Variants Of Significance Research Articles

Exploring health disparities in prostate cancer through circulating tumor DNA analysis.

1603 Background: African American men (AAM) suffer from the highest rates of prostate cancer in the world. In addition, AAM experience the highest rates of aggressive prostate cancer and prostate cancer-specific mortality of any ethnic group in the United States [1, 2]. The reasons behind the increased burden of prostate cancer poor outcomes in AAM remain unresolved. A large proportion of variation in prostate cancer mortality remains unexplained solely by epidemiologic risk factors. We investigated genomic differences between AAM and Caucasian men with metastatic castration resistant prostate cancer (mCRPC) utilizing circulating tumor DNA (ctDNA) analysis. Methods: AAM and Caucasian men with mCRPC were enrolled in this study. We collected and analyzed 88 plasma samples, including 66 from Caucasian and 22 from African American patients, with no significant age difference between the groups. The targeted sequencing libraries were prepared following the Agilent SureSelect XT HS2 target enrichment workflow coupled with the SureSelect custom prostate cancer panel (Agilent, Inc.). The final libraries were sequenced on an Illumina NextSeq 2000 to generate 150 bp paired-end reads. Results: A total of 88 men with mCRPC were enrolled. About 60% of African American patients exhibited organ metastasis at the time of diagnosis, while this proportion was 36% for Caucasian patients and they primarily reported metastasis during relapse. Analyzing 1824 variants in 138 genes meeting inclusion criteria, 281 significant mutations across 88 genes revealed racial differences. Notably, 29% of these variants were somatic. Approximately 60 cancer-related pathways, tissues, and diseases were identified from these datasets, encompassing 59 of our significant genes. Among these associated genes, the prevalence of significant variants, higher in AAM, exceeds that in Caucasians in 76% of cases. The top genes that have more contribution in considering pathways are shown (Table). Conclusions: Genomic variations were observed in ctDNA analysis between African American and Caucasian patients diagnosed with metastatic castration-resistant prostate cancer. Our findings highlight the potential inclusion of ctDNA analysis in the clinical assessment of these patients, offering valuable insights for informed therapeutic decision-making. The top genes with the most contribution to prostate-related pathways. [Table: see text]

Clinically significant variant classification resulting from the addition of RNA sequencing: Experience at high-volume cancer genetics center.

10603 Background: Germline cancer genetic testing plays an important role in prevention, early detection, and targeted therapy1. The increasing use of multi-gene panel testing (MGPT) underscores the importance of accurate variant classification. We report on the significant contribution of RNA analysis to variant classification with impact on clinical care. Methods: A total of 6343 patients who presented to the Nancy & James Grosfeld Cancer Genetics Center between 2019 to 2023 and underwent MGPT at a laboratory with RNA sequencing were evaluated. Patients underwent MGPT using standard DNA technology with added RNA sequencing. We analyzed these patients for clinically significant variant reclassification which includes upgrades from non-actionable variants of uncertain significance (VUS) to pathogenic/likely pathogenic (P/LP) or P/LP to VUS as a significant downgrade. Results: A total of 177 patients (2.8%) had a variant classification significantly impacted by RNA sequencing. Of those 177 patients 45 (25.4%) had clinically relevant change due to their reclassification which resulted in a change in medical or surgical management. The majority of these 45 patients were upgraded from a non-actionable result to P/LP and 14 were downgraded to non-actionable. The clinically significant upgrades included the following genes: BRCA1/2 (5), MSH2 (4), MSH6 (1), PMS2 (1), ATM (5), CHEK2 (5), RAD51C (5), CDH1 (2), and PALB2 (3), which lead to evidence-based early detection and prevention. The clinically significant downgrades included the following genes: BRCA2 (5), RAD50 (1), and RAD51D (8), which allowed for a reduction in medical burden. Of the 177 cases impacted by RNA sequencing, 61 uncertain variants (34.4%) were downgraded to benign polymorphisms, helping to reduce uncertainty regarding management. Conclusions: Our study demonstrates the importance and added benefit of RNA sequencing in variant classification for patients undergoing multigene panel testing for hereditary cancer. The addition of RNA sequencing improved identification of individuals at increased risk, while lowering the number of uncertain variants. This improved technology will allow for a more precise approach to cancer screening, treatment and prevention.

