Replication-based Mechanisms Research Articles

Microhomology-Mediated Break-Induced Replication: A Possible Molecular Mechanism of the Formation of a Large CNV in FBN1 Gene in a Patient with Marfan Syndrome.

Marfan syndrome (MFS) is an autosomal dominant multisystem disorder caused by mutations in the fibrillin-1 gene (FBN1). A small portion of them is copy number variations (CNVs), which can occur through recombination-based, replication-based mechanisms or retrotransposition. Not many have been characterized precisely in MFS. A female patient with suspected Marfan syndrome was referred for genetic testing at our institute. After systematic sequencing of FBN1, TGFBR1, and TGFBR2 genes, multiplex ligation-dependent probe amplification was applied. Long-range PCR, subsequent Sanger sequencing with designed primers, and preliminary in silico analysis were applied for the precise characterization of the breakpoints. Primary analysis displayed a de novo large deletion affecting exons 46 and 47 in the FBN1 gene, which resulted in the loss of the 31st and 32nd calcium-binding EGFlike domains. Further examination of the breakpoints showed a 4916 nucleotide long deletion localized in intronic regions. Surprisingly a 'TG' dinucleotide insertion was detected at the junction. We hypothesize that the CNV formation was generated by a rare event based on the known microhomology-mediated break-induced replication (MMBIR). An increasing number of CNVs are associated with Mendelian diseases and other traits. Approximately 2-7% of the cases in MFS are caused by CNVs. Up to date, hardly any model was proposed to demonstrate the formation of these genomic rearrangements in the FBN1 gene. Hereby, with the help of previous models and breakpoint analysis, we presented a potential mechanism (based on MMBIR) in the formation of this large deletion.

Replication-Based Dynamic Energy-Aware Resource Provisioning for Scientific Workflows

Distributed computing services in cloud environments are easily accessible to end users. These services are delivered to end users via a subscription-based model. The “infrastructure as a service” (IaaS) cloud model is one of the best cloud environment models for running data- and computing-intensive applications. Real-world scientific applications are the best examples of data and computing intensiveness. For their implementation, scientific workflow applications need high-performance computational resources and a large volume of storage. The workflow tasks are linked based on computational and data interdependence. Considering the high volume and variety of scientific workflows (SWs), the resources of the IaaS cloud model require managing energy efficiently and without failure or loss. Therefore, in order to address the issues of power consumption and task failure for real-world SWs, this research work proposes a replication-based dynamic energy-aware resource provisioning (R-DEAR) strategy for SWs in an IaaS cloud environment. The proposed strategy, R-DEAR, is a resource- and service-provisioning strategy that implements a replication-based fault-tolerant and load-balancing mechanism. The proposed R-DEAR strategy schedules the tasks of a scientific workflow with a replication-based fault-tolerant mechanism. The proposed R-DEAR strategy also manages the power consumption of IaaS cloud resources dynamically through a load-sharing process. Simulation results show that the proposed R-DEAR strategy reduces energy consumption, execution cost, and execution time by 9%, 15%, and 18%, respectively, as compared with the existing state-of-the-art strategy.

Evidence for high breakpoint variability in 46, XX, SRY-positive testicular disorder and frequent ARSE deletion that may be associated with short stature.

The translocation of SRY onto one of the two X chromosomes results in a 46,XX testicular disorder of sex development; this is supposedly because of non-allelic homologous recombination between the protein kinase X gene (PRKX) and the inverted protein kinase Y pseudogene (PRKY). Although 46,XX SRY-positive men are infertile, the literature data indicate that some of these individuals are of short stature (relative to the general population). We sought to determine whether short stature was linked to additional, more complex chromosomal rearrangements. Twelve laboratories gathered detailed clinical, anthropomorphic, cytogenetic and genetic data (including chromosome microarray data) on patients with 46,XX SRY-positive male syndrome. SRY was present (suggesting a der(X)t(X;Y)) in 34 of the 38 cases (89.5%). When considering only the 20 patients with chromosome microarray data, we identified several chromosomal rearrangements and breakpoints, especially on the X chromosome. In the five cases for whom the X chromosome breakpoint was located in the pseudoautosomal region, there was partial duplication of the derivate X chromosome. In contrast, in the 15 cases for whom the breakpoint was located downstream of the pseudoautosomal region, part of the derivate X chromosome had been deleted (included the arylsulfatase E [ARSE] gene in 11 patients). For patients with versus without ARSE deletion, the mean height was, respectively, 167.7±4.5 and 173.1±4.0cm; this difference was not statistically significant (p =0.1005). Although 46,XX SRY-positive male syndromes were mainly because of imbalanced crossover between the X and Y chromosome during meiosis, the breakpoints differed markedly from one patient to another (especially on the X chromosome); this suggests the presence of a replication-based mechanism for recombination between non-homologous sequences. In some patients, the translocation of SRY to the X chromosome was associated with ARSE gene deletion, which might have led to short stature. With a view to explaining this disorder of sex development, whole exome sequencing could be suggested for SRY-negative patients.

