Genetics Mid-Quarter
Review Session
October 13, 2015
TAs: Ryan, Kayla, Paige, Paula, and
Grace
Notes
1. Review materials
a. The slides from this session and review tables are
posted on coursework
b. Short videos posted on coursework
c. This session will be videotaped
2. TA office hours
a. Wednesdays in LK Cafe- see weekly email for exact
times
b. We are always happy to meet: please email us if the
scheduled times do not work for you
3. Dr. Bernstein office hours
a. Check coursework for dates/times
b. Next one is tomorrow (Wed) at 2pm in LK Cafe
Review Handouts
Available on Coursework
Agenda for Review Session
1. Mendelian Inheritance (Kayla)
2. Cytogenetic Abnormalities (Grace)
3. Cytogenetic and Molecular Testing (Ryan)
4. Non-Mendelian Inheritance (Paula)
5. Hardy-Weinberg, Bayes, and Consanguinity (Paige)
Mendelian Inheritance Patterns
● Autosomal Dominant
○
○
○
○
Penetrance
Variable expression
New mutations
Anticipation
● Autosomal Recessive
○ Consanguinity
○ Founder effect
● X-linked Recessive
● X-linked Dominant
*Optional Video Available on Coursework: Modes of Inheritance and Pedigrees
What inheritance pattern is this?
Autosomal Dominant Conditions
1.
2.
3.
4.
Seen in multiple, successive generations
Males and females equally affected
Male-to-male transmission
50% risk to offspring of affected individuals
in case of complete penetrance
Autosomal Dominant Considerations
1. Penetrance:
a. Not everyone with the mutation will develop the disease
b. Think all or none
2. Variable expression:
a. Different disease phenotypes arising from the same
mutation
b. Think spectrum
3. Anticipation:
a. Symptoms appear to become more severe and/or arise
earlier as generations pass
b. Think trinucleotide repeat expansion
4. Other considerations:
a. De novo (occurrence increases with paternal age)
b. Germline mosaicism
c. Nonpaternity
What inheritance pattern is this?
Autosomal Recessive Conditions
1.
2.
3.
4.
Usually seen in a single generation
Males and females are equally affected
Recurrence risk for two carrier parents is 25%
Unaffected sibling of an affected individual has a 67%
risk of being a carrier
5. All offspring of an affected individual are obligate
carriers; may be affected if other parent is a carrier or
affected
6. Consanguinity increases likelihood
Autosomal Recessive Considerations
1. Consanguinity:
a. Matings between close relatives
b. Increases likelihood of autosomal recessive
condition
2. Founder Effect:
a. High frequency of a specific gene mutation in a
specific population due to the presence of that
mutation in a single ancestor or a small number of
ancestors
ex. Ashkenazi Jews and Tay Sachs Disease
What inheritance pattern is this?
X-linked Recessive Conditions
1.
2.
3.
4.
Affected males always related via females
Male-to-male transmission does not occur
All daughters of affected males are obligate carriers
Sons of female carriers have 50% chance of being
affected
5. Daughters of female carriers have a 50% chance of
being a carrier
What inheritance pattern is this?
X-linked Dominant Conditions
1. Transmission from affected female is like autosomal
dominant: all children (male and female) have a
50% chance of being affected
2. Transmission from affected male is like X-linked
recessive: all females and no males will be affected
3. Rule out X-linked dominant: If a son of an affected
male is affected or a daughter of an affected male is
NOT affected
Cytogenetic Abnormalities
● Disorders of Chromosome Number
●
●
Euploidy
Aneuploidy
● Isochromosomes
● Deletions/Duplications
● Ring Chromosomes
● Translocations- videos
●
●
Reciprocal
Robertsonian
● Inversions- video
●
●
Paracentric
Pericentric
Cytogenetic Abnormalities
● Disorders of Chromosome Number
●
●
Euploidy
Aneuploidy
● Isochromosomes
● Deletions/Duplications
● Ring Chromosomes
● Translocations- videos
●
Reciprocal
● Robertsonian
● Inversions- video
●
●
Paracentric
Pericentric
http://www.larasig.com/sites/larasig.com/files/images/trisomy21_0.
jpg
Euploidy v. Aneuploidy
● Euploidy = chromosome # is an exact
multiple of the haploid number (n)*
--> Instead of (2n), zygote is (3n), (4n), (5n), ...
