1. Charles (Buck) Strom, M.D., Ph.D., H.D.L.C., F.A.A.P., F.A.C.M.G.
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

2. *Congenital bilateral absence of the vas deferens.
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.

3. 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

4. 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

5. Classical Cystic Fibrosis
Prognosis
Chronic growth failure due to
Decreased intake and absorption
Increased work of breathing
Chronic infections
Average life expectancy years
Many die in adolescence due to noncompliance with treatment regimens

6. 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

7. 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.

8. CF Disease and Carrier Frequencies
Ethnic Group
CF Incidence
Carrier Risk
Caucasian
1:3,300
1:29
Hispanic
1: ,000
1:46
African American
1:15,300
1:62
Asian American
1:32,100
1:90

9. 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

10. 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

11. 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

12. 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
Genet Med. 2001;3:

13. 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
Genet Med. 2004;6:

14. How accurate are CF reagents?
3 series
(n >1,000 patients)
with no errors
ABI / Celera / Abbott OLA ASR

15. Clinical Issues in Population Based CF Testing

16. 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

17. 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

18. 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

19. 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:
#Strom et al. Genet Med. 2004;6:

20. 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
Strom et al. Genet Med. 2004;6:

21. Rare Events: true positive versus false positive
By definition, any “ethnic specific” mutation will be found less frequently than 1:25,000 pan- ethnic 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

22. 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

23. 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

24. Extended Mutation Panels
Arguments Against (continued)
Genetic counseling will be difficult when rare mutations are detected
Increased liability possible if unaffected fetus is terminated

25. Extended Mutation Panels
Illustration #1
Mother Father Fetus
DF508 D1270N* 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

26. Extended Mutation Panels
Illustration #2
Mother Father Fetus
DF508 Rare mutation DF508/rare mutation
Couple terminates pregnancy; however, only 2 previous cases known, each of which manifested with only mild lung disease

27. 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

28. 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%

29. 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

30. 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

31. DNA Sequencing / Gene Scanning
Not indicated for population-based carrier screening

32. 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

33. Single-exon duplication Multi-exon duplication Single-intron deletion
Single-exon deletion
Multi-exon deletion
Single-exon duplication
Multi-exon duplication
Single-intron deletion
From Passarge (2001) Color Atlas of Genetics 2nd Ed. Thieme

34. Screening Samples Submitted for CFTR DNA Sequencing

35. DNA Sequencing Deletion

36. 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

37. 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

38. Conclusions re Appropriate Testing
Population-based carrier testing
ACMG panel screen
Spouse of a known carrier
Suspected CF-affected person
Sweat test
ACMG screen if above +/-
Sequencing and/or deletion/duplication test
IRT positive, sweat test equivocal
ACMG screen
Sequencing
Duplication/deletion test
IRT, immunoreactive trypsinogen.