High Allele Frequency Research Articles

EP404: Evidence supporting the pathogenicity of the NADSYN1 c.1717G>A (p.Ala573Thr) variant in individuals referred for clinical testing

Vertebral, cardiac, renal, and limb defects syndrome 3 (VCRL3; OMIM #618845) is an autosomal recessive multiple congenital anomaly syndrome caused by pathogenic variants in the NADSYN1 gene. NADSYN1 encodes a nicotinamide adenine dinucleotide (NAD) synthetase that is part of the de novo NAD synthesis pathway. Other genes involved in this pathway include HAAO and KYNU. Genetic variants in any of these genes result in reduced de novo NAD synthesis and cause congenital anomalies of the heart, kidney, and vertebrae. Of note, presence of these three features is also considered clinical evidence for vertebral defects, anal atresia, cardiac defects, tracheo-esophageal fistula, renal anomalies, and limb abnormalities (VACTERL) association, which is often a diagnosis of exclusion (ie, other genetic conditions with overlapping features must be ruled out before assigning this diagnosis). The recommended approach for genetic testing in possible VACTERL patients is to either pursue a comprehensive testing option, such as exome or genome sequencing, or a panel approach with the most likely candidates. The NADSYN1 gene is included on a VACTERL Association and Related Disorders panel and several patients were noted to be homozygous for the NADSYN1 c.1717G>A (p.Ala573Thr) variant, thus providing additional evidence for both the pathogenicity and high carrier frequency of this change. To date, we have identified five patients who are homozygous for the NADSYN1 (NM_018161.4) c.1717G>A (p.Ala573Thr) variant. One patient was identified through targeted testing, one through testing with the VACTERL Association and Related Disorders panel, and three through exome sequencing. The first four cases were from pregnancies evaluated for the differential diagnosis of VACTERL association via panel testing or exome sequencing. All four fetuses were reported to have cardiac malformation and segmental vertebral anomalies. Parental data was available for three of the four fetuses, and all parents tested were heterozygous for the NADSYN1 c.1717G>A (p.Ala573Thr) variant. All cases denied consanguinity. In contrast to the severe and early onset features of the other four patients identified, the targeted testing case was an adult patient who previously had the NADSYN1 c.1717G>A variant identified in a research study. This individual was noted to have cardiac defects, vertebral defects, and short stature. This variant is relatively common in non-Finnish Europeans with an allele frequency of 0.15% in the gnomAD v2 dataset. Five patients from four families with VCRL3 have been described in the literature. Of these patients, three had the c.1717G>A variant: two homozygous siblings and one compound heterozygote with a loss of function variant on the opposite allele. Parental sequencing data confirmed the biallelic variants in all reported patients. In addition, yeast model functional studies performed by Szot and colleagues demonstrated that the p.Ala573Thr change diminished NAD synthetase activity and reduced yeast viability, consistent with a loss of function mechanism. Taken together, although the NADSYN1 c.1717G>A (p.Ala573Thr) has a relatively high minor allele frequency, there is sufficient evidence for this variant to be classified as pathogenic using ACMG/AMP variant classifications and internal variant interpretation guidelines. The NADSYN1 c.1717G>A (p.Ala573Thr) variant should be interpreted as pathogenic given the 5 additional homozygous patients and parental segregation data presented here. Additionally, the diagnosis of VCRL3 in an adult patient suggests that although penetrance for NAD deficiency is complete, variable expressivity is observed and could be explained by other genetic or non-genetic factors modulating disease severity. Given the high population frequency of this variant, genetic testing for VACTERL association should always include evaluation of NADSYN1 to consider VCRL3 in the differential diagnosis.

Xeno-monitoring of molecular drivers of artemisinin and partner drug resistance in P. falciparum populations in malaria vectors across Cameroon

