CFTR scanning for individuals with p.I148T or [p.D1270N;p.R74W]

From 1990 to 2003, we have analysed for CFTR mutations genomic DNA from 437 families with CF and 170 with isolated azoospermia caused by CBAVD, using a combination of mutation screening for known and scanning for unknown mutations. The first step was the search of 31 CF mutations detected by the ABI oligonucleotide ligation assay and 3 common intronic mutations by using restriction analysis. The second step was the scanning of coding/flanking sequences by DGGE (Denaturing Gradient Gel Electrophoresis) using 32 GC-clamped amplimers (which in our experience detected 98% of CFTR mutants), followed by sequencing to resolve abnormal PCR products (BigDye terminator cycle sequencing on ABI 310 automate sequencer). Whenever possible, family members were assayed for the mutations and associated polymorphisms. We detected 160 different mutations in the CF group accounting for 97 % of CF alleles, and 64 different mutations in the CBAVD group accounting for 85% of CBAVD alleles, which represents one of the highest allelic heterogeneity reported so far. Usually, mutation scanning is stopped when two mutations are found to be in trans.

In this study, we analyzed by DGGE the entire coding and flanking regions of the CFTR gene of individuals who had been previously found to carry p.I148T or the complex allele [p.R74W;p.D1270N] and assayed their relatives for the additional sequence changes identified. We also re-analyzed the CFTR gene of a CF patient who had been originally described with c.394delTT in trans of c.3195del6 [12], now renamed c.3199del6 (see the Results). Studies to determine the frequency of each sequence alteration described in this report were performed on 600 chromosomes from our general population (Southern France). In addition, we also reanalyzed two additional CF patients previously found to carry p.I148T in the Lyon genetic center. The study was approved by the institutional ethical committees and informed consent was obtained from families.

Nomenclature

Gene variants and mutants are described using DNA and protein designation: intronic changes, deletions, insertions and frameshifts are reported at the cDNA level (c.) and amino acid changes at the protein level (p.), as recommended in the Human Genome Variation Society web page http://www.genomic.unimelb.edu.au/mdi/.

A CF mutation (c.3199del6 or c.3395insA) is associated in ciswith p.I148T in CF patients

Mutation c.3199del6 can also occur alone as a CF-causing allele

Mutation c.3199del6 was found to be carried without p.I148T in a young CF male with 394delTT on the other allele, diagnosed at the age of 7 years on the basis of typical pulmonary disease, pancreatic insufficiency, poor growth and positive sweat test [12]. Mutation c.3199del6 was initially described by us in 1994 in this patient as c.3195del6 [12], in accordance with the first draft of mutation nomenclature [13]. However, it occurred in the same palindromic sequence in exon 17a than mutation c.3199del6 reported in 1998 by Bozon et al. [14]. Both mutations are expected to delete either amino acids Val1022 and Ile1023, or Ile1023 and Val1024 from the CFTR protein. As it is impossible to determine at the genomic level in which part of the palindrome each of them occurred, the most 3'copy of the repeat is arbitrarily assigned to have been mutated, according to the current rule [15]. Consequently, mutations c.3195del6 and c.3199del6 should be considered as identical and reported as c.3199_3204del. The familial segregation analysis of polymorphisms covering the CFTR gene showed that p.I148T, when present in individuals with a CF phenotype, occurred on a unique haplotype carrying IVS8-9T whatever the mutation in cis, c.3395insA or c.3199del6 (table 1). By contrast, c.3199del6 without p.I148T occurred on a different haplotype carrying IVS8-7T.

Mutations p.I148T, c.3199del6 and c.3395insA were not found on 600 chromosomes from our general population.

Triple-mutant allele [p.R74W;p.V201M;p.D1270N] is found in males with CBAVD whereas double-mutant allele [p.R74W;p.D1270N] is found in asymptomatic individuals

