Changes In Allele Frequencies Research Articles

Antimalarial drug resistance profiling of Plasmodium falciparum infections in India using Ion Torrent deep sequencing

IntroductionTracking the emergence and spread of antimalarial drug resistance is critical for supporting progress towards the control and eventual elimination of malaria in South Asia, especially India. Plasmodium falciparum has evolved resistance to virtually every antimalarial drug, and significant progress has been made to identify the molecular genetic mechanisms involved in the most common types of resistance.MethodsAn amplicon sequencing protocol was used for molecular surveillance of antimalarial drug resistance in a total of 158 patient isolates collected from December 2012 to September 2015 from three sites in south, west and east India: Tamil Nadu, Gujarat, and Odisha respectively. Five full length Plasmodium falciparum genes whose mutant proteins are implicated in antimalarial drug resistance were investigated: Pfcrt for chloroquine, Pfdhfr for pyrimethamine, Pfdhps for sulfadoxine, Pfk13 for artemisinin and Pfmdr1 for resistance to multiple antimalarial drugs.ResultsWe observed a high proportion of wild-type Pfcrt and Pfdhfr haplotypes from the P. falciparum-dominant site Rourkela, while mutant Pfcrt and Pfdhfr haplotypes were fixed at the P. vivax dominant sites Chennai and Nadiad. The wild-type Pfdhps haplotype was predominant across all study sites. We identified mutations in the propeller domain of Pfk13, although they are not associated with resistance to artemisinin. Finally, using samples taken from the same patient on day 2, day 7, and day 14 after artemisinin combination treatment, we were able to observe changes in allele frequency of drug resistance genes during the course of an infection.DiscussionThis is the first high-throughput deep sequencing study of five full-length P. falciparum genes in clinical isolates from three different study sites in India with varying transmission ecologies. Amplicon sequencing based on ion-torrent has the potential to track changes in the frequency of drug resistant alleles as a patient is undergoing drug therapy, and to identify new resistance mutations as they increase in frequency in the patient. This study showed possibility of whole gene sequencing, leads to in-depth molecular surveillance of multiple antimalarial resistant candidates and furthermore suggests investigations on reversal of resistant genotypes due to usage of artemisinin combination therapy in P. falciparum prevalent areas of the country.

Abstract 6443: Enhanced detection of ctDNA molecular response for immunotherapy treated non-small cell lung cancer through analyses of cell-free and matched white blood cell DNA

Abstract Introduction: Immune checkpoint inhibitors (ICIs) have greatly improved clinical outcomes for patients with non-small cell lung cancer (NSCLC), however, programmed death-ligand 1 (PD-L1) and tumor mutation burden (TMB) have failed to consistently predict therapeutic response and outcomes in this population. There remains a critical unmet need to identify patients who are likely to experience clinical benefit from treatment, particularly as additional treatment regimens become available. Longitudinal cell-free tumor DNA (ctDNA) assessments have recently been applied as a strategy to identify molecular response (mR) and better predict long term clinical response. Through this real-world analysis of NSCLC, we explore the technical and biological considerations associated with determination of ctDNA mR through matched cell-free and white blood cell (WBC) DNA analyses and apply this optimized approach to assess therapeutic response to ICIs. Methods: Plasma from 50 patients with advanced or metastatic NSCLC who received ICIs from 2017-2019 at Hospital del Mar had detectable pre-treatment ctDNA and underwent analysis using the 521 gene PGDx elio plasma complete liquid biopsy assay. Patient blood was collected prior to the start of ICI therapy, early on-treatment (median 4 weeks; range 2.4-9 weeks) and longitudinally throughout clinical follow-up and disease progression. Matched WBCs were also analyzed for each patient to identify alterations arising from germline polymorphisms and clonal hematopoiesis (CH). mR was calculated as the percent change in mean variant allele frequency of the tumor-specific alterations assessed at the early on-treatment timepoint compared to the pre-treatment timepoint, and compared to radiographic response, progression-free, and overall survival (OS). Results: Best overall response for patients within the cohort was 4% complete response, 26% partial response, 34% stable disease, and 36% progressive disease. The removal of germline or CH variants allowed for improved characterization of tumor-specific somatic alterations and associations with radiographic and survival-based endpoints. With the integrated ctDNA and matched WBC approach, we observed a mR rate of 34% (17/50) with a median OS of 36.8 months compared to 9.2 months for patients experiencing molecular progression at the early on-treatment timepoint (HR 5.6, 95% CI 2.4-12.7, p<0.001, log-rank test). Specifically, of all patients that survived ≤12 months after initiation of treatment, 84% (21/25) were categorized as experiencing molecular progression through early on-treatment ctDNA analyses. Conclusions: This real-world study of ICI treated, advanced NSCLC highlights the opportunity afforded through integrated analyses of ctDNA and matched WBCs to accurately determine mR and the associations with radiographic response and OS. Citation Format: Jennifer B. Jackson, Pedro Rocha, James R. White, Andrew P. Georgiadis, Ellen L. Verner, Amy Greer, Rafael Bach Mora, Laura Masfarré Pintó, Nil Navarro, Álvaro Taus, Beatriz Bellosillo Paricio, Sergi Clavé, Mark T. Sausen, Edurne Arriola. Enhanced detection of ctDNA molecular response for immunotherapy treated non-small cell lung cancer through analyses of cell-free and matched white blood cell DNA [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 6443.

