Complete Duplication Research Articles

Characterization of a new BRCA1 rearrangement in an Italian woman with hereditary breast and ovarian cancer syndrome.

We report a novel BRCA1 LGR, involving the complete duplication of exon 3, in an Italian patient with a strong family history of breast and ovarian cancer. Our purpose is to provide an effective characterization of this LGR using a combination of different methods able to establish the exact breakpoints of the duplication. MAQ assay was used as primary screening method in LGRs detection. Array CGH, RT-PCR, and Long-PCR were used for a careful characterization of rearrangement and breakpoint regions. The Repeat Masker program was employed to identify Alu sequences at breakpoint junctions. RNA analysis showed that this in tandem duplication of exon 3 causes an in frame insertion of 18 amino acids within the protein. Array CGH and Long-PCR strategies revealed that the duplication (g.100411_102863dup) involves exactly 2.452 nucleotides between intron 2 and intron 3 of the gene. In addition, while an Alu Sx sequence was identified at upstream breakpoint, no Alu repeats were found at downstream junction. This supports the hypothesis that the new duplication was the result of a non-homologous recombination event between Alu and Non-Alu sequences. Our strategy, which combines a comprehensive set of methodologies, has been able to characterize the new BRCA1 duplication confirming, as previously reported, that MAQ assay represents a reliable and effective method for a primary screening of BRCA rearrangements. We underline the relevance of incorporating quantitative methods for BRCA genes dosage testing into routine diagnostic practice.

The sweet side of the cell cycle.

Cell division (mitosis) and gamete production (meiosis) are fundamental requirements for normal organismal development. The mammalian cell cycle is tightly regulated by different checkpoints ensuring complete and precise chromosomal segregation and duplication. In recent years, researchers have become increasingly interested in understanding how O-GlcNAc regulates the cell cycle. The O-GlcNAc post-translation modification is an O-glycosidic bond of a single β-N-acetylglucosamine sugar to serine/threonine residues of intracellular proteins. This modification is sensitive toward changes in nutrient levels in the cellular environment making O-GlcNAc a nutrient sensor capable of influencing cell growth and proliferation. Numerous studies have established that O-GlcNAcylation is essential in regulating mitosis and meiosis, while loss of O-GlcNAcylation is lethal in growing cells. Moreover, aberrant O-GlcNAcylation is linked with cancer and chromosomal segregation errors. In this review, we will discuss how O-GlcNAc controls different aspects of the cell cycle with a particular emphasis on mitosis and meiosis.

Bifid phallus with complete duplication and a separate scrotum in a German shepherd dog: a case report

Diphallia or penile duplication is a rare congenital abnormality thought to result from duplication of the cloacal membrane in the early developmental stages in the uterus. The present case concerned a 4-year old intact male German shepherd dog. The patient presented with a complaint of paraphimosis. Clinical, laboratory, radiological and ultrasonographic examinations were carried out. Semen samples collected prior to surgery were examined. Following amputation, the penis with paraphimosis was sent for histopathological investigation. Karyotype analysis of the patient was also performed. As well as the first report of this abnormality in Turkey, this case report represents the first report of this condition in a German shepherd dog.

Prevalence and function of Heschl's gyrus morphotypes in musicians.

