Mutations in SCNM1 cause orofaciodigital syndrome due to minor intron splicing defects affecting primary cilia.

Orofaciodigital syndrome (OFD) is a genetically heterogeneous ciliopathy characterized by anomalies of the oral cavity, face, and digits. We describe individuals with OFD from three unrelated families having bi-allelic loss-of-function variants in SCNM1 as the cause of their condition. SCNM1 encodes a protein recently shown to be a component of the human minor spliceosome. However, so far the effect of loss of SCNM1 function on human cells had not been assessed. Using a comparative transcriptome analysis between fibroblasts derived from an OFD-affected individual harboring SCNM1 mutations and control fibroblasts, we identified a set of genes with defective minor intron (U12) processing in the fibroblasts of the affected subject. These results were reproduced in SCNM1 knockout hTERT RPE-1 (RPE-1) cells engineered by CRISPR-Cas9-mediated editing and in SCNM1 siRNA-treated RPE-1 cultures. Notably, expression of TMEM107 and FAM92A encoding primary cilia and basal body proteins, respectively, and that of DERL2, ZC3H8, and C17orf75, were severely reduced in SCNM1-deficient cells. Primary fibroblasts containing SCNM1 mutations, as well as SCNM1 knockout and SCNM1 knockdown RPE-1 cells, were also found with abnormally elongated cilia. Conversely, cilia length and expression of SCNM1-regulated genes were restored in SCNM1-deficient fibroblasts following reintroduction of SCNM1 via retroviral delivery. Additionally, functional analysis in SCNM1-retrotransduced fibroblasts showed that SCNM1 is a positive mediator of Hedgehog (Hh) signaling. Our findings demonstrate that defective U12 intron splicing can lead to a typical ciliopathy such as OFD and reveal that primary cilia length and Hh signaling are regulated by the minor spliceosome through SCNM1 activity.

Germline and Mosaic Variants in PRKACA and PRKACB Cause a Multiple Congenital Malformation Syndrome.

PRKACA and PRKACB code for two catalytic subunits (Cα and Cß) of cAMP-dependent protein kinase (PKA), a pleiotropic holoenzyme that regulates numerous fundamental biological processes such as metabolism, development, memory, and immune response. We report seven unrelated individuals presenting with a multiple congenital malformation syndrome in whom we identified heterozygous germline or mosaic missense variants in PRKACA or PRKACB. Three affected individuals were found with the same PRKACA variant, and the other four had different PRKACB mutations. In most cases, the mutations arose de novo, and two individuals had offspring with the same condition. Nearly all affected individuals and their affected offspring shared an atrioventricular septal defect or a common atrium along with postaxial polydactyly. Additional features included skeletal abnormalities and ectodermal defects of variable severity in five individuals, cognitive deficit in two individuals, and various unusual tumors in one individual. We investigated the structural and functional consequences of the variants identified in PRKACA and PRKACB through the use of several computational and experimental approaches, and we found that they lead to PKA holoenzymes which are more sensitive to activation by cAMP than are the wild-type proteins. Furthermore, expression of PRKACA or PRKACB variants detected in the affected individuals inhibited hedgehog signaling in NIH 3T3 fibroblasts, thereby providing an underlying mechanism for the developmental defects observed in these cases. Our findings highlight the importance of both Cα and Cß subunits of PKA during human development.

FOSL2 truncating variants in the last exon cause a neurodevelopmental disorder with scalp and enamel defects.

PURPOSE: We aimed to investigate the molecular basis of a novel recognizable neurodevelopmental syndrome with scalp and enamel anomalies caused by truncating variants in the last exon of the gene FOSL2, encoding a subunit of the AP-1 complex. METHODS: Exome sequencing was used to identify genetic variants in all cases, recruited through Matchmaker exchange. Gene expression in blood was analyzed using reverse transcription polymerase chain reaction. In vitro coimmunoprecipitation and proteasome inhibition assays in transfected HEK293 cells were performed to explore protein and AP-1 complex stability. RESULTS: We identified 11 individuals from 10 families with mostly de novo truncating FOSL2 variants sharing a strikingly similar phenotype characterized by prenatal growth retardation, localized cutis scalp aplasia with or without skull defects, neurodevelopmental delay with autism spectrum disorder, enamel hypoplasia, and congenital cataracts. Mutant FOSL2 messenger RNAs escaped nonsense-mediated messenger RNA decay. Truncated FOSL2 interacts with c-JUN, thus mutated AP-1 complexes could be formed. CONCLUSION: Truncating variants in the last exon of FOSL2 associate a distinct clinical phenotype by altering the regulatory degradation of the AP-1 complex. These findings reveal a new role for FOSL2 in human pathology.

