COQ4 is required for the oxidative decarboxylation of the C1 carbon of coenzyme Q in eukaryotic cells.

Coenzyme Q (CoQ) is a redox lipid that fulfills critical functions in cellular bioenergetics and homeostasis. CoQ is synthesized by a multi-step pathway that involves several COQ proteins. Two steps of the eukaryotic pathway, the decarboxylation and hydroxylation of position C1, have remained uncharacterized. Here, we provide evidence that these two reactions occur in a single oxidative decarboxylation step catalyzed by COQ4. We demonstrate that COQ4 complements an Escherichia coli strain deficient for C1 decarboxylation and hydroxylation and that COQ4 displays oxidative decarboxylation activity in the non-CoQ producer Corynebacterium glutamicum. Overall, our results substantiate that COQ4 contributes to CoQ biosynthesis, not only via its previously proposed structural role but also via the oxidative decarboxylation of CoQ precursors. These findings fill a major gap in the knowledge of eukaryotic CoQ biosynthesis and shed light on the pathophysiology of human primary CoQ deficiency due to COQ4 mutations.

Increased mitophagy in the skeletal muscle of spinal and bulbar muscular atrophy patients.

Spinal and bulbar muscular atrophy (SBMA) is a neuromuscular disorder caused by polyglutamine expansion in the androgen receptor (AR) and characterized by the loss of lower motor neurons. Here we investigated pathological processes occurring in muscle biopsy specimens derived from SBMA patients and, as controls, age-matched healthy subjects and patients suffering from amyotrophic lateral sclerosis (ALS) and neurogenic atrophy. We detected atrophic fibers in the muscle of SBMA, ALS and neurogenic atrophy patients. In addition, SBMA muscle was characterized by the presence of a large number of hypertrophic fibers, with oxidative fibers having a larger size compared with glycolytic fibers. Polyglutamine-expanded AR expression was decreased in whole muscle, yet enriched in the nucleus, and localized to mitochondria. Ultrastructural analysis revealed myofibrillar disorganization and streaming in zones lacking mitochondria and degenerating mitochondria. Using molecular (mtDNA copy number), biochemical (citrate synthase and respiratory chain enzymes) and morphological (dark blue area in nicotinamide adenine dinucleotide-stained muscle cross-sections) analyses, we found a depletion of the mitochondria associated with enhanced mitophagy. Mass spectrometry analysis revealed an increase of phosphatidylethanolamines and phosphatidylserines in mitochondria isolated from SBMA muscles, as well as a 50% depletion of cardiolipin associated with decreased expression of the cardiolipin synthase gene. These observations suggest a causative link between nuclear polyglutamine-expanded AR accumulation, depletion of mitochondrial mass, increased mitophagy and altered mitochondrial membrane composition in SBMA muscle patients. Given the central role of mitochondria in cell bioenergetics, therapeutic approaches toward improving the mitochondrial network are worth considering to support SBMA patients.

Mutations in COQ8B (ADCK4) found in patients with steroid-resistant nephrotic syndrome alter COQ8B function.

Mutations in COQ8B cause steroid-resistant nephrotic syndrome with variable neurological involvement. In yeast, COQ8 encodes a protein required for coenzyme Q (CoQ) biosynthesis, whose precise role is not clear. Humans harbor two paralog genes: COQ8A and COQ8B (previously termed ADCK3 and ADCK4). We have found that COQ8B is a mitochondrial matrix protein peripherally associated with the inner membrane. COQ8B can complement a ΔCOQ8 yeast strain when its mitochondrial targeting sequence (MTS) is replaced by a yeast MTS. This model was employed to validate COQ8B mutations, and to establish genotype-phenotype correlations. All mutations affected respiratory growth, but there was no correlation between mutation type and the severity of the phenotype. In fact, contrary to the case of COQ2, where residual CoQ biosynthesis correlates with clinical severity, patients harboring hypomorphic COQ8B alleles did not display a different phenotype compared with those with null mutations. These data also suggest that the system is redundant, and that other proteins (probably COQ8A) may partially compensate for the absence of COQ8B. Finally, a COQ8B polymorphism, present in 50% of the European population (NM_024876.3:c.521A > G, p.His174Arg), affects stability of the protein and could represent a risk factor for secondary CoQ deficiencies or for other complex traits.

