Wes Goar and Melissa Marchal Present their Research on 10/10/16

Exome Sequencing of Three Israeli Families with Keratoconus

Wesley Goar

Purpose: Keratoconus (KT) is the most common corneal dystrophy with an occurrence rate of 1 in every 2,000 people. Currently, corneal transplantation is the only treatment for KT when visual acuity is no longer correctable by contact lenses. We hypothesize that KT is a genetically heterogeneous disease that is caused by mutations in one of several genes.

Methods: Samples from 3 Israeli KT families (16 samples) were genotyped using genome-wide SNP microarrays. The SNP data was analyzed for regions of autozygosity using PLINK. Three samples with KT (one from each family) were chosen for exome sequencing. The resulting variants were filtered based upon variant quality, predicted function, and population prevalence. The final variant list for each family was annotated with corneal expression (http://genome.uiowa.edu/otdb) to assist in prioritizing potential candidates.

Results: Using the autozygous regions, we have prioritized candidates and areas of the genome on which to focus our investigation. No plausible variations were found in genes previously reported to cause KT. In addition, no single gene with plausible mutations was shared across all three families. However, we identified at least 3 areas of autozygosity that are shared between the 3 families. Using CoNIFER, we also identified regions of the exomes with shared copy number variations.

Conclusion: Further work is needed to identify the causative mutations in these families. We will continue to pursue these through ascertainment of additional families and family members. This will allow us to further narrow the intervals of the genome in search of the causative mutations.



Melissa Marchal1 and Douglas Houston1

1The Interdisciplinary Program in Genetics and The Department of Biology, University of Iowa, Iowa City, IA, 52242

Dorsoventral (DV) body axis specification in Xenopus laevis requires the proper translocation of maternal mRNAs and proteins. Upon sperm penetration, these vegetally localized maternal determinants are transported to the future dorsal side of the embryo, in a microtubule dependent process called cortical rotation. An outcome of cortical rotation is the asymmetric activation of the Wnt/β-Catenin signaling pathway in dorsal nuclei. Accumulated β-Catenin then acts to transcriptionally activate dorsal-specific genes at the midblastula transition/zygotic genome activation. Although, previous studies have established a role for various Wnts and Wnt signaling components in DV axis determination, the function of other maternally expressed Wnts, their cognate receptors (Frizzleds), and co-receptors (LRP5/6) have not been thoroughly investigated. Moreover, the upstream factors and mechanisms responsible for initiating β-catenin stabilization, as well as their relationship to the timing of LRP6 activation by phosphorylation are poorly understood. In this work, we characterize the function of fzd1 in DV axis formation through a maternal overexpression/loss-of-function approach. We find that fzd 1 and fzd 7 are expressed abundantly in the oocyte, with fzd7 maintaining high expression levels until the tailbud stages. We show that overexpression of fzd1 results in morphogenetic defects and an expansion of dorsal specific markers. Additionally, we find that depletion of maternal fzd1 results in delayed and disrupted gastrulation, a ventralization phenotype, as well as defects in dorsal-specific gene expression. The specificity of these phenotypes were confirmed via maternal rescue experiments. We also show that oocytes depleted of fzd1, have significantly reduced vegetal microtubule arrays, suggesting that Frizzled-dependent signaling may play a role in cortical rotation. Additionally, we present evidence for the dynamic phosphorylation of LRP6 in cleavage stage embryos. We find that both fzd1 depletion and excessive Wnt signaling inhibit this phosphorylation pattern.


Posted on October 7, 2016, in Student Seminar. Bookmark the permalink. Leave a comment.

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