Long-Read Sequencing Identifies the First Retrotransposon Insertion and Resolves Structural Variants Causing Antithrombin Deficiency.

The identification of inherited antithrombin deficiency (ATD) is critical to prevent potentially life-threatening thrombotic events. Causal variants in SERPINC1 are identified for up to 70% of cases, the majority being single-nucleotide variants and indels. The detection and characterization of structural variants (SVs) in ATD remain challenging due to the high number of repetitive elements in SERPINC1 . Here, we performed long-read whole-genome sequencing on 10 familial and 9 singleton cases with type I ATD proven by functional and antigen assays, who were selected from a cohort of 340 patients with this rare disorder because genetic analyses were either negative, ambiguous, or not fully characterized. We developed an analysis workflow to identify disease-associated SVs. This approach resolved, independently of its size or type, all eight SVs detected by multiple ligation-dependent probe amplification, and identified for the first time a complex rearrangement previously misclassified as a deletion. Remarkably, we identified the mechanism explaining ATD in 2 out of 11 cases with previous unknown defect: the insertion of a novel 2.4 kb SINE-VNTR-Alu retroelement, which was characterized by de novo assembly and verified by specific polymerase chain reaction amplification and sequencing in the probands and affected relatives. The nucleotide-level resolution achieved for all SVs allowed breakpoint analysis, which revealed repetitive elements and microhomologies supporting a common replication-based mechanism for all the SVs. Our study underscores the utility of long-read sequencing technology as a complementary method to identify, characterize, and unveil the molecular mechanism of disease-causing SVs involved in ATD, and enlarges the catalogue of genetic disorders caused by retrotransposon insertions.

EP402: Partial trisomy of chromosome 22 mediated by chromoanasynthesis in an 8-month-old male

Chromoanasynthesis is a subtype of chromoanagenesis, a term used to describe the single-step processes by which complex chromosomal rearrangements lead to localized structural genomic reorganization of one or a few chromosomes. During chromoanasynthesis, complex chromosomal rearrangements are mediated by replication-based mechanisms including fork stalling and template switching (FoSTeS) or by microhomology-mediated break-induced replication (MMBIR). As a result, highly reorganized chromosomes with multiple copy number changes including deletions, duplications, and triplications are generated.

Xq27.1 palindrome mediated interchromosomal insertion likely causes familial congenital bilateral laryngeal abductor paralysis (Plott syndrome)

Bilateral laryngeal abductor paralysis is a rare entity and the second most common cause of stridor in newborns. So far, no conclusive genetic or chromosomal aberration has been reported for X-linked isolated bilateral vocal cord paralysis, also referred to as Plott syndrome. Via whole genome sequencing (WGS), we identified a complex interchromosomal insertion in a large family with seven affected males. The 404 kb inserted fragment originates from chromosome 10q21.3, contains no genes and is inserted inversionally into the intergenic chromosomal region Xq27.1, 82 kb centromeric to the nearest gene SOX3. The patterns found at the breakpoint junctions resemble typical characteristics that arise in replication-based mechanisms with long-distance template switching. Non protein-coding insertions into the same genomic region have been described to result in different phenotypes, indicating that the phenotypic outcome likely depends on the introduction of regulatory elements. In conclusion, our data adds Plott syndrome as another entity, likely caused by the insertion of non-coding DNA into the intergenic chromosomal region Xq27.1. In this regard, we demonstrate the importance of WGS as a powerful diagnostic test in unsolved genetic diseases, as this genomic rearrangement has not been detected by current first-line diagnostic tests, i.e., exome sequencing and chromosomal microarray analysis.