BackgroundMonitoring of drug resistance in Plasmodium populations is crucial for malaria control. This has primarily been performed in humans and rarely in mosquitoes where parasites genetic recombination occurs. Here, we characterized the Plasmodium spp populations in wild Anopheles vectors by analyzing the genetic diversity of the P. falciparum kelch13 and mdr1 gene fragments implicated in artemisinin and partner drug resistance across Cameroon in three major malaria vectors. MethodsAnopheles mosquitoes were collected across nine localities in Cameroon and dissected into the head/thorax (H/T) and abdomen (Abd) after species identification. A TaqMan assay was performed to detect Plasmodium infection. Fragments of the Kelch 13 and mdr1 genes were amplified in P. falciparum positive samples and directly sequenced to assess their drug resistance polymorphisms and genetic diversity profile. ResultsThe study revealed a high Plasmodium infection rate in the major Anopheles vectors across Cameroon. Notably, An. funestus vector recorded the highest sporozoite (8.0%) and oocyst (14.4%) infection rates. A high P. falciparum sporozoite rate (80.08%) alongside epidemiological signatures of significant P. malariae (15.9%) circulation were recorded in these vectors. Low genetic diversity with six (A578S, R575I, G450R, L663L, G453D, N458D) and eight (H53H, V62L, V77E, N86Y, G102G, L132I, H143H, Y184F) point mutations were observed in the k13 and mdr1 backbones respectively. Remarkably, the R575I (4.4%) k13 and Y184F (64.2%) mdr1 mutations were the predominant variants in the P. falciparum populations. ConclusionThe emerging signal of the R575I polymorphism in the Pfk13 propeller backbone entails the regular surveillance of molecular markers to inform evidence-based policy decisions. Moreover, the high frequency of the 86N184F allele highlights concerns on the plausible decline in efficacy of artemisinin-combination therapies (ACTs); further implying that parasite genotyping from mosquitoes can provide a more relevant scale for quantifying resistance epidemiology in the field.

Association of recurrent APOBEC3B alterations with the prognosis of gastric-type cervical adenocarcinoma

ObjectiveGastric-type cervical adenocarcinoma (GCA) is a rare and aggressive type of endocervical adenocarcinoma (ECA) with distinct histopathologic features and unfavorable treatment outcomes, but no genomic prognostic factor has been revealed. We aimed to systematically investigate the somatic alterations of GCA at genome-wide level and evaluate their prognostic value. MethodsWe performed whole-exome sequencing (WES) on 25 pairs of tumor and matched normal samples to characterize the genomic features of Chinese patients with GCA and investigated their relations to histopathological characterizations and prognosis. The prognostic value of the genomic alterations was evaluated in a total of 58 GCA patients. ResultsMutations were commonly observed in reported GCA-related driver genes, including TP53 (32%), CDKN2A (20%), SKT11 (20%), BRCA2 (12%), SMAD4 (12%), and ERBB2 (12%). Recurrent novel trunk mutations were also observed in PBRM1 (12%), FRMPD4 (12%), and NOP2 (8%) with high variant allele frequency. Moreover, enrichment of the APOBEC signature was attributed to frequent gain of somatic copy number alteration (SCNA) of APOBEC3B (20%), which perfectly matched the nuclear-positive staining of APOBEC3B through immunohistochemistry. In contrast, APOBEC3B alteration was absent in patients with conventional type of ECA (N = 52). Notably, positive APOBEC3B was consistently enriched in patients with favorable prognosis in both the discovery cohort and an additional 33 GCA patients, thus indicating a significant association with lower relapse risk of GCA independent of cancer stage (P = 0.02). ConclusionOur results can aid understanding of the molecular basis of GCA in the Chinese population by providing genomic profiles and highlighting the potential prognostic value of APOBEC3B for GCA through routine clinical IHC.

Efficiency analysis of VersaPlex™ 27PY system in Central Indian Population: First report from Indian population

In the current scenario, DNA typing is the need of forensic science field due to its ability to provide results in much shorter time. In view of advancement of forensic DNA typing and incensement in the number of STRs markers, Promega offered a new VersaPlex™ 27PY system with 27 loci (23 autosomal STR loci, Amelogenin, DYS391 and two rapidly mutating Y-STR loci (DYS570 and DYS576)). In this study, the efficacy of "23 autosomal STR loci" for paternity testing and personal identification was demonstrated in Indian population. For this, 217 central Indians were tested and all the statistical parameters of forensic and population genetic interest were calculated. In addition, sensitivity of the kit was also tested for forensic casework. During investigation with VersaPlex™ 27PY system, allele 11 at locus TPOX was observed to be most frequent with the highest allelic frequency 0.432. Studied 23 loci showed valuable together with highest value of combined power of discrimination (CPD = 1), combined power of exclusion (CPI = 0.9999999989) and lowest value of combined matching probability (CPM = 7.92x10-28).

