# Handbook of Genetic Counseling/Cystic Fibrosis Carrier Screening-2

Cystic Fibrosis

## What is CF?

* Cystic Fibrosis (CF) is one of the most common inherited diseases, affecting about 1 in 3300 live births in the United States.
* It is most common in Caucasians.
* CF is a complex multisystem disease that affects the respiratory tract, pancreas, intestine, reproductive tract, and hepatobiliary system.
* CF causes many parts of the body to produce thick mucus leading to pneumonia, diarrhea, poor growth, and infertility. This is due to the faulty transport of sodium and chloride within cells lining certain organs.

## Diagnosis

* The median age at diagnosis is 6 to 8 months; nearly two-thirds of individuals are diagnosed before 1 year of age.
* The diagnosis of CF is established in individuals with one or more characteristic phenotypic features of CF plus evidence of an abnormality in CFTR function based upon ONE of the following:
** Presence of two disease-causing mutations in the CFTR gene
** Two abnormal quantitative sweat chloride values
** An abnormal value for the transepithelial nasal potential difference (NPD)
* The sweat test has been the "gold standard" for diagnosing CF for more than 40 years.
** It is a diagnostic procedure that determines the amount of sodium and chloride (salt) in the sweat.
** It is painless, relatively inexpensive, and gives definitive answers.
** The test can be performed on individuals of any age.
** A colorless, odorless chemical known to cause sweating is applied to a small area on the arm or leg.
** An electrode is attached to stimulate a weak electrical current to the area. Patients may feel a tingling sensation in the area or a feeling or warmth. This lasts approximately 5 minutes.
** The stimulated area is then cleaned, and sweat is collected on filter paper or in a plastic coil. This is analyzed for increased amounts of sodium and chloride.
* The diagnosis of CF may be made in the absence of phenotypic features of CF in the following circumstances:
** Confirmed diagnosis of CF in a sibling and an abnormal sweat chloride value or presence of the same two disease-causing mutations in the CFTR gene as identified in the affected sibling.
** Diagnosis in a newborn screening program.
*** As of November 2001, Colorado, Wisconsin and Wyoming offer CF in the newborn screen.
*** Connecticut, Massachusetts, and Montana either test in a select population, have a pilot program, or are planning to begin CF newborn screening.
** In utero diagnosis by CFTR mutational analysis.
*** 10.3% of newly diagnosed patients in 1999 were diagnosed by newborn screening or prenatal diagnosis.

## Prognosis

* Median survival has increased from 18 years in 1976 to 30.1 years in 1995. Current approximation is 36.5 years (geneclinics.org).
* Pulmonary disease is the major cause of morbidity and mortality in CF. Patients have lower airway inflammation and chronic endobronchial infection, which often progresses to end stage lung disease characterized by extensive airway damage.
* Patients with pancreatic sufficiency (<10%) have a milder clinical course with greater median survival (56 years according to 1995 CFF Patient Registry) as compared to those with pancreatic insufficiency.
* There is a wide variability of disease expression from death in early childhood to patients presenting as young adults with a history of recurrent sinusitis and bronchitis or male infertility.

## Features of CF

* Intelligence is normal.
* Phenotypic feature of CF may include:
** Chronic sino-pulmonary disease
*** Including chronic cough, sputum production, chronic wheeze, obstructive lung disease on lung function test, chronic chest radiograph abnormalities, and digital clubbing.
** Gastrointestinal/nutritional abnormalities
*** Such as malabsorption/pancreatic insufficiency, distal intestinal obstructive syndrome, rectal prolapse, recurrent pancreatitis, chronic hepatobiliary disease, failure to thrive, hypoproteinemia, and fat-soluble vitamin deficiencies.
** Infertility
*** More than 95% of males with CF are infertile due to azoospermia resulting from altered Wolffian duct structures.
*** Most women with CF are fertile, although some have abnormal cervical mucus which may contribute to infertility
** Salt-loss syndrome

