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April 8, 2010
Timothy Uphoff PhD, DABMG, MT(ASCPCM )
Molecular Genetic CaseMolecular Genetic CaseStudy: What Does aStudy: What Does aNegative Cystic FibrosisNegative Cystic Fibrosis
Result Mean?Result Mean?
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Sam is eight years old. His brother Ethan is three weeks old and has beenexperiencing a number of health issues. He has had a chronic coughand diarrhea since they brought him home from the hospital. He stayedin the hospital a couple extra days after he was born because of acondition the doctors called meconium ileus. Ethans newbornscreening tests came back showing higher than normal levels ofimmunoreactive trypsinogen. Sweat chloride testing was inconclusive.Ethans next test will be a molecular diagnostic test for Cystic fibrosis.
How common is this disease in the U.S. population? What gene product is altered in patients with CF? How is CF inherited? What will a molecular test for CF identify?
Ethan tested positive for CF using the new molecular test, Sam has nosymptoms of the disease. What is the chance that Sam is a carrier of CF?
What is the chance of the alternative [Sam is not a CF carrier?]
If Sams parents decide to have another child what is the risk thattheir next child will have CF? Be a carrier of CF? Suppose Sam would like to have children in the future, what is the
risk that his children will have CF?
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Talk ObjectivesTalk ObjectivesDescribe the biology of cystic fibrosis
Normal and abnormal physiologySpectrum of disease
Mutations of significance, mode of inheritance,nomenclature and classification
Discuss the guidelines and indications for testingDescribe the test assay technology
Clarify interpretation of reported results
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Cystic Fibrosis Transmembrane
Conductance Regulator
CFTR
7q31.2Positionally cloned in 1989230 kbcontains 27 coding exonsmRNA is 6.5 kb
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More than
1,000
mutations in
CFTR genehave been
identified
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Classification Scheme forClassification Scheme for
CFTR MutationsCFTR MutationsMutationClass
Effect of Mutationon CFTR Protein
Mechanisms
I Reduced or absent
synthesis
Nonsense, frameshift, or
splice-junction mutations
II Block in proteinprocessing
Missense mutations,amino acid deletions
III Block in regulation of
CFTR chloride channel
Missense mutations
IV Altered conductance of CFTR chloride channel
Missense mutations
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CF Inheritance AutosomalCF Inheritance Autosomal
RecessiveRecessive
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CFTR forms a chloride permeable
channel in the cell membrane
Passage is allowed only when the 2
nucleotide binding domains dock
with and cleave ATP and the
regulatory domain becomes studded
with phosphate
CFTR Function
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What is Cystic Fibrosis?What is Cystic Fibrosis?
N Engl J Med 2002;347:439
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Improved SurvivalImproved Survival
Cystic Fibrosis Foundation. Patient
Registry Annual Data Report, 2008.
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Improved SurvivalImproved Survival
Cystic Fibrosis Foundation. Patient
Registry Annual Data Report, 2008.
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Incidence of CFIncidence of CF
Approximately 30,000 children and adults
in the United States have cystic fibrosis.
An additional ten million moreor about
one in every 31 Americansare carriers
of the defective CF gene, but do not have
the disease.
CF is most common in Caucasians, but it
can affect all races.
http://www.cff.org/8/9/2019 #30 Molecular Genetic Case Study
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source of slide: Dr. Wayne Grody presentation on http://www.ampweb.org
Original ACMG Recommendations (2001)Original ACMG Recommendations (2001)
Grody WW, Cutting GR, Klinger KW, Richards CS, Watson MS, Desnick RJ. Laboratory
standards and guidelines for population-based cystic fibrosis carrier screening.
Genet Med2001;3:149154.
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INTERPRETATION:
Result: No mutations detected
Indication for Testing: Carrier Screen
Ethnicity: Caucasian
Interpretation:
None of the listed mutations were detected. This result decreases the likelihood
but does not exclude the possibility that this individual is a carrier of Cystic
Fibrosis (CF). The residual risk of this patient carrying an unidentified mutation is
1/206 based on a Bayesian analysis using the detection rate of 88% and CF
carrier frequency of 1/25 for the American Caucasian ethnic population. If the
patient has a family history of the disease, referral for genetic counseling is
recommended. If results obtained do not match the clinical findings or the patient
has a suspected diagnosis of CF, additional testing should be considered.
