Sequencing Of Locus Research Articles

Hist-O-06 - Image-guided lymph node core-needle biopsy predicts survival in mycosis fungoides and Sézary syndrome

<h3>Background:</h3> The prognosis of Sézary syndrome (SS) and mycosis fungoides (MF) depends on lymph node (LN) involvement. The usefulness of LN image-guided core-needle biopsies (CNBs), instead of surgical sampling, has been poorly evaluated. <h3>Objectives:</h3> To determine the prognostic value of LN CNB in MF/SS. <h3>Methods:</h3> A retrospective search was conducted to identify all LN biopsy specimens of MF/SS between 2008 and 2019. Biopsies were staged according to the International Society for Cutaneous Lymphomas/European Organisation for Research and Treatment of Cancer (ISCL/EORTC) criteria. We performed immunolabelling and determined the tumour clone frequency (TCF) by high-throughput sequencing of the T-cell receptor beta locus. <h3>Results:</h3> We included 119 consecutive biopsies from 100 patients, 45 with MF and 55 with SS. N1, N2 and N3 stages were diagnosed in 34 (29%), 26 (22%) and 59 (49%) cases, respectively. The TCF, Ki67 index, and percentage of cells positive for thymocyte selection-associated high mobility group box protein (TOX), programmed cell death protein 1 (PD1), killer cell immunoglobulin-like receptor 3DL2 (KIR3DL2) and cluster of differentiation (CD)30 were all positively correlated with the N stage. Median overall survival (OS) for N1/N2 vs. N3 patients was 42 months (range 26–not reached) vs. 14 months (range 5–30), respectively (p<0.001). In univariate analyses, an age >75 years, LN short-axis diameter >15 mm, N3 stage, presence of large-cell transformation, TOX >60%, PD1 >25%, Ki67 >30%, KIR3DL2 >15%, CD30 >10% and TCF >25% were identified as adverse prognostic factors. In multivariate analyses, only an age >75 years and Ki67 index >30% were associated with reduced OS. We developed a new prognostic index associating the N stage and the Ki67 index, which better discriminates N3 patients with poor prognosis. <h3>Conclusions:</h3> CNB allows an objective assessment of the LN involvement in MF/SS, relevant for staging and prognosis.

Isolation of Metarhizium guizhouense and Metarhizium robertsii Strains from Soil-Exposed Amblyomma americanum (Acarina: Ixodidae) from Northwest Arkansas, USA

The lone star tick, Amblyomma americanum (L.) (Acarina: Ixodidae), is the most abundant tick species found in Arkansas and is involved in the transmission of pathogens of medical and veterinary importance. When not feeding, most non-nidicolous tick species shelter in the soil and leaf litter where they may be exposed to and potentially infected with entomopathogenic fungi that reside naturally in the soil. Entomopathogenic fungi in the genus Metarhizium Sorokīn (Hypocreales: Clavicipitaceae) have shown promise as biological control agents of ticks. Here, the first study to isolate and identify Arkansas-derived isolates of Metarhizium from A. americanum ticks is presented. We exposed 320 ticks artificially to native soil from Savoy. Of these soil exposed ticks, 2.5% of adults and 1.5% of nymphs displayed signs of infection with Metarhizium. Of the infected Savoy adults, 3.3% were females and 1.7% were males. Similarly exposed ticks from West Fork resulted in only 2.4% of nymphal ticks being infected with this fungus. Eight isolates of Metarhizium were cultured from infected ticks exposed to soil from these locations. Four of these Metarhizium isolates (3 from Savoy and 1 from West Fork) were identified to species by sequencing of the ITS locus and the EF1-α genes. Three Savoy strains (P10N1, P10AF1, and P2AM1) had identical sequences and were identified as Metarhizium robertsii (Bischoff, Rehner & Humber) (Hypocreales: Clavicipitaceae). The strain from West Fork (P9N2) was identified as Metarhizium guizhouense (Chen & Guo) (Hypocreales: Clavicipitaceae). The ITS and the EF1-α sequences of the Savoy strains showed 100% similarity to M. robertsii strains ARSEF 2575 and ART 500, respectively. The ITS and EF1-α sequences of the West Fork strain showed 99% similarity to M. guizhouense strains ARSEF 977 and CBS 258.90, respectively. This study demonstrates that entomopathogenic fungi M. guizhouense and M. robertsii are pathogenic to and can be isolated from A. americanum. Furthermore, the EF1-α genetic marker was shown to be a very effective tool for distinguishing different species of Metarhizium from ticks when used in conjunction with ITS sequence data. Standardizing the use of ticks in soil exposure methods for isolating entomopathogenic fungi could be useful for obtaining isolates that are highly virulent to A. americanum. The isolation and identification of Metarhizium spp. from A. americanum in Arkansas indicates that further exploration of entomopathogenic fungi as biological agents to control A. americanum is warranted.

