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PubMed2026

Functional Validation of a Novel Homozygous TTN Splice-Site Variant Reveals Aberrant Splicing in Hypertrophic Cardiomyopathy.

The TTN gene encodes a crucial structural protein within cardiac sarcomeres, and its variants may contribute to hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy; however, phenotype and genotype are different. Whole-exome sequencing (WES) was conducted on a Chinese proband diagnosed with HCM. In silico splicing prediction tools and minigene assays were employed to investigate the impact of the identified variant on mRNA splicing. A literature review was performed to retrieve and analyze previously reported splicing variants in the TTN gene associated with HCM. A 42-year-old male proband presented with nonobstructive HCM and paroxysmal atrial arrhythmias. A novel homozygous TTN variant was found, predicted to cause a 14-base pair deletion at a splice acceptor site. Two asymptomatic offspring were found to carry the heterozygous variant. Based on variant interpretation guidelines, the variant met the PM2_supporting criterion and was classified as a variant of uncertain significance (VUS). The predicted aberrant splicing effect was subsequently confirmed by the minigene splicing assay, demonstrating altered pre-mRNA splicing leading to an in-frame insertion/deletion (p.Arg32498_Glu32504delinsGln). Functional verification confirmed that this mutation conforms to the PM4 criterion, suggesting that it can be reclassified as a tepid VUS (scoring 3 points). The functional result might provide pathogenic evidence. We also reviewed 28 previously reported splicing variants in TTN associated with HCM. Of these, 57.1% (16/28) localized to the I-band region, whereas 21.4% (6/28) were situated in the A-band domain of titin. Notably, 21.4% (6/28) co-occurred with pathogenic variants in other sarcomeric genes (MYH7 or MYBPC3), correlating with more severe clinical phenotypes. Reclassification and reinterpretation of the variants revealed that none met the level of likely pathogenic or higher. The present case contributes a homozygous splice-site variant with experimentally confirmed aberrant splicing and an in-frame protein alteration in titin. The focus of this report is the Mendelian genetic basis of the proband's cardiomyopathy phenotype, and our data provide additional case-level and functional evidence for a possible role of specific TTN splicing defects in HCM.

Humans↗

PubMed2026

Bayesian Integration of Tumor Mutational Signatures and Somatic Features Refines Pathogenicity Assessment of Germline Mismatch Repair Variants.

Variants of uncertain significance (VUS) in mismatch repair (MMR) genes represent a persistent bottleneck in germline interpretation for Lynch syndrome, creating a critical opportunity to leverage tumor biology to refine pathogenicity assessment. Although tumor features such as microsatellite instability (MSI) and immunohistochemistry (IHC) are routinely evaluated, they are typically interpreted separately from germline classification, and their quantitative contribution within ACMG/AMP frameworks remains poorly defined. We therefore analyzed paired germline and tumor sequencing data from 1110 tumors across 1073 patients with colorectal or endometrial cancer to determine whether mismatch repair-deficient (MMR-d) mutational signatures can be quantitatively integrated into Bayesian germline variant interpretation. Using COSMIC single-base substitution signatures, tumors were classified as MMR-d or MMR proficient, and an empirically derived likelihood ratio (LR) quantified the association between MMR-d signatures and pathogenic germline MMR variants. The presence of an MMR-d signature increased the likelihood of an underlying pathogenic germline MMR variant approximately eightfold (LR ≈ 8; log10 LR ≈ 0.90), whereas its absence provided moderate-to-strong benign evidence (LR ≈ 0.156; log10 LR ≈ -0.81). Applying this integrative framework to 45 germline MMR VUS, joint modeling of tumor mutational signatures with additional somatic and variant-level evidence resulted in clinically significant reclassification of 38 (84.4%) variants, including three reclassified as pathogenic or likely pathogenic and 35 as likely benign. A total of 16 downgraded variants were independently downgraded by Invitae. These findings demonstrate that tumor mutational signatures can be formally incorporated into Bayesian germline interpretation, transforming tumor data into quantitative pathogenicity evidence and offering a principled strategy to reduce VUS burden in hereditary cancer genetics.

Humans↗

PubMedDate not supplied

Peroxidase reaction as a parameter for discrimination of Streptococcus mutans and Streptococcus sobrinus.

