Place of concordance-discordance model in evaluating NGS performance.

INTRODUCTION: Ideally, evaluating NGS performance requires a gold standard; in its absence, concordance between replicates is often used as substitute standard. However, the appropriateness of the concordance-discordance criterion has been rarely evaluated. This study analyzes the relationship between the probability of discordance and the probability of error under different conditions. METHODS: This study used a conditional probability approach under conditional dependence then conditional independence between two sequencing results and compares the probabilities of discordance and error in different theoretical conditions of sensitivity, specificity, and correlation between replicates, then on real results of sequencing genome NA12878. The study examines also covariate effects on discordance and error using generalized additive models with smooth functions. RESULTS: With 99% sensitivity and 99.9% specificity under conditional independence, the probability of error for a positive concordant pair of calls is 0.1%. With additional hypotheses of 0.1% prevalence and 0.9 correlation between replicates, the probability of error for a positive concordant pair is 47.4%. With real data, the estimated sensitivity, specificity, and correlation between tests for variants are around 98.98%, 99.996%, and 93%, respectively, and the error rate for positive concordant calls approximates 2.5%. In covariate effect analyses, the effects' functional form are close between discordance and error models, though the parts of deviance explained by the covariates differ between discordance and error models. CONCLUSION: With conditional independence of two sequencing results, the concordance-discordance criterion seems acceptable as substitute standard. However, with high correlation, the criterion becomes questionable because a high percentage of false concordant results appears among concordant results.

Asymptomatic Bacteriuria and Urological Surgery: Risk Factor or Not? Results From the National and Multicenter TOCUS Database.

PURPOSE: Current guidelines recommend screening and treatment of asymptomatic bacteriuria prior to all urological surgeries breaching the mucosa. But little evidence supports this recommendation. At the least, risk stratification for postoperative UTI to support this strategy is lacking. The aim of this study was to define the associated factors for postoperative febrile infectious complications (UTI or surgical site infection) in urological surgery. MATERIALS AND METHODS: We conducted a retrospective, multicentric study including all consecutive patients undergoing any urological surgery with preoperative urine culture. The primary outcome was the occurrence of a UTI or surgical site infection occurring within 30 days after surgery. RESULTS: From 2016 to 2023, in 10 centers, 2389 patients were included with 838 (35%) positive urine cultures (mono-/bi-/polymicrobial). Postoperative infections occurred in 106 cases (4.4%), of which 44 had negative urine cultures (41%), 42 had positive mono-/bimicrobial urine cultures (40%), and 20 had polymicrobial urine cultures (19%). In multivariable analysis, UTI during the previous 12 months of surgery (odds ratio [OR] 3.43; 95% CI 2.07-5.66; P < .001), monomicrobial/bimicrobial preoperative urine culture (OR 3.68; 95% CI 1.57-8.42; P = .02), polymicrobial preoperative urine culture (OR 2.85; 95% CI 1.52-5.14; P < .001), and operative time (OR 1.09; 95% CI 1.04-1.15; P < .001) were independent associated factors for postoperative febrile infections. CONCLUSIONS: Positive urine culture, including preoperative polymicrobial urine culture, prior to urological surgery was associated with postoperative infection. Additionally, patients experiencing infectious complications also had a higher incidence of other complications. The effectiveness of systematic preventive antibiotic therapy for a positive urine culture has not been conclusively established.

Bicentric retrospective study comparing the postoperative outcomes of patients treated surgically for bladder stones with or without concomitant surgery for BPH.

PURPOSE: To compare the postoperative outcomes of male patients who underwent bladder stone (BS) removal, with or without concomitant benign prostatic hyperplasia (BPH) surgery. PATIENTS AND METHODS: All men aged > 50 years who underwent BS removal at two French university hospitals between 2009 and 2018 were retrospectively reviewed. Four binary outcome criteria were identified during the follow-up: early postoperative complications, stone recurrence, subsequent surgery for BS or BPH, and late surgical complications. A composite score ranging from 0 to 4 was calculated by combining the four criteria. RESULTS: A median follow-up period of 42 months was observed in 179 patients. Of these, 107 patients were in the "concomitant surgical treatment" (CST) group and 72 in the bladder "stone removal alone" (SRA) group. The CST group presented higher baseline post-void residual volume (105 vs. 30 ml, p = 0.005). Patients who underwent CST had a significantly lower rate of BS recurrence (12% vs. 39%; p = 0,001) and underwent fewer subsequent surgeries (14% vs. 44%; p < 0.001). There was no significant difference in the early (51% vs. 35%, p = 0,168) and late (26% vs. 17%, p = 0,229) complications rates between the two groups. A better composite score was observed in the CST than in the SRA, but the difference was not significant (3.07 vs. 2.72, p = 0.078). CONCLUSION: As CST increases morbidity and decreases the risk of reoperation, each situation should be considered, taking into account patient choice and comorbidities.

