Human Genetics and Evolution

Genomics

the study of the genomics of organisms including structure, function, evolution, and mapping

Human Genome Project

a public genome project launched in 1990 by Watson, Collins, and Sulston; a worldwide effort conducted by both academic and government institutions to assemble the genome using maps

Celera Genome Project

a private genome project launched in 1998 by Craig Venter; aimed to sequence the human genome in 3 years using a 'shotgun' approach

The Bermuda Accord

1996 meeting where researchers involved in the sequencing of the human genome outlined a number of principles including automatic release of data freely

Sanger Sequencing Methodology

Double stranded template (unknown) DNA is separated, and a primer is added along with sequence complementary to the primer that is radioactive; four polymerase solutions are added, one for each base; fragments grow and are run through gel electrophoresis, then analyzed based on color to determine the sequencing

Next Generation Sequencing (NGS)

Allows parallel sequencing of millions of different DNA fragments simultaneously increasing speed and reducing costs; technique to sequence EVERYTHING, even the transcriptome; traditionally uses short-reads

454' pyrosequencing

Makes use of the pyrophosphate (PPi) released upon nucleotide incorporation by DNA polymerase

Reversible terminator sequencing

Similar in principle to Sanger Sequencing, but the terminator can be removed

Transcriptome

Full range of mRNA molecules expressed by an organism

Nanopore Sequencing

Provides portable genome analysis, faster sample prep, longer read lengths, and real-time analysis of data

Personalized medicine

Knowledge of our own genomes could allow us to take a 'one size fits all approach' and tailor it to the individual; allows researchers to make individual predictions about disease risk, develop prevention plans, and target drug therapy

Microarrays

Tool for studying the transcriptome, including genotyping and expression arrays

Antimicrobial Resistance (AMR)

occurs when bacteria, viruses, fungi and parasites change over time and no longer respond to medicines making infections harder to treat and increasing the risk of disease spread, severe illness and death

Main drivers of AMR

misuse and overuse of antimicrobials, lack of access to clean water, sanitation and hygiene for both humans and animals, poor infection and disease prevention/control in healthcare facilities and farms; poor access to quality, affordable medicines, vaccines, and diagnostics; lack of awareness and knowledge; lack of enforcement legislation

Sequencing and AMR

If we have the whole genome of an organism, we can use knowledge of the genome to look for antimicrobial resistance markers and evidence of transmission; e.g. TB

Metagenomics

Describes the study of microbial communities at the genetic level without the need for culture; rather than using traditional cultures to diagnose infections, the bacterial DNA is extracted from the clinical sample, sequenced, and then compared to databases of whole-genome sequences to identify the bacteria causing the infection

Microbiome

The microorganisms of a particular environment

The human microbiome

the collection of all microorganisms living in association with the human body; including bacteria, viruses, archaea, and fungi

Functions of the human microbiome

Nutrition, producing vitamins humans can't make and breaking down food to extract nutrients; immune system, teaching out immune system how to recognize dangerous invaders and helpful anti-inflammatory compounds that fight off disease

States of the human microbiome

Linked to different states; disease, changes to our microbiome correlate with numerous diseases; behavior, gut microbiome affects the brain, depression and autism have been linked to the microbiome

Human Microbiome Project

Initiative by the US National Institutes of Health to improve understanding of human microbial flora by generating a resource for the comprehensive characterization of the human microbiome and analysis of its role in health and disease

Pathogen

bacterium, virus, or other microorganism that can cause disease

Coevolution

The reciprocal, adaptive, genetic change in two or more species, usually refers to selective sweeps

Selective Sweeps

When new alleles occur, either by migration or mutation, and eventually become fixed in the population; results in transient polymorphism

Dynamic Polymorphisms

Fluctuations in allele frequencies caused by selection that are inherently persistent; fixation may occur by genetic drift; also known as 'Red Queen'

Conditions for co-evolution

Requires ... genetic variation in the population; reciprocal effects of the traits involved in the fitnesses of the two populations; dependence of the outcome on the combination of genotypes

