Genetic Testing Family Secrets Copyright © 2009, University of Rochester

Genetic Testing

Family Secrets

Genetic disease and testing

• The patient’s journey…

• Types of genetic tests

• Techniques used in genetic testing

• Implications of genetic testing

• The future…

Inherited disease

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Genetic disease

Symptoms

Predictive testing Tells a person if she carries a mutation that will cause, or put her at higher risk for, a disease later in life.

Newborn screening Detects common disorders in newborns, where immediate treatment can prevent dangerous symptoms

Carrier testing Tells a person whether or not he carries a mutation that could be passed on to his offspring. One can be a carrier, but not be at risk for a disease (as in recessive genes)

? ? ? ?

Genetic Testing

Predictive testing Tells a person if she carries a mutation that will cause, or put her at higher risk for, a disease later in life.

Newborn screening Detects common disorders in newborns, where immediate treatment can prevent dangerous symptoms

Carrier testing Tells a person whether or not he carries a mutation that could be passed on to his offspring. One can be a carrier, but not be at risk for a disease (as in recessive genes)

? ? ? ?

Genetic Testing

Physician

GeneticCounselor

Test family members with disease symptoms?

Test patient?

Treatment

Prevention

Reproduction

PhysicianGenetic Counselor

Genetic Testing:Molecular Techniques

Protein

Function

Protein

Function

Genetic testing

Protein

ProteinFunction

Levels of Genetic Testing

normal mutated

Analysis of whole chromosomes – for large changes; extra chromosome, very large deletions or insertions

atcgatcgatcg atcgaAcgatcgAnalysis of sequence – for small changes; mutations in the sequence, small deletions or insertions

Analysis of protein shape – for any change that may affect the folding of the protein

Analysis of protein function – if the functional protein is supposed to make something, then some tests can detect the presence or absence of the product

Protein

ProteinFunction

normal mutated

Analysis of whole chromosomes – for large changes; extra chromosome, very large deletions or insertions

atcgatcgatcg atcgaAcgatcgAnalysis of sequence – for small changes; mutations in the sequence, small deletions or insertions

Analysis of protein shape – for any change that may affect the folding of the protein

Analysis of protein function – if the functional protein is supposed to make something, then some tests can detect the presence or absence of the product

Levels of Genetic Testing

Examples of Mutations in the DNA Sequence…

aaaccatctaggctatattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcaaaggtttttttttttcagctagctggggggggggggatcgggtgtgtcgatgtgtgagcaaaatattagcaaccccccccccattactgatgtcattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcaaaggtttttttttttcagctagcttacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatcgatctagatatcgatctatcggatctatctactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcaggatctaggctatattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtacgatcagggatatcgatctatcggatctatctactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcaggatctaggctatattcggatgatctatctactagagctgatctatctactagagctgtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatc

Normal

aaaccatctaggctatattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcaaaggtttttttttttcagctagctggggggggggggatcgggtgtgtcgatgtgtgagcaaaatattagcaaccccccccccattactgatgtcattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgataatcaaaggtttttttttttcagctagcttacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatcgatctagatatcgatctatcggatctatctactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcaggatctaggctatattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtacgatcagggatatcgatctatcggatctatctactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcaggatctaggctatattcggatgatctatctactagagctgatctatctactagagctgtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatc

Single base pair mutation

(Sickle cell anemia)

Deletion

Examples of Mutations in the DNA Sequence…Examples of Mutations in the DNA Sequence…

(Cystic fibrosis)

Deletion

(Duchenne muscular dystrophy)

aaaccatctaggctatattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcaaaggtttttttttttcagctagctggggggggggggatcgggtgtgtcgatgtgtgagcaaaatattagcaaccccccccccattactgatgtcattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcaaaaaaaaaaaaaaaaaaggtttttttttttcagctagcttacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatcgatctagatatcgatctatcggatctatctactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcaggatctaggctatattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtacgatcagggatatcgatctatcggatctatctactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcaggatctaggctatattcggatgatctatctactagagctgatctatctactagagctgtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatc

Insertion

(Huntington’s disease)

aaaccatctaggctatattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcaaaggtttttttttttcagctagctggggggggggggatcgggtgtgtcgatgtgtgagcaaaatattagcaaccccccccccattactgatgtcattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatggtcaaaggtttttttttttcagctagcttacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatcgatctagatggggatctatcggatctatctactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcaggatctaggctatattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaacacagcgatctaatataaatctgatgatcgatcgacatttttttttaaaaaaaaaaaaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtacgatcagggatatcgatctatcggatctatctactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcaggatctaggctatattcggatgatctatctactagagctgatctatctactagagctgtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatc

Multiple mutations

(Diabetes, susceptibility to breast cancer)

aaaccatctaggctatattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcaaaggtttttttttttcagctagctggggggggggggatcgggtgtgtcgatgtgtgagcaaaatattagcaaccccccccccattactgatgtcattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatggtcaaaggtttttttttttcagctagcttacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatcgatctagatggggatctatcggatctatctactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcaggatctaggctatattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaacacagcgatctaatataaatctgatgatcgatcgacatttttttttaaaaaaaaaaaaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtacgatcagggatatcgatctatcggatctatctactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcaggatctaggctatattcggatgatctatctactagagctgatctatctactagagctgtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatc

Multiple mutations

(Diabetes, susceptibility to breast cancer)

How do we “look” at the DNA sequence?

