master thesis in biology.pdf

Eötvös Loránd University (ELTE) master thesis in Biology Specialty: MOLECULAR GENETICS, CELL AND DEVELOPMENTAL BIOLOGY GENETIC VARIANTS (SNPS) AND ( INDELS ) SEARCHING IN AUTOPHAGY AND AGING RELATED GENES IN DOGS USING BIOINFORMATIC METHODS...

Eötvös Loránd University (ELTE) master thesis in Biology Specialty: MOLECULAR GENETICS, CELL AND DEVELOPMENTAL BIOLOGY GENETIC VARIANTS (SNPS) AND ( INDELS ) SEARCHING IN AUTOPHAGY AND AGING RELATED GENES IN DOGS USING BIOINFORMATIC METHODS STUDENT: IZZEDDIN HALAWA SUPERVISOR: Nóra Á. Bana Research Assistant ELTE Department of Genetics Prof. Tibor Vellai Head of department, University professor ELTE Department of Genetics Academic year: 2019 Budapest, Hungary Table of content : Table of content …………………………………………………………………………….. List of abbreviation…………………………………………………………………………. 1. Introduction ……………………………………………………………………………… 2. Literature overview………………………………………………………………………. 2.1 Autophagy ……………………………………………………………………………… 2.2 Autophagy major types ……………………………………………………………….. 2.3 Autophagy mechanism and regulation ………………………………………………… 2.4 Autophagy and benefit health ………………………………………………………… 2.5 autopagy andaging ……………………………………………………………………… 2.6.1 Gnentic varient ……………………………………………………………………….. 2.6.2 Types of genetic variant ………………………………………………………………. 2.7 Illumina Next generation sequencing ………………………………………………….. 2.8 FAMILY DOG PROJECT……………………………………………………………… 3. Material and method……………………………………………………………………… 3.1 laboratory work ………………………………………………………………………… 3.2 the procedure …………………………………………………………………………… 3.3 sequencing ………………………………………………………………………………,. 3.4 Variant Calling Pipeline: FastQ to Annotated SNPs…………………………………. 3.5 Variant Effect predictor. ……………………………………………………………… 3.6 Searching aging and autophagy genes in Eensmbl ………………………………….. 4.Result………………………………………………………………………………………. 5. Discussion…………………………………………………………………………………. 6.summary…………………………………………………………………………………... 7. References ……………………………………………………………………………….. 8.Acknowledgement ……………………………………………………………………….. 9. APPENDICES ………………………………………………………………………….. 1 List of abbreviation A Adenine AMPAK AMP Activated protein kinase ATG Autophagy Related Genes AWAK Script language was named by its authors Aho, Weinberger, and Kernighan BAM Binary Alignment/Map BED Browser extensible data BLAST Base local alignment search tool Bp Base pair BQSR Base Quality Score Recalibration BWA Burrows-Wheeler Aligner C Cytosine CAM Chaperon mediated Autophagy CR Caloric restriction DNA Deoxyribo Nucleic Acid ER Endoplasmic Reticulium FIP Family interacting protein FS Fisher strand G Guanine GATAK Genome analysis tool kit GB gigabyte Gbp giga base pairs Ggplot data visualization package for the statistical programming language R IGF Insluin like growth factor INDLES INSERTION and Deletion LC3 Microtubule associated protein 1A/1B-light chain 3 MB Megabyte 2 Mbp Mega base pair MEM Maximal Exact Matches MQ Mapping quality MQR Maooing Qouality RANK NBR1 Neighbor of BRCA1 gene 1 P62 ubiquitin-binding protein p62 PE Phosphorylethanolamines PIP2 phosphatidylinositol biphosphate PIP3 phosphatidylinositol-3-phosphate QD Qoual by Depth RAB7 RAB family of RAS-related GTP-binding proteins SIRT1 sirtuin (silent mating type information regulation 2 homolog) SNARE soluble N-ethylmale-imide-sensitive factor-attachment protein receptors SOR Strand Odds Ratio T Tyrosine SNPs Single Nucleotide Polymorphisms TOR The mammalian target of rapamycin ULK1 UnCoordinated 51- like kinase 1 VCF Variant Call Format VEP Variant effect predictor VPS Vacuolar Protein Sorting WIPI WD-repeat protein Interacting with PhosphoInositides 3 1. Introduction The SNPs (Single Nucleotide Polymorphism) is a single nucleotide change at a particular position in the genome. Not all single nucleotide changes are SNPs. The researchers talk about SNP when the frequency of the variation in the population exceeds 1%. The less frequent variation is usually called mutation. More and more SNPs can be identified, and it seems that these alterations are behind several biological phenomena. Personal differences in these nucleotides result for example in elevated disease susceptibilities, that is, certain nucleotides are more frequent in patients suffering from different diseases comparing to the healthy population. SNPs may cause substantial alterations in the cells, e.g. the enzyme activity of the respective gene changes, but in other cases, the effects of the SNPs are not so pronounced. I assumed that some specific SNPs can be rendered to individuals living a longer life than the average. Perhaps these results will not directly lead to the lengthening of the maximal life span; however, genes that play an important role in the aging process could be identified. In this respect, SNPs are important factors in determining the information level of the cells of individuals which determines the maximal life span. In turn, SNP is one of the factors that determine the aging process. Since there are certain age-related