A retrospective review of multiple findings in diagnostic exome sequencing: half are distinct and half are overlapping diagnoses.

PURPOSE: We evaluated clinical and genetic features enriched in patients with multiple Mendelian conditions to determine which patients are more likely to have multiple potentially relevant genetic findings (MPRF). METHODS: Results of the first 7698 patients who underwent exome sequencing at Ambry Genetics were reviewed. Clinical and genetic features were examined and degree of phenotypic overlap between the genetic diagnoses was evaluated. RESULTS: Among patients referred for exome sequencing, 2% had MPRF. MPRF were more common in patients from consanguineous families and patients with greater clinical complexity. The difference in average number of organ systems affected is small: 4.3 (multiple findings) vs. 3.9 (single finding) and may not be distinguished in clinic. CONCLUSION: Patients with multiple genetic diagnoses had a slightly higher number of organ systems affected than patients with single genetic diagnoses, largely because the comorbid conditions affected overlapping organ systems. Exome testing may be beneficial for all cases with multiple organ systems affected. The identification of multiple relevant genetic findings in 2% of exome patients highlights the utility of a comprehensive molecular workup and updated interpretation of existing genomic data; a single definitive molecular diagnosis from analysis of a limited number of genes may not be the end of a diagnostic odyssey.

TNFα and IL-1ß but not IL-18 Suppresses Hippocampal Long-Term Potentiation Directly at the Synapse.

CNS inflammatory responses are linked to cognitive impairment in humans. Research in animal models supports this connection by showing that inflammatory cytokines suppress long-term potentiation (LTP), the best-known cellular correlate of memory. Cytokine-induced modulation of LTP has been previously studied in vivo or in brain slices, two experimental approaches containing multiple cell populations responsive to cytokines. In their target cells, cytokines commonly increase the expression of multiple cytokines, thus increasing the complexity of brain cytokine networks even after single-cytokine challenges. Whether cytokines suppress LTP by direct effects on neurons or by indirect mechanisms is still an open question. Here, we evaluated the effect of a major set of inflammatory cytokines including tumor necrosis factor-α (TNFα), interleukin-1ß (IL-1ß) and interleukin-18 (IL-18) on chemically-induced LTP (cLTP) in isolated hippocampal synaptosomes of mice, using fluorescence analysis of single-synapse long-term potentiation (FASS-LTP). We found that TNFα and IL-1ß suppress synaptosomal cLTP. In contrast, cLTP was not affected by IL-18, at a concentration previously shown to block LTP in hippocampal slices. We also found that IL-18 does not impair cLTP or brain-derived neurotrophic factor (BDNF) signaling in primary hippocampal neuronal cultures. Thus, using both synaptosomes and neuron cultures, our data suggest that IL-18 impairs LTP by indirect mechanisms, which may depend on non-neuronal cells, such as glia. Notably, our results demonstrate that TNFα and IL-1ß directly suppress hippocampal plasticity via neuron-specific mechanisms. A better understanding of the brain's cytokine networks and their final molecular effectors is crucial to identify specific targets for intervention.

Topologically associated domains enriched for lineage-specific genes reveal expression-dependent nuclear topologies during myogenesis.

The linear distribution of genes across chromosomes and the spatial localization of genes within the nucleus are related to their transcriptional regulation. The mechanistic consequences of linear gene order, and how it may relate to the functional output of genome organization, remain to be fully resolved, however. Here we tested the relationship between linear and 3D organization of gene regulation during myogenesis. Our analysis has identified a subset of topologically associated domains (TADs) that are significantly enriched for muscle-specific genes. These lineage-enriched TADs demonstrate an expression-dependent pattern of nuclear organization that influences the positioning of adjacent nonenriched TADs. Therefore, lineage-enriched TADs inform cell-specific genome organization during myogenesis. The reduction of allelic spatial distance of one of these domains, which contains Myogenin, correlates with reduced transcriptional variability, identifying a potential role for lineage-specific nuclear topology. Using a fusion-based strategy to decouple mitosis and myotube formation, we demonstrate that the cell-specific topology of syncytial nuclei is dependent on cell division. We propose that the effects of linear and spatial organization of gene loci on gene regulation are linked through TAD architecture, and that mitosis is critical for establishing nuclear topologies during cellular differentiation.