Spatial patterns of co-evolution

Assumes that different populations are at different stages of the evolution process, so space can substitute for timing; helps because temporal variation can take hundreds of generations to study

Signatures of selection

the patterns of variation that remain in the human genome; affected by the timing, strength, and direction of selection (positive, negative, stabilizing)

Signatures of positive selection over the last 250,000 years

a selected variant that increases rapidly in frequency can be detected through an unusual reduction in diversity

Signatures of positive selection in the last 5,000 to 10,000 years

can be detected through unusually large allele frequency differences between populations; unusually high frequency of newly derived variants and unusually extended LD caused by the rapid increase in frequency of a particular alle

Duffy Antigen disruptor mutation

In much of the sub-Saharan Africa, the population is Duffy-negative, meaning P. vivax cannot infect RBCs by binding to the duffy antigen; example of positive selection and selective sweep

Signatures of negative selection

Can be identified by an excess of rare alleles at one locus or a loss of functional variation

Example of negative selection

On the ganges river delta, human populations have the lowest frequency of Type O blood in the world because individuals with Type O blood have the highest risk of dying from the cholera pathogen

Signatures of balancing selection

an excess number of intermediate frequency polymorphisms near the variant

Example of balancing selection

Sickle cells - with two healthy alleles, individuals are at risk of severe malaria; with two diseased alleles, individuals develop sickle cell anemia; heterozygotes are healthiest

Heterozygote advantage

When heterozygotes are more fit than either homozygous phenotype

Major Histocompatibility Complex (MHC)

The set of genes encoding many of the proteins involved in surface structures on immune cells; great diversity (over 2,700 alleles described!) in this area allows the human immune system to evade infection from a wide range of pathogens

Zimmerman, 2017

Recent study that suggests the P.vivax parasite is evolving to infect both Duffy negative and Duffy positive individuals

Sweaty T-Shirt Experiment'

Suggested that women preferred the scent of men who had dissimilar MHC or MHC-linked genes; suggests non-random mate choice, 'evolving to evolve'

H. Influenzae Mutational Hotspots

Regions of the H. Influenzae genome that have mutational rates hundreds if not thousands of times higher than the rest of the genome; these mutational hotspots code for surface structures

Fumagalli et al., 2011

Using 55 distinct human populations, showed that the main selective pressure of genetic adaptation was pathogens, rather than environmental pressures like diet or ecology

Innate immune system

Involves a non-specific response that is immediately available to fight a wide range of pathogens without ever having been exposed to them

Adaptive immune system

Involves the production of antibodies against a particular pathogen during the lifetime of the individual as an adaptation to infection by that pathogen

Pattern Recognition Receptors (PRRs)

Receptors located on innate immune cells like macrophages, dendritic cells, or neutrophils that recognize pathogen-associated molecular patterns (PAMPs)

Pathogen-associated molecular patterns (PAMPs)

Microbial molecules that occur in repeated patterns on the pathogen surface; recognized by PRRs

Stages of the immune response

Innate Immunity:

1. When a pathogen is encountered, the PRRs of the immune cells become activated, releasing cytokines and chemokines to initiate inflammation

2. Macrophages and dendritic cells will ingest the pathogen if possible

3. Inflammation causes vasodilation and increased vascular permeability allowing more immune cells to be recruited

Adaptive Immunity:

1. After the dendritic cell phagocytosis the pathogen, it breaks down the pathogenic proteins so that they bind to the dendritic cells MHC; at this point, the dendritic cell becomes an antigen-presenting cell (APC)

2. The APC migrates to the lymph node where it begins activating pathogen specific lymphocytes

3. When a match is made, the naive lymphocyte will become activated and proliferate (clonal expansion)

4. The effector B-cells release antibodies that bind to the pathogen and neutralize them/tag them for destruction

5. The effector T cells either assist the B cells or directly attack and destroy infected cells

6. Memory cells remain in long-term circulation in case the pathogen is accounted again

Macrophages

Large leukocyte and phagocyte, play an important role in initiating adaptive immunity, found in essentially all tissues