COX16 is required for assembly of cytochrome c oxidase in human cells and is involved in copper delivery to COX2.

Cytochrome c oxidase (COX), complex IV of the mitochondrial respiratory chain, is comprised of 14 structural subunits, several prosthetic groups and metal cofactors, among which copper. Its biosynthesis involves a number of ancillary proteins, encoded by the COX-assembly genes that are required for the stabilization and membrane insertion of the nascent polypeptides, the synthesis of the prosthetic groups, and the delivery of the metal cofactors, in particular of copper. Recently, a modular model for COX assembly has been proposed, based on the sequential incorporation of different assembly modules formed by specific subunits. We have cloned and characterized the human homologue of yeast COX16. We show that human COX16 encodes a small mitochondrial transmembrane protein that faces the intermembrane space and is highly expressed in skeletal and cardiac muscle. Its knockdown in C. elegans produces COX deficiency, and its ablation in HEK293 cells impairs COX assembly. Interestingly, COX16 knockout cells retain significant COX activity, suggesting that the function of COX16 is partially redundant. Analysis of steady-state levels of COX subunits and of assembly intermediates by Blue-Native gels shows a pattern similar to that reported in cells lacking COX18, suggesting that COX16 is required for the formation of the COX2 subassembly module. Moreover, COX16 co-immunoprecipitates with COX2. Finally, we found that copper supplementation increases COX activity and restores normal steady state levels of COX subunits in COX16 knockout cells, indicating that, even in the absence of a canonical copper binding motif, COX16 could be involved in copper delivery to COX2.

The COQ2 genotype predicts the severity of coenzyme Q10 deficiency.

COQ2 (p-hydroxybenzoate polyprenyl transferase) encodes the enzyme required for the second step of the final reaction sequence of Coenzyme Q10 (CoQ) biosynthesis. Its mutations represent a frequent cause of primary CoQ deficiency and have been associated with the widest clinical spectrum, ranging from fatal neonatal multisystemic disease to late-onset encephalopathy. However, the reasons of this variability are still unknown.We have characterized the structure of human COQ2, defined its subcellular localization and developed a yeast model to validate all the mutant alleles reported so far.Our findings show that the main functional transcript of COQ2 is shorter than what was previously reported and that its protein product localizes to mitochondria with the C-terminus facing the intermembrane space. Complementation experiments in yeast showed that the residual activity of the mutant proteins correlates with the clinical phenotypes observed in patients.We defined the structure of COQ2 with relevant implications for mutation screening in patients and demonstrated that, contrary to other COQ gene defects such as ADCK3, there is a correlation between COQ2 genotype and patient's phenotype.

Coenzyme Q biosynthesis in health and disease.

Coenzyme Q (CoQ, or ubiquinone) is a remarkable lipid that plays an essential role in mitochondria as an electron shuttle between complexes I and II of the respiratory chain, and complex III. It is also a cofactor of other dehydrogenases, a modulator of the permeability transition pore and an essential antioxidant. CoQ is synthesized in mitochondria by a set of at least 12 proteins that form a multiprotein complex. The exact composition of this complex is still unclear. Most of the genes involved in CoQ biosynthesis (COQ genes) have been studied in yeast and have mammalian orthologues. Some of them encode enzymes involved in the modification of the quinone ring of CoQ, but for others the precise function is unknown. Two genes appear to have a regulatory role: COQ8 (and its human counterparts ADCK3 and ADCK4) encodes a putative kinase, while PTC7 encodes a phosphatase required for the activation of Coq7. Mutations in human COQ genes cause primary CoQ(10) deficiency, a clinically heterogeneous mitochondrial disorder with onset from birth to the seventh decade, and with clinical manifestation ranging from fatal multisystem disorders, to isolated encephalopathy or nephropathy. The pathogenesis of CoQ(10) deficiency involves deficient ATP production and excessive ROS formation, but possibly other aspects of CoQ(10) function are implicated. CoQ(10) deficiency is unique among mitochondrial disorders since an effective treatment is available. Many patients respond to oral CoQ(10) supplementation. Nevertheless, treatment is still problematic because of the low bioavailability of the compound, and novel pharmacological approaches are currently being investigated. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi.

Molecular characterization of the human COQ5 C-methyltransferase in coenzyme Q10 biosynthesis.