Genetic and Clinicopathologic Characteristics of Papillary Thyroid Carcinoma in the Chinese Population: High BRAF Mutation Allele Frequency, Multiple Driver Gene Mutations, and RET Fusion May Indicate More Advanced TN Stage.

PurposeTo describe the genetic landscape and clinical characteristics of Chinese patients diagnosed with papillary thyroid cancer (PTC) and to determine which high-risk genetic characteristics suggest a likelihood of lymph node metastasis (LNM) and lateral lymph node metastasis (LLNM).Patients and MethodsData from previously untreated patients with PTC collected between May 2018 and December 2020 from 14 hospitals in China were analyzed retrospectively. High-risk pathologic characteristics were defined as T3/T4, N(+), and N1b(+) stages. All patients were tested for 57 genes by second-generation sequencing. The t-test, chi-square test, and Fisher’s exact test were performed for statistical analysis.ResultsOverall, 395 patients were enrolled in this study. The prevalence of BRAF mutation was 78.53%. BRAF mutant allele frequency (MAF) >16.93% was associated with a significantly higher risk of LNM, LLNM, and T3 + T4 stage compared with a low-risk group, defined by a MAF <2.54% (odd ratios [ORs] for each risk=3.38, 3.46, and 8.54, respectively), and an intermediate-risk group, defined by a MAF of 2.54% to 16.93% (ORs=2.04, 2.07, and 4.07, respectively). The population with RET fusion had higher T, N, and N1b stages (ORs for each stage=10.40, 7.60, and 8.77, respectively) compared with a RET-negative population. Similar conclusions about T, N, and N1b stages were observed in relation to multiple driver gene mutations (ORs for each stage=7.48, 2.80, and 7.04, respectively) compared with population without multiple driver mutations. These genetic characteristics may be suggestive of high clinical risk. However, regardless of genetic profiles, patients younger than age 45 years had greater rates of LNM and LLNM.ConclusionThe main driver gene in this study, BRAF, differs significantly between the United States (79% vs 51%) and other countries. The Chinese population in this study that experienced more aggressive tumor biology had a BRAF MAF greater than 16.93%, exhibited RET fusion events, and had multiple driver gene mutations; thus, these traits may be considered high-risk genetic characteristics in PTC that could warrant aggressive treatment in such population.

The emerging importance and evolving understanding of clonal hematopoiesis in multiple myeloma

Multiple myeloma (MM) is the second most common hematologic malignancy diagnosed in the United States. With a growing arsenal of novel therapies, patients are living longer and hence are at increased risk of secondary cancers such as myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). While MDS-associated cytogenetic abnormalities have been described in patients with a diagnosis of for decades, clonal hematopoiesis (CH) has been described only recently. CH has been shown to correlate with inferior survival in MM due to increased risk of disease progression in patients who are treated with high-dose melphalan without lenalidomide maintenance. When involving specific high-risk genes, multiple genes, or when present at high variant allelic frequencies, CH could also potentially elevate the risk of secondary MDS and/or AML, cardiovascular events, and venous thromboembolic events. Despite growing knowledge about CH in patients with MM, many questions remain unanswered. Further studies are needed to better understand the prognostic and therapeutic significance of CH in MM and its precursor conditions, as well as the effect of specific treatments on long-term outcome.

The ALA5/ALA6/ALA7 repeat polymorphisms of the glutathione peroxidase-1 (GPx1) gene and autism spectrum disorder.

Autism is a severe neurodevelopmental disorder leading to deficits in social interaction, communication, and several activities. An increasing number of evidence suggests a role of oxidative stress in the etiology of autism spectrum disorder (ASD). Indeed, impaired antioxidant mechanisms may lead to the inadequate removal of H2O2 with a consequent increase in highly active hydroxyl radicals and other reactive oxygen species causing cellular damages. The GPx1 is one of the most important enzymes counteracting oxidative stress. In this work, we investigated a possible correlation between the GCG repeat polymorphism present in the first exon of GPx1 gene encoding a tract of five to seven alanine residues (ALA5, ALA6, and ALA7) and ASD. Our findings highlighted a high frequency of ALA5 allele in ASD subjects. Moreover, proteins corresponding to the three GPx1 variants were produced in vitro, and the evaluation of their activity showed a lower values for GPx1 having ALA5 polymorphism. The comparison of the secondary and tertiary structure predictions revealed an alpha‐helix in correspondence of alanine stretch only in the case of GPx1‐ALA7 variant. Finally, to better investigate protein structure, steady‐state fluorescence measurements of GPx1 intrinsic tryptophan were carried out and the three tested proteins exhibited a different stability under denaturing conditions. This work demonstrates the importance in adopting a multidisciplinary strategy to comprehend the role of GPx1 in ASD.Lay SummaryResults here obtained suggest a possible role of ALA5 GPx1 variant in ASD. However, given the multifactorial nature of autism, this evidence might be a piece of a more complex puzzle being the GPx1 enzyme part of a complex pathway in which several proteins are involved.