Neutrophils

Phagocyte and granulocyte, most abundant type of leukocyte in most mammals; secrete products that stimulate macrophages

Eosinophils

Granulocytes; important for combatting parasites and helminth infections; also important mediators of allergic responses and asthma

Leukocyte

white blood cell

Phagocyte

Cell capable of engulfing bacteria or other small cells or particles

Granulocyte

Type of immune cell that has granules with enzymes that are released during infections, allergic reactions, and asthma

Dendritic cells

Bridge' linking innate and adaptive immunity; specialized to capture and process pathogen antigens, present these on their surface to activate cells of the adaptive response

Cytokines and chemokines

Special signaling molecules that coordinate the immune response; released by lots of different cells; tell immune cells how to behave (e.g. migration, stimulation, and function); begin the inflammation process

T cells

Mature in the Thymus; have a T cell receptor (TCR) on the surface which recognizes the antigen; cellular immune response; important in response to extracellular pathogens

B cells

Mature in the bone marrow; have a B cell receptor (BCR) on the surface which recognizes antigen; humoral immune response; important in response to extracellular pathogens

B and T cell receptors

Each TCR and BCR has a constant region (anchors to the cell membrane) and a variable region (determines antigen specificity); it is specific to one antigen but with some cross-reactivity

Clonal deletion

Process by which the immune system prevents itself from attacking the self; lymphocytes bearing receptors for ubiquitous self molecules are deleted at an early stage of cell development

Helper T-cells

Has CD4 co-receptor of TCR; helps the activity of other immune cells by releasing cytokines; can activate other cells, or suppress/regulate immune responses

Cytotoxic (Killer) T-cells

Has CD8 co-receptor of TCR; kills infected cells; also kills cancer cells or cells damaged in other ways

CD4 co-receptor

On helper T-cells, recognizes MHC class II

CD8 co-receptor

On killer T-cells, recognizes MHC class I

Clonal expansion

After binding with the antigen, B and T cells make many copies of themselves that are either memory or effector cells

V(D)J Recombination

Process by which B and T cell receptors are made to generate lots of diversity within a small section of the genome; antibodies are copies of the receptor so also influences antibody diversity; 'Variable, diversity, joining'

Forms of diversity in V(D)J

Recombinatorial diversity - different V, D, and J regions are selected

Junction diversity - enzymes can induce a frameshift mutation through small random changes at junctions

Combinatorial diversity - the combination of the heavy and light chain also creates diversity

Somatic hypermutation (only in BCR)

Effector B cells

Activated effector B cells undergo clonal expansion to produce plasma cells that produce antibodies

Innate immunity

rapid (0-4 hours); non-specific; includes macrophages, neutrophils and dendritic cells; diversity of pathogen receptors is in the germline (PRRs)

Adaptive immunity

Slower (after 4 days); specific; includes T cells and B cells (+antibodies); includes memory-based responses; diversity of pathogen receptors is somatic

Two class of T-cell receptor

An individual T cell expresses either αβ chains or γδ chains; 95% of T cells have a TCR consisting of αβ

BCR heavy chain

contains V, D, and J segments

BCR light chain

contains only V and J segments

TCR β chain

contains V, D, and J segments

TCR α chain

contains only V and J segments

BCR Somatic Hypermutation

When a B cell recognizes its antigen, it is stimulated to proliferate; during this process, the BCR undergoes an extremely high rate of somatic mutation at hotspots regions of the DNA (concentrated in the regions encoding to the variable binding site)

HLA gene complex

human-specific MHC; highly polymorphic with much of the polymorphism in the antigen-binding cleft; all polymorphisms are co-dominant

Autoimmunity

Adaptive immune response attacks self-antigen because of cross-reactivity, genetic factors, environment/lifestyle

Hypersensitivity

Too much immune response or a response to an innocuous antigen (e.g. asthma and some allergies)

