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1
Tran, Grace. "Parents’ Perspectives: Child’s Whole Exome Sequencing (WES) Research Results of Uncertain Significance". University of Cincinnati / OhioLINK, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1397234121.
Testo completo
2
Kause-Zriouil, Franziska [Verfasser]. "Systematische Identifizierung von Krankheitsgenen für intestinale und urogenitale Fehlbildungen mittels „Whole Exome Sequencing“ (WES) / Franziska Kause-Zriouil". Bonn : Universitäts- und Landesbibliothek Bonn, 2021. http://d-nb.info/1239729774/34.
Testo completo
3
PELUSI, SERENA. "IMPACT OF WHOLE EXOME SEQUENCING (WES) ON THE CLINICAL MANAGEMENT OF PATIENTS WITH ADVANCED NONALCOHOLIC FATTY LIVER (NAFL)". Doctoral thesis, Università degli Studi di Milano, 2021. http://hdl.handle.net/2434/827810.
Testo completoAbstract (sommario):
Abstract
Non-alcoholic fatty liver disease (NAFLD) is a potentially progressive disorder, possibily leading to cirrhosis and hepatocellular carcinoma (HCC).
Both acquired and genetic risk factors play an important role in disease progression. In this respect, the presence of metabolic comorbidities such as obesity and type two diabetes mellitus has been associated to a more severe phenotype, while genetic factors modulating hepatic lipid metabolism such as the variants in PNPLA3 (patatin-like phospholipase domain-containing protein 3), TM6SF2 (transmembrane 6 superfamily member 2), MBOAT7 (membrane bound O-acyltranferase domain containing 7), GCKR (glucokinase regulatory protein) and APOB (apolipoprotein B) have been demostrated to influence disease predisposition.
Secondly, a consistent fraction of patients with liver disorders are diagnosed at advanced stage and in approximately one third of cases it is not possible to establish an etiological diagnosis (cryptogenic cirrhosis).
By exploiting Whole Exome Sequencing (WES) technology, in this work we aimed to:
1) identify the prevalence of known pathogenic mutations in candidate genes mutated in genetic liver diseases and hereditary cancer syndromes in patients with HCC and cirrhosis due to NAFLD or cryptogenic disease, to compare it to that of healthy individuals and to evaluate in the aforementioned genes the burden of rare or novel mutations of unknown significance, that determine an alteration of protein sequence, and are therefore likely pathogenic;
2) test the functional and clinical significance of the mutations identified, by bioinformatics algorithms and in vitro assays;
3) identify novel genetic risk factors predisposing to progressive NAFLD;
4) implement a genetic risk score (GRS) aiming to improve the stratification of the risk of progressive NAFLD;
5) test the impact of WES on the clinical management of six patients with cryptogenic liver disease.
Briefly, we detected an enrichment in pathogenic (p = 0.024), and likely pathogenic variants (p = 1.9*10−6), particularly in APOB (p = 0.047). APOB variants were associated with lower circulating triglycerides and higher HDL cholesterol (p < 0.01). A genetic risk score predicted NAFLD-HCC (OR 4.96, 3.29–7.55; p = 5.1*10−16), outperforming the diagnostic accuracy
of common genetic risk variants, and of clinical risk factors (p < 0.05).
Furthermore, in one third of the six patients with cryptogenic liver disease and aggressive phenotype it was possible to identify a probable genetic disorder expleining the phenotype.
In conclusion, rare pathogenic variants in genes involved in liver disease and cancer predisposition are associated with NAFLD-HCC development.
Genetic study by WES can represent a precious instrument for risk stratification and allow a Precision Medicine approach in the context of liver disease aimed at the targeted treatment of the underlying cause of the disease.
4
Mastantuono, Elisa [Verfasser], Thomas [Akademischer Betreuer] Meitinger, Heribert [Gutachter] Schunkert e Karl-Ludwig [Gutachter] Laugwitz. "Whole exome sequencing (WES) to elucidate the molecular basis of cardiac disease / Elisa Mastantuono ; Gutachter: Heribert Schunkert, Karl-Ludwig Laugwitz ; Betreuer: Thomas Meitinger". München : Universitätsbibliothek der TU München, 2018. http://d-nb.info/1169825559/34.
Testo completo
5
Licchetta, Laura <1981>. "In-Depth Clinical, Genetic and Neuropsychological Study of Familial and Sporadic Cases with Sleep-Related Hypermotor Epilepsy (SHE): Identification of New Genes by Whole Exome Sequencing (WES)". Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2017. http://amsdottorato.unibo.it/8079/1/tesi%20PhD%20_licchetta.pdf.
Testo completoAbstract (sommario):
Sleep-related Hypermotor Epilepsy (SHE) is a genetically heterogeneous epilepsy syndrome. Differences in epilepsy phenotype/endophenotype were associated with mutations in specific genes. However, the genes identified so far cumulatively account for 25% of cases. Moreover, systematic neuropsychological investigations on comprehensive SHE cohorts are lacking.
This study provides an accurate clinical, genetic and neuropsychological characterization of a large cohort of patients diagnosed with SHE according to reliable diagnostic criteria. A subgroup underwent a preliminary screening, partly performed by dHPLC to exclude mutations in CHRNA4, CHRNB2, CHRNA2.
Using a number of genetic strategies, we identified causative mutations in 10.4% of our cases. The mutation frequencies were 2.3% for KCNT1 (CI: 0.3-8.1%), 5.9% for GATOR1-complex genes (CI: 2.0-13.3%), 3.1% for CHRNA4 (CI: 0.6-8.8%) and 1.7% for SCN1A (CI 0-8.9%).
WES analysis allowed the identification of a novel epilepsy gene, NPRL2, coding a component of GATOR1, a negative regulator of mTOR pathway. Altogether, mutations in the GATOR1 complex genes DEPDC5 and NPRL2 account for the majority of our cases (6%). Genotype-phenotype correlations confirmed their association with focal cortical dysplasia, higher rates of drug-resistance, aura and seizures in wakefulness, with relevant implications in diagnostic and treatment strategies.
Moreover, we confirm that mutations in KCNT1 are implicated in severe forms with intellectual disability and psychiatric disorders.
The unexpected detection of CHRNA4 mutations highlighted the low sensitivity of dHPLC assay.
The systematic neuropsychological study showed that neurocognitive deficits are not uncommon in SHE. Mutated patients scored significantly lower at WAIS, supporting a crucial role of genetics in cognitive deficit by different biological mechanisms and molecular pathways. About 47% of patients of normal intelligence showed some degree of cognitive dysfunction. The profile of neuropsychological impairment was characterized by significant worse scores in verbal IQ, deficits in memory and in selected executive functions, with preserved shifting abilities and cognitive flexibility.
6
Agatea, Lisa. "An integrated proteomic and genomic approach to study FAP patients without APC and MutHY mutations". Doctoral thesis, Università degli studi di Padova, 2016. http://hdl.handle.net/11577/3424509.
Testo completoAbstract (sommario):
Familial Adenomatous Polyposis (FAP) is one of the most important clinical forms of inherited susceptibility to colorectal cancer, that is characterized by the development of hundreds to thousands of adenomas in the colon and rectum during the second decade of life. FAP is due to a germline mutation in the APC gene or to biallelic variations of MutYH gene. Almost all patients will develop cancer if the disease is not identified and surgically treated at an early stage.
The aim of this study was to characterize, by peptidomic and genetic approaches, 4 pa-tients that, although at the colonoscopy showed many polyps, they did not present any mutations of APC and MutYH genes (defined here unresolved FAP).
Regarding the peptidomic study, MALDI-TOF analysis was performed on mutated and unresolved FAP patients. These data were compared with the one from adenoma patients, CRC patients and healthy control subjects. The peptide fingerprint of mutated FAP patients was obtained after performing statistical analysis. A subset of 45 ionic species was found differently expressed in the four groups considered, 12 of them peculiar of FAP patients. Four ionic species were found significantly different in the switch between adenoma and malignant carcinoma. In this study, the potentially prognostic peptides identified derive mainly from circulating proteins and some of them are involved in the inflammatory response. In particular, proteins such as Complement C3 and C4 are known to be cleaved by exoproteases that seem pathology-related.
In the case of unresolved FAP patients, in order to better define a specific pattern, the data from MALDI-TOF were combined with whole exome sequencing. The peptidomics data clearly mark a substantial difference between mutated and unresolved FAP patients. Indeed, unresolved FAP patients have characteristics similar to the control subjects, adenoma patients, CRC patients but not to mutated FAP patients. To understand the possible molecular pathway involved in the unresolved FAP cases, the whole exome sequencing (WES) was performed. From WES data analysis, 285 genes present in all the four unresolved FAP patients were filtered and selected. Among them, the O-linked glycans pathway of the mucins was the most represented.
In conclusion, in this study it was defined for the first time a specific panel of peptides for mutated FAP patients, that could be useful to monitor and predict the pathological evolution of adenocarcinoma malignancy. Furthermore, it was possible to characterize a preliminary genetic variations pattern for unresolved FAP patients, in which mucin genes might represent the key of the molecular pathway involved.
However, further study are necessary to relate the identified mucin gene variations to their possible causative role in the polyposis. Future analysis of this pattern will be helpful, indeed, to better understand the interatome (the biological network that in-cludes the whole set of direct and indirect molecular interactions in a cell) of these un-resolved FAP patients.La poliposi adenomatosa familiare (FAP) è una delle più importanti forme cliniche di cancro colo-rettale ereditario ed è caratterizzata dallo sviluppo di centinaia/migliaia di polipi adenomatosi nel colon e nel retto durante la seconda decade di vita. La FAP è causata da una mutazione germinale del gene APC o da varianti bialleliche del gene MutYH. Quasi tutti i pazienti FAP sviluppano il cancro se la patologia non viene precocemente identificata e trattata chirurgicamente. Lo scopo di questo lavoro è stato caratterizzare 4 pazienti in cui, nonostante l’esame colonscopico presentasse una poliposi conclamata, non risultavano mutazioni nei gene APC e MutYH (in questa tesi definiti pazienti FAP irrisolti) utilizzando un approccio integrato di peptidomica e genomica. Riguardo la peptidomica, il MALDI-TOF è stato utilizzato per studiare il profilo peptidico plasmatico di pazienti FAP mutati ed irrisolti comparando i dati ottenuti con quelli derivanti dallo studio di pazienti con adenoma, cancro colo-rettale e soggetti sani di controllo. Dopo analisi statistica è stato ottenuto il fingerprint peptidico dei pazienti FAP mutati. Sono state ottenute 45 specie ioniche differentemente espresse nei quattro gruppi considerati, 12 delle quali peculiari per i pazienti FAP. L’intensità di segnale di quattro di queste specie ioniche è stata trovata statisticamente alterata nello switch tra adenoma e carcinoma maligno. I peptidi potenzialmente prognostici identificati in questo studio derivano principalmente da proteine circolanti, alcune delle quali implicate nella risposta infiammatoria. In particolare è noto dalla letteratura che proteine del sistema del complemento come C3 e C4 vengono tagliate da esoproteasi che sembrano essere patologia correlate. Riguardo ai pazienti FAP irrisolti, per definirne un pattern specifico, i dati derivanti dall’analisi con il MALDI-TOF sono stati combinati con quelli ottenuti dal sequenzia-mento dell’esoma. I dati di peptidomica hanno chiaramente evidenziato le differenze tra pazienti FAP mutati e FAP irrisolti. Infatti i pazienti FAP irrisolti presentano caratteristiche simili a quelle dei soggetti di controllo, dei pazienti con adenoma e cancro colo rettale ma non a quelle dei pazienti FAP mutati. Allo scopo di capire la via di trasduzione del segnale implicata, è stato quindi eseguito il sequenziamento dell'esoma dei pazienti FAP irrisolti. Da questa analisi sono stati selezionati 285 geni variati in tutti i pazienti e tra questi la via di trasduzione del segnale della O-glicosilazione delle mucine è risultata la più rappresentata. In conclusione, in questo studio è stato definito per la prima volta un set peptidico specifico per i pazienti FAP mutati che potrebbe essere utilizzato per monitorare e predire l’evoluzione patologica della malattia. Inoltre è stato possibile caratterizzare un pattern preliminare per i pazienti FAP irrisolti in cui i geni delle mucine potrebbero rappresentare la chiave della via di trasduzione del segnale implicata. Ulteriori studi saranno necessari per correlare i geni delle mucine con la poliposi e costruire l'interatoma (network biologico definito come l’insieme di tutte le interazioni molecolari dirette e indirette che ci sono all'interno di una cellula e di un organismo) di questi pazienti FAP irrisolti.
7
Bhatia, Sugandha. "EMT & MET: Underpinning the phenotypic plasticity and chemoresistance in breast cancer". Thesis, Queensland University of Technology, 2020. https://eprints.qut.edu.au/180913/1/Sugandha_Bhatia_Thesis.pdf.
Testo completoAbstract (sommario):
This dissertation aims to identify the functional characteristics and genetic factors present within breast cancers that contribute to intratumoural heterogeneity and therapy resistance. The study utilises breast cancer cell line model systems to address epithelial-mesenchymal plasticity (EMP) at the cellular and functional level and underpins its role in cancer biology and chemoresistance. This research also interrogates the EMP programme in single cell-generated clones and through shRNA mediated functional drug screening assay identifies inhibitors that provides significant synergistic drug combinations. A comprehensive review of the drugs that can clinically target EMP was also consolidated.