Interstitial telomeric loops and implications of the interaction between TRF2 and lamin A/C.

The protein-DNA complexes that compose the end of mammalian chromosomes-telomeres-serve to stabilize linear genomic DNA and are involved in cellular and organismal aging. One mechanism that protects telomeres from premature degradation is the formation of structures called t-loops, in which the single-stranded 3' overhang present at the terminal end of telomeres loops back and invades medial double-stranded telomeric DNA. We identified looped structures formed between terminal chromosome ends and interstitial telomeric sequences (ITSs), which are found throughout the human genome, that we have termed interstitial telomeric loops (ITLs). While they form in a TRF2-dependent manner similar to t-loops, ITLs further require the physical interaction of TRF2 with the nuclear intermediate filament protein lamin A/C. Our findings suggest that interactions between telomeres and the nucleoskeleton broadly impact genomic integrity, including telomere stability, chromosome structure, and chromosome fragility. Here, we review the roles of TRF2 and lamin A/C in telomere biology and consider how their interaction may relate telomere homeostasis to cellular and organismal aging.

An additional case of Hennekam lymphangiectasia-lymphedema syndrome caused by loss-of-function mutation in ADAMTS3.

Hennekam lymphangiectasia-lymphedema syndrome (HKLLS) is a genetically heterogeneous lymphatic dysplasia with characteristic of facial dysmorphism, neurocognitive impairments, and abnormalities of the pericardium, intestinal tract, and extremities. It is an autosomal recessive condition caused by biallelic mutations in CCBE1 (collagen- and calcium-binding epidermal growth factor domain-containing protein 1) (HKLLS1; OMIM 235510) or FAT4 (HKLLS2; OMIM 616006). CCBE1 acts via ADAMTS3 (a disintegrin and metalloprotease with thrombospondin motifs-3 protease) to enhance vascular endothelial growth factor C signaling. There is report of one family supporting mutations in ADAMTS3 as causative for the phenotype labeled as HKLLS3. Here, we report an additional case of HKLLS that appears to be associated with homozygous nonsense mutation of ADAMTS3.

Synapse-specific IL-1 receptor subunit reconfiguration augments vulnerability to IL-1ß in the aged hippocampus.

In the aged brain, synaptic plasticity and memory show increased vulnerability to impairment by the inflammatory cytokine interleukin 1ß (IL-1ß). In this study, we evaluated the possibility that synapses may directly undergo maladaptive changes with age that augment sensitivity to IL-1ß impairment. In hippocampal neuronal cultures, IL-1ß increased the expression of the IL-1 receptor type 1 and the accessory coreceptor AcP (proinflammatory), but not of the AcPb (prosurvival) subunit, a reconfiguration that potentiates the responsiveness of neurons to IL-1ß. To evaluate whether synapses develop a similar heightened sensitivity to IL-1ß with age, we used an assay to track long-term potentiation (LTP) in synaptosomes. We found that IL-1ß impairs LTP directly at the synapse and that sensitivity to IL-1ß is augmented in aged hippocampal synapses. The increased synaptic sensitivity to IL-1ß was due to IL-1 receptor subunit reconfiguration, characterized by a shift in the AcP/AcPb ratio, paralleling our culture data. We suggest that the age-related increase in brain IL-1ß levels drives a shift in IL-1 receptor configuration, thus heightening the sensitivity to IL-1ß. Accordingly, selective blocking of AcP-dependent signaling with Toll-IL-1 receptor domain peptidomimetics prevented IL-1ß-mediated LTP suppression and blocked the memory impairment induced in aged mice by peripheral immune challenge (bacterial lipopolysaccharide). Overall, this study demonstrates that increased AcP signaling, specifically at the synapse, underlies the augmented vulnerability to cognitive impairment by IL-1ß that occurs with age.

Classification of Genes: Standardized Clinical Validity Assessment of Gene-Disease Associations Aids Diagnostic Exome Analysis and Reclassifications.