Challenge of Clinical Diagnosis and Treatment for Proteus Syndrome: A Case Report

Proteus syndrome (PS) is an extremely rare complex disorder, and caused by PS associated somatic mosaic AKT1 gene mutation. PS is characterized by progressive asymmetric, disproportionate overgrowth of patient’s skin, soft tissue, and bone of the different body parts since infant period. There is diversity in both clinical appearances, and with or without PS associated pathogenic genetic mutation situation can be existed for each patient. That caused clinical challenges for both accurate diagnosis and proper early treatment. We report a case of 17 years-old girl she presented with significant craniofacial dysmorphism, including prominent craniosynostosis and generalized skull bone sclerotic hyperostosis changes, hearing impairment after age of 3 years-old, progressive asymmetric overgrowth of the left lower limb with leg length discrepancy in childhood. Focal lipomatous tumors grow over the abdomen, left thigh and spinal deformity developed around teenage years. Hypermenorrhea with adenomyosis and ovarian cystadenoma lesions after menarche 12 years-old. Patient was fulfilled positive clinical criteria > 17 points for clinical Proteus syndrome diagnosis, clinically strict met (mosaic distribution of lesions, sporadic occurrence, and progressive course) three general criteria for PS diagnosis. Although the whole exome sequencing test from affected thigh lipoma did not identify pathogenic/likely pathogenic variant AKT1, PTEN, PIK3CA gene. Whole genome sequencing test from affected cranial hyperostosis bone revealed MSH6 variant of uncertain significance (NM-000179.3: c.754T>G) gene mutation. The patient has received surgical treated at three different medical centers in Taiwan for craniofacial dysmorphism, hearing loss, leg length discrepancy, and hypermenorrhea problems respectively. All clinical symptoms and problems with postoperative have partial relieved and improvement. Further more complicated surgery was necessary for her. Clinically we are challenged both for early confirming PS clinical diagnosis, and make the differential diagnosis from other clinical mimic overgrowth spectrum disorder.

Screening of Mutations in Maturity-onset Diabetes of the Young-related Genes and RFX6 in Children with Autoantibody-negative Type 1 Diabetes Mellitus

Maturity-onset diabetes of the young (MODY) is the most common type of monogenic diabetes. To date, mutations have been identified in 14 different genes of patients with a clinical diagnosis of MODY. This study screened mutations in 14 MODY-related genes and the regulator factor X6 (RFX6) gene in children. The presence of clinical features of MODY and negative results for three autoantibody markers of type 1 diabetes mellitus (T1DM) in children and adolescents were used as inclusion criteria for genetic testing. The screening panel for next-generation sequencing included 14 MODY-related genes (GCK, HNF4A, HNF1A, HNF1B, PDX1, NEUROD1, KLF11, CEL, PAX4, INS, BLK, ABCC8, KCNJ11, and APPL1) and the RFX6 gene. Twenty-four different variants in MODY-related genes were identified in 49 children diagnosed with autoantibody-negative T1DM. Twelve variants were classified as pathogenic/likely pathogenic (P/LP) while 12 were interpreted as variant of unknown significance. Nine of the P/LP variants were found in GCK, two in HNF1B, and one in ABCC8. Three variants were novel, and one was a de novo variant. All but one of the variants exhibited heterozygotic inheritance. The frequencies of the MODY subtypes differed from previous reports. Although GCK-MODY was the most frequent mutation in Turkish children, similar to previous studies, the second most prevalent MODY subtype was HNF1B-MODY. This study also established three additional novel mutations in different MODY genes.

Genetic trends and common BRCA1/2 pathogenic sequence variants in black African and Indian breast cancer patients presenting at Inkosi Albert Luthuli Central Hospital, KwaZulu-Natal, South Africa.

Hereditary breast cancer is characterised by the presence of a pathogenic sequence variant passed from one generation to the next. These cancers are aggressive, develop early, and account for 5 - 10% of all breast cancer cases. In South Africa (SA), the common variants that predispose to hereditary breast cancer have been well documented among white patients and form part of screening panels during targeted testing. For non-white patients, common variants are not well understood, and as such, all populations are offered the same test optimised for white patients. This carries a risk of misdiagnosis, the consequences of which include recurrence and increased mortality. To retrospectively describe genetic trends in the black African and Indian breast cancer patients from KwaZulu-Natal Province, SA. We reviewed clinical and genetic data of breast cancer and high-risk patients who consulted at Inkosi Albert Luthuli Central Hospital between 2011 and 2021. Inclusion criteria were based on clinical and demographic characteristics as defined by SA clinical guidelines. Black African patients were young (mean 37.6 years, standard deviation 11.16) and had the majority of triple-negative tumours (37.5%). Indians represented 50% of bilateral breast cancers and of high-risk individuals. We identified 30 pathogenic BRCA1/2 sequence variants, four large genomic rearrangements and 13 variants of unknown significance. Twenty black patients carried 12, 13 white patients carried 4, 25 Indian patients carried 16, and 3 coloured patients carried 3 pathogenic sequence variants. The most frequent variants were BRCA2 c.5771_5774del, p.Ile1924fs among black patients, BRCA2 c.7934del, p.Arg2645fs among white patients, and BRCA2 c.8754+1G>A among Indian patients. None of the founder mutations common in white patients was reported in either black, Indian or coloured patients, which explains why black, Coloured and Indian SA patients consistently test negative during targeted screening. This study highlights unique genetic trends for SA populations and the need for more inclusive targeted tests that are optimal for these populations.