A Diagnostic TaqMan Real-Time PCR Assay for In Planta Detection and Quantification of Colletotrichum theobromicola, Causal Agent of Boxwood Dieback.

Boxwood dieback, caused by Colletotrichum theobromicola, is spreading at an alarming rate in the boxwood industry in the United States. Although C. theobromicola has been accepted as a distinct species within the C. gloeosporioides species complex, it is difficult to distinguish it from other closely related species based on morphology. Moreover, molecular identification of C. theobromicola requires amplification and sequencing of multiple loci, which can be expensive and time consuming. Therefore, a diagnostic TaqMan real-time PCR assay was developed for early and accurate detection and quantification of C. theobromicola in boxwood. The study involved the design of species-specific primers and a TaqMan probe to differentiate C. theobromicola from other closely related Colletotrichum species. The primers and probe discriminate between C. theobromicola and other species in the C. gloeosporioides species complex and can detect C. theobromicola at very low concentrations, illustrating the high specificity and sensitivity of the assay. This TaqMan real-time PCR assay accurately and rapidly distinguishes boxwood dieback from other diseases with similar symptomatology, including Macrophoma blight, Phytophthora root rot, and Volutella blight, as well as some disorders produced by abiotic agents.

Listeria monocytogenes Contamination Characteristics in Two Ready-to-Eat Meat Plants From 2019 to 2020 in Shanghai.

Listeria monocytogenes is a ubiquitous foodborne pathogen that causes listeriosis and is mostly linked to consumption of ready-to-eat (RTE) foods. Lack of hygiene in food processing environments may be a primary reason for contamination by L. monocytogenes isolates. In this study, L. monocytogenes strains isolated from two RTE meat processing plants in the Shanghai municipality, China, were characterized during 2019–2020 using pulsed-field gel electrophoresis and whole-genome sequencing. Results showed that 29 samples (12.2%) out of 239 were positive for L. monocytogenes, with 21 (18.9%) and 8 (6.25%) isolates from plants A and B, respectively. The packaging room at plant A had the most contamination (14, 48.3%; p < 0.05), with a peak occurrence of 76.5% in processing environments. Nineteen L. monocytogenes isolates belonging to the pulsotype (PT) 7 group were indistinguishable (≥ 95.7%). Furthermore, core-genome multiple loci sequencing typing identified up to nine allelic differences, and the closet pairwise differences among these ST5 isolates included 0–16 small nucleotide polymorphisms. Therefore, L. monocytogenes likely persisted at plant A during 2019–2020 with ongoing clone transmission. In contrast, no L. monocytogenes isolates were identified from processing environments at plant B. Most L. monocytogenes isolates were sampled from raw materials (62.5%). Several isolates (ST378, ST8, and ST120) were detected only once in 2020 and were considered as transient isolates. However, three ST121 isolates with the same PT (PT2) were detected in 2020 and should be noted for their stronger survival ability in harsh environments. These results suggest that continuous monitoring, stringent surveillance, and source tracking are crucial to guaranteeing food safety in RTE food plants.

Hybridization with mountain hares increases the functional allelic repertoire in brown hares

Brown hares (Lepus europaeus Pallas) are able to hybridize with mountain hares (L. timidus Linnaeus) and produce fertile offspring, which results in cross-species gene flow. However, not much is known about the functional significance of this genetic introgression. Using targeted sequencing of candidate loci combined with mtDNA genotyping, we found the ancestral genetic diversity in the Finnish brown hare to be small, likely due to founder effect and range expansion, while gene flow from mountain hares constitutes an important source of functional genetic variability. Some of this variability, such as the alleles of the mountain hare thermogenin (uncoupling protein 1, UCP1), might have adaptive advantage for brown hares, whereas immunity-related MHC alleles are reciprocally exchanged and maintained via balancing selection. Our study offers a rare example where an expanding species can increase its allelic variability through hybridization with a congeneric native species, offering a route to shortcut evolutionary adaptation to the local environmental conditions.

