Mean Allele Frequency Research Articles

Updates on Rare Genetic Variants, Genetic Testing, and Gene Therapy in Individuals With Obesity.

The goal of this paper is to aggregate information on monogenic contributions to obesity in the past five years and to provide guidance for genetic testing in clinical care. Advances in sequencing technologies, increasing awareness, access to testing, and new treatments have increased the utilization of genetics in clinical care. There is increasing recognition of the prevalence of rare genetic obesity from variants with mean allele frequency < 5% -new variants in known genes as well as identification of novel genes- causing monogenic obesity. While most of these genes are in the leptin melanocortin pathway, those in adipocytes may also contribute. Common variants may contribute either to higher lifetime tendency for weight gain or provide protection from monogenic obesity. While specific genetic mutations are rare, these segregate in individuals with early-onset severe obesity; thus, collectively genetic etiologies are not as rare. Some genetic conditions are amenable to targeted treatment. Research into the discovery of novel genetic causes as well as targeted treatment is growing over time. The utility of therapeutic strategies based on the genetic risk of obesity is an advancing frontier.

Abstract PO2-13-10: Predicting outcomes in locally advanced breast cancer through profiling post-neoadjuvant tissue-based minimal residual disease

Abstract Background: Neoadjuvant chemotherapy (NAC) for locally advanced breast cancer can down-stage disease prior to surgery, monitor chemo-sensitivity, and provide key prognostic information via pathologic response that guides adjuvant treatment. Greater residual disease as assessed by the residual cancer burden (RCB) index is associated with inferior outcomes but imperfectly predicts recurrence, particularly in triple negative breast cancer (TNBC) where there is heterogeneity of outcomes across RCB scores. As an alternative to RCB and PCR, blood-based minimal residual disease (MRD) from circulating tumor DNA (ctDNA) has been explored. However, poor sensitivity limits ctDNA, which is typically negative post-NAC when treatment decisions are made in patients who later recur. Methods: To address this lack of sensitivity, we developed tissue-based MRD (t-MRD) to assess molecular disease burden in post-NAC breast and lymph node resection tissue. We performed whole exome sequencing on diagnostic tumor and matched germline tissues from five pilot patients with early-stage TNBC with a range of responses to NAC. For each patient, we designed a personalized assay to track tumor-specific SNVs, combined with limited panel of genes recurrently mutated in breast cancer, to enable MRD detection. We then applied this assay to DNA from individual pathology blocks from the tumor resection via ultra-deep targeted hybrid capture. We assessed each sample for the presence of MRD using a Monte Carlo statistical framework to integrate all tumor mutations as previously described (Newman et al, Nature Biotechnology 2016). We aggregated results across all pathology blocks from each case to define each patient’s t-MRD profile and assessed the performance of t-MRD versus gold standard pathologic assessment. Results: We tracked a mean of 136 MRD reporters per case across 119 post-NAC blocks derived from five patients. Twenty-five samples had pathologic residual disease and 28 had detectable t-MRD. There was strong concordance between t-MRD mean allele frequency (AF) and tumor cellularity as assessed by pathology, defined as percent cellularity within the region containing viable tumor (Spearman r = 0.9052, p&amp;lt; 0.0001), and excellent classification of true positive pathology samples by genomic sequencing (AUC = 0.9675, p &amp;lt; 0.0001). Samples (n = 6) where t-MRD but not pathologic residual disease was detected had a lower mean AF than those where there was also histologic residual disease (p=0.0001). Among the five studied cases, we confirmed a molecular complete response with no t-MRD detected in the RCB-0 case (0/44 t-MRD positive blocks). In the two RCB-III cases, we observed more diffuse t-MRD than that assessed by pathology (n = 4 additional blocks with t-MRD but not pathologic disease detected), and in the RCB-II case, we detected minimal t-MRD in only a single block where pathology did not detect residual disease. Significance: By applying blood-based MRD assessment tools to tumor tissue, we developed t-MRD to profile molecular residual disease at the landmark post-NAC timepoint when disease burden is low and adjuvant treatment decisions are made. We demonstrated high concordance with gold standard pathologic assessment, with increased sensitivity and minimal false positive signal. Further studies assessing the additional prognostic value of t-MRD beyond stage-based prognostication and post-NAC RCB scores are ongoing. Citation Format: Julia Ransohoff, Mia Carleton, Alisha Birk, Gregory Bean, Melinda Telli, Ash Alizadeh, James Ford, George Duran, Michael Khodadoust, David Kurtz. Predicting outcomes in locally advanced breast cancer through profiling post-neoadjuvant tissue-based minimal residual disease [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-13-10.

Association of candidate alterations with primary resistance to KRAS G12D targeting in colorectal cancer.

