TOPIC 9: Chromosome

variation SEX CHROMOSOME

ANEUPLOIDY ● Usually viable & minor symptoms
→ symptoms due to increase copy of
● Aneuploidy: individual chromosomes
pseudoautosomal genes
gained or lost due to nondisjunction
● types:
→ fail to separate during meiosis →
- XXX(triplo-X): asymptotic females
eggs/sperm contain extra or less
→ defects in secondary sexual
chromosome → abnormal chromosome
characteristics, sterility
number in embryos
→ mild IQ deficit
CHROMOSOME LOSS - XXY(Klinefelter syndrome): males
AUTOSOMES → undiagnosed
● Autosomal monosomy → symptoms: tall, rudimentary testes
- mammals: lethal (infertile), depression, ADHD, autism
- other species: can survive - XYY: male
● consequences: → symptoms: above average height,
- recessive lethal/deleterious alleles lower IQ, link with antisocial behaviour,
- haploinsufficiency: insufficient gene criminality
product expressed from single wild type POLYPLOIDY
allele
● Polyploidy: more than 2 complete sets of
SEX CHROMOSOME chromosomes
● Y: not viable - triploid = 3 sets
● X: viable → Turner syndrome - tetraploid = 4 sets
- 1:2000 live female births ● Prevalence:
- symptoms: malformed ovaries, short - rare in most animals
stature, cardiovascular defects - common in fish, amphibians
- due to haploinsufficiency → only 1 copy - common in plants → seedless fruit
of genes in pseudoautosomal regions of X derived by crossing seed containing
chromosomes diploid & tetraploid varieties
CHROMOSOME GAIN ● Result from:
- nondisjunction of entire set of
● Autosomal trisomy
chromosomes in meiosis
- lethal in humans
- fail to undergo reduction division in
- in 13,18
meiosis ii
→ spontaneous miscarriages or die soon
- fertilisation of egg with 2 sperm →
after birth
triploid
→ severe developmental effects
- cytokinesis failure after fertilisation →
DOWN SYNDROME
tetraploid
● Trisomy 21 → nondisjunction during
● 2 rounds of polyploidy occurred during
maternal meiosis
vertebrate evolution → multiple copies of
● Survive till ~50 years
many genes present in genome
● symptoms:
- mental retardation
CONSEQUENCES
- flat face ● Divergent roles: multiple copies of genes
- heart disease present → allow different mutations to
● Probability increases with mother's age
 accumulate in distinct copies → obtain DOWN SYNDROME
new roles
➔ Evolution of egg myoglobin &
haemoglobin via duplication → now
different roles
● One step speciation: tetraploid individuals
can interbreed with e/o → no diploid
fertile offspring produced
ENDOPOLYPLOIDY
● Endopolyploidy: certain cell types contain
multiple sets of chromosome
→ insect salivary gland cells
DELETIONS & DUPLICATIONS
● Deletions: part of chromosome missing
● rare cases of familial
→ small or large
● Carriers:
→ large lethal
➢ translocation linking most/all of
● Duplications: repeated segment
chromosome 21 & 14
EXAMPLE: CRI DU CHAT
➢ asymptomatic → full complement of
● Deletion of 5p tip
genetic material
● Sporadic loss in gametes or early
➢ gametes:
development
- ¼ normal
● Symptoms caused by specific regions in
- ¼ contain translocation → carrier
5p
progeny
REARRANGEMENTS - ¼ lack material from chromosome 21
INVERSIONS - ¼ contain double normal chromosome
● 2 dsDNA breaks forms in chromosome → 21 material → down syndrome children
intervening sequence reorientated before
breaks are rejoined
● Asymptomatic
● Maternal & paternal chromosomes may
not align properly during meiosis I →
meiosis perturbed → abnormal gametes
& infertility
TRANSLOCATIONS
● Movement of chromosomal segment to
another region of genome
- tip of one chromosome removed & added
to another chromosome → common
- Reciprocal → tips of 2 chromosomes
swapped
● Asymptomatic → ∵ genetic material not
lost or gained
● abnormal segregation during meiosis →
duplicated/deficient gametes → reduced
fertility
 Type 2 (30%): OCA2 (africans)
TOPIC 10: Autosomal ●
→encode P protein → transport melanin
recessive precursor tyrosine into melanosomes
● Expression of one allele from 2
autosomal genes enough for function
→ wild type allele can function
● Features:
- affected individuals have unaffected
parents
- unaffected parents → asymptotic
carriers
- prevalence of parental consanguinity
- affects both sec with equal probability
- carrier parents → 25% chance of
affected offspring
- mutations in both alleles
→ same mutation (homozygous)
→ different mutations (compound
heterozygous)