High-throughput functional mapping of variants in an arrhythmia gene, KCNE1, reveals novel biology

BackgroundKCNE1 encodes a 129-residue cardiac potassium channel (IKs) subunit. KCNE1 variants are associated with long QT syndrome and atrial fibrillation. However, most variants have insufficient evidence of clinical consequences and thus limited clinical utility.MethodsIn this study, we leveraged the power of variant effect mapping, which couples saturation mutagenesis with high-throughput sequencing, to ascertain the function of thousands of protein-coding KCNE1 variants.ResultsWe comprehensively assayed KCNE1 variant cell surface expression (2554/2709 possible single-amino-acid variants) and function (2534 variants). Our study identified 470 loss- or partial loss-of-surface expression and 574 loss- or partial loss-of-function variants. Of the 574 loss- or partial loss-of-function variants, 152 (26.5%) had reduced cell surface expression, indicating that most functionally deleterious variants affect channel gating. Nonsense variants at residues 56–104 generally had WT-like trafficking scores but decreased functional scores, indicating that the latter half of the protein is dispensable for protein trafficking but essential for channel function. 22 of the 30 KCNE1 residues (73%) highly intolerant of variation (with > 70% loss-of-function variants) were in predicted close contact with binding partners KCNQ1 or calmodulin. Our functional assay data were consistent with gold standard electrophysiological data (ρ = − 0.64), population and patient cohorts (32/38 presumed benign or pathogenic variants with consistent scores), and computational predictors (ρ = − 0.62). Our data provide moderate-strength evidence for the American College of Medical Genetics/Association of Molecular Pathology functional criteria for benign and pathogenic variants.ConclusionsComprehensive variant effect maps of KCNE1 can both provide insight into IKs channel biology and help reclassify variants of uncertain significance.

Pharmacogenomics of coronary artery response to intravenous gamma globulin in kawasaki disease

Kawasaki disease (KD) is a multisystem inflammatory illness of infants and young children that can result in acute vasculitis. The mechanism of coronary artery aneurysms (CAA) in KD despite intravenous gamma globulin (IVIG) treatment is not known. We performed a Whole Genome Sequencing (WGS) association analysis in a racially diverse cohort of KD patients treated with IVIG, both using AHA guidelines. We defined coronary aneurysm (CAA) (N = 234) as coronary z ≥ 2.5 and large coronary aneurysm (CAA/L) (N = 92) as z ≥ 5.0. We conducted logistic regression models to examine the association of genetic variants with CAA/L during acute KD and with persistence >6 weeks using an additive model between cases and 238 controls with no CAA. We adjusted for age, gender and three principal components of genetic ancestry. The top significant variants associated with CAA/L were in the intergenic regions (rs62154092 p < 6.32E–08 most significant). Variants in SMAT4, LOC100127, PTPRD, TCAF2 and KLRC2 were the most significant non-intergenic SNPs. Functional mapping and annotation (FUMA) analysis identified 12 genomic risk loci with eQTL or chromatin interactions mapped to 48 genes. Of these NDUFA5 has been implicated in KD CAA and MICU and ZMAT4 has potential functional implications. Genetic risk score using these 12 genomic risk loci yielded an area under the receiver operating characteristic curve (AUC) of 0.86. This pharmacogenomics study provides insights into the pathogenesis of CAA/L in IVIG-treated KD and shows that genomics can help define the cause of CAA/L to guide management and improve risk stratification of KD patients.

LMNA p.Arg624His missense variant reduces lamin expression at mRNA level: elucidating molecular pathways toward cardiac involvement in laminopathies

