Describe the methodology of a GWAS and apply it to a disease

Genome- wide association studies (GWAS)

- The recruitment of large, ethically-matched case-control cohorts to discover differences in allelic frequency
between healthy (control) and affected cohorts.
- Various genome projects combined have mapped more than 4 million SNPs/ odds ratio which have to
alleles: C and T . GWAS doesn’t genotype all SNPs across a genome. It genotypes SNPs that identify
haplotypes. These SNPs are called tagging SNPs.
- Each Haplotype is identified by a unique sequence of alleles and tagging SNPs identify the unique sequences.
- The various SNPs present reside on haplotype and haplotype blocks. This means that the haplotype carries
alleles that influence the risk of disease.
- Scientists use GWAS to genotype several hundred thousand tagging SNPs to identify haplotypes as opposed
to 4 million SNPs.
- Scientist search for significant differences in allele frequency between cases and controls in matched
population. This allows for identification that carry risk alleles for a disease or genotype.
- Linkage disequilibrium identifies combination of alleles (haplotype) that occur in a population.

Genotyping uses chips this is a solid support that carries short oligonucleotides that correspond to tagging SNPs
across the human genome.

1. The oligonucleotide corresponds to DNA that flanks each tagging SNP;

2. Fragmented genomic DNA is hybridized to the bead chip
3. single nucleotides are added and incorporated into the hybridized genomic DNA
4. the incorporated nucleotides correspond to specific alleles and carry fluorescent tags of different colours
5. Laser light excites the tags and the light produced is read by a computer that converts this signal to a
genotype

Type 1 Diabetes Type 2 Diabetes

- An autoimmune disease (T cell destruction of
insulin producing cells in the pancreas) with
environmental input.
- Genetic association is in the MHC locus – is a
protein complex protein complex and if it is not
coded by a section of = mutation and lead to
autoimmunity.
- GWAS identified additional genes that
contribute to type 1 diabetes.
- Inheritance of risk alleles in the MHC locus as
well as PTPN22 a protein tryosin phosphate
that regulates immune signal.
- A single change in coding region of PTPN22
argine to tryptophan amino acid subsition =
change in signalling from a T cell receptor
dampening the T cell response.
- Also, CTSH associated with increased severity of
illness and poor response to treatment. = EQTL
expression trait locus (alternative SNPs lead to
change in gene expression). Alternative alleles
of rs3825932 change expression of CTSH =
change in insulin production and remission of
Type 1 diabetes.
- a progressive condition in which the body
becomes resistant to the normal effects of insulin
and/or gradually loses the capacity to produce
enough insulin in the pancrease.
- GWAS have identified many loci for example the
infinitesimal model that contains risk alleles that
contribute to Type 2 diabetes. A number of these
associations are in enhancers that control insulin
production in the pancreas. There are also
association with related phenotypes e.g. FTO
gene and body mass index/obesity.
- Type 2 is a enhancer in FTO which regulates Irx3
which is expressed in pancreatic islets and control
the number of insulin producing B-cells.

GWAS provide the opportunity to understand in more detail the molecular origins of common diseases, the
relationships between different diseases, and possibly new therapeutic interventions.
 Describe the methodology of Next Generation DNA sequencing and apply it to a disease

- Next Generation DNA sequencing like Sanger sequencing it combines the techniques used in sanger
sequencing to process millions of reactions in parallel m resulting in very high speeds and at a reduced cost.
- The genome sequencing project that initially took many years can now completed within hours using Next
Generation DNA sequencing.
- Next-generation DNA sequencing can be classified as whole-genome sequencing and exomic sequencing.
- Whole- genome sequencing is particularly valuable for identifying and mapping genetic variants across the
human genome in populations, as well as identifying new mutations in very rare (orphan) diseases.
- Exomic sequencing sequences only the exons of genes and is very valuable for cancer genetics. Exomic
sequencing is able to generate coverage of 500-1000X that means it is able to detect new mutations with
very high accuracy.

