DISEASEFunctional genomics are: genetics, cell biology and physiology and how they relate to human health and disease.

Genetics, health and environment.

Examples where our genetics affect our health:

heritability• describes how much of a phenotype is explained by geneticcauses• Single-gene Mendelian diseases have a high heritability• the heritability of a genetically complex disorder such asasthma has a much lower heritability and involves aninteraction between many polymorphisms in many genes

PenetranceThe frequency with which a genotype manifests itself in a given phenotype. The mutation that causes Huntington’s disease has a high penetrance- if you have inherited the mutation, you will exhibit the phenotypeIn contrast, the polymorphisms that are associated with asthma have low Penetrance• Over the history of human migration, different populations have settledin different regions and environments• These environments have shaped our genomes

Genetics and the Environment

The ability of adult humans to digest milk follows the domestication of cattle• About 10,000 years ago humans in Northern Europe and Central Africa domesticated cattle• The ability to digest milk is due to mutations in the lactase gene and provides a selective advantage (the mutation becomes common in a population)

Asthma and the hygiene hypothesisan imperfect fit between our genes and the environment?

Is early-life exposure to microbial agents important to “educate” our immune systems?• If we are “too clean” does this have deleterious effects on our immune system?• Are some people with a specific genotype more strongly affected by such an environmental change?• This is known as the “hygiene hypothesis”- it is still being debated and would not explain all types of asthma

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SNPs do not directly cause disease• A mutation can often be the direct cause of a disease (eg. cystic fibrosis, Huntington’s disease)• SNPs influence the likelihood that someone will develop a particular illness.• SNPs are not absolute indicators of disease development.

• A gene with an average length of 34,000bp will have between 100 and 350 SNPs• Genome-wide, there are as many as 10,000,000 SNPs where both alleles have a frequency of at least 1%• SNPs can occur anywhere within or between genes

The “common disease, common variant” hypothesis:The genetic basis of many common diseases is attributable to SNP alleles that are present at a frequency of more than 1%–5% in the population

• Our genome is a mixture of old common variants and a continuing appearance of new mutations.• The genomes of different ethnic populations will show many differences in SNP allele frequency• The differences in allele frequency can be correlated with susceptibility to Disease

• There are millions of SNPs across our genome• Combinations of different alleles of these SNPs influence our susceptibility to disease• How do we discover which alleles confer susceptibility to disease?

How is genetic diversity generated during meiosis?

• Haplotype blocks are important in genetic mapping studies as they allow geneticists to predict genotypes• For example, if you know the allele at SNP #5, then you can predict the allele at SNPs # 6-12: this reduces the number of SNPs we need to genotype• Haplotype: A series of alleles found at linked loci on a single chromosome• Locus: a unique chromosomal location defining the position of an individual gene or DNA sequence• Using haplotypes is important in genotyping studies• Instead of genotyping all 10,000,000 SNPs in the genome, geneticists can genotype a few hundred thousand that will identify all haplotypes in the humangenome• This is easily achieved with modern genotyping technologies

• Most cases of breast cancer are sporadic• Mutations (inherited or sporadic) in genes greatly increase the risk of cancer• Up to 1 in 8 women will develop breast cancer

• Generally, cancer is caused by mutations that are rare in a population.• These mutations result in a very high to certain probability (depending on the cancer) that a person will develop cancer- high penetranceMany genes are associated with common diseases such as Type 2diabetes, inflammatory bowel disease and asthmaWhat might we mean by “associated with” and how does this differ from a gene that causes cancer?• The odds ratio compares the odds of having asthma and a risk allele against

the odds of having asthma but not having the risk allele (the risk allele is the allele that is more common in asthmatics; an odds ratio of 1= no effect)• The important point is that the odds ratios are very close to 1- this means a single allele imparts only a very small increase in risk on a person of developing asthma• Asthma (and other common diseases) are the sum effect of many alleles interacting with the environmentMany health problems that confront our society are complex, involving multiple environmental, cellular and genetic influences.

