Draft Genetic Test Review

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Cystic Fibrosis
Clinical Validity

(436KB)

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CLINICAL VALIDITY

Question 18: How often is the test positive when the disorder is present?
Question 19: How often is the test negative when the disorder is not present?
Question 20: Are there methods to resolve clinical false positive results in a timely manner?
Question 21: What is the prevalence of the disorder in this setting?
Question 22: Has the test been adequately validated on all populations to which it may be offered?
Question 23: What are the positive and negative predictive values?
Question 24: What are the genotype/phenotype relationships?
Question 25: What are the genetic, environmental or other modifiers?

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CLINICAL VALIDITY

Question 24: What are the genotype/phenotype relationships?

Phenotype
Cystic fibrosis is a monogenic autosomal recessive disorder. Genotype, typically defined as the presence of two disease-causing mutations on separate alleles, is, therefore, a primary cause of the development of the clinical phenotype. The underlying cause of cystic fibrosis involves abnormal chloride and sodium ion transport resulting from dysfunction of a cell membrane protein, the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). Disease-causing mutations in the CFTR gene on chromosome 7 result in complete or partial loss of functional CFTR, and development of a cystic fibrosis phenotype.

Phenotype is defined by the natural history of the disease, including a specific configuration of signs and symptoms, their severity, and the time of presentation. While there is some variability in clinical presentation, cystic fibrosis is a serious, progressive, multi-system disease (Table 3-29). It is characterized by chronic obstructive pulmonary disease, exocrine pancreatic insufficiency, elevated sweat chloride concentration (>60 mM), and infertility in almost all males due to obstructive azoospermia (congenital bilateral absence of the vas deferens or CBAVD). Age of onset of symptoms varies but is generally early. In a minority of cases, infants are diagnosed in the neonatal period due to meconium ileus (i.e., intestinal obstruction due to inspissated secretions). The median age at diagnosis is 6 months, with about 80 percent of cases diagnosed by 3 years of age (CF Foundation 1999 Data Registry). About 5 to 15 percent of affected individuals retain some level of pancreatic function (pancreatic sufficiency). Between 1 and 2 percent individuals classified as having cystic fibrosis are less severely affected. Some have less severe pulmonary disease, or no evidence of pancreatic dysfunction and borderline or normal sweat chloride levels (Rosenstein et al., 1998; Cutting, 2001). Others are described with late onset of symptoms or unusually mild pulmonary disease.

Gap in Knowledge: Unbiased information about the genotype/phenotype relationship is currently limited.
The estimates contained in Table 3-29 are derived from multiple studies and a general consensus has not yet been achieved. Some studies were small and other may have been subject to biases of ascertainment. For example, the Cystic Fibrosis Foundation has data available to perform a survival analysis for cystic fibrosis individuals stratified by pancreatic status. However, such an analysis has not yet been reported.

Table 3-29. Clinical Characteristics of Cystic Fibrosis

Clinical Characteristics of Cystic Fibrosis	Proportion of Indivduals with Cystic Fibrosis having that Clinical Charactericstic (%)
Severe chronic pulmonary disease	98
Elevated sweat chloride levels	90-98
Male infertility	95
Pancreatic insufficiency
Total	83-94
Partial	5-15
None	1-2

 

Other medical conditions associated with cystic fibrosis mutations
Several monosymptomatic disorders have been described that are, or may be, CFTR-related, including:

• Congenital bilateral absence of the vas deferens (CBAVD)
• Idiopathic pancreatitis
• Disseminated bronchiectasis
• Allergic bronchopulmonary aspergillosis
• Atypical sinopulmonary disease

The purpose of prenatal screening is to identify couples at high risk of having a fetus with the typical features of cystic fibrosis (Table 3-29). For this reason, these unusual and, for the most part, rare conditions are unlikely to be a major consideration in prenatal cystic fibrosis screening.

