Interchromosomal Recombination Research Articles

Characterization of the complete mitogenome of Tiarella polyphylla, commonly known as Asian foamflower: insights into the multi-chromosomes structure and DNA transfers

BackgroundTiarella polyphylla D. Don has been traditionally used to cure asthma and skin eruptions. However, the sequence and the structure of the mitogenome of T. polyphylla remained elusive, limiting the genomic and evolution analysis based on the mitogenome.ResultsUsing a combination of Illumina and Nanopore sequencing reads, we de novo assembled the complete mitogenome of T. polyphylla. In addition to unveiling the major configuration of the T. polyphylla mitogenome was three circular chromosomes with lengths of 430,435 bp, 126,943 bp, and 55,269 bp, we revealed five (R01-R05) and one (R06) repetitive sequence could mediate the intra- and inter-chromosomal recombination, respectively. Furthermore, we identified 208 short and 25 long tandem segments, seven cp-derived mtDNAs, 106 segments of mtDNAs transferred to the nuclear genome, and 653 predicted RNA editing sites. Based on the sequence of the mitogenomes, we obtained the resolved phylogeny of the seven Saxifragales species.ConclusionsThese results presented the mitogenome features and expanded its potential applications in phylogenetics, species identification, and cytoplasmic male sterility (CMS) in the future.

Subtelomeric plasticity contributes to gene family expansion in the human parasitic flatworm Schistosoma mansoni

BackgroundThe genomic region that lies between the telomere and chromosome body, termed the subtelomere, is heterochromatic, repeat-rich, and frequently undergoes rearrangement. Within this region, large-scale structural changes enable gene diversification, and, as such, large multicopy gene families are often found at the subtelomere. In some parasites, genes associated with proliferation, invasion, and survival are often found in these regions, where they benefit from the subtelomere's highly plastic, rapidly changing nature. The increasing availability of complete (or near complete) parasite genomes provides an opportunity to investigate these typically poorly defined and overlooked genomic regions and potentially reveal relevant gene families necessary for the parasite’s lifestyle.ResultsUsing the latest chromosome-scale genome assembly and hallmark repeat richness observed at chromosome termini, we have identified and characterised the subtelomeres of Schistosoma mansoni, a metazoan parasitic flatworm that infects over 250 million people worldwide. Approximately 12% of the S. mansoni genome is classified as subtelomeric, and, in line with other organisms, we find these regions to be gene-poor but rich in transposable elements. We find that S. mansoni subtelomeres have undergone extensive interchromosomal recombination and that these sites disproportionately contribute to the 2.3% of the genome derived from segmental duplications. This recombination has led to the expansion of subtelomeric gene clusters containing 103 genes, including the immunomodulatory annexins and other gene families with unknown roles. The largest of these is a 49-copy plexin domain-containing protein cluster, exclusively expressed in the tegument—the tissue located at the host-parasite physical interface—of intramolluscan life stages.ConclusionsWe propose that subtelomeric regions act as a genomic playground for trial-and-error of gene duplication and subsequent divergence. Owing to the importance of subtelomeric genes in other parasites, gene families implicated in this subtelomeric expansion within S. mansoni warrant further characterisation for a potential role in parasitism.

Meiosis in an asymmetric dikaryotic genome of Tremella fuciformis Tr01 facilitates new chromosome formation

