Neonatal Screening for Cystic Fibrosis: Addition of Molecular Diagnostics to Increase Specificity

Newborn screening for cystic fibrosis (CF) has been carried out on approximately 106,000 neonates in western Pennsylvania since 1987 using the immunoreactive trypsinogen (IRT) assay on dried filter paper blood specimens (DBS). Molecular analysis utilizing a duplicate DBS from the same sample was implemented in November 1989 for newborns having elevated IRT levels. DNA is amplified directly from the DBS and the amplified products are tested for the delta F508 deletion and several common exon 11 mutations. Substituting dUTP for dTTP in the PCR reaction and an initial treatment with uracil N-glycosylase (UNG) virtually eliminates PCR carryover contamination. The number of confirmed cases of CF is 20, giving an estimated incidence of 1:5287 in the western Pennsylvania population. Eight of the CF patients are homozygous and 12 are compound heterozygotes for the delta F508 deletion and a second mutation. Two of the compound heterozygotes carry the G551D mutation and one has the R553X mutation. Twenty-one additional neonates that are heterozygous for the delta F508 mutation are normal carriers for CF. In approximately 55% of the cases, molecular analysis of the CF gene confirmed the diagnosis of CF prior to sweat testing. The incorporation of molecular analysis into our CF screening program increases the specificity of the screening strategy and has the potential to decrease the false positive and sweat test referral rate, reduce parental anxiety, and bring CF infants to the attention of physicians more rapidly.     

Cystic fibrosis mutations in the Hutterite Brethren.

The presence or absence of the major cystic fibrosis (CF) mutation, delta F508, in the general patient population was determined by Kerem et al. using allele-specific oligonucleotides for the mutant and normal sequences in the polymerase chain reaction (PCR). delta F508 was identified by Riordan et al., and it is a 3-bp deletion of the phenylalanine codon at position 508. The Hutterite Brethren are an inbred North American population who have three different DNA marker haplotypes of CF chromosomes. Genomic DNA from both a CF child and one parent from each of 10 Hutterite families was analyzed for the presence or absence of the deletion mutation. delta F508 is associated with one of the three CF haplotypes in the Hutterite population, and this is the most common haplotype in a subset of the linkage family data of Kerem et al. The other two Hutterite CF haplotypes are generally rate in Caucasian populations. Since these two CF haplotypes do not carry the deletion mutation, they must carry a different CF mutation(s). The results of the PCR analysis for the deletion mutation lend additional support to our previous conclusion that there were at least three original carriers of CF mutations among the founders of the Hutterite population and that all copies of the same CF haplotype were identical by descent. One Hutterite CF patient has both of the haplotypes which do not carry delta F508. Analysis of this individual's DNA should allow identification of two additional CF mutations in this population.     

A mouse model for the cystic fibrosis delta F508 mutation.

Most cystic fibrosis (CF) patients produce a mutant form (delta F508) of the cystic fibrosis transmembrane conductance regulator (CFTR), which is not properly processed in normal cells but is active as a chloride channel in several experimental systems. We used a double homologous recombination ('Hit and Run') procedure to generate a mouse model for the delta F508 mutation. Targeted embryonic stem (ES) cells (Hit clones) were found; of these either 80 or 20% of the clones had lost the delta F508 mutation, depending on the distance between the linearization site in the targeting construct and the delta F508 mutation. Correctly targeted clones underwent a second selection step resulting in ES cell clones (Run clones) heterozygous for the delta F508 mutation with an efficiency of 2-7%. Chimeric mice were generated and offspring homozygous for the delta F508 mutation showed electrophysiological abnormalities in nasal epithelium, gallbladder and in the intestine, and histological abnormalities in the intestine, typical of CF. Our data suggest that the delta F508 mice have residual delta F508 CFTR activity which would explain the mild pathology of the delta F508 mice. The delta F508 mouse may provide a useful model for the study of the processing defect of delta F508 CFTR and for the development of novel therapeutic approaches based on circumvention of the processing block.     

The mapping of chromosome 4q markers in relation to facioscapulohumeral muscular dystrophy (FSHD).

The Basque population is one of the oldest populations of Europe. It has been suggested that the Basques arose from a population established in western Europe during the late Paleolithic Age. The Basque language (Euskera) is a supposedly pre-Indo-European language that originates from the first settlers of Europe. The variable distribution of the major cystic fibrosis (CF) mutation (delta F508 deletion) in Europe, with higher frequencies of the mutation in northern Europe and lower frequencies in southern Europe, has suggested that the delta F508 mutation was spread by early farmers migrating from the Middle East during the Neolithic period. We have studied 45 CF families from the Basque Country, where the incidence of CF is approximately 1/4,500. The birthplaces of the parents and grandparents have been traced and are distributed according to their origin as Basque or Mixed Basque. The frequency of the delta F508 mutation in the chromosomes of Basque origin is 87%, compared with 58% in those of Mixed Basque origin. The analysis of haplotypes, both with markers closely linked to the CF gene and with intragenic markers, suggests that the delta F508 mutation was not spread by the Indo-European invasions but was already present in Europe more than 10,000 years ago, during the Paleolithic period.     

