Comprehensive metabolomics analysis reveals novel biomarkers and pathways in falsely suspected glutaric aciduria Type-1 newborns.

BACKGROUND: Glutaric aciduria type-1 (GA-1) is a rare metabolic disorder due to glutaryl coenzyme A dehydrogenase deficiency, causing elevated levels of glutaryl-CoA and its derivatives. GA-1 exhibits symptoms like macrocephaly, developmental delays, and movement disorders. Timely diagnosis through genetic testing and newborn screening is crucial. However, in some cases, transiently elevated level of glutarylcarnitine (C5DC) challenges accurate diagnosis, highlighting the need for alternative diagnostic methods, like mass spectrometry-based untargeted metabolomics, to identify additional biomarkers for distinguishing falsely suspected GA-1 from healthy newborns. METHODOLOGY: DBS samples from falsely suspected GA-1 newborns (n = 47) and matched control were collected through the NBS program. Untargeted metabolomics using liquid chromatography-high-resolution mass spectrometry (LC-HRMS) was performed to enable biomarker and pathway investigations for significantly altered metabolites. RESULTS: 582 and 546 were up- and down-regulated metabolites in transient GA-1. 155 endogenous metabolites displayed significant variations compared to the control group. Furthermore, our data identified novel altered metabolic biomarkers, such as N-palmitoylcysteine, heptacarboxyporphyrin, 3-hydroxylinoleoylcarnitine, and monoacylglyceride (MG) (0:0/20:1/0:0), along with perturbed metabolic pathways like sphingolipid and thiamine metabolism associated with the transient elevated C5DC levels in DBS samples. CONCLUSIONS: A distinct metabolic pattern linked to the transient C5DC elevation in newborns was reported to enhance the prediction of the falsely positive cases, which could help avoiding unnecessary medical treatments and minimizing the financial burdens in the health sector.

Fatal cervical myelopathy in a child with glutaric aciduria type 1.

We report the case of a Syrian female refugee with late diagnosis of glutaric aciduria type 1 characterised by massive axial hypotonia and quadriplegia who only started adequate diet upon arrival in Switzerland at the age of 4 years, after a strenuous migration journey. Soon after arrival, she died from an unexpected severe upper cervical myelopathy, heralded by acute respiratory distress after a viral infection. This was likely due to repeated strains on her hypotonic neck and precipitated by an orthotopic os odontoideum who led to atlanto-axial subluxation. This case reminds us not to omit handling patients with insufficient postural control and hypotonia with great care to avoid progressive cervical myelopathy.

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.

Modeling Glutaric Aciduria Type I in human neuroblastoma cells recapitulates neuronal damage that can be rescued by gene replacement.

Glutaric Aciduria type I (GA1) is a rare neurometabolic disorder caused by mutations in the GDCH gene encoding for glutaryl-CoA dehydrogenase (GCDH) in the catabolic pathway of lysine, hydroxylysine and tryptophan. GCDH deficiency leads to increased concentrations of glutaric acid (GA) and 3-hydroxyglutaric acid (3-OHGA) in body fluids and tissues. These metabolites are the main triggers of brain damage. Mechanistic studies supporting neurotoxicity in mouse models have been conducted. However, the different vulnerability to some stressors between mouse and human brain cells reveals the need to have a reliable human neuronal model to study GA1 pathogenesis. In the present work we generated a GCDH knockout (KO) in the human neuroblastoma cell line SH-SY5Y by CRISPR/Cas9 technology. SH-SY5Y-GCDH KO cells accumulate GA, 3-OHGA, and glutarylcarnitine when exposed to lysine overload. GA or lysine treatment triggered neuronal damage in GCDH deficient cells. SH-SY5Y-GCDH KO cells also displayed features of GA1 pathogenesis such as increased oxidative stress vulnerability. Restoration of the GCDH activity by gene replacement rescued neuronal alterations. Thus, our findings provide a human neuronal cellular model of GA1 to study this disease and show the potential of gene therapy to rescue GCDH deficiency.

Compilation of Genotype and Phenotype Data in GCDH-LOVD for Variant Classification and Further Application.

Glutaric aciduria type 1 (GA-1) is a rare but treatable autosomal-recessive neurometabolic disorder of lysin metabolism caused by biallelic pathogenic variants in glutaryl-CoA dehydrogenase gene (GCDH) that lead to deficiency of GCDH protein. Without treatment, this enzyme defect causes a neurological phenotype characterized by movement disorder and cognitive impairment. Based on a comprehensive literature search, we established a large dataset of GCDH variants using the Leiden Open Variation Database (LOVD) to summarize the known genotypes and the clinical and biochemical phenotypes associated with GA-1. With these data, we developed a GCDH-specific variation classification framework based on American College of Medical Genetics and Genomics and the Association for Molecular Pathology guidelines. We used this framework to reclassify published variants and to describe their geographic distribution, both of which have practical implications for the molecular genetic diagnosis of GA-1. The freely available GCDH-specific LOVD dataset provides a basis for diagnostic laboratories and researchers to further optimize their knowledge and molecular diagnosis of this rare disease.