Correlated Allele Frequency Changes Reveal Clonal Structure and Selection in Temporal Genetic Data.

In evolving populations where the rate of beneficial mutations is large, subpopulations of individuals with competing beneficial mutations can be maintained over long times. Evolution with this kind of clonal structure is commonly observed in a wide range of microbial and viral populations. However, it can be difficult to completely resolve clonal dynamics in data. This is due to limited read lengths in high-throughput sequencing methods, which are often insufficient to directly measure linkage disequilibrium or determine clonal structure. Here, we develop a method to infer clonal structure using correlated allele frequency changes in time-series sequence data. Simulations show that our method recovers true, underlying clonal structures when they are known and accurately estimate linkage disequilibrium. This information can then be combined with other inference methods to improve estimates of the fitness effects of individual mutations. Applications to data suggest novel clonal structures in an E. coli long-term evolution experiment, and yield improved predictions of the effects of mutations on bacterial fitness and antibiotic resistance. Moreover, our method is computationally efficient, requiring orders of magnitude less run time for large data sets than existing methods. Overall, our method provides a powerful tool to infer clonal structures from data sets where only allele frequencies are available, which can also improve downstream analyses.

A quantitative genetic model of background selection in humans.

Across the human genome, there are large-scale fluctuations in genetic diversity caused by the indirect effects of selection. This "linked selection signal" reflects the impact of selection according to the physical placement of functional regions and recombination rates along chromosomes. Previous work has shown that purifying selection acting against the steady influx of new deleterious mutations at functional portions of the genome shapes patterns of genomic variation. To date, statistical efforts to estimate purifying selection parameters from linked selection models have relied on classic Background Selection theory, which is only applicable when new mutations are so deleterious that they cannot fix in the population. Here, we develop a statistical method based on a quantitative genetics view of linked selection, that models how polygenic additive fitness variance distributed along the genome increases the rate of stochastic allele frequency change. By jointly predicting the equilibrium fitness variance and substitution rate due to both strong and weakly deleterious mutations, we estimate the distribution of fitness effects (DFE) and mutation rate across three geographically distinct human samples. While our model can accommodate weaker selection, we find evidence of strong selection operating similarly across all human samples. Although our quantitative genetic model of linked selection fits better than previous models, substitution rates of the most constrained sites disagree with observed divergence levels. We find that a model incorporating selective interference better predicts observed divergence in conserved regions, but overall our results suggest uncertainty remains about the processes generating fitness variation in humans.

Changes in capture availability due to infection can lead to detectable biases in population-level infectious disease parameters.

Correctly identifying the strength of selection that parasites impose on hosts is key to predicting epidemiological and evolutionary outcomes of host-parasite interactions. However, behavioral changes due to infection can alter the capture probability of infected hosts and thereby make selection difficult to estimate by standard sampling techniques. Mark-recapture approaches, which allow researchers to determine if some groups in a population are less likely to be captured than others, can be used to identify infection-driven capture biases. If a metric of interest directly compares infected and uninfected populations, calculated detection probabilities for both groups may be useful in identifying bias. Here, we use an individual-based simulation to test whether changes in capture rate due to infection can alter estimates of three key metrics: 1) reduction in the reproductive success of infected parents relative to uninfected parents, 2) the relative risk of infection for susceptible genotypes compared to resistant genotypes, and 3) changes in allele frequencies between generations. We explore the direction and underlying causes of the biases that emerge from these simulations. Finally, we argue that short series of mark-recapture sampling bouts, potentially implemented in under a week, can yield key data on detection bias due to infection while not adding a significantly higher burden to disease ecology studies.

The contribution of gene flow, selection, and genetic drift to five thousand years of human allele frequency change

Genomic time series from experimental evolution studies and ancient DNA datasets offer us a chance to directly observe the interplay of various evolutionary forces. We show how the genome-wide variance in allele frequency change between two time points can be decomposed into the contributions of gene flow, genetic drift, and linked selection. In closed populations, the contribution of linked selection is identifiable because it creates covariances between time intervals, and genetic drift does not. However, repeated gene flow between populations can also produce directionality in allele frequency change, creating covariances. We show how to accurately separate the fraction of variance in allele frequency change due to admixture and linked selection in a population receiving gene flow. We use two human ancient DNA datasets, spanning around 5,000 y, as time transects to quantify the contributions to the genome-wide variance in allele frequency change. We find that a large fraction of genome-wide change is due to gene flow. In both cases, after correcting for known major gene flow events, we do not observe a signal of genome-wide linked selection. Thus despite the known role of selection in shaping long-term polymorphism levels, and an increasing number of examples of strong selection on single loci and polygenic scores from ancient DNA, it appears to be gene flow and drift, and not selection, that are the main determinants of recent genome-wide allele frequency change. Our approach should be applicable to the growing number of contemporary and ancient temporal population genomics datasets.

Uncovering the architecture of selection in two Bos taurus cattle breeds.

