Cognitive flexibility, attributed to frontal cortex, is vital for navigating the complexities of everyday life. The mediodorsal thalamus (MD), interconnected to frontal cortex, may influence cognitive flexibility. Here, male rats performed an attentional set-shifting task measuring intradimensional (ID) and extradimensional (ED) shifts in sensory discriminations. MD lesion rats needed more trials to learn the rewarded sensory dimension. However, once the choice response strategy was established, learning further two-choice discriminations in the same sensory dimension, and reversals of the reward contingencies in the same dimension, were unimpaired. Critically though, MD lesion rats were impaired during the ED shift, when they must rapidly update the optimal choice response strategy. Behavioral analyses showed MD lesion rats had significantly reduced correct within-trial second choice responses. This evidence shows that transfer of information via the MD is critical when rapid within-trial updates in established choice response strategies are required after a rule change. We demonstrate for the first time that rodent mediodorsal thalamus (MD) is a critical node when choice response strategies need to change rapidly after a within-session rule change during reward-guided learning. MD interactions with orbitofrontal cortex (OFC) are critical for value-based learning, while MD interactions with medial prefrontal cortex (PFC) are critical for rapid within-trial updating of optimal choice response rules. MD interactions with the OFC are not always necessary for reversal learning. These results indicate that the MD also contributes to attentional set-shifting. Our evidence advocates for studies investigating deficits in updating optimal choice response strategies because of potentially disrupted cortico-thalamocortical transfer of information in people diagnosed with Alzheimer’s disease (AD) combined with more frontal pathology, Parkinson’s disease (PD), or schizophrenia.Cognitive flexibility describes our ability to quickly and selectively switch our thoughts, responses, or behavior to everyday dynamic situations. This capacity to rapidly update or alter one’s actions conveys evolutionary benefit and is key to survival . CognitiCognitive flexibility is not unique to primates. Studies show that rats can readily switch between attentional sets to optimize reward outcome . TypicalIn mammals, the MD is densely interconnected with areas of frontal cortex involved in various aspects of cognitive flexibility. Recent work shows MD projections to frontal cortex allow for the simultaneous sharing of information across multiple cortical regions. This distributed pattern of frontal innervation by the MD appears to be especially true for the central (MDc) and medial (MDm) subdivisions, which have projections to the orbitofrontal cortex (OFC), medial PFC, and frontal association areas . AlthougThus, in the current study, rats with excitotoxic lesions to the MD or MD sham controls were run on a well-established test of cognitive flexibility, the intradimensional (ID)/extradimensional (ED) attentional set-shifting task . To testIn the current experiment, we predicted MD lesion rats would show deficits during the initial sensory acquisition  and the ED shift. Both the SD and ED subtasks require rapid, within trial learning or updating of choice response strategies. Therefore, impaired SD and ED performance would be consistent with the selective learning deficits observed in other rodent studies after MD perturbations . In addiTwenty-five Lister hooded male rats weighing between 420 and 480 g at time of surgery (Charles River) were group housed in a temperature and humidity-controlled environment (21 ± 1°C). The housing and husbandry compiled with the ARRIVE guidelines of the European Directive (2010/63/EU) for the care and use of laboratory animals. Testing was conducted in the light phase of a 12/12 h light/dark cycle (lights on at 7 A.M.s). The rats were maintained on a controlled feeding schedule (20 g/rat/d) with water freely available in the home cage. All experimental procedures were performed in compliance with the United Kingdom Animals (Scientific Procedures) Act of 1986. A project license authorized all procedures after review by the university animal care and ethical review committee and Home Office Inspectorate.m NMDA dissolved in phosphate buffer (pH 7.20) in each hemisphere from a 1 μl-gauge Hamilton bevelled-tip syringe. The injections were performed manually, taking 3 min each, and after injection the needle was left in situ for a further 3 min to allow diffusion and to limit wicking of the NMDA. A further 10 rats (MD sham controls) received injections of sterile phosphate buffer instead of NMDA using the same injection coordinates. Injections were given bilaterally in the MD. Upon completion of surgery, wounds were sutured with Vicryl 4.0. Rats were housed singly during recovery for up to 3 d and were then returned to their presurgery housing cohort. Postoperative oral analgesia, 1 mg/kg Metacam  dissolved and set in jelly was provided in individual bowls to each rat daily for 3 d. Behavioral and physiological evidence showed that all rats recovered well, with normal eating and presurgery weights returning within 24 h. Food regulation (20 g/d/rat) started again 10d after recovery. Postoperative testing began at least 15d after surgery. The researcher was blind to the lesion group of each animal until all tests were completed.Rats were anesthetized by isoflurane and oxygen mix , and given analgesia in subcutaneous injections of 0.05 mg/kg buprenorphine  and 1 mg/kg Metacam . Rats were secured in a stereotaxic frame (Kopf) with atraumatic ear bars and the nose bar set to +3.3 mm to achieve a level skull. A subcutaneous injection of 2 mg/kg bupivacaine  was administered into the scalp in the location of the midline incision. Viscotears was applied to keep the eyes moist. During surgery, each rat was placed on a heat pad and covered in bubble wrap with an internal rectal thermometer probe to monitor and maintain normal body temperature. Warmed sterile saline (1 ml/100 g) was administered subcutaneous into the scruff of the neck after 1 h. A midline incision was performed. Bregma and λ coordinates were determined. A dental drill with a trephine head was used to create a craniotomy over the midline. To maximize lesion accuracy, anterior and posterior injection sites were calculated according to the bregma–λ distance in each rat. Coordinates were for anterior MD injection: anterior-posterior (AP) = – 0.395 mm, medial-lateral (ML) = – 0.1 , and dorsal-ventral (DV) = 0.56 mm (from dura), volume of excitotoxin = 0.20 μl; for posterior MD: AP = – 0.435 mm, ML = + 0.1 and DV = 0.57 mm (from dura), volume of excitotoxin = 0.18 μl. Fifteen rats (MD lesion) received 0.12 The test chamber consisted of a modified Plexiglas home cage (40 × 70 × 18 cm) with Perpex dividers separating the cage into a large start chamber (40 × 46 cm) and two identically sized (24 × 20 cm) choice chambers to which access was controlled by removable Perspex doors. The digging bowls were ceramic  and were placed within the choice chambers. The bowls were filled with digging media of different textures, and the digging media were scented with different spices  using either of the two sensory dimensions: odor (mint vs oregano), or digging media (shredded paper vs cotton pads), to a criterion of six consecutive correct trials. The rewarded odors or digging media were counterbalanced across the two groups, and these exemplars were not used again throughout testing. Digging was defined as active digging with both front paws or active foraging with the snout in the digging media. Sniffing or touching the media with the paws was not scored as a dig.The following day, each rat was given a series of seven two-choice discriminations (subtasks) in the following order: a new SD using either of two sensory dimension (odor or digging media); a compound discrimination (CD) using the same rewarded sensory dimension and two-choice discrimination as the SD, combined with the other irrelevant sensory dimension; first reversal (REV1), in which the exemplars remained the same as in the CD but the correct (rewarded) and incorrect exemplars were reversed; ID shift, in which new exemplars were used, but the relevant sensory dimension remained the same as in the previous subtasks; second reversal (REV2), where exemplars in the ID remained the same but the reward contingencies of the two exemplars were reversed; ED shift, where new exemplars were used and the previously irrelevant sensory dimension becomes relevant; and a third reversal (REV3), where exemplars in the ED subtask remained the same but the reward contingencies of the two exemplars were reversed.The task is self-paced and relies on the natural foraging behaviors of the rats. The first five two-choice discrimination subtasks  must be solved by discriminating between exemplars from the same sensory dimension. In this stage of the task, the rat is rewarded for choices based on specific perceptual features of the stimuli, while ignoring other features that also distinguish the stimuli. After acquiring each set of two-choice discriminations to a performance criterion of six correct consecutive trials, the rats encounter a reversal of the reward contingencies associated with the exemplars . For the final two subtasks (ED and REV3), exemplars from the previously irrelevant sensory dimension become relevant. Therefore, the rat must shift its attention (and adjust its choice response strategy) to the previously irrelevant sensory dimension and perceptual feature to receive reward (ED subtask). The attentional set-shifting cost is calculated by comparing trials to criterion during the ID shift, where the sensory dimension is consistent with previous subtasks, and the ED shift, where the now relevant sensory dimension had been previously ignored and thus requires a shift to optimize continuing to retrieve rewards.At the completion of ID/ED testing, the olfactory abilities of the rat to smell the odor of the buried half-Honey Nut Cheerio were assessed. This task consisted of 10 consecutive trials where the rat was exposed to bowls containing similar bedding. The rat was placed in the waiting area of the set-shifting apparatus. Similar bedding-filled bowls were placed, one in each of the choice chambers, only one bowl contained half a Honey Nut Cheerio at the bottom (pseudorandomly assigned to avoid the rat developing a response strategy). The barrier was raised, allowing the rat access to both bowls. The rat had to chose to dig into one bowl and had up to 15 min to make a digging response on each trial. If the rat dug in the correct bowl, the trial was recorded as correct and the rat was returned to the waiting area and the barrier lowered to block access to both choice chambers. If the rat dug in the incorrect bowl, the trial was marked as incorrect. The rat was permitted to continue to explore the incorrect bowl; the trial was terminated when the rat returned to the