● Analysis of OCA2 mutations in unrelated

Tanzanian patients
- albinism: loss of function mutations →
lack of activity of OCA2 gene product
- recessive: wild type allele encode
enough enzyme → no symptoms

CYSTIC FIBROSIS
● Symptoms:

OCULOCUTANEOUS
ALBINISM (OCA)
● Deficiency of melanin in eyes, hair & skin
● Increased risk of skin damage & cancers
GENES
e.g melanoma
● caused by mutations in cystic-fibrosis
GENES INVOLVED transmembrane regulator gene (CFTR)
● Type 1(50%): TYR (caucasians) ● RFLR analysis used to map gene →
→ encodes tyrosinase → melanin compared with cDNA → common
biosynthesis deletion
 → genotyping by hybridising SEVERITY
oligonucleotides specifically bound to
● Influenced by environmental and genetic
wild type or deleted alleles → match
factors
pedigrees
- Lung symptoms:
● CFTR: encode large protein expressed in
→ worsened by infections
membranes of epithelial cells in lungs &
- pancreatic symptoms:
other organs
→ worsened by variants in pancreatic
- ion channel → export chloride proteins
function genes
- mutations impair production, transport
within cell &/or biochemical activity →
TREATMENTS
reduction in CFTR activity → mucus build SYMPTOM-BASED TREATMENT
up in airways → unable to clear infections ● Mucus reduction:
- different levels of CFTR activity in - beat chest/back to loosen mucus →
different organs cough up
GENOTYPE/PHENOTYPE - exercise
- anti-inflammatory medicines
RELATIONSHIP - bronchodilators
F508DEL (3 BASE DELETION) ● Infection control:
● Most common mutant alleles - antibiotics
● null mutation → most proteins misfolded ● Advanced lung diseases:
& degraded, others membrane localised - oxygen
→ reduced CFTR activity - lung transplant
● CFTR F508del homozygous → severely ● Digestive symptoms:
affected - pancreatic enzymes
● 1 CFTR F508del allele + mutant allele → - vitamin supplements
cystic fibrosis symptoms & reduced life - feeding tube → extra nutrition
span ● Nasal polyps:
G551D (NON-CONSERVATIVE - surgical removal → help breathing
➔ increase life expectancy
SUBSTITUTION)
● 3rd most common
TARGETED THERAPIES
● Severe “loss-of-function” mutation →
proteins properly trafficked & reach cell
membrane but reduced channel function
● 1 CFTR G511D allele + mutant allele →
cystic fibrosis symptoms & reduced life
span
R117H (CONSERVATIVE
SUBSTITUTION)
● rare
● Most common residual activity
● mild “loss-of-function” mutation →
proteins have reduced channel function
● 1 CFTR R117H allele + second residual
activity allele → more severe symptoms
but live longer
 Most common CFTR mutations affect:
●
TOPIC 11: Autosomal
- folding
→ lead to degradation AND/OR
dominant
→ ability of protein to conduct chloride
ions once in cell membrane
POTENTIATORS
● target mutations affect channel activity
● Drug: Ivacaftor
- stimulates opening of mutant CFTR
channels
- patients with at least 1 G551D mutant ● 1 affected parent
allele given placebo or Ivacaftor → ● affect either sex
dramatic improvements → standard care ● Transmitted by either sex

