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.

Directed yeast genome evolution by controlled introduction of trans-chromosomic structural variations

Background: Naturally occurring structural variations (SVs) are a considerable source of genomic variation and can reshape chromosomes 3D architecture. The synthetic chromosome rearrangement and modification by loxP-mediated evolution (SCRaMbLE) system has been proved to generate random SVs to impact phenotypes and thus constitutes powerful drivers of directed genome evolution. However, controllable methods to introduce complex SVs and revealing the related molecular mechanism has so far remained challenging. Results: We develop a SV-prone yeast strain by using SCRaMbLE with two synthetic chromosomes, synV and synX. An heterologous biosynthesis pathway allowing a high throughput screen for increased yield of astaxanthin is used as readout and a proof of concept for the application of SV in industry. We report here that complex SVs, including a pericentric inversion and a trans-chromosomes translocation between synV and synX, result in two neochromosomes and a 2.7-fold yield of astaxanthin. We mapped genetic targets contributing to higher astaxanthin yield and demonstrated the SVs can led to large reorganization of the genetic information along the chromosomes. We also used the model learned from the aforementioned random screen and successfully harnessed the precise introduction of trans-chromosomes translocation and pericentric inversions by rational design. Conclusions: Our work provides an effective tool to not only accelerate the directed genome evolution but also reveal mechanistic insight of complex SVs for altering phenotypes.

An Apparently Balanced Complex Chromosome Rearrangement Involving Seven Breaks and Four Chromosomes in a Healthy Female and Segregation/Recombination in Her Affected Son

Duplication of the distal 1q and 4p segments are both characterized by the presence of intellectual disability/neurodevelopmental delay and dysmorphisms. Here, we describe a male with a complex chromosome rearrangement (CCR) presenting with overlapping clinical findings between these 2 syndromes. In order to better characterize this CCR, classical karyotyping, FISH, and chromosomal microarray analysis were performed on material from the patient and his parents, which revealed an unbalanced karyotype with duplications at 1q41q43 and 4p15.2p14 in the proband. The rearrangements, which were derived from a maternal balanced karyotype, included an insertion of a segment from the long to the short arm of chromosome 1, a balanced translocation involving chromosomes 14 and 18, and an insertion of a segment from the short arm of chromosome 4 into the derived chromosome 14. This study aimed to better define the clinical history and prognosis of a patient with this rare category of chromosomal aberration. Our results suggest that the frequency of CCR in the general population may be underestimated; when balanced, they may not have a phenotypic effect. Moreover, they emphasize the need for cytogenetic techniques complementary to chromosomal microarray for proper genetic counseling.

Discrepancy of Karyotype and CMA/NGS in a PGD Patient With Cryptical Complex Chromosomal Rearrangement

Introductions: Complex chromosome rearrangement (CCR) is a structural rearrangement involving more than two breakpoints. CCR carriers are at high risk for phenotypic abnormalities or reproductive failure, such as chromosomal abnormalities in fetuses and infertility. In this study, we presented a carriers with chromosome (3,18) balanced translocation, whose fetus had duplications in chromosome 3 and deletions in chromosome 10 demonstrated by chromosomal microarray analysis (CMA).By revealing the cryptical translocation, we aimed to provide CCR carriers with more accurate risk assessment of abnormal pregnancy and better assisted reproduction with CMA and next generation sequencing(NGS).Results: By using the high resolution of GTG-banding technology, a cryptical translocation in chromosome 10 was found and the karyotype of the carrier was revised as 46,XY,t(3;10;18) (p26.3;q26.1;q21.1).In the cycle of preimplantation genetic diagnosis (PGD),21 oocytes were retrieved, and 15 were fertilized. At last 7 embryos were biospied and sent to diagnosis by next generation sequencing(NGS).Unfortunately, none of the NGS results from the 7 biopsy embryos were normal. Combining previous literature and our results, we assessed the odds of a balanced embryo in a CCR carrier to be about 9.3%(28/302).The transferable embryo rate was approximately 71.4%(20/28) and healthy live born delivery rate was 55%(11/20).Conclusions: NGS and CMA featured high automation, relatively low cost, high throughput, and high repeatability, which made them commonly used during prenatal diagnosis and PGD. The multiple technology combination can provide more accurate diagnosis and better fertility services for CCR patients.

Systematic Chromosome Rearrangement Induced by CRISPR-Cas9 Reshapes the Genome and Transcriptome of Human Cells

Chromosome rearrangement plays important roles in development, carcinogenesis and evolution. However, its mechanism and subsequent effects are not fully understood. At present, large-scale chromosome rearrangement has been performed in the simple eukaryote, wine yeast, but the relative research in mammalian cells remains at the level of individual chromosome rearrangement due to technical limitations. In this study, we used CRISPR-Cas9 to target the highly repetitive human endogenous retrotransposons, LINE-1 (L1) and Alu, resulting in a large number of DNA double-strand breaks in the chromosomes. While this operation killed the majority of the cells, we eventually obtained live cell groups. Karyotype analysis and genome re-sequencing proved that we have achieved systematic chromosome rearrangement (SCR) in human cells. The copy number variations (CNVs) of the SCR genomes showed typical patterns that observed in tumor genomes. For example, the most frequent deleted region Chr9p21 containing p15 and p16 tumor suppressor, and the amplified region Chr8q24 containing MYC in tumors were all identified in both SCR cells. The ATAC-seq and RNA-seq further revealed that the epigenetic and transcriptomic landscapes were deeply reshaped by the SCR. Gene expressions related to p53 pathway, DNA repair, cell cycle and apoptosis were greatly altered to facilitate the cell survival under the severe stress induced by the large-scale chromosomal breaks. In addition, we found that the cells acquired CRISPR-Cas9 resistance after SCR by interfering with the Cas9 mRNA. Our study provided a new application of CRISPR-Cas9 and a practical approach for SCR in complex mammalian genomes.

