IMED2004 1- Development and genetics , IMED2004 11- Complex and chronic diseases , IMED2004 10- Finding mendelian disease genes , IMED2004 9- Linkage and recombination II , IMED2004 8- Linkage and recombination I , IMED2004 6 + 7- Genetic variation ,…

Name the three germ layers

Meso, endo and ectoderm

What are the three outcomes of cell lineage decisions

- Establish the axes (anterior/posterior, dorsal/ventral)
-Subdivide segments
-Produce tissues and organs

Give three examples of signals that regulate gene expression

Cell-cell contact, hormones, morphogens

What are morphogens

Molecules that specify cellular identify, many are transcription factors.

Define transcription factor

A protein which binds to a regulatory element of a gene and either acitvates or represses.

What is a monogenic disease + give example

caused by a single gene, not influenced by environment, rare. E.g. CF, sickle cell

What is a polygenic disease + give example

Caused by many genes, influenced by environment, common. E.g. hypertension

Define complex disease

Disorders that result from the contributions of multiple genomic variants and genes in conjunction with significant influences of the physical and social environment

Define chronic disease

Diseases with kong-lasting conditions and persistent effect.

How many Australians have a chronic diseae

1 in 5

What are the most prevalent chronic conditions in aus

Mental, back problems, arthritis, asthma, diabetes

How is a person who inherits a 'risk allele' for a complex diseased described

As having a genetic predisposition, they may be more likely to develop the disease but does not definitively mean they will develop it

Are Mendelian traits quantitative or qualitative

Qualitative- one phenotype or the other

Are complex traits quantitative or qualitative

Quantitative- continuous characteristics, polygenic/multifactorial inheritance

What is the relationship between number of genes involved in a trait and the contribution of each to the trait

Large amount= each gene contributes smaller effects

What is the formula for H (the proportion of phenotypic variation that is due to genetic variation)

H2 = Vg/ Vp

What does H2= 0 indicate

Phenotype variance is due to environment only

What does H2=1 indicate

Means phenotype variance is due to genotypes only (mendelian)

What does GWAS stand for

Genome wide association studies

What are the two mai aims of GWAS

1. Identify markers that can be used to predict individual disease risk.
2. Identify the molecular pathways underlying disease susceptibility and providing potential targets for therapy

GWAS cannot identify disease-causing alleles, what can it identify?

variants that are in linkage disequilibrium with the causative allele.

What Is linkage disequilibrium

Genetic association, due to shared segments of DNA from a distant common ancestor. The more distant, the smaller each shared segment but the number of people who share the segment will be greater

How can linkage disequilibrium be used to identify the chromosomal regions harbourign variants

If most affected individuals in a pop share the same variant allele, then the LD can be used to locate the chr regions

What is a haplotype

Combination of markers that are found to be commonly inherited together as a single block

What has GWAS been successful at

Identifying sites of common genetic variation associated with complex diseases. Contributed to our understanding of biology underlying some of these diseases.

What is missing heritability

Available GWAS data explains only small proportion of the overall genetic contribution to complex diseases.

What contributes to missing heritability

GWAS misses common disease variants if they have a very weak effect (due to cumulation) and it is restricted to identifying associations with common variants. It doesn't take into account gene-gene and gene-environment interactions

What is the collective incidence of Mendelian diseases world-wide

>1/3000

Why do we need to find disease genes

The genes underlying 34% of known mendelian phenotypes are still unknown and many more have yet to be recognised.

Why is a molecular diagnosis important for families

-Closure, family planning, genetic counselling, prognosis, life-style interventions, patient and family support, access to targeted treatment and clinical trials, ability to design treatments

Define mutation

An event which changes one allele to another.

Define mutant allele

A mutation that is inherited by somatic cells

Define do novo mutation

A mutation passes on through germ-line

Define polymorphism

A DNA sequence variation in which both alleles are common in the population, neither is considered normal. Population frequency of 1% or above.

