#30 Molecular Genetic Case Study

8/9/2019 #30 Molecular Genetic Case Study

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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/


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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:


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:


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

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#30 Molecular Genetic Case Study