We propose an experimental model suitable for demonstrating allele frequency change in Drosophila melanogaster populations caused by selection against an easily scorable conditional lethal, namely recessive flightless alleles such as apterous and vestigial. Homozygotes for these alleles are excluded from reproduction because the food source used to establish each generation is accessible only by flight. The observed dynamics of flightless-allele frequencies generally follows the theoretically predicted pattern, with slight deviation toward less intense selection. We also suggest observing selection against flightindependent visible marker alleles in the same population as a meaningful comparison. The proposed experiments can easily be scheduled within one semester, and the expected data provide ample opportunities for discussion of quantitative evolutionary patterns.

We report on an 8-year- old girl with severe developmental and epileptic encephalopathy due to the compound heterozygous null variants p.(Gln661*) and p.(Leu830Profs*2) in GRIN2A resulting in a knockout of the human GluN2A subunit of the N-methyl- D- aspartate receptor. Both parents had less severe GRIN2A-related phenotypes and were heterozygous carriers of the respective null variant. Functional investigations of both variants suggested a loss-of- function effect. This is the first description of an autosomal recessive, biallelic type of GRIN2A-related disorder. Nonetheless, there are marked parallels to two previously published families with severe epileptic encephalopathy due to homozygous null variants in GRIN1 as well as various knockout animal models. Compared to heterozygous null variants, biallelic knockout of either GluN1 or GluN2A is associated with markedly more severe phenotypes in both humans and mice. Furthermore, recent findings enable a potential precision medicine approach targeting GRIN-related disorders due to null variants.

Yes / Currently there is no consensus regarding services required to help families with consanguineous marriages manage their increased genetic reproductive risk. Genetic services for communities with a preference for consanguineous marriage in the UK remain patchy, often poor. Receiving two disparate explanations of the cause of recessive disorders (cousin marriage and recessive inheritance) leads to confusion among families. Further, the realisation that couples in non-consanguineous relationships have affected children leads to mistrust of professional advice. British Pakistani families at-risk for recessive disorders lack an understanding of recessive disorders and their inheritance. Such an understanding is empowering and can be shared within the extended family to enable informed choice. In a three-site qualitative study of British Pakistanis, we explored family and health professional perspectives on recessively inherited conditions. Our findings suggest, first, that family networks hold strong potential for cascading genetic information, making the adoption of a family centred approach an efficient strategy for this community. However, this is dependent on provision of high quality and timely information from health care providers. Secondly, families’ experience was of ill-coordinated and time-starved services, with few having access to specialist provision from Regional Genetics Services; these perspectives were consistent with health professionals’ views of services. Thirdly, we confirm previous findings that genetic information is difficult to communicate and comprehend, further complicated by the need to communicate the relationship between cousin marriage and recessive disorders. A communication tool we developed and piloted is described and offered as a useful resource for communicating complex genetic information. / Department of Health

Doctor of Philosophy / Department of Plant Pathology / Frank F. White / Plants have evolved sophisticated mechanisms to protect against microbial invaders of which resistance (R) genes are an important component. R genes mediate specific recognition of pathogens possessing cognate avirulence (avr) gene products, which leads to the induction of plant defense responses and the arrest of pathogen ingress. In contrast to numerous examples of R gene–avr interactions, the susceptible interaction is less well examined. Recent studies on rice and wheat indicate that host resistance to pathogens also involves genetic variability in dominant traits for susceptibility. Xanthomonas oryzae pv.oryzae (Xoo) causes bacterial blight disease in rice, a serious threat in the major rice growing regions of Asia. The pathogenicity of Xoo depends on the translocation of a cocktail of effector proteins into rice cells by a type III secretion system. The family of transcription activator like (TAL) effectors is the one of the most intriguing due to their eukaryotic features and function as major virulence determinants. The specificity of TAL effectors is determined by the nearly identical repeat units at the center of each protein. The major virulence determinant of the strain PXO99A is PthXo1, which hijacks the transcription of the host susceptibility (S) gene Os8N3, an allele of recessive resistance gene xa13. The strains that overcome xa13-mediated resistance harbor alternate major TAL effectors including PthXo2, PthXo3 and AvrXa7. Alternate effectors do not induce Os8N3. This study identified the alternate S gene Os11N3, which is dependent on the effectors AvrXa7 and PthXo3. The effectors bind to specific elements in the proximal promoter regions of the respective S genes and act as transcriptional activators. Our results indicate that rice–Xoo interactions involve gene-for-gene susceptibility to bacterial blight in addition to gene-for-gene resistance.

