Overview Capstone 2007-Joy Paul The following capstone paper is a study on the genetics of Tay-Sachs disease. This topic was chosen for two main

Transcription

1 Overview Capstone 2007-Joy Paul The following capstone paper is a study on the genetics of Tay-Sachs disease. This topic was chosen for two main reasons. First, and most importantly, I have always been fascinated by the study of genetics. Genes are nothing more than bits of chemicals. Deoxyribose sugar made of carbon, hydrogen and oxygen atoms; phosphate groups comprised of phosphorous and oxygen atoms; and some nitrogen bases, containing nitrogen, hydrogen and oxygen atoms are the atomic building blocks of genes. These atoms of carbon, nitrogen, hydrogen, oxygen and phosphorous chemically combine to determine every structure and chemical reaction in the human body. These microscopic genes; bits of DNA, can also be the source of grave illnesses. The power that these atoms hold over the human body never ceases to amaze me. To have gained a bit more understanding about genes and their effect on humans almost humbles me as a student and learner. Having completed this project has shown me how quickly scientific information changes. Current genetic knowledge is vast, yet is only the tip of the iceberg. This capstone project has shown me the necessity of trying to stay up to date with at least some of the newest current scientific information. I narrowed my studies to Tay-Sachs disease because of my teaching environment. I currently am a science teacher at Robert Saligman Middle School, a Conservative Jewish day school in suburban Philadelphia, Pennsylvania. I was raised in and continue to practice the Christian faith. Therefore, my background knowledge of Jewish culture and faith is quite small. Learning about Tay-Sachs disease adds to my knowledge of issues that can have a significant impact upon my students. By understanding some of the issues facing Jewish students, I feel I become a better teacher for them. The first section of my capstone project reviews some of the genetics of Tay- Sachs disease (TSD). TSD is a disease primarily affecting Ashkenazi Jews. It is a genetic defect in the enzyme that breaks down a lipid in the brain. This lipid, called GM2, is a normal by-product of brain activity. Accumulation of this lipid causes neurological degeneration that quickly leads to death, usually by the age of 5 years. The enzyme needed to break down this lipid is Hex-A. An insertion of four extra base pairs within a person s DNA is one way that formation of the Hex-A enzyme is interrupted. Without

2 Hex-A, GM2 accumulation occurs. Genetic and non-genetic therapies are being explored to find a cure for TSD. The second section of my capstone project is an outline for a two week long (approximately) unit on teaching genetics and inheritance to eighth grade students. The unit plan is based upon a learning/teaching strategy called the yo-yo design. Students are led up and down the biological organization levels as they study genetics to better understand the connections between genes and the human organism. Mitosis, meiosis and protein synthesis are some of the units covered. Students use a role playing activity to determine how a single change in DNA affects the entire organism. Students will design and conduct an online experiment using virtual fruit flies to investigate the concepts of inheritance, genotypes and phenotypes. Finally, students will discuss genetic testing and therapy as it relates to Jewish law.

3 Genetics of Tay-Sachs Disease Capstone 2007-Joy Paul I. Background: Tay-Sachs disease is defined as a neurodegenerative disease involving the nerve cells of the brain. It is the result of a defective enzyme that is normally responsible for breaking down a specific lipid that builds up in brain tissue. There are three types of Tay- Sachs disease; infantile, juvenile and adult-onset. This paper focuses on the most severe form, infantile Tay-Sachs. The disease was first identified by an ophthalmologist, Warren Tay who, in 1881 reported a condition showing degeneration of the retina. A cherry-red spot was visible on the macula portion of the retina of a patient. Tay also observed other symptoms characteristic of a degenerating nervous system. Around the same time, the physician Bernard Sachs published a report on a serious, progressive neurodegenerative disease that showed the same symptoms as the one Tay had noticed. Sachs reported that this disease seemed to have a familial link to Ashkenazi Jews (Kaback & Desnick, 2001). It was later realized that these two doctors were reporting on the same disease. Hence the name; Tay- Sachs disease. The genetic basis of Tay-Sachs was determined early on (reviewed by Suzuki, 1996). During post-mortem autopsies it was determined that a substance was building up in the brain cells of Tay-Sachs victims. In 1939 Klenk determined that this material was a lipid associated with neurons. He named these newly found compounds as gangliosides. Advances in biochemistry eventually identified this ganglioside as GM2- ganglioside (Suzuki, 1996). GM2 consists of the lipid ceramide linked to the sugar galactose. The general structure of GM2 is seen in figure 1 (retrieved from: Gangliosides are found on the surface of the cells within the central nervous system and contribute to the stability and formation of myelin which insulates certain nerve cells and aids in the transmission of nerve impulses (reviewed by Platt & Butters, 2000).