Complete characterisation of two new large Xq28 duplications involving F8 using whole genome sequencing in patients without haemophilia A.

Depending on the location of insertion of the gained region, F8 duplications can have variable clinical impacts from benign impact to severe haemophilia A phenotype. To characterize two large Xq28 duplications involving F8 incidentally detected by chromosome microarray analysis (CMA) in two patients presenting severe intellectual disability but no history of bleeding disorder. Whole genome sequencing (WGS) was performed in order to characterize the two large Xq28 duplications at nucleotide level. In patient 1, a 60-73kb gained region encompassing the exons 23-26 of F8 and SMIM9 was inserted at the int22h-2 locus following a non-homologous recombination between int22h-1 and int22h-2. We hypothesized that two independent events, micro-homology-mediated break-induced replication (MMBIR) and break-induced replication (BIR), could be involved in this rearrangement. In patient 2, the CMA found duplication from 101 to 116-kb long encompassing the exons 16-26 of F8 and SMIM9. The WGS analysis identified a more complex rearrangement with the presence of three genomic junctions. Due to the multiple micro-homologies observed at breakpoints, a replication-based mechanism such as fork stalling and template switching (FoSTeS) was greatly suspected. In both cases, these complex rearrangements preserved an intact copy of the F8. This study highlights the value of WGS to characterize the genomic junction at the nucleotide level and ultimately better describe the molecular mechanisms involved in Xq28 structural variations. It also emphasizes the importance of specifying the structure of the genomic gain in order to improve genotype-phenotype correlation and genetic counselling.

Cytogenetically visible inversions are formed by multiple molecular mechanisms

Cytogenetically detected inversions are generally assumed to be copy number and phenotypically neutral events. While nonallelic homologous recombination is thought to play a major role, recent data suggest the involvement of other molecular mechanisms in inversion formation. Using a combination of short‐read whole‐genome sequencing (WGS), 10X Genomics Chromium WGS, droplet digital polymerase chain reaction and array comparative genomic hybridization we investigated the genomic structure of 18 large unique cytogenetically detected chromosomal inversions and achieved nucleotide resolution of at least one chromosomal inversion junction for 13/18 (72%). Surprisingly, we observed that seemingly copy number neutral inversions can be accompanied by a copy‐number gain of up to 350 kb and local genomic complexities (3/18, 17%). In the resolved inversions, the mutational signatures are consistent with nonhomologous end‐joining (8/13, 62%) or microhomology‐mediated break‐induced replication (5/13, 38%). Our study indicates that short‐read 30x coverage WGS can detect a substantial fraction of chromosomal inversions. Moreover, replication‐based mechanisms are responsible for approximately 38% of those events leading to a significant proportion of inversions that are actually accompanied by additional copy‐number variation potentially contributing to the overall phenotypic presentation of those patients.

The involvement of U-type dicentric chromosomes in the formation of terminal deletions with or without adjacent inverted duplications.

An inverted duplication with a terminal deletion (inv-dup-del) is one of the complex constitutional structural rearrangements that can occur in a chromosome. Although breakages of dicentric chromosome have been suggested, the precise mechanism of this is yet to be fully understood. In our present study, we investigated the genomic structure of 10 inv-dup-del cases to elucidate this mechanism. Two recurrent 8p inv-dup-del cases harbored a large copy-number-neutral region between the duplication and deletion in common. Although the other non-recurrent cases did not appear to have this copy-number-neutral region, refined sequencing analysis identified that they contained a small intervening region at the junction between the inverted and non-inverted segment. The size of this small intervening region ranged from 1741 to 3728bp. Combined with a presence of microhomology at the junction, a resolution of the replication fork stalling through template switching within the same replication fork is suggested. We further observed two cases with mosaicism of the dicentric chromosome and various structural rearrangements related to the dicentric chromosome. Refined analysis allowed us to identify different breakpoints on the same chromosome in the same case, implicating multiple rounds of U-type formation and its breakage. From these results, we propose that a replication-based mechanism generates unstable dicentric chromosomes and that their breakage leads to the formation of inv-dup-dels and other related derivative chromosomes.