A dimorphic 4-bp repeat in the cystic fibrosis gene is in absolute linkage disequilibrium with the delta F508 mutation: implications for prenatal diagnosis and mutation origin.

The gene causing cystic fibrosis (CF) has been recently cloned, and the major mutation (delta F508) accounting for approximately 70% of CF chromosomes has been uncovered. We have identified at the 3' end of intron 6 in the CF gene a 4-bp tandem repeat (GATT) that exhibits interesting features. First, PCR screening of 103 normal individuals revealed that the repeat exists only in two polymorphic allelic forms, either as a hexamer or a heptamer. These two alleles are in Hardy-Weinberg equilibrium and predict a heterozygote frequency of 41% (p[seven repeats] = .71; q [six repeats] = .29). Second, the allele with six repeats was found linked to delta F508 on all 76 CF chromosomes investigated, demonstrating strong linkage disequilibrium and suggesting that delta F508 had originated on the gene bearing six repeats. Third, when the repeat alleles are linked to the DNA markers XV2c and KM19, extended haplotypes are generated. These new haplotypes become informative in situations in which prenatal diagnosis cannot be performed solely with XV2c and KM19. Since this repeat marker is located in the CF gene and would be very less likely to recombine with the gene, it can serve as a valuable DNA marker for haplotype analysis. A possible crossover, however, was identified between XV2c and KM19, transferring delta F508 to a different haplotype.     

Cystic fibrosis patients bearing both the common missense mutation, Gly→Asp at codon 551 and the ΔF508 mutation are clinically indistinguishable from ΔF508 homozygotes, except for decreased risk of meconium ileus

The glycine-to-aspartic acid missense mutation at codon 551 (G551D), which is within the first nucleotide-binding fold of the cystic fibrosis transmembrane conductance regulator (CFTR), is the third most common cystic fibrosis (CF) mutation, with a worldwide frequency of 3.1% among CF chromosomes. Regions with a high frequency correspond to areas with large populations of Celtic descent. To determine whether G551D confers a different phenotype than does delta F508, the most common CF mutation, we studied 79 compound heterozygotes for G551D/delta F508, from nine centers in Europe and North America. Each subject was matched, by age and sex, with a delta F508 homozygote from the same center. A retrospective cohort analysis was performed on the following outcome parameters: age at diagnosis, sweat chloride, meconium ileus at birth, height, weight, weight for height, FVC, FEV1, chest X-ray score, pseudomonas colonization, pancreatic sufficiency, and Shwachman clinical score. There was less meconium ileus among the G551D/delta F508 compound heterozygotes (relative risk 0.33; 95% confidence interval .13-.86), as well as a trend toward later age at diagnosis of pancreatic insufficiency. No statistically significant difference was found between the groups for any other parameter. These results suggest that the CF genotype can be a predictor of pancreatic and intestinal phenotype. Prenatal counseling for the two genotype groups should differ only with respect to probability of meconium ileus. Clinical outcome (after survival of meconium ileus) for G551D/delta F508 compound heterozygotes and delta F508 homozygotes is indistinguishable; therefore, prognostic counseling should not differ.     

Screening for cystic fibrosis gene mutations by multiplex DNA amplification

Summary We have developed a simple rapid DNA screening test that allows us simultaneously to analyze seven CF mutations (deltaF508, R347P, S549N, G551D, R553X, R334W, 444delA) that together account for about 60% of all CF mutations in the Italian population. It consists of three steps: multiplex polymerase chain reaction (PCR) amplification of exons 4, 7, 10 and 11; restriction endonuclease digestion of the PCR products; and vertical polyacrylamide gel electrophoresis analysis. We have used our multiplex assay for analyzing 15 CF chromosomes (non delta F508) and have found 3 cases of the R553X mutation; the latter have been confirmed by amplification and digestion of exon 11.     

Screening for five mutations detects 97% of cystic fibrosis (CF) chromosomes and predicts a carrier frequency of 1:29 in the Jewish Ashkenazi population.

