Lecture 6: Clinical DNA Diagnosis

Question 1

Types of Point Mutations (Single Base Substitutions)

Answer 1

1. Silent Mutation
2. Nonsense Mutations
3. Missense Mutations
4. Splice Site
   Mutations

Question 2

Silent Mutation

Answer 2

Base pair change does not alter the amino acid sequence due to redundancy (wobble) in the genetic code.

Question 3

Nonsense Mutations

Answer 3

Replace an amino acid with a stop codon.

Question 4

Missense Mutations

Answer 4

Replace one amino acid with another in the gene product.

Question 5

Splice-Site Mutation

Answer 5

Creates or destroys signals for exon/intro splicing.

Question 6

Deletion

Answer 6

Mutation that results in an absence of a portion of a DNA sequence (usually in coding DNA or regulatory sequences).

Ranges from 1 base pair to millions of base pairs.

Question 7

Insertions

Answer 7

Mutation that results in an extra DNA sequence within the coding sequence of a gene.

Ranges from 1 base pair to millions of base pairs.

Question 8

Frameshift Mutation

Answer 8

Mutation that can be produced by deletions, insertions, or splicing errors resulting in the shift of the reading frame.

Often results in a stop codon that produces a truncated polypeptide.

Question 9

Promoter Mutation

Answer 9

Mutation affecting the binding of RNA polymerase to the promoter site.

Question 10

Duplication

Answer 10

A type of insertion due to repeated regions of DNA.

Question 11

Expansion of Tandem Repeat Sequences

Answer 11

An increase of a normal, small number of repeats (20-30 copies of a short sequence such as CGC) to a dramatically larger number (100s or 1000s). Can cause genetic diseases usually with genetic anticipation.

Question 12

Loss of Function (LOF) Mutations

Answer 12

Mutations that result in protein products that have reduced function, no function, and/or may interfere with the function of the normal product.

Question 13

Three types of LOF mutations:

Answer 13

1. Classical Recessive Inheritance
2. Haploinsufficiency
3. Dominant-negative Mutations

Question 14

Classical Recessive Inheritance (LOF)

Answer 14

For many genes, the amount of protein produced by the gene is not crucial for a normal biological outcome.

Even 40-50% of normal protein production is sufficient.

Must inherit both abnormal alleles.

Question 15

Haploinsufficiency (LOF)

Answer 15

Occurs when half (haplo) of the normal gene product is insufficient for completely normal biological function.

Autosomal dominant, incompletely dominant. Two abnormal copies gives rise to more serious phenotypes.

Question 16

Dominant-Negative Mutations (LOF)

Answer 16

Mutations that result in a mutant product from one allele that not only loses its original function, but also prevents the product of the other, normal allele from functioning in a heterozygous individual.

Question 17

Gain of Function Mutations

Answer 17

Mutations that cause the protein product to be overproduced or to actively function in a new, abnormal way.

i.e. Achondroplasia (receptor turned on and stuck–no chondrocyte proliferation–short bones).

Question 18

Four benefits of DNA-based tests?

Answer 18

1. Definitive diagnosis.
2. Pre-symptomatic diagnosis.
3. Pre-implantation or pre-natal diagnosis.
4. Genotype-phenotype correlations.

Question 19

Five challenges of DNA-based tests?

Answer 19

1. Heterogeneity
2. Allelic disorders
3. Variable Expression
4. Non-paternity exposure
5. Genetic discrimination

Question 20

Direct Mutation Testing

Answer 20

The optimal, but not always practical method of DNA-based diagnosis.

Question 21

Specimen

Answer 21

DNA samples are obtained from the individual from places such as blood, skin, cheeks, semen, etc. In prenatals, CVS, amniocentesis, or maternal cell-free fetal DNA.

A type of direct DNA testing.

Question 22

Karyotype Analysis

Answer 22

The chromosomes are examined under a microscope for duplications, deletions, or translocations of large segments of DNA. Less than 3 Mbp cannot be detected.

A type of direct DNA testing.

Question 23

FISH

Answer 23

Flourescent in situ Hybridization.

Used to look for well-defined common deletion or duplication syndromes. Can confirm smaller deletions/duplications by microarray.

A type of direct DNA testing.

Question 24

Methylation Specific RT-PCR

Answer 24

Methylation specific reverse transcriptase polymerase chain reaction.

Then differentiates segments of DNA that are methylated and those that are not.

A type of direct DNA testing.

Question 25

Chip Technology

Answer 25

Automated technique that is useful for screening large genes with many different mutations.

Also called microarray or array comparative genomic hybridization.

A type of direct DNA testing.

Question 26

Direct Sequencing

Answer 26

Used to look for disease-causing mutations within a given gene.

A type of direct DNA testing.

Question 27

Allele Specific Oligonucleotide Testing

Answer 27

Commonly used to look for a panel of common mutations in a given gene or several genes.

A type of direct DNA testing.

Question 28

STRPs

Answer 28

Short Tandem Repeat Polymorphisms.

The use of just 13 STRPs allows for a match probability of one in a trillion. Used in crime scenes and paternity testing.

A type of direct DNA testing.

Question 29

MLPA

Answer 29

Multiple Ligation-Dependent Amplification.

Allows for the detection of multiple regions of DNA at the same time, using one set of PCR primers. Can be used to detect large deletions, small deletions, and even single base pair changes.

A type of direct DNA testing.

Question 30

Southern Blot

Answer 30

Useful in detecting DNA fragments of variable lengths. Tests for TNT repeat disorders.

A type of direct DNA testing.

Question 31

RFLP

Answer 31

Restriction Fragment Length Polymorphisms.

Used to test for disease that are caused by mutations that occur at specific restriction sites.

A type of direct DNA testing.

Question 32

Next-Gen Sequencing

Answer 32

Uses the latest automated sequencing to sequence the entire genome in just days for a few thousand dollars. Will revolutionize medicine.

A type of direct DNA testing.

Question 33

Indirect Mutation Testing

Answer 33

Used in clinical scenarios where the disease gene is not yet known, rendering direct DNA testing impossible. Often requires large families to be informative.

Question 34

What is the difference between screening and testing?

Answer 34

Screening is the process of identifying people who are at higher-than-usual risk for a disease, but are not affected yet.

Testing is the process of targeted disease-specific lab procedures to definitively establish that someone is or is not affected.

Question 35

Population screening can detect…

Answer 35

mutations pre-symptomatically to allow for better outcomes.