Characterization of mRNA dysfunctional mechanisms associated with the genetic disease cystic fibrosis

Tese de doutoramento, Bioquímica (Genética Molecular), Universidade de Lisboa, Faculdade de Ciências, 2018Cystic Fibrosis (CF) is the most common autosomal disease in Caucasians, with an estimated incidence of 1:6000 births in Portugal. The most relevant clinical aspect of its classic manifestation is chronic lung disease, which is the main cause of morbidity and mortality. Other symptoms include, pancreatic dysfunction, male infertility and high concentrations of chloride (Cl-) in sweat. However, even though the classic form of the disease is well defined, its pathophysiology is not completely understood with this pleiotropic disease having highly variable manifestations of clinical phenotypes. Novel therapies aim to correct the basic defect, specifically focusing on the rescue of Cystic fibrosis transmembrane conductance regulator (CFTR) function in CF airways. Most of these CFTR modulator strategies target the F508del, the most common mutation. Nevertheless, widespread evidence has demonstrated that a significant number of CFcausing mutations affect splicing efficiency and the stability of mRNA molecules. Here, we propose to elucidate the regulatory mechanisms underlying these CF-associated mutations. To this end, our aims are: 1) to identify CFTR gene mutations in individuals with non-CF chronic lung diseases, namely chronic obstructive pulmonary disease (COPD), asthma and disseminated bronchiectasis (DB); 2) to identify CFTR gene mutations in individuals with a suspicion of CF, followed by the analysis of CFTR expression in their native tissues to characterize the impact of CFTR splicing or premature termination codon (PTC) mutations in the structure and levels of mRNA; 3) to identify key factors in the nonsense/mediated decay (NMD) pathway by automated microscopy screens using a cell model expressing a novel CFTR NMD-PTC/read-through mini-gene reporter; and 4) to screen for novel compounds suppressing PTC mutations by automated microscopy screens using the previous cell-based model, as potential corrective therapies for CF. The expected results will provide knowledge on RNA-processing dysfunction and on the efficacy of novel RNA modulator compounds towards a "personalised medicine" approach. Regarding the first objective, our data show that 7 (out of 136) patients with non-CF respiratory diseases presented CFTR mutations in one allele, in contrast with the control group, in which no mutations were detected. We analysed the association of CFTR gene mutations with each of the three respiratory diseases considered. For asthma our data did not show an increase in mutation frequency when compared to the control group. For DB, we found an increase in the frequency of CFTR gene mutations, albeit with no statistical significance, which is in agreement with previous reports. For COPD however, we found a statistically significant increase in CFTR gene mutation incidence, relative to the control group. Our data reinforce the importance of characterizing CFTR gene mutations on non-CF respiratory diseases in Portuguese patients, to gain a better understanding of the epidemiology and etiology of these diseases. The results also lead to the identification of groups of patients who may benefit from the new therapeutic compounds currently under development to correct the basic CFTR protein defect in CF. Concerning the second objective, we have developed a novel RNA-based approach to detect unknown CFTR mutations [Felício et al (2017) Clin Genet 91: 476-481]. We are currently using this protocol for patients with a suspicion of CF and none or just one CFTR mutation identified. With this method we identified one mutation (711+3A>T) which had been previously reported but had not been characterized. We can conclude that this is a rapid, robust and inexpensive method to detect rare mutations, and therefore a method that can be easily used after a first screen. Regarding the third objective, we used this CFTR-NMD reporter to identify novel NMD factors by screening a previously validated shRNA library – a subset of The RNAi Consortium (TRC) – which is enriched in shRNAs targeting genes with a known or predicted involvement in transcript processing (425 genes), using HT microscopy. We selected the 24 top hits for the confirmation: 11 genes with NMD score ≥ 2 and more than 2 shRNAs with the same phenotype; 2 genes from the screen that showed read-through activity; and 11 other genes resulting from a high-throughput screen (HTS) aimed at the identification of CFTR splicing regulators (unpublished data). We chose these genes related to splicing because this is a process known to be required for NMD to occur and thus the knock-down of such genes can lead to NMD inhibition. The confirmation screen was performed using a library of siRNAs targeting the previously selected genes, however the results were inconclusive due to the low NMD score obtained. We have identified 4 genes with higher values but NMD score ≤ 1, three (eIF4A3, SREK1 and RPS19) are related to splicing, elF4A3 is directly and RPS19 is indirectly related to NMD, SREK1 is related only to splicing, with the fourth gene, ADIRF, having unknown functional properties. Some of the hits identified within this screen may be potential drug targets by their effects in inhibiting NMD, when knocked-down. With the results obtained in the confirmation screen we decided to follow the 24 genes identified in the primary screen for validation studies using two different techniques: WB and qRT-PCR (study in progress). Lastly, the fourth objective was to restore functional protein production to PTC mutations using read-through compounds, with the ultimate aim of CF patient treatment. The CFTRNMD construct used has the G542X nonsense mutation, mCherry at the N terminus and eGFP fused at the CFTR C-terminus. Through the screen of the library, we identified new small-molecule compounds that induced PTC read-through. To confirm the read-through efficiency future experiments are needed using additional techniques, such as, WB and transcript analysis by semi-quantitative PCR and qRT-PCR. Finally, to validate the top hit compounds, it is necessary to test them in patient’s materials, including nasal primary cells for functional activity and intestinal organoids to determine a dose response and to test these compounds in combination with potentiators and correctors. The studies presented in this dissertation had the overall aims of advancing the current knowledge on RNA-processing dysfunction and of identifying novel RNA modulator compounds towards a "personalised medicine" approach. The results obtained have indeed provided new insights into: 1) the relationship of other respiratory diseases with the presence of CFTR mutations; 2) new approaches to detect CFTR mutations in DNA and RNA; and 3) our understanding of key factors in NMD and read-through activity in relation to CFTR nonsense mutations