## Management

* Referral to a regional CF center is strongly recommended for individuals with CF.
* Epidemiological data show that patients followed on a regular basis in accredited CF centers have improved clinical outcome.
* Respiratory:
** Patients are regularly followed with pulmonary function studies, chest radiographs, and specific blood and urine tests.
** Intervention to treat or prevent pulmonary complications may include oral, inhaled or I*antibiotics, bronchodilators, anti-inflammatory agents, mucolytic agents, and chest physiotherapy (postural drainage with chest percussion).
* Gastrointestinal:
** Nutritional therapy may include special formulas for infants to enhance weight gain, supplemental feeding to increase caloric intake, and additional fat-soluble vitamins to prevent vitamin deficiencies.
** Pancreatic insufficiency is treated with oral pancreatic enzyme replacement.
* Pregnancy:
** With increasingly better pulmonary treatment and improved nutrition, pregnancies today are well tolerated, especially in women with mild to moderate disease.
** The risk for congenital anomalies in the fetus has not been seen to be increased.
** Breastfeeding is possible.
* Gene therapy is in a research phase only and is not able to control or treat the symptoms related to CF at this time.
** U.S. Congress appropriated $40 million to the NIH to establish nine CF gene therapy centers, which will run clinical trials for the development of gene therapy technology.
** Pilot and feasibility studies are currently active at the following gene therapy centers:
*** Cornell University Medical College, New York, New York
*** Institute for Human Gene Therapy at the University of Pennsylvania, Philadelphia
*** Johns Hopkins University School of Medicine, Baltimore, Maryland
*** University of North Carolina at Chapel Hill
*** University of Washington, Seattle
*** University of Iowa, Iowa City
*** University of Alabama at Birmingham.

## Inheritance

* CFTR gene mutations are inherited in an autosomal recessive manner.
* Parents of a proband are obligate carriers.
* Carriers are asymptomatic. They have no biochemical or physiologic alterations by which they could be readily identified.
* Siblings of a proband have a 25% chance of being affected, a 50% chance of being an unaffected carrier, and a 25% chance of being unaffected and not a carrier.

## Molecular Study

* CF's causative gene, CFTR (cystic fibrosis transmembrane conductance regulator), and its mutations were characterized in 1989.
* The normal gene product of the CFTR gene is a 1,480 amino acid integral membrane protein that functions as a regulated chloride channel in epithelial cells.
** The CFTR gene is 230 kilobases, contains 27 exons, and produces a 6.5 kilobase mRNA product.
* There are over 900 mutations known; almost all are point mutations or small (1-84 bp) deletions and are specific to an affected family.
** The most common mutation is delta F508, which accounts for about 30-80% of mutant alleles depending on the ethnic group.
** There is a panel of 25 CFTR alleles recommended by the American College of Medical Genetics for general population CF carrier screening.
* Mutations in CFTR can affect the CFTR protein quantitatively and/or qualitatively.
* The best correlation between genotype and phenotype occurs in the area of pancreatic sufficiency. The most common mutations have been classified as pancreatic-sufficient (PS) or pancreatic-insufficient (PI).
** Patients with pancreatic sufficiency usually have either one or two PS alleles.
* In contrast, genotype-phenotype correlation is generally poor for pulmonary disease in CF.
* Many mutations are sufficiently rare that genotype-phenotype predictions cannot be made on the basis of empirical studies of populations of patients.

## Population Genetics

* The CFTR mutation detection rate varies with ethnic background.
* The following chart approximates incidence and carrier frequency by ethnicity:
** Group Incidence Carrier Frequency
** Caucasian 1/3,300 1/28
** Hispanics 1/8-9,000 1/46
** African American 1/15,300 1/61
** Asian Americans 1/32,100 1/90