diseases, the discovery and the description of the SNPs as a function of age and diseases may result in a better understanding of the common roots of aging diseases. I was studying Unique (94) aging genes and (100) autophagy genes in dog sample using bioinformatic methods( see Appendix1,Appendix2). for example variant effect predictor, and one of the aims of our work was aligning the read files came from from old dog sample to the latest reference dog genome (CanFam3.1), and then identified genomic variants in the aligned read sequences, such as single nucleotide polymorphisms (SNPs) and DNA insertions and deletions (indels). I searched the Ensembl identifier of the most important aging and autophagy ortholog genes in Canis familiaris reference genome assembly (CanFam3.1), and successfully identified the genomic variants in these ortholog dog genes. I suppose they have majority role of aging and autophagy genetic background. The SNPs of genes associated with particular traits like aging and autophagy can disclose the process of these genes. SNPs that occur within a gene or in a regulatory area near a gene may directly affect that gene’s function and the resulting trait. 4 2. Literature overview 2.1 Autophagy : Autophagy is the destruction and recycling of damaged and redundant cellular components this function is absolutely necessary in the body in order to clean up cellular debris so that the cell can function properly this is also a survival and adaptation mechanism when the body is suffering from physiological stress.(Parzych &klionsky,2014). 2.2 Autophagy types : There are currently three types of autophagy in mammalian cells, first type maroautophagy the bulk degradation of different cellular components via Autophagosome. and the other type of autophagy is microautophagy and chaperon mediated autophagy and all types of autopahgy have one thing in common and that is the degradation of substrates within the lysosome whether those substrates be proteins, lipid droplets or organelles ( see figure 1). (Mizushima, 2007). that macro autopahgy is an umbrella term for various different types of autophagy that all utilize autophagosome and are degraded through bulk degradation in the lysosome and different types of macroautophagy are named according to what they actually degrade one type of macroautophgy is my mitophagy or the bulk degradation of mitochondria another type of macroautophagy is reticulophagy or the degradation of the endoplasmic reticulum in the lysosome another one is nuleophagy is the degradation of nucleus in the lysosome and all these are related to organelle degradation another type of macroautophagy is Lipophagy is the degradation of lipid droplets and another type called which is xenophagy is the degradation of foreign microorganisms such as bacteria and each process of each type of macroautophagy is named by the type of substrate that it's targeting.(Mizushima, 2007). The three main forms of autophagy are macroautophagy microautophagy and chaperone mediated autophagy and the most common form of a autophagy is macroautophagy which is best known for its formation of a double membrane structure called an autophagosome the process is induced by stimulus such as starvation resulting in the formation of an autophagosome and a zonal vesicles delivered lysosomal components such as membrane proteins or enzymes to the auto phagosome at this point the autophagosome are also called late or degradation artifact vacuoles finally Auto phagosome fuse with either late endosomes multi vesicular bodies or 5 lysosomes the autophagy body is delivered to the lumen to be degraded by enzymes such as lysosomal hydrolysis Microautophagy defers from macroautopahgy due to the absence of the autophagosome the intermediate vesicle that delivers cytosol contents in the latter process in microautophagy the cytosol contents are directly taken up through invaginations in the lysosome membrane leading to immediate degradation.(Mizushima, 2007). chaperone mediated autophagy also known as CMA is another type of autophagy that differs from macroautophagy by the specific selection of cargo and delivery process to the lysosomal lumen for disposal in CMA a chaperone and Co chaperone proteins recognize a specific pentapeptide sequence in all cytosolic proteins and form a chaperone substrate complex the chaperone substrate complex is recognized by the lysosomal membrane receptor resulting in their unfolding and degradation of the substrate protein by the lysosomal lumen.