Allele Frequencies Of Variants Research Articles

Abstract 5016: Building better contrived samples to properly evaluate Next-generation MRD and LBx assays

Abstract Circulating tumor DNA (ctDNA) is a component of cell-free DNA (cfDNA) that originates from tumor tissue in the plasma of cancer patients. Although ctDNA is a broadly applicable tumor biomarker, the absolute amount of ctDNA varies across indication and stage, with the level of ctDNA present reflective of tumor volume. As novel methods for the characterization of ctDNA develop, there is need to evaluate these assays with appropriate samples. But current commercial reference standards, generated from modified cell lines or oligonucleotides lack important features such as DNA methylation, fragmentation pattern and nucleosome positioning, whilst clinical trial samples lack the required volume to perform multiple tests and often lack proper consent. We have created a framework for generating contrived samples containing the biological features of clinical samples and the attributes of a reference standard. The plasma-in-plasma contrived samples are generated from commercially purchased and fully consented matched tumor and plasma from cancer patients with age and gender matched healthy donor plasma. Samples are characterized by an internally validated 700 gene NGS panel to identify somatic variants and copy number alterations, which establishes ‘ground truth’. A contrived plasma dilution range based on the mean variant allele frequency (VAF) of the concordant somatic variants identified is generated. Using these methods, we have been able to generate sample sets for pan-cancer and disease specific evaluations. We have tested these samples through many novel high sensitivity assays that utilize both tumor-informed and tumor agnostic methods. We have confirmed that plasma-in-plasma contrived samples are amenable to all evaluated assays to date. The FFPE tumor tissue provide results matching clinical trial samples and the extraction yields of cfDNA are within the range of plasma samples from clinical trial data. Each dilution can be generated with replicates for reproducibility metrics. Current models of the sample dilutions have been generated to increase the number of replicates at relevant levels of tumor burden (i.e., 0.01% VAF). Negative samples consisting of the background plasma were also generated for each sample used. These plasma-in-plasma contrived samples have greater commutability to clinical samples than other commercial reference standards, however there are limitations. The complexity of generating these samples restrict the number of unique patients that can be utilized. Also, due to the volume of healthy plasma used as diluent, it is difficult to generate volumes greater than 4ml. To our knowledge, this is the first report examining the generation and usage of a contrived sample set that can be applied to evaluating novel high sensitivity MRD assays with clinical sample commutability. Citation Format: Paul Labrousse, Hugh Russell, David Shera, Daniel Stetson, Barrett Nuttall, Brian Dougherty, Darren Hodgson, James Hadfield. Building better contrived samples to properly evaluate Next-generation MRD and LBx assays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5016.

Abstract 227: Novel and easy-to-use approach to triple-negative breast cancer tumoroid culture

Abstract Triple-negative breast cancer (TNBC) accounts for 15-20% of all breast cancer diagnoses in the United States. Patients with metastatic TNBC have a low survival rate, driven in part by limited treatment options for the disease. Tumoroids, also known as cancer organoids, have been shown to better maintain tumor-specific mutational and gene expression profiles over long-term culture, and evidence is growing that the functional response of tumoroids to therapeutics better recapitulates in vivo response. These improved preclinical models of TNBC may facilitate the development of new therapies, but tumoroids are yet to be widely adopted due to challenging culture methods. In this study, we show that two TNBC tumoroid models publicly available through the National Cancer Institute Patient-Derived Models Repository can be expanded in suspension culture using Gibco™ OncoPro™ Tumoroid Culture Medium supplemented with FGF10 and beta-estradiol. We compared the bulk tumoroid morphologies, growth rates, maintenance of mutational and gene expression profiles, and molecular phenotypes of cells grown in the NCI-recommended medium in embedded culture, and the OncoPro medium in embedded culture and suspension culture. We saw comparable morphologies and growth rates in all culture conditions. A multivariate analysis of the allelic frequencies of single nucleotide variants and ploidy values of copy number variants for cells expanded in all experimental conditions revealed >95% and >88% correlation, respectively, with the initial material. Interestingly, despite the change in the medium formulation, gene expression was also highly correlated between the initial material and the OncoPro medium conditions for >20,000 RefSeq genes (Pearson r>0.94) and for a cancer-gene specific panel of 1,423 genes (r>0.87). Further analysis showed unchanged expression levels in 97% of total genes analyzed and the basal molecular subtype was maintained for both lines in all conditions. The OncoPro media system was also used to derive a new TNBC tumoroid line, which demonstrates consistent growth rate and maintains the patient-specific mutational profile of the original breast tumor. Differential gene expression analysis revealed that changes in the gene expression profile were largely due to the loss of non-malignant cell populations from the original tumor. Finally, we utilized this new tumoroid line to examine donor-specific killing efficiencies of primary natural killer cells in a co-culture experiment. The discrepancy in the dynamics of killing a tumoroid line versus the standard K562 cell line indicates that tumoroids may offer an improved approach for screening new cell therapies. In summary, the easy-to-adopt OncoPro medium and culture method does not change the characteristics of existing models and may be used to derive new tumoroid lines, both of which can be leveraged for preclinical studies to develop critical new therapies for TNBC. Citation Format: Brittany Balhouse, Chris Yankaskas, Colin Paul, Shyanne Salen, Sylvia Beam, Pradip Shahi Thakuri, Matt Dallas, David Kuninger. Novel and easy-to-use approach to triple-negative breast cancer tumoroid culture [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 227.

