RL Lectures 45-46: Genetic Variation and Genetic Testing

Question 1

What are indels?

Answer 1

insertion or deletion of base-pairs that can be from a few bps up to 10,000bps
can be more serious if the number of missing/inserted
nucleotides is not a multiple of three (results in frameshift)

Question 2

What is a copy number variant (CNV)?

Answer 2

a sequence of more than 10,000 bps is present in more or less than the normal number copies

Question 3

What is a frequently occurring mechanism for generating a SNV?

Answer 3

C –> T change
methylation
via the C of a CpG hotspot

Question 4

What are VNTRs?

Answer 4

variable number of tandem repeats
a special case of indel comprises short sequence that is repeated many times in tandem most often found in non-coding regions

Question 5

What is a pseudogene?

Answer 5

DNA sequences that resemble known genes bur are nonfunctional

Question 6

List the available methods for detection of SNVs.

Answer 6

Sanger sequencing
Next-Generation
Sequencing

Question 7

What is Sanger sequencign used for?

Answer 7

detection of small changes in genome sequence directed by a the sequencing of a particular gene that is already known or suspected

Question 8

Describe the mechanism of Sanger sequencing.

Answer 8

target DNA is generated by standard PCR primer extension reaction is performed on target DNA (with high concentrations of dNTPs and low concentrations dideoxy NTPs (ddNTPs) without 3’ hydroxyl group)
target ddNTPs are tagged with different fluorophore incorportation of ddNTP which results in termination of the sequence –> series of DNA fragments of increasing size that are separated by capillary electrophoresis separation by size permits reading devices to assign a sequence call as each fragment migrates past the detector in size order

Question 9

What can next generation sequencing be used for?

Answer 9

sequencing rapidly a set of many genes that may have relevance to a disease condition or the entire set of protein-coding genes or even the whole genome

Question 10

Describe the steps for getting next generation sequencing results.

Answer 10

production of DNA fragments that can be selected for appropriate size for subsequent enzymatic attachment of adaptor sequences DNA denatured into single stands
individual fragments captured on sequencing matrix platform containing millions of wells
all short sequences in the wells are amplified by PCR into clon clusters
primer complementary to teh adaptor sequence is extended by a base in teh presence of dNTPs whose 3’ end is blocked by an azise group with a modified fluorophore tag for each cluster, one complementary base is incorporated and imaged, then based and tag are removed to access the next base and the steps are repeated to generate sequence data on a massive scale

Question 11

Describe the steps of whole exome sequencing.

Answer 11

extraction of genomic DNA sheering of DNA denaturation of DNA ligation of adaptors to sequences
capture of exons by hybridization targets entire set of known genes t(can be modified to target just disease associated genes or panels of genes associated with specific conditions)

Question 12

Provide examples of requirements and limitations for full genome sequencing via NGS.

Answer 12

computational requirements
sequencing alignment
variant identification (mostly for indels)
variant annotation and interpretation
incidental or secondary findings (counseling aspects)

Question 13

How are VNTRs detected?

Answer 13

accurate determination of repeat number is done by PCR amplification and measurement of PCR fragment length

Question 14

How are CNVs (low copy repeats) detected?

Answer 14

segmental duplications

FiSH

Question 15

Describe the process of FiSH.

Answer 15

targeting of a genomic region suspected of having a deletion via DNA fragment that is complementary to the sequence of interest is tagged with fluorophore that fluoresces when bound to target locus genes present in the normal copy number will give two fluorescent signals while deletion of a gene will reveal a single spot

Question 16

What is array comparative genomic hybridization (CGH)?

Answer 16

comparison of large numbers of DNA oligonucleotides that are complementary to specific regions of each chromosome imbedded on a glass slide (chip) DNA fragments from reference DNA are labeled with one color and the patient DNA is labeled with a different one
the two are combined and allowed to bind to the chip
ratio of flurophore-labeled molecules bound at each location on the chip is determined by a computer

Question 17

Describe the possible results of a CGH.

Answer 17

ratio of 1 indicates normal copy number
ratio of >1 indicates duplication at the region
ratio of <1 indicates deletion at the region

Question 18

What are the symptoms associated with DiGeorge Syndrome.

Answer 18

22q11 deletion syndrome/ velocardiofacial syndrome
velopharyngeal insufficiency, cleft palate, cardiac defects, mild dysmorphic features, thymic hypoplasia (immunodeficiency), learning disabilities, hypocalcemia (leading to seizures)

Question 19

What symptoms are associated with Williams Syndrome?

Answer 19

7q11.23 deletion
distinctive “elf” facies, “cocktail personality”, cardiovascular disease, intellectual disabilities, endocrine abnormalities

Question 20

What symptoms are associated with Wold-Hirschorn Syndrome?

Answer 20

4p16.3 deletion
fetal growth restriction, microcephaly, hypotonia, characteristic greek helmet facies, severe intellectual disabilities, seizures

Question 21

What symptoms are associated with Cru de Chat syndrome?

Answer 21

5p- terminal deletion
microcephaly
round face
hypertelorism
epicanthal folds
low-set ears
micrognathia
severe psychomotor and mental retardation
pulmonary and heart defects
cleft palate
very sociable (but may have maladaptive behaviors such as inattentiveness, hyperactivity, or self injury)

Question 22

What symptoms are associated with 1p36 deletion syndrome?

Answer 22

hypotonia, developmental delays, growth restriction, obesity, microcephaly, orofacial clefting, typical facial features, minor cardiac malformations