Asymptomatic bacteriuria prior to partial and radical nephrectomy: To screen or not to screen? Results from the national and multicenter TOCUS database.

INTRODUCTION: In the era of increased bacterial resistance, the main strategy is to reduce the prescription of antibiotics when possible. Nowadays, it is highly recommended to screen for asymptomatic bacteriuria (ABU), prior to urological surgery with potential mucosal breach or urine exposure. Screening and treating urinary colonization is a strategy widely adopted before radical and partial nephrectomy but without any evidence. Our main end point in this study is to analyze the relationship between preoperative urine culture and the risk of postoperative febrile urinary tract infection (UTI) or surgical-site infection (SSI) in partial or radical nephrectomy patients. METHODS: We conducted a multicenter retrospective cohort study between January 2016 and January 2023 in 11 French tertiary referral hospitals (TOCUS database). We collected the data for 269 patients including several pre-, intra-, and post-operative variables that could potentially increase the risk of postoperative UTI and SSI including preoperative urinary culture results. RESULTS: The incidence rate of postoperative UTI and SSI was 8.9% in our study. After conducting a logistic multivariate analysis, a propensity score matching analysis, and a subgroup analysis, we found no significant correlation between the urine culture and the postoperative UTI risk [OR = 1.2 (0.5-2.7) (p = 0.7)]. Only the postoperative non-infectious complications were related to a higher risk of postoperative UTI [OR = 12 (4-37), p < 0.001)]. CONCLUSION: Our research shows that screening and treating for ABU prior to radical or partial nephrectomy seems to be unnecessary to prevent postoperative UTI and SSI.

Exploring the in situ evolution of nitrofurantoin resistance in clinically derived uropathogenic Escherichia coli isolates.

BACKGROUND: Nitrofurantoin has been re-introduced as a first-choice antibiotic to treat uncomplicated acute urinary tract infections in England and Wales. Highly effective against common uropathogens such as Escherichia coli, its use is accompanied by a low incidence (<10%) of antimicrobial resistance. Resistance to nitrofurantoin is predominantly via the acquisition of loss-of-function, step-wise mutations in the nitroreductase genes nfsA and nfsB. OBJECTIVE: To explore the in situ evolution of NitR in E. coli isolates from 17 patients participating in AnTIC, a 12-month open label randomized controlled trial assessing the efficacy of antibiotic prophylaxis in reducing urinary tract infections (UTIs) incidence in clean intermittent self-catheterizing patients. METHODS: The investigation of NitR evolution in E. coli used general microbiology techniques and genetics to model known NitR mutations in NitSE. coli strains. RESULTS: Growth rate analysis identified a 2%-10% slower doubling time for nitrofurantoin resistant strains: NitS: 20.8 ±â€Š0.7 min compared to NitR: 23 ±â€Š0.8 min. Statistically, these data indicated no fitness advantage of evolved strains compared to the sensitive predecessor (P-value = 0.13). Genetic manipulation of E. coli to mimic NitR evolution, supported no fitness advantage (P-value = 0.22). In contrast, data argued that a first-step mutant gained a selective advantage, at sub-MIC (4-8 mg/L) nitrofurantoin concentrations. CONCLUSION: Correlation of these findings to nitrofurantoin pharmacokinetic data suggests that the low incidence of E. coli NitR, within the community, is driven by urine-based nitrofurantoin concentrations that selectively inhibit the growth of E. coli strains carrying the key first-step loss-of-function mutation.

Nephrometry scores to predict oncological outcomes following partial nephrectomy (UroCCR Study 70).

