Large-scale genomic investigation of pediatric cholestasis reveals a novel hepatorenal ciliopathy caused by PSKH1 mutations.

PURPOSE: Pediatric cholestasis is the phenotypic expression of clinically and genetically heterogeneous disorders of bile acid synthesis and flow. Although a growing number of monogenic causes of pediatric cholestasis have been identified, the majority of cases remain undiagnosed molecularly. METHODS: In a cohort of 299 pediatric participants (279 families) with intrahepatic cholestasis, we performed exome sequencing as a first-tier diagnostic test. RESULTS: A likely causal variant was identified in 135 families (48.56%). These comprise 135 families that harbor variants spanning 37 genes with established or tentative links to cholestasis. In addition, we propose a novel candidate gene (PSKH1) (HGNC:9529) in 4 families. PSKH1 was particularly compelling because of strong linkage in 3 consanguineous families who shared a novel hepatorenal ciliopathy phenotype. Two of the 4 families shared a founder homozygous variant, whereas the third and fourth had different homozygous variants in PSKH1. PSKH1 encodes a putative protein serine kinase of unknown function. Patient fibroblasts displayed abnormal cilia that are long and show abnormal transport. A homozygous Pskh1 mutant mouse faithfully recapitulated the human phenotype and displayed abnormally long cilia. The phenotype could be rationalized by the loss of catalytic activity observed for each recombinant PSKH1 variant using in vitro kinase assays. CONCLUSION: Our results support the use of genomics in the workup of pediatric cholestasis and reveal PSKH1-related hepatorenal ciliopathy as a novel candidate monogenic form.

Direct Infusion Mass Spectrometry to Rapidly Map Metabolic Flux of Substrates Labeled with Stable Isotopes.

Direct infusion-high-resolution mass spectrometry (DI-HRMS) allows for rapid profiling of complex mixtures of metabolites in blood, cerebrospinal fluid, tissue samples and cultured cells. Here, we present a DI-HRMS method suitable for the rapid determination of metabolic fluxes of isotopically labeled substrates in cultured cells and organoids. We adapted an automated annotation pipeline by selecting labeled adducts that best represent the majority of 13C and/or 15N-labeled glycolytic and tricarboxylic acid cycle intermediates as well as a number of their derivatives. Furthermore, valine, leucine and several of their degradation products were included. We show that DI-HRMS can determine anticipated and unanticipated alterations in metabolic fluxes along these pathways that result from the genetic alteration of single metabolic enzymes, including pyruvate dehydrogenase (PDHA1) and glutaminase (GLS). In addition, it can precisely pinpoint metabolic adaptations to the loss of methylmalonyl-CoA mutase in patient-derived liver organoids. Our results highlight the power of DI-HRMS in combination with stable isotopically labeled compounds as an efficient screening method for fluxomics.

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.

Long-term monitoring of fatty acid oxidation defects: results from a MetabERN survey.

BACKGROUND: Implementation of long-chain fatty acid oxidation defects (LCFAOD) in newborn screening (NBS) programs allows for pre-symptomatic diagnosis and treatment. The long-term natural history of NBS LCFAOD patients is largely unknown and may differ from clinically diagnosed pre-NBS patients. This complicates long-term monitoring of LCFAOD and may cause high monitoring variability. To gain insight in current clinical practice, we performed a web-based questionnaire among all metabolic members of the European Reference Network for Hereditary Metabolic Disorders (MetabERN). RESULTS: Thirty-seven colleagues representing at least 35 European metabolic centres shared their experience and results were discussed at the European Metabolic Group (EMG) meeting 2022. The centres concurred in many aspects of long-term monitoring of LCFAOD including the frequency of clinical visits, determination of laboratory parameters, cardiac monitoring and retinopathy screening. Main discrepancies comprised hepatic imaging, glucose monitoring and electrophysiological investigations. CONCLUSIONS: Discrepancies may reflect differences in local availability of monitoring tools, the inclusion of LCFAOD in NBS programs as well as differences in local genotypes and phenotypes. Because monitoring strategies are largely based on the natural disease course of clinically identified patients, there might be over-monitoring of some NBS patients. Nevertheless, we advocate long-term monitoring because resulting information is essential to further characterize the natural disease course, develop evidence-based guidelines and provide a basis for evaluation of future therapies.