Immunodeficiency

Too little immune response, can be a primary disorder or result from other conditions

Trained immunity

Functional state of the innate immune response that is characterized by long-term epigenetic reprogramming of innate immune cells; evidence of trained immunity in humans from nonspecific live virus vaccines

Clonal Selection Theory

Proposed by Burnet, a microevolutionary process amongst naive lymphocytes; Includes four postulates

1. Each lymphocyte bears a receptor of unique specificity

2. Interaction with specific pathogen leads to lymphocyte activation and differentiation (cloning)

3. Differentiated cells bear identical specificity to parent cell

4. [Clonal Deletion] Lymphocytes bearing receptors for ubiquitous self-molecules are deleted early

Hanahan and Weinberg Hallmarks of Cancer (2000)

Self-sufficiency in growth signals, insensitivity to anti-growth signals, tissue invasion and metastasis, limitless reproductive potential, sustained angiogenesis (new blood vessel growth)

Updated Hallmarks of Cancer

sustained proliferative signaling, evading growth suppressors, non mutational epigenetic reprogramming, avoiding immune destruction, enabling replicative immortality, tumor-promoting inflammation, polymorphic microbiomes, activating invasion and metastasis, inducing or accessing vasculature, senescent cells, genome instability and mutation, resisting cell death, deregulating cell metabolism, unlocking phenotypic plasticity

Oncogenes

Mutated versions of genes that generally code for proteins that when mutated can lead cells towards cancers; gain-of-function mutations

Tumor suppressor genes

Genes involved in the control of abnormal cell proliferation, usually recessive alleles; when mutated, these are loss-of-function mutations

p53

Guardian of the Genome;' more than 50% of all tumors contain a mutated version of these gene which normally stops cells with mutated or damaged DNA from dividing

Causes of cancerous mutations

Errors from cell division, environmental damage, and inheritance from one's parents

Precision oncology

molecular profiling of tumors to identify targetable alterations

Neoplastic proliferation

New growth,' e.g. the growth of tumors

Cancer

unchecked cell division caused by a breakdown of the regulation of the cell cycle; dysregulation of apoptosis

Evolution of Cancer

The result of somatic evolution; cancer is selected for on the somatic level but selected against on the germline level -- cancerous alleles do not cause problems until later in life, meaning that the deleterious genes are already passed on

Mutation-accumulation hypothesis

Most mutations that cause age-related diseases are neutral early in life but deleterious later on

Antagonistic-pleiotropy hypothesis

Most mutations that cause age-related diseases are beneficial early in life but deleterious later on; e.g. study found that women that have BRCA1 and BRCA2 have higher natural fertility

Driver mutations

Mutations that cause a selective sweep in the cancer tumor

Passenger mutations

Mutations that are 'along for the ride,' present in the same genome as a driver

Mutational hotspots

Areas identified by population studies or NGS that likely have driver mutations based on their recurrence; in the past these were limited to family studies (e.g. BRCA1 and BRCA2)

PARP Inhibitor in BRCA2 mutation

PARP inhibitor causes a synthetic lethal; in the absence of PARP1 (poly ADP ribose polymerase), single stranded DNA breaks are not repaired and converted into double strand breaks during DNA replication; BRCA2 compromises the homologous recombination machinery and the double stranded breaks accumulate in cells, becoming lethal

Basket trials

Clinical trials that group patients based on mutations, rather than stage/location/type of cancer

Acquired resistance

Patients with metastatic cancers nearly always developed this following prolonged treatment with targeted therapy; can be addressed by doing sequencing pretreatment and post-treatment to immediately identify novel mutations

Logistical challenges with NGS

Expensive, hard to find actionable mutations, and sample-specific challenges, e.g. formalin fixed, paraffin embedded (FFPE) tissue blocks alter DNA

Weak mutations concern

Balmain, 2002 - suggested that cancer is so complex as there may be hundreds of tumor susceptibility alleles and it is difficult to determine how these factors interact to drive risk and progression