Intragenic DNA inversions expand bacterial coding capacity.

Bacterial populations that originate from a single bacterium are not strictly clonal and often contain subgroups with distinct phenotypes1. Bacteria can generate heterogeneity through phase variation-a preprogrammed, reversible mechanism that alters gene expression levels across a population1. One well-studied type of phase variation involves enzyme-mediated inversion of specific regions of genomic DNA2. Frequently, these DNA inversions flip the orientation of promoters, turning transcription of adjacent coding regions on or off2. Through this mechanism, inversion can affect fitness, survival or group dynamics3,4. Here, we describe the development of PhaVa, a computational tool that identifies DNA inversions using long-read datasets. We also identify 372 'intragenic invertons', a novel class of DNA inversions found entirely within genes, in genomes of bacterial and archaeal isolates. Intragenic invertons allow a gene to encode two or more versions of a protein by flipping a DNA sequence within the coding region, thereby increasing coding capacity without increasing genome size. We validate ten intragenic invertons in the gut commensal Bacteroides thetaiotaomicron, and experimentally characterize an intragenic inverton in the thiamine biosynthesis gene thiC.

The barley pan-genome reveals the hidden legacy of mutation breeding.

Genetic diversity is key to crop improvement. Owing to pervasive genomic structural variation, a single reference genome assembly cannot capture the full complement of sequence diversity of a crop species (known as the 'pan-genome'1). Multiple high-quality sequence assemblies are an indispensable component of a pan-genome infrastructure. Barley (Hordeum vulgare L.) is an important cereal crop with a long history of cultivation that is adapted to a wide range of agro-climatic conditions2. Here we report the construction of chromosome-scale sequence assemblies for the genotypes of 20 varieties of barley-comprising landraces, cultivars and a wild barley-that were selected as representatives of global barley diversity. We catalogued genomic presence/absence variants and explored the use of structural variants for quantitative genetic analysis through whole-genome shotgun sequencing of 300 gene bank accessions. We discovered abundant large inversion polymorphisms and analysed in detail two inversions that are frequently found in current elite barley germplasm; one is probably the product of mutation breeding and the other is tightly linked to a locus that is involved in the expansion of geographical range. This first-generation barley pan-genome makes previously hidden genetic variation accessible to genetic studies and breeding.

Precise and Predictable CRISPR Chromosomal Rearrangements Reveal Principles of Cas9-Mediated Nucleotide Insertion.

Chromosomal rearrangements including large DNA-fragment inversions, deletions, and duplications by Cas9 with paired sgRNAs are important to investigate genome structural variations and developmental gene regulation, but little is known about the underlying mechanisms. Here, we report that disrupting CtIP or FANCD2, which have roles in alternative non-homologous end joining, enhances precise DNA-fragment deletion. By analyzing the inserted nucleotides at the junctions of DNA-fragment editing of deletions, inversions, and duplications and characterizing the cleaved products, we find that Cas9 endonucleolytically cleaves the noncomplementary strand with a flexible scissile profile upstream of the -3 position of the PAM site in vivo and in vitro, generating double-strand break ends with 5' overhangs of 1-3 nucleotides. Moreover, we find that engineered Cas9 nucleases have distinct cleavage profiles. Finally, Cas9-mediated nucleotide insertions are nonrandom and are equal to the combined sequences upstream of both PAM sites with predicted frequencies. Thus, precise and predictable DNA-fragment editing could be achieved by perturbing DNA repair genes and using appropriate PAM configurations.

Structural basis of direct and inverted DNA sequence repeat recognition by helix-turn-helix transcription factors.

Some transcription factors bind DNA motifs containing direct or inverted sequence repeats. Preference for each of these DNA topologies is dictated by structural constraints. Most prokaryotic regulators form symmetric oligomers, which require operators with a dyad structure. Binding to direct repeats requires breaking the internal symmetry, a property restricted to a few regulators, most of them from the AraC family. The KorA family of transcriptional repressors, involved in plasmid propagation and stability, includes members that form symmetric dimers and recognize inverted repeats. Our structural analyses show that ArdK, a member of this family, can form a symmetric dimer similar to that observed for KorA, yet it binds direct sequence repeats as a non-symmetric dimer. This is possible by the 180° rotation of one of the helix-turn-helix domains. We then probed and confirmed that ArdK shows affinity for an inverted repeat, which, surprisingly, is also recognized by a non-symmetrical dimer. Our results indicate that structural flexibility at different positions in the dimerization interface constrains transcription factors to bind DNA sequences with one of these two alternative DNA topologies.