Biallelic truncating variants in MAPKAPK5 cause a new developmental disorder involving neurological, cardiac, and facial anomalies combined with synpolydactyly.

PURPOSE: This study aimed to identify the genetic cause of a new multiple congenital anomalies syndrome observed in three individuals from two unrelated families. METHODS: Clinical assessment was conducted prenatally and at different postnatal stages. Genetic studies included exome sequencing (ES) combined with single-nucleotide polymorphism (SNP) array based homozygosity mapping and trio ES. Dermal fibroblasts were used for functional assays. RESULTS: A clinically recognizable syndrome characterized by severe developmental delay, variable brain anomalies, congenital heart defects, dysmorphic facial features, and a distinctive type of synpolydactyly with an additional hypoplastic digit between the fourth and fifth digits of hands and/or feet was identified. Additional features included eye abnormalities, hearing impairment, and electroencephalogram anomalies. ES detected different homozygous truncating variants in MAPKAPK5 in both families. Patient-derived cells showed no expression of MAPKAPK5 protein isoforms and reduced levels of the MAPKAPK5-interacting protein ERK3. F-actin recovery after latrunculin B treatment was found to be less efficient in patient-derived fibroblasts than in control cells, supporting a role of MAPKAPK5 in F-actin polymerization. CONCLUSION: Our data indicate that loss-of-function variants in MAPKAPK5 result in a severe developmental disorder and reveal a major role of this gene in human brain, heart, and limb development.

Identification of 34 novel mutations in propionic acidemia: Functional characterization of missense variants and phenotype associations.

Propionic acidemia (PA) is caused by mutations in the PCCA and PCCB genes, encoding α and ß subunits, respectively, of the mitochondrial enzyme propionyl-CoA carboxylase (PCC). Up to date, >200 pathogenic mutations have been identified, mostly missense defects. Genetic analysis in PA patients referred to the laboratory for the past 15 years identified 20 novel variants in the PCCA gene and 14 in the PCCB gene. 21 missense variants were predicted as probably disease-causing by different bioinformatics algorithms. Structural analysis in the available 3D model of the PCC enzyme indicated potential instability for most of them. Functional analysis in a eukaryotic system confirmed the pathogenic effect for the missense variants and for one amino acid deletion, as they all exhibited reduced or null PCC activity and protein levels compared to wild-type constructs. PCCB variants p.E168del, p.Q58P and p.I460T resulted in medium-high protein levels and no activity. Variants p.R230C and p.C712S in PCCA, and p.G188A, p.R272W and p.H534R in PCCB retained both partial PCC activity and medium-high protein levels. Available patients-derived fibroblasts carriers of some of these mutations were grown at 28 °C or 37 °C and a slight increase in PCC activity or protein could be detected in some cases at the folding-permissive conditions. Examination of available clinical data showed correlation of the results of the functional analysis with disease severity for most mutations, with some notable exceptions, confirming the notion that the final phenotypic outcome in PA is not easily predicted.

Treatment with antioxidants ameliorates oxidative damage in a mouse model of propionic acidemia.