Novas aplicações de métodos multivariados na análise da mortalidade : um estudo na região Norte de Portugal, 2001-2005

A análise da mortalidade é fundamental no processo de planeamento da saúde e dos serviços de saúde, sendo a taxa de mortalidade padronizada pela idade um dos principais indicadores utilizados. A aplicação da Análise em Componentes Principais e da Análise de Clusters surge, neste trabalho, com o objectivo de identificar conjuntos de causas de morte que possam estar mais correlacionadas entre si e de reunir Agrupamentos de Centros de Saúde (ACES) segundo perfis de mortalidade semelhantes

Statistical analysis of health inequalities in the Northern Region of Portugal

Different public health needs demand differentiated interventions by the Health services in order to attain efficiency and thus reducing Health inequalities. The statistical model which was developed will allow the Public Health Services to conduct a diagnostic evaluation of Health inequalities, with a specification of the death causes and age groups that most contribute to those inequalities. It will also allow a prognostic evaluation by analyzing the impact of some interventions in reducing the relative gap in life expectancy, in terms of health gains

Albuminuria Reduction after High Dose of Vitamin D in Patients with Type 1 Diabetes Mellitus: A Pilot Study

BackgroundSome studies suggest an association between diabetic kidney disease (DKD) and vitamin D (VD), but there is no data about the effect of high dose of VD on DKD in type 1 diabetes mellitus (T1DM). Our pilot study aims to evaluate albuminuria reduction in patients with T1DM supplemented with high dose of VD.Methods22 patients received doses of 4,000 and 10,000 IU/day of cholecalciferol for 12 weeks according to patient’s previous VD levels. They were submitted to continuous glucose monitoring system, 24 hours ambulatory blood pressure monitoring and urine albumin-to-creatinine ratio before and after VD supplementation.ResultsThere was a reduction of DKD prevalence at the end of the study (68 vs 32%; p = 0.05), with no changes on insulin doses, glycated hemoglobin, glycemic variability and blood pressure values. A correlation between percentage variation of VD levels (ΔVD) and albuminuria at the end of the study was presented (r = −0.5; p < 0.05). Among T1DM patients with DKD at the beginning of the study, 8/13 (62%) had their DKD stage improved, while the other five ones (38%) showed no changes (p < 0.05).ConclusionOur pilot study suggests an association between VD high dose supplementation, lower prevalence and improvement in stages of DKD in T1DM

