Bioinformatics - Retinoblastoma Disease - Assignment Example

BIOINFORMATICS REPORT Course Code BIOC 6017 Submitted By: Student Name Student ID Submitted To: Dr. Simon Worrall 1. Abstract After conducting thorough research on the matter, I have found Rb1 to be a critical gene in the human body for cell regulation and for matters relating to certain kinds of cancers associated with the gene. The protein pRb and particularly its inactivation have huge implications for the human body. There still needs to be much further scientific research for us to completely determine the role of genes in the human body because the tests conducted so far are not perfect as we will see later in the course of our discussion. The interactions of the gene with other genes, as well of the interactions of the proteins to form complexes is an area which demand further research for science to be able to completely combat by the particular health concerns raised. Retinoblastoma is a disease heavily associated with the gene and hence we will now begin by discussing it first. 2. Introduction Retinoblastoma is a disease; it is a rare form of eye cancer which develops in the cells of the retina of some children. According to the Retinoblastoma Genetic Screening Unit, “the majority of retinoblastoma patients (85%) have no family history of the disease”. [i] About 66% of children have unilateral retinoblastoma, where only one eye is affected whereas where the parent had also been affected it is more likely the disease will be bilateral. Genetic testing is helping doctors detect and cure the tumour. It is impossible to know without genetic testing whether the sporadic unilateral form will be heritable or not. The prevalence of the disease and the medical difficulties associated with curing the potential consequences particularly attracted me to further research the gene related for the disease. 3. Methods The methodology involved diverse research on the internet. The Barts and the London organization was used as a reference point for tests and data. The scientific information on the gene was acquired from the Ensembl website and the NCBI database was used extensively in this regard. The RB1 gene was compared to many other genes by using the NCBI GEO database, there are many examples of related genes. Some examples obtained from [ii] are cited as follows: 3.1. RB1 retinoblastoma 1 [Macaca mulatta] Chromosome: 17 Annotation: Chromosome 17, NC_007874.1 (27097341..27272303) Gene ID: 704871 3.2. RB1 retinoblastoma 1 (including osteosarcoma) [Pan troglodytes] Chromosome: 13 Annotation: Chromosome 13, NC_006480.2 (48185603..48362566) GeneID: 452715 3.3. RB1 Official Symbol Rb1 and Name: retinoblastoma 1 [Rattus norvegicus] Other Designations: Retinoblastoma 1 (including osteosarcoma) Chromosome: 15; Location: 15q12 Annotation: Chromosome 15, NC_005114.2 (53828881..53962099, complement) Gene ID: 24708 3.4. RB1 Official Symbol rb1 and Name: retinoblastoma 1 [Danio rerio] Other Aliases: zgc:154147 Chromosome: 21 Annotation: Chromosome 21, NC_007132.3 (9193893..9202890) Annotation: Chromosome 21, NC_007132.3 (18960985..19002134) Gene ID: 777733 3.5. RB1 retinoblastoma 1 [Bos taurus] Other Aliases: P105-RB, PP110, RB11 Other Designations: retinoblastoma 1 (including osteosarcoma); retinoblastoma-associated protein Chromosome: 12 Annotation: Chromosome 12, NC_007310.3 (17508776..17628174) Gene ID: 534712 3.6. RB1 Official Symbol RB1 and Name: retinoblastoma 1 (including osteosarcoma) [Gallus gallus] Other Designations: retinoblastoma 1 Chromosome: 1 Annotation: Chromosome 1, NC_006088.2 (173042817..173121034) Gene ID: 386582 [iii] 4. Introduction of the Gene Discussing the gene RB1 itself, we find that the retinoblastoma susceptibility gene, known as RB1, is a tumour suppressor gene located on chromosome 13q. The gene contains instructions for making a protein called pRB with the function of suppressing a tumour i.e. it regulates cell growth and prevents uncontrolled manner. The official symbol for the gene is Rb1 and its other aliases are Rb, Rb-1 and pRb. pRB can also prevent other proteins from replicating DNA subject