What is the most in 'depth' sequencing method

SNP array and targeted gene panel

What sequence technique covers the highest % of genome

Whole genome sequencing

What is a targeted panel

A select group of genes that are known to have an association with a specific disease type/variant in these genes have previously been shown to cause the phenotype of interest. E.g. muscle disease

Outline the method of targeted panel sequencing

Genomic DNA fragmented, hybridised with RNA library baits that anneal to genes, baits contain biotin label, target DNA purified by addition of streptavidin coated magnetic beads that collected target DNA, purified target DNA can then be amplified and sequenced.

Benefits of target panel sequencing

Can diagnose a single individual without family, very successful, fast, cheap, fewer targets means coverage is better

What are some limitations of target panel sequencing

Gaps in coverage, sequencing errors, if variant/gene has not previously been reported then it is not on the panel.

How does WES work

Hybridisation selection probes capture the coding portion of the genome for high-throughput sequencing. Useful when you have the whole family, need to filter variants against unaffected members.

Benefit of WES

Can pick up mutations in gene not already known to cause disease and reduced cost compared to WGS

Negative of WES

Cannot be used for a single individual

Benefits of WGS

picks up splice site variants and can be used in conjunction with RNA seq

Limitations of WGS

Determining effect of non-coding sequence variants, increased cost

What method can be used is after sequencing and variant filtering there is still no clear variant that appears to be causal for disease

Linkage mapping if a family pedigree is available

Give an example of Target panel sequencing failure

Doctor ordered targeted panel sequence for muscle panel on a child with delayed motor milestones. It came back negative and a WES found the variant on Chr14 after sanegr sequencing, It was a known diseae causing mutation associated with spinal muscular atrophy which is a 'nerve' condition.

List some challenges for disease gene discovery

Incomplete penetrance, genetic phenotypic and allelic heterogeneity, need for accurate clinical info, lack of knowledge about certain proteins, dominant genes harder to find, CNVs.

What cant sequencing capture

Copy number variants, splicing alterations in introns, regions of low coverage (telomere, centromere)

Why are 40-60% of cases unsolved

Synonymous changes, variants of unkown significance, novel non-coding genes, regulatory non-coding variants, large repetitive genes

What are the classes of variants

1- benign, 2- likely benign, 3- unknown significance, 4- likely pathogenic, 5- pathogenic

What is the relationship between gene distance and probability of crossing over

Genes closer together on chromosomes are more tightly linked and are going to be inherited together, less likely for recombination

What is RF used to measure

Recombination frequency is used to measure distance between two loci and a measure of degree of linkage

What does a RF of 50% tell us

The genes are unlinked

What does an RF of 100% tell us

The genes are completely linked

What are some limitations of RF

-No way to distinguish between genes on different chr and those far apart on same chr.
-Underestimated true physical distance because it does not reveal double crossovers

How does configuration of alleles affect linkage

If two loci are linked, the configuration of the alleles relative to each other will determine which combination of alleles segregate together in gametes

What is linkage mapping

The analysis of genetic linkage within pedigrees and the application of this information to identify disease genes

Give an example of linkage mapping being used to identify a disease location

Nail-patella syndrome does not develop in children with blood type A, therefor causative mutation for NPS is likely to be inclose proximity to the gene for blood type on Chr 9

List some advantages of DNA markers

-Phenotypes can be hard to distinguish.
-Allows for distinction between homo and hetero
-Can be typed rapidly by PCR, microarray or sequencing.

what are genetic markers

Polymorphic DNA sequences spread throughout the genome, that are usually highly variable between individuals and can be easily typed to track inheritance through a pedigree.

What is multipoint linkage mapping

Typing thousands of markers at once, allows for refinement of the genomic region that is linked to the phenotype and more precise gating of the regions within which the disease gene is likely to be located

What are some limitations to multipoint linkage mapping

-Only a few meiosis/recombinations occurring because it is used to analyse families of a few gens.
-Resolution is poor because of large chr segments being inherited
-Need lots of affected family members to say with certainty that a marker is linked to the disease locus.