Genotype
More than 900 mutations in the cystic fibrosis gene have been reported to the Cystic Fibrosis Genetic Analysis Consortium (http://www.genet.sickkids.on.ca/cftr/). Not all have been found to cause disease, and some are known to be benign polymorphisms. The most common mutation, delF508, accounts for 66 percent of cystic fibrosis chromosomes in a worldwide survey (Tsui and Durrie, 1997). Among the more than 15,000 cystic fibrosis patients in the United States genotyped through 1999, 52 percent are homozygous for delF508, and another 36 percent are compound heterozygotes having delF508 and another mutation (CF Foundation, 1999 Data Registry). The next most common mutations worldwide are G542X, G551D, 621+1G>T, N1303K and W1282X, each accounting for 1 to 2.5 percent of mutations. About 20 additional less common mutations occur at frequencies at or above 0.1 percent. All of the rest are rare, with some reported only in one case or within a single family. Mutation frequencies, and, consequently, the proportion of mutations detectable using a specific panel, vary by race and ethnicity. For more information about mutation frequencies, see Question 18.

Relationship between genotype and phenotype
Cystic fibrosis mutations can be classified by the molecular mechanisms by which they cause dysfunction (Table 3-30) (Kerem and Kerem, 1996; Rosenstein and Zeitlin, 1998; Mickle and Cutting, 1998; Mickle and Cutting, 2000; Zielensky, 2000). Mutations can result in CFTR that is absent, reduced, or abnormally functioning. These are grouped, as follows:

Class I mutations (e.g., G542X, 621+1G>T, and 711+1G>T) result in total deficiency or unstable/non-functional CFTR protein.

• Class II mutations (e.g., delF508, N1303K, and delI507) disrupt normal intracellular processing (e.g., glycosylation), causing instability of CFTR protein, or interfering with its movement to the correct cellular location.
• Class III mutations (e.g., G551D) result in a normal amount of CFTR protein being produced and positioned at the cell surface, but the protein is non-functional.
• Class IV mutations (e.g., R117H, A455E) result in a normal amount of functional CFTR at the cell membrane, but chloride conductance is reduced. These mutations are generally associated with a pancreatic sufficiency.
• Class V mutations (e.g., 3849+10KbC>T)result in reduced levels of normally functional CFTR protein at the cell membrane and are also associated with a less severe phenotype.

Table 3-30. Mutation Classification by Mechanism of CFTR Dysfunction

CFTR Mutations Included in the Recommended Panel of 25
Mutation Classes I, II, and III		Mutation Classes IV and V
delF508	N1303K	R117H
G542X	711+1G>T	R334W
621+1G>T	delI507	A455E
G551D	R1162X	R347P
W1282X	R560T	3849+10KbC>T
R553X	1078delT	IVS8-5T
1717-1G>A	I148T	G85E
3659delC	2184delA	2789+5G>A
		3120+1G>T

The phenotypic effects of mutations in the first three classes are generally more severe, resulting in chronic pulmonary disease, pancreatic exocrine dysfunction, elevated sweat chloride levels, and CBAVD. Phenotypic severity does not appear to vary significantly for any combination of two mutations from these classes. The last two classes of mutations produce phenotypic effects that are similar to those found for the first three classes with regard to pulmonary disease, but pancreatic function is more often preserved. This is consistent with clinical observations of strong concordance in pancreatic function in affected sibs. Cystic fibrosis patients with Class IV or V mutations may also develop symptoms at a later age (as adolescents or adults). In a small percentage of individuals with two mutations, the presentation is less typical (Kerem and Kerem, 1996; Rosenstein and Zeitlin, 1998; Mickle and Cutting, 1998; Zielenski, 2000). While these expectations are generally correct, the associations are far from absolute. In a summary of seven published studies, a proportion of affected individuals with Class I, II or III mutations have delayed onset of pancreatic dysfunction, and a third of affected individuals with one or two of the Class IV or V mutations suffer from pancreatic insufficiency (Murray et al., 1999; Cutting, 2001).

