MOLECULAR DIAGNOSTICS AT YNHH: CYSTIC FIBROSIS

CF is the most common lethal, autosomal recessive disorder among Caucasians, with a frequency of 1 in 2500. The rate is lower among African-American (1 in 18,000) and Asian (1 in 90,000) populations. In the U.S. there are 1000 new cases per year of CF and approximately 30,000 people live with the disorder. In the United States, 4% of Caucasians are heterozygous carriers, that is, harboring a single CF mutation; heterozygous individuals are asymptomatic. Some studies have shown that infants diagnosed early through screening programs have a more favorable prognosis than infants diagnosed later in life. A European study of patients diagnosed with CF by screening found that 88% were alive at 14 years of age, as compared to a 60% rate in those diagnosed after symptoms developed. Although CF lung disease cannot be cured, treatment programs have greatly increased median survival. Clinical management of CF consists of: promoting clearance of respiratory secretions by use of DNase (Pulmozyme), administration of fat-soluble vitamins and pancreatic enzyme supplement to prevent malnutrition, and judicious use of anti-microbial agents to control bronchopulmonary infection.

In the absence of screening, except in patients with intestinal obstruction (meconium ileus occurs in 5-10% of newborns with CF) or a positive family history of CF, delays of up to a year are common before the diagnosis of CF is made. It has been reported that the blood level of immunoreactive trypsinogen (IRT) is increased in newborns with CF because of their obstructed pancreatic ductules. Increased levels of IRT during the first few weeks after birth can occur for other reasons, however, including renal failure, hypoxic insult, and viral infection. Thus, there is a high percentage of false positives. For this reason, a positive IRT screen requires a confirmatory test, direct identification of the CF mutation.

The CF gene was identified in 1989 and is termed the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. The CFTR gene is located on the long arm of chromosome 7 (7q31). The gene contains 27 exons and spans over 230kb. The protein product is 1480 amino acids. The primary evidence that CFTR was the gene associated with CF came from mutational analysis. In 90% of individuals with CF, studies demonstrated a 3 bp deletion in the CFTR gene which removed a phenylalanine at position 508 ((F508 mutation). Subsequently, a total of 622 mutations of the CFTR gene have been identified. Some of these mutations are prominent in specific ethnic or racial groups; for example, the W1282X mutation represents 50% of all mutations in the CF-affected Ashkenazi Jewish population, whereas (F508 is present in only 30% of the affected individuals in this population. The genetic defect in CF results in impaired chloride transport caused by defective function of a salt channel in epithelial cells.

IRT screening is obtained on all newborns at Yale-New Haven Hospital. Blood from heal-sticks of all newborns is spotted onto filter paper (Guthrie card) and sent to the Prenatal Lab for IRT measurement. Newborns with IRT levels > 70ng/ml have a portion of their blood sample sent to the Molecular Diagnostics Lab where a polymerase chain reaction (PCR) analysis for the (F508 mutation is done. This test consists of two PCR reactions using patient DNA and a combination of primers specific for normal or mutant CF sequences. A normal individual generates a PCR product only in the PCR amplification using normal primers; a heterozygote yields products in both reactions; and an individual homozygous for the (F508 mutation generates a PCR reaction only with the mutant primers. The use of both reactions with internal controls ensures that false-negative results are not obtained.

If a newborn with an elevated IRT is homozygous for the (F508 mutation, no further testing is done. If the newborn is heterozygous for (F508, then a portion of the blood sample is sent to a specialty reference laboratory for testing for 70 additional CF mutations. If an additional mutation is found or if the newborn is homozygous for (F508, then the child is determined to have CF. The screening program to-date has identified three newborns with a homozygous (F508 genotype; all three were confirmed to have CF by an abnormal sweat test. The Molecular Diagnostics Lab is currently developing a testing protocol which will take into consideration racial/ethnic differences in mutation frequency and which will test for additional CF mutations.

CF genetic testing is also available to children who were not screened at YNHH and to concerned family members. CF genetic testing requires one tube of EDTA-anticoagulated blood (lavender top) which should be kept at room temperature and sent to the Immunology Laboratory at YNHH Monday through Friday. Call the Immunology Laboratory at 785-2440 or the Laboratory Medicine resident at 340-3411 with questions.

References

1. Ferrie RM et al. (1992). Development, Multiplexing, and Application of ARMS Tests for common mutations in the CFTR gene. Am J Hum Genet 51: 251-262.
2. Kirschbaum NE, Foster PA (1995). The polymerase chain reaction with sequence specific primers for the detection of the Factor V mutation associated with activated protein C resistance. Thrombos Haemostas 74: 874-878.
3. Bellissimo DB, Kirschbaum NE, Foster PA (1996). Improved method for Factor V Leiden typing by PCR-SSP. Thrombos Haemostas 75: 520-526.
4. Gregg RG et al. (1993). Application of DNA analysis in a population-screening program for neonatal diagnosis of cystic fibrosis (CF): Comparison of screening protocols. Am J Hum Genet 52:616-626.
5. Dankert-Roelse JE et al. (1989). Survival and clinical outcome in patients with cystic fibrosis with or without neonatal screening. J Peds 114: 362-367.
6. Crossley JR et al. (1979). Dried-blood spot screening for cystic fibrosis in the newborn. Lancet 1: 472-474.
7. Heeley AF, Bangert SK (1992). The neonatal detection of cystic fibrosis by measurement of immunoreactive trypsin in blood. Ann Clin Biochem 29: 361-376.

John Greg Howe, Ph.D.