Next Generation DNA sequencing involves:

1. Genomic DNA Fragmented genomic DNA is generated and short oligonucleotide called adaptors are ligated
to the DNA
2. The adapters are homologous to oligonucleotides on the amplification.
3. This creates spots of DNA across the flow cell that collectively represents the human genome (or exons if
exomic sequencing is done).
4. The DNA is then sequenced one base at a time;
5. short sequence fragments are aligned using sophisticated bioinformatics software.
- Exomic sequencing is the method of choice for analysing cancer genomes as 85% of all mutations occur in
exons of cancer genomes.

Cancer
- Disease that is characterized by the acquisition of mutations – as a cancer progresses from the day
of diagnosis to therapy to remission to relapse the genetic profile of the cancer changes.
- mutation in cancer are most often found in the coding regions of genes whereas GWAS looks for
tagging SNPs for common diseases.
- mutations that cause cancer can be assigned to different categories that are defined as hallmarks
of cancer; apoptosis, proliferation, DNA repair are examples of these hallmarks.
- A more recent addition is the hallmark of epigenetic regulation, as typified by mutations in
SMARCA4.
- SMARCA4 is part of a multi-protein chromatin remodelling complex that opens DNA, allowing
transcription to take place.
- Mutations in SMARCA4 leads to repression of gene transcription programs required for the
differentiation of cells, including self-renewing stem cells. There is increasing evidence that cancer
arises from self-renewing highly proliferative cells called cancer stem cells.
- The loss of SMARCA4 is one part of the change in epigenetic programming that is thought to
promote the formation of cancer stem cells.
- Mutations in SMARCA4 leads to repression of gene transcription programs required for the
differentiation of cells, including self-renewing stem cells. There is increasing evidence that cancer
arises from self-renewing highly proliferative cells called cancer stem cells.
- The loss of SMARCA4 is one part of the change in epigenetic programming that is thought to
promote the formation of cancer stem cells.
 Understand epigenetics- mechanisms and in the context of human health and disease

- The modification of gene expression is a natural process that occurs within the cell to adjust the type and
number of proteins expressed in a cell. The two main types of such modifications are DNA methylation and
histone methylation.
- Epigenetics describes modification of DNA and histones and effect this has on gene transcription. It controls
the way genes in genomes are turned on and off and their gene products controls the way cells differentiate
and behave.
- Epigenetics modification can be highly stable, and it is critical for normal development.
- Epigenetics Is transient and regulate gene expression in response to specific signals (e.g. hormonal) and
epigenetics programming can be modified by environmental factors.
- SMARCA4 is chromatin remodelling enzyme that is part of the SWI/SNF complex that promotes
transcription.

There are many examples of epigenetic regulation of gene expression this includes:

X- inactive
- a form of dosage compensation where one X
chromosome from a female is condensed (by
random) to form the structure known as a Barr
Bod.
- The purpose of this process is X-inactivation is
stably maintained through successive mitotic
cell divisions.
Rett syndrome
- an X-linked neurodevelopmental disorder that
occurs almost exclusively in females (X-linked
lethal in males).
- It is caused by mutations in the DNA methyl-
binding protein MeCP2. This means that the
molecular basis of Rett syndrome is linked to
changes in chromatin architecture.
Imprinting
- a type of inheritance whereby the methylation
and histone modification patterns are
imprinted into germ line (i.e. sex cells), by the
parents.
- This results in an unaltered genetic sequence
that can be passed on by further cell divisions.
The Fragile X CGG
- CGG triplet repeat expansion in the 5’ CpG
island of the gene FMR1. This results in
increased DNA methylation and FMR1 gene
repression.
- FMR1 encodes a protein that is essential for
normal cognitive development and female
reproductive

Beckwith-Widemann syndrome
- Is caused by a loss of imprinting and regulation
at specific sites on chromosomes. This gives an
insight into the molecular mechanisms of
epigenetics.
 The methodology of iPSC and its application in understanding genetics