4 Fig. 1. Top diagram shows structure of GM2 ganglioside molecule. The long chains represent a sphingosine (top chain) and a fatty acid (bottom chain). The enzyme hexosaminidase A is necessary to catalyze the removal of N-acetylgalactosamine from the GM2 ganglioside. Tay-Sachs disease (TSD) is most commonly found among the Ashkenazi Jewish population; Jews of central and Eastern Europe descent and/or western Russia (Kaback & Desnick, 2001). 1 in 25 Ashkenazi Jews is a carrier of TSD (Suzuki, 1996), a rate ten times more frequent than carriers found in the general population (Myerowitz & Costigan, 1988). Another population frequently affected by TSD is the non-jewish French Canadians, where approximately 1 in 30 is a carrier (Ensenauer, Michels & Reinke, 2005). TSD also occurs within the Cajun and Pennsylvania Dutch. Since the Ashkenazi Jews are the most affected population, that population is a focus of this paper. Babies born with infantile Tay-Sachs disease appear normal at birth. Within four to six months, parents will see a slow down of growth and the child will become less responsive to outside stimuli. The child will begin to lose motor and mental skills that had been previously acquired. Muscle tone and head control decline quickly. Eyesight degrades until blindness occurs. By two to three years of age the child has lost contact with their environment. Brain function shows progressive, continuous decline. The head enlarges due to swelling of the neural cells. Death often occurs by four years of age (Suzuki, 1996).

5 Within each neural cell the lysosomes fill with an excess of GM2 ganglioside. This excess storage of GM2 is what classifies Tay-Sachs as a lysosomal storage disease, also called GM2 gangliosidosis (Kaback & Desnick, 2001). TSD is also classified as a sphingolipidoses due to the sphingosine component of the accumulating lipid (Suzuki, 1996). A diagram of a sphingolipid is seen in figure 2 (retrieved from: As lysosomes fill with GM2, the cell nuclei and other organelles are pushed to the sides of the cell. The neural cells swell to 4-5 times their normal size. The amount of GM2 that accumulates is several hundred times that of corresponding normal neurons (Suzuki, 1996). Fig. 2. Sphingosine is attached to a fatty acid via an amide group. The R group consists of at least three sugars. See Figure 1 for R groups specific to GM2. The accumulation of GM2 results from a genetic defect that causes poor activity in the enzyme that breaks down GM2. A person only needs 10% of normal enzyme activity to be healthy. TSD patients have 0-5% (Triggs-Raine, Mahuran & Gravel, 2001). The devastating defects of TSD along with the known genetic component has meant that extensive research into further understanding the genes involved in TSD has been tremendous.