425 strains of mutans streptococci and 12 reference strains were investigated by membrane fatty acid spectra (MFAS) and peroxidase reaction (PR) after aerobic and anaerobic incubation. 423 strains were identified as Streptococcus mutans. The remaining 2 strains were identified as Streptococcus sobrinus. The PR of 29 strains was doubtful; immediately after anaerobic incubation a negative PR changed into a slightly positive PR. To test the diagnostic value of PR the strains were additionally investigated by means of species-specific polymerase chain reactions (PCR). The species-specific PCRs were developed on the basis of the respective genes of 16S rRNA of the pathogens S. mutans and S. sobrinus. Specificity and sensitivity were tested on reference strains (n = 17) and negative control strains (n = 39). The results of this investigation showed that an anaerobic incubation regime could lead to false-positive (S. mutans) or false-negative (S. sobrinus) PR. The 425 MS strains were classified as either S. mutans (n = 420) or S. sobrinus (n = 5). The findings on the reference strains required a reclassification of S. mutans V 100 into S. sobrinus V 100. Summarising, it is possible now to differentiate strains of mutans streptococci by MFAS and PR after aerobic incubation.

Aerobiosis↗

PubMed2004

Usefulness of microalbuminuria in cardiovascular risk stratification of essential hypertensive patients.

BACKGROUND/AIMS: To evaluate the influence of microalbuminuria (albumin excretion rate--AER) determination and echocardiography (ECHO) on cardiovascular risk stratification, initially performed according the 1999 WHO/ISH guidelines by using only routine diagnostic procedures with or without fundal examination. METHODS: 312 essential hypertensives attending our institution were studied retrospectively. Cardiovascular risk was assessed in a semiquantitative way using four categories of absolute cardiovascular disease risk (low, medium, high and very high risk), as proposed by the 1999 WHO/ISH guidelines, on the basis of data on the average 10-year risk of cardiovascular events among participants in the Framingham Study. RESULTS: Without the retinal data, estimating the level of global cardiovascular risk on the basis of routine work-up alone, 14% were classified as low-risk patients, 48% were as medium-risk, 20% as high-risk and 18% at very-high-risk patients. The combined use of AER and ECHO, in line with the newer ESH-ESC guidelines, determined a statistically significant reclassification of the hypertensive patients. Only 10% remained in the low-risk category, 28% were classified in the medium-, 42% in the high- and 20% in the very-high-risk classes. The overall percentage of patients that changed risk stratum (mostly shifting from the medium- to the high-risk class) was significantly different from the proportion of subjects reclassified after the addition of either microalbuminuria or echocardiography alone. No change in the distribution of risk categories was observed when AER assay and ECHO were added to routine procedures including funduscopic examination. CONCLUSIONS: Considering the questionable prognostic value of qualitative retinal examination, our results suggest that cardiovascular risk evaluation based only on simple routine work-up, ignoring the information provided by AER determination and ECHO, may underestimate the level of absolute risk.

Albuminuria↗

PubMed2006

Validation of a simplified definition of anger attacks.

BACKGROUND: Anger attacks, sudden spells of anger with vegetative hyperarousal, are highly prevalent symptoms in depression. Assessment normally requires the use of specific instruments. The aim of this study was the validation of a simplified definition for anger attacks. METHODS: Anger attacks were assessed in 203 patients suffering from major depression with the Anger Attacks Questionnaire. The first three items of the questionnaire (irritability, overreaction to minor annoyances, episodes with inappropriate anger or rage) were compared separately with the diagnosis, and their value as single screening questions to establish the diagnosis was assessed. RESULTS: Irritability was only weakly associated with the diagnosis of anger attacks (Cohen's kappa kappa = 0.214 +/- 0.058) and yielded a rather low specificity (0.302), while overreaction to minor annoyances (kappa = 0.869 +/- 0.037) and the question about episodes with inappropriate anger or rage (kappa = 0.901 +/- 0.032) had a high degree of agreement with the diagnosis of the questionnaire (specificity 0.918 and 0.935, respectively). The combination of the two later items resulted in an almost perfect reclassification of cases (kappa = 0.955 +/- 0.022; specificity = 0.971). CONCLUSIONS: As anger attacks are probably underdiagnosed in clinical practice, simplification of the diagnostic process is imperative. Our results demonstrate that asking one or two simple screening questions suffices to recognize the majority of patients with anger attacks.