waiting area. In all cases, the ability of each rat to detect the reward location was no different to chance guessing.m PBS. Brains were then removed and postfixed overnight (4°C), then incubated using 30% (w/v) sucrose in 0.1 m PBS over 48 h (4°C). Free floating sections (40 μm) were cut on a freezing microtome, the slices were then preserved in cryoprotectant (0.1 m PBS containing 24% (v/v) glycerol and 24% (v/v) ethylene glycol) and stored at −20°C until required. MD thalamus lesion locations were histologically verified from cresyl violet-stained brain sections.At the end of all experimental testing, rats were deeply anaesthetized with sodium pentobarbital , and perfused transcardially with 0.9% (w/v) saline and 4% (w/v) paraformaldehyde in 0.1 http://rsbweb.nih.gov/ij/). In each rat, the intact MD volume from both the left and the right hemisphere was assessed. The final reading was calculated according to the Cavalieri principle (Photomicrographs of the Nissl-stained MD sections were captured using a camera mounted on a freestanding Leica DMR microscope (Leica Microsystems) using a 1.6× objective so that the whole section could be visualised in the photomicrograph. MD volumes were measured using the NIH software ImageJ  or independent t tests for relevant subtasks (corrected for multiple comparisons). Univariate ANOVA was used to determine whether the Shift between the two sensory dimensions between the MD lesion or MD sham control had an effect on the number of errors performed in the ED subtask. All statistical analyses were calculated using SPSS 24 software. The significance level was set at p < 0.05.In the behavioral experiments, all rats performed a preoperative and postoperative session, and they completed the seven two-choice discrimination subtasks in an identical order. Data are expressed as mean and standard deviation. Preoperative and postoperative mean trials to criterion, shift cost, percent correct second choice within trial responses, and latency to dig (seconds) were analyzed using repeated measures ANOVAs with Stage (comprising the seven subtasks) as the within-group repeated measure and Lesion_type (MD lesion or MD sham control) as the between-group factor. For significant interactions, Of the 25 adult rats involved in this experiment, 15 sustained MD lesions and 10 were MD sham controls . All 15 MD lesion rats had extensive excitotoxic (NMDA) lesion damage  within tt test confirmed there was no significant difference between the two groups during preoperative learning of the SD, t(22) = 0.095, p > 0.05, with rats from both groups requiring a similar number of trials to reach criterion, MD lesion (mean (M) = 9.67, SD = 2.44) and MD sham control . Overall trials to criterion during the preoperative training session are presented in Preoperatively, rats were pseudo-randomly assigned to either of two groups: MD lesion or MD sham control, based on the number of trials needed to reach the learning criterion  during the SD subtask. Preoperative data from one MD sham control rat had to be discarded. An independent samples 10.1523/ENEURO.0162-21.2022.f2-1Extended Data Figure 2-1p < 0.001). MD sham control rats also needed more trials to learn the ED, as expected. The MD lesion rats required significantly fewer trials to learn REV3, the reversal occurring immediately after the ED, compared to the MD sham controls (*p < 0.001). Box shows 1st and 3rd quartile and whiskers are the minimum and maximum trials to criterion for individual rats. Download Figure 2-1, TIF file.Boxplots of the mean trials to criterion for individual rats during the postoperative session of the attentional set-shifting task. MD lesion rats took significantly more trials to learn the SD and the ED subtasks compared to MD sham controls  = 63.65, p < 0.001, a significant main effect of Stage, F = 184.73, p < 0.001, and a significant main effect of Lesion_type, F = 20.22, p < 0.001  were computed. For the SD subtask, MD lesion rats required more trials to criterion  compared with MD sham controls , indicating that the rats with the MD lesion were slower to acquire the new sensory discrimination. An independent samples t test confirmed this difference was significant, t(23) = 10.88, p < 0.001. However, once the rats with MD lesion acquired the rewarded sensory dimension , they were unimpaired in subsequent subtasks that maintained the same rewarded sensory dimension, namely, concurrent discrimination (CD): t(23) = 0.00, p =1.0, and ID shift: t(23) = 1.08, p < 0.291. MD lesion rats were also not impaired in the reversals (REV1 and REV2) of the reward contingencies associated with the learned exemplars completed after the CD subtask, REV1: t(23) = 2.31, p < 0.030, or after the ID subtask, REV2: t(23) = 1.34, p < 0.192, although, all rats were familiar with the reversal rule as they had completed the ID/ED task preoperatively . Extended Data Postoperatively, a repeated measures ANOVA with Stage  as the within-group factor and Lesion_type (MD lesion vs MD sham control) as the between-group factor revealed a significant interaction, Stage × Lesion_type,  < 0.001 B. To expF = 99.96, p < 0.001, a significant effect of Stage, F = 306.11, p < 0.001, and a significant effect of Lesion_type, F = 32.83, p < 0.001 (t(23) = 10.00, p < 0.001.For the ED subtask, the rats had to shift their attention to the previously irrelevant (unrewarded) sensory dimension. Thus, given the rats had acquired the attentional set strategy, as expected, all rats required more trials to criterion to learn the ED subtask. MD sham controls required  more trials to learn the ED when compared with trials required to learn the ID . Similarly, the MD lesion rats required more trials  compared with trials to learn the ID  = 6.51, p < 0.001, suggesting a facilitation in performance as a consequence of over-training experienced during the ED subtask (see Discussion for interpretation).Interestingly, for REV3, the reversal subtask that occurred after the ED shift, MD lesion rats required fewer trials to criterion  compared with the MD sham controls  = 44.06, p < 0.001, a significant effect of Session, F = 38.27, p < 0.001, and a significant effect of Lesion_type, F = 15.46, p =0.001 (p < 0.001). By contrast, the MD sham controls showed a similar shift cost to their preoperative performance.The additional repeated measures ANOVA of session (preoperative vs postoperative) shift cost A betweenp =0.001 A. The siGiven the observed deficits in the MD lesion group, we investigated the behavioral responses made in the MD lesion or MD sham control rats. Given the task design and a criterion of six consecutive correct responses before switching to the next stage, overall during testing, the rats do not complete many trials. Nevertheless, we could determine whether the rats rapidly adapted their choice responses in the here and now  as measured by the number of correct second choice within trial responses made, i.e., when the rat dug from the correct bowl only after visiting the incorrect bowl first but without digging in it, divided by the total number of correct responses (correct first and second choice responses combined) for each session. During each subtask of the session, we recorded whether the rat made a correct choice, either on the first attempt (“first choice response”), or on the second attempt . We did not include error trials in this analysis.F = 9.40, p = 0.006, a significant main effect of Session, F = 57.06, p < 0.001, and a significant main effect of Lesion_type, F = 7.18, p = 0.014  made fewer correct second choice within trial responses compared with the MD sham controls . This deficit suggests the MD lesion rats had a diminished ability to rapidly bind together their previous choice about what stimuli are worth sampling to update their choice response strategy within the trial, rather than a deficit in perseverative responding, or an inability to respond to negative feedback.A repeated measures ANOVA of total percent correct second choice within trial responses was conducted with session (preoperative vs postoperative) as the repeated measure × Lesion_type revealed a significant interaction of Session × Lesion_type,  = 0.014 B. To invF = 106.03, p < 0.001, but no main effect of Sensory Shift, F = 3.84, p = 0.063 and no interaction effect, F = 0.19, p = 0.666.In addition, we analyzed the rats’ errors made reaching criterion during the ED subtask, as a consequence of whether they experienced a sensory dimension shift from odor to digging medium or digging medium to odor, which indicated that the increase in errors occurs regardless of the direction of the attentional shift. The univariate ANOVA with Lesion_type and Sensory Shift as the between-subject factors and errors to criterion for the ED subtask as the dependent measure revealed a main effect of Lesion_type, Latency to dig (in seconds) for error trials and correct trials were also computed for each rat in preoperative A,C and pF = 23.99, p < 0.001, but no effect of lesion, and no interaction, Fs < 1.0. This result indicates there was no difference in response times between the pseudo-randomly assigned groups (MD lesion and MD sham controls) preoperatively and that all rats responded quicker on errors trials as they moved through subsequent subtasks within the session.For error trials C, the reF = 26.81, p < 0.001, a significant main effect of Stage, F = 22.45, p < 0.001, and a significant main effect of Lesion_type, F = 57.15, p < 0.001. To investigate the interaction effect, post hoc comparisons of the simple main effects (corrected for multiple comparisons) were computed. Postoperatively, MD lesion rats responded slower on error trials during the SD subtask, t(22) = 10.09, p < 0.001, the CD subtask, t(22) = 7.65, p < 0.001, and the REV1 subtask, t(22) = 4.44, p < 0.001. However, for the final reversal subtask, REV3, completed after the ED shift, MD lesion rats were quicker on the error trials than MD sham controls, t(22) = 3.53, p = 0.002. This result in combination with the reduced trials to criterion required to complete this subtask (ps > 0.05).In contrast, the repeated measures ANOVA of latency to dig during postoperative error trials D reveale subtask A suggest subtask . For corThis set of experiments investigated the active influence of rat MD in cognitive flexibility. More specifically, we examined whether rats with excitotoxic lesions of the central and medial MD were impaired in performing an attentional set-shifting task that measures ID and ED shifts in attention to sensory discriminations (digging media or odors). Our new results indicate that rats with MD lesions required more trials to criterion to learn the optimal response strategy during the initial SD. Although, once this stable response strategy had been learnt, MD lesion rats were able to attain an attentional set strategy as they acquired further two-choice discriminations involving the same sensory dimension during the CD and ID shifts, and during reversals (REV1 and REV2) of the reward contingencies associated to the exemplars. However, when the relevant sensory dimension