Morphological variations of the first transverse Heschl's gyrus (HG) in the human auditory cortex (AC) are common, yet little is known about their functional implication. We investigated individual morphology and function of HG variations in the AC of 41 musicians, using structural and functional magnetic resonance imaging (fMRI) as well as magnetoencephalography (MEG). Four main morphotypes of HG were (i) single HG, (ii) common stem duplication (CSD), (iii) complete posterior duplication (CPD), and (iv) multiple duplications (MD). The vast majority of musicians (90%) exhibited HG multiplications (type ii-iv) in either one (39%) or both (51%) hemispheres. In 27% of musicians, MD with up to four gyri were found. To probe the functional contribution of HG multiplications to auditory processing we performed fMRI and MEG with auditory stimulation using analogous instrumental tone paradigms. Both methods pointed to the recruitment of all parts of HG during auditory stimulation, including multiplications if present. FMRI activations extended with the degree of HG gyrification. MEG source waveform patterns were distinct for the different types of HG: (i) hemispheres with single HG and (ii) CSD exhibited dominant N1 responses, whereas hemispheres with (iii) CPD and (iv) MD exhibited dominant P1 responses. N1 dipole amplitudes correlated with the localization of the first complete Heschl's sulcus (cHS), designating the most posterior anatomical border of HG. P2 amplitudes were significantly higher in professional as compared to amateur musicians. The results suggest that HG multiplications occur much more frequently in musicians than in the general population and constitute a functional unit with HG.

Novel Presentation of Complete Coronal Urethral Duplication: a Case Report

Urethralduplication is a rare condition occurs as a congenital malformation either independently or in the setting of other congenital malformations such as caudal duplication syndrome. Its prevalence becomes even rarer if it manifests as two side-by-side tracts in coronal plan.Nonetheless, we introduce a unique presentation of complete coronal urethral duplication accompanied by astounding manifestations of gastrointestinal malformations different from what is expected for a normal hind gut or caudal duplication syndrome. A 6-year-old boy with complete coronal duplication of urethra along with duplication of appendix, sigmoid and rectum as well as developmental delay, attracted our attention to report.

Unilateral Complete Ureteral Duplication with Impacted Stone at Ectopic Opening of Upper Moiety Ureter in Posterior Urethra and Simultaneous Bladder Stone: A Rare Entity

Unilateral Complete Ureteral Duplication with Impacted Stone at Ectopic Opening of Upper Moiety Ureter in Posterior Urethra and Simultaneous Bladder Stone: A Rare Entity

Variations of planum temporale asymmetries with Heschl’s Gyri duplications and association with cognitive abilities: MRI investigation of 428 healthy volunteers

In a large sample of 428 healthy adults balanced for gender and manual preference (MP), we investigated planum temporale (PT) surface area variability in relation with Heschl's gyrus (HG) duplication pattern, MP, and familial sinistrality (FS), considering different PT definitions. In a sub-sample of 362 participants, we also investigated whether variability of PT asymmetry was associated with differences in verbal abilities. On each participant brain hemisphere MRI, we delineated a posterior PT area (PTpost), excluding the second Heschl gyrus in case of either complete posterior duplication (CPD) or common stem partial duplication (CSD). We then defined a total PT area (PTtot) as the union of PTpost and of the second HG when present, and a HGPT area as the union of PTtot and of the first HG. The HG duplication pattern of one hemisphere was found to significantly affect the PTpost surface area of the same hemisphere, a larger reduction being present in case of CPD than in case of CSD, leading to a strong impact of both left and right HG duplication patterns on PTpost asymmetry. The HG duplication pattern had no effect on PTtot surface areas, while a significant effect of the left HG duplication was present on PTtot asymmetry that was larger in case of a CSD as compared to a single HG. By contrast, the type of HG duplication did not affect HGPT and neither left nor right HG duplication pattern had an effect on HGPT asymmetry. Meanwhile, MP had no effect on PTpost, PTtot, HGPT, or their asymmetries. The absence of a left PTpost was associated with existence of FS (FS+) (7FS+ among nine without PTpost). Removing the nine individuals lacking PTpost, a lower left PTpost surface area was observed in FS+ participants with left CPD. In the sub-sample of 362 participants, we observed a significant interaction between PTpost asymmetry and cognitive abilities due to poorer lexical performances in individuals having a symmetric PTpost as compared to individuals having either a leftward or a rightward asymmetric PTpost. By contrast, there was no significant effect of PTtot or HGPT asymmetry on cognitive abilities. This study shows that HG duplication pattern mainly affects the surface area of the most posterior part of PT and its asymmetry, this PTpost area being specifically associated with variability in verbal performances. This study also shows, for the first time, an association between decreased performances and lack of PTpost anatomical asymmetry, being rightward asymmetrical having no deleterious effect on verbal abilities, thereby supporting the idea that anatomical lateralization is necessary for optimal verbal performances.

