Human Mutated MYOT and CRYAB Genes Cause a Myopathic Phenotype in Zebrafish

Myofibrillar myopathies (MFMs) are a group of hereditary neuromuscular disorders sharing common histological features, such as myofibrillar derangement, Z-disk disintegration, and accumulation of degradation products into protein aggregates. They are caused by mutations in several genes that encode either structural proteins or molecular chaperones. Nevertheless, the mechanisms by which mutated genes result in protein aggregation are still unknown. To unveil the role of myotilin and αB-crystallin in the pathogenesis of MFM, we injected zebrafish fertilized eggs at one-cell stage with expression plasmids harboring cDNA sequences of human wildtype or mutated MYOT (p.Ser95Ile) and human wildtype or mutated CRYAB (p.Gly154Ser). We evaluated the effects on fish survival, motor behavior, muscle structure and development. We found that transgenic zebrafish showed morphological defects that were more severe in those overexpressing mutant genes which developed a myopathic phenotype consistent with that of human myofibrillar myopathy including the formation of protein aggregates. Results indicate that pathogenic mutations in myotilin and αB-crystallin genes associated with MFM cause a structural and functional impairment of the skeletal muscle in zebrafish, thereby making this non-mammalian organism a powerful model to dissect disease pathogenesis and find possible druggable targets

Autosomal Dominant Tubulointerstitial Kidney Disease with Adult Onset due to a Novel Renin Mutation Mapping in the Mature Protein

Autosomal dominant tubulointerstitial kidney disease (ADTKD) is a genetically heterogeneous renal disorder leading to progressive loss of renal function. ADTKD-REN is due to rare mutations in renin, all localized in the protein leader peptide and affecting its co-translational insertion in the endoplasmic reticulum (ER). Through exome sequencing in an adult-onset ADTKD family we identified a new renin variant, p.L381P, mapping in the mature protein. To assess its pathogenicity, we combined genetic data, computational and predictive analysis and functional studies. The L381P substitution affects an evolutionary conserved residue, co-segregates with renal disease, is not found in population databases and is predicted to be deleterious by in silico tools and by structural modelling. Expression of the L381P variant leads to its ER retention and induction of the Unfolded Protein Response in cell models and to defective pronephros development in zebrafish. Our work shows that REN mutations outside of renin leader peptide can cause ADTKD and delineates an adult form of ADTKD-REN, a condition which has usually its onset in childhood. This has implications for the molecular diagnosis and the estimated prevalence of the disease and points at ER homeostasis as a common pathway affected in ADTKD-REN, and possibly more generally in ADTKD