Antigen: any material that can evoke an immune responseImmunological tolerance (hypo-responsiveness): Reduced responsiveness to antigens. It is an active process, not simply a passive lack of response.Commensal bacteria: present on body surfaces covered by epithelial cells thatare exposed to the external environment (mucosa). The adult human intestine ishome to an estimated 1014 commensal bacteriaThe mammalian intestine provides a stable microenvironment for bacteria that isrich in nutrients. Commensal bacteria are essential for normal intestinalphysiology (absorption, digestion etc.)

Insulin reduces blood glucose levelsby: Promoting the cellular uptake ofglucose and Slowing glycogen breakdown in theLiver and Promoting fat storage

Glucagon increases blood glucoselevels by: Stimulating conversion of glycogento glucose in the liver and Stimulating breakdown of fat and protein into glucose

environment-(food intake)intestinal commensal microbesimmune responsebarrier response (physical barrier of the intestinal epithleium)

Immunology and Homeostasis in the Human Intestine• The human intestine is constantly exposed to a high and highly diverse number of antigens• In the healthy intestine, mucosal immune cells must distinguish between beneficial antigens (food and commensal flora) and harmful antigens on pathogenic Bacteria• This means a state of immune tolerance must exist in a healthy human intestine• Pathways important for dampening the inflammatory response (tolerance)may be disrupted in ulcerative colitis and Crohn’s disease (inflammatorybowel disease).

Inflammatory Bowel Disease:• Crohn’s disease can arise anywhere along the gastrointestinal tract between the mouth and anus and affects the full thickness of the gut from mucosa to muscle

• Ulcerative colitis is restricted to the large intestine and affects only the superficial layer of the mucosa

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Microbial-associated molecular pattern (MAMP): a microbial structural determinant that can be recognised by pattern recognition receptors that are part of the innate immune system.• Pattern recognition receptors can be on the cell membrane: TLR (Toll-like Receptors)• Pattern receptors can be cytoplasmic: NOD (nucleotide-binding oligomerization domain)• Both types of pattern recognition receptors recognize structures that are part of bacteria: lipopolysaccheride (LPS), carbohydrates etc.

Paneth cells:• directly sense bacteria through TLR/MyD88 activation and limit bacterial penetration of the mucosal surface.• The activation of MyD88 initiates an antimicrobial response• This limits the numbers of bacteria that localize at the mucosal surface. This is also important in controlling infection.

Model of small intestinal Paneth cell function.

• Homeostasis in the human intestine involves the induction of immune tolerance toward commensal bacteria• The innate immune system, involving the secretion of anti-microbial peptides from Paneth cells, is essential for homeostasis in the intestine• What happens when homeostasis is compromised?

Inflammation is a protective attempt by the organism to remove the injurious stimuli and to initiate the healing process. It involves both the adaptive and the innate arms of the immune system.Infection leads to inflammation but not all inflammation is due to infection.

acute disease refers to the duration of the disease; successful defence by the body of acute infections will restore health.chronic diseases are long lasting and can often involve additional healthproblems that are not seen in acute diseases. This is seen in the case of asthma where chronic airway

inflammation leads to irreversible damage to the airwaysIn obese individuals a spectrum of health disorders that includes diabetes and heart disease and is linked to persistent inflammation is often seen.

• Inflammation is a result of infection, arising from bacteria or viruses crossing the epithelial barrier• The innate and adaptive arms of the immune system are activated• Paneth cells synthesize and secrete increased amounts of antimicrobial protein

• Chronic inflammation results in tissue damage, largely due to the release of highly potent immuno-modulators from activated immune cells (eg. ROS)

• Water-soluble proteins destined for secretion, and membranebound proteins are imported intothe ER• A specific signal sequence on the growing polypeptide signals entry into the ER

• In the ER protein synthesis is completed, the proteins are folded and modified (glycosylation)

• Secretory cells- such as Paneth cells in the intestine- respond to specific cues to increase their secretion of proteins• This requires increased protein synthesis and an increased amount of unfolded proteins in the ER• The imbalance between the amount of unfolded protein and the capacity of the ER to process unfolded protein is referred to as ER stress.• The UPR ensures correct protein folding and prevents an accumulation of unfolded proteins,• The UPR involves changes in translation and transcription

