Heterozygous Loss Research Articles

Whole exome sequencing make a definitive diagnosis of a Vietnamese patient with a late onset urea cycle disorder

Our report describes a female presenting with vomiting, fever, coma and right hemiplegia at 26 months of age. Biochemical tests revealed hyperammonemia, hyperlactatemia, elevated glutamine level, elevated transaminase and disorder of prothrombin time. She was priory diagnosed with urea cycle disorders (UCDs). UCDs are caused by mutations in eight genes that regulate the synthesis of enzymes and cofactors involved in urea metabolism. Singleton whole exome sequencing was applied to screen causative variants in these genes in the patient at 6 years of age. The result showed one heterozygous stop loss mutation c.1065A>G in the OTC gene as a potential disease causing in the patient. The mutation c.1065A>G leads to alternation of stop codon to tryptophan, resulting in elongation of fourteen amino acids in ornithine transcarbamylase (OTC) protein (p.Ter355TrpextTer14). Sanger sequencing in the family revealed the mutation c.1065A>G was not present in healthy parents and brother. Therefore, this mutation is considered as a de novo mutation in the patient. The mutation c.1065A>G was conferred to pathogenic according to the standards and guidelines of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology with 1 strong (PS2), 3 moderate (PM2, PM4 and PM5) and 1 support criteria (PP2). Although OTC deficiency is an X-linked recessive inheritance, approximately 15% of females carrying heterozygous variants showed the late onset OTC deficiency. Therefore, in combination of clinical presentations, laboratory findings and molecular genetic analyses, we made a definitive diagnosis of the patient with late onset OTC deficiency, a disorder of UCDs.

Identification of Genes Involved in the Differentiation of R7y and R7p Photoreceptor Cells in Drosophila.

The R7 and R8 photoreceptor cells of the Drosophila compound eye mediate color vision. Throughout the majority of the eye, these cells occur in two principal types of ommatidia. Approximately 35% of ommatidia are of the pale type and express Rh3 in R7 cells and Rh5 in R8 cells. The remaining 65% are of the yellow type and express Rh4 in R7 cells and Rh6 in R8 cells. The specification of an R8 cell in a pale or yellow ommatidium depends on the fate of the adjacent R7 cell. However, pale and yellow R7 cells are specified by a stochastic process that requires the genes spineless, tango and klumpfuss. To identify additional genes involved in this process we performed genetic screens using a collection of 480 P{EP} transposon insertion strains. We identified genes in gain of function and loss of function screens that significantly altered the percentage of Rh3 expressing R7 cells (Rh3%) from wild-type. 36 strains resulted in altered Rh3% in the gain of function screen where the P{EP} insertion strains were crossed to a sevEP-GAL4 driver line. 53 strains resulted in altered Rh3% in the heterozygous loss of function screen. 4 strains showed effects that differed between the two screens, suggesting that the effect found in the gain of function screen was either larger than, or potentially masked by, the P{EP} insertion alone. Analyses of homozygotes validated many of the candidates identified. These results suggest that R7 cell fate specification is sensitive to perturbations in mRNA transcription, splicing and localization, growth inhibition, post-translational protein modification, cleavage and secretion, hedgehog signaling, ubiquitin protease activity, GTPase activation, actin and cytoskeletal regulation, and Ser/Thr kinase activity, among other diverse signaling and cell biological processes.

Targeting of \u03b4-catenin to postsynaptic sites through interaction with the Shank3 N-terminus