The R117H mutation and reflexive testing
The R117H mutation is found in about 0.7 percent of chromosomes in non-Hispanic Caucasian individuals affected with cystic fibrosis (Question 18, Table 3-4). Among unaffected individuals in this population group, therefore, an R117H carrier would be expected once in every 3,860 individuals tested (1:27/0.007). Even fewer carriers would be expected in other populations (Question 18, Tables 3.6, 3-8, 3-10 and 3-12). Early pilot trials did not include this mutation in their screening panels. The first pilot trial to report experience with this mutation found 16 R117H carriers among 2,633 non-Hispanic Caucasians tested (carrier rate of 1:166) (Witt et al., 1996). This was nearly 20 times higher than expected. Clearly, most of these individuals were not carriers of a serious mutation. If the R117H mutation is to be used in prenatal screening, it would be necessary to identify conditions under which the mutation contributes to the classic cystic fibrosis phenotype.

Based on analyses of the CFTR gene in affected and unaffected individuals, it was determined that a gene modifier determined the phenotype associated with the R117H mutation when it was combined with another mutation (e.g., delF508) (Kiesewetter, 1993). The impact of R117H is dependent on the length of the polypyrimidine tract located in intron 8. Three length variants have been identified and designated 5T, 7T and 9T. These are found in about 5 percent, 10 percent and 85 percent of the general population, respectively. This Poly-T variant occurs in a noncoding region of the gene several exons removed from the R117H location (exon 4), but it affects gene expression by influencing splicing efficiency. The phenotypic variation is molecularly based and determined by whether the R117H mutation is located on the same (cis) or opposite (trans) chromosome 7. In order for this mutation to produce a severe phenotype, it must be 1) associated with the 5T variant on the same chromosome (in cis), and 2) the other chromosome must also carry a CF mutation that is capable of producing the phenotype. In the setting of prenatal screening, the ACMG has recommended that Poly-T testing be performed only as a reflex test for carriers shown to be heterozygous for the R117H mutation (Grody et al., 2001).

For example, a pregnant woman is found to be a carrier of the R117H mutation. Reflexive testing is performed to determine whether she carries the 5T polymorphism. In 95 percent of women tested, the polymorphism will be either 7T or 9T, and the woman can be informed that the mutation will not be associated with classic cystic fibrosis in the fetus, even if the partner if found to be a carrier. No further testing is required. It is necessary to test the parents of the remaining 5 percent of women, to determine whether the 5T polymorphism is in cis or trans with the R117H mutation. If it is in trans, no further testing is necessary and the woman can be informed that the mutation will not be associated with classic cystic fibrosis in the fetus, even if the partner if found to be a carrier. If, however, the 5T is in cis, the testing process continues to the next step; obtaining a sample from the partner. In the event that the woman's parents cannot be tested, testing of her partner could be undertaken with the knowledge that the partner will not have an identifiable mutation in approximately 29 of 30 such instances.  

Published recommendations and some laboratory practices have clouded this relatively straightforward process by including a discussion of infertility, due to congenital absence of the vas deferens (CBAVD) in otherwise healthy men (Anguiano et al., 1992; Gervais et al., 1993; Kiesewetter et al., 1993; Dork et al., 1997). Several combinations of the R117H mutation and Poly-T have been associated with CBAVD, and this finding can be helpful when investigating infertility. However, testing for the Poly-T in the absence of the R117H mutation can result in placing the couple, the laboratory and the referring physician in a difficult situation. For example, it may necessitate the discussion of possible CBAVD in male fetuses or the possibility of identifying the male partner as being infertile. In addition, misinterpretation of the penetrance of a 5T finding alone can lead to unnecessary diagnostic testing. In spite of all this, laboratories commonly test for Poly-T variants in samples known to be for prenatal screening and report them even when the R117H mutation is not present, thereby creating difficult and complex counseling situations for their clients. 

Gastrointestinal problems
Pancreatic exocrine function has been discussed above. Meconium ileus occurs in 15 to 20 percent of newborns with cystic fibrosis, nearly always in association with pancreatic insufficiency. While this complication nearly always occurs in individuals with pancreatic insufficiency, there is no association with specific mutations. Since most individuals with cystic fibrosis and pancreatic insufficiency do not develop meconium ileus, other genetic and/or environmental factors are likely to be involved. Other less common gastrointestinal problems, such as liver disease, and diabetes, are also not associated with genotype but are associated with pancreatic insufficiency. There is evidence that other genetic and/or environmental factors are involved in these diseases as well. 

