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

“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.

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.

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

Worldwide Distributions of Cystic Fibrosis Gene Mutations O'Sullivan, B, Freedman, S. Cystic Fibrosis. Lancet 2009; 373:1891-1904

Autosomal Recessive Inheritance

Family History

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

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

Clinical Phenotype Ratjen, F. Cystic fibrosis: Pathogenesis and future treatment strategies. Respir Care 2009; 54(5): 595-602.

Clinical Phenotype Respiratory GI, pancreas Reproductive Current life expectancy 31-37 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.

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

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

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

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

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): 851-868

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.

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. 60-70% of men with CBAVD carry one mutation in the CFTR gene. 5T reduces the number of functional Cl channels

Congenital Bilateral Absence of the Vas Deferens (CBAVD)

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

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

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

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

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

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

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

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

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

Resources Cystic Fibrosis Foundation http://www.cff.org http://cysticfibrosis.com GeneTests and GeneReviews http://www.genetests.org National Society of Genetic Counselors http://www.nsgc.org Mountain States Genetics Network http://www.mostgene.org

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