BackgroundNeurodevelopmental disorders such as autism spectrum disorder (ASD) may be caused by alterations in genes encoding proteins that are involved in synapse formation and function. This includes scaffold proteins such as Shank3, and synaptic adhesion proteins such as Neurexins or Neuroligins. An important question is whether the products of individual risk genes cooperate functionally (exemplified in the interaction of Neurexin with Neuroligin isoforms). This might suggest a common pathway in pathogenesis. For the SHANK3 gene, heterozygous loss of function, as well as missense mutations have been observed in ASD cases. Several missense mutations affect the N-terminal part of Shank3 which contains the highly conserved Shank/ProSAP N-terminal (SPN) and Ankyrin repeat (Ank) domains. The role of these domains and the relevance of these mutations for synaptic function of Shank3 are widely unknown.MethodsWe used purification from a synaptic protein fraction, as well as a variety of biochemical and cell biological approaches to identify proteins which associate with the Shank3 N-terminus at postsynaptic sites.ResultsWe report here that δ-catenin, which is encoded by CTNND2, an autism candidate gene, directly interacts with the Ank domain of Shank3 at postsynaptic sites through its Armadillo-repeat domain. The interaction is not affected by well-known posttranslational modifications of δ-catenin, i.e. by phosphorylation or palmitoylation. However, an ASD-associated mutation in the SPN domain of Shank3, L68P, significantly increases the interaction of Shank3 with δ-catenin. By analysis of postsynaptic fractions from mice, we show that the lack of SPN-Ank containing, large isoforms of Shank3 results in the loss of postsynaptic δ-catenin. Further, expression of Shank3 variants containing the N-terminal domains in primary cultured neurons significantly increased the presence of coexpressed δ-catenin at postsynaptic sites.LimitationsWork in model organisms such as mice, and in primary cultured neurons may not reproduce faithfully the situation in human brain neurons. Work in primary cultured neurons was also hampered by lack of a specific antibody for endogenous δ-catenin.ConclusionsOur data show that the interaction between Shank3 N-terminus and δ-catenin is required for the postsynaptic targeting of δ-catenin. Failure of proper targeting of δ-catenin to postsynaptic sites may contribute to the pathogenesis of autism spectrum disorder.

Bi-allelic pathogenic variations in DNAJB11 cause Ivemark II syndrome, a renal-hepatic-pancreatic dysplasia

DNAJB11 (DnaJ Heat Shock Protein Family (Hsp40) Member B11) heterozygous loss of function variations have been reported in autosomal dominant cystic kidney disease with extensive fibrosis, associated with maturation and trafficking defect involving both the autosomal dominant polycystic kidney disease protein polycystin-1 and the autosomal dominant tubulointerstitial kidney disease protein uromodulin. Here we show that biallelic pathogenic variations in DNAJB11 lead to a severe fetal disease including enlarged cystic kidneys, dilation and proliferation of pancreatic duct cells, and liver ductal plate malformation, an association known as Ivemark II syndrome. Cysts of the kidney were developed exclusively from uromodulin negative tubular segments. In addition, tubular cells from the affected kidneys had elongated primary cilia, a finding previously reported in ciliopathies. Thus, our data show that the recessive disease associated with DNAJB11 variations is a ciliopathy rather than a disease of the autosomal dominant tubulointerstitial kidney disease spectrum, and prompt screening of DNAJB11 in fetal hyperechogenic/cystic kidneys.

Heterozygous Loss of Yap1 in Mice Causes Progressive Cataracts.

PurposeYap1 encodes an evolutionarily conserved transcriptional coactivator and functions as a down-stream effector of the Hippo signaling pathway that controls tissue size and cell growth. Yap1 contributes to lens epithelial development. However, the effect of Yap1 haplodeficiency on the lens epithelium and its role in the development of cataracts has not been reported. The aim of the current study is to investigate Yap1 function and its regulatory mechanisms in lens epithelial cells (LECs).MethodsLens phenotypes were investigated in Yap1 heterozygous mutant mice by visual observation and histological and biochemical methods. Primary LEC cultures were used to study regulatory molecular mechanism.ResultsThe heterozygous inactivation of Yap1 in mice caused cataracts during adulthood with defective LEC phenotypes. Despite a normal early development of the eye including the lens, the majority of Yap1 heterozygotes developed cataracts in the first six months of age. Cataract was preceded by multiple morphological defects in the lens epithelium, including decreased cell density and abnormal cell junctions. The low LEC density was coincident with reduced LEC proliferation. In addition, expression of the Yap1 target gene Crim1 was reduced in the Yap1+/− LEC, and overexpression of Crim1 restored Yap1+/− LEC cell proliferation in vitro.ConclusionsHomozygosity of the Yap1 gene was critical for adequate Crim1 expression needed to maintain the constant proliferation of LEC and to maintain a normal-sized lens. Yap1 haplodeficiency leads to cataracts.