8
Spracklen, Timothy. "Whole-exome sequencing of cases with familial cardiomyopathy". Doctoral thesis, Faculty of Health Sciences, 2021. http://hdl.handle.net/11427/33999.
Testo completoAbstract (sommario):
Introduction: Cardiomyopathies are disorders of the myocardium that can lead to heart failure, arrhythmias and sudden death. Heritable forms include dilated, hypertrophic and arrhythmogenic cardiomyopathy (DCM, HCM and ACM respectively). As heterogeneous disorders, over 50 genes have been implicated in these cardiomyopathies to date. However, the yield of genetic testing ranges from less than 40% in idiopathic DCM to over 50% in ACM and HCM, indicating that many causal genes are yet to be identified. This is particularly true in African populations, where the genetics of cardiomyopathy is underexplored. In a review of the role of next-generation sequencing in gene discovery, over 20 new cardiomyopathy genes were found to have been identified through exome sequencing of cardiomyopathy patients. The literature review also highlighted the need for functional validation of newly identified disease genes. Therefore, the aims of this investigation were to utilise exome sequencing to identify disease-causing mutations in South African families with heritable cardiomyopathy, and to establish methods of variant validation through functional modelling in zebrafish. Methods: Five probands and 34 relatives were included in this investigation. The probands and their relatives were clinically examined and diagnosed with DCM, HCM or ACM at Groote Schuur Hospital, Cape Town. Exome sequencing was performed on each of the five probands as well as at least one other family member. Variants of interest were identified by filtering the exome sequencing data by allele frequency, variant quality, variant consequence, predicted deleteriousness, and the potential inheritance patterns as determined by family history analysis. Variants occurring in known cardiomyopathy genes were prioritised, but genes outside the cardiac panel were considered based on literature mining, expression in the heart, and results of prior animal models. Candidate variants were validated by Sanger sequencing and assessed using international criteria for pathogenicity. The candidate ACM gene POLG was investigated in zebrafish larvae using two genetic manipulations. Firstly, zebrafish polg was disrupted using CRISPR/Cas9 in single-cell embryos and, at three days post-fertilisation, the phenotypic effects were compared to uninjected control larvae, as well as larvae in which other known cardiomyopathy genes were disrupted. Secondly, human POLG cDNA was cloned, and the c.2942A>G variant introduced using site-directed mutagenesis; this construct was used to generate variant POLG mRNA that was injected into zebrafish embryos. Larvae were phenotypically examined at four days post-fertilisation and compared to three control groups (unmutated POLG-injected, water-injected, and uninjected embryos). Results: In three families, genotype-phenotype correlations were identified that have not yet been reported in South Africa, although this genetic overlap between cardiomyopathies has been described elsewhere. Family 1: the mutation MYH7 c.4394C>T (p.S1465L) was identified in three siblings with DCM. Although MHY7 is typically associated with HCM, mutations in this region have been reported in DCM patients in other populations. Family 2: the mutation GLA c.774_775del (p.R259Rfs*5) was found in a mother and her son, both of whom had been diagnosed with HCM. The finding of a pathogenic truncating GLA mutation in this family resulted in the genetic rediagnosis of those individuals with Fabry disease, an HCM phenocopy. Family 3: in this large DCM family consisting of three affected brothers and their nephew, no pathogenic variants were identified, but two variants of uncertain significance (VUSs) were found in the genes DSC2 and PKP2. Both variants fulfilled some criteria for pathogenicity, but have not been associated with DCM in South African patients before. In Families 4 and 5, no mutations in known cardiomyopathy-causing genes were identified. Family 4: exome sequencing revealed the variant POLG c.2492A>G (p.Y831C) in this ACM family, with a clinical phenotype consisting of arrhythmia and left ventricular fibrosis. This was a VUS, but in vivo modelling using CRISPR/Cas9 in zebrafish larvae demonstrated that disruption of the gene may impair cardiac development, while expression of the c.2492A>G variant in zebrafish larvae resulted in a significant reduction in heart rate, ventricle size and cardiac output. These results indicate that POLG variation may underly the arrythmia observed in the family, while prior mouse models reported that POLG mutations can induce cardiac fibrosis. Family 5: rare, compound heterozygous missense mutations in ITGB5 were identified as the candidate causative variants in this small family with severe paediatric DCM, possibly affecting adhesion of cardiomyocytes to the extracellular membrane. Conclusion: In total, pathogenic or likely pathogenic mutations were identified in two out of five families studied, while three VUSs with moderate or strong pathogenic potential were identified in two other families. The potential role of POLG in human cardiomyopathy and arrhythmic phenotypes is a finding that should be explored further, as should the putative role of ITGB5 in paediatric cardiomyopathy. This study indicates how exome sequencing, combined with in vivo functional analysis, can identify variants that are likely to contribute to disease in human patients. These techniques may prove useful in bridging the gap in cardiomyopathy knowledge in Africa.
9
Giri, Harirambapu Dinesh. "Whole exome sequencing in children with rare endocrine disorders". Thesis, University of Liverpool, 2017. http://livrepository.liverpool.ac.uk/3014406/.
Testo completo
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Hartill, Verity Laura. "Congenital heart disease gene identification by whole exome sequencing". Thesis, University of Leeds, 2017. http://etheses.whiterose.ac.uk/18531/.
Testo completoAbstract (sommario):
Congenital Heart Disease (CHD) is the most common congenital defect, but the genetic aetiology of a large proportion of CHD is unexplained. This project aimed to delineate novel genetic causes of CHD using Whole Exome Sequencing (WES) in a family-based approach. Sixteen families were recruited to the study. WES data analysis followed a standardized pipeline and candidate variants were prioritized on the basis of in silico pathogenicity prediction tools and review of the current literature. Known and candidate genes in CHD were successfully identified using WES. In one family, a mutation in PIGV was identified, providing a diagnosis of Hyperphosphatasia and Mental Retardation syndrome and expanding the known phenotype of this condition. In a family with early-onset cardiomyopathy, a mutation in PPA2 was identified, encoding a mitochondria-specific pyrophosphatase enzyme. Through collaboration this gene was identified to be causative in three further families and mutation pathogenicity was investigated by functional studies. In a further family, a missense mutation in DNAAF1 was associated with heterotaxy, in the absence of clinical features of Primary Ciliary Dyskinesia, the phenotype usually associated with this gene. Zebrafish studies supported the pathogenicity of this variant and functional experiments identified novel interactants of DNAAF1 to include Pontin, Reptin and IFT88. Pontin was found to be expressed on the left side of the embryonic node in mice and zebrafish, a pattern which was abolished in dnaaf1-/- mutant fish, suggesting DNAAF1 and Pontin to be involved in the development of early laterality. In two families with athelia, choanal atresia and CHD a candidate variant in KMT2D was identified. The phenotype was distinct from Kabuki syndrome and is likely to represent a novel KMT2D-related disorder. WES was a successful tool in gene identification in CHD and, coupled with functional studies, has provided novel insights into the pathogenesis of CHD.
11
Mouhlas, Danielle. "Experiences with Whole Exome Sequencing: A Collective Case Study". Case Western Reserve University School of Graduate Studies / OhioLINK, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=case1429804360.
Testo completo
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Li, Karen Christine. "Supporting decision-making in whole genome/exome sequencing : parents' perspectives". Thesis, University of British Columbia, 2014. http://hdl.handle.net/2429/50835.
Testo completoAbstract (sommario):
Whole genome sequencing/exome sequencing (WGS/ES) technology is becoming more affordable and accessible, and will become more frequently used in various clinical settings, including for diagnosing rare childhood diseases. However, its use means that parents face decisions that could uncover life-altering information, unrelated to their child’s illness that may also have personal and ethical implications for their families. The purpose of this study is to explore and describe parents’ perceptions of their decisional needs when deciding on WGS/ES for their child.
The qualitative methodological approach known as Interpretive Description, the concept of shared decision-making and the Ottawa Decision Support Framework were used to inform and guide this study. Parents of children who had previously undergone WGS/ES informed consent were invited to participate in a focus group or individual in-person or telephone interviews. Parents had children with a range of undiagnosed conditions suspected to be genetic in origin. 15 parents were interviewed and transcriptions were analyzed concurrently and iteratively. Repeat interviews were conducted with 5 of the parents to confirm, challenge or expand on the developing conceptualizations.
Participants felt that their decision to proceed with WGS/ES for their child was easy. However, they expressed many unresolved decisional needs including: a lack of knowledge about certain topics that became relevant and important to them later, unmet expectations, and a need for more support and resources. Participants also acknowledged that the high volume of information and urgency of their circumstances may have caused them to be less receptive (or even unreceptive) to information during their WGS/ES decision-making (DM) process. Additionally, participants had ongoing informational and psychosocial needs beyond the single clinical encounter where their WGS/ES DM occurred. The content and amount of information that participants considered to be important varied.
Prior to the widespread use of clinical WGS/ES, parents’ perspectives about their decisional needs should be considered in order to implement parent-tailored education, counselling, decision support and informed consent processes.Applied Science, Faculty of
Nursing, School of
Graduate
13
Skarp, S. (Sini). "Whole exome sequencing in identifying genetic factors in musculoskeletal diseases". Doctoral thesis, Oulun yliopisto, 2019. http://urn.fi/urn:isbn:9789526223315.
Testo completoAbstract (sommario):
Abstract Musculoskeletal diseases, such as osteoarthritis (OA), lumbar disc degeneration (LDD) and osteoporosis (OP), are common complex disorders affected by both environmental and genetic factors. OA and LDD are degenerative diseases affecting joints and spine and Modic changes (MC) are a specific phenotype of LDD. OP is a disorder causing bone fragility. There are families with a history of early onset cartilage degradation, disc disorders and bone fragility as well as rare, more severe disorders with these traits as part of the phenotype. The aim of this study was to identify predisposing genetic factors in Finnish families with three different musculoskeletal phenotypes and to investigate the use of whole exome sequencing (WES) as a tool. Six families were studied here, three diagnosed with hip and knee OA, two with MC and one with primary OP. Using WES together with in silico and in vitro analyses we identified new candidate genes. In the two OA families we identified family specific variants, c.-127G>T in the 5’UTR of FIP1L1 and p.Arg210Gly in OLIG3. We observed expression of these genes in human bone and cartilage. Both FIP1L1 and OLIG3 participate in the regulation of transcription. Family specific variants were also found in both families with MC: p.Gln1611fs in HSPG2 and p.Glu553Lys in MAML1. HSPG2 encodes for an important structural protein in the disc and MAML1 is a transcription factor. The family with primary OP had previously been reported to carry a heterozygous COL1A2 deletion leading to nonsense-mediated mRNA decay. In the WES we identified an additional change that may contribute to the phenotype: p.Arg428* in ZNF528. We showed experimentally that the variant leads to expression of a truncated form of ZNF528 in the nucleus. ZNF528 binding sites are located near genes associated with bone phenotypes. We identified twelve potential target genes for ZNF528 that were differentially expressed in patients’ cells compared to controls. Altogether, we identified five new candidate genes for the studied phenotypes demonstrating that WES can be used as a tool in studying complex musculoskeletal phenotypes in families. One of the identified candidate genes, HSPG2, encodes a structural protein, whereas, OLIG3, FIP1L1, MAML1 and ZNF528, participate in the regulation of transcription supporting the importance of regulatory mechanisms in the pathogenesis of musculoskeletal diseasesTiivistelmä Tuki- ja liikuntaelinsairaudet, kuten nivelrikko, välilevyrappeuma ja osteoporoosi, ovat yleisiä, monitekijäisiä sairauksia. Nivelrikko ja välilevynrappeuma ovat eteneviä nivelten ja selkärangan sairauksia. Modic muutokset ovat välilevyn ja nikaman välisten päätelevyjen muutoksia. Osteoporoosi on luuta haurastuttava sairaus. Varhaisessa iässä ilmenevää ruston haurastumista, välilevyn sairauksia tai luun haurautta tavataan myös suvuittain esiintyvinä sairauksina tai vakavien harvinaisten sairauksien oireina. Tutkimuksen tarkoitus oli tunnistaa altistavia geneettisiä tekijöitä kolmelle tuki- ja liikuntaelimistön sairaudelle suomalaisissa perheissä käyttäen eksomisekvensointi-menetelmää. Aineisto koostui kuudesta perheestä: kolmessa oli diagnosoitu lonkan ja polven nivelrikko, kahdessa selän välilevyjen Modic muutoksia ja yhdessä primaarinen vaikea selän osteoporoosi. Tunnistimme uusia ehdokasgeenejä käyttäen eksomisekvensointi-menetelmää sekä in silico ja in vitro analyysejä. Kahdessa nivelrikkoperheessä tunnistimme perhekohtaiset variantit kahdessa geenissä: c.-127G>T variantin FIP1L1 geenin säätelyalueella ja p.Arg210Gly variantin OLIG3 geenissä. Osoitimme, että nämä traskription säätelyyn osallistuvat geenit ilmenevät ihmisen luu- ja rustokudoksessa. Perhekohtaiset variantit havaittiin myös perheissä, joilla oli todettu Modic muutoksia: p.Gln1611fs HSPG2 -geenissä ja p.Glu553Lys MAML1 -geenissä. HSPG2 koodaa välilevylle tärkeää rakenneproteiinia ja MAML1 on transkriptiota säätelevä tekijä. Primaarista osteoporoosia sairastavalla perheellä oli aiemmin havaittu heterotsygootti, geenituotteen hajottamiseen johtava deleetio, COL1A2 -geenissä. Eksomisekvensoinnlla havaitsimme mahdollisesti taudin ilmiasuun lisäksi vaikuttavan muutoksen ZNF528 -geenissä. Osoitimme kokeellisesti, että havaittu variantti johtaa lyhentyneen proteiinin tuottoon solussa. ZNF528 on transkriptiotekijä, jolle tunnistimme kaksitoista mahdollista kohdegeeniä ja havaitsimme että niiden tuotto oli muuttunut potilaiden soluissa kontrollisoluihin verrattuna. Tunnistimme viisi uutta ehdokasgeeniä kolmessa eri sairaudessa eksomisekvensointi-menetelmän avulla. Yksi tunnistetuista geeneistä, HSPG2, koodaa rakenneproteiinia, ja muut osallistuvat transkription säätelyyn. Tämä tukee käsitystä säätelytekijöiden tärkeydestä TULE sairauksien synnyssä
14
Ramos, Alexis. "Cancer Genome Characterization with SNP Array and Whole-Exome Sequencing Analysis". Thesis, Harvard University, 2011. http://dissertations.umi.com/gsas.harvard:10036.
