The contribution of X-linked coding variation to severe developmental disorders

Abstract: Over 130 X-linked genes have been robustly associated with developmental disorders, and X-linked causes have been hypothesised to underlie the higher developmental disorder rates in males. Here, we evaluate the burden of X-linked coding variation in 11,044 developmental disorder patients, and find a similar rate of X-linked causes in males and females (6.0% and 6.9%, respectively), indicating that such variants do not account for the 1.4-fold male bias. We develop an improved strategy to detect X-linked developmental disorders and identify 23 significant genes, all of which were previously known, consistent with our inference that the vast majority of the X-linked burden is in known developmental disorder-associated genes. Importantly, we estimate that, in male probands, only 13% of inherited rare missense variants in known developmental disorder-associated genes are likely to be pathogenic. Our results demonstrate that statistical analysis of large datasets can refine our understanding of modes of inheritance for individual X-linked disorders

Genetic Analysis of C. elegans Metabolism

Regulation of nutrient consumption and storage is a complex process requiring numerous interconnected genetic pathways that monitor and respond to both environmental and internal conditions. Though we have a good framework understanding of some of the basic modules of this network, our grasp of the intricate regulation of these processes remains limited. We have utilized the model organism C. elegans to investigate the genetic pathways regulating nutrient storage. To monitor nutritional status of these animals, we have utilized a number of previously described assays, and have adapted and improved others, described in chapter II. By visually monitoring aspects of nutrient storage, while simultaneously manipulating genes, either individually or in combinations, we have described a novel pathway regulating lipid storage, described in chapter III, and have begun work on a second pathway which appears to regulate the cell biology of a nutrient storage compartment, described in chapter IV. In a forward genetic screen for genes regulating lipid storage, we isolated a mutation in acs-3, encoding a long chain fatty acyl-CoA synthase. These mutants exhibit elevated lipid staining intensity, and abnormally large intestinal lipid droplets. Our characterization of this gene revealed that it is expressed in a limited set of tissues, and expression of acs-3 in the hypodermal seam cells, a stem cell-like population, is sufficient to rescue the abnormal lipid storage of mutant animals. We performed both unbiased and candidate suppressor screens, and identified a set of genes that function in the acs-3 pathway to regulate nutrient storage. Our findings suggest a pathway in which acs-3, acdh-10, acdh-11 and elo-6 function to modulate fatty acyl-CoA levels, which in turn regulate nhr-25 function, perhaps as components of nhr-25 ligands. The work described here sheds light on networks of genes that likely function in different tissues to regulate nutrient storage in a concerted fashion. In addition, these findings underscore the utility of genetic interrogation of metabolic networks, and demonstrate that enzymes with well-understood enzymatic activities can function in unexpected ensembles to modulated metabolism via unpredictable mechanisms

Representations of Taste Modality in the Drosophila Brain

Gustatory receptors and peripheral taste cells have been identified in flies and mammals, revealing that sensory cells are tuned to taste modality across species. How taste modalities are processed in higher brain centers to guide feeding decisions is unresolved. Here, we developed a large-scale calcium-imaging approach coupled with cell labeling to examine how different taste modalities are processed in the fly brain. These studies reveal that sweet, bitter, and water sensory cells activate different cell populations throughout the subesophageal zone, with most cells responding to a single taste modality. Pathways for sweet and bitter tastes are segregated from sensory input to motor output, and this segregation is maintained in higher brain areas, including regions implicated in learning and neuromodulation. Our work reveals independent processing of appetitive and aversive tastes, suggesting that flies and mammals use a similar coding strategy to ensure innate responses to salient compounds

A paradigm shift in the delivery of services for diagnosis of inherited retinal disease