PURPOSE: Partial nephrectomy (PN) for large or complex renal tumors can be difficult and associated with a higher risk of recurrence than radical nephrectomy. We aim to evaluate the clinical useful of nephrometry scores for predicting oncological outcomes in a large cohort of patients who underwent PN for renal cell carcinomas. METHODS: Our analysis included patients who underwent PN for renal cell carcinoma in 21 French academic centers (2010-2020). RENAL, PADUA, and SPARE scores were calculated based on preoperative imaging. Uni- and multivariate cox models were performed to identify predictors of recurrence-free survival and overall survival. The area under the curve (AUC) was used to identify models with the highest discrimination. Decision curve analyses (DCAs) determined the net benefit associated with their use. RESULTS: A total of 1927 patients were analyzed with a median follow-up of 32 months (14-45). RENAL score (p = 0.01), age (p = 0.002), histological type (p = 0.001), high nuclear grade (p = 0.001), necrotic component (p < 0.001), and positive margins (p = 0.005) were significantly related to recurrence in multivariate analyses. The discriminative performance of the 3 radiological scores was modest (65, 63, and 63%, respectively). All 3 scores showed good calibration, which, however, deteriorated with time. Decision curve analysis of the three models for the prediction of overall and recurrence-free survival was similar for all three scores and of limited clinical relevance. CONCLUSION: The association between nephrometry scores and oncological outcomes after NP is very weak. The use of these scores for predicting oncological outcomes in routine practice is therefore of limited clinical value.

Cut-off time for surgery and prediction of orchiectomy in spermatic cord torsion: a retrospective multicentric study over 15 years.

PURPOSE: Cut-off time to avoid orchiectomy relies on small series of patients. The objective was to determine the cut-off time to avoid orchiectomy in torsion of the spermatic cord in a large cohort. METHODS: We performed a retrospective multicenter study (TORSAFUF cohort) of patients with suspected spermatic cord torsion between 2005 and 2019. All patients aged > 12 years who were suspected of having a torsion of the spermatic cord in 14 University Hospitals in France were included (n = 2986). Patients for whom data on pain duration were not available (n = 923) or for whom the final diagnosis was not torsion of the spermatic cord (n = 807) were excluded. The primary outcome was orchiectomy. The secondary outcomes were testicular survival time and the prediction of orchiectomy with the duration of pain. RESULTS: 1266 patients were included with an orchiectomy rate of 12% (150 patients). The mean age was 21.5 years old in the salvage group and 23.7 years old in the orchiectomy group (p = 0.01), respectively. The median time from the onset of pain to surgery was 5.5 (IQR = 5) hours in the salvage group and 51.1 (IQR = 70) hours in the orchiectomy group (p < 0.0001). The risk of orchiectomy increased after a time cut-off of 6 h 30. A delay of 15 h 30 in pain duration was found to predict orchiectomy (sensitivity: 0.81; specificity: 0.87). CONCLUSIONS: Pain duration can predict the probability of salvaging the testicles and performing orchiectomy. Rapid intervention should be recommended, regardless of the time elapsed from the onset of pain.

Factors associated with urinary diversion and fatality of hospitalised acute pyelonephritis patients in France: a national cross-sectional study (FUrTIHF-2).

Acute pyelonephritis (AP) epidemiology has been sparsely described. This study aimed to describe the evolution of AP patients hospitalised in France and identify the factors associated with urinary diversion and fatality, in a cross-sectional study over the 2014-2019 period. Adult patients hospitalised for AP were selected by algorithms of ICD-10 codes (PPV 90.1%) and urinary diversion procedure codes (PPV 100%). 527,671 AP patients were included (76.5% female: mean age 66.1, 48.0% Escherichia coli), with 5.9% of hospital deaths. In 2019, the AP incidence was 19.2/10,000, slightly increasing over the period (17.3/10,000 in 2014). 69,313 urinary diversions (13.1%) were performed (fatality rate 6.7%), mainly in males, increasing over the period (11.7% to 14.9%). Urolithiasis (OR [95% CI] =33.1 [32.3-34.0]), sepsis (1.73 [1.69-1.77]) and a Charlson index ≥3 (1.32 [1.29-1.35]) were significantly associated with urinary diversion, whereas E. coli (0.75 [0.74-0.77]) was less likely associated. The same factors were significantly associated with fatality, plus old age and cancer (2.38 [2.32-2.45]). This nationwide study showed an increase in urolithiasis and identified, for the first time, factors associated with urinary diversion in AP along with death risk factors, which may aid urologists in clinical decision-making.

