Unformatted text preview: Genes - Structure and Function
• Cystic fibrosis case study
– Disease characteristics, genetics, biochemistry • Genetic mutation types: synonymous, nonsense,
missense, frameshift, alternate splice site, regulatory
• Genetic tests and their limitations Learning goals:
• Look at the complexity of human disease alleles in a case
study on cystic fibrosis
• Describe the basic structure of a gene • Describe the types of mutations that can lead to allelic
• Assess risk using genetic test results Case Study – Cystic Fibrosis
Part 1: Meeting
Sarah stared blankly at the blue paisley wallpaper. Her husband Mike sat by her side, bending and
unbending a small paper clip.
“Sarah and Michael, it’s good to meet you,” welcomed the genetic counsellor, as she entered the
room. “I apologize for being late, but I was just meeting with another couple. Let’s see, you’d like
to have a child, but you’re concerned because of your family history of cystic fibrosis.” “Yes,” Sarah replied softly. “Mike and I met at a CF support group meeting a few years ago. He
had a younger brother who died of cystic fibrosis, and I had a younger sister. We saw the painful
lives they had—difficulty breathing, the constant respiratory infections. Although the treatments for
CF are better now, we just don’t know if we can…” she trailed off .
“I can certainly understand your concern,” the genetic counsellor responded sympathetically.
“That’s where I hope to help, by providing as much information and advice as I can. I’m glad that
you came to see me before you became pregnant so I can fully advise you of all options
“To start, let’s go over what we know about your case so far: What are the symptoms of cystic fibrosis? Which organs are affected? What is the common effect in targeted organs? What is the prognosis? Genotype:
Let’s refer to the gene associated with cystic fibrosis as CF
The two alleles are represented as CF = functional
cf = nonfunctional
Note: The gene is now known as CFTR and it codes for the CFTR (Cystic
Fibrosis Transmembrane Regulator) protein.
We will use the shorter designation “CF” for simplicity.
CF/CF homozygote CF/cf heterozygote = carrier cf/cf homozygote affected by symptoms CF/- no symptoms = either CF/CF or CF/cf
need more genotypic information Pedigree analysis: write out genotypes for CF gene ? ? Part 2: Genetic testing “So, what’s next?” asked Mike.
“First, we’ll collect DNA samples from both of you. We’ll then analyze your CF
genes for the most common mutations to see if you are carriers for this
recessive genetic disease.”
A few weeks later, the genetic counsellor welcomed back Sarah and Mike.
“We’ve received the results of your genetic tests for common CF mutations.
Michael, you’re a carrier of the most common disease allele, delta-F508, and
Sarah, you tested negative for the most common CF mutations.”
“Thank goodness,” Sarah replied with relief. “That means it’s safe for us to have
a child, right?” “Not exactly,” cautioned the genetic counsellor. Complete the Punnett Square: if Sarah is not a carrier
if Sarah is a carrier
gametes: CF CF cf The CFTR gene codes for a protein that acts as a channel across
the membrane of the cell.
Welsch, M.J. and Smith,
A.E. 1995. Cystic Fibrosis.
Scientific American. 273:5259.
CF-SciAmerican.pdf The CFTR gene codes for a protein that acts as a channel across
the membrane of the cell. Welsch, M.J. and Smith, A.E. 1995. Cystic Fibrosis. Scientific American. 273:52-59. How do we get from the CF gene (inheritance of an allele)
to a protein?
STEP 1: Transcription of DNA into a mRNA
STEP 2: Processing of mRNA – removing introns
STEP 3: Translation of mature (processed) mRNA into a protein
STEP 4: Processing, folding, transport of protein STEP 1:
Transcription: DNA to RNA = messenger RNA or mRNA Animation: STEP 2:
Editing the mRNA: splicing STEP 3:
Translation: mRNA to protein Animation: STEP 4:
Protein transport The CFTR protein must be at the cell membrane to function. CFTR Gene to Protein Overview:
24 exons = Protein-coding sequences
23 introns = intervening sequences 1480 amino acids: Transmembrane
membrane): Most common mutation:
Three nucleotides are missing in
the gene and as a result, a single
amino acid is missing.