CORRECTORS ● 1 affected + 1 unaffected parent → 50%

chance of affected child
● target mutations causing misfolding
● F508del affects transport & activity
● Dominant mutations cause gain of
● Drug: Lumacaftor
function change by:
- facilitate correct correct folding →
- boosting expression
traffick CFTR F508del → ↑ quantity at cell
- changing timing of expression
surface
- stimulating expression in abnormal cell
- patients with F508del mutations given
type
placebo or Lumacaftor + Ivacaftor →
- changing protein sequence → different
significant improvement
behaviour
● Dominant effects by loss of function/null
➔ Cystic fibrosis not cured → Ivacaftor +
alleles if:
lumacaftor will only modestly extend life
- haploinsufficiency: insufficient product
span
made from wild type + mutant allele for
normal function
- mutant exert dominant negative effect
→ inactive mutant protein interferes with
activity of protein encoded by wild type
allele

MARFAN SYNDROME
● connective tissue disorder
- cardiovascular: aortic dilation & rupture
- skeletal: bone overgrowth
- eye: lens dislocation, myopia, retinal
detachment
● Mostly inherited by some sporadic cases
GENOTYPE/PHENOTYPE ● Test: mutant protein should cause
phenotype even of 2 wild type alleles
RELATIONSHIP present
● Gene: Fibrillin-1/FBN1 1. mice created with C1039G transgene
- form extracellular fluids → extracellular 2. Aorta width measured
matrix structure → results:
- sequence TDF-beta - Replacement with 1 wild type
→ regulate signalling allele with C1039G → ↑ aorta
→ regulate transcription of genes width
controlling cell cycle & cell-cell - 2 wild type allele + C1039G
interaction transgene → normal phenotype
➔ Not consistent
HAPLOINSUFFICIENCY ✓
● single wild type allele may not produce
enough FBN1 for functional fibrils
● Test: mutations that prevent protein
production should produce Marfan
phenotype
- patient has Marfan phenotype →
although no mRNA made from deletion
● Mutations affect:
allele
- intercellular connection and/or
➔ Consistent
- TGF-beta signalling
➔ cause Marfan syndrome via
haploinsufficiency TREATMENT/MANAGEMENT
● Limitation in sports
CAUSE OF DOMINANCE
● Administration of beta-receptor blockers
GAIN OF FUNCTION ╳
→ reduce aortic pressure
● most mutations in coding region unlikely
● ECG every 1-2 years + periodic MRI/CT
to change expression patterns
scan
● Different mutations produce similar
● Aortic root surgery → if aortic diameter
phenotypes → unlikely to form new
increases
function
→ 70% die of acute cardiovascular events
DOMINANT NEGATIVE ╳
● Mutant monomers present → may disrupt
LI FRAUMENI SYNDROME
fibril structure → loosen connections ● Li fraumeni syndrome: families with

between cells & release TGF-ꞵ unusually high rate of cancer arising at

● Missense mutations affect cysteine young age

residues → mutant FBN1 proteins may → autosomal dominant

ruin fibril structure by forming incorrect ● Linkage analysis difficult due to early

