Optimization of statistical and bioinformatic methods for the analysis of next generation sequencing data for rare disease diagnosis

The main focus of this thesis, presented as a compendium of research articles, is the optimization of the analysis of Next Generation Sequencing data in order to facilitate the diagnosis of rare diseases. For this goal, we present an appropach to prioritize single nucleotide variants and small insertions and deletions, not only in terms of their type and genomic position, but also in terms of the mutational tolerance of the gene encompassing them. We also evaluate the strengths and weakness of the currently published copy number variation (CNV) detection tools, and develop a methodology to create sinthetic samples with artificial CNVs to test them. Finally, we present a novel CNV-detection program, optimized for gene panel assays

Retrospective study to identify risk factors for chronic kidney disease in children with congenital solitary functioning kidney detected by neonatal renal ultrasound screening

To evaluate the prognostic significance of factors frequently associated with a reduction in renal mass, such as prematurity, low birth weight, and congenital anomalies of kidney and urinary tract (CAKUT), in patients with solitary functioning kidney (SFK) and investigate signs of early renal injury due to glomerular hyperfiltration damage or dysplasia in the remaining kidney. Retrospective observational study of congenital SFK diagnosed and followed at a tertiary care hospital over a period of 10 years in which 32,900 newborns underwent routine neonatal abdominal ultrasound screening. We analyzed age at diagnosis, sex, gestational age, anthropometric measurements at birth and prenatal and neonatal ultrasound findings, in addition to follow-up data corresponding to imaging findings (ultrasound, micturating cystourethrography, dimercaptosuccinic acid renal, and scintigraphy), ipsilateral CAKUT, compensatory hypertrophy, and renal injury in the form of albuminuria, blood pressure, and estimated glomerular filtration rate (eGFR). In total, 128 congenital SFK cases were detected (1 per 257 live births). Of these, 117 (91.4%) were diagnosed by neonatal ultrasound screening and 44.5% of these had been previously identified by prenatal ultrasound. Neonatal ultrasound had a specificity of 100% and a sensitivity of 92.1%. Forty-five patients (35.2%) had ipsilateral CAKUT and the most common type was urinary collecting system anomalies (75.5%). Over a median follow-up of 6.3 years (1?10 years), compensatory renal hypertrophy was observed in 81 patients (63.7%), most of whom had ipsilateral CAKUT (76.1% vs 56.6% of patients without ipsilateral CAKUT). Albuminuria and hypertension were observed in 3.12% and 5% of patients, respectively, and both were associated with ipsilateral CAKUT (P<.05). In addition, 75% of albuminuria cases (P=.031), 83.3% of hypertension cases (P=.004), and 100% of decreased eGFRcases (P=.031) were significantly associated with CAKUT (renal parenchymal anomaly category), being the strongest predictor of GFR the presence or absence of CAKUT. Neonatal ultrasound screening is useful for the early diagnosis of SFK. The presence of ipsilateral CAKUT should be evaluated in all patients with SFK as congenital anomalies of the renal parenchyma are associated with a poorer prognosis. Because morbidity from CAKUTs may not develop until adulthood, patients should be closely followed throughout life

Similarities between acylcarnitine profiles in large for gestational age newborns and obesity

Large for gestational age (LGA) newborns have an increased risk of obesity, insulin resistance, and metabolic syndrome. Acylcarnitine profiles in obese children and adults are characterized by increased levels of C3, C5, and certain medium-chain (C12) and long-chain (C14:1 and C16) acylcarnitines. C2 is also increased in insulin-resistant states. In this 1-year observational study of 2514 newborns (246 LGA newborns, 250 small for gestational age (GA) newborns, and 2018 appropriate for GA newborns), we analyzed and compared postnatal acylcarnitine profiles in LGA newborns with profiles described for obese individuals. Acylcarnitine analysis was performed by tandem mass spectrometry on dried-blood spots collected on day 3 of life. LGA newborns had higher levels of total short-chain acylcarnitines (p < 0.001), C2 (p < 0.01) and C3 (p < 0.001) acylcarnitines, and all C12, C14, and C16 acylcarnitines except C12:1. They also had a higher tendency towards carnitine insufficiency (p < 0.05) and carnitine deficiency (p < 0.001). No significant differences were observed between LGA newborns born to mothers with or without a history of gestational diabetes. This novel study describes a postnatal acylcarnitine profile in LGA with higher levels of C2, C3, total acylcarnitines, and total short-chain acylcarnitines that is characteristic of childhood and adult obesity and linked to an unhealthy metabolic phenotype.Dr Lopez has received funding from the European Community’s Seventh Framework Programme (FP7/2007-2013) under grant agreement no. 281854 -the ObERStress project, Xunta de Galicia (2015-CP079 and 2016-PG068), MINECO co-funded by FEDER (SAF2015-71026-R and BFU2015-70454-REDT/Adipoplast)S

