Transgenic tools for targeted chromosome rearrangements allow construction of balancer chromosomes in non-melanogaster Drosophila species

Perhaps the most valuable single set of resources for genetic studies of Drosophila melanogaster is the collection of multiply-inverted chromosomes commonly known as balancer chromosomes. Balancers prevent the recovery of recombination exchange products within genomic regions included in inversions and allow perpetual maintenance of deleterious alleles in living stocks and the execution of complex genetic crosses. Balancer chromosomes have been generated traditionally by exposing animals to ionizing radiation and screening for altered chromosome structure or for unusual marker segregation patterns. These approaches are tedious and unpredictable, and have failed to produce the desired products in some species. Here I describe transgenic tools that allow targeted chromosome rearrangements in Drosophila species. The key new resources are engineered reporter genes containing introns with yeast recombination sites and enhancers that drive fluorescent reporter genes in multiple body regions. These tools were used to generate a doubly-inverted chromosome 3R in D. simulans that serves as an effective balancer chromosome.

Azoospermia and reciprocal translocations: should whole-exome sequencing be recommended?

Background Although chromosome rearrangements are responsible for spermatogenesis failure, their impact depends greatly on the chromosomes involved. At present, karyotyping and Y chromosome microdeletion screening are the first-line genetic tests for patients with non-obstructive azoospermia. Although it is generally acknowledged that X or Y chromosome rearrangements lead to meiotic arrest and thus rule out any chance of sperm retrieval after a testicular biopsy, we currently lack markers for the likelihood of testicular sperm extraction (TESE) in patients with other chromosome rearrangements. Results We investigated the use of a single nucleotide polymorphism comparative genome hybridization array (SNP-CGH) and whole-exome sequencing (WES) for two patients with non-obstructive azoospermia and testicular meiotic arrest, a reciprocal translocation: t(X;21) and t(20;22), and an unsuccessful TESE. No additional gene defects were identified for the t(X;21) carrier - suggesting that t(X;21) alone damages spermatogenesis. In contrast, the highly consanguineous t(20;22) carrier had two deleterious homozygous variants in the TMPRSS9 gene; these might have contributed to testicular meiotic arrest. Genetic defect was confirmed with Sanger sequencing and immunohistochemical assessments on testicular tissue sections. Conclusions Firstly, TMPRSS9 gene defects might impact spermatogenesis. Secondly, as a function of the chromosome breakpoints for azoospermic patients with chromosome rearrangements, provision of the best possible genetic counselling means that genetic testing should not be limited to karyotyping. Given the risks associated with TESE, it is essential to perform WES - especially for consanguineous patients.

Engineering chromosome rearrangements in cancer

ABSTRACT The identification of large chromosomal rearrangements in cancers has multiplied exponentially over the last decade. These complex and often rare genomic events have traditionally been challenging to study, in part owing to lack of tools that efficiently engineer disease-associated inversions, deletions and translocations in model systems. The emergence and refinement of genome editing technologies, such as CRISPR, have significantly expanded our ability to generate and interrogate chromosomal aberrations to better understand the networks that govern cancer growth. Here we review how existing technologies are employed to faithfully model cancer-associated chromosome rearrangements in the laboratory, with the ultimate goal of developing more accurate pre-clinical models of and therapeutic strategies for cancers driven by these genomic events.

Mechanisms Underlying the Suppression of Chromosome Rearrangements by Ataxia-Telangiectasia Mutated

Chromosome rearrangements are structural variations in chromosomes, such as inversions and translocations. Chromosome rearrangements have been implicated in a variety of human diseases. Ataxia-telangiectasia (A-T) is an autosomal recessive disorder characterized by a broad range of clinical and cellular phenotypes. At the cellular level, one of the most prominent features of A-T cells is chromosome rearrangement, especially that in T lymphocytes. The gene that is defective in A-T is ataxia-telangiectasia mutated (ATM). The ATM protein is a serine/threonine kinase and plays a central role in the cellular response to DNA damage, particularly DNA double-strand breaks. In this review, the mechanisms by which ATM suppresses chromosome rearrangements are discussed.