6 II. Genetics of TSD: A. Genes involved in causing Tay-Sachs Disease A sphingolipidoses is defined as the abnormal accumulation of lipids that contain sphingosine (Fig. 2). All sphingolipidoses are inherited via classic Mendelian recessive inheritance patterns. Both parents of an affected child must carry one abnormal gene for the disease. Those that carry one allele for a disease are termed carriers. The chance of carrier parents having an affected child is ¼. Carriers of the disease can be identified by the decrease in the enzyme activity needed to break down GM2. A carrier s enzyme activity is much lower than that of a homozygous normal person, since the defective gene inhibits the production of a normal amount of enzyme (Suzuki, 1996). The accumulation of GM2 is the result of a genetic defect in the α subunit of β hexosaminidase, the enzyme necessary for its breakdown. The enzyme removes one monosaccharide at a time to break down GM2. The product from the removal of one sugar becomes the substrate for the next enzyme reaction and so on as the neural ganglioside is hydrolyzed (Platt & Butters, 2000). The enzyme which uses GM2 as a substrate is hexosaminidase-a also known as Hex-A (Suzuki, 1996). Hex-A is used in the second step of the breakdown of gangliosides (Platt & Butters, 2000). See figure 3 for schematic of complete catabolism of glycosphingolipids in humans (retrieved from: Over 60 mutations in the gene coding for Hex-A are known. The combination of two abnormal alleles of n mutations is n (n+1)/2. This creates over 1830 possible genotypes. With over 60 gene mutations causing TSD, compound heterozygosity, where each abnormal allele is different from the other, is common (Suzuki, 1996). The gene that codes for Hex-A is found on chromosome 15. Hex-A formation depends upon the formation of a compound known as Hex-B. Those genes are found on chromosome 5. For GM2 hydrolysis, a small helper protein is needed called GM2A (Kolodny, 2001). As reported by Mahuran and Gravel (2001), the GM2A interacts with the carbohydrate and lipid portion of the ganglioside, lifting the ganglioside molecule from the membrane of a neuron and presenting it to Hex-A for hydrolysis. Without the helper protein, GM2 cannot be broken down. Therefore GM2 gangliosidosis can result from inherited defects

7 in the gene that codes for Hex-A, Hex-B or GM2A (Kolodny, 2001). This paper will focus on four of the most common genetic defects involved in the formation of Hex-A. Fig. 3. Catabolism of GSLs in humans: enzymes involved and enzyme related diseases. Tay-Sachs disease is seen in the second step of the pathway. B. Common genetic mutations affecting Hex-A formation While over 60 genetic mutations involved in Hex-A formation are known, there are four common genetic defects that occur. The first and most common defect is a four base pair insertion of TATC (thymine-adenine-thymine-cytosine) that is identical to the four bases preceding it. This insertion occurs within a protein coding region of

8 chromosome 15 known as exon 11. See Figure 4 for photograph of analysis of base pair insertion (Myerowitz & Costigan, 1988). The insertion causes a shift in the reading frame during transcription that produces a premature signal to stop encoding the mrna (Myerowitz & Costigan, 1988 and Myerowitz, 2001). Therefore, deficient mrna is formed in TSD patients (Myerowitz & Costigan, 1988). This mutation is present in approximately 70% of all carriers of TSD within the Ashkenazi Jewish population (Kolodny, 2001 and Myerowitz & Costigan, 1988). Fig. 4. Nucleotide sequence analysis of normal and mutant α -chain genes containing the insertion. The insertion is marked with asterisks, and the nucleotide sequences are listed beside the autoradiograms. The second most common genetic mutation amongst Ashkenazi Jews results from a point mutation; a change in one nitrogen base, on chromosome 15 at a non-coding portion of the chromosome, the first nucleotide of intron 12. Here the G-T (guaninethymine) base pair that serves as a splice junction during transcription changes to a C-T (cytosine-thymine) base pair. This change from G to C inactivates the necessary G-T dinucleotide at the splice junction creating poor splicing (Myerowitz, 2001). Therefore, the RNA molecule is unstable (Chavany & Jendoubi, 1998). This point mutation occurs in about 30% of the Jewish carriers of TSD (Myerowitz, 2001).

9 Within the non-jewish French Canadian population, a deletion at one end of the section of chromosome 15 coding for Hex-A is the most common genetic defect causing TSD. An alteration at site intron 9 of chromosome 15 is commonly seen in carriers of Cajun and Pennsylvania Dutch origin (Kaback & Desnick, 2001). These four genetic alterations are the most common in TSD carriers. As stated above, over 60 different genetic mutations in the genes coding for Hex-A are known. Most of these other mutations occur rarely and are spread amongst various populations. C. Genetic testing of carriers of TSD New knowledge about these genetic abnormalities has tremendously aided in the identification of carriers of Tay-Sachs disease. Heterozygous carriers are most commonly identified by an enzymatic screening. The amount of Hex-A enzyme activity is much lower than that found in a normal person due to the one defective gene limiting the production of Hex-A. This decreased amount of Hex-A is detected in an enzymatic screening and used to identify carriers of a defective Tay-Sachs gene. If the enzyme screening test is non-determinant, a genetic screening test is used. With 60 or more possible mutations in the Hex-A gene, mass genetic screening was impractical (Suzuki, 1996). New genetic screening models have simplified this process, making the genetic detection of carriers much simpler. If a defective gene is found during screening, it is very important to explain to the individual being tested that they are only a carrier of the disease. They do not actually suffer from TSD (Ensenauer, et al., 2005). Amniotic fluid or chorionic villi can be sampled to test fetuses for Tay-Sachs. The amount of Hex-A activity is determined and pregnancy termination options can be explored if positive results for TSD are found (Suzuki, 1996). Enzymatic and genetic screening, along with pre-natal testing has significantly reduced the incidence of Tay-Sachs disease in North America by 90% (McGinnis, et al., 2002). These preventative measures currently serve as the most reliable form of treatment for TSD. D. Non-genetic therapies for the treatment of Tay-Sachs disease Besides the preventative measures discussed above, there are other forms of treatment being explored. None have met with tremendous success. While genetic therapies are the focus of this paper, here I summarize several non-genetic forms of treatment. Since TSD is caused by an enzyme deficiency in Hex-A, injection of this