• There are 3 arms to the UPR response• We we be looking only at the pathway involving XBP1

IRE1α: A protein with kinase activity and an endoribonuclease activity. The endonuclease activity cuts and activates XBP1 RNA. IRE1α is important in altering gene expression via XBP1 as a response to endoplasmic reticulum-based stress signals.BiP: a chaperone that is bound to IRE1a in non-stressed cells and to unfolded protein in stressed cellsXBP1:Resolves ER stress- Turns on the transcription of genes encoding ER chaperones and enzymes that promote protein folding, maturation, secretion and ER-associated protein degradation.If the ER stress cannot be resolved (if homeostasis is not restored)-The UPR will initiate apoptosis (cell death) to protect the organism by removing the stressed cells (eg. Paneth cells).• The highest incidence rates and prevalence of ulcerative colitis and Crohn’s disease have been reported from Northern Europe, the UK, and North America.• Lower socio-economic status- absence of tap water, absence of hot water, large or poor families with several children, crowded living conditions, consumption of contaminated foods- lowers the risk of developing inflammatory bowel disease• Does excessive sanitation limit exposure to environmental antigens and impair the functional maturation of the mucosal immune system?• Does this impair the induction of immune tolerance in the gut later in life?

Experimental findings:• A mouse that lacks XBP1 develops spontaneous colitis (inflammation of the bowel)• A mouse that lacks XBP1 has a massive reduction in the number of Paneth cellsConclusions:• XBP1 is essential for intestinal Paneth cell development and survival• XBP1 in intestinal Paneth cells to promotes high level secretion of anti-microbialPeptides

Polymorphisms in XBP1 are associated with inflammatory bowel diseaseInitial genetic study: A German patient cohort of 1103 controls and 550 CD and 539 UC patients, genotyping for 20 SNPs that identified all haplotypes around XBP1. Three SNPs were significantly associated with IBD:Replication genetic studies:2 independent cohorts (1854 patients, 2042 control and 1446 patients, 2177 controls) confirmed these findingsDNA sequencing of the XBP1 gene in patients:revealed a range of polymorphisms that caused changes in amino acids in XBP1Functional tests:showed many of these variants decreased XBP1 function or expression

• Genetic studies of inflammatory bowel disease have identified common alleles that are located in or near genes that are important for innate and adaptive immunity, and for effective secretion of anti-microbial proteins from Paneth cells• These alleles have low penetrance and increase the risk of this disease by mechanisms involving gene-gene and gene-environment interactions.

Chronic inflammation results in the increased production of molecules and chemicals from activated immune cells that will damage tissues.• Lynch syndrome, a heritable form of bowel cancer, is due to mutations in genes that repair DNA• The DNA mismatch repair pathway is compromised in patients with Lynch syndrome• Bowel cancer that is linked to Inflammatory Bowel Disease is also due to errors in DNA repair• In the case of IBD, the DNA is damaged by chemicals released as part of chronic inflammation

Acute inflammatory response:elimination of an infectious agent, activation of innate and possibly adaptive immune responses, Shut-down of the inflammatory immune response, tissue repairChronic inflammation:Can arise due to failure to clear an infection, or due to tissue damage arising from an autoimmune responses generation of free radicals by macrophages that phagocytose and kill bacteria, Reactive oxygen species damage DNA and affect the mismatch repair pathway!• There is a strong link between some chronic inflammatory conditions and an accompanying increased risk of cancer in the affected tissues.• Patients who have been affected by ulcerative colitis for 10-years or more have a 20- to 30-fold greater risk of developing colorectal cancer.• Both chromosomal instability and microsatellite instability are present in ulcerative Colitis• Our DNA is damaged and in several different ways• Chemical modification of DNA can cause base changes as well as structural changes toDNA• There are DNA repair pathways to remove the damaged bases• ROS are important mediators of inflammation• ER stress generates ROS, leading to an accumulation of intracellular ROS (oxidative stress).• the use of molecular oxygen as the terminal electron recipient leads to the production of ROS.