FIRST EXPERIENCE OF THE URETERO-URETEROANASTOMOSIS (URETEROPYELOANASTOMOSIS) IN CHILDREN WITH COMPLETE URETERAL DUPLICATION

Introduction . The Duplex kidney is one of the most common anomalies of urinary system. Approaches to the surgical management of these conditions significantly differ. Taking into account the preferences of surgeons, heminephroureterectomy is offered to perform openly or laparoscopically, uretero-cysto-anastomosis of ectopic ureter or two ureters en bloc. Some publications about the possibility of ureteroureteral anastomosis have lately begun to appear. We present the experience of ureteroureteral anastomosis performed openly and laparoscopically in 9 clinics of Russia and Belarus. Objective . Improvement of treatment results of ureterohydronephrosis in children with complete ureteral duplication. Material and methods . We retrospectively reviewed medical records of 38 children who underwent ureteroureteral anastomosis surgery from 2007 to 2016. There were 10 boys (26.3%) and 28 girls (73.7%). The mean age of patients was 40.2 months. Right duplex kidney was confirmed in 15 (39.5%) children, the left one was diagnosed in 23 (60.5%) children. All the patients were divided into two groups according to the surgical technique used. Fourteen children 14 (36.8%) were included in the first group, in whom the ureteroureteral anastomosis has been performed openly, the second group comprised 24 (63.2%) patients with laparoscopically made anastomosis. Results . There weren’t any cases of conversion in this cohort. One patient in each group suffered from acute pyelonephritis. In the early postoperative period, urine leakage along the drainage occurred in 3 (12.5%) patients after laparoscopically performed ureteroureteral anastomosis, one (4.2%) child had residual ureteral stump. Thus, five (13.2%) children had complications, one (2.6%) patient required redo surgery. Conclusion . There are few clinics where ureteroureteral anastomosis is performed via open or laparoscopic approach. This operation is at the stage of development and accumulation of experience. It is a safe technique which allows to minimize the risk of loss of duplex kidney function.

Regulation of Replication Origins.

In eukaryotes, genome duplication starts concomitantly at many replication initiation sites termed replication origins. The replication initiation program is spatially and temporally coordinated to ensure accurate, efficient DNA synthesis that duplicates the entire genome while maintaining other chromatin-dependent functions. Unlike in prokaryotes, not all potential replication origins in eukaryotes are needed for complete genome duplication during each cell cycle. Instead, eukaryotic cells vary the use of initiation sites so that only a fraction of potential replication origins initiate replication each cell cycle. Flexibility in origin choice allows each eukaryotic cell type to utilize different initiation sites, corresponding to unique nuclear DNA packaging patterns. These patterns coordinate replication with gene expression and chromatin condensation. Budding yeast replication origins share a consensus sequence that marks potential initiation sites. Metazoan origins, on the other hand, lack a consensus sequence. Rather, they are associated with a collection of structural features, chromatin packaging features, histone modifications, transcription, and DNA-DNA/DNA-protein interactions. These features confer cell type-specific replication and expression and play an essential role in maintaining genomic stability.

Anatomical variations of ureter in central India: A cadaveric study

Background: Duplication of ureter is a developmental anomaly of urogenital system. The possible embryological reasons for the formation of a duplicated ureter could be splitting of ureteric bud, resulting two ureters draining into single kidney. Prevalence occurs in approximately 1% of the population. It may be associated with or without other congenital defects. Aim: To study the major anatomical variations of the ureter in routine dissection method. Materials and Methods: The present study conducted in the Department of Anatomy on 156 formalin-fixed cadavers of both sex and focus on the ureteric anomaly in routine dissection method. The ureter was followed from the renal pelvis to the uretrovesical junction, an opening into the urinary bladder (UB). Out of them, we got in one cadaver that two ureters were draining a single kidney into the UB separately and were not associated with any other congenital anomaly. There was complete duplication of ureter on unilateral side (left). Right kidney and its ureter were entirely normal. Conclusion: The knowledge of abnormality in renal collecting system is necessary for effective endo-urological applications and intrarenal surgeries. The present study adds on to the literature and will be helpful and interesting for the radiologists and urologists as it describes the ureteric anomaly in the Central Indian population.