Remember, n = 23 in humans
● Aneuploidy = chromosome # is NOT an exact multiple of the
haploid number (n)*
Monosomy: one fewer chromosome
Turner Syndrome (45, XO)
Trisomy: one extra chromosome
Klinefelter Syndrome (47, XXY), Down Syndrome (47, XX/XY, +21)
Aneuploidy
●
Abnormal chromosome # that is NOT an exact multiple of the haploid number
- Missing/extra chromosomes can be autosomes or sex chromosomes
● Due to: nondisjunction
■ Anaphase I of meiosis: homologous chromosomes fail to separate
■ Anaphase II of meiosis: sister chromatids fail to separate
parent 1
gametes:
parent 2
gametes:
zygote:
+ n = 2n + 1 ➔ trisomy
ONE EXTRA
+ n = 2n - 1 ➔ monosomy
ONE FEWER
http://www.biology.iupui.edu/biocourses/N100/2k2humancsomaldisorders.html
Cytogenetic Abnormalities
● Disorders of Chromosome Number
●
●
Euploidy
Aneuploidy
● Isochromosomes
● Deletions/Duplications
● Ring Chromosomes
● Translocations- videos
●
Reciprocal
● Robertsonian
● Inversions- video
●
●
Paracentric
Pericentric
Isochromosomes
Definition: Chromosomes with identical arms with each pair of
loci equidistant from the center
Result from: transverse
rather than longitudinal
division at the centromere
(http://www.wikilectures.eu/images/2/29/Isochromosomes.jpg)
Cytogenetic Abnormalities
● Disorders of Chromosome Number
●
●
Euploidy
Aneuploidy
● Isochromosomes
● Deletions/Duplications
● Ring Chromosomes
● Translocations- videos
●
Reciprocal
● Robertsonian
● Inversions- video
●
●
Paracentric
Pericentric
(http://upload.wikimedia.org/wikipedia/commons/5/5f/Deletion.
gif)
Deletions & Duplications
● Can be large or small (micro)
○ Larger deletions/duplications tend to have greater effects
● Location
○ Terminal - at the end of a chromosome
○ Interstitial - NOT at the end of a chromosome
Deletions: generally more harmful
○ Ex: DiGeorge Syndrome (22q11.2 deletion)
Duplications: generally less harmful
Cytogenetic Abnormalities
● Disorders of Chromosome Number
●
●
Euploidy
Aneuploidy
● Isochromosomes
● Deletions/Duplications
● Ring Chromosomes
● Translocations- videos
●
Reciprocal
● Robertsonian
● Inversions- video
●
●
Paracentric
Pericentric
ring chromosome in Turner Syndrome
Ring Chromosomes
● Double strand breaks at ends of a chromosome -->
fusion of opposite arms to form a ring
● Usually associated with terminal deletions
● Can be inherited
ring chromosome in Turner Syndrome
Cytogenetic Abnormalities
● Disorders of Chromosome Number
●
●
Euploidy
Aneuploidy
● Isochromosomes
● Deletions/Duplications
● Ring Chromosomes
● Translocations- videos
●
●
Reciprocal
Robertsonian
● Inversions- video
●
●
Paracentric
Pericentric
Translocations
● Definition: movement (“trans-location”) of a fragment of a
chromosome to another, non-homologous chromosome
● Types:
○ Balanced v. Unbalanced
■ Balanced – no loss or gain of genetic information
■ Unbalanced – loss or gain of genetic information
○ Reciprocal and Robertsonian
Reciprocal Translocations
● Non-homologous chromosomes swap chromosome segments
●
●
●
Any pair of non-homologous chromosomes
Breaks at any point along the chromosomes
Can be balanced (more common) or unbalanced
Possible Gametes - Reciprocal
Chromosomal Rearrangements
Robertsonian Translocations
● Non-homologous chromosomes swap chromosome segments
●
●
ONLY acrocentric non-homologous chromosomes (13, 14, 15, 21, 22)
Breaks ONLY at the centromeres
● This produces…
●
●
Metacentric fusion of p arms
→ lost in subsequent divisions,
contain redundant material
Metacentric fusion of q arms
→ propagated in offspring
p
p
q
q
q
p
p
q
Possible Gametes - Robertsonian
balanced carrier
trisomy/monosomy
trisomy/monosomy
Chromosomal Arrangements
Cytogenetic Abnormalities
● Disorders of Chromosome Number
●
●
Euploidy
Aneuploidy
● Isochromosomes
● Deletions/Duplications
● Ring Chromosomes
● Translocations- videos
●
Reciprocal
● Robertsonian
● Inversions- video
●
●
Paracentric
Pericentric
Inversions
● Definition: a segment of a chromosome is inverted (reversed,
end to end) and inserted back into the same position
● Types:
1) Paracentric – the inverted segment
DOES NOT include the centromere
→ Produces gametes with acentric
and dicentric chromosomes; these
offspring are non-viable
2) Pericentric – the inverted segment
DOES include the centromere
* I = INCLUDES the centromere!