Testo completoAbstract (sommario):
Cancer, the uncontrolled growth of morphologically and genetically abnormal cells in the body, is a major worldwide public health problem and there is a great need for novel insights into this disease. The majority of tumors arise from the acquisition of somatic alterations leading to changes in gene function and expression. The clinical success of targeted therapeutics in molecularly defined subsets of patients has highlighted the need for comprehensive characterization of the somatic alterations in individual cancer types. Copy number profiling using SNP arrays is a common approach for profiling the extent of copy number variation across the cancer genome. In addition, next-generation sequencing technologies now offer researchers the ability to also systematically catalog nucleotide substitutions and structural rearrangements in dramatically less time and expense. In this thesis, we describe the application of SNP arrays and whole-exome sequencing to characterize two separate cohorts of cancer samples, as well as describe the development of a software tool to aid in the annotation of mutational data. Specifically, we detailed focal amplifications of PDGFRA and KIT in a combined set of lung adenocarcinoma and squamous cell carcinomas. Furthermore, in a cohort of small bowel neuroendocrine tumors, we characterized the global genetic landscape to show that these tumors are molecularly distinct from other neuroendocrine tumors. Lastly, we report Oncotator, a novel web application and service for comprehensive annotation of point mutations and indels found in cancer. It is hoped that the knowledge gained from these studies will fuel improvements in cancer diagnosis, prognosis, and therapy.
15
Blosser, Beverly. "Meanings Parents Attribute to an Answer from Whole Exome Sequencing Research". University of Cincinnati / OhioLINK, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1406809942.
Testo completo
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Fiorini, Claudio <1993>. "Whole Exome Sequencing widens the genetic landscape of Hereditary Optic Neuropathies". Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2022. http://amsdottorato.unibo.it/10126/1/Thesis_Claudio_Fiorini.pdf.
Testo completoAbstract (sommario):
Hereditary optic neuropathies (HON) are a genetic cause of visual impairment characterized by degeneration of retinal ganglion cells. The majority of HON are caused by pathogenic variants in mtDNA genes and in gene OPA1. However, several other genes can cause optic atrophy and can only be identified by high throughput genetic analysis. Whole Exome Sequencing (WES) is becoming the primary choice in rare disease molecular diagnosis, being both cost effective and informative.
We performed WES on a cohort of 106 cases, of which 74 isolated ON patients (ON) and 32 syndromic ON patients (sON). The total diagnostic yield amounts to 27%, slightly higher for syndromic ON (31%) than for isolated ON (26%).
The majority of genes found are related to mitochondrial function and already reported for harbouring HON pathogenic variants: ACO2, AFG3L2, C19orf12, DNAJC30, FDXR, MECR, MTFMT, NDUFAF2, NDUFB11, NDUFV2, OPA1, PDSS1, SDHA, SSBP1, and WFS1. Among these OPA1, ACO2, and WFS1 were confirmed as the most relevant genetic causes of ON. Moreover, several genes were identified, especially in sON patients, with direct impairment of non-mitochondrial molecular pathways: from autophagy and ubiquitin system (LYST, SNF8, WDR45, UCHL1), to neural cells development and function (KIF1A, GFAP, EPHB2, CACNA1A, CACNA1F), but also vitamin metabolism (SLC52A2, BTD), cilia structure (USH2A), and nuclear pore shuttling (NUTF2).
Functional validation on yeast model was performed for pathogenic variants detected in MECR, MTFMT, SDHA, and UCHL1 genes. For SDHA and UCHL1 also muscle biopsy and fibroblast cell lines from patients were analysed, pointing to possible pathogenic mechanisms that will be investigated in further studies.
In conclusion, WES proved to be an efficient tool when applied to our ON cohort, for both common disease-genes identification and novel genes discovery. It is therefore recommended to consider WES in ON molecular diagnostic pipeline, as for other rare genetic diseases.
17
Jaber, Dana. "Identification et caractérisation d’un nouveau gène dont les mutations sont responsables d’Arthrogrypose Multiple Congénitale De Novo Mutations of SCN1Aare Responsible for Arthrogryposis Broadening the SCN1A– Related Phenotypes". Thesis, université Paris-Saclay, 2020. http://www.theses.fr/2020UPASL047.
Testo completoAbstract (sommario):
L’arthrogrypose multiple congénitale (AMC) est une maladie rare caractérisée par des rétractions d’au moins deux articulations distinctes présentes à la naissance. L’AMC est génétiquement hétérogène et des variants dans de très nombreux gènes ont été identifiés. Notre projet a eu pour but d’identifier de nouvelles causes génétiques de l’AMC en utilisant le séquençage de l’exome entier (WES).Dans le gène SCN1A, nous avons identifié des variants pathogéniques dominants survenus de novo chez trois patients non apparentés. SCN1A code Nav1.1, un composant des canaux sodiques voltage-dépendants exprimés au niveau de l’AIS (Axon Initial Segment) et des nœuds de Ranvier et contribue à l’initiation et la propagation des potentiels d’action. Nous avons montré que SCN1A est exprimé dans le cerveau et la moelle épinière mais pas dans le muscle à des stades du développement similaires à l’âge de l’apparition de l’AMC et que l’AMC est due à un défaut de SCN1A dans le cortex moteur.Nous montrons pour la première fois que des variants de SCN1A sont responsables d’un déficit moteur sévère provoquant l’AMC, suggérant un rôle critique de SCN1A dans le développement moteur. Ces résultats élargissent le spectre phénotypique de SCN1A allant d’une encéphalopathie épileptique sévère isolée ou associée à un défaut moteur causant l’arthrogrypose (Jaber et al. sous presse). Nos données élargissent ainsi le groupe des AMC liées à une atteinte du système nerveux central et précisément celles causées par des anomalies des canaux ioniques tels que NALCN ou CACNA1EArthrogryposis multiplex congenita (AMC) is a rare disease characterized by the presence of multiple joint contractures at birth. AMC includes a large spectrum of diseases which result from variants in genes encoding components required for neuromuscular junctions, skeletal muscle, motor neurons, peripheral nerves or connective tissues. AMC may also result from central nervous involvement. Despite important progress in this field, many AMC patients remain genetically undiagnosed. Our project aimed at identifying novel genes in which variants can cause AMC. We identified pathogenic de novo dominant variants in SCN1A gene in three unrelated patients. SCN1A encodes Nav1.1 voltage-dependent sodium channel, a critical component of axon initial segment and nodes of Ranvier. We showed that SCN1A is expressed in both brain and spinal cord but not in skeletal muscle at a developmental stage similar to that of AMC observation and showed that AMC is caused by brain involvement.We showed that SCN1A variants are responsible for early onset motor defect leading to AMC indicating a critical role of SCN1A in prenatal motor development and broadening the phenotypic spectrum of variants in SCN1A (Jaber et al. in press). Altogether, our data enlarge the group of AMC linked to central nervous system involvement and caused by variants of genes encoding channels such as NALCN or CACNA1E
18
Guffroy, Aurélien. "Etude des mécanismes de rupture de tolérance lymphocytaire au cours des déficits immunitaires primitifs de l'adulte avec manifesations auto-immunes". Thesis, Strasbourg, 2019. http://www.theses.fr/2019STRAJ012.
Testo completoAbstract (sommario):
L’association entre déficits immunitaires primitifs (DIPs) et manifestations auto-immunes peut sembler paradoxale lorsque l’on aborde les DIPs comme des défauts d’immunité opposés à l’autoimmunité vue comme excès d’immunité adaptative à l’encontre du soi. Néanmoins, loin de se résumer à un simple défaut d’une ou plusieurs composantes du système immunitaire qui prédispose aux infections par divers agents pathogènes, les DIPs sont fréquemment associés à une autoimmunité; parfois révélatrice. Ainsi, les données épidémiologiques issues de registres ou de larges séries de patients atteints de DIPs s’accordent sur une prévalence globale de 25 à 30% de complications auto-immunes (au premier rang desquelles figurent les cytopénies auto-immunes). Différentes hypothèses sont avancées pour rendre compte de l’auto-immunité dans les DIPs. On peut citer : 1°) une perturbation profonde de l’homéostasie lymphocytaire, en particulier dans les déficits immunitaires combinés sévères (CID) avec lymphopénies T et B ; 2°) des défauts intrinsèques des lymphocytes B permettant une rupture de tolérance précoce des LB auto réactifs ; 3°) un comportement aberrant des LT (défaut de maturation, excès d’activation) ; 4°) une absence de lymphocytes T ou de B régulateurs ; 5°) une production inappropriée de certaines cytokines proinflammatoires comme dans les interféronopathies. Ces hypothèses concernent surtout les DIPs pédiatriques sévères. Mon travail de thèse explore la rupture de tolérance immunitaire adaptative au cours des DIPs de l’adulte par différentes approches. Nous nous sommes en particulier attachés au plus fréquent, le DICV (Déficit Immunitaire Commun Variable), déficit immunitaire humoral pas toujours bien défini sur le plan génétique et physiopathologique qui constitue un défi thérapeutique lorsqu’il est compliqué d’une auto-immunité nécessitant un traitement immunosuppresseurThe association between primary immune deficiency (PID) and autoimmunity may seem paradoxical when PID is considered only as an immune response defect against pathogens and autoimmunity only as an excess of immunity. Nevertheless, far from being simple immune defects increasing the risk of infections, DIPs are frequently associated with autoimmunity. Even more, autoimmunes manifestations can sometimes reveal a PID. Thus, epidemiological data from registers or large series of patients with PIDs agree on an overall prevalence of 25 to 30% of autoimmune complications (with auto-immune cytopenias as first causes). Several hypotheses have been proposed with different underlying mechanisms to explain the tolerance breakdown in PIDs. We can cite : 1°) a severe disturbance of lymphocyte homeostasis, for example in severe combined immunodeficiencies ; 2°) an impaired B-cell developpement with earlystage defects of tolerance ; 3°) a dysregulation of T cells (developpement or activation impairments) ; 4°) a dysfunction of T-reg (or B-reg) ; 5°) an excess of production of proinflammatory cytokines. These hypotheses are especially true for early-onset PIDs (in infancy). In this work (PhD), we explore the mechanisms of tolerance breakdown involved in adults PIDs. We use several approaches to describe the pathways leading to autoimmunity, focusing on the most common PID in adult : CVID (common variable immunodeficiency). This syndrome is not well defined on the genetic and physiopathological level. It is still a therapeutic challenge when complicated by autoimmunity (requiring immunosuppressive therapy)
19
Tolusso, Leandra K. "Parental understanding of whole exome sequencing: A comparison of perceived and actual understanding". University of Cincinnati / OhioLINK, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1458814771.
Testo completo
20
Abdelrahman, Omar Ezzeldin Ibrahim. "Investigating the molecular basis of concussion using whole exome sequencing and bioinformatics". Thesis, Queensland University of Technology, 2021. https://eprints.qut.edu.au/211353/1/Omar_Abdelrahman_Thesis.pdf.
Testo completoAbstract (sommario):
This thesis investigated the genetic influences of concussion development and outcomes. People react differently to similar head trauma; some may recover within a few weeks and some may develop long term symptoms for months or years. This thesis used a combination of genetic sequencing methods and machine learning to create pilot models for predicting these outcomes. The long-term goal of this work will allow for personalised treatments in the wake of concussion, tailored rest recommendations, as well as potential new drugs to help with management.
21
BERTAMINO, MARTA. "NEXT GENERATION SEQUENCING FOR DIAGNOSIS IN MONOGENIC PEDIATRIC STROKE .. from NGS panel to Whole Exome Sequencing". Doctoral thesis, Università degli studi di Genova, 2019. http://hdl.handle.net/11567/945076.