A Delphi Survey Study to Formulate Statements on the Treatability of Inherited Metabolic Disorders to Decide on Eligibility for Newborn Screening.

The Wilson and Jungner (W&J) and Andermann criteria are meant to help select diseases eligible for population-based screening. With the introduction of next-generation sequencing (NGS) methods for newborn screening (NBS), more inherited metabolic diseases (IMDs) can technically be included, and a revision of the criteria was attempted. This study aimed to formulate statements and investigate whether those statements could elaborate on the criterion of treatability for IMDs to decide on eligibility for NBS. An online Delphi study was started among a panel of Dutch IMD experts (EPs). EPs evaluated, amended, and approved statements on treatability that were subsequently applied to 10 IMDs. After two rounds of Delphi, consensus was reached on 10 statements. Application of these statements selected 5 out of 10 IMDs proposed for this study as eligible for NBS, including 3 IMDs in the current Dutch NBS. The statement: 'The expected benefit/burden ratio of early treatment is positive and results in a significant health outcome' contributed most to decision-making. Our Delphi study resulted in 10 statements that can help to decide on eligibility for inclusion in NBS based on treatability, also showing that other criteria could be handled in a comparable way. Validation of the statements is required before these can be applied as guidance to authorities.

The Effect of Genetic HLA Matching on Liver Transplantation Outcome: A Systematic Review and Meta-Analysis.

Objective: We aim to investigate the effects of genetically based HLA matching on patient and graft survival, and acute and chronic rejection after liver transplantation. Background: Liver transplantation is a common treatment for patients with end-stage liver disease. In contrast to most other solid organ transplantations, there is no conclusive evidence supporting human leukocyte antigen (HLA) matching for liver transplantations. With emerging alternatives such as transplantation of bankable (stem) cells, HLA matching becomes feasible, which may decrease the need for immunosuppressive therapy and improve transplantation outcomes. Methods: We systematically searched the PubMed, Embase, and Cochrane databases and performed a meta-analysis investigating the effect of genetic HLA matching on liver transplantation outcomes (acute/chronic rejection, graft failure, and mortality). Results: We included 14 studies with 2682 patients. HLA-C mismatching significantly increased the risk of acute rejection (full mismatching: risk ratio = 1.90, 95% confidence interval = 1.08 to 3.33, P = 0.03; partial mismatching: risk ratio = 1.33, 95% confidence interval = 1.07 to 1.66, P = 0.01). We did not discern any significant effect of HLA mismatching per locus on acute rejection for HLA-A, -B, -DR, and -DQ, nor on chronic rejection, graft failure, or mortality for HLA-DR, and -DQ. Conclusions: We found evidence that genetic HLA-C matching reduces the risk of acute rejection after liver transplantation while matching for other loci does not reduce the risk of acute rejection, chronic rejection, graft failure, or mortality.

[NEXT-GENERATION SEQUENCING PERFORMED IN PATIENTS RAISING THE SUSPICION OF AN INBORN ERROR OF METABOLISM UNCOVERED A HOMOZYGOUS VARIANT IN YARS1 ALLOWING A NOVEL THERAPEUTIC TRIAL].

INTRODUCTION: Inborn-Errors of Metabolism (IEM) are genetic disorders resulting from mutations in genes encoding proteins involved in biochemical-metabolic pathways. However, some IEMs lack specific biochemical markers. Early incorporation of next-generation-sequencing (NGS) including whole exome sequencing (WES) into the diagnostic algorithm of IEMs herein provided, increases diagnostic accuracy, permits genetic counseling and improves therapeutic options. This is exemplified by diseases affecting aminoacyl-tRNA synthetases (ARSs), enzymes involved in protein translation. Recent studies showed that supplementing amino-acids to cell-culture and patients with ARSs deficiencies resulted in improvement of biochemical and clinical parameters, respectively.

Newborn screening for primary carnitine deficiency: who will benefit? - a retrospective cohort study.

