It is vital to the activities of our Center that the investigative team is positioned to a) rapidly and securely archive biological samples from patients to enable the identification of pathogenic variants, and b) use primary patient material to explore the functional consequences of those variants. Core B will accomplish both of these goals. This Core will build on the capabilities of two existing Duke School of Medicine Shared Resources: the Center for Human Genetics (CHG) Biobank, and the Duke Induced Pluripotent Stem Cell (IPS) Core facility. As patients are identified in Project 1 and recruited to the study, biospecimens will be collected from them and their willing family members. Samples can take different forms, as determined by availability and opportunities during clinical procedures without additional burden to the patient. They may include cord blood, excess blood remaining from clinical draws, or tissue samples such as chorionic villi or foreskin fibroblasts. This material will be processed to yield both extracted DNA and primary cell cultures that subsequently will be used to generate a variety of transformed cell lines, including induced pluripotent (IPS) cells. The DNA will be used for whole exome sequencing and Sanger follow-up of specific candidate variants. As candidate pathogenic variants are identified, functional analyses will be initiated. The cell lines generated will be an integral resource since they are expected, in some instances, to provide a potential source of physiologically-relevant cell types for testing gene function. Importantly, although there are currently no straightforward protocols for differentiating IPS and hESCs into kidney epithelium this may become possible in the future. Meanwhile, assays for the effects of mutations on properties such as cell polarity, cell-cell adhesion and barrier formation could be carried out using simple epithelial derived from IPS cells in culture. Through the collaboration of Core B and Project 2 or Project 3 investigators, cell lines can be propagated and the Core will assist with the implementation of protocols to differentiate iPS cells into a multiple tissue types, thus expanding the cell and tissue repertoire in which we can explore the effects of genetic variants.
The ability to biobank both DNA samples and tissue material from patients enrolled in our Center are critical to our activities. Core B will provide such support, as well as education and training on the emergent field of IPS reprogramming, a critical component to the generation of physiologically relevant assays to study genetic variation.
|Angrist, M; Jamal, L (2015) Living laboratory: whole-genome sequencing as a learning healthcare enterprise. Clin Genet 87:311-8|
|Liu, Yangfan P; Tsai, I-Chun; Morleo, Manuela et al. (2014) Ciliopathy proteins regulate paracrine signaling by modulating proteasomal degradation of mediators. J Clin Invest 124:2059-70|
|Davis, Erica E; Frangakis, Stephan; Katsanis, Nicholas (2014) Interpreting human genetic variation with in vivo zebrafish assays. Biochim Biophys Acta 1842:1960-1970|
|Gee, Heon Yung; Otto, Edgar A; Hurd, Toby W et al. (2014) Whole-exome resequencing distinguishes cystic kidney diseases from phenocopies in renal ciliopathies. Kidney Int 85:880-7|
|Margolin, David H; Kousi, Maria; Chan, Yee-Ming et al. (2013) Ataxia, dementia, and hypogonadotropism caused by disordered ubiquitination. N Engl J Med 368:1992-2003|
|Ryan, Sean; Willer, Jason; Marjoram, Lindsay et al. (2013) Rapid identification of kidney cyst mutations by whole exome sequencing in zebrafish. Development 140:4445-51|
|Katsanis, Sara Huston; Katsanis, Nicholas (2013) Molecular genetic testing and the future of clinical genomics. Nat Rev Genet 14:415-26|
|Niederriter, Adrienne R; Davis, Erica E; Golzio, Christelle et al. (2013) In vivo modeling of the morbid human genome using Danio rerio. J Vis Exp :e50338|