1. Genetic Testing for Cystic Fibrosis
Dee Quinn, MS, CGC
August, 2009

2. “Gene Testing Going Mainstream”
Cystic Fibrosis Gene Test Offered By Lauran Neergaard AP Medical Writer Monday, Oct. 1, 2001; 9:53 p.m. EDT WASHINGTON –– Gene testing is going mainstream: Starting this month, tens of thousands of white Americans will be offered testing to see if they carry a gene mutation that causes cystic fibrosis even if no one in their family has the disease.

3. Epidemiology
One of the most common autosomal recessive diseases in Caucasians
Occurs in 1 in 3300 births
30,000 affected persons in the United States
Proposed advantages cholera infection, higher fertility and resistance to a variety of infectious agents.

4. Sensitivity of Mutation Analysis
Epidemiology 97
1/29
1/3,300
Ashkenazi Jews 30
1/90
1/32,100
Asian
Americans 69
1/60-65
1/15,300
African Americans
80-90
1/52
1/3,970-1/1,500
Native Americans 57
1/46
1/8-9,000
Hispanics 90
Caucasians
(United States)
Sensitivity of Mutation Analysis
Carrier
Frequency
Incidence
Group
80-90 Sensitivity depends on ethnic background of Caucasians, N. Europeans have 90% detection rate, S. Europeans 80%
Native Americans founder effect, geographic isolation

5. Worldwide Distributions of Cystic Fibrosis Gene Mutations
O'Sullivan, B, Freedman, S. Cystic Fibrosis. Lancet 2009; 373:

6. Autosomal Recessive Inheritance

7. Family History

8. CFTR Gene CFTR - cystic fibrosis transmembrane conductance regulator
Chromosome 7q31.2
Identified in 1989
Composed of 27 exons, 250kB of genomic DNA
Gene product is a 1480-amino acid membrane protein that functions as a regulated chloride channel in epithelial cells
More than 1500 mutations have been found in this gene
Lap-Chee Tsui and Francis Collins headed team that reported the discovery of the gene, linkage analysis (a lot of families to use) and positional cloning
Name CFTR-they hypothesized the gene was involved with transport and conductance across the membrane
Large gene
Located on the long arm of chromosome 7

11. CFTR Gene Responsible for: Expressed in epithelial cells of:
respiratory tract
sweat and salivary glands
pancreas
intestine
reproductive tract
Responsible for: CF
CBAVD
Chronic sinusitis
Reason CF has the clinical presentation that it does:
CFTR forms a regulated cell membrane Cl channel with several domains, allowing chloride to be transported out of the cell to the epithelial surface, so mutations cause defective chloride transport
Look at each individually
2. Respiratory: too much chloride kept in cell, leads to dehydrated secretions, thick mucus instead of thin watery secretions
Sweat glands: high levels of NaCl (salt) not reabsorbed
Pancreas-obstruct the digestive system and prevent pancreatic enzymes from reaching the small intestine
Intestine-presence of thickened mucus and lack of digestive enzymes, lead to increased risk for bowel obstruction, can sometimes be seen by ultrasound echogenic bowel (1-13% risk)
Reproductive

12. Clinical Phenotype
Ratjen, F. Cystic fibrosis: Pathogenesis and future treatment strategies. Respir Care 2009; 54(5):

13. Clinical Phenotype Respiratory GI, pancreas Reproductive
Current life expectancy years
UK study estimated life expectancy
for child born today with CF is 50
CF is typically a multi-system disease. Most patients have both respiratory and digestive problems, while others only have respiratory problems.
Pulmonary manifestations-range from very mild pulmonary symptoms to severe progressive chronic lung disease, vast majority of patients with CF die as a result of pulmonary complications
Pancreatic insufficiency and intestinal malabsorption are present in 85% of affected individuals
Men with CF (more than 95%) are infertile due to azoospermia, because of congenital bilateral absence of the vas deferens
Also see men without any pulmonary or gastrointestinal manifestations of CF may have CBAVD resulting in azoospermia
Salt loss syndromes like salt depletion or chronic metabolic alkalosis
Intelligence and appearance are typically not affected in those with CF.

