CF mutation analysis is useful for a variety of clinical indications, including diagnosis, newborn screening, prenatal diagnosis for at-risk pregnancies, and carrier detection. The vast majority of laboratory tests are for carrier detection and risk revision.
Mutation analysis is a useful adjunct to sweat testing for diagnosis of CF, particularly for patients with borderline sweat tests, patients with atypical clinical presentations with normal sweat electrolytes, and at-risk newborns for whom sufficient quantities of sweat cannot be collected. Mutation analysis of a diagnosed proband also is useful for identifying the familial mutation for carrier testing of at-risk relatives. Both genetic and sweat testing are used as follow-up to immunoreactive trypsinogen-level testing in newborn screening programs.
Targeted mutation analysis is used for prenatal testing of pregnancies with a 1-in-4 risk for which both mutations have been identified in the two carrier parents. Prenatal testing often is offered in a lower-risk pregnancy presenting in the second trimester on ultrasound with fetal echogenic bowel (EB). EB is associated with an increased risk for CF in the absence of a positive family history. The risk for CF varies between 2% and 20% depending on the grade of the EB. Mutation analysis of the parents of a fetus with EB may reveal that both are carriers, but most often neither or only one parent is a heterozygote. Prenatal diagnosis can determine that a heterozygous parent has transmitted an identified mutation, but there is no additional testing that can further clarify the fetal CF status. In such cases, Bayesian analysis with incorporation of the grade of EB is used to modify the risk. In practice, scant clinical information, including the grade of the EB, is provided to the clinical laboratory. Furthermore, some clinicians submit a fetal sample without testing the parents, and formal genetic counseling may not be offered to the couple until the laboratory testing has been completed.
Prior to the cloning of the CFTR gene, couples learned that they were both CF carriers upon the birth of an affected child. No robust assays were available to assess a subsequent 1-in-4-risk pregnancy. Now, the most common indication for mutation analysis is direct heterozygote detection for carrier risk revision.
In 1997, a National Institutes of Health (NIH) Consensus Development Conference recommended CF carrier testing for all couples planning a pregnancy, regardless of family history. However, at that time, there was no standardization of CF testing among laboratories and no commercially available reagents or kits. The largest challenges to development of standardized mutation panels were the ethnic diversity and admixture of the US population, which complicated the selection of mutations for a standardized screening panel. Using available data from more than 20,000 CF patients, the American College of Medical Genetics (ACMG) and the American College of Obstetrics and Gynecology (ACOG) recommended a panethnic panel of 23 mutations that occur at a frequency of >0.1% in any of the major US ethnic groups, plus reflex testing for four additional sequence variants under specified conditions (see Reference 1 and http://www.acmg.net/resources/policies/pol-005.asp). Simultaneously, these organizations developed and distributed an educational document entitled "Preconceptual and Prenatal Carrier Screening for Cystic Fibrosis" to the memberships of ACMG and ACOG. Thus, CF carrier screening is now the standard of care in the United States.
The ACMG/ACOG minimum core mutation panel is shown in Table 10-1. While intended to be panethnic, it detects primarily mutations that are most frequent in the Ashkenazi Jewish and the non-Hispanic European white populations. Detection frequencies also are provided for the Hispanic (an admittedly mixed ethnic group) and black populations, but mutation detection frequency is unknown for individuals of Asian descent. The use of more extended mutation panels is not recommended by ACOG and ACMG for routine carrier screening. However, a report by Heim et al.,2 published immediately after the current testing recommendations, suggests that a panel of 64 mutations yields better coverage in all ethnic groups.
Reflex testing for the intron 8 5T/7T/9T sequence variation is specifically recommended for R117H positives, since 5T versus 7T or 9T in cis increases CF severity in a compound heterozygote with a second CF mutation. Since
Table 10-1. Recommended Core Mutation Panel for General Population CF Carrier Screening
Standard Mutation Panel
Reflex Tests intron 8 5T/7T/9T, I506V, I507V, F508C
AF508 1717(-1) G^A G542X
R560T 1898(+1) G^A 2184delA
some methodologies do not distinguish between AF508 and other alterations in close proximity, a second reflex to three exon 10 sequence variants (F508C, I506V, and I507V) is recommended when unexpected AF508 or AI507 homozygosity is detected to avoid reporting of false-positive results due to interference by these surrounding polymorphisms.
The ACMG/ACOG panel is not designed for diagnostic testing of CF patients or men with CBAVD. A larger mutation panel may provide increased detection for symptomatic patients. The intron 8 5T allele has been associated with CBAVD, but the intron 8 variation is not in the recommended screening panel because the 5T allele is no associated with classic CF.3
The ACMG/ACOG panel is expected to evolve as new data emerge, but the total number of recommended mutations will not substantially increase. As described at http://www.acmg.net/ the American College of Medical Genetics originally recommended a carrier screening panel of 25 mutations that was later decreased to 23. The I148T mutation was deleted based on non-pathogenocity and 1078delT also was dropped from the screening panel based on infrequency.
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