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Ministry of Science and Innovation LMNA is a gene encoding lamin A/C proteins of the cell nuclear lamina. Pathogenic variants in LMNA cause a group of diseases known as laminopathies, a common cause of dilated cardiomyopathy (DCM). LMNA is the second most frequently mutated gene in DCM. Although molecular mechanisms underlying the cardiac involvement are unclear, previous studies have shown a reduction of the lamin expression in LMNA heterozygous mutation carriers. We identified a missense variant in LMNA, c.1871G&amp;gt;A (p.Arg624His), in a family affected with DCM. In this study, we analysed the impact of the p.Arg624His variant on the mRNA expression through quantitative PCR (qPCR) to demonstrate its pathogenicity. RNA was isolated from patient’s peripheral blood lymphocytes. Quantitative reverse transcription PCR (RT-qPCR) with LMNA specific primers was performed to measure lamin RNA levels. Whole-exome sequencing (WES) through next-generation sequencing (NGS) yielded an heterozygous missense variant in the LMNA gene (NM_170707.4(LMNA): c.1871G&amp;gt;A; p.Arg624His; ClinVar ID 66870) classified as variant of uncertain significance (VUS) in five members of a family affected with DCM. Computational (In-Silico) programs predict a damaging effect and variant is absent from controls in population databases. RT-qPCR demonstrated a significant reduction in the LMNA mRNA expression between the control (II.1) and affected family members (II.3, III.1) (Figure 2). According to evidence of co-segregation, variant frequency, results of in-silico predictors and mRNA reduction, LMNA p.Arg624His variant has been reclassified as pathogenic. In this study we reclassified to pathogenic a variant of uncertain significance in LMNA which reduces mRNA expression and leads to cardiac laminopathy. Further investigations are needed to determine the effect on the protein.

Helicobacter pylori Treatment and Gastric Cancer Risk Among Individuals With High Genetic Risk for Gastric Cancer

Helicobacter pylori treatment and nutrition supplementation may protect against gastric cancer (GC), but whether the beneficial effects only apply to potential genetic subgroups and whether high genetic risk may be counteracted by these chemoprevention strategies remains unknown. To examine genetic variants associated with the progression of gastric lesions and GC risk and to assess the benefits of H pylori treatment and nutrition supplementation by levels of genetic risk. This cohort study used follow-up data of the Shandong Intervention Trial (SIT, 1989-2022) and China Kadoorie Biobank (CKB, 2004-2018) in China. Based on the SIT, a longitudinal genome-wide association study was conducted to identify genetic variants for gastric lesion progression. Significant variants were examined for incident GC in a randomly sampled set of CKB participants (set 1). Polygenic risk scores (PRSs) combining independent variants were assessed for GC risk in the remaining CKB participants (set 2) and in an independent case-control study in Linqu. H pylori treatment and nutrition supplementation. Primary outcomes were the progression of gastric lesions (in SIT only) and the risk of GC. The associations of H pylori treatment and nutrition supplementation with GC were evaluated among SIT participants with different levels of genetic risk. Our analyses included 2816 participants (mean [SD] age, 46.95 [9.12] years; 1429 [50.75%] women) in SIT and 100 228 participants (mean [SD] age, 53.69 [11.00] years; 57 357 [57.23%] women) in CKB, with 147 GC cases in SIT and 825 GC cases in CKB identified during follow-up. A PRS integrating 12 genomic loci associated with gastric lesion progression and incident GC risk was derived, which was associated with GC risk in CKB (highest vs lowest decile of PRS: hazard ratio [HR], 2.54; 95% CI, 1.80-3.57) and further validated in the analysis of 702 case participants and 692 control participants (mean [SD] age, 54.54 [7.66] years; 527 [37.80%] women; odds ratio, 1.83; 95% CI, 1.11-3.05). H pylori treatment was associated with reduced GC risk only for individuals with high genetic risk (top 25% of PRS: HR, 0.45; 95% CI, 0.25-0.82) but not for those with low genetic risk (HR, 0.81; 95% CI, 0.50-1.34; P for interaction = .03). Such effect modification was not found for vitamin (P for interaction = .93) or garlic (P for interaction = .41) supplementation. The findings of this cohort study indicate that a high genetic risk of GC may be counteracted by H pylori treatment, suggesting primary prevention could be tailored to genetic risk for more effective prevention.

Variants of Unknown Significance in Maturity-Onset Diabetes of the Young: High Rate of Conundrum Resolution via Variants of Unknown Significance Reanalysis

Introduction: In the era of next-generation sequencing, clinicians frequently encounter variants of unknown significance (VUS) in genetic testing. VUS may be reclassified over time as genetic knowledge grows. We know little about how best to approach VUS in the maturity-onset diabetes of the young (MODY). Therefore, our study aimed to determine the utility of reanalysis of previous VUS results in genetic confirmation of MODY. Methods: A single-center retrospective chart review identified 85 subjects with a MODY clinical diagnosis. We reanalyzed genetic testing in 10 subjects with 14 unique VUS on MODY genes that was performed >3 years before the study. Demographic, clinical, and biochemical data was collected for those individuals. Results: After reanalysis, 43% (6/14) of the gene variants were reclassified to a different category: 7% (1/14) were “likely pathogenic” and 36% (5/14) were “benign” or “likely benign.” The reclassified pathogenic variant was in HNF1A and all reclassified benign variants were in HNF1A, HNF1B and PDX1. The median time between MODY testing and reclassification was 8 years (range: 4–10 years). Conclusion: In sum, iterative reanalyzing the genetic data from VUS found during MODY testing may provide high-yield diagnostic information. Further studies are warranted to identify the optimal time and frequency for such analyses.