Elevated plasma concentration of complement factor C5 is associated with risk of future venous thromboembolism

AbstractThe role of complement in the pathogenesis of venous thromboembolism (VTE) is unclear. We wanted to investigate (1) whether plasma complement component C5 (C5) levels are influenced by genetic variants or chronic inflammation and (2) the association between plasma C5 and risk of future VTE in a nested case-control study of 415 patients with VTE and 848 age- and sex-matched controls derived from the Tromsø Study. Plasma C5 levels were measured at inclusion. Odds ratios (ORs) with 95% confidence intervals (95% CIs) for provoked and unprovoked VTE across tertiles of C5 concentrations were estimated by logistic regression. Adjustment for C-reactive protein (CRP) served as a proxy for general inflammation. Whole-exome sequencing and protein quantitative trait loci analyses were performed to assess genetic influence on C5 concentrations. There was no association between genome-wide or C5-related gene variants and C5 levels. The association between plasma C5 levels and VTE risk displayed a threshold effect, where subjects with C5 levels above the lowest tertile had increased risk of VTE. Subjects in tertile 3 (highest C5 levels) had an age- and sex-adjusted OR of 1.45 (95% CI, 1.07-1.96) compared with tertile 1 (lowest). These statistics were more pronounced for unprovoked VTE (OR, 1.70; 95% CI, 1.11-2.60). Adjustments for body mass index and CRP had minor impact on risk estimates. The OR increased substantially with shorter time between blood sampling and VTE event. In conclusion, plasma C5 was associated with risk of future VTE. C5 levels were not genetically regulated and were only slightly influenced by chronic inflammation.

P53 convergently activates Dux/DUX4 in embryonic stem cells and in facioscapulohumeral muscular dystrophy cell models.

In mammalian embryos, proper zygotic genome activation (ZGA) underlies totipotent development. Double homeobox (DUX)-family factors participate in ZGA, and mouse Dux is required for forming cultured two-cell (2C)-like cells. Remarkably, in mouse embryonic stem cells, Dux is activated by the tumor suppressor p53, and Dux expression promotes differentiation into expanded-fate cell types. Long-read sequencing and assembly of the mouse Dux locus reveals its complex chromatin regulation including putative positive and negative feedback loops. We show that the p53-DUX/DUX4 regulatory axis is conserved in humans. Furthermore, we demonstrate that cells derived from patients with facioscapulohumeral muscular dystrophy (FSHD) activate human DUX4 during p53 signaling via a p53-binding site in a primate-specific subtelomeric long terminal repeat (LTR)10C element. In summary, our work shows that p53 activation convergently evolved to couple p53 to Dux/DUX4 activation in embryonic stem cells, embryos and cells from patients with FSHD, potentially uniting the developmental and disease regulation of DUX-family factors and identifying evidence-based therapeutic opportunities for FSHD.

Characterizing Microbiomes via Sequencing of Marker Loci: Techniques To Improve Throughput, Account for Cross-Contamination, and Reduce Cost.

ABSTRACTNew approaches to characterizing microbiomes via high-throughput sequencing provide impressive gains in efficiency and cost reduction compared to approaches that were standard just a few years ago. However, the speed of method development has been such that staying abreast of the latest technological advances is challenging. Moreover, shifting laboratory protocols to include new methods can be expensive and time consuming. To facilitate adoption of new techniques, we provide a guide and review of recent advances that are relevant for single-locus sequence-based study of microbiomes—from extraction to library preparation—including a primer regarding the use of liquid-handling automation in small-scale academic settings. Additionally, we describe several amendments to published techniques to improve throughput, track contamination, and reduce cost. Notably, we suggest adding synthetic DNA molecules to each sample during nucleic acid extraction, thus providing a method of documenting incidences of cross-contamination. We also describe a dual-indexing scheme for Illumina sequencers that allows multiplexing of many thousands of samples with minimal PhiX input. Collectively, the techniques that we describe demonstrate that laboratory technology need not impose strict limitations on the scale of molecular microbial ecology studies.IMPORTANCE New methods to characterize microbiomes reduce technology-imposed limitations to study design, but many new approaches have not been widely adopted. Here, we present techniques to increase throughput and reduce contamination alongside a thorough review of current best practices.

