High Allele Frequency Research Articles

P996: UTILITY OF KIT P.D816 IN MYELOID NEOPLASM WITHOUT DOCUMENTED SYSTEMIC MASTOCYTOSIS TO DETECT HIDDEN MAST CELLS IN BONE MARROW

Background:KIT p.D816 mutation is strongly associated with systemic mastocytosis (SM). Although it is not specific for SM, it is one of the diagnostic criteria for SM since its presence in the right morphologic context (i.e. presence of atypical mast cell clusters) is a strong support for neoplastic nature of mast cells. Next-generation sequencing (NGS) is now routinely performed in almost all bone marrow sample and KIT mutations are detected from patients who are not known or suspected to have SM. Aims: Therefore, we wanted to assess if KIT mutations in this patient population are associated with unsuspected SM. Methods: We searched NGS result in our institution between 1/1/2013 and 9/30/2021 with positive result for KIT mutation from patients with known/suspected myeloid neoplasms. Patients with previously documented history of systemic mastocytosis were excluded. Bone marrow biopsies from patients with KIT mutation were assessed with immunohistochemical stains for CD117 and mast cell tryptase (MST). Results: A total of 49 patients who had KIT mutation and had available formalin-fixed paraffin embedded blocks were identified. Thirty-eight (77.6%) patients has acute myeloid leukemia (AML) and 6 patients had chronic myelomonocytic leukemia (CMML, 12.2%). The remaining patients had myelodysplastic syndrome (n=1), myelodysplastic/myeloproliferative neoplasm, unclassifiable (n=1), myeloproliferative neoplasm (MPN, n=1), AML in remission (n=1) and therapy-related myeloid neoplasm (n=1). A total of 41 patients had a single KIT mutation and 8 patients had 2 or more KIT mutations. KIT p.D816V mutation was the most common mutation (n=38, 64.4%), followed by D816Y (n=8, 13.6%), D816H (n=5, 8.5%), Y418_D419insFF (n=2, 3.4%), R815_D816insVL (n=1, 1.7%), S197L (n=1, 1.7%), K558E (n=1, 1.7%), D419del (n=1, 1.7%), N822K (n=1, 1.7%) and R815_D816insIPP (n=1, 1.7%). Immunohistochemical stains for CD117 and MST were performed in all 49 patients. The MST stain was more suitable since 10 patients (20%) had increased blasts with CD117 expression which obscured morphologic review. A total of 4 patient (8.2%) showed mast cell nodules where spindled shaped mast cells were present, meeting the criteria for SM. All four patients had KIT p.D816V mutation and had high mutant allelic frequency (~50%) except one patient (1%). These patients had MPN (n=1), AML (n=1), AML in remission (n=1) and CMML (n=1), respectively. One (2%) patient has increased (>10%) mast cells but scattered throughout the bone marrow space without nodules and mast cells were round-to-ovoid shape. Eleven patients (22.4%) showed rare scattered mast cells and 33 patients (67.3%) did not show mast cells at all. Summary/Conclusion: We discovered approximately 8% of patients who had myeloid neoplasms with unexpected KIT mutations are met for systemic mastocytosis after additional immunohistochemical studies. Our data support that application of additional immunohistochemical studies are recommended to identify underrecognized SM when KIT mutations are unexpectedly found by molecular assays.

Genetic relationships and genome selection signatures between soybean cultivars from Brazil and United States after decades of breeding

Soybean is one of the most important crops worldwide. Brazil and the United States (US) are the world’s two biggest producers of this legume. The increase of publicly available DNA sequencing data as well as high-density genotyping data of multiple soybean germplasms has made it possible to understand the genetic relationships and identify genomics regions that underwent selection pressure during soy domestication and breeding. In this study, we analyzed the genetic relationships between Brazilian (N = 235) and US soybean cultivars (N = 675) released in different decades and screened for genomic signatures between Brazilian and US cultivars. The population structure analysis demonstrated that the Brazilian germplasm has a narrower genetic base than the US germplasm. The US cultivars were grouped according to maturity groups, while Brazilian cultivars were separated according to decade of release. We found 73 SNPs that differentiate Brazilian and US soybean germplasm. Maturity-associated SNPs showed high allelic frequency differences between Brazilian and US accessions. Other important loci were identified separating cultivars released before and after 1996 in Brazil. Our data showed important genomic regions under selection during decades of soybean breeding in Brazil and the US that should be targeted to adapt lines from different origins in these countries.

Benefit of Introgression Depends on Level of Genetic Trait Variation in Cereal Breeding Programmes.

