Transcript HealthyQuest Presentation

Cystic Fibrosis
Charles (Buck) Strom, M.D., Ph.D., H.D.L.C., F.A.A.P., F.A.C.M.G.
Medical Director, Genetic Testing Center, Quest Diagnostics Nichols Institute
Classical Cystic Fibrosis
• Autosomal recessive disease
• Most common genetic disease in Caucasians
• Approximately 1:3,600 live births
• Multisystem disorder
– Viscous mucus production in the lungs
– Chronic recurrent pneumonia (Pseudomonas)
– Exocrine pancreatic insufficiency
– CBAVD*
• Semi-lethal disorder: males infertile; females fertile
*Congenital bilateral absence of the vas deferens.
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Classical Cystic Fibrosis
• Presentation
– Newborn with meconium ilius
– Infant/toddler with asthma or recurrent pneumonia
– Infant with malabsorption / failure to thrive
– Adolescent with pancreatitis
• Diagnosis– sweat chloride test
• Newborn screening
– Immunoreactive trypsinogen
– Identifies carriers as well as affecteds
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Classical Cystic Fibrosis
• Treatment
– Exocrine pancreatic insufficiency
• Oral enzyme replacement therapy
– Chronic pulmonary disease
• TID - QID chest percussion and postural drainage
• Intermittent “tune ups” in hospital or day treatment
centers (includes intensive therapy)
• Parenteral antibiotics as needed
– Heart-lung transplant is only definitive Rx
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Classical Cystic Fibrosis
• Prognosis
– Chronic growth failure due to
• Decreased intake and absorption
• Increased work of breathing
• Chronic infections
– Average life expectancy 30 - 35 years
– Many die in adolescence due to
noncompliance with treatment regimens
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Classical Cystic Fibrosis
• Prognosis (cont.)
– Adults die of chronic pulmonary disease
(pulmonary hemorrhage or congestive heart
failure due to COR pulmonale)
– Women fertile: increased incidence of
pregnancy complications so gestational
surrogacy should be considered
– Men sterile due to CBAVD, but testicular
aspiration followed by intracytoplasmic sperm
injection (ICSI) might allow reproduction
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Non-Classical Cystic Fibrosis
• Isolated CBAVD
• Isolated pancreatitis
• Dominant manifestation*
– Chronic sinusitis
– Asthma
*In people with chronic sinusitis and asthma, frequency
of CF carriers is greater than in general population.
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CF Disease and Carrier Frequencies
Ethnic Group
CF Incidence
Caucasian
1:3,300
1:29
Hispanic
1:8000 - 9,000
1:46
African American
1:15,300
1:62
Asian American
1:32,100
1:90
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Carrier Risk
Population Based CF Screening: Chronology
• 1989: cystic fibrosis transmembrane regulator
(CFTR) gene cloned
• Toronto Sick Children’s and University of
Michigan simultaneously announce:
– ΔF508 present in 60% of CF chromosomes
– >1,200 mutations currently described
• 1997: NIH Consensus Statement recommends
preconception and prenatal screening
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Population Based CF Screening: Chronology
• 1999: CF screening workshop held
– ACMG / ACOG / ELSI followed by 2 years of work
• Winter, 2000 –2001: mutation panel unofficially
announced and circulated to allow labs and IVD
manufacturers time to prepare
• April, 2001: ACMG publishes recommended
mutation panel in Genetics in Medicine
• October, 2001: ACOG recommends offering
routine testing to pregnant women and provides
educational material for physician and patients
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Population Based CF Screening: Chronology
• October, 2002: ACMG recommends testing for
3199delT when I148T is detected
• October, 2003: recommendation to drop
I148T and 1078delT from screening panel
announced at ASHG meeting
• October, 2004: recommendation to drop
I148T and 1078delT from screening panel
published in Genetics in Medicine
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ACMG Screening Panel, circa 2001
• Tests for all CF mutations predicted to have
allele frequency 0.1%
• Includes 25 mutations
• Polymorphism reflex testing
– Homozygous DF508 or DI507: test for I506V,
I507V, F508C
– R117H positive: test for IVS8 5T/7T/9T
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Genet Med. 2001;3:149-154.
ACMG Screening Panel, circa 2004
• Includes 23 mutations
• Polymorphism reflex testing (unchanged)
– Homozygous DF508 or DI507: test for
I506V, I507V, F508C
– R117H positive: test for IVS8 5T/7T/9T
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Genet Med. 2004;6:387-391.
How accurate are CF reagents?
3 series
(n >1,000 patients)
with no errors
ABI / Celera / Abbott OLA ASR
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Clinical Issues in Population
Based CF Testing
I148T Requires 3199del6 to Cause CF
• Strom et al. Genet Med. August, 2001
– I148T present >110x more frequently in carrier
screening population than in CF patients
– 2 asymptomatic adult women with DF508 /
I148T
• Rohlfs et al. Genet Med. October, 2001
– 7 of 8 CF patients with I148T and CF also had
3199del6
– 8 healthy adults were compound heterozygotes
for a CF mutation and I148T
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I148T Requires 3199del6 to Cause CF
• October, 2002: ACMG recommends testing for
3199delT when I148T is detected
• October, 2003: ACMG announces I148T to be
dropped from standard panel based on data
provided prior to publication cited below
• Buller et al. Genet Med. March, 2004
– 3199del6 present in 0.9% of 439 patients with
I148T
– Frequency of 3199del6: <0.07% of CF
mutations in Caucasian population
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Are there other I148T’s out there?