Coq5 catalyzes the only C-methylation involved in the biosynthesis of coenzyme Q (Q or ubiquinone) in humans and yeast Saccharomyces cerevisiae. As one of eleven polypeptides required for Q production in yeast, Coq5 has also been shown to assemble with the multi-subunit complex termed the CoQ-synthome. In humans, mutations in several COQ genes cause primary Q deficiency, and a decrease in Q biosynthesis is associated with mitochondrial, cardiovascular, kidney and neurodegenerative diseases. In this study, we characterize the human COQ5 polypeptide and examine its complementation of yeast coq5 point and null mutants. We show that human COQ5 RNA is expressed in all tissues and that the COQ5 polypeptide is associated with the mitochondrial inner membrane on the matrix side. Previous work in yeast has shown that point mutations within or adjacent to conserved COQ5 methyltransferase motifs result in a loss of Coq5 function but not Coq5 steady state levels. Here, we show that stabilization of the CoQ-synthome within coq5 point mutants or by over-expression of COQ8 in coq5 null mutants permits the human COQ5 homolog to partially restore coq5 mutant growth on respiratory media and Q6 content. Immunoblotting against the human COQ5 polypeptide in isolated yeast mitochondria shows that the human Coq5 polypeptide migrates in two-dimensional blue-native/SDS-PAGE at the same high molecular mass as other yeast Coq proteins. The results presented suggest that human and Escherichia coli Coq5 homologs expressed in yeast retain C-methyltransferase activity but are capable of rescuing the coq5 yeast mutants only when the CoQ-synthome is assembled.

Genetic bases and clinical manifestations of coenzyme Q10 (CoQ 10) deficiency.

Coenzyme Q(10) is a remarkable lipid involved in many cellular processes such as energy production through the mitochondrial respiratory chain (RC), beta-oxidation of fatty acids, and pyrimidine biosynthesis, but it is also one of the main cellular antioxidants. Its biosynthesis is still incompletely characterized and requires at least 15 genes. Mutations in eight of them (PDSS1, PDSS2, COQ2, COQ4, COQ6, ADCK3, ADCK4, and COQ9) cause primary CoQ(10) deficiency, a heterogeneous group of disorders with variable age of onset (from birth to the seventh decade) and associated clinical phenotypes, ranging from a fatal multisystem disease to isolated steroid resistant nephrotic syndrome (SRNS) or isolated central nervous system disease. The pathogenesis is complex and related to the different functions of CoQ(10). It involves defective ATP production and oxidative stress, but also an impairment of pyrimidine biosynthesis and increased apoptosis. CoQ(10) deficiency can also be observed in patients with defects unrelated to CoQ(10) biosynthesis, such as RC defects, multiple acyl-CoA dehydrogenase deficiency, and ataxia and oculomotor apraxia.Patients with both primary and secondary deficiencies benefit from high-dose oral supplementation with CoQ(10). In primary forms treatment can stop the progression of both SRNS and encephalopathy, hence the critical importance of a prompt diagnosis. Treatment may be beneficial also for secondary forms, although with less striking results.In this review we will focus on CoQ(10) biosynthesis in humans, on the genetic defects and the specific clinical phenotypes associated with CoQ(10) deficiency, and on the diagnostic strategies for these conditions.

A yeast based assay establishes the pathogenicity of novel missense ACTA2 variants associated with aortic aneurysms.

The ACTA2 gene codes for alpha-smooth muscle actin, a critical component of the contractile apparatus of the vascular smooth muscle cells. Autosomal dominant variants in the ACTA2 gene have been associated to familial non-syndromic thoracic aortic aneurysm/dissection (TAAD). They are thought to act through a dominant-negative mechanism. These variants display incomplete penetrance and variable expressivity, complicating the validation of ACTA2 variants pathogenicity by family segregation studies. In this study, we developed a yeast based assay to test putative TAAD-associated ACTA2 variants. We identified five new heterozygous ACTA2 missense variants in TAAD patients through next generation sequencing. We decided to test their pathogenicity in Saccharomyces cerevisiae, since yeast actin is very similar to human alpha-smooth muscle actin, and the residues at which the TAAD-associated variants occur in ACTA2 are well conserved. A wild type yeast strain was transformed with a vector expressing the different mutant alleles, to model the heterozygous condition of patients. Then, we evaluated yeast growth by spot test and cytoskeletal and mitochondrial morphology by fluorescence microscopy. We found that mutant yeast strains displayed only mild growth defects but a significant increase in the percentage of cells with abnormal mitochondrial distribution and abnormal organization of the actin cytoskeleton compared to controls. All variants appeared to interfere with the activity of wild type actin in yeast, suggesting a dominant-negative pathogenic mechanism. Our results demonstrate the utility of using the yeast actin model system to validate the pathogenicity of TAAD-associated ACTA2 variants.