Glutaryl-CoA Dehydrogenase Misfolding in Glutaric Acidemia Type 1.

Glutaric acidemia type 1 (GA1) is a neurotoxic metabolic disorder due to glutaryl-CoA dehydrogenase (GCDH) deficiency. The high number of missense variants associated with the disease and their impact on GCDH activity suggest that disturbed protein conformation can affect the biochemical phenotype. We aimed to elucidate the molecular basis of protein loss of function in GA1 by performing a parallel analysis in a large panel of GCDH missense variants using different biochemical and biophysical methodologies. Thirteen GCDH variants were investigated in regard to protein stability, hydrophobicity, oligomerization, aggregation, and activity. An altered oligomerization, loss of protein stability and solubility, as well as an augmented susceptibility to aggregation were observed. GA1 variants led to a loss of enzymatic activity, particularly when present at the N-terminal domain. The reduced cellular activity was associated with loss of tetramerization. Our results also suggest a correlation between variant sequence location and cellular protein stability (p < 0.05), with a more pronounced loss of protein observed with variant proximity to the N-terminus. The broad panel of variant-mediated conformational changes of the GCDH protein supports the classification of GA1 as a protein-misfolding disorder. This work supports research toward new therapeutic strategies that target this molecular disease phenotype.

Biochemical and molecular features of chinese patients with glutaric acidemia type 1 from Fujian Province, southeastern China.

BACKGROUND: Glutaric acidemia type 1 (GA1) is a rare autosomal recessive inherited metabolic disorder caused by variants in the gene encoding the enzyme glutaryl-CoA dehydrogenase (GCDH). The estimated prevalence of GA1 and the mutational spectrum of the GCDH gene vary widely according to race and region. The aim of this study was to assess the acylcarnitine profiles and genetic characteristics of patients with GA1 in Fujian Province, southeastern China. RESULTS: From January 2014 to December 2022, a total of 1,151,069 newborns (631,016 males and 520,053 females) were screened using MS/MS in six newborn screening (NBS) centers in Fujian Province and recruited for this study. Through NBS, 18 newborns (13 females and 5 males) were diagnosed with GA1. Thus, the estimated incidence of GA1 was 1 in 63,948 newborns in Fujian province. In addition, 17 patients with GA1 were recruited after clinical diagnosis. All but one patient with GA1 had a remarkable increase in glutarylcarnitine (C5DC) concentrations. The results of urinary organic acid analyses in 33 patients showed that the concentration of glutaric acid (GA) increased in all patients. The levels of C5DC and GA in patients identified via NBS were higher than those in patients identified via clinical diagnosis (P < 0.05). A total of 71 variants of 70 alleles were detected in patients with GA1, with 19 different pathogenic variants identified. The three most prevalent variants represented 73.23% of the total and were c.1244-2 A > C, p.(?) (63.38%), c.1261G > A, p.Ala421Thr (5.63%), and c.406G > T, p.Gly136Cys (4.22%). The most abundant genotype observed was c.[1244-2 A > C]; [1244-2 A > C] (18/35, 52.43%) and its phenotype corresponded to high excretors (HE, GA > 100 mmol/mol Cr). CONCLUSIONS: In conclusion, we investigated the biochemical and molecular features of 35 unrelated patients with GA1. C5DC concentrations in dried blood spots and urinary GA are effective indicators for a GA1 diagnosis. Our study also identified a GCDH variant spectrum in patients with GA1 from Fujian Province, southeastern China. Correlation analysis between genotypes and phenotypes provides preliminary and valuable information for genetic counseling and management.

Lysine catabolism reprograms tumour immunity through histone crotonylation.

