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.

New pathogenic variants in COQ4 cause ataxia and neurodevelopmental disorder without detectable CoQ10 deficiency in muscle or skin fibroblasts.

COQ4 is a component of an enzyme complex involved in the biosynthesis of coenzyme Q10 (CoQ10), a molecule with primary importance in cell metabolism. Mutations in the COQ4 gene are responsible for mitochondrial diseases showing heterogeneous age at onset, clinical presentations and association with CoQ10 deficiency. We herein expand the phenotypic and genetic spectrum of COQ4-related diseases, by reporting two patients harboring bi-allelic variants but not showing CoQ10 deficiency. One patient was found to harbor compound heterozygous mutations (specifically, c.577C>T/p.Pro193Ser and the previously reported c.718C>T/p.Arg240Cys) associated with progressive spasticity, while the other harbored two novel missense (c.284G>A/p.Gly95Asp and c.305G>A/p.Arg102His) associated with a neurodevelopmental disorder. Both patients presented motor impairment and ataxia. To further understand the role of COQ4, we performed functional studies in patient-derived fibroblasts, yeast and "crispant" zebrafish larvae. Micro-oxygraphy showed impaired oxygen consumption rates in one patient, while yeast complementation assays showed that all the mutations were presumably disease related. Moreover, characterization of the coq4 F0 CRISPR zebrafish line showed motor defects and cell reduction in a specific area of the hindbrain, a region reminiscent of the human cerebellum. Our expanded phenotype associated with COQ4 mutations allowed us to investigate, for the first time, the role of COQ4 in brain development in vivo.

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.

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.

Not only dominant, not only optic atrophy: expanding the clinical spectrum associated with OPA1 mutations.

BACKGROUND: Heterozygous mutations in OPA1 are a common cause of autosomal dominant optic atrophy, sometimes associated with extra-ocular manifestations. Few cases harboring compound heterozygous OPA1 mutations have been described manifesting complex neurodegenerative disorders in addition to optic atrophy. RESULTS: We report here three patients: one boy showing an early-onset mitochondrial disorder with hypotonia, ataxia and neuropathy that was severely progressive, leading to early death because of multiorgan failure; two unrelated sporadic girls manifesting a spastic ataxic syndrome associated with peripheral neuropathy and, only in one, optic atrophy. Using a targeted resequencing of 132 genes associated with mitochondrial disorders, in two probands we found compound heterozygous mutations in OPA1: in the first a 5 nucleotide deletion, causing a frameshift and insertion of a premature stop codon (p.Ser64Asnfs*7), and a missense change (p.Ile437Met), which has recently been reported to have clinical impact; in the second, a novel missense change (p.Val988Phe) co-occurred with the p.Ile437Met substitution. In the third patient a homozygous mutation, c.1180G > A (p.Ala394Thr) in OPA1 was detected by a trio-based whole exome sequencing approach. One of the patients presented also variants in mitochondrial DNA that may have contributed to the peculiar phenotype. The deleterious effect of the identified missense changes was experimentally validated in yeast model. OPA1 level was reduced in available patients' biological samples, and a clearly fragmented mitochondrial network was observed in patients' fibroblasts. CONCLUSIONS: This report provides evidence that bi-allelic OPA1 mutations may lead to complex and severe multi-system recessive mitochondrial disorders, where optic atrophy might not represent the main feature.

Infective and non-infective endocarditis in critically ill patients: a clinical-pathological study.

The aims of this study are to estimate the incidence, the outcome and the associated risk factors of infective and non-infective endocarditis (IE and NIE, respectively) in intensive care unit (ICU) patients. We studied the post-mortem findings and the clinical data of the patients who died in our ICU between 1996 and 2010. Of the 765 reviewed autopsies, 21 patients (2.7%) presented cardiac vegetations. These cases consisted of 12 IEs and 9 NIEs. Three patients with IE had a mechanical prosthetic valve, and in 11 cases invasive devices had been used. Multiple peripheral embolisms were discovered at autopsy. In particular, the brain appeared to be more affected in patients with IE, while pulmonary embolisms were commonly associated with NIE. Blood cultures were positive in nine patients with IE. The imaging diagnostics (transthoracic and transesophageal echocardiography) which were seldom performed in both groups, proved to be of little help. As a consequence, an IE was correctly diagnosed before death in three patients (25%) and suspected in two other cases (17%), while a NIE was diagnosed before death in one patient alone. In conclusions, critically ill patients admitted to general ICUs, multiple factors related both to the underlying conditions and to performed procedures can facilitate the occurrence of IE and NIE making, at the same time, their diagnosis challenging. Many cases, in fact, are diagnosed only at autopsy. Yet again, post-mortem examination proves to be an invaluable tool for the evaluation of diagnostic accuracy in critical care.

Pulmonary embolism in critically ill patients receiving antithrombotic prophylaxis: a clinical-pathologic study.

PURPOSE: The true incidence of pulmonary embolism (PE) in critically ill adult patients receiving antithrombotic prophylaxis is unknown, as well as the impact on the outcome. The aim of this study was to assess the incidence of PE in a surgical and medical intensive care unit and to evaluate the presence of risk factors that could be helpful in identifying patients at higher risk of missed diagnosis. MATERIALS AND METHODS: We retrospectively reviewed the autopsies and clinical data of all patients who died in our intensive care unit from 1996 to 2007. All patients received prophylaxis with subcutaneous low-molecular weight heparin. RESULTS: Among the 600 autopsies, the clinical diagnosis of PE was confirmed in 13 patients (true positives) and not confirmed in 20 patients; in 73 patients, the PE was discovered only at the autopsy (false negatives [FNs]). The overall incidence of PE in our patients was 14.3%. Pulmonary embolism was considered the cause of death in 45% of FNs and 77% of true positives. Among all comorbidities, only a recent abdominal surgery and the presence of acute renal failure were associated with a higher risk of missed diagnosis. In the FN group, there was a significantly higher frequency of cases of septic shock. CONCLUSIONS: Despite thromboprophylaxis, critically ill patients remain at risk for PE; and because of the difficulty in diagnosing it clinically, the death certificate diagnosis of PE underestimates the problem.

Acute myocardial infarction in non-cardiac critically ill patients: a clinical-pathological study.

BACKGROUND: In patients admitted to the Intensive Care Unit (ICU) for non cardiac disease, the diagnosis of acute coronary syndromes can be challenging. The aim of the study was to define the rate of discrepancies concerning the diagnosis of acute myocardial infarction and to evaluate the presence of risk factors that could be helpful in identifying patients at higher risk of missed diagnosis. METHODS: We compared clinical and autopsy records of 600 critically ill patients who died in our ICU in a 10-years period. We identified patients in whom acute myocardial infarction was reported as the cause of death on the clinical records or was discovered only at post-mortem examination. These subjects were subsequently divided into two Groups: patients in Group 1 underwent diagnostic evaluation for acute myocardial infarction whereas those in Group 2 were not investigated for. RESULTS: In Group 1, a definite clinical diagnosis was reached in 11 patients (14,7%) but remained undetermined in 37 patients (48%). The diagnosis was totally missed in 8 patients in Group 1 (10,6%) and in 20 patients of Group 2 (26,6%). The diagnostic discrepancy was higher in septic patients, in whom the correct diagnosis of acute myocardial infarction was established at a rate lower than 50% in respect to non-septic patients. CONCLUSIONS: Our experience strengthens the role of post-mortem examination as a source of feed-back of the overall diagnostic and therapeutic approach especially in septic patients, where the diagnostic error is more frequent.