Signaling pathways of replication stress in yeast.

Eukaryotic cells activate the S-phase checkpoint in response to a variety of events affecting the progression of replication forks, collectively referred to as replication stress. This signaling pathway is divided in two branches: the DNA damage checkpoint (DDC) and the DNA replication checkpoint (DRC). Both pathways are activated by the sensor kinase Mec1 and converge on the effector kinase Rad53. However, the DDC operates throughout the cell cycle and depends on the checkpoint mediator Rad9 to activate Rad53, whereas the DRC is specific to S phase and is mediated by Mrc1 and other fork components to signal replication impediments. In this review, we summarize current knowledge on these two pathways, with a focus on the budding yeast Saccharomyces cerevisiae, in which many important aspects of the replication stress response were discovered. We also discuss the differences and similarities between DDC and DRC and speculate on how these pathways cooperate to ensure the complete and faithful duplication of the yeast genome under various replication stress conditions.

Simultaneous Real-Time Imaging of Leading and Lagging Strand Synthesis Reveals the Coordination Dynamics of Single Replisomes

The molecular machinery responsible for DNA replication, the replisome, must efficiently coordinate DNA unwinding with priming and synthesis to complete duplication of both strands. Due to the anti-parallel nature of DNA, the leading strand is copied continuously, while the lagging strand is produced by repeated cycles of priming, DNA looping, and Okazaki-fragment synthesis. Here, we report a multidimensional single-molecule approach to visualize this coordination in the bacteriophage T7 replisome by simultaneously monitoring the kinetics of loop growth and leading-strand synthesis. We show that loops in the lagging strand predominantly occur during priming and only infrequently support subsequent Okazaki-fragment synthesis. Fluorescence imaging reveals polymerases remaining bound to the lagging strand behind the replication fork, consistent with Okazaki-fragment synthesis behind and independent of the replication complex. Individual replisomes display both looping and pausing during priming, reconciling divergent models for the regulation of primer synthesis and revealing an underlying plasticity in replisome operation.

Replication fork instability and the consequences of fork collisions from rereplication.

Replication forks encounter obstacles that must be repaired or bypassed to complete chromosome duplication before cell division. Proteomic analysis of replication forks suggests that the checkpoint and repair machinery travels with unperturbed forks, implying that they are poised to respond to stalling and collapse. However, impaired fork progression still generates aberrations, including repeat copy number instability and chromosome rearrangements. Deregulated origin firing also causes fork instability if a newer fork collides with an older one, generating double-strand breaks (DSBs) and partially rereplicated DNA. Current evidence suggests that multiple mechanisms are used to repair rereplication damage, yet these can have deleterious consequences for genome integrity.

Cell cycle proliferation decisions: the impact of single cell analyses.

Cell proliferation is a fundamental requirement for organismal development and homeostasis. The mammalian cell division cycle is tightly controlled to ensure complete and precise genome duplication and segregation of replicated chromosomes to daughter cells. The onset of DNA replication marks an irreversible commitment to cell division, and the accumulated efforts of many decades of molecular and cellular studies have probed this cellular decision, commonly called the restriction point. Despite a long-standing conceptual framework of the restriction point for progression through G1 phase into S phase or exit from G1 phase to quiescence (G0), recent technical advances in quantitative single cell analysis of mammalian cells have provided new insights. Significant intercellular heterogeneity revealed by single cell studies and the discovery of discrete subpopulations in proliferating cultures suggests the need for an even more nuanced understanding of cell proliferation decisions. In this review, we describe some of the recent developments in the cell cycle field made possible by quantitative single cell experimental approaches.