to certain conditions. DNA replication refers to a process in which the DNA makes a copy of itself; it occurs before a cell can divide hence regulation of the process can prevent growth of tumours. pRB also influences cell survival and self destruction of cells (apoptosis) by interacting with other proteins. RB1 gene is related to the disease retinoblastoma, many mutations of the gene are found in people infected with the disease. Somatic mutations, gene mutations in certain cells acquired during the lifetime, are associated with many types of cancers such as bladder cancer, lung cancer and breast cancer as well. The existence of these mutations can also increase the probability of developing other cancers like pinealoma, melanoma and osteosarcoma; making the gene a very interesting subject for an academic discourse. For the development of retinoblastoma, It is necessary that mutations must occur in both copies (alleles) of the gene. According to Barts and The London, “the spectrum of predisposing RB1 gene mutations includes large structural changes (about 10-20%) some of which are also detectable by cytogenetic analysis”. [iv] Single base substitutions and small insertions/deletions are the other changes. Most of the tumours show a loss of one copy, known as heterozygosity whereas a mutation in the other remaining copy. In almost 10% of tumours we find hypermethylation at the RB1 gene promoter, inhibiting the levels of retinoblastoma protein made by the cell instead of its function. Different types of RB1 mutation can lead to variable penetrance and expressivity, the former refers to the chance of a mutation developing into a cancer and the latter refers t the number of tumours occurring. Complete penetrance and bilateral retinoblastoma are usually associated with premature termination (truncated protein). The risk of other family members getting the disease depends on whether the victim has a germline RB1 mutation. In non-heritable retinoblastoma we usually have an absence of RB1 mutations in the blood but there is a minute risk of mosaicism. Molecular testing can show which members of a family have a risk of getting retinoblastoma. The Barts and London Retinoblastoma Genetic Screening Unit performs molecular genetic tests for predisposition to retinoblastoma. They conducted a number of genetic tests, they analyzed over 300 families and the results were compiled. The following table was obtained, RB Type Tested Mutation Found Mutation in Blood Sensitivity Bilateral & Unilateral with positive family history 206 197 196 95.6% Unilateral with no family history 139 130 25 93.5% Total 345 327 Overall Sensitivity 94.8% [v] However, the genetic tests are not a perfect representation because the techniques used are based on the current knowledge of the RB1 gene. The techniques are not perfectly sensitive and there is a possibility that mutations can be missed in any given sample. Even when the full mutation screening is negative it may not necessarily mean that the sample is completely free of RB1 mutations. It is even possible when one mutation is detected that there are others which have not been detected. Retinoblastoma also exemplifies Knudson's two-hit hypothesis. Basically, the hypothesis formed by AG Knudson in 1971 stated that two successive mutations, called hits, were necessary to turn a normal cell into a tumour cell; one of the hits was inherited in familial forms. The hypothesis was proved by a seminal study by Cavenee et al (1983); the study even established the paradigm for laboratory investigations of TS genes. The following figure depicts Knudson’s two-hit hypothesis. “Suppose there are 1 million target cells and the probability of mutation is 10-5 per cell. Sporadic retinoblastoma requires two hits and will affect 1 person in 10 000 (106 × 10-5 × 10-5 = 10-4), while the familial form requires only one hit and will be quite highly penetrant (106 × 10-5 = > 1).” [ vi] 4.1. Structure and Location of the Gene The identification of an unknown DNA market from the chromosome 13q14 region, which detected DNA rearrangements in retinoblastoma, facilitated the molecular cloning