The Recurrent Atypical e8a2 BCR::ABL1 Transcript with Insertion of an Inverted 55 Base Pair ABL1 Intron 1b Sequence: A Detailed Molecular Analysis.

Atypical BCR::ABL1 transcripts are found in approximately 2% of cases of chronic myeloid leukemia. It is important to detect them since affected patients also benefit from tyrosine kinase inhibitor therapy. In the rare e8a2 atypical BCR::ABL1 transcript, two out-of-frame exons are fused, thus, interposed nucleotides are usually found at the fusion site to restore the reading frame. In approximately half of previously reported e8a2 BCR::ABL1 cases, an inserted 55 bp sequence homologous to an inverted sequence from ABL1 intron 1b was detected. The generation of this recurrent transcript variant is not obvious. This work describes the molecular analysis of such an e8a2 BCR::ABL1 translocation from a CML patient. The genomic chromosomal breakpoint is identified, and the formation of this transcript is theoretically explained. The clinical course of the patient is reported, and recommendations are provided for the molecular analysis of future e8a2 BCR::ABL1 cases.

scoreInvHap: Inversion genotyping for genome-wide association studies.

Polymorphic inversions contribute to adaptation and phenotypic variation. However, large multi-centric association studies of inversions remain challenging. We present scoreInvHap, a method to genotype inversions from SNP data for genome-wide association studies (GWASs), overcoming important limitations of current methods and outperforming them in accuracy and applicability. scoreInvHap calls individual inversion-genotypes from a similarity score to the SNPs of experimentally validated references. It can be used on different sources of SNP data, including those with low SNP coverage such as exome sequencing, and is easily adaptable to genotype new inversions, either in humans or in other species. We present 20 human inversions that can be reliably and easily genotyped with scoreInvHap to discover their role in complex human traits, and illustrate a first genome-wide association study of experimentally-validated human inversions. scoreInvHap is implemented in R and it is freely available from Bioconductor.

The constrained architecture of mammalian Hox gene clusters.

In many animal species with a bilateral symmetry, Hox genes are clustered either at one or at several genomic loci. This organization has a functional relevance, as the transcriptional control applied to each gene depends upon its relative position within the gene cluster. It was previously noted that vertebrate Hox clusters display a much higher level of genomic organization than their invertebrate counterparts. The former are always more compact than the latter, they are generally devoid of repeats and of interspersed genes, and all genes are transcribed by the same DNA strand, suggesting that particular factors constrained these clusters toward a tighter structure during the evolution of the vertebrate lineage. Here, we investigate the importance of uniform transcriptional orientation by engineering several alleles within the HoxD cluster, such as to invert one or several transcription units, with or without a neighboring CTCF site. We observe that the association between the tight structure of mammalian Hox clusters and their regulation makes inversions likely detrimental to the proper implementation of this complex genetic system. We propose that the consolidation of Hox clusters in vertebrates, including transcriptional polarity, evolved in conjunction with the emergence of global gene regulation via the flanking regulatory landscapes, to optimize a coordinated response of selected subsets of target genes in cis.

Pathogenic 12-kb copy-neutral inversion in syndromic intellectual disability identified by high-fidelity long-read sequencing.

We report monozygotic twin girls with syndromic intellectual disability who underwent exome sequencing but with negative pathogenic variants. To search for variants that are unrecognized by exome sequencing, high-fidelity long-read genome sequencing (HiFi LR-GS) was applied. A 12-kb copy-neutral inversion was precisely identified by HiFi LR-GS after trio-based variant filtering. This inversion directly disrupted two genes, CPNE9 and BRPF1, the latter of which attracted our attention because pathogenic BRPF1 variants have been identified in autosomal dominant intellectual developmental disorder with dysmorphic facies and ptosis (IDDDFP), which later turned out to be clinically found in the twins. Trio-based HiFi LR-GS together with haplotype phasing revealed that the 12-kb inversion occurred de novo on the maternally transmitted chromosome. This study clearly indicates that submicroscopic copy-neutral inversions are important but often uncharacterized culprits in monogenic disorders and that long-read sequencing is highly advantageous for detecting such inversions involved in genetic diseases.

Higher Gene Flow in Sex-Related Chromosomes than in Autosomes during Fungal Divergence.