Meta-analysis of Epstein-Barr virus genomes in Southern Chinese identifies genetic variants and high risk viral lineage associated with nasopharyngeal carcinoma.

Genetic variants in Epstein-Barr virus (EBV) have been strongly associated with nasopharyngeal carcinoma (NPC) in South China. However, different results regarding the most significant viral variants, with polymorphisms in EBER2 and BALF2 loci, have been reported in separate studies. In this study, we newly sequenced 100 EBV genomes derived from 61 NPC cases and 39 population controls. Comprehensive genomic analyses of EBV sequences from both NPC patients and healthy carriers in South China were conducted, totaling 279 cases and 227 controls. Meta-analysis of genome-wide association study revealed a 4-bp deletion downstream of EBER2 (coordinates, 7188-7191; EBER-del) as the most significant variant associated with NPC. Furthermore, multiple viral variants were found to be genetically linked to EBER-del forming a risk haplotype, suggesting that multiple viral variants might be associated with NPC pathogenesis. Population structure and phylogenetic analyses further characterized a high risk EBV lineage for NPC revealing a panel of 38 single nucleotide polymorphisms (SNPs), including those in the EBER2 and BALF2 loci. With linkage disequilibrium clumping and feature selection algorithm, the 38 SNPs could be narrowed down to 9 SNPs which can be used to accurately detect the high risk EBV lineage. In summary, our study provides novel insight into the role of EBV genetic variation in NPC pathogenesis by defining a risk haplotype of EBV for downstream functional studies and identifying a single high risk EBV lineage characterized by 9 SNPs for potential application in population screening of NPC.

Evaluation the Application of Karyotype Analysis and Chromosome Microarray in Prenatal Diagnosis.

We aimed to compare the difference of the chromosomal abnormalities using karyotype analysis and chromosomal microarray (CMA) as well as to evaluate their application in different prenatal diagnosis indications. Overall, 3007 pregnant women with prenatal diagnosis indications from Medical Genetics Department of Linyi Women and Children's Health Care Hospital, who underwent standard G-banded karyotype analysis and CMA, were enrolled from 2018-2022. G-banded karyotype analysis and CMA were undergone simultaneously. All fetuses with genetic variants were enrolled for further analyzing. The frequency and differences of chromosomal abnormalities of the two methods were compared in different prenatal diagnosis indications groups. CMA improved 4.09% (123/3007) of genetic changes compared karyotype analysis. CMA is on par with karyotyping for detection of aneuploidies and gross unbalanced rearrangements. Serological screening and ultrasound abnormalities were the main indications of prenatal diagnosis. The detection rate of chromosomal abnormalities was highest in non-invasive prenatal testing (NIPT) abnormal group. In the ultrasound abnormality group, the detection rate of genetic variants in nuchal translucency (NT) increased group was higher than other subgroups and there was statistically significant difference in the detection rate of pCNVs. CMA can detect 5.57% (40/718) more genetic abnormalities in ultrasound abnormality group on the normal karyotype. CMA improved 0.67% (20/3007) of genetic changes with clinically significant compared karyotype, brought 3.42% (103/3007) of variants with uncertain significance (VOUS). CMA identified additional, clinically significant genetic variants on the basis of normal karyotype analysis, brought a proportion of unclear significant variants. All the pregnant women accepted amniocentesis should be informed about their characteristics of karyotype analysis and CMA by genetic counselors.

Characterization and Engineered U1 snRNA Rescue of Splicing Variants in a Turkish Neurodevelopmental Disease Cohort

Although they are rare in the population, rare neurodevelopmental disorders (RNDDs) constitute a significant portion of all rare diseases. While advancements in sequencing technologies led to improvements in diagnosing and managing rare neurodevelopmental diseases, accurate pathogenicity classification of the identified variants is still challenging. Sequence variants altering pre-mRNA splicing make up a significant part of pathogenic variants. Despite advances in the in silico prediction tools, noncanonical splice site variants are one of the groups of variants that pose a challenge in their clinical interpretation. In this study, we analyzed the effects of seven splicing variants we had previously proposed as disease-causing and demonstrated that all but one of the seven variants had a strong or moderate effect on splicing, as assessed by a minigene assay. Next, applying U1 snRNAs engineered for different splicing variants in the corresponding genes and expressed with minigene plasmids in HeLa cells provided a partial correction in four of the studied genes to varying degrees. Findings from our study highlight the importance of in vitro minigene-based assays for the reclassification of putative splice-altering variants of uncertain significance and the therapeutic potential of modified U1 snRNAs in RNDDs.