Base excision repair (BER) pathway: errors in inflammatory bowel disease• The chronic inflammation associated with inflammatory bowel disease results in very high and constant levels of reactive oxygen species (ROS)• The ROS damage DNA as well as leading to an imbalance in the synthesis of components of the BER• DNA glycosylase and the AP endonuclease are produced at very high Levels• The over-production of the DNA glycosylase and the AP endonuclease result in the accumulation of partially repaired DNA molecules• These partially repaired molecules result in mutations being incorporated into DNA after DNA replication

Lynch syndrome (hereditary nonpolyposis colorectal cancer: HNPCC)an inherited cancer of the digestive tract, particularly the colon (large intestine) and rectum.Lynch syndrome is also linked to an increased risk of cancers of the stomach, small intestine, liver, gallbladder ducts, upper urinary tract, brain, skin, and prostate.Women with Lynch syndrome have a higher risk of cancer of the endometrium (lining of the uterus) and ovaries.People with Lynch syndrome may occasionally have colon polyps: they appear at an earlier age than in the general population and they are more likely to become cancerous.Of the 160,000 new cases of colorectal cancer diagnosed each year in the USA,between 2 and 7 percent are caused by Lynch syndrome.

Mutations in mismatch repair genes: most are frameshift or nonsense mutationsMutations in the mismatch repair genes lead to microsatellite instability as well as point mutations

• 12% of colorectal carcinomas carry somatic deletions in poly(dA/dT) sequences and other simple repeats.• Cells from these tumors can carry more than 100,000 such mutations.• The mismatch repair mutator phenotype exists causes transitions and transversions in addition to microsatellite alterations.

• The intestine is a mucosal tissue and home to huge numbers of commensal bacteria• Commensal bacteria and the food we ingest contain many antigens• The immune system must develop tolerance to these antigens, while remaining able to respond to infections by pathogenic bacteria (and viruses)• A combination of barrier and immune defences maintains homeostasis in the intestine• Inflammatory bowel disease is thought to be a loss of intestinal homeostasis: it has genetic and environmental causes• Polymorphisms in genes of the innate and adaptive immune systems, and of the secretory cells, contribute to increased risk of developing this disease• Over many years, increased levels of reactive oxygen species generated by chronic inflammation increase the risk of cancer by damaging DNA and disrupting the regulation of DNA repair pathwaysThe burden of infectious diseases has been massive throughout history, with life expectancy not exceeding 20–25 years of age until the advent of Pasteur’s microbial theory of disease and the resulting control of infections by hygiene, vaccines and antibiotics

The HIV epidemic.• In 2007, there were an estimated 2.5 million new reported cases of infection and 2.1 million HIV-related deaths.• This brings with HIV Since 1981, it is estimated that 58 million people have been infected, of whom 25 million have died.• Since the mid-1990s, anti-retroviral therapy (ART) has transformed the management and prognosis of HIV infected individuals in resource-rich countries. Approximately a quarter of HIV-infected people in resource-poor countries have access to anti-retroviral therapy.HIV Set point:the viral load at which the immune system begins to fight the virus. The higher the set point the faster a person proceeds to full-blown AIDSLong-term non-progressors (LTNP):a minor subset of HIV-infected patients who do not progress to AIDS in the absence of drug therapyElite controllers (EC):a subset of LTNP who maintain their viral load below the detection limit of commercial assays (50 copies per ml of plasma)Escape mutations:mutations in the viral sequences that arise due to selective pressure from the immune response

The outcome of HIV infection in humans is defined by complex interactions between a polymorphic virus and a polymorphic host immune system.

Retroviruses

• The typical retrovirus genome is a single-stranded RNA molecule, 6000-9000 nucleotides in length• Upon entry into a cell, the genome is copied into double-stranded DNA by a few molecules of reverse transcriptase carried in the virus capsid• The double-stranded form of the retroviral genome then randomly integratesinto the host genomic DNA at a single site

Time course of a viral infectionInnate immunity:• Natural killer (NK) cellsAdaptive immunity:• Virus-specific cytotoxic• T-lymphocytes (CTLs)• Anti-viral antibodies• Re-exposure can result in aboosted adaptive immuneresponse (immune memory)Vaccines generate immune memory- no vaccine has yet been developed for HIV