BackgroundThe dikaryotic stage dominates most of the life cycle in basidiomycetes, and each cell carries two different haploid nuclei. Accurate phasing of these two nuclear genomes and their interactions have long been of interest.ResultsWe combine PacBio HiFi reads, Nanopore ultra-long reads, and Hi-C data to generate a complete, high-quality asymmetric dikaryotic genome of Tremella fuciformis Tr01, including Haplotypes A and B genomes. We assemble a meiotic haploid DBZ04 genome and detect three recombination events in these two haplotypes. We identify several chromosomal rearrangements that lead to differences in chromosome number, length, content, and sequence arrangement between these two haplotypes. Each nucleus contains a two-speed genome, harboring three accessory chromosomes and two accessory compartments that affect horizontal chromatin transfer between nuclei. We find few basidiospores are ejected from fruiting bodies of Tr01. Most monospore isolates sequenced belong to Tr01-Haplotype A genome architecture. More than one-third of monospore isolates carry one or two extra chromosomes including Chr12B and two new chromosomes ChrN1 and ChrN2. We hypothesize that homologous regions of seven sister chromatids pair into a large complex during meiosis, followed by inter-chromosomal recombination at physical contact sites and formation of new chromosomes.ConclusionWe assemble two haplotype genomes of T. fuciformis Tr01 and provide the first overview of basidiomycetous genomes with discrete genomic architecture. Meiotic activities of asymmetric dikaryotic genomes result in formation of new chromosomes, aneuploidy of some daughter cells, and inviability of most other daughter cells. We propose a new approach for breeding of sporeless mushroom.

Long-range DNA end resection supports homologous recombination by checkpoint activation rather than extensive homology generation.

Homologous recombination (HR), the high-fidelity mechanism for double-strand break (DSB) repair, relies on DNA end resection by nucleolytic degradation of the 5¢-terminated ends. However, the role of long-range resection mediated by Exo1 and/or Sgs1-Dna2 in HR is not fully understood. Here, we show that Exo1 and Sgs1 are dispensable for recombination between closely-linked repeats, but are required for interchromosomal repeat recombination in Saccharomyces cerevisiae. This context-specific requirement for long-range end resection is connected to its role in activating the DNA damage checkpoint. Consistent with this role, checkpoint mutants also show a defect specifically in interchromosomal recombination. Furthermore, artificial activation of the checkpoint partially restores interchromosomal recombination to exo1∆ sgs1∆ cells. However, cell cycle delay is insufficient to rescue the interchromosomal recombination defect of exo1∆ sgs1∆ cells, suggesting an additional role for the checkpoint. Given that the checkpoint is necessary for DNA damage-induced chromosome mobility, we propose that the importance of the checkpoint, and therefore long-range resection, in interchromosomal recombination is due to a need to increase chromosome mobility to facilitate pairing of distant sites. The need for long-range resection is circumvented when the DSB and its repair template are in close proximity.

CRISPR-Cas9 engineering in the hybrid yeast zygosaccharomyces parabailii can lead to loss of heterozygosity in target chromosomes.

The hybrid yeast Zygosaccharomyces parabailii holds potential as a cell factory mainly because of its robustness in withstanding stressors that often characterize bio-based processes. However, a complex genome and a lack of gene editing tools hinder the capacity to engineer this yeast. In this work, we developed a CRISPR-Cas9 gene editing system for Z. parabailii that allows simultaneous disruption or deletion of both alleles of a gene. We evaluated four different gRNA expression systems consisting of combinations of tRNAs, tRNA and ribozyme or ribozymes as self-cleaving flanking elements and established that the most efficient systems used an RNA Pol II promoter followed by a 5'tRNA flanking the gRNA. This gRNA system was then used to construct a strain of Z. parabailii in which both alleles of DNL4 were inactivated and so relied on homologous recombination to repair double-stranded breaks. Our system can be used for gene inactivation in a wild-type strain and precise deletion with marker insertion in a dnl4 mutant. In some cases, we observed inter-chromosomal recombination around the site of the DSB that could cause loss of heterozygosity through gene conversion or deletion. Although an additional aspect that needs to be monitored during strain engineering, this phenomenon also offers opportunities to explore genome plasticity in hybrid yeasts.

The whole-genome assembly of an endangered Salicaceae species: Chosenia arbutifolia (Pall.) A. Skv.

