Molecular genetics and metabolism最新文献

Background and objectives

Deoxyguanosine kinase deficiency is one genetic cause of mtDNA depletion syndrome. Its major phenotypes include neonatal/infantile-onset hepatocerebral disease, isolated hepatic disease and myopathic disease. In this retrospective study, we seek to describe the natural history of deoxyguanosine kinase deficiency and identify any genotype-phenotype correlations.

Methods

Retrospective literature search and collation of data from genetically confirmed cases of deoxyguanosine kinase deficiency.

Results

173 cases of DGUOK deficiency were identified. Neonatal/infantile-onset hepatocerebral disease accounted for 128 (74%) of cases. Isolated liver disease was seen in 36 (21%) and myopathic disease in 9 (5%) of cases. The most frequently involved systems were liver (98%), brain (75%), growth (46%) and gastrointestinal tract (26%). Infantile-onset disease typically presented with cholestatic jaundice and lactic acidosis. Neurological involvement included hypotonia, nystagmus and developmental delay with MRI brain abnormalities in about half of cases. Missense variants accounted for 48% of all pathogenic variants while variants resulting in truncated transcripts accounted for 39%. Prognosis was poor, especially for neonatal/ infantile-onset hepatocerebral disease for which 1 year survival was 11%. Twenty-three patients received liver transplants, of whom 12 died within 2 years of transplant. Patients with two truncating variants had a higher risk of death and were more likely to have the neonatal/infantile-onset hepatocerebral disease phenotype. No blood biomarker predictive of neurological involvement was identified. Earlier onset correlated with increased mortality.

Conclusions

There is a narrow window for therapeutic intervention. For the hepatocerebral disease phenotype, median age of onset was 1 month while the median age of death was 6.5 months implying rapid disease progression.

Over fifty years have passed since the last large scale longitudinal study of individuals with PAH deficiency in the U.S. Since then, there have been significant changes in terms of treatment recommendations as well as treatment options. The Phenylalanine Families and Researchers Exploring Evidence (PHEFREE) Consortium was recently established to collect a more up-to-date and extensive longitudinal natural history in individuals with phenylketonuria across the lifespan. In the present paper, we describe the structure and methods of the PHEFREE longitudinal study protocol and report cross-sectional data from an initial sample of 73 individuals (5 months to 54 years of age) with PAH deficiency who have enrolled. Looking forward, the study holds the promise for advancing the field on several fronts including the validation of novel neurocognitive tools for assessment in individuals with PKU as well as evaluation of the long-term effects of changes in metabolic control (e.g., effects of Phe-lowering therapies) on outcome.

Background

Due to newborn screening and early treatment, patients with phenylketonuria (PKU) and mild hyperphenylalaninemia (mHPA) develop largely normal, in terms of IQ testing and academic attainment. However, the impact of metabolic control in various stages of development on more complex cognitive abilities, i.e. executive functions (EF), is still unclear.

Methods

EFs were tested in 28 patients with PKU/mHPA, aged 8–17 years, identified by newborn screening and continuously treated. The relation to current (testing day & past 10 phenylalanine (Phe) values) and long-term metabolic control (age periods: childhood <6, 6–10, adolescence >10 years, lifetime Phe) was analyzed.

Results

EFs were in the lower normative range (IQR of T-values: 47.35–51.00). Patients reaction time was significantly slower than the population mean (divided attention/TAP: median 40, p < 0.01). Both, long-term and current metabolic control correlated with performance in EF tests: Higher current Phe impaired reaction times (Go/No-Go, r = −0.387; working memory, r = −0.425; p < 0.05) and performance in planning ability (ToL r = −0.465, p < 0.01). Higher long-term Phe values both in childhood and adolescence mainly affected attention (omissions/TAP r = −0.357 and − 0.490, respectively, both p < 0.05) as well as planning ability (ToL r = −0.422 and − 0.387, adolescence and lifetime, p < 0.05).

Conclusion

Current and long-term metabolic control in PKU/mHPA, including the adolescent period, influence EFs, especially affecting reaction time and planning abilities. This should be taken into account in patient counselling.