Testo completoAbstract (sommario):
Background and Purpose: Pediatric arterial ischemic stroke (AIS) may underlie an as yet undiagnosed syndrome often characterized by simple Mendelian inheritance. We aimed to establish and validate a targeted gene panel for AIS associated with monogenic disorders, and to determine its diagnostic yield and clinical utility.
Methods: Clinical and neuro-radiological data were collected for every patient enrolled in the study. DNA samples were tested by means of a customized gene panel including 15 genes associated with known genetic diseases related with AIS.
Results: Thirty-eight patients (23 males, mean age 6.5 years) were selected with heterogeneous AIS phenotypes, mostly multiple and asynchronous and secondary to vasculopathy. Ten out of 38 resulted to carry rare potentially causative mutations in at least one of the 15 genes analyzed. In 4 cases the analyses led to a definite genetic diagnosis while results were either controversial or null in the remaining patients.
Conclusions: While the complexity of the different clinical phenotypes associated with AIS is not fully accounted for by the genes tested in the present study, the achieved diagnoses had a great beneficial impact on patient management. A wider gene panel or an unbiased genome wide approach would be better suited to explain a greater proportion of pediatric stroke events.
22
Niemiec, Emilia <1987>. "Ethical, legal and social issues related to the offer of whole exome and whole genome sequencing". Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2018. http://amsdottorato.unibo.it/8295/1/NIEMIEC_EMILIA_tesi.pdf.
Testo completoAbstract (sommario):
Whole genome and exome sequencing (WGS and WES) raise numerous ethical, legal and social issues (ELSI), such as related to informed consent and usage of sequencing data in research. These concerns may be amplified when genomic sequencing is offered direct-to-consumer (DTC) bypassing the traditional heathcare system. This thesis discusses ELSI related to WES/WGS and DTC genetic testing, provides an overview of current DTC genetic testing market, and analyses the impact of the recently adopted Regulation of the European Parliament and of the Council on in vitro diagnostic medical devices on DTC genetic testing.
To provide insights into how ethical issues are addressed in DTC offer of WES/WGS, content analysis of websites of relevant DTC companies was conducted; the results were compared to relevant recommendations of expert groups. The analysis revealed the following concerns: lack of pre-test counselling, inadequate informed consent documents for genetic testing and/or for research activities on consumers’ samples and data, lack of relevant information and/or presence of potentially misleading descriptions in some of the companies studied. Consequently, consumers might not be aware of all the implications of undergoing WGS/WES, and their informed consent may be compromised.
Another study presented in this thesis evaluated readability of informed consent forms for clinical WGS and WES using the SMOG and the Flesch-Kincaid formulas. All 36 forms studied failed to meet the average recommended reading grade level for informed consent forms, indicating that the content of the forms may not be comprehensible to many patients.
In order to respect patients/consumers, the compliance with ethical standards when offering genetic testing should be strived for, also in the commercial DTC offer of WES and WGS. The findings presented herein indicate specific areas in which practices should be improved and provide reference and guidance for well-informed and potentially policy-relevant discussions between various stakeholders.
23
Carmichael, Heather. "Whole Exome Sequencing and Large-Scale Targeted Sequencing to Identify Novel Candidate Genes in Idiopathic Short Stature". Thesis, Harvard University, 2015. http://nrs.harvard.edu/urn-3:HUL.InstRepos:17295875.
Testo completoAbstract (sommario):
Short stature is a common problem in pediatric endocrinology. With the exception of chronic disease and overt hormonal deficiencies, patients with short stature often go without a definitive diagnosis, and are therefore classified as having idiopathic short stature. Treatment with growth hormone therapy is of limited effect in many of these individuals, highlighting the multiple pathways involved in human growth. Height has been shown to be more than 90% heritable, and a large portion of the genetic variation in height can be explained by the additive effects of many common variants; however, there is a substantial contribution of rare variants with larger effect size, particularly at the extremes of height. The chapters of this thesis describe the use of both whole exome sequencing as well as large-scale targeted sequencing in patients with extreme short stature as strategies to discover novel or rare pathogenic variants that lead to short stature phenotypes. A number of pathogenic variants were identified including novel mutations in the insulin-like growth factor 1 receptor (IGF1R) and natriuretic peptide receptor-B (NPR2). These studies also highlight the potential for the detection of a large number of candidate genes, complicating interpretation of results in both the research and clinical settings.
24
MELCHIONDA, LAURA. "New genes involved in mitochondrial and neurodegenerative diseases identified by whole exome sequencing". Doctoral thesis, Università degli Studi di Milano-Bicocca, 2014. http://hdl.handle.net/10281/50025.
Testo completoAbstract (sommario):
The scope of my thesis was the identification of genes responsible for:
1) an adult-onset neurological syndrome, with leukodystrophy and motor-neuron disease, in two half-siblings;
2) an infantile hypertrophic cardiomyopathy with lactic acidosis and mitochondrial respiratory chain defects.
Since all genetic screenings performed on the basis of clinical manifestations did not provide a diagnosis for these patients, we carried out whole exome sequencing.
My work contributed to the publications of three papers.
In the second chapter of this thesis, there is the article concerning the identification and characterization of the first GFAP-ε mutation,causing an adult form of Alexander disease in two affected siblings.
The male presented a severe motor-neuron disease whereas his sister showed a mild movement disorder with cognitive impairment. In addition to the GFAP-ε mutation, we found a variant in HDAC6 on chromosome X, present only in the male patient; HDAC6 is a candidate MND susceptibility gene and the identified missense variant is probably responsible for his different phenotype. Cellular models
were used to experimentally prove the altered functionality of mutant GFAP-ε and HDAC6. The third chapter contains the paper reporting the first mutations in MTO1, responsible for a mitochondrial disorder associated with hypertrophic cardiomyopathy, lactic acidosis and mitochondrial respiratory chain defects. Then chapter four consists of the article
where we described and characterized news MTO1 mutations found in other patients. In both articles characterization of the identified mutations and validation of their deleterious effects were assessed in
patients’ specimens (fibroblasts) and in yeast Saccharomyces cerevisiae.
25
Cox, Allison Jeanne. "Whole exome analysis of individuals and families with chronic recurrent multifocal osteomyelitis (CRMO)". Diss., University of Iowa, 2016. https://ir.uiowa.edu/etd/2199.
Testo completoAbstract (sommario):
Chronic recurrent multifocal osteomyelitis (CRMO) is a rare, pediatric, autoinflammatory disease characterized by bone pain due to sterile osteomyelitis, and is often accompanied by psoriasis or inflammatory bowel disease. There are two syndromic forms of CRMO, Majeed syndrome and DIRA, for which the genetic cause is known. However, for the majority of cases, the genetic basis is unknown. Via whole-exome sequencing and linkage analysis, we determined the most likely causative mutations in four families. While the mutations are in three different genes – FBLIM1, PLCG2 and PIP; all three genes are involved in Fcγ signaling and osteoclast activation.
In a large cohort of 61 individuals with CRMO, we performed gene and pathway based association analysis using the 1000 genomes participants of European ancestry as controls. One gene from the family-based analyses, ANO6, was significantly enriched for rare variants in our cohort of cases. ANO6 is involved in P2RX7- mediated inflammasome activation and in the regulation of bone mineralization. While no pathways were enriched for rare variants in the CRMO cohort after genome-wide correction, four pathways were significantly enriched for rare variants in the control samples, indicating a protective effect of the variants. The second most significant pathway, activation of chaperone genes by XBP1s, is relevant to CRMO pathogenesis as XBP1s is a transcription factor that attenuates ER stress, and regulates the expression of genes involved in RANKL signaling and bone remodeling.
An association analysis using a larger set of cases followed by functional validation of candidate genes is necessary to confidently declare the mutations isolated in the work presented here to be pathogenic. Our preliminary findings suggest that mutations in genes involved in both the inflammatory response and bone remodeling underlie the pathogenesis of CRMO.
26
van, Vuuren Larry Peter. "Identification of coding variants associated with familial systemic lupus erythematosus through whole exome sequencing". University of the Western Cape, 2016. http://hdl.handle.net/11394/6122.
Testo completoAbstract (sommario):
Philosophiae Doctor - PhD (Bioinformatics)Systemic lupus erythematosus (SLE) is a chronic inflammatory autoimmune disease characterized by the production of a wide range of autoantibodies directed against selfantigens. SLE can influence almost any organ system and its appearance and course are highly varied, ranging from remission to disease flare. SLE demonstrates a variety of constitutional symptoms, such as the skin, musculoskeletal and mild hematologic involvement. On the other hand, some patients present with primarily hematologic, renal or neuropsychiatric manifestations.
27
Albury, Cassie Louise. "Using whole exome sequencing and genetic association studies to investigate common and complex migraine". Thesis, Queensland University of Technology, 2018. https://eprints.qut.edu.au/122954/1/Cassie_Albury_Thesis.pdf.
Testo completoAbstract (sommario):
This dissertation focused on expanding the knowledge and understanding of common and complex migraine genetics. The central objectives of this research was to 1) identify new migraine causative genes using modern sequencing techniques and; 2) investigate potential genetic associations between common and complex migraine. This study has successfully identified 1) a susceptibility association between a potential familial hemiplegic migraine (FHM) gene and common migraine and; 2) implicated 4 new genes as potentially FHM causative. As a result of this study we foresee the development of a more comprehensive genetic test with improved diagnostic success rates.
28
GENOVESE, GIOVANNI. "SYNDROMIC HIDRADENITIS SUPPURATIVA: GENOTYPE-PHENOTYPE CORRELATION THROUGH WHOLE-EXOME SEQUENCING IN 10 UNRELATED PATIENTS". Doctoral thesis, Università degli Studi di Milano, 2022. https://hdl.handle.net/2434/945807.
Testo completoAbstract (sommario):
Background: The genetics of syndromic hidradenitis suppurativa (HS), an immune-mediated condition associated with systemic comorbidities such as inflammatory bowel diseases and arthritis, has not been completely elucidated.
Objective: To describe clinical features and genetic signature of patients with the main syndromic HS forms, i.e., PASH, PAPASH, and PASH/SAPHO overlapping.
Methods: Whole-exome sequencing (WES) approach was performed in ten patients with syndromic HS.
Results: Three clinical settings have been identified based on presence/absence of gut and joint inflammation. Four PASH patients who had also gut inflammation showed three different variants in NOD2 gene, two variants in OTULIN, and a variant in GJB2, respectively. Three PAPASH and three PASH/SAPHO overlapping patients who had also joint inflammation showed two different variants in NCSTN, one in WDR1 and PSTPIP1, and two variants in NLRC4, one of whom was present in a patient with a mixed phenotype characterized by gut and joint inflammation.
Limitations: Limited number of patients that can be counterbalanced by the rarity of syndromic HS.
Conclusion: Syndromic HS can be considered as a polygenic autoinflammatory condition; currently WES is a diagnostic tool allowing more accurate genotype-phenotype correlation.
29
Zhang, Lu, e 张璐. "Identification and prioritization of single nucleotide variation for Mendelian disorders from whole exome sequencing data". Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2012. http://hub.hku.hk/bib/B48521905.
Testo completoAbstract (sommario):
With the completion of human genome sequencing project and the rapid development of sequencing technologies, our capacity in tackling with genetic and genomic changes that underlie human diseases has never been greater. The recent successes in identifying disease causal single nucleotide variations (SNVs) for Mendelian disorders using whole exome sequencing may bring us one step further to understand the pathogenesis of Mendelian diseases. However, many hurdles need to be overcome before the promises can become widespread reality.
In this study, we investigated various strategies and designed a toolkit named PriSNV for SNV identification and prioritization, respectively. The SNV identification pipeline including read alignment, PCR duplication removal, indel realignment, base quality score recalibration, SNV and genotype calling was examined by simulation and real sequencing data. By incorporating sequencing errors and small indels, most of the read alignment software can achieve satisfied results. Nonetheless, the reads with medium size and large indels are prone to be wrongly mapped to the reference genome due to the limitation of gap opening strategies of available read alignment software. In addition, although mapping quality can only reflect certain information of the mapping error rate, it is still important to be adopted to filter out obvious read alignment errors. The PCR duplication removal, indel realignment and base quality score recalibration have proven to be necessary and can substantially reduce the false positive SNV calls. Based on the same quality criterion, Varscan performs as the most sensitive software for SNV calling, unfortunately at mean time the false positive calls are enriched in its result.
In order to prioritize the small subset of functionally important variants from tens of thousands of variants in whole human exome, we developed a toolkit called PriSNV, a systematic prioritization pipeline that makes use of information on variant quality, gene candidacy based on the number of novel nonsynonymous mutations in a gene, gene functional annotation, known involvement in the disease or relevant pathways, and location in linkage regions. Prediction of functional impact of the coding variants is also used to aid the search for causal mutations in Mendelian disorders. For the patient affected by Chron's disease, the candidate genes can be substantially reduced from 9615 to 3 by the gene selection strategies implemented in PriSNV.
In general, our results for SNV identification can help the biologists to realize the limitation of available software and shed light on the development of new strategies for accurately identifying SNV calls in the future. PriSNV, the software we developed for SNV prioritization, can provide significant help to biologists in prioritizing SNV calls in a systematic way and reducing search space for further analysis and experimental verification.published_or_final_version
Paediatrics and Adolescent Medicine
Master
Master of Philosophy
30
MEREU, ELISABETTA. "Joint whole exome sequencing and linkage analysis in a multigenerational family segregating Type 1 Diabetes". Doctoral thesis, Università degli Studi di Cagliari, 2015. http://hdl.handle.net/11584/266614.
