Author Correction: A robust benchmark for detection of germline large deletions and insertions.

Inherited retinal diseases such as autosomal dominant retinitis pigmentosa (adRP) are strikingly complex, with mutations in many different genes causing the same disease, with many different mutations in each gene, and with different clinical consequences resulting from the same mutation, even within the same family. For example, mutations in sixteen genes are known to cause adRP and an additional two adRP genes have been mapped but not identified yet (Table 1). This raises two questions: what fraction of adRP cases are accounted for by mutations in known genes, and what accounts for the remaining cases? To answer these questions we applied a step-wise screening process to a cohort of wellcharacterized adRP families, now numbering 215. 1 Methods included sequencing of known genes, detection of deletions using MLPA (multiplex ligation-dependent probe amplification), 2 linkage mapping against known loci, and genome-wide linkage mapping. By this combination of approaches we detected mutations in 58% of the families (largely Americans of European origin). Approximately 3% of these families have large deletions that cannot be detected by conventional PCR-based methods, and linkage testing against known loci revealed several additional mutations that were not detected earlier. Thus some of the remaining families are likely to have large deletions or other “hidden” mutations in known genes. However, linkage testing also confirms the existence of new adRP genes. Being able to find the cause of adRP in all or nearly all affected individuals is a difficult but achievable goal, perhaps within the next decade. 3 This information is of immediate value to patients and families, and is a necessary precursor to gene and mutation-specific therapies.

Structural rearrangements, such as inversions, translocations, duplications, and large insertions and deletions, are large-scale genomic variants that can play an important role in shaping phenotypic variation and in genome adaptation and evolution. We used chromosomal-level assemblies from eight Fusarium graminearum isolates to study structural variants and their role in fungal evolution. We generated the assemblies of four of these genomes after Oxford Nanopore sequencing. A total of 87 inversions, 159 translocations, 245 duplications, 58,489 insertions, and 34,102 deletions were detected. Regions of high recombination rate are associated with structural rearrangements, and a significant proportion of inversions, translocations, and duplications overlap with the repeat content of the genome, suggesting recombination and repeat elements are major factors in the origin of structural rearrangements in F. graminearum. Large insertions and deletions introduce presence-absence polymorphisms for many genes, including secondary metabolite biosynthesis cluster genes and predicted effectors genes. Translocation events were found to be shuffling predicted effector-rich regions of the genomes and are likely contributing to the gain and loss of effectors facilitated by recombination. Breakpoints of some structural rearrangements fall within coding sequences and are likely altering the protein products. Structural rearrangements in F. graminearum thus have an important role to play in shaping pathogen-host interactions and broader evolution through genome reorganization, the introduction of presence-absence polymorphisms, and changing protein products and gene regulation.

α-Thalassemia (α-thal) is a common hemoglobinopathy mainly caused by deletion of one or both α-globin genes. We describe an autochthonous Belgian family diagnosed with α-thal trait. Molecular analysis revealed a novel large deletion of at least 170 kb between 226.68 kb (0.2 Mb) and 402.68 kb (0.4 Mb) from the telomere of 16p, leaving the subtelomeric region intact. The deletion includes both α-globin genes (HBA1 and HBA2) but also flanking genes possibly related to non hematological effects: HBQ1, LUC7L, ITFG3, RGS11, ARHGDIG, PDIA2 and AXIN1. These genes are not contained in the region (0.9 and 1.7 Mb from the telomere of 16p) associated with α-thal intellectual disability (ATR-16) syndrome. However, further research is necessary to exclude other potential effects than α-thal in patients with a large deletion at 0.2-0.4 Mb from the telomere of 16p. Genetic counseling is important for carriers of this deletion as homozygosity for the α-globin (– –/) haplotype may lead to Hb Bart’s (γ4) hydrops fetalis syndrome.

Precision Medicine Assays Detect Novel Targetable FLT3 Fusions Amenable to Therapeutic Intervention in a Patient with Refractory Acute Myeloid Leukemia

Hypoplasies vermiennes avec atteintes extracérébrales (rétine, rein, foie) : des syndromes difficiles à classer

We developed a targeted DNA sequencing method that is capable of detecting a comprehensive panel of DNA mutations including small DNA mutations and large DNA deletions with unknown/flexible boundaries. The method directly identifies the large DNA deletions (Cat-D) without relying on sequencing coverage to make the genotype calls. We performed the method to simultaneously detect 10 small DNA mutations in β-thalassemia and 2 large genomic deletions in α-thalassemia from 10 genomic DNA samples. Cat-D was performed on 8 genomic DNA samples in duplicate. The 18 Cat-D samples were combined in one sequencing run. In total, 216 genotype calls were made, and 215 of the genotype calls were accurate. No false negative genotype calls were made. One false positive genotype call was made on one target mutation in one experimental duplicate from a genomic DNA sample. In summary, Cat-D can be developed into a robust, high-throughput and cost-effective method suitable for population-based carrier screens.

