Dissecting the genetic bases of Amyotrophic lateral sclerosis and Late-onset Pompe disease through Next-generation sequencing

Amyotrophic lateral sclerosis (ALS) is a fatal neuromuscular disorder that affects motor neurons. ALS genetic landscape continues to shift as the number of genes associated with the disease keeps on growing. Moreover, ALS clinical manifestation is complicated by genetic and phenotypic overlapping with Frontotemporal dementia (FTD), Hereditary spastic paraplegia (HSP), Parkinson’s disease (PD) and other neuromuscular diseases. ALS is now considered an oligogenic disease in which the sum of several variants with small effects in different genes may lead to disease onset. Late-onset Pompe disease (LOPD) is an autosomal recessive disorder, caused by mutations in the GAA gene. The molecular genetic testing for LOPD is based on Sanger sequencing, however, large deletions/duplications are poorly investigated. LOPD is characterized by highly variable clinical presentation, hence genetic factors other than pathogenic GAA mutations may play a role in determining it. The aims of the two Ph.D. projects were 1) the study of the genetic architecture of 248 ALS patients through Next-generation sequencing (NGS); the investigation of the correlation of known ALS-implicated with our ALS cohort; the study of oligogenic bases for ALS onset in our cohort of patients. 2) The genetic investigation through MLPA of LOPD patients which carried only one pathogenic mutation in the GAA gene; the study of the impact of three single nucleotide polymorphisms (SNPs) within the GAA gene on protein stability; the identification of LOPD genetic modifiers through the WES analysis on 30 LOPD patients. All the genetic analyses have been performed upon genomic DNA extraction. Through NGS, we identified 38 pathogenic variants and 31 likely pathogenic variants (16%) out of 423 variants retrieved in the ALS cohort of patients. Among the pathogenic and likely pathogenic variants, 25 were in ALS causative genes, followed by 5 variants in the HSP and PD associated genes respectively and the remaining three variants were in genes linked to other NDs. We established a high degree of genetic heterogeneity among our patients, with 26 different ALS-implicated variants from 14 ALS genes identified among 54,4% of cases. Among the 26 variants, two were significantly associated with our cohort of ALS patients, an in-frame deletion (p.Gly173_Gly174del) within FUS and a missense variant (p.Arg208Trp) within SIGMAR1. Additionally, 7 patients (3%) of our cohort presented more than one of the listed ALS-implicated variants, suggesting oligogenic bases for these patients. Through MLPA analysis we identified two heterozygous deletions in 2 LOPD patients, one in exon 17 and one in exon 1 of the GAA gene. Through NGS we found a total of 176 variants in 103 (34%) out of 302 genes analyzed across six virtual gene panels. Through the statistical analysis, 73 variants out of 176 (43%) resulted significantly associated with our cohort of patients, however, we lack to found genetic modifiers for the clinical presentation of the LOPD pathology. This experimental thesis has reaffirmed the heterogeneity of the genetic of ALS, which takes into consideration not only ALS-causative genes and risk factors in genes notably associated with ALS, but consider also genes associated with other NDs such as FTD, HSP and PD, as well as gene-related to other motor neuron diseases, highlighting the importance of genetic comorbidity studies. Furthermore, here we confirmed the new vision of ALS as a polygenic disease characterized by oligogenic inheritance. Additionally, here we underline also the importance of the utilization of NGS technology in genetically less complex diseases, such as LOPD, to find genetic variants associated with the disease that may at the basis of the clinical variability observed among LOPD patients. Eventually, MLPA technology should be used when only one pathogenic mutation is identified

THERAPY-RELATED MYELOID NEOPLASMS: CONSIDERATIONS FOR PATIENTS’ CLINICAL EVALUATION

Therapy-related myeloid neoplasms (t-MNs) encompass a specific sub-group of myeloid malignancies arising after exposure to radio/cytotoxic agents for the treatment of unrelated diseases. Such malignancies present unique features, including advanced age, high comorbidities burden, and unfavorable genetic profiles. All these features justify the need for a specific diagnostic work-up and dedicated treatment algorithms. However, as new classification systems recognize the unique clinical characteristics exhibited by t-MN patients, how to assess fitness status in this clinical setting is largely unexplored. Optimizing fitness assessment would be crucial in the management of t-MN patients, considering that factors usually contributing to a worse or better outcome (like age, comorbidities, and treatment history) are patient-specific.In the absence of specific tools for fitness assessment in this peculiar category of AML, the aim of this review is to describe all those factors related to patient, treatment, and disease that allow planning treatments with an optimal risk/benefit ratio

Introducing Collaboration in Single-user Applications through the Centralized Control Architecture

In this paper we describe a novel Model-View­ Controller based architecture, Centralized Control, that intro­duces collaboration in single-users applications. The architecture is able to add collaboration with no need to modify the source code of the original single-user application, and providing also the capability to introduce group semantics into the new, collab­orative application that is obtained. The architecture is shown in practice, by introducing CollabXMind, a collaborative mind map tool, that is based on a well-known single-user tool, XMind