Molecular-genetic characterization and rescue of a TSFM mutation causing childhood-onset ataxia and nonobstructive cardiomyopathy

Oxidative phosphorylation dysfunction has been found in many different disorders. This biochemical pathway depends on mitochondrial protein synthesis. Thus, mutations in components of the mitochondrial translation system can be responsible for some of these pathologies. We identified a new homozygous missense mutation in the mitochondrial translation elongation factor Ts gene in a patient suffering from slowly progressive childhood ataxia and hypertrophic cardiomyopathy. Using cell, biochemical and molecular-genetic protocols, we confirm it as the etiologic factor of this phenotype. Moreover, as an important functional confirmation, we rescued the normal molecular phenotype by expression of the wild-type TSFM cDNA in patient''s fibroblasts. Different TSFM mutations can produce the same or very different clinical phenotypes, going from abortions to moderately severe presentations. On the other hand, the same TSFM mutation can also produce same or different phenotypes within the same range of presentations, therefore suggesting the involvement of unknown factors

Diplopia Is Frequent and Associated with Motor and Non-Motor Severity in Parkinson’s Disease: Results from the COPPADIS Cohort at 2-Year Follow-Up

[Background and objective] Diplopia is relatively common in Parkinson’s disease (PD) but is still understudied. Our aim was to analyze the frequency of diplopia in PD patients from a multicenter Spanish cohort, to compare the frequency with a control group, and to identify factors associated with it.[Patients and Methods] PD patients who were recruited from January 2016 to November 2017 (baseline visit; V0) and evaluated again at a 2-year ± 30 days follow-up (V2) from 35 centers of Spain from the COPPADIS cohort were included in this longitudinal prospective study. The patients and controls were classified as “with diplopia” or “without diplopia” according to item 15 of the Non-Motor Symptoms Scale (NMSS) at V0, V1 (1-year ± 15 days), and V2 for the patients and at V0 and V2 for the controls.[Results] The frequency of diplopia in the PD patients was 13.6% (94/691) at V0 (1.9% in controls [4/206]; p < 0.0001), 14.2% (86/604) at V1, and 17.1% (86/502) at V2 (0.8% in controls [1/124]; p < 0.0001), with a period prevalence of 24.9% (120/481). Visual hallucinations at any visit from V0 to V2 (OR = 2.264; 95%CI, 1.269–4.039; p = 0.006), a higher score on the NMSS at V0 (OR = 1.009; 95%CI, 1.012–1.024; p = 0.015), and a greater increase from V0 to V2 on the Unified Parkinson’s Disease Rating Scale–III (OR = 1.039; 95%CI, 1.023–1.083; p < 0.0001) and Neuropsychiatric Inventory (OR = 1.028; 95%CI, 1.001–1.057; p = 0.049) scores were independent factors associated with diplopia (R2 = 0.25; Hosmer and Lemeshow test, p = 0.716).[Conclusions] Diplopia represents a frequent symptom in PD patients and is associated with motor and non-motor severity.Martínez-Martin P. has received honoraria from National School of Public Health (ISCIII), Editori-al Viguera and Takeda Pharmaceuticals for lecturing in courses, and from the International Parkinson and Movement Disorder Society (MDS) for management of the Program on Rating Scales. Mir P. has received honoraria from AbbVie, Abbott, Allergan, Bial, Merz, UCB, and Zambon and have received grants from the Spanish Ministry of Economy and Competitiveness [PI16/01575], co-founded by ISCIII (Subdirección General de Evaluación y Fomento de la Investigación) and by Fondo Europeo de Desarrollo Regional (FEDER), the Consejería de Economía, Innovación, Ciencia y Empleo de la Junta de Andalucía [CVI-02526, CTS-7685], the Consejería de Salud y Bienestar Social de la Junta de Andalucía [ PI-0437-2012, PI-0471-2013], the Sociedad Andaluza de Neurología, the Jacques and Gloria Gossweiler Foundation, the Fundación Alicia Koplowitz, the Fundación Mutua Madrileña.Peer reviewe