Abstract 528: MDSC suppress the T cell repertoire and contribute to a pathologic cytokine milieu in cancer patients

Abstract Myeloid derived suppressor cells (MDSC) are elevated in tumors and draining lymph nodes of cancer patients and produce a variety of immune suppressive factors that impair T cell activation and anti-tumor immunity. MDSC are also recruited from the bone marrow in response to T cell activation, including in response to immunotherapies that activate T cells. As MDSC and immune suppressive monocytes are significant producers of IL6, IL1β and TNFα, their elevation and activation in response to T cell activation can promote pathologic levels of these cytokines leading to potentially serious conditions such as cytokine release syndrome. Furthermore, patients deficient in functional antigen-specific T cells have elevated MDSC, consistent with their described suppression of antigen presentation and T cell activation. Therapeutic agents that deplete MDSC may offer both an improved therapeutic index for maintenance of T cell activation while promoting expansion of anti-tumor T cells. AMV564, a bivalent T cell engager that selectively depletes MDSC, is in clinical development in solid tumor patients. The cytokine profiles of patients treated with AMV564 as monotherapy or in combination with pembrolizumab were assessed using multiplex immunoassays, and the peripheral T cell repertoire was assessed by deep sequencing of the TCRβ locus. While CD33 has no apparent functional role on differentiated myeloid cells, on MDSC and immature monocytes, CD33 signaling promotes the production of immune-suppressive factors. Avid binding of clustered CD33 in this active signaling configuration drives the selectivity of AMV564 for these cell types. Analysis of patient samples demonstrates that AMV564 selectively depletes MDSC and promotes T cell activation and increases in effector CD8 and CD4 populations1. AMV564 promotes strong induction of IFNγ and other proinflammatory cytokines and chemokines, but with comparatively limited induction of IL6, IL1β and TNFα, consistent with the role of MDSC in producing these factors. This cytokine profile was maintained in combination with pembrolizumab. Dynamic changes in the peripheral T cell repertoire were apparent for patients treated with AMV564 monotherapy or in combination with pembrolizumab. Observations include significant expansion after one cycle of therapy (p = 0.008) including T cell clones not detectable at baseline, changes in diversity that reflect increases in middle-frequency clones, and oscillation in the peripheral T cell fraction consistent with T cell trafficking to tissues and tumors. Repertoire expansion correlated with increases in effector memory CD8 T cells. These findings in patients treated with AMV564 support the role of MDSC in both limiting the priming and activation of anti-tumor T cells as well as contributing to an immune suppressive and pathologic cytokine response to T cell activation. 1. Smith, V et. al. Cancer Res. 2020;80(16):5699 Citation Format: Sterling Eckard, Aurelien Sarde, Li Mei, Curtis Ruegg, Patrick Chun, Victoria Smith. MDSC suppress the T cell repertoire and contribute to a pathologic cytokine milieu in cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 528.

Development and Validation of a New Tool for the Characterization of Immunoglobulin Gene Rearrangements in Patients with Chronic Lymphocytic Leukemia By Deep Next Generation Sequencing

