This web page was produced as an assignment for Genetics 564, an undergraduate course at UW-Madison.
Briefing:
For my Genetics 564 course here at the University of Wisconsin - Madison, we were to brain storm, research, and put together a brief specific aims page on our gene and syndrome, as one would do for a research grant. This was great to do, for not only were we able to understand how to write concisely, but there is so much that needs and can be done when is comes to the further understanding of Larsen Syndrome and its effects. Below is the specific aims page that I wrote for Genetics 564, and what I thought could be done to further benefit the understanding of Larsen Syndrome, and how this can start to be achieved. Hopefully in the future, prenatal genetic screening and diagnosis of Larsen Syndrome can become a norm instead of the rarity that it is today. Prenatal diagnosis before the first trimester or at latest the first 20 weeks of pregnancy will allow parents several options for their pregnancy, whether that be termination, or to get proper education by a genetic counselor to develop a birthing plan. Prenatal diagnosis will also benefit physicians on how to properly deliver the child safely as well as follow up with any surgeries and check-ups that may need to be done.
My Specific Aims (5/19/2014) :
Larsen Syndrome (LS) is a rare heterozygous genetic disorder affecting 1 in every 100,000 births (1). Common features of LS patients are facial and skeletal malformations as well as large joint dislocations that often lead to intensive orthopedic surgeries (2). The resulting cause of LS, and their large joint dislocations, is because the FLNB protein is unable to organize the actin cytoskeleton of the cell properly, leading to cartilage cells inability to compact correctly in large joints (3, 4). The FLNB protein is composed of two CH domains that bind to actin filaments, and 24 repeating filamin domains which attach the actin filaments to the plasma membrane of a cell (5).With this understanding of how FLNB works at the cellular level there is a lack of information as to how the role of FLNB in humans results in severe bone deformities and constant dislocations. To better understand how these bone deformities occur as well as affect the body through time, a knowledge of what proteins interact with FLNB would be helpful. FLNB primarily interacts with several ubiquitin proteins involved in protein degradation as well as several fibronectin receptors that are involved in cartilage cell development and migration (6). There is a lack in knowledge though as to how these interacting proteins affect FLNBs basic role in the cell, and if this change over time leads to more developmental defects. A clear understanding of how FLNB operates within the cell in different locations and with time can lead to greater hope in finding a treatment for LS.
Proper diagnosis for patients with LS is not done in a timely manner. Typically diagnosis of patients with LS is made after birth by a combination of specific phenotypic defects as well as genomic sequencing, showing a variation in FLNB. With later diagnosis comes an inability to understand the disorder fully at various stages of life. Having an understanding of the wild type FLNB protein can lead to many advancements in understanding the abnormal FLNB protein and its effects. The hope is that with research, prenatal screenings can become more likely to occur as well as lead to better management for the disorder (7, 8). When examining the roles of the wild type FLNB protein, I hypothesize that FLNBs function and interactions change over time.
The primary goal of this study is to further understand to role of FLNB across species and time, in hopes to benefit those diagnosed with Larsen Syndrome, and other related filamin syndromes, in the future. The role of FLNB across various homologous species can lead to a greater understanding of how FLNB functions in species with different characteristics, such as decreased cartilage or an absence of bones. Also examining how the role of FLNB is affected over time can provide much insight as to what to expect in patients as they age.
Specific Aim 1: To determine the role of FLNB between vertebrates and non-vertebrates.
Hypothesis: FLNB has a different function in non-vertebrates than in vertebrates.
Experimental Methods: Use Clustal Omega alignment to analyze the CH domain and filmamin domain of vertebrate and non-vertebrate species filamin B protein sequences found via BLAST. Tag filamin B protein with GFP in each non-vertebrate species to determine protein localization. Continue experiments to determine the role of filamin B in non-vertebrates to determine if a difference is seen and its role.
Specific Aim 2: To determine how FLNB interacting proteins change in bone development with age.
Hypothesis: Ubiquitin interacting protein’s function will increase with age where as fibronectin receptor protein’s function will decrease with age.
Experimental Methods: Use STRING to examine interaction protein networks of homologous species to determine a good model organism. This model organism should be very similar in terms of the human FLNB interaction network. Take cartilage or bone marrow tissue from model organism at embryonic, young, and adult stages. Tap tag the specific ubiquitin and/ or fibronectin receptor proteins from this tissue and sort. Run proteins through mass spectrometry to determine the levels of tap tagged proteins in this tissue as well as identify any new proteins.
Proper diagnosis for patients with LS is not done in a timely manner. Typically diagnosis of patients with LS is made after birth by a combination of specific phenotypic defects as well as genomic sequencing, showing a variation in FLNB. With later diagnosis comes an inability to understand the disorder fully at various stages of life. Having an understanding of the wild type FLNB protein can lead to many advancements in understanding the abnormal FLNB protein and its effects. The hope is that with research, prenatal screenings can become more likely to occur as well as lead to better management for the disorder (7, 8). When examining the roles of the wild type FLNB protein, I hypothesize that FLNBs function and interactions change over time.
