Detection Of Duplications Research Articles

Chromoanagenesis of chromosome 22 in a subject with obesity and borderline cognitive performance

Chromoanagenesis events consist of complex chromosome rearrangements with multiple breakpoints in one or few chromosomes. Mechanisms of chromoanagenesis are split into three major groups: chromothripsis, chromoanasynthesis and chromoplexy. This study aims to delineate a chromoanagenesis event at the level of chromosome 22 in an individual showing obesity and borderline cognitive performance as major disturbances. The proband and his parents were subjected to conventional karyotyping, CGH array and whole genomic sequencing (WGS). By conventional karyotyping a “de novo” pericentric inversion of chromosome 22 was identified. CGH array identified several imbalances (either deletions or duplications) in the long arm of chromosome 22; the largest is a 4.5 Mb duplication at 22q12.1-22q1.3. The detection of extensive duplications would suggest the occurrence of a chromoanasynthesis event. WGS, in addition to the structural alterations identified by karyotyping and CGH array, revealed two translocations from chromosome 22 to chromosomes 6 and 21 as well as a heterozygous pathogenetic variant of ALMS1 gene; the latter could have contributed to the obesity of our patient. The pericentric inversion induces loss of initial part of TCF20 gene including the 5’ regulatory region and the first, noncoding, exon. Heterozygous loss-of-function mutations of TCF20 gene have been found in patients with autism spectrum disorder or intellectual disability, some of them presenting obesity. It is, therefore, possible that disruption of TCF20 gene structure would contribute to a fraction of the patient’s phenotype.

Experiences with using ears for stakeholder requirements deve-lopment of the safe integration of unmanned aircrafts in the airspace project

Requirements permeate the entire lifecycle of a system or product, from identifying the stakeholders’ needs to disposal, including verification and validation activities. Therefore, the process for the requirements development must be carefully observed and established in order to ensure that the requirements are complete, consistent, and correct. The use of an appropriate predefined structure can improve the requirements’ elicitation, enabling the reduction of ambiguities and the detection of duplications or omissions. Thus, this article proposes the use of syntax patterns during the development of the stakeholders’ requirements in the context of an airspace project. The result of applying the proposal of this article in a real case was a list of initial requirements in standardized language with their traceability guaranteed.

DTDHM: detection of tandem duplications based on hybrid methods using next-generation sequencing data.

Tandem duplication (TD) is a common and important type of structural variation in the human genome. TDs have been shown to play an essential role in many diseases, including cancer. However, it is difficult to accurately detect TDs due to the uneven distribution of reads and the inherent complexity of next-generation sequencing (NGS) data. This article proposes a method called DTDHM (detection of tandem duplications based on hybrid methods), which utilizes NGS data to detect TDs in a single sample. DTDHM builds a pipeline that integrates read depth (RD), split read (SR), and paired-end mapping (PEM) signals. To solve the problem of uneven distribution of normal and abnormal samples, DTDHM uses the K-nearest neighbor (KNN) algorithm for multi-feature classification prediction. Then, the qualified split reads and discordant reads are extracted and analyzed to achieve accurate localization of variation sites. This article compares DTDHM with three other methods on 450 simulated datasets and five real datasets. In 450 simulated data samples, DTDHM consistently maintained the highest F1-score. The average F1-score of DTDHM, SVIM, TARDIS, and TIDDIT were 80.0%, 56.2%, 43.4%, and 67.1%, respectively. The F1-score of DTDHM had a small variation range and its detection effect was the most stable and 1.2 times that of the suboptimal method. Most of the boundary biases of DTDHM fluctuated around 20 bp, and its boundary deviation detection ability was better than TARDIS and TIDDIT. In real data experiments, five real sequencing samples (NA19238, NA19239, NA19240, HG00266, and NA12891) were used to test DTDHM. The results showed that DTDHM had the highest overlap density score (ODS) and F1-score of the four methods. Compared with the other three methods, DTDHM achieved excellent results in terms of sensitivity, precision, F1-score, and boundary bias. These results indicate that DTDHM can be used as a reliable tool for detecting TDs from NGS data, especially in the case of low coverage depth and tumor purity samples.