Cancer cells rewire metabolism to favour the generation of specialized metabolites that support tumour growth and reshape the tumour microenvironment1,2. Lysine functions as a biosynthetic molecule, energy source and antioxidant3-5, but little is known about its pathological role in cancer. Here we show that glioblastoma stem cells (GSCs) reprogram lysine catabolism through the upregulation of lysine transporter SLC7A2 and crotonyl-coenzyme A (crotonyl-CoA)-producing enzyme glutaryl-CoA dehydrogenase (GCDH) with downregulation of the crotonyl-CoA hydratase enoyl-CoA hydratase short chain 1 (ECHS1), leading to accumulation of intracellular crotonyl-CoA and histone H4 lysine crotonylation. A reduction in histone lysine crotonylation by either genetic manipulation or lysine restriction impaired tumour growth. In the nucleus, GCDH interacts with the crotonyltransferase CBP to promote histone lysine crotonylation. Loss of histone lysine crotonylation promotes immunogenic cytosolic double-stranded RNA (dsRNA) and dsDNA generation through enhanced H3K27ac, which stimulates the RNA sensor MDA5 and DNA sensor cyclic GMP-AMP synthase (cGAS) to boost type I interferon signalling, leading to compromised GSC tumorigenic potential and elevated CD8+ T cell infiltration. A lysine-restricted diet synergized with MYC inhibition or anti-PD-1 therapy to slow tumour growth. Collectively, GSCs co-opt lysine uptake and degradation to shunt the production of crotonyl-CoA, remodelling the chromatin landscape to evade interferon-induced intrinsic effects on GSC maintenance and extrinsic effects on immune response.

Exploring genotype-phenotype correlations in glutaric aciduria type 1.

Glutaric aciduria type 1 (GA1) is a rare neurometabolic disease caused by pathogenic variants in the gene encoding the enzyme glutaryl-CoA dehydrogenase (GCDH). We performed an extensive literature search to collect data on GA1 patients, together with unpublished cases, to provide an up-to-date genetic landscape of GCDH pathogenic variants and to investigate potential genotype-phenotype correlation, as this is still poorly understood. From this search, 421 different GCDH pathogenic variants have been identified, including four novel variants; c.179T>C (p.Leu60Pro), c.214C>T (p.Arg72Cys), c.309G>C (p.Leu103Phe), and c.665T>C (p.Phe222Ser).The variants are mostly distributed across the entire gene; although variant frequency in GA1 patients is relatively high in the regions encoding for active domains of GCDH. To investigate potential genotype-phenotype correlations, phenotypic descriptions of 532 patients have been combined and evaluated using novel combinatorial analyses. To do so, various clinical phenotypes were determined for each pathogenic variant by combining the information of all GA1 patients reported with this pathogenic variant, and subsequently mapped onto the 2D and 3D GCDH protein structure. In addition, the predicted pathogenicity of missense variants was analyzed using different in silico prediction score models. Both analyses showed an almost similar distribution of the highly pathogenic variants across the GCDH protein, although some hotspots, including the active domain, were observed. Moreover, it was demonstrated that highly pathogenic variants are significantly correlated with lower residual enzyme activity and the most accurate estimation was achieved by the REVEL score. A clear correlation of the genotype and the clinical phenotype however is still lacking.

Phenotypic prediction in glutaric aciduria type 1 combining in silico and in vitro modeling with real-world data.

Glutaric aciduria type 1 (GA1) is caused by inherited deficiency of glutaryl-CoA dehydrogenase (GCDH). To further understand the unclear genotype-phenotype correlation, we transfected mutated GCDH into COS-7 cells resembling known biallelic GCDH variants of 47 individuals with GA1. In total, we modeled 36 genotypes with 32 missense variants. Spectrophotometry demonstrated an inverse correlation between residual enzyme activity and the urinary concentration of glutaric acid and 3-hydroxyglutaric acid, confirming previous studies (Pearson correlation, r = -0.34 and r = -0.49, p = 0.045 and p = 0.002, respectively). In silico modeling predicted high pathogenicity for all genotypes, which caused a low enzyme activity. Western blotting revealed a 2.6-times higher GCDH protein amount in patients with an acute encephalopathic crisis (t-test, p = 0.015), and high protein expression correlated with high in silico protein stability (Pearson correlation, r = -0.42, p = 0.011). The protein amount was not correlated with the enzyme activity (Pearson correlation, r = 0.09, p = 0.59). To further assess protein stability, proteolysis was performed, showing that the p.Arg88Cys variant stabilized a heterozygous less stable variant. We conclude that an integration of different data sources helps to predict the complex clinical phenotype in individuals with GA1.

Rescue of glutaric aciduria type I in mice by liver-directed therapies.