Paving the Way for Personalized Medicine in First Kidney Transplantation: Interest of a Creatininemia Latent Class Analysis in Early Post-transplantation.

Plasma creatinine is a marker of interest in renal transplantation but data on its kinetics in the first days following transplantation are scarce. The aim of this study was to identify clinically relevant subgroups of creatinine trajectories following renal transplantation and to test their association with graft outcome. Among 496 patients with a first kidney transplant included in the French ASTRE cohort at the Poitiers University hospital, 435 patients from donation after brain death were considered in a latent class modeling. Four distinct classes of creatinine trajectories were identified: "poor recovery" (6% of patients), "intermediate recovery" (47%), "good recovery" (10%) and "optimal recovery" (37%). Cold ischemia time was significantly lower in the "optimal recovery" class. Delayed graft function was more frequent and the number of hemodialysis sessions was higher in the "poor recovery" class. Incidence of graft loss was significantly lower in "optimal recovery" patients with an adjusted risk of graft loss 2.42 and 4.06 times higher in "intermediate recovery" and "poor recovery" patients, respectively. Our study highlights substantial heterogeneity in creatinine trajectories following renal transplantation that may help to identify patients who are more likely to experience a graft loss.

Outcome of febrile neutropenic patients treated for bacteriuria in hematology.

INTRODUCTION: Positive urine sample is a frequent finding in post-chemotherapy febrile neutropenia (FN) and can lead to prolonged antibiotic therapy. The aim of this study was to assess the outcome of bacteriuria episodes in FN patients receiving targeted antibiotic therapy. MATERIALS AND METHODS: A multi-centric retrospective study was conducted over a four-year period (2014-2019) on systematic urinalysis. All consecutive first bacteriuria episodes (≤ 2 bacteria with at least ≥ 103 CFU/mL) during FN in hospitalized adult patients for hematological malignancies were included. Relapse and recurrence were defined by fever or urinary tract symptoms (UTS) with the same bacterial subspecies in urine occurring ≤ 7 days and ≤ 30 days, respectively, after antibiotic discontinuation. Mortality rate was determined at 30 days. Targeted antibiotic therapy ≤ 10 days for women and ≤ 14 for men was considered as short course. RESULTS: Among 97 patients, 105 bacteriuria episodes on systematic urinalysis were analyzed; 67.6% occurred in women, 41.9% in AML patients, 17.1% were bacteremic, 14.2% presented with UTS, and 61.9% were treated with short-course antibiotic treatment. One death was reported. In men, no relapse/recurrence was noted, even in the short-course antibiotic group. In women, 2.8% of episodes treated with short-course antibiotic led to relapse or recurrence. CONCLUSIONS: Relapse, recurrence, and mortality were uncommon events in FN patients experiencing bacteriuria episode, whatever the antibiotic duration. To distinguish asymptomatic bacteriuria from infection remained challenging in women. In men, systematic urinalysis at onset of FN could be useful.

Predictors of ultrasound abnormalities among women treated for community-onset acute pyelonephritis: a prospective study.

OBJECTIVES: Acute pyelonephritis (AP) is a common but potentially severe infection. It may be complicated by pyelocaliceal dilatation which requires emergency urine drainage. In outpatient care, ultrasound (US) appears to be the easier way to diagnose pyelocaliceal dilatation, though most APs will not be complicated and do not require immediate ultrasound. We aimed to identify predictors of ultrasound abnormalities in an outpatient AP cohort. METHODS: A prospective study was conducted from April 2006 to September 2019 in an outpatient care network. Patients aged 15 and over treated for AP were included. Men, pregnant women, patients with solitary kidney, and patients without data on their management were excluded. A common algorithm ensured the same approach from diagnosis to treatment. Data were collected prospectively in an anonymized database. We described the characteristics of the patients. Univariate and then multivariate analyses were performed to identify predictors of ultrasound abnormalities. RESULTS: 2054 women were treated for AP. Among them, 32.5% (n = 667) had a history of urinary tract infections and 5.8% (n = 120) of uropathy. The most frequent uropathogen was E. coli (n = 1,432; 69.7%); Extended-Spectrum Beta-Lactamases (ESBLs) were found in 39 (1.9%) urine cultures. Ultrasound was abnormal in 7.3% (n = 149). Age over 55 years (OR = 2.23; 95% CI 1.58‒3.15; p < 0.0001) and uropathy (OR = 3.69; 95% CI 2.26‒6.01; p < 0.0001) were independently identified as predictors of ultrasound abnormalities. The risk increased by 1.8% (95% CI 1.0‒2.6) with each additional year of age. CONCLUSIONS: This study identified age and uropathy as independent predictors of abnormal ultrasound in women treated with community-onset AP.