We investigated the benefit from introgression of external lines into a cereal breeding programme and strategies that accelerated introgression of the favourable alleles while minimising linkage drag using stochastic computer simulation. We simulated genomic selection for disease resistance and grain yield in two environments with a high level of genotype-by-environment interaction (G × E) for the latter trait, using genomic data of a historical barley breeding programme as the base generation. Two populations (existing and external) were created from this base population with different allele frequencies for few (N = 10) major and many (N ~ 990) minor simulated disease quantitative trait loci (QTL). The major disease QTL only existed in the external population and lines from the external population were introgressed into the existing population which had minor disease QTL with low, medium and high allele frequencies. The study revealed that the benefit of introgression depended on the level of genetic variation for the target trait in the existing cereal breeding programme. Introgression of external resources into the existing population was beneficial only when the existing population lacked variation in disease resistance or when minor disease QTL were already at medium or high frequency. When minor disease QTL were at low frequencies, no extra genetic gain was achieved from introgression. More benefit in the disease trait was obtained from the introgression if the major disease QTL had larger effect sizes, more selection emphasis was applied on disease resistance, or more external lines were introgressed. While our strategies to increase introgression of major disease QTL were generally successful, most were not able to completely avoid negative impacts on selection for grain yield with the only exception being when major introgression QTL effects were very large. Breeding programmes are advised to carefully consider the level of genetic variation in a trait available in their breeding programme before deciding to introgress germplasms.

The H92R substitution in PSST is a reliable diagnostic biomarker for predicting resistance to mitochondrial electron transport inhibitors of complex I in European populations of Tetranychus urticae.

Mitochondrial Electron Transport Inhibitors of complex I (METI-I), such as tebufenpyrad and fenpyroximate, are acaricides that have been used extensively to control Tetranychus urticae Koch (Acari: Tetranychidae) for more than 20 years. Because of the ability of this spider mite to rapidly develop acaricide resistance, field (cross-) resistance monitoring and elucidation of resistance mechanisms are extremely important for resistance management (RM). In the present study, 42 European T. urticae field populations were screened for tebufenpyrad and fenpyroximate resistance, and the correlation between resistance and the H92R substitution in PSST was investigated. According to the calculated lethal concentration values that kill 90% of the population (LC90 ), tebufenpyrad and fenpyroximate would fail to control many of the collected populations at recommended field rates. Six populations exhibited high to very high resistance levels (200- to over 1950-fold) to both METI-Is. Analysis based on the LC50 values displayed a clear correlation between tebufenpyrad and fenpyroximate resistance, further supporting cross-resistance, which is of great operational importance in acaricide RM. The previously uncovered METI-I target-site mutation H92R in the PSST homologue of complex I (NADH:ubiquinone oxidoreductase) was found with high allele frequencies in populations resistant to tebufenpyrad and fenpyroximate. Synergist assays showed this mutation is not the only factor involved in METI-I resistance and additive or synergistic effects of multiple mechanisms most likely determine the phenotypic strength. The predictive value of resistance by H92R is very high in European populations and offers great potential to be used as a molecular diagnostic marker for METI-I resistance. © 2022 Society of Chemical Industry.

Pharmacogenomic Profile of Amazonian Amerindians.

Given the role of pharmacogenomics in the large variability observed in drug efficacy/safety, an assessment about the pharmacogenomic profile of patients prior to drug prescription or dose adjustment is paramount to improve adherence to treatment and prevent adverse drug reaction events. A population commonly underrepresented in pharmacogenomic studies is the Native American populations, which have a unique genetic profile due to a long process of geographic isolation and other genetic and evolutionary processes. Here, we describe the pharmacogenetic variability of Native American populations regarding 160 pharmacogenes involved in absorption, distribution, metabolism, and excretion processes and biological pathways of different therapies. Data were obtained through complete exome sequencing of individuals from 12 different Amerindian groups of the Brazilian Amazon. The study reports a total of 3311 variants; of this, 167 are exclusive to Amerindian populations, and 1183 are located in coding regions. Among these new variants, we found non-synonymous coding variants in the DPYD and the IFNL4 genes and variants with high allelic frequencies in intronic regions of the MTHFR, TYMS, GSTT1, and CYP2D6 genes. Additionally, 332 variants with either high or moderate (disruptive or non-disruptive impact in protein effectiveness, respectively) significance were found with a minimum of 1% frequency in the Amazonian Amerindian population. The data reported here serve as scientific basis for future design of specific treatment protocols for Amazonian Amerindian populations as well as for populations admixed with them, such as the Northern Brazilian population.

HLA Diversity in Saudi Population: High Frequency of Homozygous HLA Alleles and Haplotypes.

