Exam 2: Lecture 33

Question 1

What are the 2 broad types of genetic testing?

Answer 1

1) Germline testing
2) somatic (cancer) testing

Question 2

Constitutional (Germline) variants

Answer 2

• in gametes of mom or dad
• will be present in every cell in the body
• variants can be passed down to offspring

Question 3

Somatic variants

Answer 3

• not present at birth or in the gametes
• will develop later
• only present in cells where teh somatic variation occurred (“bad luck cells)
• won’t be passed on to the offspring

Question 4

Describe the categories of genetic variation

Answer 4

• single gene mutations
• chromosomal disorders - arise from structural or numerical chromosomal alterations ( aneuploidy or translocation)
• Complex multi- genic disorders (genes + environment like hypertension and diabetes)

Question 5

What is Cytogenetics?

Answer 5

• genetics of a cell
• look at structure and number of chromosomes for abnormality

Question 6

What are 2 important aspects of cytogenetic techniques?

Answer 6

• resolution (size)
• coverage ( whole genome vs target, types of chromosomal abnormalities)

Question 7

Why would you do cytogenetic testing in pregnant women?

Answer 7

• Advanced maternal age
• fetal anomalies observed on US
• Follow-up of abnormal non-invasive prenatal screening (NIPS)

Question 8

Why would you use a cytogenetic test for post-natal testing?

Answer 8

• multiple congenital anomalies
• developmental delay/ intellectual disability
• suspected chromosome abnormality ( features of down syndrome or DiGeorge)

Question 9

You would use cytogenetic testing for diagnosis and management of cancer

Question 10

How to perform a normal Karyotype?

Answer 10

1) Cultured
2) make sure cell is arrested in metaphase (when chromosomes are most condense and easy to visualize)
3) stained with Giemsa to allow visualization of bands (G-banded karyotype)

Question 11

Karyotype Benefits

Answer 11

• Whole genome analysis
• Detects all types of gross chromosomal abnormalities (aneuploidy, trisomy 21 and turner syndrome, translocations, and some deletions like DiGeorge)

Question 12

Karyotype Limitations

Answer 12

• Need actively growing cells ( not suited for frozen or fixed tissue) –> ex. colon cancer; some tissues don’t divide after birth like neurons
• low resolution (small abnormalities are not visual)
• poor lower limit of detection ( only 20 cells analyzed)
• labor intensive with TAT3 to 21 days

Question 13

What are some clinical applications of karyotype?

Answer 13

• suspected chromosomal aneuploidy ( down syndrome and turner syndrome)
• prenatal diagnosis of aneuploidy ( advanced maternal age, abnormal maternal screen/US, and abnormal NIPS)
• cancer diagnosis/treatment/prognosis (mostly for HEMATOLOGICAL MALIGNANCIES – bone marrow and peripheral blood samples that are easy to culture)

Question 14

What is FISH?

Answer 14

• targeted technique to detect and visualize specoific chromosomal regions
• used to diagnose aneuploidy, microdeletions, translocations and gene amplifications

Question 15

When would we uses FISH centromere probe vs locus specific probe?

Answer 15

• want quicker results
• Centromere probe: can look at the centromere to see how many copies we have (chromosomes); like with down syndrome
• Locus specific probe: allows us to detect specific abnormalities; like DIC and rara?

Question 16

What are the benefits of FISH?

Answer 16

• can be performed on fresh, frozen, or fixed tissue ( don’t need to culture our cells)
• better resolution than karyotype –> because we have a specific FISH probe for our specific region of interest
• Rapid TAT (<24HR)
• better lower limit of detection than karyotyping–> looking at a few things for every case, nit all 46 chromosomes; 200 cells analyzed

Question 17

What are the limitations of FISH ?

Answer 17

• need to know what you are fishing for
• only detect 3-4 abnormalities at once
• does not delineate size or genes involved –> not every patient have the same deletions (could affect the prognosis or presentation of the diagnoses)

Question 18

What are the clinical applications of FISH?

Answer 18

• rapid diagnosis of aneuploidies (13, 18,21, X, Y)
• detection of cryptic chromosomal abnormalities–> like things not detected by Karyotypes (10% of DiGeorge syndrome aren’t visible on karyotype)
• characterization of genetic abnormalities for cancer diagnosis, treatment, and prognosis (translocations and gene amplifications like HER2 amplifications)

Question 19

What is Chromosomal microarray?

Answer 19

• used to detect copy number variants[CNV] (gain of deletion)–> 10s-100s of kb
• Forst tier test for:
• developmental delay/intellectual disability
• autism spectrum disorder
• multiple congenial anomalies
• used in oncology to detect CNV, and regions of loss of heterozygosity (LOD)

Question 20

What are the types of microarray?