170 Background: KRAS-mutant colorectal cancer (CRC) has a worse prognosis and greater resistance to therapy, attracting tremendous efforts to develop both allele-specific and pan-KRAS targeting agents including KRAS G12C and G12D inhibitors. CRC has demonstrated both primary and acquired resistance to KRAS G12C inhibitors, which can be better evaluated by circulating tumor DNA (ctDNA)-based next generation sequencing (NGS) methods without tissue sampling bias. Here, we utilized clinical ctDNA data to delineate the landscape of candidate alterations associated with primary resistance to KRAS G12D targeting in CRC. Methods: Samples from advanced CRC patients with Guardant360 data in 2021 were categorized into a KRAS G12D group and comparator groups of KRAS G12C and KRAS not detected (ND) according to KRAS mutation status. Demographics, MSI-H status, and blood tumor mutational burden (bTMB) were summarized and compared between groups using t-test for continuous variables and Fisher’s exact test for categorical variables. Co-occurrence patterns of alterations between KRAS and other genes were assessed. Primary resistance mutations were inferred by acquired resistance findings to KRAS G12C inhibitors reported by Awad et al. N Engl J Med. 2021 and Tanaka et al. Cancer Discov. 2021. Results: Among approximately 2500 samples, 16.8% had KRAS G12D, the most common KRAS mutation in CRC. The mean allele frequency of KRAS G12D (11.4%) is comparable to that of KRAS G12C (10.9%). Mean age (61.6 years), MSI-H frequency (3.8%), and mean bTMB (14.2 Mut/Mb) of KRAS G12D group is comparable to those of KRAS ND group. This contrasts with KRAS G12C with significantly lower mean age (60.2 years, p=0.01), lower MSI-H frequency (0.56%, p=0.03), and higher mean bTMB (16.3 Mut/Mb, p=0.02) compared to KRAS ND. Both KRAS G12D and G12C significantly co-occur with APC, PIK3CA, and SMAD4 alterations. FBXW7, AR, and KEAP1 alterations were unique co-occurrences to KRAS G12D, while KIT was unique to KRAS G12C. Candidate alterations associated with primary resistance in KRAS G12D CRC included simultaneously present mutations of KRAS Q61H (14.1%), G12V (6.4%), G12C (5.9%), G13D (3.6%), BRAF V600E (2.7%) and others, amplification of MET (2%), MYC (1.6%), and KRAS (1.4%), and FGFR3-TACC3 translocation (Table). Conclusions: KRAS G12D is associated with unique co-occurring molecular alterations compared to KRAS G12C in CRC. ctDNA-based NGS platforms can survey candidate alterations associated with primary resistance to KRAS G12D targeting in CRC. Further validation in preclinical models is needed. [Table: see text]

The Exact Stochastic Process of the Haploid Multi-Allelic Wright-Fisher Mutation Model.

Diffusion models are widely applied in population genetics, but their approximate solutions may not accurately capture the exact stochastic process. Nevertheless, this practice was necessary due to computing limitations, particularly for large populations. In this article, we develop the exact Markov chain algebra (MCA) for a discrete haploid multi-allelic Wright-Fisher model (MA-WFM) with a full mutation matrix to address this challenge. A special case of nonzero mutations between multiple alleles have not been captured by previous bi-allelic models. We formulated the mean allele frequencies for asymptotic equilibrium analytically for the tri- and quad-allelic case. We also evaluated the exact time-dependent Markov model numerically, presenting it concisely in terms of diffusion variables. The convergence with increasing population size to a diffusion limit is demonstrated for the population composition distribution. Our model shows that there will never be exact irreversible extinction when there are nonzero mutation rates into each allele and never be an exact irreversible fixation when there are nonzero mutation rates out of each allele. We only present results where there is no complete extinction and no complete fixation. Finally, we present detailed computations for the full Markov process, exposing the behavior near the boundaries for the compositional domains, which are non-singular boundaries according to diffusion theory.

The Impact of BRAF V600E Mutation Allele Frequency on the Histopathological Characteristics of Thyroid Cancer.

A BRAF V600E mutation in papillary thyroid cancer (PTC) has been shown to be associated with aggressive behavior. Nevertheless, not all BRAF V600E PTCs behave aggressively. Allele frequency (AF) is the number of mutated molecules divided by the total number of wild-type molecules at a specific location in the genome. The relationship between BRAF V600E AF and the histopathological features of thyroid malignancies is not well understood. We hypothesized that the BRAF V600E AF will correlate directly with aggressive histopathological behavior. The aim of this study was to examine this relationship. A retrospective chart review was performed for patients treated for BRAF V600E thyroid malignancies from 2019 to 2022 at McGill University tertiary care hospitals (n = 317). Patients with BRAF V600E-positive malignancies that included information on AF were included (n = 44). The correlation between AF and tumor histopathological features was analyzed. Out of the 44 nodules with a BRAF V600E mutation, those with aggressive features of PTC had a mean AF of 25.8%, which was significantly higher than the non-aggressive group with a mean AF of 10.25% (p = 0.020). Additionally, there was a statistically significant difference in mean AF between patients with a positive sentinel LN (29%) and those with a negative sentinel LN (17.8%) (p = 0.021). Classical PTC was present in 29.5% (13/44) of nodules, with a mean AF of 15.6%. The tall cell subtype was found in 64% (28/44) of nodules, with a mean AF of 23%. Solid and hobnail subtypes were less common in this study, and there was no statistically significant relationship between AF and histopathological subtypes (p = 0.107). Nodules smaller than 1cm had a mean AF of 13.3%, while nodules ranging from 1 2cm had a mean AF of 20.6%, and those larger than 2cm had a mean AF of 27.7%. However, no statistical difference was observed between AF and nodule size (p = 0.160). In this study, BRAF V600E mutations in conjunction with AF help to determine whether thyroid malignancies will display aggressive behavior. This pre-operative finding can help thyroid specialists to determine the extent of thyroidectomy and whether lymph node dissection is required.

Genetic diversity analysis of released Indian linseed (Linum usitatissimum L.) varieties using SSR markers

A genetic diversity analysis was performed on 91 released linseed varieties of India using 20 polymorphic linseed specific SSR markers. The genotypes were categorized in to 15 clusters. The mean allele frequency was 0.7 and Shannon diversity index was 4.15, indicating that the germplasm was highly diverse.

