Introduction to development; Human genetic

Question 1

What are the 3 Mendel Laws?

Answer 1

1. Law of dominance
2. Law of segregation
3. Law of independent assortment

Question 2

What did the Law of dominance say?

Answer 2

The law says if there are two differing alleles at a a locus, then the dominant allele determines the organism’s phenotype and the recessive allele has no noticeable effect. (This is mostly true…)

Question 3

What does the Law of Segregation say?

Answer 3

Two alleles that can be inherited, seperate randomly during gamete formation and end up in different gametes.

Question 4

What does the law of independent assortment say?

Answer 4

Each pair of alleles seperate independent of another pair of alleles during gamete formation (i.e one trait cannot be inherited with another trait). This is mostly true…

Question 5

What is the gerrmline?

Answer 5

Cell line from which gametes are developed. Germ cells are set aside in the embryo, migrate to developing gonads and undergo cell division and differentiation to sperm and ova

Question 6

Is a zygote diploid or haploid?

Answer 6

Zygote is diploid, 23 paternal + 23 maternal chr

Question 7

Are gametes haploid or diploid?

Answer 7

Gametes are haploid, 23 chr. Diploid organisms can produce 2n different gametes (223 = 8 388 608)

Question 8

What does phenotype mean?

Answer 8

The expression of alleles of a gene carried by a organism

Question 9

What does genotype mean?

Answer 9

The combination of alleles of a gene carried by an organism (e.g AA or Cc)

Question 10

What does loci mean?

Answer 10

Specific positions of alleles/genes on a chromosome

Question 11

What does “alleles” mean?

Answer 11

Different versions of a gene. Dominant alleles =capital letter. Recessive alleles =lowercase letter

Question 12

What is cytogenetics?

Answer 12

Structure, properties and behaviour of chromosomes

Question 13

What do karyotypes do and how can they be used in diagnostics?

Answer 13

Used to determine chromosome number and structure. Can detect copy number aberrations and large structural rearrangement (e.g in cancer cells)

Question 14

How is the line between cytogenetics and molecular genetics blurring?

Answer 14

Molecular methods can detect cytogenetic abnormalities. E.g microarry for submicroscopic deletions and duplications

Question 15

What is “molecular genetics” the study of?

Answer 15

Studies structure & function of genes at a molecular level

Uses molecular biology & genetics

Inter-relationship between DNA, RNA & synthesis of
polypeptides

Tests typically DNA- or RNA- based

Question 16

What is epigenetics?

Answer 16

Heritable changes in gene expression that do not involve changes to the underlying DNA sequence.

= Change in phenotype without change in genotype.

Question 17

What factors can epigenetics be influenced by?

Answer 17

Age, environment,lifestyle, disease state

Question 18

How can an organism be influenced by factors such as age or environment to result in a change in phenotype without a change in genotype?

Answer 18

Epigenetics- Despite all nucleated cells containing the same complement of DNA, each cell type expresses a different complement of genes – turning genes on and off in different tissues, developmental time points and/or in response to stimuli.

Question 19

What is an example of an epigenetic modification?

Answer 19

Some examples of epigenetic modification include DNA methylation (often associated with gene silencing) and histone modification – neither changes the DNA sequence, but both change the way the gene is expressed.

Question 20

What are some examples of single gene/Mendelian disorders?

Answer 20

Down syndrome
Cystic fibrosis
Sickle cell disease

Question 21

What are some examples of complex genetic diseases?

Answer 21

Diabetes (type 2)
Alzheimer’s disease
Cardiovascular disease
Obesity

Question 22

What are some environmental diseases?

Answer 22

Allergies and asthma
Mesothelioma
Influenza

Question 23

What is a germline mutation?

Answer 23

Can be passed on to offspring (all cells - any tissue from
that patient can be sampled for genetic testing).
Genetic predisposition to certain cancers (e.g.
BRCA1, BRCA2)

Question 24

What is somatic mutation?

Answer 24

Not inherited

Mutation will only be in affected cells, not all tissues

Accumulation of somatic mutations in DNA over time — causes cancer

Question 25

What are de novo mutations?

Answer 25

De novo = new. A variant that arises in the germ cell
or the fertilized egg early in development. The variant
is not present in the parent but is present for the first
time in their offspring.

Question 26

Human traits are either:

Answer 26

Monogenic: controlled by a single gene (Mendelian traits)

• Change in 1 gene sufficient for disease
• 1 gene -> 1 disease or multiple diseases (pleitrophic)
• Multiple genes -> same disorder (heterogeneity)
• Not influenced by environment

Polygenic: controlled by many genes (Complex traits)

• Can involve changes in 1000’s of genes acting in combination
• are influenced by the environment
• Cardiovascular disease, Schizophrenia, height

Question 27

What is OMIN?

