NOG-related symphalangism spectrum disorder (NOG-SSD) is a broad diagnostic term for a family of 5 rare diseases with a wide range of related symptoms [1]. These diseases have all been linked to a gene known as NOG, which produces the protein noggin. Noggin is secreted from the cell and inhibits bone morphogenetic proteins (BMPs) by binding them and preventing their association with their receptors [2]. The interaction between BMPs and noggin impacts body patterning, apoptosis induction in the digital and interdigital regions, and middle ear formation [3]. Loss of noggin function in mice has been shown to cause an increase in BMP activity, as well as developmental abnormalities such as shorter bones, absence of joints, and bony fusion in the upper and lower extremities [1]. The symptoms associated with NOG-SSD diseases are mostly related to bone fusion, though there is a wide range of phenotypic features, including symphalangism, tarsal and carpal coalitions, and hearing loss caused by stapes fixation [1]. The role of NOG in the wide variety of bone development processes is unclear.
The primary goal of this project is to characterize the factors that lead to the variability in phenotype of NOG-SSD. I plan to use Mus musculus as a model organism because a common symptom of NOG-SSD is symphalangism, or fusion of the joints in the fingers and toes; therefore, an organism with these features should be used. Additionally, mice have previously been used to study the effects of loss of noggin function, so they have already proven to be a successful model [1]. I hypothesize that the variability in phenotypes seen in NOG-SSD is caused by a combination of genotypic and epistatic effects. My long-term goal is to identify potential targets for treatment of NOG-SSD.
Aim 1: Determine which amino acids in NOG lead to differential NOG-SSD bone development defects.
Hypothesis: I hypothesize that differences in genotype will cause some variation in phenotype observed in NOG-SSD, but that will not fully explain the phenotypic variability observed. Approach: I will use MEGA software to perform multiple sequence alignment to identify conserved amino acids in NOG between humans and model organisms. I will choose several conserved sites in different parts of the protein. I will then mutate the chosen sites in mice using CRISPR/Cas9 to generate models with various NOG mutations. Finally, I will compare the phenotype of these mice to see if the location of the mutation within the genome correlates with the observed phenotype. Rationale: This study will help determine whether the location of a mutation on NOG will cause the observed differences in phenotype in NOG-SSD.
Aim 2: Investigate the involvement of epistatic effects on differential NOG-SSD bone development defects.
Hypothesis: I hypothesize that epistatic effects will change the phenotype associated with a particular NOG mutation and show a different presentation of NOG-SSD. Approach: I will do a CRISPR screen on human cells in culture to identify other genes that interact with NOG. Once these potential modifier genes are identified, I will use the mutant mice generated in Aim 1 to validate the findings of the CRISPR screen. I will group the mice by their conserved site mutation. Within each group of mutants, I will mutate several identified modifier genes and compare the phenotypes of the mice with others in their group to see if the modifier gene impacts disease presentation. Rationale: In one paper, authors identified two mutations in NOG can cause multiple different phenotypes [4]. This suggests that epistatic modifiers can change the phenotype of a given mutation. This study will shed light on what genes are involved in the epistatic modification of NOG-SSD phenotypes.
Aim 3: Quantify proteomic differences that result from epistatic modifiers on differential NOG-SSD bone development defects.
Hypothesis: I hypothesize that different epistatic modifications will greatly alter the proteome between mice with the same NOG mutation. Approach: I will focus on one NOG mutation and the associated epistatic modifier mutant mice generated in Aim 2. From these mice, I will harvest osteoblasts on which I perform iTRAQ. The data generated from iTRAQ can be used to create heat maps with which to compare relative amounts of protein in the osteoblasts of mice with the same NOG mutation but with different epistatic modifiers. Rationale: NOG is a repressor of BMPs, which regulate genes involved in osteoblast differentiation and bone induction, among other things. [5] By using iTRAQ, I can quantify the differences in downstream BMP pathway proteins that result from epistatic modifiers.
The primary goal of this project is to characterize the factors that lead to the variability in phenotype of NOG-SSD. I plan to use Mus musculus as a model organism because a common symptom of NOG-SSD is symphalangism, or fusion of the joints in the fingers and toes; therefore, an organism with these features should be used. Additionally, mice have previously been used to study the effects of loss of noggin function, so they have already proven to be a successful model [1]. I hypothesize that the variability in phenotypes seen in NOG-SSD is caused by a combination of genotypic and epistatic effects. My long-term goal is to identify potential targets for treatment of NOG-SSD.
Aim 1: Determine which amino acids in NOG lead to differential NOG-SSD bone development defects.
Hypothesis: I hypothesize that differences in genotype will cause some variation in phenotype observed in NOG-SSD, but that will not fully explain the phenotypic variability observed. Approach: I will use MEGA software to perform multiple sequence alignment to identify conserved amino acids in NOG between humans and model organisms. I will choose several conserved sites in different parts of the protein. I will then mutate the chosen sites in mice using CRISPR/Cas9 to generate models with various NOG mutations. Finally, I will compare the phenotype of these mice to see if the location of the mutation within the genome correlates with the observed phenotype. Rationale: This study will help determine whether the location of a mutation on NOG will cause the observed differences in phenotype in NOG-SSD.