Detection of KMT2A Partial Tandem Duplications ( KMT2A-PTDs) in Healthy Donors Using Next Generation Sequencing

Introduction KMT2A ( MLL) fusions are relevant biomarkers in myeloid malignancies. KMT2A-PTD (also called MLL-PTD, partial tandem duplication), a non-fusion gene rearrangement, also occurs and serves as an important biomarker for Menin-inhibitors. Traditionally, KMT2A-PTDs are detected by RT-qPCR (quantitative real-time PCR). Here we report the detection of KMT2A-PTDs in whole blood samples taken from healthy donors using the Oncomine TM Myeloid Assay GX v2, which is a next-generation sequencing (NGS) based assay that enables efficient detection of multiple genetic alterations in parallel. KMT2A-PTD positive clinical research samples were used as a control in this study. This study was initiated based on reports from various healthcare professionals who observed several healthy blood donors identified with KMT2A-PTD using the Oncomine TM Myeloid Assay GX v2. As a result, we conducted tests on healthy controls and compared them with clinical research samples known to have myeloid malignancies to explore this genomic alteration further. Methods We sequenced 20 whole blood samples (total 104 replicates) from healthy donors at 3 different sites of Thermo Fisher Scientific TM (South San Francisco, CA; Guilford, CT; Carlsbad, CA). These whole blood samples were received from Stanford Blood Center or purchased from Discovery Life Sciences. As a control, we also sequenced 20 clinical research samples (total 85 replicates) that are known to have myeloid malignancies in both Sonora Quest Laboratories and at Thermo Fisher Scientific TM South San Francisco site. These samples were run on the Ion Torrent TM Genexus TM Software 6.6 and analyzed using the Oncomine TM Myeloid Assay GX v2 with fusion profiling, where the assay design targets 6 different KMT2A-PTD variants. Results The data set has mean read length 90 - 120bp and total mapped fusion reads 20,000 - 30,000. We have found evidence of KMT2A-PTDs in confirmed positive samples from myeloid malignancies (including: AML, acute myeloid leukemia; MPN, myeloproliferative neoplasms; MDS, myelodysplastic syndromes; CML, chronic myeloid leukemia; CMML, chronic myelomonocytic leukemia; or JMML, juvenile myelomonocytic leukemia) and healthy donor samples. The read counts of PTDs detected in the healthy donor samples are on average about 1/3 of the read counts of PTDs detected in the myeloid positive samples, but the distributions overlap. Approximately 33% of myeloid positive samples have PTD read counts higher than the maximum read counts detected in any of the healthy donor samples. The sequences of KMT2A-PTDs found in both healthy donor samples and myeloid samples were further analyzed in BLAT (BLAST-Like Alignment Tool) to confirm that they match the specific exons on the KMT2A gene. Based on this data, only samples with myeloid malignancies have >2000 reads that support KMT2A-PTDs with this assay. Conclusions We describe the detection of KMT2A-PTDs in a range of clinical research samples, including healthy controls as well as samples with myeloid malignancies. KMT2A-PTDs were detected in healthy control samples at lower levels, however, one third of myeloid samples showed significantly higher KMT2A-PTD read counts compared to healthy controls. This intriguing finding warrants further investigation, as it opens up the possibility of conducting a prospective study to follow up on these individuals and observe if they develop myeloid malignancies. Moreover, this finding presents an opportunity for conducting retrospective studies to explore whether healthy controls previously identified with this alteration several years ago after blood donation were subsequently recorded in the health national system with myeloid malignancies. (For research use only. Not for use in diagnostic procedures.)

Role of Cytogenetics and Fluorescence In Situ Hybridization in the Laboratory Workup of Acute Myeloid Leukemias