Abstract PO-139: BRCA sequence variants in breast cancer patients with African ancestry

Abstract Introduction Breast cancer is the most common cause of cancer related deaths in women worldwide; however, significant disparities are evident in the epidemiology of breast cancer subtypes and clinical outcomes of different racial/ethnic groups. African Americans are 40% more likely to die from breast cancer—a disparity that is partially explained by genetic predisposition to early onset breast cancer and triple negative breast cancer (TNBC), an aggressive subtype linked to West African ancestry and associated with BRCA-1 germline mutations. Pathogenic BRCA mutations significantly alter breast cancer treatment, informing alternate screening, chemoprevention, prophylactic resections and use of PARB inhibitors; however, AAs have higher prevalence of BRCA variants of uncertain significance (VUS) secondary to underrepresentation of African ancestry in the reference genome. The aim of this study is therefore to further characterize BRCA mutations and BRCA VUS in breast cancer patients with African ancestry. Methods RNA Sequencing platforms were used to determine somatic mutation profiles from primary tumor samples of over 70 breast cancer patients from Ethiopia and Ghana, by identifying single nucleotide polymorphisms (SNPs) in BRCA 1 and 2. Each sequence variant was evaluated for consequence on the gene, previously reported clinical significance, and allele frequency by self-reported race in comparison to global allele frequencies. Results BRCA1: 10 BRCA1 SNPs were identified: Missense: Rs1799966, rs16942, rs16941, rs799917, rs4986850, rs1799950; synonymous: rs1060915, rs16940, rs1799949; variant of 3'UTR: rs3092995. Rs1799966, rs16942, rs16941 and rs1799950 cause deleterious missense mutations, and have been reported as uncertain clinical significance. No SNPs were previously reported pathogenic, 6 had uncertain significance, and 4 were classified as either benign or likely benign. Allele frequency of Rs799917 was high among Ethiopian, Ghanaian, and 1KG African subgroups. BRCA2: 13 BRCA2 SNPs were identified: Missense: rs766173, rs144848, rs1799944, rs169547; synonymous: rs1801439, rs1801499, rs1801406, rs543304, rs206075, rs1799955, rs9590940; downstream/missense: rs1801426; variant of 5'Prime UTR: rs1799943. Rs766173 caused a deleterious missense mutation, with conflicting interpretations of pathogenicity. No SNPs were previously reported pathogenic, 4 had uncertain significance, and 8 were either benign or likely benign. No SNPs had high allele frequency in African ancestry. Conclusions The BRCA-1 SNP Rs799917 has a high allele frequency rate among Ethiopian, Ghanaian, and 1KG African ancestry, suggesting this mutation is potentially unique to African ancestry. Further investigation with RNA seq or whole genome sequencing in larger cohorts is warranted. Citation Format: Solange Bayard, Rachel Martini, Yalei Chen, Brittany Jenkins, Isra Elhussin, Esther Cheng, Syed Hoda, Paula Ginter, Jeffrey Hanover, Rozina Zeidan, Joseph Oppong, Kofi Gyan, Clayton Yates, Rick Kittles, Engida Abebe, Ishmael Kyei, Frances Aitpillah, Michael Adinky, Lisa Newman, Melissa Davis. BRCA sequence variants in breast cancer patients with African ancestry [abstract]. In: Proceedings of the AACR Virtual Conference: 14th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2021 Oct 6-8. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr PO-139.

Novel Variant Identified in the Enhancer Region of Host Transcription Factor, BRN3A, is a Significant Risk Factor for HPV-Induced Uterine Cervix Cancer.

