SNP array and FISH analysis of a proband with a 22q13.2- 22qter duplication shed light on the molecular origin of the rearrangement

Background In about one third of healthy subjects, the microscopic analysis of chromosomes reveals heteromorphisms with no clinical implications: for example changes in size of the short arm of acrocentric chromosomes. In patients with a pathological phenotype, however, a large acrocentric short arm can mask a genomic imbalance and should be investigated in more detail. We report the first case of a chromosome 22 with a large acrocentric short arm masking a partial trisomy of the distal long arm, characterized by SNP array. We suggest a possible molecular mechanism underlying the rearrangement. Case presentation We report the case of a 15-year-old dysmorphic girl with low grade psychomotor retardation characterized by a karyotype with a large acrocentric short arm of one chromosome 22. Cytogenetic analysis revealed a normal karyotype with a very intense Q-fluorescent and large satellite on the chromosome 22 short arm. Fluorescence in situ hybridisation analysis showed a de novo partial trisomy of the 22q13.2-qter chromosome region attached to the short arm of chromosome 22. SNP-array analysis showed that the duplication was 8.5 Mb long and originated from the paternal chromosome. Haplotype analysis revealed that the two paternal copies of the distal part of chromosome 22 have the same haplotype and, therefore, both originated from the same paternal chromosome 22. A possible molecular mechanism that could explain this scenario is a break-induced replication (BIR) which is involved in non-reciprocal translocation events. Conclusion The combined use of FISH and SNP arrays was crucial for a better understanding of the molecular mechanism underlying this rearrangement. This strategy could be applied for a better understanding of the molecular mechanisms underlying cryptic chromosomal rearrangements

Orexinergic neuron susceptibility to neuroinflammatory and aging-related neurodegenerative diseases

Orexins (a.k.a. hypocretins) play a crucial role in several physiological functions, including energy balance and the maintenance of wakefulness. Deficient orexin signalling is the hallmark of the sleep disorder narcolepsy. Although immune mechanisms have been hypotesized, the pathogenesis of narcolepsy remains to be clarified. Less attention has been devoted to potential orexinergic system alterations in other conditions, and their potential relationships with inflammatory signalling. Neuroinflammation has raised increasing interest in recent years, not only in relation to typical neuroinflammatory diseases, but also with regard to aging-related neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease. A role for neuroinflammatory signalling in normal, “healthy” aging is also currently debated, since several lines of evidence have pointed to aging as a chronic low-grade proinflammatory condition. We have examined neurons in the lateral hypothalamus expressing orexin A in different paradigms: i) normal aging in mice, ii) rodent models of a chronic infectious neuroinflammatory condition represented by a parasitic disease that causes sleep/wake alterations, iii) PDAAP mutant mice, a model of Alzheimer’s disease. In these paradigms, we have identified different degrees of neuronal loss in the orexinergic cell population and/or evidence of functional dysregulation of these neurons, together with glial activation in the lateral hypothalamus and sleep/wake changes. Altogether, the data point to a vulnerability of orexin to inflammatory signalling, and potentially place the neuropeptide at the center of neural-immune interactions, drawing attention on the relationships between neuroinflammation, sleep regulation, and orexin neuron damage

Experimental sleep deprivation as a tool to test memory deficits in rodents.

Paradigms of sleep deprivation (SD) and memory testing in rodents (laboratory rats and mice) are here reviewed. The vast majority of these studies have been aimed at understanding the contribution of sleep to cognition, and in particular to memory. Relatively little attention, instead, has been devoted to SD as a challenge to induce a transient memory impairment, and therefore as a tool to test cognitive enhancers in drug discovery. Studies that have accurately described methodological aspects of the SD protocol are first reviewed, followed by procedures to investigate SD-induced impairment of learning and memory consolidation in order to propose SD protocols that could be employed as cognitive challenge. Thus, a platform of knowledge is provided for laboratory protocols that could be used to assess the efficacy of drugs designed to improve memory performance in rodents, including rodent models of neurodegenerative diseases that cause cognitive deficits, and Alzheimer's disease in particular. Issues in the interpretation of such preclinical data and their predictive value for clinical translation are also discussed

Opposite Response to Vitamin K Antagonists: A Report of Two Cases and Systematic Review of Literature