Testo completoAbstract (sommario):
Backround: Type 1 diabetes (T1D) is a complex autoimmune disease with a
strong familial segregation. On the other hand, the recent and rapid increase
in incidence is a proof of the importance of environmental factor in the
etiology of disease. Linkage and Genome-wide association studies had
revealed almost 60 loci associated with the risk of T1D, explaining about 80%
of the total heritability, mostly due to HLA locus. However, many familial T1D
cases remains unexplained.
Objective: To identify rare variants contributing to T1D susceptibility, we
studied a Sardinian family with 9 individuals affected across 3 generations.
Methods: We performed exome sequencing in 3 affected members and a
healthy individual. In addition, all samples were extensively genotyped using
Illumina OmniExpress beadchips for about 750K SNPs. A combined linkage
analysis was carried out.
Results: This combined approach identified three variants predicted to be
damaging that are very rare in the general population (frequency <1%) and
that are likely causing the disease.
31
Wagner, Justin. "Whole Exome Sequencing to Identify Disease-Causing Mutations in Lower Motor Neuron Disease and Peripheral Neuropathy". Thesis, Université d'Ottawa / University of Ottawa, 2016. http://hdl.handle.net/10393/34124.
Testo completoAbstract (sommario):
Lower motor neuron diseases and peripheral neuropathies are two groups of diseases that include multiple rare disorders where many causes are unknown and definitive treatments are unavailable. Understanding the molecular etiology of these genetic diseases provides an opportunity for rapid diagnosis, preconception genetic counseling and, in a subset, direction for the development of future treatment options. The recent introduction of whole exome sequencing (WES) marks a new era in Mendelian genetic disease research as the majority of the coding region of the genome can be sequenced in a timely and cost-effective manner. In this study, WES was used to investigate the molecular etiology of a cohort of 37 patients presenting with lower motor neuron disease or peripheral neuropathy. A molecular diagnosis was determined for seven patients informing the diagnostic utility of WES. Novel phenotypes were found for three genes originally associated with a different disorder. Finally, the foundation has been laid, through the use of functional studies and large scale data-sharing, to identify novel disease-causing genes for lower motor neuron disease and peripheral neuropathy.
32
Jambaljav, Byambatseren. "Whole-exome sequencing in a Japanese family with highly aggregated diabetes identifies a candidate susceptibility mutation in ADAMTSL3". Kyoto University, 2018. http://hdl.handle.net/2433/232467.
Testo completo
33
Fisher, Rachel. "Clinical whole exome sequencing in an academic pediatric hospital: A descriptive study of the diagnostic odyssey". University of Cincinnati / OhioLINK, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1427813369.
Testo completo
34
Miossec, Matthieu Josepth. "Whole-exome capture and next-generation sequencing to discover rare variants predisposing to congenital heart disease". Thesis, University of Newcastle upon Tyne, 2016. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.701158.
Testo completoAbstract (sommario):
Congenital heart disease (CHD) is the most common congenital malformation, affecting 8 out of 1000 lives births, yet its aetiology remains largely unresolved. The rapidly growing number of point mutations implicated in isolated CHD suggests that single mutations may contribute significantly to CHD risk. This thesis presents an investigation of the genetic underpinnings of various types of CHD following different study designs. First, I designed a new approach to variant calling which I implemented as the variant caller BAMily. My aim was to develop a method of uncovering putative variants in next-generation sequencing data, shared by a subset of individuals and absent in another subset. I tested the variant caller’s performance against other known variant callers and demonstrated that it provides comparable; and often better, results. This novel variant caller was applied to a study of 8 families in which a disease trait was segregating; along with the variant caller SAMtools, leading to the discovery of likely disease-causing variants in 5 families. Second, I studied de novo mutation in 32 sporadic cases of transposition of the great arteries (TGA) in an attempt to identify genes that, when mutated, lead to TGA. The 32 patients with TGA were sequenced with their parents; as well as one unaffected sibling. To achieve this aim, three variant callers were used: SAMtools, GATK Unified Genotyper and BAMily, the latter acting as a filter. Potential de novo variants were found in GREB1, RBP5, SNX13. Results suggested a complex genetic etiology underlying TGA. Finally, I studied a large series of cases of tetralogy of Fallot (ToF). The study involved 824 patients which ToF and a comparator set of 490 patients with neurodevelopmental disorders lifted from the UK10K project. The aim of the study was to identify genes that, when mutated, play a role in the manifestation of ToF might cluster. For this, I first categorised variants according to their potential to disrupt protein function. I then compared genes in which potentially disease-causing rare variants occurred to lists of genes previously implicated in CHD in the literature. Following this, I identified the clustering of potentially deleterious rare variants across the coding region of genes and exons in ToF patients, hypothesising that variants influencing ToF would cluster in ToF patients. This study led to the discovery of candidate variants in FLT4 and NOTCH1 for non-syndromic ToF. As with TGA, the results I have obtained suggested a complex etiology for ToF.
35
Raykova, Doroteya. "Genetics of Two Mendelian Traits and Validation of Induced Pluripotent Stem Cell (iPSC) Technology for Disease Modeling". Doctoral thesis, Uppsala universitet, Medicinsk genetik och genomik, 2015. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-246228.
Testo completoAbstract (sommario):
Novel technologies for genome analysis have provided almost unlimited opportunities to uncover structural gene variants behind human disorders. Whole exome sequencing (WES) is especially useful for understanding rare Mendelian conditions, because it reduces the requirements for a priori clinical data, and can be applied on a small number of patients. However, supporting functional data on the effect of specific gene variants are often required to power these findings. A variety of methods and biological model systems exists for this purpose. Among those, induced pluripotent stem cells (iPSCs), which are capable of self-renewal and differentiation, stand out as an alternative to animal models. In papers I and II we took advantage of WES to identify gene variants underlying autosomal recessive pure hair and nail ectodermal dysplasia (AR PHNED) as well as autosomal dominant familial visceral myopathy (FVM). We identified a homozygous variant c.821T>C (p.Phe274Ser) in the KRT74 gene as the causative mutation in AR PHNED, supported by the fact that Keratin-74 was undetectable in hair follicles of an affected family member. In a family segregating FVM we found a heterozygous tandem base substitution c.806_807delinsAA (p.(Gly269Glu)) in the ACTG2 gene in the affected members. This novel variant is associated with a broad range of visceral symptoms and a variable age of onset. In Paper III we explored the similarity between clonally derived iPSC lines originating from a single parental fibroblast line and we highlighted the necessity to use lines originating from various donors in disease modeling because of biological variation. Paper IV focused on how the genomic integrity of iPSCs is affected by the choice of reprogramming methods. We described several novel cytogenetic rearrangements in iPSCs and we identified a chromosome 5q duplication as a candidate aberration for growth advantage. In summary, this doctoral thesis brings novel findings on unreported disease-causing variants, as supported by extensive genetic analysis and functional data. A novel molecular mechanism behind AR PHNED is presented and the phenotypic spectrum associated with FVM is expanded. In addition, the thesis brings novel understanding of benefits and limitations of the iPSC technology to be considered for disease modeling.
36
Cox, Brian James. "EMS Mutagenesis in Quinoa: Developing a Genetic Resource". BYU ScholarsArchive, 2020. https://scholarsarchive.byu.edu/etd/9080.
Testo completoAbstract (sommario):
Chenopodium quinoa, a South American pseudocereal, has valuable agricultural traits such as salt tolerance and drought tolerance, and it has beneficial nutritional properties such as high protein content and a complete amino acid profile. However, problems including disease susceptibility, low harvest index, lodging, seed shattering, low heat tolerance, and saponin content plague quinoa. Genetic resources for quinoa are needed to fix these problems and make quinoa more available throughout the world. We used ethyl methanesulfonate (EMS) to create a mutant population of QQ74 quinoa (USDA GRIN PI 614886) of 5,030 mutant families. We did whole exome sequencing (WES) on 44 mutant families. Using the recently published quinoa reference genome and MAPS, a mutation detection pipeline, we found a mutation rate of 11.35 mutations/Mb in these families. We also used whole genome sequencing (WGS) to calculate a mutation rate of 21.67 mutations/Mb in an additional nine mutant families. To demonstrate the utility of this population as a genetic resource, we found an EMS-induced nonsense mutation in the betalain synthesis pathway that prevents red betacyanins from accumulating in the hypocotyl of quinoa. With the mutation rates in our population, we calculate that analysis of 300 mutant families will yield 3-7 mutations in any gene of interest, which will facilitate forward and reverse genetic studies in quinoa.
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Ikeda, Atsuyuki. "Leptin Receptor Somatic Mutations are Frequent in HCV-Infected Cirrhotic Liver and Associate with Hepatocellular Carcinoma". Kyoto University, 2014. http://hdl.handle.net/2433/188668.
Testo completo
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Leão, Delva Pereira. "Sequenciamento de nova geração : explorando aplicações clínicas de dados de Targeted Gene Panel e Whole Exome Sequencing". reponame:Biblioteca Digital de Teses e Dissertações da UFRGS, 2017. http://hdl.handle.net/10183/173625.
Testo completoAbstract (sommario):
A tecnologia de sequenciamento de nova geração (next-generation sequencing – NGS) e suas aplicações tem sido cada vez mais utilizada na prática médica para elucidar a base molecular de doenças Mendelianas. Embora seja uma poderosa ferramenta de pesquisa, ainda existe uma importante transição quanto à análise dos dados entre as tecnologias tradicionais de sequenciamento e o NGS. A primeira parte deste trabalho aborda aspectos analíticos envolvidos nesta mudança, com foco na plataforma Ion Torrent Personal Genome Machine. Esta é uma plataforma amplamente utilizada para sequenciar painéis de genes, já que esta aplicação requer menor rendimento de dados. Este trabalho demonstra indicadores adequados para avaliar a qualidade de corridas de sequenciamento e também uma estratégia baseada em valores de profundidade de cobertura para avaliar a performance de amplicons em diferentes cenários. Por outro lado, o NGS permitiu a realização de estudos populacionais em larga escala que estão mudando nossa compreensão sobre as variações genéticas humanas. Um desses exemplos são as mutações até então chamadas de silenciosas, que estão sendo implicadas como causadoras de doenças humanas. A segunda parte deste trabalho investiga a patogenicidade de polimorfismos de núcleotídeo único sinônimos (synonymous single nucleotide polymorphisms – sSNP) baseado em dados públicos obtidos do Exome Aggregation Consortium (ExAC) (exac.broadinstitute.org/) utilizando o software Silent Variant Analysis (SilVA) (compbio.cs.toronto.edu/silva/) e outros recursos para reunir informações adicionais sobre consequências funcionais visando fornecer um panorama dos efeitos patogênicos de sSNP em mais de 60.000 exomas humanos. Nós demonstramos que de 1,691,045 variantes sinônimas, um total de 26,034 foram classificadas como patogênicas pelo SilVA, com frequência alélica menor que 0,05. Análises funcionais in silico revelaram que as variantes sinônimas patogênicas estão envolvidas em processos biológicos importantes, como regulação celular, metabolismo e transporte. Ao expor um cenário de variações sinônimas patogênicas em exomas humanos, nós concluímos que filtrar sSNP em workflows de priorização é razoável, no entanto em situações específicas os sSNP podem ser considerados. Pesquisas futuras neste campo poderão fornecer uma imagem clara do papel de tais variações em doenças genéticas.Next-generation sequencing (NGS) technologies and its applications are increasingly used in medicine to elucidate the molecular basis of Mendelian diseases. Although it is a powerful research tool, there is still an important transition regarding data analysis between traditional sequencing techniques and NGS. The first part of this work addresses analytical aspects involved on this switch-over, focusing on the Ion Torrent Personal Genome Machine platform. This is a widely used platform for sequencing gene panels, as this application demands lower throughput of data. We present indicators suitable to evaluate quality of sequencing runs and also a strategy based on depth of coverage values to evaluate amplicon performance on different scenarios. On the other hand, NGS enabled large-scale population studies that are changing our understanding about human genetic variations. One of these examples are the so-called silent mutations, that are being implied as causative of human diseases. The second part of this work investigates the pathogenicity of synonymous single nucleotide polymorphisms (sSNP) based on public data obtained from the Exome Aggregation Consortium (ExAC) (exac.broadinstitute.org/) using the software Silent Variant Analysis (SilVA) (compbio.cs.toronto.edu/silva/) and other sources to gather additional information about affected protein domains, mRNA folding and functional consequences aiming to provide a landscape of harmfulness of sSNP on more than 60,000 human exomes. We show that from 1,691,045 synonymous variants a total of 26,034 were classified as pathogenic and by SilVA, with allele frequency lower than 0.05. In silico functional analysis revealed that pathogenic synonymous variants found are involved in important biological process, such as cellular regulation, metabolism and transport. By exposing a scenario of pathogenic synonymous variants on human exomes we conclude that filtering out sSNP on prioritization workflows is reasonable, although in some specific cases sSNP should be considered. Future research on this field will provide a clear picture of such variations on genetic diseases.
