CIAO1 and MMS19 deficiency: A lethal neurodegenerative phenotype caused by cytosolic Fe-S cluster protein assembly disorders.

PURPOSE: The functionality of many cellular proteins depends on cofactors; yet, they have only been implicated in a minority of Mendelian diseases. Here, we describe the first 2 inherited disorders of the cytosolic iron-sulfur protein assembly system. METHODS: Genetic testing via genome sequencing was applied to identify the underlying disease cause in 3 patients with microcephaly, congenital brain malformations, progressive developmental and neurologic impairments, recurrent infections, and a fatal outcome. Studies in patient-derived skin fibroblasts and zebrafish models were performed to investigate the biochemical and cellular consequences. RESULTS: Metabolic analysis showed elevated uracil and thymine levels in body fluids but no pathogenic variants in DPYD, encoding dihydropyrimidine dehydrogenase. Genome sequencing identified compound heterozygosity in 2 patients for missense variants in CIAO1, encoding cytosolic iron-sulfur assembly component 1, and homozygosity for an in-frame 3-nucleotide deletion in MMS19, encoding the MMS19 homolog, cytosolic iron-sulfur assembly component, in the third patient. Profound alterations in the proteome, metabolome, and lipidome were observed in patient-derived fibroblasts. We confirmed the detrimental effect of deficiencies in CIAO1 and MMS19 in zebrafish models. CONCLUSION: A general failure of cytosolic and nuclear iron-sulfur protein maturation caused pleiotropic effects. The critical function of the cytosolic iron-sulfur protein assembly machinery for antiviral host defense may well explain the recurrent severe infections occurring in our patients.

Tracer-based lipidomics enables the discovery of disease-specific candidate biomarkers in mitochondrial ß-oxidation disorders.

Carnitine derivatives of disease-specific acyl-CoAs are the diagnostic hallmark for long-chain fatty acid ß-oxidation disorders (lcFAOD), including carnitine shuttle deficiencies, very-long-chain acyl-CoA dehydrogenase deficiency (VLCADD), long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD) and mitochondrial trifunctional protein deficiency (MPTD). The exact consequence of accumulating lcFAO-intermediates and their influence on cellular lipid homeostasis is, however, still unknown. To investigate the fate and cellular effects of the accumulating lcFAO-intermediates and to explore the presence of disease-specific markers, we used tracer-based lipidomics with deuterium-labeled oleic acid (D9-C18:1) in lcFAOD patient-derived fibroblasts. In line with previous studies, we observed a trend towards neutral lipid accumulation in lcFAOD. In addition, we detected a direct connection between the chain length and patterns of (un)saturation of accumulating acylcarnitines and the various enzyme deficiencies. Our results also identified two disease-specific candidate biomarkers. Lysophosphatidylcholine(14:1) (LPC(14:1)) was specifically increased in severe VLCADD compared to mild VLCADD and control samples. This was confirmed in plasma samples showing an inverse correlation with enzyme activity, which was better than the classic diagnostic marker C14:1-carnitine. The second candidate biomarker was an unknown lipid class, which we identified as S-(3-hydroxyacyl)cysteamines. We hypothesized that these were degradation products of the CoA moiety of accumulating 3-hydroxyacyl-CoAs. S-(3-hydroxyacyl)cysteamines were significantly increased in LCHADD compared to controls and other lcFAOD, including MTPD. Our findings suggest extensive alternative lipid metabolism in lcFAOD and confirm that lcFAOD accumulate neutral lipid species. In addition, we present two disease-specific candidate biomarkers for VLCADD and LCHADD, that may have significant relevance for disease diagnosis, prognosis, and monitoring.

Sex-specific newborn screening for X-linked adrenoleukodystrophy.

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.

Polar metabolomics in human muscle biopsies using a liquid-liquid extraction and full-scan LC-MS.

We describe here a user-friendly analysis protocol for semi-targeted polar metabolomics in human muscle biopsies using Zwitterionic Hydrophilic Interaction Liquid Chromatography and high-resolution full-scan mass spectrometry. Previously, this protocol has been used for Caenorhabditis elegans. Here we show that it can be successfully applied to human muscle biopsies with minor adjustments. Summarized instructions for other matrices are also provided. As peak integration in metabolomics can be challenging, we provide expected retention times and extensive peak descriptions to aid this process. For complete details on the use and execution of this protocol, please refer to Molenaars et al. (2021).

Molecular Biomarkers for Adrenoleukodystrophy: An Unmet Need.

X-linked adrenoleukodystrophy (ALD) is an inherited progressive neurometabolic disease caused by mutations in the ABCD1 gene and the accumulation of very long-chain fatty acids in plasma and tissues. Patients present with heterogeneous clinical manifestations which can include adrenal insufficiency, myelopathy, and/or cerebral demyelination. In the absence of a genotype-phenotype correlation, the clinical outcome of an individual cannot be predicted and currently there are no molecular markers available to quantify disease severity. Therefore, there is an unmet clinical need for sensitive biomarkers to monitor and/or predict disease progression and evaluate therapy efficacy. The increasing amount of biological sample repositories ('biobanking') as well as the introduction of newborn screening creates a unique opportunity for identification and evaluation of new or existing biomarkers. Here we summarize and review the many studies that have been performed to identify and improve knowledge surrounding candidate molecular biomarkers for ALD. We also highlight several shortcomings of ALD biomarker studies, which often include a limited sample size, no collection of longitudinal data, and no validation of findings in an external cohort. Nonetheless, these studies have generated a list of interesting biomarker candidates and this review aspires to direct future biomarker research.