The primary goal of this study is to further understand to role of FLNB across species and time, in hopes to benefit those diagnosed with Larsen Syndrome, and other related filamin syndromes, in the future. The role of FLNB across various homologous species can lead to a greater understanding of how FLNB functions in species with different characteristics, such as decreased cartilage or an absence of bones. Also examining how the role of FLNB is affected over time can provide much insight as to what to expect in patients as they age.
Specific Aim 1: To determine the role of FLNB between vertebrates and non-vertebrates.
Hypothesis: FLNB has a different function in non-vertebrates than in vertebrates.
Experimental Methods: Use Clustal Omega alignment to analyze the CH domain and filmamin domain of vertebrate and non-vertebrate species filamin B protein sequences found via BLAST. Tag filamin B protein with GFP in each non-vertebrate species to determine protein localization. Continue experiments to determine the role of filamin B in non-vertebrates to determine if a difference is seen and its role.
Specific Aim 2: To determine how FLNB interacting proteins change in bone development with age.
Hypothesis: Ubiquitin interacting protein’s function will increase with age where as fibronectin receptor protein’s function will decrease with age.
Experimental Methods: Use STRING to examine interaction protein networks of homologous species to determine a good model organism. This model organism should be very similar in terms of the human FLNB interaction network. Take cartilage or bone marrow tissue from model organism at embryonic, young, and adult stages. Tap tag the specific ubiquitin and/ or fibronectin receptor proteins from this tissue and sort. Run proteins through mass spectrometry to determine the levels of tap tagged proteins in this tissue as well as identify any new proteins.
mary-kaytjones5-19-14.pdf | |
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References:
1.) Zhang, D. et. al., (2006). "Mutations Responsible for Larsen Syndrome Cluster in the FLNB Protein." Journal of Medical Genetics, 43(24), doi: 10.1136/jmg.2005.038695
2.) Dobbs, M. B., Boehm, S., Grange, D. K. and Gurnett, C. A., (2007). "Congenital Knee Dislocation in a patient with Larsen Syndrome and a Novel Filamin B Mutation." Clinical Orthopaedics and Related Research. 446(6), doi: 10.1007/s11999-008-0196-5
3.) "Genetics Home Reference: Larsen Syndrome". Web. January 26, 2014. http://ghr.nlm.nih.gov/condition/larsen-syndrome
4.) "BCC: The skeleton, bones, and joints". Web. April 15, 2014. http://www.bbc.co.uk/schools/gcsebitesize/pe/appliedanatomy/2_anatomy_skeleton_rev4.shtml
5.) Krakow, D., et al., (2004). Mutations in the gene encoding filamin B disrupt vertebral segmentation, joint formation and skeletogenesis. Nature Genetics, 36(4), doi: 10.1038/ng1319
6.) "Fibronectin, an extracellular adhesion molecule". Web. April 24, 2014. http://biology.kenyon.edu/BMB/Chime/Fibronectin/fibro.htm
7.) Shih, J. C., et. al., (2004). Three-dimensional ultrasound diagnosis of Larsen Syndrome with further characterization of neurological sequelae. Ultrasound in Obstetrics & Gynecology, 24(1), doi:10.1002/uog.1080
8.) Kulkami, M. B., et. al., (2010). Antenatal Diagnosis of Larsen Syndrome. Indian Journal of Pediatrics. 77(7), doi: 10.1007/s12098-010-0110-5
2.) Dobbs, M. B., Boehm, S., Grange, D. K. and Gurnett, C. A., (2007). "Congenital Knee Dislocation in a patient with Larsen Syndrome and a Novel Filamin B Mutation." Clinical Orthopaedics and Related Research. 446(6), doi: 10.1007/s11999-008-0196-5
3.) "Genetics Home Reference: Larsen Syndrome". Web. January 26, 2014. http://ghr.nlm.nih.gov/condition/larsen-syndrome
4.) "BCC: The skeleton, bones, and joints". Web. April 15, 2014. http://www.bbc.co.uk/schools/gcsebitesize/pe/appliedanatomy/2_anatomy_skeleton_rev4.shtml
5.) Krakow, D., et al., (2004). Mutations in the gene encoding filamin B disrupt vertebral segmentation, joint formation and skeletogenesis. Nature Genetics, 36(4), doi: 10.1038/ng1319
6.) "Fibronectin, an extracellular adhesion molecule". Web. April 24, 2014. http://biology.kenyon.edu/BMB/Chime/Fibronectin/fibro.htm
7.) Shih, J. C., et. al., (2004). Three-dimensional ultrasound diagnosis of Larsen Syndrome with further characterization of neurological sequelae. Ultrasound in Obstetrics & Gynecology, 24(1), doi:10.1002/uog.1080
8.) Kulkami, M. B., et. al., (2010). Antenatal Diagnosis of Larsen Syndrome. Indian Journal of Pediatrics. 77(7), doi: 10.1007/s12098-010-0110-5