Directional selection alters the genome via hard sweeps, soft sweeps, and polygenic selection. However, mapping polygenic selection is difficult because it does not leave clear signatures on the genome like a selective sweep. In populations with temporally stratified genotypes, the Generation Proxy Selection Mapping (GPSM) method identifies variants associated with generation number (or appropriate proxy) and thus variants undergoing directional allele frequency changes. Here, we use GPSM on two large datasets of beef cattle to detect associations between an animal's generation and 11 million imputed SNPs. Using these datasets with high power and dense mapping resolution, GPSM detected a total of 294 unique loci actively under selection in two cattle breeds. We observed that GPSM has a high power to detect selection in the very recent past (<10 years), even when allele frequency changes are small. Variants identified by GPSM reside in genomic regions associated with known breed-specific selection objectives, such as fertility and maternal ability in Red Angus, and carcass merit and coat color in Simmental. Over 60% of the selected loci reside in or near (<50 kb) annotated genes. Using haplotype-based and composite selective sweep statistics, we identify hundreds of putative selective sweeps that likely occurred earlier in the evolution of these breeds; however, these sweeps have little overlap with recent polygenic selection. This makes GPSM a complementary approach to sweep detection methods when temporal genotype data are available. The selected loci that we identify across methods demonstrate the complex architecture of selection in domesticated cattle.

Suggested mechanism of CCR5Δ32, CCR2-64I and SDF 1-3'A allele frequency change in Polish and Lithuanian gene pools from the perspective of passing time.

The study aimed to determine the frequency of the alleles associated with hereditary immune response in 16 historical populations and assess which evolutionary forces may have contributed to the observed frequency fluctuation. The analysed polymorphic sites are located in three genes - CCR5, CCR2 and SDF 1 (CXCL12). Protein products are involved in the innate immune response and are also involved in various types of infections, autoimmune diseases and tumours. The frequency of the alleles found in the DNA of the studied individuals was determined by the Sanger methodology and was compared with the data obtained for modern populations. To confirm the authenticity of the obtained results, mtDNA HVRI haplotypes of all the studied samples were obtained and compared with the genetic database of the laboratory personnel who came into contact with the studied material. Based on the variability of allele frequency, advanced biostatistical analysis was used to distinguish the effect of natural selection from genetic drift, i.e. the forces operating on the polymorphic sites studied. All procedures were performed according to the guidelines for working with ancient DNA to avoid contamination with modern DNA molecules. 681 samples from 39 archaeological sites in Poland and Lithuania dated to the 40th century BC and the 19th century were studied. The biostatistical analysis showed that the fluctuations in the frequency of CCR5Δ32 in the analysed time interval could be mainly the effect of genetic drift. Nevertheless, for CCR2-64I and SDF 1-3'A, the results confirm the suggestion of negative selection as the mechanism involved. Since all the polymorphic sites encode the elements of innate immune response that are indirectly associated with the process of an HPV infection and the development of cervical cancer, the human papillomavirus may be a good candidate for a selection coefficient affecting the frequency of CCR2-64I and SDF 1-3'A. However, for CCR5Δ32, selection was not detected despite its proven role in the molecular mechanism involved in the response to an HPV infection. The presented work seems to be the first in which the problem of the pattern of CCR5Δ32, CCR2-64I and SDF 1-3'A frequency fluctuations in a temporal perspective was discussed, proposing HPV as a factor influencing the occurrence of the CCR2 and SDF1 alleles.

Evolution is more repeatable in the introduction than range expansion phase of colonization

Abstract How repeatable is evolution at genomic and phenotypic scales? We studied the repeatability of evolution during 8 generations of colonization using replicated microcosm experiments with the red flour beetle, Tribolium castaneum. Based on the patterns of shared allele frequency changes that occurred in populations from the same generation or experimental location, we found adaptive evolution to be more repeatable in the introduction and establishment phases of colonization than in the spread phase, when populations expand their range. Lastly, by studying changes in allele frequencies at conserved loci, we found evidence for the theoretical prediction that range expansion reduces the efficiency of selection to purge deleterious alleles. Overall, our results increase our understanding of adaptive evolution during colonization, demonstrating that evolution can be highly repeatable while also showing that stochasticity still plays an important role.

Effects of Recurrent Selection on Population Structure and Allele Frequencies in the M3S Maize Population

The effects of four cycles of recurrent selection on the allele frequencies of simple sequence repeat (SSR) markers and population structure were examined in the Maksimir 3 Synthetic (M3S) maize population (Zea mays L.). Genotyping of 32 plants from each selection cycle at 38 SSR loci revealed that the mean number of alleles per locus and the mean expected heterozygosity were preserved across selection cycles, indicating the maintenance of sufficient genetic variability in the population required for future genetic gain. The Waples test of selective neutrality revealed that genetic drift was the main force in changing allele frequencies in the population. The proportion of selectively non-neutral loci in single cycles of selection varied between 16% and 37%. Some non-neutral loci shared the same genomic locations with previously published QTLs controlling important agronomic traits. An analysis of molecular variance revealed that 5.6% of the genetic variation occurred among and 94.4% within cycle populations. Between 5% and 29% of loci were found to be in a significant Hardy–Weinberg (HW) disequilibrium, with the majority showing an excess of homozygosity. The excess of homozygosity at several loci was highly consistent across cycle populations, suggesting positive assortative mating as a possible cause of the observed HW disequilibrium. Linkage disequilibrium (LD) tests revealed that the M3S population was essentially in linkage equilibrium. The proportion of pairs of loci in significant LD varied from 0.1% to 1.8% across selection cycles, probably due to the effects of genetic drift and epistatic selection.

