Genetics Block Bev's Cards WEEK ONE

Week slide info with all the practice problems from workshop, quiz and practice problem worksheet

Central dogma

DNA to RNA to Protein

how many autosome pairs do humans have

22

Over half of the genome is

highly repetitive

How many of our genes code protein?

<2%

DNA sequence of any 2 individuals is

99.9% identical

tandem repeats

repeated DNA sequences are next to each other (adjacent).

Example: CAG-CAG-CAG-CAG

Dispersed repeats

Interspersed/dispersed repeats: repeated sequences are spread out in different locations in the genome.

Example: CAG-XXXXXX-CAG-XXXXXXXX-CAG

single copy sequence

• Protein coding sequence: ~1.5% genome

• Introns and noncoding RNAs: ~40%

• Gene Families

• Pseudogenes

Microsatellites

aka Short tandem repeats (STRs)

Clusters of very short tandem repeats, 2-6 bp

• (CA)n, where n= 5 to several hundred

Highly polymorphic (means that the number of repeat units can vary a lot between different people) – many possible numbers of repeats

• Analyze by PCR using primers

Dispersed repeats

• SINEs: Short repeated sequences scattered throughout genome

• LINE: Long repeated sequences scattered throughout genome

• Low copy repeats (LCR): Large DNA sequences repeated

adenine

Thymine

Cytosine

Guanine

Uracil

Chargaff’s Rules

%A ≈ %T and %G ≈ %C

The discovery of the double helix nature of DNA

Rosalind Franklin had been using x-ray crystallography to characterize the

structure of DNA

How does this DNA fit into such a small volume?

DNA is organized by histones & nucleosomes

These subunits can be modified post-translationally to affect chromatin accessibility and gene expression

• 8 histone subunits make up a nucleosome

RNA primase synthesizes

short priming sequences for DNA replication

DNA polymerase I

adds nucleotides in the 5’ -> 3’ direction

lagging strand requires formation of

Okazaki fragments and ligation activity to seal the nicks

RNA can be hydrolyzed due to

the 2’ OH

Regulation of transcription and production of mRNA (What determines whether a gene gets turned ON and an mRNA gets made?)

• Enhancers

• Core promoter

• TATA Box

• Transcription factors

• Transcript start site

Enhancers

An enhancer is a region of DNA that can increase transcription of a gene

Enhancers contain binding sites for transcription factors

an enhancer doesn't necessarily have to be right next to the gene

Core promoter

the region of DNA right around the beginning of a gene where the transcription machinery assembles.

TATA box

a specific DNA sequence found in the core promoter of some genes.

Proteins recognize this region and help position RNA Polymerase II correctly.

Transcription factors

Transcription factors are proteins that regulate transcription.

They bind to specific DNA sequences and can either:

Increase transcription → activators

Decrease transcription → repressors

mRNAs

make up ~5% of the total RNA in the cell and encode proteins

mRNAs are transcribed by

RNA polymerase II

mRNAs typically have (2 things)

• Almost all mRNAs in human undergo (blank)

mRNAs typically have a 5’ cap and a poly-adenine tail

• Almost all mRNAs in human undergo splicing

The 5’ cap protects the 5’ end of mRNA from degradation, and also recruits

translation initiation factors

Alternative splicing

Same DNA/gene → same initial pre-mRNA → different splicing → different mature mRNAs → different proteins

Nonsense-mediated decay

NMD detects certain mRNAs that contain a premature stop codon (>55 nucleotides away from the next splice junction) and destroys the mRNA

Splicing can be dysregulated in disease (disease example)

Hutchinson-Gilford progeria syndrome is caused by a genetic typo in the LMNA gene

Splicing can also be manipulated to treat diease

SMA patient treated with nusinersen (modifies the SMN2 gene)

RNA processing – cleavage& polyadenylation

mRNAs are cleaved and then polyadenylated by protein complexes

The polyA tail protects the 3’ end of the RNA from degradation

Ribosomal RNA makes of (% of RNA) and is transcribed by what?