HIV/AIDS: variation in the host response and resistance/susceptibility to diseaseHaemophilia:• a vast majority of severe haemophilia A patients born before 1979 became HIV-1 seropositive, due to virtually universal exposure to contaminated batches of factor VIII concentrates.• About 5% of these exposed individuals remained seronegative

• Of those people who are resistant to infection, is resistance due to innate or adaptive immunity, or to some other mechanism?• What are the cellular pathways that allow apparently permanent control of the virus amongst a subset of those that do become infected?• These are important questions in the effort to develop effective strategies to combat HIV-1 and to understand the genetics of infection.Infection:• Individuals completely resistant to HIV infection are known as elite controllers.Progression:• Individuals who show a very slow progression to AIDS are known as long-termnon-progressors.• Other individuals succumb very quickly to viral infection. Variation in HIV Infection and Progression to AIDSAll of these results suggest that our genetic makeup contributes to controlling HIV infection and disease progression.

Course of HIV infection

• The eclipse phase is the start of HIV infection• After several days the virus reaches draining lymph nodes and can be detected in plasma.• This is followed by rapid viral replication in activated CD4 T cells and systemic spread, with an exponential increase in plasma viral load,• Plasma viral load peaks at 3–4 weeks after infection.

• At this time, reservoirs of latent virus are established, and eradication becomesvirtually impossible.

The course of HIV Infection: the acute phase, viral load and CD8 CTLs

• Viral load increases to about 1 x 107 HIV RNA copies per ml• Viral load then declines over the next few weeks to a median viral set point of 30,000 RNA copies per ml of plasma• The decrease in viral load is accompanied by the appearance of an HIV-specific CD8+ cytotoxic T lymphocyte (CTL) response• This suggests that CTLs are important for reducing the levels of virus at this early stage of infection• Peak plasma viremia is followed by a decrease to a relatively stable level called• “set point” – this marks the end of the acute phase of infection• Long-term non-progressors maintain viral loads at near-undetectable levels of about 50 copies of virus/ml of plasma• The viral copy number at viral set point is an important indicator of disease ProgressionThe asymptomatic chronic phase can last for years: viral load gradually increases and CD4 T-cell counts decrease to the point where the immune system can no longer protect the body from opportunistic infections or certain cancers.

A CD4 T-cell count of 200 cells/ml of blood is the point of AIDS onset.

• CD4 T cells are the principal targets for HIV• HIV infects a cell through cell surface receptors (CCR5); once a cell is infected an anti-viral program of gene expression is initiated by the cell.• A 32–base pair deletion in the CCR5 chemokine receptor (CCR5Δ32) variant has been found in homozygous form in about 1% of Europeans.• There is also a much rarer point mutation (m303T) that results in a defective CCR5 protein product that is not expressed at the cell surface.Heterozygosity for CCR5 variants, as well as variants of the CCR5 promoter region have been consistently shown to associate with differences in viral load and/ or disease progressionIn homozygotes (CCR5Δ32/CCR5Δ32; m303T/m303T) or combined heterozygous(CCR5Δ32/m303T) form these genetic variants in CCR5 confer complete resistance to infection by HIV-1 viruses that use CCR5 as co-receptor.Note: some strains of HIV infect T-cells by the CXCR4 receptors, and individualswith mutations in CCR5 are therefore not protected from infection.Chemokine receptor gene polymorphisms and HIV Control

1. Specific alleles of MHC class I molecules are consistently associated withparticular HIV disease outcomes2. More rapid disease progression is observed in individuals with MHC class I homozygosity.3. The appearance of viral mutants that escape CD8+ T-cell recognition are accompanied by a loss of immune control of infection.