Anger↗

PubMedDate not supplied

Prion diseases in humans and their relevance to other neurodegenerative diseases.

Molecular genetics has led to considerable advances in our understanding of the transmissible spongiform encephalopathies. The identification of pathogenic mutations in the prion protein gene has enabled a molecular reclassification of the familial forms of these diseases, which may now be referred to as inherited prion diseases. Prion diseases of both humans and animals are associated with deposition of an abnormal isoform of a host-encoded protein, the prion protein (PrP). Human prion diseases have inherited, sporadic and acquired forms. A considerable body of evidence now supports the idea that the transmissible agent in these diseases may be an abnormal isoform of the prion protein. The identification of pathogenic mutations in the PrP gene has enabled the identification of cases of inherited prion disease that would not have been recognised using existing clinical and pathological diagnostic criteria. Since marked clinical and neuropathological overlap between the different neurodegenerative disorders is well recognised, PrP gene analysis is of increasing importance in differential diagnosis. Frontal lobe dementia of non-Alzheimer type and Pick's disease share a number of important clinical and pathological features with prion diseases, and could be considered as candidate prion diseases. However, we have not been able to demonstrate either PrP mutations or the presence of the disease-associated isoform of prion protein in several well-characterised families with these disorders.

Humans↗

PubMed1987

COMSTAT rule for vigilance classification based on spontaneous EEG activity.

For the classification of sleep stages, international standards based on visual EEG analysis have been established and are in common use, although we are well aware of their limitations. Several authors have suggested different procedures for classifying the stages of vigilance during the waking stages. No universally accepted paradigm, however, has yet been developed. The proposed vigilance classification procedures are based either on visual or automatic analysis procedures. Even though the EEG activity and patterns that reflect vigilance changes have been identified and described as indicators of the state of alertness, opinion is divided on how these should be combined in a vigilance classification rule. Automatic methods, on the other hand, have up to now used only part of the information available, the relationship of which to vigilance indicators has only been partially explored. The COMSTAT (Dept. of Computation and Statistics, AFB-Arzneimittelforschung, Berlin, FRG) rule combines visual and automatic analysis procedures. Different vigilance-dependent EEG patterns, such as the proportion of occipital background rhythm under resting conditions and its replacement by either faster or slower waves, the frequency range of the occipital rhythm and the anteriorization phenomena, have been used as information for a latent class analysis (LCA5) with 5 classes (stages of vigilance). There is a high correlation between the results of the LCA5 with visual classification rules made by experts. Using a robust discriminant analysis function which takes into account prior probabilities of the classes, and with a linear cost function for misclassification, an automatic rule with power spectrum variables was fitted to the results of the LCA5. Reclassification and split-half classification showed a high overlap between LCA5 and automatic classification. The result of this procedure is a new vigilance classification rule that is based on an objective mathematical rationale for the combination of different vigilance-indicative EEG activities and patterns but which can be applied to power-spectral estimators in an automatic EEG analysis procedure.

Aged↗

PubMed1989

Computerized analysis of the in vitro activation of the plasmatic clotting system.

We analyzed the kinetics of the clotting system by a chromogenic substrate method in plasma with isolated factor deficiency. The sigmoidal extinction curve obtained was mathematically described by an equation with four constants K(i) relating to the activity of clotting factors and to the concentration of fibrinogen. The analysis of constants obtained from dilution series of factor-deficient plasma revealed a distribution pattern differing from one factor to another both for the extrinsic and the intrinsic system. The observations indicated that isolated factor deficiency may be identified through the analysis of 2 of the 4 constants, K(1) being the time value of the point of inflection of the extinction curve and K(2) a measure for the slope of the curve at the point of inflection. The observation was confirmed by a reclassification through nearest-neighbor discriminant analysis of K(1) and K(2) which revealed a correct classification in the pathological range for all factor deficiencies investigated with the exception of factors VIII and IX, the distribution patterns of which were superposed within the limits of distribution.

