Plasma C24:0- and C26:0-lysophosphatidylcholines are reliable biomarkers for the diagnosis of peroxisomal ß-oxidation disorders.

The gold-standard diagnostic test for peroxisomal disorders (PDs) is plasma concentration analysis of very long-chain fatty acids (VLCFAs). However, this method's time-consuming nature and limitations in cases which present normal VLCFA levels necessitates alternative approaches. The analysis of C26:0-lysophosphatydylcholine (C26:0-LPC) in dried blood spot samples by tandem-mass spectrometry (MS/MS) has successfully been implemented in certain newborn screening programs to diagnose X-linked adrenoleukodystrophy (ALD). However, the diagnostic potential of very long-chain LPCs concentrations in plasma remains poorly understood. This study sought to evaluate the diagnostic performance of C26:0-LPC and other very long-chain LPCs, comparing them to VLCFA analysis in plasma. The study, which included 330 individuals affected by a peroxisomal ß-oxidation deficiency and 407 control individuals, revealed that C26:0- and C24:0-LPC concentrations demonstrated the highest diagnostic accuracy (98.8% and 98.4%, respectively), outperforming VLCFA when C26:0/C22:0 and C24:0/C22:0 ratios were combined (98.1%). Combining C24:0- and C26:0-LPC gave the highest sensitivity (99.7%), with ALD females exhibiting notably higher sensitivity compared with the VLCFA ratio combination (98.7% vs. 93.5%, respectively). In contrast, C22:0-LPC exhibited suboptimal performance, primarily due to its low sensitivity (75%), but we identified a potential use to help distinguish between ALD and Zellweger spectrum disorders. In summary, MS/MS analysis of plasma C24:0- and C26:0-LPC concentrations represents a rapid and straightforward approach to diagnose PDs, demonstrating superior diagnostic accuracy, particularly in ALD females, compared with conventional VLCFA biomarkers. We strongly recommend integrating very-long chain LPC plasma analysis in the diagnostic evaluation of individuals suspected of having a PD.

Genome and RNA sequencing were essential to reveal cryptic intronic variants associated to defective ATP6AP1 mRNA processing.

The diagnosis of Mendelian disorders has notably advanced with integration of whole exome and genome sequencing (WES and WGS) in clinical practice. However, challenges in variant interpretation and uncovered variants by WES still leave a substantial percentage of patients undiagnosed. In this context, integrating RNA sequencing (RNA-seq) improves diagnostic workflows, particularly for WES inconclusive cases. Additionally, functional studies are often necessary to elucidate the impact of prioritized variants on gene expression and protein function. Our study focused on three unrelated male patients (P1-P3) with ATP6AP1-CDG (congenital disorder of glycosylation), presenting with intellectual disability and varying degrees of hepatopathy, glycosylation defects, and an initially inconclusive diagnosis through WES. Subsequent RNA-seq was pivotal in identifying the underlying genetic causes in P1 and P2, detecting ATP6AP1 underexpression and aberrant splicing. Molecular studies in fibroblasts confirmed these findings and identified the rare intronic variants c.289-233C > T and c.289-289G > A in P1 and P2, respectively. Trio-WGS also revealed the variant c.289-289G > A in P3, which was a de novo change in both patients. Functional assays expressing the mutant alleles in HAP1 cells demonstrated the pathogenic impact of these variants by reproducing the splicing alterations observed in patients. Our study underscores the role of RNA-seq and WGS in enhancing diagnostic rates for genetic diseases such as CDG, providing new insights into ATP6AP1-CDG molecular bases by identifying the first two deep intronic variants in this X-linked gene. Additionally, our study highlights the need to integrate RNA-seq and WGS, followed by functional validation, in routine diagnostics for a comprehensive evaluation of patients with an unidentified molecular etiology.

Targeted ultra performance liquid chromatography tandem mass spectrometry procedures for the diagnosis of inborn errors of metabolism: validation through ERNDIM external quality assessment schemes.