BACKGROUND: Newborn screening (NBS) programmes identify a wide range of disease phenotypes, which raises the question whether early identification and treatment is beneficial for all. This study aims to answer this question for primary carnitine deficiency (PCD) taking into account that NBS for PCD identifies newborns with PCD and also until then undiagnosed mothers. METHODS: We investigated clinical, genetic (variants in SLC22A5 gene) and functional (carnitine transport activity in fibroblasts) characteristics of all referred individuals through NBS (newborns and mothers) and clinically diagnosed patients with PCD (not through NBS). Disease phenotype in newborns was predicted using data from PCD mothers and cases published in literature with identical SLC22A5 variants. RESULTS: PCD was confirmed in 19/131 referred newborns, 37/82 referred mothers and 5 clinically diagnosed patients. Severe symptoms were observed in all clinically diagnosed patients, 1 newborn and none of the mothers identified by NBS. PCD was classified as severe in all 5 clinically diagnosed patients, 3/19 newborns and 1/37 mothers; as benign in 8/19 newborns and 36/37 mothers and as unknown in 8/19 newborns. Carnitine transport activity completely separated severe phenotype from benign phenotype (median (range): 4.0% (3.5-5.0)] vs 26% (9.5-42.5), respectively). CONCLUSION: The majority of mothers and a significant proportion of newborns with PCD identified through NBS are likely to remain asymptomatic without early treatment. Conversely, a small proportion of newborns with predicted severe PCD could greatly benefit from early treatment. Genetic variants and carnitine transport activity can be used to distinguish between these groups.

Bi-allelic variants in NAE1 cause intellectual disability, ischiopubic hypoplasia, stress-mediated lymphopenia and neurodegeneration.

Neddylation has been implicated in various cellular pathways and in the pathophysiology of numerous diseases. We identified four individuals with bi-allelic variants in NAE1, which encodes the neddylation E1 enzyme. Pathogenicity was supported by decreased NAE1 abundance and overlapping clinical and cellular phenotypes. To delineate how cellular consequences of NAE1 deficiency would lead to the clinical phenotype, we focused primarily on the rarest phenotypic features, based on the assumption that these would best reflect the pathophysiology at stake. Two of the rarest features, neuronal loss and lymphopenia worsening during infections, suggest that NAE1 is required during cellular stress caused by infections to protect against cell death. In support, we found that stressing the proteasome system with MG132-requiring upregulation of neddylation to restore proteasomal function and proteasomal stress-led to increased cell death in fibroblasts of individuals with NAE1 genetic variants. Additionally, we found decreased lymphocyte counts after CD3/CD28 stimulation and decreased NF-κB translocation in individuals with NAE1 variants. The rarest phenotypic feature-delayed closure of the ischiopubic rami-correlated with significant downregulation of RUN2X and SOX9 expression in transcriptomic data of fibroblasts. Both genes are involved in the pathophysiology of ischiopubic hypoplasia. Thus, we show that NAE1 plays a major role in (skeletal) development and cellular homeostasis during stress. Our approach suggests that a focus on rare phenotypic features is able to provide significant pathophysiological insights in diseases caused by mutations in genes with pleiotropic effects.

A comprehensive transcriptomic comparison of hepatocyte model systems improves selection of models for experimental use.

The myriad of available hepatocyte in vitro models provides researchers the possibility to select hepatocyte-like cells (HLCs) for specific research goals. However, direct comparison of hepatocyte models is currently challenging. We systematically searched the literature and compared different HLCs, but reported functions were limited to a small subset of hepatic functions. To enable a more comprehensive comparison, we developed an algorithm to compare transcriptomic data across studies that tested HLCs derived from hepatocytes, biliary cells, fibroblasts, and pluripotent stem cells, alongside primary human hepatocytes (PHHs). This revealed that no HLC covered the complete hepatic transcriptome, highlighting the importance of HLC selection. HLCs derived from hepatocytes had the highest transcriptional resemblance to PHHs regardless of the protocol, whereas the quality of fibroblasts and PSC derived HLCs varied depending on the protocol used. Finally, we developed and validated a web application (HLCompR) enabling comparison for specific pathways and addition of new HLCs. In conclusion, our comprehensive transcriptomic comparison of HLCs allows selection of HLCs for specific research questions and can guide improvements in culturing conditions.

Neonatal Long-Chain 3-Ketoacyl-CoA Thiolase deficiency: Clinical-biochemical phenotype, sodium-D,L-3-hydroxybutyrate treatment experience and cardiac evaluation using speckle echocardiography.