Introduction: Chronic Lymphocytic Leukemia (CLL) is a deeply heterogeneous disease from both biological and clinical points of view. This heterogeneity is partly reflected by differences in the mutation status of immunoglobulin variable heavy chain (IgHV) genes. According to this criterion, two main subsets are distinguished by whether CLL cells express an unmutated or mutated, reflecting the stage of normal B cell differentiation from which they originate. These groups also have important clinical differences in terms of clinical outcome. Thus, patients with unmutated IgHV (UM; ≥98% of identity to the germline) genes have a more aggressive disease course and develop more frequently unfavourable genetic deletions or mutations than patients with mutated IgHV (M; <98%).Currently, IgHV Somatic Hypermutation testing is assessed by Sanger sequencing (Sseq), which allows defining the main clone. However, it is not always possible to identify different clones in subclonal patients, which accounts for 20% of cases. Deep next generation sequencing (NGS) of the IgHV locus using consensus primers could broad the availability of deciphering the heterogeneity of CLL.Objective: Validation of a deep next generation sequencing (NGS) tool to analyze the IgHV locus and determinate the presence of multiple productive rearrangements in a series of patients with CLL.Methods: We included 140 samples extracted from peripheral blood of patients diagnosed of CLL according to the National Cancer Institute Working Group guidelines in our institution between 1986 and 2017 (median absolute lymphocytes 11.4x109/L [2,8-239,5x109/L]). Sseq amplification and sequence analysis of IgHV rearrangements were performed on either DNA or cDNA conforming to the updated ERIC recommendations. In any cases we were able to determinate the IGVH identity (68 M and 72 UM) and the stereotypic subset.For IgHV-NGS analysis gDNA was amplified using the Sequencing Multiplex Kit based on based on IGH leader (forward primers) and consensus JH (reverse primer), master mixes and PCR products were purified using magnetic beads, normalized and pooled to create a library for sequencing using a MiSeq equipment. Sequencing data were analyzed using our bioinformatics pipeline.Results:The median of: a) total sequence count analyzed per patient was 2492 (111-17445); b) most abundant unique sequence was 984 (111-7029); c) homology of variable region (IgHV) was 99.3% (87.22-100). We excluded unproductive rearrangement, and found 81.42% (114/140) of patients with unique clone and 18.6% (26/140) with multiple productive rearrangements (Figure 1). The median frequency of the second most abundant productive clone was 9.16% (3.33%-50%) (Table 1).Our tool led to identify of a dominant clonotypic IgHV in all cases (140/140, 100%). When we compared the NGS sequence/mutational status for the most abundant clone with Sseq and for the IgHV status determination, only 1 of 140 (0,7%) showed a major clone with productive rearrangement mutated by Sseq but unmutated by NGS.On the basis of IgVH-NGS subclonal profiles recently described, we identified the following distribution: 5.71% (8/140) of patients presented multiple M clones; 35% (49/140) 1 M clone; 13.6% (19/140) mix of M-UM clones; 37.85% (53/140) 1 UM clone; and 7.85% (11/140) multiple UM clones (Figure 2).Conclusion:The results indicate that our tool is useful to approach IgVH rearrangements since we were able both classified correctly according to IgVH mutational status, and V gene concordant. Moreover it was permitted to provide both the complete V-D-J sequence as well as a relative quantification of different subclones.Acknowledgments: Samples provided by the INCLIVA Biobank (PT13/0010/0004). Supported by: FISS PI 10/02095 and FISS PI 14/02018 [Display omitted] DisclosuresSolano:Neovii: Honoraria.

Combining QTL-seq and linkage mapping to fine map a candidate gene in qCTS6 for cold tolerance at the seedling stage in rice

BackgroundCold stress caused by low temperatures is an important factor restricting rice production. Identification of cold-tolerance genes that can stably express in cold environments is crucial for molecular rice breeding.ResultsIn this study, we employed high-throughput quantitative trait locus sequencing (QTL-seq) analyses in a 460-individual F2:3 mapping population to identify major QTL genomic regions governing cold tolerance at the seedling stage in rice. A novel major QTL (qCTS6) controlling the survival rate (SR) under low-temperature conditions of 9°C/10 days was mapped on the 2.60-Mb interval on chromosome 6. Twenty-seven single-nucleotide polymorphism (SNP) markers were designed for the qCST6 region based on re-sequencing data, and local QTL mapping was conducted using traditional linkage analysis. Eventually, we mapped qCTS6 to a 96.6-kb region containing 13 annotated genes, of which seven predicted genes contained 13 non-synonymous SNP loci. Quantitative reverse transcription PCR analysis revealed that only Os06g0719500, an OsbZIP54 transcription factor, was strongly induced by cold stress. Haplotype analysis confirmed that +376 bp (T>A) in the OsbZIP54 coding region played a key role in regulating cold tolerance in rice.ConclusionWe identified OsbZIP54 as a novel regulatory gene associated with rice cold-responsive traits, with its Dongfu-104 allele showing specific cold-induction expression serving as an important molecular variation for rice improvement. This result is expected to further exploration of the genetic mechanism of rice cold tolerance at the seedling stage and improve cold tolerance in rice varieties by marker-assisted selection.

Resolving species boundaries in a recent radiation with the Angiosperms353 probe set: the Lomatium packardiae/L. anomalum clade of the L. triternatum (Apiaceae) complex.

PremiseSpeciation not associated with morphological shifts is challenging to detect unless molecular data are employed. Using Sanger‐sequencing approaches, the Lomatium packardiae/L. anomalum subcomplex within the larger Lomatium triternatum complex could not be resolved. Therefore, we attempt to resolve these boundaries here.MethodsThe Angiosperms353 probe set was employed to resolve the ambiguity within Lomatium triternatum species complex using 48 accessions assigned to L. packardiae, L. anomalum, or L. triternatum. In addition to exon data, 54 nuclear introns were extracted and were complete for all samples. Three approaches were used to estimate evolutionary relationships and define species boundaries: STACEY, a Bayesian coalescent‐based species tree analysis that takes incomplete lineage sorting into account; ASTRAL‐III, another coalescent‐based species tree analysis; and a concatenated approach using MrBayes. Climatic factors, morphological characters, and soil variables were measured and analyzed to provide additional support for recovered groups.ResultsThe STACEY analysis recovered three major clades and seven subclades, all of which are geographically structured, and some correspond to previously named taxa. No other analysis had full agreement between recovered clades and other parameters. Climatic niche and leaflet width and length provide some predictive ability for the major clades.ConclusionsThe results suggest that these groups are in the process of incipient speciation and incomplete lineage sorting has been a major barrier to resolving boundaries within this lineage previously. These results are hypothesized through sequencing of multiple loci and analyzing data using coalescent‐based processes.