Answer 20

• single nucleotide polymorphism –> you can figure out how many copies of the gene people have
• Array comparative genomic hybridization–> use label patient and control DNA compete to bind to DNA probes; allows you to figure out if there’s a copy gain or loss
• if patient is normal, will get equal hybridization
• if loss–> signaling will be off ( not 2 copies of each)
• looking at fluorescent lights

Question 21

What is molecular genetics?

Answer 21

• uses the techniques of molecular biology to investigate DNA sequence alterations
• PCR
• Sanger sequencing
• Next Generation sequencing

Question 22

What are indications of molecular genetic testing?

Answer 22

• Prenatal testing: carrier testing and non-invasive prenatal screening (NIPS)
• post-natal testing: investigation of suspected inherited disease; pre-symptomatic genetic testing (potentially a pathogenic variant in a family member); pharmacogenomics (might be trying to look at enzyme variants that affects how they metabolize a drug)
• diagnosis of cancer

Question 23

What are examples of single known pathogenic variant diseases?

Answer 23

• sickle cell disease –> different than Beta thalassemia where you have to sequence the entire gene
• Huntington disease (trinucleotide repeat disorder)

Question 24

Describe mutation hotspots

Answer 24

• genes that have places that are typically mutated
• ex. JAK2 gene ( Y-axis ( frequency of described mutations; X-axis is aa of protein positions- entire length of protein) –>V617F position

Question 25

PCR

Answer 25

- need sequence of interest to copy - need primers to bind to the sequence of interest (dNTP) - will form a template to copy the DNA - denaturation; annealing; extension; amplification cycles

Question 26

Describe targeted mutation analysis

Answer 26

- used when there is limited heterogeneity (use to diagnosis JAK2 V617F)--> hotspot mutations like CF, FX, HD, MD - Detects single nucleotide variants, small insertions. deletions (5-50 bp) - different techniques: PCR-RFLP, allele specific PCR, single nucleotide extension, PCR fragment analysis. digital droplet PCR

Question 27

Describe single-base extension analysis

Answer 27

- useful for identifying specific mutations at nucleotide positions - primer that targets region of interest; line them up next to each other; detect what lights up (40 min) - ex. BRAF V600E and Factor V Leiden - targeted technique... will miss it if the mutation is in 601

Question 28

Describe Fragment Analysis

Answer 28

- can use this for Fragile X (CGG repeat region expanded) - can tell us the size of the region (smaller size to left, larger to right; full mutation is in the red zone) - >200 repeats (full mutation)

Question 29

What is sanger Sequencing?

Answer 29

- uses a modified PCR technique--> take labeled ntp + fluorescent tag and put in PCR reaction - will be grabbing labeled nucleotide and add the to the chain - will get sanger products that detect sequence of DNA strand - can be used to identify single nucleotide changes, small insertions/deletions throughout the gene

Question 30

What are examples of scenarios where you'd use Sanger Sequencing?

Answer 30

- Beta Thalassemia - Rett Syndrome - Cystic Fibrosis

Question 31

How do you assess Sanger Sequencing graph?

Answer 31

- need reference genome - patient genome sequence - will compare the 2... are there regions that are different in the patient? (common snp section) - single color--> homozygous - multiple color at the position--> heterozygous - must look to see if the protein changes to not misdiagnose

Question 32

Describe Next- Generation Sequencing

Answer 32

- capable of producing very large amounts of sequence data - can sequence multiple genes at once --> can order a gene panel like assessing multiple genes that may contribute to hearing loss

Question 33

What is happening with Next-generation sequencing?

Answer 33

- template strand.. figure out with NTP binds based off of color - cameral captures it - each dot is an individual sequence

Question 34

Compare whole genome and targeted sequencing

Answer 34

- whole genome sequencing includes both introns and exons - 85% of known pathogenic variants occur in exons

Question 35

What are the limitations of Next Generation Sequencing?

Answer 35

- relatively long routine turn-around time (2-8 weeks) - substantial bioinformatics processing required (need an algorithm to filter out benign vs pathogenic mutations; interpretation/storage of large amounts of data - variants of uncertain significance--> may run across a mutation, but don't know the significance - not an ideal tech for certain alterations/genes--> repeat expansions, variants in genes with pseudogenes, structural variants, insertions/deletions > 20-50bp

Question 35

How to be pull out our targets? (Hybridization based targets)

Answer 35

- probes/ baits binds specifically to region of interest - attach them to magnets to pull out exons (NGS panels?)