was switched, for the ED subtask, the MD lesion rats were markedly impaired and required many more trials to update their choices using a new response strategy, as measured by the significant ED shift cost. Further, for REV3, the third reversal performed after the ED shift, the MD lesion rats showed facilitated learning, suggesting they had received “overtraining” as a consequence of completing many more trials to reach criterion in the ED subtask (see below). The analyses of behavioral responses indicated that the MD lesion rats made significantly fewer correct second choice within-trial responses suggesting that information transfer via the MD is critical for the rapid, within-trial monitoring and updating of an optimal choice response. Interestingly, similar updating deficits in choice responding have been previously reported in rhesus macaques with magnocellular MD excitotoxic lesions .These behavioral data accord somewhat with previous studies conducted in different species. For example, marmosets with lateral PFC lesions  or rats As already indicated, with more trials, our rats with MD lesions were able to learn the SD subtask to criterion and apply the optimal rule during subsequent subtasks. For the SD subtask, trials revert back to presenting only one sensory dimension and two stimulus exemplars. Other studies also confirm MD lesion rats are impaired in acquisition of an initial task rule . Other rHowever, during the ED subtask, our MD lesion rats were markedly impaired at rapidly adapting this now well-established choice response strategy when they had to attend to the previously irrelevant (nonrewarded) sensory dimension. The demands on cognitive flexibility, response inhibition, and adapting behavioral responses after negative feedback are increased when the animals must make an ED shift . The incEvidence from studies in mice suggests the MD supports the frontal cortex to sustain intracortical attentional control without transferring categorical information about a particular task rule , or thatFor the reversal subtasks (REV1 and REV2), somewhat surprisingly, our MD lesion rats were unimpaired during reversals in the reward contingencies. Rats and marmosets with comparable medial PFC damage are also not impaired in reversal learning subtasks . InsteadMD is reciprocally connected to the OFC and medial PFC . Thus, tIntriguingly, our additional analyses used to investigate the types of behavioral deficits occurring after MD lesions showed that our rats made fewer correct second choice within-trial responses. Given that our MD lesion rats required more trials to criterion to learn the SD and ED subtasks, this change in behavioral responses suggests that without the MD thalamus, our rats could not rapidly adapt their choice responses as the trial was progressing  after the changed task demands. However, this reduced correct second choice within-trial responding does not suggest the rats adopted perseverative responding  or were not learning about the stimulus and associated rewards. We can conclude this because, after acquiring the ED shift, the MD lesion rats required fewer trials to criterion to learn the final reversal subtask, REV3. This facilitation of learning during the final reversal subtask suggests that the MD lesion rats had experienced “overtraining” on all of the stimulus features associated with each sensory dimension as they required more trials to learn the ED subtask . It is wHowever, in the There may be other explanations for the SD and ED impairments observed in our MD lesion rats. With the ID/ED task, it is important to establish that the animals understand the relevant sensory dimension that is rewarded across the related subtasks within the session. Fortunately, in our animals, this transfer of knowledge was evident as the MD lesion rats showed reliable learning in the subtasks, CD, REV1, ID, and REV2, that required the implementation of choice responses (follow the same rule) to the same sensory dimension as the SD to receive reward. Further, both sham controls and MD lesion rats showed similar numbers of trials to criterion during the REV1 and REV2 subtask, which indicates that they did not favor one feature of the stimulus dimension more than the other or that they were insensitive to negative feedback. However, we only used male rats in this current study so the results might not transfer to female rats. Additionally, the MD is a subcortical node in the olfactory neural circuitry that also includes the OFC . HoweverFinally, the diffuse influence of the MD thalamocortical inputs to several frontal cortex structures  supportsThe ID/ED task is analogous to the Wisconsin Card Sorting Test (WCST) in humans. In healthy humans performing the WCST during neuroimaging, the MD is activated during responding to negative feedback after the choice has been executed . UnfortuTo conclude, excitotoxic damage to the rodent central and medial MD selectively increased the trials to criterion on the ED subtask, producing a shift cost. This selective performance deficit is similar to monkeys with lateral PFC inactivation , 1997 an


Arabidopsis thaliana has been extensively used to understand evolutionary processes in natural populations, mainly focusing on single-nucleotide polymorphisms. Conversely, structural variation has been largely ignored in spite of its potential to dramatically affect phenotype. Here, we identify 155,440 indels and structural variants ranging in size from 1 bp to 10 kb, including presence/absence variants (PAVs), inversions, and tandem duplications in 1,301 A. thaliana natural accessions from Morocco, Madeira, Europe, Asia, and North America. We show evidence for strong purifying selection on PAVs in genes, in particular for housekeeping genes and homeobox genes, and we find that PAVs are concentrated in defense-related genes  and F-box genes. This implies the presence of a “core” genome underlying basic cellular processes and a “flexible” genome that includes genes that may be important in spatially or temporally varying selection. Further, we find an excess of intermediate frequency PAVs in defense response genes in nearly all populations studied, consistent with a history of balancing selection on this class of genes. Finally, we find that PAVs in genes involved in the cold requirement for flowering  and drought response are strongly associated with temperature at the sites of origin.Genomic variation in the model plant  Arabidopsis thaliana has been important for deciphering core physiological, developmental, and adaptive processes  (The model plant rocesses . Arabidos (SNPs) . Howevers (SNPs) . Compares (SNPs) . Structus (SNPs) .SVs are common in humans  and only a subset has been associated with phenotypes, including disease . For exaA. thaliana  smaller than 10 kb. We identify structural variation in A. thaliana natural accessions examine their global patterns of polymorphism. We identify sets of “core” highly conserved genes and variable “dispensable” genes, a subset of which show evidence for balancing selection. Finally, we examine evidence of local adaptation on PAVs based on correlations with environmental variables. This analysis reveals strong correlations for PAVs in three genes involved in the vernalization (cold) response for flowering as well as genes involved in drought and heat tolerance. We make our variant calls available together with information about the level of support for each call, which can be used for flexible integration of SVs into existing analysis pipelines.In this study, we identify SVs in 1,301 Identifying and calling SVs in short-read data is not a trivial task. To identify the best software for calling SVs, we compared the performance of three popular tools: PINDEL, DELLY, and LUMPYEXPRESS . We condFor the simulation-based approach, LUMPYEXPRESS outperformed DELLY and PINDEL . Both prWe considered the results from long-read calls (PacBio) to represent a “high confidence” set and then assessed how well calls from short-read data using LUMPYEXPRESS, DELLY, and PINDEL agreed with this high confidence set. Although PacBio data are much more reliable for large SVs, error rates in PacBio data are high for individual SNPs and short INDELs. Therefore, in an effort to maximize the true positive rate for calls with the PacBio data, we only considered SVs between 50 bp and 10 kb in length in this comparative analysis. Given the discrepancy between the very high sensitivity and precision, we observed in simulations compared with the lower levels with PacBio data, we were interested in better understanding what drives the concordant and discordant calls in short- and long-read data. We focused on the LUMPYEXPRESS software and examined alignments in randomly chosen regions where calls based on PacBio data (using SNIFFLES) and Illumina data (using LUMPYEXPRESS) agreed and where they differed. A. thaliana accessions. Next, we examined the patterns of variation in structural polymorphisms across diverse wild We examined the global pattern of polymorphism in our total set of PAVs in order to assess whether these variants recapitulated the signals found in SNP data. We consider this to be a further test to validate the PAV genotyping because if the quality of PAV genotypes is high, we expect to see global patterns that are similar to those found using SNP data. We clustered a representative subset of diverse accessions using PAVs and compared this with results obtained previously from SNP data . We foun. At a broad scale, PAVs were nearly uniformly distributed across chromosomes, except in pericentromeric regions where they were more common , consistent with stronger purifying selection in genic regions  and translational elongation . These categories include tRNAs and rRNAs, which are classical examples of housekeeping genes . Homeobox genes play an important role in body plan specification of higher organisms during early stages of embryogenesis  enrichment analysis. We tested significance using both Fisher’s exact tests (FET) and a more conservative permutation-based approach that corrects for clustering of signals in the genome (Gowinda) . Categorng genes . In addiogenesis . Additioogenesis  online.Although genes show a deficit of PAVs relative to nongenic regions overall, some types of genes may be more likely to contain PAVs than others. To identify these, we extracted locations of all genes that overlap at least one PAV among the 1,301 samples and performed GO enrichment analysis using both FET and the Gowinda permutation-based approach . The resP = 2E-2), but the enrichment was not significant with Benjamini correction. This discrepancy is likely due to the extreme clustering of defense genes across the genome.Signal transduction was the most significantly enriched class with FET, and the enrichment was driven largely by defense-related genes mainly consisting of TIR-NBS, TIR-NBS-LRR, LLR, and TIR classes and secondary metabolites. Other enriched classes included the more specific defense response category and a category related to the production of Lignans, a class of secondary metabolites . In addiNext, we examined the patterns within populations. We extracted all PAVs for each population and performed GO term enrichment. D statistic. This statistic summarizes the information in the SFS such that an excess of intermediate frequency variants results in a more positive Tajima’s D, and an excess of low-frequency variants results in a more negative Tajima’s D   and Northern Sweden, where the significance was marginal (P = 0.0254)   online.  