Ribosomal RNA makes of >90% of the RNA in the cell

Ribosomal RNA is transcribed by RNA polymerase I

tRNA

• tRNAs are “charged” with amino acids

• tRNAs have an anti-codon loop that is complementary to mRNA

• tRNAs are transcribed by RNA polymerase III

• Some tRNAs must be spliced to be functional

Mutations in tRNA splicing machinery can cause

pontocerebellar hypoplasia and other diseases

Speed of translation can be influenced by

codon identity and tRNA abundance (which is why silent mutations can still be harmful)

Translation initiation

• Initiation factors recognize the 5’ cap

• The 40s subunit is recruited and begins scanning for AUG

• Upon reaching an AUG with a suitable Kozak sequence, the 60s subunit joins

Translation Elongation

• The tRNA in the peptidyl (P) site is the one that is charged with the growing polypeptide chain

• An amino-acylated tRNA is delivered to the aminoacyl (A) site

• A new peptide bond is formed between the polypeptide chain and the charged tRNA in the A site

• tRNAs in the P and A site translocate into the exit (E) and P sites, respectively

Translation termination

• Eukaryotic release factor 1 (eRF1) recognizes the stop codon (UAG, UGA, UAA)

• Together with eRF3, it facilitates hydrolysis to release the nascent peptide chain

Proteins can be regulated post-translationally

• Ubiquitination

• Phosphorylation

• Sumoylation

• Dimerization

• Protein complex formation

MicroRNA

microRNAs are ~21 nucleotide long non-coding RNAs that exert regulatory activities on other RNA

Missense variant

different amino acid

Nonsense variant

stop codon

Frame-shift

change reading frame of message

DNA → RNA

transcription

RNA → protein

translation

“Variant” versus “Mutation”

avoid negative connotations of the word “mutation”

5 Classifiers are used to describe the effect of a variant

Pathogenic (P)

Likely

Pathogenic (LP)

Variant of Uncertain Significance (VUS)

Likely Benign (LB)

Benign (B)

SNVs

Single Nucleotide Variant

Genomic nomenclature

based on sequencing ALL DNA (intergenic, intragenic, exons, and introns

NC- Nomenclature Chromosome

LMNA NC_000001.11:g.156138613C>T

cDNA nomenclature

based on exons OR exons + some intronic (+/-100bp)(exome sequencing)

NM- Nomenclature mRNA

Example: LMNA NM_170707.4:c.1824C>T

Protein nomenclature

based on amino acid change resulting from DNA variant(s)

Example: LMNA NM_170707.4:c.1824C>T p.(G608G) OR p.(G608=)

cDNA – what is it?

DNA complementary to the mRNA sequence (rna degrades too quickly)

+ (plus) Nomenclature rule

indicate intronic variants;

c.123+45A>G = 45 nucleotide after the previous exon’s last nucleotide

(You are MOVING FORWARD)

- (minus) Nomenclature rule

used in nucleotide numbering, indicate intronic OR promoter/5’UTR variants;

c.124-56C>T = 56 nucleotides before the next exon’s first nucleotide

(YOU ARE MOVING BACK)

+1 or +2 in nomenclature (general area)

5′ splice donor site

-1 or -2 in nomenclature (general area)

3′ splice acceptor site

* (asterisk) nomenclature

to indicate a translation termination (stop)

examples:

c.*32G>A A G was changed to an A, 32 DNA letters after the stop codon

p.Trp41*

c.*32G>A

A G was changed to an A, 32 DNA letters after the stop codon

p.Trp41*

At position 41 of the protein, the amino acid tryptophan (Trp) was changed into a STOP signal

_ (underscore) nomenclature

is used to indicate a range; g.12345_12678del

fs nomenclature

indicates a frameshift

p.Arg456Glyfs*17

At amino acid 456, the sequence gets shifted, changing Arg to Gly, and the new incorrect sequence continues for 17 amino acids before hitting a STOP

Variant Allele Frequency (VAF)

% of cells in which a variant is found

VAF of 0.5 or 50%

means the variant is heterozygous in 100% of cells

VAF >50%

may indicate homozygosity and/or deletion of the other allele

VAF ~30-60%

may also be germline in a neoplastic assay

haploinsufficiency

happens when a single working copy of a gene is not enough to keep the body working normally

NONSENSE VARIANTS

Create premature stop codons

Abnormal mRNA may undergo (blank) IF the variant is >55nt upstream of the natural stop codon

nonsense-mediated decay (NMD) ,

this means no protein will be made

<55nt to natural STOP

truncated protein (abnormal, shortened protein) could lack function, may not be localized properly, or cause other problems