T cell receptor beta locus sequencing early post-allogeneic stem cell transplant identifies patients at risk of initial and recurrent cytomegalovirus infection.

Identification of patients at risk of initial & recurrent cytomegalovirus (CMV) reactivation following allogeneic stem cell transplant (alloSCT) may help guide prophylactic strategies. T-cell receptor beta (TRB) deep sequencing was used to identify and enumerate the T-cell repertoire harbouring TRB sequences with annotated specificity to CMV (pubCMVrep), as well as the overall T-cell receptor (TCR) repertoire diversity at day +30 & day +60 post-alloSCT for 65 patients. T-cells harbouring TRB sequences with annotated specificity for CMV were identifiable in all patients. 56% of patients required CMV treatment and 23% of the cohort developed recurrent CMV. PubCMVrep size at day +30 was not associated with reactivation, however amongst patients with antecedent CMV viremia a low day +60 pubCMVrep was associated with a greater incidence of recurrent CMV (75% vs. 21%, HR 6.16, 95% CI 1.29-29.40, P = 0.0008). Moreover, patients with high pubCMVrep only developed recurrent CMV in the setting of GVHD. Low TCR diversity at day +30 was associated with a greater incidence of initial CMV reactivation (71% vs. 22%, HR 5.39, 95% CI 1.70-17.09, p = 0.0002). pubCMVrep and TCR diversity are promising biomarkers to identify patients at risk of initial & recurrent CMV who may benefit from novel prophylactic strategies.

Novel Cysteine-Sparing Hypomorphic NOTCH3 A1604T Mutation Observed in a Family With Migraine and White Matter Lesions.

ObjectiveTo conduct a clinical study of a family with neurologic symptoms and findings carrying a novel NOTCH3 mutation and to analyze the molecular consequences of the mutation.MethodsWe analyzed a family with complex neurologic symptoms by MRI and neurologic examinations. Exome sequencing of the NOTCH3 locus was conducted, and whole-genome sequencing was performed to identify COL4A1, COL4A2, and HTRA1 mutations. Cell lines expressing the normal or NOTCH3A1604T receptors were analyzed to assess proteolytic processing, cell morphology, receptor routing, and receptor signaling.ResultsCerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is the most common hereditary form of cerebral small vessel disease (SVD) and caused by mutations in the NOTCH3 gene. Most CADASIL mutations alter the number of cysteine residues in the extracellular domain of the NOTCH3 receptor, but in this article, we describe a family in which some members carry a novel cysteine-sparing NOTCH3 mutation (c.4810 G>A, p.Ala1604Thr). Two of 3 siblings heterozygous for the NOTCH3A1604T mutation presented with migraine and white matter lesions (WMLs), the latter of a type related to but distinct from what is normally observed in CADASIL. Two other members instead carried a novel COL4A1 missense mutation (c.4795 G>A; p.(Ala1599Thr)). The NOTCH3A1604T receptor was aberrantly processed, showed reduced presence at the cell surface, and less efficiently activated Notch downstream target genes.ConclusionsWe identify a family with migraine and WML in which some members carry a cysteine-sparing hypomorphic NOTCH3 mutation. Although a causal relationship is not established, we believe that the observations contribute to the discussion on dysregulated Notch signaling in cerebral SVDs.

Natural variation identifies a Pxy gene controlling vascular organisation and formation of nodules and lateral roots in Lotus japonicus.