An overload of missense variants in the OTOG gene may drive a higher prevalence of familial Meniere disease in the European population

Meniere disease is a complex inner ear disorder with significant familial aggregation. A differential prevalence of familial MD (FMD) has been reported, being 9–10% in Europeans compared to 6% in East Asians. A broad genetic heterogeneity in FMD has been described, OTOG being the most common mutated gene, with a compound heterozygous recessive inheritance. We hypothesize that an OTOG-related founder effect may explain the higher prevalence of FMD in the European population. Therefore, the present study aimed to compare the allele frequency (AF) and distribution of OTOG rare variants across different populations. For this purpose, the coding regions with high constraint (low density of rare variants) were retrieved in the OTOG coding sequence in Non-Finnish European (NFE).. Missense variants (AF < 0.01) were selected from a 100 FMD patient cohort, and their population AF was annotated using gnomAD v2.1. A linkage analysis was performed, and odds ratios were calculated to compare AF between NFE and other populations. Thirteen rare missense variants were observed in 13 FMD patients, with 2 variants (rs61978648 and rs61736002) shared by 5 individuals and another variant (rs117315845) shared by two individuals. The results confirm the observed enrichment of OTOG rare missense variants in FMD. Furthermore, eight variants were enriched in the NFE population, and six of them were in constrained regions. Structural modeling predicts five missense variants that could alter the otogelin stability. We conclude that several variants reported in FMD are in constraint regions, and they may have a founder effect and explain the burden of FMD in the European population.

Relationship of calcium-sensing receptor gene polymorphism CASR rs 1042636, CASR rs 1802757, and cinacalcet response among Egyptian hemodialysis patients with secondary hyperparathyroidism

Introduction: Secondary hyperparathyroidism (SHPT) is a common complication associated with morbidity and mortality among hemodialysis (HD) patients. Objectives: The current study aims to evaluate the frequency of CASR gene polymorphism variants related to parathyroid hormone (PTH) regulation (CASR rs1042636 and CASR rs1802757) and to test the hypothesis that single nucleotide polymorphisms (SNPs) in the CASR gene alter the response to cinacalcet among Egyptian HD patients with SHPT. Patients and Methods: A case-control study that included 50 HD patients with intact parathyroid hormone (iPTH) ≥300 pg/mL treated with cinacalcet for a 6-month duration and 40 healthy volunteers as a control group. Eligible patients were recruited from Ain Shams university hospitals. Blood samples were collected from patients and controls to assess allele frequencies of CASR gene polymorphism variants using real-time polymerase chain reaction (PCR), corrected calcium (Ca), phosphorus (P), Ca×P product, iPTH level, and alkaline phosphatase before and after treatment. HD patients were categorized into two groups based on the reduction percentage; responders’ patients (iPTH ≥20%) and non-responders to cinacalcet treatment. Results: Of 50 HD patients, 48 (96%) carried the rs1042636 AA wild gene, while only two (4%) carried the rs1042636 AG mutant gene, 42 (87.5%) carried the rs1802757 CC wild genotype, and 6 (12.5%) carried the CT mutant genotype. The minor alleles T and G were (6.3% and 2%) respectively, with no statistically significant difference between the patient and control groups regarding the CASR genotypes or alleles distribution. There was no significant difference between responders and non-responder’s patient groups regarding CASR genotypes or allele frequencies. Moreover, no significant correlation between CASR genotypes or alleles to delta change of Ca, P, Ca×P product, or PTH was seen. However, CASR rs1802757 CT mutant genotype was associated with a significant reduction in alkaline phosphatase levels after treatment. Conclusion: There is no significant association between the gene polymorphism CASR rs1042636 or CASR rs1802757 and the reduction in PTH levels as a response to cinacalcet treatment among Egyptian HD patients with SHPT.

Gene variants for the WNT pathway are associated with severity in periodontal disease

ObjectiveStudies of Wnt variants-related to bone resorption in periodontitis are limited. The aim of this study was to establish the genotype and allele frequency of gene variants associated with the Wnt pathway in systemically healthy individuals with and without periodontitis (PD).Materials and methodsOne hundred fifty-seven systemically healthy individuals were evaluated, 90 with PD and 67 without PD. Periodontal clinical indexes, serological and clinical indices of inflammation, and the following variants associated with the Wnt pathway: DKK, SOST, LRP5, and KREMEN were analyzed by high resolution melting and confirmed by Sanger sequencing.ResultsIn the PD-free group, 67.2% of the individuals presented the variant for DKKrs1896367 (p = 0.008) and 82.6% had the variant for KREMEN rs132274 (p = 0.016). The heterozygous variant for the DKK rs1896367 polymorphism was associated with the absence of PD and lower severity OR: 0.33 (CI95% 0.15–0.70) and OR: 0.24 (CI95% 0.11–0.53), respectively. Similarly, KREMEN rs132274 was the homozygous variant associated with the absence of PD (OR: 0.33 (CI95% 0.13–0.88)). On the contrary, 85.6% of individuals with PD presented a variant for DKK rs1896368 (p = 0.042), all suffering severe forms of periodontitis.ConclusionThe presence of DKKrs1896367 and KREMENrs132274 variants in individuals without PD suggests that these single nucleotide polymorphisms could be protective factors for bone loss in PD. A very interesting finding is that the DKKrs1896368 variant was found in a high percentage of severe cases, suggesting that the presence of this variant may be related to the severe bone loss observed in PD.