Ascertaining a diagnosis through exome sequencing can provide potential benefits to patients, insurance companies, and the healthcare system. Yet, as diagnostic sequencing is increasingly employed, vast amounts of human genetic data are produced that need careful curation. We discuss methods for accurately assessing the clinical validity of gene-disease relationships to interpret new research findings in a clinical context and increase the diagnostic rate. The specifics of a gene-disease scoring system adapted for use in a clinical laboratory are described. In turn, clinical validity scoring of gene-disease relationships can inform exome reporting for the identification of new or the upgrade of previous, clinically relevant gene findings. Our retrospective analysis of all reclassification reports from the first 4 years of diagnostic exome sequencing showed that 78% were due to new gene-disease discoveries published in the literature. Among all exome positive/likely positive findings in characterized genes, 32% were in genetic etiologies that were discovered after 2010. Our data underscore the importance and benefits of active and up-to-date curation of a gene-disease database combined with critical clinical validity scoring and proactive reanalysis in the clinical genomics era.

Rapamycin and interleukin-1ß impair brain-derived neurotrophic factor-dependent neuron survival by modulating autophagy.

The mammalian target of rapamycin (mTOR) pathway has multiple important physiological functions, including regulation of protein synthesis, cell growth, autophagy, and synaptic plasticity. Activation of mTOR is necessary for the many beneficial effects of brain-derived neurotrophic factor (BDNF), including dendritic translation and memory formation in the hippocampus. At present, however, the role of mTOR in BDNF's support of survival is not clear. We report that mTOR activation is necessary for BDNF-dependent survival of primary rat hippocampal neurons, as either mTOR inhibition by rapamycin or genetic manipulation of the downstream molecule p70S6K specifically blocked BDNF rescue. Surprisingly, however, BDNF did not promote neuron survival by up-regulating mTOR-dependent protein synthesis or through mTOR-dependent suppression of caspase-3 activation. Instead, activated mTOR was responsible for BDNF's suppression of autophagic flux. shRNA against the autophagic machinery Atg7 or Atg5 prolonged the survival of neurons co-treated with BDNF and rapamycin, suggesting that suppression of mTOR in BDNF-treated cells resulted in excessive autophagy. Finally, acting as a physiological analog of rapamycin, IL-1ß impaired BDNF signaling by way of inhibiting mTOR activation as follows: the cytokine induced caspase-independent neuronal death and accelerated autophagic flux in BDNF-treated cells. These findings reveal a novel mechanism of BDNF neuroprotection; BDNF not only prevents apoptosis through inhibiting caspase activation but also promotes neuron survival through modulation of autophagy. This protection mechanism is vulnerable under chronic inflammation, which deregulates autophagy through impairing mTOR signaling. These results may be relevant to age-related changes observed in neurodegenerative diseases.

Using a chat-based informed consent tool in large-scale genomic research.

OBJECTIVE: We implemented a chatbot consent tool to shift the time burden from study staff in support of a national genomics research study. MATERIALS AND METHODS: We created an Institutional Review Board-approved script for automated chat-based consent. We compared data from prospective participants who used the tool or had traditional consent conversations with study staff. RESULTS: Chat-based consent, completed on a user's schedule, was shorter than the traditional conversation. This did not lead to a significant change in affirmative consents. Within affirmative consents and declines, more prospective participants completed the chat-based process. A quiz to assess chat-based consent user understanding had a high pass rate with no reported negative experiences. CONCLUSION: Our report shows that a structured script can convey important information while realizing the benefits of automation and burden shifting. Analysis suggests that it may be advantageous to use chatbots to scale this rate-limiting step in large research projects.

Brain-derived neurotrophic factor-dependent synaptic plasticity is suppressed by interleukin-1ß via p38 mitogen-activated protein kinase.