Your Favorite Diagram! Pericentric Inversions
Normal
TAKE-HOME POINTS:
Inverted
* 4 possible meiotic products
* If you suspect two family
members have the same condition
but their phenotypes differ...
* If you see a duplication & deletion
on the same chromosome...
THINK PERICENTRIC INVERSION
Helpful OPTIONAL video on
Coursework!
Cytogenetic and Molecular Techniques
● Cytogenetic techniques:
○ Karyotype - video
○ FISH - video
○ array CGH - video
●
Molecular Techniques:
○ Sanger sequencing - video
○ Next generation sequencing - video
○ SNP genotyping - videos
○ MLPA - video
○ Methylation studies - video
Helpful OPTIONAL videos on
Coursework!
Karyotype
● What is it?
○
A cytogenetic method of surveying the
entire genome for numerical or structural
chromosome abnormalities
● How is it done?
● What types of tissue sources are
used?
○
○
○
○
T lymphocytes from blood (easily induced
to divide in cell culture)
Fibroblasts from skin biopsy
Amniotic fluid and chorionic villi (prenatal)
Bone marrow, solid tumors (cancer)
All living cells!
Karyotype: Final Result
chromosomes are placed in order from the
images
Karyotype resolution
Karyotype
●
Typically used for:
○ Numerical chromosomal
abnormalities
○ Balanced translocations
○ Inversions (peri- and
para-)
●
Limitations:
○ Do not detect
submicroscopic
deletions and duplications
Fluorescence in situ hybridization (FISH)
● What is it?
○
A method to look for presence or absence, copy number, or
chromosomal location of a particular DNA sequence in the context
of a chromosome
● How is it done?
○
○
○
○
○
○
Produce an interphase cell nucleus or metaphase chromosome
preparation; attach cells/chromosomes to glass slide
Construct a cloned DNA sequence (aka ‘probe’)
Tag with fluorescent dye
Denature cell DNA in place (in situ) to expose single strands; denature
probe
Allow probe to hybridize to the chromosomal DNA
The hybridized probe will fluoresce when viewed with wavelength of
light that excites the fluorescent dye
View under microscope
Fluorescence in situ hybridization (FISH)
Metaphase versus Interphase FISH
●
Metaphase FISH
○
○
●
Chromosomes look like those in a karyotype
Provides better information on the position of a
probe target sequences
Interphase FISH
○
○
Can aid in identifying closely spaced duplications of
genetic material as the chromosomes are less
condensed
Can be done more quickly since the cells do not
have to be in any specific stage of the cell cycle.
Fluorescence in situ hybridization (FISH)
●
Typically used for:
○ Testing for specific microduplication or microdeletion
○ Confirming array CGH results/confirming the location
●
Limitations:
○ Each FISH probe tests for deletion or duplication at
one locus
●
Alternatives:
○ MLPA
Array Comparative Genomic
Hybridization (CGH)
● What is it?
○
Method to quantitatively test for presence or absence of many
specific DNA sequences simultaneously (can test the entire genome
at once)
● How is it done?
Sample aCGH data-deletion chromosome 22
duplication chromosome 16
* More dots (DNA elements on array) = higher resolution
* Smallest detectable anomalies: ~50 kb
Array Comparative Genomic
Hybridization (CGH)
● Typically used for:
○ Simultaneous testing for microduplications and
microdeletions across the genome
○ Can detect changes too small to be seen under
microscope
○ Does not require prior knowledge of chromosomal
location of any variant
● Limitations:
○ Does not detect balanced rearrangements
○ Resolution depends on the number of elements on
array
○ Does not provide information on position of
Sanger Sequencing
●
What is it?
○
○
●
Most commonly used method for assessment of
DNA sequence in clinical practice
Most clinical sequencing focused on exons and exon
boundary sites
How is it done?
Sanger Sequencing
● Typically used for:
○ Identification of single base pair change
○ Small insertions and deletions
● Limitations:
○ Does not reliably detect large deletions / duplications
○ Not quantitative
○ Allele drop-out (one of two alleles is not detected in the
assay because presence of deletion prevents primer
binding)
○ Does not distinguish whether two sequences are
present in cis versus trans configuration
Next Generation Sequencing
Next Generation Sequencing
●
●
●
●
●
●
●
DNA is fragmented
Linker is ligated, DNA is conjugated to flow
cell
Bridge amplification of DNA
First round of amplification with reversible
terminator
Laser excite and image
Repeat cycles
Image and reconstruct data
Next Generation Sequencing
●
●
●
●
●
Typically used for screening of multiple
genes
In combination with capture approaches can
be used to sequence a specific panel of
genes
Higher error rate than Sanger sequencing
(this is improving)
High chance of incidental findings
Relatively expensive
Multiplex Ligation-Dependent Probe
Amplification Assay (MLPA)
●
What is it?
