Analysis of variants of Dnmbp, a Putative Modifier of Cecr2 and Neural Tube Defect Susceptibility Gene

• Author / Creator

  Singh, Parmveer

• One of the most common groups of birth defects are neural tube defects (NTDs), affecting about 1 out of 1000 established pregnancies. NTDs arise during neurulation in vertebrates, preventing proper formation of the brain and/or spina cord. NTDs are characterized by an unfused neural tube, which leads to neurodegeneration due to exposure of neural tissue to the uterine environment. Both genetic and environmental factors contribute to the formation of NTDs. In mice, hundreds of genes have been associated with NTDs. One of these genes is Cecr2, which encodes a protein involved in chromatin remodelling. Mutation to Cecr2 leads to the lethal cranial NTD exencephaly in mice. However, the penetrance of exencephaly in homozygous Cecr2 mutants differs between strains. In BALB/cCrl mice, a homozygous hypomorphic Cecr2 gene-trap mutation causes exencephaly in 54% of embryos while the same mutation on an FVB/N background does not lead to exencephaly. This difference in penetrance is likely due to modifier genes having an effect on the Cecr2 mutant phenotype. Further genetic analyses revealed a major modifier locus on chromosome 19 thought to contain multiple modifiers of Cecr2. In a previous study, coding variants of genes in the modifier region that differed between the two strains of mice were identified along with expression differences. Candidate mouse genes hypothesized to be modifiers of Cecr2 were then sequenced in a cohort of 156 unrelated human cranial NTD fetuses to uncover human variants of the genes that may lead to modification of Cecr2. Human and mouse variants were ranked based on in silico analyses, rarity, and predicted function. One of the top candidates was Dnmbp, a scaffold protein involved in actin and dynamin dynamics as well as CDC42 activation. In this thesis, I hypothesized that Dnmbp modifies the penetrance of exencephaly in mice through Cecr2. Eight human variants of DNMBP and one mouse variant underwent functional testing for the preservation of protein-protein interactions, CDC42 activation, and cellular localization. Each variant was produced through site-directed mutagenesis and cloned in an expression vector with an N-terminus HA-tag. Variants were transiently transfected into cell lines for analyses. VASP and MENA are two proteins that interact with DNMBP and have been associated with NTDs. I was unable to determine if DNMBP variants maintain their interaction with VASP or MENA due to inconsistency with the co-immunoprecipitation experiment. An assay that measured the number of dense-core vesicles in neuroblastoma cells was conducted to test the level of activated CDC42. Over expression of DNMBP should lead to increased dense-core vesicles due to increased activation of CDC42. However, when the mean fluorescence intensity of a dense-core vesicle marker was measured, there was much variation. Thus, this assay did not provide information on the ability of each variant to activate CDC42. To determine if there was a genetic interaction between Cdc42 and Cecr2, a mouse model was produced on a FVB/N background containing a heterozygous deletion of Cdc42 and homozygous mutation to Cecr2. Exencephaly was not observed in mice with these two mutations, thus, Dnmbp may not be modifying Cecr2 through Cdc42. Irregularities in localization of DNMBP protein were observed with one of the variants (R1024X) in epithelial cells. Caco-2 cells transfected with R1024X did not show localization of DNMBP to the apical cell membrane, confirming that this variant is damaging and giving evidence that Dnmbp is an NTD modifier gene. While other variants did show localization to the apical membrane of epithelial cells, it is possible that they may not localize correctly to specific regions of other cell types. Thus, it would be worth investigating localization patterns of each variant in other cellular regions DNMBP is known to concentrate. To further associate Dnmbp as a modifier of Cecr2 and a NTD susceptibility gene, the best strategy would be to knockout one allele of Dnmbp in the resistant FVB/N strain of mice with a homozygous Cecr2 mutation. If penetrance of exencephaly increases, it would be well supported that Dnmbp is a modifier of Cecr2. Characterizing the function and localization of Dnmbp variants from susceptible mice and human NTD fetuses could be beneficial in revealing a possible NTD susceptibility gene as well as further elucidating the complexity of neural system development.

• Subjects / Keywords

  • DNMBP
  • Tuba
  • neural tube defects
  • Cecr2
  • NTD
  • modifier genes
  • CDC42

• Graduation date

  Spring 2017

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/R3T727Z0X

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.