Among the HPV-mediated cervical cancers, cellular factor BRN3A has gained considerable attention due to its role in promoting an anti-apoptotic cellular environment and in facilitating epitheliotropic transformations of the host. The majority of previous studies looked at BRN3A's molecular characteristics; however, the possibility of genetic variations in BRN3A's auto-regulatory region in relation to cervical cancer risk has been underestimated until now. In a retrospective study in the Eastern UP population, India, we detected genetic variations in the cis-regulatory proximal enhancer region located around 5.6 kb upstream of transcription start site of BRN3A. Our analysis of PCR and DNA sequencing confirmed this novel SNP (BRN3A g.60163379A>G) within the auto-regulatory region of BRN3A. As compared to control subjects, cancer cases exhibited a 1.32-fold higher allele frequency (χ2 = 6.315, p = 0.012). In homozygous (GG) but not in heterozygous conditions, odds ratio (OR) analysis suggests a significant association of cancer risk with the SNP (OR = 2.60, p ≤ 0.004). We further confirmed using the functional analysis that this SNP increased the luciferase gene activity in HPV-positive cervical cancer SiHa cells that were exposed to progesterone. As a result of the association of polymorphisms in a non-coding region of an oncogene with increased cancer risks, we are suggesting that this genetic variation in non-coding region can be used in prediction, diagnosis, or predicting the progression of the disease.

A1/A2 Milk Research in Indian Cattle

Bovine beta casein A1 and A2 are the most common variants in cattle breeds. Because of the bioactive peptide beta casomorphin-7 (BCM7) produced by raw or processed A1-milk, which has high affinity for opioid receptors and can exert regulatory activities, A1 variant is considered a risk factor for many human diseases, but not by A2-milk, during digestion. The frequency of A1/A2 allele of beta casein is breed dependent and can be very easily checked with simple PCR-RFLP technique. The aim of this article was to review the different studies and assess the status of A1/A2 in Indian native cattle breeds as well as crossbred/taurine populations and breeding bulls being used at different AI centres in India. Analysis of frequency pattern clearly indicated that all Indian native cattle breeds have high frequency of A2 allele and homozygous A2A2 genotype and hence are very good resource for A2 milk. Crossbred, taurine cattle populations and breeding bulls being used at different AI centres have a higher frequency of A2 allele and A1A2 genotype. The percentage of animals with homozygous A2A2 genotype is low. This suggests the need for screening of crossbred/exotic bulls being used in A1 and modify the existing breeding policy so as to drift the herds towards A2.

Analysis of the Frequency of 10 Polymorphic Markers of CDKN2A and RB1 Genes in Russian Populations.

The study of population frequencies of rare clinically significant alleles is a prerequisite of the development of personalized medicine. We performed genotyping of 1785 DNA samples from representatives of Russian populations according to 10 benign polymorphic markers of two genes involved in oncogenesis: 3 variants of the CDKN2A gene (rs3731249, rs116150891, and rs6413464) and 7 markers of the RB1 gene (rs149800437, rs147754935, rs183898408, rs146897002, rs4151539, rs187912365, and rs144668210). Genotyping was performed using the Illumina biochip test system. The sample covered 28 populations of the Russian Federation and neighboring countries, which were later combined into 3 groups (Asian, European, and Caucasian). The information from the ALFA (NCBI) project was used as reference for the frequencies of these polymorphisms in the Asian and European populations. It was shown that rare alleles in 8 of 10 studied polymorphic markers are presented in Russian populations of European and Caucasian origin with frequencies that are tens and hundreds of times higher than the available data for Western European populations, and in Russian Asian populations, alternative alleles of 5 markers absent in the Asian population of the ALFA project were found. In the subpopulation of Astrakhan Tatars, exceptionally high frequencies of rare alleles were identified; this requires further study.

Determination of the frequency of ABCB1 gene polymorphisms (C1236T, C3435T) in the population of the Tashkent region of Uzbekistan

This work aimed to determine the frequency of 2 polymorphisms in the ABCB1 gene (С1236T, С3435T) in healthy volunteers from the population of Tashkent region of Uzbekistan to assess their prevalence and improve the quality of personalized therapy and treatment efficacy. The study included 121 conditionally healthy volunteers from the Tashkent region of Uzbekistan aged 18 to 54 years. Genotyping was performed by polymerase chain reaction (PCR) and restriction fragment length polymorphism. It was found that for C1236T, the T allele had a higher frequency (0.63) than the C allele (0.37). Regarding genotypes, CT and TT were found significantly more often than CC. The frequency of the С3435Т C allele was 41%, and that of the T allele was 59%; the CT and TT genotypes were detected equally and had a frequency higher than that of CC. In conclusion, we observed a high frequency of altered alleles of C1236T and C3435T polymorphisms of the ABCB1 gene among healthy volunteers of the Uzbek population. Thus, such research needs to be scaled up to select the optimal individual therapy for many diseases. Keywords: P-glycoprotein, SNP, genotype.