10 deficient enzyme has been investigated. Success has been limited due to the difficulty of the injected enzyme crossing over into the brain where it is needed. Also, Hex-A is rapidly cleared by the liver after an intravenous injection. The implantation of neural stem cells that can integrate into the affected neurons of adults with adult-onset Tay- Sachs has shown some promise (Chavany & Jendoubi, 1998). Bone marrow transplantation has also been explored in an effort to increase the amount of Hex-A in the body (Platt & Butters, 2000). Substrate deprivation therapy is also under investigation. This therapy aims to reduce the amount of substrate, or GM2, that the enzyme acts upon. The hope is that the minimal Hex-A enzyme activity seen in Tay-Sachs patients can keep up with a lower level of GM2 production preventing excess storage of the lipid within the lysosomes. There has been some success with substrate deprivation using an orally administered drug that minimizes the production and therefore storage of GM2 without destroying myelin production (Platt & Butters, 2000). Hopefully one of these therapies, either alone or in combination with genetic therapies, will provide an effective treatment for Tay-Sachs disease. E. Genetic therapies for the treatment of Tay-Sachs disease via craniotomy Genetic therapies attempt to correct defective genes by inserting genes that will function normally. Most often viral vectors are used to insert and deliver the normal genes into the affected organ. The virus inserts these genes into the cells that contain the abnormal genes. These corrective genes are implanted into the cell s DNA as it undergoes mitosis. However, adult brain cells are post-mitotic and do not replicate easily. The brain repairs itself very poorly. For infantile TSD, the GM2 has already begun building up in-utero. Therefore, affected fetuses have already incurred extensive neural damage that is difficult to correct after birth with traditional gene therapy (Suzuki, 1996). One of the limiting problems in gene therapy for TSD is delivery of the corrective gene to the brain. Many viral vectors that are injected intravenously will not cross over into the brain from the blood. This barrier between the blood and the brain is termed the blood-brain barrier (BBB) (Schlachetzki, Zhang, Broado & Pardridge, 2004). Since the viral vectors do not cross the BBB, previous research with genetic therapy has required a craniotomy and a localized injection of the viral vectors containing the corrective genes. Schlachetzki, et al (2004) has reported that this method of delivery limited the