Human leukocyte antigens (HLA) diversity has a tremendous impact on shaping the transplantation practices, transfusion-associated graft versus host disease prevention strategies, and host–pathogen interactions. Here, we conducted a retrospective study of HLA class I and class II homozygosity at allelic and haplotype levels in unrelated individuals genotyped from 2012 to 2016 in a tertiary hospital in the capital of Saudi Arabia. Among 5,000 individuals, 2,773 individuals meet inclusion criteria and were retrospectively analyzed for HLA-A, -B, -C–DRB1, and -DQB1 homozygosity at allelic and haplotype levels. HLA molecular typing was performed using a commercial reverse sequence-specific oligonucleotide (rSSO) kit. We were able to identify 15 HLA-A, 20 HLA-B, 11 HLA-C, 13 HLA-DRB1, and five HLA-DQB1 homozygous alleles demonstrating a very low genetic diversity in the Saudi population. The highest homozygosity in HLA class I was found in locus C followed by A and B (20.3% > 16.1% > 15.5%; p < 0.001) where the most homozygote alleles were A*02 (9.2%), B*51 and B*50 (5.7% and 3.7%), and C*07, C*06, and C*15 (7.2%, 5.48%, and 3.3%) and in HLA class II, the highest homozygosity was found in locus DQB1 compared to DRB1 (31.71% > 19.2%; p < 0.001), with the most common homozygote alleles being DRB1*07 and DRB1*04 (5.33% and 4.2%) and DQB1*02, DQB1*06, and DQB1*03 (13.55%, 7.92%, and 7.64%). The frequency of finding an individual with one homozygote allele was (24.6%), two homozygote alleles (13.5%), three homozygote alleles (4.7%), four homozygote alleles (3.4%), and five alleles were (4.8%). The most frequent homozygote haplotypes are A*23∼C*06∼B*50∼DRB1*07∼DQB1*02 and A*02∼C*06∼B*50∼DRB1*07∼DQB1*02. This study shows low diversity of both class I and II alleles and haplotypes in the Saudi population, which would have a significant impact on shaping the transplantation practices, transfusion-associated graft versus host disease prevention strategies, and host–pathogen interactions.

2P Spectrum of BRCA1, BRCA1, ATM and PALB2 alleles in ovarian cancer patients from North Caucasus

BackgroundNorth Caucasus hosts several large ethnic groups, which preserved their national identity through the course of history. These populations are likely to have a unique pattern of disease-predisposing alleles reflecting the genetic background of their ancestors.MethodsThis study involved 180 ovarian cancer (OC) patients from Chechnya (n = 68), Kabardino-Balkaria (n = 49), North Ossetia (n = 32), Ingushetia (n = 16) and Dagestan (n = 15). The entire coding sequences of BRCA1, BRCA2, ATM and PALB2 genes were analyzed by next-generation sequencing.ResultsOC patients belonging to various ethnic groups had high frequency of BRCA1/2 mutations ranging from 18% to 33%. There were founder pathogenic alleles detected in Chechens (BRCA1 c.3629_3630delAG; 9 out of 15 BRCA1/2 mutations) and North Ossetians (BRCA2 c.6341delC; 6 out 8 BRCA1/2 mutations). Interestingly, Chechen BRCA1 c.3629_3630delAG allele was not present among patients of Ingush ethnicity, despite these nations are believed to have common roots. BRCA2 Q3299X mutation was repeatedly observed across several ethnic groups. Patients from Kabardino-Balkaria had unusually high frequency of germ-line ATM truncating alleles (3/49, 6%); all 3 ATM mutations were represented by distinct ATM pathogenic variants. There were no instances of PALB2 germ-line mutations.ConclusionsGenetic analysis of ovarian cancer patients is efficient in revealing ethnicity-specific BRCA1/2 mutations. Contribution of BRCA1/2 pathogenic alleles in OC morbidity is high across various ethnic groups. Founder BRCA1/2 alleles are characteristic for some but not all North Caucasus nations.Legal entity responsible for the studyThe authors.FundingThis study has been supported by the Russian Science Foundation, grant 21-75-30015.DisclosureAll authors have declared no conflicts of interest. BackgroundNorth Caucasus hosts several large ethnic groups, which preserved their national identity through the course of history. These populations are likely to have a unique pattern of disease-predisposing alleles reflecting the genetic background of their ancestors. North Caucasus hosts several large ethnic groups, which preserved their national identity through the course of history. These populations are likely to have a unique pattern of disease-predisposing alleles reflecting the genetic background of their ancestors. MethodsThis study involved 180 ovarian cancer (OC) patients from Chechnya (n = 68), Kabardino-Balkaria (n = 49), North Ossetia (n = 32), Ingushetia (n = 16) and Dagestan (n = 15). The entire coding sequences of BRCA1, BRCA2, ATM and PALB2 genes were analyzed by next-generation sequencing. This study involved 180 ovarian cancer (OC) patients from Chechnya (n = 68), Kabardino-Balkaria (n = 49), North Ossetia (n = 32), Ingushetia (n = 16) and Dagestan (n = 15). The entire coding sequences of BRCA1, BRCA2, ATM and PALB2 genes were analyzed by next-generation sequencing. ResultsOC patients belonging to various ethnic groups had high frequency of BRCA1/2 mutations ranging from 18% to 33%. There were founder pathogenic alleles detected in Chechens (BRCA1 c.3629_3630delAG; 9 out of 15 BRCA1/2 mutations) and North Ossetians (BRCA2 c.6341delC; 6 out 8 BRCA1/2 mutations). Interestingly, Chechen BRCA1 c.3629_3630delAG allele was not present among patients of Ingush ethnicity, despite these nations are believed to have common roots. BRCA2 Q3299X mutation was repeatedly observed across several ethnic groups. Patients from Kabardino-Balkaria had unusually high frequency of germ-line ATM truncating alleles (3/49, 6%); all 3 ATM mutations were represented by distinct ATM pathogenic variants. There were no instances of PALB2 germ-line mutations. OC patients belonging to various ethnic groups had high frequency of BRCA1/2 mutations ranging from 18% to 33%. There were founder pathogenic alleles detected in Chechens (BRCA1 c.3629_3630delAG; 9 out of 15 BRCA1/2 mutations) and North Ossetians (BRCA2 c.6341delC; 6 out 8 BRCA1/2 mutations). Interestingly, Chechen BRCA1 c.3629_3630delAG allele was not present among patients of Ingush ethnicity, despite these nations are believed to have common roots. BRCA2 Q3299X mutation was repeatedly observed across several ethnic groups. Patients from Kabardino-Balkaria had unusually high frequency of germ-line ATM truncating alleles (3/49, 6%); all 3 ATM mutations were represented by distinct ATM pathogenic variants. There were no instances of PALB2 germ-line mutations. ConclusionsGenetic analysis of ovarian cancer patients is efficient in revealing ethnicity-specific BRCA1/2 mutations. Contribution of BRCA1/2 pathogenic alleles in OC morbidity is high across various ethnic groups. Founder BRCA1/2 alleles are characteristic for some but not all North Caucasus nations. Genetic analysis of ovarian cancer patients is efficient in revealing ethnicity-specific BRCA1/2 mutations. Contribution of BRCA1/2 pathogenic alleles in OC morbidity is high across various ethnic groups. Founder BRCA1/2 alleles are characteristic for some but not all North Caucasus nations.