Modeling C19orf12 deficiency in mammalian cells and zebrafish

La categoria Neurodegenerazione con Accumulo di Ferro nel Cervello (NBIA) comprende un gruppo di malattie neurodegenerative caratterizzate dall'accumulo eponimo di ferro nel parenchima cerebrale, specialmente nei gangli della base. Le mutazioni nel gene C19orf12 causano l’NBIA4 nota anche come Neurodegenerazione Associata alla Proteina della Membrana Mitocondriale (MPAN). La funzione biologica del gene è finora scarsamente definita; i dati disponibili suggeriscono il suo coinvolgimento nella funzione mitocondriale, nell'autofagia e nel metabolismo lipidico. Utilizzando un approccio di editing del genoma abbiamo generato modelli cellulari e animali con mutazioni deleterie nel gene C19orf12, per indagare il ruolo del gene e i meccanismi coinvolti nello sviluppo del processo neurodegenerativo. Abbiamo valutato diversi processi biologici tra cui l'omeostasi del ferro, la respirazione mitocondriale, l'autofagia e il metabolismo lipidico. Il modello cellulare knockout (KO) non ha mostrato differenze significative nella respirazione mitocondriale, tuttavia ha evidenziato lievi alterazioni nell’induzione del processo autofagico, un aumento del ferro libero e un aumento del contenuto totale di ferro in seguito all'esposizione al metallo, misurato mediante ICP-MS. Da un’analisi preliminare le cellule KO hanno presentato profili lipidici alterati, suggerendo il possibile coinvolgimento del gene nel metabolismo lipidico. La riduzione dell’espressione dell'ortologo umano in zebrafish, c19orf12a, mediante iniezione di un morfolino specifico, ha indotto significative alterazioni morfologiche nelle fasi iniziali dello sviluppo del pesce, confermando la possibilità di utilizzare tale modello per studiare in modo sistematico gli effetti indotti dal deficit di c19orf12a. Tramite genome editing, abbiamo ottenuto linee KO con mutazioni deleterie in omozigosi nel gene c19orf12a e indagato lo sviluppo neuronale tramite WISH con diversi marcatori specifici senza riscontrare anomalie significative. L'analisi del comportamento dei mutanti a diverse età ha mostrato differenze rispetto agli animali di controllo. In particolare, si è osservato un aumento del numero di avvolgimenti della coda a 24 ore dopo la fecondazione, un aumento della distanza percorsa a 120 ore dopo la fertilizzazione e una diminuzione della velocità e della distanza percorsa nei mutanti adulti. Inoltre, indagini preliminari hanno mostrato cambiamenti nel contenuto totale di lipidi e nel loro profilo di distribuzione nelle larve mutanti. Per concludere, in questo studio sono stati generati modelli KO cellulari e animali per C19orf12, la cui caratterizzazione ha evidenziato la presenza di alterazioni fenotipiche e biologiche che potrebbero essere utili per comprendere meglio il ruolo biologico del gene, i meccanismi alla base di MPAN ed essere utilizzati per screening di molecole con potenziale terapeutico.Neurodegeneration with Brain Iron Accumulation (NBIA) is a group of neurodegenerative diseases characterized by the eponymous accumulation of iron in the brain parenchyma, especially in the basal ganglia. Mutations in the gene C19orf12 cause NBIA4, a subtype of NBIA, also called Mitochondrial-membrane Protein-associated Neurodegeneration (MPAN). The biological function of the gene is poorly defined so far; available data suggest its involvement in mitochondrial function, autophagy and lipid metabolism. This study aimed to generate cellular and animal models carrying C19orf12 mutant alleles, using a genome editing approach, to investigate the role of the gene and understand the mechanism underpinning the development of neurodegeneration. We evaluated different biological processes including iron homeostasis, mitochondrial respiration, autophagy, and lipid metabolism. The cellular loss-of-function (LOF) model did not display significant differences in mitochondrial respiration, however, it showed impaired autophagy initiation, a high level of labile iron pool and increased total iron content following exposure to iron, as measured by ICP-MS. Preliminary results on C19orf12 knockout (KO) cells displayed changes in the lipidomic profiles, suggesting a possible involvement of the gene in lipid metabolism. The transient downregulation of the zebrafish orthologue of the human C19orf12, c19orf12a, resulted in significant morphological alterations in the early stages of development, confirming the potential of zebrafish KO models to investigate thoroughly the effects of c19orf12a deficiency. We generated KO lines using the CRISPR-Cas9 technology and followed their development, with a particular focus on neurogenesis. According to WISH analysis, we did not find significant defects in the development of the main neuronal regions in embryos. The analysis of the mutant’s behavior at different developmental stages showed differences compared to wild-type siblings with an increase in the number of tail coiling events at 24 hours-post-fertilization (hpf), an increase in the distance traveled at 120 hpf in the light-dark locomotion test and a decrease in velocity and distance covered by adult mutants in a novel-tank diving test. Additionally, preliminary results showed changes in lipid content and profile in mutant larvae. In conclusion, we generated both in vitro and in vivo models for C19orf12 deficiency. Their characterization highlights different phenotypes and altered pathways related to the absence of the gene that could be useful to better understand the mechanisms underlying this disorder and could be used to screen molecules with therapeutic potential

Coenzyme a Biochemistry: From Neurodevelopment to Neurodegeneration

Coenzyme A (CoA) is an essential cofactor in all living organisms. It is involved in a large number of biochemical processes functioning either as an activator of molecules with carbonyl groups or as a carrier of acyl moieties. Together with its thioester derivatives, it plays a central role in cell metabolism, post-translational modification, and gene expression. Furthermore, recent studies revealed a role for CoA in the redox regulation by the S-thiolation of cysteine residues in cellular proteins. The intracellular concentration and distribution in different cellular compartments of CoA and its derivatives are controlled by several extracellular stimuli such as nutrients, hormones, metabolites, and cellular stresses. Perturbations of the biosynthesis and homeostasis of CoA and/or acyl-CoA are connected with several pathological conditions, including cancer, myopathies, and cardiomyopathies. In the most recent years, defects in genes involved in CoA production and distribution have been found in patients affected by rare forms of neurodegenerative and neurodevelopmental disorders. In this review, we will summarize the most relevant aspects of CoA cellular metabolism, their role in the pathogenesis of selected neurodevelopmental and neurodegenerative disorders, and recent advancements in the search for therapeutic approaches for such diseases

