M2C Exam 1

Genotype

The genetic constitution of an individual organism

phenotype

set of observable characteristics of an individual resulting from the interaction of its genotype with the environment

-includes disease states (cancer, heart disease)

dysmorphology

study of human birth defects, particularly those affecting the morphology of the individual

minor malformations

- do not impact function

- common in population

-most people have 1-2

- presence of 3+ may indicate syndrome or presence of major malformation

major malformations

- impacts function/health

-more indicated of genetic, but not always (ex: if they have a cleft palate but nothing else)

karyotype

the number and visual appearance of the chromosomes in the cell nuclei of an organism

- confirms diagnosis in something like down syndrome

Exome sequencing

A strategy of sequencing only the coding regions of a genome

- genotype leads to clarified phenotype and prognosis information

allele

one of two or more alternative forms of a gene that arise by mutation and are found at the same place on a chromosome

compound heterozygous (in trans)

-two different variants in the same gene.

-The two variants are on opposite copies of the chromosome—one inherited from mom and one from dad.

compound heterozygous (in cis)

-two different variants in the same gene.

-The two variants are on the same copy of the chromosome—both inherited from either mom OR dad

-more rare

pleiotropy

The ability of a single variant to cause many different traits

Epistasis

the interaction of genes that are not alleles, in particular the suppression of the effect of one such gene by another.

-ex: baldness masks hair color

Locus heterogeneity

mutations at different loci can produce the same phenotype

allelic heterogeneity

Different mutations in the same locus produce the same phenotype

polygenic/ multifactorial

traits that are influenced by a combination of multiple genes/alleles as well as environmental factors

Narrow expressivity

When there is only a small range of possible phenotypes for a particular genotype

variable expressivity

individuals with the same genotype have related phenotypes that vary in intensity

complete penetrance

All people with the allele display the disease phenotype

incomplete penetrance

Not all individuals with a mutant genotype show the mutant phenotype

nuclear DNA

-found in nucleus

- makes up chromosomes

- two copies of each gene

-inherited from both parents

Mitochondrial DNA

DNA found in the mitochondria that is inherited only through mothers

- only contains genes for mitochondrial function

pseudogenes

resemble functional genes but are nonfunctional

- may lack sequences needed for transcription or translation start

-may be due to frameshift variants, splicing variants, nonsense variants

human karyotype

46 chromosomes, 23 pairs

-22 the same between male and female (autosomes)

- arranged by size and by location of the centromere

-21 is actually smaller than 22

what are the acrocentric chromosomes?

13, 14, 15, 21, 22

What is required for transcription?

A promoter and multiple transcription factors

RNA polymerase II does what?

uses DNA as a template to create mRNA

▪ Reads along the anti-sense/non-coding/template strand to create an RNA copy of the sense/coding strand; mRNA synthesized from 5' to 3' (reads the template from 3' to 5').

what is a transcript?

Parts/sequence of a gene that will end up being translated into a protein

• genes can produce more than one transcript, due to alternative splicing

• different transcripts can be influenced due to time in development, tissue type, and environmental factors

Canonical transcript

predominant/common transcript in the body

alternate transcripts

alternatively spliced RNA sequences that are less common, expressed in specific tissues, or at specific timepoints in development

what transcript is used for genetic testing reports?

canonical transcript. They will report variants where the patient’s DNA sequence varies from the canonical RNA transcript

SHANK3: PHELAN-MCDERMID SYNDROME

• caused by a point variant or deletion of SHANK3

• intellectual developmental disorder

• significant behavior phenotypes

• autism w/ regression

• poor speech

• dysmorphic features

• less commonly: seizures and birth defects

• 4 potentially protein-coding transcripts of different lengths (1 canonical, 3 alternate)

GC role with transcripts and variants

• not responsible for determining the appropriate way to look a gene’s sequence

• need to know that different transcripts exist and have changed over time

• need to know that theoretical possibilities exist where a pt may have a variant in a gene that the lab can miss

• need to know to use a lab you trust to navigate these nuances

point mutations

silent

nonsense

missense

insertion-deletions

can be out-of-frame (involving any number of bases other than 3

• causes frameshift

• usually causes a premature stop codon

can be in-frame

What do you think the most deleterious (disease-causing/damaging) types of variants are?

GENERALLY nonsense and frameshift

When may a nonsense or frameshift variant be benign?

if they happen at the end

Is there ever a time a silent mutation can cause disease?

yes, can destroy a splice site

effects of variants in transcription factors

• lack of transcription

• excess transcription

• multi-system disease

• incompatibility with life if responsible for enough gene expression

conditions associated with them: congenital heart disease, DiGeorge syndrome (TBX1), Holt-Oram syndrome (TBX5), PAX6-related disorders, pituitary hypoplasia

CONGENITAL HEART DEFECTS

• most common birth defect

• my by isolated or syndromic

• multifactorial (mostly)

HOLT-ORAM SYNDROME (TBX5)

• congenital heart defect

  • atrial septal defect is most common

• cardiac arrhythmias

• limb anomalies

  • asymmetrical, absent thumb or bifid thumb, radius/ulna abnormalities

• may have other skeletal abnormalities

TBX5 INTERACTIONS - "THE INTERACTOME"

