This web page was produced as an assignment for Genetics 564, an undergraduate capstone course at UW-Madison.
Final Specific Aims
Classic Galactosemia is characterized by the inability to metabolize the monosaccharide galactose, prompting a buildup of galactose in the bloodstream [1]. Galactosemia is caused by a variety of loss of function mutations in the GALT gene, which encodes a galactose-1-phosphate uridylyltransferase, which transfers a UDP between UDP-galalactose and UDP-glucose. [1]. Phenotypes of galactosemia are manifested in a wide variety of symptoms, including premature ovarian insufficiency in females [2]. Galactosemia is typically managed by excluding galactose from the diet, however, many symptoms of galactosemia can surface later in life. In fact, nearly 87% of female galactosemia sufferers displayed premature ovarian insufficiency, despite following a galactose restricted diet from birth [2]. Furthermore, known biochemical markers of galactosemia, namely elevated levels of erythrocyte galactose-1-phosphate and urinary galactitol, do not correlate with the long term outcome of premature ovarian insufficiency. Further, it is unknown how perturbations in carbon metabolism caused by GALT deficiency cause premature ovarian insufficiency to arise despite elimination of galactose from the diet.
My primary goal is to elucidate how GALT deficiency can lead to premature ovarian insufficiency and to illuminate the broader role of GALT outside its well-known role in galactose catabolism. My hypothesis is that previously overlooked metabolic roles of GALT, including glycosylation, play a central role in the pathology of classic galactosemia. I will use both the common mouse (Mus musculus) and Saccharomyces cerevisiae as model systems, due to their similar disease phenotypes to humans and ease of rapid analysis, respectively.
Aim 1: Characterize and identify conserved amino acids of GALT that are critical for ovary development.
Approach: I will screen the functionality of known Human GALT mutant alleles using a growth assay with S. cerevisiae. Next I will use sequence alignment methods to determine if loss of function mutations occur in evolutionary conserved sites. Finally, I will select of subset (mutated in conserved sites vs. non-conserved sites and functional vs. non-functional) of Human GALT mutants and use CRISPR/Cas9 to create transgenic mice lines with the various Human GALT disease alleles. I will then screen female mice for those that exhibit premature ovarian insufficiency.
Rationale: Not all females with galactosemia develop premature ovarian insufficiency, thus determining the mutations in GALT that lead to premature ovarian insufficiency will allow for better correlation of genotype to phenotype.
Hypothesis: I expect GALT alleles with mutations in evolutionary conserved sites will display the phenotype of premature ovarian insufficiency, while mutations in non-conserved sites will not.
Aim 2: Characterize deferentially expressed genes across ovarian development in GALT deficient mice.
Approach: I will perform RNA-seq on the ovaries of wild type and GALT deficient mice throughout ovarian development and into adulthood, mice will be fed a galactose free diet. RNA-seq data will be sorted using GO terminology and compared between both WT and GALT deficient mice and between the sampled time points.
Rationale: Genes that are deferentially regulated in the ovaries in the absence of GALT are possible targets for identifying novel processes that GALT may modulate. Further, determining the time-point of gene dysregulation will help elucidate the pathology of premature ovarian insufficiency.
Hypothesis: Since males with galactosemia do not exhibit infertility, I expect gene dysregulation to occur after sex determination happens in development. Further I expect genes involved in N- and O-glycosylation, ER stress, and various carbon metabolic pathways to be deferentially regulated.
Aim 3: Characterize the ovarian glycoproteome of mice and the effects of GALT deficiency on the former.
Approach: I will perform glycoproteomics by using mass spectrometry on ovarian tissue of adult WT and GALT deficient mice. Proteins that have altered glycosylation will be identified by comparing the glycoproteomes of WT vs. GALT deficient mice. Identified proteins will be sorted by their biological process and molecular functions.
Rationale: UDP-gal and UDP-glc are common carbohydrate donors for numerous galacto-/glycoproteins and galacto-/glycolipids, thus deficiency in GALT will result in aberrant glycosylation and metabolic dysregulation.
Therefore, identifying proteins that have altered glycosylation in ovarian cells of GALT deficient mice will provide insight to the pathology of premature ovarian insufficiency and other late life effects of galactosemia.
Hypothesis: I expect to see abnormal (hypo, hyper, or newly) glycosylation in the GALT deficient mice. This will lead to ER stress and changes of the glycosylation receptors as the cell deals with defective glycosylation.
References:
1. Isselbacher KJ, Anderson EP, Kurahashi K, et al. Congenital Galactosemia, a single enzymatic block in galactose metabolism. Science 1956;13:635–6.