Integrated mutational landscape analysis of uterine leiomyosarcomas

Uterine leiomyosarcomas (uLMS) are aggressive tumors arising from the smooth muscle layer of the uterus. We analyzed 83 uLMS sample genetics, including 56 from Yale and 27 from The Cancer Genome Atlas (TCGA). Among them, a total of 55 Yale samples including two patient-derived xenografts (PDXs) and 27 TCGA samples have whole-exome sequencing (WES) data; 10 Yale and 27 TCGA samples have RNA-sequencing (RNA-Seq) data; and 11 Yale and 10 TCGA samples have whole-genome sequencing (WGS) data. We found recurrent somatic mutations in TP53, MED12, and PTEN genes. Top somatic mutated genes included TP53, ATRX, PTEN, and MEN1 genes. Somatic copy number variation (CNV) analysis identified 8 copy-number gains, including 5p15.33 (TERT), 8q24.21 (C-MYC), and 17p11.2 (MYOCD, MAP2K4) amplifications and 29 copy-number losses. Fusions involving tumor suppressors or on-cogenes were deetected, with most fusions disrupting RB1, TP53, and ATRX/DAXX, and one fusion (ACTG2-ALK) being po-tentially targetable. WGS results demonstrated that 76% (16 of 21) of the samples harbored chromoplexy and/or chromothrip-sis. Clinically actionable mutational signatures of homologous-recombination DNA-repair deficiency (HRD) and microsatellite instability (MSI) were identified in 25% (12 of 48) and 2% (1 of 48) of fresh frozen uLMS, respectively. Finally, we found olaparib (PARPi; P = 0.002), GS-62 6510 (C-MYC/BETi; P < 0.000001 and P = 0.0005), and copanlisib (PIK3CAi; P = 0.0001) monotherapy to significantly inhibit uLMS-PDXs harboring de-rangements in C-MYC and PTEN/PIK3CA/AKT genes (LEY11) and/ or HRD signatures (LEY16) compared to vehicle-treated mice. These findings define the genetic landscape of uLMS and sug-gest that a subset of uLMS may benefit from existing PARP-, PIK3CA-, and C-MYC/BET-targeted drugs

Rare single nucleotide and copy number variants and the etiology of congenital obstructive uropathy : implications for genetic diagnosis

Significance statement: Congenital obstructive uropathy (COU) is a prevalent human developmental defect with highly heterogeneous clinical presentations and outcomes. Genetics may refine diagnosis, prognosis, and treatment, but the genomic architecture of COU is largely unknown. Comprehensive genomic screening study of 733 cases with three distinct COU subphenotypes revealed disease etiology in 10.0% of them. We detected no significant differences in the overall diagnostic yield among COU subphenotypes, with characteristic variable expressivity of several mutant genes. Our findings therefore may legitimize a genetic first diagnostic approach for COU, especially when burdening clinical and imaging characterization is not complete or available. Background: Congenital obstructive uropathy (COU) is a common cause of developmental defects of the urinary tract, with heterogeneous clinical presentation and outcome. Genetic analysis has the potential to elucidate the underlying diagnosis and help risk stratification. Methods: We performed a comprehensive genomic screen of 733 independent COU cases, which consisted of individuals with ureteropelvic junction obstruction ( n =321), ureterovesical junction obstruction/congenital megaureter ( n =178), and COU not otherwise specified (COU-NOS; n =234). Results: We identified pathogenic single nucleotide variants (SNVs) in 53 (7.2%) cases and genomic disorders (GDs) in 23 (3.1%) cases. We detected no significant differences in the overall diagnostic yield between COU sub-phenotypes, and pathogenic SNVs in several genes were associated to any of the three categories. Hence, although COU may appear phenotypically heterogeneous, COU phenotypes are likely to share common molecular bases. On the other hand, mutations in TNXB were more often identified in COU-NOS cases, demonstrating the diagnostic challenge in discriminating COU from hydronephrosis secondary to vesicoureteral reflux, particularly when diagnostic imaging is incomplete. Pathogenic SNVs in only six genes were found in more than one individual, supporting high genetic heterogeneity. Finally, convergence between data on SNVs and GDs suggest MYH11 as a dosage-sensitive gene possibly correlating with severity of COU. Conclusions: We established a genomic diagnosis in 10.0% of COU individuals. The findings underscore the urgent need to identify novel genetic susceptibility factors to COU to better define the natural history of the remaining 90% of cases without a molecular diagnosis