Genetic Variation and Genetic Testing- Lectures 45-46 Flashcards
About ______ genes are associated with human diseases.
5000
We acquire about ____ different SNVs de novo, however this number can increase with ______.
50-80
increased paternal age
A frequently-occurring mechanism for generation of a SNV is a ______ change via ____ of the ___ residue. Such variants are said to occur in ______.
C –> T change
methylation
C
CpG hotspots
What are indels?
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)
What is a copy number variant (CNV)?
a sequence of more than 10,000 bps is present in more or less than the normal number of copies
Describe the gene dosage effect.
duplications can sometimes give rise to disease if the cell is sensitive to a change in expression level caused by the duplicated gene
What are VNTRs?
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
What is a pseudogene?
DNA sequences that resemble known genes but are nonfunctional
List the available methods for detection of SNVs.
Sanger sequencing
Next-Generation Sequencing
What is Sanger sequencing used for?
detection of small changes in genome sequence directed by a the sequencing of a particular gene that is already known or suspected
Describe the mechanism of Sanger sequencing.
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
What can next generation sequencing be used for?
sequencing rapidly a set of many genes that may have relevance to a disease condition or the entire set of protien-coding genes or even the whole genome
Describe the steps for getting next generation sequencing results.
production of DNA fragments that can be selected for appropriate size for subsequent enzymatic attachment of adaptor sequences
DNA denatured into single strands
individual fragments captured on sequencing matrix platform containing millions of wells
all short sequences in the wells are amplified by PCR into clone clusters
primer complementary to the adaptor sequence is extended by on base in the presence of dNTPs whose 3’ end is blocked by an azide group with a modified fluorophore tag
for each cluster, one complementary base is incorporated and imaged, then base and tag are removed to access the next base and the steps are repeated to generate sequence data on a massive scale
Describe the steps of whole exome sequencing.
extraction of genomic DNA sheering of DNA denaturation of DNA ligaton of adaptors to sequences capture of exons by hybridization targets entire set of known genes (can be modified to target just disease associated genes or panels of genes associated with specific conditions)
Provide examples of requirements and limitations for full genome sequencing via NGS.
computational requirements
sequencing alignment
variant identification (mostly for indels)
variant annotation and interpretation
incidental or secondary findings (counseling aspects)
How are VNTRs detected?
accurate determination of repeat number is done by PCR amplification and measurement of PCR fragment length
How are CNVs (low copy repeats) detected?
segmental duplications
FiSH (fluorescence in situ hybridization)
Describe the process of FiSH.
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
What is array comparative genomic hybridization (CGH)?
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 regerence 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-labled molecules bound at each location on the chip is determined by a computer
ratio of 1 indicates normal copy number
ratio of >1 indicates duplication at the region
ratio of <1 indicates deletion at the region
Describe DiGeorge deletion syndrome.
22q11.2
velopharyngeal insufficiency, cleft palate, cardiac defects, mild dysmorphic features, thymic hypoplasia (immunodeficiency), learning disabilities, hypocalcemia (leading to seizures
1 in 4,000 births
Describe Williams Syndrome
deletion 7q11.23
distinctive “elf”facies, “cocktail personality”, cardiovascular disease, intellectual disabilities, endocrine abnormalities
1 in 7,500 to 20,000 births
Describe Wolf-Hirschhorn syndrome.
4p16.3 deletion
fetal growth restriction, microcephaly, hypotonia, characteristic greek helmet facies, severe intellectual disabilities, seizures
1 in 50,000 births
Describe Cri du Chat.
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)
Describe 1p36 deletion syndrome.
hypotonia, developmental delays, growth restriction, obesity, microcephaly, orofacial cleftign, typical facial features, minor cardiac malformations