A long-term responding epidermal growth factor receptor mutated non-small cell lung cancer patient with extremely high mutation allele frequency.

ENWEndNote BIBJabRef, Mendeley RISPapers, Reference Manager, RefWorks, Zotero AMA Miyazaki K, Sato Y, Satoh H, Hizawa N. A long-term responding epidermal growth factor receptor mutated non-small cell lung cancer patient with extremely high mutation allele frequency. Contemporary Oncology/Współczesna Onkologia. 2022;26(1):88-89. doi:10.5114/wo.2022.115447. APA Miyazaki, K., Sato, Y., Satoh, H., & Hizawa, N. (2022). A long-term responding epidermal growth factor receptor mutated non-small cell lung cancer patient with extremely high mutation allele frequency. Contemporary Oncology/Współczesna Onkologia, 26(1), 88-89. https://doi.org/10.5114/wo.2022.115447 Chicago Miyazaki, Kunihiko, Yoshiharu Sato, Hiroaki Satoh, and Nobuyuki Hizawa. 2022. "A long-term responding epidermal growth factor receptor mutated non-small cell lung cancer patient with extremely high mutation allele frequency". Contemporary Oncology/Współczesna Onkologia 26 (1): 88-89. doi:10.5114/wo.2022.115447. Harvard Miyazaki, K., Sato, Y., Satoh, H., and Hizawa, N. (2022). A long-term responding epidermal growth factor receptor mutated non-small cell lung cancer patient with extremely high mutation allele frequency. Contemporary Oncology/Współczesna Onkologia, 26(1), pp.88-89. https://doi.org/10.5114/wo.2022.115447 MLA Miyazaki, Kunihiko et al. "A long-term responding epidermal growth factor receptor mutated non-small cell lung cancer patient with extremely high mutation allele frequency." Contemporary Oncology/Współczesna Onkologia, vol. 26, no. 1, 2022, pp. 88-89. doi:10.5114/wo.2022.115447. Vancouver Miyazaki K, Sato Y, Satoh H, Hizawa N. A long-term responding epidermal growth factor receptor mutated non-small cell lung cancer patient with extremely high mutation allele frequency. Contemporary Oncology/Współczesna Onkologia. 2022;26(1):88-89. doi:10.5114/wo.2022.115447.

Peptide ancestry informative markers in uterine neoplasms from women of European, African, and Asian ancestry

SummaryCharacterization of ancestry-linked peptide variants in disease-relevant patient tissues represents a foundational step to connect patient ancestry with disease pathogenesis. Nonsynonymous single-nucleotide polymorphisms encoding missense substitutions within tryptic peptides exhibiting high allele frequencies in European, African, and East Asian populations, termed peptide ancestry informative markers (pAIMs), were prioritized from 1000 genomes. In silico analysis identified that as few as 20 pAIMs can determine ancestry proportions similarly to >260K SNPs (R2 = 0.99). Multiplexed proteomic analysis of >100 human endometrial cancer cell lines and uterine leiomyoma tissues combined resulted in the quantitation of 62 pAIMs that correlate with patient race and genotype-confirmed ancestry. Candidates include a D451E substitution in GC vitamin D-binding protein previously associated with altered vitamin D levels in African and European populations. pAIMs will support generalized proteoancestry assessment as well as efforts investigating the impact of ancestry on the human proteome and how this relates to the pathogenesis of uterine neoplasms.

SNP-Based Analysis Reveals Authenticity and Genetic Similarity of Russian Indigenous V. vinifera Grape Cultivars.

9 Russian Vitis vinifera grape varieties and the European variety Muscat Hamburg were sequenced and genotyped using 527 SNPs (single nucleotide polymorphisms) with high minor allele frequency for the first time. The data were coupled with previously identified genotypes of 783 varieties and subjected to parentage and population analysis. As a result, contrary to the historical and ampelographic data published in many sources from 1800 to 2012, only two of the nine Russian varieties (Pukhlyakovskiy Belyi and Sibirkovyi) were related to foreign ones and were obviously imported from Europe to the Russian Empire. The remaining seven varieties, led by Krasnostop Zolotovskiy, are not directly related either in the Caucasus or in Europe, they form separate clusters on the genetic distance-based dendrogram and the world parentage network of V. vinifera. The resulting pedigree of Muscat Hamburg and its descendants is in accordance with SSR-based (simple sequence repeats) studies and the described pedigree of this variety which confirms the use of the reduced SNP set for further studies.