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.

Longitudinal Analysis of Circulating Tumor DNA (ctDNA) in Diffuse Large B-Cell Lymphoma (DLBCL) Using KAPA HyperCap Design Share Non-Hodgkin Lymphoma (KAPA HyperCap DS NHL) Next-Generation Sequencing (NGS) Panel

Introduction Studies have shown that dynamic changes in lymphoma ctDNA levels are associated with patient outcomes and that monitoring lymphoma ctDNA may help identify patients who are at risk of refractory disease or relapse. NGS panels can successfully identify patient-specific tumor reporter variants in plasma specimens using a tissue-naïve approach that avoids limitations of patient tissue availability. Recently, using the AVENIO Oncology Assay (AOA) NHL Test* validated by the Roche Molecular CAP/CLIA Laboratory, a pre-specified analysis plan using samples from ~800 DLBCL patients from the POLARIX study validated ctDNA as an early prognostic biomarker (Herrera et al., Blood 2022). To further support clinical research on early molecular response (EMR) to treatment and minimal residual disease (MRD) in NHL, we introduce a KAPA HyperCap DS NHL panel designed to cover 100% of the target regions in the AOA NHL Test used for the POLARIX study. We describe the NGS workflow and bioinformatics analysis suitable for identification and monitoring of ctDNA using a tissue-naïve approach. We further describe performance characterization studies and feasibility of ctDNA detection using plasma specimens from patients with DLBCL. Methods KAPA HyperCap DS NHL panel covers coding and/or untranslated regions of 383 genes, plus additional intergenic regions, for a total of 341 Kb. This panel is used in combination with KAPA HyperCap workflows and KAPA reagents on Illumina® platforms, to sequence plasma cfDNA and matched genomic (g)DNA to identify tumor-specific single-nucleotide variants (SNVs) and monitor the dynamics of ctDNA. ctDNA detection and monitoring are supported by open-source bioinformatics tools for a fully integrated MRD analysis solution. Contrived samples for workflow characterization were comprised of commercially available reference materials with known SNV allele frequencies (AF) mimicking cfDNA (SeraSeq® Complete Mutation Mix and Twist Pan-cancer Reference Standard), as well as pre-characterized healthy donor cfDNA. Clinically annotated DLBCL samples were characterized and serially diluted to demonstrate feasibility of longitudinal mutation analysis. Results Serially diluted cfDNA samples and high molecular weight gDNA samples were used to assess the performance of the plasma cfDNA and germline workflows. Library quality control metrics met the yield and size distribution criteria for sequencing. A median of 88 M raw reads were obtained across all libraries. De-duplication yielded a median coverage depth range from 5000-9100 across samples. Median on-target rate (% selected bases) was between 74% and 80%. Average error rate was between 0.00024 or 0.00031 mismatches/read depth. 9 and 3 variants were detected after germline and blocklist filtering in the two commercial reference samples. Variant calling sensitivity was 100% across replicates at 5, 1 and 0.5% AF. In the serial dilution analysis, ctDNA was detected in all replicates at 5, 0.1 and 0.05% mean AF. For samples at 0.01% mean AF, ctDNA detection sensitivity was 83%. Initial data demonstrate that 131 SNV reporters were detected at 23% mean AF in a plasma sample from a treatment-naïve DLBCL patient. 4 SNV reporters were detected at 1.9% mean AF in a plasma sample from an immunochemotherapy-treated DLBCL patient. After 10x and 500x dilution, ctDNA positivity was accurately called at 2.3% and 0.05% mean AF levels for the patient with 131 reporters, and at 0.19% mean AF level for the patient with 4 reporters (Monte Carlo p-value &amp;lt;0.0001). Conclusion The use of KAPA HyperCap DS NHL panel with KAPA HyperCap workflows and open-source bioinformatics tools enables the detection and monitoring of ctDNA for NHL research applications. In contrived samples, variants were detected with high reproducibility at AF as low as 0.05% and with good reproducibility at AF of 0.01%. For a DLBCL patient with 131 SNV reporters in plasma, ctDNA was detectable down to 0.05% mean AF levels, while for a DLBCL patient with 4 SNV reporters, ctDNA was detectable down to 0.19% mean AF levels, demonstrating that sensitivity of Monte Carlo-based ctDNA detection depends on the number of reporter variants. *AVENIO Oncology Assay (AOA) NHL Test and KAPA HyperCap Design Share panels are for Research Use Only, not for use in diagnostic procedures. AVENIO and KAPA are trademarks of Roche. All other product names and trademarks are the property of their respective owners.

Early Prediction of Treatment Response By Circulating Tumor DNA Profiling in Patients with Diffuse Large B-Cell Lymphoma Receiving CAR T-Cell Therapy