Identification of pharmacogenomic variants associated with oncology treatments in Brazilian Amazonian Amerindians.

e15082 Background: Adverse drug reactions are an important cause of morbidity and mortality commonly found in different therapeutic regimens, particularly in oncologic. About 20-30% of these reactions are due to the individual genetic variability of patients. Pharmacogenomics focuses on the identification of genetic variants that influence drug efficacy, response, and/or toxicity by changes in pharmacokinetics/pharmacodynamics. Pharmacogenomic investigations are known to have a population bias. There is a gap in the accumulated about pharmacogenomic variants in poorly studied populations, such as the Amerindian population from the Brazilian Amazon region. The objective of this work is to describe the pharmacogenetic variability regarding 160 pharmacogenes involved in pharmacokinetic processes and biological pathways of different therapies, including oncology treatments, based on data obtained through complete exome sequencing of 64 individuals from different Amerindian groups of the Brazilian Amazon. Methods: The present study was approved by the National Committee for Ethics in Research and the Research Ethics Committee of the UFPA Tropical Medicine Center, under CAAE number 20654313.6.0000.5172. All participants signed a free-informed consent. The DNA extraction was performed using phenol-chloroform. Libraries were prepared using the Nextera Rapid Capture Exome and SureSelect Human All Exon V6 kits. Bioinformatic analysis was performed by ViVa software. Results: Our results show a total of 3,311 variants found in the study subjects. Of this, 167 are exclusive variants found in the Amerindians. Among these new variants, we found a non-synonymous coding variant in the DPYD gene with an allelic frequency of 3%, and variants with high allelic frequencies in intronic regions, which may regulate gene expression: MTHFR (5.56%), TYMS (11.5%) GSTT1 (54.1%), and three variants in the CYP2D6 gene with frequencies of 54%, 62.3%, and 65.6%. The DPYD is a pivotal gene involved with the efficacy of fluoropyrimidine-based treatment; the MTHFR and TYMS genes also participate in the biological pathways of these drugs, responsible for variation in response rates. The GSTT1 is a gene associated with platinum-based treatments, such as carboplatin and cisplatin. And, finally, CYP2D6 is one of the main pharmacogene described, involved in several drug schemes, including tamoxifen and gefitinib, used to treat breast and lung cancer, respectively. Conclusions: Understanding the diversity of genetic markers in Amazonian Amerindian is crucial to the implementation of pharmacogenomic-guided oncology treatment protocols, since pharmacogenomic data validated in other ancestral groups may not be fully applicable in these populations due to their unique genetic profile. Keywords: Exome, Native American populations, fluoropyrimidine, platinum-based drugs, tamoxifen, gefitinib.

Benchmarking Low-Frequency Variant Calling With Long-Read Data on Mitochondrial DNA.