Retinoic acid receptor activation reprograms senescence response and enhances anti-tumor activity of natural killer cells.

Cellular senescence can exert dual effects in tumors, either suppressing or promoting tumor progression. The senescence-associated secretory phenotype (SASP), released by senescent cells, plays a crucial role in this dichotomy. Consequently, the clinical challenge lies in developing therapies that safely enhance senescence in cancer, favoring tumor-suppressive SASP factors over tumor-promoting ones. Here, we identify the retinoic-acid-receptor (RAR) agonist adapalene as an effective pro-senescence compound in prostate cancer (PCa). Reactivation of RARs triggers a robust senescence response and a tumor-suppressive SASP. In preclinical mouse models of PCa, the combination of adapalene and docetaxel promotes a tumor-suppressive SASP that enhances natural killer (NK) cell-mediated tumor clearance more effectively than either agent alone. This approach increases the efficacy of the allogenic infusion of human NK cells in mice injected with human PCa cells, suggesting an alternative therapeutic strategy to stimulate the anti-tumor immune response in "immunologically cold" tumors.

Functional analysis of missense mutations of OAT, causing gyrate atrophy of choroid and retina.

We studied eight kindreds with gyrate atrophy of choroid and retina (GA), a rare autosomal recessive disorder caused by mutations of the OAT gene, encoding the homoexameric enzyme ornithine-delta-aminotransferase. We identified four novel and five previously reported mutations. Missense alleles were expressed in yeast strain carrying a deletion of the orthologous of human OAT. All mutations markedly reduced enzymatic activity. However, the effect on the yeast growth was variable, suggesting that some mutations retain residual activity, below the threshold of the enzymatic assay. Mutant proteins were either highly unstable and rapidly degraded, or failed to assemble to form the active OAT hexamer. Where possible, fibroblast analysis confirmed these data. We found no correlation between the residual enzymatic activity and the age of onset, or the severity of symptoms. Moreover, the response to B6 was apparently not related to the specific mutations carried by patients. Overall these data suggest that other factors besides the specific OAT genotype modulate (GA) phenotype in patients. Finally, we found that 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), an AMPK activator known to increase mitochondrial biogenesis, markedly stimulates OAT expression, thus representing a possible treatment for a subset of GA patients with hypomorphic alleles.

Haploinsufficiency of COQ4 causes coenzyme Q10 deficiency.

BACKGROUND: COQ4 encodes a protein that organises the multienzyme complex for the synthesis of coenzyme Q(10) (CoQ(10)). A 3.9 Mb deletion of chromosome 9q34.13 was identified in a 3-year-old boy with mental retardation, encephalomyopathy and dysmorphic features. Because the deletion encompassed COQ4, the patient was screened for CoQ(10) deficiency. METHODS: A complete molecular and biochemical characterisation of the patient's fibroblasts and of a yeast model were performed. RESULTS: The study found reduced COQ4 expression (48% of controls), CoQ(10) content and biosynthetic rate (44% and 43% of controls), and activities of respiratory chain complex II+III. Cells displayed a growth defect that was corrected by the addition of CoQ(10) to the culture medium. Knockdown of COQ4 in HeLa cells also resulted in a reduction of CoQ(10.) Diploid yeast haploinsufficient for COQ4 displayed similar CoQ deficiency. Haploinsufficency of other genes involved in CoQ(10) biosynthesis does not cause CoQ deficiency, underscoring the critical role of COQ4. Oral CoQ(10) supplementation resulted in a significant improvement of neuromuscular symptoms, which reappeared after supplementation was temporarily discontinued. CONCLUSION: Mutations of COQ4 should be searched for in patients with CoQ(10) deficiency and encephalomyopathy; patients with genomic rearrangements involving COQ4 should be screened for CoQ(10) deficiency, as they could benefit from supplementation.