Canagliflozin and renal outcomes in type 2 diabetes and nephropathy

BACKGROUND Type 2 diabetes mellitus is the leading cause of kidney failure worldwide, but few effective long-term treatments are available. In cardiovascular trials of inhibitors of sodium–glucose cotransporter 2 (SGLT2), exploratory results have suggested that such drugs may improve renal outcomes in patients with type 2 diabetes. METHODS In this double-blind, randomized trial, we assigned patients with type 2 diabetes and albuminuric chronic kidney disease to receive canagliflozin, an oral SGLT2 inhibitor, at a dose of 100 mg daily or placebo. All the patients had an estimated glomerular filtration rate (GFR) of 30 to <90 ml per minute per 1.73 m2 of body-surface area and albuminuria (ratio of albumin [mg] to creatinine [g], >300 to 5000) and were treated with renin–angiotensin system blockade. The primary outcome was a composite of end-stage kidney disease (dialysis, transplantation, or a sustained estimated GFR of <15 ml per minute per 1.73 m2), a doubling of the serum creatinine level, or death from renal or cardiovascular causes. Prespecified secondary outcomes were tested hierarchically. RESULTS The trial was stopped early after a planned interim analysis on the recommendation of the data and safety monitoring committee. At that time, 4401 patients had undergone randomization, with a median follow-up of 2.62 years. The relative risk of the primary outcome was 30% lower in the canagliflozin group than in the placebo group, with event rates of 43.2 and 61.2 per 1000 patient-years, respectively (hazard ratio, 0.70; 95% confidence interval [CI], 0.59 to 0.82; P=0.00001). The relative risk of the renal-specific composite of end-stage kidney disease, a doubling of the creatinine level, or death from renal causes was lower by 34% (hazard ratio, 0.66; 95% CI, 0.53 to 0.81; P<0.001), and the relative risk of end-stage kidney disease was lower by 32% (hazard ratio, 0.68; 95% CI, 0.54 to 0.86; P=0.002). The canagliflozin group also had a lower risk of cardiovascular death, myocardial infarction, or stroke (hazard ratio, 0.80; 95% CI, 0.67 to 0.95; P=0.01) and hospitalization for heart failure (hazard ratio, 0.61; 95% CI, 0.47 to 0.80; P<0.001). There were no significant differences in rates of amputation or fracture. CONCLUSIONS In patients with type 2 diabetes and kidney disease, the risk of kidney failure and cardiovascular events was lower in the canagliflozin group than in the placebo group at a median follow-up of 2.62 years

Novas aplicações de métodos multivariados na análise da mortalidade: um estudo na região norte de Portugal, 2001-2005

A análise da mortalidade é fundamental no processo de planeamento da saúde e dos serviços de saúde, sendo a taxa de mortalidade padronizada pela idade um dos principais indicadores utilizados. A aplicação da Análise em Componentes Principais e da Análise de Clusters surge, neste trabalho, com o objectivo de identificar conjuntos de causas de morte que possam estar mais correlacionadas entre si e de reunir Agrupamentos de Centros de Saúde segundo perfis de mortalidade semelhantes.Este trabalho foi parcialmente financiado pelo Centro de Matemática da Universidade do Minho por Fundos Nacionais através da FCT - “Fundação para a Ciência e a Tecnologia”, no âmbito do projecto PEstOE/MAT/UI0013/2014.info:eu-repo/semantics/publishedVersio

Saúde da população da região norte: construção e análise de indicadores

A Saúde é um fator muito importante no dia-a-dia de uma pessoa, por isso é essencial estar informado sobre ela. O conhecimento da Saúde de uma população é fundamental para uma melhor intervenção dos serviços de Saúde e afetação dos seus recursos. Existem diversos indicadores de Saúde e com a ajuda destes é possível ter essa informação. O principal objetivo deste trabalho foi recolher, construir, organizar e analisar indicadores de saúde da população e seus determinantes.Fundação para a Ciência e a Tecnologia (FCT

