Lecture 6

Question 1

what is the advantage to Sanger sequencing?

Answer 1

very high accuracy

Question 2

what is a big advantage of NGS?

Answer 2

if further analysis of the interpretation is necessary, we can re-start our studies with the analysis of the FASTQ file in which we have all the data about the sequencing of the sample analyzed

Question 3

what is the core disease gene list?

Answer 3

the list in which all genes are associated with a clinical phenotype and they could be named as “causative genes” - they have demonstrated a pathogenic role in many patients

Question 4

in what situation is it better to perform whole exome sequencing?

Answer 4

to explore a causative mutation or a correlation with a disease

Question 5

in what situation is it better to use the core disease gene list?

Answer 5

for diagnosis and clinical utility

Question 6

what is clinical utility?

Answer 6

a conformation, by the genetic testing, of diagnostic hypothesis and the utility of the results is not only for the proband, but also for the other affected family members or for a family member at risk to develop the phenotype

Question 7

what are the criteria that we have to remember in our “filtering process” when analyzing mutation classification?

Answer 7

1. the frequency of the mutation in the general population
2. the localization of the mutation on the gene
3. the previous reported cases
4. databases

Question 8

what qualifies a mutation to be class V, or very strong?

Answer 8

if the mutation is a nonsenses mutation, a mutation localized on the information codon and a single or a multi-gene deletion → it is very impactive for the disease so the score will be high

Question 9

what qualifies a mutation to be class IV, or a strong mutation?

Answer 9

• if we have the same amino acidic charge that previously was established by a pathogenic variant regardless of the nucleotide change
• de novo in a patient with disease and no family history
• well established in vivo and in vitro functional studies that are supportive of a damaging effect on the gene or gene product

Question 10

what qualifies a mutation to be a class III mutation?

Answer 10

mutation localized in a hotspot / critical functional domain of a protein, also assigned for recessive disorders or detected in trans with a pathogenic variant

Question 11

what is one thing to keep in mind when considering classes of mutations?

Answer 11

it is a subjective evaluation of the possible role that the variant plays considering different aspects of that mutation

Question 12

what types of things are considering when deciding on the final score of classification of a mutation?

Answer 12

• frequency
• localization
• functional studies
• family segregation
• conservation of the variant among the species

Question 13

how are class III variants handled differently among labs?

Answer 13

some choose not to report these variants because there is no solid relation to a specific disease

Question 14

how does San Raffaele deal with class III variants?

Answer 14

class III variants are reported, but only class IV and V are confirmed by Sanger

Question 15

how might the view of a class II variant change if a family member is also affected?

Answer 15

the variant score is higher if the mutation is present in more than one individual in a family, so the mutation is probably a pathogenic variant

Question 16

what must a final genetic analysis report contain?

Answer 16

1. the approach used for analysis and the limitation of the procedure
2. the reference sequence
3. the list of genes analyzed

Question 17

what are cardiomyopathies characterized by?

Answer 17

a heterogeneity between a clinical and genetic point of view

Question 18

what are the two main groups of cardiomyopathy patients?

Answer 18

those with structural alterations and those with alteration of the electrical activity

Question 19

which form of cardiomyopathy occurs when there is an alteration of the electrical activity without the structural alteration of the heart muscle?

Answer 19

arrhythmogenic forms

Question 20

can the structural form of cardiomyopathy lead to electrical issues?

Answer 20

yes

Question 21

what are the four forms of structural disease?

Answer 21

1. hypertrophic cardiomyopathy
2. dilated cardiomyopathy
3. restrictive cardiomyopathy
4. arrhythmogenic right ventricular dysplasia

Question 22

what are the four forms of arrhythmogenic disease?

Answer 22

1. Brugada syndrome (BrS)
2. long QT syndrome
3. short QT syndrome
4. CPVT (catecholaminergic polymorphic ventricular tachycardia)

Question 23

what are the disorder characterized by?

Answer 23

an overlapping of clinical symptoms - the same symptom can be present in multiple diseases

Question 24

what does incomplete penetrance mean?

Answer 24

even if a subject carries a mutation, he might not have the disease, so there are no causative effects and the affected individual couldn’t sho the mutation’s related phenotype

Question 25

what is variable expressivity related with?

Answer 25

the severity of the disease

Question 26

what are cardiomyopic disorders characterized as?

Answer 26

oligogenic disorders - different genes can be associated with the same phenotype

Question 27

what is the detection rate?

Answer 27

the percentage of cases that remains genetically unsolved (the cases that are negative)

Question 28

for example in Brugada syndrome, can the patients have one of the 16 genes or more than one of the identified 16?

Answer 28

one mutation on one of these genes can be the main mutation associated with the clinical phenotype → recent hypothesis stated that maybe there is more than one of these genes associated

Question 29

describe the presence of genetic overlapping:

Answer 29

occurs when the same gene causes different phenotypes depending on the kind of mutation → different phenotypes not only in structural disorders, but we can also have overlapping of the same genes that cause arrhythomgenic forms and the DCM for example

Question 30

what is a crucial downside for NGS in regards to genetic overlapping?

Answer 30

we can sequence the genes and detect the mutation, but based on the genetic results we don’t know if that mutation is causative or one of the forms of arrhythmogenic disorders or dilated cardiomyopathy

Question 31

why is it important to start with a clear clinical diagnosis before genetic testing is performed?

Answer 31

if the genetic testing cannot distinguish between the causative mutation and another form, there is no clinical utility because we can have variation in one of those genes, but that variation can be associated with many different clinical phenotypes

Question 32

what is the SCN5A gene associated with?

Answer 32

the sodium voltage-gated channel

Question 33

what happens if there is a loss of function mutation in the SCN5A gene?

Answer 33

can cause Brugada syndrome

Question 34

what happens if there is a gain of function mutation in the SCN5A gene?

Answer 34

there are many options

Question 35

what happens if there is both a gain and loss of function mutation in the SCN5A gene?

Answer 35

atrial fibrillation

Question 36

how can genetic testing affect the patient?

Answer 36

based on the gene altered it is possible to suggest different treatments for the patient or different lifestyle changes

Question 37

when 130 patients were analyzed for about 200 genes, there were 103 genetic variants and 38 genes detected, however there was a positive genetic testing for only 23 of these cases……. why?

Answer 37

the same genes are mutated in different clinical phenotypes

Question 38

what is a phenocopy?

Answer 38

a variation in phenotype, which is caused by environmental conditions (often but not necessarily during an organisms development) such that the organism’s phenotype matches a phenotype which is redetermined by genetic factors →non-hereditary

Question 39

what is a gene that encodes for the ryanodine receptor and is a very well-known causative gene in CPVT?

Answer 39

RYR2 gene

Question 40

what is the Ryanodine receptor important for?

Answer 40

the regulation of calcium flow from the sarcoplasm reticulum to the cytosol → an alteration of this receptor might induce an electrical abnormality due to the abnormal increase of the cytosolic calcium during the excitation-contraction coupling and this can lead to ventricular fibrillation and sudden death