CDH1 and CTNNA1 Genetic Screening in Tunisian Patients with Hereditary Diffuse Gastric Carcinoma

Background: Mutational screening of the CDH1 gene is a standard treatment for patients who meet the criteria for Hereditary Diffuse Gastric Cancer (HDGC). In this framework, the classification of variants found in this gene is a crucial step for the clinical management of patients at high risk for HDGC. The aim of this study was to identify CDH1 as well as CTNNA1 mutation profiles predisposing to HDGC from Tunisia. Methods: Thirty four cases were included for this purpose with a mean age at diagnosis of 48 years old. We performed Sanger Sequencing for the entire coding sequence of both genes and MLPA (Multiplex Ligation Probe Amplification) assay to investigate large rearrangements of the CDH1 gene. Results: As a result, three cases (8.82%) carried probably pathogenic variants in the CDH1 gene. These variants involves a novel splice alteration, a missense located in exon 14 detected by Sanger Sequencing and a large rearrangement detected by MLPA assay.Conclusion: Our results suggest that the CDH1 p.G761R variant is probably pathogenic and involved in the conformational space shift of the protein. Molecular modeling analysis highlights a putative influence on the conformational properties of the Juxta-Membrane Domain core (JMD) that could result in destabilizing the protein-protein complexes and therefore impacting the downstream pathways. Also, a large deletion from the 5' locus including exons 1 and 2 of the CDH1 gene implicating the signal peptide and a part of the precursor domain of the protein. These findings highlight the critical importance of screening for large CDH1 rearrangements as well as mutations for the management of HDGC families and individuals at high risk for more personalised medicine. We therefore suggest a revision of the status of p.G761R mutation from Variant of Unknown Significance (VUS) to likely pathogenic.

Molecular Basis of Mismatch Repair Protein Deficiency in Tumors from Lynch Suspected Cases with Negative Germline Test Results

Some 10–50% of Lynch-suspected cases with abnormal immunohistochemical (IHC) staining remain without any identifiable germline mutation of DNA mismatch repair (MMR) genes. MMR proteins form heterodimeric complexes, giving rise to distinct IHC patterns when mutant. Potential reasons for not finding a germline mutation include involvement of an MMR gene not predicted by the IHC pattern, epigenetic mechanism of predisposition, primary mutation in another DNA repair or replication-associated gene, and double somatic MMR gene mutations. We addressed these possibilities by germline and tumor studies in 60 Lynch-suspected cases ascertained through diagnostics (n = 55) or research (n = 5). All cases had abnormal MMR protein staining in tumors but no point mutation or large rearrangement of the suspected MMR genes in the germline. In diagnostic practice, MSH2/MSH6 (MutS Homolog 2/MutS Homolog 6) deficiency prompts MSH2 mutation screening; in our study, 3/11 index individuals (27%) with this IHC pattern revealed pathogenic germline mutations in MSH6. Individuals with isolated absence of MSH6 are routinely screened for MSH6 mutations alone; we found a predisposing mutation in MSH2 in 1/7 such cases (14%). Somatic deletion of the MSH2-MSH6 region, joint loss of MSH6 and MSH3 (MutS Homolog 3) proteins, and hindered MSH2/MSH6 dimerization offered explanations to misleading IHC patterns. Constitutional epimutation hypothesis was pursued in the MSH2 and/or MSH6-deficient cases plus 38 cases with MLH1 (MutL Homolog 1)-deficient tumors; a primary MLH1 epimutation was identified in one case with an MLH1-deficient tumor. We conclude that both MSH2 and MSH6 should be screened in MSH2/6- and MSH6-deficient cases. In MLH1-deficient cases, constitutional epimutations of MLH1 warrant consideration.

The flagellar motor of Vibrio alginolyticus undergoes major structural remodeling during rotational switching

ABSTRACTThe bacterial flagellar motor is an intricate nanomachine that switches rotational directions between counterclockwise (CCW) and clockwise (CW) to direct the migration of the cell. The cytoplasmic ring (C-ring) of the motor, which is composed of FliG, FliM, and FliN, is known for controlling the rotational sense of the flagellum. However, the mechanism underlying rotational switching remains elusive. Here, we deployed cryo-electron tomography to visualize the C-ring in two rotational biased mutants (CCW-biased fliG-G214S and CW-locked fliG-G215A) in Vibrio alginolyticus. Sub-tomogram averaging was utilized to resolve two distinct conformations of the C-ring. Comparison of the C-ring structures in two rotational senses provide direct evidence that the C-ring undergoes major structural remodeling during rotational switch. Specifically, FliG conformational changes elicit a large rearrangement of the C-ring that coincides with rotational switching, whereas FliM and FliN form a spiral-shaped base of the C-ring, likely stabilizing the C-ring during the conformational remodeling.