As a fast-growing tree species, Chosenia arbutifolia has a unique but controversial taxonomic status in the family Salicaceae. Despite its importance as an industrial material, in ecological protection, and in landscaping, C. arbutifolia is seriously endangered in Northeast China because of artificial destruction and its low reproductive capability. To clarify its phylogenetic relationships with other Salicaceae species, we assembled a high-quality chromosome-level genome of C. arbutifolia using PacBio High-Fidelity reads and Hi-C sequencing data, with a total size of 338.93 Mb and contig N50 of 1.68 Mb. Repetitive sequences, which accounted for 42.34% of the assembly length, were identified. In total, 33,229 protein-coding genes and 11,474 small noncoding RNAs were predicted. Phylogenetic analysis suggested that C. arbutifolia and poplars diverged approximately 15.3 million years ago, and a large interchromosomal recombination between C. arbutifolia and other Salicaceae species was discovered. Our study provides insights into the genome architecture and systematic evolution of C. arbutifolia, as well as comprehensive information for germplasm protection and future functional genomic studies.

Validation and Data-Integration of Yeast-Based Assays for Functional Classification of BRCA1 Missense Variants.

Germline mutations in the BRCA1 gene have been reported to increase the lifetime risk of developing breast and/or ovarian cancer (BOC). By new sequencing technologies, numerous variants of uncertain significance (VUS) are identified. It is mandatory to develop new tools to evaluate their functional impact and pathogenicity. As the expression of pathogenic BRCA1 variants in Saccharomyces cerevisiae increases the frequency of intra- and inter-chromosomal homologous recombination (HR), and gene reversion (GR), we validated the two HR and the GR assays by testing 23 benign and 23 pathogenic variants and compared the results with those that were obtained in the small colony phenotype (SCP) assay, an additional yeast-based assay, that was validated previously. We demonstrated that they scored high accuracy, sensitivity, and sensibility. By using a classifier that was based on majority of voting, we have integrated data from HR, GR, and SCP assays and developed a reliable method, named yBRCA1, with high sensitivity to obtain an accurate VUS functional classification (benign or pathogenic). The classification of BRCA1 variants, important for assessing the risk of developing BOC, is often difficult to establish with genetic methods because they occur rarely in the population. This study provides a new tool to get insights on the functional impact of the BRCA1 variants.

Metal exposure causes rDNA copy number to fluctuate in mutation accumulation lines of Daphnia pulex

Ribosomal (r)DNA is a highly dynamic, conserved, multigene family whose sequence homogeneity is thought to be maintained by intra- and interchromosomal recombination, which are capable of changing rDNA copy number. It is generally not known how environmental stress such as sublethal exposure to environmentally relevant concentrations of metals impacts rDNA copy number. To determine how chronic metal exposure affects rDNA, we measured copy number of the 18S rRNA gene in 355 copper and nickel-exposed samples and 132 metal-free samples derived from 325 mutation accumulation (MA) lines of two genetically distinct Daphnia pulex lineages. The MA lines were sampled at four time points over 100+ generations of clonal propagation. The copy number of rDNA was also measured in 15 individuals sampled from a metal-free non-MA control population established from the same progenitor as one of the MA lineages. We found that mean rDNA copy number fluctuated across lines exposed to metals with a tendency to decrease over time. In contrast, mean rDNA copy number in the metal-free control lines and the non-MA population remained stable over time. It is generally accepted that extreme rDNA loss results in the loss of organism fitness. Thus, fluctuations in rDNA copy number, including losses, could affect the long-term viability of natural populations of Daphnia in metal-contaminated habitats.

Lineage Tracing and Clonal Analysis in Developing Cerebral Cortex Using Mosaic Analysis with Double Markers (MADM)

Beginning from a limited pool of progenitors, the mammalian cerebral cortex forms highly organized functional neural circuits. However, the underlying cellular and molecular mechanisms regulating lineage transitions of neural stem cells (NSCs) and eventual production of neurons and glia in the developing neuroepithelium remains unclear. Methods to trace NSC division patterns and map the lineage of clonally related cells have advanced dramatically. However, many contemporary lineage tracing techniques suffer from the lack of cellular resolution of progeny cell fate, which is essential for deciphering progenitor cell division patterns. Presented is a protocol using mosaic analysis with double markers (MADM) to perform in vivo clonal analysis. MADM concomitantly manipulates individual progenitor cells and visualizes precise division patterns and lineage progression at unprecedented single cell resolution. MADM-based interchromosomal recombination events during the G2-X phase of mitosis, together with temporally inducible CreERT2, provide exact information on the birth dates of clones and their division patterns. Thus, MADM lineage tracing provides unprecedented qualitative and quantitative optical readouts of the proliferation mode of stem cell progenitors at the single cell level. MADM also allows for examination of the mechanisms and functional requirements of candidate genes in NSC lineage progression. This method is unique in that comparative analysis of control and mutant subclones can be performed in the same tissue environment in vivo. Here, the protocol is described in detail, and experimental paradigms to employ MADM for clonal analysis and lineage tracing in the developing cerebral cortex are demonstrated. Importantly, this protocol can be adapted to perform MADM clonal analysis in any murine stem cell niche, as long as the CreERT2 driver is present.

