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The Human Genome The human genome is the sum of all of the genetic material in an organism a.k the sum of DNA. the human genome includes the coding regions of DNA, which encode all of the genes (between 20,000-25,000) of the human organism, as well as the non-coding regions of DNA which do not encod...

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The Human Genome The human genome is the sum of all of the genetic material in an organism a.k.a the sum of DNA. the human genome includes the coding regions of DNA, which encode all of the genes (between 20,000-25,000) of the human organism, as well as the non-coding regions of DNA which do not encode any genes. The nuclear genome comprises approx 3 billion base pairs of DNA that make up the entire set of chromosomes of the human organism. Each of the approx 10^13 cells in the adult human body has its own copy of the genome, exept cells such as RBC as they do not contain a nucleus. The vast majority of cells are diploid and so have 2 copies of each autosome plus 2 sex chromosomes - 46 chromosomes which contain the DNA (somatic cells). In contrast, gametes or sex cells are haploid and have 23 chromosomes. The human genome is a collection of long polymers of DNA. these polymers are maintained in duplicate copy in the form of chromosomes in every human cell and encode in their sequence of constituent bases (guanine (G), adenine (A), thymine (T) cytosine ©) the details of molecular and physical characteristics that form the organism. Genes are made up of DNA and each chromosomes has multiple genes. The sequence of DNA polymers, their organisation and structure and the chemical modifications they contain, not only provide the machinery needed to express the info held within the genome but also provide the genome with the capability to replicated, repair, package and maintain itself. DNA replication? The correct transfer of genomes to daughter cells is essential for the successful propagation of humans/organisms. One process that is involved in the maintenance of propagation of genetic information is DNA replication. The replication of the genome is a conserved mechanism that restricts DNA replication to occur once per cell cycle. Proper control of DNA replication is critical to ensure genomic integrity and maintenance during proliferation. DNA replication involves two primary tasks (1) the unwinding of DNA and (2) the semiconservative replication of DNA. the initiation of DNA replication occurs in G1 phase at replication origins across the genome. The human genome contained within the chromosome has thousands of bidirectional origins. The initiation of DNA replication involves 4 steps (1) the separation of start sites by the ORC complex and the cdc6 helicase loader, (2) the formation of the pre-RC by the loading of the inactive MCM2-7 helicase by orc.cdc6 and ctd1 (3) the activation of the helicase by the formation of the CMG cdc45.MCM2-7.GINS complex and finally (4) the establishment of a bidirectional replication fork. DNA replication is a highly regulated event and each protein is loaded onto origins in a timely and coordinated manner. ORC binds to origins first and forms a complex with cdc6. Together these recruit MCM2-7 in a complex with cdt1 to origins and the inactive MCM2-7 double hexamer surrounds dsDNA. As cells pass from G1 to S phase the inactive helicase is transformed into an active replication fork by the formation of the CMG complex which has robust helicase activity. The kinases CDK and DDK regulate this process. The active CMG helicase then unwinds dsDNA ahead of the recruitment of DNA polymerases at the replication fork, which now contains to ss template known as the leading and lagging strands. The leading strand is replicated by DNA pol-ε in the same direction as DNA unwinding and the lagging strand is synthesised by DNA pol-δ in a fragmented discontinous manner, producing replication products known as Okazaki fragments. The replication of the leading and lagging strands is highly regulated and is mediated by the help

of several proteins. The base pairing and chain formation reactions that make up the double helix are mediated by dna polymerases that move along the ss templates, reading them and incorporating the appropriate nucleobases. DNA pol-a primase provides dna rna primers required by the two DNA polymerases. RPA is ss DNA binding protein that stabilizes ss templates. Sliding clamps, PCNA, strengthen the association between dna polymerases and the templates and increase processivity 1,000 fold. RPC is needed to the repeated loading of PCNA at the initiation of each okazaki fragment synthesis and clamp loaders remove sliding clamps at replication termination. The maturation of okazaki fragments requires further activity of exonucleases such as Fen1 and also DNA ligase I. It has long been recognised that each of the replication proteins have an essential role in ensuring accurate dna replication, however much of their biochemical roles have remained elusive. A study conducted by Pellegrini et al which was published in nature on the 18th of may 2016, looked into the structure of cdc45 and its implications in the CMG helicase. They presented a crystallographic model of human cdc45, along with functional analysis. The uncovered that during its association with MCM2-7, cdc45 wedges its CID between adjacent A-subdomains of mcm5 and mcm2. It fastens and further stablizes the N-terminal ring of the MCM2-7 hexamer, leaving the C-terminal ring of AAA+ ATPase domain free to move as required for helicase activity. They reported that a remarkable feature of cdc45s association with GINs and MCMs is the large extent of its surface area e.g the helical protrusion comprising a long helix a6. This mode of cdc45 association within the CMG is compatible with the reported involvement of cdc45 with several additional replisome factors. They also proposed that cdc45 might act as a molecular brake by holding onto the unwound dna behind the fork and preventing ss DNA generation during replication stress, while mediating rapid CMG reassembly once the replication road block is resolved.

