Browsing by Author "Bech-Hansen, N. Torben"

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    Channeling Vision: Voltage-Gated Calcium Channels of Rods and Cones
    (2018-11-27) Waldner, Derek; Stell, William K.; Bech-Hansen, N. Torben; McFarlane, Sarah; Altier, Christophe
    Congenital stationary night blindness (CSNB) is a set of inherited diseases characterized by defects in neurotransmission from photoreceptors to second-order neurons in the retina. CSNB2A, specifically, is caused by mutations in Cacna1f – the pore-forming sub-unit of the voltage-gated calcium channel (VGCC), CaV1.4, which is responsible for calcium-mediated glutamate release at photoreceptor ribbon synapses. In this work, we initially expanded the characterization of the Cacna1f-KO mouse retina with a particular focus on cones using our own Cacna1fG305X mouse line. We have comprehensively characterized cone morphology and viability throughout the murine lifespan, and also verified several findings reported in an alternative Cacna1f-KO model which suggest possibility of late-stage rescue. We also identified ectopic, synapse-like cone-rod bipolar cell contacts, which have been described in no other model of retinal disease. We then sought to investigate whether late-stage rescue of retinal morphology and function is feasible in the Cacna1f-KO retina. To this end, we have designed a strategy employing transgenic mice that, with appropriate gene combinations, will allow for inducible expression of Cacna1f. We have characterized several transgenic mice relevant to these experimental aims, and provided some proof-of-concept for future experiments which may provide insight into the plausibility of therapeutic interventions. We also sought to establish an alternative model of CSNB2A in the post-embryonic chick. The relative lack of tools for genetic manipulation of this model led to us developing a novel means – avian adeno-associated viral (A3V) vectors. Using A3V vectors we were able to achieve highly efficient local transduction of photoreceptors following sub-retinal injection, thus providing us with a new tool for investigation of chicken retinal circuitry. Finally, in an attempt to replicate CSNB2A in the chicken retina, we sought to characterize VGCC expression to definitively establish the target for gene knockdown. We were able to definitively prove expression of a Cacna1f orthologue in the chicken retina, and establish its sequence and mRNA expression patterns. Unfortunately, shRNA-encoding A3Vs were unable to achieve significant knockdown, but we have established a powerful framework for future investigations in this model.
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    Characterization of murine NYX
    (2003) Sarna, Magdalena A.; Bech-Hansen, N. Torben
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    Isolation and characterization of human DNA clones from region XpII.4-XpII.22
    (1993) Moore, Brenda J.; Bech-Hansen, N. Torben
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    Isolation of human DNA clones from bacteriophage lambda libraries that identify variable number tandem repeat loci
    (1989) Kraus, Steven L.; Bech-Hansen, N. Torben
    Gene mapping is a very active area of research in human genetics. This effort is still in its infancy, with less than 2% of the expected gene complement having been mapped to chromosomes. Recent advances in DNA technology that permit the detection of DNA sequence variations, should provide the large number of highly polymorphic loci necessary to map the total human genome with high resolution. Isolation of new DNA clones that recognize highly polymorphic loci constitutes an important component of the gene mapping process. Based on its size, chromosome 9 is relatively underrepresented in terms of the available number of DNA markers. This fact, coupled with the tentative localization of the gene responsible for tuberous sclerosis, provided the incentive to attempt to isolate new chromosome 9 DNA markers. Library screening methods using oligonucleotide probes and competitive hybridization similar to those used for cosmid libraries were successfully employed to isolate a highly polymorphic marker from an unamplif ied human total genomic lambda library. Though this study failed in its attempt to isolate new highly polymorphic chromosome 9 markers from two chromosome 9-enriched lambda libraries, these studies reiterated the importance of constructing and maintaining lambda libraries with recombination deficient hosts which do not select against the repetitive sequences associated with these loci. This study demonstrates that unamplified lambda libraries can be used as a source of DNA clones that recognize variable number tandem repeat loci and defines parameters for the selection of appropriate libraries and efficient isolation of new highly polymorphic DNA markers.