AbstractA new understanding of acute myeloid leukemia as a varied group of unique biologic entity has emerged, as a result of the identification of various chromosomal aberrations and their association with clinical prognosis and diagnosis. Following induction treatment, cytogenetic examination can establish the presence of any residual malignant cells, it's recurrence, clonal evolution if any, or the formation of novel abnormalities. The G-banded karyotype has been the gold standard method for detecting all of these aberrations for years. The capacity to examine the entire genome through karyotype analysis quickly enabled the detection of deletions, duplications, and structural rearrangements across every chromosome, and the more frequent ones were associated with particular aberrant clinical symptoms. Fluorescence in situ hybridization (FISH) is a sensitive technology that aids in differential diagnosis or therapeutic planning and provides rapid results. Furthermore, the combination of cytogenetic and molecular profiling enables a more precise evaluation of disease prognosis, diagnosis, classification, risk stratification, and patient treatment. Interphase FISH analysis, in conjunction with G-banded chromosomal analysis, can be used as a major testing tool for the evaluation of hematological neoplasms. For accurate and consistent descriptions of genomic changes identified by karyotyping and FISH, a specified terminology is necessary. The International System for Human Cytogenomic Nomenclature is the main source and provides instructions for documenting cytogenetic and molecular findings in laboratory reports. This review discusses the two methods, karyotyping and FISH, their advantages and limitations, sample requirements, various FISH probes that are used, nomenclature for results reporting, and the necessary quality control measures.

Persistence of FLT3-TKD in Blood Prior to Allogeneic Transplant Is Associated with Increased Relapse and Death in Adults with AML in First Remission

Introduction: Next-generation sequencing (NGS) represents a promising modality for measurable residual disease (MRD) testing in patients with acute myeloid leukemia (AML), but systematic efforts are required to define the appropriate targets, test characteristics, and clinical implications. We recently reported that detection of persistent NPM1 or FLT3 internal tandem duplication (ITD) mutations in adults with AML in first complete remission (CR1) prior to allogeneic hematopoietic cell transplantation (alloHCT) is associated with increased relapse and death compared with those testing negative (JAMA 2023, PMID: 36881031). FLT3 tyrosine kinase domain (TKD) variants are present in 7-10% of adult patients with AML but the utility of this target for AML MRD testing in CR1 prior to alloHCT was previously unknown. Methods: Patients aged 18 or older who underwent alloHCT for AML in CR1 between 2013 and 2019 reported to have a FLT3-TKD variant at diagnosis and a pre-conditioning remission blood sample available in the CIBMTR biorepository were eligible for this study. Single-amplicon NGS (SA-NGS) library generation utilizing UMI-tagged primers targeting the D835 and I836 codons of FLT3 was performed on 400ng genomic DNA sequenced with unique dual indices, with error-corrected variant calling. Primary outcomes were overall survival (OS) and cumulative incidence of relapse (CIR). Secondary outcomes were relapse-free survival (RFS) and non-relapse mortality (NRM), with NRM being considered as a competing risk. Results: A total of351 patients met inclusion criteria for this study, and 342 (97.4%) had sufficient DNA for SA-NGS and were analyzed for clinical outcomes. Of these patients, 81 (23.7%) experienced relapse at a median of 5 months post-alloHCT. Using the current ELN-recommended variant allele fraction (VAF) threshold of 0.1% for NGS-based AML MRD, 14 patients (4.1%) tested positive. Patients with FLT3-TKD MRD VAF≥0.1% experienced higher rates of relapse (73.8% vs. 20.5%, p < 0.0001) and decreased OS (11.4% vs. 66.8%, p < 0.001) compared to VAF<0.1%. The highly sensitive SA-NGS method allowed for exploration to determine if a deeper VAF threshold of 0.01% (previously validated for NPM1 and FLT3-ITD in the Pre-MEASURE study) was prognostic for FLT3-TKD as an MRD target. This led to an additional 20 patients being reclassified as positive (n = 34 with VAF above 0.01%, =9.9%). However, there was no significant difference in CIR (33.6% vs. 19.7%, p = 0.089) or OS (65.2% vs. 66.9%, p = 0.267) between patients with 0.01%≤VAF<0.1% and VAF<0.01% ( Figure). Out of 28 SA-NGS positive calls, 6 could not be validated by orthogonal digital droplet PCR testing, and interestingly, none of those 6 patients experienced relapse. Site-reported pre-transplant remission multiparametric flow cytometry (MFC) was available for 335 patients (98%). Results of MFC were not associated with differences in CIR (HR 1.2, 95% CI 0.52 - 2.76, p = 0.67) or OS (HR 0.78, 95% CI 0.34 - 1.77, p = 0.551).In multivariable analysis, FLT3-TKD NGS-MRD VAF≥0.1% remained prognostic for CIR (HR 6.1, 95% CI 3.1 - 12.0, p < 0.001) and OS (HR 3.2, 95% CI 1.7 - 5.9, p < 0.001). Conclusions: Detectable persistence of FLT3-TKD variants in blood from AML patients in first remission prior to first alloHCT is rare, but at a VAF level of 0.1% was strongly associated with increased relapse and death after transplant compared to those testing negative. In contrast to FLT3-ITD NGS-MRD, no evidence was found to support lowering the NGS-MRD VAF threshold from 0.1% to 0.01% for FLT3-TKD. Alternative methodology may further improve NGS-MRD test performance. Site-reported MFC prior to alloHCT did not stratify for post-transplant relapse or survival. Optimal NGS-MRD testing for patients with AML will likely require a multi-target approach and testing for persistence of FLT3-TKD MRD is shown here to represent an important component of this strategy.

