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Summary NWI-BM072 Translational genomics + SSA notes
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Summary of all lectures of translational genomics + SSA notes
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Week 1LE: Genome architecture
There is DNA in the nucleus and in the mitochondria. There are 22 pairs of autosomes, 2
sex chromosomes, 20,000 genes that are coding and 25,000 non-coding genes.
First exon of a gene has a lot of G-C hydrogen bonds, that is why it is difficult to sequence
the first codon. It contains a lot of G-C bindings, because it needs a spot for the ribosome to
bind on the mRNA. In functional DNA there are protein coding genes, non-coding genes and
regulatory elements. One gene can have different isoforms, this is caused by MAPT splicing.
5’ UTR is a sequence that is recognized by the ribosome which allows the ribosome to bind
and initiate translation. The 3' UTR is found immediately following the translation stop codon.
The 5’ UTR is shorter than the 3’ UTR.
Small ncRNA
Long gene that encodes small precursors, like miRNA precursors. Gene is transcribed in
mRNA and cut in smaller pieces that can form hairpins like miRNA precursors and siRNA
are mostly double stranded or hairpin.Processed by dicer to 22 nt miRNA and 21 nt siRNA.
miRNA are used to recognize other RNAs and DNAs that they can bind to and are guided by
Argonaute.
→ Inhibition of translation, inhibition of translation elongation and mRNA deadenylation.
Long non-coding RNA
NAT is a natural antisense transcript on the antisense strand. They influence expression of
the sense strand. Also have intronic, intergenic, decoy, guiding and scaffolding lncRNA.
They can bind to proteins, DNA and RNA (also miRNA).
Regulatory elements are like a core promoter, which has different elements. There are also
elements that are after the promoter. Lnr is where transcription starts.
Non-coding RNA
Transposable elements (TEs) are 45% of the human genome and only 0.05% is active. The
most abundant one is Alu elements. They are around the genes that are brought together
and they are controlled together in expression. → structure of the genome. They alter
regulatory networks, gene expression, and to rearrange genomes as a result of their
transposition.
Genomic imprinting the most extreme example of the effect on gene expression is
X-chromosome inactivation, this is an epigenetic modification.. This is at the 3000 cell
stage you get inactivation of half of the genome. In the end you get only the X of the mother
is inactivated → Barr body. DNA methylation causes genomic imprinting, which then causes
inactivation.
Genomic imprinting is essential for normal development and deregulation results in
diseases, like Pradar -Willi syndrome. Same deletion on a chromosome can cause different
syndromes, depending on if the deletion is on the maternal or paternal chromosome.
When the line in the genome browser is thicker it codes for protein.
,LE: Genome variation
Polymorphism: variation >1% of the alleles in a population
Mutation: variation <1% of the alleles in a population
SNVs and Indels
Silent changes: substitution but amino acid stays the same c.6T>C and p.(=)
Missense changes: substitution that changes amino acid c.4T>C and p.Tyr2His)
Nonsense changes: substitution causes stop codon c.10T>C and p.(Arg4*)
Frameshift changes: deletion causes reading frame c.13delA and p.(Met5fs*)
Frameshift changes: insertion causes reading frame c.13dupA and p.(Met5Asnfs*)
In frame deletion: deletion that is in frame c.10_12delCGA and p.(Arg4del)
Splicing: splicing of an intron c.33-1G>A and p.(?)
c stands for coding level and p stands for protein level.
Acceptor splice site is at 3’ of an intron, mostly AG.
Donor splice site is at 5’ of intron, mostly GT.
Down syndrome has an extra chromosome 21. Can be causes by:
- An extra isolated chromosome 21
- Roberstonian translocation: chromosome 21 attaches e.g. to chromosome 13 or 14
- Reciprocal translocations: only if a big part of chromosome 21 is exchanges for a
small nonessential part of another chromosome
- Duplication of the Down syndrome critical region on chromosome 21 (rarely)
Chromosome 21 has fewer genes than chromosome 13 and 18, so it is better to have a
trisomy of 21 than the other two, because they have more severe consequences.
Translocations
Example of a translocation notation: t(14,21)(q21;p31). P is the short arm
and q the long arm. They exchange DNA.
Robertsonian translocation is on the acrocentric chromosomes, so
13/14/15/21/22. Chromosomes don’t have a real P-arm, no protein coding
genes on there. So two chromosomes can be merged together. Fused P
arms won’t be visible, because they are so small.
Reciprocal translocation can be on each chromosome and parts of the q
or p arms can be switched on chromosomes.
Deletions and inversions
Interstitial deletions which lack genes.
Terminal deletions: ring chromosomes → telomeres deleted, no protective
mechanism at the end of the chromosome.
Pericentric inversion: is chromosomal aberration at both sides of the centromere.
Paracentric inversion: is on 2 p or q arms.
The Isochromosome consists of 2 copies of the long or short arms, so two centromeres.
ESAC/SMC are small and difficult to identify and can result in severe intellectual disability.
, Week 2
LE: Determination of variation
Genome-wide determination of variation
Karyotyping (CNV → del/dup >1 kb)
For karyotyping you need dividing cells, such as prenatally (fluid of the womb). These come
from the embryo itself, like fibroblasts. Can also take cells from the placenta, but this can be
mosaic. So the placenta can have trisomy 21 for example and the embryo can be normal.
Other options are postnatally, like blood cells (whole blood cells) and a skin biopsy
(fibroblasts), these are also dividing cells.
Three types of chromosomes
- Metacentric: short arm and long arm similar (chromosome 1/3/16/19/20)
- Submetacentric: Centromere is a bit more higher
- Acrocentric: only a long arm (with protein coding genes) and satellite arms that code
for ribosomal RNA (chromosome 13,14,15,21,22). All these satellite arms are in the
nucleus → these chromosomes can form robertsonian translocations.
Chromosomal microarray analysis CMA (CNV)
Patient DNA and reference DNA both are labeled with a
fluorophore, combine them and hybridize DNA.
It can detect whole chromosome aneuploidies and CNVs but
can’t detect balanced translocations.
Exome sequencing (CNV/SNV/indel/SCA/aneuploidies)
This sequences the exons, small fragments of 100-200 bp but can be used when disease
causing mutation is unknown. Difficult to use when the deletions are big, because the reads
are short.
Local determination of variation
FISH (CNV/aneuploidies/translocations → need dividing cells (metaphase))
It can detect aneuploidies, CNVs but also translocations. They separate by
denaturation of the DNA and then hybridize the DNA with a probe.
Derivatives (translocations) can be detected by small dots on other
chromosomes.
In this case chromosome X gets activated, so that there are active copies of
chromosome 7 and one of chromosome X.
Sanger sequencing (SNV)
Template DNA with different reagents and the primers anneal to the template DNA and
cause chain extension. At the end there will be chain termination. Fluorescent labels will bind
to the DNA sample (short fragments are each labeled). Separate the DNA by electrophoresis
and look at the sequence by analysis.
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