OBJECTIVES: Early diagnosis of inborn errors of metabolism (IEM) is crucial to ensure early detection of conditions which are treatable. This study reports on targeted metabolomic procedures for the diagnosis of IEM of amino acids, acylcarnitines, creatine/guanidinoacetate, purines/pyrimidines and oligosaccharides, and describes its validation through external quality assessment schemes (EQA). METHODS: Analysis was performed on a Waters ACQUITY UPLC H-class system coupled to a Waters Xevo triple-quadrupole (TQD) mass spectrometer, operating in both positive and negative electrospray ionization mode. Chromatographic separation was performed on a CORTECS C18 column (2.1 × 150, 1.6 µm). Data were collected by multiple reaction monitoring. RESULTS: The internal and EQA results were generally adequate, with a few exceptions. We calculated the relative measurement error (RME) and only a few metabolites displayed a RME higher than 30 % (asparagine and some acylcarnitine species). For oligosaccharides, semi-quantitative analysis of an educational panel clearly identified the 8 different diseases included. CONCLUSIONS: Overall, we have validated our analytical system through an external quality control assessment. This validation will contribute to harmonization between laboratories, thus improving identification and management of patients with IEM.

Analysis of a second-tier test panel in dried blood spot samples using liquid chromatography-tandem mass spectrometry in Catalonia's newborn screening programme.

OBJECTIVES: Acylcarnitine and amino acid analyses of dried blood spot (DBS) samples using tandem mass spectrometry in newborn screening (NBS) programmes can generate false positive (FP) results. Therefore, implementation of second-tier tests (2TTs) using DBS samples has become increasingly important to avoid FPs. The most widely used 2TT metabolites include methylmalonic acid, 3-hydroxypropionic acid, methylcitric acid, and homocysteine. METHODS: We simultaneously measured 46 underivatised metabolites, including organic acids, acylglycine and acylcarnitine isomers, homocysteine, and orotic acid, in DBS samples using tandem mass spectrometry. To validate this method, we analysed samples from 147 healthy newborns, 160 patients with genetic disorders diagnosed via NBS, 20 patients with acquired vitamin B12 deficiency, 10 newborns receiving antibiotic treatment, and nine external quality control samples. RESULTS: The validation study revealed that 31 metabolites showed good analytical performance. Furthermore, this method detected key metabolites for all diseases associated with increased levels of the following acylcarnitines: C3, C4, C5, C4DC/C5OH, and C5DC. The sensitivity of this method to detect all diseases was 100 %, and the specificity was 74-99 %, except for glutaric aciduria type 1. This method can also be used to diagnose mitochondrial fatty acid ß-oxidation disorders (FAODs) and urea cycle defects (UCDs). CONCLUSIONS: We have described a 2TT panel of 31 metabolites in DBS samples based on an easy and rapid method without derivatisation. Its implementation allowed us to distinguish between different organic acidurias, some FAODs, and UCDs. This new strategy has increased the efficiency of our NBS programme by reducing FP and false negative results, second sample requests, and the time required for diagnosis.

CRISPR/Cas9-based functional genomics strategy to decipher the pathogenicity of genetic variants in inherited metabolic disorders.

The determination of the functional impact of variants of uncertain significance (VUS) is one of the major bottlenecks in the diagnostic workflow of inherited genetic diseases. To face this problem, we set up a CRISPR/Cas9-based strategy for knock-in cellular model generation, focusing on inherited metabolic disorders (IMDs). We selected variants in seven IMD-associated genes, including seven reported disease-causing variants and four benign/likely benign variants. Overall, 11 knock-in cell models were generated via homology-directed repair in HAP1 haploid cells using CRISPR/Cas9. The functional impact of the variants was determined by analyzing the characteristic biochemical alterations of each disorder. Functional studies performed in knock-in cell models showed that our approach accurately distinguished the functional effect of pathogenic from non-pathogenic variants in a reliable manner in a wide range of IMDs. Our study provides a generic approach to assess the functional impact of genetic variants to improve IMD diagnosis and this tool could emerge as a promising alternative to invasive tests, such as muscular or skin biopsies. Although the study has been performed only in IMDs, this strategy is generic and could be applied to other genetic disorders.