(Mizushima, 2007). Macroautophagy and CMA have distinct processes but communicate with each other in the autophagy lysosomal degradation process during certain conditions one process can act as a backup mechanism when the other is defective our understanding of the relationship between these two processes is very limited more research in the advantages and limitations of these two autophagy pathways is crucial to better develop effective therapeutic interventions and autophagy associated human disorders mediate a degradation of damaged organelles proteins and lipids plays a critical role in the development of many eight related diseases as a result the process of autophagy serves as a novel pharmaceutical target in the development of therapies certain conditions can be improved with enhanced autophagy such as neuro degradation and the pathogenesis of neurodegenerative Diseases. (Mizushima, 2007) 6 Figure 1: the three types of autophagy , the first type macroautophagy and the second miroautophagy and third chaperon mediated autophagy.(Parzych & Klionsky, 2014). 2.3 Autophagy mechanism and regulation : all types of macroautophagy begin at the end of plasmic reticulum and it starts by the formation of something called a pre Autophagosome a budding of membrane off of the endoplasmic reticulum and this pre autophagosome it's a it's a developing cup shaped membrane and it is it requires protein complex with proteins ULK1 and ATG 13 stands for autophagy related gene or autophagy protein so ATG 13 it requires FIP 200 and ATG 101. (Glick, Barth, & Macleod, 2010). that ULk1 is important for initiating processes of pre auto phagosome formation and initiating steps of macroautophagy and the next step that occurs as this pre auto phagosome is becoming larger and becoming more elongated there's another protein complex that gets involved and this protein complex involves vacuole ER protein sorting 34 bps 34 VPS 15 becklin 1 proteins now ULK1 actually activates becklin 1 through phosphorylation and this complex with VPS 34 VPS 15 and beckclin 1 all act like a pip3 kinase and they actually produce PIP3 from PIP2 this increasing concentration of PIP3 actually leads to the recruitment of WIPI proteins to the preautophagysome membrane , once those WIPI proteins have been recruited other proteins are recruited as well some of these proteins include P62 which is ubiquitin cargo binding protein NBR1 is a related protein that acts similarly to P62 and these act as partial selective receptors for different substrates in the cell , another protein that's very important in this process as the pre autophagysome elongates and matures and becomes larger is called LC3-1 , another protein complex that's important in this process as well is the protein complex that consists of ATG5 ,ATG16,ATG12 and this complex actually helps to target particular substrates and particular proteins to the developing autophagosome , LC3-1 is crucially important in all this process ,LC3-1 actually comes from LC 3 or pro LC 3 they call it and procede three is their pro LC 3 is actually cleaved by a cysteine protease ATG 4 into LC 3-1 so once LC 3 one has been targeted to the autophagsome membrane it actually gets activated by ATG 7 and bound to ATG3 and then conjugated to PE or faso ethanol amine to actually formLC3- 2 so as this conversion from LC 3 -1 to LC3 -2 occurs the autophagosome actually continues to elongate it and actually becomes a full-fledged autophagsome vesicle this is when we call it a mature autophagsome and then once you have a mature autophagosome with its contents it'll actually 7 fuse to the lysosome via SNARE proteins and Rab 7 once it fuses to the lysosome it becomes an auto lysosome and then what happens is the membranes fuse together the autophagosome releases its contents into the lysosome and then those substrates are actually degraded in the acidic lysosome by particular proteases such as cathepsine B and there are other cathepsine as well (Yu, Chen, & Tooze, 2018). first process is typically referred to as initiation the second process whereby we're having form a of pip3 this is what we call nucleation another process as the auto phagosome actually becomes larger through lc3 -1 conjugation processes this is what we call elongation then once the actual Autophagsome vesicle actually forms and closes this is what we call maturation and in the final step is fusion (see figure 2).(Ravikumar et al., 2009). Figure2: the main steps of autophagy. begin with stimuli like starvation and first step is initiation and after that vesicle elongation and then vesicle maturation and the fusion and docking and final step vesicle breakdown and degradation(Choi, Ryter, & Levine, 2013). 2.4 Autophagy and aging : Aging is a complex process characterized by the progressive failure of maintenance and repair pathways important for cellular homeostasis, which results in a gradual accumulation of aberrant macromolecules and organelles. The accumulation of such oxidized, misfolded, cross-linked, or aggregated molecules has deleterious effects on cellular homeostasis and on tissue and organ integrity. The defective molecules can disrupt homeostasis directly or by interfering with the activity of functional molecules and organelles, which leads to further dysfunction. This progressive decline in cellular integrity leads to aging, disease, and ultimately, to death. Although our understanding of the biology of aging has increased over the past century, the molecular events underlying this