TOPIC 16: Human Variation
NEANDERTHALS &
DENISOVANS
NEANDERTHALS
● Southern Europe/Asia shared with
ancestors
DENISOVANS
● Sequences hominin
→ neither human nor Neanderthal
⇒ evidence of interbreeding with archaic humans
after sequencing fossils
→ modern & archaic people mated several times
→ 3 encounters with Neanderthals → left DNA
in current humans
→ Denisovans mated with ancestors of today’s
Melanesians
⇒ genomic distribution:
- DNA concentrated in particular regions of
chromosomes in people of non-African ancestry
- X chromosomes → little archaic DNA
- mitochondrial genomes in all humans →
exclusively humans
ADMIXING OF
NEANDERTHAL/DENISOVANS
● Retention of some
Neanderthal/Denisovan sequences in
human genome → due to selective
pressure
→ but most DNA lost
● Positive selection:
→ Neanderthal alleles:
- variants of genes that strengthen skin,
hair nails in East Asians
- pale skin colour & freckling in Europeans
→ fair skin allow extra production of
vitamin D from limited sunlight
- particular HLA variants → confer
resistance against various pathogens
→ Denisovan alleles:
- adaptation of high altitude through
lower haemoglobin levels in Tibetans
HIGH ALTITUDE LIFE IN TIBETANS
● Particular region of chromosome 2
→ 9 SNPs with genome wide significance
→ most significant : EPAS-1 locus
⇒ derived from denisovans
● Gene retained to help Tibetans who
settled on high-altitude Tibetan plateau
to adapt
● In high altitude:
- other humans:
⇒ hypoxia
→ produce more RBCs to
overcompensate for low O2 levels
→ constricts pulmonary vascular →
hypertension → high BP → heart failure,
stroke, pre-eclampsia
- tibetans:
⇒ muted response to hypoxia
→ adapted by having mutated response
to hypoxia
FUNCTIONAL CONSEQUENCE OF
EPAS-1 VARIANT
● EPAS-1 → encode transcription factor →
induce erythropoietin (EPO) in response
to hypoxia → stimulated red blood cell
production
- Hypoxic conditions:
→ Tibentan variant of EPAS-1 locus →
increase expression → EPO production
much less dramatically
→ accounts for observations no increase
in RBC in response to hypoxia in Tibetans
LACTASE TOLERANCE
● All infant mammals → express lactase in
small intestine → generates glucose from
milk sugar lactose → dietary lactase
cannot be absorbed
● All non-human mammals & most humans
→ lactase expression transcriptionally
down-regulated after weaning
 ● In adult mammals → consume lactose →
passes into large intestine where colonic
bacteria metabolise it → lactose
intolerant symptoms
⇒ 35% inherit ability to express lactase
→ lactase tolerant
CONVERGENT EVOLUTION
● lactose tolerant trait
- in many ancestral population
- dominant (gain of function) trait
- Lactase gene (LCT)
→ identified through STR marker assays
& linkage analysis
→ polymorphisms at 2 sites correlate
with phenotype
GENOTYPE TO PHENOTYPE
● Variants for lactose tolerance → in
enhancer upstream of LCT
MOUSE EXPERIMENT
● tested C to T change in enhancer on
transcription of LCT gene after weaning
→ lactose tolerance during adulthood
● transgenic mice → expressed LCT
reporter in intestine
⇒ enhancer with T or C at -13910
upstream of lactase promoter + luciferase
gene
- C: only expressed luciferase when
young
- T: expressed luciferase as adults
● Luciferase activity
- high → transcription of lactase high →
lactase break down lactose → lactose
tolerant
- low → transcription of lactase low → no
lactose breakdown by lactase → lactose
intolerant
SELECTIVE PRESSURE
● In arid areas
→ drinking milk in adulthood →
additional nutrients & hydration
HUMAN VARIATION IN IDENTICAL
TWINS
● mutations
- occur early after separation →
distinguish identical twins
- occur in germline cells → differences
inherited
● 2 genomic differences on average →
mosaic state
TOPIC 17:Gene therapy APPROACHES
approaches