Introduction Patients with relapsed or refractory diffuse large B-cell lymphoma (r/r DLBCL) are characterized by a particularly poor prognosis. Anti-CD19 chimeric antigen receptor (CAR) T-cell therapy has improved outcomes of patients with r/r DLBCL, yet a substantial proportion of patients still experiences relapse or progression after CAR T-cell treatment. Identifying patients at high risk of CAR T-cell therapy resistance or future lymphoma progression remains challenging. Here, we explored the value of circulating tumor DNA (ctDNA) as a prognostic biomarker at baseline and early into treatment in DLBCL patients undergoing standard-of-care CAR T-cell therapy, without the need for matched tumor genotypes. We further investigated associations between ctDNA and other known clinical risk factors. Methods We applied targeted next-generation sequencing (NGS) to a total of 53 samples from r/r DLBCL patients receiving CAR T-cell therapy (axicabtagene ciloleucel and tisagenlecleucel, n=16) at the University Medical Center Freiburg (Germany), using a modified version of the AVENIO ctDNA analysis workflow (Roche; Research Use Only) that covered ~314kb and targeted 466 distinct genes recurrently mutated in DLBCL (based on the CAPP-Seq workflow, Kurtz DM et al. J Clin Oncol 2018). Tumor genotypes were assessed noninvasively from plasma samples obtained at baseline before lymphodepletion with matched germline controls. Concentrations of ctDNA were quantified at baseline and early after CAR T-cell therapy at day 7-10. Initial treatment response was assessed by conventional PET-CT obtained 4-6 weeks after treatment based on the Lugano criteria. Results At baseline prior to lymphodepletion, we detected somatic mutations in 100% of plasma samples by noninvasive genotyping, with a median of 19.5 mutations per patient (range: 3-97). The most frequently mutated genes were IGHV, IGLL5, BCL2, MYC, BTG1, CREBBP, and TP53. Patients with high baseline ctDNA concentrations (mean allele frequency [AF] &amp;gt;= 1%) showed shorter progression-free survivial (PFS) and overall survival (OS) than patients with ctDNA levels below this threshold (median PFS: 86.5 days vs. not reached, p=0.04, HR: 6.3, 95% CI: 1.7-23.6; median OS: 219 days vs. not reached, p=0.048, HR: 4.1, 95% CI 1.0-16.6, Figure 1). Pretreatment ctDNA levels were significantly correlated with LDH concentrations ( p=0.049, r=0.5). We further found significantly higher ctDNA concentrations in patients with transformed DLBCL (vs. non-transformed, p=0.008) and in those with 3 or more prior treatment lines (vs. 1-2, p=0.04). At day 7-10 after CAR T-cell therapy, ctDNA levels dropped a median of 10-fold compared to the pretreatment time point ( p=0.008). Patients achieving a complete (CMR) or partial metabolic response (PMR) by PET-CT after 4-6 weeks showed a significant decrease of ctDNA ( p=0.001), while those with a stable (SD) or progressive disease (PD) had no significant ctDNA reduction at day 7-10 after CAR T-cell infusion. Vice versa, while 100% of patients with a &amp;gt;1.5-log-fold drop of ctDNA level between the pretreatment and interim time point revealed a CMR or PMR in early PET-CT scans, only 62% of patients with a smaller ctDNA decline responded to treatment according to PET-CT. Conclusions Our data suggests, together with previously published studies (Sworder BJ et al., Cancer Cell, 2023; Frank MJ et al. J Clin Oncol, 2021), that ctDNA profiling may allow robust noninvasive genotyping, accurate risk-stratification, and early prediction of clinical outcomes in r/r DLBCL patients undergoing CAR T-cell therapy. Although limited by small sample size, this pilot study highlights a potential future role of ctDNA measurements for personalized treatment selection and guidance in clinical trials.

Characterization of Clonal Hematopoietic of Indeterminate Potential (CHIP) Mutations in an Imid-Naïve Multiple Myeloma (MM) Autologous Stem Cell Transplant (ASCT) Population: First Results from a Pre-Transplant Time Point in a Prospective, Longitudinal Study

Background: Clonal Hematopoiesis of Indeterminate Potential (CHIP) is defined by the acquisition of ≥ 1 somatic mutations in the blood of healthy adults, but can also be detected at higher prevalence in patients with malignancies, particularly after DNA damaging therapy. The presence of CHIP mutations in MM confers a reduced overall survival (Mouhieddine, JCO 2020) but high quality, prospective evaluation of CHIP mutation evolution over the transplant sequence, including the impact of high-dose melphalan and immunomodulatory (IMiD)-based therapy, is largely under-studied. Serial assessment of CHIP over the transplant course has been reported from the coMMpass dataset, demonstrating a 4-fold increase in mutation prevalence from 5.8% at diagnosis to 25% after 3.1 years (Mouhieddine, Blood, 2021). In this data set, most patients were exposed to prior IMiD-containing at some point during therapy. Whether the survival benefit reported with IMiD-based maintenance therapy post-transplant is associated with CHIP mutation modulation is unclear. We are therefore investigating the longitudinal evolution of CHIP in a cohort of transplant-eligible, largely IMiD-naïve patients (pts) over the pre- and post-ASCT course in an ongoing study. Here, we present preliminary findings from the pre-transplant time-point after non-IMiD-containing induction but before high-dose melphalan and ASCT. Methods: The ongoing Princess Margaret Cancer Center ARCH 001 trial is a prospective, longitudinal study evaluating evolution of CHIP in a transplant-eligible MM population. Mutation testing is performed using the highly sensitive single-molecule molecular inversion probe (SmMIP) next-generation sequencing (NGS) technique (Abelson, Bioinformatics, 2022). After sequencing with a minimum sequencing depth of 4000x, the list of mutation calls produced by smMIP-tools is subject to various filters to reduce the likelihood of false positives. Synonymous mutations, as well as any variants falling within introns or splice regions are removed. The minor allele frequency of the variant (if available) is required to be above 0.1% so as to exclude mutations that are common SNPs. VAF frequency threshold of 1-30 % is set for calling somatic mutations, with a view to minimize false positives and germ-line mutations. Multiple time points for testing pertinent to therapy include: within 1 month pre-transplant (after induction and stem cell collection), 3 months post-transplant, and two subsequent samples 12 and 24 months post-sample 2. Here we report preliminary results of CHIP mutation testing in the first 66 pts at the pre-transplant time point. Results: Patient, disease and treatment characteristics for all pts and their stratification by the presence of CHIP mutations are outlined in Table 1. The total cohort is typical for a transplant-eligible population with median age 65 years, male predominance (M 59%/F 41%), 60% IgG subtype, 23% high-risk FISH cytogenetics. Most (90%) received a non-IMiD containing induction regimen CyBorD; only 6% received an IMiD pre-ASCT. CHIP mutations were identified in 28/66 pts, for a mutation prevalence rate of 43%; 6 patients (9%) carried ≥ 1 mutation. The median age at transplant as well as other baseline demographics did not differ between those with and without mutations (Table 1). Mutation profiles are shown in Figure 1, and are consistent with those described in the literature with the most commonly mutated genes being DNMT3a, TET2, ASXL1 and PPM1D, and the mutation prevalence rate highest for DNMT3a at 21%. 6 pts (9%) had ≥ 1 mutation (range 2-3) identified, with 3 of 6 involving the DNMT3a gene. Other genes involved in patients with multiple mutations were TET2 (n=3), ASXL1 (n=2), PPM1D (n=2), SRSF2 (n=1), SF3B1 (n=1). Mean allele frequency was 4%, ranging from 1-19. Conclusion: Our results using deep SmMIP sequencing show a mutation prevalence rate of 43% in a cohort of transplant-eligible, largely IMiD-naive MM pts studied after induction. These rates are higher than those generally described in the literature, which may relate to the high sensitivity of the assay used, variation in filtering between institutions, and the deliberate use of a relatively low VAF threshold with the intent to capture clinically significant variants for longitudinal tracking during the course of this ongoing study. Further studies during lenalidomide maintenance will be forthcoming.