process have only recently begun to be explored. Interestingly, research in the last couple of decades focused on unraveling the molecular underpinnings of aging has shown that, in many model organisms, the rate of aging can be 8 modulated by altering conserved signaling pathways and processes, suggesting that the aging process itself may ultimately be amenable to therapeutic manipulation. We provide a short overview of these conserved longevity paradigms below(Gelino & Hansen, 2012). Increased Autophagy Delays Aging and Extends Longevity:The first indication that increased autophagy may contribute to longevity comes from the seminal observation that inhibition of the insulin-like growth factor pathway causes autophagy in C. elegans and that inhibition of autophagy by mutation of essential Atg genes prevents the gain of longevity. Caloric restriction (CR), that is reduced food intake without malnutrition, is the key anti-aging intervention that extends life span in most animals so far tested, including in rhesus monkeys, in which it reduces the incidence of diabetes, cardiovascular disease, cancer, and brain atrophy. Epidemiological studies suggest that CR is also beneficial to human health. CR is the most physiological inducer of autophagy , and inhibition of autophagy prevents the anti-aging effects of CR in all species investigated in this respect. Depending on the way that CR is imposed—by intermittent feeding versus chronically reduced intake, its onset (juvenile versus adult), or its intensity—CR induces autophagy through the activation of either of two energy sensors, AMPK and Sirtuin 1 (SIRT1), which engage in a positive forward loop of mutual activation. Moreover, CR can induce autophagy through the inhibition of insulin/insulin-like growth (IGF) factor signaling, which also results in TOR inhibition. CR does not further increase life span when TOR signaling is already reduced in yeast, worms, or flies, suggesting that common mechanisms mediate both of these anti-aging interventions(Rubinsztein, Mariño, & Kroemer, 2011). 2.5 Autophagy and health benefit : at a cellular level as to what is happening there's something called the autophagsome what it does is it goes around the cell it picks up damaged cell parts and then combines with something called the lysosome after combining with the lysosome it creates new cell parts by recycling the old stuff by recycling the damaged stuff it creates new cell components and energy now when we look at times in physiological stress such as fasting(Martinez-Lopez et al., 2017). some of the different health benefits of autophagy so the first benefit is a decrease in inflammation this is super powerful because inflammation or chronic inflammation I should say is really the root cause of disease when we look at cancer,and heart disease all these different chronic conditions they started with a chronic inflammatory problem in the body so decreasing inflammation is 9 going to be really powerful for avoiding disease in your future it also is going to boost your immune system in some Research shows us that with a four day water fast as four days will actually act as a complete reset to your immune system this is very powerful because many many people today suffer from autoimmune conditions and then it's also going to improve the anti- aging effect on your body so its is really support your skin and help keep your skin healthy it's gonna help reduce wrinkles it's also gonna support you if you're someone who has lost a lot of weight activating our autophagy in your body is gonna be very powerful for reducing that loose skin it's also going to improve your brain function some research shows that it's going to improve your brain function but also act as a protective mechanism for the brain to protect you from neurodegenerative diseases such as Alzheimer's and Parkinson's(Jaeger & Coray, 2009). it's going to decrease pathogens and bacteria which is very powerful in order to keep you healthy and it's also going to support longevity okay you know as I mentioned up here decreasing inflammation preventing cancer preventing heart disease preventing serious diseases in your future is very powerful as a matter 18 hour fast is going to boost our autophagy in the body and help cleaning up those cellular debris and keep your cells healthy now the other way that you can actually boost our autophagy in the body is by doing high-intensity interval training so doing short-term exercise that is very high in intensity and that is going to also boost autopsy so the two best ways to activate autophagy in the body is by doing fasting and by doing high-intensity short duration exercise the benefits here are clear the benefits here are really amazing at autopahgy is an incredible tool that we can use in order to prevent disease and keep us healthy.