2. Guerrero NV, Singh RH, Manatunga A, et al. Risk factors for premature ovarian failure in females with galactosemia. J Pediatr 2000;137:833–41
3. Berry GT. Classic Galactosemia and Clinical Variant Galactosemia. Retrived from: https://www.ncbi.nlm.nih.gov/books/NBK1518/
4. Berry GT, Moate PJ, Reynolds RA, et al. The rate of de novo galactose synthesis in patients with galactose-1-phosphate uridyltransferase deficiency. Mol Genet Metab 2004;81:22–30
My primary goal is to elucidate how GALT deficiency can lead to premature ovarian insufficiency and to illuminate the broader role of GALT outside its well-known role in galactose catabolism. My hypothesis is that previously overlooked metabolic roles of GALT, including glycosylation, play a central role in the pathology of classic galactosemia. I will use both the common mouse (Mus musculus) and Saccharomyces cerevisiae as model systems, due to their similar disease phenotypes to humans and ease of rapid analysis, respectively.
Aim 1: Characterize and identify conserved amino acids of GALT that are critical for ovary development.
Approach: I will screen the functionality of known Human GALT mutant alleles using a growth assay with S. cerevisiae. Next I will use sequence alignment methods to determine if loss of function mutations occur in evolutionary conserved sites. Finally, I will select of subset (mutated in conserved sites vs. non-conserved sites and functional vs. non-functional) of Human GALT mutants and use CRISPR/Cas9 to create transgenic mice lines with the various Human GALT disease alleles. I will then screen female mice for those that exhibit premature ovarian insufficiency.
Rationale: Not all females with galactosemia develop premature ovarian insufficiency, thus determining the mutations in GALT that lead to premature ovarian insufficiency will allow for better correlation of genotype to phenotype.
Hypothesis: I expect GALT alleles with mutations in evolutionary conserved sites will display the phenotype of premature ovarian insufficiency, while mutations in non-conserved sites will not.
Aim 2: Characterize deferentially expressed genes across ovarian development in GALT deficient mice.
Approach: I will perform RNA-seq on the ovaries of wild type and GALT deficient mice throughout ovarian development and into adulthood, mice will be fed a galactose free diet. RNA-seq data will be sorted using GO terminology and compared between both WT and GALT deficient mice and between the sampled time points.
Rationale: Genes that are deferentially regulated in the ovaries in the absence of GALT are possible targets for identifying novel processes that GALT may modulate. Further, determining the time-point of gene dysregulation will help elucidate the pathology of premature ovarian insufficiency.
Hypothesis: Since males with galactosemia do not exhibit infertility, I expect gene dysregulation to occur after sex determination happens in development. Further I expect genes involved in N- and O-glycosylation, ER stress, and various carbon metabolic pathways to be deferentially regulated.
Aim 3: Characterize the ovarian glycoproteome of mice and the effects of GALT deficiency on the former.
Approach: I will perform glycoproteomics by using mass spectrometry on ovarian tissue of adult WT and GALT deficient mice. Proteins that have altered glycosylation will be identified by comparing the glycoproteomes of WT vs. GALT deficient mice. Identified proteins will be sorted by their biological process and molecular functions.
Rationale: UDP-gal and UDP-glc are common carbohydrate donors for numerous galacto-/glycoproteins and galacto-/glycolipids, thus deficiency in GALT will result in aberrant glycosylation and metabolic dysregulation.
Therefore, identifying proteins that have altered glycosylation in ovarian cells of GALT deficient mice will provide insight to the pathology of premature ovarian insufficiency and other late life effects of galactosemia.
Hypothesis: I expect to see abnormal (hypo, hyper, or newly) glycosylation in the GALT deficient mice. This will lead to ER stress and changes of the glycosylation receptors as the cell deals with defective glycosylation.
References:
1. Isselbacher KJ, Anderson EP, Kurahashi K, et al. Congenital Galactosemia, a single enzymatic block in galactose metabolism. Science 1956;13:635–6.
2. Guerrero NV, Singh RH, Manatunga A, et al. Risk factors for premature ovarian failure in females with galactosemia. J Pediatr 2000;137:833–41
3. Berry GT. Classic Galactosemia and Clinical Variant Galactosemia. Retrived from: https://www.ncbi.nlm.nih.gov/books/NBK1518/
4. Berry GT, Moate PJ, Reynolds RA, et al. The rate of de novo galactose synthesis in patients with galactose-1-phosphate uridyltransferase deficiency. Mol Genet Metab 2004;81:22–30
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Specific Aims
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