Predictors of Loss of Functional Independence in Parkinson’s Disease: Results from the COPPADIS Cohort at 2-Year Follow-Up and Comparison with a Control Group

COPPADIS Study Group.[Background and objective] The aim of this study was to compare the progression of independence in activities of daily living (ADL) in Parkinson’s disease (PD) patients versus a control group, as well as to identify predictors of disability progression and functional dependency (FD).[Patients and Methods] PD patients and control subjects, who were recruited from 35 centers of Spain from the COPPADIS cohort between January 2016 and November 2017 (V0), were included. Patients and subjects were then evaluated again at the 2-year follow-up (V2). Disability was assessed with the Schwab & England Activities of Daily Living Scale (S&E-ADLS) at V0 and V2. FD was defined as an S&E-ADLS score less than 80%.[Results] In the PD group, a significant decrease in the S&E-ADLS score from V0 to V2 (N = 507; from 88.58 ± 10.19 to 84.26 ± 13.38; p < 0.0001; Cohen’s effect size = −0.519) was observed but not in controls (N = 124; from 98.87 ± 6.52 to 99.52 ± 2.15; p = 0.238). When only patients considered functional independent at baseline were included, 55 out of 463 (11.9%) converted to functional dependent at V2. To be a female (OR = 2.908; p = 0.009), have longer disease duration (OR = 1.152; p = 0.002), have a non-tremoric motor phenotype at baseline (OR = 3.574; p = 0.004), have a higher score at baseline in FOGQ (OR = 1.244; p < 0.0001) and BDI-II (OR = 1.080; p = 0.008), have a lower score at baseline in PD-CRS (OR = 0.963; p = 0.008), and have a greater increase in the score from V0 to V2 in UPDRS-IV (OR = 1.168; p = 0.0.29), FOGQ (OR = 1.348; p < 0.0001) and VAFS-Mental (OR = 1.177; p = 0.013) (adjusted R-squared 0.52; Hosmer and Lemeshow test = 0.94) were all found to be independent predictors of FD at V2.[Conclusions] In conclusion, autonomy for ADL worsens in PD patients compared to controls. Cognitive impairment, gait problems, fatigue, depressive symptoms, more advanced disease, and a non-tremor phenotype are independent predictors of FD in the short-term.Fundación Curemos el Parkinson (www.curemoselparkinson.org).Peer reviewe

Prioritization of Variants Detected by Next Generation Sequencing According to the Mutation Tolerance and Mutational Architecture of the Corresponding Genes

The biggest challenge geneticists face when applying next-generation sequencing technology to the diagnosis of rare diseases is determining which rare variants, from the dozens or hundreds detected, are potentially implicated in the patient&rsquo;s phenotype. Thus, variant prioritization is an essential step in the process of rare disease diagnosis. In addition to conducting the usual in-silico analyses to predict variant pathogenicity (based on nucleotide/amino-acid conservation and the differences between the physicochemical features of the amino-acid change), three important concepts should be borne in mind. The first is the &ldquo;mutation tolerance&rdquo; of the genes in which variants are located. This describes the susceptibility of a given gene to any functional mutation and depends on the strength of purifying selection acting against it. The second is the &ldquo;mutational architecture&rdquo; of each gene. This describes the type and location of mutations previously identified in the gene, and their association with different phenotypes or degrees of severity. The third is the mode of inheritance (inherited vs. de novo) of the variants detected. Here, we discuss the importance of each of these concepts for variant prioritization in the diagnosis of rare diseases. Using real data, we show how genes, rather than variants, can be prioritized by calculating a gene-specific mutation tolerance score. We also illustrate the influence of mutational architecture on variant prioritization using five paradigmatic examples. Finally, we discuss the importance of familial variant analysis as final step in variant prioritization

Fomento del desarrollo sostenible a través de la educación financiera. Un estudio práctico a través de un proyecto de Aprendizaje-Servicio