Aneuploidy Correct transfer of genetic information to daughter cells is essential for successful propagation of any organism. Three processes are involved in maintenance and propagation of genetic information: DNA replication, DNA damage repair and chromosome segregation. Error in any of these processes might result in cell death, or, in another scenario, in survival of cells with altered genetic information. This might be reflected either by single nucleotide changes as well as small insertions and deletions; or it might lead to larger alterations in the structure and number of chromosomes, together called aneuploidy. Meiosis is a specialised cell division which results in the production of eggs and sperm. In female meiosis (more so), chromosomes frequently segregate incorrectly, resulting in eggs with an abnormal number of chromosomes. When fertilized, these eggs give rise to aneuploid embryos that usually fail to develop. These abnormalities in meiosis are surprisingly common, but are not widely observed in populations are the consequences are very severe and often lead to fatal death, particularly in the first semester of pregnancy. Aneuploidy is defined as the loss or gain of one or more chromosomes during cell division.

COULD ALSO MENTION IN GENOME TECHNOLOGY Q: There are a variety of different methodologies for the detection of aneuploidy in human

oocytes/embryos and polar bodies. One of these methodologies is fluorescence in situ hybridization (FISH) which is a molecular cytogenetic technique. The chromosome specific FISH probes recognise a subset of clinically relevant trisomies (13, 16, 18, 21, X and Y abnormalities) which are hybridized to DNA and the FISH signals are counted to infer the chromosome constitution of the embryo. FISH only detects a subset of chromosomes and diagnosis is often inaccurate, although it is still used widely for embryo selection. Newer more sensitive methods such as array comparative genome hybridization (aCGH) and next generation sequencing (NGS) platforms provide improved statistics for the prevalence of aneuploidy and better characterization of segregation errors. These findings have enhanced the understanding of how chromosome segregation errors arise in meiosis. In aCGH, DNA from individual cells is amplified, fluorescently labelled and hybridized to microchip arrays decorated with thousands of probes that cover the entire genome. The analysis reveals chromosome gains or loses. It is a fast and sensitive method. Single whole cell genome amplification (WGA) analysis by NGS characterises the entire genomic content of polar bodies and oocytes - accurate, precise, however expensive and complex. The uses of such methodologies include its role in assisted reproductive technology (ART). Studies of live births conducted in the 1960/70’s demonstrated that approx 0.3% of newborn infants were trisomic or monosomic, whereas subsequent studies of spontaneous abortions identified a much higher incidence at approx 35%. These together established aneuploidy as the leading cause of congenital birth defects and miscarriage. Aneuploidies involving autosomes include nullisomies (lacking pair of homologs), monosomies (missing one chromosome) and trisomies (extra chromosomes). Nullisomy is lethal at preimplantation stage. Trisomy is usually lethal during embryonic/fetal stages, however individuals with trisomy 13 (Patau syndrome) and trisomy 18 (edwards syndrome) may survive to term and those with trisomy 21 (Down syndrome) may survive beyond 40. Down syndrome provided the first link between a clinical disorder and chromosome abnormality. It is caused by a meiotic error involving an autosome - usually result from failure of homologous chromosomes to separate properly (nondisjunction) in meiosis I in the ovary. Aneuploidies involving sex chromosomes in males include 47, XXY (extra X chromosome) or Klinefelter syndrome, 47, XYY (extra Y) and in females 47, XXX or 45, X. Most aneuploidies are attribute to errors in meiosis I, and in particular maternal meiosis. Two classical pathways that have been suggested to account for chromosome segregation errors in meiosis are nondisjunction (NDJ) and the premature separation of sister chromatids (PSSC). For NDJ, homologous chromosomes or sister chromatids fail to segregate in meiosis I or meiosis II respectively. However several studies have reported that many aneuploidies in meiosis I comprise losses or gains of single chromatids, but not pairs of chromatids that would indicate NDJ, this finding established PSSC as a model where sister chromatid pairs split from one another to independently segregate during anaphase I.

