Single Gene Disorders

define Mendelian disorders

define Mendelian disorders

genetic disease where one gene is involved and its mutation causes disease

APOE4 allele

Increases risk of sporadic AD. 25% of people have 1 copy (increases risk 2x), 2 to 3% have 2 copies (increases risk 12 to 15x)

Why is it important to identify genes responsible for hereditary conditions/disorders?

May allow disrupted biological processes to be understood → treatment/cures/gene therapy. Also allows diagnosis prior to symptoms eg BRCA1 identification

example autosomal dominant disorders and their prevalence

familial hypercholesterolaemia (1/500) and Huntington’s (1/15,000)

example autosomal recessive disorders and their prevalence

cystic fibrosis (1/2000 caucasians) and Tay Sachs (1/3000 American Jews)

X linked Mendelian disorders and their prevalence

Duchenne muscular dystrophy (1/7000) and hemophilia (1/10,000)

How is approach to identifying affected gene decided on?

Based on what is already known about gene/disease. Whatever approach is chosen, DNA samples from patient, family, population and healthy controls are needed

20th century gene identification techniques used

chromosomal damage and positional cloning

gene identification 21st century

patient genome/exome sequenced and causative mutations searched for

visible chromosomal damage/cytogenetic rearrangements

hypothesised to be responsible for disrupting/deleting gene - marker for gene location

Duchenne muscular dystrophy (DMD)

presents in boys around 3-5 years old. Patients show progressive muscle weakness/atrophy and sarcopenia

DMD gene identification

Initially identified by chromosomal damage. Xp21 completely sequenced in 1987

dystrophin

• mutated DMD protein

• 3685 amino acids long

• anchors moving muscle to solid ECM

When was positional cloning used?

when no visible evidence of chromosomal damage

positional cloning step 1

• large multigenerational families w/ extensive disorder history identified

• pedigree analysis

• linkage analysis w/ genetic markers

positional cloning step 2

identify overlapping DNA clones from the chromosomal locus

positional cloning step 3

identify genes found in the affected chromosomal locus and determine exons and introns

positional cloning step 4

analysed to determine if mutation(s) are in patient samples and not in healthy family members

what happens in Huntington’s

Neurons in basal ganglia and deep cortical layers (responsible for motor control) killed → dementia, psychosis, chorea and premature death

Who did the HD collaborative research group study?

Venezuelan village with very high (1/50) HD rate - founder effect

How was the HD gene cloned?

• RFLP polymorphic markers - 8th, G8, showed linkage - region 4p16.3

• 4 candidate genes in region characterised

• repeat expansion in IT15 found → Huntingtin cloned

trinucleotide repeat expansion mutations

polymorphic repeats of a specific trinucleotide sequence. Unstable in meiosis so number of repeats changes between generation due to incorrect melting and reannealing of DNA followed by incorrect repair

observations in HD families

number of repeats increases over generations and thus severity increases and age of onset is earlier

HTT gene repeats

• trinucleotide repeat CAG variation → changes in glutamine (Q) expression

• normal HTT 11 - 34 repeats (median 19)

• Disease HTT has 37 to 86 repeats (median 45)

Huntingtons gain in function

increased inflammatory response, mitochondrial oxidative stress, apoptosis and transcriptional misregulation → cell death

HTT gene function

unknown - proteins healthy and disease gene binds have roles in transcription, vesicle transport and cytoskeleton/synapse

current targets of disease gene research

very rare mutants/variants that cause specific conditions being targeted now that genome sequencing is more accessible

Describe the process of modern Sanger chain termination

primer, DNA polymerase, dNTPs and small amounts of fluorescently labelled ddNTPs added. Primer extension, chain termination and product recovery take place followed by electrophoresis

How does Sanger sequencing work?

Fluorescent dyes emit light at different wavelengths, different base emission during electrophoresis

what does whole genome sequencing rely on?

Next generation sequencing aka high throughput sequencing

issues with WGS

• massive data production/storage

• analysis of assembly of reads into a genome

• differentiating disease mutation vs benign polymorphism vs benign rare variant

Huntingtons treatment

Has no cure or treatment to stop/slow down neurodegeneration but can take drugs to relieve symptoms

medicines to treat chorea caused by Huntingtons

tetrabenazine, antipsychotics eg haloperidol which suppress movement as side effect, amantadine

drugs to treat mental health issues associated with Huntingtons

antidepressants eg citalopram, antipsychotics eg olanzapine

non-drug treatments for Huntingtons

psychotherapy, speech therapy, physical and occupational therapy