disulfide bonds patient death → difficult to distinguish

LFS cancer from spontaneous cancer →
no obvious chromosome rearrangement
GENES INVOLVED
● P53 gene
- heterozygous p53 mutation → cancer in
skin cells
→ dominant inheritance
- homozygous wildtype → no cancer
- some heterozygous → yet to have
cancer
DOMINANT NEGATIVE P53
MUTATIONS
● p53
- transcription factor → induces genes →
stimulate anti-cancer processes (cell
death, cell cycle arrest, repair DNA
damage)
➔ Missense mutation → prevent DNA
binding → promote cancer development
despite wild type p53 present → ∴
dominant
- function as tetramer
➔ mutant monomers → prevent
transcription factor function (TOPIC 12)
Mendelian vs non-mendelian
inheritance
● Mendelian inheritance:
- 2 alleles per gene
- dominance/recessive
- alleles segregate during gamete
formation
- independent assortment of genes
● Non-mendelian inheritance: traits not
inherited in dominant/recessive,
independently segregating,
sex-independent manner
HETEROZYGOUS CELLS → e.g:
● p53 tetramers inactive - linked genes (recombination frequency
● cells express wild type 53 or DNA - X/Y linked genes
binding domain mutant or neither or both - mitochondrial/chloroplast inheritance
→ DBD mutant expression → impair - incomplete penetrance (genetic or
ability of wild type p53 to induce target environmental factors)
genes → dominant negative - anticipation
- imprinting
TOPIC 12: X-linked recessive
● Mostly affect males
● Mostly unaffected parents
● Heterozygous females → carriers
DUCHENNE MUSCULAR
DYSTROPHY (DMD)
● Progressive muscle weakness
→ arm, leg muscles affected initially
● Early teens → heart, respiratory muscles
affected → death
● Milder form → becker muscular dystrophy
(BMD)
TREATMENT/MANAGEMENT
● daily corticosteroids
→ improve muscle strength
→ limit use due to side effects
→ mechanism unknown
● baseline ECG at diagnosis → regular
screening
● Cardiomyopathy drugs
● Nocturnal ventilation → also daytime
later
● Stretching, night splints → delay
contracture progression
GENE INVOLVED (DMD)
● linkage analysis using Restriction
Fragment Length Polymorphism (RFLP)
markers
- work out recombination frequencies
between disease locus & each marker
- LOD scores → show gene most closely
linked to marker “754”
● Gene: DMD → encode dystrophin protein
● Type of mutations:
1. Deletion of exons (68%)
2. Duplication of exons (11%)
➔ cause frameshift
3. Small mutations (20%)
- Nonsense mutations (10%)
- Small mutation within exon
(insertion,deletion) (7%)
- splice site mutation (3%)
GENOTYPE → PHENOTYPE
● DMD mutations → deletion including
exon 50 or 52 → disrupt reading frame →
encode non-functional proteins →
degraded
● BMD mutations → delete exons 48 &/or
49 → maintain reading frame → protein
retains some function → muscle degrade
overtime
● Dystrophin → links muscle cell
membrane to actin filaments → protects
from damage as muscle contract
RECESSIVE
● minority of heterozygous females →
symptoms → vary sensitivity
➔ ∵ skewed X activity: X chromosome with
wildtype DMD allele inactivated in most
muscle cells → by chance
TARGETED TREATMENT
EXON SKIPPING
● agents promote alternative splicing →
remove exons flanking
deletions/mutations → yield inframe
truncated protein
● Drug: Eteplirsen
→ modified oligonucleotide → binds at
start of exon 51 of pre-mRNA → skipped
during splicing → correct reading frame in
cells which dystrophin genes lack exon
50 → produce functional DMD which
contains N & C termini but lacks middle
section