To determine the distribution and frequency of cystic fibrosis (CF) mutations in the Israeli population, we have screened 96 patients for 11 relatively common mutations. Five mutations--delta F508, G542X, W1282X, N1303K, and 3849 + 10kb C-->T--were found to account for 97% of the CF alleles in the Ashkenazi Jews. In contrast, of the 11 mutations tested, only delta F508 was detected in Jewish patients of Sephardic or Oriental origin, accounting for 43% of the CF alleles. Four mutations--delta F508, G542X, W1282X, and N1303K--accounted for 55% of the CF alleles in Arab patients. In a pilot screening study, a random sample of 424 Ashkenazi individuals was analyzed for three mutations--delta F508, W1282X, and G542X. Thirteen individuals were detected as heterozygotes (six for delta F508 and seven for W1282X), predicting a heterozygote frequency of 1:29. This is similar to the frequency of carriers in the Caucasian population of northern European ancestry. On the basis of these data, the Ashkenazi population is considered to be a candidate for CF heterozygote screening.     

The mutation ΔF508 on Dutch cystic fibrosis chromosomes: frequency and relation to patients age at diagnosis

Summary We tested 190 chromosomes from Dutch cystic fibrosis (CF) patients and carriers for the presence or absence of the major CF mutation ΔF508. This mutation was found on 77% of the Dutch CF chromosomes. We observed a significant difference in the distribution of the ages at diagnosis between homozygotes for ΔF508 and the other patients. ΔF508 homozygotes tend to be identified as patients at neonatal or infantile age. The age at diagnosis of patients with at least one unknown allele, on the other hand, ranged between neonatal and young adult age.     

Mutation analysis of the cystic fibrosis transmembrane regulator gene in native American populations of the southwest

We report DNA and clinical analyses of cystic fibrosis (CF) in two previously unstudied, genetically isolated populations: Pueblo and Navajo Native Americans. Direct mutation analysis of six mutations of the CFTR gene--namely, delta F508, G542X, G551D, R553X, N1303K, and W1282X--was performed on PCR-amplified genomic DNA extracted from blood samples. Haplotype analyses with marker/enzyme pairs XV2c/TaqI and KM19/PstI were performed as well. Of the 12 affected individuals studied, no delta F508 mutation was detected; only one G542X mutation was found. None of the other mutations was detected. All affected individuals have either an AA, AC, or CC haplotype, except for the one carrying the G542X mutation, who has the haplotype AB. Clinically, six of the affected individuals examined exhibit growth deficiency, and five (all from the Zuni Pueblo) have a severe CF phenotype. Four of the six Zunis with CF are also microcephalic, a finding not previously noted in CF patients. Our DNA data have serious implications for risk assessment of CF carrier status for these people.     

Molecular basis of cystic fibrosis in Lithuania: incomplete CFTR mutation detection by PCR-based screening protocols

Mutational analysis of the cystic fibrosis transmembrane regulator (CFTR) gene was performed in 98 unrelated CF chromosomes from 49 Lithuanian CF patients through a combined approach in which the p.F508del mutation was first screened by allele-specific PCR while CFTR mutations in nonp.F508del chromosomes have been screened for by denaturing gradient gel electrophoresis analysis. A CFTR mutation was characterized in 62.2% of CF chromosomes, two of which (2.0%) have been previously shown to carry a large gene deletion CFTRdele2,3(21 kb). The most frequent Lithuanian CF mutation is p.F508del (52.0%). Seven CFTR mutations, p.N1303K (2.0%), p.R75Q (1.0%), p.G314R (1.0%), p.R553X (4.2%), p.W1282X (1.0%), and g.3944delGT (1.0%), accounted for 10.1% of Lithuanian CF chromosomes. It was not possible to characterize 35.8% of the CF Lithuanian chromosomes. Analysis of intron 8 (TG)mTn and M470V polymorphic loci did not permit the characterization of the CFTR dysfunction underlying the CF phenotype in the patients for which no CFTR mutation was identified. Thus, screening of the eight CFTR mutations identified in this study and of the large deletion CFTRdele2,3(21 kb) allows the implementation of an early molecular or confirmatory CF diagnosis for 65% of Lithuanian CF chromosomes.     

Identification of carriers by screening for delta F508 deletion in a multi-generation cystic fibrosis family

Samples from 30 members of a french cystic fibrosis (CF) family had to be typed with probes for restriction fragment length polymorphisms (RFLPs) known to be linked to the CF gene, to fulfill the expectations of twenty-two low-risk relatives who were asking for carrier testing. Classical linkage-disequilibrium data between KM-19 and XV-2c polymorphisms and the CF locus were not informative enough for some individuals, and other RFLPs had to be analyzed to determine which chromosomes carried the deficient gene in the family. We report the retrospective screening for delta F508 mutation in this extended family to illustrate the drastic improvements that the direct detection of the major mutation responsible for CF has on genetic counselling of relatives of patients with cystic fibrosis.     