Real-time digital polymerase chain reaction (PCR) as a novel technology improves limit of detection for rare allele assays.

BackgroundTumor heterogeneity may lead to false negative test results for tissue biopsy-based companion diagnostic tests. Real-time polymerase chain reaction (PCR) and digital PCR assays are used to detect rare alleles in cell-free circulating DNA for liquid biopsies; however, those tests lack strong sensitivity at low allele frequencies. We show here a novel real-time digital PCR instrument that utilizes cycle-based amplification curves to further improve the sensitivity and quantification accuracy of digital PCR.MethodsThe novel real-time digital PCR instrument was compared to an endpoint digital PCR system to determine the sensitivity and quantification accuracy of both instruments. Samples were all thermal cycled on the real-time digital PCR instrument but were analyzed on both endpoint and real-time digital PCR instruments to compare the performance without introducing other variables. Contrived samples for epidermal growth factor receptor (EGFR) exon 19 deletion, T790M, and L858R point mutations as well as human epidermal growth factor receptor 2 (HER2) amplification were tested. Different mutant allele frequencies and wildtype to mutant gene copy number ratios were tested for EGFR and HER2, respectively.ResultsBy removing false positive datapoints using real-time amplification curves, real-time digital PCR improved sensitivity by lowering the baseline for wildtype samples. For EGFR 19del assay, samples with 2 or more fluorescein amidite (FAM) labeled positive wells are determined positive by real-time digital PCR, while a minimum of 5 FAM positive datapoints is needed by endpoint digital PCR. Improved limit of detection for EGFR 19del mutation was also observed. Real-time digital PCR also had better quantification accuracy and sensitivity, resulting in the mutant allele frequencies being closer to the expected values for all EGFR mutations, especially at very low allele frequencies. However, at high allele frequencies or for gene amplification assays, real-time digital PCR is comparable with endpoint digital PCR.ConclusionsThis novel technology with improved sensitivity is important and needed because it addresses current issues with liquid biopsy tests. Due to limited amounts of circulating tumor DNA (ctDNA) obtained for liquid biopsy tests, few copies of mutant alleles are expected. With the lower baseline of real-time digital PCR, false negative test results from tissue biopsy would be more effectively reduced, leading to more patients receiving the targeted therapy they need for better survival.

Protein-associated chemical switches differentially modulate the binding activities of human apolipoprotein E isoforms with their receptors and with amyloid beta peptides.

Apolipoprotein E (ApoE) acts as a major cholesterol carrier supporting lipid transport and tissue repair in the brain. The ApoE gene is polymorphic, having three prominent alleles which are the main genetic determinants of Alzheimer Disease (AD) risk in humans. ApoE ε3 has the highest allele frequency in the general population. ApoE ε4 carriers have an increased risk of AD relative to ApoE ε3 carriers, while ApoE ε2 carriers have a decreased risk of developing AD. ApoE proteins bind to several cell surface receptors including low-density lipoprotein receptor (LDLR) and very low-density lipoprotein receptor (VLDLR) to deliver lipids. ApoE binds to Sortilin, which mediates uptake of ApoE containing lipoproteins into neurons, and to amyloid-b (Ab) peptide, which is thought to play important roles in the pathogenesis of AD. We recently discovered that physiological chemicals (such as bicarbonate and sodium hydrogen sulfide) modulate the binding activities of antibodies as a function of the differential external cellular pH between the acidic tumor microenvironment (pH 5.8-6.7 resulting from glycolysis) and the alkaline environment of normal tissues and blood (≥pH7.4). This enables the use of these physiological chemicals as Protein-associated Chemical Switches (PaCS) in cancer therapies1 . Inflamed and senescent cells are glycolytic and are suspected components in the manifestation of AD. We investigated the effects of PaCS molecules on receptor binding as a function of pH. At physiological concentrations, bicarbonate and sodium hydrogen sulfide were observed to differentially affect the binding activities of the ApoE isoforms to LDLR, VLDLR, Sortilin and Aβ peptide. We observed reduced binding of ApoE under alkaline conditions versus acidic conditions, with ApoE ε4 showing the highest binding activity under acidic conditions relative to the other isoforms. Our data indicate that PaCS molecules can conditionally modulate the interactions of different ApoE isoforms with cell surface receptors. These results provide potential mechanistic insights into the differential activity of the ApoE isoforms in normal blood versus acidic tissues associated with inflammation, as well as insights into the potential isoform-dependent protection from PaCS molecules in the pathogenesis of neurodegenerative diseases. Reference: Chang, et al. (2021), PNAS 118(9): 1-10, Suppl 1-19.