Development, Evaluation, and Implementation of a House-Made Targeted Next-Generation Sequencing Spoligotyping in a French Laboratory.

Epidemiological studies investigating transmission chains of tuberculosis are undertaken worldwide to tackle its spread. CRISPR locus diversity, called spoligotyping, is a widely used genotyping assay for Mycobacterium tuberculosis complex (MTBC) characterization. Herein, we developed a house-made targeted next-generation sequencing (tNGS) spoligotyping, and compared its outputs with those of membrane-based spoligotyping. A total of 144 clinical MTBC strains were retrospectively selected to be representative of the local epidemiology. Data analysis of a training set allowed for the setting of "presence"/"absence" thresholds for each spacer to maximize the sensibility and specificity related to the membrane-based spoligotyping. The thresholds above, in which the spacer was considered present, were 50 read per millions for spacers 10 and 14, 20,000 for spacers 20, 21, and 31, and 1000 for the other spacers. The confirmation of these thresholds was performed using a validation set. The overall agreement on the training and validation sets was 97.5% and 93.8%, respectively. The discrepancies concerned six strains: Two for spacer 14, two for spacer 31, and two for spacer 32. The tNGS spoligotyping, whose thresholds were finely-tuned during a careful bioinformatics pipeline development process, appears be a technique that is reliable, inexpensive, free of handling errors, and automatable through automatic transfer into the laboratory computer system.

Epidemiology and risk factors for ureteral stent-associated urinary tract infections in non-transplanted renal patients: a systematic review of the literature.

PURPOSE: Pathophysiology and risk factors for Ureteral Stent-Associated Urinary Tract Infection (USAUTI) have been poorly investigated. This situation results in highly diverse practices regarding USAUTI prevention, diagnosis and treatment. The aim of the present study was to describe the epidemiology and risk factors for USAUTI in non-transplanted patients. METHODS: We conducted a systematic literature review based on a comprehensive PubMed® bibliographic strategy, between October 1998 and March 2020. The methodological quality of the studies included was analyzed according to dedicated grids. The main endpoints were the correlation between different potential risk factors and infection ureteral stent-associated urinary tract infection or colonization rate. Conclusions and their level of evidence were reported on the basis of a critical analysis of the best available scientific evidence. This work has been submitted to a national review, which enabled the potentially divergent opinions of experts to be collected, thereby ensuring adequate quality of data. RESULTS AND CONCLUSION: Twenty-six studies out of the 505 articles identified, were included in the final analysis. Staphylococcus spp, E. coli, Klebsiella spp, Pseudomona aeruginosa, Enterococcus spp. and Candida spp. were the microorganisms most often responsible for asymptomatic bacteriuria (ABU) or USAUTI. Longer indwelling time, diabetes mellitus, female gender, chronic renal failure, diabetic nephropathy and cancer were identified as risk factors for ABU and ureteral stent colonization. No specific risk factor for UTI was identified in the literature studied. A causal relationship between ureteral stent colonization and USAUTI or urosepsis remains to be demonstrated.

Associated measures to antibiotic prophylaxis in urology.

PURPOSE: Urinary tract infections are amongst the most frequent nosocomial infections followed by surgical site infections (SSI). Antibiotic prophylaxis is only one way to reduce the risk of post-operative infection. Our aim was to establish a review of the literature on measures to reduce post-operative infections outside antibiotic prophylaxis. METHODS: We performed a review of the literature in February 2019 using Medline database to identify relevant studies published in the last 10 years. We arbitrarily decided to fix our limit to the 10 last years considering that the resistance rate of bacteria to antibiotics and the surgical technics evolve over time. This search identified 229 studies. We included 53 studies selected upon the title or the abstract. RESULTS: Forty-four studies were finally included. We evaluated the efficacy to prevent SSI by skin antisepsis, surgical hand hygiene and technique, type of surgeons' caps, preservation of normothermia, hair removal, increased oxygen delivery, wound protectors, bathing, normoglycemia and immunonutrition. CONCLUSIONS: Antibiotic prophylaxis represents one way to prevent SSI but various other measures should be known and considered by anesthesiologists and surgeons. Some measures to reduce post-operative infections are not clearly established yet and more studies are needed.