Isolated long-chain 3-keto-acyl CoA thiolase (LCKAT) deficiency is a rare long-chain fatty acid oxidation disorder caused by mutations in HADHB. LCKAT is part of a multi-enzyme complex called the mitochondrial trifunctional protein (MTP) which catalyzes the last three steps in the long-chain fatty acid oxidation. Until now, only three cases of isolated LCKAT deficiency have been described. All patients developed a severe cardiomyopathy and died before the age of 7 weeks. Here, we describe a newborn with isolated LCKAT deficiency, presenting with neonatal-onset cardiomyopathy, rhabdomyolysis, hypoglycemia and lactic acidosis. Bi-allelic 185G > A (p.Arg62His) and c1292T > C (p.Phe431Ser) mutations were found in HADHB. Enzymatic analysis in both lymphocytes and cultured fibroblasts revealed LCKAT deficiency with a normal long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD, also part of MTP) enzyme activity. Clinically, the patient showed recurrent cardiomyopathy, which was monitored by speckle tracking echocardiography. Subsequent treatment with special low-fat formula, low in long chain triglycerides (LCT) and supplemented with medium chain triglycerides (MCT) and ketone body therapy in (sodium-D,L-3-hydroxybutyrate) was well tolerated and resulted in improved carnitine profiles and cardiac function. Resveratrol, a natural polyphenol that has been shown to increase fatty acid oxidation, was also considered as a potential treatment option but showed no in vitro benefits in the patient's fibroblasts. Even though our patient deceased at the age of 13 months, early diagnosis and prompt initiation of dietary management with addition of sodium-D,L-3-hydroxybutyrate may have contributed to improved cardiac function and a much longer survival when compared to the previously reported cases of isolated LCKAT-deficiency.

Genetic, biochemical, and clinical spectrum of patients with mitochondrial trifunctional protein deficiency identified after the introduction of newborn screening in the Netherlands.

Long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD) is included in many newborn screening (NBS) programs. Acylcarnitine-based NBS for LCHADD not only identifies LCHADD, but also the other deficiencies of the mitochondrial trifunctional protein (MTP), a multi-enzyme complex involved in long-chain fatty acid ß-oxidation. Besides LCHAD, MTP harbors two additional enzyme activities: long-chain enoyl-CoA hydratase (LCEH) and long-chain ketoacyl-CoA thiolase (LCKAT). Deficiency of one or more MTP activities causes generalized MTP deficiency (MTPD), LCHADD, LCEH deficiency (not yet reported), or LCKAT deficiency (LCKATD). To gain insight in the outcomes of MTP-deficient patients diagnosed after the introduction of NBS for LCHADD in the Netherlands, a retrospective evaluation of genetic, biochemical, and clinical characteristics of MTP-deficient patients, identified since 2007, was carried out. Thirteen patients were identified: seven with LCHADD, five with MTPD, and one with LCKATD. All LCHADD patients (one missed by NBS, clinical diagnosis) and one MTPD patient (clinical diagnosis) were alive. Four MTPD patients and one LCKATD patient developed cardiomyopathy and died within 1 month and 13 months of life, respectively. Surviving patients did not develop symptomatic hypoglycemia, but experienced reversible cardiomyopathy and rhabdomyolysis. Five LCHADD patients developed subclinical neuropathy and/or retinopathy. In conclusion, patient outcomes were highly variable, stressing the need for accurate classification of and discrimination between the MTP deficiencies to improve insight in the yield of NBS for LCHADD. NBS allowed the prevention of symptomatic hypoglycemia, but current treatment options failed to treat cardiomyopathy and prevent long-term complications. Moreover, milder patients, who might benefit from NBS, were missed due to normal acylcarnitine profiles.

Thermo-sensitive mitochondrial trifunctional protein deficiency presenting with episodic myopathy.