Circadian clock- and temperature-associated genes contribute to overall genomic differentiation along elevation in lichenized fungi.

Circadian regulation is linked to local environmental adaptation, and many species with broad climatic niches display variation in circadian genes. Here, we hypothesize that lichenizing fungi occupying different climate zones tune their metabolism to local environmental conditions with the help of their circadian systems. We study two species of the genus Umbilicaria occupying similar climatic niches (Mediterranean and the cold temperate) in different continents. Using homology to Neurospora crassa genes, we identify gene sets associated with circadian rhythms (11 core, 39 peripheral genes) as well as temperature response (37 genes). Nucleotide diversity of these genes is significantly correlated with mean annual temperature, minimum temperature of the coldest month and mean temperature of the coldest quarter. Furthermore, we identify altitudinal clines in allele frequencies in several non-synonymous substitutions in core clock components, for example, white collar-like, frh-like and various ccg-like genes. A dN/dS approach revealed a few significant peripheral clock- and temperature-associated genes (e.g. ras-1-like, gna-1-like) that may play a role in fine-tuning the circadian clock and temperature-response machinery. An analysis of allele frequency changes demonstrated the strongest evidence for differentiation above the genomic background in the clock-associated genes in U. pustulata. These results highlight the likely relevance of the circadian clock in environmental adaptation, particularly frost tolerance, of lichens. Whether or not the fungal clock modulates the symbiotic interaction within the lichen consortium remains to be investigated. We corroborate the finding of genetic variation in clock components along altitude-not only latitude-as has been reported in other species.

Recurrent selection effects on the genetic structure of maize landrace assessed by SSR markers

Recurrent selection method was applied in one maize landrace (Zea mays L.) population, Macaco, for one cycle of selection. In the present study the changes in genetic structure were investigated at 18 microsatellite loci. A total of 81 alleles were observed in cycle 0 (C0) population which 79 were maintained in cycle 1 (C1) population. Thus, a reduction of 2.5% in the allele frequency was observed after one cycle of selection in C1 population. The heterozygozity reduction was also observed after one cycle of selection. In nearly 44% and 55% of the loci evaluated in C0 and C1 populations, respectivelly, the changes in allelic frequencies in populations were not due exclusively by genetic drift effects. Although few changes have been observed in allelic frequencies, the grain yield increase was observed after one cycle of recurrent selection. The genetic differentiation estimated by GST and RST values, for microsatellite data between C0 and C1 populations indicate that genetic variability degree was maintained in C1 population. This result suggest that C1 population can be used in a new recurrent selection cycles.

A cosmopolitan inversion facilitates seasonal adaptation in overwintering Drosophila.

Fluctuations in the strength and direction of natural selection through time are a ubiquitous feature of life on Earth. One evolutionary outcome of such fluctuations is adaptive tracking, wherein populations rapidly adapt from standing genetic variation. In certain circumstances, adaptive tracking can lead to the long-term maintenance of functional polymorphism despite allele frequency change due to selection. Although adaptive tracking is likely a common process, we still have a limited understanding of aspects of its genetic architecture and its strength relative to other evolutionary forces such as drift. Drosophila melanogaster living in temperate regions evolve to track seasonal fluctuations and are an excellent system to tackle these gaps in knowledge. By sequencing orchard populations collected across multiple years, we characterized the genomic signal of seasonal demography and identified that the cosmopolitan inversion In(2L)t facilitates seasonal adaptive tracking and shows molecular footprints of selection. A meta-analysis of phenotypic studies shows that seasonal loci within In(2L)t are associated with behavior, life history, physiology, and morphological traits. We identify candidate loci and experimentally link them to phenotype. Our work contributes to our general understanding of fluctuating selection and highlights the evolutionary outcome and dynamics of contemporary selection on inversions.

Abstract PR006: Initial results from first-in-human study of AMG 193, an MTA-cooperative PRMT5 inhibitor, in biomarker-selected solid tumors