Vitamin K antagonists (VKAs) are used in the prophylaxis and treatment of thromboembolic disorders. Despite a high efficacy, their narrow therapeutic window and high response variability hamper their management. Several patients experience fluctuations in dose–response and are at increased risk of over- or under-anticoagulation. Therefore, it is essential to monitor the prothrombin time/international normalized ratio to determine the so-called stable dose and to adjust the dosage accordingly. Three polymorphisms, CYP2C9∗2, CYP2C9∗3 and VKORC1-1639G>A, are associated with increased sensitivity to VKAs. Other polymorphisms are associated with a request for a higher dose and VKA resistance. We described the clinical cases of two patients who were referred to the Clinical Pharmacology and Pharmacogenetics Unit of the University Hospital of Salerno for pharmacological counseling. One of them showed hypersensitivity and the other one was resistant to VKAs. A systematic review was performed to identify randomized clinical trials investigating the impact of pharmacogenetic testing on increased sensitivity and resistance to VKAs. Although international guidelines are available and information on the genotype-guided dosing approach has been included in VKA drug labels, VKA pharmacogenetic testing is not commonly required. The clinical cases and the results of the systematically reviewed RCTs demonstrate that the pharmacogenetic-based VKA dosing model represents a valuable resource for reducing VKA-associated adverse events

The aging brain, neuroinflammatory signaling and sleep-wake regulation

Tissues and organs change over time, regulated by intrinsic (genetic) determinants and environmental (and microenvironmental) adaptation. Brain changes during lifetime are especially critical, as the brain is the effector of cognition and the vast majority of neurons live throughout the life of the individual. In addition, brain aging mechanisms are especially critical for disease vulnerability, given the aging­related prevalence of pathologies that include neurodegenerative diseases. In this context, the present contribution concisely highlights data yielded by recent trends of research on the normal aging brain, and specifically: the occurrence of synaptic changes (rather than neuronal loss) and the altered regulation of adult neurogenesis (which represents a novel exciting field of knowledge); the development of a low­grade chronic inflammatory state which primes glial cells and may lead to changes in intercellular crosstalk, thus playing a potential role in the brain susceptibility to neurodegeneration; changes occurring in state­dependent behavior, sleep and wake, which are products of global brain functioning and underlie consciousness and cognitive performance; changes in the biological clock, the hypothalamic suprachiasmatic nucleus, which regulates sleep­wake alternation and other endogenous rhythms. Altogether, the present synopsis of recent studies at the molecular, cellular, and functional levels emphasizes the idea that the normal aging brain should be viewed as an example of adaptation and plasticity rather than as an obligatory decline

Generation of induced pluripotent stem cells (iPSC) from an atrial fibrillation patient carrying a PITX2 p.M200V mutation

Atrial fibrillation (AF) is the most common sustained arrhythmia associated with several cardiac risk factors, but increasing evidences indicated a genetic component. Indeed, genetic variations of the specific PITX2 gene have been identified in patients with early-onset AF. To investigate the molecular mechanisms underlying AF, we reprogrammed to pluripotency polymorphonucleated leukocytes isolated from the blood of a patient carrying a PITX2 p.M200V mutation, using a commercially available non-integrating expression system. The generated iPSCs expressed pluripotency markers and differentiated toward cells belonging to the three embryonic germ layers. Moreover, the cells showed a normal karyotype and retained the PITX2 p.M200V mutation

Neonatal treatment with clomipramine induces morphological and cellular changes in the adult rat brain

Clomipramine (CLI) is a tricyclic serotonin reuptake blocker, widely used to treat depression, obsessive compulsive disorder (OCD), and other psychiatric conditions in human patients. Chronic CLI administration in the neonate rodent alters serotonergic circuits and serotonine levels in the brain, and has been reported to cause a complex pattern of behavioral changes in the adult life, including abnormalities of rapid eye movement sleep, decreased aggression and sexual behavior, anhaedonia and helplessness. Such symptoms suggest a parallel with humans endogenous depression and have been proposed as a novel animal model of OCD. The present study was aimed at identifying morphological and cellular changes after chronic neonatal treatment with clomipramine (daily i.p. injections, 20 mg/kg, from P5 to P21) in the brain of 5 month-old male Sprague-Dawley rats, compared to saline-treated littermates, using three distinct experimental approaches. 1) In vivo volumetric analyses based on structural MRI scans performed at 4.7T on 6 CLI-treated and 6 control rats revealed a significant reduction in total brain and hippocampal volume, as well as enlarged ventricles in CLI-treated rats, compared to saline-treated cohorts. 2) In order to investigate treatment-related developmental disorders, we studied the dendritic arborization of newly generated cells in the hippocampus of 7 CLI and 7 control rats. Two-dimensional dendritic tracing diagrams were reconstructed with Neurolucida, and quantitative analyses of total dendritic length and arborization indices in the two groups are still ongoing. 3) Brain-derived neurotrophic factor (BDNF) levels were assessed in the hippocampus and neocortex of 5 CLI and 5 control rats by ELISA assay. Interestingly, we found significant region-specific, between-group differences. In particular, BDNF levels, important for neurogenesis, differentiation and neuronal survival, and highly expressed in brain areas involved in cognitive and emotional behavior, were significantly decreased in the hippocampus of CLI rats compared to controls, whereas no differences were found in the cortex. Taken together, the data suggest that interfering with serotonergic regulation during early postnatal development can produce permanent brain changes. These resemble brain abnormalities repeatedly observed not only in human depression but also in schizophrenia. Further morphological analyses as well as experiments aimed at characterizing the behavioral correlates of early CLI administration are in progress

