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Mutation and haplotype analysis of the CFTR gene in atypically mild cystic fibrosis patients from Northern Ireland

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Editor—Although cystic fibrosis (CF) is a monogenic disorder of autosomal recessive inheritance, it displays a diverse clinical phenotype. Over 1000 molecular lesions in the cystic fibrosis transmembrane conductance regulator (CFTR) gene together with the action of modifying genes can result in the variable expression of CF.1 2

Classical CF causes dysfunction of the lung, sweat glands, testis, ovary, intestine, and pancreas.3 However, there is considerable variation in measurements of the onset age, presence of pancreatic insufficiency, sweat electrolyte levels, and progression of lung disease. Particularly mild manifestations of cystic fibrosis are conveniently grouped as “atypical CF” and result from a differentCFTR mutation spectrum from classical CF patients.4-8 Recognition of the wide range of disease presentation in CF is important for appropriate treatment and effective counselling of those at risk. The repertoire of other disorders associated with CFTR variants include various respiratory afflictions such as asthma,9 chronic bronchitis, disseminated bronchiectasis,6 7 10 allergic bronchopulmonary aspergillosis,11 nasal polyposis,12 chronic pancreatitis,13 and certain forms of male infertility characterised as congenital absence of the vas deferens (CAVD) and obstructive azoospermia.8 14

This report details the CFTR variants, characterised by fluorescent sequencing after screening the entire coding and surrounding intronic sequences by denaturing gradient gel electrophoresis (DGGE), in a panel of 31 unrelated atypical CF patients from Northern Ireland. The frequencies of these variants in the normal population was also examined. Automated detection of fluorescently labelled multiplex PCR fragments was used to type three intragenicCFTR dinucleotide repeats for assigning microsatellite haplotypes to the atypical CF alleles.15The genotypes for a functionally important polythymidine branch/acceptor site of intron 8 in the CFTRgene (Tn=IVS8-6(T)n)16 were also typed for our CF, atypical CF, and normal chromosomes.

The affected subjects from Northern Ireland were diagnosed during neonatal CF screening by two increased levels of immunoreactive trypsin from Guthrie card samples, followed by two sweat tests at one week intervals.17 Older patients (those currently over 14 years) were diagnosed by sweat testing after referral on clinical suspicion. Diagnosis of “classical” CF is generally based on a pathological sweat test, a CFTR mutation on each CF allele, and typical clinical symptoms of gastrointestinal and pulmonary disease.3 Diagnostic problems arise when one or more of the above criteria are not fulfilled. For example, Highsmithet al 18 reported CF gene mutations in patients with normal sweat chloride concentrations. The highest sweat electrolyte results for the atypical CF group were in the diagnostic range for CF (table 1), although they tended to provide borderline results which required repeating before diagnosis was made. The overall clinical course is much milder than for our CF patients, as shown by slower decline on chest x ray appearances, Shwachman scores, deviation of height and weight below the mean, and decline in pulmonary function. They did not present with meconium ileus and only a few have hadPseudomonas aeruginosa infections (table 1). Only about half of the patients receive pancreatic enzyme supplements (Aileen Redmond, Royal Victoria Hospital, Belfast, personal communication).

Table 1

The CFTR genotypes and clinical details for a panel of 31 atypical CF patients from Northern Ireland

DNA samples from the atypical CF patients were analysed by DGGE and variants characterised by fluorescent sequencing as for our CF cohort.19 This screening procedure enabled detection of all 80 control CFTR variants spread through all the exons.

The six CFTR variants and their associated Tn genotypes found in the atypical CF samples are shown in table 1. They were present on 22 of the 62CFTR alleles examined (35%). Only four patients (Nos 1-4 in table 1) had CFTRdefects detected on both chromosomes. Of the 31 subjects tested 18 had at least one CFTR defect characterised (58%), which is significantly different from both normal and cystic fibrosis populations from Northern Ireland (the 30CFTR mutations found in our CF population account for 94% of the CF alleles in Northern Ireland19). Twelve of the chromosomes carried one of three pathogenic mutations typically found in CF: ΔF508 (eight alleles), R117H (two), and S1235R (two). The latter defect is also associated with disseminated bronchiectasis.6 Three of the amino acid changes detected, R75Q, R297Q, and S1235R, were not observed in CF patients from Northern Ireland.19

A 5T tract in the splice acceptor site of intron 8 (Tn), which greatly decreases the amount of available full lengthCFTR mRNA,16 20 21 was typed from eight alleles. There is a wide clinical variation, from no disease to CAVD,22-25 chronic idiopathic pancreatitis,26 bronchiectasis, aspergillosis, and moderate CF,6 27 in association with this Tn allele that depends on the differences in levels of normalCFTR mRNA. The T5 allele effect on exon 9 skipping has been shown to increase the phenotypic severity of the R117H mutation (R117H occurs on two chromosomal backgrounds, one associated with T5 and the other with T7).28 The frequency of the T5 allele is 12.9% in the atypical cohort and has been estimated to be some 5% in the general population.14 22After typing the Tn genotype in 200 normal chromosomes from Northern Ireland in this study, T5 was found to occur at a frequency of 7.1%.