Abstract Background: AMG 193 is a second-generation protein arginine methyltransferase 5 (PRMT5) inhibitor that targets the MTA-bound state of PRMT5 in methylthioadenosine phosphorylase (MTAP)-null tumors. PRMT5 is responsible for methylation and gene silencing of cell-essential proteins dysregulated in cancer and is partially inhibited in tumors harboring MTAP deletion, which occurs in ~15% of solid tumors. First generation PRMT5 inhibitors were intolerable due to indiscriminate inhibition of PRMT5 leading to dose-limiting myelosuppression. In preclinical studies, AMG 193 demonstrated selective antitumor activity in MTAP-null models by further suppressing PRMT5 function while sparing normal function, thereby improving upon first generation molecules. We report the initial clinical results from dose-escalation in the ongoing first-in-human (FIH) study. Methods: AMG193 was orally administered in continuous 28-day cycles to patients (pts) with advanced MTAP-null solid tumors. Dose escalation proceeded via a BLRM method. The primary objectives include safety, tolerability, and identification of the maximum tolerated dose (MTD). Secondary objectives include preliminary antitumor activity by investigator-assessed RECIST, pharmacokinetics (PK) and pharmacodynamic (PD) effects. Results: As of August 8, 2023, 47 pts with MTAP-null cancer (PDAC n = 10; NSCLC n = 6; CCA = 5; MESO n = 3; others n = 23) were enrolled in seven escalating cohorts. Five pts had DLTs, and exploration continues per protocol to identify the MTD. The most common TRAEs were nausea (45%), fatigue (26%), decreased appetite (17%), and vomiting (17%). Preliminary PK analyses showed dose-proportional systemic exposure with a half-life of 7–11 hrs. Among 31 pts who had at least one postbaseline scan, there were 5 with confirmed PRs [PDAC (–100%), ovarian Sertoli-Leydig (–59%), RCC (–58%), esophageal (–46%), and gallbladder cancer (–63%), 1 each], 14 with stable disease (including 9 with some degree of tumor shrinkage), and 12 with disease progression. All PRs were ongoing at the data cutoff with treatment durations of 140–275 days. PD effects demonstrated dose-dependent reduction in serum total SDMA levels and complete PRMT5 inhibition was confirmed in five pts with on-treatment biopsies spanning multiple dose levels. Exploratory analysis of changes in variant allele frequency by ctDNA demonstrated rapid treatment effects that was predictive and correlated with response. Conclusion: AMG 193 is an MTA-cooperative PRMT5 inhibitor designed to induce synthetic lethality in MTAP-null solid tumors while sparing hematologic toxicity. The initial results of the FIH study demonstrate proof-of-concept with encouraging signs of preliminary clinical activity without evidence of myelosuppression. Dose escalation continues to proceed to establish the MTD. AMG 193 has demonstrated promise as a potential new therapeutic for pts with tumors that have MTAP loss. Citation Format: Jordi Rodon, Noboru Yamamoto, Toshihiko Doi, François Ghiringhelli, Maria-Elisabeth Goebeler, Yutaka Fujuwara, Miguel Villalona-Calero, Nicolas Penel, Amita Patnaik, Jean-Pascal Machiels, Alfredo Addeo, James Abbruzzese, Sophie Postel-Vinay, Sylvie Rottey, Chunxu Liu, Chen-Hua Chuang, William Kormany, Paul Hughes, Kiana Keyvanjah, Bert O'Neil. Initial results from first-in-human study of AMG 193, an MTA-cooperative PRMT5 inhibitor, in biomarker-selected solid tumors [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 PR006.

Bomedemstat (IMG-7289), an LSD1 Inhibitor, Manages the Signs and Symptoms of Essential Thrombocythemia (ET) While Reducing the Burden of Cells Homozygous for Driver Mutations

The ultimate goal in the treatment of ET is to eliminate mutant cells with the capacity to both proliferate and self-renew. Lysine-specific demethylase-1, LSD1, is an enzyme critical for regulating the proliferation of hematopoietic stem cells and the maturation of progenitors (Sprussel et al. 2012). Bomedemstat is an orally active LSD1 inhibitor that reduces platelets, inflammatory cytokines, and the mutant cell burden in mouse models of myeloproliferative neoplasms (Jutzi et. al., 2019). All current treatments - hydroxyurea, interferon, or anagrelide - have their limitations and do not substantially affect the natural history of ET. IMG-7289-CTP-201 was a global, open-label, Phase 2b study of bomedemstat (IMG-7289, MK-3543) taken once daily for 24+ weeks in patients with ET resistant to or intolerant of at least one standard treatment (NCT04254978). The primary endpoint was reduction of platelets to &amp;lt;400 x 10 9/L in the absence of new thromboembolic events. Exploratory endpoints included changes in frequencies of mutant alleles (VAF) assessed by deep sequencing (median exonic depth =1784 reads) of 261 genes of germline and somatic DNA. Homozygosity of mutant alleles was imputed when VAF &amp;gt;50% and/or loss-of-heterozygosity (LOH) was evident based on the difference between the minor allele frequency of flanking single nucleotide polymorphisms (“MAF method”) in germline versus granulocyteDNA. Single cell genotypes (scDNA) (Tapestri ®) were determined in stem/progenitor (CD34+) and monocytes (CD14+) cells in patients with LOH (using the MAF method) showing a decrease in LOH in whom follow-up samples were available. For patients treated ≥24 wks, 95% (61/64) had reduced platelet counts to ≤400 x 10 9/L without new thromboembolic events, in a median time of 10 wks. In the first 48 weeks, four patients experienced a thromboembolic event ( per PI) (1 PE, 2 TIAs, and 1 transient global amnesia). Of the 14/73 (19%) patients with Day 1 WBC count &amp;gt;10 x 10 9/L, 100% treated for ≥24 wks had reduced the WBC count to ≤10 x 10 9/L; mean Hb remained stable. In patients with baseline MPN-SAF TSS &amp;gt;20 (28/73), at Wk 24, 78% (18/23) experienced an improvement with 52% (12/23) improving ≥10 points. The median baseline score for worst fatigue was 4.0 (N=73); by Wk 24, the score had improved to 3.0 (N=62). Among all patients (N=73), the most common AEs regardless of causality were dysgeusia (55%), constipation (38%), thrombocytopenia (34%), and arthralgia (27%). Twenty-four patients reported serious AEs (SAE), with related events ( per PI) in 4 patients. Twenty-one patients discontinued treatment, 11 due to AEs (3 for dysgeusia), 7 subject's decisions, 1 PI decision, 1 disease progression, and 1 unrelated death due to aspiration pneumonia. There have been no safety signals or deaths related to drug in patients treated for up to 979 days. At Wk 24, 86% patients remained on study and 75% at Wk 48. At ~Wk 24, 39 of 46 (85%) of evaluable patients had a decrease in driver VAF (Fig. 1). The greatest changes in VAF occurred in patients with cells homozygous for a driver mutation (Fig. 1). Nine of ten patients with homozygous driver clones had follow-up data: by bulk sequencing of germline+granulocyte DNA, 7 showed a reduction in the proportion of homozygous cells. In all cases tested, scDNA of CD14+ (monocytes) and CD34+ (stem/progenitor) cells were qualitatively similar to results obtained with bulk granulocyte sequencing (Fig.2). By bulk sequencing, the proportion of homozygous cells dropped by a mean of 66% (-4% to -100% (-100% = homozygosity not detectable)). Decreases in LOH by bulk and sc sequencing during treatment with bomedemstat were identified in patients with homozygosity at JAK2, CALR, and MPL. This study demonstrates that treatment with bomedemstat results in a consistent hematologic response in the majority of patients with ET. Further, deep bulk sequencing of germline+granulocyte DNA reveals LOH ( i.e., homozygosity) events with high accuracy that enables determination of the distribution of homozygote, heterozygote and wild-type genotypes. Furthermore, this study shows that sequencing germline+granulocyte DNA provides a reliable qualitative view of the impact of treatment on the genotypic distribution among CD34+ cells as revealed by scDNA genotyping. Finally, bomedemstat showed activity reducing stem/progenitor cells homozygous for mutations in JAK2, CALR, and MPL.