• D1270N: 205x more frequent in screened
population than in CF patients – we found by
accident
• Published: Strom et al. Genet Med. August,
2001
• Probably others; need more data to know
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Rare Events: 1078delT
• 1078delT not seen in initial 20,103 CF
screens*
• 1078delT seen 6 times in >335,000 CF
screens#
– 0.06% of CF mutations
– 1:55,867 screens
• ACMG decides to drop it from panel
*Strom et al. Genet Med. 2002;4:289-296.
#Strom et al. Genet Med. 2004;6:136-140.
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Rare Events: 2184delA
• 2184delA has next lowest frequency
– Seen 14 times in >335,000 CF screens
– 0.14% of CF mutations
– 1:23,943 tests
– Still on ACMG panel
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Strom et al. Genet Med. 2004;6:136-140.
Rare Events:
true positive versus false positive
• By definition, any “ethnic specific” mutation will
be found less frequently than 1:25,000 panethnic tests
• If found in significantly higher frequency, probably
a polymorphism and not a mutation
• False-positive rate of CF screening tests not
likely to be <1:20,000
• When assaying rare mutations, the false-positive
rate may approach or exceed the true-positive
rate
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Extended Mutation Panels
Arguments in favor of
• Increased detection rate for some ethnic
minorities
• Improved cost/benefit
• Decreased liability if baby is born with CF
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Extended Mutation Panels
Arguments Against
• How many mutations is enough? 40? 80? 87?
1,000?
• Low increased pick up rate, even in ethnic
minorities
• There may be other “I148T” and “D1270N” out
there
• Additional mutations will be rare; difficulties with
rare event detection (eg, false-positives) will
become an issue
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Extended Mutation Panels
Arguments Against (continued)
• Genetic counseling will be difficult when rare
mutations are detected
• Increased liability possible if unaffected fetus
is terminated
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Extended Mutation Panels
Illustration #1
Mother
DF508
Father
D1270N*
Fetus
DF508/ D1270N
Fetus considered affected; pregnancy subsequently
terminated, but no evidence of CF on autopsy
Two years later, couple is pregnant again and told
D1270N is not a CF mutation because intervening
publication† demonstrated D1270 is a polymorphism,
not a mutation. Couple told new fetus is not at risk;
couple is angry, upset, and threatening litigation.
*D1270N part of extended panel, but not ACMG standard panel.
†Strom et al. Genet Med. 2001.
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Extended Mutation Panels
Illustration #2
Mother
DF508
Father
Rare mutation
Fetus
DF508/rare mutation
Couple terminates pregnancy; however, only 2
previous cases known, each of which manifested
with only mild lung disease
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Quest Diagnostics’ Policy
Screening panel to include
• Only mutations shown to be real CF mutation
(based on published literature)
• Only mutations for which we have genomic
controls
• Currently 31
• ~ 50 by 2007
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Other CF Tests
• Sweat chloride: the gold standard of CF
diagnosis
• Immunoreactive trypsinogen (IRT) testing of
newborn blood spots (ie, newborn screening)
• DNA scanning
– Uses conformational changes to detect
heterozygous amplicons followed by DNA
sequencing of abnormal amplicon
– Sensitivity not established; specificity ~100%
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Other CF Tests - Continued
• DNA Sequencing
– Determines exact DNA sequence for all
coding regions and splice junctions in CFTR
gene
• Deletion / Duplication
– Analyzes for presence of large deletions and
duplications
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CF Complete™
• Sequences entire coding region, promotor
region and 250 bp into each exon
• Detects >99% of all described CF mutations
• Detects novel mutations and polymorphisms
• Indicated only for CF patients and relatives
• Cost >$2,000 / test
• Single and 2 exon sequencing available for
family members and prenatal diagnosis when
mutation(s) is known
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DNA Sequencing / Gene Scanning
Not indicated for population-based
carrier screening
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Interpret with Caution,
Do No Harm
• More than half of all mutations found by CF
Complete testing are novel
• More than ¾ have been described fewer than
2 times
• Clinical significance of novel missense
mutations is carrier screening is unknown
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Single-exon deletion
Multi-exon deletion
Single-exon duplication
Multi-exon duplication
Single-intron deletion
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From Passarge (2001) Color Atlas of Genetics 2nd Ed. Thieme
Screening Samples Submitted for
CFTR DNA Sequencing
6%
N=2
N=16
N=16
47%
47%
Two Mutations
No Mutations
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One Mutation
6%
Neg,
n=2
One
mutation
n=7
One Mutation
N=16
47%
Two mutations
n=16
47%
21%
DNA Sequencing
Promoter
/Exon
Deletions
N=9
Deletion
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26%
Patients with Classical CF
• Approximately 60% of affected Caucasians
will have 2 mutations identified by ACMG
panel screening
• If remaining 40% are submitted for
comprehensive analysis (sequencing and
deletion analysis), 84% will have 2 mutations
identified
• Therefore, 94% of all affected patients will
have both CF mutations identified
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Newborn CF Screening
• Immunoreactive trypsinogen will identify ~90%
of CF-affected newborns as well as some
carriers
• Sweat tests often equivocal or false-negative in
newborn period
• Tremendous anxiety in parents whose children
test IRT positive, have equivocal sweat tests,
and only 1 CF mutation identified. Child affected
or carrier?
Role for CF Complete & duplication/deletion test
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Conclusions re Appropriate Testing
Population-based carrier
testing
ACMG panel screen
Spouse of a known carrier
ACMG panel screen
Suspected CF-affected
person
1. Sweat test
2. ACMG screen if above +/3. Sequencing and/or
deletion/duplication test
IRT positive, sweat test
equivocal
1. ACMG screen
2. Sequencing
3. Duplication/deletion test
IRT, immunoreactive trypsinogen.
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