BACKGROUND: No F8 genetic abnormality is detected in approximately 1% to 2% of patients with severe hemophilia A (HA) using conventional genetic approaches. In these patients, deep intronic variation or F8 disrupting genomic rearrangement could be causal.
OBJECTIVE: The study aimed to identify the causal variation in families with a history of severe HA for whom genetic investigations failed.
METHODS: We performed whole F8 gene sequencing in 8 propositi. Genomic rearrangements were confirmed by Sanger sequencing of breakpoint junctions and/or quantitative polymerase chain reaction.
RESULTS: A structural variant disrupting F8 was found in each propositus, so that all the 815 families with a history of severe HA registered in our laboratory received a conclusive genetic diagnosis. These structural variants consisted of 3 balanced inversions, 3 large insertions of gained regions, and 1 retrotransposition of a mobile element. The 3 inversions were 105 Mb, 1.97 Mb, and 0.362 Mb in size. Among the insertions of gained regions, one corresponded to the insertion of a 34 kb gained region from chromosome 6q27 in F8 intron 6, another was the insertion of a 447 kb duplicated region from chromosome 9p22.1 in F8 intron 14, and the last one was the insertion of an Xq28 349 kb gained in F8 intron 5.
CONCLUSIONS: All the genetically unsolved cases of severe HA in this cohort were due to structural variants disrupting F8. This study highlights the effectiveness of whole F8 sequencing to improve the molecular diagnosis of HA when the conventional approach fails.

Site-specific integration (SSI) cell line systems are gaining popularity for biotherapeutic development and production. Despite the proven advantages for these expression hosts, the SSI system is still susceptible to rare off-target events and potential vector rearrangements. Here we describe the development process of an SSI cell line for production of an IgG1 monoclonal antibody (mAb-086). During cell line generational studies to assess suitability of clone C10 for commercial purposes, restriction fragment lengths of genomic DNA harboring the light chain (LC) were not in agreement with the predicted size. We first confirmed that the SSI landing-pad achieved occupancy of the desired expression plasmid. Additional investigation revealed that random integration had occurred, resulting in the acquisition of a partial copy of the LC and a full-length copy of the heavy chain (HC) at a different locus in the host genome. This off-target event had no impact on the genotypic consistency and phenotypic stability of the cell line, the production process, or the drug substance product quality. Given the genetic, phenotypic, and process consistency of the cell line, clone C10 was deemed suitable as a manufacturing cell line.

Cardiac Contractility Modulation (CCM) has been proposed for inpatients affected by heart failure with reduced ejection fraction (HFrEF), with relapsing HF symptoms. We present a case of a patient treated with percutaneous coronary intervention (PCI) in the setting of acute coronary syndrome without persistent ST-segment elevation, with the best medical therapy for decompensated HF. The patient refused the implantable cardioverter-defibrillator (ICD), and to reduce the increasing number of hospitalizations for HF exacerbations, we proposed the use of the cardiac contractility modulation device. After the implant, the patient demonstrated a marked improvement in exercise effort and quality of life (QOL) with a six-minute walk test (SMWT), Minnesota Living with Heart Failure Questionnaire (MLWHFQ), and echocardiographic parameters. At 9 months after discharge, no hospital admissions for HF were recorded. We showed with the speckle tracking imaging how the improvement in global longitudinal strain (GLS) correlates with the remodeling effects on myocardial cells.

Integrons are bacterial genetic elements notorious for their role in spreading antibiotic resistance in clinical settings. In the natural environment, integrons present a wide and hidden diversity, raising questions as to their broader role in bacterial adaptation. From the One Health perspective, they must be considered a threatening pool of resistance determinants.

Multidrug-resistant Gram-negative carriers of Klebsiella pneumoniae carbapenemases (KPCs) often subvert antibiotic therapy due to inadequate sensitivity in laboratory detection. Although unstable gene amplification has been recognized to crucially contribute to underestimation or misestimation of antimicrobial resistance in clinical isolates, the precise mechanisms underlying carbapenem resistance driven by amplification of blaKPC-2 remain obscure. Here, we reported that IS26-mediated amplification of blaKPC-2 rapidly and robustly gave rise to carbapenem hyperresistant phenotypes in an Escherichia coli clinical strain following sublethal meropenem or tobramycin preexposure. Intriguingly, IS26 also underpinned amplification of a 47 kb multiple drug resistance (MDR) region encompassing nine antibiotic resistance genes and six IS26 insertion sequences. Tandem-repeat analysis and experimental validation demonstrated that blaKPC-2 amplification was indeed mediated by IS26, which was further experimentally shown to involve intricate genetic rearrangement. Such gene amplification arose dynamically under antibiotic stress and subsided upon antibiotic withdrawal. Instead of reducing the amplification of the IS26-flanked MDR region, drug combinations in vitro exacerbated it. Our study, thus, provides valuable insights into how dynamic gene amplification processes can precipitously transform resistance status and complicate diagnosis. IMPORTANCE Klebsiella pneumoniae carbapenemases (KPCs) are powerful β-lactamases that enable Gram-negative pathogens to destroy clinically important carbapenems in antibiotic therapies. In particular, KPC-2 is difficult to detect due to a lack of instrument sensitivity in regular laboratory screens, which leads to misdiagnosis and poor treatment outcomes. It remains unclear how blaKPC-2 rapidly induces exceedingly high-level resistance against carbapenems following the challenges of sublethal antibiotics. Here, we demonstrated that, under sublethal doses of antibiotics, insertion sequence IS26 mediated rapid amplification of multiple resistance determinants, including blaKPC-2 and a multiple drug resistance (MDR) region, which was accompanied by intricate genetic rearrangement.