DELIVERY VECTORS
● Mostly modified lipid particle or virus

● type used depends on which type of

molecules to be deliver
→ hydrophilic or hydrophobic
● Advantages:
- Less immunogenic
GENE THERAPY - easier to produce
● Introducing new or modified genes into ● Disadvantage:
patient cells to treat or prevent a genetic - Less efficient
disease
● Can be used for genetic diseases
- causative genes must be identified
- cells affected by genetic disease
conditions must be accessible for
treatment
 ● General mode of action: OTC(ornithine transcarbamylase)
1. Entry into the cell
2. Delivery of genetic material
DEFICIENCY
3. Expression of gene ● Defect in OTC gene → impedes liver
4. Integration metabolism of ammonia → affect urea
cycle
⇒ excess elevation of ammonium ion in
brain → result in life-threatening
hyperammonemia encephalopathy, coma
and brain damage
GENE THERAPY WITH ADENOVIRUS

● Adenovirus → replication-defective
● Transient side effects(fever & flu-like
symptoms) → stimulate immune
response
CASE STUDIES ● Poor transgene expression in hepatocytes

ADA-SCID → relies on introduction of functional

copy of OTC gene into patient’s liver cells
● Significant metabolic correction did not
occur

● One individual in cohort 6 (jesse

gelsinger) → died from massive
inflammatory response trigger by virus
- first person to die in gene therapy
clinical trial
- given adenovirus vector with normal
OTC gene
- died 4 days later → safety & ethic of
gene therapy
X-LINKED SEVERE COMBINED
IMMUNODEFICIENCY (X-SCID)
● IL2-Ry gene located on X-chromosome →
essential role in immune system
development & function
CRISPR-Cas9 GENE EDITING
⇒ mutation → IL2 receptors dont
● CRISPR: clustered regularly interspaced
function → immunodeficiency
short palindromic repeats
GENE THERAPY WITH RETROVIRUS - cas proteins: CRISPR associated
● Treatment: Patients treated with proteins
autologous hematopoietic stem cells - CRISPR RNA(crRNA): act as guide to
(harvested from own bone marrow) → target site for cut
transduced ex vivo with recombinant ● Cas9 → cuts up virus DNA at specific seq
retrovirus expressing gamma chain of IL2 specified by CRISPR element
receptor → transduced hematopoietic - uses 2 short RNA linked to guide RNA
precursor cells transplanted back into (gRNA) → direct Cas9 to sequence of
patient choosing by adding appropriate gDNA
● Results :
- 9/11 children → dramatic improvements
with almost fully restored immune
system
- 5/9 children → developed leukaemia
between 3-6 years after treatment
● Viral DNA (Viral RNA reverse transcribed
to DNA) → disrupted:
- proto-oncogene LMO2 gene
● Current CRISPR trials:
→ involved in development of leukaemia
- Beta-Thalassemia
- LMO2 + proto-oncogene BMI1 ( patient
- Lung cancer
10)
- LCA
→ cell proliferation & differentiation of
- type 1 diabetes
stem cells
- HIV/AIDS
- proto oncogene CCND2 (patient 7(
→ cell cycle
● 27 trials with retroviral vectors
immediately halted
RETINAL BLINDNESS
● Successful
● Affected by mutations affecting photo
effector cells → 18 genes impacted
● Treatment: package RPE65 genes into
AAV cells → directly injected into retina
GENE EDITING
● DNA binding protein delivered to directly
cut & edit DNA
→ instead of providing exogenous copy of
gene to patient cells
TOPIC 18:PERSONALISED EFFECT OF CYP2D6 VARIATION ON
MEDICINE METABOLISM OF NORTRIPTYLINE
● most treatments designed for average
patient
- variable successes due to variable
patient factors
- individualised approach to treating
diseases
● Classify patients based on molecular data
→ enables:
- optimised drug dosage
- targeted drug design
- immunotherapies

DEVELOPING TARGET DRUGS

● design drugs that are targeted to genetic
profile
HERCEPTIN
● Monoclonal antibody
PHARMACOGENOMICS ● Regulates cell growth & division
● how genetics makeup influences
response to drugs
● help to optimise drug dosage for
individuals
GENETIC FACTORS INFLUENCING
DRUG METABOLISM

TARGET CANCER IMMUNOTHERAPY

● use patient immune system to kill cancers
● Use cytotoxic T cells(CTLs)
CYTOTOXIC T-CELLS
● T cells mature in thymus → migrate to
blood & lymph
OPTIMISING DRUG RESPONSE ● Activated by antigen presenting cells
through MHC/antigen-TCR interaction
● Lead to differentiation functional T cell
class
● Activation by APC with tumour antigen →
lead to CTL activation
1. ADOPTIVE CELL TRANSFER
● Harvesting & expanding patient’s own T
cells
● Extract tumour-infiltrating lymphocytes
CHALLENGES
(TILs) → amplify them in vitro &
● Technical challenges
reintroduce into patient
→ vast amount of data generated by
genomic sequencing
● Data privacy and rights to access
● Training of medical & health practitioners
to understand & utilise this data
→ patient must understand risks &
benefits