Delta F508 = deletion of
phenylalanine (F) at amino acid
position 508. Part 3: CFTR alleles
“Since Sarah tested negative for CF, it seems we don’t have much to worry about, right?”
said Michael, pointing to the pedigree in which Sarah is not a carrier. “So, what did you
mean when you said not exactly?”
The genetic counsellor grabbed her laptop computer and positioned it in front of the
three of them. Her fingers quickly typed out a web address and the words “OMIM Online Mendelian Inheritance of Man” stretched across the browser window. “We can
learn a lot more about this disease from this site. It will begin to explain why I said not
exactly when Sarah asked if it was safe to conceive a child.” How many variant alleles are there? What is the effect of
Go to: OMIM.org More than 1900* alleles of the CFTR gene have been identified.
Some have no effect on the protein and show no symptoms.
Others show varying degrees of symptoms. Types of Genetic Mutations
AA_ TGC TAT GGT
Coding 5’ – TCA GAG GTG AAA TGC TAT GGT – 3’
Template 3’ – AGT CTC CAC TTT ACG ATA CCA – 5’
mRNA 5’ – UCA GAG GUG AAA UGC UAU GGU – 3’
AA_ UGC UAU GGU
Protein N – Ser-Glu-Val-Lys-Cys-Tyr-Gly – C
N – Ser-Glu-Val-Lys-Cys-Tyr-Gly – C Synonymous N – Ser-Glu-Val-Lys-Cys-STOP-Gly – C Nonsense N – Ser-Gly-Val-Lys-Cys-Tyr-Gly – C Missense N – Ser-Glu-Val-Asn-Ala-Met- – C Frameshift (12)70057-7 Go to: OMIM.org
.0001 CYSTIC FIBROSIS [CFTR, PHE508DEL] Loss of a single codon in mRNA;
loss of single amino acid in protein
.0003 CYSTIC FIBROSIS [CFTR, GLN493TER] Nonsense mutation in mRNA;
premature termination of translation yields shorter protein
.0004 CYSTIC FIBROSIS [CFTR, ASP110HIS] Missense mutation in mRNA; nonconservative amino acid change may disrupt protein structure/function
.0019 CYSTIC FIBROSIS [CFTR, 2-BP INS, 2566AT] Frameshift mutation in
mRNA causes all amino acids encoded after this site to be altered; probably
shorter protein due to out of frame stop codon Mutation
processing .0008 CYSTIC FIBROSIS [CFTR, IVS10, G-A, -1] Altered splice site cause introns
10 to be retained, so longer mRNA; this inserts extra amino acids and may also
result in shorter protein due to out of frame stop codon
.0064 CYSTIC FIBROSIS [CFTR, IVS12, G-A, +1] Altered splice site causes exon
12 to be removed, so shorter mRNA; shorter protein because amino acids missing
.0123 CYSTIC FIBROSIS [CFTR, 21-KB DEL] Deletion of exons results in shorter
mRNA; out of frame stop codon produces shorter protein What do all of these mutations have in common? Sarah wondered how all these different mutations can cause the
same disease. As the genetic counselor, how would you explain this
to Sarah and Michael? Part 4: Interpreting risk Sarah and Michael looked confused.“But….” Michael started to say.
“How can the test say I’m not a carrier, but you say that that I could still be a
carrier?” Sarah finished.
