From whole chromosomes to single bases

Question 1

How is a chromosome recognised?

Answer 1

Banding pattern with specific stains
Length
Position of centromere

Question 2

Acrocentric chromosomes

Answer 2

Most chromosomes have a telomere at both ends of the chromosome, a short arm, a long arm and a centromere.
The short arm doesn’t really matter
Some chromosomes only have a long arm these are celled acrocentric chromosomes e.g. satellite ribosomal genes, tRNAs etc. Therefore they only have a centromere and long arm.

Question 3

What are some chromosome changes that cause disease?

Answer 3

Balanced chromosome rearrangement is where all the chromosomal material is present.
Unbalanced chromosome rearrangement is where there is extra or missing chromosomal material. Usually 1 or 3 copies of some of the genome.
(Having 1 or 3 copies of a part of your genome is developmentally bad news).

Question 4

what is the definition of Robertsonian Translocation?

Answer 4

This is where two acrocentric chromosomes are stuck end to end.
There is an increased risk of trisomy in a pregnancy.
If the father is considered normal and the mother has balanced robertsonian translocation chromosomes there are 4 possible outcomes,
Normal
Balanced Translocation
Trisomy 14 (Miscarriage)
Trisomy 21 (Down Syndrome)

Question 5

What does Trisomy 18 result in?

Answer 5

Edwards Syndrome.
It’s rarer than down syndrome and is a fatal condition and so all with the condition either miscarry or normally die within the first month of life.

Question 6

Why is X chromosome aneuploidy is better tolerated?

Answer 6

Because of X chromosome inactivation.
45X Turner syndrome
47XXX Triple X
47XXY Klinefelter syndrome

Question 7

Why is having a big translocation possibly better than having a small translocation?

Answer 7

If an embryo has a big translocation causing a big imbalance, it will most likely miscarry.
If an embryo has a small translocation, there is a greater chance of the embryo surviving but resulting in a child with a developmental abnormality.

Question 8

If the mother is considered normal and the father has reciprocal translocations in his chromosomes, what are possible outcomes for offspring?

Answer 8

Normal
Balanced 1:9 Translocation
Partial Trisomy 9 + Monosomy 1
Partial Trisomy 1 + Monosomy 9

Question 9

What are the reproductive risks for someone with Reciprocal Translocations?

Answer 9

For most translocations estimated 50% of concepts will either have normal chromosomes or the balanced translocation.
Unbalanced products result in
- miscarriage (large segments)
- dysmorphic delayed child (small segments)

Question 10

If the translocation is smaller than 5 million bases, It’s too small to see what is this called?

Answer 10

This is microdeletion

Question 11

Molecular Cytogenetics

Answer 11

You no longer need to see chromosomes to count them - molecular techniques are quicker, cheaper and more sensitive.
You will only detect imbalance.
You can detect tiny changes.
Some small changes are polymorphisms.

Question 12

What is Array CGH (aCGH)?

Answer 12

Now the first line chromosome test
It is genome wide
It also finds lots of polymorphisms
Technique is “DNA based” - reflects underlying chromosomes.
In aCGH - if there is half as much DNA present it indicates a deletion.
aCGH can detect any size of imbalance.
aCGH does not detect balanced rearrangements.

Question 13

What is Mosaicism?

Answer 13

Different cells have a different genetic constitution.

This could be a mosaic chromosome abnormality or mosaicism for a point mutation.

Question 14

What is Somatic Mosaicism?

Answer 14

(Everyone starts off as one cell which then proliferates and the cells differentiate. if one cell develops a mutation, it will then pass it onto it’s daughter cells and so on causing somatic mosaicism).
All cells suffer mutations as they divide
- At meiosis and at mitosis
- Approx. 10 to the power of -6 per gene per cell division.
However, repair mechanisms exist.

Question 15

Chromosome changes could cause what?

Answer 15

Chromosome changes could

• Activate an oncogene (a cell becomes cancerous)
• Delete a tumour suppressor (a tumour suppressor stops a cancerous cell dividing so by deleting it this would cause cancer).

Question 16

What is Philadelphia chromosome?

Answer 16

This is where chromosomes 9 and 22 translocate.