Evidence for the importance of MHC Class I Molecules and CD8 CTLs

1. High-frequency HIV-specific CD8+ T-cell responses are usually detectable in HIV-infected individuals who have developed- despite a CD8 response, viral control is not achieved2. Most MHC class I alleles have little impact on the disease outcome of HIV infection- MHC Class I alleles poorly present HIV antigens3. Most new viral escape mutants do not significantly affect viral load- failure to be controlled by CD8 response.Evidence that different people will have CD8 CTLs that differ in their effectiveness to fight HIV infection

The MHC, Antigen Presentation and CD8 Cytotoxic T-cellsThe elimination of virus often requires the destruction of virus infected cellsThe destruction of virus infected cells is the job of cytotoxic CD8 T-cells, calledcytotoxic T-lymphocytes (CTLs). CD8 is a cell surface glycoprotein co-receptor that is required to recognize class I MHC molecules

How do Cytotoxic (Killer) T-lymphocytes (CTLs) work?1. CTLs are isolated from a mouse infected with influenza2. Target T-cells are loaded with radioactive chromium and then either left uninfected or infected with influenza virus3. The target cells and CTLs are mixed4. The T-cell receptor on the surface of CTLs will recognise antigen presented by class I MHC on infected cells only5. The CTL will lyse and destroy the infected cell: this will be seen by the release of radioactivity into the supernatant

Cytotoxic T-cell activity is virus specific and MHC restricted

• CTLs from an infected mouse will only kill cells that have been infected with the same virus• CTLs will only kill infected cells from a second mouse that is genetically identical for the MHC Class I gene clusterMHC and Antigen PresentationThe MHC cell surface molecules present antigen (in the form of short peptides) to a receptor on T-cellsMHC class I:• CD8 T-cells – kill infected cells through recognition of viral epitopes• Regulate Natural Killer (NK) cell activity via interactions with NK cell receptors, including killer cell immunoglobulin-like receptor (KIR).MHC Class II:• CD4 T-cells- recognize antigens in the context of MHC class II, and producecytokines that help other immune cells to respond.

The complex between MHC molecule, T-cell receptor and antigen peptide isincredibly specific A T-cell receptor will recognise a specific combination of antigen and MHC molecule

The CD8+ CTL T-cell receptor will only ever recognise antigen with class I MHC

MHC (major histocompatibility complex) and Antigen PresentationMHC molecules play an essential role in the recognition of virus-infected cells by cytotoxic T-cells called cytotoxic T-lymphocytes (CTLs).The destruction of virus-infected cells is carried out by cytotoxic T-cells called cytotoxic T-lymphocytes (CTLs)Antigen receptors on T-cells recognize short fragments of processed antigen that are presented to the T-cells by membrane glycoproteins encoded by the MHCMHC-A, -B and -C are extremely polymorphic and encode protein products that are fundamental in the immune recognition process: expressed at the cell surface, they present antigenic epitopes including processed viral peptides to CD8+ T lymphocytes, thereby initiating a cytotoxic T cell (CTL) response.

• CD8 T-cell receptors only recognise antigens that are presented by class I MHC molecules• The class I MHC molecules are HLA-A, HLA-B and HLA-C

• The MHC gene cluster is extremely polymorphic• Variation across MHC alleles is concentrated within the regions encoding sites that bind peptides and the T cell receptor.• The MHC Class I gene HLA-B is the most polymorphic gene in the human genome, with 1,800 alleles• A single amino acid change can influence structural and functional properties of MHC antigensPolymorphism of the MHC Gene Complex

Most of the allelic variants in the genes of the MHC complex are found in the region that interacts with antigen peptide and T-cell receptorThe defining feature of MHC class I and class II genes is their extreme polymorphism.Why might a high number of polymorphisms in the MHC genes be an advantage?Human leukocyte antigen (MHC) class I variation and HIVResults from Genetic StudiesRecent European ancestry and HLA-B*5701:Protective effect, the largest contributor to inter-individual variability for both viral set point and CD4+ T cell.

African-Americans, and HLA-B*5703: the most important determinant of viral control.Other HLA-B types:HIV-1 control is better in the presence of MHC-B*27, B*51, and B*5801, HIV-1 control is poorer in the presence of MHC-B*5802 and of alleles from the MHCB35Px group.Homozygosity for class I alleles: also leads to faster progression and higher viremia, presumably because it reduces the diversity of the epitope recognitionmachinery, thereby impairing antiretroviral CTL response.