From gene editing to genome engineering: restructuring plant chromosomes via CRISPR/Cas.

In the last years, tremendous progress has been achieved in the field of gene editing in plants. By the induction of single site-specific double-strand breaks (DSBs), the knockout of genes by non-homologous end joining has become routine in many plant species. Recently, the efficiency of inducing pre-planned mutations by homologous recombination has also been improved considerably. However, very little effort has been undertaken until now to achieve more complex changes in plant genomes by the simultaneous induction of several DSBs. Several reports have been published on the efficient induction of deletions. However, the induction of intrachromosomal inversions and interchromosomal recombination by the use of CRISPR/Cas has only recently been reported. In this review, we want to sum up these results and put them into context with regards to what is known about natural chromosome rearrangements in plants. Moreover, we review the recent progress in CRISPR/Cas-based mammalian chromosomal rearrangements, which might be inspiring for plant biologists. In the long run, the controlled restructuring of plant genomes should enable us to link or break linkage of traits at will, thus defining a new area of plant breeding.

Chromosome rearrangements shape the diversification of secondary metabolism in the cyclosporin producing fungus Tolypocladium inflatum

BackgroundGenes involved in production of secondary metabolites (SMs) in fungi are exceptionally diverse. Even strains of the same species may exhibit differences in metabolite production, a finding that has important implications for drug discovery. Unlike in other eukaryotes, genes producing SMs are often clustered and co-expressed in fungal genomes, but the genetic mechanisms involved in the creation and maintenance of these secondary metabolite biosynthetic gene clusters (SMBGCs) remains poorly understood.ResultsIn order to address the role of genome architecture and chromosome scale structural variation in generating diversity of SMBGCs, we generated chromosome scale assemblies of six geographically diverse isolates of the insect pathogenic fungus Tolypocladium inflatum, producer of the multi-billion dollar lifesaving immunosuppressant drug cyclosporin, and utilized a Hi-C chromosome conformation capture approach to address the role of genome architecture and structural variation in generating intraspecific diversity in SMBGCs. Our results demonstrate that the exchange of DNA between heterologous chromosomes plays an important role in generating novelty in SMBGCs in fungi. In particular, we demonstrate movement of a polyketide synthase (PKS) and several adjacent genes by translocation to a new chromosome and genomic context, potentially generating a novel PKS cluster. We also provide evidence for inter-chromosomal recombination between nonribosomal peptide synthetases located within subtelomeres and uncover a polymorphic cluster present in only two strains that is closely related to the cluster responsible for biosynthesis of the mycotoxin aflatoxin (AF), a highly carcinogenic compound that is a major public health concern worldwide. In contrast, the cyclosporin cluster, located internally on chromosomes, was conserved across strains, suggesting selective maintenance of this important virulence factor for infection of insects.ConclusionsThis research places the evolution of SMBGCs within the context of whole genome evolution and suggests a role for recombination between chromosomes in generating novel SMBGCs in the medicinal fungus Tolypocladium inflatum.