Patterns of somatic structural variation in human cancer genomes

A key mutational process in cancer is structural variation, in which rearrangements delete, amplify or reorder genomic segments that range in size from kilobases to whole chromosomes1–7. Here we develop methods to group, classify and describe somatic structural variants, using data from the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium of the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA), which aggregated whole-genome sequencing data from 2,658 cancers across 38 tumour types8. Sixteen signatures of structural variation emerged. Deletions have a multimodal size distribution, assort unevenly across tumour types and patients, are enriched in late-replicating regions and correlate with inversions. Tandem duplications also have a multimodal size distribution, but are enriched in early-replicating regions—as are unbalanced translocations. Replication-based mechanisms of rearrangement generate varied chromosomal structures with low-level copy-number gains and frequent inverted rearrangements. One prominent structure consists of 2–7 templates copied from distinct regions of the genome strung together within one locus. Such cycles of templated insertions correlate with tandem duplications, and—in liver cancer—frequently activate the telomerase gene TERT. A wide variety of rearrangement processes are active in cancer, which generate complex configurations of the genome upon which selection can act.

Distinct patterns of complex rearrangements and a mutational signature of microhomeology are frequently observed in PLP1 copy number gain structural variants

BackgroundWe investigated the features of the genomic rearrangements in a cohort of 50 male individuals with proteolipid protein 1 (PLP1) copy number gain events who were ascertained with Pelizaeus-Merzbacher disease (PMD; MIM: 312080). We then compared our new data to previous structural variant mutagenesis studies involving the Xq22 region of the human genome. The aggregate data from 159 sequenced join-points (discontinuous sequences in the reference genome that are joined during the rearrangement process) were studied. Analysis of these data from 150 individuals enabled the spectrum and relative distribution of the underlying genomic mutational signatures to be delineated.MethodsGenomic rearrangements in PMD individuals with PLP1 copy number gain events were investigated by high-density customized array or clinical chromosomal microarray analysis and breakpoint junction sequence analysis.ResultsHigh-density customized array showed that the majority of cases (33/50; ~ 66%) present with single duplications, although complex genomic rearrangements (CGRs) are also frequent (17/50; ~ 34%). Breakpoint mapping to nucleotide resolution revealed further previously unknown structural and sequence complexities, even in single duplications. Meta-analysis of all studied rearrangements that occur at the PLP1 locus showed that single duplications were found in ~ 54% of individuals and that, among all CGR cases, triplication flanked by duplications is the most frequent CGR array CGH pattern observed. Importantly, in ~ 32% of join-points, there is evidence for a mutational signature of microhomeology (highly similar yet imperfect sequence matches).ConclusionsThese data reveal a high frequency of CGRs at the PLP1 locus and support the assertion that replication-based mechanisms are prominent contributors to the formation of CGRs at Xq22. We propose that microhomeology can facilitate template switching, by stabilizing strand annealing of the primer using W-C base complementarity, and is a mutational signature for replicative repair.

Genomic landscape and chronological reconstruction of driver events in multiple myeloma

The multiple myeloma (MM) genome is heterogeneous and evolves through preclinical and post-diagnosis phases. Here we report a catalog and hierarchy of driver lesions using sequences from 67 MM genomes serially collected from 30 patients together with public exome datasets. Bayesian clustering defines at least 7 genomic subgroups with distinct sets of co-operating events. Focusing on whole genome sequencing data, complex structural events emerge as major drivers, including chromothripsis and a novel replication-based mechanism of templated insertions, which typically occur early. Hyperdiploidy also occurs early, with individual trisomies often acquired in different chronological windows during evolution, and with a preferred order of acquisition. Conversely, positively selected point mutations, whole genome duplication and chromoplexy events occur in later disease phases. Thus, initiating driver events, drawn from a limited repertoire of structural and numerical chromosomal changes, shape preferred trajectories of evolution that are biologically relevant but heterogeneous across patients.

Atypical chromosome 22q11.2 deletions are complex rearrangements and have different mechanistic origins.