KIR genes are clustered on human chromosome 19q13.4• There are multiple genes that span approximately 100–200 kb• The KIR gene cluster is divided into haplotypes• The receptors can activate or inhibit NK cells• Although not as polymorphic as the MHC genes, the KIR genes do contain a large number of polymorphisms

MHC and KIR class I molecules• A key role in the induction of CTL responses,• Ligands for the killer cell immunoglobulin-like receptors (KIRs), expressed at the surface of natural killer (NK) cells.• KIRs increase or decrease NK cell activation and directly regulate the innate immune response to HIV-1 infection.• Combinations of KIR genes and MHC class I alleles have epistatic influences on the outcome of HIV-1 infection:• KIR3DL1 and KIR3DS1 have been associated with better control of HIV-1 when they are found in patients that have MHC-B alleles with a Bw4 specificity.Evidence suggests that even a single amino acid change can influence structural and functional properties of MHC antigens and can influence susceptibility to HIV disease• The extreme polymorphism of MHC genes parallels the diversity of the functionally related KIR genes, which encode receptors that regulate NK cell function.• NK cells are key mediators of innate immunity that kill aberrant target cells or secrete cytokines without prior exposure to the target.• The different alleles of the MHC Class I genes interact with the many genes and polymorphisms of the NK KIR receptors to change the effectiveness of the NK innate immune response to HIV infectionThe strength of the CD8 T-cell response during the acute phase of infection is related to the ability of the CD8 T-cells to recognise HIV antigens

A strong CD8 T-cell response during the acute phase will result in a lower viral set-point, slower progression during the chronic phase and higher CD4 T-cell countsHIV-specific CD8 T cells from elite controllers are more potent (higher cytotoxicty, increased production of immune molecules such as cytokines) compared with non-controllersThe HLA-B locus is the most polymorphic of all the MHC loci and shows thestrongest and most consistent genetic effects on outcomes of HIV infection.• Most CD8+ T cell responses to HIV are HLA-B restricted• A specific HLA-B allele (HLA-B∗57) has the strongest protective effect against HIV, controlling viral load control and delaying progression to AIDS. The HLA-B∗57 allele carriers are over-represented among elite controllers• The HLA-B∗57 allele carriers most strongly associated with a reduction in time to CD4 counts of <200 (AIDS)The HIV virus will constantly acquire new mutations to evade detection by the MHC Class I moleculeThese new mutants are called escape mutants because they evade or weaken the immune response to HIV

• CD8+ T cells targeting epitopes in Gag are associated with lower viral• load than CD8+ T cells targeting the more mutable proteins in HIV• Escape mutations in the Gag protein reduce the fitness of HIV• In people able to control HIV replication to undetectable levels without medication (elite controllers), the role of the HLA B*57 allele appears to be to drive the generation of fitness-impairing epitope mutations that cannot easily be compensated• In other words, the HLA-B*57 allele is very effective in presenting viral epitopes to the CTL• The CTL cells also elicit a very strong anti-viral immune response• Escape mutants of HIV are in the Gag gene, a region that does not tolerate mutations and results in reduced viral fitnessAn example of a biological arms race between the host and the HIV virusWhy might the HLA-B∗57 allele be so effective in controlling HIV?

Most of the common variants important in the control of HIV-1 have now been identified, particularly in individuals of European ancestry.However, a great deal of the inter-individual differences in viral control remain tobe explained.The confirmed host genetic determinants of HIV-1 control are only able to explainabout 20% of the observed variation in viral load or disease progression.

The outcome of HIV infection in humans is defined by complex interactions between a polymorphic virus and a polymorphic host immune system.

If we could scan the entire transcriptome of these two cells,could we:• Use differences in gene expression to identify healthy and unwell individuals?• Monitor response to drug therapy?• Understand how genetics and the environment affects gene expression?

if a gene is highly expressed there will be more copies of RNA in the cell, more oligonucleotides corresponding to that gene will by bound by labelled sequence and the signal will be higher. The opposite is true if a gene is expressed at low levels• Microarray analysis simultaneously analyses the expression of thousands of genes• We can “see” whether a specific RNA is hybridized to an oligonucleotide because the RNA is labelled• In microarray experiments the label is a fluorescent probe that can be read automatically in a scanner by a laser Microarray experiments can be expressed as a “heatmap”. A heatmap is a way of organizing and presenting gene expression patterns• A heatmap is a graphical way of displaying a table of numbers by using colors to represent numerical values.• Low values are cooler colours (blue, green) while higher values tend to hotter yellow, orange and red colours.• Heatmaps re-arrange the rows and columns of the table so that similar rows, and similar columns, are grouped together.

