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.

Egg components reverse the atrophy induced by injury in skeletal muscles.

Skeletal muscle atrophy is the loss of muscle tissue caused by factors such as inactivity, malnutrition, aging, and injury. In this study, we aimed to investigate whether egg components exert inhibitory effects on muscle atrophy. An egg mix solution was orally administered for 10 consecutive days to male C57BL/6J mice injected with cardiotoxin in the tibialis anterior (TA) muscle. The administration of egg mixture significantly decreased the atrogin-1 and MuRF-1 protein levels, key factors in muscle atrophy, as observed by western blotting. Furthermore, we investigated the effects of egg components such as avidin, lecithin, biotin, 3-sn-phosphatidylcholine, and L-α-phosphatidylcholine on dexamethasone (DEX)-treated C2C12 myotubes. Lecithin, biotin, 3-sn-phosphatidylcholine, and L-α-phosphatidylcholine in egg yolk significantly recovered the diameters of C2C12 myotubes decreased upon DEX application. Avidin did not show such reversal. Biotin, 3-sn-phosphatidylcholine, and L-α-phosphatidylcholine also attenuated atrogin-1 protein expression enhanced by DEX. Our findings reveal that egg yolk components could contribute to the reversal of skeletal muscle atrophy induced by muscle injury.

A 5-Minute Delay From Bolus Injection to Infusion Halves Plasma Concentration of Tissue Plasminogen Activator.

BACKGROUND: Reducing the time between onset of cerebral infarction and treatment with tissue plasminogen activator improves the prognosis of patients with cerebral infarction. Diverse dosing protocols have been developed with the aim of reducing the time to bolus injection; however, only a few studies have investigated the methods and effects of the interrupted time between bolus and post-bolus infusion. OBJECTIVE: We evaluated the impact of the interrupted time on pharmacokinetic parameters. METHODS: We calculated the changes in alteplase concentration after a bolus injection with high precision, in relation to different interval times. Simulations were performed using the linpk package of the statistical analysis software R. Post-bolus infusion was initiated at 0-, 5-, 15-, and 30-min intervals after bolus dosing. The calculation interval was set as 6 s. RESULTS: Alteplase concentration rose to 1.23 mg/mL after bolus dosing. However, it dropped to 0.53 mg/mL (43.4%) during a 5-min interval, 0.27 mg/mL (22.23%) during a 15-min interval, and 0.10 mg/mL (8.38%) during a 30-min interval. CONCLUSIONS: Because of the short half-life of alteplase, even a short delay in initiating post-bolus infusion can cause a significant reduction in serum alteplase concentration.

Scattered cerebral infarction in the corona radiata predicts worse outcomes.

OBJECTIVES: Intracranial branch atheromatous disease often results in progressive motor deficits in the lenticulostriate arteries (LSA). In some patients with LSA infarction, magnetic resonance imaging (MRI) revealed single lesions at the LSA origin from the middle cerebral artery spreading in a scattered manner toward the distal area. This study aimed to elucidate the clinical characteristics of such cases. MATERIALS AND METHODS: This was a single-center, retrospective study comprising 1,840 consecutive patients admitted to the Ina Central Hospital, Japan. Two neurologists selected patients with LSA infarctions on the basis of MRI data. Patients with a single mass of infarct lesion from the origin were classified as the single group, whereas patients with infarct lesions as a single mass at LSA origin but divided and independent as the infarct area extended distally were classified as the scattered group. We compared the clinical characteristics and outcomes in these groups. RESULTS: The single and scattered groups included 119 and 35 patients, respectively. We defined worsening as an increase of one point or more on the National Institute of Health Stroke Scale. Univariate analysis demonstrated that patients in the scattered group showed significantly more worsening after hospitalization compared with those in the single group (48.6% vs. 28.6%; p < .05). Moreover, this can easily lead to increased disease severity (p < .016). In a multivariate analysis, group (odds ratio, 2.5 [95% CI, 1.11-5.74], p < .03) was an independent predictor of symptom worsening. CONCLUSIONS: Scattered infarction in the corona radiata is an aggravating factor leading to worse outcomes.