A parish level study in the northern region of Portugal

Introduction: Diabetes and hypertension are highly prevalent conditions in Portugal. Little is known about the geographical and social patterning of these diseases, which precludes the design of targeted health policies. This study aimed to measure the geographical and socioeconomic distribution of type 2 diabetes and hypertension prevalence in the population resident in the Northern region of Portugal, for the year 2013. Material and Methods: An ecological correlation study analyzed the 2,028 parishes of the region. Prevalence data were obtained from the Regional Health Administration information system. Socioeconomic data were also obtained from this administrative database and from the 2011 national census. The association between each socioeconomic indicator and age-standardized prevalence was measured using the difference in prevalence, population attributable risk, relative inequality index, and regression coefficient. Results: The prevalence of type 2 diabetes and hypertension was 6.16% and 19.35%, respectively, and varied across parishes. These prevalences were significantly associated with low educational level, low tertiary sector weight, unemployment, and low-income rate (with prevalence differences between the most and least advantaged deciles up to 1.3% and 5.3%, respectively). Socioeconomic factors accounted for up to 20% of prevalence. Discussion: This study design did not allow us to evaluate causality and it may underestimate these diseases prevalence or its association with socioeconomic factors, but its results are in line with the evidence from other countries. Conclusion: These results emphasize the socioeconomic and geographical patterning of major diseases associated with a high mortality, and the need of health policies targeting the most deprived parishes.publishe

The Ankle-Arm Index As a Predictive Risk Factor For Peripheral Arterial Disease In Patients With In Patients With Type 2 Diabetes Mellitus

Background: Peripheral arterial disease in patients with type 2 diabetes mellitus is an important risk factor for vascular events. Recommendations about whether ankle-brachial index should be performed differ depending on the source; therefore, it is necessary to re-evaluate the most important risk factors associated with peripheral arterial disease and whether it is useful to perform ankle-brachial index in newly diagnosed and drug-naïve patients with diabetes, independent of age or peripheral arterial disease symptoms. Methods: A total of 711 subjects were divided into groups: group 1, 600 type 2 diabetes mellitus patients, symptomatic or not for peripheral arterial disease; group 2, 61 type 2 diabetes mellitus patients newly diagnosed and drug naïve; and group 3, 50 subjects without diabetes. Ankle-brachial index, medical records and physical examination were performed in all patients, accessing cardiovascular risk factors. Results: Analysing group 1 asymptomatic patient to peripheral arterial disease, we found abnormal ankle-brachial index in 49% (77/156) ⩾50years and 42% (16/38) <50years (p=not significant). Considering drug-naïve patients, a peripheral arterial disease prevalence of 39% (24/61) was found; among these, 48% (13/27) were <50years and 32% (11/34) were ⩾50years (p=not significant). A forward stepwise regression model was developed, with type 2 diabetes mellitus duration (r2=0.12) and sedentary lifestyle (r2=0.14) found as independent variable predictors of severity of peripheral arterial disease, related to ankle-brachial index. Conclusion: We suggest that, in type 2 diabetes mellitus, ankle-brachial index should be measured at diagnosis. In addition, sedentary lifestyle was strongly associated with presence and severity of peripheral arterial disease

Cochlear dysfunction and microvascular complications in patients with type 1 diabetes mellitus

Abstract Sensorineural hearing impairment has been associated with DM, and it is probably linked to the same pathophysiological mechanisms as well-established in microvascular diabetes complications. The study of otoacoustic emissions (OAEs) is useful to identify subclinical cochlear dysfunction. Therefore, the aim of this study was to evaluate the association between abnormal OAEs responses, diabetic kidney disease (DKD) and diabetic cardiac autonomic neuropathy (CAN). We performed a cross-sectional study with 37 type 1 DM patients without auditory symptoms, submitted to the study of Distortion Product Otoacoustic Emissions (DPOAEs) and screened for DKD and CAN. The otoacoustic emissions responses were considered abnormal in 27/37 (73%) patients. A correlation was found between abnormal OAEs responses and presence of DKD (r = 0.36, p < 0.05), and 14/16 (88%) patients with a lower amplitude of OAEs in 8 kHz frequency band presented DKD. Abnormal OAEs responses in the 6 kHz frequency band were correlated with the presence (r = 0.41, p = 0.01) and severity of CAN (r = 0.44, p < 0.001). Additionally, 7/9 (78%) patients with abnormal OAE responses in this frequency also presented abnormal CAN scores. Our results suggest that abnormal otoacoustic emissions responses in high frequency bands are associated with diabetes microvascular complications and could be a risk marker for DKD and CAN, presenting low sensitivity and high specificity. Therefore, assuming that hearing impairment is a pre-clinical stage of hearing loss, performing distortion product otoacoustic emissions in T1DM patients with microvascular complications could be useful to identify those who would be benefit with regular audiologic follow up and tighter diabetes control