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.

Consensus guidelines for the diagnosis and management of succinic semialdehyde dehydrogenase deficiency.

Succinic semialdehyde dehydrogenase deficiency (SSADHD) (OMIM #271980) is a rare autosomal recessive metabolic disorder caused by pathogenic variants of ALDH5A1. Deficiency of SSADH results in accumulation of γ-aminobutyric acid (GABA) and other GABA-related metabolites. The clinical phenotype of SSADHD includes a broad spectrum of non-pathognomonic symptoms such as cognitive disabilities, communication and language deficits, movement disorders, epilepsy, sleep disturbances, attention problems, anxiety, and obsessive-compulsive traits. Current treatment options for SSADHD remain supportive, but there are ongoing attempts to develop targeted genetic therapies. This study aimed to create consensus guidelines for the diagnosis and management of SSADHD. Thirty relevant statements were initially addressed by a systematic literature review, resulting in different evidence levels of strength according to the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) criteria. The highest level of evidence (level A), based on randomized controlled trials, was unavailable for any of the statements. Based on cohort studies, Level B evidence was available for 12 (40%) of the statements. Thereupon, through a process following the Delphi Method and directed by the Appraisal of Guidelines for Research and Evaluation (AGREE II) criteria, expert opinion was sought, and members of an SSADHD Consensus Group evaluated all the statements. The group consisted of neurologists, epileptologists, neuropsychologists, neurophysiologists, metabolic disease specialists, clinical and biochemical geneticists, and laboratory scientists affiliated with 19 institutions from 11 countries who have clinical experience with SSADHD patients and have studied the disorder. Representatives from parent groups were also included in the Consensus Group. An analysis of the survey's results yielded 25 (83%) strong and 5 (17%) weak agreement strengths. These first-of-their-kind consensus guidelines intend to consolidate and unify the optimal care that can be provided to individuals with SSADHD.

Discovery of novel diagnostic biomarkers for Sjögren-Larsson syndrome by untargeted lipidomics.

AIM: Sjögren-Larsson syndrome (SLS) is a rare neurometabolic disorder that mainly affects brain, eye and skin and is caused by deficiency of fatty aldehyde dehydrogenase. Our recent finding of a profoundly disturbed brain tissue lipidome in SLS prompted us to search for similar biomarkers in plasma as no functional test in blood is available for SLS. METHODS AND RESULTS: We performed plasma lipidomics and used a newly developed bioinformatics tool to mine the untargeted part of the SLS plasma and brain lipidome to search for SLS biomarkers. Plasma lipidomics showed disturbed ether lipid metabolism in known lipid classes. Untargeted lipidomics of both plasma and brain (white and grey matter) uncovered two new endogenous lipid classes highly elevated in SLS. The first biomarker group were alkylphosphocholines/ethanolamines containing different lengths of alkyl-chains where some alkylphosphocholines were > 600-fold elevated in SLS plasma. The second group of biomarkers were a set of 5 features of unknown structure. Fragmentation studies suggested that they contain ubiquinol and phosphocholine and one feature was also found as a glucuronide conjugate in plasma. The plasma features were highly distinctive for SLS with levels >100-1000-fold the level in controls, if present at all. We speculate on the origin of the alkylphosphocholines/ethanolamines and the nature of the ubiquinol-containing metabolites. CONCLUSIONS: The metabolites identified in this study represent novel endogenous lipid classes thus far unknown in humans. They represent the first plasma metabolite SLS-biomarkers and may also yield more insight into SLS pathophysiology.

A spectroscopic test suggests that fragment ion structure annotations in MS/MS libraries are frequently incorrect.

Modern untargeted mass spectrometry (MS) analyses quickly detect and resolve thousands of molecular compounds. Although features are readily annotated with a molecular formula in high-resolution small-molecule MS applications, the large majority of them remains unidentified in terms of their full molecular structure. Collision-induced dissociation tandem mass spectrometry (CID-MS2) provides a diagnostic molecular fingerprint to resolve the molecular structure through a library search. However, for de novo identifications, one must often rely on in silico generated MS2 spectra as reference. The ability of different in silico algorithms to correctly predict MS2 spectra and thus to retrieve correct molecular structures is a topic of lively debate, for instance in the CASMI contest. Underlying the predicted MS2 spectra are the in silico generated product ion structures, which are normally not used in de novo identification, but which can serve to critically assess the fragmentation algorithms. Here we evaluate in silico generated MSn product ion structures by comparison with structures established experimentally by infrared ion spectroscopy (IRIS). For a set of three dozen product ion structures from five precursor molecules, we find that virtually all fragment ion structure annotations in three major in silico MS2 libraries (HMDB, METLIN, mzCloud) are incorrect and caution the reader against their use for structure annotation of MS/MS ions.

CYP2U1: An emerging treatable neurometabolic disease with cerebral folate deficiency in 2 Chinese brothers.

With the rapid advancement of medical technologies in genomic and molecular medicine, the number of treatable neurometabolic diseases is quickly expanding. Spastic paraplegia 56 (SPG56), one of the severe autosomal recessive forms of neurodegenerative disorders caused by pathogenic variants in the CYP2U1 gene, has no reported specific targeted treatment yet. Here we report 2 Chinese brothers with CYP2U1 bi-allelic pathogenic variants with cerebral folate deficiency who were treated for over a decade with folinic acid supplement. Patients have remained stable under therapy.

Clinical, Neuroimaging, and Metabolic Footprint of the Neurodevelopmental Disorder Caused by Monoallelic HK1 Variants.

Background and Objectives: Hexokinase 1 (encoded by HK1) catalyzes the first step of glycolysis, the adenosine triphosphate-dependent phosphorylation of glucose to glucose-6-phosphate. Monoallelic HK1 variants causing a neurodevelopmental disorder (NDD) have been reported in 12 individuals. Methods: We investigated clinical phenotypes, brain MRIs, and the CSF of 15 previously unpublished individuals with monoallelic HK1 variants and an NDD phenotype. Results: All individuals had recurrent variants likely causing gain-of-function, representing mutational hot spots. Eight individuals (c.1370C>T) had a developmental and epileptic encephalopathy with infantile onset and virtually no development. Of the other 7 individuals (n = 6: c.1334C>T; n = 1: c.1240G>A), 3 adults showed a biphasic course of disease with a mild static encephalopathy since early childhood and an unanticipated progressive deterioration with, e.g., movement disorder, psychiatric disease, and stroke-like episodes, epilepsy, starting in adulthood. Individuals who clinically presented in the first months of life had (near)-normal initial neuroimaging and severe cerebral atrophy during follow-up. In older children and adults, we noted progressive involvement of basal ganglia including Leigh-like MRI patterns and cerebellar atrophy, with remarkable intraindividual variability. The CSF glucose and the CSF/blood glucose ratio were below the 5th percentile of normal in almost all CSF samples, while blood glucose was unremarkable. This biomarker profile resembles glucose transporter type 1 deficiency syndrome; however, in HK1-related NDD, CSF lactate was significantly increased in all patients resulting in a substantially different biomarker profile. Discussion: Genotype-phenotype correlations appear to exist for HK1 variants and can aid in counseling. A CSF biomarker profile with low glucose, low CSF/blood glucose, and high CSF lactate may point toward monoallelic HK1 variants causing an NDD. This can help in variant interpretation and may aid in understanding the pathomechanism. We hypothesize that progressive intoxication and/or ongoing energy deficiency lead to the clinical phenotypes and progressive neuroimaging findings.