• Sequencing

• RFLP analysis

• Probes– Dot blot– Microarray

• Gel electrophoresis • PCR

• Sequencing

• RFLP analysis

• Gel electrophoresis• PCR

Gel ElectrophoresisSeparates DNA fragments on the basis of size

Large fragments take longer than small fragments to migrate

through an agarose gel

• Sequencing

• RFLP analysis

• Probes– Southern blot– Dot blot– Microarray

•Gel electrophoresis•PCR

PCR (Polymerase Chain Reaction)

aatcgaatgtgcccgtacgattcgatgcgaaactaggagccctatcgatttagcttacacgggcatgctaagctacgctttgatcctcgggatagcta

aatcgaatgtgcccgtacgattcgatgcgaaactaggagccctatcgat

ttagcttacacgggcatgctaagctacgctttgatcctcgggatagcta

Melting

Annealing

cgggcat

aactagg

Elongation

cgggcat

aactaggaatcgaatgtgcccgtacgattcgatgcga

gctaagctacgctttgatcctcgggatagcta

Melting

cgggcat

Annealing

cgggcat

aactagg

Elongation

cgggcat

aactagg

gcccgtacgattcgatgcga

aatcgaatgtgcccgtacgattcgatgcga

gctaagctacgctttgatcc

PCRMakes a lot of copies of a specific portion of DNA

PCR test for deletion

Electrophoresis

• Sequencing

• RFLP analysis

•PCR•Gel electrophoresis

Sequencing

aaaaaaaatgatgatgatgatgatgatgatgatgatgatgatg

“Known gene sequence:”

*******************************************

Patient DNA sequence

How do we find out if the patient’s sequence is different?

Sequencing

*******************************************ttttttttactactactac*

tttttttt

*******************************************

*******************************************tttttttt

actac*

actactactactag*

actactactactacgactact*

actactactacta*

Sequencing

*******************************************tttttttta*

*******************************************

ttttttttactac*

ttttttttact*

ttttttttac*

ttttttttacta*

Sequencing

*******************************************tttttttta*

*******************************************

ttttttttactac*

ttttttttact*

ttttttttac*

ttttttttacta*

Sequencing

*******************************************tttttttta*

*******************************************

ttttttttactac*

ttttttttact*

ttttttttac*

ttttttttacta*

Sequencing

*******************************************tttttttta*

*******************************************

ttttttttactac*

ttttttttact*

ttttttttac*

ttttttttacta*

Sequencing

*******************************************tttttttta*

*******************************************

ttttttttactac*

ttttttttact*

ttttttttac*

ttttttttacta*

Sequencing

*******************************************tttttttta*

*******************************************

ttttttttactac*

ttttttttact*

ttttttttac*

ttttttttacta*

Sequencing

*******************************************tttttttta*

*******************************************

ttttttttactac*

ttttttttact*

ttttttttac*

ttttttttacta*

Sequencing

*******************************************tttttttta*

*******************************************

ttttttttactac*

ttttttttact*

ttttttttac*

ttttttttacta*

ta c a

Sequencing

*******************************************tttttttta*

*******************************************

ttttttttactac*

ttttttttact*

ttttttttac*

ttttttttacta*

ta c a c…

Sequencing

actactactactagtactactactactactactac*******************************************

aaaaaaaatgatgatgatgatgatgatgatgatgatgatgatg“Known gene sequence:”

Sequencing

actactactactagtactactactactactactacaaaaaaaatgatgatgatgatcatgatgatgatgatgatgatg

aaaaaaaatgatgatgatgatgatgatgatgatgatgatgatg“Known gene sequence:”

Mutation

Issues with Sequencing

• Informative• Time consuming

• Expensive

• Sequencing

• RFLP analysis

RFLP Analysis

RFLP (Restriction Fragment Length Polymorphism) analysis relies on the use of restriction enzymes

These enzymes recognize specific DNA sequences (usually palindromes) and cut them…

RFLP AnalysisRestriction Fragment Length Polymorphism means that there are polymorphisms (differences) between people in the number of restriction sites (and therefore the length of the cut fragments). This person has 4 fragments after restriction digest.

