Chromosome Abnormalities, Mutations and Genetic Analysis

Question 1

What are some types of chromosomal abnormalities?

Answer 1

• Numerical - error in number of chromosomes, gross abnormality
• Structural – error in rearrangement, gross abnormality
• Mutational – error in DNA sequence, minute changes

Question 2

What is non-disjunction?

Answer 2

Nondisjunction is the failure of homologous chromosomes or sister chromatids to separate properly during cell division. There are three forms of nondisjunction: failure of a pair of homologous chromosomes to separate in meiosis I, failure of sister chromatids to separate during meiosis II, and failure of sister chromatids to separate during mitosis. Nondisjunction results in daughter cells with abnormal chromosome numbers (aneuploidy)

Question 3

What disorders are caused by autosomal aneuploidy?

Answer 3

• Trisomy 21 (Down syndrome)
• Trisomy 13 (Patau syndrome)
• Trisomy 18 (Edwards syndrome)

Question 4

Describe the origin of Down syndrome

Answer 4

Trisomy 21 (Down syndrome)
– Incidence: 1 in 650 to 1 in 700
• Increases with advancing maternal age
– Characteristic facial dysmorphologies
– IQ less than 50
– Average life expectancy (50-60 years)
– Alzheimer’s disease in later life
– Chromosomal findings
• Trisomy 21: non-dysjunction (95%), usually maternal origin
• Unbalanced Robertsonian translocation (4%)
• Mosaicism (1%) – differences in karyotypes in different tissues

Question 5

Describe the origin of Patau syndrome

Answer 5

Trisomy 13 (Patau syndrome)
– Incidence: 1 in 5000
– Multiple dysmorphic features and mental retardation
– About 5% die within first month, very few survive beyond first year
– Non-dysjunction (90%), maternal origin
– Unbalanced Robertsonian translocation (10%)

Question 6

Describe the origin of Edwards syndrome

Answer 6

Trisomy 18 (Edwards syndrome)
– Incidence: 1 in 3000
– Severe developmental problems; most patients die within first year, many within first month
– Non-disjunction (90%), maternal origin

Question 7

What diseases are caused by sex chromosome aneuploidy?

Answer 7

• 45,X (Turner syndrome)

* 47,XXY (Klinefelter syndrome)

Question 8

Describe the origin of Turner syndrome

Answer 8

45,X (Turner syndrome)
– Incidence: 1 in 5000 to 1 in 10000 (liveborn)
– Incidence at conception much greater, about 97% result in spontaneous loss
– Females of short stature and infertile
– Neck webbing and widely spaced nipples
– Intelligence and lifespan is normal

Question 9

Describe the origin of Klinefelters disease

Answer 9

47,XXY (Klinefelter syndrome)
–	Incidence: 1 in 1000
–	Tall stature, long limbs
–	Male but infertile, small testes, about 50% gynaecomastia (abnormal breast development)
–	Mild learning difficulties

Question 10

What are the two different types of translocation reactions?

Answer 10

Reciprocal: involving breaks in two chromosomes with formation of two new derivative chromosomes

Robertsonian: fusion of two acrocentric chromosomes to form one short chromosome and one long chromosome

Question 11

What is a balanced translocation?

Answer 11

Balanced translocation – if two simultaneous breaks occur, and repair results in translocation of the genetic material between two chromosome, ∴ all genetic material is still present following reciprocal translocation

Question 12

What is deletion?

Answer 12

Deletion - a deletion is a mutation (a genetic aberration) in which a part of a chromosome or a sequence of DNA is lost during DNA replication.

Question 13

What is unbalanced translocation?

Answer 13

Called unbalanced when the exchange of chromosome material is unequal resulting in extra or missing genes

Question 14

Describe different types of mutations

Answer 14

Non-coding
Coding single mutations
– Silent – synonymous e.g. CGA (Arg) to CGC (Arg)
– Missense - CGA (Arg) to GGA (Gly), incorrect protein
– Nonsense - CGA (Arg) to TGA (Stop), truncated protein
– Frameshift – deletion / insertion, incorrect/faulty/truncated protein

Question 15

What are the different types of point mutation?

Answer 15

Transitions
•	Purine to purine e.g. A -> G
•	Pyrimidine to pyrimidine e.g. C -> T
Transversions
•	Purine to pyrimidine e.g. A -> C
•	Pyrimidine to purine e.g. C -> G

Question 16

Name the purines

Answer 16

adenine and guanine

Question 17

Name the pyrimidines

Answer 17

cytosine and guanine

Question 18

What methods are used to detect mutations in the genome?

Answer 18

• Polymerase chain reaction (PCR)
• Gel electrophoresis
• Restriction fragment length polymorphism (RFLP) analysis
• Amplification refractory mutation system (ARMS)
• DNA sequencing

Question 19

What do you need to carry out PCR?

Answer 19

• Sequence information
• Oligonucleotide primers
• DNA
• Nucleotides
• DNA polymerase

Question 20

What is PCR?

Answer 20

Polymerase chain reaction (PCR) is a technique used in molecular biology to amplify a single copy or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence.

Question 21

Describe the PCR procedure

Answer 21

Step 1 - denature dsDNA 93-95C
Step 2 -anneal primers at 50-70C
Step 3 - extend new chains at 70-75C

Question 22

What is gel electrophoresis?

Answer 22

• Separates DNA fragments by size
• Apply an electric field
• DNA is negatively charged
• Separate through agarose gel matrix
• Visualise DNA fragments against standards

Question 23

What are the advantages of PCR-gel electrophoresis?