Respiratory problems
Because lung disease is the primary cause of morbidity and mortality in affected individuals, much attention has been paid to possible associations between pulmonary phenotype and CFTR mutations. No significant correlation with genotype, or concordance within sibships, has been demonstrated for pulmonary disease. For most genotypes, including those involving delF508, there is considerable variability in pulmonary phenotype expression. However, the vast majority of individuals with two mutations have serious, progressive lung disease. About 24 percent of children and 64 percent of adults with cystic fibrosis have moderate to severe respiratory compromise, as defined by FEV₁ less than 70 percent of predicted. About 80 percent of cystic fibrosis patients are infected with Pseudomonas aeruginosa by 18-24 years of age (CF Foundation, 1999 Data Registry). One mutation, A455E, was associated with milder lung disease and late age of onset in Dutch cystic fibrosis patients (Mickle and Cutting, 1998). Population studies show less severe lung disease in patients with mutations associated with pancreatic sufficiency (Table 3-30, Class IV and V), as compared with other mutations (Zielenski, 2000). Overall, however, genotype is a poor predictor of pulmonary outcome. 

Reproductive problems
The male reproductive tract is very sensitive to the effects of CFTR mutations. Approximately 95 percent of males with cystic fibrosis are infertile (Welsh et al., 1995; Cutting, 2001). Fertility in females is reduced, but a reliable estimate is difficult to determine. In a survey of cystic fibrosis centers in 1980, 129 pregnancies were documented in 100 patients resulting in 86 live births (67 percent); one child was affected with cystic fibrosis (Cohen et al., 1980). In 1994, 135 of female cystic fibrosis patients in the United States between the ages of 15 and 41 (3.7 percent) were pregnant (CF Patient Registry 1994). Of these, 58 (43 percent) had already resulted in a live birth, 14 pregnancies were selectively terminated (10 percent), 11 were spontaneously aborted (8 percent), 2 were lost to follow-up (2 percent) and the remaining pregnancies were still ongoing.

References

Anguiano A, Oates RD, Amos JA, Dean M, Gerrard B, Stewart C, Maher TA, White MTS, Milunsky A. 1992. Congenital bilateral absence of the vas deferens: a primarily genital form of cystic fibrosis. JAMA 267:1794-1797.

Chillon M, Casals T, Mercier B, Bassas L, Lissens W, Silber S, Romey MC, Ruiz-Romera J, Verlingue C, Claustres M. 1995. Mutations in the cystic fibrosis gene in patients with congenital absence of the vas deferens. N Engl J Med 332:1475-1480.

Cohen LF, di Sant'Agnese PA, Frielander J. 1980 Cystic fibrosis and pregnancy: A national survey. Lancet 2:842-844.

Cutting GR. Cystic Fibrosis. In Principles and Practice of Medical Genetics, Churchill Livingstone, New York, DL Rimoin, JM Connor, RE Pyeritz, Eds., 4^th Edition, In press.

Cystic Fibrosis Foundation. Patient Registry 1994 Annual Data Report. 1997. Bethesda, MD.

Cystic Fibrosis Foundation. Patient Registry 1996 Annual Data Report. 1997. Bethesda, MD.

Cystic Fibrosis Foundation. Patient Registry 1998 Annual Data Report. 1999. Bethesda, MD.

Cystic Fibrosis Foundation. Patient Registry 1999 Annual Data Report. 2000. Bethesda, MD.

Dork T, Dworniczak B, Aulehis-Schotz C, Wieczorek D, Bhm I, Mayerova A, Seydewitz H, Nieschlag E, Meschede D, Horst J. 1997. Distinct spectrum of CFTR gene mutations in congenital absence of vas deferens. Hum Genet 100:367-377.

Gervais R, Dumur V, Rigot M-M, Lafite J-J, Roussel P, Claustres M, Demaille J. 1993. High frequency of the R117H cystic fibrosis mutation in patients with congenital absence of the vas deferens. N Engl J Med 328:446-447.

Grody et al. 2001. Laboratory standards and guidelines for population-based cystic fibrosis carrier screening. Genet Med 3:149-154.