26P Impact of 17q21 locus gene co-amplification on overall survival in HER2-positive breast cancer

Marked heterogeneity in outcome and response to therapy has been demonstrated in HER2+ breast cancer. A significant number of patients are over and undertreated as a result, due to a lack of biomarkers. The advent of next-generation sequencing technology is providing better insights into tumour biology. As HER2+ disease is characterised by the 17q21 amplicon, the roles of co-amplified or altered genes at this locus were evaluated and related to cancer outcome. Genomic data from 2509 breast cancers from the METABRIC study training set and 1060 from the TCGA cancers validation set were evaluated to assess the OS relationship between the HER2 gene and other co-altered genes on 17q21, including NSRP1, MAPT, BRCA1, NF1 and TMC6. OS varied according to the status of gene co-alterations with HER2 on the 17q21 amplicon in 298 eligible samples. NSRP1, NF1 and MAPT co-amplification was associated with improved OS compared to no alteration, and copy number loss was associated with poor outcome. BRCA1 heterozygous loss was predictive of a poor outcome and TMC6 did not have any effect. MAPT amplified tumours were associated with better overall survival (log-rank, p<0.03). In a Cox regression multi-variate analysis consisting of grade, stage and MAPT status, MAPT remained significant (p=2.9x10 -9). These data were validated in the 119 TCGA samples where MAPT status was found to have discriminatory potential in HER2+ patients in this data set as well (p<0.01). These data contribute to emerging research on HER2 heterogeneity, suggesting that co-altered genes associated with HER2 gene aberrations on the 17q21 locus, especially MAPT, has a significant effect on OS. This research is hypothesis-generating in developing future escalation and de-escalation strategies in patients with HER2+ breast cancer and provokes the need for preclinical exploration of these findings.

Neuronal defects in a human cellular model of 22q11.2 deletion syndrome.

22q11.2 deletion syndrome (22q11DS) is a highly penetrant and common genetic cause of neuropsychiatric disease. Here we generated induced pluripotent stem cells from 15 individuals with 22q11DS and 15 control individuals and differentiated them into three-dimensional (3D) cerebral cortical organoids. Transcriptional profiling across 100 days showed high reliability of differentiation and revealed changes in neuronal excitability-related genes. Using electrophysiology and live imaging, we identified defects in spontaneous neuronal activity and calcium signaling in both organoid- and 2D-derived cortical neurons. The calcium deficit was related to resting membrane potential changes that led to abnormal inactivation of voltage-gated calcium channels. Heterozygous loss of DGCR8 recapitulated the excitability and calcium phenotypes and its overexpression rescued these defects. Moreover, the 22q11DS calcium abnormality could also be restored by application of antipsychotics. Taken together, our study illustrates how stem cell derived models can be used to uncover and rescue cellular phenotypes associated with genetic forms of neuropsychiatric disease.

Bi-allelic SHOC1 loss-of-function mutations cause meiotic arrest and non-obstructive azoospermia

BackgroundThe genetic causes of human idiopathic non-obstructive azoospermia (NOA) with meiotic arrest remain unclear.MethodsTwo Chinese families with infertility participated in the study. In family 1, two brothers were affected by...

Influence of beta-cluster haplotypes, alpha-gene status and UGTA1 polymorphism on clinical and hematological data in sickle-cell disease children from French Guiana.