Glutaric aciduria type I (GA-1) is an inborn error of metabolism with a severe neurological phenotype caused by the deficiency of glutaryl-coenzyme A dehydrogenase (GCDH), the last enzyme of lysine catabolism. Current literature suggests that toxic catabolites in the brain are produced locally and do not cross the blood-brain barrier. In a series of experiments using knockout mice of the lysine catabolic pathway and liver cell transplantation, we uncovered that toxic GA-1 catabolites in the brain originated from the liver. Moreover, the characteristic brain and lethal phenotype of the GA-1 mouse model was rescued by two different liver-directed gene therapy approaches: Using an adeno-associated virus, we replaced the defective Gcdh gene or we prevented flux through the lysine degradation pathway by CRISPR deletion of the aminoadipate-semialdehyde synthase (Aass) gene. Our findings question the current pathophysiological understanding of GA-1 and reveal a targeted therapy for this devastating disorder.

Two novel compound heterozygous variants of the GCDH gene in two Chinese families with glutaric acidaemia type I identified by high-throughput sequencing and a literature review.

Autosomal recessive glutaric acidaemia type I (GA-I) is a rare hereditary metabolic disease characterized by increased organic acids and neurologic symptoms. Although numerous variants in the GCDH gene have been identified to be connected with the pathogenesis of GA-I, the relationship between genotype and phenotype remains uncertain. In this study, we evaluated genetic data for two GA-I patients from Hubei, China, and we reviewed the previous research findings to clarify the genetic heterogeneity of GA-I and identify the potential causative variants. After we extracted genomic DNA from peripheral blood samples obtained from two unrelated Chinese families, we used target capture high-throughput sequencing combined with Sanger sequencing to determine likely pathogenic variants in the two probands. Electronic databases were also searched for the literature review. The genetic analysis revealed two compound heterozygous variants in the GCDH gene expected to lead to GA-I in the two probands (P1 and P2), with P1 carrying two known variants (c.892G > A/p. A298T and c.1244-2A > C/IVS10-2A > C) and P2 harbouring two novel variants (c.370G > T/p.G124W and c.473A > G/p.E158G). In the literature review, the most common alleles in low excretors (i.e., individuals with low excretion of GA) were R227P, V400M, M405V, and A298T, with variation in the severity of clinical phenotypes. Overall, we identified two novel GCDH gene candidate pathogenic variants in a Chinese patient, enriching the GCDH gene mutational spectrum and providing a solid foundation for the early diagnosis of GA-I patients with low excretion.

2-Methylglutaconic acid as a biomarker in routine urine organic acids leading to the diagnosis of glutaric acidemia type I in a low excretor.

GA1 (OMIM# 231670) is an organic aciduria caused by defective Glutaryl-CoA dehydrogenase (GCDH), encoded by GCDH. Early detection of GA1 is crucial to prevent patients from developing acute encephalopathic crisis and subsequent neurologic sequelae. Diagnosis of GA1 relies on elevated glutarylcarnitine (C5DC) in plasma acylcarnitine analysis and hyperexcretion of glutaric acid (GA) and 3-hydroxyglutaric acid (3HG) in urine organic acid (UOA) analysis. Low excretors (LE), however, exhibit subtly elevated or even normal plasma C5DC and urinary GA levels, leading to screening and diagnostic challenges. The measurement of 3HG in UOA is thus often used as the 1st tier test for GA1. We described a case of LE detected via newborn screen with normal excretion of GA, absent of 3HG and increased 2-methylglutaconic acid (2MGA), which was detected at 3 mg/g creatinine (reference interval <1 mg/g creatinine) without appreciable ketones. We retrospectively examined UOA of 8 other GA1 patients and the 2MGA level ranged from 2.5 to 27.39 mg/g creatinine, which is significantly higher than normal controls (0.05-1.61 mg/g creatinine). Although the underlying mechanism of 2MGA formation in GA1 is unclear, our study suggests 2MGA is a biomarker for GA1 and should be monitored by routine UOA to evaluate its diagnostic and prognostic value.

DeepDN_iGlu: prediction of lysine glutarylation sites based on attention residual learning method and DenseNet.

As a key issue in orchestrating various biological processes and functions, protein post-translational modification (PTM) occurs widely in the mechanism of protein's function of animals and plants. Glutarylation is a type of protein-translational modification that occurs at active ε-amino groups of specific lysine residues in proteins, which is associated with various human diseases, including diabetes, cancer, and glutaric aciduria type I. Therefore, the issue of prediction for glutarylation sites is particularly important. This study developed a brand-new deep learning-based prediction model for glutarylation sites named DeepDN_iGlu via adopting attention residual learning method and DenseNet. The focal loss function is utilized in this study in place of the traditional cross-entropy loss function to address the issue of a substantial imbalance in the number of positive and negative samples. It can be noted that DeepDN_iGlu based on the deep learning model offers a greater potential for the glutarylation site prediction after employing the straightforward one hot encoding method, with Sensitivity (Sn), Specificity (Sp), Accuracy (ACC), Mathews Correlation Coefficient (MCC), and Area Under Curve (AUC) of 89.29%, 61.97%, 65.15%, 0.33 and 0.80 accordingly on the independent test set. To the best of the authors' knowledge, this is the first time that DenseNet has been used for the prediction of glutarylation sites. DeepDN_iGlu has been deployed as a web server (https://bioinfo.wugenqiang.top/~smw/DeepDN_iGlu/) that is available to make glutarylation site prediction data more accessible.

