Scalable approaches for generating, validating and incorporating data from high-throughput functional assays to improve clinical variant classification.

As the adoption and scope of genetic testing continue to expand, interpreting the clinical significance of DNA sequence variants at scale remains a formidable challenge, with a high proportion classified as variants of uncertain significance (VUSs). Genetic testing laboratories have historically relied, in part, on functional data from academic literature to support variant classification. High-throughput functional assays or multiplex assays of variant effect (MAVEs), designed to assess the effects of DNA variants on protein stability and function, represent an important and increasingly available source of evidence for variant classification, but their potential is just beginning to be realized in clinical lab settings. Here, we describe a framework for generating, validating and incorporating data from MAVEs into a semi-quantitative variant classification method applied to clinical genetic testing. Using single-cell gene expression measurements, cellular evidence models were built to assess the effects of DNA variation in 44 genes of clinical interest. This framework was also applied to models for an additional 22 genes with previously published MAVE datasets. In total, modeling data was incorporated from 24 genes into our variant classification method. These data contributed evidence for classifying 4043 observed variants in over 57,000 individuals. Genetic testing laboratories are uniquely positioned to generate, analyze, validate, and incorporate evidence from high-throughput functional data and ultimately enable the use of these data to provide definitive clinical variant classifications for more patients.

Applications of artificial intelligence in clinical laboratory genomics.

The transition from analog to digital technologies in clinical laboratory genomics is ushering in an era of "big data" in ways that will exceed human capacity to rapidly and reproducibly analyze those data using conventional approaches. Accurately evaluating complex molecular data to facilitate timely diagnosis and management of genomic disorders will require supportive artificial intelligence methods. These are already being introduced into clinical laboratory genomics to identify variants in DNA sequencing data, predict the effects of DNA variants on protein structure and function to inform clinical interpretation of pathogenicity, link phenotype ontologies to genetic variants identified through exome or genome sequencing to help clinicians reach diagnostic answers faster, correlate genomic data with tumor staging and treatment approaches, utilize natural language processing to identify critical published medical literature during analysis of genomic data, and use interactive chatbots to identify individuals who qualify for genetic testing or to provide pre-test and post-test education. With careful and ethical development and validation of artificial intelligence for clinical laboratory genomics, these advances are expected to significantly enhance the abilities of geneticists to translate complex data into clearly synthesized information for clinicians to use in managing the care of their patients at scale.

Activation of corticostriatal circuitry relieves chronic neuropathic pain.

Neural circuits that determine the perception and modulation of pain remain poorly understood. The prefrontal cortex (PFC) provides top-down control of sensory and affective processes. While animal and human imaging studies have shown that the PFC is involved in pain regulation, its exact role in pain states remains incompletely understood. A key output target for the PFC is the nucleus accumbens (NAc), an important component of the reward circuitry. Interestingly, recent human imaging studies suggest that the projection from the PFC to the NAc is altered in chronic pain. The function of this corticostriatal projection in pain states, however, is not known. Here we show that optogenetic activation of the PFC produces strong antinociceptive effects in a rat model (spared nerve injury model) of persistent neuropathic pain. PFC activation also reduces the affective symptoms of pain. Furthermore, we show that this pain-relieving function of the PFC is likely mediated by projections to the NAc. Thus, our results support a novel role for corticostriatal circuitry in pain regulation.

Neuroplasticity underlying the comorbidity of pain and depression.

Acute pain induces depressed mood, and chronic pain is known to cause depression. Depression, meanwhile, can also adversely affect pain behaviors ranging from symptomology to treatment response. Pain and depression independently induce long-term plasticity in the central nervous system (CNS). Comorbid conditions, however, have distinct patterns of neural activation. We performed a review of the changes in neural circuitry and molecular signaling pathways that may underlie this complex relationship between pain and depression. We also discussed some of the current and future therapies that are based on this understanding of the CNS plasticity that occurs with pain and depression.

Deciphering neuronal population codes for acute thermal pain.