Ropeginterferon Alfa-2b for Pre-Fibrotic Primary Myelofibrosis and DIPSS Low/Intermediate-1 Risk Myelofibrosis: Durable Responses and Evidence of Disease Modification

Background Most patients with primary myelofibrosis (PMF) in pre-/early fibrotic stage (pre-PMF) and at low or intermediate-1 risk by the dynamic international prognostic scoring system (DIPSS) will progress to higher risks or overt MF. There is currently no consensus on the optimal treatment for these patients. Ropeginterferon alfa 2b (ropeg) is a next-generation monopegylated interferon alfa-2b developed specifically to treat myeloproliferative neoplasms (MPN). Aims P1101MF is an on-going multicenter phase 2 study of ropeg in patients with pre-PMF and DIPSS low/intermediate-risk PMF. Methods Key eligibility included morphologically confirmed pre-PMF, overt PMF, post-polycythemia vera MF (PPV-MF) or post-essential thrombocythaemia MF (PET-MF), DIPSS low/intermediate-1 risk, and the need for cytoreduction. The primary outcome were responses in haemoglobin (Hb, from 10 g/dL to upper reference range), white blood cell (WBC, &amp;lt; 10 x 10 9/L) and platelet (≤ 400 x 10 9/L) at 24 and 48 weeks. Secondary outcomes included adverse events (AEs), changes in mutant allele frequencies (MAF) of driver and non-driver genes as assessed by droplet digital polymerase chain reaction (ddPCR), quality-of-life (QOL), cytokine profiles and bone marrow morphology. Patients received ropeg at a dose of 250 mcg at week 1, followed by 350 mcg at week 2 and 500 mcg every 2 weeks from week 4 onwards. Results At the data cut-off of 30 June 2023, 37 men and 29 women with a median age of 59 (range: 30-86) years were enrolled. The diagnoses were pre-PMF (=46, 69.6%); overt PMF (N=6, 9.1%); PPV-MF (N=5, 7.5%); and PET-MF (N=9, 13.6%). Mutational profile of driver genes was JAK2V617F (N=46, 69.6%); CALR mutations (type 1/type-1 like, N=13, 19.7%; type 2/type 2-like, N=4, 6.1%); MPL mutation (N=1, 1.5%); and triple-negative (N=3, 4.5%). The median time from diagnosis to treatment was 5.8 (1-271) months. The median baseline parameters were white blood cell count (WBC): 6.49 (3.25-33.95) x 10 9/L; haemoglobin (Hb): 12.5 (7.1-15.9) g/dL; platelet count: 495 (113-1114) x 10 9/L; and lactate dehydrogenase: 277 (136-1146) IU/L. The median follow-up was 69 (4-69) weeks, with 61 patients (92%) and 51 patients (77%) having completed 24 and 48 weeks of treatment respectively. Responses in Hb, WBC and platelet were 75%, 82% and 74% respectively at 24 weeks, and 82%, 80% and 71% respectively at 48 weeks. Reduction of MAF at 24 and 48 weeks was achieved for JAK2V617F in 50% (19/38) of patients (≥50%, N=10, 26%; complete, N=3, 8%) and 79% (22/28) of patients (≥50%, N=6, 21%; complete, N=1, 4%) respectively; and for CALR in 44% (7/16) of patients (≥50%, N=1, 6%) and 50% (5/10) of patients (≥50%, N=1, 10%) respectively. Forty-six patients (69.6%) had bone marrow biopsies serially performed, with 42 patients (91%) having stable/improved marrow fibrosis. Eight patients (17.4%) had resolution of marrow fibrosis by 48 weeks. There were 5 discontinuations (personal reasons, N=1; intractable pruritus, N=1; symptom/spleen size progression, N=3). Disease progression (pre-PMF to overt PMF; development of accelerated/blast phase) was not observed. Non-haematological AEs included transaminitis (grade 1-2, N=34); malaise (grade 1-2, N=27; grade 3-4, N=1) and hair loss (grade 1-2, N=21). The most common haematological AE was anaemia (grade 1-2, N=15; grade 3-4, N=6). Conclusion: Ropeg was well-tolerated, effective in cytoreduction and induced molecular and morphologic responses in patients with pre-PMF and low/intermediate-1-risk MF.

