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 the 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 the 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.

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.

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.

DHES0815A, a novel antibody-drug conjugate targeting HER2/neu, is highly active against uterine serous carcinomas in vitro and in vivo.

OBJECTIVE: Uterine serous carcinoma (USC) is an aggressive histologic variant of endometrial cancer which portends a poor prognosis. DHES0815A is a novel antibody-drug-conjugate (ADC) which binds specifically to HER2 overexpressing tumors at a distinct epitope from that bound by trastuzumab and pertuzumab after which it delivers the toxic payload, PBD-MA, a DNA mono-alkylating agent. The objective of this study was to evaluate the preclinical activity of DHES0815A against primary USC cell lines and xenografts. METHODS: Twelve primary USC cell lines were assessed by immunohistochemistry (IHC) for HER2 protein expression and for C-erbB2 gene amplification using fluorescent in situ hybridization (FISH) analysis. Cell viability and bystander killing in USC cell lines after exposure to DHES0815A, the non-targeted ADC, and the unconjugated antibody (i.e. MHES0488A) were evaluated using flow cytometry-based-assays. In vivo activity of DHES0815A was tested against HER2/neu overexpressing USC xenografts. RESULTS: High HER2/neu protein expression was seen in 25% (3/12) of the primary USC cell lines. USC cell lines overexpressing HER2/neu were significantly more sensitive to DHES0815A when compared to the non-targeted control ADC (p < 0.001). DHES0815A did not induce significant bystander killing of HER2/neu negative tumors when admixed with HER2/neu positive tumors. DHES0815A caused growth-inhibition and increased survival in USC HER2/neu overexpressing xenografts when compared to controls (p < 0.01). CONCLUSIONS: DHES0815A is both highly selective and toxic to USC tumors overexpressing HER2/neu both in vitro and in vivo. HER2-directed ADCs, alone or in combination with other HER2/neu targeted agents may represent a novel treatment option for patients with tumors harboring HER2/neu overexpression refractory to trastuzumab and traditional chemotherapy.

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 oncogenes were deetected, with most fusions disrupting RB1, TP53, and ATRX/DAXX, and one fusion (ACTG2-ALK) being potentially targetable. WGS results demonstrated that 76% (16 of 21) of the samples harbored chromoplexy and/or chromothripsis. 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-626510 (C-MYC/BETi; P < 0.000001 and P = 0.0005), and copanlisib (PIK3CAi; P = 0.0001) monotherapy to significantly inhibit uLMS-PDXs harboring derangements 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 suggest that a subset of uLMS may benefit from existing PARP-, PIK3CA-, and C-MYC/BET-targeted drugs.

Derangements in HUWE1/c-MYC pathway confer sensitivity to the BET bromodomain inhibitor GS-626510 in uterine cervical carcinoma.

OBJECTIVE: Whole-exome-sequencing (WES) studies reported c-MYC gene-amplification and HUWE1 gene deletion/mutations in a significant number of cervical-cancer-patients (CC) suggesting HUWE1/c-MYC pathway as potential therapeutic target. We investigated HUWE1/c-MYC expression in fresh-frozen-CC and the activity of the novel BET inhibitor GS-626510 (Gilead-Science-Inc) against primary WES CC-cultures and CC-xenografts. METHODS: HUWE1 and c-MYC expression were evaluated by qRT-PCR in 23 CC including 12 fresh-frozen-tumor-tissues and 11 primary-cell-lines. c-Myc expression was also evaluated by Western-Blot (WB) and fluorescence-in-situ-hybridization (FISH) in all 11 fully sequenced primary-CC-cell-lines. Primary tumors were evaluated for sensitivity to GS-626510 in-vitro using proliferation and viability-assays. siRNA experiments were used to evaluate the effect of HUWE1 silencing on primary-CC-cell-line growth and sensitivity to GS-626510. Finally, the in-vivo activity of GS-626510 was studied in CC-CVX8-mouse-xenografts. RESULTS: Fresh-frozen-CC and primary-CC-cell-lines overexpressed c-MYC when compared to normal tissues (p = .01). FISH demonstrated amplification of c-MYC in 9/11 (82%) of the primary-CC-cell-lines. Cell-lines with derangements in HUWE1/c-MYC pathway were highly sensitive to GS-626510, with a dose-response decrease in cell proliferation and viability. siRNA silencing of HUWE1 significantly increased c-MYC expression and CC cell-proliferation and enhanced the in-vitro sensitivity to GS-626510. Twice-daily oral doses of GS-626510 were well tolerated in-vivo and highly effective in decreasing tumor-growth (p = .004) and increasing survival (p = .004) of CC-CVX8 xenografts. CONCLUSIONS: Downregulation/inactivation of HUWE1 may increase c-MYC expression and proliferation in primary-CC-cell-lines. GS-626510 may represent a novel, potentially highly effective therapeutic agent against CC overexpressing c-MYC and/or harboring HUWE1 mutations. Clinical studies with BET inhibitor in CC-patients harboring radiation/chemotherapy-resistant disease are warranted.

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.