0.0254)  and accon method , 2020. Wn method  online. We next asked whether there was evidence for involvement of specific PAVs in local adaptation based on associations with environmental variables. For this, we accessed data for four representative environmental variables  from the WorldClim database . To idenMADS AFFECTING FLOWERING (MAF) gene cluster at the bottom of chromosome 5, including a Bonferroni significant association with a PAV that impacts MAF3 as well as several less significantly correlated  PAVs that affect other genes in this cluster. MAF3 and other MAF cluster genes work together with FLM to repress expression of FT and inhibit flowering and play a role in flowering in response to cold  (AT1G22700 (tetratricopeptide repeat [TPR]-like superfamily protein) , AT1G22710 (SUC2) , AT1G43560 (THIOREDOXIN Y2) , AT5G09600 (Succinate dehydrogenase 3-1) , and AT5G22140 (FAD/NAD(P)-binding oxidoreductase family protein) .Several genic PAVs (PAVs within 10 kb of genes) strongly associated with minimum temperature in the coldest month (Bio6) were related to the timing of flowering and photosynthesis B. There ization) . Photosyization) . SeveralELONGATED HYPOCOTYL 5) , AT5G42350 (CFK1) , and AT5G58140 (PHOTOTROPIN 2) . Among the strongest correlations with precipitation in the wettest month (Bio13) were PAVs within 10 kb of several genes and gene clusters involved in cell wall morphogenesis: RGXT1 (AT4G01770) and RGXT2 (AT4G01750) , Walls Are Thin 1 (AT1G75500) , and EXPA18 (AT1G62980) and KNAT7 (AT1G62990)   (For maximum temperature in the warmest month (Bio5) PAVs at three genes involved in photomorphogenesis (hypocotyl elongation), a phenotype with a plastic temperature-mediated effect, were among the strongest correlations A. These  × 10−5) C. A PAV  × 10−5) D.P value <0.001, we identified the SNP with the highest LD (r2) within a 10-kb window. The mean and SD of r2 between a PAV with this significance level and the most correlated SNP for each of the four traits were as follows: Bio5: mean = 0.70, SD = 0.25; Bio6: mean = 0.75, SD = 0.24; Bio13: mean = 0.70, SD = 0.23; and Bio14: mean = 0.76, SD = 0.25. The full distributions of these most correlated SNPs are shown in Although having information about PAVs can be specifically useful to identify candidates in GWAS for functional follow-up analysis and therefore creates added value compared with SNPs alone, some information may be partially redundant with SNP results in the sense that LD between PAVs and SNPs will result in statistical associations with both types of variants. To examine this, we compared the signals we found with PAVs to those with SNPs in the same regions. Overall, there are many fewer PAVs than SNPs in the genome (124905 vs. 8033502). The average distance between two any SNPs is 14.4 bp and the average distance between PAVs is 771 bp, and the average distance from any PAV to the nearest SNP is 332 bp so that LD between any two SNPs tends to be higher than LD between any two PAVs. To determine the extent to which we identified novel variation by including PAVs in our analysis, we examined how well a SNP represents each strongly correlated PAV. To this end, for each PAV with a GWAS A. thaliana accessions using publicly available NGS data and we make the entire set available, which includes 124,905 PAVs, 25,061 tandem duplications, and 5,474 inversions. For researchers interested in using these data, we note that the data set includes information about the strength of evidence for SVs based on the number of reads supporting each SV as well as the type of evidence (split read and/or discordant reads), which could be used for further filtering. Here, we focused our population genetic analyses on PAVs because these were called with the highest accuracy based on our analyses.In this study, we identified SVs among 1,301 diverse Given that the genic PAVs we identify are expected to often disrupt gene function, our results were consistent with the idea that a genome is made up of an essential “core” set of genes as well as a “flexible” set of genes, which may be dispensable depending on the specific local selection pressures faced by an organism. In our analyses, we found evidence for purifying selection based on the low occurrence of PAVs in genic compared with nongenic regions, and the enrichment of core housekeeping genes in genomic regions that were most deficient in PAVs . CompareA. thaliana and related plant species  and F-box genes have an excess of PAVs compared with other genes. R-genes and F-box genes are multigene families known to be rapidly evolving . FurtherA. thaliana is sometimes more complex , AGL31(AT5G65050), and MAF3 (AT5G65050)  . This islowering .A. thaliana evolution and trait architecture using GWAS or recombinant populations.The set of SVs identified and genotyped here will be useful alone or in combination with available SNP data to investigate We retrieved Illumina short-read data for 1,327 samples from four studies . We exclWe tested three popular tools designed for SV identification from short-read data to compare their performance. These included LUMPYEXPRESS (v0.2.13) , DELLY , and PINA. thaliana TAIR10 genome for each kind including deletions, tandem duplications, and inversions. We simulated NGS reads by WGSIM (v1.9)  with SURVIVOR (v1.0.6)  on the AM (v1.9)  using thM (v1.9)  with defFor the PacBio approach, we first mapped PacBio reads to TAIR10 using NGMLR (v0.2.7)  with defhttps://github.com/HancockLab/SVS_A.thaliana) that consists of LUMPYEXPRESS (v0.2.13), SVTYPER (v0.7.0) . Our pipeline is forked and modified from . After calling SVs , we performed two genotyping steps. First, we conducted individual genotyping with SVTYPER (v0.7.0), and then we conducted joint genotyping with SVTOOLS (v0.4.0). This final genotyping allowed us to differentiate missing genotypes from matches to the reference. The discovery set can have an influence on the power to identify variants across populations. Therefore, we included all individuals in the discovery panel to reduce the false-negative rate overall; however, this likely results in a bias towards higher SV discovery rates in deeply sampled populations and regions.We identified SVs among 1,301 accessions using the pipeline ((v0.7.0) , and SVTOur pipeline failed for 26 samples, which were thus excluded from the analysis. There was no clear reason why these samples failed, and as they are not clustered into any one population removing them is not expected to bias the data set. These samples include  are BRR4 (MPI Tübingen), BRR12 (MPI Tübingen), BRR57 (MPI Tübingen), BRR107 (MPI Tübingen), Bur-0 , Can-0 , Dem-4 , KBS-Mac-74 (MPI Tübingen), LI-SET-036 (MPI Tübingen), MSGA-61 (MPI Tübingen), Oy-0 (Mott), Sf-2 , Paw-13 (MPI Tübingen), Paw-20 (MPI Tübingen), Rsch-4 , Yng-53 (MPI Tübingen), Tsu-0 (Mott), Uod-7 , Yng-4 (MPI Tübingen), Zu-0 , 11PNA1.14 (MPI Tübingen), 328PNA062 (MPI Tübingen), 87 , 36-31 , 36-17 , and 27-9.The VCF file used for subsequent population genetic analysis was generated by setting an upper size limit of 10 kb, including only polymorphic PAVs. Therefore, the sizes of variants included in this analysis range from 1 bp to 10 kb. The nature of short-read data prevented us from identifying large SVs. We provide two VCF files: one with filtering applied with only PAV variants included, and one with all raw calls including PAVs, inversions, and tandem duplications.In addition to PAVs, we also called biallelic SNPs for all 1,301 samples following the same pipeline we used previously .We produced a whole-genome neighbor-joining (NJ) tree in R with the ape (v5.0) package  using th2 test. We set the expected genic region to 60,104,494 bases  and expected intergenic region to 59,041,854 bases . If the distribution of PAVs throughout the genome was random, we would not expect to see any difference between genic and intergenic regions. Our observations for genic regions and intergenic regions overlapping with PAVs were 8,920,166 bases and 30,766,220 bases, respectively.We defined genic and nongenic regions based on TAIR10 annotation. Any region that overlaps with a gene is treated as a genic region and the rest is treated as a nongenic region. We compared genic and nongenic regions to see if there is any difference based on PAVs. We found more PAVs in intergenic regions compared with genic regions. To test the significance of finding a higher proportion of PAVs in intergenic regions than genic regions, we used the χLater we focused on the genic regions to be able to see the gene sets with excesses or deficits of PAVs. Genes that overlap with PAVs were extracted and performed enrichment analysis. Same enrichment analysis was also done for the genes that never overlap with a PAV. We tested for GO term  enrichmeA. thaliana lineages including  based on analyses in previous study  region. For each PAV, we calculated allele frequency among these five accessions. The highest frequency allele for each locus was used as the ancestral state. NA was assigned to the ancestral state where data were missing for more than one individual or when the allele frequency is equal to 0.5.We calculated unfolded SFS for each population and genomic class . To produce the frequency spectrum for each population, we calculated proportion of variants that were present once in the population , twice , etc. and these are plotted in us study . This seThe samples that we retrieved from the 1001 Genomes project were separated into populations based on their admixture groups . Other sD statistic for the set of defense genes in each population and compared this with the Tajima’s D statistic for the entire genome. We found that for each population, Tajima’s D was more positive for defense genes compared with the total set of genes. To assess significance for this result, we randomly subsampled genic PAVs to match the number of defense response PAVs 100,000 times and calculated an empirical P value based on the distribution of Tajima’s D in the resampled data sets. We further examined evidence for balancing selection using BetaScan, which relies on a signal of high variation at the haplotype level  with the command –r2 –ld-window-kb 10 –ld-window 999999 and filtered for the PAVs of interest for each environmental GWAS. For each PAV, we identified the SNP with the highest r2, created histograms for these, and used the distribution to estimate the mean and SD of r2 between these PAVs and the most correlated SNPs.We used the linear mixed model association method, GEMMA (v0.98.1) , to asseMolecular Biology and Evolution online.msaa309_Supplementary_DataClick here for additional data file.