Exploring the Mitogenomes of Mantodea: New Insights from Structural Diversity and Higher-Level Phylogenomic Analyses.

The recently reorganized classification of Mantodea has made significant progress in resolving past homoplasy problems, although some relationships among higher taxa remain uncertain. In the present study, we utilized newly sequenced mitogenomes and nuclear gene sequences of 23 mantid species, along with published data of 53 mantises, to perform familial-sampling structural comparisons of mantodean mitogenomes and phylogenomic studies. Our rstructural analysis revealed generally conserved mitogenome organizations, with a few cases of tRNA gene rearrangements, including the detection of trnL2 duplication for the first time. In our phylogenetic analysis, we found a high degree of compositional heterogeneity and lineage-specific evolutionary rates among mantodean mitogenomes, which frequently corresponded to several unexpected groupings in the topologies under site-homogeneous models. In contrast, the topologies obtained using the site-heterogeneous mixture model fit the currently accepted phylogeny of Mantodea better. Topology tests and four-cluster likelihood mapping analyses further determined the preferred topologies. Our phylogenetic results confirm the monophyly of superfamilial groups Schizomantodea, Amerimantodea, Heteromantodea, Promantidea, and Mantidea and recover the early-branching relationships as (Mantoidoidea + (Amerimantodea + (Metallyticoidea + Cernomantodea))). Additionally, the results suggest that the long-unresolved phylogenetic position of Majangidae should be placed within Mantidea, close to Mantoidea, rather than within Epaphroditoidea. Our findings contribute to understanding the compositional and structural diversity in mantodean mitogenomes, underscore the importance of evolutionary model selection in phylogenomic studies, and provide new insights into the high-level phylogeny of Mantodea.

Additional Detection of Clones Using Locally Sensitive Hashing

Today, there are many methods for detecting blocks with repetitions and redundancy in the program code. But mostly they turn out to be dependent on the programming language in which the software is developed and try to detect complex types of repeating blocks. Therefore, the goal of the research was to develop a language-independent repetition detector and expand its capabilities. In the development and operation of the language-independent incremental repeater detector, it was decided to conduct experiments for five open source systems for evaluation using the industrial detector SIG (Software Improvement Group), including the use of a tool syntactic analysis. But there was the question of extending the algorithm for additional detection of duplication and redundancy in the code, which was proposed by Hammel, and how improvements can be made to achieve independence from the programming language. Particular attention was paid to the empirical results presented in the original study, as their effectiveness is questionable. The main parameters that were considered when creating the index for LIIRD (Language-independent incremental repeat detector) and its expansion of the LSH (locally sensitive hashing): measuring time, memory and creating an incremental step. Based on the results of experiments conducted by the authors of Hammel’s work, there was a motivation to develop an extended approach. The idea of this approach is that according to the original study, the operation of calculating the entire block index with repeats and redundancy from scratch is very time consuming. Therefore, it is proposed to use LSH to obtain an effective assessment of the similarity of software project files.

Tandem and inverted duplications in haemophilia A: Breakpoint characterisation provides insight into possible rearrangement mechanisms.