RH genotypes and red cell alloimmunization rates in chronically transfused patients with sickle cell disease: A multisite study in the USA.

Red cell alloimmunization remains a challenge for individuals with sickle cell disease (SCD) and contributes to increased risk of hemolytic transfusion reactions and associated comorbidities. Despite prophylactic serological matching for ABO, Rh, and K, red cell alloimmunization persists, in part, due to a high frequency of variant RH alleles in patients with SCD and Black blood donors. We compared RH genotypes and rates of alloimmunization in 342 pediatric and young adult patients with SCD on chronic transfusion therapy exposed to >90,000 red cell units at five sites across the USA. Genotyping was performed with RHD and RHCE BeadChip arrays and targeted assays. Prevalence of overall and Rh-specific alloimmunization varied among institutions, ranging from 5% to 41% (p = .0035) and 5%-33% (p = .0002), respectively. RH genotyping demonstrated that 33% RHD and 57% RHCE alleles were variant in this cohort. Patients with RHCE alleles encoding partial e antigens had higher rates of anti-e identified than those encoding at least one conventional e antigen (p = .0007). There was no difference in anti-D, anti-C, or anti-E formation among patients with predicted partial or altered antigen expression compared to those with conventional antigens, suggesting that variant Rh on donor cells may also stimulate alloimmunization to these antigens. These results highlight variability in alloimmunization rates and suggest that a molecular approach to Rh antigen matching may be necessary for optimal prevention of alloimmunization given the high prevalence of variant RH alleles among both patients and Black donors.

Examining the Association of Rare Allelic Variants in Urate Transporters SLC22A11, SLC22A13, and SLC17A1 with Hyperuricemia and Gout.

Genetic variations in urate transporters play a significant role in determining human urate levels and have been implicated in developing hyperuricemia or gout. Polymorphism in the key urate transporters, such as ABCG2, URAT1, or GLUT9 was well-documented in the literature. Therefore in this study, our objective was to determine the frequency and effect of rare nonsynonymous allelic variants of SLC22A11, SLC22A13, and SLC17A1 on urate transport. In a cohort of 150 Czech patients with primary hyperuricemia and gout, we examined all coding regions and exon-intron boundaries of SLC22A11, SLC22A13, and SLC17A1 using PCR amplification and Sanger sequencing. For comparison, we used a control group consisting of 115 normouricemic subjects. To examine the effects of the rare allelic nonsynonymous variants on the expression, intracellular processing, and urate transporter protein function, we performed a functional characterization using the HEK293A cell line, immunoblotting, fluorescent microscopy, and site directed mutagenesis for preparing variants in vitro. Variants p.V202M (rs201209258), p.R343L (rs75933978), and p.P519L (rs144573306) were identified in the SLC22A11 gene (OAT4 transporter); variants p.R16H (rs72542450), and p.R102H (rs113229654) in the SLC22A13 gene (OAT10 transporter); and the p.W75C variant in the SLC17A1 gene (NPT1 transporter). All variants minimally affected protein levels and cytoplasmic/plasma membrane localization. The functional in vitro assay revealed that contrary to the native proteins, variants p.P519L in OAT4 (p ≤ 0.05), p.R16H in OAT10 (p ≤ 0.05), and p.W75C in the NPT1 transporter (p ≤ 0.01) significantly limited urate transport activity. Our findings contribute to a better understanding of (1) the risk of urate transporter-related hyperuricemia/gout and (2) uric acid handling in the kidneys.

Association of the Single Nucleotide Polymorphisms in the Renin-Angiotensin-Aldosterone System with Hypertension in the Uzbek Population.

This research aims to identify the association between the nine polymorphic variants (rs4961, rs699, rs4762, rs5186, rs1403543, rs1799998, rs5443, rs2070744, rs1799983) and the occurrence of hypertension and its clinical manifestations in the Uzbek population. The study included 227 individuals, comprising 179 patients with hypertension and 48 controls. Clinical parameters such as age, weight, blood glucose, triglycerides, total cholesterol, low-density lipoprotein and high-density lipoprotein, blood urea nitrogen, creatinine, pulse wave velocity, left ventricular mass, and microalbuminuria levels were identified. We assessed the distribution of allele frequencies of these polymorphic variants in the Uzbek population to establish their association with cardiovascular diseases and their clinical manifestations. Genetic analysis of the polymorphic variants demonstrated a significant association of the AGT 521 C>T variant with arterial hypertension [P ≤ 0.01; Odds Ratio (OR) = 2.91]. The NOS3 -786 T>C variant correlated with left ventricular hypertrophy (P ≤ 0.05; OR = 0.35) and increased pulse wave velocity (P ≤ 0.01; OR = 0.21). The correlations of the AGTR2 1675 G>A variant with left ventricular hypertrophy (P ≤ 0.01; OR = 1.59) and increased pulse wave velocity (P ≤ 0.01; OR = 0.33) were identified. The AGT 704 T>C variant showed a significant association with increased pulse wave velocity (P ≤ 0.05; OR = 2.73). Four of the nine studied polymorphic variants were associated with clinical manifestations of hypertension in the Uzbek population. These variants can be used as genetic biomarkers to identify the risks of developing cardiovascular diseases and hypertension in the Uzbek population.