OBJECTIVES: Current technologies for delivering gene testing are labour-intensive and expensive. Over the last 3 years, new high-throughput DNA sequencing techniques (next generation sequencing; NGS), with the capability to analyse multiple genes or entire genomes, have been rapidly adopted into research. This study examines the possibility of incorporating NGS into a clinical UK service context. METHODS: The study applied NGS of 105 genes to 50 patients known to be affected by inherited forms of blindness in the setting of a UK National Health Service-accredited diagnostic molecular genetics laboratory. The study assessed the ability of an NGS protocol to identify likely disease-causing genetic variants when compared with current methodologies available through UK diagnostic laboratories. RESULTS: Conventional testing is only applicable to the minority of patients with inherited retinal disease and identifies mutations in fewer than one in four of those patients tested. By contrast, the NGS assay is directed at all patients with such disorders and identifies disease-causing mutations in 50--55%, which is a dramatic increase. This includes patients with apparently 'sporadic' disease, and those for whom clinical management and prognosis are altered as a consequence of defining their disease at a molecular level. CONCLUSIONS: The new NGS approach delivers a step change in the diagnosis of inherited eye disease, provides precise diagnostic information and extends the possibility of targeted treatments including gene therapy. The approach represents an exemplar that illustrates the opportunity that NGS provides for broadening the availability of genetic testing. The technology will be applied to many conditions that are associated with high levels of genetic heterogeneity

Defects in the C. elegans acyl-CoA synthase, acs-3, and nuclear hormone receptor, nhr-25, cause sensitivity to distinct, but overlapping stresses.

Metazoan transcription factors control distinct networks of genes in specific tissues, yet understanding how these networks are integrated into physiology, development, and homeostasis remains challenging. Inactivation of the nuclear hormone receptor nhr-25 ameliorates developmental and metabolic phenotypes associated with loss of function of an acyl-CoA synthetase gene, acs-3. ACS-3 activity prevents aberrantly high NHR-25 activity. Here, we investigated this relationship further by examining gene expression patterns following acs-3 and nhr-25 inactivation. Unexpectedly, we found that the acs-3 mutation or nhr-25 RNAi resulted in similar transcriptomes with enrichment in innate immunity and stress response gene expression. Mutants of either gene exhibited distinct sensitivities to pathogens and environmental stresses. Only nhr-25 was required for wild-type levels of resistance to the bacterial pathogen P. aeruginosa and only acs-3 was required for wild-type levels of resistance to osmotic stress and the oxidative stress generator, juglone. Inactivation of either acs-3 or nhr-25 compromised lifespan and resistance to the fungal pathogen D. coniospora. Double mutants exhibited more severe defects in the lifespan and P. aeruginosa assays, but were similar to the single mutants in other assays. Finally, acs-3 mutants displayed defects in their epidermal surface barrier, potentially accounting for the observed sensitivities. Together, these data indicate that inactivation of either acs-3 or nhr-25 causes stress sensitivity and increased expression of innate immunity/stress genes, most likely by different mechanisms. Elevated expression of these immune/stress genes appears to abrogate the transcriptional signatures relevant to metabolism and development

Malignant undifferentiated epithelioid neoplasms with MAML2 rearrangements: A clinicopathologic study of seven cases demonstrating a heterogenous entity

Among mesenchymal tumors, MAML2 gene rearrangements have been described in a subset of composite hemangioendothelioma and myxoinflammatory fibroblastic sarcoma (MIFS). However, we have recently encountered MAML2-related fusions in a group of seven undifferentiated malignant epithelioid neoplasms that do not fit well to any established pathologic entities. The patients included five males and two female, aged 41–71 years old (median 65 years). The tumors involved the deep soft tissue of extremities (hip, knee, arm, hand), abdominal wall, and the retroperitoneum. Microscopically, the tumors consisted of solid sheets of atypical epithelioid to histiocytoid cells with abundant cytoplasm. Prominent mitotic activity and necrosis were present in 4 cases. In 3 cases, the cells displayed hyperchromatic nuclei or conspicuous macronucleoli, and were admixed with background histiocytoid cells and a lymphoplasmacytic infiltrate. By immunohistochemistry (IHC), the neoplastic cells had a nonspecific phenotype. On targeted RNA sequencing, MAML2 was the 3′ partner and fused to YAP1 (4 cases), ARHGAP42 (2 cases), and ENDOD1 (1 case). Two cases with YAP1::MAML2 harbored concurrent RAF kinase fusions (RBMS3::RAF1 and AGK::BRAF, respectively). In 2 cases with targeted DNA sequencing, mutations in TP53, RB1 and PTEN were detected in 1 case, and PDGFRB mutations, CCNE1 amplifications and CDKN2A/2B deletion were detected in another case, which showed strong and diffuse PDGFRB expression by IHC. Of the 4 cases with detailed clinical history (median follow-up period 8 months), three developed distant metastatic disease (one of which died of disease); one case remained free of disease 3 years following surgical excision. In conclusion, we describe a heterogeneous series of MAML2-rearranged undifferentiated malignant epithelioid neoplasms, a subset of which may overlap with a recently described MIFS variant with YAP1::MAML2 fusions, further expanding the clinicopathologic spectrum of mesenchymal neoplasms with recurrent MAML2 gene rearrangements