Background: Sequencing quality has improved over the last decade for long-reads, allowing for more accurate detection of somatic low-frequency variants. In this study, we used mixtures of mitochondrial samples with different haplogroups (i.e., a specific set of mitochondrial variants) to investigate the applicability of nanopore sequencing for low-frequency single nucleotide variant detection. Methods: We investigated the impact of base-calling, alignment/mapping, quality control steps, and variant calling by comparing the results to a previously derived short-read gold standard generated on the Illumina NextSeq. For nanopore sequencing, six mixtures of four different haplotypes were prepared, allowing us to reliably check for expected variants at the predefined 5%, 2%, and 1% mixture levels. We used two different versions of Guppy for base-calling, two aligners (i.e., Minimap2 and Ngmlr), and three variant callers (i.e., Mutserve2, Freebayes, and Nanopanel2) to compare low-frequency variants. We used F1 score measurements to assess the performance of variant calling. Results: We observed a mean read length of 11 kb and a mean overall read quality of 15. Ngmlr showed not only higher F1 scores but also higher allele frequencies (AF) of false-positive calls across the mixtures (mean F1 score = 0.83; false-positive allele frequencies < 0.17) compared to Minimap2 (mean F1 score = 0.82; false-positive AF < 0.06). Mutserve2 had the highest F1 scores (5% level: F1 score >0.99, 2% level: F1 score >0.54, and 1% level: F1 score >0.70) across all callers and mixture levels. Conclusion: We here present the benchmarking for low-frequency variant calling with nanopore sequencing by identifying current limitations.

Rare Alleles and Signatures of Selection on the Immunodominant Domains of Pfs230 and Pfs48/45 in Malaria Parasites From Western Kenya.

Background: Malaria elimination and eradication efforts can be advanced by including transmission-blocking or reducing vaccines (TBVs) alongside existing interventions. Key transmission-blocking vaccine candidates, such as Pfs230 domain one and Pfs48/45 domain 3, should be genetically stable to avoid developing ineffective vaccines due to antigenic polymorphisms. We evaluated genetic polymorphism and temporal stability of Pfs230 domain one and Pfs48/45 domain three in Plasmodium falciparum parasites from western Kenya. Methods: Dry blood spots on filter paper were collected from febrile malaria patients reporting to community health facilities in endemic areas of Homa Bay and Kisumu Counties and an epidemic-prone area of Kisii County in 2018 and 2019. Plasmodium speciation was performed using eluted DNA and real-time PCR. Amplification of the target domains of the two Pfs genes was performed on P. falciparum positive samples. We sequenced Pfs230 domain one on 156 clinical isolates and Pfs48/45 domain three on 118 clinical isolates to infer the levels of genetic variability, signatures of selection, genetic diversity indices and perform other evolutionary analyses. Results: Pfs230 domain one had low nucleotide diversity (π = 0.15 × 10–2) with slight variation per study site. Six polymorphic sites with nonsynonymous mutations and eight haplotypes were discovered. I539T was a novel variant, whereas G605S was nearing fixation. Pfs48/45 domain three had a low π (0.063 × 10–2), high conservation index, and three segregating sites, resulting in nonsynonymous mutation and four haplotypes. Some loci of Pfs230 D1 were in positive or negative linkage disequilibrium, had negative or positive selection signatures, and others (1813, 1955) and (1813, 1983) had a history of recombination. Mutated loci pairs in Pfs48/45 domain three had negative linkage disequilibrium, and some had negative and positive Tajima’s D values with no history of recombination events. Conclusion: The two transmission blocking vaccine candidates have low nucleotide diversity, a small number of zone-specific variants, high nucleotide conservation index, and high frequency of rare alleles. With the near fixation a polymorphic site and the proximity of mutated codons to antibody binding epitopes, it will be necessary to continue monitoring sequence modifications of these domains when designing TBVs that include Pfs230 and Pfs48/45 antigens.

Monitoring of Dynamic Changes and Clonal Evolution in Circulating Tumor DNA From Patients With IDH-Mutated Cholangiocarcinoma Treated With Isocitrate Dehydrogenase Inhibitors.

IDH mutations occur in about 30% of patients with cholangiocarcinoma. Analysis of mutations in circulating tumor DNA (ctDNA) can be performed by droplet digital polymerase chain reaction (ddPCR). The analysis of ctDNA is a feasible approach to detect IDH mutations. We isolated ctDNA from the blood of patients with IDH-mutated advanced cholangiocarcinoma collected at baseline, on therapy, and at progression to isocitrate dehydrogenase (IDH) inhibitors. Of 31 patients with IDH1R132 (n = 26) or IDH2R172 mutations (n = 5) in the tumor, IDH mutations were detected in 84% of ctDNA samples analyzed by ddPCR and in 83% of ctDNA samples analyzed by next-generation sequencing (NGS). Patients with a low variant allele frequency of ctDNA detected by NGS at baseline had a longer median time to treatment failure compared to patients with high variant allele frequency of ctDNA (3.6 v 1.5 months; P = .008). Patients with a decrease in IDH-mutated ctDNA on therapy by ddPCR compared with no change/increase had a trend to a longer median survival (P = .07). Most frequent emergent alterations in ctDNA by NGS at progression were ARID1A (n = 3) and TP53 mutations (n = 3). Detection of IDH mutations in ctDNA in patients with advanced cholangiocarcinoma is feasible, and dynamic changes in ctDNA can correspond with the clinical course and clonal evolution.