14. CFTR Mutation Testing Most labs test for 25-97 mutations
10 of the identified CF mutations occur in more than 1% of CF chromosomes
Most common mutation (Δ F508) found in 66% of individuals with CF
5 classes of CFTR mutations
Genotype/phenotype correlations may be helpful in predicting pancreatic sufficiency
With that many mutations, how could screening possibly be accomplished?
Most of the remaining mutations occur infrequently, usually only in a particular family
Helpful to organize into 5 classes

15. Class I premature termination signals and splicing abnormalities, no protein production
Class II- defective folding and disruption of protein biosynthesis, can’t make to membrane
Class III- generate protein that reaches the plasma membrane but channels show defective regulation
Class IV- generate correcting localized CFTR with defective pore properties, reduced Cl current, less Cl in and out and the channel is open a shorter time
Class V-reduced protein production
In general Class 1,2 or 3 mutations are expected to have more serious phenotypic consequences than class 4 or 5
Call attention to deltaF508 and R117h

16. Effects of Gene Mutations
Class I, II and III mutations are more common and associated with pancreatic insufficiency
Class IV and V are less common and usually associated with pancreatic sufficiency
CFTR gene interacts with other intracellular proteins which may affect clinical symptoms

17. CFTR Mutations- ΔF508 Most common mutation in North America
Deletion of phenylalanine in codon 508  misfolded protein
Protein product still functioning as Cl channel, but is degraded in the endoplasmic reticulum
ΔF508 (homozygosity): classical phenotype
Varies from ethnic group
Classical phenotype-typically have severe pancreatic insufficiency combined with variable pulmonary function

18. Effects of Common Mutations
Moskowitz, SM, et.al. Clinical practice and genetic counseling for cystic fobrosis and CFTR_related disorders. Genet Med 2008; 10(12):

19. Gene-based Therapies Gene therapy Ataluren trial uses viral vector
Still being developed
Ataluren trial
Drug which “reads through” stop codons
Currently beginning phase 3 trials
Can only be used for individuals who have CF mutation as result of nonsense mutations (Class I)
Ataluren is designed to allow the ribosome to ignore the premature stop signal and continue translation of the mRNA, resulting in formation of a functioning protein.

20. Clinical Phenotype - Congenital Bilateral Absence of the Vas Deferens (CBAVD)
Vas deferens – carries sperm from the epididymis to the ejaculatory ducts
Absence of vas occurs in 95% of males with CF
CBAVD: distinct genetic disorder which overlaps with CF and causes infertility
Noncoding region of CFTR gene involved: intron 8 with thymidine tracts (5T/7T/9T)
60-70% of men with CBAVD carry one mutation in the CFTR gene.
5T reduces the number of functional Cl channels
Intricately tied to CF
CBAVD is a distinct genetic disorder. Condition overlaps with CF, but also represents a distinct entity so it occurs outside of the CF phenotype and is responsible for 1-2% of infertility due to azoospermia
Mutations in the CFTR gene have also been identified in patients with CBAVD which suggests this condition is a genital form of CF % of men with CBAVD carry one mutation in the CFTR gene.
5T reduces the number of functional Cl channels

21. Congenital Bilateral Absence of the Vas Deferens (CBAVD)

22. Newborn Screening Screening program should include:
Specific provider and patient educational materials
Protocol for addressing positive screening results
Development of systems in collaboration with specialty care providers to track short-term and long-term child outcomes and identify resources to support this activity
Blood spots from infants taken within days of birth to identify infants at increased risk for a specific genetic disorders

23. Newborn Screening for Cystic Fibrosis
Tests for immunoreactive trypsinogen (IRT), repeat IRT if elevated
Trypsinogen is synthesized in the pancreas
Individuals with CF have elevated levels of trypsinogen, regardless of pancreatic sufficiency
Currently offered in most states – AZ began
newborn screening in 11/07
Important for:
Early treatment of respiratory illnesses
Evidence for nutritional benefit
Diagnostic test is sweat chloride test
DNA testing is important for family planning and new treatments

24. ACOG/ACMG Recommendations For Prenatal Screening
Offer screening to:
Individuals with a family history of CF
Reproductive partners of individuals with CF
Couples in whom one or both are Caucasian and are planning a pregnancy or seeking prenatal care
Other individuals must be given written information
Additional indications for screening
Echogenic bowel detected on prenatal ultrasound (15% of infants with CF have meconium ileus)
Infertility in males
Elicit family history, determine who in family has CF, refer to genetics professional because of interpretation of test results and estimation of risk may be more complex than in the general population (bring up referral to genetic professional, clearly states so that obs and PCPs will not have to provide these services and so that insurance will cover)
Clarify couple’s risk of having a child with CF, majority are aware of their increased risk for having a child with CF, again referral to genetics professional
Offered when both the frequency of carriers and the detection rate of the carrier test are relatively high in the ethnic groups of the partners
Made available to other ethnic and racial groups, informed of their delectability through educational brochures. For example, Asian Americans w/o significant Caucasian admixture should be informed of the rarity of the disease and the very low yield of the test in their population.
Problem may be difficult to classify patients ethnic background, especially in a busy clinical setting, advocate universal screening, most often happen in the PCP’s office or the OB’s office

