Date of Award
Master of Science (MS)
College of Science and Mathematics
Thesis Sponsor/Dissertation Chair/Project Chair
Carlos A. Molina
Emre U. Seli
Fragile X Syndrome (FXS) is an X-linked disorder characterized by a CGG trinucleotide repeat located within the 5’ untranslated region of the Fragile X Mental Retardation 1 gene (FMR1). The FMR1 gene is further categorized into classifications of protein function such as normal, intermediate, premutation or full mutation depending on the number of CGG repeats present23. A normal FMR1 gene exhibits anywhere between 5 and 44 repeats. An allele in intermediate range displays 45-54 repeats. A premutation allele occurs when 55-200 CGG repeats are present and levels of the FMR1 gene product, Fragile X Mental Retardation Protein (FMRP) start to deplete. When alleles fall into this range, they are subject to expansion when transmitted into subsequent generations. Additionally, female premutation carriers are at risk for inheriting Fragile X-associated Primary Ovarian Insufficiency (FXPOI), which causes the ovaries to not work correctly. A full mutation displays complete methylation of the FMR1 gene, no FMRP production, and greater than 200 repeats. An individual diagnosed with a full mutation for FXS exhibits developmental and behavioral issues that include, but are not limited to, intellectual impairment, failure to meet milestones, and lack of impulse control20.
Due to the severity of a full mutation of FXS, and risk of FXPOI and expansion, female patients seek reproductive assistance from infertility specialists. A current method of testing for FXS involves the use of quantitative Polymerase Chain Reaction (qPCR)-based single nucleotide polymorphism (SNP) genotyping for linkage analysis. The inheritance pattern of the affected allele is tracked throughout this process; however, the number of repeats present is not observed. This project aims to establish a method for detecting Fragile X CGG repeats within embryo trophectoderm (TE) biopsies. The determination of CGG repeats is a crucial addition to current methods of testing to avoid the risk of expansion and to assist patients in obtaining a healthy pregnancy12.
This study was broken down into the completion of four phases. It began with the validation of Asuragen’s Amplidex PCR/CE FMR1 reagent kit to detect CGG repeats within the FMR1 gene of genomic DNA (gDNA) samples. The use of gDNA with this kit served as a gold standard for comparisons throughout this project. The next phase included, cell lines with known CGG repeat sizes to mimic TE biopsies. They were then tested on four different amplification methods. Those methods included GenomePlex WGA4, SurePlex DNA, REPLI-g Single Cell Kit, and targeted pre-amplification. Next, arrested whole embryos were used to test the ability of the chosen amplification method on embryonic samples. Lastly, TE biopsies from discarded aneuploid whole embryos to observe the developed methodology’s accuracy on clinical samples.
The gDNA results were as expected and provided a source for the validation of this methodology. Although three out of the four amplification methods being tested did not provide usable results, the REPLI-g Single Cell Kit showed very promising results. It showed 100% consistency with the expected results from the cell lines used. This amplification method was then used on arrested whole embryos and showed CGG repeat sizes as expected from parental genotypes. The TE biopsies showed encouraging results, suggesting that this procedure has potential for clinically implementation.
CGG repeat sizes and expansion can be observed using this new methodology. It has the potential application to assess the amount of expansion for patients with limited numbers of usable embryos. Transferring embryos with FMR1 premutation alleles will encompass more diligent genetic counseling and detailed consents.
Bedard, Jenna, "Establishing a Method to Detect Fragile X CGG Repeats within the FMR1 Gene of Embryo Trophectoderm Biopsies" (2018). Theses, Dissertations and Culminating Projects. 122.