Mutation Analysis of the CFTR Gene in 225 Children: Identification of Five Novel Severe and Seven Reported Severe Mutations

Introduction

Cystic fibrosis (CF) is a common life-threatening autosomal recessive disorder of the Western World (Welsh et al., 1995). CF has been observed in India but with a lower frequency than that observed in the Caucasian population—1 pathogenic allele per 14,321 people as against 1 in 29 people in the West (Stephens, 2001; Kapoor et al., 2006). It is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene on chromosome 7, which encodes a membrane-associated protein of 1480 amino-acid residues functioning as a cAMP-regulated and ATP-gated chloride channel (Riordan et al., 1989). Patients with CF have abnormal chloride conduction across the apical membrane of epithelial cells, causing inspissated secretions in the airways, pancreas, intestines, and vas deferens (Kerem et al., 1989).

The main clinical symptomatology of the disease consists of

1. Lung disease characterized by bronchiectasis, atelectasis, hyper-inflation, airway obstruction, recurrent/chronic pneumonia, and chronic Pseudomonas sp. infections.

Molecular studies in cases of CF have important clinical implications. Siblings of a CF patient have a 25% chance of being affected, a 50% chance of being asymptomatic carriers, and a 25% chance of being unaffected. This makes genetic counseling mandatory for all cases of CF and their families. Accurate genetic counseling is most often based on the type and distribution of mutations that are prevalent in a particular ethnic group. The molecular mutations in CF have been poorly characterized in countries with a low frequency of CF, such as India (Kabra et al., 1996, 2003; Kapoor et al., 2006; Sharma et al., 2008; Shastri et al., 2008). The aim of this study was to establish and improve methods of detection of CFTR mutations in Indian CF patients. We also aimed at calculating a new frequency for CFTR mutations that would enable the development of a suitable testing and counseling protocol in countries with a low frequency of CF.

Materials

Blood samples were collected with informed consent from 225 unrelated Indian CF patients visiting the Center of Medical Genetics from 1997 to 2010 at Sir Ganga Ram Hospital. Diagnostic criteria were based on repeated positive sweat chloride tests (>60 mM) as well as typical clinical findings of chronic airway, pancreatic, and gastrointestinal disease. One hundred age-matched children with no family history of CF were selected as controls.

Methods

Genomic deoxyribonucleic acid (DNA) was extracted from the peripheral blood leukocytes by a salt precipitation method (Miller et al., 1988). The genomic DNA from the patients with CF was initially analyzed for one mutation (F508del) by using previously published protocols (Kerem et al., 1989). We also used the Elucigene^™ CF29 kit, which can identify 29 common CFTR mutations. Elucigene CF29 uses Amplification Refractory Mutation System Technology, which detects point mutations or small deletions in DNA. To identify the other mutations in the CFTR gene, each of the 27 exons and their flanking sequences were amplified by the polymerase chain reaction (PCR) using primer sequences published earlier (Montogomery et al., 2007). The primer sequences were for high-resolution melt curve analysis using real-time PCR, but we used these for single-strand conformation polymorphism analysis. Briefly, the reaction mixture contained 50-250 ng of the DNA sample, 2.5 mL of PCR buffer, 1.5 mMMgCl2, 200 mM deoxynucleotidetriphosphates, 50 pmol of each primer pair, and 1 U Taq DNA polymerase. After initial denaturation at 95°C for 5 min, Taq polymerase was added. This was followed by 35 cycles of denaturation at 95°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 1 min and 30 s. There was a final extension step at 72°C for 5 min. After PCR amplification, all the exons were analyzed by single-strand conformation polymorphism. One volume (3 mL) of PCR product was added to three volumes (9 mL) of loading buffer. The mixture was denatured at 95°C for 10 min in the PCR machine and cooled immediately on ice. The samples were loaded into wells, along with control samples. Electrophoresis was done at constant 100 V for 24 h at 4°C. The exons showing a band pattern different from that of the control samples were sequenced with the Big Dye Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems) and run in an ABI PRISM 310 genetic analyzer, and results were evaluated.