Evolving evidence suggests that brain inflammation and the buildup of proinflammatory cytokine increases the risk for cognitive decline and cognitive dysfunction. Interleukin-1ß (IL-1ß), acting via poorly understood mechanisms, appears to be a key cytokine in causing these deleterious effects along with a presumably related loss of long-term potentiation (LTP)-type synaptic plasticity. We hypothesized that IL-1ß disrupts brain-derived neurotrophic factor (BDNF) signaling cascades and thereby impairs the formation of filamentous actin (F-actin) in dendritic spines, an event that is essential for the stabilization of LTP. Actin polymerization in spines requires phosphorylation of the filament severing protein cofilin and is modulated by expression of the immediate early gene product Arc. Using rat organotypic hippocampal cultures, we found that IL-1ß suppressed BDNF-dependent regulation of Arc and phosphorylation of cofilin and cAMP response element-binding protein (CREB), a transcription factor regulating Arc expression. IL-1ß appears to act on BDNF signal transduction by impairing the phosphorylation of insulin receptor substrate 1, a protein that couples activation of the BDNF receptor TrkB to downstream signaling pathways regulating CREB, Arc, and cofilin. IL-1ß upregulated p38 mitogen-activated protein kinase (MAPK), and inhibiting p38 MAPK prevented IL-1ß from disrupting BDNF signaling. IL-1ß also prevented the formation of F-actin in spines and impaired the consolidation, but not the induction, of BDNF-dependent LTP in acute hippocampal slices. The suppressive effect of IL-1ß on F-actin and LTP was prevented by inhibiting p38 MAPK. These findings define a new mechanism for the action of IL-1ß on LTP and point to a potential therapeutic target to restore synaptic plasticity.

"Development and Implementation of Novel Chatbot-based Genomic Research Consent".

Objective: To conduct a retrospective analysis comparing traditional human-based consenting to an automated chat-based consenting process. Materials and Methods: We developed a new chat-based consent using our IRB-approved consent forms. We leveraged a previously developed platform (GiaⓇ, or "Genetic Information Assistant") to deliver the chat content to candidate participants. The content included information about the study, educational information, and a quiz to assess understanding. We analyzed 144 families referred to our study during a 6-month time period. A total of 37 families completed consent using the traditional process, while 35 families completed consent using Gia. Results: Engagement rates were similar between both consenting methods. The median length of the consent conversation was shorter for Gia users compared to traditional (44 vs. 76 minutes). Additionally, the total time from referral to consent completion was faster with Gia (5 vs. 16 days). Within Gia, understanding was assessed with a 10-question quiz that most participants (96%) passed. Feedback about the chat consent indicated that 86% of participants had a positive experience. Discussion: Using Gia resulted in time savings for both the participant and study staff. The chatbot enables studies to reach more potential candidates. We identified five key features related to human-centered design for developing a consent chat. Conclusion: This analysis suggests that it is feasible to use an automated chatbot to scale obtaining informed consent for a genomics research study. We further identify a number of advantages when using a chatbot.

Interferon regulatory factor 4 (IRF4) interacts with NFATc2 to modulate interleukin 4 gene expression.

Proteins of the nuclear factor of activated T cells (NFAT) family of transcription factors are critical for lymphocyte activation in the immune system. In particular, NFATs are important regulators of inducible IL-4 gene expression. Interferon regulatory factor 4 (IRF4) is an immune system-restricted interferon regulatory factor that is required for lymphocyte activation, but its molecular functions in the T lineage remain to be elucidated. We demonstrate that IRF4 potently synergizes with NFATc2 to specifically enhance NFATc2-driven transcriptional activation of the IL-4 promoter. This function is dependent on the physical interaction of IRF4 with NFATc2. IRF4 synergizes with NFATc2 and the IL-4-inducing transcription factor, c-maf, to augment IL-4 promoter activity as well as to elicit significant levels of endogenous IL-4 production. Furthermore, naïve T helper cells from mice lacking IRF4 are compromised severely for the production of IL-4 and other Th2 cytokines. The identification of IRF4 as a partner for NFATc2 in IL-4 gene regulation provides an important molecular function for IRF4 in T helper cell differentiation.

A-type nuclear lamins act as transcriptional repressors when targeted to promoters.

Regions of heterochromatin are often found at the periphery of the mammalian nucleus, juxtaposed to the nuclear lamina. Genes in these regions are likely maintained in a transcriptionally silent state, although other locations at the nuclear periphery associated with nuclear pores are sites of active transcription. As primary components of the nuclear lamina, A- and B-type nuclear lamins are intermediate filament proteins that interact with DNA, histones and known transcriptional repressors, leading to speculation that they may promote establishment of repressive domains. However, no direct evidence of a role for nuclear lamins in transcriptional repression has been reported. Here we find that human lamin A, when expressed in yeast and cultured human cells as a fusion protein to the Gal4 DNA-binding domain (DBD), can mediate robust transcriptional repression of promoters with Gal4 binding sites. Full repression by lamin A requires both the coiled-coil rod domain and the C-terminal tail domain. In human cells, other intermediate filament proteins such as lamin B and vimentin are unable to confer robust repression as Gal4-DBD fusions, indicating that this property is specific to A-type nuclear lamins. These findings indicate that A-type lamins can promote transcriptional repression when in proximity of a promoter.