Nonrecombining sex chromosomes are widely found to be more differentiated than autosomes among closely related species, due to smaller effective population size and/or to a disproportionally large-X effect in reproductive isolation. Although fungal mating-type chromosomes can also display large nonrecombining regions, their levels of differentiation compared with autosomes have been little studied. Anther-smut fungi from the Microbotryum genus are castrating pathogens of Caryophyllaceae plants with largely nonrecombining mating-type chromosomes. Using whole genome sequences of 40 fungal strains, we quantified genetic differentiation among strains isolated from the geographically overlapping North American species and subspecies of Silene virginica and S. caroliniana. We inferred that gene flow likely occurred at the early stages of divergence and then completely stopped. We identified large autosomal genomic regions with chromosomal inversions, with higher genetic divergence than the rest of the genomes and highly enriched in selective sweeps, supporting a role of rearrangements in preventing gene flow in genomic regions involved in ecological divergence. Unexpectedly, the nonrecombining mating-type chromosomes showed lower divergence than autosomes due to higher gene flow, which may be promoted by adaptive introgressions of less degenerated mating-type chromosomes. The fact that both mating-type chromosomes are always heterozygous and nonrecombining may explain such patterns that oppose to those found for XY or ZW sex chromosomes. The specific features of mating-type chromosomes may also apply to the UV sex chromosomes determining sexes at the haploid stage in algae and bryophytes and may help test general hypotheses on the evolutionary specificities of sex-related chromosomes.

Inversion variants in human and primate genomes.

For many years, inversions have been proposed to be a direct driving force in speciation since they suppress recombination when heterozygous. Inversions are the most common large-scale differences among humans and great apes. Nevertheless, they represent large events easily distinguishable by classical cytogenetics, whose resolution, however, is limited. Here, we performed a genome-wide comparison between human, great ape, and macaque genomes using the net alignments for the most recent releases of genome assemblies. We identified a total of 156 putative inversions, between 103 kb and 91 Mb, corresponding to 136 human loci. Combining literature, sequence, and experimental analyses, we analyzed 109 of these loci and found 67 regions inverted in one or multiple primates, including 28 newly identified inversions. These events overlap with 81 human genes at their breakpoints, and seven correspond to sites of recurrent rearrangements associated with human disease. This work doubles the number of validated primate inversions larger than 100 kb, beyond what was previously documented. We identified 74 sites of errors, where the sequence has been assembled in the wrong orientation, in the reference genomes analyzed. Our data serve two purposes: First, we generated a map of evolutionary inversions in these genomes representing a resource for interrogating differences among these species at a functional level; second, we provide a list of misassembled regions in these primate genomes, involving over 300 Mb of DNA and 1978 human genes. Accurately annotating these regions in the genome references has immediate applications for evolutionary and biomedical studies on primates.

Functional differences in the hypothalamic-pituitary-gonadal axis are associated with alternative reproductive tactics based on an inversion polymorphism.

The evolution of social behavior depends on genetic changes, yet, how genomic variation manifests itself in behavioral diversity is still largely unresolved. Chromosomal inversions can play a pivotal role in producing distinct behavioral phenotypes, in particular, when inversion genes are functionally associated with hormone synthesis and signaling. Male ruffs exhibit alternative reproductive tactics (ARTs) with an autosomal inversion determining two alternative morphs with clear behavioral and hormonal differences from the ancestral morph. We investigated hormonal and transcriptomic differences in the pituitary and gonads. Using a GnRH challenge, we found that the ability to synthesize testosterone in inversion carriers is severely constrained, whereas the synthesis of androstenedione, a testosterone precursor, is not. Inversion morphs were able to produce a transient increase in androstenedione following the GnRH injection, supporting the view that pituitary sensitivity to GnRH is comparable to that of the ancestral morph. We then performed gene expression analyses in a second set of untreated birds and found no evidence of alterations to pituitary sensitivity, gonadotropin production or gonad sensitivity to luteinizing hormone or follicle-stimulating hormone across morphs. Inversion morphs also showed reduced progesterone receptor expression in the pituitary. Strikingly, in the gonads, inversion morphs over-expressed STAR, a gene that is located outside of the inversion and responsible for providing the cholesterol substrate required for the synthesis of sex hormones. In conclusion, our results suggest that the gonads determine morph-specific differences in hormonal regulation.

