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A novel mutation in the CFTR gene correlates with severe clinical phenotype in seven Hispanic patients
  1. *Institute for Molecular and Human Genetics, Georgetown University Medical Center, M4000, 3800 Reservoir Road NW, Washington, DC 20007, USA
  2. Department of Pediatrics, Mail Stop 83, Children's Hospital Los Angeles, 4650 Sunset Boulevard, Los Angeles, California 90027, USA
  1. Dr Wong, wonglj{at}

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Editor—Cystic fibrosis (CF) is one of the most common autosomal recessive disorders, with an incidence of one in every 2000 to 3000 white people. The disease is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene.1 Over 800CFTR mutations have been identified.2 Five mutations in Ashkenazi Jews,3 4 24 in whites,5 and 15 in African-Americans6 account for 97%, 90%, and 75% of CF chromosomes in the respective populations. In contrast, 90% of CF Spanish alleles were contributed by as many as 75 mutations, indicating a heterogeneous CF genotype in Spain.7 The overall mutation spectrum and the frequency of common mutations in a particular population depend on the ethnic background. For example, the frequency of the W1282X mutation is 1.2% in the white population, but it is as high as 60% in Ashkenazi Jews.8 That of ΔF508 is 70% in northern Europeans but it is less than 50% in Spanish and Hispanics.7 9 In order to provide accurate genetic counselling, it is necessary to determine the prevalent mutations in each ethnic group. A study of Hispanic CF patients from the south western United States showed that only 58% of Hispanic CF alleles were detected by screening 23 recurrent mutations.9 Thus, there is a need to identify the mutations accounting for the remaining Hispanic CF chromosomes. In addition, on identification of mutations genotype-phenotype correlation studies can be facilitated.

The clinical diagnosis of CF has been recently reviewed.10 Although the structure and function of ΔF508 and W1282X mutant CFTR have been studied, there is a shortage of genotype-phenotype correlation studies of rare mutations, particularly the ones that appear to be unique to Hispanic CF patients. This is partly because of the lack of an effective method for screening the mutations in the largeCFTR gene and because a significant proportion (about 40%) of Hispanic CF mutations have not been identified.9 We recently described the clinical features of a patient homozygous for R1066C and a group of Hispanic patients with the 3849+10kbC>T mutation.11 12 In an effort to continue searching for unknown CF mutations in Hispanic patients, we have developed an effective method, temporal temperature gradient gel electrophoresis (TTGE), to screen DNA abnormalities in CF chromosomes and have identified several novel mutations. Here we report the discovery of 3876delA and its clinical presentation in seven Hispanic patients who were heterozygous for this novel mutation.

DNA samples of 44 CF patients, from 43 unrelated families, attending the Children's Hospital at Los Angeles (CHLA) Cystic Fibrosis Clinic were selected for mutational analysis by TTGE, according to the CCI (Committee on Clinical Investigations) approved protocol No 90-117. Thirty of the 44 patients were Hispanics, including two sibs. In this study, “Hispanics” refers to people originally from Latin America13 and “Spanish” refers to people living in Spain. These patients have been genotyped by DNA diagnostic laboratories at the University of California San Francisco, Baylor College of Medicine, or Genzyme Corporation, and had either one or both CF alleles unidentified. Clinical information was obtained from CHLA CF Clinic records and by reviewing the patients' medical charts and consulting clinicians familiar with the patients' disease progression.

DNA was extracted from patients' blood samples. All 27 exons were PCR amplified and analysed with TTGE.14 Exon 20, which contains the 3876delA mutation and its flanking intron regions were PCR amplified by using primers 5′GGTCACCATTGAAAGTGT3′ (forward) and 5′ATGAGAAAACTGCACTGGA3′ (reverse). The PCR reactions were performed under standard conditions as described previously.15 PCR products were denatured at 95°C for 30 seconds and slowly cooled down to 45°C for a period of 45 minutes at a ramp of 1.1°C/minute. The 450 bp PCR product containing the entire exon 20 was analysed by the TTGE method using the Bio-Rad DCodeTM mutation detection system according to the published procedures.15Electrophoresis was carried out at 130 V at a constant temperature increment of 1.3°C/hour. The temperature range (48°C to 56.5°C) for this exon 20 PCR fragment was determined empirically with the aid of computer simulation (MacMelt, Bio-Rad Laboratories).