ObjectivesThis cross-sectional study aimed to investigate the influence of haplotypes, alpha-gene status and UGTA1 polymorphism on the severity of sickle cell disease in children.MethodsThis cross-sectional study was conducted between 2012 and 2014 at the Cayenne Hospital, in French Guiana. Acute clinical complications were grouped into (i) severe SCD defined by the presence of stroke and/or abnormal-transcranial Doppler (TCD), (ii) moderate SCD defined by the presence of at least three annual events requiring hospitalization and/or at least one acute chest syndrome, (iii) no severe SCD (in the absence of the precited events).ResultsAmong the 86 patients, 33.7% were female with a median age of 10 years (range: 6–12 years). The vast majority of patients had SCA (HbSS) phenotype (74.4%; n = 64). The severe haplotype was found in 40% of patients. 30% were BEN/BEN. Analysis of α-globin gene deletions revealed that 32 patients (37.2%) were heterozygous (loss of 2 genes in 2 cases and loss of 1 gene in 30 cases) for α-thalassemia (3.7 kb deletion). Homozygous (TA) n TA7/7 was found in 24 (28%). In the multivariate analysis, the factors associated with the severity of sickle cell disease were the first vaso-occlusive crisis before one year of age (OR 25, [95% CI = 6.0–107.0], p<0.001) and a baseline MCV >80 fL (OR 0.20 [95% CI = 0.04–0.96], p = 0.04). The area of the ROC curve was 0.90.ConclusionProspective studies with greater statistical power would provide more knowledge on the relationship between UGT1A1 mutations and the clinical and hematological manifestations of SCA.

Abstract 1620: MDM2 P1 and P2 promoters have different roles in tumorigenesis

Abstract The murine double minute 2 (MDM2) protein is an E3 ubiquitin ligase most notable for priming tumour-suppressor p53 for degradation. MDM2 contains two promoters - the constitutive P1 and the inducible P2. The p53-independent P1 promoter is responsible for basal levels of MDM2, whilst the p53-dependent P2 promoter is activated upon stress responses. Although the oncogenic functions of MDM2 are well understood, less is known about the individual roles of the promoters and their contribution to tumorigenesis and stress responses. To tease apart the roles of the promoters, we have used genetically engineered mouse (GEM) models null for the P1-promoter and mutated for the P2-promoter in the context of intestinal cancer, lymphoma and hepatocellular carcinoma (HCC). Here, we have shown that mice with heterozygous or homozygous loss of the P1-promoter in the APCMin/+ intestinal model have extended survival and reduced intestinal tumour burden in comparison to the P1- wild type (WT), APCMin/+ mice. Surprisingly, none of the P1-null APCMin/+ mice succumbed to intestinal tumours, but rather to thymic lymphomas. In the P1-null APCMin/+ lymphoma samples, we investigated the p53 functionality by examining the expression levels of p21, a p53 downstream target. Immunohistochemistry analysis clearly revealed that p53 is non-functional as p21 was not expressed, despite variations in p53 expression levels itself. This uncommon occurrence in the APCMin/+ model led us to investigate the role of the P1 and P2 promoters in the Eµ-Myc lymphoma model. Interestingly, in this model, heterozygous loss of the P1 promoter improves survival, while heterozygous loss of the P2 promoter accelerates lymphomagenesis. Our findings thus far indicate a differential role for the P1 and P2 MDM2 promoters in tumorigenesis. In addition to using GEM models, we are investigating the role of the MDM2 promoters in stress-induced HCC. These studies will allow for a better understanding of MDM2 functions, but also perhaps provide an alternative therapeutic strategy for cancers with aberrations in the p53-MDM2 signalling axis. Citation Format: Narisa Phinichkusolchit, Thomas Jamieson, Rachel Ridgway, Dimitris Athineos, Kerryanne Crawford, Nikolina Vlatkovic, Mark Boyd, Owen Sansom, Karen Blyth. MDM2 P1 and P2 promoters have different roles in tumorigenesis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1620.

Abstract 1083: Heterozygous knockout of the histone modifier MLL4 promotes Ptch+/−-driven medulloblastoma