of RB1. To help understand the nature of the experiments already mentioned above, we will now have a look at the structure of the gene. “The RB1 gene has a complex organization with 27 exons, spanning greater than 200 kilobases (kb) of DNA, and an RNA transcript of about 4.7 kb”. (http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=cmed6.section.1570) There is a need for a detailed characterization of the sequence of the gene because despite gross deletions of the sequence observed in some cases of retinoblastomas, there is a express full-length RB1 transcript in most tumours. Additionally, they do not have detectable gene rearrangements making detection of inherited and somatic mutations of the gene very difficult especially for the purpose of molecular cloning. Mostly in patients with a single retinoblastoma there are two somatic mutations in their tumours and two normal alleles in their constitutional cells. In cases of multiple tumours arising in an individual patient we find the same germ line mutation and inherited retinoblastoma. This gives some evidence pointing towards the fact that the cloned gene and its inactivity is the essential and deterministic step in the formation of a tumour. Further evidence supporting this critical growth regulatory function of the gene can be found in experiments where the restoration of the function of RB1 has managed to suppress some aspects of retinoblastoma tumorigenesis. Once a cloned copy of the RB1 has been transferred it affects a number of properties, including including morphology and differentiated phenotype, growth rate in culture, and the ability of the cells to form colonies in soft agar and progressive tumors in nude mice. However, the results of the studies are limited to expressing an exogenous gene at nonphysiological levels and their significance can be questioned. 4.2. Results Having already discussed the role of RB1 in relation to the disease and certain experiments, we must now look at the basic scientific and biological function of the gene. We must also understand the function in relation to the interaction of proteins. The studies by Harlow and colleagues were the first to provide breakthroughs in the investigation of the gene; their findings were concurrent with Knudson's two-hit hypothesis already discussed. Basically, discussing the protein created by the RB1 gene we find that it is a nuclear phosphoprotein with a molecular weight of about 105,000 Daltons, referred to as p105-Rb or pRb.39. Scientists have determined the crystal structure of pRbAB bound to E2F(409-426) by conducting a series of biochemical experiments. At Proc Natl Acad Sci U S A, crystallography was administered. Using the hanging drop vapour diffusion method at 4°C they grew Plate-like crystals and the crystal structure of pRbAB bound to E2F was replicated. The crystals formed were frozen in a liquid which contained 25% glycerol. The European Synchrotron Research Facility used a microfocus diffractometer for calculations. Isothermal Titration Calorimetry is a method that they employed to mesure the binding by using a MicroCal Omega VP-ITC machine. The expressed His-6-RbABC obtained was phosphorylated using cyclin/cdk preparations obtained from insect cells that had been infected with recombinant baculoviruses. As a result of all these methods, the following structure was determined: [vii] The two orthogonal views in Ribbons representations show the crystal like structure of pRb/E2F. The red cylinders and the blue cylinders signify the helices of the A and B domains respectively. The yellow and green worms depict the main-chain trace of E2F and E7. The residues of the C terminus of E2F family have been highlighted in red and the other residues of pRb mutations are starred. There is a schematic representation of the interactions between E2F(409–426) and RbAB. The broken green lines are Hydrogen-bond interactions and the red spoked arcs are where hydrophobic contacts take place. pRb also forms a complex with the E1A oncoprotein encoded by murine DNa tumour virus adenovirus type. The ability of E1A to affect cell growth had already been discovered and it was now hypothesized that the functional inactivation of pRb and its interaction with E1A helped in the transforming abilities. 