(Choi et al., 2013) 2.6.1Genetic variant : Variants are differences in our DNA which we all carry. These differences give us unique characteristics, such as hair color or predisposition to a disease. You may have heard the term mutation, that's the same thing as a variant. There are many kinds of variants. You'll hear terms such as: missense, nonsense, insertion, deletion, duplication; these are terms that indicate how the genetic code changed to make it a variant. A deletion variant means that a letter or several letters in the code are missing. For example, the sequence "A A T T" could become "A A T" in a missense variant, a single letter gets changed to another letter. If our hypothetical sequence "A A T T" became "A A A T", that would be a missense variant. Variants influence how our genes function, or don't function. A tiny change could mean that a necessary protein did not get made, 10 and that can have a huge impact. Some variants are protective, and can help us fend off certain diseases, others may make us more susceptible. Certain variants determine how our bodies respond to certain medicines or which diseases we may pass along to our children, and genetic variants can tell us a lot about where our ancestors came from.(Syvänen, 2001) 2.6.2 Types of genetics variant : 1)Single nucleotide poly morphism (SNPs) an entire set of 23 human chromosomes is called a genome the human genome is composed of 3 billion base pairs variation at a single base pair is called a SNP or single nucleotide polymorphism when the body makes new cells it doesn't make many mistakes sometimes when the genome is copied to make a new cell a single base pair gets left out added or substituted single base pair substitutions create slips there are around 10 million SNPS in the human genome which account for many of the genetic differences between you and everyone else on the planet some snips account for differences in appearance others can affect how we develop diseases or respond to drugs most snips however seem to lead to no observable differences between people at all since variants are passed on from one generation to the next the number of differences between your DNA and your neighbors can tell you how closely you are related to each other.(Zhao et al., 2017) 2)Indles ( insertion and deletion ) : a frameshift mutation is when an insertion or deletion causes a disruption in the reading frame the reading frame describes how the nucleotides are broken up into sets of codons that are each three nucleotides long each codon codes for one amino acid in a protein you can see in the original DNA strand that all of the codons are CAT which codes for the amino acid histidine but if a single nucleotide is deleted the reading frame shifts causing all of the codons to change this results in incorrect amino acids being added to the protein once again we start with the same original strand of DNA we have the codons CIT which code for the amino acid histidine this time we will insert a single nucleotide but you'll notice that this also shifts the reading frame changing all of the subsequent codons and causing the incorrect amino acids to be placed into this protein as well whenever there is a disruption in the reading frame of a gene whether it is caused by an insertion or a deletion it is a frameshift mutation frameshift mutations can cause drastic changes to the amino acid sequence of a protein disrupting the protein structure and its ability to function correctly(Mills et al., 2011). 11 2.7 Illumina sequencing : the Illumina sequencing workflow is composed of four basic steps sample prep cluster generation sequencing and data analysis there are a number of different ways to prepare samples all preparation methods add adapters to the ends of the DNA fragments through reduced cycle amplification additional motifs are introduced such as the sequencing binding site indices and regions complimentary to the flow cell oligo clustering is a process where each fragment molecule is isothermally amplified the flow cell is a glass slide with lanes each lane is a channel coated with a lawn composed of two types of oligo hybridization is enabled by the first of the two types of oligo on the surface this all ago is complementary to the adapter region on one of the fragment strands a polymerase creates a complement of the hybridized fragment.(TH & MA, 2016). the double-stranded molecule is denatured and the original template is washed away the strands are clonally amplified through bridge amplification in this process the Strand folds over and the adapter region hybridizes to the second type of oligo on the flow cell polymerases generate the complementary strand and forming a double-stranded bridge this bridge is denatured resulting in two single-stranded copies of the molecule that are tethered to the flow Cell the process is then repeated over and over and occurs simultaneously for millions of clusters resulting in clonal amplification of all the fragments after bridge amplification the reverse strands are cleaved and washed off leaving only the forward strands the three prime ends are blocked to prevent unwanted priming sequencing begins with the extension of