual outcome of children with TBI in a range of important developmental domains. In addition, the potential added value of advanced neuroimaging data and the use of machine learning algorithms in the development of prognostic models will be assessed.

X-linked adrenoleukodystrophy (X-ALD) is the most common peroxisomal disorder. The disease is caused by mutations in the ABCD1 gene that encodes the peroxisomal membrane protein ALDP which is involved in the transmembrane transport of very long-chain fatty acids (VLCFA; ≥ C22). A defect in ALDP results in elevated levels of VLCFA in plasma and tissues. The clinical spectrum in males with X-ALD ranges from isolated adrenocortical insufficiency and slowly progressive myelopathy to devastating cerebral demyelination. The majority of heterozygous females will develop symptoms by the age of 60 years. In individual patients the disease course remains unpredictable. This review focuses on the diagnosis and management of patients with X-ALD and provides a guideline for clinicians that encounter patients with this highly complex disorder.