[Evaluation of French practices in 2019 regarding prevention and treatment of urinary tract infections related to ureteral stent]. / Évaluation des pratiques professionnelles françaises par questionnaire en 2019 concernant la prévention et le traitement des infections urinaires sur matériel endo-urétéral.

INTRODUCTION: Urological recommendations never focused on prevention and treatment of urinary tract infections related to endo-ureteral material. METHODS: We conducted an evaluation of French professional practices in May 2019 in the aim of highlighting the important heterogeneity of practices using a Survey Monkey inquiry. RESULTS: One-hundred-and-seventy-five urologists answered the inquiry, as to say 13% of French urologists. Questions regarding the management of pre-surgical polymicrobial urine sample, medical and surgical management of pyelonephritis on endo-ureteral material and regarding the need to diagnose and treat asymptomatic bacteriuria before endo-ureteral stent removal are the main points a majority of French urologists felt uncomfortable with. CONCLUSION: This study evaluated French practices in 2019. The diversity of the answers highlights the need for new recommendations on these subjects of daily practice. Future recommendations should allow their homogenization based on the existing evidence-based data.

Adding In Silico Assessment of Potential Splice Aberration to the Integrated Evaluation of BRCA Gene Unclassified Variants.

Clinical mutation screening of the cancer susceptibility genes BRCA1 and BRCA2 generates many unclassified variants (UVs). Most of these UVs are either rare missense substitutions or nucleotide substitutions near the splice junctions of the protein coding exons. Previously, we developed a quantitative method for evaluation of BRCA gene UVs-the "integrated evaluation"-that combines a sequence analysis-based prior probability of pathogenicity with patient and/or tumor observational data to arrive at a posterior probability of pathogenicity. One limitation of the sequence analysis-based prior has been that it evaluates UVs from the perspective of missense substitution severity but not probability to disrupt normal mRNA splicing. Here, we calibrated output from the splice-site fitness program MaxEntScan to generate spliceogenicity-based prior probabilities of pathogenicity for BRCA gene variants; these range from 0.97 for variants with high probability to damage a donor or acceptor to 0.02 for exonic variants that do not impact a splice junction and are unlikely to create a de novo donor. We created a database http://priors.hci.utah.edu/PRIORS/ that provides the combined missense substitution severity and spliceogenicity-based probability of pathogenicity for BRCA gene single-nucleotide substitutions. We also updated the BRCA gene Ex-UV LOVD, available at http://hci-exlovd.hci.utah.edu, with 77 re-evaluable variants.

BRCA1 Circos: a visualisation resource for functional analysis of missense variants.

BACKGROUND: Inactivating germline mutations in the tumour suppressor gene BRCA1 are associated with a significantly increased risk of developing breast and ovarian cancer. A large number (>1500) of unique BRCA1 variants have been identified in the population and can be classified as pathogenic, non-pathogenic or as variants of unknown significance (VUS). Many VUS are rare missense variants leading to single amino acid changes. Their impact on protein function cannot be directly inferred from sequence information, precluding assessment of their pathogenicity. Thus, functional assays are critical to assess the impact of these VUS on protein activity. BRCA1 is a multifunctional protein and different assays have been used to assess the impact of variants on different biochemical activities and biological processes. METHODS AND RESULTS: To facilitate VUS analysis, we have developed a visualisation resource that compiles and displays functional data on all documented BRCA1 missense variants. BRCA1 Circos is a web-based visualisation tool based on the freely available Circos software package. The BRCA1 Circos web tool (http://research.nhgri.nih.gov/bic/circos/) aggregates data from all published BRCA1 missense variants for functional studies, harmonises their results and presents various functionalities to search and interpret individual-level functional information for each BRCA1 missense variant. CONCLUSIONS: This research visualisation tool will serve as a quick one-stop publically available reference for all the BRCA1 missense variants that have been functionally assessed. It will facilitate meta-analysis of functional data and improve assessment of pathogenicity of VUS.