A second cohort of CHD3 patients expands the molecular mechanisms known to cause Snijders Blok-Campeau syndrome.

There has been one previous report of a cohort of patients with variants in Chromodomain Helicase DNA-binding 3 (CHD3), now recognized as Snijders Blok-Campeau syndrome. However, with only three previously-reported patients with variants outside the ATPase/helicase domain, it was unclear if variants outside of this domain caused a clinically similar phenotype. We have analyzed 24 new patients with CHD3 variants, including nine outside the ATPase/helicase domain. All patients were detected with unbiased molecular genetic methods. There is not a significant difference in the clinical or facial features of patients with variants in or outside this domain. These additional patients further expand the clinical and molecular data associated with CHD3 variants. Importantly we conclude that there is not a significant difference in the phenotypic features of patients with various molecular disruptions, including whole gene deletions and duplications, and missense variants outside the ATPase/helicase domain. This data will aid both clinical geneticists and molecular geneticists in the diagnosis of this emerging syndrome.

Inhibition of LTP-Induced Translation by IL-1ß Reduces the Level of Newly Synthesized Proteins in Hippocampal Dendrites.

In rodent hippocampus, the inflammatory cytokine interleukin-1ß (IL-1ß) impairs memory and long-term potentiation (LTP), a major form of plasticity that depends on protein synthesis. A better understanding of the mechanisms by which IL-1ß impairs LTP may help identify targets for preventing cognitive deterioration. We tested whether IL-1ß inhibits protein synthesis in hippocampal neuron cultures following chemically induced LTP (cLTP). Fluorescent-tagging using click-chemistry showed that IL-1ß reduces the level of newly synthesized proteins in proximal dendrites of cLTP stimulated neurons. Relative to controls, in cLTP stimulated neurons, IL-1ß inhibited Akt/mTOR signaling, as well as the upregulation of GluA1, an AMPA receptor subunit, and LIMK1, a kinase that promotes actin polymerization. Notably, a novel TIR domain peptidomimetic (EM163) blocked both the activation of p38 and the suppression of cLTP-dependent protein synthesis by IL-1ß. Our data support a model where IL-1ß suppresses LTP directly in neurons by inhibiting mTOR-dependent translation.

Clinical validity assessment of genes for inclusion in multi-gene panel testing: A systematic approach.

BACKGROUND: Advances in sequencing technology have led to expanded use of multi-gene panel tests (MGPTs) for clinical diagnostics. Well-designed MGPTs must balance increased detection of clinically significant findings while mitigating the increase in variants of uncertain significance (VUS). To maximize clinical utililty, design of such panels should include comprehensive gene vetting using a standardized clinical validity (CV) scoring system. METHODS: To assess the impact of CV-based gene vetting on MGPT results, data from MGPTs for cardiovascular indications were retrospectively analyzed. Using our CV scoring system, genes were categorized as having definitive, strong, moderate, or limited evidence. The rates of reported pathogenic or likely pathogenic variants and VUS were then determined for each CV category. RESULTS: Of 106 total genes, 42% had definitive, 17% had strong, 29% had moderate, and 12% had limited CV. The detection rate of variants classified as pathogenic or likely pathogenic was higher for genes with greater CV, while the VUS rate showed an inverse relationship with CV score. No pathogenic or likely pathogenic findings were observed in genes with a limited CV. CONCLUSION: These results demonstrate the importance of a standardized, evidence-based vetting process to establish CV for genes on MGPTs. Using our proposed system may help to increase the detection rate while mitigating higher VUS rates.