The counselor pursed her lips and said, “Well, because of cost, no test is as
comprehensive as we would like. This research article describes the genetic
test used to determine your CF genotypes and explains its limitations,” she
continued, handing them a printed article. How many of these CFTR mutations can be detected
by the xTAG® CFTR assay by Luminex? What criteria might researchers use when determining
which mutations to include in the xTAG® test? What is the chance that Sarah is actually a carrier for a
CFTR mutation, even though her xTAG® test results
came back negative? We are going to need more information… Since Sarah tests negative, there are two possibilities:
(1) she truly is homozygous normal (AA) or
(2) this is a false negative and she is a carrier (Aa)
(the test simply could not detect her allele). Since most North Americans of European descent (27/28) are not
carriers, usually the odds that she is a carrier are 1/28. Given her family history, Sarah has a higher chance of being a
carrier (more than the 1/28) Point 1:
Screening with this assay will only identify 88% of carriers , so
although Sarah tests negative, there is still a notable chance
that Sarah is a carrier. The frequency of false negatives for this assay (identified as
normal, but truly carrier) is about 12% (100% – 88%) Point 2:
What prior genetic information do we have about Sarah? We know for sure that she is not affected, not cf/cf.
She is either CF/CF or CF/cf.
We know both her parents were carriers (since Sarah’s sister
was affected), so before the test, the probability that she is a
carrier was 2/3. Now let’s put this all together:
For 100 people like Sarah with an affected sibling, about 33 will
be normal and about 67 will be carriers (2:3).
Of the 33 that are truly CF/CF, virtually all will test negative as
Of the 67 that are truly CF/cf, about 8 (12%) will also test
negative. Of the 33 plus 8 = 41 individuals that receive a negative test, …
8 are likely to be carriers that were undetected (i.e. false negative). So the chances that Sarah is a carrier although testing negative is
8/41 or about 20%. Explain why the genetic counselor, said “not exactly” when
Sarah asked if it was safe to conceive a child. Part 5: Further analysis:
After hearing the counselor explain the limitations of the test that was used, Sarah and
Michael discussed whether to receive further testing. While the cost was significantly
more, they felt the added knowledge would help them make an informed decision and
so they elected to have Sarah’s DNA further analyzed. A week later they met again
with the genetic counselor to discuss the test result.
“So do I have a mutation in my CF gene?” asked Sarah
“Not exactly,” the genetic counselor replied. “You have a mutation in the region just
before the part of the gene that codes for the CF protein.
The subsequent analysis of Sarah’s sample determined the DNA sequence of the
CFTR introns and the regions upstream and downstream of the exons. The DNA
sequence indicates that Sarah has a single base pair difference about 100 bases
before the ATG start codon of the CFTR gene. Second genetic assay looked at mutations in the gene sequence that disrupt
non-coding sequence – introns, upstream, and downstream sequences. First genetic assay looked at mutations in the gene sequence that disrupt
the protein coding sequence. Second genetic assay looked at mutations in the gene sequence that disrupt
non-coding sequence: 5’
before coding sequence;
5’ to coding sequence 3’
after coding sequence;
3’ to coding sequence Regulatory sequences: sequences controlling how much and when a
gene is transcribed lie upstream, downstream, and within non-coding
introns. As the genetic counselor, explain fully to Sarah and
Michael how this mutation before the protein-coding
gene raises the possibility that they could have a
child with cystic fibrosis. Part 6: Final decision.
While the discussion about the implications of the mutation Sarah harbored
was complicated, the young couple was able to grasp the essential elements.
The counselor summed up the discussion when she said, “Well, I’m
disappointed to inform you that a future child of yours could have cystic
Both Michael and Sarah were visibly upset, but the counselor continued, “But
you do still have some options. After natural or in vitro conception, we could
test any embryos to determine the genotype, and then you could choose not to
proceed with the pregnancy if needed.” “What are we going to do?” asked Sarah and Michael to each other. What is the overall risk of the couple having a child
with cystic fibrosis given all of the information that
you have? If you were Sarah or Michael, what would you choose to do? Why? (a) Not have children.
(b) Adopt children. (c) Get pregnant and continue with the pregnancy no matter what.
(d) Get pregnant, test the embryo within 9 weeks via chorionic villus
sampling (CVS), and terminate if the embryo has CF. (e) Undergo in vitro fertilization by harvesting eggs, fertilizing them, and
screening the embryos for CF prior to implantation.
What other information would you want to have before making this
decision? Follow-up questions: misconceptions
• A negative result on a genetic test removes uncertainty. • Genetics tests are uninformative. ...
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