Sulfate transport in normal and cystic fibrosis fibroblasts.

The glycoconjugate component of cystic fibrosis (CF) epithelial secretions is abnormally sulfated. Previous studies have suggested that some but not all CF fibroblasts express this secondary defect. We tested the hypothesis that the major CF mutation (delta F508/delta F508) is correlated with elevated sulfate transport, by measuring the rates of saturable and nonsaturable [35S]SO4(2-) uptake in skin fibroblasts isolated from CF patients of known genotype. No significant differences were apparent between normal and CF fibroblasts.     

Application of DNA analysis in a population-screening program for neonatal diagnosis of cystic fibrosis (CF): comparison of screening protocols.

We compare two protocols for newborn screening for cystic fibrosis (CF). The first uses the immunoreactive trypsinogen (IRT) assay with a cutoff of > or = 180 ng/ml and a sweat test to identify CF patients. The second uses the IRT assay with a 100 ng/ml cutoff in conjunction with direct analysis for the delta F508 CF transmembrane conductance regulator (CFTR) mutation in a two-tiered (i.e., IRT/DNA) protocol, followed by a sweat test. We screened 220,865 newborns from Wisconsin for CF, using the IRT protocol identifying 369 infants with an elevated IRT, of whom 46 were found to have CF. Another 7 CF patients were identified who had a false-negative IRT level. The CF incidence in the white population was 1 in 3,431 (carrier incidence of 1 in 30). The IRT protocol had a sensitivity of 87% and a positive predictive value of 12.5%. We subsequently used the IRT/DNA protocol to screen 21,258 infants. Of 518 infants with an IRT level > or = 100 ng/ml, 24 carried at least one copy of the delta F508 CFTR mutation, and 4 of these infants were found to have CF, yielding a positive predictive value for this protocol of 16.7%. Direct comparison of the positive predictive value of the two protocols is not valid, because of the different populations screened. However, had the IRT protocol been used on the IRT/DNA cohort, 50 infants, including the 4 with CF, would have received sweat tests, yielding a positive predictive value of 8%. Because of the small sample size, this positive predictive value is not significantly different from that obtained for the IRT/DNA test. However, from a practical point of view the IRT/DNA approach does decrease considerably the number of sweat tests that must be undertaken. The number of false positives for the IRT protocol (46 in 21,258) is increased significantly compared with that for the IRT/DNA approach (20 in 21,258; P < .001). The incidence of delta F508 carriers detected in cohorts with an elevated IRT level was increased compared with the incidence in the general population. The direct costs for the IRT/DNA approach (100 ng/ml) were $11,374 per CF patient detected, compared with $10,187 per CF patient detected for the IRT protocol. Therefore, we conclude that the IRT/DNA approach to CF newborn screening decreases the number of false-positive subjects contacted, without a significant increase in cost.     

Cystic fibrosis in the population of Reunion Island

The frequencies of the delta F 508 mutation and haplotypes linked to the cystic fibrosis (CF) gene and detected with DNA probes XV-2C and KM-19 have been studied in the population of Reunion Island, a French province located in the Indian Ocean. The deletion was present in 41.3% of CF chromosomes, whereas this proportion is about 70% in the French population. The delta F 508 mutation was associated with the haplotype B defined by the DNA markers XV-2C (allele 1) and KM-19 (allele 2) in 76.4% of CF chromosomes, while this proportion is over 90% in the French population. Founder effect, genetic drift and admixture can explain these differences.     

Identification of the M1101K mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene and complete detection of cystic fibrosis mutations in the Hutterite population.

The Hutterite population is a genetic isolate with an increased incidence of cystic fibrosis (CF). Previously we identified three CF haplotypes defined by polymorphisms flanking the CF transmembrane conductance regulator (CFTR) gene. delta F508 was present on one of the haplotypes in only 35% of CF chromosomes. We hypothesized that the other two CF haplotypes, one of which was the most common and the other of which is rare, each harbored different non-delta F508 mutations. Single-strand conformation polymorphism analysis detected a missense mutation, M1101K, in both chromosomes of a Hutterite patient carrying the two non-delta F508 haplotypes. M1101K appears to have originated on an uncommon CFTR allele and to be infrequent outside the Hutterite population. The presence of M1101K on two haplotypes is likely the result of a CFTR intragenic recombination which occurred since the founding, 10-12 generations ago, of the Hutterite population. The crossover was located between exons 14a and 17b, an interval of approximately 15 kbp. delta F508 and M1101K accounted for all of the CF mutations in patients from 16 CF families representing the three subdivisions of the Hutterite population.     