11 distribution of the corrective gene. The genes were only being expressed at the tip of the needle at the injection site, or had integrated randomly within the brain tissue. This random integration and expression, mutagenesis, can cause many problems, including malignancy. There is also concern with the possible toxicity of the viral vectors, which may cause brain inflammation (Schlachetzki, et al 2004). There has been success with a viral vector delivery system in mice that have Sandhoff disease, a disease closely related to Tay-Sachs. A study conducted by Cachon- Gonzalez et al., (2006) reports using recombinant adeno-associated viral vectors to encapsulate the genes to promote the expression of the Hex-A enzyme. They are injected into the brain via a craniotomy. After a single injection, GM2 storage was reduced throughout the brain and spinal cord. Four week old mice that received a single injection of viral vectors containing the genes for Hex-A showed reduced GM2 storage. Multiple injections led to a further reduction in GM2 storage. These viral vectors were able to transfer their genetic material into the neurons to promote the secretion of Hex-A. The Hex-A was either transported via the axons or recaptured and used by neurons far from the injection site. It seems very likely that the hexosaminidase is taken up by cerebrospinal fluid and distributed. There was an extended period of Hex-A expression without toxicity. Unfortunately, neurological deterioration was only delayed. The disease free interval was increased by 90%. However, the eventual neurological deterioration has yet to be explained. This study strongly suggests the use of gene therapy using recombinant adeno-associated viral vectors in combination with substrate inhibitors and/or bone marrow transplants to be a promising application for human Tay-Sachs disease (Cachon-Gonzalez et al., 2006). Martino, et al. (2005) reported the use of a non-replicating Herpes simplex viral vector that had been encoded with the Hex-A DNA. The Herpes simplex virus (HSV1) was used because it can easily infect non-replicating cells such as neurons. This viral vector is also easily transported to other nerve cells following injection and allows for an extended transgene expression. The HSV1 was injected into the internal capsule of the brain of a Tay-Sachs animal model. To reach the internal capsule, a craniotomy was required. The internal capsule is part of the corpora striata, located in the lower wall of each hemisphere of the brain. The internal capsule was used as an injection site since it

12 contains numerous fiber bundles connecting many parts of the brain at once. By injecting the HSV1 into the internal capsule, many areas of the central nervous system can be reached with just one injection. These strategies have seen some promising success. Hex- A activity was restored to a normal range throughout the brain hemispheres, cerebellum and spinal cords of infected mice. It is suspected that the therapeutic vector was distributed throughout the brain, in part, via cerebral fluid. There was total removal of GM2 storage with no adverse effects due to the viral vectors, injection site or gene expression after only one injection. This approach may hopefully slow the progression of TSD. This study also supports using the brain s structure as another tool in the treatment of TSD. While showing promising results, administering either the RAAV or HSV1 vectors to deliver the transgenes requires an intrusive craniotomy. F. Genetic therapies for the treatment of Tay-Sachs disease using non-invasive approaches Less invasive approaches providing delivery of therapeutic genes is currently under extensive research. A non-viral noninvasive approach to delivering corrective genes uses genetically engineered Trojan horses to bring the therapeutic genes to the brain after being administered intravenously ( reviewed by Schlachetzki, et al., 2004). Certain blood proteins, such as albumin are toxic to brain cells. Therefore, a delivery system is needed that will not disrupt the barrier between the blood tissue and the brain (BBB). The Trojan horses discussed in this report can cross the blood/brain barrier safely. Every neuron in the brain is virtually surrounded by its own blood vessel. There are approximately 400 miles of capillaries in the human brain forming the blood/brain barrier. If the therapeutic genes are delivered intravenously and can cross the BBB, these genes will be delivered to every neuron in the brain (Schlachetzki, et al., 2004). Certain proteins that enter the brain, such as insulin, use a delivery system called receptor-mediated transcytosis, or RMT (Schlachetzki, et al., 2004). The RMT delivery system has three steps. First, a receptor-mediated endocytosis of the peptide into the capillary endothelium occurs. This is followed by movement of the peptide through the endothelial cytoplasm. Finally, there is exocytosis of the peptide into the brain interstitial fluid. This movement of the peptide through the endothelial wall of the capillary takes place at certain receptor mediated sites (Pardridge, 2002). These peptides, such as insulin,