5. Introduction to the Protein We have already started talking about the protein pRb but before we move on, we must have a closer look at the structure of the protein. Retinoblastoma-associated proteinAdomain [viii] Here is a picture of the Retinoblastoma-associated protein A domain, which has the cyclin fold as predicted. The Sequence alignment is as follows, 1O9K_E 2 TPVRTVMNTIQQLMMILN.[2].SDQPSENLISYF.[8].ESILKRVKDIGYIF.[1].EKFAKA.[ 4].CVEIGSQRY 75 gi 75219106 274 TPVTSAMTTAKWLREVIS.[2].PDKPSSKLQQFL.[8].NAVTERVSIVLEAI.[1].PTKSSA.[14].DIPWAEARK 357 gi 74956083 511 KIIGSWKLENSKLEEVCG.[2].SDSPMATILLKS.[1].EMTNKFERTLSAEL.[1].ETINEN.[ 1].PKYHYNVRK 574 gi 85702751 376 EPVRNATNNVKQLSAFGR ITEPTDFVKQAG EEVIAKLLSIIEEI EQKFLA.[ 2].PSTEAKSRF 436 gi 82173896 353 TPVRTAMNTIQNLLNSLA.[2].SDQPSPTLKAYF.[8].RAIQERENNLGQIF.[1].QKFAEA.[ 4].CEEIGYQRF 426 gi 75220294 403 TPVSTAMTTAKWLRTYIS.[2].PSKPSPQLEKYL.[8].SEVVRRAHIIMEAI.[1].PNSALG.[14].DNIWAQQRR 486 gi 2498835 385 TPVASATQSVSRLQSIVA.[2].KSAPSEQLLNIF.[8].GNIIKIVKGIGETF.[1].QHYTQS.[ 7].HIDFAVNRL 461 gi 116242746 417 TPVSTATHSLSRLHTMLT.[2].RNAPSEKLEQIL.[8].QAIANRLKEMFEIY.[1].QHFQPD.[ 6].AKEIASKHF 492 1O9K_E 76 KLGVRLYYRVMESMLKSEEERLSIQNFSKLLNDNI FHMSLLACALEVVMATYS.[12].LSFPWILNVLNLKAFD 156 gi 75219106 358 VEASKLYYRVLEAICRAELQNSNVNNLTPLLSNER FHRCLIACSADLVLATHK.[ 3].MMFPAVLESTGLTAFD 429 gi 74956083 575 ELELVFLIFMEKIIVAELKKKVREEDLLNVIRREE FLDSVFCFCVELILVSNG.[ 2].RPFPWSAELCGVHPFM 645 gi 85702751 437 QLAKSFFFYLLDQILQAEIRNKPDIDLKRLLVQKV.[4].FNITLMACCVELVLEAYK.[ 2].LKFPWVLDCFSISAFE 511 gi 82173896 427 KVGVRLAYRVMEAMLKSEEKRLSVQNFSNLLNNSA FHESLLACSIEVVMATYG.[ 7].LSFPWILEVFKIEPYD 502 gi 75220294 487 LEALKLYYRVLETMCTAEAQLLHANNLTSLLTNER FHRCMLACSAELVLATHK.[ 3].MLFPAVLEKTGITAFD 558 gi 2498835 462 KLAEILYYKILETIMVQETRRLHGMDMSVLLEQDI FHKSLMACCLEIVLFAYS.[ 2].RTFPWIIEVLDLQPFY 532 gi 116242746 493 RFAEMLYYKVLESVIEQEQKRLGDMDLSGILEQDA FHRSLLACCLEVVTFSYK.[ 2].GNFPFITEIFDVPLYH 563 1O9K_E 157 FYKVIESFIKA EGNLTREMIKHLERCEHRIMESLAWLSDSPLFDLI 202 gi 75219106 430 LSKIIENFVRH EETLPRELKRHLNSLEEQLLESMAWEKGSSLYNSL 475 gi 74956083 646 FHKVIDLMITH EKQLSRQMVQHFSRIEETVIEYFSWKSDSPLWPMV 691 gi 85702751 512 FQKIIEIVVRH.[3].EGCLNRSLIKHLNSIEETCLERLAWARNSTVWEMI 560 gi 82173896 503 FYKVIESFIKD EPSLTREMIKHLESCEHRIMESLAWRSESPIYELI 548 gi 75220294 559 LSKVIESFIRH EDSLPRELRRHLNSLEERLLESMAWEKGSSMDNSL 604 gi 2498835 533 FYKVIEVVIRS EEGLSRDMVKHLNSIEEQILESLAWTNNSALWEAL 578 gi 116242746 564 FYKVIEVFIRA EDGLCREVVKHLNQIEEQILDHLAWKPESPLWEKI 609 (Marchler-Bauer A et al. (2009), "CDD: specific functional annotation with the conserved Domain Database.", Nucleic Acids Res. 37(D)205-10.) Briefly comparing pRb inactivation and effects in between mouse and human we find through studies and experiments that no phenotypes in vivo are induced by pRb in E7 mutants that fail to bind or inactivate pRb. In a study conducted by the American Society of Microbiology in which the effects of somatic pRB inactivation in murine skin was analyzed by using a Cre0lox based system, which allowed the scientists to compare the E7- expressing tissue in adult mice to pRb-deleted issue to determine which effects of E7 can be attributed to pRb inactivation. They have the following to say, “We demonstrate that pRb inactivation recapitulates all known phenotypes associated with E7 expression in murine skin. However, pRb-independent activities of E7 are detectable when E7 is expressed and Rb inactivated in the same tissue.” [ix] Two examples of proteins acquired from [x] serve as examples of proteins in the mouse and the human family. TIP47 protein isoform 2 Protein: ABB69732 Organism: Mouse Protein class: Soluble, non-secreted protein Subcellular Location Summary Endosomes literature data cytoplasm other SCL data endosomes other SCL data Detailed SCL data below Observed Isoforms   mPA9589.2 [325 aa]   mPA9589.1 [437 aa]    This isoform has SCL data:] Plasma retinol-binding protein precursor (PRBP) (RBP) [Contains: Plasma retinol-binding protein(1-182); Plasma retinol-binding