p.I148T is a low penetrance CF mutation or a neutral polymorphism

Since its initial description in a CF Canadian patient with pancreatic insufficiency [16], the mutation p.I148T, which changes a conserved amino acid and occurs in the first cytoplasmic loop of the CFTR protein, has been considered as a severe CF allele in many countries. It was thought to be the second most common CF mutation in the French Canadian population, accounting for 9.1% of the French Canadian chromosomes [17], whereas in France, p.I148T accounted for only 0.11 % of the CF alleles in a sample of 3,710 patients affected with the disease [5]. p.I148T can now be detected by several commercially available kits developed for routine screening of CF carriers and for CF neonatal screening, and recently it has been included in the core panel of 25 CF mutations recommended by the American College of Medical Genetics (ACMG) [18]. Thousands of individuals are being screened for this mutation worldwide and it is possible that several prenatal diagnosis have been or will be performed. However there are now several lines of evidence that question the role of p.I148T by itself in causing disease. First, compound heterozygosity for p.I148T and a severe CF mutation was recently identified in several healthy individuals [9, 19]. When affected and unaffected individuals carrying apparently the same mutational genotype were re-analyzed for additional changes that could explain the different phenotypes, p.I148T was found to be associated in cis with another mutation, c.3199del6, in patients with a classic CF phenotype, whereas healthy adults who were compound heterozygous for p.I148T and a severe CF mutation or homozygous for p.I148T did not carry the deletion [8]. In a recent study, the p.I148T mutation has been further documented to be linked with the 3199del6 mutation in all 24 CF patients of French Canadian descent originally identified as compound heterozygous for the p.I148T mutation and a second severe CFTR mutation [20]. Second, p.I148T was found to be over 100 times more common in two independent U.S. carrier screening programmes than in CF patients: it accounted for 6.4 to 7.7% of chromosomes detected in the screened populations versus 0.06 to 0.068% of CF chromosomes in CF patients [8. 9]. This discrepancy suggests that p.I148T is either a poorly penetrant mutation or a neutral polymorphism. Third, when transiently expressed in epithelial cells, p.I148T mutant protein is normally processed and is able to mediate normal chloride transport with properties identical with those of wild-type cells [21]. As the mutant seems to suppress the ability of CFTR to support HCO3- transport, it has been hypothesized that p.I148T may contribute to disease through Cl- coupled HCO3- altered transport; however, the major CFTR functions are retained by the mutant [21]. Fourth, we show in this study for the first time that p.I148T can be associated with a frameshift mutation c.3395insA in exon 17b instead of in-frame deletion c.3199del6 in exon 17a. Insertion c.3395insA (designated as c.3395_3396insA) is a previously unreported mutation that is predictive of premature termination of translation at amino acid residue 1155. The truncated protein lacking the 325 last amino acids is believed to be not functional and be degraded rapidly, generating no detectable protein. A CFTR alteration producing a premature termination signal is a class I mutation, considered severe enough to cause CF by itself and exclude the contribution of any other sequence change on the same allele. Fifth, in contrast with other studies that stated that only the complex allele [p.I148T;9T;c.3199del6] appeared to be associated with a classic CF phenotype [8], we demonstrate that c.3199del6 is associated with a CF phenotype even if the deletion occurs on a chromosome that does not carry p.I148T, which adds further value to the consideration that p.I148T is not a true mutation but simply a polymorphism. Although no functional test was performed to prove its contribution to the severe phenotype, mutation c.3199del6 has been considered as a defective allele as it results in the loss of two amino acid residues in the TM10 domain of the CFTR protein and has not been detected in non-CF alleles. Our data fully support the recent recommendation that p.I148T should not be included in the mutation panel selected for prenatal screening strategy [22].

The complex allele [p.R74W;p.D1270N] may be not enough to cause disease

We and others had initially described p.R74W [23] and p.D1270N [24] in isolation but they have since been found in association in many CBAVD or CF patients [10, 11] and these two changes were thought to be deleterious, alone or in combination. A few complex alleles have been expressed in heterologous systems to evaluate the impact on CFTR processing and Cl- channel activity and better understand the contribution of each missense mutation on phenotype. When expressed in HeLa cells, mutant p.R74W, p.D1270N and [p.R74W;p.D1270N] did not affect CFTR processing, however a lower cAMP-responsive anion conductance was observed with the double mutant [p.R74W;p.D1270N] [3]. The assay suggested that p.R74W alone should be considered as a polymorphism, p.D1270N alone could generate a CBAVD phenotype while the complex allele could produce a more severe phenotype as p.R74W could enhance the effect of p.D1270N [3]. However these findings have not yet been confirmed by other studies.

We have found here that a triple-mutant [p.R74W;p.V201M;p.D1270N] allele was carried in all three patients with CBAVD whereas only the double mutant [p.R74W;p.D1270N] allele was present in two asymptomatic individuals including an obligate carrier who was compound heterozygous for a CF mutation. Another mother carrying [p.R74W;p.D1270N] in trans of a CF mutation has been described previously; despite two positive sweat tests she was absolutely asymptomatic [25]. Missense p.V201M in exon 6a changes a valine for a methionine in the third transmembrane domain; it was initially reported alone in a French patient with CBAVD [26], then in Brazilian patients with CF [27].

Recent large scale screening for CF carrier showed that p.D1270N was present 205 times more commonly in the screened population than in the CF patients (frequency of 14% versus 0.068%); in addition, a completely asymptomatic adult compound heterozygote for p.D1270N and p.I148T has been identified [9]. Although it is not known whether these alleles are associated or not with the third change p.V201M, there are now enough evidence to question the role of the complex allele [p.R74W;p.D1270N] as being a CF or CBAVD mutation. Further experimental and genetic investigations will be necessary to demonstrate the role of p.V201M in causing disease.