Approximately half of patients with severe haemophilia A are caused by structural variants in the F8 gene. Unlike inversions or deletions directly impairing the integrity of F8, some duplications do not completely disrupt the open reading frame or even retain an intact F8 copy. Currently, only a few duplication breakpoints were precisely characterized, and the corresponding rearrangement mechanisms and clinical outcomes remain to be further investigated. Establishing an effective strategy for breakpoint characterization of duplications and revealing their rearrangement mechanisms. AccuCopy is used for the detection of duplications, long-distance PCR for the characterization of tandem duplications, genome walking technique and whole genome sequencing for the characterization of inverted duplications. Four F8 duplication rearrangements were successfully characterized at the nucleotide level: one tandem duplication (exons 7-11) and three inverted duplications (exons 7-22, exons 2-26, and exons 15-22). Two shared features of inverted duplication were found after carefully analysing our results and breakpoint information in the literature: 1, an inverted fragment was inserted into the original chromosome via two junctions; 2, one junction is mediated by a pair of inverted repetitive elements, while the other consists of two breakpoints with microhomology. Similar breakpoint features motivated us to propose a DNA replication-based model to explain the formation of duplication rearrangements. Based on our model, we further divide the inverted duplications into three basic types: type I with a DEL-NOR/INV-DUP pattern, type II with a DUP-NOR/INV-DUP pattern and type III with a DUP-TRP/INV-DUP pattern.

63. Comparative analysis of testing methods used for the detection of internal tandem duplications in the KMT2A/MLL gene

The Lysine (K)-Specific Methyltransferase 2A (KMT2A) gene (formerly MLL) that resides on human chromosome 11q23 is frequently rearranged in de novo and therapy related leukemia. KMT2A translocations with various partner genes are often easily detected by classic molecular and cytogenetic approaches. However, the detection of small intragenic insertions, the partial tandem duplications of the KMT2A gene (KMT2A-PTD), is challenging. Additional co-occurring alterations, such as Trisomy 11 which alerts one to the PTD, may further hamper the identification of KMT2A-PTD. KMT2A-PTDs occur in 3-10% of adult AML and the presence of a KMT2A-PTD is associated with a poorer AML outcome. As such, accurate detection of KMT2A-PTDs in AML routine clinical diagnostic workup is important. Here we assessed the ability of three orthogonal technologies for KMT2A-PTD analysis:1) an hybridization-capture based Next Generation Sequencing targeting the KMT2A gene region, 2) an MLPA based assay covering exons 4 and 36 of KMT2A and 3) Optical Genome Mapping. We analyzed tumor samples of ten AML patients with KMT2A-PTD and evaluated the relative performance, strengths and weaknesses of these technologies. KMT2A-PTDs are in-frame duplications that typically encompass exons 3 through 9. However, our results revealed the presence of unsuspected variants of KMT2A. Specifically, copy number based approaches, in the absence of structural data, can lead to false positive and negative calls due to genomic alterations, such as unbalanced translocations, involving KMT2A. Therefore, our data suggest that a combined structural and copy number approach are required to accurately detect KMT2A PTDs.

An overview of online resources for intra-species detection of gene duplications.

Gene duplication plays an important role in evolutionary mechanism, which can act as a new source of genetic material in genome evolution. However, detecting duplicate genes from genomic data can be challenging. Various bioinformatics resources have been developed to identify duplicate genes from single and/or multiple species. Here, we summarize the metrics used to measure sequence identity among gene duplicates within species, compare several computational approaches that have been used to predict gene duplicates, and review recent advancements of a Basic Local Alignment Search Tool (BLAST)-based web tool and database, allowing future researchers to easily identify intra-species gene duplications. This article is a quick reference guide for research tools used for detecting gene duplicates.

An interview with Miryung Kim - 2022 SIGSOFT Awardee

Miryung Kim received the 2022 SIGSOFT Influential Educator Award for outstanding achievements in undergraduate and graduate mentoring with emphasis on research excellence in software engineering, diversity, and inclusion. She received a Ph.D. in computer science from the University of Washington, Seattle (USA), and she is a Professor and a Vice Chair of graduate studies at UCLA Computer Science. Her research interests include the detection of code clones and code duplication, as well as solutions for their management and removal. She has helped to define the field of software engineering for data intensive computing.