Forward and reverse genetics using the model legumes Lotus japonicus and Medicago truncatula have been instrumental in identifying the essential genes governing legume-rhizobia symbiosis. However, little information is known about the effects of intraspecific variation on symbiotic signalling. Here, we use quantitative trait locus sequencing (QTL-seq) to investigate the genetic basis of the differentiated phenotypic responses shown by the Lotus accessions Gifu and MG20 to inoculation with the Mesorhizobium loti exoU mutant that produces truncated exopolysaccharides. We identified through genetic complementation the Pxy gene as a component of this differential exoU response. Lotus Pxy encodes a leucine-rich repeat receptor-like kinase similar to Arabidopsis thaliana PXY, which regulates stem vascular development. We show that Lotus pxy insertion mutants displayed defects in root and stem vascular organisation, as well as lateral root and nodule formation. Our work links Pxy to de novo organogenesis in the root, highlights the genetic overlap between regulation of lateral root and nodule formation, and demonstrates that natural variation in Pxy affects nodulation signalling.

Mapping of a major QTL controlling plant height using a high-density genetic map and QTL-seq methods based on whole-genome resequencing in Brassica napus.

Plant height is a crucial element related to plant architecture that influences the seed yield of oilseed rape (Brassica napus L.). In this study, we isolated a natural B. napus mutant, namely a semi-dwarf mutant (sdw-e), which exhibits a 30% reduction in plant height compared with Zhongshuang 11-HP (ZS11-HP). Quantitative trait locus sequencing (QTL-seq) was conducted using two extreme DNA bulks in F2 populations in Wuchang-2017 derived from ZS11-HP × sdw-e to identify QTLs associated with plant height. The result suggested that two QTL intervals were located on chromosome A10. The F2 population consisting of 200 individuals in Yangluo-2018 derived from ZS11-HP × sdw-e was used to construct a high-density linkage map using whole-genome resequencing. The high-density linkage map harbored 4323 bin markers and covered a total distance of 2026.52 cM with an average marker interval of 0.47 cM. The major QTL for plant height named qPHA10 was identified on linkage group A10 by interval mapping and composite interval mapping methods. The major QTL qPHA10 was highly consistent with the QTL-seq results. And then, we integrated the variation sites and expression levels of genes in the major QTL interval to predict the candidate genes. Thus, the identified QTL and candidate genes could be used in marker-assisted selection for B. napus breeding in the future.

A New Species of Bridled Darter Endemic to the Etowah River System in Georgia (Percidae: Etheostomatinae: Percina)

Percina freemanorum, the Etowah Bridled Darter, is described as a new species endemic to the Etowah River system in Georgia, specifically in Long Swamp Creek, Amicalola Creek, and the upper portion of the Etowah River. The earliest collection records for Percina freemanorum date to 1948 and in 2007 the species was delimited as populations of Percina kusha. Our investigation into the systematics of Percina kusha is motivated by the uncertain status of populations in the Coosawattee River system and observed morphological disparity in several meristic traits between populations in the Conasauga and Etowah River systems. Our analyses of morphological divergence, nuclear genotypes, and mitochondrial DNA (mtDNA) haplotype networks confirm the distinctiveness of Percina freemanorum. Morphologically, Percina freemanorum is distinguished from Percina kusha through lower average numbers of lateral line scales (65.4 vs. 72.3); rows of transverse scales (18.0 vs. 21.4); scales around the caudal peduncle (22.1 vs. 24.9); and modally more pectoral fin rays (14 vs. 13). The two species are not reciprocally monophyletic in phylogenetic analysis of mtDNA sequences, but the two species do not share mtDNA haplotypes. Analysis of up to 158,000 double digest restriction-site associated DNA (ddRAD) sequencing loci resolve each of the two species as reciprocally monophyletic and genomic clustering analysis of single nucleotide polymorphisms identifies two genetic clusters that correspond to the morphologically delimited Percina freemanorum and Percina kusha.

Identification of common predisposing loci to hematopoietic cancers in four dog breeds.

Histiocytic sarcoma (HS) is a rare but aggressive cancer in both humans and dogs. The spontaneous canine model, which has clinical, epidemiological, and histological similarities with human HS and specific breed predispositions, provides a unique opportunity to unravel the genetic basis of this cancer. In this study, we aimed to identify germline risk factors associated with the development of HS in canine-predisposed breeds. We used a methodology that combined several genome-wide association studies in a multi-breed and multi-cancer approach as well as targeted next-generation sequencing, and imputation We combined several dog breeds (Bernese mountain dogs, Rottweilers, flat-coated retrievers, and golden retrievers), and three hematopoietic cancers (HS, lymphoma, and mast cell tumor). Results showed that we not only refined the previously identified HS risk CDKN2A locus, but also identified new loci on canine chromosomes 2, 5, 14, and 20. Capture and targeted sequencing of specific loci suggested the existence of regulatory variants in non-coding regions and methylation mechanisms linked to risk haplotypes, which lead to strong cancer predisposition in specific dog breeds. We also showed that these canine cancer predisposing loci appeared to be due to the additive effect of several risk haplotypes involved in other hematopoietic cancers such as lymphoma or mast cell tumors as well. This illustrates the pleiotropic nature of these canine cancer loci as observed in human oncology, thereby reinforcing the interest of predisposed dog breeds to study cancer initiation and progression.

