Genome-wide identification of functional polymorphisms modulating individual risk and prognosis of lung cancer

Lung cancer is a leading cause of cancer death in Western countries. Although most cases are due to tobacco smoking, complex genetics may modulate this disease, as suggested by epidemiological studies and findings obtained in mouse models. Systematic population-based association studies testing several thousands of genetic markers dispersed genome-wide have recently become a powerful and widely-used approach to identify genetic factors affecting common diseases. In this thesis, the role of genetic polymorphisms and risk of cancer were investigated through a case-control association study in Italian lung adenocarcinoma (ADCA) patients and unrelated controls from general population and through a case-control association family-based study in lung cancer patients and unaffected sibs as controls. I confirmed the relevance of a polygenic model characterized by additive and interchangeable effects of rare alleles in the modulation of individual risk of lung ADCA identifying multiple inherited susceptibility alleles linked to lung cancer. Additionally, I studied the role of genetic polymorphisms modulating individual lung cancer prognosis through a case-only association study in lung ADCA patients with clinical stage I versus higher clinical stage. In particular, I identify two genes (FCN3 and TMEM100) down-regulated up to 1.8-fold in normal lung of stage >I as compared to stage I patients. These results suggest that clinical stage may be genetically determined as reflected in germ-line variations as well as in the transcriptional profile of normal lung tissue. Although clinical application of these results awaits replication in independent and large populations, I found that genetic variants may be involved in the modulation of not only individual risk of lung cancer but also clinical staging. The newly identified individual genetic profiles associated with risk and/or prognosis of lung cancer may thus represent new diagnostic tools and suggest molecular targets for the development of new therapies against lung cancer

Liquid Biopsy: A New Strategy for Future Directions in Lung Cancer Treatment

The gold standard for cancer diagnosis has always been based on radiological imaging followed by surgical tissue biopsies for molecular testing and pathological examination and surgical resection to remove the tumoral mass when possible. However, the resulting information is a limited snapshot in space and time, which poorly reflects clonal heterogeneity or tumor evolution and metastasis. Over a decade since its inception, the ability to use non-invasive methods such as a liquid biopsy to analyze tumor biomarkers has transformed the vision of future cancer care into a better patient experience thanks to real-time monitoring and early diagnosis. The liquid biopsy essay is an effective tool for detecting cancers at an early stage, when there are very few tumor-derived materials circulating in the bloodstream, being a very sensitive technique. For this reason, liquid biopsy is particularly suitable for early-stage diagnosis (stage I or II) of lung cancer whose diagnosis often occurs in the final stages of the disease as well as monitoring cancer progression and driving target therapies

A 5'-region polymorphism modulates promoter activity of the tumor suppressor gene MFSD2A

<p>Abstract</p> <p>Background</p> <p>The MFSD2A gene maps within a linkage disequilibrium block containing the MYCL1-<it>EcoRI </it>polymorphism associated with prognosis and survival in lung cancer patients. Survival discrepancies between Asians and Caucasians point to ethnic differences in allelic frequencies of the functional genetic variations.</p> <p>Results</p> <p>Analysis of three single-nucleotide polymorphisms (SNPs) mapping in the MFSD2A 5'-regulatory region using a luciferase reporter system showed that SNP rs12072037, in linkage disequilibrium with the MYCL1-<it>EcoRI </it>polymorphism and polymorphic in Asians but not in Caucasians, modulated transcriptional activity of the MFSD2A promoter in cell lines expressing AHR and ARNT transcription factors, which potentially bind to the SNP site.</p> <p>Conclusion</p> <p>SNP rs12072037 modulates MFSD2A promoter activity and thus might affect MFSD2A levels in normal lung and in lung tumors, representing a candidate ethnically specific genetic factor underlying the association between the MYCL1 locus and lung cancer patients' survival.</p

Modelling PCDH19 clustering epilepsy by Neurogenin 2 induction of patient-derived induced pluripotent stem cells

Background: Loss of function mutations in PCDH19 gene causes an X-linked, infant-onset clustering epilepsy, associated with intellectual disability and autistic features. The unique pattern of inheritance includes random X-chromosome inactivation, which leads to pathological tissue mosaicism. Females carrying PCDH19 mutations are affected, while males have a normal phenotype. No cure is presently available for this disease.Methods: Fibroblasts from a female patient carrying frameshift mutation were reprogrammed into human induced pluripotent stem cells (hiPSCs). To create a cell model of PCDH19-clustering epilepsy (PCDH19-CE) where both cell populations co-exist, we created mosaic neurons by mixing wild-type (WT) and mutated (mut) hiPSC clones, and differentiated them into mature neurons with overexpression of the transcriptional factor Neurogenin 2.Results: We generated functional neurons from patient-derived iPSC using a rapid and efficient method of differentiation through overexpression of Neurogenin 2. Was revealed an accelerated maturation and higher arborisation in the mutated neurons, while the mosaic neurons showed the highest frequency of action potential firing and hyperexcitability features, compared to mutated and WT neurons.Conclusions: Our findings provide evidence that PCDH19 c.2133delG mutation affects proper metaphases with increased numbers of centrosomes in stem cells and accelerates neuronal maturation in premature cells. PCDH19 mosaic neurons showed elevated excitability, representing the situation in PCDH19-CE brain. We suggest Ngn2 hiPSC-derived PCDH19 neurons as an informative experimental tool for understanding the pathogenesis of PCDH19-CE and a suitable approach for use in targeted drug screening strategies

Case report: PIK3CA somatic mutation leading to Klippel Trenaunay Syndrome and multiple tumors