Oxidative stress contributes to the pathogenesis of propionic acidemia (PA), a life threatening disease caused by the deficiency of propionyl CoA-carboxylase, in the catabolic pathway of branched-chain amino acids, odd-number chain fatty acids and cholesterol. Patients develop multisystemic complications including seizures, extrapyramidal symptoms, basal ganglia deterioration, pancreatitis and cardiomyopathy. The accumulation of toxic metabolites results in mitochondrial dysfunction, increased reactive oxygen species and oxidative damage, all of which have been documented in patients' samples and in a hypomorphic mouse model. Here we set out to investigate whether treatment with a mitochondria-targeted antioxidant, MitoQ, or with the natural polyphenol resveratrol, which is reported to have antioxidant and mitochondrial activation properties, could ameliorate the altered redox status and its functional consequences in the PA mouse model. The results show that oral treatment with MitoQ or resveratrol decreases lipid peroxidation and the expression levels of DNA repair enzyme OGG1 in PA mouse liver, as well as inducing tissue-specific changes in the expression of antioxidant enzymes. Notably, treatment decreased the cardiac hypertrophy marker BNP that is found upregulated in the PA mouse heart. Overall, the results provide in vivo evidence to justify more in depth investigations of antioxidants as adjuvant therapy in PA.

Role of miRNAs in human disease and inborn errors of metabolism.

MicroRNAs (miRNAs) are short, noncoding RNAs that regulate gene expression posttranscriptionally by base pairing with target messenger RNAs (mRNAs). They are estimated to target ∼60% of all human protein-coding genes and are involved in regulating key physiological processes and intracellular signaling pathways. They also exhibit tissue specificity, and their dysregulation is linked to the progression of pathology. Identifying disease associated miRNAs and their respective targets provides novel molecular insight into disease, enabling the design of new therapeutic strategies. Notably, miRNAs are present in stable form in biological fluids, making them amenable to routine clinical processing and analysis, which has paved the way for their use as novel biomarkers of disease and response to therapy. One of the most relevant findings in miRNA research concerns the therapeutic modulation of specific miRNA levels in vitro and in vivo, which has led to miRNA-based drugs entering clinical trials. Most studies relative to miRNA profiling, association with pathology, and therapeutical modulation have been conducted for cancer, cardiovascular and neurodegenerative diseases. However, for different monogenic diseases, including inborn errors of metabolism (IEM), research contributing to alterations to physiopathology caused by miRNAs is steadily increasing. Herein, we review the biogenesis pathway and mode of miRNA action, their known roles in disease states, and use of circulating miRNAs as biomarkers, describing the available research tools for basic and clinical studies. In addition, we summarize recent literature on miRNA studies in inherited metabolic diseases.

Pathogenic implications of dysregulated miRNAs in propionic acidemia related cardiomyopathy.

Cardiac alterations (hypertrophic/dilated cardiomyopathy, and rhythm alterations) are one of the major causes of mortality and morbidity in propionic acidemia (PA), caused by the deficiency of the mitochondrial enzyme propionyl-CoA carboxylase (PCC), involved in the catabolism of branched-chain amino acids, cholesterol, and odd-chain fatty acids. Impaired mitochondrial oxidative phosphorylation has been documented in heart biopsies of PA patients, as well as in the hypomorphic Pcca-/-(A138T) mouse model, in the latter correlating with increased oxidative damage and elevated expression of cardiac dysfunction biomarkers atrial and brain natriuretic peptides (ANP and BNP) and beta-myosin heavy chain (ß-MHC). Here we characterize the cardiac phenotype in the PA mouse model by histological and echocardiography studies and identify a series of upregulated cardiac-enriched microRNAs (miRNAs) in the PA mouse heart, some of them also altered as circulating miRNAs in PA patients' plasma samples. In PA mice hearts, we show alterations in signaling pathways regulated by the identified miRNAs, which could be contributing to cardiac remodeling and dysfunction; notably, an activation of the mammalian target of rapamycin (mTOR) pathway and a decrease in autophagy, which are reverted by rapamycin treatment. In vitro studies in HL-1 cardiomyocytes indicate that propionate, the major toxic metabolite accumulating in the disease, triggers the increase in expression levels of miRNAs, BNP, and ß-MHC, concomitant with an increase in reactive oxygen species. Our results highlight miRNAs and signaling alterations in the PCC-deficient heart which may contribute to the development of PA-associated cardiomyopathy and provide a basis to identify new targets for therapeutic intervention.

Altered Redox Homeostasis in Branched-Chain Amino Acid Disorders, Organic Acidurias, and Homocystinuria.