Generation of induced pluripotent stem cells (iPSC) from an atrial fibrillation patient carrying a KCNA5 p.D322H mutation

Atrial fibrillation (AF) is the most common sustained arrhythmia associated with several cardiac risk factors, but increasing evidences indicated a genetic component. Indeed, genetic variations of the atrial specific KCNA5 gene have been identified in patients with early-onset lone AF. To investigate the molecular mechanisms underlying AF, we reprogrammed to pluripotency polymorphonucleated leukocytes isolated from the blood of a patient carrying a KCNA5 p.D322H mutation, using a commercially available non-integrating system. The generated iPSCs expressed pluripotency markers and differentiated toward cells belonging to the three embryonic germ layers. Moreover, the cells showed a normal karyotype and retained the p.D322H mutation

Assessment of clinical and radiological response to sorafenib in hepatocellular carcinoma patients

Sorafenib is an effective anti-angiogenic treatment for hepatocellular carcinoma (HCC). The assessment of tumor progression in patients treated with sorafenib is crucial to help identify potentially-resistant patients, avoiding unnecessary toxicities. Traditional methods to assess tumor progression are based on variations in tumor size and provide unreliable results in patients treated with sorafenib. New methods to assess tumor progression such as the modified Response Evaluation Criteria in Solid Tumors or European Association for the Study of Liver criteria are based on imaging to measure the vascularization and tumor volume (viable or necrotic). These however fail especially when the tumor response results in irregular development of necrotic tissue. Newer assessment techniques focus on the evaluation of tumor volume, density or perfusion. Perfusion computed tomography and Dynamic Contrast-Enhanced-UltraSound can measure the vascularization of HCC lesions and help predict tumor response to anti-angiogenic therapies. Mean Transit Time is a possible predictive biomarker to measure tumor response. Volumetric techniques are reliable, reproducible and time-efficient and can help measure minimal changes in viable tumor or necrotic tissue, allowing the prompt identification of non-responders. Volume ratio may be a reproducible biomarker for tumor response. Larger trials are needed to confirm the use of these techniques in the prediction of response to sorafenib

Concomitant Administration of Capecitabine and Folate Supplements: Need to Encourage Medication Reconciliation

Hand-Foot syndrome (HFS) and diarrhoea are dose-limiting Adverse Drug Reactions (ADRs) of capecitabine-based chemotherapy. Four polymorphisms in the dihydropyrimidine dehydrogenase (DPYD) gene, encoding the DPD enzyme responsible for the metabolism of fluoropyrimidines, such as capecitabine, are strongly associated with severe ADRs, and their screening should be performed before starting treatment. Moreover, capecitabine-related toxicity may worsen due to drug-drug and drug-supplement interactions. Here we investigated factors responsible for severe HFS and diarrhoea presented by two patients, non-carriers of the recommended DPYD single nucleotide polymorphisms (SNPs) but carriers of other genetic variants suggested to increase the risk of capecitabine-related ADRs. Through careful therapy recognition, we demonstrated that, unbeknownst to the oncologists, the patients were taking folic acid during the treatment with capecitabine at a dosage higher than 2000 mg/m(2), which is the maximum tolerated dose when folate is administered. To resolve the ADRs, the therapy had to be drastically changed. In one case, dose reduction of capecitabine and discontinuation of lipid-lowering agents were carried out. In the other case, discontinuation of capecitabine and folic acid and capecitabine re-administration were performed after a month. Genetic and environmental factors should be considered good predictors of severe capecitabine-related toxicity. Medication reconciliation should be encouraged to avoid the harmful consequences of inappropriate treatments