Measuring the DNA Content of Cells in Apoptosis and at Different Cell-Cycle Stages by Propidium Iodide Staining and Flow Cytometry.

All cells are created from preexisting cells. This involves complete duplication of the parent cell to create two daughter cells by a process known as the cell cycle. For this process to be successful, the DNA of the parent cell must be faithfully replicated so that each daughter cell receives a full copy of the genetic information. During the cell cycle, the DNA content of the parent cell increases as new DNA is synthesized (S phase). When there are two full copies of the DNA (G2/M phase), the cell splits to form two new cells (G0/G1 phase). As such, cells in different stages of the cell cycle have different DNA contents. The cell cycle is tightly regulated to safeguard the integrity of the cell and any cell that is defective or unable to complete the cell cycle is programmed to die by apoptosis. When this occurs, the DNA is fragmented into oligonucleosomal-sized fragments that are disposed of when the dead cell is removed by phagocytosis. Consequently apoptotic cells have reduced DNA content compared with living cells. This can be measured by staining cells with propidium iodide (PI), a fluorescent molecule that intercalates with DNA at a specific ratio. The level of PI fluorescence in a cell is, therefore, directly proportional to the DNA content of that cell. This protocol describes the use of PI staining to determine the percentage of cells in each phase of the cell cycle and the percentage of apoptotic cells in a sample.

Inducing a Site Specific Replication Blockage in E. coli Using a Fluorescent Repressor Operator System.

Obstacles present on DNA, including tightly-bound proteins and various lesions, can severely inhibit the progression of the cell's replication machinery. The stalling of a replisome can lead to its dissociation from the chromosome, either in part or its entirety, leading to the collapse of the replication fork. The recovery from this collapse is a necessity for the cell to accurately complete chromosomal duplication and subsequently divide. Therefore, when the collapse occurs, the cell has evolved diverse mechanisms that take place to restore the DNA fork and allow replication to be completed with high fidelity. Previously, these replication repair pathways in bacteria have been studied using UV damage, which has the disadvantage of not being localized to a known site. This manuscript describes a system utilizing a Fluorescence Repressor Operator System (FROS) to create a site-specific protein block that can induce the stalling and collapse of replication forks in Escherichia coli. Protocols detail how the status of replication can be visualized in single living cells using fluorescence microscopy and DNA replication intermediates can be analyzed by 2-dimensional agarose gel electrophoresis. Temperature sensitive mutants of replisome components (e.g. DnaBts) can be incorporated into the system to induce a synchronous collapse of the replication forks. Furthermore, the roles of the recombination proteins and helicases that are involved in these processes can be studied using genetic knockouts within this system.

Innate lymphoid cells: models of plasticity for immune homeostasis and rapid responsiveness in protection.

Innate lymphoid cells (ILCs) have stormed onto the immune landscape as "newly discovered" cell types. These tissue-resident sentinels are enriched at mucosal surfaces and engage in complex cross talk with elements of the adaptive immune system and microenvironment to orchestrate immune homeostasis. Many parallels exist between innate cells and T cells leading to the initial partitioning of ILCs into rather rigid subsets that reflect their "adaptive-like" effector cytokines profiles. ILCs themselves, however, have unique attributes that are only just beginning to be elucidated. These features result in complementarity with, rather than complete duplication of, functions of the adaptive immune system. Key transcription factors determine the pathway of differentiation of progenitors towards an ILC1, ILC2, or ILC3 subset. Once formed, flexibility in the responses of these subsets to stimuli unexpectedly allows transdifferentation between the different subsets and the acquisition of altered phenotypes and function. This provides a mechanism for rapid innate immune responsiveness. Here, we discuss the models of differentiation for maintenance and activation of tissue-resident ILCs in maintaining immune homeostasis and protection.