OBJECTIVE: Pain is defined as an unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage. Current pain research mostly focuses on molecular and synaptic changes at the spinal and peripheral levels. However, a complete understanding of pain mechanisms requires the physiological study of the neocortex. Our goal is to apply a neural decoding approach to read out the onset of acute thermal pain signals, which can be used for brain-machine interface. APPROACH: We used micro wire arrays to record ensemble neuronal activities from the primary somatosensory cortex (S1) and anterior cingulate cortex (ACC) in freely behaving rats. We further investigated neural codes for acute thermal pain at both single-cell and population levels. To detect the onset of acute thermal pain signals, we developed a novel latent state-space framework to decipher the sorted or unsorted S1 and ACC ensemble spike activities, which reveal information about the onset of pain signals. MAIN RESULTS: The state space analysis allows us to uncover a latent state process that drives the observed ensemble spike activity, and to further detect the 'neuronal threshold' for acute thermal pain on a single-trial basis. Our method achieved good detection performance in sensitivity and specificity. In addition, our results suggested that an optimal strategy for detecting the onset of acute thermal pain signals may be based on combined evidence from S1 and ACC population codes. SIGNIFICANCE: Our study is the first to detect the onset of acute pain signals based on neuronal ensemble spike activity. It is important from a mechanistic viewpoint as it relates to the significance of S1 and ACC activities in the regulation of the acute pain onset.

Chronic pain induces generalized enhancement of aversion.

A hallmark feature of chronic pain is its ability to impact other sensory and affective experiences. It is notably associated with hypersensitivity at the site of tissue injury. It is less clear, however, if chronic pain can also induce a generalized site-nonspecific enhancement in the aversive response to nociceptive inputs. Here, we showed that chronic pain in one limb in rats increased the aversive response to acute pain stimuli in the opposite limb, as assessed by conditioned place aversion. Interestingly, neural activities in the anterior cingulate cortex (ACC) correlated with noxious intensities, and optogenetic modulation of ACC neurons showed bidirectional control of the aversive response to acute pain. Chronic pain, however, altered acute pain intensity representation in the ACC to increase the aversive response to noxious stimuli at anatomically unrelated sites. Thus, chronic pain can disrupt cortical circuitry to enhance the aversive experience in a generalized anatomically nonspecific manner.

Persistent neuropathic pain increases synaptic GluA1 subunit levels in core and shell subregions of the nucleus accumbens.

The nucleus accumbens (NAc) is a key component of the brain reward system, and it is composed of core and shell subregions. Glutamate transmission through AMPA-type receptors in both core and shell of the NAc has been shown to regulate reward- and aversion-type behaviors. Previous studies have additionally demonstrated a role for AMPA receptor signaling in the NAc in chronic pain states. Here, we show that persistent neuropathic pain, modeled by spared nerve injury (SNI), selectively increases the numbers of GluA1 subunits of AMPA receptors at the synapse of both core and shell subregions. Such increases are not observed, however, for the GluA2 subunits. Furthermore, we find that phosphorylation at Ser845-GluA1 is increased by SNI at both core and shell subregions. These results demonstrate that persistent neuropathic pain increases AMPA receptor delivery to the synapse in both NAc core and shell, implying a role for AMPA receptor signaling in these regions in pain states.

Persistent pain alters AMPA receptor subunit levels in the nucleus accumbens.

BACKGROUND: A variety of pain conditions have been found to be associated with depressed mood in clinical studies. Depression-like behaviors have also been described in animal models of persistent or chronic pain. In rodent chronic neuropathic pain models, elevated levels of GluA1 subunits of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in the nucleus accumbens (NAc) have been found to inhibit depressive symptoms. However, the effect of reversible post-surgical pain or inflammatory pain on affective behaviors such as depression has not been well characterized in animal models. Neither is it known what time frame is required to elicit AMPA receptor subunit changes in the NAc in various pain conditions. RESULTS: In this study, we compared behavioral and biochemical changes in three pain models: the paw incision (PI) model for post-incisional pain, the Complete Freund's Adjuvant (CFA) model for persistent but reversible inflammatory pain, and the spared nerve injury (SNI) model for chronic postoperative neuropathic pain. In all three models, rats developed depressive symptoms that were concurrent with the presentation of sensory allodynia. GluA1 levels at the synapses of the NAc, however, differed in these three models. The level of GluA1 subunits of AMPA-type receptors at NAc synapses was not altered in the PI model. GluA1 levels were elevated in the CFA model after a period (7 d) of persistent pain, leading to the formation of GluA2-lacking AMPA receptors. As pain symptoms began to resolve, however, GluA1 levels returned to baseline. Meanwhile, in the SNI model, in which pain persisted beyond 14 days, GluA1 levels began to rise after pain became persistent and remained elevated. In addition, we found that blocking GluA2-lacking AMPA receptors in the NAc further decreased the depressive symptoms only in persistent pain models. CONCLUSION: Our study shows that while both short-term and persistent pain can trigger depression-like behaviors, GluA1 upregulation in the NAc likely represents a unique adaptive response to minimize depressive symptoms in persistent pain states.