These differences were also observed between patients with CKD stage 5 and those with CKD stage 5 D (p < 0.017), but not between patients with CKD stage 4 and those with CKD stage 5, suggesting that the bone microstructure rapidly changed at the start of hemodialysis. Patients with CKD stage 5 D exhibited tibial microstructural impairment compared with those with CKD stage 4–5. HR-pQCT is useful for elucidation of the pathology of bone microstructures in patients with renal failure.Chronic kidney disease (CKD) negatively affects bone strength; however, the osteoporotic conditions in patients with CKD are not fully understood. Moreover, the changes in bone microstructure between pre-dialysis and dialysis are unknown. High-resolution peripheral quantitative computed tomography (HR-pQCT) reveals the three-dimensional microstructures of the bone. We aimed to evaluate bone microstructures in patients with different stages of CKD. This study included 119 healthy men and 40 men admitted to Nagasaki University Hospital for inpatient education or the initiation of hemodialysis. The distal radius and tibia were scanned with HR-pQCT. Patient clinical characteristics and bone microstructures were evaluated within 3 months of initiation of hemodialysis (in patients with CKD stage 5 D), patients with CKD stage 4–5, and healthy volunteers. Cortical bone parameters were lower in the CKD group than in healthy controls. Tibial cortical and trabecular bone parameters  differed between patients with CKD stage 5 D and those with CKD stage 4–5 ( For instance, patients with CKD have an increased risk of hip fractures, and the hazard ratios for hip fracture were 1.89  and 3.75 (95% CI: 2.30–6.11) among patients with estimated glomerular filtration rates (eGFR) of 30–44 and 15–29 mL/min/1.73 m/1.73 m2 .Bone strength in patients with CKD is strongly associated with bone mineral density (BMD) and other factors such as bone quality. Structural properties include bone microstructure and geometry. Material properties include collagen, degree of calcification, and microdamage . In patiHigh-resolution peripheral quantitative computed tomography (HR-pQCT) is used for the in-vivo analysis of human limb bones and enables noninvasive, three-dimensional analysis of bone microstructures ,8. This Patients with CKD, especially those on dialysis, have an increased risk of fracture, and it is thought that their bone microstructures are affected by several factors, such as metabolic disorders. We hypothesized that a higher CKD stage would be associated with a higher degree of impairment in the bone microstructure. In this study, we aimed to evaluate bone microstructures by using HR-pQCT in patients with CKD according to their renal function.In this study, patients admitted to Nagasaki University Hospital for inpatient education or the initiation of maintenance hemodialysis between April 2017 and July 2019 were evaluated. Among them, patients for whom BMD and HR-pQCT data were available were included. As the bone quality in women is affected by menopause, we excluded all female patients.2, and CKD stage 5 was defined as having an eGFR <15 mL/min/1.73 m2  [In this study, we focused on the differences in bone microstructure among patients with different stages of CKD before and after starting hemodialysis. Additionally, we compared them with a healthy control group that consisted of age-matched volunteers without CKD to investigate the normal range of bone microstructural parameters. Patients were divided into two or three groups based on CKD stage and initiation of hemodialysis . CKD stage 4 was defined as having an eGFR of 15–29 mL/min/1.73 mideline) . AdditioThe educational program comprised the assessment of risk factors for CKD progression, clinical medication review, dietary and exercise guidance, and the introduction to renal replacement therapy. Patients with CKD stage 5 D received the same drug and dietary guidance, except for precautions during dialysis. The educational program was provided by a team of healthcare professionals comprising physicians, nurses, pharmacists, dietitians, and physical therapists. The decision of initiating hemodialysis was made by doctors who engaged in patients’ management, according to the general standards in Japan . Patient® assay . All blood examinations were performed at the same time as HR-pQCT and DXA.In addition to the general blood examinations for patients with renal failure, we evaluated bone turnover markers, such as tartrate-resistant acid phosphatase-5b (TRACP-5b) and procollagen type I N-terminal propeptide (PINP). Blood sampling was performed in the morning on the day of admittance for patients with CKD stage 4–5 (around 10 am) and when dialysis started for patients with CKD stage 5 D (around 9 am). Intact PTH, TRACP-5b, and PINP concentrations were measured with electrochemiluminescence immunoassay (ECLIA), enzyme immunoassay, and ECLIA, respectively. The TRACP-5b concentration was measured using the Osteolinks® TRACP-5b assay . PINP was measured using the ECLUSIS SIYAKU total PINPAreal BMD (aBMD) and the T-score in the lumbar spine (L1 to L4) and the proximal femur  were evaluated by using DXA .Bone microstructures of the distal radius and tibia of the non-dominant arm and leg were evaluated using HR-pQCT . If a patient had an arteriovenous fistula on the non-dominant arm, the evaluation was performed on the dominant arm. We used the data within three months after initiation of hemodialysis in the CKD 5 D group. The radial scan site was an area of the distal radius, 10.2 mm in width, 4% of the forearm length proximal from the hand joint. Furthermore, the tibial scan site was an area of the distal tibia, 10.2 mm in width, and 7.3% of the lower leg length proximal from the talocrural joint. The scanning conditions were as follows : voltageThe following microstructure parameters were measured: cortical volumetric BMD (Ct.vBMD), cortical thickness (Ct.Th), cortical area (Ct.Ar), cortical perimeter, cortical porosity (Ct.Po), cortical pore diameter, trabecular volumetric BMD (Tb.vBMD), trabecular bone volume fraction (Tb.BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp).This study was conducted at a single center and evaluated with a single HR-pQCT. Phantoms were scanned every day for quality control.p < 0.05 statistically significant; however, a p < 0.017 was considered statistically significant in the post-hoc analysis. All statistical analyses were performed by using JMP Pro 14 .Categorical data are presented as numbers and percentages, and data of continuous parameters as means and standard deviations or medians and interquartile ranges. Continuous values were analyzed using the Wilcoxon rank-sum test, and categorical values were evaluated using Fisher’s exact test. The Kruskal–Wallis test was used for comparisons among three groups , and post-hoc Bonferroni correction was applied. Patient clinical characteristics and bone microstructures were analyzed among three groups . In addition, bone microstructural parameters were compared between two groups (CKD stages 4–5 and 5 D) to investigate whether renal failure affected the bone microstructure. We considered n = 25) and 5 D (n = 15) groups were 66.5 ± 9.9 and 61.1 ± 13.9 years (p = 0.16), respectively. No patient had undergone kidney transplantation. The prevalence of diabetes was 56% and 33% in the CKD stage 4–5 and 5 D groups (p = 0.16), respectively. According to interviews, none of the patients had a history of bone fractures.Among the 40 patients with CKD enrolled in this study, 11 and 14 had CKD stages 4 and 5, respectively, and 15 had CKD stage 5 D . p < 0.017). However, of those differences, only serum creatinine differed between the CKD stage 5 D and CKD stage 5 group (p < 0.017). There were no statistically significant differences in phosphate binders or bisphosphonate use among the groups. Furthermore, serum calcium, intact PTH, and TRACP-5b concentrations did not differ among the groups. Two patients were prescribed corticosteroids: one because of cholesterol embolism (CKD stage 4) and one because of anti-glomerular basement membrane disease (CKD stage 5 D). DXA aBMDs were lower in the CKD stage 5 D group than in the CKD stage 4 group  and in the CKD stage 5 group . DXA lumbar spinal aBMD did not differ between the CKD stage 4 group and the CKD stage 5 D group (p = 0.05).There were several statistically significant differences in the patient background characteristics among the three groups  and between the two groups (CKD stage 4–5 and 5 D). For example, activated vitamin D analogs were prescribed more commonly in the CKD stage 5 D group (80%) than in the CKD stage 4–5 group (24%). Additionally, hemoglobin concentration, serum albumin concentration, and body mass index were lower, while serum creatinine and phosphorus concentrations were higher in the CKD stage 5 D group than in the CKD stage 4 group (both n = 40) and the healthy control groups (aged 64.8 ± 9.9 years) (n = 119) to investigate the normal range of bone microstructural parameters  and low . Intact PTH level was not measured in one patient. Most bone microstructural parameters did not differ between these groups and only Tb.Sp was lower in patients with high concentrations of intact PTH (p = 0.01).In order to evaluate the association between secondary hyperparathyroidism and bone microstructural parameters, patients with CKD were divided into two groups according to the median concentration of intact PTH (179 pg/mL): high . There were no statistically significant differences