Interpretation
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New ACMG Mutation Panel, 2004 Rev.New ACMG Mutation Panel, 2004 Rev.
source of slide: Dr. Wayne Grody presentation on http://www.ampweb.org
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Reference ValuesReference ValuesRacial or EthnicGroup
MutationDetection Rate
Carrier Risk Priorto Screening
Carrier Risk with aNegative Result
Ashkenazi Jewish 94% 1/24 ~1/384
Caucasian American 88% 1/25 ~1/206
Hispanic American 72% 1/58 ~1/203
African American 65% 1/61 ~1/171
Asian American 49% 1/94 ~ 1/183
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Cystic Fibrosis AssayCystic Fibrosis Assay
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Target AmplificationTarget Amplification
CFTR gene
~250kb
27 exons
1480 AA
Multiplex PCR
x
x
x
x
x
x
x
x
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Indications for TestingIndications for Testing CS Reproductive couples
CS Partners of individuals w/ + FHx
CS Partners of CBAVD males
CS Family history of CF
CS Fetal echogenic bowel Dx Possible Dx of CF
Dx Confirm or rule out Dx of CF
Dx Infants w/ meconium ileus
Dx CBAVD in males
CS=Carrier Screen
DX=Diagnosis
CBAVD= Congenital Bilateral Absence of the Vas Deferens
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R117H and 5T, 7T, 9TR117H and 5T, 7T, 9T
R117H is a weak mutation that produces a CFTR
mutation with less activity.
With all R117H positive samples we reflex test for the
intron 8 polyT allele The # of Ts in this region of intron 8 affects the mRNA
splicing efficiency
5Ts leads to less efficient splicing and expression than 7 or 9 Ts
When R117H is present in conjunction with 5Ts in intron 8there is less of this poorly functioning CFTR being made
and the phenotype is worse.
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Intron 8 Poly T allelesIntron 8 Poly T alleles
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CisCis ororTransTrans ??
R117H
7T
5T
Maternal
Paternal R117H
5T
7T
Paternal
Maternal
5T in Cis w/R117H* 5T Trans w/R117H
*More severe phenotype
5T/7T/9T- polymorphism (a.k.a IVS 8 polyT stretch)
Classic CF Mutation + R117H + 5T in cis -> CF
Classic CF Mutation + R117H + 7T in cis -> CBAVD or Mild CF
5T/5T homozygous -> CBAVD
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Human CF males are sterile butHuman CF males are sterile but
CF male mice are notCF male mice are not!!!!!! The human epididymis and vas deferens are more vulnerable
than any other organ to the destructive effects of CF lesions,
Males with very mild CF mutations may present with congenital
bilateral absence of the vas deferens (CBAVD), but no other
obvious signs of CF. The exceptional vulnerability of the vas
deferens is not understood, but it does appear to be secondaryto lost secretion.
The loss of the vas deferens in CF males is not the result of
failure to develop, but is instead caused by degeneration
secondary to obstruction. The mouse epididymus relies largely on calcium-mediated Cl-
secretion that doesnt require CFTR, which may explain why the
vas deferens is spared in CF mice.
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INTERPRETATION:
Result: No mutations detected
Indication for Testing: Carrier Screen: Patient has a family history of cystic fibrosis
Ethnicity: Ashkenazi Jewish
Interpretation:
Having excluded the presence of the listed mutations, the risk that this individual isa carrier of another Cystic Fibrosis mutation is approximately 1/18. This
calculation is based on the information provided that a niece of the patient is
affected with CF and thus homozygous for CF mutations that have not been
identified. If the relative's mutations are known, this information should be
provided for inclusion in this interpretation. Additionally, the risk calculation was
based on a Bayesian analysis using a detection rate of 94% for the AshkenaziJewish population. If results obtained do not match the clinical findings or the
patient has a suspected diagnosis of CF, additional testing should be considered.
Referral for genetic counseling is recommended.
Other Interpretations?
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Inheritance= Male
= Female
= Carrier
= Affected
= Fetus
= Deceased= Marriage
= Children
Used to DescribeFamily Relationships
and Diseases
FYI
Recessive or Dominant?
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Carrier Risk and RelationshipsCarrier Risk and Relationships
AffectedIndividual Carrier Risk Carrier Probability
Son/Daughter 1 in 1 100%
Brother/Sister 2 in 3 67%
Niece/Nephew 1 in 2 50%
Aunt/Uncle 1 in 3 33%
First Cousin 1 in 4 25%
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INTERPRETATION:
Result: No mutations detected
Indication for Testing: Carrier Screen: Patient has a family history of cystic fibrosis
Ethnicity: Ashkenazi Jewish
Interpretation:
Having excluded the presence of the listed mutations, the risk that this individual isa carrier of another Cystic Fibrosis mutation is approximately 1/18. This
calculation is based on the information provided that a niece of the patient is
affected with CF and thus homozygous for CF mutations that have not been
identified. If the relative's mutations are known, this information should be
provided for inclusion in this interpretation. Additionally, the risk calculation was
based on a Bayesian analysis using a detection rate of 94% for the AshkenaziJewish population. If results obtained do not match the clinical findings or the
patient has a suspected diagnosis of CF, additional testing should be considered.
Referral for genetic counseling is recommended.
Other Interpretations?
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If Johns brothers daughter has fibrosis what is the chance
that Johns child will have CF?Before testing: 1/2 * 1/2 * 1/25 * 1/2 = 1/200
?
John
After testing: 1/18 * 1/2 * 1/206 * 1/2 = 1/14,832