Mitochondrial trifunctional protein (MTP) is involved in long-chain fatty acid ß-oxidation (lcFAO). Deficiency of one or more of the enzyme activities as catalyzed by MTP causes generalized MTP deficiency (MTPD), long-chain hydroxyacyl-CoA dehydrogenase deficiency (LCHADD), or long-chain ketoacyl-CoA thiolase deficiency (LCKATD). When genetic variants result in thermo-sensitive enzymes, increased body temperature (e.g. fever) can reduce enzyme activity and be a risk factor for clinical decompensation. This is the first description of five patients with a thermo-sensitive MTP deficiency. Clinical and genetic information was obtained from clinical files. Measurement of LCHAD and LCKAT activities, lcFAO-flux studies and palmitate loading tests were performed in skin fibroblasts cultured at 37°C and 40°C. In all patients (four MTPD, one LCKATD), disease manifested during childhood (manifestation age: 2-10 years) with myopathic symptoms triggered by fever or exercise. In four patients, signs of retinopathy or neuropathy were present. Plasma long-chain acylcarnitines were normal or slightly increased. HADHB variants were identified (at age: 6-18 years) by whole exome sequencing or gene panel analyses. At 37°C, LCHAD and LCKAT activities were mildly impaired and lcFAO-fluxes were normal. Remarkably, enzyme activities and lcFAO-fluxes were markedly diminished at 40°C. Preventive (dietary) measures improved symptoms for most. In conclusion, all patients with thermo-sensitive MTP deficiency had a long diagnostic trajectory and both genetic and enzymatic testing were required for diagnosis. The frequent absence of characteristic acylcarnitine abnormalities poses a risk for a diagnostic delay. Given the positive treatment effects, upfront genetic screening may be beneficial to enhance early recognition.

[Possible applications of organoids in medicine]. / Mogelijke toepassingen van organoïden in de geneeskunde.

With the development of organoids as three-dimensional model organs it is now possible to mimic the growth of human organs in a culture dish. As these model organs can be generated from patients' (diseased) tissue and capture the (genetic) properties thereof, they are more representative disease models than cell lines and animal models. The use of organoids in pathophysiological research has already increased our understanding of many human diseases. Furthermore, organoids are used for patient-specific drug tests for cystic fibrosis, and this will soon be possible for other genetic diseases. Also, transplantation of (own genetically corrected) organoids could become a new treatment option. To fully employ the potential of organoids in medicine, cultures need to be standardized and further optimized for better organ/disease representation. With this, organoids hold the promise to quickly revolutionize personalized and regenerative medicine.

Assessment of human leukocyte antigen matching algorithm PIRCHE-II on liver transplantation outcomes.

For liver transplantations, human leukocyte antigen (HLA) matching is not routinely performed because observed effects have been inconsistent. Nevertheless, long-term liver transplantation outcomes remain suboptimal. The availability of a more precise HLA-matching algorithm, Predicted Indirectly Recognizable HLA Epitopes II (PIRCHE-II), now enables robust assessment of the association between HLA matching and liver transplantation outcomes. We performed a single-center retrospective cohort study of 736 liver transplantation patients. Associations between PIRCHE-II and HLAMatchmaker scores and mortality, graft loss, acute and chronic rejection, ischemic cholangiopathy, and disease recurrence were evaluated with Cox proportional hazards models. Associations between PIRCHE-II with 1-year, 2-year, and 5-year outcomes and severity of acute rejection were assessed with logistic and linear regression analyses, respectively. Subgroup analyses were performed for autoimmune and nonautoimmune indications, and patients aged 30 years and younger, and older than 30 years. PIRCHE-II and HLAMatchmaker scores were not associated with any of the outcomes. However, patients who received transplants for autoimmune disease showed more acute rejection and graft loss, and these risks negatively associated with age. Rhesus mismatch more than doubled the risk of disease recurrence. Moreover, PIRCHE-II was inversely associated with graft loss in the subgroup of patients aged 30 years and younger with autoimmune indications. The absence of associations between PIRCHE-II and HLAMatchmaker scores and the studied outcomes refutes the need for HLA matching for liver (stem cell) transplantations for nonautoimmune disease. For autoimmune disease, the activated immune system seems to increase risks of acute rejection and graft loss. Our results may suggest the benefits of transplantations with rhesus matched but PIRCHE-II mismatched donor livers.

Delivery of modified mRNA to damaged myocardium by systemic administration of lipid nanoparticles.