Dynamics of insecticide resistance in Anopheles gambiae sensu lato populations from Bohicon in Sudano-guinean area of Benin, West Africa

The current study was aimed to investigate the dynamics of insecticide resistance in Anopheles gambiae sensu lato populations from Bohicon in Sudano-guinean area of Benin, West Africa. Larvae and pupae of Anopheles gambiae s.l. populations were collected from the breeding sites in Zou department in 2015 and 2020. WHO susceptibility tests were conducted on unfed female mosquitoes aged 2-5 days old. WHO bioassays were performed with impregnated papers of permethrin (0.75%) and dichlorodiphenyltrichloroethane (DDT) (4%). An. gambiae mosquitoes were identified to species using PCR techniques in 2015. Molecular assays were also carried out to identify kdr mutations in individual mosquitoes. An. Gambiae s.l. populations from Bohicon were resistant to permethrin and DDT in 2015 and still remained resistant to these products in 2020. There is cross-resistance to both insecticides. PCR revealed 100% of mosquitoes tested were Anopheles gambiae s.s. The L1014F kdr mutation was found in An. Gambiae s.s. Bohicon at high allelic frequency.

Molecular Epidemiology of Na+-Taurocholate Cotransporting Polypeptide Deficiency in Guangdong Province, China: A Pilot Study by Screening for Four Prevalent Variants of the Causative Gene SLC10A1.

Na+-taurocholate cotransporting polypeptide deficiency (NTCPD) is an autosomal recessive disorder arising from biallelic SLC10A1 mutations. As a newly-described inborn error of bile acid metabolism, the epidemiology of this condition remains largely unclear in Chinese population so far. In this study, a total of 2,828 peripheral blood samples were collected from 12 cities in Guangdong, a province with the largest population in China, and the four prevalent SLC10A1 variants c.800C > T (p.Ser267Phe), c.263T > C (p.Ile88Thr), c.595A > C (p.Ser199Arg) and c.665T > C (p.Leu222Ser) were screened for by using polymerase chain reaction (PCR)- restriction fragment length polymorphism (RFLP). As a result, 663 mutated SLC10A1 alleles were detected, and the mutated allele frequency was calculated to be 11.72% (663/5,656), with a carrier frequency 20.69% (1/5) and a theoretical morbidity rate 1.37% (1/73) of NTCPD in Guangdong province. The variant c.800C > T (p.Ser267Phe) exhibited highest allele frequency among the four prevalent variants (χ2 = 1501.27, p < 0.0001) as well as higher allele frequency in the peripheral region than that within the Pearl River Delta (χ2 = 4.834, p < 0.05). The results suggested that NTCPD might be a disorder rather common in Guangdong province. The findings depicted the molecular epidemiologic features of NTCPD, providing preliminary but significant laboratory evidences for the subsequent NTCPD diagnosis and management in Guangdong population.

Composition of mosquito fauna and insecticide resistance status of Anopheles gambiae sensu lato\xa0in Itang special district, Gambella, Southwestern Ethiopia

BackgroundAnopheles arabiensis, member species of the Anopheles gambiae complex, is the primary vector of malaria and is widely distributed in Ethiopia. Anopheles funestus, Anopheles pharoensis and Anopheles nili are secondary vectors occurring with limited distribution in the country. Indoor residual spraying (IRS) and long-lasting insecticidal nets (LLINs) are pillars for the interventions against malaria control and elimination efforts in Ethiopia. However, the emergence and widespread of insecticide resistance in An. gambiae sensu lato (s.l.), might compromise the control efforts of the country. The aim of this study was to investigate composition of mosquito fauna and insecticide resistance status of An. gambiae s.l. in Itang special district ( woreda), Gambella, southwestern Ethiopia.MethodsAdult mosquitoes were sampled from September 2020 to February 2021 using the CDC light trap and pyrethrum spray catch (PSC). CDC light traps were placed in three selected houses for two consecutive days per month to collect mosquitoes indoor and outdoor from 6:00 P.M. to 06:00 A.M. and PSC was used to collect indoor resting mosquitoes from ten selected houses once in a month from October 2020 to February 2021. Moreover, mosquito larvae were also collected from different breeding sites and reared to adults to assess susceptibility status of populations of An. gambiae s.l. in the study area. Susceptibility tests were conducted on two to three days old non blood fed female An. gambiae s.l. using insecticide impregnated papers with deltamethrin (0.05%), alpha-cypermethrin (0.05%), propoxur (0.1%), pirimiphos-methyl (0.25%) and bendiocarb (0.1%) following World Health Organization (WHO) standard susceptibility test procedure. Molecular diagnostics were done for the identification of member species of An. gambiae s.l. and detection of knockdown resistance (kdr) allele using species specific polymerase chain reaction (PCR) and allele specific PCR.ResultsIn total, 468 adult mosquitoes were collected from different houses. Culex mosquitoes were the most dominant (80.4%) followed by Anopheles mosquitoes. Three species of Anopheles (Anopheles coustani, An. pharoensis, and An. gambiae s.l.) were identified, of which An. coustani was the dominant (8.1%) species. Higher number of mosquitoes (231) were collected outdoor by CDC light traps. Out of 468 adult mosquitoes, 294 were blood fed, 46 were half-gravid and gravid whereas the remaining 128 were unfed. WHO bioassay tests revealed that the populations of An. gambiae s.l. in the study area are resistant against alpha-cypermethrin and deltamethrin, but susceptible to bendiocarb, pirimiphos-methyl and propoxur. Of the total 86 An. gambiae s.l. specimens assayed, 79 (92%) successfully amplified and identified as An. arabiensis. West African kdr (L1014F) mutation was detected with high kdr allele frequency ranging from 67 to 88%.ConclusionThe detection of target site mutation, kdr L1014F allele, coupled with the phenotypic resistance against alpha-cypermethrin and deltamethrin call for continuous resistance monitoring.