GENE THERAPY
➔ Clinical trial data:
- drug slightly boosted dystrophin levels
& improved muscle structure
- treatment significantly improved decline
in walking → but did not stop disease
progression
READ-THROUGH
● nonsense mutations → overcome by
agents promoting stop codon read
through
→ release factor detect stop codon in
mRNA → stimulate dissociation of
ribosome, protein & mRNA → tRNA that
matches stop codon incorporated →
translation continues
● drugs promoting read through → slowed
walking decline in moderately affect
patients
● Introducing wild type version of DMD
gene → challenging due to size
● Gene editing more feasible →
CRISPR/Cas9 allows site specific
cleavage to modify splice site → facilitate
exon splicing
→ achieved in dogs with deletion of exon
50
⇒ adenovirus infection used to introduce
Cas9 → guide RNA targeting splicing
acceptor site for exon 51 into muscle cells
→ IV boosted dystrophin protein levels in
muscles
TOPIC 13: X-linked GENE INVOLVED
incomplete dominant ● extra CGG repeats in fragile X mental
retardation-1 (FMR1) gene → encodes
FMRP → RNA-binding protein → role
unknown
● rare fragile X syndrome → caused by
intragenic mutations → loss of function in
FMR1→ cause symptoms
⇒ repeats + small intragenic mutations
→ loss of protein
INHERITANCE
● number of repeated correlated with
disease severity
- <50 repeats : healthy
- ~50-200 repeats: premutation
→ normal or mild symptoms (carriers)
- >200 repeats: disease
→ more repeats → more symptoms
● more severe symptoms in males than in
female heterozygous
ANTICIPATION
● Anticipation: tendency for some
conditions to become progressively more
severe in successive generations
→ Disease caused by repeats
● affect both sexes, mostly females ● Further repeat expansion
● at least one parent affected → no carrier → during oogenesis in women with
● Affected male: permutation → children with disorder
- all daughters affected → does not occur during
- no sons affected spermatogenesis
● Affected female: ⇒ fathers transmit premutation alleles
- half son and half daughters affected ⇒ expansion only with maternal
● Incomplete dominance → milder inheritance
symptoms in female GENOTYPE → PHENOTYPE
FRAGILE X SYNDROME ● Extra repeats in FMR1 → cytosine
methylation & histone deacetylation
● symptoms:
spreads to include promoter → local
- delayed development of speech &
chromatin condensed → transcription is
language
silenced
- mild to moderate intellectual disability
● Over methylation in patients → specific to
- anxiety, hyperactive behaviour
FMR1 genomic region
- autism
● less FMRP expressed
- seizures
→ intermediate levels of FMRP in
- distinctive facial shape
heterozygous females → X chromosome
- muscle weakness
with mutant FMR1 gene inactivated in
half of cells
 -active X → FMRP expressed at normal - deletions near 3’ end of coding region →
levels X-linked dominant disease
- inactivated X with wild type allele → no → overproduction of protoporphyrin,
FMRP expressed haem in RBC → cause photosensitivity &
INCOMPLETE DOMINANCE liver disease
● Autosomal genes in heterozygous INHERITANCE
individuals: 1 wildtype + 1 mutant
⇒ X linked conditions female heterozygous:
1 active wild type allele or 1 active mutant allele
● dominance/recessive in X-linked
conditions apply at whole organism level
not cell level
- symptoms present if half of neurons
lack FRMP protein → heterozygous
females with random X-inactivation
- milder symptoms in females than males
with equal mutations → ∵ normal FMRP ● affected mother → half son & daughters
protein expressed in other half of neurons ● affected father → daughters only
in females ● X-linked dominant
- severity of symptom in heterozygous GENOTYPE → PHENOTYPE
females → influenced by X-inactivation ● Gain of function mutations → cause
patterns C-terminal frameshifts in ALAS2 gene
TARGETED TREATMENTS (NOT ● In vitro experiments with purified
recombinant proteins → showed
YET)
truncated mutants more enzymatically
● drugs provoking demethylation of DNA
active than wild type protein
→ increase expression of FMR1 from cells
- overproduction of protoporphyrin →
with large repeat expansions in vitro
irradiation → photosensitivity
● clinical translation require:
- overproduction of haem in erythrocytes
- long term administration
→ haem accumulation → liver damage
→ ∵ effect in methylation & expression
● Heterozygous females → half of cells
transient
produce too much protoporphyrin & haem
- safety
→ exhibit symptoms → dominant
→ global demethylation for extended
⇒ skewed X inactivation affect severity of
periods → affect expression of other
disease in heterozygous females
genes & cause unwanted adverse effects
→ methylation status of wild type &
- agent given in utero or provoke
mutant alleles determined in blood of
demethylation in non-dividing cells
heterozygous females → proportion of
X-LINKED DOMINANT cells which wild type allele methylated
correlated with symptom severity
PROTOPORPHYRIA (XLDPP)
● X-linked gene ALAS2 → encode
aminolevulinate synthase 2 → haem
biosynthesis in RBC
- loss of function ALAS2 mutation →
recessive anaemia
 Y-LINKED TRAIT
PENTRANCE
● Y chromosome → non-essential genes
● penetrance: probability that person with
→ determine male-sepecific features
genotype will manifest character
● mutations only affect males
● dominant trait which shows phenotype →
→ affect fertility → no inheritance
100% penetrance
● All sons of affect male affected → if
● Incomplete penetrance
infertile no sons produced
→ caused by factors:
● Hypothetical
- other genes
- environment
- age MITOCHONDRIAL
- skewed X-inactivation INHERITANCE
→ prevent females from normally
● maternal inheritance
dominant X-linked diseases
● affect both sexes with equal frequency
→ lead to heterozygous females
● cell have wild type + mutant
experiencing symptom from normally
mitochondria → vary in proportion
recessive X-linked diseases
⇒ incompletely penetrant
PSEUDOAUTOSOMAL ● Minority of proteins in mitochondrial
encoded by mitochondrial genes
INHERITANCE
→ most protein encoded by nuclear
● Tips of X & Y chromosomes contain
genes, translated in cytosol & imported
homologous sequences → interact &
into mitochondria
recombine during meiosis & not subjected
to X-inactivation
⇒ lead to unusual segregation of genes
→ recombination between
pseudoautosomal regions of X & Y during
male meiosis → shift inheritance between
X-linked & Y-linked
 symptoms:
TOPIC 14 ●
- excessive appetite
IMPRINTING - hormonal defects
● imprinted : differentially transcribed from - intellectual disability
maternal vs paternal chromosome ● Deletions in many genes → deleted
→ controlled by differential methylation region in SNORD116
GAMETE SPECIFIC METHYLATION ● gene product of SNORD116 → regulate
splicing → unknown relation to
symptoms
ANGELMAN SYNDROME
● UBE3A mutation on maternal
chromosome