Superoxide Dismutase (rs2070424, rs4880, rs2536512) and Catalase (rs794316, rs1001179) SNPs and their Association with Breast Cancer Risk: Findings from a Hospital Based Case-Control Study.

The antioxidant enzymes are important cellular components involved in detoxification of reactive oxygen species (ROS) and protect cells from ROS induced oxidative damage. Single nucleotide polymorphisms (SNPs) of antioxidant enzyme coding genes such as superoxide dismutase (SOD) and catalase (CAT) may alter the enzyme activity which can influence susceptibility towards carcinogenesis. Therefore, the present study was planned to investigate possible SNPs of SOD (SOD1 (Cu,Zn-SOD), SOD2(Mn-SOD), SOD3(EC-SOD) and CAT genes and their possible association with breast cancer risk in rural Indian women. In this case-control study, the association of SOD and CAT gene polymorphism was studied by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. The study was conducted among 400 clinically breast cancer patients and 400 healthy women in a population of South-Western Maharashtra. The logistic regression analysis was carried out to calculate Odds ratio (OR) with 95% confidence interval and p-value, where p ≤0.05 was considered as statistically significant. The results of analysis of genotype frequency distribution showed significant association of rs4880 SNP of Mn-SOD with BC risk at homozygous variant (CC/CC) genotype (OR 2.46; 95%CI, 1.61-3.75; p<0.0001) and corresponding frequency of variant (C) allele (OR 1.53; 95%CI, 1.25-1.86; p<0.0001). In CAT gene polymorphisms the variant (T/T) was increased significantly in BC cases as compared to controls (OR 3.45; 95%CI, 2.17-5.50; p<0.0001) along with its variant (T) allele (OR 2.01; 95%CI, 1.63-2.48; p<0.0001). The results implied that, C/C genotype of SOD2-1183T/C polymorphism and T/T genotype of CAT-262 C/T polymorphism may be associated with an increased breast cancer risk. However, SOD1-251 A/G and SOD3-172 G/A polymorphisms did not show any significant difference in variant homozygous genotypes of patients compared to controls.

Allele frequencies and genotype distribution of three metformin transporter polymorphisms in Mexican population and their application in pharmacogenomics of type 2 diabetes.

Metformin is the first-line antidiabetic therapy for type 2 diabetes in Mexico, despite recent recommendations highlighting alternatives like GLP-1 receptor agonists for individuals with obesity. Metformin elimination is reliant on liver and kidney function, and variants in transport proteins such as Multidrug and Toxin Extrusion Protein 1 (MATE1), MATE2, and Organic Cation Transporter 2 (OCT2) can influence its pharmacokinetics. Understanding these variants' frequencies in the Mexican population is crucial for tailoring personalized treatment strategies. This study aimed to determine the genotypic and allelic frequencies of key variants in metformin transporters within a Mexican population, addressing the interindividual variability in drug response. Genetic analysis was conducted on 101 healthy, unrelated Mexican subjects who were genotyped for the MATE1, MATE2, and OCT2 variants using allele-specific real-time PCR assays. The allele frequencies were 0.07 for OCT2, 0.23 for MATE1, and 0.67 for MATE2. The g.-66T→C variant was found only in wild-type and heterozygous forms. Comparative analysis indicated significant differences in allele frequencies between this Mexican population and other ethnic groups, highlighting potential implications for metformin efficacy and safety. This study provides crucial insights into the genetic variability of metformin transporter genes in a Mexican population, offering a foundation for personalized therapeutic approaches in type 2 diabetes management.

PVUII (RS2234693) POLYMORPHISM OF THE ESTROGEN RECEPTOR ALPHA GENE IN WOMEN FROM SUMY OBLAST, UKRAINE, WITH ENDOMETRIAL HYPERPLASTIC PROCESS