Lineage-Specific Variation in IR Boundary Shift Events, Inversions, and Substitution Rates among Caprifoliaceae s.l. (Dipsacales) Plastomes.

Caprifoliaceae s.l. plastid genomes (plastomes) show that one inversion and two inverted repeat boundary shifts occurred in the common ancestor of this family, after which the plastomes are generally conserved. This study reports plastome sequences of five additional species, Fedia cornucopiae, Valeriana fauriei, and Valerianella locusta from the subfamily Valerianoideae, as well as Dipsacus japonicus and Scabiosa comosa from the subfamily Dipsacoideae. Combined with the published plastomes, these plastomes provide new insights into the structural evolution of plastomes within the family. Moreover, the three plastomes from the subfamily Valerianoideae exhibited accelerated nucleotide substitution rates, particularly at synonymous sites, across the family. The patterns of accD sequence divergence in the family are dynamic with structural changes, including interruption of the conserved domain and increases in nonsynonymous substitution rates. In particular, the Valeriana accD gene harbors a large insertion of amino acid repeat (AAR) motifs, and intraspecific polymorphism with a variable number of AARs in the Valeriana accD gene was detected. We found a correlation between intron losses and increased ratios of nonsynonymous to synonymous substitution rates in the clpP gene with intensified positive selection. In addition, two Dipsacoideae plastomes revealed the loss of the plastid-encoded rps15, and a potential functional gene transfer to the nucleus was confirmed.

Construction and comparison of three reference-quality genome assemblies for soybean.

We report reference-quality genome assemblies and annotations for two accessions of soybean (Glycine max) and for one accession of Glycine soja, the closest wild relative of G. max. The G. max assemblies provided are for widely used US cultivars: the northern line Williams 82 (Wm82) and the southern line Lee. The Wm82 assembly improves the prior published assembly, and the Lee and G. soja assemblies are new for these accessions. Comparisons among the three accessions show generally high structural conservation, but nucleotide difference of 1.7 single-nucleotide polymorphisms (snps) per kb between Wm82 and Lee, and 4.7 snps per kb between these lines and G. soja. snp distributions and comparisons with genotypes of the Lee and Wm82 parents highlight patterns of introgression and haplotype structure. Comparisons against the US germplasm collection show placement of the sequenced accessions relative to global soybean diversity. Analysis of a pan-gene collection shows generally high conservation, with variation occurring primarily in genomically clustered gene families. We found approximately 40-42 inversions per chromosome between either Lee or Wm82v4 and G. soja, and approximately 32 inversions per chromosome between Wm82 and Lee. We also investigated five domestication loci. For each locus, we found two different alleles with functional differences between G. soja and the two domesticated accessions. The genome assemblies for multiple cultivated accessions and for the closest wild ancestor of soybean provides a valuable set of resources for identifying causal variants that underlie traits for the domestication and improvement of soybean, serving as a basis for future research and crop improvement efforts for this important crop species.

InvBFM: finding genomic inversions from high-throughput sequence data based on feature mining.

BACKGROUND: Genomic inversion is one type of structural variations (SVs) and is known to play an important biological role. An established problem in sequence data analysis is calling inversions from high-throughput sequence data. It is more difficult to detect inversions because they are surrounded by duplication or other types of SVs in the inversion areas. Existing inversion detection tools are mainly based on three approaches: paired-end reads, split-mapped reads, and assembly. However, existing tools suffer from unsatisfying precision or sensitivity (eg: only 50~60% sensitivity) and it needs to be improved. RESULT: In this paper, we present a new inversion calling method called InvBFM. InvBFM calls inversions based on feature mining. InvBFM first gathers the results of existing inversion detection tools as candidates for inversions. It then extracts features from the inversions. Finally, it calls the true inversions by a trained support vector machine (SVM) classifier. CONCLUSIONS: Our results on real sequence data from the 1000 Genomes Project show that by combining feature mining and a machine learning model, InvBFM outperforms existing tools. InvBFM is written in Python and Shell and is available for download at https://github.com/wzj1234/InvBFM.

The effects of common structural variants on 3D chromatin structure.