Visualization of thrombus using iterative reconstruction and maximum intensity projection of thin-slice CT images.

OBJECTIVE: Iterative reconstruction (IR) is a noise reduction method that facilitates the synthesis of maximum intensity projection (MIP) from a larger number of slices while maintaining resolution. The present study aimed to analyze whether CT evaluation using IR and MIP is ideal for thrombus evaluation of large vessel occlusions in patients with acute ischemic stroke. METHODS: Three types of images for each patient were reconstructed and categorized into three groups: the "conventional group," evaluated using 0.5-mm slice CT, the "MIP group," evaluated using 0.5-mm slice CT processed with MIP, and the "IR + MIP group," evaluated with 0.5-mm slice CT processed with IR and MIP. Noise and image quality were evaluated with noise standard deviation (Noise SD) and contrast-to-noise ratio (CNR). Three experts evaluated the thrombus edge coordinates, made a visual assessment, and compared the data with the digital subtraction angiography (DSA) of the mechanical thrombectomy. RESULTS: Twenty-nine patients with cerebral infarction having large vessel occlusion were included in this study. The IR + MIP group had a lower Noise SD and a statistically higher CNR, leading to more favorable image evaluations. The thrombus assessment showed no inter-rater variability in thrombus edge identification, and the visual assessment and comparison with DSA were statistically better in the IR + MIP group. CONCLUSIONS: IR reduces noise and improves resolution. MIP in combination with IR facilitates visualization of thrombus.

A case of encephalitis following COVID-19 vaccine.

We describe the first case of encephalitis following coronavirus disease 2019 (COVID-19) vaccination. Our patient was a 46-year-old Japanese woman who presented with acute onset diplopia. Subsequent magnetic resonance imaging revealed brain stem encephalitis that was rapidly responsive to high dosage steroid therapy and completely improved. Although the occurrence of encephalitis after vaccination could have just been a casual temporal association, her symptoms were temporally correlated with two vaccinations. Our case suggests caution and indicates treatment and prognosis, despite no evidence of a causal relationship. Nonetheless, this report emphasizes the enormous benefits of vaccination, which should not be undermined.

Correction: Sherloc: a comprehensive refinement of the ACMG-AMP variant classification criteria.

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

Proximal Common Carotid Artery Stenosis Induced by Repetitive Mechanical Compression: A Case Report.

OBJECTIVES: Arterial branches and curvatures, such as the common carotid artery (CCA) origin and carotid bifurcation, are usual sites of atherosclerosis, which leads to carotid artery stenosis. Atherosclerosis may occur due to repetitive compression. Stenosis localized to the proximal segment away from the CCA origin and bifurcation is rare. Here, we describe the case of a patient with right proximal CCA stenosis induced by repetitive compression. METHODS: We studied an acute stroke patient who worked for a long time as a geographical surveyor carrying a tripod on his right shoulder. We found severe eccentric stenosis composed of thick plaque in the right proximal CCA, away from the right CCA origin. However, there was no finding of CCA dissection or vasculitis. RESULTS: The patient was diagnosed with ischemic stroke due to artery-to-artery embolization from the right CCA stenosis, which we believed was due to repetitive compression by the tripod. CONCLUSIONS: Repetitive mechanical stimuli can cause stenotic lesions at atypical vascular sites.

Corrigendum: Sources of discordance among germ-line variant classifications in ClinVar.

This corrects the article DOI: 10.1038/gim.2017.60.

Sources of discordance among germ-line variant classifications in ClinVar.