Introduction. The endometrial hyperplastic process is an estrogen-dependent benign condition of the uterus, which is frequently a cause of infertility, ovarian-menstrual cycle disorders, and malignant transformation into uterine cancer. The effect of estrogen on the endometrium is realized through the estrogen receptor alpha. It is manifested by a whole range of biological changes, including cell reproduction and growth, tissue development, etc. Estrogen receptor alpha is encoded by the ESR1 gene, which is located on chromosome 6q25 and has eight exons and seven introns. Today, about 9,000 polymorphisms of the ESR1 gene have been described, some of which are associated with gene activity changes. Such functional polymorphisms include the PvuII polymorphism (rs2234693), which occurs due to the substitution of thymine (T) for cytosine (C) in the first intron of the gene. Objective. The purpose of this study was to assess the frequency of allelic variants of the estrogen receptor alpha gene for the PvuII polymorphism in patients with endometrial hyperplastic process living in the Sumy Oblast of Ukraine and to study its relationship with the histological variant of endometrial hyperplastic process, anthropometric parameters, age categories, medical history, and concomitant diseases. Materials and Methods. Genomic DNA was isolated from blood samples of 95 women with endometrial hyperplastic processes and 80 healthy women. The rs2234693 polymorphism was studied using a polymerase chain reaction with subsequent restriction fragment length polymorphism analysis (PCR-RFLP). The data were processed and statistically analyzed with Microsoft Excel and SPSS Statistics 29.0 for Windows software package. Descriptive statistics, Student's test, ANOVA method, and Pearson's chi-squared test were used in this study. Results with P &lt; 0.05 were considered statistically significant. Results. The distribution of genotype variants for the PvuII polymorphism of the ESR1 gene in women with endometrial hyperplastic process was: homozygotes for the major allele (T/T) – 31.6%, heterozygotes (T/C) – 49.5%, homozygotes for the minor allele (C/C) – 18.9%; in the control group, these values were 30%, 52.5%, and 17.5%, respectively. There was no difference in the distribution of genotypes in patients with endometrial hyperplastic processes and healthy women (χ2 = 0.163, P = 0.922). No associations were found between the PvuII polymorphism and the histological variant of endometrial hyperplastic processes (χ2 = 4.14, P = 0.387), anthropometric parameters (P &gt; 0.05), age (χ2 = 2.98, P = 0.560), medical history (P &gt; 0.05), or concomitant genital and extragenital conditions (P &gt; 0.05). Conclusions. There was no difference in the distribution of T/T, T/C, and C/C genotypes for the PvuII polymorphism in the estrogen receptor alpha gene between patients with endometrial hyperplastic process and the control group. There was no correlation between the genotype variant for the studied polymorphism and the histological variant of the endometrial hyperplastic process, anthropometric parameters, age, medical history, concomitant genital and extragenital conditions in patients of the Sumy Oblast, Ukraine.

Phenome-wide association study of population-differentiating genetic variants around gene ACSL1.

Demographic dynamics and natural selection during human evolution shaped the present-day patterns of genetic variations, and geographically varying genetic factors contribute to different disease prevalences across human populations. This study aims to evaluate the presence of positive selection on the gene encoding long-chain fatty acyl-CoA synthetase 1 (ACSL1) and the phenotypic impacts of population-differentiating genetic variants around this gene. Three types of statistical tests for positive selection, based on site frequency spectrum, extended haplotype homozygosity and population differentiation, were applied to the whole-genome sequencing data from the 1000 Genomes Project. A phenome-wide association study of ACSL1 was performed with published genome-wide association studies (GWAS) and transcriptome-wide association studies, including phenome-wide studies in biobanks. Genetic variants associated with ACSL1 expression in various tissues exhibit geographically varying allele frequencies. Three types of statistical tests consistently supported the presence of positive selection on the coding and regulatory regions of ACSL1 in African, European, South Asian and East Asian populations. A phenome-wide association study of ACSL1 revealed associations with type 2 diabetes, blood glucose, age at menopause, mean platelet volume and mean reticulocyte volume. The top allele associated with lower diabetes risk has the highest frequency in European populations, whereas the top allele associated with later menopause has the highest frequency in African populations. Positive selection on ACSL1 resulted in geographically varying genetic variants, which may contribute to differential phenotypes across human populations, including type 2 diabetes and age at menopause.

Russian Regional Differences in Allele Frequencies of CFTR Gene Variants: Genetic Monitoring of Infertile Couples.

A male factor, commonly associated with poor semen quality, is revealed in about 50% of infertile couples. CFTR gene (Cystic Fibrosis Transmembrane Conduction Regulator) variants are one of the common genetic causes of azoospermia-related male infertility. Notably, the spectrum and frequency of pathogenic CFTR variants vary between populations and geographical regions. In this work, we made an attempt to evaluate the allele frequency (AF) of 12 common CFTR variants in infertile Russian men and healthy individuals from different districts of Russia. Because of the limited number of population-based studies on Russian individuals, we characterized the population AFs based on data from the Registry of Russian cystic fibrosis (CF) patients. In addition to the CF patient registry, we estimated the local frequencies of the same set of variants based on the results of genotyping of CF patients in local biocollections (from St. Petersburg and Yugra regions). AFs of common CFTR variants calculated based on registry and biocollection data showed good concordance with directly measured population AFs. The estimated region-specific frequencies of CFTR variants allowed us to uncover statistically significant regional differences in the frequencies of the F508del (c.1521_1523del; p.Phe508del) and CFTRdele2,3(21kb) (c.54-5940_273+10250del21kb; p.Ser18ArgfsX) variants. The data from population-based studies confirmed previous observations that F508del, CFTRdele2,3(21kb), and L138ins (c.413_415dup; p.Leu138dup)variants are the most abundant among infertile patients, and their frequencies are significantly lower in healthy individuals and should be taken into account during genetic monitoring of the reproductive health of Russian individuals.