The majority (99%) of individuals with 22q11.2 deletion syndrome (22q11.2DS) have a deletion that is caused by non-allelic homologous recombination between two of four low copy repeat clusters on chromosome 22q11.2 (LCR22s). However, in a small subset of patients, atypical deletions are observed with at least one deletion breakpoint within unique sequence between the LCR22s. The position of the chromosome breakpoints and the mechanisms driving those atypical deletions remain poorly studied. Our large-scale, whole genome sequencing study of >1500 subjects with 22q11.2DS identified six unrelated individuals with atypical deletions of different types. Using a combination of whole genome sequencing data and fiber-fluorescence in situ hybridization, we mapped the rearranged alleles in these subjects. In four of them, the distal breakpoints mapped within one of the LCR22s and we found that the deletions likely occurred by replication-based mechanisms. Interestingly, in two of them, an inversion probably preceded inter-chromosomal 'allelic' homologous recombination between differently oriented LCR22-D alleles. Inversion associated allelic homologous recombination (AHR) may well be a common mechanism driving (atypical) deletions on 22q11.2.

Interchromosomal template-switching as a novel molecular mechanism for imprinting perturbations associated with Temple syndrome

BackgroundIntrachromosomal triplications (TRP) can contribute to disease etiology via gene dosage effects, gene disruption, position effects, or fusion gene formation. Recently, post-zygotic de novo triplications adjacent to copy-number neutral genomic intervals with runs of homozygosity (ROH) have been shown to result in uniparental isodisomy (UPD). The genomic structure of these complex genomic rearrangements (CGRs) shows a consistent pattern of an inverted triplication flanked by duplications (DUP-TRP/INV-DUP) formed by an iterative DNA replisome template-switching mechanism during replicative repair of a single-ended, double-stranded DNA (seDNA), the ROH results from an interhomolog or nonsister chromatid template switch. It has been postulated that these CGRs may lead to genetic abnormalities in carriers due to dosage-sensitive genes mapping within the copy-number variant regions, homozygosity for alleles at a locus causing an autosomal recessive (AR) disease trait within the ROH region, or imprinting-associated diseases.MethodsHere, we report a family wherein the affected subject carries a de novo 2.2-Mb TRP followed by 42.2 Mb of ROH and manifests clinical features overlapping with those observed in association with chromosome 14 maternal UPD (UPD(14)mat). UPD(14)mat can cause clinical phenotypic features enabling a diagnosis of Temple syndrome. This CGR was then molecularly characterized by high-density custom aCGH, genome-wide single-nucleotide polymorphism (SNP) and methylation arrays, exome sequencing (ES), and the Oxford Nanopore long-read sequencing technology.ResultsWe confirmed the postulated DUP-TRP/INV-DUP structure by multiple orthogonal genomic technologies in the proband. The methylation status of known differentially methylated regions (DMRs) on chromosome 14 revealed that the subject shows the typical methylation pattern of UPD(14)mat. Consistent with these molecular findings, the clinical features overlap with those observed in Temple syndrome, including speech delay.ConclusionsThese data provide experimental evidence that, in humans, triplication can lead to segmental UPD and imprinting disease. Importantly, genotype/phenotype analyses further reveal how a post-zygotically generated complex structural variant, resulting from a replication-based mutational mechanism, contributes to expanding the clinical phenotype of known genetic syndromes. Mechanistically, such events can distort transmission genetics resulting in homozygosity at a locus for which only one parent is a carrier as well as cause imprinting diseases.

Single-cell copy number variant detection reveals the dynamics and diversity of adaptation.

Copy number variants (CNVs) are a pervasive source of genetic variation and evolutionary potential, but the dynamics and diversity of CNVs within evolving populations remain unclear. Long-term evolution experiments in chemostats provide an ideal system for studying the molecular processes underlying CNV formation and the temporal dynamics with which they are generated, selected, and maintained. Here, we developed a fluorescent CNV reporter to detect de novo gene amplifications and deletions in individual cells. We used the CNV reporter in Saccharomyces cerevisiae to study CNV formation at the GAP1 locus, which encodes the general amino acid permease, in different nutrient-limited chemostat conditions. We find that under strong selection, GAP1 CNVs are repeatedly generated and selected during the early stages of adaptive evolution, resulting in predictable dynamics. Molecular characterization of CNV-containing lineages shows that the CNV reporter detects different classes of CNVs, including aneuploidies, nonreciprocal translocations, tandem duplications, and complex CNVs. Despite GAP1’s proximity to repeat sequences that facilitate intrachromosomal recombination, breakpoint analysis revealed that short inverted repeat sequences mediate formation of at least 50% of GAP1 CNVs. Inverted repeat sequences are also found at breakpoints at the DUR3 locus, where CNVs are selected in urea-limited chemostats. Analysis of 28 CNV breakpoints indicates that inverted repeats are typically 8 nucleotides in length and separated by 40 bases. The features of these CNVs are consistent with origin-dependent inverted-repeat amplification (ODIRA), suggesting that replication-based mechanisms of CNV formation may be a common source of gene amplification. We combined the CNV reporter with barcode lineage tracking and found that 102–104 independent CNV-containing lineages initially compete within populations, resulting in extreme clonal interference. However, only a small number (18–21) of CNV lineages ever constitute more than 1% of the CNV subpopulation, and as selection progresses, the diversity of CNV lineages declines. Our study introduces a novel means of studying CNVs in heterogeneous cell populations and provides insight into their dynamics, diversity, and formation mechanisms in the context of adaptive evolution.