○
A method to detect
medium to large
deletions/duplications
(kilobase to
megabase)
●
How is it done?
○
Combination of DNA
hybridization and
PCR amplification
Multiplex Ligation-Dependent Probe
Amplification Assay (MLPA)
MLPA sample result: deletion in
dystrophin gene
Multiplex Ligation-Dependent Probe
Amplification Assay (MLPA)
● Typically used for:
○ Assessment for a specific set of microdeletions /
microduplications
● Limitations:
○ Does not provide information on large-scale
architecture
○ Does not indicate presence or absence of
cytogenetic abnormalities
○ Does not provide position information
● Alternatives:
○ high resolution CGH
Methylation studies
● Typically used for:
○ Assessment of methylation status
○ Why? DNA sequencing methods and the PCR
reaction do not distinguish methylated from
unmethylated DNA
● Methylation in DNA is present on cytosine
Methylation studies: bisulfite
treatment
methylation-sensitive PCR, MLPA
Methylation studies
Method 2: Restriction enzymes
Sample with
Sample with
No
methylation site
HpaII
HpaII
These techniques are used in
combination
● aCGH + FISH
● NGS + Sanger sequencing
● Karyotype + FISH
Non-Mendelian Inheritance
● Epigenetic
● Imprinting
○ Methylation testing
● Mitochondrial
○ Heteroplasmy
● Multifactorial
○ Threshold Model
○ Empirical Risk Studies
○ GWAS
Imprinting
What is imprinting?
A. DNA methylation at locations rich in CG dinucleotides (CG
motifs), associated with gene silencing (blocks transcription
factors, recruit repressors)
Genomic imprinting:
A. Sex of the parent can cause distinguishable methylation
during meiosis
Example of Imprinting Disorders
Prader-Willi Syndrome (PWS)
Angelman Syndrome (AS)
● Both due to defects in the same area of chr15 (15q1113)
(Pagon et al., 2013)
Angelman vs. Prader-Willi
1. Angelman Syndrome:
a. Due to deletion/inactivation of information on
maternal chromosome
b. UBE3A gene dysregulated
c. Phenotype: hypotonia, poor feeding, microcephaly,
seizures, poor speech, ataxia, happy demeanor
2. Prader-Willi Syndrome:
a. Deletion/inactivation of information on
chromosome
b. A snoRNA cluster of genes affected
c. Phenotype: hypotonia, initial poor feeding, later
excessive feeding, motor and language delay,
hypogonadism
paterna
Causes of Angelman Syndrome
(Adams 2008)
Causes of Prader-Willi Syndrome
(Cassidy & Driscoll 2009)
Detecting DNA Methylation
Mitochondrial Inheritance
(Lemire 2005)
Mitochondrial Inheritance
= Heteroplasmy
Mitochondrial
(Lemire 2005)
Multifactorial Inheritance
Liability
Environmental
Factors
Empirical Risk Model
Concordance: presence of the same trait in both twins
Heritability: portion of a trait attributable to genetic variance
(Smoller & Finn 2003)
Hardy-Weinberg
p2+2pq+q2=1
● Assumptions: random mating, no migration, no new
mutations, no selection
● 3 possible genotypes: AA, Aa, aA, aa
● Allele frequencies
○ p= frequency of dominant allele, A
○ q=frequency of recessive allele, a
● Genotype frequencies
○ p2 =chance of being AA
○ q2 =chance of being aa
○ 2pq=carrier frequency
● 2pq≈2 x 1 √(incidence of the condition)
Population and Quantitative Genetics
● Autosomal recessive
● Incidence is 1/4900
● P (Mother is Carrier) ?
● P (Father is Carrier) ?
● P (Child would be affected) ?
Bayes Theorem
● Update a risk estimate
based on new info
○ Family history
○ Test results
○ Age dependent
penetrance
● P (Child would be
affected) ?
*Optional Videos Available on Coursework: Probability and Bayes Theorem
Consanguinity
● 2nd cousins or closer
● Risk of recessive disease
● Identity by descent
○ When both copies of an allele are identical because
they were inherited from a common ancestor
● Coefficient of inbreeding
○ Fraction of a person’s genes that are identical by
descent
○ Probability that a person is identical by descent at a
particular locus
Consanguinity
● P (Mother is Carrier) ?
● P (Father is Carrier) ?
● P (Child is affected) ?
● Increased risk for AR
conditions
Thanks!
Please feel free to email the TAs if you have
any questions.
Ryan: rmgallag@stanford.edu
Kayla: kaylah@stanford.edu
Paige: pqin@stanford.edu
Paula: ptrepman@stanford.edu
Grace: gxiong@stanford.edu