[Resumen]: La toma de decisiones de carácter económico acompaña nuestros pasos prácticamente a diario, razón por la que la educación financiera desempeña un papel fundamental en nuestro bienestar individual y colectivo, siendo esencial para lograr un desarrollo sostenible. En las últimas décadas diversos organismos multilaterales, encabezados por la OCDE, han mostrado una creciente preocupación por esta materia conscientes de su importancia y sus beneficios. Esta circunstancia ha dado lugar al lanzamiento de multitud de iniciativas en decenas de países con las que se pretende sensibilizar a la población sobre la importancia de contar con una base sólida de conceptos financieros y hacer llegar a los ciudadanos las herramientas necesarias para adquirir dicha base. Este Trabajo de Fin de Grado se enmarca en un proyecto de Aprendizaje-Servicio (ApS) con el que se pretende analizar, por un lado, el nivel de conocimientos financieros de un grupo de estudiantes de Educación Secundaria tras haber visionado una serie de vídeos cortos en los que se explican de forma teórica-práctica un conjunto de conceptos económicos que les serán muy útiles a lo largo de su vida y, por otro lado, las características sociodemográficas que pueden influir en su alfabetización financiera. Asimismo, también se plantean una serie de cuestiones acerca de su comportamiento financiero, a fin de detectar cómo es y en qué aspectos se debería hacer hincapié. En una sociedad cada más digitalizada, la educación en diferido, mediante vídeos y herramientas accesibles en la red, se encuentra a la orden del día. Esta circunstancia es la motivadora de la comparativa que se efectúa en la parte final de este proyecto entre los resultados obtenidos por los estudiantes en la formación realizada con los resultados conseguidos en una formación idéntica a esta pero realizada presencialmente en las aulas.[Abstract]: The decision-making process in economic matters accompanies our steps almost every day, which is why financial education plays a fundamental role in our individual and collective well-being, and it’s essential to achieve sustainable development. In recent decades, various multilateral organisations, led by the OECD, have shown a growing concern for this issue, aware of its importance and benefits. This circumstance has led to the launch of a multitude of initiatives in dozens of countries aimed at raising awareness among the population about the importance of having a solid foundation in financial concepts and providing citizens with the necessary tools to acquire such knowledge. This Final Degree Project is part of a Service-Learning project (SL) which aims to analyse, on the one hand, the level of financial knowledge of a group of Secondary Education students after having watched a series of short videos in which a set of economic concepts that will be very useful to them throughout their lives are explained in a theoretical-practical way; and, on the other hand, the socio-demographic characteristics that may influence their financial literacy. It also raises a series of questions about their financial behaviour, to identify their current practices and highlight aspects thar should be emphasised. In an increasingly digitalized society, education on demand through videos and tools accessible on the Internet is commonplace. This is the reason for the comparison made in the final part of this project between the results obtained by students in the training course using this method and the results achieved in the same training course but carried out in the classroom.Traballo fin de grao (UDC.ECO). ADE. Curso 2022/202

Prioritization of Variants Detected by Next Generation Sequencing According to the Mutation Tolerance and Mutational Architecture of the Corresponding Genes

The biggest challenge geneticists face when applying next-generation sequencing technology to the diagnosis of rare diseases is determining which rare variants, from the dozens or hundreds detected, are potentially implicated in the patient&rsquo;s phenotype. Thus, variant prioritization is an essential step in the process of rare disease diagnosis. In addition to conducting the usual in-silico analyses to predict variant pathogenicity (based on nucleotide/amino-acid conservation and the differences between the physicochemical features of the amino-acid change), three important concepts should be borne in mind. The first is the &ldquo;mutation tolerance&rdquo; of the genes in which variants are located. This describes the susceptibility of a given gene to any functional mutation and depends on the strength of purifying selection acting against it. The second is the &ldquo;mutational architecture&rdquo; of each gene. This describes the type and location of mutations previously identified in the gene, and their association with different phenotypes or degrees of severity. The third is the mode of inheritance (inherited vs. de novo) of the variants detected. Here, we discuss the importance of each of these concepts for variant prioritization in the diagnosis of rare diseases. Using real data, we show how genes, rather than variants, can be prioritized by calculating a gene-specific mutation tolerance score. We also illustrate the influence of mutational architecture on variant prioritization using five paradigmatic examples. Finally, we discuss the importance of familial variant analysis as final step in variant prioritization