BACKGROUND: Three-dimensional spatial organization of chromosomes is defined by highly self-interacting regions 0.1-1 Mb in size termed Topological Associating Domains (TADs). Genetic factors that explain dynamic variation in TAD structure are not understood. We hypothesize that common structural variation (SV) in the human population can disrupt regulatory sequences and thereby influence TAD formation. To determine the effects of SVs on 3D chromatin organization, we performed chromosome conformation capture sequencing (Hi-C) of lymphoblastoid cell lines from 19 subjects for which SVs had been previously characterized in the 1000 genomes project. We tested the effects of common deletion polymorphisms on TAD structure by linear regression analysis of nearby quantitative chromatin interactions (contacts) within 240 kb of the deletion, and we specifically tested the hypothesis that deletions at TAD boundaries (TBs) could result in large-scale alterations in chromatin conformation. RESULTS: Large (> 10 kb) deletions had significant effects on long-range chromatin interactions. Deletions were associated with increased contacts that span the deleted region and this effect was driven by large deletions that were not located within a TAD boundary (nonTB). Some deletions at TBs, including a 80 kb deletion of the genes CFHR1 and CFHR3, had detectable effects on chromatin contacts. However for TB deletions overall, we did not detect a pattern of effects that was consistent in magnitude or direction. Large inversions in the population had a distinguishable signature characterized by a rearrangement of contacts that span its breakpoints. CONCLUSIONS: Our study demonstrates that common SVs in the population impact long-range chromatin structure, and deletions and inversions have distinct signatures. However, the effects that we observe are subtle and variable between loci. Genome-wide analysis of chromatin conformation in large cohorts will be needed to quantify the influence of common SVs on chromatin structure.

CRISPR/Cas9-Mediated Knockout and In Situ Inversion of the ORF57 Gene from All Copies of the Kaposi's Sarcoma-Associated Herpesvirus Genome in BCBL-1 Cells.

Kaposi's sarcoma-associated herpesvirus (KSHV)-transformed primary effusion lymphoma cell lines contain ∼70 to 150 copies of episomal KSHV genomes per cell and have been widely used for studying the mechanisms of KSHV latency and lytic reactivation. Here, we report the first complete knockout (KO) of viral ORF57 gene from all ∼100 copies of KSHV genome per cell in BCBL-1 cells. This was achieved by a modified CRISPR/Cas9 technology to simultaneously express two guide RNAs (gRNAs) and Cas9 from a single expression vector in transfected cells in combination with multiple rounds of cell selection and single-cell cloning. CRISPR/Cas9-mediated genome engineering induces the targeted gene deletion and inversion in situ We found the inverted ORF57 gene in the targeted site in the KSHV genome in one of two characterized single cell clones. Knockout of ORF57 from the KSHV genome led to viral genome instability, thereby reducing viral genome copies and expression of viral lytic genes in BCBL-1-derived single-cell clones. The modified CRISPR/Cas9 technology was very efficient in knocking out the ORF57 gene in iSLK/Bac16 and HEK293/Bac36 cells, where each cell contains only a few copies of the KSHV genome. The ORF57 KO genome was stable in iSLK/Bac16 cells, and, upon lytic induction, was partially rescued by ectopic ORF57 to express viral lytic gene ORF59 and produce infectious virions. Together, the technology developed in this study has paved the way to express two separate gRNAs and the Cas9 enzyme simultaneously in the same cell and could be efficiently applied to any genetic alterations from various genomes, including those in extreme high copy numbers.IMPORTANCE This study provides the first evidence that CRISPR/Cas9 technology can be applied to knock out the ORF57 gene from all ∼100 copies of the KSHV genome in primary effusion lymphoma (PEL) cells by coexpressing two guide RNAs (gRNAs) and Cas9 from a single expression vector in combination with single-cell cloning. The gene knockout efficiency in this system was evaluated rapidly using a direct cell PCR screening. The current CRISPR/Cas9 technology also mediated ORF57 inversion in situ in the targeted site of the KSHV genome. The successful rescue of viral lytic gene expression and infectious virion production from the ORF57 knockout (KO) genome further reiterates the essential role of ORF57 in KSHV infection and multiplication. This modified technology should be useful for knocking out any viral genes from a genome to dissect functions of individual viral genes in the context of the virus genome and to understand their contributions to viral genetics and the virus life cycle.

A Closed-Tube Nested Quantitative PCR Assay for Rapid Detection of Intron 22 Inversions in the Factor VIII Gene.