Abstract Medulloblastoma (MB) is the most common primary childhood brain tumor. MB development frequently results from the dysregulation of sonic hedgehog and wingless pathways. In contrast to our understanding of these pathways, the pathogenic roles of aberrant epigenetic modifiers in MB remain little known. MLL4 is one of the most commonly mutated genes in MB. In our recent study, we showed that brain-specific knockout of the gene encoding the histone H3 lysine 4 methyltransferase MLL4 (also called COMPASS-like methyltransferase, KMT2D, MLL2, and ALR) induced spontaneous MBs. Mll4 loss highly upregulated several oncogenic signaling programs, including expression of several oncogenic Ras activators (e.g., Rasgrp1, Rasgrf1, and Rasgef1a). In contrast, Mll4 loss downregulated expression of tumor-suppressive genes, such as Dnmt3a. In this study, we sought to determine whether Mll4 loss cooperates with other oncogenic event in MB genesis. Particularly, PTCH (also known as PTCH1) is one of the most frequently mutated genes in MB, and its loss induces MB in mouse models. Thus, we examined the effect of Mll4 loss on of Ptch+/−-driven MB genesis. A heterozygous loss of Mll4 increased the incidence and progression of Ptch+/−-driven MB genesis. MBs were confirmed by H &amp; E staining, and immunohistochemical staining using anti-Ki-67 showed that cell proliferation was markedly increased. Calbindin signals were undetectable in Nestin-Cre Mll4f/+ Ptch+/−, and NeuN levels were decreased in Nestin-Cre Mll4f/+ Ptch+/− cerebellum compared to Ptch+/− cerebellum, suggesting the impairment of Purkinje and granule cells. Mll4 loss reduced cerebellar granule cell differentiation. These findings indicate that a haploinsufficiency of MLL4 promotes Ptch+/−-driven MB genesis. This mouse model may be useful for future preclinical therapeutic experiments for the treatment of MB patients with MLL4 mutations. Citation Format: Shilpa S. Dhar, Dongyu Zhao, Tao Lin, Roy V. Sillitoe, Kaifu Chen, Min Gyu Lee. Heterozygous knockout of the histone modifier MLL4 promotes Ptch+/−-driven medulloblastoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1083.

Abstract 261: Evaluating MTCH2 as a Modifier of Cardiomyopathy

Background: Cardiomyopathy is a highly heritable disorder that carries a significant risk for heart failure and arrhythmias. The onset, severity, and progression of cardiomyopathy is influenced by genetic variation, with the most common form of inheritance in familial cases being autosomal dominant. All inherited cardiomyopathies are characterized by variable penetrance and expressivity, which in part arises from additional genetic variation, known as genetic modifiers. Methods and Results: Broad genomic profiling of human cardiomyopathy cases identified enriched genetic variation in the gene MTCH2. Specifically, a loss of function variant was found to be enriched in patients with cardiomyopathy. MTCH2 single nucleotide polymorphisms have also been linked to obesity, underscoring a critical role for MTCH2 in metabolic regulation. MTCH2 encodes a mitochondrial carrier protein that has a role in regulating oxidative phosphorylation. In order to investigate fundamental mechanisms by which MTCH2 contributes to cardiac and metabolic phenotypes, we generated a knockdown model of the Drosophila MTCH2 ortholog, Mtch. This knockdown model mimics what is seen in human carriers with the heterozygous loss of function allele. In the Drosophila model, Mtch RNA was reduced by approximately half. We found that cardiac-specific Mtch deficiency in flies produced heart tube dilated and reduced function as well as a shortened life span, documenting a clear role for cardiac Mtch. Cardiac deficiency of Mtch increased circulating lactate levels in flies. Oxygen consumption was reduced in cardiac Mtch deficiency flies in the presence of glucose, but not palmitate. Thus, loss of Mtch2 alters oxygen consumption in a substrate dependent manner. Conclusions: We identified MTCH2 as a modifier of the cardiomyopathy phenotype in humans. Reduction of Mtch in flies resulted in impaired cardiac function and reduced oxygen consumption under certain metabolic condition. As failed hearts are more dependent on glycolysis, these data support that reduction of MTCH2 promotes heart failure and provides a mechanism by which MTCH2 acts as a deleterious genetic modifier in heart failure.

Disruption of foxc1 genes in zebrafish results in dosage-dependent phenotypes overlapping Axenfeld-Rieger syndrome