PurposeClinVar is increasingly used as a resource for both genetic variant interpretation and clinical practice. However, controversies exist regarding the consistency of classifications in ClinVar, and questions remain about how best to use these data. Our study systematically examined ClinVar to identify common sources of discordance and thus inform ongoing practices.MethodsWe analyzed variants that had multiple classifications in ClinVar, excluding benign polymorphisms. Classifications were categorized by potential actionability and pathogenicity. Consensus interpretations were calculated for each variant, and the properties of the discordant outlier classifications were summarized.ResultsOur study included 74,065 classifications of 27,224 unique variants in 1,713 genes. We found that (i) concordance rates differed among clinical areas and variant types; (ii) clinical testing methods had much higher concordance than basic literature curation and research efforts; (iii) older classifications had greater discordance than newer ones; and (iv) low-penetrance variants had particularly high discordance.ConclusionRecent variant classifications from clinical testing laboratories have high overall concordance in many (but not all) clinical areas. ClinVar can be a reliable resource supporting variant interpretation, quality assessment, and clinical practice when factors uncovered in this study are taken into account. Ongoing improvements to ClinVar may make it easier to use, particularly for nonexpert users.

Sherloc: a comprehensive refinement of the ACMG-AMP variant classification criteria.

PurposeThe 2015 American College of Medical Genetics and Genomics-Association for Molecular Pathology (ACMG-AMP) guidelines were a major step toward establishing a common framework for variant classification. In practice, however, several aspects of the guidelines lack specificity, are subject to varied interpretations, or fail to capture relevant aspects of clinical molecular genetics. A simple implementation of the guidelines in their current form is insufficient for consistent and comprehensive variant classification.MethodsWe undertook an iterative process of refining the ACMG-AMP guidelines. We used the guidelines to classify more than 40,000 clinically observed variants, assessed the outcome, and refined the classification criteria to capture exceptions and edge cases. During this process, the criteria evolved through eight major and minor revisions.ResultsOur implementation: (i) separated ambiguous ACMG-AMP criteria into a set of discrete but related rules with refined weights; (ii) grouped certain criteria to protect against the overcounting of conceptually related evidence; and (iii) replaced the "clinical criteria" style of the guidelines with additive, semiquantitative criteria.ConclusionSherloc builds on the strong framework of 33 rules established by the ACMG-AMP guidelines and introduces 108 detailed refinements, which support a more consistent and transparent approach to variant classification.

Left Upper Lung Lobectomy Is an Embolic Risk Factor for Cerebral Infarction.

Cerebral embolism is typically caused by a cardiogenic thrombus. The patent foramen ovale is a well-known cause of paradoxical embolism. However, some idiopathic cases of stroke have been reported. Such strokes are designated as embolic stroke of undetermined sources. Among them, lung lobectomy may be a new embolic risk factor for cerebral embolism. The risk of thrombus formation is high at the pulmonary vein stump after lung lobectomy, especially in the left upper lobe. Interestingly, the risk remains several years after surgery. This condition is mostly overlooked, and reported cases of this condition are rare. Methods of early detection, prevention, and treatment have not been established. Here we report the case of a 66-year-old man who suffered a cerebral infarction 2 days after left upper lobectomy. Three-dimensional computed tomography scan clearly revealed the structural feature of the pulmonary vein stump. The stump of patients with cerebral infarction after lung lobectomy should be checked.

DNA methylation profiling reveals novel biomarkers and important roles for DNA methyltransferases in prostate cancer.