Hamosh A, FitzSimmons SC, Macek M, Knowles MR, Rosenstein BJ, Cutting GR. 1998. Comparison of the clinical manifestations of cystic fibrosis in black and white patients J Peds 132:255-259.

Kerem B, Kerem E. 1996. The molecular basis for disease variability in cystic fibrosis. Eur J Hum Genet 4:65-73.

Kiesewetter S, Macek M, Davis C, et al.. 1993. A mutation in CFTR produces different phenotypes depending on chromosomal background. Nat Genet 5:274-277.

Mickle JE, Cutting GR. 1998. Clinical implications of cystic fibrosis transmembrane conductance regulator mutations. Clin Chest Med 19:443-459.

Mickle JE, Cutting GR. 2000. Genotype-phenotype relationships in cystic fibrosis. Med Clin North Am 84 :597-607.

Murray J, Cuckle H, Taylor G, Littlewood OBE, Hewison J. 1999. Screening for cystic fibrosis. Health Technol Assess 3 :1-101.

Rosenstein BJ, Zeitlin PL. 1998. Cystic fibrosis. Lancet 351 :277-282.

Rosenstein BJ, Cutting GR. 1998. The diagnosis of cystic fibrosis: A consensus statement. J Peds 132 :589-595.

The Cystic Fibrosis Genotype-Phenotype Consortium. 1993. Correlation between genotype and phenotype in patients with cystic fibrosis. N Eng J Med 329 :1308-1313.

Tsui L, Durie P. 1997. Genotype and phenotype in cystic fibrosis. Hosp Prac 32:115-142.

Welsh MJ, Tsui LC, Boat TF, Beaudet AL. 1995 Cystic Fibrosis in The metabolic and molecular bases of inherited disease (seventh edition, Vol III), McGraw-Hill, New York

Zielenski J. 2000. Genotype and phenotype in cystic fibrosis. Respiration 67:117-133.

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CLINICAL VALIDITY

Question 25: What are the genetic, environmental or other modifiers?

The described variability in age of onset, progression of pulmonary disease, and survival in individuals with cystic fibrosis suggests that factors other than CFTR genotype, particularly genetic background and environmental influences, are likely to play a role in the natural history of the disease in individual patients (Mickle and Cutting, 1998; Mickle and Cutting, 2000). In particular, little correlation has been demonstrated between specific CFTR genotypes and the severity of lung disease, a key determinant for prognosis and survival. For that reason, environmental and other genetic factors may play an important role (Mickle and Cutting, 1998; Mickle and Cutting, 2000). Lungs are directly exposed to a variety of environmental factors, including pollutants (e.g., cigarette smoke) and pathogens. Genetically determined factors could influence key events in progression of cystic fibrosis lung disease. Susceptibility to bacterial infections, for example, could be influenced by genes or regions associated with immunity (e.g., MBL, TNFα) and inflammation (e.g., HLA region). These genetic components, as well as other candidate modifier genes implicated in mouse models and human studies, have been targeted for study (Zielenski, 2000). Genetic factors that influence response to therapeutic intervention are also being explored.

In the context of prenatal screening for cystic fibrosis, future knowledge of important modifiers that could significantly affect prognosis or response to treatment might influence parental decision-making. For example, if a specific gene were identified that influenced an affected individual's response to a new effective treatment, prenatal testing for that gene might provide additional information about prognosis.

References

Cutting GR. Cystic Fibrosis. In Principles and Practice of Medical Genetics, Churchill Livingstone, New York, DL Rimoin, JM Connor, RE Pyeritz, Eds., 4^th Edition, In press.

Mickle JE, Cutting GR. 1998. Clinical implications of cystic fibrosis transmembrane conductance regulator mutations. Clin Chest Med 19:443-459.

Mickle JE, Cutting GR. 2000. Genotype-phenotype relationships in cystic fibrosis. Med Clin North Am 84 :597-607.

Murray J, Cuckle H, Taylor G, Littlewood OBE, Hewison J. 1999. Screening for cystic fibrosis. Health Technol Assess 3:1-101.