Dissertationen zum Thema „Genetic screening“

Thesis (MSc)--Stellenbosch University, 2012.<br>ENGLISH ABSTRACT: Haliotis midae (commonly referred to as perlemoen) is the only one of five endemic species in South Africa that is commercially valued both locally and internationally. Unfortunately, natural perlemoen populations have become a dwindling resource due to commercial exploitation, poaching and the influx of natural threats, such as the West Coast rock lobster, Jasus lalandii. To preserve the natural diversity and sustainability of natural populations as well as commercial stocks, genetic management and improvement of perlemoen is critical. Genetic management requires the utilisation of molecular markers, which aid in the construction of linkage maps and the identification of quantitative trait loci (QTL) associated with economically significant traits. This will allow improvement of commercial stock management in terms of broodstock selection as well as provide valuable insight into natural population dynamics. Single Nucleotide Polymorphisms (SNPs) were selected as the marker of choice due to their successful employment as molecular markers and their wide distribution and abundance within the genomes of various marine species. This study focuses on the characterisation of novel SNPs from transcript sequences generated by Next Generation Sequencing technology. Approximately 40% of the transcripts facilitated the isolation of 105 putative markers, indicating a SNP frequency of ~1% within the H. midae genome. A subset of 24 markers, in addition to 24 previously developed markers, was characterised using the Illumina GoldenGate genotyping assay with the VeraCode technology, a medium to high-throughput genotyping technology. This is the first reported medium- to highthroughput characterisation of SNPs in H. midae. The selected markers were used to determine the efficiency and overall success rate of the GoldenGate platform. Marker characterisation was completed in both natural and commercial populations to determine the utility of these markers for genetic diversity and population structure inference. An 85% genotyping success rate was achieved with the platform. Statistical analysis indicated that the markers developed in this study are suitable for applications including population genetic structure inference, genetic diversity estimation and possibly other downstream applications such as linkage mapping. These markers are considered to be invaluable for future work regarding the genetic management and conservation of H. midae.<br>AFRIKAANSE OPSOMMING: Haliotis midae (ook bekend as perlemoen) is die enigste van vyf inheemse spesies in Suid-Afrika wat noemenswaardige kommersiële waarde toon plaaslik sowel as internasionaal. Ongelukkig het kommersiële uitbuiting, wildstropery en natuurlike bedreiging (bv. die Weskus kreef Jasus lalandii), wilde perlemoen populasies noemenswaardig verminder. Dus, om natuurlike diversiteit en die voortbestaan van beide wilde en kommersiële populasies te beskerm, is genetiese bestuur en verbetering absoluut noodsaaklik. Genetiese bestuur vereis die gebruik van molekulêre merkers as ’n hulpmiddel in die opstellingvan koppelingskaarte, en die identifisering van die relevante kwantitatiewe eienskap loki (QTL) tipies geassosieer met ekonomies belangrike eienskappe. Die laasgenoemde beoog om kommersiële voorraad bestuur te verbeter, kragtens deur broeidier seleksie sowel as om insig te verskaf m.b.t. wilde bevolking dinamika. Enkel Nukleotied Polimorfismes (SNPs) is gekies as die toepaslike merker vanweë die omvattende toepaslikheid van hierdie merkers binne die genome van verskeie mariene spesies. Hierdie studie fokus op die karakterisering van nuwe SNPs vanuit transkript volgordes ontwikkel deur middel van Volgende Generasie Volgordebepaling (“Next Generation Sequencing”). ’n Beraamde 40% van transkripte het gelei tot die ontwikkeling van 105 potensiëlemerkers, aanduidend van ’n SNP frekwensie van ~1% binne die H. midae genoom. ’n Sub-versameling van 24 merkers, tesame met 24 bestaande merkers, is gekarakteriseer deur die Illumina GoldenGate genotiperings toets met die VeraCode tegnologie, ’n medium tot hoë deurvloei genotiperingstegnologie. Hierdie is die eerste berig van medium tot hoë deurvloei karakterisering van SNPs in H. midae. Die geselekteerde merkers is gebruik om die doeltreffendheid van die GoldenGate platform te bepaal. Merker karakterisering is uitgevoer in beide wilde en kommersiële bevolkings om die effektiewe bruikbaarheid van hierdie merkers m.b.t. genetiese diversiteit, en bevolking struktuur bepaling, te ondersoek. Die platform het ’n 85% genotiperingsukses syfer getoon. Statistiese analise dui daarop dat merkers ontwikkel tydens hierdie studie toepaslik is vir bevolking genetiese struktuur bepaling, genetiese diversiteitberaming en moontlik ook genetiese koppelingskartering. Hierdie merkers word bestempel as onmisbaar vir toekomstige navorsing in genetiese bestuur en bewaring van H. midae.

In vitro fertilization (IVF) is used to help infertile couples achieve a live birth. Clinical studies have suggested that multiple, consecutive cycles of IVF can increase live birth rate significantly. Others have documented improved live birth rates from the use of new laboratory techniques for preimplantation genetic screening (PGS). This genetic screening technique seeks to determine the ploidy of the embryo prior to implantation into the woman. To date, no study has examined the cost-effectiveness of using IVF in conjunction with PGS compared to that of IVF alone for 3 consecutive cycles in achieving a live birth. This study compared the incremental cost-effectiveness ratios (ICER) from each intervention arm based on the clinical probabilities for each outcome and this study was grounded in the protection motivation theory. Costs were obtained from secondary sources, such as the literature and government databases. The model was constructed using a decision-analytical approach that allowed for z test statistical analysis of the outcomes, where the ICER is the dependent variable and the independent variables are the 2 interventions. The robustness of the model was tested through univariate and probabilistic sensitivity analysis and stratified by age groups. The results showed that PGS with IVF was cost-effective for women aged under 40 and women aged 40-42, but not for women over 42. Based on a willingness-to-pay threshold of $100,000, IVF with PGS was the most cost-effective strategy in all age groups. The positive social change implication of this study is such that understanding the costs associated with a new technology to achieve a live birth is significant for society to help guide clinical treatment of these patients.

Thesis (MSc (Genetics))--University of Stellenbosch, 2006.<br>Pre-eclampsia is a multisystemic disorder with an incidence of ~6-8% in non-Caucasian women in the Western Cape. Trophoblast invasion is vital for adequate anchorage of the placenta to the uterine wall as well as for the optimisation of utero-placental blood flow in uncomplicated pregnancies. This process is facilitated by the fetal trophoblast cells that digest the extracellular matrix of the uterus by secreting various molecules, including the metalloproteinases (MMP), of which MMP-9 has an increased production during the first trimester. Leptin, an autocrine regulator of MMP-9 secretion, functions via the leptin receptor to prevent over-invasion of maternal tissues. The aim of this study was to investigate the role of the leptin (ob) and leptin receptor (obR) genes in predisposition to pre-eclampsia and involved screening the genes in South African non-Caucasian cohorts and performing statistical analysis to determine whether any variants contributed to the disease profile.

Research Project (M.B.A.) - Simon Fraser University, 2007.<br>Theses (Faculty of Business Administration) / Simon Fraser University. Senior supervisor: Dr. Aidan Vining -- Faculty of Business Administration. MBA-MOT Program. Also issued in digital format and available on the World Wide Web.

Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiac disease. Clinical presentation is heterogeneous, outcome ranging from benign asymptomatic forms to more malignant expressions resulting in sudden or heart failure death. To date, more than 450 mutations have been reported in genes encoding sarcomeric proteins, proteins of the Z-disc, intercalated discs and in genes involved in cardiac metabolism. DNA testing is helpful for confirming diagnosis in ambiguous situations, can give some prognostic information and represents the gold standard for preclinical diagnosis in family members. However, mutation screening in HCM allows positive results in a small percentage of probands and is expensive and time-consuming. Aim of this study was to identify pathogenic mutations in the most common HCM related genes and to correlate molecular defect with clinical-morphological phenotypic pattern in a large cohort of HCM probands from a single centre. Since the mutation in the index case has been identified, a cascade screening has been performed in the first degree family members searching for carriers. Genetic screening for the 4 most commonly HCM-related sarcomeric genes (i.e. MYBPC3, MYH7, TNNT2, TNNI3) was performed by denaturing high performance liquid chromatography (DHPLC) and direct sequencing in 83 HCM index-cases from large and/or “malignant” families. A subgroup of 30 probands underwent more extensive mutation screening by 12 genes (i.e. MYH7, MYBPC3, MYL2, MYL3, TNNT2, TNNI3, TNNC1, TPM1, ACTC, CSRP3, PLN and PRKAG2) array-based DNA resequencing assay. If the mutations found were novel, these were searched in a healthy control population to rule out the possibility that they represent single nucleotide polymorphisms (SNPs). Including both screening methods, our population consisted of 99 index cases (age at diagnosis 31±17 years, age at last control 45±17 years, 70% males, 65% familial cases, 38% with obstruction). Twenty-seven pathogenic HCM-causing mutations were found in 30 probands (30%). Percentage of mutation positive patients was not different in probands with HCM family history (21/64 probands, 33%) versus sporadic cases (9/35 probands, 26%; p=0.46), and irrespective of the screening method used (25/83 probands, 30%, by DHPLC and direct sequencing vs 8/30 probands, 27%, by DNA resequencing array; p=0.72). In 14 index-cases screened both by DHPLC and DNA resequencing array, there was agreement between the two different screening methods (3 mutations in 3 patients found by both methods, and just an intronic mutation “missed” by DNA resequencing array). Genes more frequently involved were MYH7 coding for beta-myosin heavy chain (11 mutations in 11 patients) and MYBPC3 for cardiac myosin-binding protein C (8 mutations in 12 patients). In a minority of probands HCM-causing genes were TNNI3, TNNT2 and MYL3, respectively in 4, 3 and one patients. Two patients had double mutation in compound heterozygous. Wide heterogeneity in clinical presentation and evolution was present in spite of genotype characterization, but when multiple mutations were detected, they were associated with particularly severe phenotype. Fifty-one members from 16 different families were screened for the mutation(s) found in their family proband and 23 (45%) resulted carriers. Eight carriers had phenotypic expression fulfilling diagnostic criteria for HCM (i.e. maximal left ventricular wall thickness, MLVWT ≥ 13 mm), whereas 10 had only minor signs suggestive of HCM (such as ECG abnormalities, MLVWT=12-13 mm, abnormal left ventricular filling pattern at echo-Doppler), and 5 were healthy carriers. Tissue Doppler Imaging seemed to be useful for preclinical diagnosis, but a multiparametric evaluation is needed to identify mutation carriers before phenotypic expression of HCM. Nowadays, mutation screening is becoming part of diagnostic and clinical management of HCM patients and family members. The spectrum of HCM-associated genes has moved outside the myofilaments of the sarcomere to encompass additional subgroups of proteins involved in the pathogenesis of HCM. Mutation carriers without HCM phenotype represent a new subgroup of patients at risk for developing disease, whose clinical and prognostic profile remains unresolved, but of particular interest as possible target for preventive therapeutic strategies that can change the natural history of this disease.<br>La cardiomiopatia ipertrofica (CMI) rappresenta la più frequente malattia cardiaca geneticamente determinata. È caratterizzata da un decorso clinico estremamente eterogeneo, che può variare da forme benigne ed asintomatiche a quadri particolarmente severi culminanti con morte improvvisa o per insufficienza cardiaca. Fino ad oggi sono state identificate più di 450 diverse mutazioni a carico di oltre 20 geni codificanti non solo proteine del sarcomero, ma anche altre strutture cellulari quali il disco Z e i dischi intercari o geni implicati nel metabolismo cardiaco. L’analisi genetica è un importante strumento diagnostico nelle situazioni dubbie, può dare talora indicazioni prognostiche, ma soprattutto consente di porre una diagnosi preclinica nei familiari di probandi affetti da CMI. Tuttavia essa consente di ottenere dei risultati conclusivi sono in una limitata percentuale di soggetti e rappresenta una metodica costosa, laboriosa ed ancora prerogativa di pochi centri specializzati. Lo scopo di questo studio è stata l’identificazione di mutazioni patogene nei geni sarcomerici più frequentemente implicati nella CMI e la ricerca di correlazioni genotipo-fenotipo in un’ampia popolazione di pazienti con CMI seguiti presso l’ambulatorio specialistico della Clinica Cardiologica dell’Università di Padova. Una volta individuata la mutazione patogena nel probando, questa è stata ricercata nei familiari di primo grado al fine di consentire una diagnosi precoce e di programmare un adeguato follow-up clinico. Data l’impossibilità di sottoporre sistematicamente l’intera popolazione seguita presso il nostro ambulatorio specialistico a tale indagine, si è deciso di procedere con un approccio razionale “a cascata” selezionando 83 casi-indice con forme fenotipicamente più severe o appartenenti a grandi famiglie in cui si erano verificati numerosi eventi maggiori e sottoponendo questi ad analisi genetica per screening di mutazioni nei 4 geni sarcomerici noti dalla letteratura essere i più frequentemente implicati nella CMI (MYBPC3, MYH7, TNNT2, TNNI3) mediante denaturing high performance liquid chromatography (DHPLC) e sequenziamento diretto. Un sottogruppo di 30 probandi è stato sottoposto ad analisi per ricerca di mutazioni in 12 geni sarcomerici e non (MYH7, MYBPC3, MYL2, MYL3, TNNT2, TNNI3, TNNC1, TPM1, ACTC, CSRP3, PLN e PRKAG2) mediante tecnica di DNA resequencing array. Qualora la mutazione trovata non fosse già nota in letteratura, questa è stata testata in una popolazione di controllo di soggetti sani, per confermare che non si trattasse di un polimorfismo. Comprendendo entrambi i metodi di screening la nostra popolazione è risultata composta da 99 casi-indice (età media alla diagnosi 31±17 anni, età all’ultimo controllo 45±17 anni, 70% maschi, 65% con familiarità per CMI, 38% forme ostruttive). Sono state identificate 27 mutazioni patogene in 30 probandi (30%). La percentuale di probandi con mutazione è risultata non variare a seconda del metodo di screening utilizzato (25/83 probandi, 30%, identificati mediante DHPLC e sequenziamento diretto contro 8/30 probandi, 27%, mediante DNA resequencing array, p=0,72), né sulla base della storia familiare di CMI (21/64, 33%, nelle forme familiari, contro 9/35, 26%, nei casi sporadici, p=0,46). Dei 14 probandi indagati con entrambe le tecniche, in 3 casi le stesse mutazioni sono state identificate con entrambi i metodi, mostrando una buona concordanza diagnostica. Una sola mutazione in una regione intronica è stata identifica al DHPLC, ma “mancata” al DNA resequencing array. I geni più frequentemente implicati sono risultati MYH7 codificante la catena pesante della beta-miosina con 11 mutazioni in 11 probandi e MYBPC3 codificante la proteina C legante la miosina con 8 mutazioni in 12 probandi. Meno frequentemente sono state riscontrate mutazioni nei geni per le troponine cardiache I e T (rispettivamente in 4 e 3 pazienti) ed in un caso è stata riscontrata una mutazione nel gene MYL3 codificante la catena leggera essenziale della miosina. In due pazienti erano presenti doppie mutazioni. Pazienti con mutazioni a carico dello stesso gene presentavano quadri clinici e decorso estremamente variabile, particolarmente severo nei pazienti con mutazioni multiple. In 51 familiari, provenienti da 16 famiglie, è stata ricercata la mutazione patogena trovata nel probando, e di questi 23 (45%) sono risultati portatori. Mentre in 8 pazienti alla presenza di mutazione corrispondeva espressione clinica di malattia, in 5 non vi erano segni di CMI e nei restanti 10 solamente alterazioni minori non ancora diagnostiche (alterazioni aspecifiche dell’ECG, spessore parietale del ventricolo sinistro tra 12 e 13 mm, alterato rilasciamento all’eco-Doppler). L’analisi con Doppler Tissutale si è dimostrata sensibile nell’identificazione di anomalie precoci nei portatori di mutazioni e, se inserita in un approccio diagnostico multiparametrico, potrebbe consentire una diagnosi preclinica. In conclusione, sebbene rappresenti un’indagine costosa e che consente l’identificazione di mutazioni patogene solo in una percentuale di pazienti variabile (circa 30%) l’analisi genetica è entrata a far parte del percorso clinico-diagnostico della cardiomiopatia ipertrofica. Il numero di geni candidati e di mutazioni è in continuo sviluppo comprendendo anche numerosi geni non-sarcomerici. Ampia variabilità clinica e fenotipica è presente nei pazienti con singola mutazione, mentre i pazienti con doppie mutazioni vanno incontro ad un decorso particolarmente severo. Lo screening genetico nei familiari per la ricerca della mutazione identificata nel probando rappresenta il gold-standard per la diagnosi precoce e può guidare il follow-up clinico (stretta sorveglianza clinica per i portatori, rassicurazione e controlli dilazionati nei negativi). Il sottogruppo di portatori sani, il cui decorso clinico rimane ancora da chiarire, rappresenta inoltre un’interessante popolazione per studiare fenomeni precoci di comparsa della malattia ed eventuali strategie preventive.

Thesis (Master of Health Policy and Administration)--Washington State University, May 2009.<br>Title from PDF title page (viewed on June 5, 2009). "Department of Health Policy and Administration." Includes bibliographical references (p. 34-39).