• Microarrays permit the simultaneous analysis of thousands of transcripts• Microarrays are important for searching for patterns of gene expression that distinguish between healthy and unwell individuals• This may have useful clinical applications

Inflammatory bowel disease and Tumour Necrosis Factor (TNF)• TNF is produced in response to infection from innate immune cells such as macrophages• It activates an inflammatory response to counter the infection• Uncontrolled production of TNF can harm the body: sepsis, inflammatory bowel disease

• The key role of TNF in the inflammatory response of inflammatory bowel disease has led to the development of anti-TNF therapies• There are, however, potentially severe side effects with anti-TNF, including severe infections and cancer• Induction of IEC apoptosis by TNF sustains chronic inflammation• Protecting the intestine against TNF-induced apoptosis is crucial for protection against inflammatory bowel disease.• Drugs that target TNF confer protection against inflammatory bowel disease by inhibiting apoptosis in the gut.• TNF-blocking antibodies inhibit IEC apoptosis and restore barrier function in patients with Crohn's disease, reducing the inflammatory response.• A major challenge for geneticists and biologists is to understand how our genotype influences gene expression• Many of the common alleles that are associated with complex genetic diseases such as inflammatory bowel disease are located in regulatory sequences such as enhancers• These alleles can subtly change the binding of transcription factors, changing the activity of an enhancer and influencing gene expression• Initial studies show that the expression of many genes of the immune system and of genes involved in anti-viral response are influenced by common alleles

• different people show a different response to

HIV viral infection: those peoplewhose immune cells have a low viral load show a different pattern of gene expression than those people with a high viral load• The most significant group of differentially expressed genes between individuals with high and low viral loads was in the interferon pathway- a keyantiviral pathway activated early during infection and involving cells such as NK cellsInterferon acts via cell surface receptors to initiate gene transcription.There are many interferon-inducible genes that are critical for the immune response

• Poor viral control is associated with increased expression of genes involved in the interferon pathway• This means that in individuals who cannot control the virus, their immune systems are constantly activated and fighting the virus• This causes long-term damage to the immune systemMicroarray analysis of RNA from CD4 T-cells from individuals from individuals not receiving treatment showed differences in expression profiles between high and low viral set points. Many of the genes showing the greatest difference in expression between these two groups were involved in interferon-dependent anti-viral defences.What happens to gene expression following anti-retroviral therapy?

To assess the effects of anti-retroviral treatment on CD4 gene expression, the following groups were examined:1. Non-infected individuals2. Elite controllers (viral set-point <50copies/ml plasma)3. Individuals successfully responding to therapy (pre- and post-therapy)

successful anti-retroviral treatment changes gene expression in T-cells to a pattern more similar to that seen in healthy non-infected individuals the gene expression profile of elite controllers is also very similar to healthy noninfected individuals.successful anti-retroviral treatment changes gene expression in T-cells to a pattern more similar to that seen in healthy non-infected individuals the gene expression profile of elite controllers is also very similar to healthy noninfected individuals.SummaryInfection of T-cells with the HIV virus results in a specific pattern of gene expressionThere is some heterogeneity in the gene expression response to infectionAre differences in gene expression due to genetic variants that control theexpression of genes that are turned on to fight the virus?orAre differences in gene expression due to genetic variants in genes that controlviral infection and replication?Mutations in CCR5 and polymorphisms in the HLA genes are the most important host genetic factors that determine phenotypes such as viral set point.

We do not yet understand the genetics that would account for differences in the gene expression programs in individuals with low or high viral setpoints.The “common disease, common variant” hypothesis:The genetic basis of many common diseases is attributable to SNP alleles that are present at a frequency of more than 1%–5% in the population