Autosomal recessive spastic ataxia of Charlevoix Saguenay (ARSACS) is an early onset neurodegenerative disorder resulting from mutations in the SACS gene that encodes the protein sacsin. Sacsin is a 520kDa multi-domain protein localised at the cytosolic face of the outer mitochondrial membrane with suggested roles in proteostasis and most recently in the regulation of mitochondrial morphology. An excessively interconnected mitochondrial network was observed as a consequence of reduced levels of sacsin protein following SACS knockdown in neuroblastoma cells as well as in an ARSACS patient carrying the common Quebec homozygous SACS mutation 8844delT. Moreover, it was suggested that sacsin has a role in mitochondrial fission as it was found to interact with mitochondrial fission protein Dynamin related protein 1 (Drp1). The aim of this thesis was to explore sacsin’s role in the regulation of mitochondrial morphology and dynamics in non-Quebec ARSACS patients and sacsin knockdown fibroblasts. This study shows that loss of sacsin function promotes a more interconnected mitochondrial network in non-Quebec ARSACS patients and in sacsin knockdown fibroblasts. Moreover, recruitment of the essential mitochondrial fission protein Drp1 to the mitochondria was significantly reduced in ARSACS patient cells and in sacsin knockdown fibroblasts. This reduced recruitment of Drp1 to mitochondria also occurred when cells were treated to induce mitochondrial fission. Furthermore, both the size and intensity of Drp1 foci localised to the mitochondria were significantly reduced in both sacsin knockdown and patient fibroblasts. Finally, reduced ATP production, decreased respiratory capacity of mitochondria and an increase in mitochondrial reactive oxygen species demonstrated impaired mitochondrial function in ARSACS patient and sacsin knockdown fibroblasts. These results suggest a role for sacsin in the stabilisation or recruitment of cytoplasmic Drp1 to prospective sites of mitochondrial fission similar to that observed by other mitochondrial fission accessory proteins.

Sacsin, which is mutated in the neurodegenerative disease Autosomal Recessive Spastic Ataxia of Charlevoix-Saguenay (ARSACS), is a 520 kDa modular protein with regions of homology to molecular chaperones and domains linking to the ubiquitin proteasome system. This suggests a role in proteostasis. Previously, sacsin has been shown to partially localise with mitochondria, and loss of sacsin results in elongated and dysfunctional mitochondria. Moreover, alterations in neurofilaments have recently been reported in a mouse model of ARSACS. Despite these findings, pathophysiological mechanisms of ARSACS are poorly understood. The aim of this thesis was to elucidate the cellular role of sacsin by determining how loss of its function leads to the observed mitochondrial and intermediate filament defects. This hoped to shed light on the mechanism of disease in ARSACS. The results indicate that the mitochondrial elongation seen in ARSACS is likely due to reduced mitochondrial localisation of the essential fission factor DRP1. This may be mediated by loss of function of a complex involving sacsin and dynactin-6, a subunit of the dynein-dynactin motor complex, which has previously been shown to be required for DRP1 mitochondrial recruitment. DRP1-mediated mitochondrial fission is necessary for mitochondrial quality control; hence a disruption to mitochondrial quality control is likely to occur in sacsin deficient cells, which may explain the mitochondrial dysfunction in ARSACS. Furthermore, sacsin null cells display a dramatic collapse and perinuclear bundling of the vimentin intermediate filament network. This is coupled with the displacement of cellular organelles, particularly mitochondria, early endosomes and the Golgi, which accumulate at the periphery of the vimentin bundle. These are characteristic features of aggresome formation, indicating an aggregation of misfolded protein, which occurs due to disrupted proteostasis. Further supporting this, the proteostasis components ubiquitin, HSP70, LAMP2 and p62 are recruited to the perinuclear vimentin bundles. In summary, the findings of this thesis indicate a role for sacsin in mitochondrial and protein quality control, the dysfunction of which is likely to be particularly detrimental in neurons. Mitochondrial dysfunction along with protein misfolding and aggregation are implicated in many neurodegenerative diseases, and ARSACS is no exception.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2007. / Title from PDF title page (viewed on Feb. 19, 2010). Includes bibliographical references.