2. ENGINEERED T CELLS
● Manipulate T-cell (TCR) to specially
recognise cancer antigen
→ Chimeric Antigen Receptor (CAR) T
cell therapy
 Subset usd for DNA profiling
TOPIC 19: DNA Forensics ●
1. Crime scenes (CODIS(combined DNA index system)
2. Missing persons loci)
3. Wildlife forensics → specific genetic markers
→ FBI use set of 20
SOURCES
● Saliva
METHOD
● Blood
● DNA on other objects

VNTR PROFILING
● Variable number of tandem repeats =
minisatellites
● DNA sequences: 15-100bp
→ vary in length between individuals
● Non-coding regions of genome
● Number of repeats vary between
individuals
● Length of VNTR: 1-20kb
● Combine analysis of multiple VNTRs to 1. DNA extraction
create profile 2. PCR amplification
→ e.g 4 different VNTRs with 20 alleles 3. Capillary electrophoresis
each: 420 possible genotypes STR ANALYSIS
● 24-locus STR profile
● Uses electropherogram
● Heterozygous = double peaks
→ inherited 2 different alleles for specific
STR locus from parents
● Homozygous = single peaks
→ inherited identical alleles for specific
STR locus from parents
● Assign peaks from sample to profiles →
allow ID
● Issues:
● DNA extracted → digested using - complex/mixed samples
restriction enzyme → DNA fragment cut → similar DNA profiles
→ size of DNA fragment corresponds to - contamination
number of alleles → incorrect results
- degradation
STR DNA PROFILING
→ partial profiles
● PCR amplify microsatellites or short
→ due to heat, humidity, UV radiation,
tandem repeats
enzymes, chemicals
● Similar to VNTRs but shorter (2-9 bp)
● PCR primers tagged with specific
fluorescent dye → amplified → separated
based on size
 TOPIC 20: Mitochondrial
disease & therapy
MITOCHONDRIA
● Oxidative phosphorylation

● Represent number of repeat units for

specific allele at given STR locus HUMAN mtDNA
PROFILE PROBABILITY ● 16569 bp
● Probability that random person has a ● 37 genes (all essential)
DNA profile - 13 peptide genes (Complex I, III, IV)
→ decreases with more loci in analysis → oxidative phosphorylation
● Match probability → statistical measure - 22 tRNA genes
● Multiply allele frequency for each loci = → protein synthesis
genotype frequency - 2 rRNA genes
→ 20 loci set frequency = ~1 x 10 -28 → mitochondrial ribosome
● No histones
→ high mutation rate
● No DNA repair
● Hundreds of mt per cell
→ each with 2-10 copies of mtDNA
MUTATIONS
● Created by reactive oxygen species (ROS)
from ETC:
- some cells more mutations than others

DNA PHENOTYPING → lack of DNA repair

- when cells divide, daughter cells have
● use DNA seq to infer physical feature &
same mutations as parent cells
ancestry
- some cells divide often (e.g epithelial)
- Hair, eye & skin colour
while other stagnant during adulthood
- Biological sex
(e.g myocytes)
- geographic ancestry
● Each cell contains many mitochondria
● Composite images generated via SNPs
with different amount of mutation
based on GWAS
 ● Cell division may result in heteroplasmy
(mixture of both mutated & WT mtDNA)
→ level of heteroplasmy may vary among
siblings → ∵ distribution of mutated &
WT mtDNa in mother’s egg is random
PREVENTING MT DISEASE
TRANSMISSION
MITOCHONDRIAL REPLACEMENT
THERAPY (MRT) “3 parent IVF”
1. PRONUCLEAR TRANSFER (PNT)

2. MATERNAL SPINDLE TRANSFER

MITOCHONDRIAL DISEASES
● Affects 1 in 4300 live births
● Severe in children
● Lack of effective treatments
→ relieve symptoms & improve quality of
life
● Symptoms: affect many organs
- ataxia
→ loss of coordination & balance
- dystonia
→ involuntary spasms & contractions MRT ISSUES
- myoclonic epilepsy ● Incomplete removal/elimination of mutant
→ seizure mt
→ possibility of mt disease transmission
with reduced severity
● Embryo destruction
→ unused embryos discarded or
destroyed