Question 17

How do Chromosome changes indicate treatment in cancer?

Answer 17

HER2 amplification:
Monoclonal antibody: Trastuzamab
Philadelphia Chromosome:
Tyrosine Kinase inhibitor: Imatinib

Question 18

How else do we analyse DNA?

Answer 18

aCGH - for deletions/ duplications (Chromosome/ Karyotype analysis for balanced rearrangements only)
Polymerase chain reaction (PCR) and Sanger sequencing or Next Generation Sequencing (NGS).

Question 19

How is Next Generation Sequencing carried out?

Answer 19

1. extracted gDNA
2. gDNA is fragmented into a library of small segments that are each sequenced I parallel.
3. Individual sequence reads are reassembled by aligning to a reference genome.
4. The whole-genome sequence is derived from the consensus of aligned reads.
   Alignment of multiple short reads shows that there is a different base in half the reads. This is because half the reads come from a variant allele which contains either a polymorphism or a disease causing mutation.
   You can look at a person’s entire genome which costs about £1000 ($1000 genome).
   The way to identify mutations is to line them up with the reference strand.
   (NGS machines, are large, boring and need VERY big computers).

Question 20

What is the final technique that can be used to look at the genome?

Answer 20

The sock technique.

Question 21

What is the definition of Aneuploidy?

Answer 21

Whole extra or missing chromosome.

Question 22

What is the definition of translocation?

Answer 22

Rearrangement of chromosomes.

Question 23

What is the definition of insertions and deletions?

Answer 23

This is where there is missing or duplicated material.

Question 24

What is the definition of microdeletions?

Answer 24

Microdeletions are chromosomal deletions that are too small to be detected by a microscope.

Question 25

How does FISH work?

Answer 25

FISH is a type of DNA hybridisation, which uses the ability of DNA to stick to a ‘complementary copy’ of itself. The chemical letter A always pairs up with T, and C with G. To carry out FISH, scientists first unzip the DNA that makes up both the tagged gene and the chromosomes into two separate strands. So when the fluorescently tagged copy of the gene is put on to the microscope slide, it will stick to its ‘complementary copy’ on one of the chromosomes.

Question 26

Rearrangements can be balanced or unbalanced what are the implications of this?

Answer 26

If the rearrangement is balanced, there will be no change in the phenotype of the person.
Unbalance rearrangements cause multiple malformations or miscarriage.

Question 27

Where do mutations come from?

Answer 27

If one parent has a mutation it can be passed down to the next generation and this is called a germ line mutation.
If neither parent has a mutation, a mutation can occur in gametogenesis.
If one parent is mosaic, they can pass a mutations onto offspring.
If mutation is post-zygotic - the child is mosaic.

Question 28

What is the role of Polymerase chain reaction (PCR) a type of focused testing?

Answer 28

It allows us to select one small piece of the human genome from a patient (100 to 10000 bases) and amplify it - i.e. make lots of copies of one short stretch of the genome, therefore is on allele has a mutation it will be amplified many times.

Question 29

Only 2-3% of the genome is exons, what does the rest do?

Answer 29

Regulation if genes.
Spaces genes out - insulates genes from promotors.
Provides substrate to expand genome / add genes.

Question 30

What is a balanced rearrangement?

Answer 30

A balanced rearrangement has all the genes, but in a different arrangement.

Question 31

What is an unbalanced rearrangement?

Answer 31

An unbalanced rearrangement has missing or additional genetic material, which is developmentally bad news.

Question 32

What is now the first line for chromosome imbalance?

Answer 32

Array Genomic Hybridisation (aCGH) but it doesn’t detect balanced rearrangements.

Question 33

When do mutations arise?

Answer 33

They arise during sell division at any point in the human life cycle.

Question 34

What is one genetic change that contributes to cancer?

Answer 34

Somatic mosaicism for a chromosomal abnormality is one genetic change that contributes to cancer.

Question 35

What can analyse individual bits of DNA?

Answer 35

PCR and sequencing.

Question 36

What allows large scale sequencing cheaply?

Answer 36

NGS allows large scale sequencing cheaply - whole genome or whole exon.

Question 37

What is polymorphism?

Answer 37

The occurrence of different forms of the same thing.