Common Bean Subtelomeres Are Hot Spots of Recombination and Favor Resistance Gene Evolution

Subtelomeres of most eukaryotes contain fast-evolving genes usually involved in adaptive processes. In common bean (Phaseolus vulgaris), the Co-2 anthracnose resistance (R) locus corresponds to a cluster of nucleotide-binding-site leucine-rich-repeat (NL) encoding sequences, the prevalent class of plant R genes. To study the recent evolution of this R gene cluster, we used a combination of sequence, genetic and cytogenetic comparative analyses between common bean genotypes from two distinct gene pools (Andean and Mesoamerican) that diverged 0.165 million years ago. Co-2 is a large subtelomeric cluster on chromosome 11 comprising from 32 (Mesoamerican) to 52 (Andean) NL sequences embedded within khipu satellite repeats. Since the recent split between Andean and Mesoamerican gene pools, the Co-2 cluster has experienced numerous gene-pool specific NL losses, leading to distinct NL repertoires. The high proportion of solo-LTR retrotransposons indicates that the Co-2 cluster is located in a hot spot of unequal intra-strand homologous recombination. Furthermore, we observe large segmental duplications involving both Non-Homologous End Joining and Homologous Recombination double-strand break repair pathways. Finally, the identification of a Mesoamerican-specific subtelomeric sequence reveals frequent interchromosomal recombinations between common bean subtelomeres. Altogether, our results highlight that common bean subtelomeres are hot spots of recombination and favor the rapid evolution of R genes. We propose that chromosome ends could act as R gene incubators in many plant genomes.

Double-stranded DNA break polarity skews repair pathway choice during intrachromosomal and interchromosomal recombination

Activation-induced cytidine deaminase (AID) inflicts DNA damage at Ig genes to initiate class switch recombination (CSR) and chromosomal translocations. However, the DNA lesions formed during these processes retain an element of randomness, and thus knowledge of the relationship between specific DNA lesions and AID-mediated processes remains incomplete. To identify necessary and sufficient DNA lesions in CSR, the Cas9 endonuclease and nickase variants were used to program DNA lesions at a greater degree of predictability than is achievable with conventional induction of CSR. Here we show that Cas9-mediated nicks separated by up to 250 nucleotides on opposite strands can mediate CSR. Staggered double-stranded breaks (DSBs) result in more end resection and junctional microhomology than blunt DSBs. Moreover, Myc-Igh chromosomal translocations, which are carried out primarily by alternative end joining (A-EJ), were preferentially induced by 5' DSBs. These data indicate that DSBs with 5' overhangs skew intrachromosomal and interchromosomal end-joining toward A-EJ. In addition to lending potential insight to AID-mediated phenomena, this work has broader carryover implications in DNA repair and lymphomagenesis.

Position effects influencing intrachromosomal repair of a double-strand break in budding yeast.

Repair of a double-strand break (DSB) by an ectopic homologous donor sequence is subject to the three-dimensional arrangement of chromosomes in the nucleus of haploid budding yeast. The data for interchromosomal recombination suggest that searching for homology is accomplished by a random collision process, strongly influenced by the contact probability of the donor and recipient sequences. Here we explore how recombination occurs on the same chromosome and whether there are additional constraints imposed on repair. Specifically, we examined how intrachromosomal repair is affected by the location of the donor sequence along the 813-kb chromosome 2 (Chr2), with a site-specific DSB created on the right arm (position 625 kb). Repair correlates well with contact frequencies determined by chromosome conformation capture-based studies (r = 0.85). Moreover, there is a profound constraint imposed by the anchoring of the centromere (CEN2, position 238 kb) to the spindle pole body. Sequences at the same distance on either side of CEN2 are equivalently constrained in recombining with a DSB located more distally on one arm, suggesting that sequences on the opposite arm from the DSB are not otherwise constrained in their interaction with the DSB. The centromere constraint can be partially relieved by inducing transcription through the centromere to inactivate CEN2 tethering. In diploid cells, repair of a DSB via its allelic donor is strongly influenced by the presence and the position of an ectopic intrachromosomal donor.