Candidate gene-based studies have identified a handful of aberrant CpG DNA methylation events in prostate cancer. However, DNA methylation profiles have not been compared on a large scale between prostate tumor and normal prostate, and the mechanisms behind these alterations are unknown. In this study, we quantitatively profiled 95 primary prostate tumors and 86 benign adjacent prostate tissue samples for their DNA methylation levels at 26,333 CpGs representing 14,104 gene promoters by using the Illumina HumanMethylation27 platform. A 2-class Significance Analysis of this data set revealed 5912 CpG sites with increased DNA methylation and 2151 CpG sites with decreased DNA methylation in tumors (FDR < 0.8%). Prediction Analysis of this data set identified 87 CpGs that are the most predictive diagnostic methylation biomarkers of prostate cancer. By integrating available clinical follow-up data, we also identified 69 prognostic DNA methylation alterations that correlate with biochemical recurrence of the tumor. To identify the mechanisms responsible for these genome-wide DNA methylation alterations, we measured the gene expression levels of several DNA methyltransferases (DNMTs) and their interacting proteins by TaqMan qPCR and observed increased expression of DNMT3A2, DNMT3B, and EZH2 in tumors. Subsequent transient transfection assays in cultured primary prostate cells revealed that DNMT3B1 and DNMT3B2 overexpression resulted in increased methylation of a substantial subset of CpG sites that showed tumor-specific increased methylation.

Impact of Prefracture Cognitive Impairment and Postoperative Delirium on Recovery after Hip Fracture Surgery.

OBJECTIVES: This study aimed to examine the impact of prefracture cognitive impairment (CI) severity and postoperative delirium on recovery after hip fracture surgery in older patients. DESIGN: Prospective study with a 1-year follow-up. SETTING AND PARTICIPANTS: We included 355 patients aged ≥80 years from 2 acute hospitals in Japan. METHODS: Barthel Index (BI) ambulation scores were assessed prefracture and at 1, 3, 6, and 12 months postoperatively. The score at each time point minus the prefracture score was used as the ambulation recovery variable. The 21-item Dementia Assessment Sheet for the Community-based Care System (DASC-21) and Confusion Assessment Method were used to assess CI severity and delirium, respectively. The impacts of CI severity and delirium on recovery at 1 month and by 12 months postoperatively were examined. Linear multiple regression and linear mixed effects models were used. RESULTS: BI ambulation scores remained the same or improved from prefracture levels in 26.8%, 34.4%, 33.0%, and 30.4% of patients at 1, 3, 6, and 12 months, respectively. Ten patients (2.8%) had fall-related hip fractures, 20 (5.6%) were rehospitalized, and 43 (12.1%) died during this period. Although DASC-21 CI severity significantly affected the recovery both at 1 month and by 12 months postoperatively [standardized ß (Stdß) = -0.39, P < .0001, and Stdß = -0.37, P < .0001, respectively], delirium did not. Other variables affecting recovery by 12 months postoperatively included prefracture BI ambulation scores, Mini Mental State Examination scores, age, fracture type, place of residence, and time. CONCLUSIONS AND IMPLICATIONS: Postoperative ambulation recovery, excluding the effect of death and other poor outcomes, is influenced by prefracture CI severity, and the presence of delirium itself may not be the moderating variable. These results emphasize the importance of treatment planning based on prefracture CI severity and indicate that assessments such as the DASC-21 may be useful in implementing such a plan.

COVID-19 Infection Raising Suspicions of a Connection between Vascular Damage and Thrombus Formation.

Although endothelial damage has been hypothesized to be associated with coronavirus disease 2019 (COVID-19)-related cerebral infarction based on the specificity of the viral cellular invasion pathway, no case has been reported to date. We herein report a 51-year-old Japanese woman who presented with neck pain one week after COVID-19 infection. Computed tomography and magnetic resonance imaging revealed inflammation of the carotid and vertebral arteries. Ultrasonography revealed multiple flap-like structures that were assumed to be thrombi. Although the patient had no cerebral infarction, this could be an important case of vascular damage and thrombus formation in a COVID-19 patient.

An Extremely Delayed Onset of Frey Syndrome: A Case Report and Literature Review.

We report the case of an 80-year-old man with Frey syndrome that developed 30 years postoperatively, which is an exceptionally long period before its occurrence. Sweating and flushing occurred on only the side of his face where the surgery was performed, and he had no other causative abnormalities. Following treatment with botulinum toxin, the patient's symptoms resolved. Extremely early- and late-onset cases do not fit the conventional paradigm of this pathology. Various surgical methods to prevent this syndrome have been explored, but complete prevention has not yet been achieved. These findings suggest that the underlying pathophysiology of Frey syndrome may be more complex than previously recognized.

