Mendelism and Sequencing - Dr. Markie Flashcards
Mitochondrial Inheritance
- Inherited from a single gene
- Matrilineal - cytoplasmic inheritance
- Both males and females are affected
- Variable expressivity and incomplete penetrance (a consequence of heteroplasmy)
- Predominant phenotypes are muscular, retinal, and hearing
Homozygosity
two identical alleles at a (disomic) genetic locus
Heterozygosity
two distinguishable alleles at a (disomic) genetic locus
Hemizygosity
the presence of only one allele
ex. monosomy – loci on the X and Y chromosomes in males are monosomic
a large or small deletion can cause hemizygosity - may be unaware b/c its benign
Compound heterozygosity
two different recessive alleles for the same gene. Inheritance of both cause genetic disease state but they are heterozygous b/c alleles are different.
Codominance
both traits can be present in the same individual when heterozygous
Ex. ABO blood system – AB phenotype
Red Flower + White flower == Red and white flower)
Partial Dominance
The heterozygote demonstrates a phenotype intermediate between the two homozygotes
examples
* Achondroplasia - classical “dominance” inheritance of short stature. Very rare homozygotes – more severe skeletal abnormalities with early death due to respiratory difficulties
* Red flower + white flower = pink
Mendel’s First Law –
Law of Segregation
only one of the two gene copies present in an organism is distributed to each gamete (egg or sperm cell) that it makes, and the allocation of the gene copies is random
De novo mutation
New mutation
* A child suddenly gets a dominant disorder that parents don’t have
* Need to consider recurrence risk but different types carry different risk
Types of de novo mutations:
* Gametic Mutation
* Somatic Mutation
* Gonadal Mutation
Gametic Mutation
- Occurs during meiosis in formation of single sperm or egg (Parents)
- Recurrance risk is extremely low for next child
- But - the affected person’s child will have a 1 in 2 risk
Somatic Mosaic
- Occurs post-conception in developing embryo (mosaic b/c not all cells will inherit it)
- Recurrence risk is extremely low for original couple
- Risk to affected person’s children is unpredictable as it depends on is mutation occured early enough to affect germ cells
- Some cells will have the gene and others won’t – there is a threshold for how many cells have the disorder = phenotype
Gonadal Mosaic
- Parent sustained a mutation during development that contributed to germ cells
- Essentally the child of somatic or gametic mutation becomes parent
- This parent may not actually show the disorder b/c low ratio of mutated cells cannot reach threshold
- Recurrance risk depends on the proportion of cells in the gonad affected
- The risk to a second child is unpredictable but risk to grandchild is predictable
Pleiotropism
- Multiple effects of a single gene - seemingly unconnected
- Underlines our limited understanding of molecular biology
- Many genes/proteins are “multi-functional” – more than one pathway, multiple functional domains, or complete function yet to be described
Variable Expression
- Describes changes in the severity of a disorder
- Variation in the severity/age of onset
- Almost all inherited disorders show this to some extent
- Suggests the influence of other factors (genetic, environmental or random) on the development of the disorder - but not well understood
Differences within families - may have different underlying factors
Differences between families - suggests it is allelic
Anticipation
Special kind of variable expression
* Some disorders manifest more severely with each generation
* For many years it was rationalized as ascertainment bias – severe cases are presented to physicians and parents are found to have milder cases
* Still difficult to seperate ascertainment bias from anticipation in many disorders
Mechanism was identified in triplet repeat expansion disorders
* Effects protein or expression of gene
* Severity/age of onset is determined by length of repeat
* Repeat length unstable, tends to expand
Penetrance
- Probability that a person with appropriate genotype will manifest the phenotype
- Single genes predispose to some disorders but may not be the only factors (ex. modifying genes, environment, chance)
Penetrance = 1 (everyone who inherits gene gets disorder = complete penetrance)
Penetrance = 0.5 (50% of those who inherit the gene get the disorder = incomplete penetrance)
Incomplete Penetrance
Not all those who inherit a gene inherit the disorder
* Can complicate determination of mode of inheritance and diagnosis
* Most evident in dominant disorders, applicable to recessive disorders but harder to identify
* May be related to age or sex
Ex. inherited breast cancer (men have less breast tissue – less chance of inheriting
This can be relative or absolute (mainly affect women vs only affects women)
BUT NOT X OR Y LINKED – Only that your underlying biology effects risk — Disorders affecting primary or secondary sex characteristics
Imprinting
Parent of Origin Effect
– special kind of incomplete penetrance
* a minority of genes are silenced during gametogenesis in one parent or another
* For these genes we are functionally hemizygous b/c we only express one allele
* maternal and paternal imprinting
* If the allele that expressed has a mutation that leads to a non functioning allele and can often be genetically lethal
* Imprinting may be relaxed in some tissues
* Imprinted genes are not common
* Often arrise from de novo mutation b/c of genetic lethality
Maternal Imprinting
Express paternal allele
Maternal allele imprinted
* if inherit mutated allele from father you get the disorder
Paternal Imprinting
Express maternal allele
Paternal allele imprinted
* if you inherit mutated allele from mother you express the disorder
Allelic Heterogeneity
One gene – many diseases
More than one variant can produce the same or similar “disease” phenotype
* when different mutations in a single gene cause the same disease or condition
* May produce variable expressivity (between families)
In extreme cases it may produce seemingly unrelated phenotypes – clinical heterogeneity
Locus Heterogeneity
one disease, many genes
More than one gene can produce the “disease” phenotype
* Mutiple steps of biochemical pathway, multiple genes contributing to development
* Not polygeneic, rather a collection of single gene disorders that can not be easily clinically differentiated
Genocopies
When other loci can give rise to the same phenotype
Phenocopies
Aquired disorders that can give rise to the same phenotype as inherited disorders