DISEASES DUE TO MUTATIONS IN

IMPRINTED GENES
● mutations → result in diseases with
atypical inheritance patterns →
phenotype depend on parental origin of
mutant allele
● site of imprinting diseases in humans →
chromosome 15 (SNORD116 & UBE3A)

● UBE3A encodes ubiquitin ligase → critical

substates unknown
● Neurological symptoms:
- intellectual disability
- happy personality
PRADER-WILLI SYNDROME
→ ∵ loss of UBE3A function in neurons
● SNORD116 mutation on paternal
chromosome EPIGENETIC MARKS
● in utero (maternal) exposure to chemicals
→ modify DNA methylation patterns &
gene expression in offspring → modify
phenotypes
LAMARCKISM?
● Epigenetic marks not transgenerationally
inherited
→ not expressed in F3 generation &
beyond
→ no adaptive link between
environmental exposure & phenotype
⇒ no contribution to evolution
GENE-ENVIRONMENT
INTERACTIONS
● phenotypes
PHENYLKETONURIA (PKU) ● Clinical trial → effective
● autosomal recessive
● excreted phenylpyruvic acid
GENE INVOLVED
● Defect in phenylalanine metabolism
● biochemical pathway:

● loss of function mutations in PAH → gene

encodes phenylalanine hydroxylase →
cannot convert phenylalanine to tyrosine
→ phenylalanine accumulates & some
converted to phenylpyruvic acid →
phenylalanine levels ↑ in cerebrospinal
fluid & brain → mental retardation
DIAGNOSIS
● blood taken from newborn babies →
phenylalanine levels quantitated

