05 Disease Gene Identification Strategies

Question 1

All the strategies for disease gene identification rely on what?

Answer 1

The mapped human genome, and detailed clinical phenotyping

Question 2

Why do we want to identify disease genes?

Answer 2

To improve diagnosis, prognosis, treatment and management of disease.W

Question 3

What’s an example of how disease gene identification is proving beneficial?

Answer 3

Now using gene therapy on retinal genes to stop disease process to prevent the loss of vision

Question 4

What used to be the rate limiting step in disease gene identification? and what is it now?

Answer 4

Used to be data generation, now its in data interpretation

Question 5

What are the 3 big themes in disease gene identification?

Answer 5

Gene position - mapping and linkage to identify candidate regions
Segregation - How a gene segregates within and between families
Function - Of the gene and the variant
(Whilst also remembering inheritance patterns, disease prevalence, variant penetrance, and population frequencies)

Question 6

What shifted the rate limiting step from data generation to data interpretation?

Answer 6

NGS allowing massively parallel sequencing

Question 7

Who makes up the 100,000 genome project cohort?

Answer 7

cancer and rare disease patients

Question 8

Fill in the gaps:
We have __________ variants per genome.
20-30,000 of these are in ___________
_______ are de novo variants, of which 0-4 are in _________
Trios allow you to_________

Answer 8

We have 3-4 million variants per genome.
20-30,000 of these are in coding regions.
50-100 are de novo variants, of which 0-4 are in coding regions
Trios allow you to prioritise what variants to look at.

Question 9

Technique 1: Chromosome Rearrangement detection. This is like what, but with higher resolution?

Answer 9

Karyotyping and FISH

Question 10

What is the cause of WAGR syndrome?

Answer 10

11p deletion

Question 11

What are the symptoms of WAGR syndrome?

Answer 11

Renal cancers and aniridia (no iris). But a wide clinical spectrum.

Question 12

What homeobox transcription factor on 11p13 is responsible for causing aniridia in WAGR syndrom?

Answer 12

Pax6

Question 13

What causes predisposition to renal tumours in WAGR syndrome?

Answer 13

WT1 deletion from 11p13

Question 14

Do 11p deletions always cause renal cancers and aniridia?

Answer 14

No, need to see the borders

Question 15

What should you do if de novo, sporadic aniridia occurs in someone?

Answer 15

Look at their chr11 to see if WT1 has been affected, as then renal tumours would be likely.

Question 16

Why is DNA microarray / array CGH better than karyotyping and FISH?

Answer 16

You can look at smaller CNVs with array CGH.

Question 17

What is the ‘critical region’

Answer 17

Looking at individuals with the same phenotype, then narrowing down the region that they all have affected by a CNV to identify and follow up on whats in their smaller overlap.

Question 18

Technique 2 - Linkage Maps. Finish this: The closer the genes, the….

Answer 18

Lower the change of a recombination event between the two loci.

Question 19

Finish this: Alleles close together do not…….

Answer 19

Obey Mendel’s law of independent assortment, and instead show genetic linkage.

Question 20

What do we need to help us measure linkage of loci?

Answer 20

We need actual genetic markers that differ between people with a known position on specific chromosomes. This can be dinucleotide repeats, or CNVs that are polymorphic etc.

Question 21

How do you detect polymorphisms of dinucleotide repeats in linkage studies?

Answer 21

PCR product length

Question 22

What follow up studies are there when you determine through linkage studies, regions that segregate with the disease phenotype?

Answer 22

You need to target specific genes for analysis

Question 23

Name two genes that were found using linkage maps

Answer 23

CFTR gene and BRCA1

Question 24

What genes have been found through linkage using NGS data?

Answer 24

DHDDS in retinitis pigmentosa, POP1 in skeletal dysplasia and PIGV in hyperphosphatasia mental retardation syndrome.

Question 25

A special case of linkage mapping is autozygosity mapping, what is this? (Autozygosity mapping is a subset of homozygosity mapping)

Answer 25

Its used to identify recessive causes of disease that are common in consanguineous families. looking for haplotypes on both alleles that segregate with the disease.

Question 26

3M syndrome is defined by short stature, facial abnormalities and skeletal abnormalities. Microarrays were used to identify autozygous reiongs within and between families. What was then used to discover the exact molecular change behind it? And what was that change?

Answer 26

WES was used to identify the variant in CCDC8

Question 27

Where techniques are there for identifying homozygous causes of disease?

Answer 27

As well as homozygosity mapping to identify the causes of recessive disorders, there is also looking at the molecular consequence of variants, and looking at the variant frequency in the population.

Question 28

Technique 3 - Homology and Function. What knowledge do you need before doing this?

Answer 28

Knowledge of biological pathways and networks, and evolutionary genetics.

Question 29

What data does the International Mice Phenotype Consortium have?

Answer 29

data from knockout mice and their phenotypes to help us make connections with human gene functions.

Question 30

The International Mice Phenotype Consortium can tell use about mice genes that model mendelian disease, and also, what?

Answer 30

Identify novel mendelian disease gene candidates to then look at the human homologues for.

Question 31

We know of several proteins required by cilia and molecule transport in photo receptors, why is this important when we see dysfunction in photo receptors?

Answer 31

It gives us a list of candidate genes to look into.

Question 32

What can we use to help look for genes near an area associated with linkage?

Answer 32

UCSC genome browser

Question 33

Tell me how linkage mapping and functional information helped to unravel the cause behind Fuch's endothelial corneal dystrophy. A disease why there is fluid build up in the eye until it swells, and light is reflected differently, leading to blurred vision.

Answer 33

Linkage techniques were used to survey genetic markers on chr1 to find a region that segregated in the family with the disease phenotype. Collagen VIII was found to be in that area and we already know from studying the endothelium that this is in high levels in that tissue. A missense variant was found there that affected how collagen formed a heterotrimer.

Question 34

Technique 4 - Identifying non-coding variants that cause disease. What do we need to accommodate WGS compared to WES?

Answer 34

Robust informatic strategies, knowledge, and it also might benefit from a multi-omics approach.

Question 35

What does CFTR c.3874-4522A>G do, and why was it pathogenic with a recessive variant on the other allele.

Answer 35

This introduces a cryptic splice site so a pseudo exon is included that is 125bp long. This causes a frameshift, leading to a premature stop codon, so loss of function takes place.

Question 36

What does DNAse hypersensitivity sequencing do?

Answer 36

It selectively sequences areas that are naturally a bit more open and so important for gene expression.

Question 37

PRDM13 mutations in the promoter cause what?

Answer 37

A macular dystrophy

Question 38

What effect does BRCA1 c.-107 A>T have on the allele and how was it proved?

Answer 38

Changes the regulatory region methylation, leading to a switching off of the allele and thus a LOF. It was proved because there was a 50/50 split between A and T in DNA sequencing, but only the A was present in the transcriptomic sequences.

Question 39

What forms the boundaries of TADs?

Answer 39

Cohesin and CTCF binding sites.

Question 40

What happens if TAD boundaries are disrupted?

Answer 40

Genes may be expressed in cells where they aren't meant to, or at the wrong time.