REVIEW OF UNIVERSAL NEWBORN HEARING SCREENING: RATIONAL PROSPECTS OF GENETIC SCREENING FOR DFNB1 RELATED DEAFNESS Introduction: Hearing loss (HL) is the most common human birth defect occuring in 1 to 3 per thousand infants. Recent evidence for a critical period for language acquisition promoted the adoption of Universal Newborn Hearing Screening (UNHS) in many countries. In Italy UNHS was first introduced in 1997; in 2002, some hospitals of Veneto region were involved in a pilot project on UNHS and now almost all hospital birth centers has adopted it. Concurrent with the enforcement of UNHS programs, an explosion of knowledge occurred in the field of genetics with the identification of over 100 genes associated with hearing loss. However, more than half of infants with nonsyndromic sensorineural hearing loss have identifiable mutations in only two genes: GJB2 and GJB6, located on DFNB1 locus. As GJB2 and GJB6 mutations are responsible up to 50% of autosomal recessively inherited hearing loss (the so called: DFNB1 sensorineural hearing loss, DFNB1 SNHL), even if with changes in prevalence and in specific mutation frequencies in different populations, the association of genetic screening for DFNB1 SNHL to UNHS may have several clinical and financial advantages. The use of genetic testing for DFNB1 SNHL as an adjunct to bedside UNHS has been discussed but there are open questions limiting its implementation: what type of testing should be done: common mutations or sequencing the entire gene? How will the results be explained (e.g. the genetic test finds out a single GJB2 mutation)? What is the cost/benefit ratio? Purpose: The aim of this thesis is to perform a critical analysis of the UNHS done in five hospitals in the region of Veneto and to discuss the advantages of the possible implementation of genetic screening for DFNB1 SNHL. Methods: Between 2008 and 2010, a survey of five well-baby nurseries in Veneto region has been conducted to evaluate the progress of the UNHS in achieving goals established by the Joint Committee on Infant Hearing (JCIH) and to identify critical aspects of the UNHS program. Particulary, the analysis aims to describe: percentage screening coverage, type of UNHS methodological protocol, number of infants affected by SNHL identified and cases of DFNB1 SNHL, extent of the population “lost to follow up”. The hospitals envolved were Padova, Thiene (VI), Castelfranco Veneto (TV), Abano Terme (PD) and Monselice (PD). The UNHS program in these centers is organized as follows: all the nurseries screened newborns by automated transient evoked otoacoustic emissions (A-TEOAEs), the ones resulted REFER at A-TEOAEs are evaluated by automated auditory brainstem response (A-ABR) in the Pediatric Clinic of Padova within the first three months of life, if this test confirms the suspicion of hearing loss, at five months of age, the infant undergo the audiological evaluation at the Pediatric Audiology Unit of Padova Hospital. Cases of SNHL identified are addressed to the Rare Disease Laboratory of the Pediatric Clinic of Padova to perform genetic testing consisting of the complete sequencing of GJB2 and GJB6. Results: .The coverage rate for all the centers is about 100%. Each hospital has his own UNHS protocol based on JCIH guidelines, adapted to the characteristics of the birth center (birthrate, personnel responsible for the UNHS, neonatal intensive care level). The prevalence of HL detected is 0.8%. About a half of patients with SNHL underwent DFNB1 SNHL genetic test. The percentage of patients “lost to follow up” is about 30% for infants born in the Pediatric Clinic of Padova and about 46% for the ones born in Thiene and Castelfranco Veneto. Conclusions: All the centers perform UNHS according to JCIH guidelines, the prevalence of HL detected is concordant with the one reported in Italian infants. Characteristics of the HL as the degree of hearing loss and the bilaterality are concordant with literature. Patients affected by DFNB1 SNHL present the phenotypic variability reported by literature. Percentage of infants “lost to follow up” is considerable but slightly lower in comparison to the American reality of UNHS. Newborn genetic testing in this setting may be helpful in reducing the percentage of patients “lost to follow up”. However, as the above mentioned questions on DFNB1 SNHL genetic testing have not yet univocal answers, before its implementation, further studies are needed.<br>CRITICITA’ DELLO SCREENING UDITIVO NEONATALE: PROSPETTIVE RAZIONALI DELLO SCREENING GENETICO DELL’IPOACUSIA Introduzione: L’ipoacusia è il deficit neurosensoriale più comune alla nascita, la sua prevalenza è stimata 1-3 ogni 1000 nati vivi. Da studi recenti emerge come vi sia un periodo critico per l’acquisizione del linguaggio, tale evidenza ha sostenuto l’adozione dello screening uditivo neonatale universale (Universal Newborn Hearing Screening, UNHS) in molti paesi. In Italia lo UNHS è stato introdotto per la prima volta nel 1997; in 2002, alcuni ospedali della regione Veneto hanno aderito a un progetto pilota sullo UNHS e oggi la quasi totalità dei centri nascita veneti lo ha adottato. Parallelamente all’adozione dei programmi di UNHS e all’identificazione precoce dei casi di ipoacusia, si è verificato un corrispondente ampliamento delle conoscenze nel campo della genetica dell’ipoacusia preverbale. In alcune popolazioni, più della metà dei casi di deficit uditivo è dovuta a mutazioni unicamente a carico di GJB2 e GJB6, poste sul locus DFNB1. Il fatto che mutazioni dei geni GJB2 e GJB6 rendono conto di almeno il 50% delle perdite uditive autosomiche recessive (chiamate DFNB1 sensorineural hearing loss, DFNB1 SNHL), benché con variazioni di prevalenza e variazioni nella frequenza di mutazioni specifiche nelle diverse popolazioni studiate, ha indotto a considerare che l’associazione dello screening genetico di DFNB1 a UNHS può presentare numerosi vantaggi da un punto di vista clinico ed economico-sanitario. Molti studi hanno valutato l’opportunità di affiancare lo screening genetico di DFNB1 a UNHS, ma vi sono ancora molte questioni non risolte: che tipo di test andrebbe eseguito: la ricerca delle mutazioni più comuni o il sequenziamento completo? Come verrebbero interpretati determinati risultati (nel caso, per esempio lo screening genetico individuasse solo una mutazione di GJB2) Il rapporto costo beneficio è favorevole? Scopo: lo scopo di questa tesi è di analizzare la modalità di esecuzione di UNHS in cinque centri nascita della regione Veneto e di valutare il razionale dell’eventuale attuazione dello screening genetico di DFNB1 SNHL. Metodi: tra il 2008 e iI 2010, è stata condotta un’indagine in 5 punti nascita della regione Veneto al fine di valutare se le modalità di esecuzione di UNHS rispondevano ai criteri stabiliti dalle linee guida internazionali stese dal Joint Committee on Infant Hearing (JCIH) e di identificare le criticità del programma di UNHS. In particolare sono stati valutati i seguenti parametri: copertura dello screening, tipo di protocollo per l’esecuzione di UNHS, numero di soggetti con ipoacusia neurosensoriale identificati e casi di DFNB1 SNHL, percentuale di pazienti che non hanno interrotto l’iter di valutazioni audiologiche (lost to follow up). I centri nascita coinvolti nello studio sono stati Padova, Thiene (VI), Castelfranco Veneto (TV), Abano Terme (PD) and Monselice (PD). Il programma di UNHS in questi ospedali è così organizzato: presso ciascun centro di accoglienza neonatale, il personale paramedico eseguiva lo screening su tutti i neonati mediante le emissioni otocusiche transienti automatiche (A-TEOAEs), coloro che risultano REFER vengono sottoposti al test dei potenziali evocati uditivi del tronco (A-ABR) presso gli ambulatori della Clinica Pediatrica di Padova entro i tre mesi di vita, se questo test conferma il sospetto di un deficit uditivo, attorno al quinto mese di vita, il bambino esegue una valutazione audiologica presso il servizio di Audiologia pediatrica dell’Ospedale di Padova. I casi di ipoacusia identificati vengono inviati al Laboratorio di Malattie Rare della Clinica Pediatrica di Padova per eseguire il test genetico che consiste nel sequenziamento completo di GJB2 e GJB6. Risultati: la copertura in tutti i centri raggiunge circa il 100%. Ogni ospedale ha redatto un protocollo per l’esecuzione del UNHS basandosi sulle linee guida internazionali del JCIH e adattandole alle peculiarità della propria realtà (natalità, personale addetto all’esecuzione di UNHS, livello di terapia intensiva neonatale). La prevalenza di ipoacusia è stata stimata 0,8%. Circa la metà dei soggetti si è sottoposta al test genetico per mutazioni di DFNB1. La percentule di pazienti che non ha seguito l’iter delle valutazioni audiologiche è circa del 30% nella popolazione nata presso la Clinica Pediatrica di Padova e di circa il 46% di coloro che provengono dai centri di Thiene e Castelfranco Veneto Conclusioni: tutti i centri eseguono UNHS secondo le linee guida interazionali di JCIH, la prevalenza di ipoacusia rilevata è nel range di quella riportata in alcuni lavori sulla popolazione italiana. Anche le caratteristiche della perdita uditiva in termini di entità del deficit e bilateralità sono concordi con quanto riportato in letteratura. Per quanto riguarda i soggetti affetti da DFNB1 SNHL, possiamo affermare che, come riportato in numerosi studi vi è variabilità fenotipica per quanto concerne la gravità della perdita. La percentuale di pazienti che interrompono il percorso diagnostico-terapeutico è importante ma minore rispetto a quella rilevata nella realtà americana. L’introduzione dello screening genetico neonatale potrebbe ridurre tale percentuale. Tuttavia, poiché le questioni precedentemente sollevate in merito allo screening genetico di DFNB1 SNHL non hanno ancora ottenuto risposte univoche, prima della sua attuazione, sono necessari ulteriori studi.