Corrigendum: Adrenoleukodystrophy Newborn Screening in the Netherlands (SCAN Study): The X-Factor.

[This corrects the article DOI: 10.3389/fcell.2020.00499.].

Comparison of the Diagnostic Performance of C26:0-Lysophosphatidylcholine and Very Long-Chain Fatty Acids Analysis for Peroxisomal Disorders.

Peroxisomes are subcellular organelles that are involved in various important physiological processes such as the oxidation of fatty acids and the biosynthesis of bile acids and plasmalogens. The gold standard in the diagnostic work-up for patients with peroxisomal disorders is the analysis of very long-chain fatty acid (VLCFA) levels in plasma. Alternatively, C26:0-lysophosphatidylcholine (C26:0-LPC) can be measured in dried blood spots (DBS) using liquid chromatography tandem mass spectrometry (LC-MS/MS); a fast and easy method but not yet widely used. Currently, little is known about the correlation of C26:0-LPC in DBS and C26:0-LPC in plasma, and how C26:0-LPC analysis compares to VLCFA analysis in diagnostic performance. We investigated the correlation between C26:0-LPC levels measured in DBS and plasma prepared from the same blood sample. For this analysis we included 43 controls and 38 adrenoleukodystrophy (ALD) (21 males and 17 females) and 33 Zellweger spectrum disorder (ZSD) patients. In combined control and patient samples there was a strong positive correlation between DBS C26:0-LPC and plasma C26:0-LPC, with a Spearman's rank correlation coefficient of r (114) = 0.962, p < 0.001. These data show that both plasma and DBS are suitable to determine blood C26:0-LPC levels and that there is a strong correlation between C26:0-LPC levels in both matrices. Following this, we investigated how VLCFA and C26:0-LPC analysis compare in diagnostic performance for 67 controls, 26 ALD males, 19 ALD females, and 35 ZSD patients. For C26:0-LPC, all ALD and ZSD samples had C26:0-LPC levels above the upper limit of the reference range. For C26:0, one out of 67 controls had C26:0 levels above the upper reference range. For 1 out of 26 (1/26) ALD males, 1/19 ALD females and 3/35 ZSD patients, the C26:0 concentration was within the reference range. The C26:0/C22:0 ratio was within the reference range for 0/26 ALD males, 1/19 ALD females and 2/35 ZSD patients. Overall, these data demonstrate that C26:0-LPC analysis has a superior diagnostic performance compared to VLCFA analysis (C26:0 and C26:0/C22:0 ratio) in all patient groups. Based on our results we recommend implementation of C26:0-LPC analysis in DBS and/or plasma in the diagnostic work-up for peroxisomal disorders.

Adrenoleukodystrophy Newborn Screening in the Netherlands (SCAN Study): The X-Factor.

X-linked adrenoleukodystrophy (ALD) is a devastating metabolic disorder affecting the adrenal glands, brain and spinal cord. Males with ALD are at high risk for developing adrenal insufficiency or progressive cerebral white matter lesions (cerebral ALD) at an early age. If untreated, cerebral ALD is often fatal. Women with ALD are not at risk for adrenal insufficiency or cerebral ALD. Newborn screening for ALD in males enables prospective monitoring and timely therapeutic intervention, thereby preventing irreparable damage and saving lives. The Dutch Ministry of Health adopted the advice of the Dutch Health Council to add a boys-only screen for ALD to the newborn screening panel. The recommendation made by the Dutch Health Council to only screen boys, without gathering any unsolicited findings, posed a challenge. We were invited to set up a prospective pilot study that became known as the SCAN study (SCreening for ALD in the Netherlands). The objectives of the SCAN study are: (1) designing a boys-only screening algorithm that identifies males with ALD and without unsolicited findings; (2) integrating this algorithm into the structure of the Dutch newborn screening program without harming the current newborn screening; (3) assessing the practical and ethical implications of screening only boys for ALD; and (4) setting up a comprehensive follow-up that is both patient- and parent-friendly. We successfully developed and validated a screening algorithm that can be integrated into the Dutch newborn screening program. The core of this algorithm is the "X-counter." The X-counter determines the number of X chromosomes without assessing the presence of a Y chromosome. The X-counter is integrated as second tier in our 4-tier screening algorithm. Furthermore, we ensured that our screening algorithm does not result in unsolicited findings. Finally, we developed a patient- and parent-friendly, multidisciplinary, centralized follow-up protocol. Our boys-only ALD screening algorithm offers a solution for countries that encounter similar ethical considerations, for ALD as well as for other X-linked diseases. For ALD, this alternative boys-only screening algorithm may result in a more rapid inclusion of ALD in newborn screening programs worldwide.