MCDB Notes for Project 2 PDF

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Project 2...

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Assignment 2: Neutral Lipid Storage Disease There is a family of diseases associated with abnormal lipid storage and with altered lipid structures or altered lipid metabolism. As a group they are known as lipid storage diseases and many of them have been identified. This project focuses on a relatively rare condition called Neutral Lipid Storage Disease With Myopathy. Myopathy is a general term for muscle weakness. 1. (2 points) Please give a brief (2-3 sentences maximum) description of the symptoms of this disease. Name any specific molecule(s) that are related to the symptoms, but not the root cause of the disease. - Neutral Lipid Storage Disease With Myopathy is a condition in which lipids are stored abnormally in organs and tissues throughout the body. Symptoms include muscle weakness due to accumulation of fats in muscle tissues. Other symptoms include fatty liver, enlarged and weakened heart, pancreas inflammation, and type 2 diabetes. Molecules related to the symptoms of the disease include triglycerides (1). 2. (2 points) The root cause of this disease is genetic, leading to a defect in a specific protein that ultimately causes the symptoms you listed above. Describe the specific gene associated with this problem, the specific protein that is mutated, and the function of that protein. - This disease is caused by mutations in the PNPLA2 gene which provides the sequence to make the enzyme adipose triglyceride lipase (ATGL). ATGL is responsible for the first step of hydrolysis of triglycerides so that this stored form of fat can be used for energy. The mutation to PNPLA2 impairs the ATGL’s ability to break down triglycerides, causing the accumulation of them in muscle and tissues throughout the body (2). 3. (2 points) Given your answer to question 2 above, describe a way using laboratory methods that would confirm that a patient had the genetic (i.e. DNA) problem. - One laboratory method that would confirm that a patient had the genetic problem is to use DNA microarray. First, you’d need to obtain a genetic sample from the patient and run PCR on it in order to amplify the sample. Next to do the microarray you would first denature the DNA, cutting the strands of DNA into smaller fragments and labeling each fragment with a fluorescent dye. When you add the labeled DNA to the array the probes hybridize through complementary DNA pairing. If there is no mutation for the gene, the samples will bind on the chip that represents the sequence without the mutation. If there is a mutation for the gene, the DNA will not bind properly to the unmutated DNA sequences and will bind to the sequence that represents mutated DNA

4. (1 point) Because this is a disease associated with lipids, and there are many of these diseases known, you need to positively identify the specific lipid(s) affected by the genetic problem. You

obtained a biopsy of muscle tissue from a patient. Describe laboratory methods that will positively identify the specific lipid that is accumulating in the muscle cells. - In order to positively identify the specific lipid that is accumulating in the muscle cells, you can extract them using organic solvents to perform a phase extraction using a separation funnel - Homogenize the tissue in chloroform/methanol/water which is the one phase that extracts all lipids from the membranes - After extracting them in one phase, now we have to extract them into two phases so we can add water to continue the extraction - Then, we would do 2D TLC and compare it to the 2D TLC of known lipids. 5. (2 points) The protein of the mutated gene is known and its function is also known. Another lab working on this problem has given you a small amount of the purified mutated protein from another biopsy plus some of the normal human protein. Given your new knowledge of proteins and enzymes, how would you show that the function of the mutated protein was abnormal compared to the mutated protein? - I would put each protein in a separate vial with triglycerides. If the protein is in the mutant form then it shouldn’t be able to break down the triglycerides. Could then do enzyme kinetics using a spectrophotometric assay and calculate the Vmax and Km in each vial. These numbers should then exhibit whether the mutant form is functioning. The mutant should have a greater Km. - You could also extract the triglycerides from the the vials after incubating with the enzyme and separately analyse them using gas-liquid chromatography which separates lipids based on solubility and volatility which would be different for the triglycerides and the broken down triglycerides. 6. (1 point) The genetic problem is autosomal recessive, meaning each parent carries one allele for the disease. Research projects can safely focus on solutions for these genetic problems at the cellular level using tissue culture, but some of those solutions are now controversial and even unethical. Even so, at some point in the future it may be possible to consider a method to correct Neutral Lipid Storage Disease with Myopathy in future offspring of affected parents in humans and other animals. Name one method that could actually serve that role, to correct the genetic problem in future offspring (although this solution may never be accepted socially). - You could use CRISPR-Cas9 system to edit the genome of an embryo, fixing the mutations in the embryo’s PNPLA2 gene.

BONUS (1 point) There is fast and simple diagnostic method to positively identify this disease and it involves a small blood sample. Describe that diagnostic method that is actually used. - A peripheral blood smear is used to look for triglyceride in white blood cells. When triglycerides are found it is called Jordan’s anomaly. These fats appear as vacuoles in the

cytosol of peripheral white blood cells from patients with Neutral Lipid Storage Disease With Myopathy. The smear can be done by collecting a small sample of a patient’s blood, which you place on a slide, and then use a Wright-Giemsa stain to identify the presence of vacuoles in white blood cells (3).

References 1. https://rarediseases.info.nih.gov/diseases/10288/neutral-lipid-storage-disease-with-myop athy 2. https://ghr.nlm.nih.gov/condition/neutral-lipid-storage-disease-with-myopathy#genes 3. http://www.bloodjournal.org/content/131/7/837?sso-checked=true...