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Fewings, Eleanor Rose. "The use of whole exome sequencing data to identify candidate genes involved in cancer and benign tumour predisposition". Thesis, University of Cambridge, 2019. https://www.repository.cam.ac.uk/handle/1810/285963.
Testo completoAbstract (sommario):
The development of whole exome sequencing has transformed the study of disease predisposition. The sequencing of both large disease sets and smaller rare disease families enables the identification of new predisposition variants and potentially provide clinical insight into disease management. There is no standard protocol for analysing exome sequencing data. Outside of extremely large sequencing studies including thousands of individuals, statistical approaches are often underpowered to detect rare disease associated variants. Aggregation of variants into functionally related regions, including genes, gene clusters, and pathways, allows for the detection of biological processes that, when interrupted, may impact disease risk. In silico functional studies can also be utilised to further understand how variants disrupt biological processes and identify genotype-phenotype relationships. This study describes the exploration of sequencing datasets from cancers and benign tumour diseases including: i) hereditary diffuse gastric cancer, ii) sweat duct proliferation tumours, iii) adrenocortical carcinoma, and iv) breast cancer. Each set underwent germline whole exome sequencing followed by additional tumour or targeted sequencing to identify associated predisposition genes. Variants within a cluster of risk genes that are involved in double strand break repair were identified as associated with hereditary diffuse gastric cancer risk via gene ontology enrichment analysis. This cluster included PALB2 within which, using externally collated data, loss of function variants were identified as significantly associated with hereditary diffuse gastric cancer risk. Germline protein-affecting variants in the myosin gene MYH9 were identified in all individuals with a rare sweat duct proliferative syndrome, suggesting a role for MYH9 in skin development, regulation and tumorigenesis. These MYH9 variants were analysed in silico to identify a genotype-phenotype relationship between the clinical presentation and variants in the ATP binding pocket of the protein. Tumour matched normal sequence data from adrenocortical carcinoma cases was used to elucidate the role of Lynch syndrome genes in disease pathogenesis. Within the breast cancer set, candidate genes were selected to undergo targeted sequencing in a larger set of cases to further explore their role in breast cancer risk. Risk associated genes identified within this study may ultimately aid in diagnosis and management of disease. This thesis has also generated multiple novel tools and sequencing analysis techniques that may be of use for further studies by aiding in the prioritisation of candidate variants. The described techniques will provide support to researchers working on rare, statistically underpowered datasets and to provide standard analysis pipelines for a range of dataset sizes and types, including familial data and unrelated individuals.
40
Manyisa, Noluthando. "Whole exome sequencing to investigate genetic variants of non-syndromic hearing impairment in a population of African ancestry". Master's thesis, University of Cape Town, 2018. http://hdl.handle.net/11427/29272.
Testo completoAbstract (sommario):
Introduction: Hearing impairment occurs when a child has hearing loss greater than 30dB in their better hearing ear and an adult cannot detect sound lower than 40dB in the better hearing ear. It is a common sensory disorder that affecting approximately 360 million worldwide, with an incidence of 6 in 1000 live births in developing countries such as those in Sub-Saharan Africa. 50 % of hearing impairment, in developed countries, is due to genetic factors, with 70% of genetic hearing impairment being classified as non-syndromic hearing impairment, which occurs when the hearing impairment presents with no other clinical manifestations. Hearing impairment is associated with over 150 genes, of which two connexin genes, GJB2 and GJB6, are the most prevalent genes associated with hearing impairment in European, Asian and North American of European ancestries populations. These genes have however been shown to be insignificant causes of Hearing Impairment in African populations. Aim: The aim of this study is to determine the rates for putative pathogenic variants in 172 hearing impairment associated genes, among Cameroonian patients affected by hearing impairment, and non-hearing-impaired controls. Methods: Patients and controls Patients were recruited from various schools of the Deaf and Ear, Nose and Throat (ENT) clinics in Cameroon. The patients were examined by qualified medical geneticists and ophthalmologist and detailed family history and medical history was obtained from the patients and their parents. 19 patients, who were negative for GJB2 and GJB6 mutations and presented with putative non-syndromic hearing impairment, were selected from a cohort of 582 patients for the present study. The control population consisted of 130 ethnically matched groups without any personal or familial history of hearing impairment. The controls were recruited from Yaoundé Central Hospital and Laquintinie Hospital in Cameroon. Whole exome sequencing DNA was extracted from whole blood using the salting out procedure and the Puregene Blood kit®. The DNA was subjected to spectrometry and gel electrophoresis to determine the quantity and quality of the DNA samples. The samples were then subjected to whole exome sequencing on the Illumina platform using the Nextera Rapid Capture Exome Kit at an average read depth of 30X, whereby only 18 patients were successfully sequenced. The exomes were then subjected to FastQC and SolexaQC++ for quality control measures and aligned to the hg19 reference genome using GATK and VariantMetaCaller. Bioinformatics analysis Variant annotation was performed using Annovar and the annotated variants were filtered based in rarity and pathogenicity. Tests for genetic differentiation and principle component analysis was performed on the combined patient exomes and combine control exomes. The first principle component analysis included data from African populations from the 1000 Genomes Phase 3 as well as six control samples from the Democratic Republic of Congo; and the second principle component analysis analysed on the Cameroonian patients and control population. Population structure analysis was followed by protein-protein interaction analysis using custom python and R script and pathway enrichment analysis using Enrichr combined with a second custom R script. The proportion of derived and ancestral alleles was computed by downloading the SNP ancestral alleles from Ensembl and verifying the presence of the SNPs in dbSNP database. The combined patient and control exomes were annotated using the VCFtools “fillOaa” script. The ancestral alleles were computed by dividing the number of times the alternative allele matched the ancestral allele with the number of copies of all the alternative alleles across all samples at the particular position. The ancestral alleles were categorised into six bins, based on their minor allele frequency, in the patient and control populations and this was used to contrast their proportions of derived and ancestral alleles. Furthermore, the proportion of ancestral and derived alleles in hearing impairment associated genes was computed at SNP based level for the Cameroonian population and contrasted with population from the Democratic Republic of Congo. Variants validation by Sanger sequencing Primers were designed to amplify the fragment surrounding the purported SNPs in MYO15A, MYO3A, and COL9A3 as well as for the fragments surrounding the population specific SNPs in VTN, RPL3L and DHRS4L2. Polymerase chain reaction was performed for the MYO15A, and MYO3A fragments. This was followed by purification of the PCR products and direct cycle Sanger sequencing of the PCR products. The sequencing products were then purified through ethanol precipitation and the fragments were suspended in HiDi Formamide and run on the capillary electrophoresis. The variants in MYO3A, MYO15A and COL9A3 were viewed in Integrated Genomics Viewer using the Bam files as well. Results Putative deleterious variants Single nucleotide polymorphism (SNPs) in MYO3A, MYO15A and COL9A3, were filtered out as putative causative mutations for three, four and two patients respectively. Direct Sanger Sequencing and viewing the patients BAM files did not confirm the presence of any of these putative pathogenic in the patients. Variations in USH2A, HSD17B4 and MYO1A were also filtered out but these variants were not considered disease causing, after a careful genotype to phenotypes correlations. Population genetics variants differentiations At a population level, specific variations were identified in FOXD4L2, DHRS2L6, RPL3L and VTN. Significant genetic differentiation was shown to exist between the control population and the patients’ population with regard to specific variants in VTN and RPL3L; furthermore, it was shown that these variants in VTN and RPL3L interact with other hearing impairment associated proteins with evidences that that VTN is hub protein for a hearing impairment associated pathway along with nine other genes. Conversely, this was not the case for variants described in FOXD4L2 and DHRS2L6. In known hearing Impairment genes, the proportion of ancestral alleles was lowest for the patients’ population for variations with minor allele frequencies between 0.0 and 0.1. The proportion of derived and ancestral alleles was also shown to differ between the Cameroonian and the population from the Democratic Republic of Congo, indication possible regional differences in aetiology of Hearing impairment amongst multiple African populations. Discussion Low putative pathogenic variants in known hearing impairment genes among Africans The low pick up rate for putative pathogenic variants in our patients follows a similar trend observed in the African American populations, with hearing impairment, as well as data from targeted exome sequencing from South African and Nigerian populations. This result is also in agreeance with other studies that interrogated hearing impairment in African populations utilising other means besides next generation sequencing. This result also highlights the importance of validating any results obtained from next generation sequencing through traditional approaches such as Sanger Sequencing or viewing the BAM files on IGV, specifically in African population, poorly represented in Exome databases. Bioinformatics Analysis Exhibited some Specific Variants among Cameroonian Protein-protein interactions and enrichment analysis indicated that VTN and RPL3L, and their interacting proteins, are significantly associated with osteoclast differentiation, which is associated with hearing impairment in osteogenesis imperfecta. VTN was further shown as a hub protein of a protein subnetwork, along ATPB2. The presence of a second protein acting as a hub protein may account for why aberrations in VTN have not been associated with a disease; whereby ATPB2 may ameliorate the pathogenic phenotype that ought to be observed in the presence of null mutations in VTN. Evolutionary adaptation of human hearing Data indicates the patient population carried a higher proportion of derived alleles in known hearing impairment genes, at low minor allele frequencies; possibly indicating, the interactive modifiers capacities of multiple hearing impairment genes, or alternatively, the polygenetic nature of hearing impairment in some patients. The proportion of ancestral and derived alleles was contrasted in the Cameroonian and the population from the Democratic Republic of Congo and it indicated that the variations that may result in hearing impairment in the one population may not be the same variations that result in hearing impairment in the other population Due to this, it is necessary to determine the causative variants resulting in disease in each of these populations independently. Conclusion and perspectives The results support a low pick up rate of putative variants in 172 known genes in groups of Cameroonian patients with HI, underscoring the current Targeted panel sequencing for HI may not be relevant for some African populations. The result also support the need of confirmation of variants found in WES, as well careful genotype to phenotypes correlations, particularly among African, whose sequences exome is relatively low in Exomes databases, and as a result could lead to more false positive results. Population genetic analysis has provided novel insight in the genetic architecture of HI among this group of Africans; particularly, the differential frequencies of ancestral alleles vs derived alleles in HI genes among patients vs controls underline the possibility of multigenic influence on the phenotype of Hearing Impairment that have not been well investigated, and may also signal evolutionary enrichment of some variants of HI genes in the populations as the result of natural selections, that deserve further investigation. The result supports the need of intensive familial studies in multiple African populations in order to unravel the novel genes and those variants that are relevant in clinical practice for people of African ancestry.
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Ferrarini, Margherita. "From exome to whole genome sequencing: mining for inconsistencies and functional elements in coding and non-coding regions". Doctoral thesis, Università degli studi di Padova, 2018. http://hdl.handle.net/11577/3424933.
Testo completoAbstract (sommario):
Over the last two decades the advancement in DNA sequencing technologies has enormously increased the amount of sequencing data available to researchers and geneticists. This has been accompanied by the development of tools for sequencing data analysis, including the human reference genome, that is undoubtedly an indispensable resource. It is known that the reference genome does not always represent the real consensus sequence of the human population, due to the inclusion of rare alleles and sequencing errors. Moreover, genomic duplications are often misassembled and, as a result, they may be found in the reference genome as a collapsed consensus, thus generating false variants. In this work I performed a thorough search for conflicting information between the human reference genome (GRCh37 and GRCh38) and some of the most popular human genetic resources such as the 1000 Genomes Project, to disclose minor alleles and to mine genetic inconsistencies. To search for unreported genomic duplications, I performed a genome wide screening for unbalanced heterozygosity. I found that inaccuracies and errors are much higher than expected. Minor alleles occurring with a frequency <10% are found on average every ~7,000 bases and include many rare variants that are never found elsewhere, producing high numbers of false positives as well as possible false negatives. The systematic screening for unbalanced heterozygosity revealed ~86,000 variants that are likely the result of unreported genomic duplications, involving functionally relevant genes such as MAP2K3 and KCNJ12. My findings may help the ongoing quest to obtain a highly accurate human genome reference sequence. Moreover, the results presented in this thesis will be useful to human geneticists in the process of filtering and selecting causative variants.