in Ct.vBMD, Ct.Po, and Tb.N between the two groups. Additionally, there was statistically significant difference in the Ct Ar of the radius between these two groups . p < 0.017).Furthermore, we compared the tibial parameters among the CKD stages 4, 5, and 5 D groups. The results are summarized in Patients with CKD, especially those on dialysis, have an increased risk of fracture, and it is thought that their bone microstructure is affected by several factors such as metabolic disorders. In this study, we evaluated the bone microstructure by using HR-pQCT in patients with renal failure according to their renal function. Consequently, patients with CKD stage 5 D had lower tibial values for Ct.Th, Tb.vBMD, Tb.BV/TV, and Tb.Th than those with CKD stage 4–5, indicating microstructural impairments in both the cortical and trabecular bones. These differences were observed only in the tibia, a weight-bearing bone, but not in the radius.2) [2 was 2.63 (95% CI: 1.74–3.98) compared with those in healthy controls [A recent study revealed that the risk of bone fractures increases with the severity of renal dysfunction and is pronounced even in the earlier stages of CKD (eGFR of 45–59 mL/min/1.73 m2) . In one 2) . Moreove2) . In an i2) . A meta-controls .Generally, in addition to BMD, bone strength in patients with CKD may be strongly affected by other factors, such as bone quality; the latter is defined by both structural and material strength. In contrast, a previous report revealed no statistically significant association between PTH concentration and hip fractures . It is bThe most important predictive factor for bone fracture in a previous study was Tb.BV/TV of the tibia . In anotRemarkably, there were no statistically significant differences in Tb.vBMD, Tb.BV/TV, and Tb.Th in the tibia between the healthy control group and CKD group. This might have been because, even in healthy people, the trabecular bone in the tibia exhibits a more pronounced age-dependent decrease than that in the radius . These tCortical bone parameters in the radius are associated with bone fractures ,21. CompThis study has several limitations. First, the sample size was relatively small. Unfortunately, the in-hospital education program was suspended at the start of the COVID-19 pandemic, and we could not increase the number of patients. This was a proof-of-concept study, and its results should be validated in a larger cohort as HR-pQCT becomes more widely used. Second, this was a cross-sectional study, which is a critical drawback. Hence, we could not observe the time-dependent deterioration of bone quality in the same patients. Moreover, we could not evaluate the association between the duration of the abnormal altered laboratory parameters and the bone microstructure. Therefore, longitudinal studies are needed in the future. Third, although HR-pQCT can be used to assess bone microarchitecture, it cannot be used to distinguish between the many forms of CKD-mineral and bone disorders, including osteoporosis, osteomalacia, secondary hyperparathyroidism, and adynamic bone disease. Furthermore, the risk factors for osteoporosis in men, including physical activities, testosterone concentration, smoking, excess consumption of alcohol, serum vitamin D levels, and total intake of calcium, were not evaluated ,53. GeneBone microstructural changes were evaluated using HR-pQCT in patients with different stages of CKD. Patients with CKD stage 5 D had statistically significant impairments in Ct.Th, Tb.vBMD, Tb.BV/TV, and Tb.Th in the tibia compared with those with CKD stages 4 and 5. The microstructures in both cortical and trabecular bone in the tibia were altered in patients with more advanced stages of CKD relative to those with earlier stages of CKD. The changes in bone quality in patients with renal failure may be determined by renal failure-specific factors, such as secondary hyperparathyroidism, uremia, and their duration. Longitudinal studies with HR-pQCT are needed to elucidate the pathology of bone microstructures in patients with renal failure.


The focus was on CNV carriers and PRS extremes who do not have an SNPD diagnosis. We identified high-risk CNVs  in 744 study participants (2.66%), 36 (4.8%) of whom had a diagnosed SNPD. In the remaining 708 unaffected carriers, we observed lower educational attainment  and lower household income (OR = 0.77 [0.66–0.89]). Income-associated CNVs also lowered household income (OR = 0.50 [0.38–0.66]), and CNVs with medical consequences lowered subjective health (OR = 0.48 [0.32–0.72]). The impact of PRSs was broader. At the lowest extreme of PRS for EA, we observed lower EA (OR = 0.31 [0.26–0.37]), lower-income (OR = 0.66 [0.57–0.77]), lower subjective health (OR = 0.72 [0.61–0.83]), and increased mortality (Cox’s HR = 1.55 [1.21–1.98]). PRS for intelligence had a similar impact, whereas PRS for schizophrenia did not affect these traits. We conclude that the majority of working-age individuals carrying high-risk CNVs without SNPD diagnosis have a modest impact on morbidity and mortality, as well as the limited impact on income and educational attainment, compared to individuals at the extreme end of common genetic variation. Our findings highlight that the contribution of traditional high-risk variants such as CNVs should be analyzed in a broader genetic context, rather than evaluated in isolation.Copy number variants (CNVs) are associated with syndromic and severe neurological and psychiatric disorders (SNPDs), such as intellectual disability, epilepsy, schizophrenia, and bipolar disorder. Although considered high-impact, CNVs are also observed in the general population. This presents a diagnostic challenge in evaluating their clinical significance. To estimate the phenotypic differences between CNV carriers and non-carriers regarding general health and well-being, we compared the impact of SNPD-associated CNVs on health, cognition, and socioeconomic phenotypes to the impact of three genome-wide polygenic risk score (PRS) in two Finnish cohorts (FINRISK,  Large genomic rearrangements, called copy number variants (CNVs), have been identified as causative for a range of syndromes with neuropsychiatric traits –5. WhileAlthough the literature is still modest, previous work , 13–15 hPolygenic risk scores (PRSs) have shown promise in investigating the complex genetic architecture of neuropsychiatric disorders. We  and otheBoth CNVs and high PRS are observed in the general population in individuals without obvious neurodevelopmental or neuropsychiatric disorders. Especially, given the expected high-risk nature of CNVs, the clinical evaluation and interpretation of their impact are challenging due to their relatively high frequency in unaffected individuals. So, if an adult with no history of severe neurological and psychiatric disorders (SNPDs) is observed to carry a disease-associated CNV, how much impact would that potentially have on the life trajectory?We hypothesized that even if the majority of individuals carrying CNVs do not have a diagnosis of neurodevelopmental or neuropsychiatric diseases, CNVs might still contribute to the overall health and socioeconomic outcome. Thus, in participants without SNPD, we compared the impact of CNVs to the impact of the PRSs for educational attainment , schizopWe obtained phenotypic information on 35,231 individuals from the national FINRISK study , an on-gWe detected CNVs using a custom-built pipeline powered by PennCNV  and iPsyvisualize_cnv.pl script distributed via the PennCNV package, and manually curated for obvious artefacts.CNV calls were included only if they had a minimum of ten consecutive probes supporting the call and were 100 kb or greater in length. We joined adjacent CNVs with similar copy number if the adjoining region was at most 20% of the full joined CNV. We identified as probable or potential artefacts any CNVs that overlapped an HLA- or immunoglobulin region by at least 50%, or that was within 500 kb of telomere or centromere region. Finally, we visualized all remaining CNV calls using the DECIPHER CNV if at least 50% of the CNV overlapped a region associated with a CNV syndrome by the DECIPHER database .In FINRISK, there were 22,210 individuals (96.3%), and in NFBC1966 4644 individuals (94.9%), who had no diagnosed SNPD (not counting depression). We wanted specifically to analyze high-risk CNV carriers that had no record of SNPD to establish whether there was any impact on the general quality of life by analyzing overall health, education, and socioeconomic outcomes in these individuals . Our analysis indicated lower odds for the subsequent level of education among high-risk CNV carriers (OR = 0.77 [0.66–0.89]). Previously identified education-associated CNVs were not significantly associated with lower education (OR = 0.81 [0.64–1.01]). However, odds for subsequent level of education were even lower at the matched lowest extreme of PRSEA (OR = 0.31 [0.26–0.37]) and PRSIQ (OR = 0.51 [0.44–0.60]). The impact of high-risk CNVs was observed particularly with DECIPHER CNVs (OR = 0.51 [0.34–0.75]), large deletions (OR = 0.56 [0.41–0.76]), and high pLI gene deletions (OR = 0.72 [0.58–0.89]). The level of education was not significantly lower among individuals with a high PRSSZ.PRS had a higher impact on education than high-risk CNVs Fig. . We modeEA or PRSIQ distributions had a stronger effect on education (βEA = −19.3 [−2.22 to −1.65] and βIQ = −1.17 [−1.46 to −0.88]) than high-risk CNVs  or education-associated CNVs . On average, each additional +1 SD