Exonic duplication of the OTC gene by a complex rearrangement that likely occurred via a replication-based mechanism: a case report

BackgroundOrnithine transcarbamylase deficiency (OTCD) is an X-linked recessive disorder involving a defect in the urea cycle caused by OTC gene mutations. Although a total of 417 disease-causing mutations in OTC have been reported, structural abnormalities in this gene are rare. We here describe a female OTCD case caused by an exonic duplication of the OTC gene (exons 1–6).Case presentationA 23-year-old woman with late-onset OTCD diagnosed by biochemical testing was subjected to subsequent genetic testing. Sanger sequencing revealed no pathogenic mutation throughout the coding exons of the OTC gene, but multiplex ligation-dependent probe amplification (MLPA) revealed duplication of exons 1–6. Further genetic analyses revealed an inversion of duplicated exon 1 and a tandem duplication of exons 2–6. Each of the junctions of the inversion harbored a microhomology and non-templated microinsertion, respectively, suggesting a replication-based mechanism. The duplication was also of de novo origin but segregation analysis indicated that it took place in the paternal chromosome.ConclusionWe report the first OTCD case harboring an exonic duplication in the OTC gene. The functional defects caused by this anomaly were determined via structural analysis of its complex rearrangements.

Complex structural variants in Mendelian disorders: identification and breakpoint resolution using short- and long-read genome sequencing

BackgroundStudies have shown that complex structural variants (cxSVs) contribute to human genomic variation and can cause Mendelian disease. We aimed to identify cxSVs relevant to Mendelian disease using short-read whole-genome sequencing (WGS), resolve the precise variant configuration and investigate possible mechanisms of cxSV formation.MethodsWe performed short-read WGS and analysis of breakpoint junctions to identify cxSVs in a cohort of 1324 undiagnosed rare disease patients. Long-read WGS and gene expression analysis were used to resolve one case.ResultsWe identified three pathogenic cxSVs: a de novo duplication-inversion-inversion-deletion affecting ARID1B, a de novo deletion-inversion-duplication affecting HNRNPU and a homozygous deletion-inversion-deletion affecting CEP78. Additionally, a de novo duplication-inversion-duplication overlapping CDKL5 was resolved by long-read WGS demonstrating the presence of both a disrupted and an intact copy of CDKL5 on the same allele, and gene expression analysis showed both parental alleles of CDKL5 were expressed. Breakpoint analysis in all the cxSVs revealed both microhomology and longer repetitive elements.ConclusionsOur results corroborate that cxSVs cause Mendelian disease, and we recommend their consideration during clinical investigations. We show that resolution of breakpoints can be critical to interpret pathogenicity and present evidence of replication-based mechanisms in cxSV formation.

MYCN is amplified during S phase, and c‑myb is involved in controlling MYCN expression and amplification in MYCN‑amplified neuroblastoma cell lines.