Forward genetic screening allows for the identification of any genes important for a particular biological process or phenotype. While the power of this approach is broadly agreed on, the efficacy of currently available tools limits the strength of conclusions drawn from these experiments. This thesis describes a method to optimize molecular tools for high-throughput screening, both for shRNA and sgRNA based reagents. Using large shRNA efficacy datasets, we first designed an algorithm predicting the potency of shRNAs based on sequence determinants. Combined with a novel shRNA backbone that further improves the processing of synthetic shRNAs, we built a library of potent shRNAs to reliably and efficiently knock-down any gene in the human and mouse genomes. We then went on to apply a similar approach to identify sgRNAs with increased activity. We complemented this with conservation and repair prediction to increase the likelihood of generating functional knock-outs. With these tools in hand, we constructed, sequence-verified and validated arrayed shRNA and sgRNA libraries targeting any protein coding gene in the human genome. These resources allow large-scale screens to be performed in a multiplexed or arrayed format in a variety of biological contexts. I have also applied these tools to identify therapeutic targets to circumvent cancer resistance to treatment in two different contexts. To overcome the shortfalls of single target therapy, I have developed multiplexed multidimensional shRNA screening strategy, where two genes are knocked down simultaneously in each cell. This strategy allows the identification of gene pairs that could be targeted in tandem to maximize therapeutic benefits. As a proof of concept, I have used it with a subset of druggable genes in melanoma cell lines. Moreover, we have applied our genome wide shRNA libraries to a different resistance context, stroma-mediated resistance to gemcitabine in PDAC. In this project, we performed screens in a PDAC-CAF coculture setting to try and identify cancer vulnerabilities specifically in the presence of stroma. Overall, the tools developed in this thesis allow for the efficient knockdown or knockout of any gene, both in an individual or combinatorial setting. Apart from providing a resource that will be useful for many fields, we have performed several proof-of-concept studies where we have applied our tools to identify potential cancer drug targets.

G-quadruplexes are non-canonical nucleic acid secondary structures of increasing biological and medicinal interest due to their proposed physiological functions in transcription, replication, translation and telomere biology. Aberrant G4 formation and stabilisation have been linked to genome instability, cancer and other diseases. However, the specific genes and pathways involved are largely unknown, and the work within this thesis aims to investigate this. Stabilisation of G4s by small molecules can perturb G4-mediated processes and initial studies suggest that this approach has chemotherapeutic potential. I therefore also aimed to identify cell genotypes sensitive to G4-ligand treatment that may offer further therapeutic opportunities. To address these aims, I present the first unbiased genome-wide genetic screen in cells where genes were silenced via short-hairpin RNAs (shRNAs) whilst being treated with either PDS or PhenDC3, two independent G4-stabilising small molecules. I explored gene deficiencies that enhance cell death (sensitisation) or provide a growth advantage (resistance) in the presence of these G4-ligands. Additionally, I present a validation screen, comprising hits uncovered via genome-wide screening, and also the use of this in another cell line of different origin. Sensitivities were enriched in DNA replication, cell cycle, DNA damage repair, splicing and ubiquitin-mediated proteolysis proteins and pathways. Ultimately, I uncovered four synthetic lethalities BRCA1, TOP1, DDX42, GAR1, independent of cell line and ligand. These were validated with three G4-stabilising ligands (PDS, PhenDC3 and CX-5461) using an independent siRNA approach. The latter siRNA methodology was used to screen 12 PDS derivatives with improved medicinal chemistry properties and ultimately identified SA-100-128, as a lead compound. The mechanism behind synthetic lethality with G4-stabilising ligands was explored further for DDX42, which I show has in vitro affinity for both RNA- and DNA-G4s and may represent a previously unknown G4-helicase. Also within this thesis, gene deficiencies that provided a growth advantage to PDS and/or PhenDC3 as uncovered by genome-wide and focused screening were explored. These showed enrichment in transcription, chromatin and lysosome-associated genes. The resistance phenotype of three gene deficiencies, TAF1, DDX39A and ZNF217 was further supported by additional siRNA experiments. Overall, I satisfied the primary aims and established many novel synthetic lethal and resistance interactions that may represent new therapeutic possibilities. Additionally, the results expand our knowledge of G4-biology by identifying genes, functions and subcellular locations previously not known to involve or regulate G4s.

Thesis (M.S.)--University of California, San Diego, 2010.<br>Title from first page of PDF file (viewed April 15, 2010). Available via ProQuest Digital Dissertations. Includes bibliographical references (p. 65-67).

In the post-genomic era, the question of how genes give rise to the observable diversity of morphology, physiology, behaviour and disease susceptibility is becoming of one central importance. Even in a model system as well studied as the fruit fly Drosophila melanogaster, the function of the vast majority of genes, and the mechanisms by which they give rise to such diversity, remains unknown. Drosophila behaviour represents a sensitive system in which to evaluate novel methods of determining gene function. Traditionally the analysis of behavioural phenotypes has represented a time consuming, highly subjective process. I have developed a suite of automated analysis tools (the BeFly! package) that has not only made such analyses both quicker and more objective, but has also allowed data to be examined in greater depth by making complex algorithms more accessible to users. The BeFly! package was initially used to characterise a serendipitously identified circadian mutant strain provisionally named Party on. As the Party on gene could not be conclusively mapped, and a meta analysis of existing circadian microarray data suggested that many circadian genes remained to be identified, BeFly!’s high throughput tools were employed in a novel systems biology screen in which phenotypic analysis was combined with gene expression data to identify likely gene function. This approach generated a number of novel candidate clock genes, the roles of which were further analysed using RNAi knockdown, confirming that the neuropeptide gene Adipokinetic hormone-like played a role in the clock mechanism. Given the success of our new strategy, it was widened to identify genes controlling sleep in Drosophila, leading to the identification of several genes associated with distinct aspects of sleep. In conclusion, the tools and methods developed in this thesis represent a novel, sensitive method for determining gene function applicable beyond the Drosophila model.

The Roma (Gypsies) are a European people composed of a mosaic of culturally heterogeneous populations. Linguistic analyses point to their origins in the Indian subcontinent. Cultural diversity in extant Romani populations suggests that they are descended from a mixture of Indian populations. Previous population genetic studies of the Roma have supported this claim by demonstrating the genetic heterogeneity of Romani populations. More recently, medical genetic research has detected identical founder mutations in separated Romani populations, which provides evidence of their relatedness. In this thesis, the genetic heritage of the Roma and its significance for genetic disease and research is investigated. Male and female lineages were analysed in eight traditionally endogamous Romani populations. Asian specific Y chromosome haplogroup VI-68 and mitochondrial DNA (mtDNA) haplogroup M were detected in all populations and accounted for 39% and 25% of all lineages respectively. Diversity within haplogroups was assessed by genotyping Y chromosome short tandem repeats (YSTRs) and sequencing the mtDNA hypervariable segment 1 (HVSl). Lineages within haplogroups VI-68 and M were found to be closely related suggesting that Romani populations are predominantly descended from a single Indian ethnic population. The differing historical legacies of Romani populations and adherence to endogamous practices have resulted in genetic substructure and limited diversity within populations. Thus, the Roma are shown to comprise a conglomerate of related admixed population isolates. The unique genetic heritage of the Roma provides a powerful tool for the positional cloning of monogenic disease genes. This is demonstrated through the reduction of the critical chromosomal region for a novel genetic disorder, hereditary motor and sensory neuropathy type Lom (HMSNL). In the initial report, the HMSNL disease locus was defined as a 3cM region on chromosome 8q24. In this study, refined genetic mapping utilising historical and parental recombinations observed in Romani individuals from different populations reduced the HMSNL critical interval to 202kb. Sequence analysis of two genes contained within this genomic interval found all affected individuals to be homozygous for a CT mutation in codon 148 of N-myc downstream regulated gene 1 (NDRGJ), resulting in a truncating Rl48X mutation. Investigation of the population distribution of the R148X disease allele shows that it occurs in six of eight separated Romani populations. Another founder mutation, C283Y in the y-sarcoglycan gene (SGCG), which causes limb girdle muscular dystrophy type 2C (LGMD2C), was found in two of eight Romani populations. Profound founder effects are apparent within Romani populations with a carrier frequency of 19.5% determined for the R148X mutation in the Lom population, and 6.25% for the C283Y allele in the Turgovzi population. High carrier frequencies for autosomal recessive diseases can be expected to pose a significant health risk for these communities. Thus, community-wide carrier testing represents a potential means of addressing this health problem. A pilot community based carrier-testing program was implemented in a Romani community of north eastern Bulgaria and relevant attitudes assessed by means of a questionnaire. Community-based carrier screening was demonstrated to be an appropriate approach to improving health amongst the Roma.