Recurrence Prediction by Circulating Tumor DNA in the Patient with Colorectal Liver Metastases After Hepatectomy: A Prospective Biomarker Study.

The recurrence rate after hepatic resection of colorectal liver metastases (CRLM) remains high. This study aimed to investigate postoperative circulating tumor DNA (ctDNA) based on ultra-deep next-generation sequencing (NGS) to predict patient recurrence and survival. Using the high-throughput NGS method tagged with a dual-indexed unique molecular identifier, named the CRLM-specific 25-gene panel (J25), this study sequenced ctDNA in peripheral blood samples collected from 134 CRLM patients who underwent hepatectomy after postoperative day 6. Of 134 samples, 42 (31.3%) were shown to be ctDNA-positive, and 37 resulted in recurrence. Kaplan-Meier survival analysis showed that disease-free survival (DFS) in the ctDNA-positive subgroup was significantly shorter than in the ctDNA-negative subgroup (hazard ratio [HR], 2.96; 95% confidence interval [CI], 1.91-4.6; p < 0.05). When the 42 ctDNA-positive samples were further divided by the median of the mean allele frequency (AF, 0.1034%), the subgroup with higher AFs showed a significantly shorter DFS than the subgroup with lower AFs (HR, 1.98; 95% CI, 1.02-3.85; p < 0.05). The ctDNA-positivepatients who received adjuvant chemotherapy longer than 2 months showed a significantly longer DFS than those who received treatment for 2 months or less (HR, 0.377; 95% CI, 0.189-0.751; p < 0.05). Uni- and multivariable Cox regression indicated two factors independently correlated with prognosis: ctDNA positivity and no preoperative chemotherapy. The study demonstrated that ctDNA status 6 days postoperatively could sensitively and accurately predict recurrence for patients with CRLM using the J25 panel.

Abstract 212: True2 duplex sequencing reveals a diverse landscape of unique somatic mutations beyond the surgical margins of high- and low-grade diffuse gliomas

Abstract Introduction: High- and low-grade diffuse gliomas commonly recur regardless of therapy. Here, we sought to detect and profile molecular evidence of disease in nearby brain tissue absent of tumor by standard-of-care clinical imaging. Methods: Twenty-two patients presenting to the University of Utah with radiographic evidence of a new high- to low-grade intrinsic brain tumor were enrolled after providing written informed consent. Using intraoperative stereotactic guidance, tissue was acquired from bulky tumor (36 samples) and beyond the surgical margins in peritumoral edema (29 samples). All samples underwent somatic mutation discovery using a custom-designed 114 kb panel (115 genes) and True2 duplex sequencing. True2 is a duplex adapter-based technology developed and empirically validated to achieve &amp;gt;95% sensitivity for an allele frequency ≥0.1% at a false positive rate of &amp;lt;1 per 100 kb panel positions. Results: Somatic mutations were detected in all samples regardless of radiographic location. The mean number of mutations was similar between bulky tumor and peritumoral edema (5.6±2.4 vs. 5.1±2.9 mutations/sample, respectively; P=0.48). Although the mean allele frequency of variants was higher in tumor compared to edema (16.0±21.2% vs. 11.2±17.4%, respectively; P=0.017), there was a strong overlap in the allele frequency ranges (tumor: 0.07% to 90.3% vs. edema: 0.10% to 75.6%). Notably, one-third of all the somatic mutations detected had an allele frequency &amp;lt;0.5%. Common diagnostic mutations specific to each tumor type were present in both tumor and edema - TERT promoter mutations in glioblastoma and oligodendroglioma; IDH1/2 mutations in astrocytoma and oligodendroglioma. In edema, mutations associated with glioblastoma but unique to a single sample occurred in TP53, EGFR, PTEN, NF1, ATRX, SCN9A, and MTOR at an allele frequency ranging from 0.10% to 20.22%. The edema of astrocytomas similarly harbored unique mutations in TP53, PTEN, and NF1, but mutations in EGFR, ATRX, SCN9A, and MTOR were absent. In contrast to both glioblastoma and astrocytoma, oligodendrogliomas exhibited a diverse collection of unique CIC and PAFAH1B3 mutations in edema at an allele frequency between 0.13% and 3.63%. Conclusions: The application of True2 technology to conduct a discovery search for very-low frequency somatic mutations found the molecular burden between tumor and adjacent edema to be remarkably similar. Our findings indicate an extensive tumor presence beyond the surgical margins in diffuse gliomas with unique and abundant somatic mutations that may form the foundation of recurrent lesions. Molecular analysis of adjacent tissue identified as radiographically unaffected by bulky tumor to better characterize the mutational landscape of migratory subclones may directly impact personalized medicine for high- and low-grade diffuse glioma patients. Citation Format: Hunter Underhill, Michael Karsy, Christian Davidson, Sabine Hellwig, Samuel Stevenson, Sydney Vincenti, Charlie Dean, Brion Harrison, Eric Goold, Mary Bronner, Howard Colman, Randy Jensen. True2 duplex sequencing reveals a diverse landscape of unique somatic mutations beyond the surgical margins of high- and low-grade diffuse gliomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 212.