Prospective Evaluation of Universal BRCA Testing for Women With Triple-Negative Breast Cancer

Background Limited published literature exists on women with triple-negative breast cancer (TNBC) diagnosed over the age of 60 years with breast cancer gene (BRCA) pathogenic variants. Our study determined whether the rate of BRCA pathogenic variants in a prospective cohort of TNBC patients outside the definition of current clinical genetic testing (GT) guidelines warrants a change in recommendations. Methods A prospective study of 395 women with TNBC underwent genetic counseling and 380 (96.2%) underwent clinical BRCA GT regardless of age of diagnosis beginning January 2014 to October 2015 at The University of Texas MD Anderson Cancer Center, Houston. TNBC patients older than 60 years who did not meet clinical GT guidelines had comprehensive sequencing and large rearrangement GT as part of the research protocol. Results Fifty-one of 380 (13.4%) women with TNBC who underwent clinical BRCA GT were BRCA positive. Of the 86 patients diagnosed at age over 60 years and underwent GT, only two (2.3%) were positive for BRCA. These two patients would have met clinical testing criteria due to family or ancestral history. Conclusions Our study does not support universal BRCA testing for TNBC patients diagnosed older than 60 years as their only risk factor for a BRCA pathogenic variant. Both of the positive BRCA patients older than 60 years identified would have met current National Comprehensive Cancer Network criteria for testing. Therefore, our study demonstrates that the National Comprehensive Cancer Network guidelines provide sufficient criteria for identifying BRCA pathogenic variants in women with TNBC at 60 years or younger.

Possibility of BKV-Associated Nephropathy in Hospitalized Burn Patients

Although renal failure in burn patients results from some defined reasons, there are various causes which are still unclear. BK virus is a human polyomavirus, which, in case of reactivation, can cause late-onset renal dysfunction and cystitis among immunodeficient patients such as transplant, pregnant, diabetic, and HIV patients. Regarding the related challenges, Polyomavirus BK (BKV), as a ubiquitous virus, is considered as one of the potential threats in the occurrence of Polyomavirus-associated nephropathy (PAN). Hypovolemia, occurring due to the weakness of the immune system, may be regarded as the major reason for the possibility of PAN as a risk factor in burn patients. Accordingly, this study was designed to evaluate the reactivation of BKV as a probable risk factor for renal failure or a problem in the future life of burn patients. This case–control study was conducted from October 2014 to September 2016, during which 270 patients were admitted to the burn unit. The patients were divided into two groups of case and control according to the inclusion criteria, and 20 patients were assigned to each group. The serum samples were first assessed for BKV-IgG and then were quantified by specific quantitative real-time polymerase chain reaction for BKV load. Positive samples were assessed for changes in noncoding regulatory region (NCRR) compared to Archetype strain by PCR sequencing method. Amplified sequences were analyzed for NCRR arrangement while the reactivation was assessed through these changes in NCRR. In both groups, patients were seropositive for BKV-IgG. Eight patients (40%) in the case group and two patients (10%) in the control group were found to be positive for BKV DNA with a load of ≥1000 and ≥100 copies/ml, respectively. There was a significant association between BKV DNA and kidney injury in the case group. The NCRR of DNA-positive samples had a large rearrangement compared to standard strain, but they showed relatively high similarity. Compared with other patients, burn patients are among the most susceptible ones to PAN, which can be considered as a major risk factor in the treatment of burn patients and optimizing their therapy.

NMDA Receptor Opening and Closing—Transitions of a Molecular Machine Revealed by Molecular Dynamics

We report the first complete description of the molecular mechanisms behind the transition of the N-methyl-d-aspartate (NMDA) receptor from the state where the transmembrane domain (TMD) and the ion channel are in the open configuration to the relaxed unliganded state where the channel is closed. Using an aggregate of nearly 1 µs of unbiased all-atom implicit membrane and solvent molecular dynamics (MD) simulations we identified distinct structural states of the NMDA receptor and revealed functionally important residues (GluN1/Glu522, GluN1/Arg695, and GluN2B/Asp786). The role of the “clamshell” motion of the ligand binding domain (LBD) lobes in the structural transition is supplemented by the observed structural similarity at the level of protein domains during the structural transition, combined with the overall large rearrangement necessary for the opening and closing of the receptor. The activated and open states of the receptor are structurally similar to the liganded crystal structure, while in the unliganded receptor the extracellular domains perform rearrangements leading to a clockwise rotation of up to 45 degrees around the longitudinal axis of the receptor, which closes the ion channel. The ligand-induced rotation of extracellular domains transferred by LBD–TMD linkers to the membrane-anchored ion channel is responsible for the opening and closing of the transmembrane ion channel, revealing the properties of NMDA receptor as a finely tuned molecular machine.