What is a Lod score (Z)

A statistic that describes the strength of evidence for linkage, given the family data available. Estimates whether the likelihood that two loci are linked is greater than the likelihood that the two are unlinked.

What is the minimum cut-off of Lod score to say with statistical certainty that there is evidence for linkage

Lod of 3 That means it is 1000 times more likely that they are linked than unlinked.

List some factors that confound linkage mapping

Genetic heterogeneity, incomplete penetrance, need for detailed and accurate clinical info about the phenotype

What is the phenotypic ratio when single genes are crossed by two heterozygous parents at one locus

3:1

What is the phenotypic ratio when two genes are crossed that separate independently

9:3:3:1

List the steps in meiosis I

Interphase -> prophase -> metaphase -> anaphase -> telophase

In what phase of meiosis does independent assortment occur

Anaphase I

The further apart on a chromosome genes are the more likely they are to undergo...

Homologous recombination (crossing over)

What is inter-chromsomal recombination

Segregation of homologous chromosomes into different daughter cells during cell division in anaphase/telophase I

What is intra-chromosomal recombination

Crossing over between homologous chromosome pairs during prophase I

Define tetrad

Structure containing 2 homologous chromosomes

Define chiasma

Non-sister chromatids cross-over at these sites

Give an example of molecular crossing-over

B cell class switching to produce different immunoglobulins

Outline the holliday model of crossing over

1. Homologous Chr aline and form tetrads.
2. Endonuclease cleaves one DNA strand from each non-sister
3. Broken strand invades other chromatid and finds areas of sequence complementary with base pairs.
4. DNA is covalently joined by dna ligase forming holliday junction
5. Holliday junction moves along Che.
6. Holliday junction resolved to produce recombinant chromatid

What can go wrong in homologous recombination

-Homologous chromosomes can misalign or non-homologous chr can pair because of repetitive sequences

Give an example of a disease caused by misalignment during crossing over

Cri du chat syndrome caused by deletions on short arm of chr 5

What is balanced (reciprocal) translocation

When non-homologous chromatids line up and cross over but it causes a balanced translocation. The individual is usually phenotypically normal

What is the risk of being a carrier of a balanced translocation

Have a risk of creating gametes with unbalanced translocations (monosomic in one regions and trisomic in another). Can lead to infertility, miscarriage and other abnormalities

Give an example of a somatic translocation

CML caused by t(9;22)

What did the human genome project generate + when

The first complete sequence of the human genome in 2002

What percentage of the human genome is protein coding regions

1.5%

What are the forms of genetic changes that do not affect DNA content

Translocation and inversions or single nt change

What are the forms of genetic changes that do affect DNA content

Change in copy number abnormal chr segregation, indels

Define DNA variants

Mutations that result as alternative forms of DNA

Define allele

Alternative forms of gene sequence found at the same location on a chromosome

What is the value for a genetic variant to be considered polymorphic

above 0.01 frequency in a population

What is a SNIP

A single nucleotide polymorphism

What causes the human patterns of single nucleotide polymorphisms

Regional intolerance to variation, mt DNA being higher than nuclear, excess of C-T substitutions and evolutionary ancestry.

Define copy number variation

Change in copy number of sequences that reuslts in larger indels

What size events does indels describe

Small events 1-100nt in length

Satellite polymorphism- length + location

20-100kb- centromere and heterochromatic regions

Minisatellite polymorphism- length + location

100bp-20kb- telomeres and subtelomeric regions

Microsatellite polymorphism- length + location

Fewer than 100bp- euchromatin

Short tandem repeats- length

1-6bp

If replication slippage occurs with the loop on the new strand what type of variation does it cause

insertion

If replication slippage occurs with the loop on the template strand what type of variation does it cause

Deletion

What is unequal crossing over

Gene duplication or deletion events that deletes a sequence in one strand and replaces it with a duplication from its sister chromatid in mitosis or from its homologous chromosome during meiosis.