The advancement in DNA sequencing technologies also accounts for the increasing usage of Whole Genome Sequencing approaches both in the research and clinical fields, thus revealing that the large majority of disease-associated SNPs are located in non-coding regions of the human genome. However, the functional interpretation of non-coding variants is still challenging. Part of my work also addressed this problem, aiming to develop a method for non-coding variant prioritization. The method, presented in the last chapter of this thesis, is based on a comparative genomics approach for the identification of functional constraints in primate orthologous genes. The first steps of my approach have proved to be powerful in identifying orthologous genes, but further work is necessary to optimize the multiple sequence alignment step and the identification of conserved domains.Nel corso dell’ultimo ventennio l’avanzamento tecnologico nel campo del sequenziamento del DNA ha portato a un enorme aumento della quantità di dati di sequenziamento accessibili a ricercatori e genetisti. Questa crescita è stata accompagnata dallo sviluppo di strumenti necessari all’analisi dei dati; tra questi il genoma umano di riferimento è senza dubbio una risorsa indispensabile. È noto che il genoma di riferimento non sempre rappresenta la reale sequenza consenso della popolazione umana, poiché alleli rari ed errori di sequenziamento sono stati inclusi in essa. Inoltre, duplicazioni genomiche sono spesso mal assemblate e, di conseguenza, possono essere trovate nel genoma di riferimento come collassate, generando così false varianti. In questa tesi è descritta la ricerca approfondita di incongruenze tra il genoma umano di riferimento (GRCh37 e GRCh38) e alcune delle più popolari risorse di genetica umana, come il 1000 Genomes Project, per scovare alleli minori e inconsistenze genetiche. Per identificare duplicazioni genomiche non riportate nel genoma, è stata poi condotta un’ampia ricerca di eterozigosità sbilanciata. Questa analisi ha dimostrato che incongruenze ed errori sono molto più frequenti di quanto atteso. Infatti, alleli minori con una frequenza <10% sono stati trovati in media ogni ~7,000 basi e tra essi sono presenti molte varianti rare mai riportate nei database. Lo screening sistematico per l’eterozigosità sbilanciata ha mostrato inoltre che ~86,000 varianti possono derivare da duplicazioni genomiche non riportate nella sequenza di riferimento e che alcune di esse coinvolgono geni importanti come MAP2K3 e KCNJ12. I risultati descritti in questo lavoro possono contribuire alla definizione di una sequenza di riferimento del genoma umano altamente accurata. Inoltre, questi stessi risultati potranno essere utili ai genetisti umani nel processo di filtraggio e selezione delle varianti potenzialmente associate a malattie. L’avanzamento nel settore del sequenziamento del DNA ha condotto inoltre dell’utilizzo sempre maggiore degli approcci di sequenziamento dell’intero genoma, sia nel campo della ricerca sia nella diagnosi clinica, rivelando così che la gran parte degli SNP associati a malattia è localizzata nelle regioni non codificanti del genoma umano. Tuttavia, l’interpretazione funzionale delle varianti non codificanti è ancora una questione problematica. Parte del mio lavoro ha riguardato anche questo aspetto, con lo scopo di sviluppare un metodo per la prioritizzazione delle varianti non codificanti. Questo metodo, descritto nell’ultimo capitolo della tesi, si basa su un approccio di genomica comparata per l’identificazione di domini funzionali in geni ortologhi di organismi primati. I primi passaggi di questo approccio hanno dimostrato essere molto buoni per l’identificazione dei geni ortologhi, ma ulteriore lavoro è necessario per ottimizzare il processo di allineamento multiplo delle sequenze e l’identificazione dei domini conservati.
42
O'Grady, Gina Louise. "Improving the Diagnosis of Congenital Muscular Dystrophy with Next Generation Sequencing Technology". Thesis, The University of Sydney, 2016. http://hdl.handle.net/2123/16490.
Testo completoAbstract (sommario):
The congenital muscular dystrophies (CMDs) are inherited disorders of skeletal muscle characterized by early onset muscle weakness and dystrophic changes on muscle biopsy. The traditional approach to diagnosis included muscle biopsy, protein-based studies of muscle specimens, and Sanger sequencing of candidate genes. The diagnostic yield was less than 25%. As Next Generation Sequencing (NGS) moves rapidly into clinical practice, this thesis evaluates the efficacy of NGS in the diagnosis of CMD, identifies new genetic causes of congenital myopathy, evaluates the cost efficacy of a NGS approach, and re-evaluates the diagnostic algorithm. 123 CMD patients were investigated with a traditional approach, yielding a genetic diagnosis in 32% of patients. Undiagnosed patients available for further testing, were investigated using NGS techniques. Of the 85 patients who presented in the last 20 years, 28 of the 51 who lacked a confirmed genetic diagnosis consented to NGS studies. This confirmed diagnoses in a further 11 patients. Using the combination of approaches, a genetic diagnosis was achieved in 51% (43/85). In the cohort three new genetic causes of disease were identified: Early onset myopathy due to variants in PYROXD1; multisystem disease, including congenital muscular dystrophy, due to variants in PIGY; and pre-synaptic congenital myasthenic syndrome due to variants in SLC18A3. The spectrum of ACTA1, CHD7 and COL1A1-related disease was expanded. A detailed cost benefit analysis was performed on a combined CMD and nemaline myopathy cohort. Compared with the traditional diagnostic algorithm, both the NMD panel and WES, had significantly increased diagnostic yields (from 46% to 75% for the NMD panel, and 79% for WES), reduced the cost per diagnosis from AUD$22,596 to AUD$5,077 for the NMD panel and AUD$7,734 for WES. This thesis supports NGS as an effective, non-invasive first-tier investigation which can be performed in patients of any age. Muscle biopsy should be reserved as a second-tier investigation for patients undiagnosed by a NGS neuromuscular gene panel. The challenge of neuromuscular genetic research now lies with families undiagnosed after first tier NGS investigations.
43
Pathak, Vaibhav Sanjay. "IDENTIFYING SOMATIC COPY NUMBER ABERRATIONS WITHIN GLIOBLASTOMA MULTIFORME AND LOW GRADE GLIOMAS USING BIOINFORMATICS TOOLS EXCAVATOR AND XHMM". Case Western Reserve University School of Graduate Studies / OhioLINK, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=case1481394117039479.
Testo completo
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MUSTAFA, NOOR HUSSEIN MOHAMMAD. "Whole Exome Sequencing in Hereditary Ocular Disease Leads to Recognition of Novel and Recurrent Disease-Causing Mutations: Pros and Cons". Doctoral thesis, Università degli studi di Pavia, 2017. http://hdl.handle.net/11571/1203328.
Testo completoAbstract (sommario):
Hereditary ocular diseases show an extreme genetic and phenotypic heterogeneity,
ranging from mild retinal dysfunctions to severe congenital forms of blindness. Most of
the many genes associated with these diseases encode signaling and structural
proteins are involved in the developing eye. Another category of serious visual
impairments concerns the degeneration of neurons as photoreptors, related to the
various forms of retinitis pigmentosa. All modes of Mendelian inheritance occur and
many are sporadic cases. Identification of the underlying genetic basis for families and
affected individuals, understanding genotype-phenotype correlations and developing
therapeutic approaches are therefore highly challenging. In this study whole exome
sequencing (WES) was performed on Hiseq2500 platform (Illumina) as a powerful
strategy for assessment of multiple candidate disease genes (> 450) at the same time in
11 unrelated families (16 patients) with different eye defects, including microcornea/
coloboma / microphthalmia / anophthalmia, Axenfeld-Rieger syndrome, Retinitis
Pigmentosa, Leber congenital amaurosis and Stargardt macular degeneration.
Afterward, Sanger sequencing was performed to confirm and determine whether any of
the candidate variants co-segregated with the disease phenotype in the families. We
evaluated the diagnostic yield, the spectrum of clinical referrals, the challenge of
variants’ interpretation and the genetic heterogeneity of such diseases.Our data indicate
that this approach enables us to genetically diagnose approximately 80 % of the
patients (n = 13) with variant(s) in known disease-associated genes. We revealed four
pathogenic variants in RAB3GAP1 (p.(Tyr958*), CHD7 (p.Ala1347Glnfs*25), KCNK9
(p.Gly266Arg), CDH23 (p.Leu1343Phe) and TULP1 (p.Gly266Val, four of them are
novel and not reported in the literature or dbSNP: RAB3GAP1, CHD7, CDH23, and
TULP1 . In addition, we identified 6 known disease-associated variants, previously
reported, in USH2A (p.Asp347Gly & p.Leu3606Pro), FOXC1 (p.Asp261Argfs*45),
STRA6 (p.Arg655His), GUCY2D (p.Pro130Leufs*36), and ABCA4 (p.Ser1696Asn). The
identified mutation spectrum involved novel variants and previously described recurrent
mutations Further, we pointed out two variants probably explaining the abnormal ocular
phenotype in: TULP1 (c.823-17G>C) and CDH23 (p.Cys1045Phe). The vast majority of
mutations have not been reported in the Italian population (only USH2A; p.Asp347Gly)
.We also identified a novel phenotype for mutations in KCNK9. In one family, identified
phenotypes were different from the previously reported clinical findings associated with
the causative gene in STRA6.WES can rapidly identify variants in various families
affected by different forms of hereditary ocular diseases in contrast to Sanger
sequencing of candidate genes. In fact the genetic and phenotypic heterogeneity of eye
diseases impair straightforward genotype-phenotype relationships . Without WES, we
would not have been able to arrive at a molecular diagnosis in 13 cases, even by using
commercial panels of genes involved in eye defects that, although continuously
upadated, do not included all the possible causative genes.We discussed the
challenges experienced in data analysis and showed examples of cases where the
detection of the causative variant required further investigation due to the low coverage
of some genes, or would be valued unlikely applying the standard WES analysis. For
instance, in two families, we were not able to detect mutations in genes currently
associated with inherited eye diseases in humans or in animal models.
45
Borgström, Erik. "Technologies for Single Cell Genome Analysis". Doctoral thesis, KTH, Genteknologi, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-181059.
Testo completoAbstract (sommario):
During the last decade high throughput DNA sequencing of single cells has evolved from an idea to one of the most high profile fields of research. Much of this development has been possible due to the dramatic reduction in costs for massively parallel sequencing. The four papers included in this thesis describe or evaluate technological advancements for high throughput DNA sequencing of single cells and single molecules. As the sequencing technologies improve, more samples are analyzed in parallel. In paper 1, an automated procedure for preparation of samples prior to massively parallel sequencing is presented. The method has been applied to several projects and further development by others has enabled even higher sample throughputs. Amplification of single cell genomes is a prerequisite for sequence analysis. Paper 2 evaluates four commercially available kits for whole genome amplification of single cells. The results show that coverage of the genome differs significantly among the protocols and as expected this has impact on the downstream analysis. In Paper 3, single cell genotyping by exome sequencing is used to confirm the presence of fat cells derived from donated bone marrow within the recipients’ fat tissue. Close to hundred single cells were exome sequenced and a subset was validated by whole genome sequencing. In the last paper, a new method for phasing (i.e. determining the physical connection of variant alleles) is presented. The method barcodes amplicons from single molecules in emulsion droplets. The barcodes can then be used to determine which variants were present on the same original DNA molecule. The method is applied to two variable regions in the bacterial 16S gene in a metagenomic sample. Thus, two of the papers (1 and 4) present development of new methods for increasing the throughput and information content of data from massively parallel sequencing. Paper 2 evaluates and compares currently available methods and in paper 3, a biological question is answered using some of these tools.QC 20160127
46
Maksemous, Neven. "Development of high through-put neurogenetics diagnostics". Thesis, Queensland University of Technology, 2016. https://eprints.qut.edu.au/101097/1/Neven_Maksemous_Thesis.pdf.
Testo completoAbstract (sommario):
This project was an effort to improve the speed and affordability of genetic testing for neurological disorders. The research involved the development of a genetic diagnostic testing technique using Next Generation Sequencing technologies to investigate Familial Hemiplegic Migraine and related neurological conditions. The method successfully expanded the scope of testing, resulting in the ability to detect genetic changes that would have gone unnoticed in previous tests and significantly reduced the cost of testing for patients and the healthcare system.
47
Carraro, Marco. "Development of bioinformatics tools to predict disease predisposition from Next Generation Sequencing (NGS) data". Doctoral thesis, Università degli studi di Padova, 2018. http://hdl.handle.net/11577/3426807.