Allele-informed copy number evaluation of plasma DNA samples from metastatic prostate cancer patients: the PCF_SELECT consortium assay.

Sequencing of cell-free DNA (cfDNA) in cancer patients’ plasma offers a minimally-invasive solution to detect tumor cell genomic alterations to aid real-time clinical decision-making. The reliability of copy number detection decreases at lower cfDNA tumor fractions, limiting utility at earlier stages of the disease. To test a novel strategy for detection of allelic imbalance, we developed a prostate cancer bespoke assay, PCF_SELECT, that includes an innovative sequencing panel covering ∼25 000 high minor allele frequency SNPs and tailored analytical solutions to enable allele-informed evaluation. First, we assessed it on plasma samples from 50 advanced prostate cancer patients. We then confirmed improved detection of genomic alterations in samples with <10% tumor fractions when compared against an independent assay. Finally, we applied PCF_SELECT to serial plasma samples intensively collected from three patients previously characterized as harboring alterations involving DNA repair genes and consequently offered PARP inhibition. We identified more extensive pan-genome allelic imbalance than previously recognized in prostate cancer. We confirmed high sensitivity detection of BRCA2 allelic imbalance with decreasing tumor fractions resultant from treatment and identified complex ATM genomic states that may be incongruent with protein losses. Overall, we present a framework for sensitive detection of allele-specific copy number changes in cfDNA.

Next-generation T cell-activating vaccination increases influenza virus mutation prevalence.

To determine the potential for viral adaptation to T cell responses, we probed the full influenza virus genome by next-generation sequencing directly ex vivo from infected mice, in the context of an experimental T cell–based vaccine, an H5N1-based viral vectored vaccinia vaccine Wyeth/IL-15/5Flu, versus the current standard-of-care, seasonal inactivated influenza vaccine (IIV) and unvaccinated conditions. Wyeth/IL-15/5Flu vaccination was coincident with increased mutation incidence and frequency across the influenza genome; however, mutations were not enriched within T cell epitope regions, but high allele frequency mutations within conserved hemagglutinin stem regions and PB2 mammalian adaptive mutations arose. Depletion of CD4+ and CD8+ T cell subsets led to reduced frequency of mutants in vaccinated mice; therefore, vaccine-mediated T cell responses were important drivers of virus diversification. Our findings suggest that Wyeth/IL-15/5Flu does not generate T cell escape mutants but increases stochastic events for virus adaptation by stringent bottlenecks.

High frequency of complex CFTR alleles associated with c.1521_1523delCTT (F508del) in Russian cystic fibrosis patients

Cystic fibrosis (CF, MIM# 219,700) is an autosomal recessive disease caused by pathogenic variants within the CFTR gene. It was shown that genetic variants located in cis can affect disease severity or treatment response because of additive or epistatic effects. Studies on the prevalence of complex alleles in Russian CF patients have just begun.AimTo evaluate frequencies and genetic background of complex alleles carrying c.1521_1523delCTT (F508del) and c.1399C>T (L467F), c.2562T>G (T854=) or c.4389G>A (Q1463=) in cis; to determine clinical consequences of complex allele c.[1399C>T;1521_1523delCTT] ([L467;F508del]) in Russian CF patients.MethodsSequencing of coding regions of CFTR gene and analysis of polymorphic markers in CF patients carrying F508del variant. Comparing of clinical features in two groups patients having genotypes [L467F;F508del];[F508del] (group 1) and [F508del];[F508del] (group 2).ResultsFrequency of [L467F;F508del] allele linked to 2–2–21–6–17–13 haplotype was 4.42%, of [F508del;T854=;Q1463=] allele linked to haplotype 1–2–21–6–17–13 – 2.2% in F508del chromosomes. No differences in disease severity in patients carrying complex allele [L467F;F508del] and patients homozygous for F508del was found.ConclusionThe frequency of complex alleles associated with F508del was at least 6.6% in Russian CF patients, which should be taken into account for the decision on optimal treatment options with CFTR modulators.