BACKGROUND: An inversion of intron 22 in the Factor VIII gene (Inv22) is the causative mutation for 45% of severe hemophilia A cases. Available methods for molecular diagnosis of Inv22 are generally tedious and not ideal for routine clinical use. METHODS: We report here a new method using a single closed-tube nested quantitative PCR (CN-qPCR) for rapid detection of Inv22. This method combines a 12-cycle long-distance PCR (LD-PCR) amplifying the int22h regions, followed by a duplex qPCR targeting two specific regions close to the int22h regions. All reagents were added to a single PCR mixture for the closed-tube assay. Sequential LD-PCR and qPCR was achieved by designing primers at substantially different melting temperatures and optimizing PCR conditions. RESULTS: Seventy-nine male hemophilia A patients of different disease severity were tested by both the CN-qPCR assay and the standard LD-PCR assay. CN-qPCR successfully made calls for all samples, whereas LD-PCR failed in eight samples. For the 71 samples where both methods made calls, the concordance was 100%. Inv22 was detected in 17 out of the 79 samples. Additionally, CN-qPCR achieved clear separation for 10 female carriers and 10 non-Inv22 females, suggesting the assay may also be useful for molecular diagnosis of female carriers. CONCLUSIONS: This new CN-qPCR method may provide a convenient and accurate F8 Inv22 test suitable for clinical use.

First submicroscopic inversion of the OPA1 gene identified in dominant optic atrophy - a case report.

BACKGROUND: Dominant optic atrophy (DOA) is an inherited optic neuropathy that mainly affects visual acuity, central visual fields and color vision due to a progressive loss of retinal ganglion cells and their axons that form the optic nerve. Approximately 45-90% of affected individuals with DOA harbor pathogenic variants in the OPA1 gene. The mutation spectrum of OPA1 comprises nonsense, canonical and non-canonical splice site, frameshift and missense as well as copy number variants, but intragenic inversions have not been reported so far. CASE PRESENTATION: We report a 33-year-old male with characteristic clinical features of DOA. Whole-genome sequencing identified a structural variant of 2.4 kb comprising an inversion of 937 bp at the OPA1 locus. Fine mapping of the breakpoints to single nucleotide level revealed that the structural variation was an inversion flanked by two deletions. As this rearrangement inverts the entire first exon of OPA1, it was classified as likely pathogenic. CONCLUSIONS: We report the first DOA case harboring an inversion in the OPA1 gene. Our study demonstrates that copy-neutral genomic rearrangements have to be considered as a possible cause of disease in DOA cases.

Long-read sequencing for rare human genetic diseases.

During the past decade, the search for pathogenic mutations in rare human genetic diseases has involved huge efforts to sequence coding regions, or the entire genome, using massively parallel short-read sequencers. However, the approximate current diagnostic rate is <50% using these approaches, and there remain many rare genetic diseases with unknown cause. There may be many reasons for this, but one plausible explanation is that the responsible mutations are in regions of the genome that are difficult to sequence using conventional technologies (e.g., tandem-repeat expansion or complex chromosomal structural aberrations). Despite the drawbacks of high cost and a shortage of standard analytical methods, several studies have analyzed pathogenic changes in the genome using long-read sequencers. The results of these studies provide hope that further application of long-read sequencers to identify the causative mutations in unsolved genetic diseases may expand our understanding of the human genome and diseases. Such approaches may also be applied to molecular diagnosis and therapeutic strategies for patients with genetic diseases in the future.

Analyses of breakpoint junctions of complex genomic rearrangements comprising multiple consecutive microdeletions by nanopore sequencing.

The widespread use of genomic copy number analysis has revealed many previously unknown genomic structural variations, including some which are more complex. In this study, three consecutive microdeletions were identified in the same chromosome by microarray-based comparative genomic hybridization (aCGH) analysis for a patient with a neurodevelopmental disorder. Subsequent fluorescence in situ hybridization (FISH) analyses unexpectedly suggested complicated translocations and inversions. For better understanding of the mechanism, breakpoint junctions were analyzed by nanopore sequencing, as a new long-read whole-genome sequencing (WGS) tool. The results revealed a new chromosomal disruption, giving rise to four junctions in chromosome 7. According the sequencing results of breakpoint junctions, all junctions were considered as the consequence of multiple double-strand breaks and the reassembly of DNA fragments by nonhomologous end-joining, indicating chromothripsis. KMT2E, located within the deletion region, was considered as the gene responsible for the clinical features of the patient. Combinatory usage of aCGH and FISH analyses would be recommended for interpretation of structural variations analyzed through WGS.