Clonal architecture predicts clinical outcomes and drug sensitivity in acute myeloid leukemia

The impact of clonal heterogeneity on disease behavior or drug response in acute myeloid leukemia remains poorly understood. Using a cohort of 2,829 patients, we identify features of clonality associated with clinical features and drug sensitivities. High variant allele frequency for 7 mutations (including NRAS and TET2) associate with dismal prognosis; elevated GATA2 variant allele frequency correlates with better outcomes. Clinical features such as white blood cell count and blast percentage correlate with the subclonal abundance of mutations such as TP53 and IDH1. Furthermore, patients with cohesin mutations occurring before NPM1, or transcription factor mutations occurring before splicing factor mutations, show shorter survival. Surprisingly, a branched pattern of clonal evolution is associated with superior clinical outcomes. Finally, several mutations (including NRAS and IDH1) predict drug sensitivity based on their subclonal abundance. Together, these results demonstrate the importance of assessing clonal heterogeneity with implications for prognosis and actionable biomarkers for therapy.

Both Binding Strength and Evolutionary Accessibility Affect the Population Frequency of Transcription Factor Binding Sequences in Arabidopsis thaliana.

Mutations in DNA sequences that bind transcription factors and thus modulate gene expression are a source of adaptive variation in gene expression. To understand how transcription factor binding sequences evolve in natural populations of the thale cress Arabidopsis thaliana, we integrated genomic polymorphism data for loci bound by transcription factors with in vitro data on binding affinity for these transcription factors. Specifically, we studied 19 different transcription factors, and the allele frequencies of 8,333 genomic loci bound in vivo by these transcription factors in 1,135 A. thaliana accessions. We find that transcription factor binding sequences show very low genetic diversity, suggesting that they are subject to purifying selection. High frequency alleles of such binding sequences tend to bind transcription factors strongly. Conversely, alleles that are absent from the population tend to bind them weakly. In addition, alleles with high frequencies also tend to be the endpoints of many accessible evolutionary paths leading to these alleles. We show that both high affinity and high evolutionary accessibility contribute to high allele frequency for at least some transcription factors. Although binding sequences with stronger affinity are more frequent, we did not find them to be associated with higher gene expression levels. Epistatic interactions among individual mutations that alter binding affinity are pervasive and can help explain variation in accessibility among binding sequences. In summary, combining in vitro binding affinity data with in vivo binding sequence data can help understand the forces that affect the evolution of transcription factor binding sequences in natural populations.

Estimating genetic diversity in Sorghum bicolor using molecular markers

Aim: The present study aimed at measuring the genetic diversity of a set of 219 sorghum accessions differing in their grain colour. Methodology: About 219 diverse sorghum lines differing in their grain colour were obtained from National Bureau of Plant Genetics Resources (NBPGR), New Delhi and genotyped using 17 SSR markers. Polymorphism information content (PIC) and allele frequency were determined using PowerMarker V3.25. Clustering and factorial analysis were performed using DARwin 6.0. GenAlex version 6.5 was used to perform Principal Coordinates Analysis (PCoA) and AMOVA. Diversity analysis was performed by using Darwin. Results: Genotyping of 219 sorghum accessions using 17 SSR markers produced a total of 399 alleles with an average PIC value of 0.85 and gene diversity of 0.87. Highest allele frequency was observed for the marker, Xtxp 265 whereas highest major allele frequency was observed in 196 accessions for the marker, Xtxp 278. Diversity analysis divided the 219 accessions into three clusters (1, 2 and 3) and genotypes belonging to same geographical origin were found to be clustered together. Interpretation: SSR marker based genetic diversity analysis grouped 219 sorghum accessions into three clusters. Grouping and clustering of accessions was mostly based on the geographical origins with some exceptions which may be due to cross hybridisation of accessions between countries paving a way for cross gene flow.