13 are delivered to the neurons without disrupting the BBB. In the study reviewed by Schlachetzki, et al (2004), a cloned antibody produced in the lab can mimic a peptide that uses RMT to cross the blood/brain barrier. The antibody binds to a portion of the endothelial receptor and piggybacks across the BBB using the RMT system. The mab binds to a part of the endothelium receptor not used by the peptide it is mimicking such as insulin. Therefore, the antibody does not interfere with the uptake of the necessary peptide (Pardridge, Kang, Buciak, & Yang, 1995). This monoclonal antibody, mab, can be used as the Trojan horse to deliver the therapeutic genes to the brain. To deliver the therapeutic genes, the mab must be merged with a nonviral gene delivery system using liposomes (Schlachetzki, et al., 2004). Schlachetzki (2004) discusses the use of nonviral delivery systems using liposomes. The corrective DNA can be encapsulated within a very small liposome, which is a sac surrounded by phospholipid layers. These liposomes are about the size of many viral gene delivery systems. The outside of the liposome is combined with several thousand strands of polyethyleneglycol (PEG), which makes the liposome stable. The PEGylated liposome will not cross the BBB. Therefore, about 1-2% of these PEGs are combined with the monoclonal antibody (mab) which does cross the BBB. The cloned antibodies bound to the PEGylated liposomes are injected intravenously. Since the mab mimics peptides that can use the RMT to cross the blood/brain barrier, the therapeutic genes encapsulated within the liposomes cross the BBB (Schlachetzki, et al., 2004). Since there are insulin receptors on neural cell membranes, the mab, which mimics peptides such as insulin, will mediate the passage of the therapeutic genes encased within the liposomes into the neural cells for expression (Coloma, Lee, Kurihara, et al., 2000). Results have been promising. Rhesus monkeys that were injected intravenously with a liposome containing the genes for β galactosidase were found to have been expressed throughout the entire brain. The transgenes were able to cross the BBB and integrate within the neurons. A concern is whether or not these genes may be expressed in other organs that also have insulin receptors, since the mab is injected intravenously and circulates throughout the entire body (Schlachetzki, et al., 2004). Using a therapeutic gene that is under the control of a brain specific gene promoter has shown some success and may alleviate this concern (Shi, Zhang, Boado, Zhu & Pardridge, 2001).

14 Since the therapeutic DNA is contained within liposomes instead of viruses, it does not integrate into the nuclear DNA. This reduces the chance of mutagenesis occurring, reducing the risk of cancer. Instead, the corrective DNA is episomal. It functions independently of the nuclear DNA, as a type of mini chromosome. Eventually this episomal DNA gets degenerated resulting in a loss of therapeutic gene expression. Therefore this therapy must be repeated. A challenge in developing this therapy of noninvasive transgene delivery is to develop plasmid formulas of DNA that do not become degraded by nuclear enzymes (Schlachetzki, et al. 2004). Also, the long term effect of gene therapy in humans is not known. III. Conclusion: Infantile Tay-Sachs disease is a degenerative, fatal neurological disease. It most commonly strikes the Ashkenazi Jewish population. Carrier identification has been the best prevention against TSD thus far. While the most common gene mutations causing this disease are known, many more possible mutations are unknown. Non-genetic and genetic therapies are being studied, discovered and scrutinized. Hopefully, one treatment or a combination of treatments will soon become a cure for Tay-Sachs disease. Resources: Cachon-Gonzalez, M.B., Wang, S.Z., Lynch, A., Ziegler, R., Cheng, S.H., & Cox, T.M. (2006). Effective gene therapy in an authentic model of Tay-Sachs-related diseases. [Electronic version]. Proceedings of the National Academy of Sciences of the United States of America, 103(27), Chavany, C., & Jendoubi, M. (1998). Biology and potential strategies for the treatment of GM2 gangliosidoses. [Electronic version]. Molecular Medicine Today, April, 157. Coloma, J., Lee, H. J., Kurihara, A., Landaw, E., Boado, R., Morrison, S., et al. (2000). Transport across the primate blood-brain barrier of a genetically engineered chimeric monoclonal antibody to the human insulin receptor. [Electronic version]. Pharmaceutical Research, 17(3), 266. Ensenauer, Regina E. MD, Michels, Virginia V. MD, & Reinke, Shanda S. MS. (2005). Genetic testing: Practical, ethical, and counseling considerations. [Electronic version]. Mayo Clinic Proceedings, 80(1), 63.

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16 Shi, N., Zhang, Y., Boado, R. J., & Zhu, C. Pardridge, W.M. (2001). Brain-specific expression of an exogenous gene after I.V. administration. Proceedings of the National Academy of Sciences, 98(22), Suzuki, K. (1996). The genetic lysosomal diseases: Tay-Sachs disease as the prototype. Mental Retardation and Developmental Disabilities Research Reviews, 2, 167. Triggs-Raine, B., Mahuran, D., & Gravel, R. (2001). Naturally occurring mutations in GM2 gangliosidosis: A compendium. In R. Desnick & M. Kaback (Eds.), Advances in Genetics, Vol.44 (pp ). San Diego, California: Academic Press.