Genomic architecture of phenotypic extremes in a wild cervid

Identifying the genes underlying fitness-related traits such as body size and male ornamentation can provide tools for conservation and management and are often subject to various selective pressures. Here we performed high-depth whole genome re-sequencing of pools of individuals representing the phenotypic extremes for antler and body size in white-tailed deer (Odocoileus virginianus). Samples were selected from a tissue repository containing phenotypic data for 4,466 male white-tailed deer from Anticosti Island, Quebec, with four pools representing the extreme phenotypes for antler and body size after controlling for age. Our results revealed a largely homogenous population but detected highly divergent windows between pools for both traits, with the mean allele frequency difference of 14% for and 13% for antler and body SNPs in outlier windows, respectively. Genes in outlier antler windows were enriched for pathways associated with cell death and protein metabolism and some of the most differentiated windows included genes associated with oncogenic pathways and reproduction, processes consistent with antler evolution and growth. Genes associated with body size were more nuanced, suggestive of a highly complex trait. Overall, this study revealed the complex genomic make-up of both antler morphology and body size in free-ranging white-tailed deer and identified target loci for additional analyses.

Metacommunities, fitness and gradual evolution

We analyze the evolution of a multidimensional quantitative trait in a class-structured focal species interacting with other species in a wider metacommunity. The evolutionary dynamics in the focal species as well as the ecological dynamics of the whole metacommunity is described as a continuous-time process with birth, physiological development, dispersal, and death given as rates that can depend on the state of the whole metacommunity. This can accommodate complex local community and global metacommunity environmental feedbacks owing to inter- and intra-specific interactions, as well as local environmental stochastic fluctuations. For the focal species, we derive a fitness measure for a mutant allele affecting class-specific trait expression. Using classical results from geometric singular perturbation theory, we provide a detailed proof that if the effect of the mutation on phenotypic expression is small (“weak selection”), the large system of dynamical equations needed to describe selection on the mutant allele in the metacommunity can be reduced to a single ordinary differential equation on the arithmetic mean mutant allele frequency that is of constant sign. This invariance on allele frequency entails the mutant either dies out or will out-compete the ancestral resident (or wild) type. Moreover, the directional selection coefficient driving arithmetic mean allele frequency can be expressed as an inclusive fitness effect calculated from the resident metacommunity alone, and depends, as expected, on individual fitness differentials, relatedness, and reproductive values. This formalizes the Darwinian process of gradual evolution driven by random mutation and natural selection in spatially and physiologically class-structured metacommunities.

Population structure and phylogenetic relationships in Brassica rapa L. subspecies by using isozyme markers.

The present study aimed to assess population structure and phylogenetic relationships of nine subspecies of Brassica rapa L. represented with thirty-five accessions cover a wide range of species distribution area using isozyme analysis in order to select more diverse accessions as supplementary resources that can be utilized for improvement of B. napus. Enzyme analysis resulted in detecting 14 putative polymorphic loci with 27 alleles. Mean allele frequency 0.04 (rare alleles) was observed in Cat4A and Cat4B in sub species Oleifera accession CR 2204/79 and in subspecies trilocularis accessions CR 2215/88 and CR 2244/88. The highest genetic diversity measures were observed in subspecies dichotoma, accession CR 1585/96 (the highest average of observed (H0) and expected heterozygosity (He), and number of alleles per locus (Ae)). These observations make this accession valuable genetic resource to be included in breeding programs for the improvement of oilseed B. napus. The average fixation index (F) is significantly higher than zero for the analysis accessions indicating a significant deficiency of heteozygosity. The divergence among subspecies indicated very great genetic differentiation (FST = 0.8972) which means that about 90% of genetic diversity is distributed among subspecies, while 10% of the diversity is distributed within subspecies. This coincides with low value of gene flow (Nm = 0.0287). B. rapa ssp. oleifera (turnip rape) and B. rapa ssp. trilocularis (sarson) were grouped under one cluster which coincides with the morphological classification.