Complex rearrangement in acute myeloid leukemia M2 with RUNX1/RUNX1T1 fusion involving chromosomes 8, 17 and 21

Background The translocation t(8;21)(q22;q22) is one of the most frequent chromosomal abnormalities associated with acute myeloid leukemia (AML) sub type M2. About 3–5 % of cases with additional chromosomal abnormalities, including structural and numerical ones, are reported to include a complex translocation t(8;21;N). Case presentation Here we report a chromosome rearrangement observed in a 19 years-old female diagnosed with AML-M2. When subjected to (molecular) cytogenetic analyses a complex three-way translocation involving chromosomes 8, 17 and 21 was detected, forming not a t(8;21;17) as one would expect. Real time-polymerase chain reaction analysis using 6 AML specific markers showed the presence of RUNX1/RUNX1T1 fusion gene transcripts identical to those found in classical translocation t(8;21) coupled with presence of FLT3-ITD mutation identified by fragment analysis. Conclusions The present case highlights importance of complex rearrangements rarely encountered in AML, suggesting that all involved regions harbor critical candidate genes regulating the pathogenesis of AML, leading to novel as well as well-known leukemia associated chromosomal aberrations.

Identification of satellited markers by microdissection and fluorescence in situ hybridization: a clinical case of isodicentric chromosome 22

Background The presence of small supernumerary marker chromosomes (sSMCs) in a karyotype leads to diagnostic questions because the resulting extra material may cause abnormal development depending on the origin of the duplication/triplication. Because SMCs are so small, their origin cannot be determined by conventional cytogenetic techniques, and new molecular cytogenetic methods are necessary. Here, we applied a target approach to chromosome rearrangement analysis by isolating a chromosome of interest via microdissection and using it in fluorescence in situ hybridization (FISH) as a probe in combination with whole-chromosome painting probes. This approach allows to identify origins of both the euchromatin and repeat-rich regions of a marker. Case presentation We report a case of an adult male with congenital atresia of the rectum and anus, anotia, and atresia of the external auditory canal along with hearing loss. Karyotyping and FISH analysis with whole-chromosome painting probes of acrocentric chromosomes and the constructed microdissection library of the marker chromosome reliably identified an additional chromosome in some metaphases: mos 47,XY,+idic(22)(q11.2)[14]/46,XY [23]. Conclusion We propose to use whole-chromosome libraries and microdissected chromosomes in FISH to identify SMCs enriched with repeated sequences. We show that the methodology is successful in identifying the composition of a satellited marker chromosome.

When the Ends Justify the Means: Regulation of Telomere Addition at Double-Strand Breaks in Yeast

Telomeres, repetitive sequences located at the ends of most eukaryotic chromosomes, provide a mechanism to replenish terminal sequences lost during DNA replication, limit nucleolytic resection, and protect chromosome ends from engaging in double-strand break (DSB) repair. The ribonucleoprotein telomerase contains an RNA subunit that serves as the template for the synthesis of telomeric DNA. While telomere elongation is typically primed by a 3′ overhang at existing chromosome ends, telomerase can act upon internal non-telomeric sequences. Such de novo telomere addition can be programmed (for example, during chromosome fragmentation in ciliated protozoa) or can occur spontaneously in response to a chromosome break. Telomerase action at a DSB can interfere with conservative mechanisms of DNA repair and results in loss of distal sequences but may prevent additional nucleolytic resection and/or chromosome rearrangement through formation of a functional telomere (termed “chromosome healing”). Here, we review studies of spontaneous and induced DSBs in the yeast Saccharomyces cerevisiae that shed light on mechanisms that negatively regulate de novo telomere addition, in particular how the cell prevents telomerase action at DSBs while facilitating elongation of critically short telomeres. Much of our understanding comes from the use of perfect artificial telomeric tracts to “seed” de novo telomere addition. However, endogenous sequences that are enriched in thymine and guanine nucleotides on one strand (TG-rich) but do not perfectly match the telomere consensus sequence can also stimulate unusually high frequencies of telomere formation following a DSB. These observations suggest that some internal sites may fully or partially escape mechanisms that normally negatively regulate de novo telomere addition.

Discrepancy of Karyotype and CMA /NGS in PGD Patient with Cryptical Complex Chromosomal Rearrangement

Background: Complex chromosome rearrangement (CCR) is a structural rearrangement involving more than two breakpoints. CCR carriers are at high risk for phenotypic abnormalities or reproductive failure, such as chromosomal abnormalities in fetuses and infertility.Methods: We presented a carriers with chromosome (3,18) apparent balanced translocation diagnosed in eleswhere, whose fetus had duplications in chromosome 3 and deletions in chromosome 10 demonstrated by chromosome microarray analysis(CMA). Results: Through the high resolution of GTG-banding, a cryptical translocation in chromosome 10 was found and the karyotype of the carrier was revised as 46,XY,t(3;10;18) (p26.3;q26.1;q21.1).In the cycle of preimplantation genetic diagnosis (PGD),21 oocytes were retrieved, and 15 were fertilized. At last 7 embryos were biospied and sent to diagnosis by next generation sequencing(NGS).Unfortunately, none of the NGS results from the 7 biopsy embryos were normal. Combining previous literature and our results, we assessed the odds of a balanced embryo in a CCR carrier to be about 9.3%(28/302).The transferable embryo rate was approximately 71.4%(20/28) and healthy live born delivery rate was 55%(11/20).Conclusions: NGS and CMA featured high automation, relatively low cost, high throughput, and high repeatability, which made them commonly used during prenatal diagnosis and PGD. The multiple technology combination can provide more accurate diagnosis and better fertility services for CCR patients.