Induced pluripotent stems cells (iPSC)

- reprogramming somatic cells to a pluripotent state via overexpression of a key set of pluripotency-related
transcription factors.
- The differentiation of stem cells requires extensive chromatin remodelling and the induction of
heterochromatin through recruitment of proteins such as HP1 to methylated histones.
- Transcription factors for reprogramming somatic cells in pluripotent stem cells include:
o OCT4 required for the maintenance of embryonic stem cells pluripotency,
o SOX2 required to maintain self-renewal of undifferentiated embryonic stem cells,
o KLF4 involved in the proliferation, differentiation and survival
o MYC (cMYC)-TF that control hundreds of target genes, many of which are also oncogenes or tumour
suppressors.

The reprogramming of somatic cell into a pluripotent stem cells involves:

1. The expression of transcription factors OCT-4, Soc-2, KLF4, Myc ; this can be done by viral infection , or by
using plasmid expression vectors to trigger reprogramming of the somatic cell.
2. Reprogramming is defined as a reversal of the epigenetic programming of the endogenous genes that are
only turned on in the pluripotent stem cells.
3. Once the epigenetic reprogramming these genes has taken place, the cell can be now considered
reprogrammed.
4. In a reprogrammed cell the endogenous stem cell specific genes are once again turned on. The exogenous
transcription factors are no longer required.
5. Conversely, many genes that were turned on in the differentiated somatic cell must now be turned off.
- Results in a change of differentiated adult somatic cell into an undifferentiated pluripotent stem cell which
generates specific cell lines that can be tested for new medication and the study of the function of specific
mutations.

Alzheimer’s disease
- The generation of amyloid-B (AB) peptides, from amyloid precursor protein (APP), and their
subsequent accumulation, aggregation, and deposition in the brain as senile plaques are defining
events in the pathogenesis of Alzheimer’s disease.
- Late onset Alzheimer’s disease is a multi-genic disease, with the major association found in the
ApoE4 allele. Early onset Alzheimer’s disease can be caused by mutations in the gene PSEN1 and
PSEN2, or APP.
 The clinical application of GWAS and Next Generation DNA sequencing

Genome wide associate studies (GWAS)

- identify genetic relationships between different inflammatory diseases and this knowledge can be put to
use to investigate new therapies that treat not just one disease but several diseases.
- The power of GWAS is understanding the relationships between common diseases in populations and
looking for new ways of treating these diseases.

Inflammatory bowel disease

- Crohn’s disease a chronic inflammatory bowel disease that affects the lining of the digestive tract.
- Ulcerative colitis a long-term condition that results in inflammation and ulcers of the colon and
rectum.
- GWAS has shown that a number of inflammatory diseases can co-occur in a single person. This is
called co-morbidity.

Next generation DNA sequencing

- Has shown that lung cancer can be classified according to the expression and mutation of epigenetic
remodelling complexes.

Lung cancer
- The genetic profiling of lung can be used to predict response to therapy (e.g. TPMT), as well as
identifying new drug targets.
- TPMT is thiopurine S-methyltransferase decrease in thiopurine can cause myelosuppression a
condition in which bone marrow activity is decreased = fewer red blood cells, white cells and
platelets.
- Tumours high in EZH2, expression but carrying inactivating mutations in SMARCA4 respond well to
etoposide, an inhibitor of topoisomerase II.
- Topoisomerase 2A is essential for resolving concatenated sister chromatids during cell division which
is dependent on the ATPase activity of SMARCA4.
- E2H2 inhibits gene expression genes responsible for suppressing tumour development
- SMARCA4 is part of the BAF (SWI/SNF) remodelling chromatin complex that promotes transcription.
SWI/SNF also promotes binding topoisomerase 2A binding to chromatin.
- Tumours high in EXZH2 expression but not carrying SMARCA4 mutations respond poorly to
etoposide. Etoposide inhibits topoisomerase 2A inhibiting separation of chromatids and cell division.