Abstract A123: Circulating tumor DNA (ctDNA) genomic and epigenomic profiling (GuardantINFINITY) for diagnosis of DNA damage repair (DDR) loss of function (LOF) and response monitoring in the TRESR and ATTACC trials

Abstract Background First-generation panel-based ctDNA sequencing is widely used to identify actionable genomic alterations, but limited coverage and sensitivity hinder broader clinical utility, especially in cancers driven by DDR LOF. Further, tissue-free, variant-based approaches for response monitoring have several challenges, including the limited number and low variant allele frequencies (VAF) of monitorable variants and contamination from non-tumor-derived sources (eg, clonal hematopoiesis [CH]). Newer panels may address these challenges with broader coverage, optimized bioinformatics, and the integration of epigenetic analysis for molecular response (MR). Methods Blood samples were collected from 2 phase 1 studies (TRESR; NCT04497116 and ATTACC; NCT04972110) of the ataxia telangiectasia and Rad3-related (ATR) inhibitor camonsertib in combination with gemcitabine or PARPi (talazoparib, niraparib, or olaparib). Cell-free DNA and PBMCs were sequenced using the combined genomic (&amp;gt;800 genes) and methylation (15Mb) GuardantINFINITY platform. Results The study included 117 patients (pts) with ovarian (n=29), breast (n=18), pancreatic (n=18), prostate (n=16) and other (n=36) cancers, enrolled with pathogenic alterations in BRCA2 (n=41), ATM (n=32), BRCA1 (n=23), PALB2 (n=8) and other genes (n=13). Enrollment alterations identified by local NGS were confirmed in 83% (94/114) of pts excluding 3 enrolled by protein loss. 50% (5/10) of complex alterations (large deletions or rearrangements) with sufficient circulating tumor fraction (cTF) were confirmed. Among pts with breast, ovarian, prostate, or pancreatic cancers previously treated with PARPi and/or platinum and harboring BRCA1/2 or PALB2 alterations, reversions were detected in 31% (17/54), with 6 pts reverted by 1 or more large intragenic deletions. A CRC pt with a clonal biallelic sATM alteration had 2 missense reversions in cis, an event not previously reported to occur in ATM. In pts with matched PBMCs, confirmed CH alterations were detected in 79% (44/56), including 59% (33/56) with at least 1 CH variant over 1% VAF. 106 pts had evaluable paired samples at baseline and on-treatment (taken between 2-12 weeks on-treatment [median, 3 weeks]). Using mVAF of the Guardant360 74-gene subset or methylation-based cTF, 72% or 86% were monitorable, respectively. In the subset of evaluable pts with matched PBMC sequencing (n=54), after PBMC-informed CH filtering, monitorability was reduced from 69% to 61% between G360 mVAF and G360 mVAF-CH, respectively. PBMC-informed CH filtering improved the correlation between variant-based and methylation-based monitoring (G360 mVAF, R=0.89, p=1.1e-11 vs G360 mVAF-CH, R=0.95, p=3e-15; Pearson correlation). Correlation of ctDNA monitoring metrics with clinical outcomes is ongoing. Conclusions Broad panel coverage enabled accurate diagnosis of complex DDR LOF and detection of reversion alterations. Methylation-based cTF allows monitoring of most pts in this cohort and consistent with previous observations may avoid signal contamination from CH for robust MR analysis. Citation Format: Ezra Rosen, Joseph D Schonhoft, Ian M Silverman, Arielle Yablonovitch, Sunantha Sethuraman, Parham Nejad, Danielle Ulanet, Julia Yang, Insil Kim, Kezhen Fei, Yi Xu, Errin Lagow, Shile Zhang, Mingyang Cai, Maria Koehler, Benedito A Carneiro, Stephanie Lheureux, Michael Cecchini, Benjamin Herzberg, Jorge S Reis-Filho, Victoria Rimkunas, Timothy A Yap. Circulating tumor DNA (ctDNA) genomic and epigenomic profiling (GuardantINFINITY) for diagnosis of DNA damage repair (DDR) loss of function (LOF) and response monitoring in the TRESR and ATTACC trials [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A123.

Detecting Protective Rare Variants for Alzheimer’s Disease from Whole‐Genome Sequencing in the Amish

AbstractBackgroundA shift in focus from risk to resilience for Alzheimer’s disease (AD) encourages efforts to uncover novel AD biological mechanisms. Rare variants identified through whole genome sequencing (WGS) may represent an important and understudied component of complex trait genetics. While population‐based studies are powered to discover associations with common genetic variants, founder populations are better powered for discovery of previously unknown rare alleles that have risen to higher frequency due to genetic drift.MethodWe examined WGS data from the Mid‐Western Amish population in a genome‐wide search for rare coding variants shared only among cognitively‐unimpaired (CU) individuals. The cognitive status of each individual was assigned via consensus review of medical history and neuropsychological testing. Allele frequencies were calculated across all samples and for CU and cognitively‐impaired (CI) groups separately. We defined rare variants as having an allele frequency &lt;0.05 across all samples. Variants with a minor allele count (MAC)&gt;10 in the CU group, and 0 in the CI group were annotated to determine likely loss of function.ResultAfter extensive QC, 1,048 samples were available to estimate overall allele frequency. Allele frequencies of rare variants (n = 11,854,817) within 634 CU (mean age = 81.53±6.10, 60% female) and within 184 CI (mean age = 84.77±6.60, 62% female) individuals were compared and 51,616 variants with MAC &gt;10 were found only in CU. There were 316 unique coding variants (288 missense, 4 inframe_insertion, 6 inframe_deletion, 11 frameshift, 4 stop_gained, 3 splice_donor) and 180 synonymous variants located within 425 unique genes. Among them, 7 missense variants and 7 synonymous variants had MAC &gt;20 and the remaining variants had MAC of 11‐20. The mean allele frequency of the 7 missense variants was 0.0038 in TOPMed compared to 0.0154 in our data. Two synonymous variants were located within proposed AD genes (NYAP1 and ZNF423). Additionally, several untranslated region or nonsense‐mediated decay transcript variants existed within 26 known AD gene regions, among which 8 of them are reported to harbor protective variants for AD.ConclusionNumerous rare variants potentially impacting gene function were found only in CU individuals, providing a rich resource for further investigation of genes that may provide protection from cognitive impairment.