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    ROHHAD and Prader-Willi syndrome (PWS): clinical and genetic comparison
    (2018-07-20) Barclay, Sarah F; Rand, Casey M; Nguyen, Lisa; Wilson, Richard J A; Wevrick, Rachel; Gibson, William T; Bech-Hansen, N. Torben; Weese-Mayer, Debra E
    Abstract Background Rapid-onset obesity with hypothalamic dysfunction, hypoventilation, and autonomic dysregulation (ROHHAD) is a very rare and potentially fatal pediatric disorder, the cause of which is presently unknown. ROHHAD is often compared to Prader-Willi syndrome (PWS) because both share childhood obesity as one of their most prominent and recognizable signs, and because other symptoms such as hypoventilation and autonomic dysfunction are seen in both. These phenotypic similarities suggest they might be etiologically related conditions. We performed an in-depth clinical comparison of the phenotypes of ROHHAD and PWS and used NGS and Sanger sequencing to analyze the coding regions of genes in the PWS region among seven ROHHAD probands. Results Detailed clinical comparison of ROHHAD and PWS patients revealed many important differences between the phenotypes. In particular, we highlight the fact that the areas of apparent overlap (childhood-onset obesity, hypoventilation, autonomic dysfunction) actually differ in fundamental ways, including different forms and severity of hypoventilation, different rates of obesity onset, and different manifestations of autonomic dysfunction. We did not detect any disease-causing mutations within PWS candidate genes in ROHHAD probands. Conclusions ROHHAD and PWS are clinically distinct conditions, and do not share a genetic etiology. Our detailed clinical comparison and genetic analyses should assist physicians in timely distinction between the two disorders in obese children. Of particular importance, ROHHAD patients will have had a normal and healthy first year of life; something that is never seen in infants with PWS.
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    Studies of the retina in the Cacna1f G305X mutant mouse
    (2006) Orton, Noelle Catharine; Bech-Hansen, N. Torben
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    The characterization of nyctalopin
    (2006) Blaker, Jessica Jade; Bech-Hansen, N. Torben
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    Using Next-Generation Sequencing to Identify Genes Mutated in Human Disorders
    (2017) Barclay, Sarah; Bech-Hansen, N. Torben; Iaria, Giuseppe; Parboosingh, Jillian; Wilson, Richard; Innes, Micheil; Wasmuth, James; Brooks-Wilson, Angela
    Next-generation sequencing – and, in particular, exome sequencing, the targeted application of next-generation sequencing to the coding portion of the genome – appeared on the scene, in the last decade, as a promising new way of efficiently searching for and successfully identifying causative mutations in human disease. I have applied exome sequencing to the study of two human traits. The first is ROHHAD (Rapid-Onset Obesity with Hypoventilation, Hypothalamic Dysfunction and Autonomic Dysregulation), a rare, complex, and potentially fatal pediatric syndrome. The second is DTD (Developmental Topographical Disorientation), a novel behavioural phenotype in which individuals are unable to effectively navigate their surroundings. Though the two phenotypes themselves have little in common, they are united in the fact that both are believed to have a genetic basis, and are therefore candidates for the application of exome sequencing to search for causative mutations. The research described in this thesis has applied exome sequencing to both ROHHAD and DTD to search for causative mutations. In neither case were we able to identify causative genes, but in both cases we were able to rule out several genetic hypotheses (rare coding mutations across most of the genome), and, perhaps most importantly, we were able to rule out exome sequencing as a means of identifying disease-causing mutations for these traits. In addition to ruling out major hypotheses as to the underlying bases of these two traits, thus clearing the way for the exploration of additional hypotheses, the ROHHAD and DTD studies also provided informative illustrations of next-generation sequencing studies that do not identify causative genes. Together, they provided a framework for reporting negative results from next-generation sequencing studies, highlighting the importance of clearly reporting the data-quality, as well as ways to limit the number of false positive variant calls and considerations for the effective filtering of DNA sequence variants to ensure that the search for disease-causing candidates has been as thorough as possible.