Blood Coagulation↗

PubMed1994

Diagnosis of narcissistic self-esteem regulation in patients with factitious illness (Munchausen syndrome).

A quantitative taxonomy for the identification of patients with narcissistic pathology and with borderline personality disorders based on test results is presented. The quantitative identification of these subgroups was produced using a Q-factor analysis. Based on the correlation of the subjects by means of the 241 questions from the narcissistic inventory of Deneke and Müller [27], three subgroups could be defined. Two of these groups exhibited a pathology of the self-system which corresponded to the pathology described by Kernberg [24] for narcissistic and borderline personality disorders. The third group is characterized by reduced observable self-pathology traits from the narcissistic inventory. By means of the reclassification of these three taxonomical groups with the discriminant analysis, two discriminant functions could be calculated, using the weighting of the single test scales for a classification of new patients. These classification functions were used to examine 18 patients suffering from factitious disorders. The evaluation of test profiles with the reduced narcissistic inventory of Deneke and Müller [11] in order to identify the three taxonomical groups showed that 9 patients (50%) had a borderline personality disorder and 6 patients (33%) a narcissistic personality disorder, while 3 patients (17%) could be assigned to the subgroup without self-pathology. In summary, 83% of the examined patients with factitious disorders exhibited a disorder in self-regulation. The previous clinical observations of self-regulation for patients with factitious disorders could thus be confirmed. It becomes clear that different high levels of disorder in self-regulation (position in the sphere of the discriminant function) correspond to varying degrees of prognostic significance.

Adult↗

PubMedDate not supplied

Is it possible to define a better ASCUS class in cervicovaginal screening? A review of 187 cases.

OBJECTIVE: To try to better define the cytologic diagnosis of atypical squamous cells of undetermined significance (ASCUS) in a cervical screening protocol. STUDY DESIGN: Smears from 187 patients with cytologic diagnoses of ASCUS and histologic or two years' cytologic/colposcopic follow-up were reviewed. When an ASCUS diagnosis was confirmed, it was done strictly on the basis of the morphologic criteria recommended by the Regione Emilia Romagna Screening Protocol in 1997, trying also to subclassify ASCUS into favor reactive or favor neoplasia. RESULTS: Seventy ASCUS cases were negative (37.4%). Three cases (1.6%) were low grade squamous intraepithelial lesion, and seven (3.8%) were high grade squamous intraepithelial lesion. One hundred seven ASCUS cases (57.2%) were confirmed. Among the 70 negative cases, 36 (51.4%) had reactive changes on biopsy, 30 (42.9%) koilocytosis, 3 cervical intraepithelial neoplasia (CIN 1) and one CIN not otherwise specified (5.7% total). CONCLUSION: Reclassification of ASCUS cases using tighter criteria reduced them to a rate of 57.2% but missed 30 patients with histologic diagnoses of koilocytosis and 4 with histologic diagnoses of CIN.

Biopsy↗

PubMedDate not supplied

Computer-assisted rescreening of clinically important false negative cervical smears using the PAPNET Testing System.

OBJECTIVE: To investigate the efficacy of the PAPNET Testing System and its ability to detect significant areas on clinically important false negative gynecologic smears. STUDY DESIGN: Sixty-two gynecologic smears that had been obtained from women studied in a previous case-control investigation, completed in 1987, and had originally been interpreted as negative were rescreened by two independent, blind cytotechnologist-cytopathologist teams. Twenty-nine of these "negative" smears were from 19 women who had been subsequently diagnosed with invasive squamous cell carcinoma and had self-reported a history of only negative gynecologic smears. Thirty-three smears were from 33 control women who did not develop cervical cancer. One team, at the University of Southern California (USC), manually rescreened the smears as part of the original study. The other team, at the University of California at Los Angeles (UCLA), recently used the PAPNET Testing System to rescreen the same smears. This computer-assisted system utilizes neural network technology to recognize and select potentially abnormal cell scenes on a conventionally prepared gynecologic smear. The PAPNET-selected scenes are displayed for review by trained cytologists, who ultimately diagnose the smear. RESULTS: Manual reevaluation of the smears by the USC team in 1987 resulted in the reclassification of 9 of the 29 case smears (31%) and 2 of 33 control smears (6%) as class II to V (atypical squamous cells of undetermined significance to invasive carcinoma). Using the PAPNET System to scan and review the same smears, the cytotechnologist at UCLA referred 24 case smears to the cytopathologist, who ultimately reclassified 12 of the 29 case smears (41%) and 5 of the 33 control smears (15%) as abnormal. CONCLUSION: This study supports the use of the PAPNET System as an effective, routine rescreener for the detection of clinically significant false negative gynecologic smears.