Aim 2: Investigate the involvement of epistatic effects on differential NOG-SSD bone development defects.
Hypothesis: I hypothesize that epistatic effects will change the phenotype associated with a particular NOG mutation and show a different presentation of NOG-SSD. Approach: I will do a CRISPR screen on human cells in culture to identify other genes that interact with NOG. Once these potential modifier genes are identified, I will use the mutant mice generated in Aim 1 to validate the findings of the CRISPR screen. I will group the mice by their conserved site mutation. Within each group of mutants, I will mutate several identified modifier genes and compare the phenotypes of the mice with others in their group to see if the modifier gene impacts disease presentation. Rationale: In one paper, authors identified two mutations in NOG can cause multiple different phenotypes [4]. This suggests that epistatic modifiers can change the phenotype of a given mutation. This study will shed light on what genes are involved in the epistatic modification of NOG-SSD phenotypes.
Aim 3: Quantify proteomic differences that result from epistatic modifiers on differential NOG-SSD bone development defects.
Hypothesis: I hypothesize that different epistatic modifications will greatly alter the proteome between mice with the same NOG mutation. Approach: I will focus on one NOG mutation and the associated epistatic modifier mutant mice generated in Aim 2. From these mice, I will harvest osteoblasts on which I perform iTRAQ. The data generated from iTRAQ can be used to create heat maps with which to compare relative amounts of protein in the osteoblasts of mice with the same NOG mutation but with different epistatic modifiers. Rationale: NOG is a repressor of BMPs, which regulate genes involved in osteoblast differentiation and bone induction, among other things. [5] By using iTRAQ, I can quantify the differences in downstream BMP pathway proteins that result from epistatic modifiers.
First Draft:
wright_specificaims_3-12-24.docx | |
File Size: | 17 kb |
File Type: | docx |
Second Draft:
wright_specificaims_4-16-24.docx | |
File Size: | 20 kb |
File Type: | docx |
Final Draft:
wright_specificaims_final_5-9-24.docx | |
File Size: | 20 kb |
File Type: | docx |
References
[1] Potti TA, Petty EM, Lesperance MM. A comprehensive review of reported heritable noggin-associated syndromes and proposed clinical utility of one broadly inclusive diagnostic term: NOG-related-symphalangism spectrum disorder (NOG-SSD). Hum Mutat. 2011 Aug;32(8):877-86. doi: 10.1002/humu.21515. Epub 2011 Jun 21. PMID: 21538686.
[2] Canalis E, Economides AN, Gazzerro E. Bone morphogenetic proteins, their antagonists, and the skeleton. Endocr Rev. 2003 Apr;24(2):218-35. doi: 10.1210/er.2002-0023. PMID: 12700180.
[3] Carlson RJ, Quesnel A, Wells D, Brownstein Z, Gilony D, Gulsuner S, Leppig KA, Avraham KB, King MC, Walsh T, Rubinstein J. Genetic Heterogeneity and Core Clinical Features of NOG-Related-Symphalangism Spectrum Disorder. Otol Neurotol. 2021 Sep 1;42(8):e1143-e1151. doi: 10.1097/MAO.0000000000003176. PMID: 34049328; PMCID: PMC8486042.
[4] Dixon, M., Armstrong, P., Stevens, D. et al. Identical mutations in NOG can cause either tarsal/carpal coalition syndrome or proximal symphalangism. Genet Med 3, 349–353 (2001). https://doi.org/10.1097/00125817-200109000-00004
[5] Ahmed, S., Metpally, R., Sangadala, S., & Reddy, B.V. (2009). Computational Design of Inhibitory Agents of BMP-Noggin Interaction to Promote Osteogenesis. World Academy of Science, Engineering and Technology, International Journal of Medical, Health, Biomedical, Bioengineering and Pharmaceutical Engineering, 3, 329-333.
[2] Canalis E, Economides AN, Gazzerro E. Bone morphogenetic proteins, their antagonists, and the skeleton. Endocr Rev. 2003 Apr;24(2):218-35. doi: 10.1210/er.2002-0023. PMID: 12700180.
[3] Carlson RJ, Quesnel A, Wells D, Brownstein Z, Gilony D, Gulsuner S, Leppig KA, Avraham KB, King MC, Walsh T, Rubinstein J. Genetic Heterogeneity and Core Clinical Features of NOG-Related-Symphalangism Spectrum Disorder. Otol Neurotol. 2021 Sep 1;42(8):e1143-e1151. doi: 10.1097/MAO.0000000000003176. PMID: 34049328; PMCID: PMC8486042.
[4] Dixon, M., Armstrong, P., Stevens, D. et al. Identical mutations in NOG can cause either tarsal/carpal coalition syndrome or proximal symphalangism. Genet Med 3, 349–353 (2001). https://doi.org/10.1097/00125817-200109000-00004
[5] Ahmed, S., Metpally, R., Sangadala, S., & Reddy, B.V. (2009). Computational Design of Inhibitory Agents of BMP-Noggin Interaction to Promote Osteogenesis. World Academy of Science, Engineering and Technology, International Journal of Medical, Health, Biomedical, Bioengineering and Pharmaceutical Engineering, 3, 329-333.
This web page was produced as an assignment for Genetics 564, a capstone course at UW-Madison.