Recommendations for diagnosing and managing individuals with glutaric aciduria type 1: Third revision.

Glutaric aciduria type 1 is a rare inherited neurometabolic disorder of lysine metabolism caused by pathogenic gene variations in GCDH (cytogenic location: 19p13.13), resulting in deficiency of mitochondrial glutaryl-CoA dehydrogenase (GCDH) and, consequently, accumulation of glutaric acid, 3-hydroxyglutaric acid, glutaconic acid and glutarylcarnitine detectable by gas chromatography/mass spectrometry (organic acids) and tandem mass spectrometry (acylcarnitines). Depending on residual GCDH activity, biochemical high and low excreting phenotypes have been defined. Most untreated individuals present with acute onset of striatal damage before age 3 (to 6) years, precipitated by infectious diseases, fever or surgery, resulting in irreversible, mostly dystonic movement disorder with limited life expectancy. In some patients, striatal damage develops insidiously. In recent years, the clinical phenotype has been extended by the finding of extrastriatal abnormalities and cognitive dysfunction, preferably in the high excreter group, as well as chronic kidney failure. Newborn screening is the prerequisite for pre-symptomatic start of metabolic treatment with low lysine diet, carnitine supplementation and intensified emergency treatment during catabolic episodes, which, in combination, have substantially improved neurologic outcome. In contrast, start of treatment after onset of symptoms cannot reverse existing motor dysfunction caused by striatal damage. Dietary treatment can be relaxed after the vulnerable period for striatal damage, that is, age 6 years. However, impact of dietary relaxation on long-term outcomes is still unclear. This third revision of evidence-based recommendations aims to re-evaluate previous recommendations (Boy et al., J Inherit Metab Dis, 2017;40(1):75-101; Kolker et al., J Inherit Metab Dis 2011;34(3):677-694; Kolker et al., J Inherit Metab Dis, 2007;30(1):5-22) and to implement new research findings on the evolving phenotypic diversity as well as the impact of non-interventional variables and treatment quality on clinical outcomes.

Characterization and structure of the human lysine-2-oxoglutarate reductase domain, a novel therapeutic target for treatment of glutaric aciduria type 1.

In humans, a single enzyme 2-aminoadipic semialdehyde synthase (AASS) catalyses the initial two critical reactions in the lysine degradation pathway. This enzyme evolved to be a bifunctional enzyme with both lysine-2-oxoglutarate reductase (LOR) and saccharopine dehydrogenase domains (SDH). Moreover, AASS is a unique drug target for inborn errors of metabolism such as glutaric aciduria type 1 that arise from deficiencies downstream in the lysine degradation pathway. While work has been done to elucidate the SDH domain structurally and to develop inhibitors, neither has been done for the LOR domain. Here, we purify and characterize LOR and show that it is activated by alkylation of cysteine 414 by N-ethylmaleimide. We also provide evidence that AASS is rate-limiting upon high lysine exposure of mice. Finally, we present the crystal structure of the human LOR domain. Our combined work should enable future efforts to identify inhibitors of this novel drug target.

NRF2 mediates melanoma addiction to GCDH by modulating apoptotic signalling.

Tumour dependency on specific metabolic signals has been demonstrated and often guided numerous therapeutic approaches. We identify melanoma addiction to the mitochondrial protein glutaryl-CoA dehydrogenase (GCDH), which functions in lysine metabolism and controls protein glutarylation. GCDH knockdown induced cell death programmes in melanoma cells, an activity blocked by inhibition of the upstream lysine catabolism enzyme DHTKD1. The transcription factor NRF2 mediates GCDH-dependent melanoma cell death programmes. Mechanistically, GCDH knockdown induces NRF2 glutarylation, increasing its stability and DNA binding activity, with a concomitant transcriptional upregulation of ATF4, ATF3, DDIT3 and CHAC1, resulting in cell death. In vivo, inducible inactivation of GCDH effectively inhibited melanoma tumour growth. Correspondingly, reduced GCDH expression correlated with improved survival of patients with melanoma. These findings identify melanoma cell addiction to GCDH, limiting apoptotic signalling by controlling NRF2 glutarylation. Inhibiting the GCDH pathway could thus represent a therapeutic approach to treat melanoma.