Genomic time-series data show that gene flow maintains high genetic diversity despite substantial genetic drift in a butterfly species.

Effective population size affects the efficacy of selection, rate of evolution by drift and neutral diversity levels. When species are subdivided into multiple populations connected by gene flow, evolutionary processes can depend on global or local effective population sizes. Theory predicts that high levels of diversity might be maintained by gene flow, even very low levels of gene flow, consistent with species long-term effective population size, but tests of this idea are mostly lacking. Here, we show that Lycaeides butterfly populations maintain low contemporary (variance) effective population sizes (e.g. ~200 individuals) and thus evolve rapidly by genetic drift. However, populations harboured high levels of genetic diversity consistent with an effective population size several orders of magnitude larger. We hypothesized that the differences in the magnitude and variability of contemporary versus long-term effective population sizes were caused by gene flow of sufficient magnitude to maintain diversity but only subtly affect evolution on generational timescales. Consistent with this hypothesis, we detected low but nontrivial gene flow among populations. Furthermore, using short-term population-genomic time-series data, we documented patterns consistent with predictions from this hypothesis, including a weak but detectable excess of evolutionary change in the direction of the mean (migrant gene pool) allele frequencies across populations and consistency in the direction of allele frequency change over time. The documented decoupling of diversity levels and short-term change by drift in Lycaeides has implications for our understanding of contemporary evolution and the maintenance of genetic variation in the wild.

Hearing loss and tinnitus: association studies for complex-hearing disorders in mouse and man.

Genome-wide association studies (GWAS) provide an unbiased first look at genetic loci involved in aging and noise-induced sensorineural hearing loss and tinnitus. The hearing phenotype, whether audiogram-based or self-report, is regressed against genotyped information at representative single nucleotide polymorphisms (SNPs) across the genome. Findings include the fact that both hearing loss and tinnitus are polygenic disorders, with up to thousands of genes, each of effect size of < 0.02. Smaller human GWAS' were able to use objective measures and identified a few loci; however, hundreds of thousands of participants have been required for the statistical power to identify significant variants, and GWAS is unable to assess rare variants with mean allele frequency < 1%. Animal studies are required as well because of inability to access the human cochlea. Mouse GWAS builds on linkage techniques and the known phenotypic differences in auditory function between inbred strains. With the advantage that the laboratory environment can be controlled for noise and aging, the Hybrid Mouse Diversity Panel (HDMP) combines 100 strains sequenced at high resolution. Lift-over regions between mice and humans have identified over 17,000 homologous genes. Since most significant SNPs are either intergenic or in introns, and binding sites between species are poorly preserved between species, expression quantitative trait locus information is required to bring humans and mice into agreement. Transcriptome-wide analysis studies (TWAS) can prioritize putative causal genes and tissues. Diverse species, each making a distinct contribution, carry a synergistic advantage in the quest for treatment and ultimate cure of sensorineural hearing difficulties.

Detection of Clonal Hematopoiesis Is a Rare Event in Patients with Dyskeratosis Congenita

Introduction: Telomeres shorten with each cell division and reflect the replicative history of a cell. Cells undergo replicative senescence when reaching a critically short telomere length (TL) or alternatively, may undergo clonal evolution due to increasing genetic instability. Thus, dependent of the integrity of the respective checkpoints, telomere shortening on the one hand represents an established predisposing factor for malignant transformation while on the other hand it is a known tumor suppressor mechanism.Dyskeratosis Congenita (DKC) is a multisystem disorder characterized by bone marrow failure, increased risk of cancer development and critically short telomeres. Premature telomere shortening is frequently caused by mutations in telomerase or related genes leading to reduced functional telomerase activity. In various acquired bone marrow failure syndromes, such as aplastic anemia, premature telomere shortening was linked to mutations in genes typically found in clonal disorders such as myelodysplastic syndromes (MDS). For patients with inherited DKC, no data is available about the frequency of gene mutations related to clonal hematopoiesis. In this study, we aimed to analyze MDS-related genes in patients with molecularly confirmed DKC.Methods and Patients: Analysis included 13 patients from the Aachen Telomeropathy Registry and the Freiburg pediatric MDS registry. All patients had molecularly confirmed mutations in DKC related genes (TERC n=6, TERT n=6, DKC1 n=1). TL was measured by flow-FISH. Next-generation sequencing (NGS) was carried out on peripheral blood due to the hypoplastic/aplastic bone marrow. A self-customized NGS panel (“telomere-panel”) for telomeropathy-causing genes (CTC1, DKC1, NHP2, NOP10, RTEL1, TERC, TERT, TCAB1, TINF2, USB1) was used to detect DKC-associated gene mutations and especially, the allele burden of each mutation to identify any deviation (of expected 50%) in the allele ratio of the underlying DKC causing mutation. Furthermore, we used a self-designed NGS-panel (“MDS/MPN-panel”) including genes harboring mutations which are associated with myelodysplastic syndrome, e.g. as markers for clonal hematopoesis (ABL, ASXL1, BARD, CALR, CBL, CEBPA, CHEK2, CSF3R, DNMT3A, ETNK1, ETV6, EZH2, IDH1, IDH2, JAK2, KIT, KRAS, MPL, NFE2, NRAS, PDGFRA, PTPN11, RUNX1, SETBP1, SF3A1, SF3B1, SH3B2 (LNK), SRSF2, TCF12, TET2, TP53, U2AF1). Again, allele burden was analyzed for all detected mutations.Results: Telomere lengthwas found to be below the first percentile in the lymphocytes of the peripheral blood in all 13 patients. Mean TL was 4.77 kb±0.66SD in the lymphocytes and 5.28kb±0.92SD in the granulocyte subpopulation, respectively. Regarding the performance of the NGS analysis, quality parameters were good (Q30>85%) and for both panels 95% of the expected area was covered with a coverage of >300 (MDS/MPN-panel) and >100 (telomere-panel). Using the “telomere-panel”, all detected mutations showed a mutational allele burden of about 50%, most likely according to heterozygosity of the detected mutations. Applying the “MDS-panel”, only 2 out of 13 patients revealed relevant MDS-linked mutations. One patient harbored a U2AF1 gene mutation (c.101C>T, p.Ser34Phe; allele burden 16%), recently described in MDS as acquired mutation and thus classified as pathogenic without further confirmation in addition to a DKC-causing TERT mutation (c.2915G>A; p.Arg972His). In another patient with an inherited TERC mutation, the ASXL1 variant (c.3244_3246CTGdel; freq.: 48% [4708/9347]; p.Leu1082del) was detected. This variant is being discussed to be pathogenic as bystander or rare germline variant (mean allele frequency /dbsnp: 0,002%). Finally, mean telomere length in granulocytes did not differ between patients with detected MDS-linked mutations (4.77 kb) and without mutations (4.78kb).Conclusions: In patients with DKC, clonal hematopoiesis with detection of MDS-related genes can be detected but is a rare event compared to other myeloid diseases associated with short telomeres, suggesting independence of clonal hematopoiesis from telomere length. Genetic mosaicism or epigenetic factors (e.g. genomic imprinting) can be discussed as a mechanism to allow clonal expansion in telomeropathies, but further studies are needed to elucidate the mechanism how cells carrying impaired telomerase activity can progress to clonality. DisclosuresCorbacioglu:Jazz Pharmaceuticals: Consultancy, Honoraria. Koschmieder:BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; Roche: Other: Clinical Trial participation; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support. Brummendorf:Takeda Pharmaceuticals International Co: Consultancy, Research Funding.