The development of new drug therapies traditionally requires mass screening of thousands if not millions of substances to identify lead compounds. They are then further optimised to increase potency. The screening of the large pharmaceutical compound libraries can be incredibly expensive, with the industry responding by miniaturising the assays to smaller formats, enabling the compound screening to be automated and, importantly, eliminating assay reagents that are a major contributing cost for running large screens. A potential target for such an approach is the genetic recoding site of viruses like HIV-1 and SARS. They use programmed recoding of the genetic code to regulate the translation of necessary proteins required for viable virus production. For example HIV-1 uses a -1 frameshift mechanism to regulate the ratio of the Gag to the Pol proteins, crucial for viable virus formation. The study of recoding, including readthrough of premature termination codons have most recently used bicistronic reporters with different combinations of enzymes. The most widely used plasmid bicistronic reporter utilises a dual luciferase arrangement comprised of firefly luciferase and Renilla luciferase reporters flanking the DNA being studied. Both of the luciferase enzymatic reporters emit light in response to their respective substrates. The cost of these substrates is the major issue to using luciferase reporters for high throughput screening. My study aimed at designing and developing a bicistronic assay suitable for genetic recoding that was amenable to high throughput screening. The luciferase reporters were replaced with Green Fluorescent Protein (GFP) reporters that do not require the addition of substrates. The development of a dual GFP assay required the appropriate selection of GFP fluorophores, the best arrangement of the GFPs to maximise the ratio of relative fluorescence intensity signal to background, the optimisation of the cells and growth conditions, DNA transfection, plate reader selection, and optical filter sets. Cassettes encoding protein linkers were also incorporated into the design of the constructs to separate the fluorescent proteins spatially to facilitate unimpaired folding into their functional units within the fusion protein. The assay was further improved by moving from transient transfection to stably expressing cell lines. A viable assay was almost achieved for 96 (and 384) well plates with a Z� factor compatible with the assay being suitable for high throughput screening. The assay was used to test a small collection of compounds known to interact with the ribosome and compounds known in the literature to affect frameshifting. This proof of concept was important, since it showed that the assay, with the various modifications, optimisations and miniaturisation steps, still retained the capability of correctly measuring the -1 frameshifting efficiency at the HIV-1 recoding site, and recording compound-induced modulations to the frameshifting efficiency. The compounds cycloheximide and anisomycin, for example, were shown to decrease -1 frameshifting albeit at some expense to overall protein synthesis. The dual GFP assay was also shown to be able to measure accurately changes in the frameshift efficiency brought about by mutations to the frameshift element, and additionally, it would be suitable for the detection and study of compounds, like the recently reported PTC-124 (currently undergoing phase II clinical trial for Duchenne Muscular Dystrophy and cystic fibrosis) that increases readthrough of a UGA premature stop codon mutation. The dual GFP assay developed in this study is at most only 1/10th of the cost of a comparable dual luciferase assay, largely due to removal of assay substrates and transfection reagents. The assay has a robust Z� factor comparable to that of the dual luciferase assay, and would substantially decrease the costs of high throughput screening in situations where a bicistronic reporter is required. The HIV-1 frameshift element is such a site.

Genetic and chemical biology screens of C. elegans have been of enormous benefit in providing fundamental insight into neural function and neuroactive drugs. Recently the exploitation of microfluidic devices has added greater power to this experimental approach providing more discrete and higher throughput phenotypic analysis of neural systems. This repertoire is extended through the design of a semi-automated microfluidic device, NeuroChip, which has been optimised for selecting worms based on the electrophysiological features of the pharyngeal neural network. This device has the capability to sort mutant from wild-type worms based on high definition extracellular electrophysiological recordings. NeuroChip resolves discrete differences in excitatory, inhibitory and neuromodulatory components of the neural network from individual animals. Worms may be fed into the device consecutively from a reservoir and recovered unharmed. It combines microfluidics with integrated electrode recording for sequential trapping, restraining, recording, releasing and recovering of C. elegans. Thus mutant worms may be selected, recovered and propagated enabling mutagenesis screens based on an electrophysiological phenotype. Drugs may be rapidly applied during the recording thus permitting compound screening. For toxicology, this analysis can provide a precise description of sub-lethal effects on neural function. The chamber has been modified to accommodate L2 larval stages C. elegans and J2 stage G. pallida showing applicability for small size nematodes including parasitic species which otherwise are not tractable to this experimental approach. NeuroChip may be combined with optogenetics for targeted interrogation of the function of the neural circuit. NeuroChip thus adds a new tool for exploitation of C. elegans and G. pallida and has applications in neurogenetics, drug discovery and neurotoxicology.

The psychological response of 64 women identified as CF carriers and their partners who received a negative test result were assessed together with selected controls on 4 further occasions: 10 on receiving the carrier's positive test result; 2) on receiving the partner's negative test result; 3) six weeks later; 4) six weeks after delivery. Knowledge of the genetics of CF and attitude to having been screened were measured by self-administered questionnaire. Compared to control subjects, carriers showed a significant increase in generalised psychological disturbance attributed specifically to symptoms of anxiety and depression during the period awaiting their partner's test result but returned to control levels on receipt of a partner's negative test result. Although there was no significant difference in generalised psychological disturbance between partners and their selected controls, partners did become significantly more anxious and manifested signs of inadequacy while awaiting their own test result. All four groups were well informed about the genetics of CF and the significance of being a gene carrier, although 23% of carriers felt information given at the booking clinic was insufficient. 20% of carriers felt regret or ambivalence about having been screened. There was a consensus that screening should be routinely offered to pregnant women but should also be made available in family planning clinics and GP centres. Results showed that the implications for midwifery practice focus on 3 areas of care: information giving; counselling; and emotional support.

Considerable development in the field of nanotechnology is increasingly yielding novel applications of nanoparticles. The unique properties of nanoparticles in particular their high aspect ratio (length : width ratio), however could pose potential risks to the user. A high throughput genetic screening platform, RISCI (robotic single cDNA investigation), was previously established for the systematic evaluation of single gene activities. Here, RISCI was utilised to identify pro-apoptotic genes as well as genes involved in the positive and negative regulation of silica nanoparticle-induced cell death. This project describes the further development of the screening platform by harnessing its capability to screen a cDNA library comprising approximately 30,000 full length, completely annotated, and sequenced human genes for novel regulators of apoptosis. It integrates an extensive skill sets and is broadly organised into three major phases: Setup, Screen and Analysis. The integration of a pro-apoptosis treatment to screen for inhibitors and sensitizers is a novel aspect of the current experimental setup, along with the low redundancy library. The extensive setup phase focused on technical aspects. The cDNA library, acquired as plasmid DNA, was transformed into a bacterial host for replication and subsequent DNA isolation. A new high-throughput process was developed encompassing the production of competent bacteria and a heat shock transformation protocol, which was subsequently transferred onto the robotic platform. In parallel, the software controlling the robots was redeveloped to allow for execution of user-defined protocols while novel transfection protocols were adapted for automation. The screen identified 699 apoptosis inducers, 1,141 inhibitors and 626 sensitizers. Bioinformatics analysis revealed that the inducers were highly enriched for cell death associated terms, while the inhibitors were strongly associated with cancer profiles. Both inducers and sensitizers were predominantly achieving the functional effect on the protein level, but inhibitors were mainly transcription based. Enriched metal response genes also suggest that the silica nanoparticles were causing their toxicity through reactive oxygen species generation. Intriguingly, the screen identified many noncoding sequences as being functionally capable of regulating apoptosis. These noncoding candidates are capable of regulating the protein coding counterparts identified from the screen. The truly interesting part of the project outcome remains those unknown candidates that were implicated in apoptosis regulation for the first time. Dissemination of the consolidated candidate list would help accelerate the experimental validation of these candidates and aid other researchers in deriving novel hypotheses when the candidates are placed in their research context. [For supplementary files please contact author].