Efficacy of the Allosteric MEK Inhibitor Trametinib in Relapsed and Refractory Juvenile Myelomonocytic Leukemia: A Report from the Children's Oncology Group

Background: Juvenile myelomonocytic leukemia (JMML) is an aggressive myeloproliferative neoplasm of infants and toddlers. Upfront therapies typically include high-dose cytarabine or azacitidine, but the only definitive treatment is hematopoietic stem cell transplantation (HSCT). While HSCT cures ~50% of patients, the prognosis is dismal for those who relapse. In the absence of a second HSCT, patients who relapse have a 2-year overall survival of ~10%. JMML is initiated by germline and somatic driver mutations in NF1, KRAS, NRAS, PTPN11, and CBL. These mutations converge on Ras signaling, leading to elevated levels of active Ras-GTP in specific cell lineages. Genetically engineered mouse (GEM) models accurately model key molecular, biologic, and biochemical features of JMML. Preclinical trials of the allosteric MEK inhibitors in Kras and Nf1 mutant mice demonstrated dramatic phenotypic responses with reduction in white blood cell counts, resolution of splenomegaly, and reversion to normal erythropoiesis. Based on the promising efficacy signal in GEM models, we evaluated trametinib, an orally bioavailable allosteric inhibitor of MEK1/2, in a prospective clinical trial in children with relapsed or refractory JMML to determine the overall response rate to trametinib. Results: Ten infants and children with JMML (median age 23.6 months) were enrolled and all were evaluable for safety and efficacy. Patients received age-adjusted dosing of trametinib for 28-day cycles and could remain on study for up to 12 cycles in the absence of disease progression or toxicity. A clonal Ras pathway mutation was confirmed in the blood and/or bone marrow of all patients. The objective response rate was 50% (two complete and three partial clinical responses). Four patients proceeded to HSCT after receiving protocol therapy and remain alive in complete remission with undetectable levels of the Ras pathway mutation identified at enrollment. Three additional patients completed 12 cycles of trametinib and continue to receive off-protocol therapy 6-24 months later with no change in the variant allele frequency of the underlying Ras pathway mutation. The remaining three patients had progressive disease with two demonstrating molecular evolution by the end of cycle 2. Paired pre- and post-trametinib RNASeq and proteomic analyses confirmed on-target biochemical effects of trametinib with down-regulation of both Ras/MAPK pathway related gene expression and MEK1/2 kinase activity, respectively. To gain deeper insight into how trametinib might affect distinct populations of hematopoietic cells, we generated single cell RNASeq data before and after treatment. The most prominent finding was a reduction in the proportions of classical and non-classical monocytes and broad downregulation of immune-related pathways in all cell populations. However, the downregulation of MAPK signaling genes was confined to specific cell types including, macrophages, classical monocytes, and granulocyte-monocyte progenitors, which uniquely displayed downregulated KRAS-related signatures following treatment. High DNA methylation and the presence of &amp;gt;1 mutation at enrollment correlated with lack of response to trametinib. Conclusions: We conducted an open label, phase 2 trial of trametinib in children with relapsed or refractory JMML. This is the first completed study of a MEK inhibitor in any hematologic malignancy in children. The trial met its primary objective and demonstrated a 50% objective response rate with 70% of patients bridging to a successful HSCT or completing the maximum 12 cycles permitted on study. The three patients who completed 12 cycles continue to receive trametinib off study for as long as 2 years. Although limited by a small number of patients, the long-term survival, and correlative molecular analyses from this trial raise provocative questions about whether certain patients with JMML might be spared the long-term adverse health risks of genotoxic HSCT conditioning regimens based on favorable molecular characteristics at diagnosis. This hypothesis will now be tested in a national, risk-stratified therapeutic trial (NCT05849662) of trametinib in combination with azacitidine in newly diagnosed patients. The authors would like to acknowledge funding and support from: NCTN Operations Center Grant (U10CA180886), NCTN Statistics &amp; Data Center Grant (U10CA180899), and the St. Baldrick's Foundation.

Global Frequency Analyses of Canine Progressive Rod-Cone Degeneration-Progressive Retinal Atrophy and Collie Eye Anomaly Using Commercial Genetic Testing Data.