TREM2 Burden associates solely with Alzheimer’s Disease, and decreases the Chance to become a Centenarian

AbstractBackgroundRare TREM2 variants are major risk factors for Alzheimer’s disease (AD), but TREM2 variants may also confer a higher risk for other neurodegenerative diseases and a lower probability for cognitively healthy aging. We studied the TREM2 burden associated with different types of dementia and cognitively healthy centenarians (CHCs).MethodWe included participants of the 100‐Plus Study, Amsterdam Dementia Cohort, Longitudinal Aging Study Amsterdam and various Dutch memory clinics. TREM2 variants (R47H, R62H, Q33X, R62C, T96K) were genotyped with a high density array or imputed using standard methods (TOPMED reference panel). To avoid family bias and ethnic differences, related individuals and those of non‐European ancestry were excluded. Frequencies of individual variants and the burden of TREM2 variants were compared in AD dementia, dementia with Lewy Bodies (DLB), frontotemporal dementia (FTD), cognitively normal controls and CHCs (correcting for 5 principal components).ResultWe identified 242 carriers of TREM2 mutations and 6,683 controls without a TREM2 mutation with a mean age of 69 years (SD ±14 years), 50% female (Table 1). TREM2 variants were significantly associated with AD dementia (OR 1.4, 95%CI 1.1‐1.8, p = 0.01) (Table 2). R47H conferred the highest risk for AD (OR 2.8, 95%CI 1.4‐5.6, p = 0.004), compared to 1.3‐fold increased risk of R62H (95%CI 0.9‐1.7, p = 0.11). The carrier frequency was not increased in DLB (OR 1.4, 95%CI 0.7‐2.6, p = 0.34) and was not increased in FTD compared to controls (OR 0.59, 95%CI 0.3‐1.3, p = 0.18). Of interest, the allele frequency of TREM2 variants in centenarians is half of the expected frequency (OR = 0.46, 95%CI 0.1‐1.1, p = 0.10), and only concerns R62H.ConclusionWe replicate the association of TREM2 with increased risk of AD. In contrast to previous reports no association was observed with DLB or FTD. Cognitively healthy centenarians show a negative trend for TREM2 allele frequency suggesting that carrying a TREM2 variant decreases chances of survival to extreme age.

Role of the redox state of the Pirin-bound cofactor on interaction with the master regulators of inflammation and other pathways.

Persistent cellular stress induced perpetuation and uncontrolled amplification of inflammatory response results in a shift from tissue repair toward collateral damage, significant alterations of tissue functions, and derangements of homeostasis which in turn can lead to a large number of acute and chronic pathological conditions, such as chronic heart failure, atherosclerosis, myocardial infarction, neurodegenerative diseases, diabetes, rheumatoid arthritis, and cancer. Keeping the vital role of balanced inflammation in maintaining tissue integrity in mind, the way to combating inflammatory diseases may be through identification and characterization of mediators of inflammation that can be targeted without hampering normal body function. Pirin (PIR) is a non-heme iron containing protein having two different conformations depending on the oxidation state of the iron. Through exploration of the Pirin interactome and using molecular docking approaches, we identified that the Fe2+-bound Pirin directly interacts with BCL3, NFKBIA, NFIX and SMAD9 with more resemblance to the native binding pose and higher affinity than the Fe3+-bound form. In addition, Pirin appears to have a function in the regulation of inflammation, the transition between the canonical and non-canonical NF-κB pathways, and the remodeling of the actin cytoskeleton. Moreover, Pirin signaling appears to have a critical role in tumor invasion and metastasis, as well as metabolic and neuro-pathological complications. There are regulatory variants in PIR that can influence expression of not only PIR but also other genes, including VEGFD and ACE2. Disparity exists between South Asian and European populations in the frequencies of variant alleles at some of these regulatory loci that may lead to differential occurrence of Pirin-mediated pathogenic conditions.

Genotypic and allelic frequencies of progressive rod-cone degeneration and other main variants associated with progressive retinal atrophy in Italian dogs.