Author Correction: Varying levels of X chromosome coalescence in female somatic cells alters the balance of X-linked dosage compensation and is implicated in female-dominant systemic lupus erythematosus.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Varying levels of X chromosome coalescence in female somatic cells alters the balance of X-linked dosage compensation and is implicated in female-dominant systemic lupus erythematosus.

The three-dimensional organization of the genome in mammalian interphase nuclei is intrinsically linked to the regulation of gene expression. Whole chromosome territories and their encoded gene loci occupy preferential positions within the nucleus that changes according to the expression profile of a given cell lineage or stage. To further illuminate the relationship between chromosome organization, epigenetic environment, and gene expression, here we examine the functional organization of chromosome X and corresponding X-linked genes in a variety of healthy human and disease state X diploid (XX) cells. We observe high frequencies of homologous chromosome X colocalization (or coalescence), typically associated with initiation of X-chromosome inactivation, occurring in XX cells outside of early embryogenesis. Moreover, during chromosome X coalescence significant changes in Xist, H3K27me3, and X-linked gene expression occur, suggesting the potential exchange of gene regulatory information between the active and inactive X chromosomes. We also observe significant differences in chromosome X coalescence in disease-implicated lymphocytes isolated from systemic lupus erythematosus (SLE) patients compared to healthy controls. These results demonstrate that X chromosomes can functionally interact outside of embryogenesis when X inactivation is initiated and suggest a potential gene regulatory mechanism aberration underlying the increased frequency of autoimmunity in XX individuals.

Age- and calorie-independent life span extension from dietary restriction by bacterial deprivation in Caenorhabditis elegans.

BACKGROUND: Dietary restriction (DR) increases life span and delays age-associated disease in many organisms. The mechanism by which DR enhances longevity is not well understood. RESULTS: Using bacterial food deprivation as a means of DR in C. elegans, we show that transient DR confers long-term benefits including stress resistance and increased longevity. Consistent with studies in the fruit fly and in mice, we demonstrate that DR also enhances survival when initiated late in life. DR by bacterial food deprivation significantly increases life span in worms when initiated as late as 24 days of adulthood, an age at which greater than 50% of the cohort have died. These survival benefits are, at least partially, independent of food consumption, as control fed animals are no longer consuming bacterial food at this advanced age. Animals separated from the bacterial lawn by a barrier of solid agar have a life span intermediate between control fed and food restricted animals. Thus, we find that life span extension from bacterial deprivation can be partially suppressed by a diffusible component of the bacterial food source, suggesting a calorie-independent mechanism for life span extension by dietary restriction. CONCLUSION: Based on these findings, we propose that dietary restriction by bacterial deprivation increases longevity in C. elegans by a combination of reduced food consumption and decreased food sensing.

Clinical Epigenetic Therapies Disrupt Sex Chromosome Dosage Compensation in Human Female Cells.

Sex chromosome gene dosage compensation is required to ensure equivalent levels of X-linked gene expression between males (46, XY) and females (46, XX). To achieve similar expression, X-chromosome inactivation (XCI) is initiated in female cells during early stages of embryogenesis. Within each cell, either the maternal or paternal X chromosome is selected for whole chromosome transcriptional silencing, which is initiated and maintained by epigenetic and chromatin conformation mechanisms. With the emergence of small-molecule epigenetic inhibitors for the treatment of disease, such as cancer, the epigenetic mechanism underlying XCI may be inadvertently targeted. Here, we test 2 small-molecule epigenetic inhibitors being used clinically, GSK126 (a histone H3 lysine 27 methyltransferase inhibitor) and suberoylanilide hydroxamic acid (a histone deacetylase inhibitor), on their effects of the inactive X (Xi) in healthy human female fibroblasts. The combination of these modifiers, at subcancer therapeutic levels, leads to the inability to detect the repressive H3K27me3 modification characteristic of XCI in the majority of the cells. Importantly, genes positioned near the X-inactivation center (Xic), where inactivation is initiated, exhibit robust expression with treatment of the inhibitors, while genes located near the distal ends of the X chromosome intriguingly exhibit significant downregulation. These results demonstrate that small-molecule epigenetic inhibitors can have profound consequences on XCI in human cells, and they underscore the importance of considering gender when developing and clinically testing small-molecule epigenetic inhibitors, in particular those that target the well-characterized mechanisms of X inactivation.