Neuroblastoma derived from primitive sympathetic neural precursors is a common type of solid tumor in infants. MYCN proto-oncogene bHLH transcription factor (MYCN) amplification and 1p36 deletion are important factors associated with the poor prognosis of neuroblastoma. Expression levels of MYCN and c-MYB proto-oncogene transcription factor (c-myb) decline during the differentiation of neuroblastoma cells; E2F transcription factor 1 (E2F1) activates the MYCN promoter. However, the underlying mechanism of MYCN overexpression and amplification requires further investigation. In the present study, potential c-Myb target genes, and the effect of c-myb RNA interference (RNAi) on MYCN expression and amplification were investigated in MYCN-amplified neuroblastoma cell lines. The mRNA expression levels and MYCN gene copy number in five neuroblastoma cell lines were determined by quantitative polymerase chain reaction. In addition, variations in potential target gene expression and MYCN gene copy number between pre- and post-c-myb RNAi treatment groups in MYCN-amplified Kelly, IMR32, SIMA and MHH-NB-11 cell lines, normalized to those of non-MYCN-amplified SH-SY5Y, were examined. To determine the associations between gene expression levels and chromosomal aberrations, MYCN amplification and 1p36 alterations in interphases/metaphases were analyzed using fluorescence in situ hybridization. Statistical analyses revealed correlations between 1p36 alterations and the expression of c-myb, MYB proto-oncogene like 2 (B-myb) and cyclin dependent kinase inhibitor 1A (p21). Additionally, the results of the present study also demonstrated that c-myb may be associated with E2F1 and L3MBTL1 histone methyl-lysine binding protein (L3MBTL1) expression, and that E2F1 may contribute to MYCN, B-myb, p21 and chromatin licensing and DNA replication factor 1 (hCdt1) expression, but to the repression of geminin (GMNN). On c-myb RNAi treatment, L3MBTL1 expression was silenced, while GMNN was upregulated, indicating G2/M arrest. In addition, MYCN gene copy number increased following treatment with c-myb RNAi. Notably, the present study also reported a 43.545% sequence identity between upstream of MYCN and Drosophila melanogaster amplification control element 3, suggesting that expression and/or amplification mechanisms of developmentally-regulated genes may be evolutionarily conserved. In conclusion, c-myb may be associated with regulating MYCN expression and amplification. c-myb, B-myb and p21 may also serve a role against chromosome 1p aberrations. Together, it was concluded that MYCN gene is amplified during S phase, potentially via a replication-based mechanism.

Identification and characterization of a novel 43-bp deletion mutation of the ATP7B gene in a Chinese patient with Wilson\u2019s disease: a case report

BackgroundWilson’s disease (WD) is an autosomal recessive disorder characterized by copper accumulation. ATP7B gene mutations lead to ATP7B protein dysfunction, which in turn causes Wilson’s disease.Case presentationWe describe a male case of Wilson’s disease diagnosed at 10 years after routine biochemical test that showed low serum ceruloplasmin levels and Kayser–Fleischer rings in both corneas. Analysis of the ATP7B gene revealed compound heterozygous mutations in the proband, including the reported c.3517G > A mutation and a novel c.532_574del mutation. The c.532_574del mutation covered a 43-bp region in exon 2, and resulted in a frameshift mutation (p.Leu178PhefsX10). By base sequence analysis, two microhomologies (TCTCA) were observed on both deletion breakpoints in the ATP7B gene. Meanwhile, the presence of some sequence motifs associated with DNA breakage near the deletion region promoted DNA strand break.ConclusionsBy comparison, a replication-based mechanism named fork stalling and template switching/ microhomology-mediated break-induced replication (FoSTeS/MMBIR) was used to explain the formation of this novel deletion mutation.

Doublet-Mediated DNA Rearrangement-A Novel and Potentially Underestimated Mechanism for the Formation of Recurrent Pathogenic Deletions.

Deletions and duplications of genomic DNA contribute to evolution, phenotypic diversity, and human disease. The underlying mechanisms are incompletely understood. We identified deletions of exon 10 of the SPAST gene in two unrelated families with hereditary spastic paraplegia. We excluded a founder event, but observed that the breakpoints map to identical repeat regions. These regions likely represent an intragenic "doublet," that is, an enigmatic class of local duplications. The fusion sequences for both deletions are compatible with recombination-based as well as with replication-based mechanisms. Searching the literature, we identified a partial SLC24A4 deletion that involved two copies of another doublet, and was likely formed in an analogous way. Comparing the SPAST and the SLC24A4 doublets with doublets identified previously suggested that many additional doublets have a high potential for triggering rearrangements. Considering that doublets are still being formed in the human genome, and that they likely create high local instability, we suggest that a two-step mechanism consisting of doublet generation and subsequent doublet-mediated deletion/duplication may underlie certain copy-number changes for which other mechanisms are currently assumed. Further studies are necessary to delineate the significance of the thus-far understudied doublets for the formation of copy-number variation.