of PRSEA added 0.84 years [0.79–0.89] of education, and each additional +1 SD of PRSIQ 0.54 years [0.50–0.59] of education in FINRISK. Educational attainment in individuals at the high extreme of PRSSZ was not lower than in controls (β = 0.03 [−0.25 to +0.32]).We employed a linear regression model in FINRISK to estimate years of education Fig. , correctEA and PRSIQ both reported lower household income  than controls, though the confidence intervals overlapped with the OR of high-risk CNVs. Each additional +1 SD of PRSEA in FINRISK increased household income (OREA = 1.22 [1.19–1.25]); the same was true for +1 SD of PRSIQ in FINRISK (ORIQ = 1.13 [1.10–1.16]). In high-risk CNV subgroups, household income was lower for carriers of large deletions (OR = 0.51 [0.38–0.70]) and high pLI gene deletions (OR = 0.66 [0.54–0.81])  whereas household income was not significantly lower in PRS extremes than in controls.Household income was conversely lower for carriers of income-associated CNVs than for individuals at the PRS extremes or for high-risk CNV carriers Fig. . We analI2 = 59.9%), associating with lower health in NFBC1966 . The impact of PRSIQ (OR = 0.76 [0.56–1.02]) differed between the cohorts (I2 = 60.1%); low PRSIQ was not associated with lower subjective health in NFBC1966 (p = 0.56) whereas it was in FINRISK .CNVs with reported medical consequences and PRS extremes were associated with lower subjective health, while high-risk CNVs reported similar health as controls Fig. . We analEA extreme, but not in high-risk CNV carriers  compared to individuals within the middle 20–80% of the PRSEA distribution. PRSEA had no effect on mortality when taking lifestyle factors  into account. We did not observe higher mortality among individuals at the extremes of PRSIQ  or PRSSZ (HR = 1.06 [0.82–1.37]), or among high-risk CNV carriers  or CNVs with medical consequences (HR = 1.71 [0.85–3.43]). We did not estimate mortality in NFBC1966 due to the young age of the participants.Mortality was higher among individuals at the lowest PRSers Fig. . Estimatpadj = 0.0097; Supplementary Fig. Only a small subgroup of high-risk CNVs showed higher general morbidity Fig. . To estiEA in particular, to have a more substantial impact than high-risk CNVs . We tested variant impacts in a multinomial logistic regression model using group 2 as a reference, with sex, age, year of enrollment, and PCs 1–10 as covariates. We observed no group 1 or group 3 enrichment in individuals carrying high-risk CNVs  nor in any CNV subgroup  and at lower odds of high SES and good health . Individuals at the highest extreme of the PRSSZ distribution did not show enrichment or depletion in any of the assigned groups. These analyzes suggest that the polygenic component is likely to have a higher predictive value for socioeconomic status and general well-being than the different CNV classes.The effect of PRS was clearer. Individuals at the lowest extreme of PRSHere we find that the majority of working-age individuals in Finland carrying high-risk CNVs have a modest, if any, increased risk for major health or socioeconomic consequences. Only 4.8% of carriers had an associated neuropsychiatric disease, but this is not an accurate reflection of the true population frequency, as the most severe cases are underrepresented. We furthermore show no increase in overall mortality or morbidity in most high-risk CNVs. Unlike the relatively mild effect of most CNV classes, we observed a clear polygenic effect on socioeconomic outcome with educational attainment and IQ PRS scores. Belonging to the matched lowest PRS extremes (lowest 2.66%) of educational attainment or IQ had an overall stronger impact on the socioeconomic outcome than belonging to most high-risk CNV groups, and a generally stronger impact on health and survival, with the exception of household income-associated CNVs. These results imply that while on an individual level, high-risk variants can show a significant burden on specific neuropsychiatric disease risk and personal health, for carriers without such disease, the quality of life is expected to be comparable to that of the general population.In general, the effect of deleterious rare variants (CNVs and protein truncating variants) on cognition and functional outcomes is well-established , 38, 39.It is important to highlight that the rare deleterious CNVs studied here do not represent the full spectrum of the categories used. Many of the CNVs highlighted by the DECIPHER study  have onlThe broad impact of CNVs, pleiotropic both clinically and subclinically, has been observed in numerous previous studies –16. CaseThe recent study by Männik et al.  in the EAs stated above, the observed effect of polygenic scores was broader than that of structural variants. We observed strong effects in PRSs for intelligence and educational attainment on education, income and socioeconomic status. In line with previous studies, PRS for schizophrenia is mostly associated with schizophrenia with little effect on other traits, including subclinical or socioeconomic effect and educational attainment , 23. ManThe study design has some limitations. Firstly, as an observational and epidemiological study, no causal inference can be made. Second, analyzing CNVs in broad categories does not provide an insight into the effect of individual variants or loci, and can dilute the effect of these variants and loci. Third, with the chosen CNV calling method, we cannot separate between germline and somatic variants. Fourth, the calling accuracy of CNVs (Supplementary Methods) is outpaced by that of SNPs, potentially biasing the observed CNV impact. Fifth, as a large proportion of CNVs, are de novo, estimating their impact has somewhat different confounding factors than common variant-based polygenic scores. Sixth, diseases were mainly captured from hospital records without primary care data, potentially biasing the range of diseases captured. Finally, due to the underrepresentation of severe and highly penetrant CNVs, the observed frequency of 4.8% affected individuals does not accurately reflect the true disease risk , 6, 48. There are also some differences between the two cohorts used. The 5-yearly collected data of the FINRISK cohort provides a good representation of the adult age spectrum from most geographical regions of Finland from different time points, different vocations, and different socioeconomic backgrounds. However, it also ensures that differences in the income and education distributions in the population are subject to long-term trends and fluctuations in the economical state and educational developments in the country –51. WhilThe finding that the polygenic association with both education and the socioeconomic outcome is stronger than for most structural variants when cases with SNPD were excluded, highlights the polygenic background of these traits. The polygenic contribution in many complex traits has become evident in the wide GWAS literature. It seems that the genetic background of socioeconomic outcome has a strong polygenic component as well, which also modifies the effect of rare, stronger effect variants , 41.We conclude that while contributing significantly to the risk for the development of neurological and psychiatric disorders, the majority of working-age individuals carrying high-risk CNVs have modest or no impact on morbidity and mortality, as well as limited impact on income and educational attainment. The vast majority of working-age individuals with observable high-risk CNVs have no associated SNPD. These unaffected carriers report on average lower subjective health, educational attainment, or income, but this impact is generally more modest than that observed among individuals at the extreme end of common genetic variation, highlighting the polygenic genetic background.Supplemental Material DocumentationSupplementary Table 1: Counts and Frequencies of Phenotype-associated CNVs in FINRISK and NFBCSupplementary Table 2: Disease endpoints consideredSupplementary Table 3: DECIPHER disease-associated CNV regions and frequenciesSupplementary Table 4: Charlson Comorbidity PhenotypesSupplementary Table 5: Socioeconomic status groupingsSupplementary Figure 1: Correlation between neuropsychiatric disorders in FINRISKSupplementary Figure 2: Meta-analysis of SNPD association with CNV subgroupsSupplementary Figure 3: SNPD association with CNV subgroups in FINRISKSupplementary Figure 4: SNPD association with CNV subgroups in NFBCSupplementary Figure 5: Spearman’s correlation between categorical socioeconomic endpoints in FINRISKSupplementary Figure 6: Meta-analysis of level of household income in CNV subgroupsSupplementary Figure 7: Subjective health in different CNV subgroups in NFBCSupplementary Figure 8: Subjective health in different CNV subgroups in FINRISKSupplementary Figure 9: Charlson Comorbidity Index in different CNV subgroups in FINRISKSupplementary Figure 10: Meta-analysis of impact of high-risk CNVs and PRS outlier status on socioeconomic groupingSupplementary Figure 11: PRS_EA distribution in FINRISK CNV carriers vs non-carriers not affected by SNPD, by level of educationSupplementary Figure 12: PRS_EA distribution in FINRISK CNV carriers vs non-carriers not affected by SNPD, by years of education