OPA1 drives macrophage metabolism and functional commitment via p65 signaling.

Macrophages are essential players for the host response against pathogens, regulation of inflammation and tissue regeneration. The wide range of macrophage functions rely on their heterogeneity and plasticity that enable a dynamic adaptation of their responses according to the surrounding environmental cues. Recent studies suggest that metabolism provides synergistic support for macrophage activation and elicitation of desirable immune responses; however, the metabolic pathways orchestrating macrophage activation are still under scrutiny. Optic atrophy 1 (OPA1) is a mitochondria-shaping protein controlling mitochondrial fusion, cristae biogenesis and respiration; clear evidence shows that the lack or dysfunctional activity of this protein triggers the accumulation of metabolic intermediates of the TCA cycle. In this study, we show that OPA1 has a crucial role in macrophage activation. Selective Opa1 deletion in myeloid cells impairs M1-macrophage commitment. Mechanistically, Opa1 deletion leads to TCA cycle metabolite accumulation and defective NF-κB signaling activation. In an in vivo model of muscle regeneration upon injury, Opa1 knockout macrophages persist within the damaged tissue, leading to excess collagen deposition and impairment in muscle regeneration. Collectively, our data indicate that OPA1 is a key metabolic driver of macrophage functions.

COQ4 is required for the oxidative decarboxylation of the C1 carbon of Coenzyme Q in eukaryotic cells.

Coenzyme Q (CoQ) is a redox lipid that fulfills critical functions in cellular bioenergetics and homeostasis. CoQ is synthesized by a multi-step pathway that involves several COQ proteins. Two steps of the eukaryotic pathway, the decarboxylation and hydroxylation of position C1, have remained uncharacterized. Here, we provide evidence that these two reactions occur in a single oxidative decarboxylation step catalyzed by COQ4. We demonstrate that COQ4 complements an Escherichia coli strain deficient for C1 decarboxylation and hydroxylation and that COQ4 displays oxidative decarboxylation activity in the non-CoQ producer Corynebacterium glutamicum. Overall, our results substantiate that COQ4 contributes to CoQ biosynthesis, not only via its previously proposed structural role, but also via oxidative decarboxylation of CoQ precursors. These findings fill a major gap in the knowledge of eukaryotic CoQ biosynthesis, and shed new light on the pathophysiology of human primary CoQ deficiency due to COQ4 mutations.

The multiple roles of coenzyme Q in cellular homeostasis and their relevance for the pathogenesis of coenzyme Q deficiency.

Coenzyme Q (CoQ) is a redox active lipid that plays a central role in cellular homeostasis. It was discovered more than 60 years ago because of its role as electron transporter in the mitochondrial respiratory chain. Since then it has become evident that CoQ has many other functions, not directly related to bioenergetics. It is a cofactor of several mitochondrial dehydrogenases involved in the metabolism of lipids, amino acids, and nucleotides, and in sulfide detoxification. It is a powerful antioxidant and it is involved in the control of programmed cell death by modulating both apoptosis and ferroptosis. CoQ deficiency is a clinically and genetically heterogeneous group of disorders characterized by the impairment of CoQ biosynthesis. CoQ deficient patients display defects in cellular bioenergetics, but also in the other pathways in which CoQ is involved. In this review we will focus on the functions of CoQ not directly related to the respiratory chain, and on how their impairment is relevant for the pathophysiology of CoQ deficiency. A better understanding of the complex set of events triggered by CoQ deficiency will allow to design novel approaches for the treatment of this condition.

Deficit of human ornithine aminotransferase in gyrate atrophy: Molecular, cellular, and clinical aspects.

Gyrate Atrophy (GA) of the choroid and retina (MIM# 258870) is an autosomal recessive disorder due to mutations of the OAT gene encoding ornithine-delta-aminotransferase (OAT), associated with progressive retinal deterioration and blindness. The disease has a theoretical global incidence of approximately 1:1,500,000. OAT is mainly involved in ornithine catabolism in adults, thus explaining the hyperornithinemia as hallmark of the disease. Patients are treated with an arginine-restricted diet, to limit ornithine load, or the administration of Vitamin B6, a precursor of the OAT coenzyme pyridoxal phosphate. Although the clinical and genetic aspects of GA are known for many years, the enzymatic phenotype of pathogenic variants and their response to Vitamin B6, as well as the molecular mechanisms explaining retinal damage, are poorly clarified. Herein, we provide an overview of the current knowledge on the biochemical properties of human OAT and on the molecular, cellular, and clinical aspects of GA.