ReALLEN: structural variation discovery in cancer genome by sensitive analysis of single-end reads

BackgroundThe structural abnormalities in chromosomes are important issues in cancer genomics. Next generation sequencing technologies have big potentials to detect the structural variations precisely and comprehensively. Nevertheless, it is still difficult problem to detect large structural variations from short read sequence data. Major efforts have been achieved with paired-end reads, since discordant pairs directly reflect the existence of large rearrangement. Furthermore, approaches to detect structural variations from single-end reads are still worthwhile challenge because they allow wide choices of sequencing platforms.ResultWe present ReALLEN, a series of tools to detect genomic rearrangement with base-pair resolution from single-end reads provided by next generation sequencing. We examined the performance of ReALLEN using simulated dataset and real dataset sequenced by Ion Torrent systems. In most cases on the simulated dataset, ReALLEN showed nearly 100% precision and better sensitivity than other major tools. Notably, ReALLEN showed stable scores even if it was on some unfavorable conditions, for example, low coverage or small variant size. On the real dataset sequenced by Ion Torrent systems, ReALLEN accurately found an insertional translocation that was crucial for the diagnosis of chronic myeloid leukemia.ConclusionReALLEN is useful to researchers in finding genomic rearrangements. It will contribute to discovery of cancer-specific fusion proteins, precise diagnosis of known types of cancers, and understanding of genetic diseases caused by abnormal chromosomes.

Study of Some Genetic Variants for Cancer in Women with Breast Cancer In the East Azarbaijan Region by MLPA Method

Breast cancer is one of the main factors in the mortality of Iranian women. A large rearrangement genome is observed in most genes, especially in BRCA1 / BRCA2 genes lacking small mutations in breast cancer. Therefore, methods are needed to detect one or more exon deletions or their duplication. Therefore, the aim of this study was to determine the change in the number of copies of ATM, BRCA1, CHEK2, PTEN, and P53 genes in women with breast cancer in the East Azarbaijan region by MLPA method. This research is a descriptive study that was conducted randomly among 150 Azeri women with breast cancer who were referred to Tabriz Nour Najat Hospital; sixteen healthy people were selected as control samples. Deletion and duplication of ATM, BRCA1, P53, CHEK2 and PTEN genes were investigated using the MLPA method. The results showed that there was no pathogenicity mutation in these five genes. Therefore, it can be said that a large rearrangement genome in the East Azarbaijan province is very unlikely to lead to breast cancer in the area.

Structure of NDP-forming Acetyl-CoA synthetase ACD1 reveals a large rearrangement for phosphoryl transfer

The NDP-forming acyl-CoA synthetases (ACDs) catalyze the conversion of various CoA thioesters to the corresponding acids, conserving their chemical energy in form of ATP. The ACDs are the major energy-conserving enzymes in sugar and peptide fermentation of hyperthermophilic archaea. They are considered to be primordial enzymes of ATP synthesis in the early evolution of life. We present the first crystal structures, to our knowledge, of an ACD from the hyperthermophilic archaeonCandidatus Korachaeum cryptofilum.These structures reveal a unique arrangement of the ACD subunits alpha and beta within an α2β2-heterotetrameric complex. This arrangement significantly differs from other members of the superfamily. To transmit an activated phosphoryl moiety from the Ac-CoA binding site (within the alpha subunit) to the NDP-binding site (within the beta subunit), a distance of 51 Å has to be bridged. This transmission requires a larger rearrangement within the protein complex involving a 21-aa-long phosphohistidine-containing segment of the alpha subunit. Spatial restraints of the interaction of this segment with the beta subunit explain the necessity for a second highly conserved His residue within the beta subunit. The data support the proposed four-step reaction mechanism of ACDs, coupling acyl-CoA thioesters with ATP synthesis. Furthermore, the determined crystal structure of the complex with bound Ac-CoA allows first insight, to our knowledge, into the determinants for acyl-CoA substrate specificity. The composition and size of loops protruding into the binding pocket of acyl-CoA are determined by the individual arrangement of the characteristic subdomains.

Cloud and Radiative Balance Changes in Response to ENSO in Observations and Models

The authors use observations and four GFDL AGCMs to analyze the relation between variations in spatial patterns and area-averaged quantities in the top-of-the-atmosphere radiative fluxes, cloud amount, and precipitation related to El Niño over the period 1979–2008. El Niño is associated with an increase in tropical average sea surface temperature of order +0.1 K (with a maxima of +0.5 K), large local anomalies of +2 K (maxima +6 K), and tropical tropospheric warming of +0.5 K (maxima +1 K). The authors find that model-to-observation biases in the base state translate into corresponding biases in anomalies in response to El Niño. The pattern and amplitude of model biases in reflected shortwave (SW) and outgoing longwave radiation (OLR) follows expectations based on their biases in cloud amount: models with a positive cloud amount bias, compared to observations, have too strong local responses to El Niño in cloud amount, SW, OLR, and precipitation. Tropical average OLR increases in response to El Niño in observations and models [correlation coefficients (r) with Niño-3.4 index in the range 0.4–0.6]. Weaker correlations are found for SW (r: −0.6 to 0), cloud amount (r: −0.2 to +0.1), and precipitation (r: −0.2 to 0). Compositing El Niño events over the period 2001–07 yields similar results. These results are consistent with El Niño periods being warmer due to a heat pulse from the ocean, and a weak response in clouds and their radiative effect. These weak responses occur despite a large rearrangement in the spatial structure of the tropical circulation, and despite substantial differences in the mean state of observations and models.