Combined home and clinic-based therapy versus home-based exercise programme after distal radial fracture: a randomized controlled study.

We investigated outcomes of clinic-based hand therapy combined with a home-based exercise programme after anterior plating for distal radial fractures. A total of 102 patients were randomly assigned to one of three groups: a home-based exercise programme alone; a home-based exercise programme combined with four hand therapy sessions in the clinic; and a home-based exercise programme with seven sessions in the clinic. Mean Patient-Rated Wrist Evaluation scores at 6 weeks were significantly better for the group of patients with seven sessions in the clinic than in those with only home exercises (12 vs. 30), but the difference was no longer significant at 12 weeks. Grip strength was significantly better at 6 and 12 weeks. Combined home- and clinic-based hand therapy may facilitate an earlier return of function after anterior plating for distal radius fractures.Level of evidence: II.

HTRA1-related cerebral small-vessel disease causes cerebral microbleeds on the brainstem surface.

BACKGROUND AND OBJECTIVES: Cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL) has recently been known as HTRA1-related cerebral small-vessel disease (CSVD), it is caused by variants in HTRA1. Recently, it has been reported to develop in heterozygotes with some variants of the gene. Multiple prospective studies have reported that the frequency of heterozygous HTRA1 variants developing CSVD is 2 - 6.5 % in CARASIL. Heterozygous variant cases lack unique clinical features, have an older age of onset, and are difficult to detect. Characteristic findings are required to identify such cases. METHOD: Magnetic resonance imaging (MRI) images of cases that experienced cerebral infarction and carried heterozygous variants in HTRA1 were reviewed. RESULTS: Four cases of heterozygous HTRA1-related CSVD in two families (Family 1: c.754G > A, p.A252T; three males. Family 2: c.497G > T, p.R166L, one female). In all cases, white matter lesions with lacunar infarcts were observed in the periventricular and basal ganglia, external capsule, and brainstem. Moreover, T2 star weighted image (T2*WI) low presented dot-like lesions were present along the surface of the brainstem, which have only been reported in one homozygous case. Susceptibility-weighted imaging (SWI) was performed in two cases, and the dot-like lesions on T2*WI resembled a pearly tiara along the surface of the brainstem. CONCLUSION: Brainstem surface on T2*WI low showed dot-like lesions, which are not generally observed in patients with stroke and can be characteristic of HTRA1-CSVD associated with heterozygous variant. The pathology requires further investigation for diagnosis.

Tissue plasminogen activator for acute branch atheromatous disease exhibits transient improvement and worsening.

BACKGROUND: Tissue plasminogen activator (tPA) is an effective treatment for acute ischemic stroke. Although initial improvement is observed when administered for branch atheromatous disease (BAD), some cases subsequently worsen. Clinical data on the characteristics of these patients is lacking, and the benefits of tPA are unclear. OBJECTIVE: To analyze rebound cases and elucidate the clinical characteristics and outcomes associated with tPA administration in BAD. METHODS: This multicenter retrospective study was conducted in Japan. Worsening after initial improvement of a condition is termed as rebound, and such cases were compared with other types of ischemic stroke in patients with and without rebound. The characteristics of patients with BAD who rebounded were examined. RESULTS: The study included 93 patients. Among the patients who were administered tPA, the NIHSS scores at 24 h and 7 days post-tPA were significantly higher in patients with BAD than in patients with other types of infarcts. The group with BAD exhibited a significantly higher rate of rebound than other groups (37.5 % vs. 0 %, P < 0.001). However, no differences were observed in outcomes between patients who experienced rebound after tPA administration and those who did not. CONCLUSIONS: Reevaluation and changing the strategy of tPA use in patients with BAD may be necessary. However, this study does not totally discourage its use, as specific patients can benefit.