Multilocus Genotyping of Sympatric Hepatozoon Species Infecting the Blood of Ontario Ranid Frogs Reinforces Species Differentiation and Identifies an Unnamed Hepatozoon Species.

Intraerythrocytic gamonts of at least 2 named Hepatozoon species have been reported to infect the erythrocytes of ranid frogs in Ontario, Canada. Although gamonts of both species are morphometrically similar, the cytopathological changes that 1 of these species, Hepatozoon clamatae, causes to host erythrocytes, manifested by nuclear fragmentation, was used historically to distinguish this parasite from Hepatozoon catesbianae. Molecular characterization of these 2 Hepatozoon species has been equivocal in correlating genotype with gamont morphotype. Amplification and sequencing of multiple potential genotyping loci within the nuclear (18S ribosomal deoxyribonucleic acid [rDNA]; internal transcribed spacer 1), apicoplast (23S rDNA), and mitochondrial genomes (complete genomes, cytochrome c oxidase subunits I and III [COI and COIII], and cytochrome b) were conducted on Hepatozoon species that infect ranid frogs in Ontario. Sequence data were then used to evaluate the diversity of parasites present in these amphibian hosts and to assign genotypes to gamont morphotypes, if possible. Three distinct genotypes were identified at all loci; the data permitted the discovery of a third, formerly unrecognized Hepatozoon species in ranid frogs from Ontario. Although all genetic loci demonstrated differences between Hepatozoon species, mitochondrial COIII sequences were most suitable for genotypic differentiation of these parasites of frogs. Linking genotypes to gamont morphotypes proved impossible; genotypes identified as H. catesbianae and H. clamatae were found in infections with or without nuclear fragmentation of their host erythrocytes. This suggests that differentiating these species must rely on suitable genotyping methods for identification in the blood of their amphibian intermediate hosts.

Breast Cancer Patient Prognosis Is Determined by the Interplay between TP53 Mutation and Alternative Transcript Expression: Insights from TP53 Long Amplicon Digital PCR Assays

Simple SummaryThe TP53 gene, the most commonly mutated gene in human cancers, is capable of producing multiple RNAs (transcripts). The aim of our study was to measure the abundance of each TP53 transcript, combined with TP53 gene mutation information, to determine the interplay between these in a cohort of breast tumors from New Zealand patients. To do this, we devised a new assay which then enabled the measurement of all known TP53 transcripts. We showed how TP53 gene mutations influenced the levels of specific TP53 transcripts in breast tumors. We evaluated whether a combination of TP53 tumor information, including TP53 mutation status and the levels of certain TP53 transcripts, with standard clinical and pathological information, was associated with breast cancer patient outcome. We recommend that a truly comprehensive analysis of TP53 needs to incorporate data about both TP53 DNA mutations and the expression of the alternative TP53 transcripts.The TP53 gene locus is capable of producing multiple RNA transcripts encoding the different p53 protein isoforms. We recently described multiplex long amplicon droplet digital PCR (ddPCR) assays to quantify seven of eight TP53 reference transcripts in human tumors. Here, we describe a new long amplicon ddPCR assay to quantify expression of the eighth TP53 reference transcript encoding ∆40p53α. We then applied these assays, alongside DNA sequencing of the TP53 gene locus, to tumors from a cohort of New Zealand (NZ) breast cancer patients. We found a high prevalence of mutations at TP53 splice sites in the NZ breast cancer cohort. Mutations at TP53 intron 4 splice sites were associated with overexpression of ∆133TP53 transcripts. Cox proportional hazards survival analysis showed that interplay between TP53 mutation status and expression of TP53 transcript variants was significantly associated with patient outcome, over and above standard clinical and pathological information. In particular, patients with no TP53 mutation and a low ratio of TP53 transcripts t2 to t1, which derive from alternative intron 1 acceptor splice sites, had a remarkably good outcome. We suggest that this type of analysis, integrating mutation and transcript expression, provides a step-change in our understanding of TP53 in cancer.