Phosphorus Deficiency Inhibits Cell Division But Not Growth in the Dinoflagellate Amphidinium carterae.

Phosphorus (P) is an essential nutrient element for the growth of phytoplankton. How P deficiency affects population growth and the cell division cycle in dinoflagellates has only been studied in some species, and how it affects photosynthesis and cell growth remains poorly understood. In the present study, we investigated the impact of P deficiency on the cell division cycle, the abundance of the carbon-fixing enzyme Rubisco, and other cellular characteristics in the Gymnodiniales peridinin-plastid species Amphidinium carterae. We found that under P-replete condition, the cell cycle actively progressed in the culture in a 24-h diel cycle with daily growth rates markedly higher than the P-deficient cultures, in which cells were arrested in the G1 phase and cell size significantly enlarged. The results suggest that, as in previously studied dinoflagellates, P deficiency likely disenables A. carterae to complete DNA duplication or check-point protein phosphorylation. We further found that under P-deficient condition, overall photosystem II quantum efficiency (Fv/Fm ratio) and Rubisco abundance decreased but not significantly, while cellular contents of carbon, nitrogen, and proteins increased significantly. These observations indicated that under P-deficiency, this dinoflagellate was able to continue photosynthesis and carbon fixation, such that proteins and photosynthetically fixed carbon could accumulate resulting in continued cell growth in the absence of division. This is likely an adaptive strategy thereby P-limited cells can be ready to resume the cell division cycle upon resupply of phosphorus.

Functions of Ubiquitin and SUMO in DNA Replication and Replication Stress.

Complete and faithful duplication of its entire genetic material is one of the essential prerequisites for a proliferating cell to maintain genome stability. Yet, during replication DNA is particularly vulnerable to insults. On the one hand, lesions in replicating DNA frequently cause a stalling of the replication machinery, as most DNA polymerases cannot cope with defective templates. This situation is aggravated by the fact that strand separation in preparation for DNA synthesis prevents common repair mechanisms relying on strand complementarity, such as base and nucleotide excision repair, from working properly. On the other hand, the replication process itself subjects the DNA to a series of hazardous transformations, ranging from the exposure of single-stranded DNA to topological contortions and the generation of nicks and fragments, which all bear the risk of inducing genomic instability. Dealing with these problems requires rapid and flexible responses, for which posttranslational protein modifications that act independently of protein synthesis are particularly well suited. Hence, it is not surprising that members of the ubiquitin family, particularly ubiquitin itself and SUMO, feature prominently in controlling many of the defensive and restorative measures involved in the protection of DNA during replication. In this review we will discuss the contributions of ubiquitin and SUMO to genome maintenance specifically as they relate to DNA replication. We will consider cases where the modifiers act during regular, i.e., unperturbed stages of replication, such as initiation, fork progression, and termination, but also give an account of their functions in dealing with lesions, replication stalling and fork collapse.

Anatomy of trisomy 12.

Trisomy 12 is a rare aneuploidy and fetuses with this defect tend to spontaneously abort. However, mosaicism allows this anomaly to manifest itself in live births. Due to the fact that mosaicism represents a common genetic abnormality, trisomy 12 is encountered more frequently than expected at a rate of 1 in 500 live births. Thus, it is imperative that medical practitioners are aware of this aneuploidy. Moreover, this genetic disorder may result from a complete or partial duplication of chromosome 12. A partial duplication may refer to a specific segment on the chromosome, or one of the arms. On the other hand, a complete duplication refers to duplication of both arms of chromosome 12. The combination of mosaicism and the variable duplication sites has led to variable phenotypes ranging from normal phenotype to Potter sequence to gross physical defects of the various organ systems. This article provides a review of the common anatomical variation of the different types of trisomy 12. This review revealed that further documentation is needed for trisomy 12q and complete trisomy 12 to clearly delineate the constellation of anomalies that characterize each genetic defect. Clin. Anat. 29:633-637, 2016. © 2016 Wiley Periodicals, Inc.