Viral genomic variation and the severity of genital HSV-2 infection as quantified by shedding rate: a viral genome-wide association study.

The clinical severity of genital HSV-2 infection varies widely among infected persons with some experiencing frequent genital lesions while others are asymptomatic. The viral genital shedding rate is closely associated with and has been established as a surrogate marker of clinical severity. To assess the relationship between viral genetics and shedding, we assembled a set of 145 persons who had the severity of their genital herpes quantified through determination of their HSV genital shedding rate. An HSV-2 sample from each person was sequenced and biallelic variants among these genomes were identified. We found no association between metrics of genome-wide variation in HSV-2 and shedding rate. A viral genome-wide association study (vGWAS) identified the minor alleles of three individual unlinked variants as significantly associated with higher shedding rate (p<8.4x10-5): C44973T (A512T), a non-synonymous variant in UL22 (glycoprotein H); A74534G, a synonymous variant in UL36 (large tegument protein); and T119283C, an intergenic variant. We also found an association between the total number of minor alleles for the significant variants and shedding rate (p=6.6x10-7). These results add to a growing body of literature for HSV suggesting a connection between viral genetic variation and clinically important phenotypes of infection.

APF2: an improved ensemble method for pharmacogenomic variant effect prediction

Lack of efficacy or adverse drug response are common phenomena in pharmacological therapy causing considerable morbidity and mortality. It is estimated that 20–30% of this variability in drug response stems from variations in genes encoding drug targets or factors involved in drug disposition. Leveraging such pharmacogenomic information for the preemptive identification of patients who would benefit from dose adjustments or alternative medications thus constitutes an important frontier of precision medicine. Computational methods can be used to predict the functional effects of variant of unknown significance. However, their performance on pharmacogenomic variant data has been lackluster. To overcome this limitation, we previously developed an ensemble classifier, termed APF, specifically designed for pharmacogenomic variant prediction. Here, we aimed to further improve predictions by leveraging recent key advances in the prediction of protein folding based on deep neural networks. Benchmarking of 28 variant effect predictors on 530 pharmacogenetic missense variants revealed that structural predictions using AlphaMissense were most specific, whereas APF exhibited the most balanced performance. We then developed a new tool, APF2, by optimizing algorithm parametrization of the top performing algorithms for pharmacogenomic variations and aggregating their predictions into a unified ensemble score. Importantly, APF2 provides quantitative variant effect estimates that correlate well with experimental results (R2 = 0.91, p = 0.003) and predicts the functional impact of pharmacogenomic variants with higher accuracy than previous methods, particularly for clinically relevant variations with actionable pharmacogenomic guidelines. We furthermore demonstrate better performance (92% accuracy) on an independent test set of 146 variants across 61 pharmacogenes not used for model training or validation. Application of APF2 to population-scale sequencing data from over 800,000 individuals revealed drastic ethnogeographic differences with important implications for pharmacotherapy. We thus think that APF2 holds the potential to improve the translation of genetic information into pharmacogenetic recommendations, thereby facilitating the use of Next-Generation Sequencing data for stratified medicine.

Novel gene-specific Bayesian Gaussian mixture model to predict the missense variants pathogenicity of Sanfilippo syndrome.

MPS III is an autosomal recessive lysosomal storage disease caused mainly by missense variants in theNAGLU, GNS, HGSNAT, and SGSH genes. Thepathogenicity interpretation of missense variants is still challenging. We aimed to develop unsupervised clustering-based pathogenicity predictor scores using extracted features from eight in silico predictors to predict the impact of novel missense variants of Sanfilippo syndrome. The model was trained on a dataset consisting of 415 uncertain significant (VUS) missense NAGLU variants. Performance The SanfilippoPred tool was evaluated by validation and test datasets consisting of 197-labelled NAGLU missense variants, and its performance was compared versus individual pathogenicity predictors using receiver operating characteristic (ROC) analysis. Moreover, we tested theSanfilippoPred tool using extra-labelled 427 missense variants to assess its specificity and sensitivity threshold. Application of the trained machine learning (ML) model on the test dataset of labelled NAGLU missense variants showed that SanfilippoPred has an accuracy of 0.93 (0.86-0.97 at CI 95%), sensitivity of 0.93, and specificity of 0.92. The comparative performance of theSanfilippoPred showed better performance (AUC = 0.908) than the individual predictors SIFT (AUC = 0.756), Polyphen-2 (AUC = 0.788), CADD (AUC = 0.568), REVEL (AUC = 0.548), MetaLR (AUC = 0.751), and AlphMissense (AUC = 0.885). Using high-confidence labelled NAGLU variants, showed that SanfilippoPred has an85.7% sensitivity threshold. The poor correlation between the Sanfilippo syndrome phenotype and genotype represents a demand for a new tool to classify its missense variants. This study provides asignificant tool forpreventing themisinterpretation of missense variants of the Sanfilippo syndrome-relevant genes. Finally, it seems that ML-based pathogenicity predictors and Sanfilippo syndrome-specific prediction tools could be feasible andefficient pathogenicity predictors in the future.