On the 26th June 2000, the US President, Bill Clinton and the United Kingdom (UK) Prime Minister Tony Blair, announced in a joint satellite broadcast that the first working draft map of the human genome had been completed ahead of the 2003 schedule of the Human Genome Project (HGP). Less than a year later on the 12th February 2001, the full sequence of the human genome was made public on the Internet by the Human Genome Project team. The primary goal of the HGP is to acquire the fundamental information needed to further our basic scientific understanding of human genetics and the role various genes play in health and disease. The potential use of genetic diagnostic technology for population screening could potentially improve the predictability of common and rare diseases that affect the community, and could signal the beginning of a new era in public health. However public health practitioners and policy makers must be cognisant of the complex social, ethical and legal implications of genetic medicine and consider the role of genetics in public health beyond the tradition of surveillance and medical intervention. Public health policy is yet to clearly articulate the benefits and disadvantages of gene technology in population health. In this context, the aim of the study was to gain a critical insight into perceptions of self-help group members and to describe the attitudes and beliefs of genetic health service providers about genetic screening and testing and make recommendation for public health policy. The findings of this study are from eighty-three (83) people from thirteen genetic self-help groups in South-east Queensland who were interviewed or participated in focus groups, and a survey of eighty-six (86) genetic health service providers. The study methodology was based on the principles of constructivism whereby the findings of the inquiry are presented as multiple, holistic and sometimes competing realities. A hermeneutic dialectic process was used to explore the perceptions and understanding from genetic self-help groups about genetic testing and screening in combination with a survey of health professionals from clinical genetic services from around Australia. Genetic self-help group members are in a unique position to share their perceptions about genetic screening and testing. Much of their experiences and understandings closely relate to the disease or disorder that impact on their lives. They share the experience of genetic disease, but each experience is different. Being a parent, partner, sibling, carer or sufferer shapes these perceptions. Drawing from the sociological literature, two themes were revealed. These were 'Risk' and 'Civil Society'. The theme of 'Risk' revealed a number of sub-themes that included: • Having a Choice • Burden of Decision Making for Women • Preparing Parents The theme of 'Civil Society' revealed a number of sub-themes that included: • The New Eugenics • Valuing Diversity • Seeking Perfection The narratives demonstrated a high degree of awareness and understanding of the complexity of issues that surround gene technology and how there is no singular means of interpreting them. Risk and Civil Society were useful sociological lens through which to consider the implications of genetic screening and testing. Findings from the national survey of genetic health professionals revealed a greater number of women working in the area of genetic counselling and administration. Consumers were poorly represented in the development of policy and services however health professionals indicated strongly that there was a need for close collaboration and coordination of services between self-help groups and clinical services. The majority of respondents indicated that their organisation had either formal or informal networks with self-help groups. The stated attitude of the patient toward self-help, the severity of the disease and the patients' geographical accessibility of the group influenced the main reasons for referral to self-help groups. On issues of privacy, 40 percent of the respondents felt that current legislation did not provide adequate protection of personal genetic information and that genetic information should only be provided to interested third parties with the consent of the individual. The need for community education at various levels was considered essential, particularly to address the issues of discrimination and stigmatisation. Health professionals also had differing levels of acceptance of mild and severe mental and physical disability. Individuals with severe mental and physical disabilities were considered to contribute less to the richness and diversity in society. A number of recommendations have been made as a result of this study and these relate to consumer participation in public health policy and clinical service development and shaping the future direction of genetic technology in public and population health.

Introduction: Tay Sachs Disease (TSD) is a fatal genetic disorder with autosomal recessive inheritance, occurring more frequently in Ashkenazi Jewish (AJ) populations. No cure or effective treatment exists. Carrier couples have a 1 in 4 risk that a baby will have TSD. The desire to prevent TSD inspired the first pre-conception genetic screening programs. The development of assisted reproductive technologies and pre-implantation genetic diagnosis of embryos has broadened reproductive options available for carrier-couples to avoid having a child with a serious genetic condition. Aim: My aims were to discover: • The allelic distribution of HEXA mutations in the Australian AJ community • The accuracy of TSD carrier risk self-determination in AJ individuals aged 20 to 40 years when questioned by health professionals • Evidence that TSD carrier screening prevents cases • Evidence of reported access to TSD screening • Evidence to make recommendations for primary care clinicians about TSD screening. • Clinical impacts and ethical issues raised by expanded genetic screening strategies. Methods: This thesis by publication represents my research work with Australian TSD screening programs (2008-2013). Results: A high TSD carrier frequency exists amongst Australian AJ individuals currently aged 20-40 years. Disclosure of AJ heritage can accurately predict TSD carrier risk. Screening programs have been effective in preventing TSD cases. Systematic review of the international literature on TSD screening formed NHMRC graded evidence-based best practice recommendations for primary care clinicians. Australasian clinical practice guidelines for genetic screening in Ashkenazi Jews are presented. Conclusion: Preconception genetic screening is effective in rare recessive disease prevention, with important implications for Australian health-economic policy.

Wheat diseases such as rusts and powdery mildews are among the most important and ancient diseases that affect wheat cultivation worldwide. The pathogen race specific resistance genes cannot maintain long lasting resistance. On the other hand, the presence of genes confers the non-race specific resistance last much longer. The durable resistance phenotypes in wheat against various rust and powdery mildew diseases were reported as Lr34, Yr18, and Pm38 separately<br>nevertheless, they were known to locate very close to each other based on linkage analysis. Recently, it was shown that all of these resistance phenotypes are indeed conferred by the same gene, encoding Adenosine triphosphate&ndash<br>binding cassette transporter (ABC-transporter) type protein. The way with which this transporter is functioning to maintain the durable resistance against different types of pathogens is still unknown. v The presence of LR34 (or ABC-transporter) gene resistant allele can be detected by specially designed markers. In spite of the few DNA sequence differences between the resistant and the susceptible alleles, the easily applicable PCR based markers allow the detection of the presence of this durable resistant allele on any given variety or cultivar. In our study, 62 different Turkish bread wheat varieties were screened by the gene specific molecular markers, developed from those LR34 gene mutation sites. The 11 cultivars determined to contain the resistant allele. This study is the first screening of Turkish cultivars for the presence of LR34 gene.

It is increasingly apparent that target-based drug discovery is not delivering adequate numbers of clinical candidates for treatment of malignancies. The main problems encountered are target identification, validation and late stage attrition. New complementary techniques are required to increase the success rate of cancer drug discovery. Phenotype-guided discovery, using chemicai genetic screens, provides an alternative strategy that circumvents many of the issues associated with target-based discovery. Zebrafish is a vertebrate model uniquely suited to such high throughput screens, and establishing relevant neoplasia models would enable screening for novel anti-cancer therapeutics.

The aim of a newborn screening programme is to provide the early detection of infants with a disorder resulting in a reduction in morbidity and mortality using tests that have an optimal balance between sensitivity and specificity. In most instances screening tests using DNA mutational analysis have been used to provide confirmation of abnormal levels of biochemical markers. However with high throughput automated instruments, DNA from dried blood spot samples, can not only be used as a second tier screening test for disorders such as cystic fibrosis (CF), but possibly also provide a primary screen for a large number of disorders, for example fragile X syndrome (FXS). This study evaluates the feasibility and reliability of DNA based testing on dried blood spots for CF and FXS. CF is a severe recessively inherited disorder which mainly affects the lungs and pancreas. The current protocol for the screening for CF in NSW, initially tests for a pancreatic protein (immunoreactive trypsin, IRT). Samples with an elevated IRT are further tested for DNA mutations. If 2 mutations are detected the patient is referred to a CF clinic for treatment; if only one mutation is found, a sweat test is performed; and if no mutations are found there is no further follow-up. There are over 1500 disease causing mutations in the cystic fibrosis transmembrane conductance regulator gene with p.F508del, the most common, being the only one tested for in routine newborn screening in NSW. The next most common mutations in the NSW CF population are p.6551 D and p.G542X, both found on the same exon. The CF screening protocol was expanded to include p.G551 D, p.G542X and p.R553X found in the same CFTR exon using high-resolution DNA melting (HRM). HRM is mainly a scanning assay but with the addition of an unlabelled probe can be used as a genotyping assay. Using HRM, 522 consecutive specimens with raised IRT and heterozygous for p.F508del were analysed. Of the 522, 77(14.8%) were confirmed to have CF by sweat testing. Using the HRM assay there were 20 babies identified with CF who were compound heterozygotes: 12 with p.G551 D; 1 with p.R553X and 7 with p.G542X. This would allow these 20/77 babies to receive appropriate treatment in less than half the time of waiting for sweat test results, thus improving morbidity. FXS is the most prevalent heritable cause of cognitive impairment and is primarily a triplet CGG repeat disorder in the FMR1 gene. As a FXS pilot study 2094 women delivering at John Hunter Hospital were approached by a genetic counsellor in the postnatal period, and asked if they were willing to have the routine NBS sample also tested for FXS. The standard PCR screening assays for FXS are not able to identify large premutations and full mutations which fail to amplify or to differentiate between homozygous normal females and females with one expanded allele. A published method for the detection of fragile X alleles in both males and females in the normal, premutation, and full mutation ranges using a PCR assay with a chimeric CGG-targeted primer was modified to provide improved reliability when analysing routine newborn screening dried blood spot samples. Both a standard PCR-based FXS assay and the chimeric primer assay were performed in parallel to assess the results. Babies with fragile X allele sizes greater than 58 were formally reported and familial studies were undertaken to assess FMR1 allele stability upon transmission from generation to generation. Of 2000 (1013 males and 987 females) newborn screening dried blood spot samples analysed: 1986 had a CGG repeat number only within the normal range; 8 had intermediate alleles, 50 — 58 CGG repeats, (1/165 females and 1/507 males); 6 had premutation alleles, 59 - 200 CGG repeats (1/197 females and 1/1013 males). There were no samples with a CGG repeat number within the full mutation range, >200 CGG repeats. Whilst no samples had persistent PCR amplification failure, 3% required repeat testing which included 56 PCR failures, where the results were too faint to verify thus requiring re-analysis, and 5 extraction failures. For this small cohort there was complete concordance between the results for the standard fragile X assay and the chimeric assay. In conclusion, the HRM unlabelled probe genotyping CF assay provides a rapid, reliable assay for use in newborn screening and the technique could easily be expanded to screen for additional mutations. Similarly the chimeric FXS assay can reliably identify, from routine dried blood spot samples, FMR1 alleles in the normal, premutation and full mutation ranges in both males and females. However, despite the automation used, the person time and cost required to test for FXS currently does not make it feasible for routine primary screening of newborns. At present DNA mutational analysis is useful for second tier testing on the initial sample reducing time to diagnosis and parental anxiety.

