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Genetic Diseases and their treatment

A quick reminder

DNA = deoxyribonucleic Acid, a double helix

Strands linked via chemicals called nucleotide bases

Thymine always links with Adenosine

Guanine always links with Cytosine

This is the genetic code

CCGATTCGACCGATTCGA

There are FOUR such bases

Thymine, Adenosine, Guanine and Cytosine

Each set of 3 bases = codon

Thousands of genes are collected together on CHROMOSOMES

DNA strands in chromosome

Genes represented by

coloured bands in chromosome

Humans have 23 pairs of chromosomes – 22 pairs autosomal and one pair sex chromosomes (called “X” & “Y” because of shape)

1 - 3 4 - 5

6 - 12

13 - 15 16 - 18

19 - 20 21 - 22

23

1 - 3 4 - 5

6 - 12

13 - 15 16 - 18

19 - 20 21 - 22

23

GENES

GENES

...CCGATTCGA......CCGATTCGA...

Part of a gene sequence could be

Mary had a little limb

Mary hid a little lamb

Mary hid a little limb

Mary had a little lamb

This could mean...

A defect in the gene coding for the letter “a” (i.e. a POINT MUTATION)

could result in

Each human cell has > 3 billion base pairs

Although we have > 90 % genes in common with chimps and mice, a 1-2% difference = 10s millions combinations

Genes are not necessarily contiguous sections of a chromosome.

Coding bits (codons) are interspersed with “non-functional” DNA (so-called Junk DNA).

Might provide protection against harmful mutations – disputed.

MAJOR change in “output” from apparently

trivial error

(humans = 1% of genome code for proteins)

GENES and INHERITANCE

Single genes RARELY code for a single effect – but they are much easier to study

Between genes are codons that control “START” reading of a gene and “STOP” reading

Mutations (errors in replication ; UV radiation etc)

Gene for eye colour

Both copies code

for BROWN eyes (homozygous)

INHERITANCE - Dominant and Recessive Genes

BROWN eyes BLUE eyes

Both copies code

for BLUE eyes (homozygous)

BROWN eyes

BROWN eye colour is DOMINANT

One copy codes BROWN, other copy codes BLUE

(heterzygous)

Genetic Diseases

● AUTOSOMAL RECESSIVE ● AUTOSOMAL DOMINANT Only one mutated gene = HETEROZYGOUS

Autosomal recessive inheritance

Have to have TWO aberrant genes i.e. be HOMOZYGOUS

If only one mutated gene i.e. HETEROZYGOUS CARRIER

● X-LINKED RECESSIVE (Female is CARRIER but MALE

is affected)

Autosomal RECESSIVE - Cystic Fibrosis

● Signs &Symptoms➢ Mucous build up in LUNGS (pancreas, liver, kidney, intestines)

➢ Breathing difficulties➢ Repeated lung & sinus infections (prolonged antibiotic treatment)

➢ Poor food absorption ➢ Poor growth

Most common in Northern Europeans (1 in 3000 births; 1 in 25 = carrier)Least common in African/Asians

Life Expectancy 40 – 50 yrs in developed world

Treatment

Usually due to DELETION of 3 bases on gene 7 – results in faulty protein. This results in secretions that should be thin being THICK (but ~ 300 mutations identified in CF)

Autosomal RECESSIVE - Sickle Cell Anaemia

● Signs & Symptoms● Impaired oxygen carrying● Tendency to block blood vessels● Pain● Stroke● Swollen hands & feet● Infections

80% of cases in SubSaharan Africa

In 2015 4.4 million people affected43 million = CARRIERS 114,800 deaths

TREATMENT

Heterozygy confers resistance to malaria in early childhood

Distorted shape of Red Blood Cells

Autosomal DOMINANT – Huntington's Disease (Chorea)

Signs & Symptoms➢ NONE (apart from occasional mood swings/unsociability) in 1st 30-40yrs

– ie until AFTER had children (hence inheritance)➢ Twitches, jerks, spasms➢ Objects hard to grasp➢ Hands & legs uncontrollable writhing (“giant puppet show”)

➢ Finally deep cognitive decline with near complete loss motor function

➢ Malnourishment, dementia, infections - DEATH

TREATMENT

Neurological disease

Woody Guthrie

Autosomal DOMINANT – Marfan's Syndrome

● Can have norm life span● M and F equally affected● 1 in 5000 (¾ = inherited' ¼ = spontaneous

mutation)● No ethnicity

Connective Tissue disease

Signs & Symptoms➢ Defects in heart valves, lungs, eyes,

bones & meninges➢ People tend to be TALL, THIN with

disproportionally LONG LEGS, TOES, ARMS,FINGERS

TREATMENT

X-linked RECESSIVE - Haemophilia

● Signs & Symptoms➢ Minor injuries (bruise)

cause uncontrolled bleed under skin and into joint

➢ Pain➢ Distortion of joint➢ Death

Queen Victoria's family tree – inheritance of Haemophilia

Haemophilia (due to missing clotting factor) is (usually) due to faulty, recessive gene on X chromosome – so

very rare in women (women have TWO X chromosomes) but they can be carriers (men rarely carriers

as do not live long enough to have children)