Case-Control Studies↗

PubMed1990

C4 reference typing report.

Human C4 is most polymorphic at the protein level, distinction between allotypes of the C4A and C4B proteins resting on electrophoretic migration patterns and difference in hemolytic activity. The aim of the C4 reference typing has been the definition of reference variants, the assignment of rare variants, and the investigation of duplicated, deleted, or non-expressed and hybrid genes. Samples from 136 individuals, predominantly with known segregation, from 16 laboratories were investigated by standard electrophoretic techniques, for their relative hemolytic activity, reactivity with monoclonal antibodies and Rg/Ch reagents, alpha-, and beta-chain types, relative electrophoretic migration distance, as well as the C4/21-OH-TaqI RFLPs. The results were evaluated in three groups; they consisted in the definition of the eight most common C4 alleles, and the ten Rg/Ch standard phenotypes in group I. In group II twelve C4A and fourteen C4B duplications among 96 complotypes, as well as eighteen deleted/non-expressed C4A and twenty-two C4B alleles, and hybrid alleles were seen by correlation of lytic activity, electrophoretic mobility, and monoclonal and/or Rg/Ch reactivity. Group III consisted of the newly defined allotypes A 8, A 7, A 58, A 55, A 45, B 45, B 35, and B 22, furthermore of alleles subdividing the A 1/A 91, and the B 13/B 12/B 11 regions. The reference typing has allowed reclassification of the majority of described C4 allotypes and resulted in a revision of the C4 nomenclature.

Blood Grouping and Crossmatching↗

PubMed1979

Therapy of urothelial bladder tumors. Results of a retrospective study of 615 cases.

615 urothelial bladder tumors are evaluated retrospectively after reclassification of histological and cytological specimens according to the recommendations of the World Health Organization (1973) and International Union Against Cancer (1974). The reason for a subdivision of the T1 category for exophytic noninfiltrating carcinomas is discussed. The importance of stage and grade is demonstrated as well as the different prognosis of patients with primary or recurrent tumors of the same T category. The results of the study demand a differentiated therapeutic regimen for urothelial bladder tumors.

Follow-Up Studies↗

PubMed1999

Independent effects of Apo E phenotype and plasma triglyceride on lipoprotein particle sizes in the fasting and postprandial states.

LDL particle sizes and Apo E phenotypes were determined in 212 subjects of whom 51 had angina. LDL diameter was significantly less in subjects with an epsilon2 allele (24.76+/-0.08 vs 24.94+/-0.02 nm, P=0.02), and this was evident for both E2/E3 (24.77+/-0.09 nm) and E2/E4 (24.69+/-0.08 nm) phenotypes. Although there was a negative relation between LDL diameter and plasma triglyceride, the effect of apo E2 was still evident with adjustment for triglyceride. In multiple regression analysis, the significant determinants of LDL diameter were gender (with females having larger particles than males), body mass index, and the presence (or absence) of E2. HDL particle sizes and compositions were determined on fasting samples and, additionally, 5 and 8 hours after a fat-rich meal for 48 coronary heart disease cases and 49 control subjects. Fasting HDL particle sizes were not related to the presence of E2 but were significantly smaller for subjects possessing an epsilon4 allele (8. 09+/-0.08 vs 8.39+/-0.05 nm, P=0.003) and were negatively related to plasma triglyceride. However, the effect of E4 persisted after adjustment for triglyceride. In a multiple regression analysis, the only significant determinant of fasting HDL diameter was the presence (or absence) of E4 with fasting plasma triglyceride just failing to reach significance (P=0.06). There was a postprandial increase in HDL diameter that was less marked in subjects with coronary heart disease. The postprandial increase in HDL diameter was of sufficient magnitude to result in size reclassification of HDL particles. The influence of E4 was also evident at both postprandial time points. Compositional analysis demonstrated that the increase in HDL diameters postprandially could be attributed to triglyceride enrichment, with an accompanying fall in cholesterol ester content. Phospholipid changes postprandially were biphasic with an initial fall followed by a rise in concentration. The increase in triglyceride content was significantly less in those subjects with angina despite an equivalent rise in plasma triglyceride. The present study demonstrates significant, but different, effects of variation in apo E phenotype on the particle sizes of both HDL and LDL. Such effects were still evident with adjustment for differences in plasma triglyceride and suggests that variation in apo E phenotype exerts effects on lipoprotein particle sizes by mechanisms additional to those dependent on change in plasma triglyceride.