We report a case of Klippel Trenaunay Syndrome that was monitored both clinically and molecularly over a period of 9 years. A somatic mosaic mutation of PIK3CA (p(E545G)) was identified using both cfDNA NGS liquid biopsy and tissue biopsy. At the age of 56, due to intervening clonal mutations in PIK3CA background, she developed a squamous cell carcinoma in the right affected leg which was treated surgically. Nine years later, lung bilateral adenocarcinoma arose on PIK3CA mutated tissues supported by different clonal mutations. One year later, the patient died from metastases led by a new FGFR3 clone unresponsive to standard-of-care, immunotherapy-based. Our results highlight the presence of a molecular hallmark underlying neoplastic transformation that occurs upon an angiodysplastic process and support the view that PIK3CA mutated tissues must be treated as precancerous lesions. Importantly, they remark the effectiveness of combining cfDNA NGS liquid and tissue biopsies to monitor disease evolution as well as to identify aggressive clones targetable by tailored therapy, which is more efficient than conventional protocols

Omic Approach in Non-Smoker Female with Lung Squamous Cell Carcinoma Pinpoints to Germline Susceptibility and Personalized Medicine

Lung cancer is strongly associated to tobacco smoking. However, global statistics estimate that in females the proportion of lung cancer cases that is unrelated to tobacco smoking reaches fifty percent, making questionable the etiology of the disease

The polymorphism L412F in TLR3 inhibits autophagy and is a marker of severe COVID-19 in males

The polymorphism L412F in TLR3 has been associated with several infectious diseases. However, the mechanism underlying this association is still unexplored. Here, we show that the L412F polymorphism in TLR3 is a marker of severity in COVID-19. This association increases in the sub-cohort of males. Impaired macroautophagy/autophagy and reduced TNF/TNFα production was demonstrated in HEK293 cells transfected with TLR3L412F-encoding plasmid and stimulated with specific agonist poly(I:C). A statistically significant reduced survival at 28 days was shown in L412F COVID-19 patients treated with the autophagy-inhibitor hydroxychloroquine (p = 0.038). An increased frequency of autoimmune disorders such as co-morbidity was found in L412F COVID-19 males with specific class II HLA haplotypes prone to autoantigen presentation. Our analyses indicate that L412F polymorphism makes males at risk of severe COVID-19 and provides a rationale for reinterpreting clinical trials considering autophagy pathways.publishedVersio

Shorter androgen receptor polyQ alleles protect against life-threatening COVID-19 disease in European males

Background: While SARS-CoV-2 similarly infects men and women, COVID-19 outcome is less favorable in men. Variability in COVID-19 severity may be explained by differences in the host genome. Methods: We compared poly-amino acids variability from WES data in severely affected COVID-19 patients versus SARS-CoV-2 PCR-positive oligo-asymptomatic subjects. Findings: Shorter polyQ alleles (≤22) in the androgen receptor (AR) conferred protection against severe outcome in COVID-19 in the first tested cohort (both males and females) of 638 Italian subjects. The association between long polyQ alleles (≥23) and severe clinical outcome (p = 0.024) was also validated in an independent cohort of Spanish men <60 years of age (p = 0.014). Testosterone was higher in subjects with AR long-polyQ, possibly indicating receptor resistance (p = 0.042 Mann-Whitney U test). Inappropriately low serum testosterone level among carriers of the long-polyQ alleles (p = 0.0004 Mann-Whitney U test) predicted the need for intensive care in COVID-19 infected men. In agreement with the known anti-inflammatory action of testosterone, patients with long-polyQ and age ≥60 years had increased levels of CRP (p = 0.018, not accounting for multiple testing). Interpretation: We identify the first genetic polymorphism that appears to predispose some men to develop more severe disease. Failure of the endocrine feedback to overcome AR signaling defects by increasing testosterone levels during the infection leads to the polyQ tract becoming dominant to serum testosterone levels for the clinical outcome. These results may contribute to designing reliable clinical and public health measures and provide a rationale to test testosterone as adjuvant therapy in men with COVID-19 expressing long AR polyQ repeats. Funding: MIUR project "Dipartimenti di Eccellenza 2018-2020" to Department of Medical Biotechnologies University of Siena, Italy (Italian D.L. n.18 March 17, 2020) and "Bando Ricerca COVID-19 Toscana" project to Azienda Ospedaliero-Universitaria Senese. Private donors for COVID-19 research and charity funds from Intesa San Paolo

Analysis of the phenotypes in the Rett Networked Database

Rett spectrum disorder is a progressive neurological disease and the most common genetic cause of intellectual disability in females. MECP2 is the major causative gene. In addition, CDKL5 and FOXG1 mutations have been reported in Rett patients, especially with the atypical presentation. Each gene and different mutations within each gene contribute to variability in clinical presentation, and several groups worldwide performed genotype-phenotype correlation studies using cohorts of patients with classic and atypical forms of Rett spectrum disorder. The Rett Networked Database is a unified registry of clinical and molecular data of Rett patients, and it is currently one of the largest Rett registries worldwide with several hundred records provided by Rett expert clinicians from 13 countries. Collected data revealed that the majority of MECP2-mutated patients present with the classic form, the majority of CDKL5-mutated patients with the early-onset seizure variant, and the majority of FOXG1-mutated patients with the congenital form. A computation of severity scores further revealed significant differences between groups of patients and correlation with mutation types. The highly detailed phenotypic information contained in the Rett Networked Database allows the grouping of patients presenting specific clinical and genetic characteristics for studies by the Rett community and beyond. These data will also serve for the development of clinical trials involving homogeneous groups of patient