Results

Mutations in the CFTR gene were found in 79/225 (35.1%) of CF patients. However, no mutation except F508del was detected by the Elucigene CF29 kit.

Discussion

CF is a complex, recessive disorder caused by mutations in the CFTR gene, resulting in defective epithelial transport of chloride through the CFTR channel. Classic CF is clinically characterized by respiratory, pancreatic, and nutritional abnormalities, infertility in men, and elevated sweat chloride and sodium levels (Mickle and Cutting, 1998). Patients with CF carry severe mutations in both the alleles. The presence of severe mutation in one allele reduces the activity of the CFTR protein to about half of normal. However, to cause CF, the CFTR activity should be <3% of normal (Wong et al., 2004; Disset et al., 2005).

In the present study, of 225 patients with CF, 54 (24%) patients had the mutation in the homozygous state. The commonest severe mutations observed in a homozygous state were F508 deletion (31.1%), R1162× (2.2%), M1T (0.8%), and S549N (0.8%). The F508del mutation has been reported in 19.1%-33% of cases of CF in India (Kabra et al., 2003; Sharma et al., 2008; Shastri et al., 2008), 72.4% in Albania (Macek et al., 1997), 62.9% in Austria (Estiville et al., 1997), 67.7% in France (Chevalier-Porst et al., 1994), 75.5% in the United Kingdom (Schwartz et al., 1995), and 48% in America (Carles et al., 1996). The other three mutations observed in the homozygous state were p.R1162×, p.M1T, and p.S549N; these mutations have been reported in 1.9% in Austria (Estiville et al., 1997), 0.5% in Belgium (Cuppens et al., 1993), 0.8% in France (Claustres et al., 1993), 9.8% in Italy (Bonizzato et al., 1995), 1.6% in America (Grebe et al., 1994), 0.5% in Belarus (Dork et al., 2000), 0.5% in the United Kingdom (Cheadle et al., 1993), and 0.7% in America (Carles et al., 1996).

Severe mutation in a compound heterozygote state was observed in 8/225 (3.5%). The frequency of mutations (F508del/p.C343×, F508/M469V, F508del/p.I1000×, F508del/p.R75G, F508del/Y577×, F508del/p.Y808Yfs*10, F508del/p.R80N11fs*11, and F508del/I331×) was detected as being 0.4%. Three of these mutations (p.C343×, p.I1000×, and M469V) have been previously reported from India, the United Kingdom, France, and Sweden (Schwartz et al., 1993; Malone et al., 1998; Girodon, 2003; Sharma et al., 2008; Shastri et al., 2008). The latter five (p.I331×, p.R80N11Fs*11, p.R75G, p.Y577×, and p.Y808YFs*10) are novel mutations. In 17 patients, only one mutation F508del was present in one allele, and a second mutation was not identified. Failure to identify the mutation in these patients with CF in this study may be due to the presence of mutations in noncoding regions of the CFTR gene that would reduce the function of the gene.

In the present study, a total of 12 mutations were identified in the 225 CF cases. Of the 12 mutations, 7 were previously reported (Cutting, 1990; Gasparini et al., 1991; Parveen et al., 1992; Schwartz et al., 1993; Girodon et al., 1999; Girodon, 2003) and 5 were novel. High numbers of undetected mutations (66.6%) creates the need for continuing the study using alternative methods of detection such as DNA direct sequencing or denaturing gradient gel elecrophoresis. The Elucigene CF29 kit was able to detect only one mutation (F508del), which leads us to conclude that use of such commercial kits may not suffice for detecting a significant percent (>90%) of the mutations that appear in patients with CF in countries with a low frequency of CF. While creating a mutation panel for CFTR screening in CF cases, it will be desirable to have more population-specific mutations included in the panel. This would help overcome the underdiagnosis of CF and other CFTR-related diseases in those countries, where the mutational distribution does not match that of the western countries.