The fall armyworm (Spodoptera frugiperda) is a highly polyphagous lepidopteran pest of relevant food and fiber staple crops. In the Americas, transgenic corn and cotton producing insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) have controlled and reduced the damage caused by S. frugiperda. However, cases of field-evolved S. frugiperda resistance to Bt corn producing the Cry1F insecticidal protein have been documented in North and South America. When characterized, field resistance to Cry1F is linked to insertions and mutations resulting in a modified or truncated ABC transporter subfamily C2 (SfABCC2) protein that serves as Cry1F receptor in susceptible S. frugiperda. In this work, we present detection of a large genomic deletion (~ 8 kb) affecting the SfABCC2 and an ABC transporter gene subfamily 3 –like gene (SfABCC3) as linked to resistance to Cry1F corn in a S. frugiperda strain from Florida (FL39). Monitoring for this genomic deletion using a discriminatory PCR reaction in field-collected S. frugiperda moths detected individuals carrying this allele in Florida, but not in surrounding states. This is the first report of a large genomic deletion being involved in resistance to a Bt insecticidal protein.

An Escherichia coli plasmid-based, mutational system in which supF mutants are selectable: Insertion elements dominate the spontaneous spectra

The recent CRISPR revolution has provided researchers with powerful tools to perform genome editing in a variety of organisms. However, recent reports indicate widespread occurrence of unintended CRISPR-induced on-target effects (OnTEs) at the edited site in mice and human induced pluripotent stem cells (iPSCs) that escape standard quality controls. By altering gene expression of targeted or neighbouring genes, OnTEs can severely affect phenotypes of CRISPR-edited cells and organisms and thus lead to data misinterpretation, which can undermine the reliability of CRISPR-based studies. Here we describe a broadly applicable framework for detecting OnTEs in genome-edited cells and organisms after non-homologous end joining-mediated and homology-directed repair-mediated editing. Our protocol enables identification of OnTEs such as large deletions, large insertions, rearrangements or loss of heterozygosity (LOH). This is achieved by subjecting genomic DNA first to quantitative genotyping PCR (qgPCR), which determines the number of intact alleles at the target site using the same PCR amplicon that has been optimized for genotyping. This combination of genotyping and quantitation makes it possible to exclude clones with monoallelic OnTEs and hemizygous editing, which are often mischaracterized as correctly edited in standard Sanger sequencing. Second, occurrence of LOH around the edited locus is detected by genotyping neighbouring single-nucleotide polymorphisms (SNPs), using either a Sanger sequencing-based method or SNP microarrays. All steps are optimized to maximize simplicity and minimize cost to promote wide dissemination and applicability across the field. The entire protocol from genomic DNA extraction to OnTE exclusion can be performed in 6-9 d.

A biopsy specimen from a patient with follicular lymphoma was divided into two fragments. DNA was extracted from one fragment and a 1.2 kb region of the functional heavy chain (IgH) gene was amplified, cloned and sequenced (eight clones). From the other fragment a cell line (HF-1) was started. The IgH gene region was amplified from the cell line, and sequenced without cloning. The nine sequences obtained could be arranged into a genealogical tree where the individual sequences differed from the deduced ancestor by 16-29 single nucleotide changes, some also by an insertion and/or a deletion. It is apparent that the sequence alterations were caused by somatic mutations during the growth of the lymphoma. The comparison of the sequences with two published (allelic) germline sequences of the human JH region showed approximately 20% non-homology. The differences included five additional multinucleotide insertion/deletion changes, the longest of them a 101-nucleotide insertion. Two long insertions were homologous to the adjacent germline sequences. We propose that most of the changes observed, including long deletions and insertions, represent or are linked to somatic hypermutation events of the Ig gene type. Although in a few cases large deletions and insertions (> 2 bp) have been found in mutated immunoglobulin genes, our results, for the first time, firmly link these deletions/insertions to somatic hypermutations; their frequency was found to be 2.2% of the observed mutational events in the non-translated gene regions. HF-1 is the first follicular lymphoma line successfully established from a lymphoma known to have hypermutated its Ig genes during the malignant growth. It is a candidate cell line to be studied for its ability to generate mutations of B cell type in cell cultures.

