Testing for Mandelian disorders

Question 1

What is a gene?

Answer 1

A distinct sequence of nucleotides forming part of a chromosome, the order of which determines the order of monomers in a polypeptide or nucleic acid molecule which a cell may synthesize

Question 2

What is a reference genome?

Answer 2

• A nucleic acid sequence database, which is a representative example of a set of genes
• They are assembled from the sequencing of DNA from a number of donors
• Reference genome does not accurately represent the set of genes of any single person but rather provides a haploid mosaic of different DNA sequence from each donor
• Analysis of the whole genome or parts therefore will identify differences or variants between individuals in parts/whole genome

Question 3

T/F: abnormal variation cannot create a disease state by changing the protein structure/ function

Answer 3

False, an abnormal variation can create a disease state by changing the protein structure or function

Question 4

Define polymorphism

Answer 4

A single nucleotide change / substitution that does not result in a change in a codon thus does not affect the synthesis of a protein

Question 5

Define missense mutation

Answer 5

A substitution in a nucleotide sequence that results in a different amino acid resulting in a different protein

Question 6

Define nonsense mutation

Answer 6

A substitution in a nucleotide sequence that results in a premature stop codon

Question 7

Define a frameshift mutation

Answer 7

A mutation caused by the deletion / addition of a nucleotide ,resulting in a frameshift reading of the nucleotide sequence

Question 8

Define inversion

Answer 8

When part of a chromosome ,breaks ,flips and reattaches

Question 9

List the classification of genetic variation

Answer 9

class 1: benign 
class 2: likely benign 
class 3: uncertain significant
class 4; likely pathogenic 
class 5; pathogenic

Question 10

How do we test for variation in a single gene

Answer 10

1. Traditional Sanger sequencing :
   * Directed: you need to know what you are looking for
   * testing for specific sequence changes

Benefits and downfalls of Sanger sequencing :

• Analyse gene or two at a time
• high certainty of results
• predictable outcomes
• Time and cost consuming
• Relatively easy to compare labs and tests offered
• limited y size of the DNA fragment which can be a sequence

Question 11

Development of NGS

Answer 11

• Rapidly sequence advancing technology
• Cost reducing rapidly but still expensive
• Able sequence multiple genes simultaneously
• It is less hypothesis drive ,you do not need to suspect a condition

Question 12

The process of NGS involves what?

Answer 12

1. Sequence Mapping: Alignment of sequences (reads) on a reference genome
2. Variants calling: Identification of difference with regards to the reference genome.
3. Variants filtering: Selection of pathogenic variants based on their characteristics
4. Variants ranking: Prioritization of potential pathogenic based on their characteristics and correlation to the disease

Question 13

what is depth sequencing

Answer 13

The number of times that each DNA is sequenced

Question 14

True or false: the better the depth sequencing and coverage, the more accurate the sequencing will be

Answer 14

false

Question 15

Benefits of the NGS approach

Answer 15

1.Increasing ability to make accurate diagnoses
>Ability to do multiple analyses simultaneously allows us to :
*identify mutations in rarer genes
*identify disease modifiers
*identify gene interactions

> Opportunities to identify new disease-associated genes

> Opportunities to characterize disease more precisely with improved family risk and improved understanding of the genotype-phenotype relationship

Question 16

Discuss the NGS gene panel testing

Answer 16

> Gene panel is designed to test multiple related genes simultaneously (in a range of 2-100genes )
*There can be a high yield if the genes are chosen correctly with less ambiguous and unwanted results

> It is useful if the is relatively distinct or multiple genes are known to cause the same phenotype

Question 17

Exome sequencing

Answer 17

Sequence exons ,with no analysis of non-coding DNA but rather only analyses 1,2% of the genome but 85%of ;pathogenic mutations are in the exons

When to use Exome sequencing :
>Poorly defined phenotype(new syndrome )
> Negative panel ( likelihood of positive finding )

Disadvantage : Interpretation of new data is challenging and often generates new data thus difficult to prove causation

Question 18

What is whole genome sequencing?

Answer 18

• Sequencing all the DNA of an individual(300Mb)
• Deep intronic mutation, breakpoints, and structural rearrangement may be detected

> it is very expensive and it is not widely used in the clinical sphere

Question 19

What are the NGS challenges

Answer 19

Class 3 variants : Variants of unknown significance, after considering the genetic test results ,gives uncertain results whether the variation is the cause of patients disease

it is not wise to make a reproductive / clinical recommendation on uncertain results

2.Incidental or secondary findings
>unrelated to the phenotype(disease)
>May be of medical value to patient
> is usually not part of reason for testing

> Reportable variants :

• 1% of tests have a reportable incidental variants
• ACMG list of 59 genes
• Pathogenic /likely pathogenic variants must be reported to patients