the first sequencing primer to produce the first read with each cycle fluorescently tagged nucleotides compete for addition to the growing chain only one is incorporated based on the sequence of the template after the addition of each nucleotide the clusters are excited by a light source and a characteristic fluorescent signal is emitted this proprietary process is called sequencing by synthesis the number of cycles determines the length of the read the emission wavelength along with the signal intensity determines the base call for a given cluster all identical strands are read simultaneously hundreds of millions of clusters are sequenced in a massively parallel process this image represents a small fraction of the flow cell after the completion of the first read the read product is washed away in this step the index one read primer is introduced and hybridized to the template the read is generated similar to the first read after completion of the index read the read product is washed off and the three prime ends of the template are protected the template now folds over and binds the second oligo on the flow cell index to is read in the same 12 manner as index one polymerases extend the second flow cell all ago forming a double-stranded bridge this double stranded DNA is then linearized and the three prime ends are blocked the original forward strand is cleaved off and washed away leaving only the reverse Strand begins with a sequencing primer as with read. one the sequencing steps are repeated until the desired read length is achieved product is then washed away this entire process generates millions of reads representing all the fragments sequences from pooled sample libraries are separated based on the unique indices introduced during the sample preparation for each sample reads with similar stretches of base calls are locally clustered forward and reverse reads are paired creating contiguous sequences these contiguous sequences are aligned back to the reference genome for variant identification the paired and information is used to resolve ambiguous alignments genomic data can be securely transferred stored analyzed and shared in base base sequence hub discover the possibilities of next-generation sequencing ( see figure 3).(Buermans & den Dunnen, 2014). Figure 3: Illumina sequencing the four major steps first it is library preparation and second step cluster formation and third step sequencing and fourth step sequences identification.(Zhou & Li, 2015). 13 2.8 The family dog project : The family dog project established in the Department of ethology in ELTE university where we took our dog sample from them to analyse the dog genome and searching about the genetics variant (SNPs,Indles) using the bioinformatics method The aim of this project is to explore ageing of family dogs, using an approach with the most advanced Behavioral , neuroscientific and genetic testing methods. They are expected to provide guidelines for a healthy lifestyle toward better understanding for ageing in Dogs. Dogs’ mean life time is around 13.Senescence is a naturally occurring and its complex biological process, and it is one of the most relevant problems to understand how active and healthy ageing can be achieved. The study of dog ageing is important to know more about variant if had some relation to dogs longevity or aging. , very little is known about the actual prevalence and risk factors of age-related changes in dogs. The relative life span in dogs offered by the protective human environment artificially.their Research could facilitate the early recognition and treatment of certain conditions, as well as provide a way for a predictive approach. And More life experiences dogs living in families provide a good natural model for human ageing. Dogs sharing their lives with humans gained from this alliance by doubling their mean life span in Dogs.. (https://familydogproject.elte.hu/about-us/our-research/). 14 3. Materials and methods Basic information The DNA sample came from 22 years old female mixed ( greyhound, pointer, german shepherd, etc.) dog. She lived in an asylum, but before that, she had lived in the countryside. We suppose that she was healthy because she seems to be healthy. Her name was Kedves (Darling). 3.1 Laboratory work Before our bioinformatic work, the laboratory researcher team executed the DNA preparation. They took 1 ml blood out of this dog and put the sample into a tube which contained EDTA This protocol is for purification of total (genomic, mitochondrial, and viral) DNA from whole blood, plasma, serum, buffy coat, lymphocytes, and body fluids using a microcentrifuge. buffer after that froze the sample. Sample collection was performed according to standard veterinary medical practice and it was in accordance with Hungarian Veterinary Chamber [Hungarian Animal Rights Law (243/1998, XII.31)]. 3.1.1 The materials All centrifugation steps are carried out at room temperature (15–25°C). They used Use carrier DNA if the sample contained