Answer 27

A database- Online Mendelian Inheritance in Man (OMIM): https://omim.org
>16,112 genes
>6,452 phenotypes described, molecular basis known
>3,000 phenotype/locus, molecular basis unknown

Question 28

Single gene disorders are often cause by:

Answer 28

Mutations (rare in population)

Question 29

For complex diseases we are often dealing with:

Answer 29

polymorphisms (common in the population >1% frequency). Once a polymorphisms has been linked with or associated with a complex disease it is called a risk allele. On it’s own it does not cause disease, but contributes a small increase in risk of developing disease. (example?)

Question 30

What is an example of a risk allele?

Answer 30

Example: Risk of developing deep vein thrombosis (DVT) is
modified by the copy number of the a mutation in the Factor V gene, termed Factor V Lieden (FVL).

Population risk of DVT = 1/1000

Risk if you are heterozygous for FVL = 1/200.

About 5% of Caucasians are FVL heterozygous

Risk if you are homozygous for FVL = 1/20

The relative risk of developing a DVT seems high but the absolute risk of having a DVT is still quite low. Having the variant doesn’t guarantee you will have a DVT, but you have an increased risk compared to the general population.

Question 31

Why is gene expression fundamental to cell differentiation and development?

Answer 31

We go from 2 haploid germ cells to a whole organism. Cells have to differentiate. Each somatic cell (except a few) has same DNA but express different RNA and protein. Gene expression and its regulation is fundamental to this and cell differentiation.

Question 32

Why is transcriptional regulation important?

Answer 32

• Basic biology- new insights into human development and regulation (can be used for novel application)
• Genetic diagnosis: enables us to pinpoint the cause when things go wrong
• New treatments for disease: insights to cause and pathobiology of disease enables the rational design of treatment which target the underlying molecular mechanism.

Question 33

Explain why very few mutations occur?

Answer 33

DNA is stable, near perfect fidelity but mistakes do happen- causing mutations and evolution

Question 34

The central dogma:

Answer 34

DNA—mRNA—protein

(The same genes are present in (almost) every somatic cell type BUT each cell type is very different. Each cell type expresses a unique set of genes)

Question 35

Give two examples of proteins/enzymes that are only expressed in certain cells due to gene expression in different cell types.

Answer 35

• Beta-actin= major component of cytoskeleton. Expressed in all cells (housekeeping gene)

Enzyme Tyrosine aminotransferase. Expressed in Liver Hepatocytes not others

(In order to precisely turn on and off the correct sets of genes for each cell type, gene expression is regulated at many levels.)

Question 36

Differential gene expression from the same nuclear repertoire is accomplished by regulation at several levels. What are these six ways?

Answer 36

1. Transcriptional control
2. RNA processing control
3. RNA transport and localisation control
4. Translation control
5. mrna degradation control
6. protein activity control

Question 37

What are some factors influencing gene expression

Answer 37

-Signals

(cell-cell contact, hormones morphogènes- man are TF factors)

• Transcription factors
• (often synthesised in response to external stimuli )
• (two main categories)- general or cell/tissue specific

Question 38

What are transcription factors?

Answer 38

• A transcription factor is a protein that binds to the regulatory element (e.g promoter or enhancer or both) of a gene to either contribute to activation or repression of that gene.

During development, TFs can act in hierarchical cascades to direct appropriate lineage restriction.

Question 39

What are the modes of action of transcription factors?

Answer 39

1. Feed Forward
2. Single input
3. Simple cascade

Question 40

What are master regulators?

Answer 40

• Key drivers of of particular cell fates ( e.g Myod- master regulator for muscle cells)
• Often act by activating the transcription of additional key transcription factors, thereby initiating a cascade of transcriptional events
• can also be auto-regulatory - they promote their own transcription which reinforces and strengthens the commitment to the specific lineage they regulate.

Question 41

Give an example of when transcriptional regulation could go wrong

Answer 41

During development transcriptional program must strike a balance between proliferation and differentiation

Mutation in MYOD1 causes embryonal rhabdomyosarcoma!

• MYOD1 Leu122Arg mutant binds to MYOD1 consensus
sites but does not activate transcription, thereby competing
with wild-type MYOD1 and preventing differentiation

• The Leu122Arg mutant also makes MYOD1 more similar to
MYC (another transcription factor in the same family, which
is associated with increased proliferation) and a shift to a
MYC-like transcriptional program is observed

Question 42

Master regulators are highly conserved. These master regulatory circuits are frequently highly conserved. Give an example of this:

Answer 42

Pax-6 gene.