Methylation status of genes escaping from X-chromosome inactivation in patients with X-chromosome rearrangements

Background X-chromosome inactivation (XCI) is a mechanism in which one of two X chromosomes in females is randomly inactivated in order to compensate for imbalance of gene dosage between sexes. However, about 15% of genes on the inactivated X chromosome (Xi) escape from XCI. The methylation level of the promoter region of the escape gene is lower than that of the inactivated genes. Dxz4 and/or Firre have critical roles for forming the three-dimensional (3D) structure of Xi. In mice, disrupting the 3D structure of Xi by deleting both Dxz4 and Firre genes led to changing of the escape genes list. To estimate the impact for escape genes by X-chromosome rearrangements, including DXZ4 and FIRRE, we examined the methylation status of escape gene promoters in patients with various X-chromosome rearrangements. Results To detect the breakpoints, we first performed array-based comparative genomic hybridization and whole-genome sequencing in four patients with X-chromosome rearrangements. Subsequently, we conducted array-based methylation analysis and reduced representation bisulfite sequencing in the four patients with X-chromosome rearrangements and controls. Of genes reported as escape genes by gene expression analysis using human hybrid cells in a previous study, 32 genes showed hypomethylation of the promoter region in both male controls and female controls. Three patients with X-chromosome rearrangements had no escape genes with abnormal methylation of the promoter region. One of four patients with the most complicated rearrangements exhibited abnormal methylation in three escape genes. Furthermore, in the patient with the deletion of the FIRRE gene and the duplication of DXZ4, most escape genes remained hypomethylated. Conclusion X-chromosome rearrangements are unlikely to affect the methylation status of the promoter regions of escape genes, except for a specific case with highly complex rearrangements, including the deletion of the FIRRE gene and the duplication of DXZ4.

Classical, Molecular, and Genomic Cytogenetics of the Pig, a Clinical Perspective

The chromosomes of the domestic pig (Sus scrofa domesticus) are known to be prone to reciprocal chromosome translocations and other balanced chromosome rearrangements with concomitant fertility impairment of carriers. In response to the remarkable prevalence of chromosome rearrangements in swine herds, clinical cytogenetics laboratories have been established in several countries in order to screen young boars for chromosome rearrangements prior to service. At present, clinical cytogenetics laboratories typically apply classical cytogenetics techniques such as giemsa-trypsin (GTG)-banding to produce high-quality karyotypes and reveal large-scale chromosome ectopic exchanges. Further refinements to clinical cytogenetics practices have led to the implementation of molecular cytogenetics techniques such as fluorescent in-situ hybridization (FISH), allowing for rearrangements to be visualized and breakpoints refined using fluorescently labelled painting probes. The next-generation of clinical cytogenetics include the implementation of DNA microarrays, and next-generation sequencing (NGS) technologies such as DNA sequencing to better explore tentative genome architecture changes. The implementation of these cytogenomics techniques allow the genomes of rearrangement carriers to be deciphered at the highest resolution, allowing rearrangements to be detected; breakpoints to be delineated; and, most importantly, potential gene implications of those chromosome rearrangements to be interrogated. Clinical cytogenetics has become an integral tool in the livestock industry, identifying rearrangements and allowing breeders to make informed breeding decisions.

SINE-B1 Distribution and Chromosome Rearrangements in the South American Proechimys gr. goeldii (Echimyidae, Rodentia)

<i>Proechimys</i> species are remarkable for their extensive chromosome rearrangements, representing a good model to understand genome evolution. Herein, we cytogenetically analyzed 3 different cytotypes of <i>Proechimys</i> gr. <i>goeldii</i> to assess their evolutionary relationship. We also mapped the transposable element SINE-B1 on the chromosomes of <i>P.</i> gr. <i>goeldii</i> in order to investigate its distribution among individuals and evaluate its possible contribution to karyotype remodeling in this species. SINE-B1 showed a dispersed distribution along chromosome arms and was also detected at the pericentromeric regions of some chromosomes, including pair 1 and the sex chromosomes, which are involved in chromosome rearrangements. In addition, we describe a new cytotype for <i>P.</i> gr. <i>goeldii</i>, reinforcing the significant role of gross chromosomal rearrangements during the evolution of the genus. The results of FISH with SINE-B1 suggest that this issue should be more deeply investigated for a better understanding of its role in the mechanisms involved in the wide variety of <i>Proechimys</i> karyotypes.