protein(1-181); Plasma retinol-binding protein(1-179); Plasma retinol- binding protein(1-176)]. Protein: ENSP00000360518 Organism: Human Protein class: Soluble, non-secreted protein Subcellular Location Summary Extracellular literature data Detailed SCL data below Observed Isoforms   hPA3412.3 [199 aa]   hPA3412.1 [201 aa]    This isoform has SCL data:]   hPA3412.4 [199 aa]    This isoform has SCL data:   hPA3412.5 [199 aa]    This isoform has SCL data:] [xi] Further research showed that pRb als formed complexes with oncoproteins forming proteins such as including SV40 T antigen and the E7 proteins of human papillomavirus (HPV) types 16 and 18. This shows how significant pRb physical interaction with other proteins is as these proteins and the role of pRb is linked to cancer development. Cells are transformed partly by DNA tumour viruses by the inactivation of pRb hence we find that normally the role of pRb is to regulate and control cell growth by its interaction with other proteins. This functional activity of pRb, it is regulated by phosphorylation during normal progression through the cell cycle. “Accordingly, pRb appears to be predominantly unphosphorylated or hypophosphorylated in the G1 phase of the cell cycle and maximally phosphorylated in G2” [ xii] “Phosphorylation regulates the function of pRb during the cell cycle. The pRb protein is hypophosphorylated in the G1 phase of the cell cycle, and phosphorylation (P) of specific sites appears to increase during progression through the cell cycle.” [ xiii] The retinoblastoma protein shares similarities with two proteins known as p107 and p130. Even they form complexes with certain DNA tumour virus proteins. These proteins are even called the cousins of pRb because of their similarities. However, there is not enough evidence about the mutations of the other proteins, there have been no germ line mutations of these proteins in humans whereas there have only been some somatic mutations in a small fraction of small cell lung and nasopharyngeal cancers in the cases of p130 and somatic mutations are even more rarer in p107. There is not enough evidence for us to complete know the functions of these proteins. It is fair to suggest the hypothesis at this stage that pRb has a role to play in regulating cell genes in terms of its interaction with certain E2F family members but the role and functions of its “cousins” needs further experimentation. Discussing proteins that have a similar domain structure we find the following information after using the Conserved Domain Architecture Retrieval Tool found on the NCBI website. [xiv] We have already discussed the role of RB1 and the protein pRb in Homo sapiens in preventing apoptosis and cell generation. In the Mus Musculus family, the tumour suppressor protein plays a very critical role in controlling cellular proliferation. It does so by majoring regulating and controlling E2F activities. RB1 retinoblastoma has a huge gene family, the gene belonging to the Homo sapiens category has a gene ID 5925 and its lineage is Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Primates; Haplorrhini; Catarrhini; Hominidae; Homo (provided by HGNC). It was the first tumour suppressor gene found. From the many aliases from the Homo sapiens family is RB1CC1 RB1-inducible coiled-coil 1. An alias of gene from the mus musculus family is “Rb1cc1 RB1-inducible coiled-coil 1”, its official symbol is Rb1cc1 and its lineage is Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Sciurognathi; Muroidea; Muridae; Murinae; Mus (provided by MGI). The Rb1 gene also interacts with chemical compounds like the Abl C-terminus. It interacts with the retinoblastoma protein to bind DNA in the cell nucleus. It can be regarded as a potential mediator of the growth-inhibitory effect. Histone H3 lysine and methylation are also known to regulate some euchromatic genes targeted by pRb. The functional capacity of RB as a growth suppressor has a close relation to its ability to bind E2F as far as the physical interactions of Rb1 are concerned. JMJ and HBP1 are also known to interect with RB1. The gene also plays a major role in neuronal