CRISPR-Cas9 targeted disruption of the yellow ortholog in the housefly identifies the brown body locus

The classic brown body (bwb) mutation in the housefly Musca domestica impairs normal melanization of the adult cuticle. In Drosophila melanogaster, a reminiscent pigmentation defect results from mutations in the yellow gene encoding dopachrome conversion enzyme (DCE). Here, we demonstrate that the bwb locus structurally and functionally represents the yellow ortholog of Musca domestica, MdY. In bwb Musca strains, we identified two mutant MdY alleles that contain lesions predicted to result in premature truncation of the MdY open reading frame. We targeted wildtype MdY by CRISPR-Cas9 RNPs and generated new mutant alleles that fail to complement existing MdY alleles, genetically confirming that MdY is the bwb locus. We further found evidence for Cas9-mediated interchromosomal recombination between wildtype and mutant bwb alleles. Our work resolves the molecular identity of the classic bwb mutation in Musca domestica and establishes the feasibility of Cas9-mediated genome editing in the Musca model.

FLT3 and JAK2 Mutations in Acute Myeloid Leukemia Promote Interchromosomal Homologous Recombination and the Potential for Copy Neutral Loss of Heterozygosity.

Acquired copy neutral LOH (CN-LOH) is a frequent occurrence in myeloid malignancies and is often associated with resistance to standard therapeutic modalities and poor survival. Here, we show that constitutive signaling driven by mutated FLT3 and JAK2 confers interchromosomal homologous recombination (iHR), a precedent for CN-LOH. Using a targeted recombination assay, we determined significant iHR activity in internal tandem duplication FLT3 (FLT3-ITD) and JAK2V617F-mutated cells. Sister chromatid exchanges, a surrogate measure of iHR, was significantly elevated in primary FLT3-ITD normal karyotype acute myeloid leukemia (NK-AML) compared with wild-type FLT3 NK-AML. HR was harmonized to S phase of the cell cycle to repair broken chromatids and prevent iHR. Increased HR activity in G0 arrested primary FLT3-ITD NK-AML in contrast to wild-type FLT3 NK-AML. Cells expressing mutated FLT3-ITD demonstrated a relative increase in mutation frequency as detected by thymidine kinase (TK) gene mutation assay. Moreover, resistance was associated with CN-LOH at the TK locus. Treatment of FLT3-ITD- and JAK2V617F-mutant cells with the antioxidant N-acetylcysteine diminished reactive oxygen species (ROS), restoring iHR and HR levels. Our findings show that mutated FLT3-ITD and JAK2 augment ROS production and HR, shifting the cellular milieu toward illegitimate recombination events such as iHR and CN-LOH. Therapeutic reduction of ROS may thus prevent leukemic progression and relapse in myeloid malignancies. Cancer Res; 77(7); 1697-708. ©2017 AACR.

Insertion DNA Accelerates Meiotic Interchromosomal Recombination in Arabidopsis thaliana.

Nucleotide insertions/deletions are ubiquitous in eukaryotic genomes, and the resulting hemizygous (unpaired) DNA has significant, heritable effects on adjacent DNA. However, little is known about the genetic behavior of insertion DNA. Here, we describe a binary transgenic system to study the behavior of insertion DNA during meiosis. Transgenic Arabidopsis lines were generated to carry two different defective reporter genes on nonhomologous chromosomes, designated as "recipient" and "donor" lines. Double hemizygous plants (harboring unpaired DNA) were produced by crossing between the recipient and the donor, and double homozygous lines (harboring paired DNA) via self-pollination. The transfer of the donor's unmutated sequence to the recipient generated a functional β-glucuronidase gene, which could be visualized by histochemical staining and corroborated by polymerase chain reaction amplification and sequencing. More than 673 million seedlings were screened, and the results showed that meiotic ectopic recombination in the hemizygous lines occurred at a frequency >6.49-fold higher than that in the homozygous lines. Gene conversion might have been exclusively or predominantly responsible for the gene correction events. The direct measurement of ectopic recombination events provided evidence that an insertion, in the absence of an allelic counterpart, could scan the entire genome for homologous counterparts with which to pair. Furthermore, the unpaired (hemizygous) architectures could accelerate ectopic recombination between itself and interchromosomal counterparts. We suggest that the ectopic recombination accelerated by hemizygous architectures may be a general mechanism for interchromosomal recombination through ubiquitously dispersed repeat sequences in plants, ultimately contributing to genetic renovation and eukaryotic evolution.

Multicopy gene family evolution on primate Y chromosomes.