The emergence and rapid spread of artemisinin-resistant Plasmodium falciparum in southeast Asia highlights the importance of identifying genetic determinants of drug response and discovering novel potent antimalarials. In support of these efforts, the parental lines and progeny of the 803xGB4 genetic cross were characterized by pharmaceutical compound screening and whole-genome sequencing (WGS). Results of the compound screen identified 52 highly-active compounds with potential for development as antimalarials. A confirmatory screen was performed to more accurately define parasite responses to 384 of the 2,816 compounds in the primary screen. Linkage analysis revealed 25 genetic loci significantly associated with parasite response to 61 compounds. A locus on chromosome 5 containing pfmdr1 was linked to response to six compounds, including the widely-used antimalarial mefloquine and the potential antimalarial triclosan. Progeny bearing the 803 allele at this locus were less sensitive to all six of these compounds. Results from RT-PCR experiments indicated increased transcription of pfmdr1 in 803 in the absence of copy number variation (CNV), implicating a possible promoter polymorphism. Analysis of WGS data suggested the presence of several possible CNVs, including a deletion of Rh2b in 803 inherited by a proportion of progeny, and a non-Mendelian amplification of Rh2a and Rh2b in three progeny. Results from PCR and RT-PCR experiments confirmed the deletion event, and indicated increased Rh2a and Rh2b transcript levels in one of the progeny with the amplification. High-throughput compound screening and WGS enable the discovery of parasite biological responses that have the potential to further malaria elimination efforts.

More than a decade after the completion of the human genome project, the function of a large number of genes remains to be elucidated. Forward and reverse genetic approaches have proven to be powerful tools to study gene function and have provided insights into fundamental biological processes. Furthermore, functional genetic screening can lead to a better understanding of the action of endogenous and exogenous stimuli such as hormones or drugs on biological systems. Thus far, systematic and unbiased studies have largely been limited to model organisms. However, complex disease-relevant genotypes and phenotypes cannot be studied in entirety in lower organisms creating a need for systematic approaches in human cells. This thesis describes a series of studies using forward and reverse genetic approaches combined with state-of-the-art technology in haploid human cells. The first chapter describes the development of a quantitative phenotypic read-out using a novel application of RNA-sequencing that allows the functional annotation of genes in signalling pathways. The presented data demonstrate that the employed shallow RNA-sequencing method is scalable and suitable as a read-out for reverse genetic screening. The second chapter focuses on the implementation of this method in a large reverse genetic study in human cells to functionally annotate tyrosine kinases in signalling pathways upon stimulation with a set of ten polypeptides and small molecules. The screens revealed known and unexpected interactions between different signalling molecules and pathways, validating the technical approach in a biological context. The third chapter presents a pilot study describing the set-up of a forward genetic technique for compound mode-of-action screening using a pooled human mutant cell line collection. The chemical genetic approach displayed sufficient sensitivity and allowed to monitor thousands of gene-drug interactions simultaneously. Together, this thesis combines elements to advance technological and biological aspects of functional genomics and chemical genetics.

Polycomb Group (PcG) proteins are responsible for post-transcriptional modifications in histone tails leading to chromatin condensation and changes in gene expression. In Arabidopsis thaliana, curly leaf (CLF) is a member of the Polycomb Reporssive Complex 2 (PRC2), which cnfers a repressive epigenetic mark, namely trimethylation of histone H3 at lysine 27 (H3K27me3). In the clf mutant, the expression of the floral organ identity gene AGAMOUS (AG) is derepressed in vegetative stages and coincides with loss of H3K27me3 at the AG locus. Recent whole genome prfiling studies have suggested that PcG genes regulate mang more developmental regulators than AG (about 15% of Arabidopis genes). However, it remains unclear what the relevance of PcG regulation of these targets is for plant development; in addition, it is not known how changes in J3K27me3 casue gene repression in plants. To unravel the role of CLFcin A. thaliana, a T-DNA mutagenesis in the clf background was performed to identify mutations enhancing or suppressing the Clf- phenotype, as these may identify additional PcG genes and targets. Firstly, I screened an A. thaliana T-DNA mutagenized population and identified four mutations suppressing the Clf- phenotype: suppressor of polycomb 1 to 4 (sop1, sop2, sop3 and sop4). Secondly, I characterized these four mutants. The sop1 mutant had normal flowering time and the suppressed phenotype is due to a loss of function mutation in SEPALLATA3 (SEP3). I establied the SEP3 is an activator and a co-factor of AG. Also, I found that SEP3 is stronlgy mis-expressed in clf mutants and SEP3 chromatin is enriched the H3K27me3, which stronly suggests that SEP3 is a direct target of CLF. In addition, I showed that a mutation in Flowering Locus T (FT), which is a positive regulator of SEP3, suppreesed the Clf- phenotype suggesting the FT is also a target of CLF. Suppressors sop3, sop3 and sop4 are late flowering, unlike sop1, and show increased expression of Flowering Locus C (FLC), a MADS-box transcription factor gene that represses flowering. I found that the sop4 mutation in likely casued by disruption of FPA, a predicted RNA binding protein that promotes flowering time by repressing FLC. Consistent with this, sop4 mutants show hight levels of FLC. Unexpectedly, fpa clf (sop4) mutatns are much later flowering than clf FRI mutants, which have similarly high levels of FLC. This suggests that FPA may regulate other genes controlling flowering thant FLC. The genes involved in sop2 and sop3 mutants remain to be identified. In this thesis I brought genetic and molecular evidence showing that CLF, though the PRC2, control floral induction (FLC), floral integration (FT) and floral organ formation (SEP3 and AG) in A. thaliana.

Congenital bicuspid aortic valve (BAV) is the commonest cardiac defect occurring in 2% of the general population causing aortic valve disease and is associated with ascending aortic aneurysms. Different genetic, metabolic and haemodynamic studies have tried to explain the aetiology of BA V and the development of aortic aneurysm. However, to date, the aetiology of BA V is not fully understood. 1.1.2 Aims To determine whether BAV is due to a genetic mutation in GATA5, fibrillin-l (FBN]) or transforming growth factor beta receptor-2 (TGFBR2), genes recognised as essential for valvulogenesis and aortogenesis. In addition, to perform a comprehensive transcriptomics analysis of the extracellular matrix (ECM) of BA V aortas with or without aneurysm. A third aim was to determine the dilatation of the aorta following aortic valve and/or root replacement. Our aims can aid us in understanding the different pathologies involved in aortic aneurysm development and its outcome. 1.1.3 Methods A consecutive series of 65 patients were included in the study. Thirty-five patients with BAV and 30 with tricuspid aortic valve (TA V) with and without aortic aneurysm were studied prospectively. Twenty millilitres of venous blood was collected from patients and a small aortic sample was taken from the aortotomy site during surgery. Different methods were used to analyse the samples; blood was used for genomic DNA analysis to determine the presence of genetic mutations. Gene microarray was performed on 24 aortic tissue samples to determine the differentially expressed genes amongst the different groups and real time polymerase chain reaction (qPCR) analysis was performed to validate the results. 9 • In addition 395 BAV and TAV patients with and without aortic aneurysms were studied retrospectively by collecting 2D echo cardiograms and CT scan data to determine dilatation of the remaining aorta following aortic valve and/or root replacement at 5 years follow-up. 1.1.4 Results In the cohort of 35 patients with confirmed BA V at surgery, one patient exhibited a missense mutation in GATA5 in the coding position 698 T>C; therefore, demonstrating for the first time in human cardiac disease that mutation in GATA5 might be causative of BAV. Gene expression data revealed impaired expression of VEGFA and COL3AI genes in TAV with aneurysm; a finding not present in the BA V group. Our clinical data showed no significant dilatation of the ascending aorta following A VR when aortic diameter <4.5cm at the time of surgery. Similarly, dilatation of the arch was not seen in patients with ascending aorta diameter 24.5cm following ARR comparing BA V with TAV patients at 5 years following surgery. This supports intervention with ascending aorta 24.5cm in BA V patients with concomitant valvular disease. 1.1.5 Conclusion -. Our findings can help a better understanding of the aetiology, pathology and progression of aortic aneurysms and a better identification of this phenotype and risk prediction. 10 •