The Forkhead Box C1 (FOXC1) gene encodes a forkhead/winged helix transcription factor involved in embryonic development. Mutations in this gene cause dysgenesis of the anterior segment of the eye, most commonly Axenfeld-Rieger syndrome (ARS), often with other systemic features. The developmental mechanisms and pathways regulated by FOXC1 remain largely unknown. There are two conserved orthologs of FOXC1 in zebrafish, foxc1a and foxc1b. To further examine the role of FOXC1 in vertebrates, we generated foxc1a and foxc1b single knockout zebrafish lines and bred them to obtain various allelic combinations. Three genotypes demonstrated visible phenotypes: foxc1a-/- single homozygous and foxc1-/- double knockout homozygous embryos presented with similar characteristics comprised of severe global vascular defects and early lethality, as well as microphthalmia, periocular edema and absence of the anterior chamber of the eye; additionally, fish with heterozygous loss of foxc1a combined with homozygosity for foxc1b (foxc1a+/-;foxc1b-/-) demonstrated craniofacial defects, heart anomalies and scoliosis. All other single and combined genotypes appeared normal. Analysis of foxc1 expression detected a significant increase in foxc1a levels in homozygous and heterozygous mutant eyes, suggesting a mechanism for foxc1a upregulation when its function is compromised; interestingly, the expression of another ARS-associated gene, pitx2, was responsive to the estimated level of wild-type Foxc1a, indicating a possible role for this protein in the regulation of pitx2 expression. Altogether, our results support a conserved role for foxc1 in the formation of many organs, consistent with the features observed in human patients, and highlight the importance of correct FOXC1/foxc1 dosage for vertebrate development.

Genotype-phenotype correlation at codon 1740 of SETD2.

The SET domain containing 2, histone lysine methyltransferase encoded by SETD2 is a dual-function methyltransferase for histones and microtubules and plays an important role for transcriptional regulation, genomic stability, and cytoskeletal functions. Specifically, SETD2 is associated with trimethylation of histone H3 at lysine 36 (H3K36me3) and methylation of α-tubulin at lysine 40. Heterozygous loss of function and missense variants have previously been described with Luscan-Lumish syndrome (LLS), which is characterized by overgrowth, neurodevelopmental features, and absence of overt congenital anomalies. We have identified 15 individuals with de novo variants in codon 1740 of SETD2 whose features differ from those with LLS. Group 1 consists of 12 individuals with heterozygous variant c.5218C>T p.(Arg1740Trp) and Group 2 consists of 3 individuals with heterozygous variant c.5219G>A p.(Arg1740Gln). The phenotype of Group 1 includes microcephaly, profound intellectual disability, congenital anomalies affecting several organ systems, and similar facial features. Individuals in Group 2 had moderate to severe intellectual disability, low normal head circumference, and absence of additional major congenital anomalies. While LLS is likely due to loss of function of SETD2, the clinical features seen in individuals with variants affecting codon 1740 are more severe suggesting an alternative mechanism, such as gain of function, effects on epigenetic regulation, or posttranslational modification of the cytoskeleton. Our report is a prime example of different mutations in the same gene causing diverging phenotypes and the features observed in Group 1 suggest a new clinically recognizable syndrome uniquely associated with the heterozygous variant c.5218C>T p.(Arg1740Trp) in SETD2.

Incorporating Spinal Muscular Atrophy Analysis by Next-Generation Sequencing into a Comprehensive Multigene Panel for Neuromuscular Disorders.

Background: Spinal muscular atrophy (SMA) is traditionally molecularly diagnosed by multiplex ligation-dependent probe amplification or quantitative polymerase chain reaction (qPCR). SMA analyses are not routinely incorporated into gene panel analyses for individuals with suspected SMA or broader neuromuscular indications. Aim: We sought to determine whether a next-generation sequencing (NGS) approach that integrates SMA analyses into a multigene neuromuscular disorders panel could detect undiagnosed SMA. Materials and Methods: Sequence and copy number variants of the SMN1/SMN2 genes were simultaneously analyzed in samples from 5304 unselected individuals referred for testing using an NGS-based 122-gene neuromuscular panel. This diagnostic approach was validated using DNA from 68 individuals who had been previously diagnosed with SMA via quantitative PCR for SMN1/SMN2. Results: Homozygous loss of SMN1 was detected in 47 unselected individuals. Heterozygous loss of SMN1 was detected in 118 individuals; 8 had an indeterminate variant in "SMN1 or SMN2" that supported an SMA diagnosis but required additional disambiguation. Of the remaining SMA carriers, 44 had pathogenic variants in other genes. Concordance rates between NGS and qPCR were 100% and 93% for SMN1 and SMN2 copy numbers, respectively. Where there was disagreement, phenotypes were more consistent with the SMN2 results from NGS. Conclusion: Integrating NGS-based SMA testing into a multigene neuromuscular panel allows a single assay to diagnose SMA while comprehensively assessing the spectrum of variants that can occur in individuals with broad differential diagnoses or nonspecific/overlapping neuromuscular features.