Inborn errors of metabolism (IEMs) are a group of monogenic disorders characterized by dysregulation of the metabolic networks that underlie development and homeostasis. Emerging evidence points to oxidative stress and mitochondrial dysfunction as major contributors to the multiorgan alterations observed in several IEMs. The accumulation of toxic metabolites in organic acidurias, respiratory chain, and fatty acid oxidation disorders inhibits mitochondrial enzymes and processes resulting in elevated levels of reactive oxygen species (ROS). In other IEMs, as in homocystinuria, different sources of ROS have been proposed. In patients' samples, as well as in cellular and animal models, several studies have identified significant increases in ROS levels along with decreases in antioxidant defences, correlating with oxidative damage to proteins, lipids, and DNA. Elevated ROS disturb redox-signaling pathways regulating biological processes such as cell growth, differentiation, or cell death; however, there are few studies investigating these processes in IEMs. In this review, we describe the published data on mitochondrial dysfunction, oxidative stress, and impaired redox signaling in branched-chain amino acid disorders, other organic acidurias, and homocystinuria, along with recent studies exploring the efficiency of antioxidants and mitochondria-targeted therapies as therapeutic compounds in these diseases.

Dysregulated miRNAs and their pathogenic implications for the neurometabolic disease propionic acidemia.

miRNome expression profiling was performed in a mouse model of propionic acidemia (PA) and in patients' plasma samples to investigate the role of miRNAs in the pathophysiology of the disease and to identify novel biomarkers and therapeutic targets. PA is a potentially lethal neurometabolic disease with patients developing neurological deficits and cardiomyopathy in the long-term, among other complications. In the PA mouse liver we identified 14 significantly dysregulated miRNAs. Three selected miRNAs, miR-34a-5p, miR-338-3p and miR-350, were found upregulated in brain and heart tissues. Predicted targets involved in apoptosis, stress-signaling and mitochondrial function, were inversely found down-regulated. Functional analysis with miRNA mimics in cellular models confirmed these findings. miRNA profiling in plasma samples from neonatal PA patients and age-matched control individuals identified a set of differentially expressed miRNAs, several were coincident with those identified in the PA mouse, among them miR-34a-5p and miR-338-3p. These two miRNAs were also found dysregulated in childhood and adult PA patients' cohorts. Taken together, the results reveal miRNA signatures in PA useful to identify potential biomarkers, to refine the understanding of the molecular mechanisms of this rare disease and, eventually, to improve the management of patients.

Functional characterization of the spf/ash splicing variation in OTC deficiency of mice and man.

The spf/ash mouse model of ornithine transcarbamylase (OTC) deficiency, a severe urea cycle disorder, is caused by a mutation (c.386G>A; p.R129H) in the last nucleotide of exon 4 of the Otc gene, affecting the 5' splice site and resulting in partial use of a cryptic splice site 48 bp into the adjacent intron. The equivalent nucleotide change and predicted amino acid change is found in OTC deficient patients. Here we have used liver tissue and minigene assays to dissect the transcriptional profile resulting from the "spf/ash" mutation in mice and man. For the mutant mouse, we confirmed liver transcripts corresponding to partial intron 4 retention by the use of the c.386+48 cryptic site and to normally spliced transcripts, with exon 4 always containing the c.386G>A (p.R129H) variant. In contrast, the OTC patient exhibited exon 4 skipping or c.386G>A (p.R129H)-variant exon 4 retention by using the natural or a cryptic splice site at nucleotide position c.386+4. The corresponding OTC tissue enzyme activities were between 3-6% of normal control in mouse and human liver. The use of the cryptic splice sites was reproduced in minigenes carrying murine or human mutant sequences. Some normally spliced transcripts could be detected in minigenes in both cases. Antisense oligonucleotides designed to block the murine cryptic +48 site were used in minigenes in an attempt to redirect splicing to the natural site. The results highlight the relevance of in depth investigations of the molecular mechanisms of splicing mutations and potential therapeutic approaches. Notably, they emphasize the fact that findings in animal models may not be applicable for human patients due to the different genomic context of the mutations.