Genomic landscape of comprehensive genomic profiling in patients with malignant solid tumors in Japan.

Comprehensive genomic profiling (CGP) can aid the discovery of clinically useful, candidate antitumor agents; however, the variant annotations sometimes differ among the various types of CGP tests as well as the public database. The aim of this study is to clarify the genomic landscape of evaluating detected variants in patients with a malignant solid tumor. The present, cross-sectional study used data from 57,084 patients with a malignant solid tumor who underwent CGP at the Center for Cancer Genomics and Advanced Therapeutics (C-CAT) between June 1, 2019 and August 18, 2023. The pathogenicity of the variants was annotated using public databases. As a result of re-annotation of the detected variants, 20.1% were pathogenic and 1.4% were benign. The mean number of pathogenic variants was 4.30 (95% confidence interval: 4.27-4.32) per patient. Of the entire cohort, 5.7% had no pathogenic variant. The co-occurrence of the genes depended on the tumor type. Germline findings were detected in 6.2%, 8.8%, and 15.8% of the patients using a tumor/normal panel, tumor-only panel, and liquid panel, respectively, with the most common gene being BRCA2 followed by TP53 and BRCA1. The detected variants should be re-annotated because several benign variants or variants of unknown significance were included in the CGP, and the genomic landscape derived from these results will help researchers and physicians interpret the results of CGP tests. The method of extracting presumptive, germline, pathogenic variants from patients using a tumor-only panel or circulating tumor DNA panel requires improvement.

Impact of functional gene groups on outcomes after ablation of ventricular tachycardia in patients with inherited left ventricular cardiomyopathy: an international multicentre study

Abstract Background Advances in genetic testing has enabled identification of pathogenic variants of different functional gene groups associated with non-dilated left ventricular cardiomyopathy (NDLCM)/dilated cardiomyopathy (DCM) and ventricular tachycardia (VT). However, there is limited data on the impact of functional groups on VT ablation outcome. Aim We aimed to assess the impact of likely pathogenic/pathogenic variants (LP/Pv) of different functional gene groups on heart failure (HF) outcomes and VT recurrence in patients with NDLCM/DCM after VT ablation. Methods Patients with NDLCM/DCM who underwent genetic testing (next-generation sequencing) and were referred for VT ablation were enrolled at 6 participating centers. Patients were followed for VT recurrence, HF-related adverse outcomes (death, left ventricular assist device [LVAD], or heart transplantation due to HF), and all-cause mortality. Results A total of 239 patients (age 55±14yrs; male n=193; LVEF 40±12%; amiodarone at admission n=103; ICD n=180) were included. LP/Pv were identified in 128 patients (54%), predominantly found in genes encoding sarcomeric (n=36/128, [28%]; titin n=20, myosin binding protein C n=12), followed by nuclear membrane (n=27/128, [22%]; lamin A/C; n=26), desmosomal (n=23/128, [18%]; desmoplakin n=11, plakophilin n=9), cytoskeleton (n=12/128, [9%]; filamin C n=8), and sarcoplasmic reticulum proteins (n=12/128, [9%]; phospholamban n=10). Variants of unknown significance (n=32) were grouped with patients who tested negative. Acute complete ablation success, defined as non-VT inducibility of any sustained monomorphic VT, was achieved in 113 patients (47%), 63 (56%) with LP/Pv. During a median follow-up of 32 months, 108 patients (45%) had VT recurrence, 41 (17%) had HF-related adverse outcomes (death with/without LVAD in 34, LVAD in 2, heart transplantation in 5) and 47 (20%) died. Patients with LP/Pv were more likely to have VT recurrence, HF-related adverse outcomes and had a higher mortality compared to those without (VT, 68 [53%] vs. 40 [36%]; HF-related adverse outcomes, 30 [23%] vs. 11 [10%]; mortality, 30 [23%] vs. 17 [15%]; log-rank, P&amp;lt;0.01 for all). As compared to patients without LP/Pv, those with LP/Pv in genes encoding nuclear membrane or sarcomeric proteins had significantly worse (P&amp;lt;0.01 for both) and those with LP/Pv in genes encoding desmosomal proteins had similar HF and VT recurrence outcomes (Figure). Of interest, patients with LP/Pv in genes encoding cytoskeleton proteins had favorable HF-related adverse outcomes, but worse VT recurrence (P=0.02) compared to those without LP/Pv (Figure). Conclusion In patients with NDLCM/DCM and VT, the impact of LP/Pv on HF and VT-recurrence outcomes after ablation differs across functional groups. Integrating genetic testing in the comprehensive evaluation of patients with NDLCM/DCM undergoing VT ablation may facilitate personalized ablation strategies and early consideration of alternative HF treatments.