SNP g.1117G&gt;A HSP70 Gene Polymorphism in Domestic Beef Cattle using PCR-RFLP Method

Abstract Temperature increases have detrimental impacts on livestock, such as decreased metabolism, reproduction, and performance. The response of livestock to heat stress can be shown by the expression of HSP70 gene. This study aims to identify the SNP g.1117G&gt;A HSP70 gene polymorphism (HSP70|BanI) using the PCR-RFLP method. This study used 118 DNA samples of 5 breeds of cattle consisting of 20 heads of PO cattle, 20 heads of Madura cattle, 12 heads of Kuantan cattle, 53 heads of Bali cattle, and 13 heads of Pesisir cattle. The PCR-RFLP approach was used to analyze the diversity of the HSP70 gene. The enzyme used for cutting was BanI with forwarding primer 5‘-GAA AGG AAA AAA GAG ACA GA-3’ and reverse primers 5‘-AAT ACG CAG GAG TAG GTG GT-3’. The variation of the HSP gene was analyzed using allele frequency, genotype frequency, and level of heterozygosity. The Hardy-Weinberg equilibrium was evaluated using the Chi-Square test on PopGen 32. HSP70 gene amplification resulted in 539 bp fragments and 2 alleles (A and G). The HSP70 gene consisted of three genotypes i.e., AA (475 bp), AG (475 bp, 369 bp, and 106 bp), and GG (369 bp and 106 bp). The highest allele frequency of the A allele was found in PO and G allele in Bali cattle. Based on the results, it can be concluded that the HSP70 gene in 5’ UTR (untranslated region) in local beef cattle was polymorphic.

Novel alleles gained during the Beringian isolation period

During the Last Glacial Maximum, a small band of Siberians entered the Beringian corridor, where they persisted, isolated from gene flow, for several thousand years before expansion into the Americas. The ecological features of the Beringian environment, coupled with an extended period of isolation at small population size, would have provided evolutionary opportunity for novel genetic variation to arise as both rare standing variants and new mutations were driven to high frequency through both neutral and directed processes. Here we perform a full genome investigation of Native American populations in the Thousand Genomes Project Phase 3 to identify unique high frequency alleles that can be dated to an origin in Beringia. Our analyses demonstrate that descendant populations of Native Americans harbor 20,424 such variants, which is on a scale comparable only to Africa and the Out of Africa bottleneck. This is consistent with simulations of a serial founder effects model. Tests for selection reveal that some of these Beringian variants were likely driven to high frequency by adaptive processes, and bioinformatic analyses suggest possible phenotypic pathways that were under selection during the Beringian Isolation period. Specifically, pathways related to cardiac processes and melanocyte function appear to be enriched for selected Beringian variants.

Genomic miscellany and allelic frequencies of Plasmodium falciparum msp-1, msp-2 and glurp in parasite isolates

IntroductionThe genomic miscellany of malaria parasites can help inform the intensity of malaria transmission and identify potential deficiencies in malaria control programs. This study was aimed at investigating the genomic miscellany, allele frequencies, and MOI of P. falciparum infection.MethodsA total of 85 P. falciparum confirmed isolates out of 100 were included in this study that were collected from P. falciparum patients aged 4 months to 60 years in nine districts of Khyber Pakhtunkhwa Province. Parasite DNA was extracted from 200µL whole blood samples using the Qiagen DNA extraction kit following the manufacturer’s instructions. The polymorphic regions of msp-1, msp-2 and glurp loci were genotyped using nested PCR followed by gel electrophoresis for amplified fragments identification and subsequent data analysis.ResultsOut of 85 P. falciparum infections detected, 30 were msp-1 and 32 were msp-2 alleles specific. Successful amplification occurred in 88.23% (75/85) isolates for msp-1, 78.9% (67/85) for msp-2 and 70% (60/85) for glurp gene. In msp-1, the K1 allelic family was predominantly prevalent as 66.66% (50/75), followed by RO33 and MAD20. The frequency of samples with single infection having only K1, MAD20 and RO33 were 21.34% (16/75), 8% (6/75), and 10.67% (8/75), respectively. In msp-2, both the FC27 and 3D7 allelic families revealed almost the same frequencies as 70.14% (47/67) and 67.16% (45/67), respectively. Nine glurp RII region alleles were identified in 60 isolates. The overall mean multiplicity of infection for msp genes was 1.6 with 1.8 for msp-1 and 1.4 for msp-2, while for glurp the MOI was 1.03. There was no significant association between multiplicity of infection and age groups (Spearman’s rank coefficient = 0.050; P = 0.6) while MOI and parasite density correlated for only msp-2 allelic marker.ConclusionsThe study showed high genetic diversity and allelic frequency with multiple clones of msp-1, msp-2 and glurp in P. falciparum isolates in Khyber Pakhtunkhwa, Pakistan. In the present study the genotype data may provide valuable information essential for monitoring the impact of malaria eradication efforts in this region.