Corrigendum to “Gene mapping strategy for Alu elements rearrangements: Detection of new large deletions in the SERPING1 gene causing hereditary angioedema in Brazilian families” [Gene 685C (2019) 179–185

Next generation molecular diagnosis of mitochondrial disorders

Factor V (FV) deficiency is a rare autosomal recessive bleeding disorder caused by mutations in the F5 gene. FV-deficient patients in whom no mutation or only one mutation is found may harbour large gene rearrangements, which are not detected by conventional mutation screening strategies. The aim of this study was to develop and validate a multiplex ligation-dependent probe amplification (MLPA) assay for the detection of large deletions and duplications in the F5 gene. Twenty-two MLPA probes targeting 19 of the 25 exons and the upstream and downstream regions of the F5 gene were designed and tested in 10 normal controls, a patient with a known heterozygous deletion of F5 exons 1-7 (positive control) and 14 genetically unexplained FV-deficient patients. MLPA results were confirmed by digital PCR on a QuantStudio(™) 3D Digital PCR System. The F5-specific probes yielded a reproducible peak profile in normal controls, correctly detected the known deletion in the positive control and suggested the presence of a novel deletion of exons 9-10 in a patient with undetectable FV levels and only one identified mutation. Follow-up by chip-based digital PCR, long-range PCR and direct sequencing confirmed that this patient carried a heterozygous F5 deletion of 1823 bp extending from intron 8 to intron 10. Bioinformatics sequence analysis pinpointed repetitive elements that might have originated the deletion. In conclusion, we have developed and validated an MLPA assay for the detection of gross F5 gene rearrangements. This assay may represent a valuable tool for the molecular diagnosis of FV deficiency.

beta-Thalassemia is mainly caused by mutations involving single base substitution and small deletions. However, a considerable number of carriers are suspected to have large deletions in beta-globin gene cluster. Common strategy for identifying deletions with definite breakpoints is based on Gap PCR. There are, however, some cases with indefinite breakpoints which usually cannot be detected by this method. We developed and optimized a quantitative real-time PCR assay for copy number analysis of beta-globin gene cluster. The copy number of target fragments (i.e. beta, delta or (G)gamma-globin genes) was determined using comparative threshold cycle method. In addition, gene dosage was analyzed using multiplex ligation-dependent probe amplification (MLPA) method in all suspected carriers. Using these relative quantitative assays, normal or carrier statuses of all 26 unknown samples were successfully determined according to the ranges obtained from the ratios of normal and definite carrier samples. Interestingly, large deletions involving the entire beta-globin gene cluster were observed in six carrier individuals. This study showed that the MLPA as a preliminary screening test can be followed by SYBR Green real-time PCR for analysis of copy number variations in beta-globin gene cluster. Combination of these relative quantitative PCR methods could be an appropriate approach for accurate diagnosis of unknown beta-thalassemia deletions in routine diagnosis of beta-thalassemia mutations.

Transposable Elements (TEs) or jumping genes are DNA sequences that have an intrinsic capability to move within a host genome from one genomic location to another. Studies show that the presence of a TE within or adjacent to a functional gene may alter its expression. TEs can also cause an increase in the rate of mutation and can even mediate duplications and large insertions and deletions in the genome, promoting gross genetic rearrangements. The proper classification of identified jumping genes is important for analyzing their genetic and evolutionary effects. An effective classifier, which can explain the role of TEs in germline and somatic evolution more accurately, is needed. In this study, we examine the performance of a variety of machine learning (ML) techniques and propose a robust method, ClassifyTE, for the hierarchical classification of TEs with high accuracy, using a stacking-based ML method. We propose a stacking-based approach for the hierarchical classification of TEs. When trained on three different benchmark datasets, our proposed system achieved 4%, 10.68% and 10.13% average percentage improvement (using the hF measure) compared to several state-of-the-art methods. We developed an end-to-end automated hierarchical classification tool based on the proposed approach, ClassifyTE, to classify TEs up to the super-family level. We further evaluated our method on a new TE library generated by a homology-based classification method and found relatively high concordance at higher taxonomic levels. Thus, ClassifyTE paves the way for a more accurate analysis of the role of TEs. The source code and data are available at https://github.com/manisa/ClassifyTE. Supplementary data are available at Bioinformatics online.