25. ACOG/ACMG Recommendations
Provider’s Role:
Purpose of screening
Voluntary nature of screening
Symptoms of CF, treatment and
prognosis
Genetics of CF and population
frequencies
Meaning of positive and negative test
results
Factors to consider in deciding to have
or not to have screening
Common patient belief that any test offered by a physician must be necessary and advantageous, see this with amnio
Booklet address these issues, makes explicit the optional nature of the testing and presents information in written form so that the patient and partner can consider their concerns and formulate questions about testing before discussing it with the support staff or provider, suggested that booklet be sent to a woman before her initial clinic visit, reinforces idea that CF screening is a personal decision between the couple
Factors for screening, if risk seems high and insurance covers, against doesn’t detect all carriers
Booklets are intended to supplement not replace communication with providers
Informed consent: counseling and offer of screening and the couple’s decision about screening should be discussed and documented in the medical record, written informed consent should be obtained only after the woman and her partner have opportunity to review the educational material and receive pretest counseling.
Lower risk ethnic groups, CF screening should be made available, printed information provided, screening and counseling provided upon request

26. Diagnostic Prenatal Tests
Testing offered when:
When both members of a couple are carriers, ie: 25% risk of having a baby with CF
When one member of a couple is carrier and other member not available for testing
Testing options:
Chorionic villus sampling (CVS)
9-11 weeks
Amniocentesis
After 14 weeks

27. Other Approaches Procedure Caveats In vitro fertilization
One cell removed from early embryo to test for mutations which were found in parents
Cell without a CF genotype transferred to mother’s uterus
Caveats
Technically demanding and complex procedure
Available on a limited basis
Expensive: $4,000 - $12,000
Ethical implications

28. Limitations of CF Screening
Does not detect all carriers
Estimate of residual risk applies only when family history is negative and to the current pregnancy
Cannot make reliable predictions for outcome based on mutations
Non-paternity
Not all CF mutations can be detected in screening panels. Impt to go over residual risk based on the racial or ethnic group of the partner, the specific mutations the test covered, and whether one or both partners were tested like we just looked at
At end: All of these issues should be discussed with couple before testing and reviewed after results given

29. Genetic Counseling
Various outcomes of prenatal and newborn testing will generate need for genetic counseling:
Newly diagnosed child with CF
Healthy males who carry mutations associated with infertility
Identification of positive/negative couples who request additional mutational analyses or counseling to clarify residual risk
Positive/positive couples

30. Ethical, Legal, and Social Implications of CF Screening
Unnecessary anxiety created
Inadequate pretest information
Legal
Informed consent
Insurance discrimination
Social
Expense swell health costs
Societal pressure not to bear affected offspring
NIH conducted pilot studies before issuing consensus statement. Ethical, legal and social issues were looked at in deciding whether CF screening should be implemented.
Ethical:
Anxiety levels: no increase in anxiety levels by CF education or DNA testing
Less than half (44%) whose result was negative understood that they could still have a child with CF, providers in that study spent little time explaining CF carrier screening and generally relied in patient comprehending written material (article)
Legal:
Without proper education, can’t really be considered an informed consent to testing, suggestions have been made to use quizzes that would test patient understanding, provider cannot be held liable for couples that have children with CF
1/3 of those express concern about insurance discrimination, laws in place that prevent discrimination, most of the concern with insurance discrimination is with predictive gene tests and insurance companies reducing benefits on the basis of predictive gene test.
Social:
57% offered screening accepted, factors against religious beliefs, 1/3 insurance fears
Most decided on their own rather than on the basis of physician, reasons were logical, most women who would not terminate a pregnancy for CF chose not to be tested
Overall, NIH pilot studies have not shown results that substantiate concern over any of these implications, except for adequate pretest education. Several studies have been done since then and these same issue has arisen, that providers aren’t familiar with the information about CF and in turn do not pass along to their patients

31. Resources Cystic Fibrosis Foundation http://www.cff.org
GeneTests and GeneReviews
National Society of Genetic Counselors
Mountain States Genetics Network

32. Conclusions
“It will be very important to see how this goes. Certainly it requires primary care providers to become more familiar with genetics than many of them have previously had occasion to do.” -Francis Collins
Implications for other gene testing