TREATMENT
● Manage diets → keep blood Phe low →
develop normal mental function
⇒ low Phe regular food
⇒ supplements: essential amino acids,
vitamins, minerals, trace nutrients
TARGETED DRUG TREATMENT
● enzyme administered → break down
phenylalanine into harmless compounds
→ processed & excreted
 stabilised chemical environment → kept
TOPIC 15: Human Evolution
ribozymes close to products
ORIGINS OF LIFE TO ● Mechanisms which primitive cell
MULTICELLULARITY membranes formed → high speculative
⇒ early enzymes scavenge chemically
RNA formed lipids from environment or
● encode genetic information synthesised them
● Ribozymes: RNAs with enzymatic activity
RIBONUCLEOTIDES
● could be generated from available
chemicals using energy from UV light
RIBOZYMES
● coenzymes that can polymerise RNA from
nucleotides created based on naturally
occurring ribozymes

● primitive RNA polymerising ribozyme

extents RNA primer from template strand
EUKARYOTIC EVOLUTION
→ end base pairs with ribozyme
● evolved with subcellular organelles
- mitochondria: make energy from oxygen
- chloroplast: photosynthesis
- nucleus: house genome
- membrane structures: Golgi, ER,
lysosomes

PROTEINS, DNA, CELLS

● ribosome precursors made of RNA →
protein enzymes evolved to replace RNA
● Deoxyribose more complex than ribose →
DNA replaced RNA genome after
evolution of protein enzymes synthesising
deoxyribose → evolution favoured ● Engulfment of bacterial cells → by
enhanced stability of DNA as genetic archeal cell & bacterial cell → acquisition
material of mitochondria
● Compartmentalisation of biomolecules ● Development of nucleus &
within phospholipid membrane → endomembrane → unknown if before or
after acquisition of mitochondria
MULTICELLULAR ORGANISMS
● Multicellularity → arise independently
numerous times during evolution of
plants, fungi & animals
→ molecular structures & processes
developed → allow cell-cell contact
● Important steps:
- cell to cell adhesion
- cell to cell communication & cooperation
- specialisation of cells
MAMMALS TO HUMANS
FIRST PLACENTAL MAMMAL
● Morphology: >4500 characters across
126 species
● Genomic analysis(27 genes)
● Proposed features:
● Conclusion:
- evolved around 65 myo
- four-legged
- insectivore
- weight: 6-245g
- lifestyle: general scampering
- cerebral cortex convoluted
EVOLUTION OF LACTATION
● Ancestors of reptile/birds & mammals →
nutrition via egg yolk vitellogenin protein
→ encoded by 3 VIT genes
⇒ Mammalian evolution → VIT genes
disabled
- monotremes → retain 1 functional VIT
gene
- marsupials & placental mammals → 3
VIT genes inactivated
● Mammals secrete milk from modified hair
follicles
→ casein → major milk protein
● gene duplication & divergence → distinct
genes for casein, saliva proteins & tooth
enamel matrix proteins in mammals
APE-HUMAN EVOLUTION
HUMAN/CHIMP/GORILLA
DIVERGENCE
● human & chimps more closely related to
each other than to gorillas
DIFFERENCE IN GENOMES &
PROTEOMES BETWEEN CHIMPS &
HUMANS
● ~4% genome difference
● ~1% proteome difference
● ⅓ protein sequences identical
SPEECH DEVELOPMENT IN
HUMANS
● gene: FOXP2
→ distinct to humans
→ 2 a.a identical in apes but different in
humans → change contributed to
evolution of human speech
FOXP2 MUTATION
● Mutations cause speech defects
→ difficulty organising sounds into words
& poor mouth control
● FOXP2 → family of transcription factors
→ cause dominant phenotype through
haploinsufficiency
ANIMAL MODELS OF FOXP2
FUNCTION
● FOXP2 highly expressed in regions of
brain important for speech
● Mice with homozygous deficiency in
FOXP2 → severe defects in vocalisation
⇒ heterozygous mice → less vocal than
wild type mice
● Mice with humanised FOXP2 genes →
learn tasks faster than wild type mice
● In birds → FOXP2 expression upregulated
when learning new songs
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