## Genetic Testing

* Molecular genetic testing for disease causing mutations in the CFTR gene (7q31) is used:
** To establish or confirm the diagnosis of CF in symptomatic individuals.
** For carrier detection in population screening programs, in at-risk relatives, and their reproductive partners.
** In prenatal testing of at-risk pregnancies and for pregnancies in which fetal echogenic bowel has been identified.
* The GeneTests-GeneClinics Laboratory Directory lists 64 worldwide labs that offer clinical testing for CF.
** This list is not comprehensive because laboratory participation is voluntary.
** There are additional laboratory sites that offer research testing for CF.
* Genzyme Genetics: http://www.genzymegenetics.com/Our-Services/Reproductive-Testing/cf-plus.aspx
** Genzyme has a mutation panel containing 97 of the most common mutations, including those found in diverse ethnic populations or associated with mild disease.
** Many commercial testing facilities offer only the recommended panel of 25 mutations.
** Specimen Requirements:
*** 1. Blood: ACD-A (yellow-top) or EDTA (lavender-top) tube. Adult: 10 ml whole blood; 30 ml whole blood if ordering multiple tests.
*** 2. Dried Blood Spot: Dried blood spots collected on Genzyme provided filter paper/test requisition. Fill a minimum of three circles completely with blood. Please Note: If prenatal carrier screening for cystic fibrosis is ordered in conjunction with first trimester screening all six circles should be filled.
*** 3. Mouthwash: Please Note: Patient should not eat, drink, smoke or chew gum for at least one hour prior to collection. (See specimen kit for detailed instructions). Mouthwash samples can be sent when ordering up to two tests. When ordering more than two tests, blood samples are required. 10 ml of Scope® mouthwash that has been swished vigorously for at least 1 minute. Collect in orange-top 50 ml polypropylene tube.
*** 4. Prenatal: Please Note: Call a laboratory genetic coordinator prior to obtaining any specimens. In some circumstances, specimens from other family members may be required. All prenatal specimens (including cord blood) must be accompanied by a maternal blood or mouthwash specimen for analysis of possible maternal cell contamination. Discard first 2 ml; then 15 ml amniotic fluid in 15 ml orange-top polypropylene tube, 10-15 mg chorionic villi in laboratory-provided screw-top tubes with sterile transport medium or 1 T-25 flask of confluent cells. Additional sample must be obtained for back-up culture at one of our cytogenetics laboratories or another facility. If additional testing is desired, more amniotic fluid is needed. For example, chromosome analysis requires an additional 15-25 ml (see Cytogenetics – Amniotic Fluid Chromosome Analysis test page: http://www.genzymegenetics.com/Our-Services/Reproductive-Testing/amniotic-fluid-testing.aspx) and AFAFP requires an additional 2 ml (see Amniotic Fluid Alpha-Fetoprotein - AFAFP test page: http://www.genzymegenetics.com/Our-Services/Reproductive-Testing/afafp-test.aspx).
** Genzyme provides a brief informed consent for DNA testing for the patient: http://www.genzymegenetics.com/For-Clients/informed-consent-forms-reproductive.aspx
* Ambry Genetics Corporation
** On 10/17/01, a press release from Ambry Genetics reported the development of "The Ambry Test: CF, the most comprehensive genetic test analyzing the CF gene."
** This test is a PCR based assay analyzing exonic and surrounding intronic regions of the patient's genomic DNA.
** Sequence analysis of the full CFTR gene allow for detection of novel mutations.
** As of 01/20/02 Ambry reports having found 64 different mutations, and seven of those are novel variants found in patients with CF.
** The cost is $285, and results are available in 1-4 weeks.
** This test may be useful for patients diagnosed with CF who have received a negative test result from the Genzyme panel analysis.
* CHMCC
** The Children's Hospital has a contract agreement with Genzyme; so standard CF testing is done with them.
** The cost of the blood test is $270, but 60% of that cost is in added charges from the CHMCC laboratory. Genzyme offers the test for $110.
** Blood specimens are typically used for carrier testing; 20 cc of blood in a purple top (EDTA) tube is sent at room temperature.
** Patients can be sent to the test referral center with the Genzyme test requisition form: http://www.genzymegenetics.com/For-Clients/test-requisitions.aspx, and the test referral center will take care of all processing.
** Samples should be drawn on Monday through Wednesday or Thursday before noon.
** Results typically take 2 weeks and are faxed to the lab unless you write in the fax number for the Human Genetics Department.
** The original report is mailed to the referring physician, and the patient is typically phoned with the results.
** Don't forget that the referring physician MUST sign the test requisition form!

## Prenatal Diagnosis

* Prenatal testing is available for pregnancies at 25% risk in which the disease-causing mutations of the CFTR gene have been identified in both parents.
* It is also available for pregnancies in which fetal echogenic bowel and/or dilated bowel is observed on ultrasound examination.

## NIH Consensus Statement

* A NIH Consensus Development Conference was held in 1997 to address the issue of genetic testing for CF.
* The conclusion was that genetic testing for CF should be offered to adults with a positive family history of CF, to partners of people with CF, to couples currently planning a pregnancy, and to couples seeking prenatal care.
* The panel did not recommend offering CF genetic testing to the general population or newborn infants.
* They felt that the offering of CF carrier testing should be phased in over a period of time to ensure that adequate education and appropriate genetic testing and counseling services are available to all people being tested.

## Resources

* Cystic Fibrosis Foundation
    Phone: (800) 344 4823
    Email: info@cff.org
    Website: www.cff.org
* NCBI Genes and Disease Webpage at www.ncbi.nlm.nih.gov/disease/CF

## References

* Grody, W.W., and R. J. Desnick. "Cystic Fibrosis Population Carrier Screening: Here at Last-Are We Ready?" Genetics in Medicine. March/April 2001. Vol 3, No 2, pp87-90.
* NIH Consensus Statement, Genetic testing for Cystic Fibrosis, Volume 15, Number 4, April 14-16, 1997.
* www.geneclinics.org
* http://www.GenzymeGenetics.com/
* www.cff.org

## Notes

The information in this outline was last updated in 2002.