Molecular and Cellular Studies Reveal Folding Defects of Human Ornithine Aminotransferase Variants Associated With Gyrate Atrophy of the Choroid and Retina.

The deficit of human ornithine aminotransferase (hOAT) is responsible for gyrate atrophy (GA), a rare recessive inherited disorder. Although more than 60 disease-associated mutations have been identified to date, the molecular mechanisms explaining how each mutation leads to the deficit of OAT are mostly unknown. To fill this gap, we considered six representative missense mutations present in homozygous patients concerning residues spread over the hOAT structure. E. coli expression, spectroscopic, kinetic and bioinformatic analyses, reveal that the R154L and G237D mutations induce a catalytic more than a folding defect, the Q90E and R271K mutations mainly impact folding efficiency, while the E318K and C394Y mutations give rise to both folding and catalytic defects. In a human cellular model of disease folding-defective variants, although at a different extent, display reduced protein levels and/or specific activity, due to increased aggregation and/or degradation propensity. The supplementation with Vitamin B6, to mimic a treatment strategy available for GA patients, does not significantly improve the expression/activity of folding-defective variants, in contrast with the clinical responsiveness of patients bearing the E318K mutation. Thus, we speculate that the action of vitamin B6 could be also independent of hOAT. Overall, these data represent a further effort toward a comprehensive analysis of GA pathogenesis at molecular and cellular level, with important relapses for the improvement of genotype/phenotype correlations and the development of novel treatments.

Cholesterol Metabolic Reprogramming in Cancer and Its Pharmacological Modulation as Therapeutic Strategy.

Cholesterol is a ubiquitous sterol with many biological functions, which are crucial for proper cellular signaling and physiology. Indeed, cholesterol is essential in maintaining membrane physical properties, while its metabolism is involved in bile acid production and steroid hormone biosynthesis. Additionally, isoprenoids metabolites of the mevalonate pathway support protein-prenylation and dolichol, ubiquinone and the heme a biosynthesis. Cancer cells rely on cholesterol to satisfy their increased nutrient demands and to support their uncontrolled growth, thus promoting tumor development and progression. Indeed, transformed cells reprogram cholesterol metabolism either by increasing its uptake and de novo biosynthesis, or deregulating the efflux. Alternatively, tumor can efficiently accumulate cholesterol into lipid droplets and deeply modify the activity of key cholesterol homeostasis regulators. In light of these considerations, altered pathways of cholesterol metabolism might represent intriguing pharmacological targets for the development of exploitable strategies in the context of cancer therapy. Thus, this work aims to discuss the emerging evidence of in vitro and in vivo studies, as well as clinical trials, on the role of cholesterol pathways in the treatment of cancer, starting from already available cholesterol-lowering drugs (statins or fibrates), and moving towards novel potential pharmacological inhibitors or selective target modulators.

Hybrid Minigene Assay: An Efficient Tool to Characterize mRNA Splicing Profiles of NF1 Variants.

Neurofibromatosis type 1 (NF1) is caused by heterozygous loss of function mutations in the NF1 gene. Although patients are diagnosed according to clinical criteria and few genotype-phenotype correlations are known, molecular analysis remains important. NF1 displays allelic heterogeneity, with a high proportion of variants affecting splicing, including deep intronic alleles and changes outside the canonical splice sites, making validation problematic. Next Generation Sequencing (NGS) technologies integrated with multiplex ligation-dependent probe amplification (MLPA) have largely overcome RNA-based techniques but do not detect splicing defects. A rapid minigene-based system was set up to test the effects of NF1 variants on splicing. We investigated 29 intronic and exonic NF1 variants identified in patients during the diagnostic process. The minigene assay showed the coexistence of multiple mechanisms of splicing alterations for seven variants. A leaky effect on splicing was documented in one de novo substitution detected in a sporadic patient with a specific phenotype without neurofibromas. Our splicing assay proved to be a reliable and fast method to validate novel NF1 variants potentially affecting splicing and to detect hypomorphic effects that might have phenotypic consequences, avoiding the requirement of patient's RNA.