Treatment

Faulty blood clotting

X-linked RECESSIVE – Duchenne's Muscular Dystrophy

● Signs & Symptoms➢ Progressive leg weakness, baby boy using parents' legs to pull self

up when learning to walk, not able to walk by 12 years➢ Eventually all voluntary muscles affected, then heart and

breathing later on➢ Paralysed from neck down by 21st year

Life Expectancy ~ 25 years

1 in 7000 live birthsTREATMENT

Progressive muscular weakness & wasting

ALSO● Higher risk ADHD● Learning Disorders (eg

dyslexia)● Short term memory impairment

Genetic therapies

● Deliver functioning genes to adult (ie already affected) cells● Deliver functioning genes to embryos – then implant● Delete aberrant genes from (preimplant) embryonic / foetal cells OR adult cells

AIM

PROBLEM➢ How to get editing system to target➢ How to change ALL affected cells➢ How to ensure correction is permanent (heritable) not just adding a functional gene

Genetic therapies

● MANIPULATION SYSTEMS➢ CRISPR cas9➢ Base editing➢ (older systems (eg zinc-finger

nucleases & TALENs, seem redundant now)

● DELIVERY SYSTEMS

Direct injection into organ (eg functioning

gene into lungs into CF patients) or into fertilised ovum (before 1st division)

Nanoparticles (ie 1-100nm) silica, gold (injected into muscle of mice with MD, only 5% muscles repaired but enough to improve muscle strength)

Fatty particles (used to treat hepatitis B in mice

VirusesBacteria

Own Cells white blood cells (leukaemia)

T- cells (cancer)

red blood cells (sickle cell anaemia)

Stem cells (vast potential applications)

Side Step.......

● Viruses ● “Average virus” is 1/100th size of “average”

bacteria ● Packet of DNA or RNA in protein coat =

VIRION● Invade cells ● Commandeer cell's chemical machinery to

keep virus going & replicate virus● Viruses readily mutate in host cells● Provide horizontal gene transfer – ie

movement of genetic material between unicellular and/or multicellular organisms (vertical is parent to offspring)

Capsid = protein coat, sometimes enveloped in lipid

VIRUS particle = virion

DNA or RNA strands in core

BacteriaOne celled organism lacking nucleus or membrane bound cellular organelles

Gene Editing

CRISPRPart of bacterial (Streptococcus

pyogenes) defence against viruses

Exposure to virus means bacterium copies a bit of viral DNA so it can recognise the virus in the future

A repeat exposure to the virus means the bacterium can use a special RNA molecule to guide a protein (Cas 9) to that part of the viral DNA, and disable it by cutting

For therapeutic use, guide RNA (gRNA) is created that matches the gene of interest.

gRNA plus Cas9 are put into a plasmid, and the plasmid put into target cell

gRNA locates target gene in the cell's DNA and Cas9 cuts both strands of DNA

Cell repairs break by joining the cleaved DNA strands thus disabling target gene (non homologous end joining NHEJ

After cleavage, a functional gene (=mutation)can be inserted (homology directed repair HMD

Guide RNA

Gene Editing

CRISPR vs previous gene editing techniques

● Easier● Cheaper ● Quicker (1-2 weeks vs 1-2 years)● More efficient

CRISPR good at disabling gene (65% of HD gene in key areas animal brain disabled)Less good at repairing genes (repair is more useful in embryos)

Mosaics : - embryos where only SOME of the cells are “fixed”. Adult MAY be Ok – but faulty gene still present – might become active; if in germ cells will pass to next generation Difficult to screen for in pre-implantation embryos (usual screen might not work)

How to switch off CRISPR after desired change made and before potentially deleterious change happens (already achieved, “kamikaze” CRISPR)

CRISPR targets a sequence of bases – not individual ones (cf base editing)

DNA repair mechanism not perfect – may incorporate wrong bits of DNA as part of the “patch”Some problems

Base Editing (modified version of CRISPR)

Has been used experimentally to correct blood disease fault in non-viable human embryos

DISEASE VS DISORDER

Bipolar disorder

Asperger's

Schizophrenia

Van Gogh

Elly Simmonds Steve Jobs

Philip K DickDwarfism

Criminal Behaviour

Bonnie & Clyde

SOME ETHICAL CONSIDERATIONS

● Would-be parents who want unaffected children can have IVF with donor egg / sperm OR select from pre-implantation embryos

● Are lives of people affected by genetic diseases “less”

● Are “healthy” “normal” people entitled to make decisions of behalf of those with life limiting diseases

● Should genetic manipulation be only targeted at the post-natal (curing existing condition) not the preimplantation

● Is this EUGENICS by another route?

Sources

● Biologist Magazine● The Gene (S. Mukherjee)● New Scientist● BBC.co.uk● BBC R4● Royal Soc Biology Blogs and News Items● Shutterstock● Wikipedia● Www.jax.org

THANK YOU!!