Lipid Nanoparticles (LNPs) are a promising drug delivery vehicle for clinical siRNA delivery. Modified mRNA (modRNA) has recently gained great attention as a therapeutic molecule in cardiac regeneration. However, for mRNA to be functional, it must first reach the diseased myocardium, enter the target cell, escape from the endosomal compartment into the cytosol and be translated into a functional protein. However, it is unknown if LNPs can effectively deliver mRNA, which is much larger than siRNA, to the ischemic myocardium. Here, we evaluated the ability of LNPs to deliver mRNA to the myocardium upon ischemia-reperfusion injury functionally. By exploring the bio-distribution of fluorescently labeled LNPs, we observed that, upon reperfusion, LNPs accumulated in the infarct area of the heart. Subsequently, the functional delivery of modRNA was evaluated by the administration of firefly luciferase encoding modRNA. Concomitantly, a significant increase in firefly luciferase expression was observed in the heart upon myocardial reperfusion when compared to sham-operated animals. To characterize the targeted cells within the myocardium, we injected LNPs loaded with Cre modRNA into Cre-reporter mice. Upon LNP infusion, Tdtomato+ cells, derived from Cre mediated recombination, were observed in the infarct region as well as the epicardial layer upon LNP infusion. Within the infarct area, most targeted cells were cardiac fibroblasts but also some cardiomyocytes and macrophages were found. Although the expression levels were low compared to LNP-modRNA delivery into the liver, our data show the ability of LNPs to functionally deliver modRNA therapeutics to the damaged myocardium, which holds great promise for modRNA-based cardiac therapies.

The potential and limitations of intrahepatic cholangiocyte organoids to study inborn errors of metabolism.

Inborn errors of metabolism (IEMs) comprise a diverse group of individually rare monogenic disorders that affect metabolic pathways. Mutations lead to enzymatic deficiency or dysfunction, which results in intermediate metabolite accumulation or deficit leading to disease phenotypes. Currently, treatment options for many IEMs are insufficient. Rarity of individual IEMs hampers therapy development and phenotypic and genetic heterogeneity suggest beneficial effects of personalized approaches. Recently, cultures of patient-own liver-derived intrahepatic cholangiocyte organoids (ICOs) have been established. Since most metabolic genes are expressed in the liver, patient-derived ICOs represent exciting possibilities for in vitro modeling and personalized drug testing for IEMs. However, the exact application range of ICOs remains unclear. To address this, we examined which metabolic pathways can be studied with ICOs and what the potential and limitations of patient-derived ICOs are to model metabolic functions. We present functional assays in patient ICOs with defects in branched-chain amino acid metabolism (methylmalonic acidemia), copper metabolism (Wilson disease), and transporter defects (cystic fibrosis). We discuss the broad range of functional assays that can be applied to ICOs, but also address the limitations of these patient-specific cell models. In doing so, we aim to guide the selection of the appropriate cell model for studies of a specific disease or metabolic process.

NAA80 bi-allelic missense variants result in high-frequency hearing loss, muscle weakness and developmental delay.

The recent identification of NAA80/NAT6 as the enzyme that acetylates actins generated new insight into the process of post-translational actin modifications; however, the role of NAA80 in human physiology and pathology has not been clarified yet. We report two individuals from a single family harbouring a homozygous c.389T>C, p.(Leu130Pro) NAA80 genetic variant. Both individuals show progressive high-frequency sensorineural hearing loss, craniofacial dysmorphisms, developmental delay and mild proximal and axial muscle weakness. Based on the molecular structure, we predicted and confirmed the NAA80 c.389T>C, p.(Leu130Pro) variant to result in protein destabilization, causing severely decreased NAA80 protein availability. Concurrently, individuals exhibited a ∼50% decrease of actin acetylation. NAA80 individual derived fibroblasts and peripheral blood mononuclear cells showed increased migration, increased filopodia counts and increased levels of polymerized actin, in agreement with previous observations in NAA80 knock-out cells. Furthermore, the significant clinical overlap between NAA80 individuals and individuals with pathogenic variants in several actin subtypes reflects the general importance of controlled actin dynamics for the inner ear, brain and muscle. Taken together, we describe a new syndrome, caused by NAA80 genetic variants leading to decreased actin acetylation and disrupted associated molecular functions. Our work suggests a crucial role for NAA80-mediated actin dynamics in neuronal health, muscle health and hearing.