Abstract A34: Targeting Protein Phosphatase 2A in Combination with PARP inhibitors for the treatment of high-grade serous epithelial ovarian cancer

Abstract Ovarian cancer (OvC) is the deadliest of all female gynecologic malignancies, ranking as the fifth cause of cancer-related deaths among women. High-grade serous epithelial ovarian cancer (HGSOC) accounts for almost 90% of all forms of OvC, remaining as the most lethal subtype of ovarian malignancies. HGSOC is usually diagnosed at a later stage at which the disease has already progressed into metastatic, having a 5-year survival rate of only 17%. Treatment of HGSOC relies on aggressive approaches including surgery and platinum-based chemotherapies, with the majority of cases requiring combination of both. The molecular mechanism driving ovarian tumorigenesis is not fully understood, and its investigation is essential to provide better and novel therapeutic options for the treatment and cure of this disease. Targeted therapies have been vastly explored in the cancer research field due to their promise in reducing side effects by specifically targeting oncogenic pathways predominantly deregulated in human malignancies. DNA damage and repair (DDR) pathway aberrations and genomic instability are two main hallmarks of OvC. Extensive efforts in targeting this pathway have been devoted to introduce new drugs in the clinic that can target the DDR and homologous recombination (HR) pathways as therapeutic means for treatment of HGSOC. The use of PARP inhibitors (PARPi) to achieve synthetic lethality in OvC has been shown to have great promise in efficiently reducing tumor burden by specifically targeting tumor cells for cell death. However, PARPi’s antitumoral efficacy alone is limited by the status of the BRCA1/2 genes, narrowing down the patient population that can benefit from such therapies. Eventually, patients treated with PARPi alone relapse and develop resistant OvC, for which treatment options available are extremely limited. Our preliminary data show that first-in-class small-molecule activators of PP2A (SMAPs) in combination with PARPi can effectively reduce tumor burden in PDX models, promoting a synergistic effect in platinum-sensitive and -resistant OvC tumors, despite the BRCA gene status. Protein Phosphatase 2A (PP2A) and Protein Phosphatase 1 (PP1) account for 90% of all serine/threonine phosphatase activity in the cell and act as two main tumor suppressors. These phosphatases tightly regulate signaling pathways involved with cell cycle, growth, migration, metabolism, survival and many oncogenic pathways by counteracting kinases. HGSOC tumors express inactivated PP2A due to PPP2RA1 heterozygous loss, although lacking mutations, making it an ideal target for reactivation in these cancers. Moreover, we have shown that reactivation of PP2A via SMAPs leads to potent inhibition of several key DDR proteins. We have found that SMAPs treatment in BRCA1/2 WT tumors confers a “BRCAness” phenotype, which sensitizes tumors to DNA repair inhibitors such as PARPi. In sum, SMAPs can allow us to expand the patient population that can benefit from PARPi therapies and possibly overcome drug resistance. Citation Format: Rita A. Avelar, Amy Armstrong, Goutham Narla, Analisa DiFeo. Targeting Protein Phosphatase 2A in Combination with PARP inhibitors for the treatment of high-grade serous epithelial ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A34.

OPA1 deficiency accelerates hippocampal synaptic remodelling and age-related deficits in learning and memory.

A healthy mitochondrial network is essential for the maintenance of neuronal synaptic integrity. Mitochondrial and metabolic dysfunction contributes to the pathogenesis of many neurodegenerative diseases including dementia. OPA1 is the master regulator of mitochondrial fusion and fission and is likely to play an important role during neurodegenerative events. To explore this, we quantified hippocampal dendritic and synaptic integrity and the learning and memory performance of aged Opa1 haploinsufficient mice carrying the Opa1Q285X mutation (B6; C3-Opa1Q285STOP; Opa1+/−). We demonstrate that heterozygous loss of Opa1 results in premature age-related loss of spines in hippocampal pyramidal CA1 neurons and a reduction in synaptic density in the hippocampus. This loss is associated with subtle memory deficits in both spatial novelty and object recognition. We hypothesize that metabolic failure to maintain normal neuronal activity at the level of a single spine leads to premature age-related memory deficits. These results highlight the importance of mitochondrial homeostasis for maintenance of neuronal function during ageing.