(non-age related causes) So why is aneuploidy more prevalent in females? Altered recombination is a serious cause of aneuploidy (maternal and paternal), but as most aneuploidy arises during oogenesis, the female is clearly at greater risk. On examination of crossovers it was found that in the male almost all chromosomes are joined by at least one crossover, whereas in females, over 10% of oocytes contain at least one ‘crossover-less’ bivalent. This failure to recombine and/or suboptimally located crossover e,g, too close to centromeres or telomeres is a huge cause of trisomy. Moreover the spindle assembly checkpoint (SAC) is less robust in mammalian oocytes. The SAC senses when kinetochores are unattached to spindle microtubules, and only allows the cell to progress to anaphase I if all kinetochores are attached to MT. however oocytes can proceed through meiosis despite chromosomes being misaligned. Another reason is that the length of oocyte development predisposes eggs to chromosome missegregation errors: advanced maternal age ----------> Age related causes of aneuploidy Fertility declines with advancing maternal age. Infertility is often reached from the age of 35 to 10 years later. Meiotic segregation errors increase sharply in this age window. A FISH study conducted examining over 20,000 oocytes reported aneuploidy in 20% of oocytes from 35 year old women and 60% in oocytes from women over 43. WHY? = in meiosis I (diplotene) human oocytes arrest which may last several decades. This occurs following the formation of bivalent chromosomes. The oocytes remain surrounded by a follicle to prepare the oocyte to mature into an egg that can give rise to an embryo after fertilization. Oocytes exist arrest after puberty and at ovulation meiosis I resumes. One hypothesis to explain the age related decline in female fertility is that ageing leads to the progressive deterioration of chromosome structures. The integrity of bivalents is essential for accurate chromosome segregation. However, evidence shows that cohesion is degraded during the protracted meiotic arrest. Bivalents experience two major structural defects with age. First sister kinetochores separate by large distances which correlates to the incorrect attachments of the sister kinetochores to spindle fibres. Second bivalents from ages oocytes more frequently separate into individual chromosomes called univalents. Pairs of univalents can

segregate in an uncoordinated manner and may contribute to aneuploidy. Tension is compromised (loss of biorientation).

A recent study published in oncogene in May 2017 by Dawar et al identified that caspase-2 mediated cell death is required for deleting aneuploid cells. They generated caspase-2 deficient mice and showed that when challenged by certain stressors that they succumb to enhanced tumour development and aneuploidy. Acute silencing in of caspase-2 in cultured human cells recapitulates the results that caspase-2 is required for the deletion of mitotically aberrant cells. They further generated Casp2C320S mutant mice to demonstrate that caspase 2 catalytic activity is essential for its function in limiting aneuploidy. These results provide direct evidence that the apoptotic activity of caspase-2 is necessary for deleting cells with mitotic aberrations to limit aneuploidy. HGP, GWAS, gene sequencing - human genome/technologies Q By 2003 the DNA sequence of the entire human genome was uncovered. Knowledge of the human genome provides and understanding of the origin of homosapiens, the relationships between subpopulations and the health tendencies or disease risks of individuals. In the past 20 years knowledge of the sequence and structure of the human genome revolutionized many fields of study. The human genome project was an international research project. The human genome was deciphered in 2 major ways: determining the sequence of all the bases, making maps to show the location of genes for major sections of all our chromosomes and producing what are called linkage maps, through which inherited traits can be tracked over generations. The genome published was a mosaic of a variety of different individuals, as it is thought that any two individuals at 99.9% identical. Analysis from the HGP first revealed SNPs as the source of genetic and phenotypic variation. SNPs are the most common variation, but various technologies have uncovered ‘structural variation’ or CNVs in the form of duplication and deletions, which affect the dosage of many genes. SNPs along with CNVs have made an important contribution into human diversity and disease susceptibility. The discovery of PCR, improvement in DNA sequencing, bioinformatics and methodologies like FISH and CGH have enabled the detection of the organisation and copy numbers of specific sequences in the genome. Moreover in the past 10 years GWAS studies have emerged as a powerful tool for investigating the makeup of complex traits, especially those of polygenic nature e.g

