Cytogenetics 2

Whats G banding and what are its pros and cons?

• It’s the old school way of looking at chromosomes

• It’s good to js look over an entire genome but bad for small things cuz it has low resolution

What’s FISH and what’s its use?

• Fluorescence in situ hybridization

• Uses specific fluorescent probes to hybridize and visualize specific points in genome

• Useful for specific situations and hypotheses

• Has high resolution, wherever it binds to DNA will fluoresce

What’re microsyndromes seen by, and what’s the general idea behind them?

• Can be seen by FISH

• Specific features of syndrome may occur as single Mendelian disorders

• Involved multiple, unrelated loci physically continuous in critical region

• They are submicroscopic effects which suggests not all patients have visible deletions

Whats an example of micro syndromes?

• Microdeletion and microduplication

• Occurs due to unequal crossing over due to repeat regions

• Explain diagram

Explain what microdeletion 22q11.2 is and related disorders

• It’s the most common chromosomal disorder

• Related disorders: DiGeorge syndrome, autosomal dominant Opitz, Sedlackova syndrome, Sphrintzen syndrome, VCF

Explain the idea of VCF

What was DiGeorge syndrome used to to be thought to be caused by?

• Environmental factors like alcohol or retinoids acid exposure due to craniofacial defects As well as immune deficiency, brain and behavioural abnormalities

• With modern techniques known to be micro deletion syndrome

What’s the most amount of genes affected by 22q11.2 deletion syndrome?

• 60 genes affected

Whats the cause of 22q11.2 deletion syndrome and explain diagram that goes with it

• Caused by non-allelic homologous recombination (unequal crossing over) at a series of low copy repeats

• Depending on how far/ distant alleles that recombined are dictates how bad/ severe it is

What kind of inheritance is in 22q11.2 deletion syndrome?

• Autosomal dominant inheritance (Mostly on one copy, people aren’t really heterozygous for it)

• Incidence of 1/4000 births

• 10% inherited - 90% de novo

• No bias for sex of ethnicity

• 1/1000 fetuses carry this deletion, but most (3/4) terminate naturally

Explain the expressivity of 22q11.2 deletion syndrome

• Variable expressivity depending on the size of the deletions which causes variable severities. This is due to variations in gene expression levels (epigenetics) and modifier genes

• Has variable expressivity even among people with the same deletion

What genes are involved in the deleted region of 22q11.2 deletion syndrome?

Several of them are involved in facial development, covering gene dosage for a bunch of genes involved in facial development

• TBX1 - Involved in neural crest cell migrations and craniofacial development

  • TBX1 -/- knockout mice have similar facial defects to 22q11.2 DS

  • CRKL and ERK2 also cause facial defects in -/- knockout mice

Combined haploinsufficiency of multiple genes causes the defect

Explain contiguous gene syndrome

• When a micro deletion results in the loss of a cluster of genes on a chromosome. This results in heterozygosity for these loci

• But the problems start arising when theres heterozygosity for many loci which is like an autosomal monopsony

• Genes that function together often cluster together so CGS can affect multiple loci acting in one developmental pathway which is like combined haploinsuffciency (ex: facial development in 22q11.2)

Provide an example of FISH used to test a specific hypothesis

Whats an exception in FISH that looks at the entire genome?

• SKY (but is rlly intensive as it requires a lot of filters)

Whats the difference between FISH and Microarrays?

• FISH is for specific questions/ chromosomes and you usually have to know what you’re looking for cuz you need sequence specific probes (except for SKY which looks at entire genome)

• Microarrays let you take a general look at the genome without initial bias. Like G-banding but with very high resolution

They both use high resolution

What’re the 2 types of cytogenetic microarrays?

1. Copy number or comparative genomic hybridization array (aCGH)

2. SNP (single nucleotide polymorphism)

What does comparative genomic hybridization (CGH) do and explain how it works?

• Looks at copy number variation

• Has 2 samples (patient and control) so 2 copies of everything. Compares between the patient and control to see If patient has same amount of DNA as control

• If patient as a control have same amount of DNA = yellow

• If not will have some spots red and/or green depending on what has more

What are microarrays packaged into?

• Gene chips

Explain the 2 examples on the back

What does + and - mean on the log scale for CGH?

• +/ up = Extra copy/ duplication

• -/ down = Deletion

What’re Copy number variants or copy number change (CNC)?

• A DNA segment with a variable copy number or copy number change compared to a reference genome

• Although present in a normal person, may be associated with disease susceptibility (reduced penetrance and variable expression)

  • Ex: how we process drugs/ caffeine depending on copy # of enzymes

Can CGH microarrays be used to detect a reciprocal translocation or inversion and why?

• No cuz the copy number in a reciprocal translocation or inversion doesnt get affected/changed cuz theres no net gain or loss of copy number

What do SNPs do?

• Analyze single base pair variation between samples

• Can track allelic ratios through the whole genome

• Any SNP locus allele can be scored as either A or B

• They don’t need a control

How many genotype possibilities at each SNP locus if disomy?

• 3 (AA, AB, BB)

How many genotype possibilities at each SNP locus if monopsony?

• 2 (A or B)

How many genotype possibilities at each SNP locus if trisomy?

• 4 (AAA, AAB, ABB, BBB)

Explain the following example

What’re the 2 ways to diagnose 22q11.2 deletion?

1. If the patient signs and symptoms suggest it, FISH could be used as a direct test of a deletion (ex: facial features)

2. CGH or SNP microarray can reveal a deletion (would show where it is and its size)

Whats QF-PCR and its function?

Quantitative Fluorescent-PCR

• Amplify STR loci from the chromosomes of interest. You can separate the alleles by size and also compare relative amounts of DNA

• Normally there’ll be 2 alleles with equal amounts of DNA

• In a trisomy there will be 3 alleles (triallelic) or 2 alleles with one having double the amount of DNA (diallelic)

Explain trisomy triallelic and trisomy diallelic in QF-PCR

Whats imprinting?

• Expression of some genes is determined by parental origin

• Expression in the zygote/ offspring is mono-allelic (expressed only from the maternal or paternal chromosome)

• Ex: the maternal or paternal copy of an imprinted locus is silenced through germline methylation

How is imprinting determined?

• Via methylation, its determined in the maternal and paternal germlines and maintained in the somatic tissues

Explain genomic imprinting in somatic and germline tissues

Explain the different parental origin effects if an allele is silenced

Explain what would happen from an XX individual if methylation was rewritten for maternal imprint

Explain what would happen from an XY individual if methylation was rewritten for paternal imprint

What’re some examples of imprinting disorders?

• Uniparental disomy (UPD)

• Monoallelic expression

• Prader-Willi chr 15

Explain Prader-Willi syndrome

Whats the mechanism of silencing?

• DNA methylation silences transcription, but not directly on the imprinted locus

• DNA methylation can silence lncRNA loci that act to silence the imprinted loci (like Xist and X-inactivation)

• So the methylated chromosome can be the one with the active allele

Whats the parental conflict hypothesis?