A substitution of arginine for glutamine at codon 75 in exon 3 was found on five chromosomes. This R75Q variant is not thought to cause multiorgan CF but has been found to be associated, like R117H, S1235R, and T5, with atypical CF manifestations, such as CAVD14 22 29 and bronchiectasis.6 This amino acid variant was also found in two out of 200 normal alleles but was not detected on 412 CF chromosomes. Thus, the R75Q allele is significantly more frequent in our group of atypical CF patients than in the general population (p=0.01, Fisher's exact text). This observation suggests an association of the R75Q substitution with mild CF symptoms, as proposed by Zielenski and Tsui.30

Another Arg to Gln change, in codon 297 of exon 7, was previously reported as the CF mutation R297Q.31 The female sibs (aged 14 and 17) carrying this variant are now classified in the atypical group as they present with a quite mild CF phenotype (NB, only one of the R297Q alleles is counted in the cohort). R297Q was found in cis with T5 which would probably augment any deleterious effect of the amino acid change. Previously, R297Q was also reported in two healthy French sibs who both carry CF mutations on their other chromosomes.32 However, in this case the mutation occurs on a different haplotype associated with T7. Other evidence that R297Q may contribute to disease, in conjunction with a second site variation like T5, is that no further mutations were found after DGGE screening of the entire CFTR coding and flanking regions. R297Q was not detected in 206 CF or 100 normal subjects after DGGE analysis. Interestingly, the ovine equivalent of this variant was reported as the first putative CF mutation to be detected in another species.33

Three intragenic CFTR microsatellites, IVS8CA, IVS17bTA, and IVS17bCA , were also typed for the atypical CF cohort. These haplotypes plus the Tn scores (table 2) help to postulate whether chromosomal background would modulate the effect of variants that are found in both CF and atypical CF. Microsatellite haplotypes associated with ΔF508, R117H, and S1235R are equivalent, and thus genetic background is probably similar to those obtained from CF patients with these mutations in Ireland and Britain.15 34

Table 2

Haplotypes defined for 62 atypical CF alleles and their associated CFTR variants

The R75Q and T5 defects were associated with several haplotypes and so would appear to be carried on different genetic backgrounds that might regulate their severity. R75Q was associated with two main haplotype backgrounds; 16-T7-31 or 46-13 and 17-T5-07-17. In addition to R117H (the two R117H alleles occur on the more pathogenic T5 background), T5 occurs in cis with both R75Q and R297Q respectively. Examination of the Tn genotypes for the 30 mutations in our independent CF cohort showed that only R117H was associated with T5 (12 out of the 16 R117H CF alleles). All the other CF mutations were associated with T7 or T9. There were 23 different haplotypes typed from the 40 uncharacterised atypical CF alleles, which suggests that there could be many other variants still to be found in the non-coding region of theCFTR gene. Recent reports suggest that some cases of atypical CF also could result from other genetic or environmental factors.2 35

The CFTR gene has been shown to be a predisposing factor for the development of various atypical manifestations of CF. Examination of the entire coding and flanking sequences of the CFTR gene by denaturing gradient gel electrophoresis was undertaken to screen for mutations in 31 patients who express a markedly mild form of CF. They have borderline or increased sweat electrolyte levels with pulmonary dysfunction but no substantial bacterial colonisation. Three intragenicCFTR microsatellites and a Tn tract, whose T5 allelic variant causes aberrant splicing and is strongly associated with atypical CF, were typed to construct haplotypes for the disease alleles. There were six variants (including T5) characterised on 22 of the 62 alleles tested (35%), though only 12 of these chromosomes harboured mutations commonly found in CF. While only four patients were found to be compound heterozygotes for CFTRdefects, 18 (58%) had at least one mutantCFTR allele. TheCFTR genotypes for the atypical cohort are markedly different from both normal and cystic fibrosis populations from Northern Ireland.

The apparatus and optimisation of DGGE for analysis of theCFTR gene is described by Hugheset al.19 Briefly, samples were loaded in a 6% polyacrylamide gel with a linearly increasing concentration of denaturing gradient from 0% to 80% denaturant (100% denaturant: 6 mol/l urea, 40% formamide v/v) and analysed on a modified Biorad Protean 11 vertical electrophoresis system at a constant temperature of 60°C. Fragments displaying altered gel mobilities were visualised by ultraviolet light after ethidium bromide staining. The corresponding PCR products were sequenced in both directions with fluorescent dyedeoxy chain terminator nucleotides on an Perkin Elmer ABI 373A sequencer. For each DGGE amplicon, control samples carrying known mutations were analysed under the same conditions and in parallel with the uncharacterised samples.

IVS8CA, IVS17bTA, and IVS17bCA were amplified by fluorescent multiplex PCR.15 34 The reverse primers were 5′ end labelled with fluorescent phosphoramidites (Perkin Elmer). Samples were loaded onto 6% polyacrylamide gels (Sequagel, National Diagnostics) and analysed on a Perkin Elmer ABI 373A DNA sequencer. Markers of known size, labelled with a different phosphoramidite, were run with the samples to determine allele sizes using the Genescan 672 software according to the manufacturer's instructions. IVS8GT and IVS8-6(T)n were typed by a combination of SSCP, restriction digests, and direct sequencing as described by Dörk et al.36


We wish to thank the Cystic Fibrosis Research Trust for providing DH with a short term postdoctoral travelling fellowship to the medical school in Hanover (grant No UK02). We thank Aileen Redmond, Royal Victoria Hospital, Belfast, for the clinical data on the atypical CF patients. We warmly thank all our patients for always being eager to help us in our research.