A 5-HT1D -receptor agonist protects Dravet syndrome mice from seizure and early death.

Mutations in the SCN1A gene encoding the Nav1.1 sodium channel cause several forms of epilepsy, the most severe is Dravet syndrome (DS). DS patients are at high risk for sudden death and seizures are often triggered by fever or hyperthermia. To improve understanding of how serotonergic ligands might influence DS in this study, we tested several drugs for their effect on hyperthermia-induced seizure using a mouse model of DS consisting of a heterozygous loss of function of Scn1A. We found that a mixed 5-HT2A/2C receptor agonist had no effect while a mixed 5-HT1B/D receptor agonist had a modest effect reducing the severity of seizures. Hypothesizing selective agonists may be more effective, we tested selective 5-HT1B and 5-HT1D receptor agonists, CP-93129 and GR-46611, respectively. Of these GR-46611 significantly increased the threshold of hyperthermia-induced seizure and lowered seizure severity. Given chronically at 1mgkg-1 day-1 , GR-46611 also significantly improved survival of DS mice. Thus, 5-HT1D -receptors may represent a meaningful target for pharmacotherapy for DS with potential relevance for related forms of epilepsy, particularly those with a known sensory trigger such as heat.

An assessment of the role of vinculin loss of function variants in inherited cardiomyopathy.

The ACMG/AMP variant classification framework was intended for highly penetrant Mendelian conditions. While it is appreciated that clinically relevant variants exhibit a wide spectrum of penetrance, accurately assessing and expressing the pathogenicity of variants with lower penetrance can be challenging. The vinculin (VCL) gene illustrates these challenges. Model organism data provideevidence that loss of function of VCL may play a role in cardiomyopathy and aggregate case-control studies suggest low penetrance.VCL loss of function variants, however, are rarely identified in affected probands and therefore there is a paucity of family studies clarifying the clinical significance of individual variants. This study, which aggregated data from >18,000 individuals who underwent gene panel or exome testing for inherited cardiomyopathies, identified 32 probands with VCL loss-of-function variants and confirmed enrichment in probands with dilated cardiomyopathy (odds ratio [OR] = 9.01; confidence interval [CI] = 4.93-16.45). Our data revealed that the majority of these individuals (89.5%) had pediatric onset of disease. Family studies demonstrated that heterozygous loss of function of VCL alone is insufficient to cause cardiomyopathy but that these variants do contribute to disease risk. In conclusion, VCL loss-of-function variants should be reported in a diagnostic setting but need to be clearly distinguished as having lower penetrance.

DEPDC5 haploinsufficiency drives increased mTORC1 signaling and abnormal morphology in human iPSC-derived cortical neurons

Mutations in the DEPDC5 gene can cause epilepsy, including forms with and without brain malformations. The goal of this study was to investigate the contribution of DEPDC5 gene dosage to the underlying neuropathology of DEPDC5-related epilepsies. We generated induced pluripotent stem cells (iPSCs) from epilepsy patients harboring heterozygous loss of function mutations in DEPDC5. Patient iPSCs displayed increases in both phosphorylation of ribosomal protein S6 and proliferation rate, consistent with elevated mTORC1 activation. In line with these findings, we observed increased soma size in patient iPSC-derived cortical neurons that was rescued with rapamycin treatment. These data indicate that human cells heterozygous for DEPDC5 loss-of-function mutations are haploinsufficient for control of mTORC1 signaling. Our findings suggest that human pathology differs from mouse models of DEPDC5-related epilepsies, which do not show consistent phenotypic differences in heterozygous neurons, and support the need for human-based models to affirm and augment the findings from animal models of DEPDC5-related epilepsy.