LMNA-related dilated cardiomyopathy (DCM) driven by

haploinsufficiency (AD) occurs when a single functional copy of the LMNA gene is insufficient to produce normal levels of nuclear lamin A/C proteins

nuclear lamina

which is involved in maintaining nuclear structure, chromatin organization, and gene regulation

INDELS

insertion/deletion

Can cause FRAMESHIFT variants

DMD/BMD (X-linked)

Most patients have large deletions of multiple exons

In-frame = Becker Muscular Dystrophy (milder symptoms)

Out-of-frame = Duchenne Muscular Dystrophy

CNVs

Copy Number Variants-the number of copies of a DNA segment varies between people.

Deletion or duplication

Copy Number Variants (CNVs): Deletion Disease example

Deletion of one or more copies of HBA or HBB can result in thalassemia traits or disease

Copy Number Variants (CNVs): Duplication Diseased Example

MECP2 Duplication Syndrome (X-linked)

How can synonymous variants cause phenotypic effects if the amino acid does not change?

Affect splicing & mRNA stability/regulation & Affect translation efficiency

SMA (Spinal Muscular Atrophy)

In people with SMA, both copies of the SMN1 gene are missing or faulty

SMA is an autosomal recessive condition

Our “backup” SMNA2, the more copies an SMA patient has, the better the outcomes

SNPs

Single Nucleotide Polymorphisms

To be polymorphic, at least 1% of the population has a variant allele (major vs minor) without obvious phenotypic effect

MISSENSE variants can have many effects

Loss of Function

Gain of Function

Dominant Negative

Stop-Loss

Loss-of-function

(Autosomal Recessive/Autosomal Dominant)

if one copy of a functional gene is sufficient, Heterozygotes (carriers) are NOT affected

Gain of function

Autosomal Dominant

results in protein with a new function OR excessive normal function (hypermorph)

Dominant negative

Autosomal Dominant

results in abnormal gene product affecting the function of the NORMAL gene product from the other allele

Stop-loss

Autosomal Dominant

results in loss of a stop codon, causing readthrough; may or may not result in an abnormal gene product

NF1

Neurofibromatosis type 1 (NF1)

MISSENSE AD Loss Of Function (LOF)

one of the most common AD genetic conditions

Familial Hypercholesterolemia

MISSENSE AD Loss Of Function (LOF)

Co-dominance – Both alleles in a trait are fully expressed but heterozygous and homozygous carriers may have different phenotypes

Sickle Cell Anemia

MISSENSE Autosomal Recessive LOF

(p.Glu7Val)

Achondroplasia

MISSENSE AD Gain-of-function (GOF)

FGFR3 gene

DOMINANT NEGATIVE Variants

4 Mechanisms:

1. Structural Disruption: Abnormal protein integrates into a multi-protein

complex and weakens the integrity of the complex

2. Competitive Inhibition: competes with the WT protein for binding, but does not

function properly, if at all

3. Sequestration: Abnormal protein prevents the normal protein (or another protein)

from functioning

4. Multimer interference: Abnormal protein subunit can still form multimer, but it

prevents the complex from functioning

Osteogenesis Imperfecta

COL1A1

MISSENSE - Dominant Negative - Structural Disruption

Pathogenic missense SNVs alter the structural integrity of the collagen triple helix

Hutchison-Guilford Progeria syndrome

MISSENSE - Dominant Negative - Structural Disruption

C to T missense

Cryptic Splicing

von Willebrand Disease

MISSENSE - Dominant Negative- Competitive Inhibition

The most common inherited bleeding disorder

Abnormal vWF and wild type vWF both compete for platelet (PLT) binding

PCSK9-mediated familial hypercholesterolemia

MISSENSE - Dominant Negative- Sequestration

variants make abnormal pro-PCSK9 that is not cleaved and sequesters the normal PCSK9, preventing both from leaving

the Endoplasmic Reticulum

Abnormal p53 protein

MISSENSE - Dominant Negative - Multimer Interference

forms the tetramer with wild type and prevents p53 tumor suppressor activity

Can evolve to homozygous into neoplasia

Li-Fraumeni Syndrome