The search for south European cystic fibrosis mutations: identification of two new mutations, four variants, and intronic sequences

The major mutation in the cystic fibrosis (CF) gene is a 3-bp deletion (delta F508) in exon 10. About 50% of the CF chromosomes in Southern Europe carry this mutation, while other previously described mutations account for less than 4%. To identify other common mutations in CF patients from the Mediterranean area, we have sequenced, exon by exon, 16 chromosomes that did not show the delta F508 deletion from a selected panel of eight unrelated CF patients. We describe here one missense and one nonsense mutation, and four sequence polymorphisms. We have also found two previously reported mutations in three chromosomes. Overall, these mutations may account for about 20% of CF alleles in the Italian and Spanish populations. No other mutations were detected in 10 out of 16 CF chromosomes after analyzing about 90% of the coding region of the CF gene, and 39 out of 54 intron/exon boundaries. Therefore, about 26% of CF mutations remain to be identified. In addition we provide the intron/exon boundary sequences for exons 4 to 9. These results together with previously reported linkage data suggest that in the Mediterranean populations further mutations may lie in the promoter region, or in intron sequences not yet analyzed.     

Frequency of the ΔF508 mutation and flanking marker haplotypes at the CF locus from 167 Czech families

Summary This study analyses distribution patterns of the ΔF508 mutation of the cystic fibrosis transmembrane conductance regulator gene (CFTR) gene and the cystic fibrosis (CF)-linked marker loci MET, D7S23, D7S399, and D7S8 in a sample of 167 (116 complete) CF families from Bohemia and Moravia (Czechoslovakia). DNA typing was performed by polymerase chain reaction amplification, restriction analysis, and agarose or polyacrylamide gel electrophoresis. The frequency of the ΔF508 mutation in this sample is 67% and the frequency of the B haplotype is 77.6% on CF chromosomes. Linkage disequilibrium was found between ΔF508 and all markers tested.     

Spectrum of cystic fibrosis mutations in Serbia and Montenegro and strategy for prenatal diagnosis

We have screened 175 patients for molecular defects in the cystic fibrosis transmembrane conductance regulator (CFTR) gene using nondenaturing polyacrylamide gel electrophoresis (PAGE), denaturing gradient gel electrophoresis (DGGE), and sequencing. Six different mutations (F508del, G542X, 621+1G --> T, 2789+5G --> A, R1070Q, and S466X) accounted for 79.71% of CF alleles, with the F508del mutation showing a frequency of 72.28%. Another 12 mutations (R334W, 2184insA, I507del, 1525-1G --> A, E585X, R75X, M1I, 457TAT --> G, 574delA, 2723delTT, A120T, and 2907delTT) covered an additional 3.36%. A novel mutation (2723delTT) was found in one CF patient (F508del/2723delTT). Thus, a total of 18 mutations cover 82.57% of CF alleles. During our study, 72% of families at risk for having a CF child were found to be fully informative for prenatal diagnosis. Prenatal diagnosis was performed on 56 families; 76 analyses resulting in 16 affected, 38 carriers, and 22 healthy fetuses. These results imply that the molecular basis of CF in Serbia and Montenegro is highly heterogeneous, as is observed in other eastern and southern European populations. Because we detected more then 80% of CFTR alleles, results could be used for planning future screening and appropriate genetic counseling programs in our country.     

CA/GT microsatellite alleles within the cystic fibrosis transmembrane conductance regulator (CFTR) gene are not generated by unequal crossingover.

The gene responsible for cystic fibrosis (CF) has recently been identified, and a three-nucleotide deletion (delta F508 mutation) that results in the loss of a phenylalanine residue in the first putative ATP-binding domain of the predicted protein (CF transmembrane conductance regulator, CFTR) has been found to be the major CF mutation. Although several other mutations have been identified in the CFTR gene, most of them are very rare, making their application to genetic diagnosis difficult. While characterizing the genomic region encompassing the CF locus, we have identified three CA/GT blocks that flank exon 9 of the CF gene. One of the CA/GT blocks exhibits a highly informative variable number of dinucleotide repeats (VNDR) polymorphism. This intragenic VNDR microsatellite should, by itself, provide full information for genetic analysis in approximately 80% of CF families and will help elucidate the associations between DNA polymorphism haplotypes and specific gene mutations. Haplotype analyses of CF chromosomes with and without the delta F508 mutation suggest that the different alleles are generated by slipped-strand mispairing within the dinucleotide repeat during DNA replication, rather than by unequal crossingover within a recombination hot spot.