BackgroundThe primate Y chromosome is distinguished by a lack of inter-chromosomal recombination along most of its length, extensive gene loss, and a prevalence of repetitive elements. A group of genes on the male-specific portion of the Y chromosome known as the “ampliconic genes” are present in multiple copies that are sometimes part of palindromes, and that undergo a form of intra-chromosomal recombination called gene conversion, wherein the nucleotides of one copy are homogenized by those of another. With the aim of further understanding gene family evolution of these genes, we collected nucleotide sequence and gene copy number information for several species of papionin monkey. We then tested for evidence of gene conversion, and developed a novel statistical framework to evaluate alternative models of gene family evolution using our data combined with other information from a human, a chimpanzee, and a rhesus macaque.ResultsOur results (i) recovered evidence for several novel examples of gene conversion in papionin monkeys and indicate that (ii) ampliconic gene families evolve faster than autosomal gene families and than single-copy genes on the Y chromosome and that (iii) Y-linked singleton and autosomal gene families evolved faster in humans and chimps than they do in the other Old World Monkey lineages we studied.ConclusionsRapid evolution of ampliconic genes cannot be attributed solely to residence on the Y chromosome, nor to variation between primate lineages in the rate of gene family evolution. Instead other factors, such as natural selection and gene conversion, appear to play a role in driving temporal and genomic evolutionary heterogeneity in primate gene families.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2187-8) contains supplementary material, which is available to authorized users.

Internal Tandem Duplication Mutation of FLT3 (FLT3/ITD) Significantly Increases Inter Chromosomal Homologous Recombination Providing a Direct Link Between Constitutive FLT3-ITD Activity and Propagation of Copy Neutral Loss of Heterozygosity (CN-LOH) in Acute Myeloid Leukaemia

The majority of patients with adult acute myeloid leukaemia (AML) that present with an apparently normal karyotype (NK-AML) are grouped together in the “intermediate” risk category and constitute 40-45% of all adult AML patients. Mutations in the FMS-like tyrosine kinase 3 (FLT3) receptor whether it is internal tandem duplication (ITD) of its juxtamembrane domain or point mutations in its kinase domain are one of the most common mutations in NK-AML. The presence of FLT3-ITD mutation in NK-AML results in a more aggressive disease, resistance to therapy and poor survival. Acquired copy neutral loss of heterozygosity (CN-LOH) also referred to as uniparental disomy (UPD) is a common phenomenon of myeloid malignancies where an oncogenic allele is duplicated on the other chromosome. The use of single nucleotide phenotype analysis (SNP-A) karyotyping detects CN-LOH in 20% and 40% of newly diagnosed and relapsed AML respectively. CN-LOH at 6p or at the FLT3 locus 13q associated with a FLT3-ITD mutation in NK-AML results in an even more aggressive disease compared to NK-AML + FLT3-ITD without CN-LOH. It has been proposed that CN-LOH is the result of a homologous recombination (HR) DNA repair event. However, the underlying mechanisms that confer CN-LOH have yet to be determined.To elucidate the mechanisms that produce CN-LOH in NK-AML we determined whether oncogenes such as FLT3-ITD have the propensity to generate CN-LOH through up-regulation of inter chromosomal (between maternal and paternal chromosomes) HR DNA repair. We have showed previously that constitutional FLT3-ITD kinase activity increased reactive oxygen species resulting in elevated double strand breaks. Moreover, FLT3-ITD mutation increases homologous recombination activity through transcriptional augmentation of HR factor, RAD51 expression.Firstly, to evaluate FLT3-ITD induced genomic instability we measured sister chromatid exchanges (SCE) that result from HR mediated chromosome cross-over. It has been shown previously in the pre-leukaemic chromosomal instability disorder, Blooms syndrome is characterised by increased SCE and an increased propensity for CN-LOH. FLT3/ITD primary AML (n=17), MOLM-13 and FLT3-ITD transfected U937 cells show a significant increase in SCE compared to WT FLT3 primary AML cells, U937 cells transfected with empty vector, (10 vs. 6 SCE per metaphase, p<0.001) (Figure 1A-B). Importantly, primary AML with CN-LOH at 13q + FLT3-ITD mutation gives significantly higher HR activity and SCE than primary AML + FLT3-ITD mutation without CN-LOH (p<0.05). Moreover, treatment of FLT3/ITD primary AML, MOLM-13 and FLT3-ITD transfected U937 cells with the FLT3 inhibitor, CEP701 and the anti-oxidant agent, N-acetyl cysteine restored SCE to levels observed for WT FLT3. Next, we investigated whether inappropriate HR activity at G0 rather than at S phase of the cell cycle could result in illegitimate HR resulting in inter chromosomal HR. Western blot studies showed that G0 primary AML with FLT3/ITD, synchronised G0 MOLM 13 and FLT3-ITD transfected U937 cells showed augmented RAD51 expression compared to non-mutated FLT3 primary AML cells, cell lines and KG-1 transfected with empty vector (Figure 1C). Lastly, we used a thymidine kinase (TK) gene mutation assay system to screen for FLT3-ITD derived CN-LOH. The human lymphoblastoid cell line, TK6 is heterozygous for TK and becomes resistant to trifluorothymidine (TFT) upon loss of the wild type TK allele on 17q. FLT3-ITD transfected TK6 cells (using 2 different expression constructs) demonstrated significantly increased mutation frequency (grey bars) and mutation ate (Black bars) and TFT resistance compared to WT FLT3 and emptor vector control transfection (p<0.001) (Figure 1D). Colony PCR of TFT resistant colonies revealed that resistance was due primarily (80%) to inter chromosomal HR at 17q in contrast to empty vector (20%) and cisplatin treated (45%) TK6 cells (Figure 1E). The findings that FLT3-ITD mutation induces increased ROS production inducing DNA double strand breaks and the concomitant increase in the rad51 dependent inter homologous recombination provide a strong proof of principle that constitutive FLT3-ITD kinase activity directly confers CN-LOH that furthers our understanding of leukaemic progression and relapse in NK-AML. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Screening of copy number variants in the 22q11.2 region of congenital heart disease patients from the São Miguel Island, Azores, revealed the second patient with a triplication.