Classic Sanger Sequencing
Sanger di-deoxy chain termination sequencing
* Clone a singal fragment into cloning vector, purify it, add primer that allows for polymerase to add bases
* Add a small amount of dideoxynucleotides will stop the reaction at random points preventing elongation
* incorporate radioactivity/flourescence and separate reactivity on a gel to see where a single nucleotide is in a sequence
Steps of Human Genome Seqeuncing
Obtaining Reads
* Library construction
* Target capture
* Sequence run
Processing Reads
* Alignment to reference
* Variant Calling
Finding Disease Variants
* Filtering
* Evaluation
Genome Sequencing
What to filter
- Inheritance models (de novo, autosomal recessive, autosomal dominant, X-linked, mitochondrial, genes in common, parental non-contribution)
- Quality (truth sensitivity, genotype liklihoods, depth of coverage, allele bias)
- Location (Condidate gene lists, genomic regions)
- Platform artifacts
- Population frequency
- Effect prediction (Gene impact, protein prediction tools)
How to evaluate disease causing candidate variant
Gene
* Is the gene a disease causing gene (clinically), is there reccurance (research)
* Is it plausably a disease causing gene through pathway knowledge)
Variant
* Is the variant previously described to cause a disease, does it have the same amino acid substitution as another variant
Functional information
* experimental (model systems, biochemical, cell culture, whole animal)
Strong functional predictions based on conservation scores
Problem with short read sequencing
- repetitive sequences can map to multiple locations
- many reference sequences miss out centromeres or regions of long repeats (missing areas that may have functioning genes)
- Gene families and psuedogenes – the same gene is repeated multiple times but have different functions and regions that structurally appear like genes but cant code for protein
- Centromeres and ribosomal RNA gene arrays on acrocentric short arms are invisible
- Cant really handle structural variants
Structural variance in short reads
Discordant pairs
- map to different chromosomes or too far apart
Structural variance in short reads
Split reads
Part of the read aligns to one part and the other part to a different region
* Strongly suggests there is some issue in that region of the individual’s genome
Structural variance in short reads
Allelic Fraction
Is it arond 50% or does it vary for any single nucleotide variance in the region
(the proportion of sequencing reads that contain a mutation.)
Structural variance in short reads
Depth of Coverage
Could be heterogenous deletion – use depth of coverage to see
* can also provide confidence to variant calls
Advantages of Long Read Sequencing
- Resolving misalignment through repeats, gene clusters
- Disambiguating pseudogene misreads
- Improved structural variant calling
- Phasing over longer distances
- Potential for assembly rather than alignment
Difference between Alignment and Assembly
Alignment
* Take reads and find the best alignment against the standard reference sequence
Assembly
* Building an individual’s genome based on looking at overlap through long reads
* Gives a better view at individual genome which in some ways will be different from the reference sequence
What affects structural compartmentalisation to form chromosomes
- Proximity of teh chromosomal territories changes the ability to interact and alter function
- Topologically associated domains (TAD) - form compartments and sit within the nucleus (not just DNA soup but residences for DNA)
- Chomosomal domains in functioning interphase nucleus
- Spatial topology can potentially change over time
Molecular techniques to detect submicroscopic chromosomal anomalies
- FISH - flouresence in situ hybridization
- Locus specific FISH
- Interphase FISH
- Chromosomal microarray
- Comparative Genomic Hybridization
FISH
Flourescence in situ hybridization (locus-specific probes)
Used for detection of submicroscopic chromosomal anomalies
CAN ANSWER - “Is something present”, “where is it located” and “are there duplications”
- Hybridization of complementary, single stranded nucleic acid prob to a fixed target
- Flourescently labelled probs find location in the DNA and fluoresces
- Can only target one loci at a given time
Widely used in diagnosis of:
* Microdeletions: contiguous gene syndromes
* Specific chromosomal translocations
* “Counting” chromosomal or locus copy number (e.g in cancer states)
Interphase FISH
Can detect
* Chromosomal mosaicism
* Intercellular genomic variations (aneuploidy or polyploidy)
* Structural chromosome aberrations
Chromosomal Microarray
- High resolution technique
- screens the genome for small genetic changes, such as missing or extra pieces of chromosomes.
- diagnose chromosomal imbalances
Can tell you - deletion in sample at that locus, duplication in sample at that locus, or balanced at that locus
Comparative Genomic Hybridization
- global overview of chromosomal gains and losses throughout the whole genome
- method for analysing copy number variations (CNVs) relative to ploidy
Detecting deletions or duplications below the resolution of the karyotype
- PCR (ver small deletions – up to 3 kb)
- FISH (probes are large – deletions of >50 kb)
- To kayrotype - deletions must be >5 -10 mb to be detectable
Methods to diagnostically assess differentially imprinted alleles at a locus
- Need parental information
- need to measure methylation
- need to measure expression
Measuring methylation and demonstrate differing methylation patterns across alleles
* methylation specific PCR, Bisulphite conversion, nanopore sequencing
Demonstrating that its parental specific
* Look at polymorphisms to identify what allele comes from what parent
Measuring expression
Demonstrating that coding polymorphisms are reliably present from one allele from another
* transcription is only coming from one allele
Detecting Structural variants in cancer with whole genome sequencing
- Split reads indicate fragments have origins at different chromosomal sites
– TRANSLOCATION
Techniques that can assay for the presence or absence of a deletion of any size
- qPCR - qualitative PCR
- MLPA - multiplex ligation dependent probe amplification
- Whole genome sequencing using short read sequencing (not perfect)
- Long read sequencing
qPCR
Qualitative PCR
* determines dosage of template at any one locus
Detecting heterozygous deletions
- PCR approaches won’t work
Quantitative approach needed - qPCR
- MLPA
- Quantitative sequencing