Question 43

Transcriptional control is considered the primary means by which gene expression is regulated. But Transcription is also regulated at various levels. Name these:

Answer 43

1, Availability/concertation/localisation of TFs

2. Competition between positive and negative regulatory factors
3. Local chromatin conformation (and epigenetics)
4. Large-scale chromatin conformation
5. The nuclear architecture

The promoter integrates all the internal and external signals to determine the probability that the gene will be transcribed.

Question 44

Demonstration of transcription factor availability:

Answer 44

Rapid translocation of a calcium responsive transcription factor NFATc1 within minutes of addition of ionomycin which increases calcium concerntration in the cell.

Question 45

Eukaryotic genes are controlled by combinations of transcription factors binding to DNA binding sites (cis-elements ) in the promoter and other regulatory regions called “Combinatorial control”. This is dependent on:

Answer 45

1. The DNA sequence present
2. The transcription factors present
3. The interactions between regulatory proteins

Question 46

Transcription is also regulated by chromatin architecture. Explain how this works

Answer 46

Before TFs can bind to the DNA and exert their regulatory function, they must first be able to access the DNA i.e the chromatin must be in an open conformation

The position of histone proteins relative to the DNA sequence can influence the ability of a TF to bind to DNA

Question 47

Chromatin conformation is also regulated at many levels!

Answer 47

Local: Enhancer-promoter looping, boundary elements, epigenetics

Large-scale: Topologically Associating Domains (TADs), Nuclear positioning (chromosome territories)

Question 48

3D chromosome architecture

Answer 48

Larger scale (Megabases) loops also form which cluster genes into different compartment called Topologically Associating Domains (TAD)

Within a single TAD, multiple enhancer -promoter loops can form. Few interactions are observed between TADs

Tad boundaries prevent the spread of epigenetic marks between TADs

Question 49

What are chromosome territories?

Answer 49

TADs are organised within the nucleus into active and repressed compartments and chromosomes are organised into territories.

Question 50

Why is 3D structure of the chromatin important?

Answer 50

Important to restrict which enhancers can come into contact with which genes at specific times during development and in specific locations in the developing embryo. Tu ensure the correct pattern of gene expression in each cell.

It is now becoming recognised in that mutations in enhancers and regulatory regions also cause disease- not just mutations in protein-coding sequences. ( E.g 3D chromatin architecture and limb morphogenesis)

Question 51

Describe the example where alteration in 3D chromatin architecture can result in limb malformation?

Answer 51

• The Pitx1 transcription factor is important in limb
morphogenesis

• Expression of Pitx1 is controlled by an enhancer (Pen)
• Pitx1 is expressed only in the hindlimbs

• But the enhancer Pen is active in both forelimbs and
hindlimbs

• In forelimbs, Pitx1 and Pen are physically separated
• In hindlimbs, Pitx1 and Pen are in physical proximity
• If you invert the chromosomal region containing the Pen enhancer, Pitx1 is expressed in forelimbs
• This causes the forelimb to develop morphological characteristics of the hindlimb

This mimics a human condition, Liebenberg syndrome, in which bones and other tissues in the elbows, forearms, wrists, and hands have characteristics of related structures in the lower limbs. Human patients have a deletion next to Pen which disrupts chromatin organisation at the Pitx1 locus

Question 52

What are the requirements for gene expression?

Answer 52

1. Transcription factor/s transcribed in response to stimuli
2. Transcription factor/s activated/transported to the nucleus in
   response to stimuli
3. Large chromosomal segment containing target gene/s is
   sequestered into an active chromosomal territory
4. MB sized chromosomal segment containing target gene/s is
   localised within an ‘active’ TAD
5. Within the ‘active’ TAD, histone modifications are deposited
   on local chromatin and regulatory regions becomes
   accessible
6. Permissive enhancer-promoter looping brings regulatory
   regions together
7. If the correct combination of transcription factors assembles
   on the gene promoter and enhancer/s transcription may
   occur

Question 53

Gene expression is a symphony

Answer 53

• Within a specific cellular niche, extracellular signals
are transmitted to the nucleus

• large functional units of chromatin are sequestered to
the edge of the cell or located in active territories close
to the centre of the nucleus, MB sized TADs
compartmentalise and organise sets of genes to direct
specific expression of lineage-driving factors that
subsequently act at sets of specific promotors to
activate lineage-appropriate genes and repress
alternative cell fates.

• From the whole cell to each individual molecule within
it, the coordination and precision is astounding.