BackgroundThe rearrangements in the 22q11.2 chromosomal region, responsible for the 22q11.2 deletion and microduplication syndromes, are frequently associated with congenital heart disease (CHD). The present work aimed to identify the genetic basis of CHD in 87 patients from the São Miguel Island, Azores, through the detection of copy number variants (CNVs) in the 22q11.2 region. These structural variants were searched using multiplex ligation-dependent probe amplification (MLPA). In patients with CNVs, we additionally performed fluorescent in situ hybridization (FISH) for the assessment of the exact number of 22q11.2 copies among each chromosome, and array comparative genomic hybridization (array-CGH) for the determination of the exact length of CNVs.ResultsWe found that four patients (4.6%; A to D) carried CNVs. Patients A and D, both affected with a ventricular septal defect, carried a de novo 2.5 Mb deletion of the 22q11.2 region, which was probably originated by inter-chromosomal (inter-chromatid) non-allelic homologous recombination (NAHR) events in the regions containing low-copy repeats (LCRs). Patient C, with an atrial septal defect, carried a de novo 2.5 Mb duplication of 22q11.2 region, which could have been probably generated during gametogenesis by NAHR or by unequal crossing-over; additionally, this patient presented a benign 288 Kb duplication, which included the TOP3B gene inherited from her healthy mother. Finally, patient B showed a 3 Mb triplication associated with dysmorphic facial features, cognitive deficit and heart defects, a clinical feature not reported in the only case described so far in the literature. The evaluation of patient B’s parents revealed a 2.5 Mb duplication in her father, suggesting a paternal inheritance with an extra copy.ConclusionsThis report allowed the identification of rare deletion and microduplication syndromes in Azorean CHD patients. Moreover, we report the second patient with a 22q11.2 triplication, and we suggest that patients with triplications of chromosome 22q11.2, although they share some characteristic features with the deletion and microduplication syndromes, present a more severe phenotype probably due to the major dosage of implicated genes.