Genetic diversity and population structure among indigenous and imported goat breeds in Kenya

Population structure and relationship information among goats is critical for genetic improvement, utilization, and conservation. This study explored population structure and level of introgression among four goat breeds in Kenya: the indigenous Galla (n = 12) and three imported breeds, the Alpine (n = 29), Toggenburg (n = 31), and Saanen (n = 24). Genetic diversity was analyzed using four indices (polymorphic SNPs, mean allele frequency, observed and expected heterozygosity and inbreeding coefficient) within each breed. Population structure assessed using model-based clustering (ADMIXTURE) revealed four breeds according to their geographic regions in Kenya. Kenyan Alpine goats were the most admixed breed with about 10 % of its genome derived from Galla, 10 % and 6 % from Saanen and Toggenburg respectively. The association of Galla with other breeds was anticipated since the Galla breed was used as the founder population for crossbreeding with Saanen, Alpine and Toggenburg breeds. The relationship information evaluated by computing Reynolds genetic distance revealed five distinctive clusters: Alpine, Galla, Saanen, Toggenburg and some mixture of Alpine and Toggenburg. Saanen and Galla breeds seem to be the most genetically distinct among the sampled populations. The genetic variation among the goat populations observed will provide a good opportunity for sustainable utilization, conservation, and future genetic resource improvement programmes in goat breeds in Kenya.

Genetic variability in COVID-19-related genes in the Brazilian population

SARS-CoV-2 utilizes the angiotensin-converting enzyme 2 (ACE2) receptor and transmembrane serine protease (TMPRSS2) to infect human lung cells. Previous studies have suggested that different host ACE2 and TMPRSS2 genetic backgrounds might contribute to differences in the rate of SARS-CoV-2 infection or COVID-19 severity. Recent studies have also shown that variants in 15 genes related to type I interferon immunity to influenza virus might predispose patients toward life-threatening COVID-19 pneumonia. Other genes (SLC6A20, LZTFL1, CCR9, FYCO1, CXCR6, XCR1, IL6, CTSL, ABO, and FURIN) and HLA alleles have also been implicated in the response to infection with SARS-CoV-2. Currently, Brazil has recorded the third-highest number of COVID-19 cases worldwide. We aimed to investigate the genetic variation present in COVID-19-related genes in the Brazilian population. We analyzed 27 candidate genes and HLA alleles in 954 admixed Brazilian exomes. We used the information available in two public databases (http://www.bipmed.org and http://abraom.ib.usp.br/) and additional exomes from individuals born in southeast Brazil, the region of the country with the highest number of COVID-19 patients. Variant allele frequencies were compared with the 1000 Genomes Project phase 3 (1KGP) and gnomAD databases. We detected 395 nonsynonymous variants; of these, 325 were also found in the 1KGP and/or gnomAD. Six of these variants were previously reported to influence the rate of infection or clinical prognosis of COVID-19. The remaining 70 variants were identified exclusively in the Brazilian sample, with a mean allele frequency of 0.0025. In silico analysis revealed that seven of these variants are predicted to affect protein function. Furthermore, we identified HLA alleles previously associated with the COVID-19 response at loci DQB1 and DRB1. Our results showed genetic variability common to other populations and rare and ultrarare variants exclusively found in the Brazilian population. These findings might lead to differences in the rate of infection or response to infection by SARS-CoV-2 and should be further investigated in patients with this disease.