Phenylketonuria (PKU, OMIM 261600) is a genetic disorder caused by a deficiency of the hepatic enzyme phenylalanine hydroxylase (PAH). If left untreated, PKU leads to systemic phenylalanine (Phe) accumulation, which can result in irreversible brain damage and intellectual disabilities. In the last 60 years, early and strict dietary restriction of phenylalanine (Phe) intake proved to prevent the severe clinical phenotype of untreated PKU. While the specific mechanisms through which phenylalanine causes brain damage are still poorly understood, preclinical models have been deeply explored to characterize the neurotoxic effect of Phe on neurodevelopmental processes. At the same time, that on the aging brain still needs to be explored. In the brain of untreated PAH^{Enu2(−/−)} mouse, we previously reported a reduction of myelin basic protein (MBP) during postnatal development up to 60 PND. Later in the diseased mouse's life, a spontaneous and persistent restoration of MBP was detected. In this present longitudinal study, ranging from 14 to 540 post-natal days (PND) of untreated PAH^{Enu2(−/−)} mice, we further investigated: a) the long-life consistency of two Phe-related brain metabolic alterations, such as large neutral amino acids (LNAA) and biogenic amine neurotransmitters' depletion; b) the outcome of locomotor functions during the same life span; c) the integrity of myelin as assessed ex vivo by central (hippocampus) and peripheral (extensor digitorum longus-sciatic nerve) action potential conduction velocities. In contrast with the results of other studies, brain Leu, Ile, and Val concentrations were not significantly altered in the brain PAH^{Enu2(−/−)} mouse. On the other hand, 3-O-Methyldopa (3-OMD, a biomarker of L-DOPA), serotonin, and its associated metabolites were reduced throughout most of the considered time points, with consistent reductions observed prevalently from 14 to 60 PND. Normal saltatory conduction was restored after 60 PND and remained normal at the last examination at 360 PND, resulting nonetheless in a persistent locomotor impairment throughout a lifetime. These new findings contribute to laying the foundations for the preclinical characterization of aging in PKU, confirming neurotransmitter defects as consistent metabolic traits. LNAAs have a minor role, if any, in brain damage pathogenesis. Transient myelin synthesis failure may impact brain connectivity during postnatal development but not nervous signal conduction.

Gyrate atrophy of the choroid and retina (GACR) is caused by pathogenic biallelic variants in the gene encoding ornithine-δ-aminotransferase (OAT), and is characterized by progressive vision loss leading to blindness. OAT is a pyridoxal-5′-phosphate (PLP) dependent enzyme that is mainly involved in ornithine catabolism, and patients with a deficiency develop profound hyperornithinemia. Therapy is aimed at lowering ornithine levels through dietary arginine restriction and, in some cases, through enhancement of OAT activity via supraphysiological dosages of pyridoxine. In this study, we aimed to extend diagnostic practices in GACR by extensively characterizing the consequences of pathogenic variants on the enzymatic function of OAT, both at the level of the enzyme itself as well as the flux through the ornithine degradative pathway. In addition, we developed an in vitro pyridoxine responsiveness assay. We identified 14 different pathogenic variants, of which one variant was present in all patients of Dutch ancestry (p.(Gly353Asp)). In most patients the enzymatic activity of OAT as well as the rate of [¹⁴C]-ornithine flux was below the limit of quantification (LOQ). Apart from our positive control, only one patient cell line showed responsiveness to pyridoxine in vitro, which is in line with the reported in vivo pyridoxine responsiveness in this patient. None of the patients harboring the p.(Gly353Asp) substitution were responsive to pyridoxine in vivo or in vitro. In silico analysis and small-scale expression experiments showed that this variant causes a folding defect, leading to increased aggregation properties that could not be rescued by PLP. Using these results, we developed a diagnostic pipeline for new patients suspected of having GACR. Adding OAT enzymatic analyses and in vitro pyridoxine responsiveness to diagnostic practices will not only increase knowledge on the consequences of pathogenic variants in OAT, but will also enable expectation management for therapeutic modalities, thus eventually improving clinical care.

Phosphomannomutase 2 deficiency (PMM2-CDG), the most frequent congenital disorder of glycosylation, is an autosomal recessive disease caused by biallelic pathogenic variants in the PMM2 gene. There is no cure for this multisystemic syndrome. Some of the therapeutic approaches that are currently in development include mannose-1-phosphate replacement therapy, drug repurposing, and the use of small chemical molecules to correct folding defects. Preclinical models are needed to evaluate the efficacy of treatments to overcome the high lethality of the available animal model. In addition, the number of variants with unknown significance is increasing in clinical settings. This study presents the generation of a cellular disease model by knocking out the PMM2 gene in the hepatoma HepG2 cell line using CRISPR-Cas9 gene editing. The HepG2 knockout model accurately replicates the PMM2-CDG phenotype, exhibiting a complete absence of PMM2 protein and mRNA, a 90% decrease in PMM enzymatic activity, and altered ICAM-1, LAMP1 and A1AT glycoprotein patterns. The evaluation of PMM2 disease-causing variants validates the model's utility for studying new PMM2 clinical variants, providing insights for diagnosis and potentially for evaluating therapies. A CRISPR-Cas9-generated HepG2 knockout model accurately recapitulates the PMM2-CDG phenotype, providing a valuable tool for assessing disease-causing variants and advancing therapeutic strategies.