cell cycle control and apoptosis, where it is cleaved by caspase. Cyclin dependent kinase (cdk) 4 and cdk6 also are enzymes that interact with pRb to phosphorylate it in the nucleus to regulate G1 phase of the cell cycle. Finally, we will now look at the effects of some drugs on the protein pRb and some difficulties faced by the medical world. “Even though ginsenoside Rb1 is effective in the direct intracerebroventricular infusion, however, it appears impossible to apply ginsenoside Rb1 to human transient cerebral ischemic attack (TIA) and cerebral infarction due to the problems in the route of administration, similarly to other peptide growth factors” (Sakanaka M., 4635-4640, 1998; Wen, 635-649, 1998). “Examining the neuroprotective effect of ginsenoside Rb1 we see that in some culture experiments the high concentrations (0.11-11 μg/ml) of ginsenoside reduce glutamate-mediated neurotoxicity to prevent neuronal cell death reduce” (Kim Y. -C., 426-432, 1998). On the other hand, higher concentrations can possibility to prevent apoptosis. It is still not clear as to exactly how the mechanism of neuroprotection effects the protein pRb. The medical world is trying to find drugs which can have a therapeutic effect on patients who have cerebral infarction. They are also trying to find a way to protect cells and ways to effectively administer ginsenoside Rb1 by providing pharmaceutical compositions containing it or its salt to inhibit apoptosis. 6. Conclusion In conclusion, we have already seen how important the protein pRb and its function are for the development of many cancers. Science has still not managed to completely understand the role and function of this miraculous gene, further medical and genetical testing will give us more insights into the biological world and will hopefully elucidate the exact role of the gene Rb1. Based on the experiments and the interactions of the protein with other proteins and efforts to produce therapeutic drugs we can hope further experimentation can at least allow us to successfully administer the drug to protect the cell and prevent the death of cell caused by the inactivity of Rb1 and other diseases particularly related to the gene. We have found how the gene interacts with other proteins and it gives us a great insight into the functionalities of the protein, looking at the figures and relying on the hypothesis mentioned we can use the power of science to administer the gene in laboratory experiments. These experiments may in the future give us the ability to prevent the growth of tumour, development of cancer and hereditary retinoblastoma in people specifically related to the gene Rb1. In the end, the purpose of the entire research is to make sure we can make breakthroughs in the world of medicine to prevent people from suffering due to diseases by providing methods and ways of artificially administering drugs. 7. References 1) (http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&list_uids=19645) -Used to obtain scientific information about Rb1. 2) (http://www.bartsandthelondon.nhs.uk/Retinoblastoma/tests.asp) -Used for the results of testing on families with retinoblastoma 3) (http://mcb.asm.org/cgi/content/full/23/24/9094) -Comparing the protein across Human and Mice 4) (http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=151346&rendertype=figure&id=F2) -Picture of the structure of pRb/E2F crystal formed 5) (http://www.ncbi.nlm.nih.gov/Structure/cdd/cddsrv.cgi?ascbin=8&maxaln=10&seltype=2&uid=110825) -Picture of the Protein 6) (http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=hmg&part=A2342&rendertype=figure&id=A2382) -Used for figures and calculations of Knudson's two-hit hypothesis. 7) (http://www.ihop-net.org/UniPub/iHOP/gs/91639.html) -Used to obtain uses of the proteins 8) Sakanaka M. et al., Proc. Natl. Acad. Sci. USA, 95, 4635-4640, 1998; Wen T. -C. et al., J. Exp. Med., 188, 635-649, 1998 9) Kim Y. -C., et al., J. Neurosci. Res., 53, 426-432, 1998 10) Marchler-Bauer A et al. (2009), "CDD: specific functional annotation with the Conserved Domain Database.", Nucleic Acids Res. 37(D)205-10. 8. BIBLIOGRAPHY Read More