• GATA4 and NKX2.5 - associated with cardiac formation

• NKX2.5 and Id2 – formation of cardiac conduction system

• SHOX2 and BMP4 - cardiac conduction system

• EMT - associated with limb development and differentiation

• SALL4 - upper limb and heart defects

• SCN5A – cardiac conduction defects

Classes of Amino Acids

• Neutral and non-polar (hydrophobic)

• neutrals and polar (hydrophilic)

• charged (also hydrophilic)

what are the branched amino acids

leucine, isoleucine, valine (nonpolar)

which amino acid is the only one capable of making strong disuldfide bonds

cysteine

which amino acid is a common target for post-translational modifications

serine, due to free hydroxyl group

True or false: if one amino acid is substituted for another, it is less likely to be problematic if the amino acids have similar properties

true

Protein Structures

• Primary Structure – sequence of amino acids

• Secondary structure – local structure of amino acids arranged in a repeating pattern, formed by hydrogen bonds (alpha helix, beta sheets)

• Tertiary structure – three-dimensional folding, due to interactions of the different amino acid side chains

• Quaternary structure (if applicable) - multiple chains of amino acids/subunits interacting with each other

What else affects protein folding?

• bonds (disulfide stronger than ionic)

• allosteric effects (how bulky are side chains?)

• environment: acidic, basic, hot, cold?

• binding of co-factors

• chaperone proteins

collagen amino make up

combination of glycine, and a larger molecule such as proline or hydroxyproline

osteogenesis imperfecta (OI)

• brittle bone disease

• dominant condition

• most commonly due to variants in COL1A1 or COL1A2 (collagen making genes)

• can range from mild to neonatally lethal

• besides easy fractures, can cause hearing loss, blue sclera, brittle teeth, short stature

genotype-phenotype correlation of OI

• depends on type of variant

• missense variant= 100% of chains made, 50% made incorrectly

• nonsense variant= 50% of chains made, 100% made correctly

MISSENSE IS WORSE

dominant negative variants

a variant in one allele (and its resulting protein) can interfere with the function of the healthy copy

• OI missense variant

mechanisms of disease

• complete loss of protein or loss of its normal function (recessive disorders, X-linked)

• loss of half the amount of protein/half of its normal function (dominant disorder, haploinsufficiency)

• abnormal protein poisoning the function of the normal copy (dominant negative)

• a new function of the protein (gain of function, hyperactivity)

Nonsense, whole-gene deletions, frameshifts, and indels are almost always going to be more deleterious than missense variants. HOWEVER, what are the exceptions?

• dominant negative

• gain-of-function

• triplosensitivity (having too much protein product)- duplications of genes

domain rules

genetic variants in domains lead to a higher chance that the primary function of the protein is compromised

• outside of domains may disrupt overall protein shape or regulatory interactions, which may stop domains from interacting with their targets

motifs

• recurring tertiary structure of a protein

• proteins of differing functions can have the same motif

• a single protein can have multiple motifs

• not stable on their own

• common: barrels, zinc-fingers

what are domains?

conserved sequences in tertiary structure

G0 phase

cells are at rest, not dividing

G1 phase

cells increase in size, synthesis of RNA and proteins takes place to prepare for replication

S phase

DNA synthesis replicates the genetic material

• each chromosome consists of two sister chromatids

G2 phase

further cell growth, some DNA repair, metabolic changes assemble the cytoplasmic materials necessary for mitosis and cytokinesis

M phase

nuclear division (mitosis) followed by a cell division (cytokinesis)

how is the cell cycle regulated?

proto-oncogenes

tumor suppressor genes

proto-oncogenes

promotes cells growth and survival

• inhibits apoptosis

• growth factors

tumor suppressor genes

prevent uncontrolled cell growth

• restricts cell division

• repairs DNA errors

• activates apoptosis

Cell Cycle Regulation: Check points

• cell size

• DNA replication

• DNA damage mitotic check points

Cell Cycle Regulation: proteins

• cyclins

• cyclin-dependent kinases (CDKs)

• p53

mosaicism

the presence of two or more cell lineages with different genotypes arising from a single zygote in a single individual

• random, hard to predict which parts of body will be affected

• earlier on in development mutation occurs, the greater percentage of the body that will be affects

• due to mutation during DNA replication or aneuploidy during cell division

sister chromatids

two identical copies of one chromosome produced during DNA replication

euploid

containing the normal entire set of chromosomes for an organism

diploid

two sets of every chromosome (default state for somatic cells)

haploid

having one set of every chromosome (default for germline cells/gametes)

hallmarks of cancer

• sustaining proliferative signaling

• genome instability & mutation

• resisting cell death

• evading growth suppressors

• avoiding immune destruction

True or false: all cancer is genetic

true.

but not all cancers are due to inherited mutations… but all are due to mutations in cell cycle

when does meiosis occur

• occurs continuously in males in testes

• in females, begins as a fetus, but eggs are frozen after meiosis 1 until puberty