This person has a mutation that eliminates one of the sites that the restriction enzyme cuts at. Therefore, this person has 3 bands, one of them being much larger than the rest. If this mutation was associated with a disease, a restriction digest would show that a carrier of the mutation had 3 fragments instead of 4

RFLP Analysis

Genomic DNA is very long and contains a lot of restriction enzyme cut sites

PCR makes many copies of a small region of DNA, better for RFLP analysis

RFLP Analysis

Issues with RFLP analysis

• Can not detect all mutations – the mutation has to coincide with a restriction enzyme cut site

• Sequencing

• RFLP analysis

Probes

DNA bases pair with complementary bases

Probes

A probe is an a short fragment of DNA that is complementary to part of a longer DNA sequence.

DNA strands can be separated with high heat.

As the temperature is lowered, smaller fragments that have complementary sequences (probes) will base pair faster than the long original strands of DNA

ProbesThe ability of a probe to bind depends on:

*Its complementarity to the DNA strand it’s binding to…

-Single base pair differences can affect binding depending on…

*The stringency of the binding conditions

-Temperature

-Buffer conditions

Low stringency

High stringency

• Sequencing

• RFLP analysis

Dot Blot(A Southern blot without the gel…)

A patient’s DNA (genomic DNA or PCR products) is denatured and spotted to a membrane…

Dot Blot

Membrane is washed in a solution that contains radioactively labeled probe

Probe binds to complementary sequence

Dot Blot

Membrane is washed in a solution that contains radioactively labeled probe

Excess probe is washed off – only probe bound to DNA on the membrane remains

Probe binds to complementary sequence

Dot Blot

The membrane is exposed to autoradiography film.. Therefore, wherever there is radioactive probe, the film will be exposed…

Dot Blot

The membrane is exposed to autoradiography film.. Therefore, wherever there is radioactive probe, the film will be exposed…

And a black dot will be developed where the probe was…

Dot blot to diagnose cystic fibrosis (CF)

Normal

Normal probe

Mutant probe

CF patient (homozygous mutant)

CF carrier (heterozygous mutant)

DNA from a patient is spotted out twice, one will be used with a probe complementary to the normal sequence, the other will be used with a probe that is complementary to a mutated sequence.

Since a heterozygote has one copy of the normal gene and one copy of the abnormal gene, both probes can bind, but only half as much binds, making the dot lighter.

Issues with dot blots

• Can be used to test patient for multiple mutations

• Single base pair mutations may be hard to detect with dot blots

• Sequencing

• RFLP analysis

Microarray(High throughput dot blots. Allows for testing of many different mutations…)

Microarrays start with a “chip” on which is spotted many different probes for different mutations. Each dot represents a different probe.

If this were a dot blot, it would be called a “reverse dot blot” because the PROBE is spotted, rather than the DNA.

Microarray(High throughput dot blots. Allows for testing of many different mutations…)

The chip is treated much like a southern or a dot blot, except it is washed with a patient’s DNA (fragmented with restriction enzymes, denatured and labeled with a flourescent dye), and it automated.

Microarray

Binds to a probe for a CF mutation

Binds to a probe for a colon-cancer susceptibility mutation

Binds to a probe for a breast-cancer susceptibility mutation

If a patient’s DNA binds to spotted probes, a computer detects this by the measuring the intensity of the flourescence emanating from that spot. A homozygote for a CF mutation will have a more intense spot than a heterozygote for the same mutation

Issues with Microarrays

• Expensive

Available Predictive Tests

Cystic Fibrosis

Tay Sachs Disease

Lou Gherig’s Disease (ALS)

Huntington’s Disease

Catastrophically high cholesterol

Rare Cancers

Inherited susceptibilty to cancerBreast Cancer Colon Cancer Thyroid Cancer

Benefits of Genetic Testing

If Negative

Relief

Fewer health check-ups and tests that go with being in a family that tends to have a higher risk of a particular genetic diseases

If Positive

Able to make informed decisions

May be possible to reduce the risk of coming down with severe symptoms

Limitations of Genetic Testing

• Mutations may not always lead to disease• Existing tests only look for the most

common mutations, some disease causing mutations will not be detected by conventional tests

• Small chance of errors in testing procedure

• Testing is not always matched by treatment

Limitations of Genetic Testing

• Mutations may not always lead to disease• Existing tests only look for the most

common mutations, some disease causing mutations will not be detected by conventional tests

• Small chance of errors in testing procedure

• Testing is not always matched by treatment

Should I get tested?

What will I do if it’s positive?

What will we do if he’s positive?

Testing is not always matched by treatment:Confidentiality

Health InsuranceEmployers

Family

Where do we go now?

• Try to resolve ethical, legal and social issues– Experts in science, ethics, law.– Policy makers

• Develop new, cheap, simple, effective and accurate tests– Testing for all

• Advance research in disease treatment– Gene therapy– Drug treatment

Genetic Testing Family Secrets Copyright © 2009, University of Rochester

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