Progressive retinal atrophy (PRA) is a group of canine inherited retinal disorders affecting up to 100 breeds. Genetic tests are available. The aim of this study was to retrospectively evaluate the genetic variants associated with PRA among dogs residing in Italy. Genetic data of 20 variants associated with different forms of PRA were collected through DNA tests over a 10-year period for several dog breeds in the Italian canine population. Allelic and genotypic frequencies were calculated. A total of 1467 DNA tests were conducted for 1180 dogs. Progressive rod-cone degeneration (PRCD) was the most tested form of PRA, with 58.15% (n = 853) of the DNA tests. Among the widespread breeds in Italy, Labrador retrievers and toy poodles showed a prevalence of heterozygous carriers higher than 15%. Among the others, 175 DNA tests for golden retrievers (GR) showed a prevalence of heterozygous carriers of 13.04% (n = 12) for GR-PRA1 and 8.43% (n = 7) for GR-PRA2. The zwergschnauzer breed was tested for the type B and/or the type B1 forms of PRA with 25.32% (n = 20) heterozygous carriers and 0%, respectively. The study offers an overview of the prevalence of PRCD and other PRA forms within some of the most popular breeds in Italy.

A Next-Generation Sequencing Study in a Cohort of Sicilian Patients with Parkinson's Disease.

Parkinson's disease (PD) is a multisystem and multifactorial disorder and, therefore, the application of modern genetic techniques may assist in unraveling its complex pathophysiology. We conducted a clinical-demographic evaluation of 126 patients with PD, all of whom were Caucasian and of Sicilian ancestry. DNA was extracted from the peripheral blood for each patient, followed by sequencing using a Next-Generation Sequencing system. This system was based on a custom gene panel comprising 162 genes. The sample underwent further filtering, taking into account the allele frequencies of genetic variants, their presence in the Human Gene Mutation Database, and their association in the literature with PD or other movement/neurodegenerative disorders. The largest number of variants was identified in the leucine-rich repeat kinase 2 (LRRK2) gene. However, variants in other genes, such as acid beta-glucosidase (GBA), DNA polymerase gamma catalytic subunit (POLG), and parkin RBR E3 ubiquitin protein ligase (PRKN), were also discovered. Interestingly, some of these variants had not been previously associated with PD. Enhancing our understanding of the genetic basis of PD and identifying new variants possibly linked to the disease will contribute to improved diagnostic accuracy, therapeutic developments, and prognostic insights for affected individuals.

SYNE1 gene novel variant is associated with myocardial infarction in young people with a family history of premature atherosclerosis

Abstract Introduction Genetic background is thought to be responsible for a family occurred premature atherosclerosis, including myocardial infarction (MI). Purpose The aim of the study was to investigate the role of genetic variants in young patients (aged &amp;lt;50 years) with MI and family history of premature atherosclerosis. Methods The studied group consisted of 70 patients aged 26-49 (mean 43.1, SD ±4.3), 17 women and 53 men, with MI and with family history of premature atherosclerosis, defined as MI or ischaemic stroke in first-degree relatives at age &amp;lt;65 years in women or &amp;lt;55 years in men. The mean BMI in the studied group was 28.8 kg/m2 (SD ±3.5), total cholesterol level 202.3 mg/dl (SD ±3.5), HDL 40.6 mg/dl (SD ±9.6), LDL 124.0 mg/dl (SD ±28.9), triglycerides 164.4 mg/dl (SD ±63.5), glucose 108.3 mg/dl (SD ±19.9) and creatinine 0.9 mg/dl (SD ±0.2). Regarding other risk factors 63 patients (90%) were smokers (mean 24.1 pack-years, SD ±13.0), 44 (62.8%) had hypertension, 11 (15.7%) had diabetes mellitus, and 8 (11.4%) had depression. All patients had diagnosed MI including MI with ST elevation (STEMI) in 18 cases (25.7%) and MI without ST elevation (NSTEMI) in 52 cases (74.3%). All patients, except 2 (no consent), had performed coronary angiography revealing 1-vessel coronary artery disease (VCAD) in 33 patients (47.1%), 2-VCAD in 17 patients (24.3%), and 3-VCAD in 20 patients (28.6%). The mean left ventricular ejection fraction (LVEF) was 51.8% (SD ±8.0). The total DNA has been extracted from whole peripheral blood samples. The targeted enrichment library was prepared and analyzed using New Generation Sequencing method on Hiseq4000, 2x150 cycles. Statistical analyses were performed using the R software package (www.r-project.org/). The results of sequencing were compared to data from the reference control population consisting of 597 healthy people (418 women, 179 men) aged 18-83 (mean 40.5, SD ± 12,4) as a whole and after matching with a studied group by age and gender in a proportion 1:3 (210 people, 51 women and 159 men, aged 18-77, mean 42.1, SD ±10.6) using Propensity Score Matching. Risks associated with detected variants were evaluated using Fisher’s exact test based on the allelic frequencies of variants in both groups. Results SYNE1 gene variant rs36215567 (NM_182961.4: c.20396+22A&amp;gt;G) has been detected in statistically significant higher incidence in the studied group in comparison to the control population (p=0.005) with OR=4,80 (95%CI 1,43-14,45) as well as when compared to the control population matched by age and gender (p=0.004) OR=9,31 (95%CI 1,64-95,41). There were no statistically significant differences in the incidence of familial hypercholesterolemia related gene variants: LDLR c.667G&amp;gt;A, PCSK9 c.658-36G&amp;gt;A, PCSK9 c.996+55C&amp;gt;A and APOB c.12382G&amp;gt;A between both cohorts. Conclusion A novel variant of SYNE1 gene is associated with myocardial infarction in young age patients with a family history of premature atherosclerosis.