The DNA samples that showed abnormal banding patterns by TTGE analysis were sequenced using the BigDye terminator cycle sequencing kit (Perkin-Elmer, Applied Biosystems) and analysed on ABI Prism 377 DNA Sequencer (Perkin-Elmer, Applied Biosystems) according to the manufacturer's protocols. The sequencing data were analysed with ABI DNA sequencing analysis software (version 3.0).

TTGE analysis of exon 20 of the CFTR gene showed an abnormal banding pattern in seven, including two sibs, out of 29 unrelated Hispanic patients. Fig 1 (above) illustrates the results of TTGE analysis. Normal subjects showed a single band and patients showed three bands. Sequencing analysis showed a deletion of an A at nucleotide position 3876 of the CFTR gene (fig 1, below). All seven patients were heterozygous for 3876delA. Two of these patients were sibs (patients 3 and 4). The remaining 28 patients screened were from unrelated families. Thus, this novel mutant allele represents a frequency of 10.3% (6/(29 × 2)) in this Hispanic patient group. The 3876delA mutation was not detected in 14 unrelated non-Hispanic CF patients examined by TTGE.

Figure 1

(Above) TTGE analysis of the DNA fragment containing CFTR exon 20. Lanes 1 and 4, fragments containing wild type exon 20. Lanes 3 and 4, exon 20 containing fragments amplified from patients 5 and 6. (Below) The upper panel shows the wild type DNA sequence surrounding the nucleotide position 3876 in the CFTR gene. The underlined “A” is deleted in heterozygous mutant DNA (lower panel).

Table 1 summarises the clinical findings of these patients. Five out of the seven patients with 3876delA mutation were females. Five of them had ΔF508 as the other mutant allele, one had 1949del84, and one had an unidentified mutant allele. The current age of the five surviving patients ranged from 4 to 26 years; two had died at 19 and 20 years. The age of diagnosis ranged from 3 weeks to 7 months, except for patient 6 who was diagnosed at 16 months. In all cases, high levels of sweat chloride (84-142 mmol/l) were detected.

Table 1

Clinical presentations of Hispanic cystic fibrosis patients with 3876delA frameshift mutation in the CFTR gene

The pulmonary function was measured by percent predicted FEV1 and percent predicted FVC at different ages. Patients 1, 5, and 6 showed a drastic drop in both FEV1 and FVC values within four years, indicating a rapid deterioration in pulmonary function. Patient 4 was too young to be tested for FEV1 and FVC. Pulmonary function of patients 2 and 3 has remained normal (>90% predicted values). All patients had infections ofPseudomonas and Staphylococcus (table1). Five of the seven patients who carried this 3876delA mutation developed the syndrome of allergic bronchopulmonary aspergillosis (ABPA). ABPA is an immune mediated pulmonary disorder triggered by fungal colonisation, associated with signs of lung infiltrates, increased cough, respiratory distress, and wheezing. These five patients were from unrelated families. The two without ABPA were the sibs, who had similar clinical symptoms, including liver disease and gastro-oesophageal reflux. Overall, these patients showed very similar respiratory symptoms and common, yet somewhat variable, bronchopulmonary impairment.

All seven patients were pancreatic insufficient (PI) and required daily treatment of pancreatic enzyme supplement. Since the other alleles, ΔF508 and 1949del84, render a PI phenotype,5 10 these results suggest that 3876delA is also a deleterious mutation with a PI phenotype. Consistently, all patients had poor growth. Although patient 3 recovered from the earlier low growth centile at the age of 10, she suffered respiratory distress as a newborn, portal hypertension, and hyponatraemia, in addition to liver disease and gastro-oesophageal reflux. Patient 6 developed insulin dependent diabetes. Patient 5 had cor pulmonale, a late terminal event of CF.5 She had poor weight gain and died at the age of 20 of pulmonary failure as did patient 1. In summary, these observations are consistent with severe phenotypes, which correlate with the predicted disruption of NBD2 by 3876delA.