Testo completoAbstract (sommario):
The sequencing of the human genome has opened up completely new avenues in research and the notion of personalized medicine has become common. DNA Sequencing technology has evolved by several orders of magnitude, coming into the range of $1,000 for a complete human genome. The promise of identifying genetic variants that influence our lifestyles and make us susceptible to diseases is now becoming reality. However, genome interpretation remains one the most challenging problems of modern biology. The focus of my PhD project is the development of bioinformatics tools to predict diseases predisposition from sequencing data. Several of these methods have been tested in the context of the Critical Assessment of Genome Interpretation (CAGI), always achieving good prediction performances. During my PhD project I faced the complete spectrum of challenges to be address in order to translate the sequencing revolution into clinical practice. One of the biggest problem when dealing with sequencing data is the interpretation of variants pathogenic effect. Dozens of bioinformatics tools have been created to separate mutations that could be involved in a pathogenic phenotype from neutral variants. In this context the problem of benchmarking is critical, as prediction performance are usually tested on different sets of variants, making the comparison among these tools impossible. To address this problem I performed a blinded comparison of pathogenicity predictors in the context of CAGI, realizing the most complete performance assessment among all the iterations of this collaborative experiment. Another challenge that needs to be address to realize the personalized medicine revolution is the phenotype prediction. During my PhD I had the opportunity to develop several methods for the complex phenotype prediction from targeted enrichment and exome sequencing data. In this context challenges like misinterpretation or overinterpretation of variants pathogenicity have emerged, like in the case of phenotype prediction from the Hopkins Clinical Panel. In addition, other complementary issues of phenotype predictions, like the possible presence of incidental findings have to be considered. Ad hoc prediction strategies have been defined while facing with different kinds of sequencing data. A clear example is the case of Crohn’s disease risk prediction. Always in the context of the CAGI experiment, three iterations of this prediction challenge have been run so far. Analysis of datasets revealed how population structure and bias in data preparation and sequencing could affect prediction performance, leading to inflated results. For this reason a completely new prediction strategy has been defined for the last edition of the Crohn’s disease challenge, exploiting data from Genome Wide Association Studies and Protein Protein Interaction network, to address the problem of missing heritability. Good prediction performance have been achieved, especially for individuals with an extreme predicted risk score. Last, my work has been focused on the prediction of a health related trait: the blood group phenotype. The accuracy of serological tests is very poor for minor blood groups or weak phenotypes. Blood groups incompatibilities can be harmful for critical individuals like oncohematological patients. BOOGIE exploits haplotype tables, and the nearest neighbor algorithm to identify the correct phenotype of a patient. The accuracy of our method has been tested in ABO and RhD systems achieving good results. In addition, our analyses paved the way for a further increase in performance, moving towards a prediction system that in the future could become a real alternative to wet lab experiments.Il completamento del progetto genoma umano ha aperto numerosi nuovi orizzonti di ricerca. Tra questi, la possibilità di conoscere le basi genetiche che rendono ogni individuo suscettibile alle diverse malattie ha aperto la strada ad una nuova rivoluzione: l’avvento della medicina personalizzata. Le tecnologie di sequenziamento del DNA hanno subito una notevole evoluzione, ed oggi il prezzo per sequenziare un genoma è ormai prossimo alla soglia psicologica dei $ 1 000. La promessa di identificare varianti genetiche che influenzano il nostro stile di vita e che ci rendono suscettibili alle malattie sta quindi diventando realtà. Tuttavia, molto lavoro è ancora necessario perché questo nuovo tipo di medicina possa trasformarsi in realtà. In particolare la sfida oggi non è più data dalla generazione dei dati di sequenziamento, ma è rappresentata invece dalla loro interpretazione. L'obiettivo del mio progetto di dottorato è lo sviluppo di metodi bioinformatici per predire la predisposizione a patologie, a partire da dati di sequenziamento. Molti di questi metodi sono stati testati nel contesto del Critical Assessment of Genome Interpretation (CAGI), una competizione internazionale focalizzata nel definire lo stato dell’arte per l’interpretazione del genoma, ottenendo sempre buoni risultati. Durante il mio progetto di dottorato ho avuto l'opportunità di affrontare l’intero spettro delle sfide che devono essere gestite per tradurre le nuove capacità di sequenziamento del genoma in pratica clinica. Uno dei problemi principali che si devono gestire quando si ha a che fare con dati di sequenziamento è l'interpretazione della patogenicità delle mutazioni. Decine di predittori sono stati creati per separare varianti neutrali dalle mutazioni che possono essere causa di un fenotipo patologico. In questo contesto il problema del benchmarking è fondamentale, in quanto le prestazioni di questi tool sono di solito testate su diversi dataset di varianti, rendendo impossibile un confronto di performance. Per affrontare questo problema, una comparazione dell’accuratezza di questi predittori è stata effettuata su un set di mutazioni con fenotipo ignoto nel contesto del CAGI, realizzando la valutazione per predittori di patogenicità più completa tra tutte le edizioni di questo esperimento collaborativo. La previsione di fenotipi a partire da dati di sequenziamento è un'altra sfida che deve essere affrontata per realizzare le promesse della medicina personalizzata. Durante il mio dottorato ho avuto l'opportunità di sviluppare diversi predittori per fenotipi complessi utilizzando dati provenienti da pannelli genici ed esomi. In questo contesto sono stati affrontati problemi come errori di interpretazione o la sovra interpretazione della patogenicità della varianti, come nel caso della sfida focalizzata sulla predizione di fenotipi a partire dall’Hopkins Clinical Panel. Sono inoltre emersi altri problemi complementari alla previsione di fenotipo, come per esempio la possibile presenza di risultati accidentali. Specifiche strategie di predizione sono state definite lavorando con diversi tipi di dati di sequenziamento. Un esempio è dato dal morbo di Crohn. Tre edizioni del CAGI hanno proposto la sfida di identificare individui sani o affetti da questa patologia infiammatoria utilizzando unicamente dati di sequenziamento dell’esoma. L'analisi dei dataset ha rivelato come la presenza di struttura di popolazione e problemi nella preparazione e sequenziamento degli esomi abbiano compromesso le predizioni per questo fenotipo, generando una sovrastima delle performance di predizione. Tenendo in considerazione questo dato è stata definita una strategia di predizione completamente nuova per questo fenotipo, testata in occasione dell'ultima edizione del CAGI. Dati provenienti da studi di associazione GWAS e l’analisi delle reti di interazione proteica sono stati utilizzati per definire liste di geni coinvolti nell’insorgenza della malattia. Buone performance di predizione sono state ottenute in particolare per gli individui a cui era stata assegnata una elevata probabilità di essere affetti. In ultima istanza, il mio lavoro è stato focalizzato sulla predizione di gruppi sanguigni, sempre a partire da dati di sequenziamento. L'accuratezza dei test sierologici, infatti, è ridotta in caso di gruppi di sangue minori o fenotipi deboli. Incompatibilità per tali gruppi sanguigni possono essere critiche per alcune classi di individui, come nel caso dei pazienti oncoematologici. La nostra strategia di predizione ha sfruttato i dati genotipici per geni che codificano per gruppi sanguigni, presenti in database dedicati, e il principio di nearest neighbour per effettuare le predizioni. L’accuratezza del nostro metodo è stata testata sui sistemi ABO e RhD ottenendo buone performance di predizione. Inoltre le nostre analisi hanno aperto la strada ad un ulteriore aumento delle prestazioni per questo tool.
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Lebeko, Kamogelo. "Genetic aetiology of autosomal recessive non-syndromic hearing loss in sub-Saharan African patients: evaluation using targeted and whole exome sequencing". Doctoral thesis, Faculty of Health Sciences, 2019. http://hdl.handle.net/11427/30376.
Testo completoAbstract (sommario):
Hearing Loss (HL) is one of the highest contributors to disability worldwide. The highest incidence of the disease is seen in developing countries, such as those in subSaharan Africa (SSA). Patients affected with disabling HL are reported to be more than 466 million worldwide. The causes of HL can either be environmental or genetic with each contributing about 50% towards all cases, in many settings. In developing countries, the environment might contribute more due to poor health services and infrastructure available to the population. In the absence of environmental causes, there is a genetic component at play, that is largely unknown in African populations. Up to 70% of HL of genetic origin are non-syndromic (NS). The mode of inheritance is recessive in nearly 77% of non-syndromic HL. Up to date, more than 100 genes have been associated with HL harbouring more than 1000 causative variants. In many populations of European and Asian descent, pathogenic variants in GJB2 (connexin gene 26) and GJB6 (connexin gene 30) are a major contributor to autosomal recessive non-syndromic hearing loss (ARNSHL). Comprehensive hearing health care programs should cover genetic causes by providing molecular testing, and genetic counselling, specifically SSA where genes and mutations causing HL remain largely unknown. The aim of this project was thus to uncover the genetic causes of HL among patients’ cohorts from Cameroon and South Africa. This was addressed by 1) sequencing common variants in the most relevant genes in other populations (GJB2 and GJB6), 2) using a targeted gene panel to resolve HL in 10 multiplex families from Cameroon presenting with ARNSHL and negative for GJB2 and GJB6 mutations screening, 3) screening novel variants found in known genes in a cohort of 82 singleplex HL cases from Cameroon and South Africa, and lastly, 4) using Whole Exome sequencing to explore the two unresolved multiplex cases with and subsequent findings confirmed by functional studies, and also screened in 80 singleplex HL cases. The following findings are reported: GJB6, GJA1 mutations screening and literature review No GJA1 or GJB6 mutation was not found in multiplex and simplex cases of HL in both Cameroonians and South Africans. The review of the literature confirms that the prevalence of GJB2- or GJB6-related NSHL is approximating to zero in most subSaharan African populations. Targeted Exome Sequencing (OtoSCOPE) The targeted genes, panel that included 116 genes, was able to resolve 7 of 9 families (77.8%) which were successfully sequenced, with one family failing to be sequenced. The causative variants identified in the 7 resolved families were : 1) compound heterozygous c.5806_5808delCTC and c.5880_5882delCTT in MYO7A; 2) compound heterozygous c.646T>A (p.Phe216Ile) and c.38G>A (p.Arg13His) in LOXHD1; 3) homozygous c.766-2A>G in OTOF; 4) a deletion and a complex copy number variation in STRC; 5) compound heterozygous c.1678G>A (p.Asp560Asn) and c.2007C>A(p.Asp669Glu) in SLC26A4; 6) Homozygous c.1996C>T(p.Arg666Stop) in MYO7A; 7) compound heterozygous c.6399C>A(p.Asp2133Glu) and c.2000T>C (p.Met667Thr) in CDH23. Five out of 12 variants were novel. Screening of these causative variants in known genes, in 82 singleplex HL cases from Cameroon and South Africa was unable to resolve any of the cases: the variants were in either heterozygous in low frequency or absent. Bioinformatic pathways exploration of SNP data of known HL genes revealed an extensive network within the HL genes, with 10 identified as important nodes, including MYO7A. Most HL genes were found to be involved in two biological processes which were sensory perception of mechanical stimulus (GO: 0050954, p= 1.430e-8) and sound (GO: 0007605, p = 1.246e-8). The molecular functions of variants found within these genes were found to mostly fall within the binding (GO: 0005488) and/or structural molecule activity (GO: 0005198). Whole Exome sequencing Whole exome sequencing was performed on four of the nine multiplex families: the two families that were unresolved by targeted panel sequencing, and two previously resolved families that were used as positive controls for the variant annotation and filtering pipeline. The results were the resolution of 3/4 families, including the two- positive control. The previously unresolved “family 8” was found to harbour a novel variant within the GRXCR2 gene, a gene only associated with HL once before. The c.251delC variant was revealed through in silico studies to cause a premature stop codon at position 116 due to its frameshift effect. The screening of this variant in our cohort of 80 singleplex cases revealed one other unrelated HL patient harbouring this causative variant. Due to the limited literature on the gene and its protein, in silico studies were used to show the predicted secondary structure folding of the protein as well as potential protein binding regions. Analysis showed that the predicted loss of a stable region of the protein as well as that of a putative binding domain could explain the pathogenic nature of the variant. In vitro studies showed that the variant hindered the detection of the protein by way of a DDK tag downstream in the plasmid. Additionally, GFP-Tagged GRXCR2 showed altered expression pattern in the variant when compared to the wildtype. In summary, our data has revealed the efficacy of using next generation sequencing tools in resolving HL among sub-Saharan African patients as opposed to the single candidate gene approach. In our quest, we have employed two widely used strategies, targeted panel and whole exome sequencing (WES), both of which have had great successes in various populations. The targeted approach was able to resolve 77.8% of our families but did not detect variants for two of the families revealing the presence of other variants harboured in rarely associated gene not captured or included on the panel. This prompted for the use of a more comprehensive approach such as WES. These results corroborated with those of two families previously resolved by targeted exome sequencing. Additionally, one of the previously unresolved family was now resolved. This showed that WES was sensitive enough to detect variants in known HL genes but comprehensive enough to detect variants in other regions of the exome which have not been associated with HL or rarely associated with HL. The benefit of WES also extends to the contribution of exomic data from patients of African descent as there is an underrepresentation of this group in exome repositories as well as genomic or SNP databases. To the best of our knowledge, this is the first study to use WES to resolve HL in patients of African descent. The other benefit of such a venture is the use of this data not only for patients in SSA but also those in the diaspora. In conclusion, we have successfully demonstrated the feasibility of using NGS tools in identifying causative variants in HL patients in SSA. Additionally, we have shown that WES is a more suitable approach to trying to resolve HL in Africa. Therefore, the data strongly support that genetic studies on families segregating HL in SSA could be the next frontier of HL genetic research, of global importance through discovering novel variants in known genes, and potentially novel genes. These studies will improve HL genetic diagnosis, retrospective counselling and testing, prevention and care including future prediction of treatment outcomes in sub-Saharan Africans, and in people of African descent.
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Matias, Margret. "Comparison of medical management and genetic counseling options pre- and post-whole exome sequencing for patients with positive and negative results". University of Cincinnati / OhioLINK, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1490352906282189.
Testo completo
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Rymer, Karen. "Identification of Candidate Genes for Craniosynostosis". VCU Scholars Compass, 2015. http://scholarscompass.vcu.edu/etd/3782.
Testo completoAbstract (sommario):
Craniosynostosis is a disorder characterized by the premature fusing of cranial sutures in an infant. Premature closure of these sutures can lead to detrimental consequences on the development of a child. The two broad categories of craniosynostosis are classified as syndromic and nonsyndromic. Nonsyndromic craniosynostosis involves only the fusion of one or more sutures, whereas syndromic craniosynostosis involves other abnormalities throughout the body of the affected individual. Two of the families analyzed in this study were of the syndromic nature, and known FGFR mutations were discovered. However, phenotypical features documented in association with these mutations differed from our individuals. Two families affected with nonsyndromic sagittal synostosis were also analyzed. Within one of these families, three candidate mutations were identified as possible disease causing mutations. These mutations were found in the genes ITGAV, SLC30A9, and BAMBI. Here we analyze the function of these proteins and determine the significance of the role they may play in nonsyndromic craniosynostosis.