Differential effects of natural rewards and pain on vesicular glutamate transporter expression in the nucleus accumbens.

BACKGROUND: Pain and natural rewards such as food elicit different behavioral effects. Both pain and rewards, however, have been shown to alter synaptic activities in the nucleus accumbens (NAc), a key component of the brain reward system. Mechanisms by which external stimuli regulate plasticity at NAc synapses are largely unexplored. Medium spiny neurons (MSNs) from the NAc receive excitatory glutamatergic inputs and modulatory dopaminergic and cholinergic inputs from a variety of cortical and subcortical structures. Glutamate inputs to the NAc arise primarily from prefrontal cortex, thalamus, amygdala, and hippocampus, and different glutamate projections provide distinct synaptic and ultimately behavioral functions. The family of vesicular glutamate transporters (VGLUTs 1-3) plays a key role in the uploading of glutamate into synaptic vesicles. VGLUT1-3 isoforms have distinct expression patterns in the brain, but the effects of external stimuli on their expression patterns have not been studied. RESULTS: In this study, we use a sucrose self-administration paradigm for natural rewards, and spared nerve injury (SNI) model for chronic pain. We examine the levels of VGLUTs (1-3) in synaptoneurosomes of the NAc in these two behavioral models. We find that chronic pain leads to a decrease of VGLUT1, likely reflecting decreased projections from the cortex. Pain also decreases VGLUT3 levels, likely representing a decrease in projections from GABAergic, serotonergic, and/or cholinergic interneurons. In contrast, chronic consumption of sucrose increases VGLUT3 in the NAc, possibly reflecting an increase from these interneuron projections. CONCLUSION: Our study shows that natural rewards and pain have distinct effects on the VGLUT expression pattern in the NAc, indicating that glutamate inputs to the NAc are differentially modulated by rewards and pain.

Development of a humanized in vitro blood-brain barrier model to screen for brain penetration of antiepileptic drugs.

PURPOSE: A biotechnologic breakthrough for the study of drug permeability across the blood-brain barrier (BBB) would be the use of a reproducible in vitro model that recapitulates the functional, structural, and pathologic properties of the BBB in situ. We developed a humanized dynamic in vitro BBB model (DIV-BBB) based on cocultures of human microvascular endothelial cells (HBMECs) from "normal" and drug-resistant epileptic brain tissue with human brain astrocytes (HAs) from epilepsy patients or controls. METHODS: HBMECs and HAs were cocultured for 28 days in polypropylene capillaries. HBMECs were exposed to physiologic levels of shear stress generated by intraluminal flow. Permeability to [3H]sucrose, [14C]phenytoin, and [14C]diazepam was measured in control and drug-resistant DIV-BBB with and without pretreatment with the MDR1 inhibitor XR9576. BBB integrity was monitored by transendothelial electrical resistance measurements (TEERs). Cell growth and viability were assessed by measurement of glucose consumption and lactate production. RESULTS: PSucrose and TEER values did not depend on the origin of the endothelium used (epileptic or normal). PPhenytoin was 10-fold less (1.54 x 10(-6) cm/s) in drug-resistant BBB models than in controls (1.74 x 10(-5) cm/s). MDR1 blockade with XR9576 was effective (3.5-fold increase) only in drug-resistant cultures. PDiazepam in control and drug-resistant DIV-BBB was not affected by XR9576 and did not depend on the epileptic or control origin of endothelia. The overall contribution of epileptic glia to pharmacoresistance was negligible. CONCLUSIONS: These results show that, for the substances used, the humanized DIV-BBB recapitulates the physiologic permeability properties of the BBB in vivo and is also capable of mimicking a drug-resistant BBB phenotype.