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Anastasia Marie Fedick
B. S., Seton Hall University - 2010
Thesis Advisor: Nathan R. Treff, Ph.D.
Graduate Program in Microbiology & Molecular Genetics
Nelson Labs, Room B-228
Wednesday, January 8, 2014
Due to continual advances in the fields of genetics and reproductive research, it is now possible to prevent the inheritance of genetic diseases at numerous steps along the pathways that lead to new life. The purpose of this thesis was to further such developments at the preconception, pre-implantation, and post-implantation stages of reproduction. At the preconception stage, new diagnostic tests were developed, with the first utilizing TaqMan assays to accurately genotype in a high-throughput manner, and the second using digital PCR to provide detailed genotypes for deletions where copy number analysis alone would be insufficient. Carrier frequency experiments for population-specific mutations were also performed to determine if such mutations should be included in ethnic screening panels, and also to reaffirm the likelihood of pathogenicity. The completion of these experiments has resulted in the addition of mutations to existing screening panels by major companies and medical centers, including Dor Yeshorim - Committee for Prevention of Jewish Genetic Diseases and New York Presbyterian Hospital / Columbia University Medical Center.
The etiology of infertility was then examined at the pre and post-implantation stages of conception. Next generation sequencing was successfully investigated in a pilot study for the diagnosis of single gene disorders in patients undergoing fertility treatment. Accurate results were obtained from embryo biopsies for a variety of mutation types, including SNPs, compound deletions, and a single nucleotide insertion in a homopolymer stretch. The cause of azoospermia was also investigated in a cohort on infertile men by performing genetic association studies and next generation sequencing of the KATNAL1 gene. Finally, euploid products of conception were retrospectively analyzed using microarray data to look for the presence of small copy number variations. The absence of any insertions or deletions in the samples reduced the likelihood that small copy number variants are causative of such miscarriages, further narrowing the etiological possibilities behind such events.
The diagnostic tests developed throughout this thesis utilize universally applicable technologies, thereby allowing for future expansion and the possibility of application in other fields. The tests also increase the number of mutations that can be screened simultaneously and reduce the cost and turnaround time for accommodating newly identified and clinically relevant mutations. Therefore the translational research developed in this thesis has resulted in diagnostic advances that are beneficial in the fields of both genetics and infertility.