Hundreds of genetic variants associated with canine traits and disorders have been identified, with commercial tests offered. However, the geographic distributions and changes in allele and genotype frequencies over prolonged, continuous periods of time are lacking. This study utilized a large set of genotypes from dogs tested for the progressive rod-cone degeneration-progressive retinal atrophy (prcd-PRA) G>A missense PRCD variant (n = 86,667) and the collie eye anomaly (CEA)-associated NHEJ1 deletion (n = 33,834) provided by the commercial genetic testing company (Optigen/Wisdom Panel, Mars Petcare Science & Diagnostics). These data were analyzed using the chi-square goodness-of-fit test, time-trend graphical analysis, and regression modeling in order to evaluate how test results changed over time. The results span fifteen years, representing 82 countries and 67 breeds/breed mixes. Both diseases exhibited significant differences in genotype frequencies (p = 2.7 × 10-152 for prcd-PRA and 0.023 for CEA) with opposing graphical trends. Regression modeling showed time progression to significantly affect the odds of a dog being homozygous or heterozygous for either disease, as do variables including breed and breed popularity. This study shows that genetic testing informed breeding decisions to produce fewer affected dogs. However, the presence of dogs homozygous for the disease variant, especially for prcd-PRA, was still observed fourteen years after test availability, potentially due to crosses of unknown carriers. This suggests that genetic testing of dog populations should continue.

Molecular surveillance of insecticide resistance in Phlebotomus argentipes targeted by indoor residual spraying for visceral leishmaniasis elimination in India.

Molecular surveillance of resistance is an increasingly important part of vector borne disease control programmes that utilise insecticides. The visceral leishmaniasis (VL) elimination programme in India uses indoor residual spraying (IRS) with the pyrethroid, alpha-cypermethrin to control Phlebotomus argentipes the vector of Leishmania donovani, the causative agent of VL. Prior long-term use of DDT may have selected for knockdown resistance (kdr) mutants (1014F and S) at the shared DDT and pyrethroid target site, which are common in India and can also cause pyrethroid cross-resistance. We monitored the frequency of these marker mutations over five years from 2017-2021 in sentinel sites in eight districts of north-eastern India covered by IRS. Frequencies varied markedly among the districts, though finer scale variation, among villages within districts, was limited. A pronounced and highly significant increase in resistance-associated genotypes occurred between 2017 and 2018, but with relative stability thereafter, and some reversion toward more susceptible genotypes in 2021. Analyses linked IRS with mutant frequencies suggesting an advantage to more resistant genotypes, especially when pyrethroid was under-sprayed in IRS. However, this advantage did not translate into sustained allele frequency changes over the study period, potentially because of a relatively greater net advantage under field conditions for a wild-type/mutant genotype than projected from laboratory studies and/or high costs of the most resistant genotype. Further work is required to improve calibration of each 1014 genotype with resistance, preferably using operationally relevant measures. The lack of change in resistance mechanism over the span of the study period, coupled with available bioassay data suggesting susceptibility, suggests that resistance has yet to emerge despite intensive IRS. Nevertheless, the advantage of resistance-associated genotypes with IRS and under spraying, suggest that measures to continue monitoring and improvement of spray quality are vital, and consideration of future alternatives to pyrethroids for IRS would be advisable.

119 Pedigree and Population-Based Genomic Inbreeding Trends Over Time in Five Commercial Swine Breeding Populations

Abstract Controlling inbreeding within a population is critical to maintain additive genetic variance and reduce negative consequences such as inbreeding depression in small, closed herds. Some breeding programs may utilize subjective standard operating procedures (SOPs) such as setting the minimum number of boars, limiting the number of matings per boar, and limiting the relationships among active boars. Software programs such as Matesel can control inbreeding within a population more precisely using evolutionary algorithms by optimizing the mating and selection process to constrain inbreeding at a given target degree or other metric specified (e.g., coancestry). The main problem has been the choice of what relationship matrix to use in constraining inbreeding in these programs. Furthermore, there are many different genomic inbreeding metrics that can be used to quantify inbreeding rates over time. The objective of the current research was to investigate the trends in the pedigree [PED, (A diagonal – 1) * 100] and two population-based genomic inbreeding metrics. One based on expected homozygosity (2pq, Ghom) and one based on drift (squared allele frequency changes, Gdrift). Data included nucleus animals born from 2017 through 2022 from five different genetic lines (lines A-E). Most parents and male selection candidates were genotyped using the commercial 50k SNP panel from GeneSeek. Pedigree was traced back at least 3 generations. A total of 80k animals were sampled from the total number of genotyped animals for computational reasons across line and birth year. Results show that in general, the maternal lines (A and B) showed the least rate of inbreeding over time (0.30 and 0.43% / year for A and B, respectively), while terminal lines were greater, especially line D (0.36 to 0.76% / year). Pedigree inbreeding in 2022 was between 1.48% (line A) to 3.80% (line D) after subtracting the average pedigree inbreeding in 2017. Population-based genomic measures of inbreeding showed a very small difference in line A (0.09%), larger Gdrift than Ghom in line B (1.37%), and larger Ghom than Gdrift in lines C, D, and E (2.87% to 4.38%). In general, the trend for PED and Gdrift was very similar for all lines, while Ghom tended to increase rapidly out of control in the three terminal lines (C-E) in later years. Future research will focus on integrating results from the diagonals of a variety of G matrices on an individual basis (e.g., VanRaden 1 and 2, runs-of-homozygosity, and a linkage-analysis matrix). Results showed the pedigree and genomic drift trends over time were similar, however, the genomic homozygosity trend was much greater in terminal lines.