The deleterious frameshift mutation, 3876delA (L1258X), was detected in six out of 29 unrelated Hispanic CF patients attending the CHLA CF clinic. This deletion accounts for about 10% of the total CF alleles in this group. The frequency of this mutation in the Hispanic CF patients attending CHLA is similar to that of G542X, only second to ΔF508. The severity of this group of CF patients is shown by the early age of diagnosis (average 6 months), the high sweat chloride (average 105 mmol/l), ABPA, PI, liver disease, cor pulmonale, and early death. Like those with ΔF508/ΔF508, ΔF508/W1282X, and W1282X/W1282X, patients with the 3876delA/ΔF508 mutation had variable pulmonary function. This indicates that other factors may be involved in this phenotype.8 16 It will be important to follow up patient 4 on her later development of portal hypertension and more severe respiratory problems. Meconium ileus, distal intestinal obstruction syndrome (DIOS), and nasal polyps were not found in this group of patients. However, the observed poor growth and cholestasis suggest that 3876delA might be associated with a variable hepatointestinal status.

Patient 1 was a compound heterozygote for 3876delA and 1949del84. She had a high sweat chloride concentration of 142 mmol/l, early age of onset (2 months), and death at the age of 19. The 1949del84 mutation, which results in an in frame deletion of 26 amino acids located in the R domain, was originally discovered in a 6 month old Spanish patient who had ΔF508 on the other mutant chromosome.17 This patient had a severe clinical course as well: a sweat chloride concentration of 80 mmol/l, PI, and both respiratory and digestive problems.17

It is intriguing that after extensive search for CF mutations in Spanish,7 European,5 Ashkenazi Jewish,3 and African-American6 populations, the 3876delA mutation was not discovered. Since most of our Hispanic CF patients are descendants of people from Mexico and South America, the 3876delA mutation might be derived from native Mexicans. Further haplotype studies will be necessary to support this hypothesis. It is concluded that 3876delA is one of the common mutations in Hispanic CF patients and should be included in the routine mutational analysis of Hispanic CF patients. This mutation does not result in change of restriction site. The allele specific oligonucleotide (ASO) dot blot analysis would be a simple method for quick diagnosis of this mutation. The genotype-phenotype correlation will greatly assist genetic counselling regarding the prognosis of CF patients.

Like other ABC transporters, the CFTR chloride channel contains two nucleotide binding domains, NBD1 and NBD2, both of which have the consensus sequences for Walker A and Walker B motifs.18Between the two NBDs is a highly positively charged regulatory (R) domain, whose phosphorylation by protein kinase A modulates the interaction between the NBDs.19 The 3876delA mutation resides in the very centre of the Walker A motif in the NBD2, resulting in the alteration of the highly conserved amino acid sequence in the second half of this motif, starting at amino acid S1248 (fig 2). It also causes a premature protein termination at L1258, 10 amino acids downstream of the mutation site, resulting in the loss of six conserved sequence blocks including the Walker B motif which hosts the γ phosphate binding pocket.1 According to the proposed secondary structure assignment for NBD2,20 the 3876delA mutation would abolish all the seven helices in NBD2. Since ATP hydrolysis at NBD2 terminates a burst of activities associated with opening the channel, loss of NBD2 would confer a loss of the gating control.21

Figure 2

3876delA changes the conserved sequences in Walker A motif of the NBD2 domain of CFTR.1 The arrow indicates the corresponding position of the single adenylic acid deletion. Conserved nucleotides are in bold. X, translation termination codon; hmdr, human multidrug resistance protein; mmdr, mouse multidrug resistance protein. N and C indicate the N-terminal and C-terminal domains of the gene where the Walker A motif is located.

A recent study shows that the Walker A motif in NBD2 is more solvent accessible than that in NBD1, suggesting a difference in structure and function for the two NBDs.22 In addition to 3876delA, a few other mutations in NBD2, including G1244E, S1255P, S1255X, 3905insT, W1282X, N1303K, and G1349D, all result in a PI phenotype.5 23 It should be noted that S1255P, S1255X, 3905insT, and 3876delA are all clustered around the Walker A motif. However, whether the PI phenotype is caused by the loss of gating control in NBD2 is not clear. Evidence is emerging for the involvement of the C-terminus of CFTR in some other biological processes. It has been shown that there is a specific and tight binding between the CFTR C-terminus and the Na+-H+ exchanger regulatory factor.15 The 3876delA mutation offers a good opportunity for understanding the molecular mechanisms of CF pathogenesis.


This study is part of the project “Molecular and Clinical Studies of CF Patients in Southern California” supported by the Webb Berger Foundation.