Alleles↗

PubMed1998

Low-density lipoprotein oxidation and vitamins E and C in sustained and white-coat hypertension.

Low-density lipoprotein oxidation and antioxidant vitamins E and C were investigated in white-coat hypertension in comparison with sustained hypertension and normotension. We selected 21 sustained hypertensive subjects, 21 white-coat hypertensive subjects, and 21 normotensive subjects matched for gender, age, and body mass index. White-coat hypertension was defined as clinical hypertension and daytime ambulatory blood pressure <139/90 (subjects were also reclassified using 134/90 and 135/85 mm Hg as cutoff points for daytime blood pressure). Blood samples were drawn for lipid profile determination, assessment of fluorescent products of lipid peroxidation in native LDL, evaluation of susceptibility to LDL oxidation in vitro (lag phase and propagation rate), and determination of LDL vitamin E and plasma vitamins E and C contents. Compared with sustained hypertensive subjects, white-coat hypertensives had significantly lower fluorescent products of lipid peroxidation (15.4+/-3.4 versus 10.2+/-3 units of relative fluorescence/mg LDL protein, P<.05), longer lag phase (54+/-10 versus 88+/-10 minutes, P<.05), lower propagation rate (8.2+/-2.5 versus 5.95+/-2.1 nmol diene/min per mg LDL cholesterol, P<.05), higher LDL vitamin E content (8.3+/-1.1 versus 10.1+/-1.8 nmol/mg LDL cholesterol, P<.05), and plasma vitamin C content (40+/-13 versus 57+9 micromol/L, P<. 05). No significant difference was observed between white-coat hypertensive and normotensive subjects. The results did not change after reclassification of subjects. Our data show that white-coat hypertensive subjects do not show an enhanced propensity to LDL oxidation or reduction in antioxidant vitamins. Given the role of LDL oxidation in the development of atherosclerosis and that of vitamin E and C in protecting against it, these findings suggest that white-coat hypertension per se carries a low atherogenic risk.

Adult↗

PubMed1996

Interobserver agreement in the classification of stroke in the physicians' health study.

BACKGROUND AND PURPOSE: The evaluation of cerebrovascular end points in prospective studies is often based exclusively on medical record examination and may be made by more than one observer over time. To address the issues of adequacy of medical record information and consistency in diagnosis over time, we evaluated interobserver agreement for the main items of the stroke classification system used in the Physicians' Health Study. This trial included 22,071 physicians randomly assigned in 1982 to receive either aspirin or placebo to assess the subsequent risk of cardiovascular events, including stroke. METHODS: Stroke subtype, stroke severity, and certainty of diagnosis were first classified from medical records from the years 1982 through 1988. The 216 stroke events reported in this period were independently reclassified in 1994 and compared with the initial classification using kappa statistics. RESULTS: Overall agreement in major stroke types (hemorrhagic, ischemic, undetermined stroke) as well as in hemorrhagic stroke subtypes was excellent (kappa = 0.81 and kappa = 0.95, respectively). A wide range of values for the ischemic stroke subtypes (kappa = 0.13 to kappa = 0.96) was obtained. Agreement was substantial in assessment of stroke severity (kappa = 0.71), and it was fair (kappa = 0.33) for certainty of diagnosis. CONCLUSIONS: Interobserver agreement is high for major stroke types as well as for categories of hemorrhagic stroke on the basis of review of medical records and results of imaging data. The classification of ischemic stroke subtypes, however, is subject to substantial interobserver disagreement. Periodic reclassification of random samples of end points might be considered in long-term prospective studies to assess potential misclassification of events by different observers.