In this thesis we explore the applicability of the X.laevis chemical genetic screening model towards drug discovery and drug development. The NCI diversity set II compound library was screened to identify abnormal pigmentation generating phenotypes that may have therapeutic application towards the treatment of melanoma cancer. 13 hit compounds identified were shown to have significantly lower IC50’s in the A375 melanoma cell line when compared to two control cell lines. Using the structural data of compounds screened (combined with the phenotypic data generated by the X.laevis screen), a report in which targets were predicted for each phenotypic category is described. Of the 10 targets predicted to generate an abnormal melanophore migration phenotype, six presented abnormal pigmentation phenotypes by compound antagonists. Two of these targets had no known previous link towards melanoma cancer. Many of the identified targets were also predicted to be targeted by nine out of 13 of the identified NCI compounds in the library screen. Thus, through a combination of forward chemical genetic screening, appropriate cell based assays and chemoinformatical analysis we have developed an efficient and effective screening strategy for the rapid identification of hit compounds that are likely to be acting through either well known or novel targets that may have possible implications towards the treatment of melanoma cancer. To assess the applicability of the X.laevis model towards drug development, in collaboration with AstraZeneca we designed a renal function toxicity assay. Renal toxicity is a serious concern in the pharmaceutical industry, being responsible for 7% of preclinical compound dropouts. I developed a biochemical assay in which renal function would be monitored by quantfying the concentration of ammonia excreted by embryos into media. A decrease in ammonia detected in the presence of nephrotoxic compounds was hypothesised to iii represent a decrease in renal function, and therefore indicate toxicity. Despite promising preliminary experiments, the original salicylic acid ammonia assay detection method was inhibited by the presence of the compound solvant DMSO. A second assay (the glutamate dehydrogenase assay (GDH)) was trialled which could not detect a change in renal function in response to nephrotoxic compounds when compared to the vehicle control. In its current form, the X.laevis renal function assay is not capable of identifying nephrotoxic compounds and so further work is required.

Introduction: In recent years, the average age of first reproduction has risen significantly, the mean now standing at around 30 years in many countries. The adverse effects of maternal age on fertility and reproduction have been well documented. However, the influence of paternal age on fertility, reproduction and postnatal health is relatively poorly understood, and 50% of all male infertility cases are classed as idiopathic or unexplained infertility. Methods: The aim of this study was to investigate factors that contribute to male infertility, split into two main parts. The first part focused on analysing data collected from patients who had undergone fertility treatment to assess the influence of different factors on infertility, especially at the genome level. The second part attempted to deal with some of the technical challenges of screening and diagnostic methods to study the genome, with the aim of providing tools that would assist future studies in pinpointing genetic factors responsible for infertility, especially in cases of idiopathic infertility. Results: Based on data from the first part of the study, it was determined that advanced paternal age can affect sperm progressive motility, sperm DNA integrity and the fertilisation rate of in vitro fertilisation (IVF) cycles, as well as the development of embryos. Direct analysis of sperm DNA fragmentation (SDF) and degradation levels revealed an association between elevated SDF and impaired embryo development. Furthermore, a correlation was shown between chromosome aneuploidy and variance in SDF and sperm DNA degradation. Moreover, aneuploidy can influence abnormal sperm morphology and consequently also progressive motility. Also, embryo development rate of IVF cycles on day three, demonstrated a significant decline in cycles where the sperm used for fertilisation had a high aneuploidy rate, which can highlight the reduced developmental capacity of aneuploid embryos. From the lifestyle factors assessed, only alcohol consumption significantly correlated with the sperm DNA damage. Therefore, poor semen quality may highlight damage that has been incurred by the sperm DNA. When the semen quality is suboptimal, the intracytoplasmic sperm injection (ICSI) technique is suggested as a standard strategy to improve the prognosis of ART. However, when the progressive motility is poor, the ICSI approach is not as effective. Based on our findings and in line with other studies, the only sperm parameter that can be affected by paternal age is sperm motility, which could be an indicator of SDF. Therefore, the decline in ICSI fertilisation rate in patients with impaired sperm progressive motility could be due to sperm DNA damage, and even ICSI cannot improve the fertilisation rate considerably. Discussion: The aim of the second part of this project was to establish a robust workflow for whole- genome amplification (WGA) and whole-genome sequencing of single cells to improve the coverage rate and fidelity, with the aim of providing means of detecting any mutation in the genome that might be responsible for reduced embryonic developmental competence. Towards this end, the efficiencies of two different WGA protocols (REPLI-g and TruePrime) were compared. Multiple technical factors required optimisation in order to create a suitable protocol. Our results demonstrated the overall superiority of REPLI-g compared to TruePrime in almost all the assessed parameters. The amplification rate of REPLI-g was much faster than that of TruePrime, and prolonged incubation led to overamplification and an increased duplication rate. However, the TruePrime method has a slower amplification rate and therefore, by increasing the incubation time, it was possible to improve the quality of the data. The modified protocol with reduced volume also had the most promising outcome in terms of the data produced, and could fulfil our expectations by being fast, cost-effective and efficient. Conclusion: In conclusion, the results from the first part of this study confirmed the negative impact of male age on assisted reproductive treatments, which can result in decreased success rates of fertilisation. Other factors such as sperm DNA damage may also contribute to this age effect, suggesting that assessing this parameter prior to fertility treatment, and attempting to mitigate elevated levels of sperm DNA damage, may be of value to older patients. Additionally, overcoming the technical challenges in studying genetic contributory factors in infertility is a promising step toward better understanding of the mutations and variations that are involved in this phenomenon.

Cell-free systems allow to perform in-vitro transcription-translation reactions without requiring living organisms, revolutionising scientific research over the last decade. This allows to easily synthesise a variety of molecular components for genetic editing applications without requiring expensive and time-consuming procedures such as cell culture, animal maintenance etc. In this work, I aimed to develop a high-throughput platform for the rapid, flexible and scalable in-vitro testing of various genetic editors, such as those part of the CRISPR/Cas repertoire. I used the commercially available E. coli cell extract (MyTXTL) in combination with a fully customisable design to generate fluorescent reporters, that allow standardised testing of various CRISPR components against any predesigned target or protospacer adjacent motif (PAM) sequences. In order to increase the scalability of this screening platform, I utilised automated liquid handling technologies (Echo 525) and explored the possibility to introduce a high throughput cloning method (BASIC assembly). I believe that this approach will be highly valuable for the screening of CRISPR components prior than their final application in in-vivo systems, such as humans or animals. These genetic editors could then be used in many biological and artificial systems, such as gene editing, metabolomics and genetic engineering.

<p>Web breaks are considered as one of the most significant runnability problems</p><p>in a pressroom. This work concerns the analysis of relation between various</p><p>parameters (variables) characterizing the paper, printing press, the printing</p><p>process and the web break occurrence. A large number of variables, 61 in</p><p>total, obtained off-line as well as measured online during the printing process</p><p>are used in the investigation. Each paper reel is characterized by a vector x</p><p>of 61 components.</p><p>Two main approaches are explored. The first one treats the problem as a</p><p>data classification task into "break" and "non break" classes. The procedures</p><p>of classifier training, the selection of relevant input variables and the selection</p><p>of hyper-parameters of the classifier are aggregated into one process based on</p><p>genetic search. The second approach combines procedures of genetic search</p><p>based variable selection and data mapping into a low dimensional space. The</p><p>genetic search process results into a variable set providing the best mapping</p><p>according to some quality function.</p><p>The empirical study was performed using data collected at a pressroom</p><p>in Sweden. The total number of data points available for the experiments</p><p>was equal to 309. Amongst those, only 37 data points represent the web</p><p>break cases. The results of the investigations have shown that the linear</p><p>relations between the independent variables and the web break frequency</p><p>are not strong.</p><p>Three important groups of variables were identified, namely Lab data</p><p>(variables characterizing paper properties and measured off-line in a paper</p><p>mill lab), Ink registry (variables characterizing operator actions aimed to</p><p>adjust ink registry) and Web tension. We found that the most important</p><p>variables are: Ink registry Y LS MD (adjustments of yellow ink registry</p><p>in machine direction on the lower paper side), Air permeability (character-</p><p>izes paper porosity), Paper grammage, Elongation MD, and four variables</p><p>characterizing web tension: Moment mean, Min sliding Mean, Web tension</p><p>variance, and Web tension mean.</p><p>The proposed methods were helpful in finding the variables influencing </p><p>the occurrence of web breaks and can also be used for solving other industrial</p><p>problems.</p>