schizophrenia or autism. With the completion of the HGP and also the HapMap project researchers were provided with the tools to allow them to find the genetic contributions to common diseases. It typically focuses on investigating SNPs that occur across the entire genome and deciphering the association of these SNPs with major human diseases. An example of how dissecting the genome has benefited the investigation of risk loci in complex traits is a study which was published by the psychiatric genomic consortium in 2014. The PGC conducted the most comprehensive GWAS study on schizophrenia to date, which included up to 36989 cases and 113075 controls. The study successfully identified 108 distinct genetic risk loci that were of genome wide significance, 83 of which had not been previously reported. Notable findings from the GWAS study included associations at DRD2 and several genes involved in glutamatergic neurotransmission which highlighted molecules of known and potential therapeutic relevance to SZ, and also associations at gene which encode voltage gated calcium proteins involved in neuronal calcium signalling. The study also uncovered the MHC region as the strongest susceptibility candidate. This implication that immune genes are of the strongest genetic risk factors for SZ is a novel finding. SZ is a highly complex and multifactorial disorder, however the novel findings from large scale genomic studies provide an aetiologically relevant foundation for treatment development studies. Moreover the largest meta-analysis of GWAS studies on parkinsons disease was published in october 2017 by Chang et al. the study presented the largest meta-analysis of PD so far, involving a total of 26,035 cases and 403,190 controls. they identified 17 novel PD loci and, using a neurocentric candidate-gene nomination pipeline, found that several of the newly identified PD risk genes have a role in lysosomal biology and autophagy. The identification of these candidate genes allows for the prioritization of functional studies to determine causal genes for PD and possible therapeutic targets. As previously mentioned, knowledge of the makeup of human genomes also allows us to understand the relationships between subpopulations. A study conducted by cavellri et al set out to describe the genetic structure within the irish traveller population, the relationship between the irish travellers and other european populations and estimate the time of divergence between the travellers and settled irish, as well as the levels of autozygosity within the irish traveller population. They did this using high density genome-wide SNP analysis. They reported that the irish Traveller population has an ancestral Irish origin, closely resembling the wider Irish population. They also investigated the hypothesis that Irish travellers are a hybrid population between the settled irish and the Roma which they found to be untrue from their ADMIXTURE analysis.

Balance between cell proliferation and cell death.

Cell proliferation produces two cells from one, and it requires cell growth followed by cell division. Cell number is dependent not only on cell proliferation, but also on cell death. Programmed cell death, or apoptosis, is the process by which excess or damaged cells in the body are removed. Apoptosis is an extensive, ongoing process in our bodies. It is the balance between the production of new cells and cell death that maintains the appropriate number of cells in a tissue (referred to as homeostasis). Apoptosis is also a key mechanism by which cancer-prone cells are eliminated. Both normal apoptotic processes and normal cell mechanisms that control proliferation usually need to be altered to produce enough abnormal cell proliferation to cause cancer. The cell cycle is divided into interphase, and mitosis. During interphase cells undergo a growth phase G1 and a DNA replication phase S phase. After S phase cells undergo an additional growth phase G2 and prepare to enter mitosis M phase. Mitosis is the division of a somatic cell into 2 identical daughter cells. Prophase is the first stage of mitosis. During prophase chromosomes begin to condense, duplicated centrosomes separate and the Nucleoli breakdown. Prometaphase involves microtubule capture by assembled kinetochores and bipolar orientation is initiated as each kinetochore captures microtubules from opposite spindle poles and the nuclear envelope breaks down. During metaphase all chromosomes have made bipolar attachments and chromosomes align at the metaphase plate. The spindle assembly checkpoints ensures all chromosomes have achieved biorientation. In anaphase chromatids separate and move towards the opposite spindle poles. Nuclear envelope reassembly commences. During telophase the nuclear envelope reassembles around sister chromatids and the poleward movement of chromosomes continues. ...