Answer 23

• Speed
• Ease of use
• Sensitive
• Robust

Question 24

What is ARMS?

Answer 24

The amplification-refractory mutation system (ARMS) is a simple method for detecting any mutation involving single base changes or small deletions. ARMS is based on the use of sequence-specific PCR primers that allow amplification of test DNA only when the target allele is contained within the sample. Following an ARMS reaction the presence or absence of a PCR product is diagnostic for the presence or absence of the target allele.

Question 25

What are the advantages of ARMS?

Answer 25

• Cheap
• Labelling not required
• Electrophoresis required

Question 26

What are the limitations of ARMS?

Answer 26

Primer design critical - may miss other unknown mutations associated with a disease (unknown variants)

Question 27

What are the disadvantages of ARMS?

Answer 27

• Need sequence information
• Limited amplification size
• Limited amounts of product
• Infidelity of DNA replication

Question 28

Whats the primary function of restriction endonucleases?

Answer 28

• Enzymes from bacterial cells involved in a protective mechanism which degrade DNA of invading viruses
• Recognise specific DNA sequences, usually 4-8 bp

Question 29

What is RFLP analysis?

Answer 29

In RFLP analysis (restriction fragment length polymorphism), the DNA sample is broken into pieces and digested by restriction enzymes and the resulting restriction fragments are separated according to their lengths by gel electrophoresis.
If a base is missing or has been changed, the fragment wont be recognized and therefore cut

Question 30

Give an example of a disease that is diagnosed using RFLP analysis

Answer 30

Sickle cell anaemia
In normal Hg gene, MST II cleaves the Hb genet create two bands representing smaller proteins
In sickle cell anaemia, the cleavage point is mutated and therefore isn’t cleaved, producing a band of a larger protein on the gel, suggesting they are affected.
If both bands are present, suggests the individual is a carrier of both the healthy and sickle cell gene.

Question 31

What are the advantages and disadvantages of RFLP analysis?

Answer 31

• Simple
• Cheap
• Non-radioactive
• Requires gel electrophoresis
• Not always feasible

Question 32

Describe the chain termination method (Sanger sequencing) of DNA sequencing

Answer 32

The classical chain-termination method requires a single-stranded DNA template, a DNA primer, a DNA polymerase, normal deoxynucleosidetriphosphates (dNTPs), and modified di-deoxynucleosidetriphosphates (ddNTPs), the latter of which terminate DNA strand elongation. These chain-terminating nucleotides lack a 3’-OH group required for the formation of a phosphodiester bond between two nucleotides, causing DNA polymerase to cease extension of DNA when a modified ddNTP is incorporated. The ddNTPs may be radioactively or fluorescently labeled for detection in automated sequencing machines

Question 33

What are the advantages and limitations of DNA sequencing?

Answer 33

• Gold standard for mutation detection
• Automation and high throughput
• Expensive equipment
• Poor quality sequence read
• Can be hard to determine between disease causing, disease predisposing and unrelated gene mutations when looking at all mutations in a genome.

Question 34

What conditions would make the ideal genetic test?

Answer 34

Depends on the abnormality, ideally it would need to be:
•	Direct test
•	Quick and easy
•	Cheap
•	Sensitivity
•	Specificity

Question 35

Describe the origin and diagnosis of disease due to abnormalities in chromosome number (Aneuploidy).

Answer 35

Trisomy 21 (Down’s Syndrome)
•	90% caused by non-disjunction (Chromatid sisters don’t split, and both pairs are carried into one gamete)
•	Chances increase with advancing maternal age
•	Distinct facial characteristic
•	IQ less than 50
Trisomy 13 (Patau Syndrome)
•	Usually caused by non-disjunction (90%)
•	Multiple dysmorphic features
•	Very few survive beyond first year of life
Trisomy 18 (Edwards Syndrome)
•	90% Non-disjunction
•	Most die within first year or month of life
X – (Turner Syndrome)
•	Short stature and infertile
•	Neck webbing and widely spaced nipples
•	Normal intelligence and lifespan
•	97% zygotes terminate at conception
XXY (Klinefelter Syndrome)
•	Tall stature and long limbs
•	Infertile, small testes
•	50% develop breasts (gynaecomastia)
•	Mild learning difficulties

Question 36

What are the different types of chromosomal anomalies?

Answer 36

o Robertsonian Translocation - This occurs in Acrocentric chromosomes (i.e. One arm is shorter than the other). This gives one chromosome with both long arms, while the other has both the short ones.

o Deletions - This occurs when sections of chromosomes just seem to disappear from one of the arms, if not both.

o Inversions - Paracentric (when a section of the DNA in the arm of the chromosome is inverted) or Pericentric (when the DNA around the centromere of the chromosome is inverted)

Question 37

Describe the types and effects of specific mutations

Answer 37

o Germline Mutation - An inheritable mutation of gametes, i.e. leads to genetic disorders in families.

o Somatic Mutations - Mutations of the other body cells which won’t be passed to future generations.

o Silent Mutations - Since most amino acids can be coded for by more than one Codon, sometimes changing 1 base won’t change much and the correct amino acid will still be used.

o Missense Mutations - In these mutations, the correct amino acid is replaced by an incorrect one in the finished protein product

o Nonsense Mutations - Mutations that replace the correct amino acid with a stop sequence, resulting in the protein being finished prematurely

o Frameshifts (Deletions/insertions) - The code is read in groups of three bases, so if one base is added or deleted, the whole sequence is changed.