Hereditary alpha tryptasemia determination by digital droplet PCR

Hereditary alpha tryptasemia (HαT) is a recently described genetically inherited trait present in about 5% of the general population. It is associated with higher basal serum tryptase levels, and over-represented among patients with severe anaphylactic reactions to insect stings and patients with mastocytosis. To developed a method to determine tryptase genotypes in humans. The protocol described by Lyons et al. [1] was adapted and employed to determine tryptase genotypes in different individuals and clinical settings by digital droplet PCR (ddPCR). The general principle of ddPCR will be presented, as well as its advantages and application to the specific case of tryptase genotyping and detection of tandem duplications of TPSAB1. The association between different tryptase genotypes and ddPCR profiles will be presented, based on clinical cases encountered since the recent implementation of the technique at St-Antoine, Tenon, and Trousseau hospitals. Digital dPCR is an innovative method to determine tryptase genotypes and diagnose HαT.

Simple and Robust Detection of CYP2D6 Gene Deletions and Duplications Using CYP2D8P as Reference.

Genotyping of the CYP2D6 gene is the most commonly applied pharmacogenetic test globally. Significant economic interests have led to the development of a plurality of assays, available for almost any genotyping platform or DNA detection chemistry. Of all the genetic variants, copy number variations are particular difficult to detect by polymerase chain reaction. Here, we present two simple novel approaches for the identification of samples carrying either deletions or duplications of the CYP2D6 gene; by relative quantification using a singleplex 5′nuclease real-time PCR assay, and by high-resolution melting of PCR products. These methods make use of universal primers, targeting both the CYP2D6 and the reference gene CYP2D8P, which is necessary for the analysis. The assays were validated against a reference method using a large set of samples. The singleplex nature of the 5′nuclease real-time PCR ensures that the primers anneal with equal affinity to both the sequence of the CYP2D6 and the reference gene. This facilitates robust identification of gene deletions and duplications based on the cycle threshold value. In contrast, the high-resolution melting assay is an end-point PCR, where the identification relies on variations between the amount of product generated from each of the two genes.

A comparative study of next-generation sequencing and fragment analysis for the detection and allelic ratio determination of FLT3 internal tandem duplication

BackgroundCurrently, FLT3 internal tandem duplication (ITD) is tested by fragment analysis. With next-generation sequencing (NGS), however, not only FLT3 ITD but also other mutations can be detected, which can provide more genetic information on disease.MethodsWe retrospectively reviewed the results of two tests—fragment analysis and a custom-designed, hybridization capture-based, targeted NGS panel—performed simultaneously. We used the Pindel algorithm to detect FLT3 ITD mutations.ResultsAmong 277 bone marrow aspirate samples tested by NGS and fragment analysis, the results revealed 99.6% concordance in FLT3 ITD detection. Overall, the allele frequency (AF) attained by NGS positively correlated with the standard allelic ratio (AR) attained by fragment analysis, with a Spearman correlation coefficient (r) of 0.757 (95% confidence interval: 0.627–0.846; p < 0.001). It was concluded that an AF of 0.11 attained by NGS is the most appropriate cutoff value (with 85.3% sensitivity and 86.7% specificity) for high mutation burden criterion presented by guidelines.ConclusionSensitive FLT3 ITD detection with comprehensive information of other mutation offered by NGS could be a useful tool in clinical laboratories. Future studies will be needed to evaluate and standardize NGS AF cutoff to predict actual clinical outcomes.

CRISPR-Cas9-mediated editing of starch branching enzymes results in altered starch structure in Brassica napus.

Starch branching enzymes (SBEs) are one of the major classes of enzymes that catalyze starch biosynthesis in plants. Here, we utilized the clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9 (CRISPR-Cas9)-mediated gene editing system to investigate the effects of SBE mutation on starch structure and turnover in the oilseed crop Brassica napus. Multiple single-guide RNA (sgRNA) expression cassettes were assembled into a binary vector and two rounds of transformation were employed to edit all six BnaSBE genes. All mutations were heterozygous monoallelic or biallelic, and no chimeric mutations were detected from a total of 216 editing events. Previously unannotated gene duplication events associated with two BnaSBE genes were characterized through analysis of DNA sequencing chromatograms, reflecting the complexity of genetic information in B. napus. Five Cas9-free homozygous mutant lines carrying two to six mutations of BnaSBE were obtained, allowing us to compare the effect of editing different BnaSBE isoforms. We also found that in the sextuple sbe mutant, although indels were introduced at the genomic DNA level, an alternate transcript of one BnaSBE2.1 gene bypassed the indel-induced frame shift and was translated to a modified full-length protein. Subsequent analyses showed that the sextuple mutant possesses much lower SBE enzyme activity and starch branching frequency, higher starch-bound phosphate content, and altered pattern of amylopectin chain length distribution relative to wild-type (WT) plants. In the sextuple mutant, irregular starch granules and a slower rate of starch degradation during darkness were observed in rosette leaves. At the pod-filling stage, the sextuple mutant was distinguishable from WT plants by its thick main stem. This work demonstrates the applicability of the CRISPR-Cas9 system for the study of multi-gene families and for investigation of gene-dosage effects in the oil crop B. napus. It also highlights the need for rigorous analysis of CRISPR-Cas9-mutated plants, particularly with higher levels of ploidy, to ensure detection of gene duplications.