Abstract Stickler and Marshall syndromes are genetic disorders both inherited in an autosomal dominant manner. The genotype-phenotype correlation was performed in ten Stickler/Marshall syndrome patients with mutations in the COL11A1 gene. Four patients had a phenotype classified as Marshall syndrome based on early-onset severe hearing loss and characteristic facial dysmorphism. A splice site mutation in intron 50 of COL11A1 was found in these patients, while the remaining six patients had an overlapping Marshall-Stickler phenotype with a mutation elsewhere in the gene. These results indicate exon 50 as a hot spot for splice site mutations leading to a phenotype of Marshall syndrome rather than Stickler syndrome. Collagen II (COL2A1) precursor mRNA undergoes alternative splicing resulting in two different isoforms, IIA including exon 2 and IIB excluding exon 2. Recent evidence indicates that premature termination codon mutations in exon 2 cause Stickler syndrome with no or minimal extraocular manifestations. Two mutations were observed in this study: Cys64Stop, and a novel structural mutation, Cys57Tyr. Results from the COL2A1 mini-gene studies suggested that both mutations altered positive cis elements for splicing resulting in a lower IIA:IIB ratio. The results further emphasize the importance of exon 2 in the development and normal function of the eye. In addition, patients displaying eye phenotypes in the absence of extraocular manifestations should be analyzed first for exon 2 mutations. Linkage analysis identified a new locus for autosomal recessive nonsyndromic hearing loss (DFNB32) on chromosome 1p13.3-22.1 in a Tunisian family with congenital profound autosomal recessive deafness. The COL11A1 gene is located in this region and was analyzed as a candidate gene. No disease causing sequence variation was observed. The analysis of 85 English and 40 Finnish subjects with high myopia resulted in the identification 23 sequence variations in the SLRP genes LUM, FMOD, PRELP, and OPTC. The two intronic variations and seven amino acid changes, one synonymous and six non-synonymous, were not found in the 308 controls analyzed. Five changes were detected in opticin, and all but one were shown to co-segregate with high myopia in families with incomplete penetrance. The results suggested that sequence variations in the SLRP genes expressed in the eye are genetic risk factors underlying the pathogenesis of high myopia.

Au cours des dernières années, la génétique a subi une progression phénoménale suite au développement de nouvelles technologies de séquençage. En effet, le séquençage de l’exome entier chez des familles a permis l’identification de nouveaux gènes impliqués pour plusieurs maladies. La neurologie a d’ailleurs bénéficié de ces avancées et plusieurs gènes ont été mis en évidence comme causatifs pour différents désordres neurologiques. Dans ce travail il sera question de deux désordres du mouvement pour lequel nous avons utilisés des technologies de séquençage traditionnelles, en l’occurrence le séquençage par Sanger, ainsi que de nouvelles technologies pour le séquençage de l’exome entier afin d’identifier de nouveaux gènes causatifs. Le premier désordre du mouvement qui sera décrit est l’ataxie, où ne seront abordées que les ataxies de cause génétiques, à transmission récessive. Le premier chapitre relatera les nouvelles mutations qui ont été trouvées chez des canadiens-français souffrant de l’ataxie de Beauce. Il sera aussi question de nouvelles mutations retrouvées dans deux autres populations, confirmant l’implication du gène SYNE1 dans les cas d’ataxie cérébelleuse à travers le monde. Le second chapitre fera la démonstration qu’il est souhaitable d’utiliser le séquençage de l’exome entier dans le but de poser un diagnostic clinique. En effet, il a été possible de trouver la cause génétique d’une famille comportant deux membres atteints d’atrophie congénitale du cervelet, où le symptôme prédominant est l’ataxie. Le séquençage de l’exome a permis la mise en évidence de mutations dans le gène PMM2, déjà connues pour cause le syndrome des glycoprotéines déficientes en hydrates de carbone. Dans un second temps, il sera question d’un autre désordre du mouvement la paraplégie spastique familiale (PSF). Le chapitre 3 relatera les mutations trouvées dans le gène CYP7B1 dans notre cohorte de patients PSF. / Over the past years, genetics has undergone a phenomenal growth due to the development of new sequencing technologies. Indeed, whole exome sequencing in families led to the identification of new genes involved in many diseases. Neurosciences were also able to benefit from these discoveries, where several new genes have been identified in several neurological diseases. This thesis will covered two different movement disorders for which we have used traditional sequencing technology, in this case by Sanger sequencing, combined with whole exome sequencing for new gene discovery. The first movement disorder that is described is ataxia, which will be focused on autosomal recessive mode of inheritance. The first chapter will relate the new mutations found in French-Canadian with ataxia of Beauce. We will also discuss new mutations found in two other populations, confirming the involvement of the gene SYNE1 in worldwide cases of cerebellar ataxia. The second chapter will demonstrate that it is desirable to use whole exome sequencing in order to make a clinical diagnosis. Indeed, it has been possible to find the genetic cause for a family with two members with congenital cerebellar atrophy, which the predominant symptom is ataxia. Exome sequencing allowed the identification of mutations in the PMM2 gene, already known to cause a syndrome leading to glycosylation deficit of glycoprotein. For the second part, we will cover another movement disorder call hereditary spastic paraplegia (HSP). Chapter 3 relates the mutations found in the CYP7B1 gene in our cohort of HSP patients.