We previously expressed a chimeric protein in which the small heat-shock protein αB-crystallin (αBC) is fused at its N-terminus to the C-terminus of the first transmembrane segment of the endoplasmic reticulum (ER) protein mitsugumin 23 and confirmed its localization to the ER. Moreover, overexpression of this N-terminally modified αBC was shown to prevent the aggregation of the coexpressed R120G αBC variant, which is highly aggregation-prone and associated with the hereditary myopathy αB-crystallinopathy. To uncover a molecular mechanism by which the ER-anchored αBC negatively regulates the protein aggregation, we isolated proteins that bind to the ER-anchored αBC and identified the lysosomal protease cathepsin D (CTSD) as one such interacting protein. Proteolytically active CTSD is produced by multi-step processing of pro-cathepsin D (proCTSD), which is initially synthesized in the ER and delivered to lysosomes. When overexpressed, CTSD itself prevented the coexpressed R120G αBC variant from aggregating. This anti-aggregate activity was also elicited upon overexpression of the W383C CTSD variant, which is predominantly sequestered in the ER and consequently remains unprocessed, suggesting that proCTSD, rather than mature CTSD, serves to suppress the aggregation of the R120G αBC variant. Meanwhile, overexpression of the A58V CTSD variant, which is identical to wild-type CTSD except for the Ala58Val substitution within the pro-peptide, did not suppress the protein aggregation, indicating that the integrity of the pro-peptide is required for proCTSD to exert its anti-aggregate activity. Based on our previous finding that overexpression of the ER transmembrane protein CLN6 (ceroid-lipofuscinosis, neuronal 6), identified as an interacting protein of the ER-anchored αBC, prevents the R120G αBC variant from aggregating, the CLN6-proCTSD coupling was hypothesized to underpin the functionality of proCTSD within the ER. Indeed, CTSD, when overexpressed in CLN6-depleted cells, was unable to exert its anti-aggregate activity, supporting our view. Collectively, we show here that proCTSD prevents the protein aggregation through the functional association with CLN6 in the microenvironment surrounding the ER membrane, shedding light on a novel aspect of proCTSD and its potential involvement in CTSD-related disorders characterized by the accumulation of aberrant protein aggregates.

The pyruvate dehydrogenase complex (PDC) is remarkable for its size and structure as well as for its physiological and pathological importance. Its canonical location is in the mitochondrial matrix, where it primes the tricarboxylic acid (TCA) cycle by decarboxylating glycolytically-derived pyruvate to acetyl-CoA. Less well appreciated is its role in helping to shape the epigenetic landscape, from early development throughout mammalian life by its ability to “moonlight” in the nucleus, with major repercussions for human healthspan and lifespan. The PDC's influence on two crucial modifiers of the epigenome, acetylation and lactylation, is the focus of this brief review.

Infantile neuronal ceroid lipofuscinosis (CLN1 Batten Disease) is a devastating pediatric lysosomal storage disease caused by pathogenic variants in the CLN1 gene, which encodes the depalmitoylation enzyme, palmitoyl-protein thioesterase 1 (PPT1). CLN1 patients present with visual deterioration, psychomotor dysfunction, and recurrent seizures until neurodegeneration results in death, typically before fifteen years of age. Histopathological features of CLN1 include aggregation of lysosomal autofluorescent storage material (AFSM), as well as profound gliosis. The current management of CLN1 is relegated to palliative care. Here, we examine the therapeutic potential of a small molecule PPT1 mimetic, N-tert-butyl hydroxylamine (NtBuHA), in a Cln1^−/− mouse model. Treatment with NtBuHA reduced AFSM accumulation both in vitro and in vivo. Importantly, NtBuHA treatment in Cln1^−/− mice reduced neuroinflammation, mitigated epileptic episodes, and normalized motor function. Live cell imaging of Cln1^−/− primary cortical neurons treated with NtBuHA partially rescued aberrant synaptic calcium dynamics, suggesting a potential mechanism contributing to the therapeutic effects of NtBuHA in vivo. Taken together, our findings provide supporting evidence for NtBuHA as a potential treatment for CLN1 Batten Disease.