Functional Evidence of CCDC186 as a New Disease-Associated Gene with Endocrine and Central Nervous System Alterations.

CCDC186 protein is involved in the maturation of dense-core vesicles (DCVs) in the trans-Golgi network in neurons and endocrine cells. Mutations in genes involved in DCV regulation, other than CCDC186, have been described in patients with neurodevelopmental disorders. To date, only one patient, within a large sequencing study of 1000 cases, and a single case report with variants in CCDC186, had previously been described. However, no functional studies in any of these two cases had been performed. We identified three patients from two gypsy families, unrelated to each other, with mutations in the CCDC186 gene. Clinically, all patients presented with seizures, frontotemporal atrophy, hypomyelination, recurrent infections, and endocrine disturbances such as severe non-ketotic hypoglycemia. Low levels of cortisol, insulin, or growth hormone could only be verified in one patient. All of them had a neonatal onset and died between 7 months and 4 years of age. Whole exome sequencing identified a homozygous variant in the CCDC186 gene (c.2215C>T, p.Arg739Ter) in the index patients of both families. Protein expression studies demonstrated that CCDC186 was almost undetectable in fibroblasts and muscle tissue. These observations correlated with the transcriptomic analysis performed in fibroblasts in one of the patients, which showed a significant reduction of CCDC186 mRNA levels. Our study provides functional evidence that mutations in this gene have a pathogenic effect on the protein and reinforces CCDC186 as a new disease-associated gene. In addition, mutations in CCDC186 could explain the combined endocrine and neurologic alterations detected in our patients.

Diagnostic Odyssey in an Adult Patient with Ophthalmologic Abnormalities and Hearing Loss: Contribution of RNA-Seq to the Diagnosis of a PEX1 Deficiency.

Peroxisomal biogenesis disorders (PBDs) are a heterogeneous group of genetic diseases. Multiple peroxisomal pathways are impaired, and very long chain fatty acids (VLCFA) are the first line biomarkers for the diagnosis. The clinical presentation of PBDs may range from severe, lethal multisystemic disorders to milder, late-onset disease. The vast majority of PBDs belong to Zellweger Spectrum Disordes (ZSDs) and represents a continuum of overlapping clinical symptoms, with Zellweger syndrome being the most severe and Heimler syndrome the less severe disease. Mild clinical conditions frequently present normal or slight biochemical alterations, making the diagnosis of these patients challenging. In the present study we used a combined WES and RNA-seq strategy to diagnose a patient presenting with retinal dystrophy as the main clinical symptom. Results showed the patient was compound heterozygous for mutations in PEX1. VLCFA were normal, but retrospective analysis of lysosphosphatidylcholines (LPC) containing C22:0-C26:0 species was altered. This simple test could avoid the diagnostic odyssey of patients with mild phenotype, such as the individual described here, who was diagnosed very late in adult life. We provide functional data in cell line models that may explain the mild phenotype of the patient by demonstrating the hypomorphic nature of a deep intronic variant altering PEX1 mRNA processing.

Dysregulation of homocysteine homeostasis in acute intermittent porphyria patients receiving heme arginate or givosiran.

Acute intermittent porphyria (AIP) is a rare metabolic disease caused by mutations within the hydroxymethylbilane synthase gene. Previous studies have reported increased levels of plasma total homocysteine (tHcy) in symptomatic AIP patients. In this study, we present long-term data for tHcy and related parameters for an AIP patient cohort (n = 37) in different clinical disease-states. In total, 25 patients (68%) presented with hyperhomocysteinemia (HHcy; tHcy > 15 µmol/L) during the observation period. HHcy was more frequent in AIP patients with recurrent disease receiving heme arginate, than in nonrecurrent (median tHcy: 21.6 µmol/L; range: 10-129 vs median tHcy: 14.5 µmol/L; range 6-77). Long-term serial analyses showed a high within-person tHcy variation, especially among the recurrent patients (coefficient of variation: 16.4%-78.8%). HHcy was frequently associated with low blood concentrations of pyridoxal-5'-phosphate and folate, while cobalamin concentration and the allele distribution of the methylene-tetrahydrofolate-reductase gene were normal. Strikingly, 6 out of the 9 recurrent patients who were later included in a regime of givosiran, a small-interfering RNA that effectively reduced recurrent attacks, showed further increased tHcy (median tHcy in 9 patients: 105 µmol/L; range 16-212). Screening of amino acids in plasma by liquid-chromatography showed co-increased levels of methionine (median 71 µmol/L; range 23-616; normal <40), suggestive of acquired deficiency of cystathionine-ß-synthase. The kynunerine/tryptophan ratio in plasma was, however, normal, indicating a regular metabolism of tryptophan by heme-dependent enzymes. In conclusion, even if HHcy was observed in AIP patients receiving heme arginate, givosiran induced an aggravation of the dysregulation, causing a co-increase of tHcy and methionine resembling classic homocystinuria.