Le Rice yellow mottle virus (RYMV) est un virus émergeant qui constitue actuellement une contrainte majeure à la riziculture sur le continent africain. Quelques rares variétés de riz, issues des espèces cultivées de riz africain et asiatique (respectivement Oryza glaberrima et O. sativa), ont récemment été identifiées comme hautement résistantes au RYMV. Ce phénotype de résistance est dû à un gène récessif RYMV1 codant le facteur d'initiation de la traduction eIF(iso)4G1 du riz.Les objectifs de cette thèse sont (i) d'étudier la durabilité de la résistance élevée du riz contre le RYMV avant son déploiement à large échelle, (ii) de caractériser les mécanismes d'émergence de génotypes contournants et (iii) d'identifier des signatures moléculaires influençant ces processus d'adaptation. Ainsi, le contournement de deux allèles de résistance, identifiés chez les deux espèces de riz cultivés, a été relié à l'émergence de mutations dans la protéine virale VPg qui permettent de rétablir l'interaction avec le facteur eIF(iso)4G1 de l'hôte résistant. Un site sous sélection diversificatrice de la VPg influence directement la capacité de contournement de la résistance élevée en fonction de l'espèce hôte. Ce site, proche des mutations de contournement, est impliqué dans l'adaptation du virus à l'espèce O. glaberrima au cours de son histoire évolutive. La démarche employée au cours de ce travail combine des études d'évolution expérimentale à des analyses fonctionnelles. Les résultats obtenus par cette approche intégrative participeront à la mise en place de stratégies de lutte intégrée à la fois efficaces et durables face à la maladie de la panachure jaune du riz en Afrique. / The Rice yellow mottle virus (RYMV) is an emerging virus currently considered as the major constraint to rice production in Africa. Some varieties of African and Asian cultivated rice (Oryza glaberrima and O. sativa, respectively), have recently been identified as highly resistant to RYMV. This resistance phenotype is caused by a recessive gene RYMV1 encoding the translation initiation factor eIF(iso)4G1 of rice.The objectives of this thesis are (i) to investigate the durability of the high resistance of rice against RYMV before broadly deployment in fields, (ii) to characterize the mechanisms of emergence of resistance-breaking (RB) genotypes and (iii) to identify molecular signatures that influence these processes of adaptation. The resistance-breaking of two resistance alleles, identified in both cultivated rice species, is mainly associated with the emergence of mutations in the viral protein VPg that restore in resistant hosts the interaction with the factor eIF(iso)4G1. A site of VPg under diversifying selection directly affects the ability to overcome the high resistance depending on the host species. This site, near the RB mutations, is involved in the adaptation of the RYMV to O. glaberrima species during its evolutionary history. The approach used during this work combines experimental evolution and functional analyses. The results of this integrative study will participate in the development of effective and sustainable control strategies toward the Rice yellow mottle virus in Africa.