Ischaemic stroke or cerebrovascular accident (CVA) due to occult atrial fibrillation (AF) may cause severe morbidity and mortality. Diagnosing occult AF can be challenging and there is no consensus regarding the optimal duration of screening. A 24-hour Holter electrocardiogram (ECG) is frequently employed to detect occult AF following ischaemic CVA.Demonstration of occult AF detection rate using a 24-hour Holter ECG in a primary care setting with descriptive analyses of independent variables to compare AF detected and non-detected patients.This retrospective cross-sectional study utilised primary care data and included patients 50 years and older with a new CVA or transient ischaemic attack (TIA) diagnosis followed by a 24-hour Holter examination within 6 months, between 01 January 2013 and 01 June 2019. The analyses included descriptive statistics comparing demographics and clinical characteristics in patients who had AF or Atrial Flutter (AFL) detection to those who did not.p = 0.050), had a higher prevalence of hypertension  and chronic kidney disease (CKD) .Out of 5015 eligible patients, 66 (1.3%) were diagnosed with AF/AFL, with a number needed to screen of 88.5. Compared with those without AF/AFL detection, those diagnosed were older (75.42 ± 7.89 vs. 69.89 ± 9.88, 24-hour Holter has a low AF/AFL detection rate. Older persons and those with hypertension or CKD are more likely to be detected with AF/AFL using this method. KEY MESSAGESA 24-hour Holter electrocardiogram (ECG) is frequently employed to detect occult AF following ischaemic CVA or TIA.Real-world data demonstrates detection rates of 1.3% using this method.24-hour Holter monitoring serves as an initial screening tool, yet a more efficient method for prolonged monitoring should be applied.Ischaemic stroke due to occult atrial fibrillation (AF) may cause severe morbidity and mortality ,2. DiagnThis study aims to demonstrate the real-world AF detection rate in a primary care setting for patients after a cerebrovascular accident (CVA) or TIA and compare clinical aspects between AF patients and patients without AF diagnosis.The study used de-identified data from the Maccabi Healthcare Services (MHS) central computerised database. MHS is the second-largest state-mandated healthcare provider in Israel, serving more than 2.5 million members  and is a representative sample of the Israeli population. This fully computerised database captures all information on patient interaction . The MHS database contains clinical data and is continuously updated and monitored internally by the medical informatics department.International Classification of Diseases version 9 (ICD-9) codes were used for data extraction and to identify patients with CVA, TIA, or APS. The 24-hour Holter examination results were automatically recorded and analysed, followed by a manual revision by an MHS cardiologist.This retrospective cross-sectional study included primary care patients 50 years and older with a new CVA or TIA diagnosis followed by a 24-hour Holter examination (index date) within the next 6 months, between 01 January 2013 and 01 June 2019. Patients included in this study were outpatients visiting an MHS clinic for the administration of a 24-hour Holter ECG post CVA or TIA diagnosis. All patients with haemorrhagic stroke or other known reasons for stroke such as disseminated intravascular coagulopathy syndrome, subdural haemorrhage, brain aneurysm and subarachnoid haemorrhage, and patients with prior AF, Atrial flutter (AFL) or antiphospholipid syndrome (APS) diagnoses were excluded from the study. The Furthermore, patients who were not MHS members for at least 12 months before the 24-hour Holter examination or those who left MHS during the study period were excluded.To ensure a complete and clear report, the ‘Strengthening the Reporting of Observational Studies in Epidemiology’ (STROBE) checklist was applied as an assessment tool for methodological quality . The STRAll variables describing chronic diseases, such as diabetes, coronary heart disease (CHD), cancer, hypertension, osteoporosis, chronic obstructive pulmonary disease (COPD), chronic kidney disease (CKD), asthma, and dyslipidemia were extracted from the patient records in the MHS data registries or from medical records that included a disease diagnosis code and diagnosis date. Chronic diseases were measured at Holter examination date and those who had any prior record were categorised as ‘ever’ since 1998.Age was calculated from the 24-hour Holter date and smoking was divided into ‘ever’ and ‘never’ categories, defining patients who were considered smokers at least once in their medical visits or those who were never considered smokers. Body Mass Index (BMI) was calculated by patients’ records of height and weight; patients were categorised as either underweight (BMI < 18.5), normal weight (BMI = 18.5–25), overweight (BMI = 25–30), or obese (BMI > 30).Smoking and BMI variables were measurements taken during physician examinations or hospital visits, therefore, data were not recorded for all patients and was categorised as ‘missing’. These measurements were taken within 12 months prior to the Holter examination date.t-test was used for normally distributed continuous variables, such as age and days to Holter administration, and a Pearson’s Chi-squared test was used for categorical variables.The analyses included descriptive statistics comparing adult patients, ages 50 years and older, who had AF or AFL detection to those who did not. Patient characteristics such as age, sex, and other comorbidities were presented and compared. To evaluate the statistical significance between the two groups, a Student’s The ‘missing’ values were included as a category for Smoking and BMI, where Chi-square analyses were also applied. Less than 1% of the cohort had ‘missing’ data in both variables, likely due to recorded measurements during patients’ recent hospitalisations or clinician visits for CVA/TIA or Holter administration.Approval was obtained from the Institutional Review Board (IRB) and Ethics Committee of MHS for the purposes of accessing and analysing the data. Individual patient informed consent was not required because of the anonymized nature of the patient records.A total of 5,102 patients in MHS had records of CVA or TIA with a 24-hour Holter ECG in the following 6 months. However, 56 patients were excluded due to a record of APS and 31 patients were excluded due to leaving the MHS healthcare system or having less than 12 months of prior registration. Thus, 5015 patients were eligible for this study.p = 0.05) and had a higher prevalence of hypertension  and chronic kidney disease . The mean number of days from CVA or TIA diagnosis to 24-hour Holter exam was 63.85 days in the AF/AFL group and 51.46 days in patients who were not diagnosed with AF/AFL (p = 0.044).Sixty-six of these patients (1.3%) were diagnosed with AF or AFL, with a number needed to screen of 88.5. Patient demographics and comorbidities, presented in Among 5,015 patients in MHS with records of CVA or TIA followed by a 24-hour Holter ECG, we detected 66 patients (1.3%) with AF/AFL. Those detected were older (75.4 vs. 69.9) and more often suffered from hypertension (80.3% vs. 66.8%) and CKD (71.2% vs. 44.2%) as compared to patients in whom no AF/AFL was detected.Prior small-scale studies showed similar results, suggesting that this method has a low detection rate. Doliwa et al., showed a detection rate of 2.0% using 24-hour Holter, in a study including 249 patients . Two addIn contrast, results are conflicted with a study performed by Alhadramy et al. with a detection rate of 9.2% . Their sFurthermore, the results from this study showed significant statistical differences between the patients who were diagnosed with AF and the patients who did not receive such diagnosis – specifically older age, hypertension and CKD. These are all known risk factors for AF/AFL and are supported by previous studies , suggestThis study demonstrates a statistically significant difference in the mean days between CVA or TIA diagnosis and 24-hour Holter examination (63.85 vs. 51.46 days) compared to patients without AF/AFL detection. A study performed by Sanna et al., using an insertable cardiac monitor demonstrated a median of 84 days from randomisation to AF detection . Newly pRecent studies propose that the implementation of loop recorders should be used to detect occult AF, where their results demonstrate high detection rates ,22, howeAdditional AF screening studies in primary care found higher rates of AF detection ,30. ThisThis study’s main limitation is the data collection and interpretation method for AF and AFL diagnosis, which was based on the free text typed by physicians. Thus, if there were any mistakes in the free text or if physicians failed to enter their notes correctly, MHS would have no record in the database. It is possible that some patients who had AF or AFL within the 7-year study period were excluded from this research due to this limitation in the data. On the contrary, using real-world data permitted this study’s population to consist of over 5000 patients with the specific inclusion criteria in a specific study period over several years.This study’s results suggest that the 24-hour ECG is not sufficient to detect AF or AFL in a primary care setting, as the rate of detection is significantly lower than the rate reported for more extended monitoring periods. A 24-hr monitoring period has a low sensitivity, which is why current European and American guidelines recommend longer monitoring periods ,12. PatiIn conclusion, the 24-hour ECG Holter has a low AF/AFL detection rate and certain high-risk patients, namely older persons and those with CKD and hypertension, are more likely to be diagnosed with AF/AFL using this method.