A hidden structural variation in a known IRD gene: a cautionary tale of two new disease candidate genes.

Rod-cone dystrophy (RCD), also known as retinitis pigmentosa, is an inherited condition leading to vision loss, affecting 1 in 3500 people. More than 270 genes are known to be implicated in the inherited retinal degenerations (IRDs), yet genetic diagnosis for ∼30% of IRD of patients remains elusive despite advances in sequencing technologies. The goal of this study was to determine the genetic causality in a family with RCD. Family members were given a full ophthalmic exam at the Retinal Service at Massachusetts Eye and Ear and consented to genetic testing. Whole-exome sequencing (WES) was performed and variants of interest were Sanger-validated. Functional assays were conducted in zebrafish along with splicing assays in relevant cell lines to determine the impact on retinal function. WES identified variants in two potential candidate genes that segregated with disease: GNL3 (G Protein Nucleolar 3) c.1187 + 3A > C and c.1568-8C > A; and PDE4DIP (Phosphodiester 4D Interacting Protein) c.3868G > A (p.Glu1290Lys) and c.4603G > A (p.Ala1535Thr). Both genes were promising candidates based on their retinal involvement (development and interactions with IRD-associated proteins); however, the functional assays did not validate either gene. Subsequent WES reanalysis with an updated bioinformatics pipeline and widened search parameters led to the detection of a 94-bp duplication in PRPF31 (pre-mRNA Processing Factor 31) c.73_266dup (p.Asp56GlyfsTer33) as the causal variant. Our study demonstrates the importance of thorough functional characterization of new disease candidate genes and the value of reanalyzing next-generation sequencing sequence data, which in our case led to identification of a hidden pathogenic variant in a known IRD gene.

Accurate Detection and Quantification of FLT3 Internal Tandem Duplications in Clinical Hybrid Capture Next-Generation Sequencing Data

FLT3 internal tandem duplications (ITDs) are found in approximately one-third of patients with acute myeloid leukemia and have important prognostic and therapeutic implications that have supported their assessment in routine clinical practice. Conventional methods for assessing FLT3-ITD status and allele burden have been primarily limited to PCR fragment size analysis because of the inherent difficulty in detecting large ITD variants by next-generation sequencing (NGS). In this study, we assess the performance of publicly available bioinformatic tools for the detection and quantification of FLT3-ITDs in clinical hybridization-capture NGS data. We found that FLT3_ITD_ext had the highest overall accuracy for detecting FLT3-ITDs and was able to accurately quantify allele burden. Although all other tools evaluated were able to detect FLT3-ITDs reasonably well, allele burden was consistently underestimated. We were able to significantly improve quantification of FLT3-ITD allelic burden independent of the detection method by utilizing soft-clipped reads and/or ITD junctional sequences. In addition, we show that identifying mutant reads by previously identified junctional sequences further improves the sensitivity of detecting FLT3-ITDs in post-treatment samples. Our results demonstrate that FLT3-ITDs can be reliably detected in clinical NGS data using available bioinformatic tools. We further describe how accurate quantification of FLT3-ITD allele burden can be added on to existing clinical NGS pipelines for routineassessment of FLT3-ITD status in patients with acute myeloid leukemia.

ACMG SF v3.0 list for reporting of secondary findings in clinical exome and genome sequencing: a policy statement of the American College of Medical Genetics and Genomics (ACMG)

ACMG SF v3.0 list for reporting of secondary findings in clinical exome and genome sequencing: a policy statement of the American College of Medical Genetics and Genomics (ACMG)

Disorders of Sex Development.

Disorders of Sex Development.