The detection of a strong episignature for Chung–Jansen syndrome, partially overlapping with Börjeson–Forssman–Lehmann and White–Kernohan syndromes

Chung-Jansen syndrome is a neurodevelopmental disorder characterized by intellectual disability, behavioral problems, obesity and dysmorphic features. It is caused by pathogenic variants in the PHIP gene that encodes for the Pleckstrin homology domain-interacting protein, which is part of an epigenetic modifier protein complex. Therefore, we hypothesized that PHIP haploinsufficiency may impact genome-wide DNA methylation (DNAm). We assessed the DNAm profiles of affected individuals with pathogenic and likely pathogenic PHIP variants with Infinium Methylation EPIC arrays and report a specific and sensitive DNAm episignature biomarker for Chung–Jansen syndrome. In addition, we observed similarities between the methylation profile of Chung–Jansen syndrome and that of functionally related and clinically partially overlapping genetic disorders, White–Kernohan syndrome (caused by variants in DDB1 gene) and Börjeson–Forssman–Lehmann syndrome (caused by variants in PHF6 gene). Based on these observations we also proceeded to develop a common episignature biomarker for these disorders. These newly defined episignatures can be used as part of a multiclass episignature classifier for screening of affected individuals with rare disorders and interpretation of genetic variants of unknown clinical significance, and provide further insights into the common molecular pathophysiology of the clinically-related Chung–Jansen, Börjeson–Forssman–Lehmann and White–Kernohan syndromes.

Current real-world evidence of genetic testing diagnostic yield in a large cohort of long QT syndrome

Abstract Background Diagnostic yield in long QT syndrome (LQTS) ranges between 30-70%, depending on the studied population. LQTS can lead to an unexpected sudden cardiac death. For this reason and due to its wider current availability, genetic testing in patients with suspicion of LQTS has spread. Purpose The aim of this study is to investigate the genetic testing yield among a large cohort of heterogeneous index cases with LQTS phenotype. Methods Diagnostic yield was restrospectively evaluated in a cohort of index cases referred to our laboratory for genetic study with LQTS phenotype. Genetic study was performed with customized libraries through next generation sequencing (NGS), which included copy number variations (CNVs) analysis. Patients with LQTS phenotype studied both with "long QT panels" and with broader cardiovascular panels were included, but those with additional cardiac phenotype (cardiomyopathies, heart defects or catecholaminergic polymorphic ventricular tachycardia) were excluded. Positive genetic reports were considered when a likely pathogenic (LP) or pathogenic (P) variant could explain patient´s phenotype. Inconclusive reports were described when a variant considered risk factor (RF) or a variant of uncertain significance variant (VUS) or a hot-VUS was identified in a clinically relevant gene. Results 1183 index cases were genotyped for LQTS phenotype. Only 164 index cases (13,8%) were studied with broader panels which included between 77 and 368 genes. The rest (n=1019) were studied with LQTS panels (either with small panels which included KCNQ1, KCNH2, KCNE1, KCNE2, SCN5A, KCNJ2, CACNA1C and with up to 36 genes-QT panels). Around 27% (n=321) of the index cases had a positive result. They carried LP/P variants in the following genes: KCNQ1 (n=166, 52%), KCNH2 (n=112), SCN5A (n=25), RYR2 (n=5), CACNA1C (n=3), HCN4 (n=4), KCNJ2 (n=2), KCNE2 (n=2) and CALM2 (n=1). 53,3% of the reports were negative. However, there were some incidental findings: 2 index cases carried P variants in LDLR, 2 carried P variants in PKP2, 2 carried a P variant in MYBPC3 and 1 index case each carried a P variant in DES, TTN and DSG2. From the 231 inconclusive reports, 37 index cases carried the RF variant in KCNE1 (p.Asp85Asn), 2 carried the RF variant in SCN5A (p.Arg1193Gln) and 50 patients carried hot-VUS in KCNQ1, KCNH2, CACNA1C, SCN5a or RYR2 gene. Conclusions In our large cohort of index cases referred for genetic testing for LQTS, diagnostic yield was around 27% when considering only LP/P variants in LQTS or arrhythmic phenotype related genes. Even by including the inconclusive but potentially relevant genetic results (RF and hot VUS), the diagnostic yield in our cohort reaches 35%. This is lower than expected for LQTS cohort. It should be taken into consideration that our cohort consists of heterogeneous patients referred for LQTS genetic testing, which would probably reflects better the real world patients seen in every day practice.