Defects in the C. elegans acyl-CoA synthase, acs-3, and nuclear hormone receptor, nhr-25, cause sensitivity to distinct, but overlapping stresses.

Metazoan transcription factors control distinct networks of genes in specific tissues, yet understanding how these networks are integrated into physiology, development, and homeostasis remains challenging. Inactivation of the nuclear hormone receptor nhr-25 ameliorates developmental and metabolic phenotypes associated with loss of function of an acyl-CoA synthetase gene, acs-3. ACS-3 activity prevents aberrantly high NHR-25 activity. Here, we investigated this relationship further by examining gene expression patterns following acs-3 and nhr-25 inactivation. Unexpectedly, we found that the acs-3 mutation or nhr-25 RNAi resulted in similar transcriptomes with enrichment in innate immunity and stress response gene expression. Mutants of either gene exhibited distinct sensitivities to pathogens and environmental stresses. Only nhr-25 was required for wild-type levels of resistance to the bacterial pathogen P. aeruginosa and only acs-3 was required for wild-type levels of resistance to osmotic stress and the oxidative stress generator, juglone. Inactivation of either acs-3 or nhr-25 compromised lifespan and resistance to the fungal pathogen D. coniospora. Double mutants exhibited more severe defects in the lifespan and P. aeruginosa assays, but were similar to the single mutants in other assays. Finally, acs-3 mutants displayed defects in their epidermal surface barrier, potentially accounting for the observed sensitivities. Together, these data indicate that inactivation of either acs-3 or nhr-25 causes stress sensitivity and increased expression of innate immunity/stress genes, most likely by different mechanisms. Elevated expression of these immune/stress genes appears to abrogate the transcriptional signatures relevant to metabolism and development

<i>acs-3(ft5)</i> and <i>nhr-25(ku217)</i> mutants have reduced lifespans and are hypersensitive to <i>D. coniospora</i> and <i>P. aerugninosa</i>, respectively.

<p>(A) Lifespan of the indicated strains on plates supplemented with FUDR and seeded OP50. All lifespan curves are significantly different from one another [n>100; p<0.0001, log-rank (Mantel-Cox) test]. B) Survival of L4 larvae of the indicated genotype on <i>Pseudomonas aeruginosa</i> strain PA14. All lifespan curves are significantly different from one another [n>100; p<4.40E-05, log-rank (Mantel-Cox)], except WT and <i>acs-3(ft5)</i> [n>100; p = 0.33, log-rank (Mantel-Cox) test]. (C) Survival of L4 larvae of the indicated genotype on <i>D. coniospora</i>. The graph is representative of two experiments showing the same results. All strain lifespans are significantly different from one another ([n>100; p≤0.0202, log-rank (Mantel-Cox) test], except <i>acs-3(ft5)</i> and <i>acs-3(ft5);nhr-25 (ku217)</i> [n>100; p = 0.38, log-rank (Mantel-Cox) test]. Detailed statistical analyses are presented in Figure S8.</p

<i>acs-3(ft5)</i> and <i>nhr-25(ku217)</i> mutant cuticles are not permeable to Hoechst 33258 dye.

<p>(A) Permeability to Hoechst 33258 dye. Mixed stage animals of the indicated genotypes were incubated with the membrane impermeable Hoechst 33258 dye and scored for nuclear staining. Error bars represent standard deviation from at least three biological replicates with >50 animals assayed in each experiment (* two-tailed T-test p<0.005). B) Representative DIC and Hoechst staining images for animals of each indicated genotype. Scale bar = 50 μm. Detailed statistical analyses, including all pair-wise comparisons are presented in Figure S10A.</p