  • each month after, one egg resumes meiosis and goes through meiosis 2 to create an egg that is ovulated

survivable aneuploidy

trisomy: 13, 18, 21, X, Y

monosomy: X

what is aneuploidy caused by?

nondisjunction

• the failure of one or more pairs of homologous chromosomes or sister chromatids to separate normally during nuclear division, usually resulting in an abnormal distribution of chromosomes in the daughter nuclei (mitosis→ mosaicism)

Germline/Gonadal Mosaicism

• variants occur at a higher number in the sperm or egg cells but not in the rest of the body (parent’s blood sample is negative)

• results in higher risk of having multiple children with a “de novo” condition

• this is the reason that we can never give a 0% recurrence risk

• not really possible to test for

mosaicism in families

• in a parent= risk for constitutional (present in every cell) condition in the child

• in child= implies variant occurred after conception and is not present in the parent

Turner syndrome

• short stature, skeletal difference

• heart problems

• learning disabilities

• delayed or absent puberty, infertility

• horseshoe kidney, webbed neck, lymphedema

• increased risk for autoimmune disease

Causes

• 45, X – 50%

• 45,X/46,XX or 45,X/46,XY or 45,X/47, XXX – 20-30%

• Structural abnormalities of the X chromosome (ring, Xq, etc) – 20-30%

Mosaicism in Turner syndrome

• can be as severely affected as those who are fully monosomy X

• myth that boys cannot have Turner syndrome

• no way of telling which organs carry a high or low burden

recombination

“genetic shuffling”

pairs of homologous chromosomes exchange info

important in maintaining genetic diversity

recombination rates

a cross over typically happens at least once per chromosome

• more frequent in females

• hotspots in the genome where crossing over is more likely

• frequency of recombination between two genes is used to determine how close they are to each other (the closer, the less likely crossing over will occur between them)

Chromosome deletions and duplications – aka Copy Number Variants (CNVs)

• occurs due to wrong alignment between homologous regions of chromosomes

• ranges in size

• recurrent/hotspot areas prone to deletions and duplications

• may be at the end of chromosome (terminal) or in the middle (interstitial)

• we all have benign CNVs

syndromes of CNVs

22q11.2 deletion syndrome, Smith Magenis syndrome (deletion of 17p11.2), 1p36 syndrome, Williams syndrome (deletion of 7q11.23), Wolf-Hirschhorn syndrome

• developmental delays, autism, birth defects, congenital anomalies

• behavioral differences, poor growth, endocrine abnormalities, dysmorphic features

why are CNVs inherited in a dominant manner?

because whole chromosomes are passed from parent to child, or X-linked

balanced translocation

occurs between two non-homologous chromosomes

• usually do not have health consequences

• occurs in about 0.18% people

• at risk of having children with unbalanced translocations

unbalanced translocations

when you see a duplication of the terminal END of a chromosome and a deletion of the END of another is a big red flag

• oftentimes results in large genetic imbalances

• usually unique

robertsonian translocations

results in the q arms of 2 acrocentric chromosomes joining at the centromere

p arms lost

• balanced, 45 chromosomes

Uniparental Disomy (UPD)

both copies of a chromosome come from the same parents (2 from egg or 2 from sperm)

UPD: heterodisomy

child inherits both chromosome copies from the same parent, but they are different homologs

UPD: isodisomy

child inherits both chromosome copies from the same parent, and they are the sister chromatids

UPD consequences

imprinting disorders (prader-willi, angelman, beckwith-weidemann)

• sometimes you need a copy from egg AND sperm in order for genes to function correctly. even if there are no variants in the gene sequence themselves

increased risk for recessive disorders

how does UPD occur?

Monosomy rescue: results in isodisomy

• initially, only had one copy of a chromosome and the cell copies its one copy

trisomy rescue: results in isodisomy or heterodisomy

• initially, had three copies of a chromosome and the cell kicked out one copy out at random

paracentric inversion

does not include centromere

• crossover occurs between one normal homolog and one inverted one

• recombinant chromosomes contain both a deletion and duplication

• may have no centromere or may have two centromeres

pericentric inversions

involves centromere

• crossover occurs between one normal homolog and one inverted homolog

• recombinant chromosomes contain both a deletion and duplication

• each recombinant contains one centromere

What type of inversion has a higher chance of a child with a birth defect?

Pericentric inversions

• paracentric are more likely to miscarry and not be brought to term

exogenous agents

DNA damage caused by external exposures

• ionizing radiation

• ultraviolet radiation

• pollutants in air, water, and food

• chemical carcinogens in tobacco products, pesticides, and toxic metals

endogenous agents

DNA damage caused by internal reactions during oxidative stress, metabolic processes, and the inflammatory response

• reactive oxygen species (ROS)

• reactive nitrogen species (RNS)

• depurination, depyrimidination, deamination

DNA damage response (DDR)

1. ALERT: DNA damage sensor proteins

2. RESPOND: proteins act as signal transducers and mediators to the repair machinery

3. RESULT: effector pathways include DNA repair, cell cycle arrest, senescence, apoptosis