Molecular Pathology

Question 1

What is a loss of function mutation?

Answer 1

A single base or multiple bases that are either mutated or deleted.

Question 2

Where are loss of function mutations found?

Answer 2

Frequently within the coding region but also within splice sites or gene promoter regions.

Question 3

Does a mutation always have an effect?

Answer 3

No. Mutations may sometimes represent a polymorphism and have no effect on the function of the encoded protein.

Question 4

What does a mutation in one of two alleles lead to?

Answer 4

Haplotype Insufficiency and a Dominant Negative Effect

Question 5

What are DNA changes associated with?

Answer 5

Loss of function mutations, usually very heterogeneous. Recessive Diseases

Question 6

What are gain of function mutations usually associated with?

Answer 6

Dominant Phenotypes and an increase in number of tri-nucleotide repeats

Question 7

What are the key attributes of micro satellite repeats?

Answer 7

Highly Polymorphic

Question 8

Where are micro satellite repeats usually found?

Answer 8

Throughout the genome, fairly evenly spaced along each chromosomal arm

Question 9

What form do micro satellite repeats usually take?

Answer 9

(CA)n or (CGG)n

Question 10

What is gene tracking?

Answer 10

A method for determining the inheritance of a particular gene in a family?

Question 11

How is gene tracking carried out?

Answer 11

RFLP’s situated in or near the locus of interest and are identified using gene probes.

Question 12

When would a marker not be clinically useful?

Answer 12

When everyone has the same heterogeneous genotype

Question 13

What are the features of the Duchenne Muscular Dystrophy gene?

Answer 13

79 Exons
2400kb Genomic DNA
X-linked Recessive
Passed on to males but females can only be carriers

Question 14

Why are micro sattelite markers special in Duchenne Muscular Dystrophy?

Answer 14

They lie within the gene itself

Question 15

What are the major mechanisms for loss of function in Duchenne Muscular Dystrophy?

Answer 15

Deletion of one or more exons

Question 16

What is linkage disequilibrium?

Answer 16

Linkage disequilibrium is the non-random association of alleles at two or more loci, that descend from single, ancestral chromosomes. Linkage disequilibrium is wholly a measurement of proximal genomic space

Question 17

What is phase?

Answer 17

The allele inherited from a grandparent

Question 18

What is essential for gene tracking for LOAD disease?

Answer 18

Must have a tightly linked marker

Question 19

What are the steps for gene tracking of LOAD disease?

Answer 19

1. Tell the parents of the consultand apart to identify who has the diseased gene
2. Identify phase to identify which allele the diseased gene is on
3. Genotype the affected parent and their parent who has been identified as diseased
4. Identify which alleles the consultand has recieved and make prediciton

Question 20

What samples can be taken for direct testing?

Answer 20

Blood DNA
Mouthwash and Buccal scrapes
Chorionic villus biopsy (fetal DNA)

Question 21

What is direct dideoxy sequencing?

Answer 21

The gold standard for genetic testing which can tell which base is mutated and whether it is a deletion or insertion

Question 22

What are the problems associated with direct dideoxy sequencing approach?

Answer 22

Genes are frequently large and mutations often occur in introns (splice donor sites)

Question 23

What are the problems associated with loss of function mutations and direct dideoxy sequencing?

Answer 23

Spectrum of mutations are often extremely heterogeneous and complexity may be reduced by sequencing the RNA transcript

Question 24

What are the advantages of direct dideoxy sequencing?

Answer 24

It looks at the actual sequence that codes for the protein and does not contain introns that have been spliced out.

Question 25

How is RNA converted to cDNA?

Answer 25

Reverse Transcription PCR

Question 26

Give some examples of diseases that have the same mutation or one of a limited number of mutations

Answer 26

SCA, Hutingdon's, CF and alpha- and beta- thalassaemia

Question 27

What is Restriction digest of PCR amplified DNA; check size on gel?

Answer 27

Only when the mutation creates or destroys a natural restriction site

Question 28

What is Hybridize PCR-amplified DNA to allele specific oligonucleotides (ASO)?

Answer 28

General method for specific point mutations; allows use of large arrays

Question 29

What is PCR using allele specific primers (ARMS test)?

Answer 29

General method for point mutations;primer design critical. Can be adopted to chip technology

Question 30

What are Oligonucleotide Ligation Assays?

Answer 30

Uses the enzyme DNA ligase Relies on the two primers forming perfect hybrids on the target Point Mutations

Question 31

What is PCR with primers located on either side of a translocation breakpoint?

Answer 31

Successful amplification shows presence of the specific deletion or specific rearrangement Point Mutations

Question 32

What is Check size of expanded repeat?

Answer 32

Dealt with when discussing Huntington disease | May also be used for Fragile X syndrome

Question 33

What is Dideoxy Fingerprinting?

Answer 33

Similar to conventional sequencing but run just two lanes-for example C and T Less expensive than conventional sequencing, it has high sensitivity but can be complicated to interpret

Question 34

What are Gene Chips?

Answer 34

Quick, high throughput can define all changes. New technology, that is expensive and used only for a limited number of genes “CHIP” is arrayed with 20mer sequences that correspond to wild type and all known mutations within a particular gene Simply PCR sample and allow to hybridise (PCR product is labelled with a fluorescent dye Possible to array 300 000 oligonucleotides within a 1.28 X 1.28 cm array

Question 35

What is a Southern Blot?

Answer 35

detects major deletions and rearrangements. It is laborious expensive and lots of DNA

Question 36

What is Heteroduplex Gel Mobilty?

Answer 36

Simple-Cheap Sequence <200bp, limited sensitivity, does not reveal position. Relies on the fact that most mutations occur as hetroduplex. DNA subject to PCR heated and denatured. Then subject to nondenaturing polyacrylamide gel electrophoresis

Question 37

What is Single Strand Conformation Polymorphism (SSCP)?

Answer 37

Similar advantages disadvantages as Hetroduplex analysis. PCR molecule will have its own migration properties dependent on its base composition.

Question 38

What is Denaturing gradient Gel Elecrophoresis(DGGE)?

Answer 38

``` High sensitivity (note than SSCP and hetroduplex analysis do generate false positive and false negative results) Requires careful initial optimisation (primer design) and conditions and does not reveal position of mutation ```

Question 39

What is Denaturing High Pressure Liquid Chromatography (dHPLC)?

Answer 39

Similar principle to DGGE but a lot faster, with higher throughput and is quantitative The disadvantages are that it again does not reveal the position of the change and the equipment is expensive

Question 40

What is Mismatch cleavage (Chemical or Enymatic)?

Answer 40

Both have high sensitivity and show position of the change Problems chemical method toxic both experimentally difficult and often poor results These methods rely on the formation of a hetroduplex which is recognised by chemicals or restriction enzymes-usually those that are sensitive to single stranded DNA in the hetroduplex. Generated products can then be resolved on a gel or sequenced