Biallelic mutations in NDUFA8 cause complex I deficiency in two siblings with favorable clinical evolution.

Isolated complex I (CI) deficiency is the most common cause of oxidative phosphorylation (OXPHOS) dysfunction. Whole-exome sequencing identified biallelic mutations in NDUFA8 (c.[293G > T]; [293G > T], encoding for an accessory subunit of CI, in two siblings with a favorable clinical evolution. The individuals reported here are practically asymptomatic, with the exception of slight failure to thrive and some language difficulties at the age of 6 and 9 years, respectively. These observations are remarkable since the vast majority of patients with CI deficiency, including the only NDUFA8 patient reported so far, showed an extremely poor clinical outcome. Western blot studies demonstrated that NDUFA8 protein was strongly reduced in the patients' fibroblasts and muscle extracts. In addition, there was a marked and specific decrease in the steady-state levels of CI subunits. BN-PAGE demonstrated an isolated defect in the assembly and the activity of CI with impaired supercomplexes formation and abnormal accumulation of CI subassemblies. Confocal microscopy analysis in fibroblasts showed rounder mitochondria and diminished branching degree of the mitochondrial network. Functional complementation studies demonstrated disease-causality for the identified mutation as lentiviral transduction with wild-type NDUFA8 cDNA restored the steady-state levels of CI subunits and completely recovered the deficient enzymatic activity in immortalized mutant fibroblasts. In summary, we provide additional evidence of the involvement of NDUFA8 as a mitochondrial disease-causing gene associated with altered mitochondrial morphology, CI deficiency, impaired supercomplexes formation, and very mild progression of the disease.

Mutations in TIMM50 cause severe mitochondrial dysfunction by targeting key aspects of mitochondrial physiology.

3-Methylglutaconic aciduria (3-MGA-uria) syndromes comprise a heterogeneous group of diseases associated with mitochondrial membrane defects. Whole-exome sequencing identified compound heterozygous mutations in TIMM50 (c.[341 G>A];[805 G>A]) in a boy with West syndrome, optic atrophy, neutropenia, cardiomyopathy, Leigh syndrome, and persistent 3-MGA-uria. A comprehensive analysis of the mitochondrial function was performed in fibroblasts of the patient to elucidate the molecular basis of the disease. TIMM50 protein was severely reduced in the patient fibroblasts, regardless of the normal mRNA levels, suggesting that the mutated residues might be important for TIMM50 protein stability. Severe morphological defects and ultrastructural abnormalities with aberrant mitochondrial cristae organization in muscle and fibroblasts were found. The levels of fully assembled OXPHOS complexes and supercomplexes were strongly reduced in fibroblasts from this patient. High-resolution respirometry demonstrated a significant reduction of the maximum respiratory capacity. A TIMM50-deficient HEK293T cell line that we generated using CRISPR/Cas9 mimicked the respiratory defect observed in the patient fibroblasts; notably, this defect was rescued by transfection with a plasmid encoding the TIMM50 wild-type protein. In summary, we demonstrated that TIMM50 deficiency causes a severe mitochondrial dysfunction by targeting key aspects of mitochondrial physiology, such as the maintenance of proper mitochondrial morphology, OXPHOS assembly, and mitochondrial respiratory capacity.