Mitochondrial Elongation and OPA1 Play Crucial Roles during the Stemness Acquisition Process in Pancreatic Ductal Adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic cancer with an overall 5-year survival rate of less than 9%. The high aggressiveness of PDAC is linked to the presence of a subpopulation of cancer cells with a greater tumorigenic capacity, generically called cancer stem cells (CSCs). CSCs present a heterogeneous metabolic profile that might be supported by an adaptation of mitochondrial function; however, the role of this organelle in the development and maintenance of CSCs remains controversial. To determine the role of mitochondria in CSCs over longer periods, which may reflect more accurately their quiescent state, we studied the mitochondrial physiology in CSCs at short-, medium-, and long-term culture periods. We found that CSCs show a significant increase in mitochondrial mass, more mitochondrial fusion, and higher mRNA expression of genes involved in mitochondrial biogenesis than parental cells. These changes are accompanied by a regulation of the activities of OXPHOS complexes II and IV. Furthermore, the protein OPA1, which is involved in mitochondrial dynamics, is overexpressed in CSCs and modulates the tumorsphere formation. Our findings indicate that CSCs undergo mitochondrial remodeling during the stemness acquisition process, which could be exploited as a promising therapeutic target against pancreatic CSCs

Massive hepatic angiomyolipoma in a young woman with tuberous sclerosis complex: Significant clinical improvement during tamoxifen treatment

Background/AimsIsolated liver angiomyolipomas (AMLs) occur in about 40% of TSC patients. Because of their slow growth, these tumors are often asymptomatic. Since AMLs express estrogen and progesteron receptors we suggest the possible benefits of tamoxifen for the treatment of liver AMLs.MethodsWe report the case of a 26-year-old female affected by tuberous sclerosis (TSC2) with cerebral, renal and hepatic involvement admitted to the Liver Unit for severe malnutrition, anorexia and abdominal pain. MRI showed a grossly enlarged liver, causing severe gastric compression. The liver was entirely filled with multiple nodular lesions of different sizes. Liver biopsy showed tumoral tissue with microscopic and ultrastructural features of angiomyolipoma. All liver function tests were repeatedly normal. Prior to considering the patient for partial hepatectomy, she was administered tamoxifen (20mg b.i.d).ResultsAfter 6 months of tamoxifen treatment a greatly improved quality of life and a significant weight gain were observed. After 12 months the clinical conditions further improved and the MRI showed a significant reduction of the largest lesion with a liquid central area and a diminished compression of the stomach.ConclusionsThis is to our knowledge the first report in which tamoxifen has been successfully used in a TSC patient with multiple liver angiomyolipomas

Attività antiproliferativa e proapoptotica di bifenili relazionabili alla struttura della curcumina su cellule di melanoma maligno

La curcumina è l’ingrediente attivo delle radici della Curcuma Longa ed è uno dei fitochimici più studiati al mondo per le sue proprietà antiossidanti, antinfiammatorie ed antitumorali. Nell'ambito della ricerca di nuovi agenti terapeutici contro il melanoma maligno (MM) abbiamo caratterizzato l’attività antiproliferativa sia della curcumina (D1) che di alcuni bifenili, strutturalmente riconducibili ad essa, su colture cellulari primarie di MM

Time for Dynamic Assessment of Fitness in Acute Myeloid Leukemia

Informed treatment decision-making in acute myeloid leukemia (AML) requires a comprehensive evaluation of all clinical and biological features that may affect the outcome with any given type or intensity of therapy [...

Would You Prescribe Mobile Health Apps for Heart Failure Self-care? An Integrated Review of Commercially Available Mobile Technology for Heart Failure Patients

Treatment of chronic diseases, such as heart failure, requires complex protocols based on early diagnosis; self-monitoring of symptoms, vital signs and physical activity; regular medication intake; and education of patients and caregivers about relevant aspects of the disease. Smartphones and mobile health applications could be very helpful in improving the efficacy of such protocols, but several barriers make it difficult to fully exploit their technological potential and produce clear clinical evidence of their effectiveness. App suppliers do not help users distinguish between useless/dangerous apps and valid solutions. The latter are few and often characterised by rapid obsolescence, lack of interactivity and lack of authoritative information. Systematic reviews can help physicians and researchers find and assess the 'best candidate solutions' in a repeatable manner and pave the way for well-grounded and fruitful discussion on their clinical effectiveness. To this purpose, the authors assess 10 apps for heart failure self-care using the Intercontinental Marketing Statistics score and other criteria, discuss the clinical effectiveness of existing solutions and identify barriers to their use in practice and drivers for change

First characterization of the CD4 and CD8 T-cell responses to QuantiFERON-TB Plus

Summary Introduction QuantiFERON ® -TB Gold Plus (QFT-Plus) is the new generation of QuantiFERON-TB Gold In-Tube test to identify latent tuberculosis infection (LTBI). QFT-Plus includes TB1 and TB2 tubes which contain selected Mycobacterium tuberculosis (Mtb) peptides designed to stimulate both CD4 and CD8 T-cells. Aim of this study is the flow cytometric characterization of the specific CD4 and CD8 T-cell responses to Mtb antigens contained within QFT-Plus. Methods We enrolled 27 active tuberculosis (TB) patients and 30 LTBI individuals. Following stimulation with TB1 and TB2, antigen-specific T-cells were characterized by flow cytometry. Data were also correlated with the grade of TB severity. Results TB1 mainly elicited a CD4 T-cell response while TB2 induced both CD4 and CD8 responses. Moreover, the TB2-specific CD4 response was detected for both active TB and LTBI patients, whereas the TB2-specific CD8 response was primarily associated with active TB (p = 0.01). Conclusions To our knowledge, we report the first characterization of the CD4 and CD8 T-cell response to QFT-Plus. CD8 T-cell response is mainly due to TB2 stimulation which is largely associated to active TB. These results provide a better knowledge on the use of this assay