Adult↗

PubMed2026

LDLR Variant Classification Through Activity-Normalized Prime Editing Screening.

BACKGROUND: Inherited variants in the LDL (low-density lipoprotein) receptor (LDLR) gene are the most common cause of familial hypercholesterolemia, significantly increasing coronary artery disease risk. Early identification of pathogenic LDLR variants enables prompt lipid-lowering therapy and cascade testing of at-risk relatives; however, most LDLR variants observed in the population have uncertain or absent clinical classifications, leaving many patients without actionable information. METHODS: We developed the first activity-normalized prime editing screening pipeline to measure the impact of 5184 LDLR coding variants on LDL-cholesterol (LDL-C) uptake. Each prime editing guide RNA is paired with a genotypic outcome reporter to correct for variable editing efficiency, overcoming a key limitation of previous pooled genome editing screens. A statistical framework further improves variant effect estimates by jointly analyzing all missense variants at each amino acid position. RESULTS: We show that prime editing of the reporter construct correlates with endogenous variant installation frequency, validating the activity normalization approach. The resulting scores capture a continuous spectrum of functional effects, robustly separate pathogenic versus benign ClinVar variants, and show concordance with LDL-C levels in UK Biobank participants. We calibrate functional evidence strengths to the ACMG/AMP variant interpretation framework, enabling integration into a clinical variant classification workflow. By combining functional, computational, population, and contextual evidence, 322 of 434 LDLR variants currently classified as variants of uncertain significance, conflicting, or absent from ClinVar appear to meet evidence thresholds for reclassification and can be prioritized for expert review, substantially expanding the pool of actionable variant classifications. The screen also reveals a cluster of gain-of-function variants in LDLR class A repeat 5, at least some of which enhance LDL-C uptake through increased apolipoprotein B interaction, with implications for therapeutic genome editing. Last, prime editing uniquely detects splice-altering coding variants missed by cDNA-based screens and pathogenicity predictors, revealing an advantage of endogenous variant installation. CONCLUSIONS: Altogether, activity-normalized prime editing provides a scalable framework for LDLR variant classification that substantially expands the proportion of variants with evidence for genetic diagnosis and reveals novel biology with therapeutic relevance.

CRISPR screening↗

PubMed2002

Value of stress myocardial perfusion single photon emission computed tomography in patients with normal resting electrocardiograms: an evaluation of incremental prognostic value and cost-effectiveness.

BACKGROUND: The incremental value and cost-effectiveness of stress single photon emission computed tomography (SPECT) is of unclear added value over clinical and exercise treadmill testing data in patients with normal resting ECGs, a patient subset known to be at relatively lower risk. METHODS AND RESULTS: We identified 3058 consecutive patients who underwent exercise dual isotope SPECT, who on follow-up (mean, 1.6+/-0.5 years; 3.6% lost to follow-up) were found to have 70 hard events (2.3% hard-event rate). Survival analysis used a Cox proportional hazards model, and cost-effectiveness was determined by the cost per hard event identified by strategies with versus without the use of SPECT. In this cohort, a normal study was associated with an exceedingly low hard-event rate (0.4% per year) that increased significantly as a function of the SPECT result. After adjusting for pre-SPECT information, exercise stress SPECT yielded incremental value for the prediction of hard events (chi2 52 to 85, P<0.001) and significantly stratified patients. In patients with intermediate to high likelihood of coronary artery disease after exercise treadmill testing, a cost-effectiveness ratio of $25 134 per hard event identified and a cost of $5417 per reclassification of patient risk were found. Subset analyses revealed similar prognostic, and cost results were present in men, women, and patients with and without prior histories of coronary artery disease. CONCLUSIONS: Stress SPECT yields incremental prognostic value and enhanced risk stratification in patients with normal resting ECGs in a cost-effective manner. These findings are opposite those of previous studies examining anatomic end points in this same population and thus, if confirmed, have significant implications for patient management.

Cohort Studies↗