Cholangiocarcinoma is an aggressive cancer with a poor prognosis and limited treatment. Gene sequencing studies have identified genetic alterations in fibroblast growth factor receptor (FGFR) in a significant proportion of cholangiocarcinoma (CCA) patients. This review will discuss the FGFR signaling pathway\'s role in CCA and highlight the development of therapeutic strategies targeting this pathway.
The development of highly potent and selective FGFR inhibitors has led to the approval of pemigatinib for FGFR2 fusion or rearranged CCA. Other selective FGFR inhibitors are currently under clinical investigation and show promising activity. Despite encouraging results, the emergence of resistance is inevitable. Studies using circulating tumor DNA and on-treatment tissue biopsies have elucidated underlying mechanisms of intrinsic and acquired resistance. There is a critical need to not only develop more effective compounds, but also innovative sequencing strategies and combinations to overcome resistance to selective FGFR inhibition. Therapeutic development of precision medicine for FGFR-altered CCA is a dynamic process of involving a comprehensive understanding of tumor biology, rational clinical trial design, and therapeutic optimization. Alterations in FGFR represent a valid therapeutic target in CCA and selective FGFR inhibitors are treatment options for this patient population.

Chronic hepatitis B virus (HBV) causes serious clinical problems, such as liver cirrhosis and hepatocellular carcinoma. Current antiviral treatments suppress HBV; however, the clinical cure rate remains low. Basic research on HBV is indispensable to eradicate and cure HBV. Genetic alterations are defined by nucleotide substitutions and canonical forms of structural variations (SVs), such as insertion, deletion and duplication. Additionally, genetic changes inconsistent with the canonical forms have been reported, and these have been termed complex SVs. Detailed analyses of HBV using bioinformatical applications have detected complex SVs in HBV genomes. Sequence gaps and low sequence similarity have been observed in the region containing complex SVs. Additionally, insertional motif sequences have been observed in HBV strains with complex SVs. Following the analyses of complex SVs in the HBV genome, the role of SVs in the genetic diversity of orthohepadnavirus has been investigated. SV polymorphisms have been detected in comparisons of several species of orthohepadnaviruses. As mentioned, complex SVs are composed of multiple SVs. On the contrary, SV polymorphisms are observed as insertions of different SVs. Up to a certain point, nucleotide substitutions cause genetic differences. However, at some point, the nucleotide sequences are split into several particular patterns. These SVs have been observed as polymorphic changes. Different species of orthohepadnaviruses possess SVs which are unique and specific to a certain host of the virus. Studies have shown that SVs play an important role in the HBV genome. Further studies are required to elucidate their virologic and clinical roles.

Patients with intrahepatic cholangiocarcinoma (iCCA) face a highly dismal prognosis, due to late stage diagnosis, the relative chemoresistance of the disease, and an overall limited portfolio of established therapeutic concepts. In recent years, a number of next generation sequencing studies have provided detailed information on the molecular landscape of biliary malignancies, and have laid the groundwork for the evaluation of novel, targeted therapeutic opportunities. Although nearly 40% of patients harbor genetic alterations for which targeted options exist, rapid translation into clinical trials is hampered by the overall low patient numbers. One of the most frequent genetic events in patients with iCCAs are fusions that involve the fibroblast growth factor receptor 2 (FGFR2). Impressive results from pivotal phase II studies in pre-treated patients have confirmed that FGFR-inhibitors are a promising therapeutic option for this genetic subgroup, and the rapid pace with which these inhibitors are being clinically developed is clearly justified by the imminent benefit for the patients. However, the success of these agents should not blind us to key challenges that need to be addressed to optimize FGFR-directed therapies in the future. A better understanding of mechanisms that convey primary and secondary resistance will be crucial to improve up-front patient stratification, to prolong the duration of response, and to implement reasonable co-treatment approaches. In this review, we provide background information on the pathobiology of oncogenic FGFR fusions and selected genetic testing strategies, summarize the latest clinical data, and discuss future directions of FGFR-directed therapies in patients with iCCA.

Middle East respiratory syndrome coronavirus (MERS-CoV) is a recently reported virus that is associated with severe, life threatening and rapidly spreading primarily respiratory illness called the Middle East respiratory syndrome. MERS-CoV possesses a unique positive-sense single-stranded RNA and can undergo rapid mutation in the viral genome. This results in antigenic switching and genetic variation, finally leading to the emergence of novel and new MERS-CoV subtypes which are uncontrollable by vaccines. Researchers are also finding difficulties to sort out therapeutic intervention strategies for MERS-CoV. This virus can spread from human to human, but transmission from dromedary camels to humans plays a crucial epidemiological significance. Dromedary camel acts as \"gene mixing vessels\" for MERS-CoV and these virus particles undergo rapid change in them. Viral receptors called dipeptidyl peptidase-4 are important receptors for attachment and spread of MERS-CoV in humans. The current method of laboratory confirmation is through real-time polymerase chain reaction on bronchoalveolar lavage, sputum and tracheal aspirates. Unfortunately, till today there are no definite anti-viral drugs available for MERS-CoV.

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