Journal of Genetic Disorders & Genetic Reports ISSN: 2327-5790

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Research Article, J Genet Disor Genet Rep Vol: 5 Issue: 1

Spectrum of CFTR Mutations in the Algerian Population: Molecular and Computational Analysis

Fatima Zohra Sediki1*, Abdelkarim Radoui2, Abdallah Boudjema1, Meriem Abdi1, Faouzia Zemani-Fodil1, Nadhira Saidi-Mehtar1 and Faiza Cabet3
1Laboratoire de Génétique Moléculaire et Cellulaire, Université des Sciences et de la Technologie d’Oran- Mohamed Boudiaf (USTO-MB), Algérie
2Service de Pneumologie et Allergologie pédiatriques, Etablissement hospitalier spécialisé (EHS) Canastel, Oran, Algérie
3Service d’endocrinologie moléculaire et maladies rares, Hôpital Femme-Mère-Enfant, Bron-Lyon, France
Corresponding author : Fatima Zohra Sediki
Laboratoire de Génétique Moléculaire et Cellulaire, Université des Sciences et de la Technologie d’Oran-Mohamed Boudiaf (USTO-MB), Algérie
Tel: (+213) 550861008
E-mail: sediki. [email protected]
Received: November 12, 2015 Accepted: December 28, 2015 Published: December 31, 2015
Citation: Sediki FZ, Radoui A, Boudjema A, Abdi M, Zemani-Fodil F, et al. (2016) Spectrum of CFTR Mutations in the Algerian Population: Molecular and Computational Analysis. J Genet Disor Genet Rep 5:1. doi:10.4172/2327-5790.1000130

Abstract

Little has been reported on the occurrence of cystic fibrosis in Algerian population. In order to contribute to the few existing data we undertook this study. The aim was in first instance to detect genetics alteration in the CFTR gene of 21 CF Algerian patients by sequencing. 14 different mutations were detected one of them
has never been described. Among these mutations the c.680T>G (L227R) which seems to be specific to the Algerian population, it was in silico studied to determine its impact at a molecular level. This is the first study that combined a molecular and computational analysis. These findings will assist in genetic counseling, prenatal diagnosis and future screening of CF in Algeria.

Keywords: CFTR gene; Mutation; Algerian population; Analysis in Silico

Keywords

CFTR gene; Mutation; Algerian population; Analysis in silico

Introduction

Cystic fibrosis (CF) [MIM # 219700] is the most common autosomal recessive genetic disease in Caucasian populations. It is due to the alteration of the CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) gene [MIM # 602421] which encodes a chloride channel protein essential for ion transport and epithelial cells homeostasis [1].
CFTR protein is classified as ATP Binding Cassette transporter, or an ABC transporter. The name refers to the fact that the channel uses ATP (Adenosine triphosphate) binding to open and close normally [2]. It is made up of five domains: two membrane spanning domains (MSD1 and MSD2) that form the chloride ion channel, two nucleotide-binding domains (NBD1 and NBD2) that bind and hydrolyze ATP, and a regulatory (R) domain [3].
Wrongly considered as a European disease, CF is also found in Algeria. Indeed, previous studies showed that it is present in this population [4-6]. The literature data on the clinical profile of CF and the spectrum of CFTR gene mutations in Algeria are poor because of a lack of studies. It is in this perspective and through this work we contribute to genotypic diagnosis to enrich the few existing data on Algerian population.
The molecular studies of the CF show that the c.680T>G (L227R) mutation seem to be specific to the Algerian population [7].
Knowledge of the 3D structure of a protein is of major assistance in understanding the function and its role in causing disease. Proteins with mutations do not always have 3D structures that are analyzed and deposited in Protein data bank (PDB). Therefore, it is necessary to construct 3D models by locating the mutation in 3D structures. This is a simple way of detecting what kind of adverse effects that a mutation can have on a protein function.
In order, to understand the impact of those mutations in a molecular level, mutation that is described as specific to the Algerian population and found in our cohort will be analyzed.

Materials and Methods

Sample composition
A total of 21 Algerian CF patients (14 boys and 7 girls), aged 1-14 years were investigated. The patients were referred to us by Pneumology and Allergology department of the specialized hospital center, Oran (Algeria). CF diagnosis was based on clinical findings and repeated positive sweat chloride tests (>60 mmol/L). A written consent to the genetic study was obtained from all subjects.
Mutation analysis
Genomic DNA was extracted from peripheral blood leukocytes using the salting out precipitation method [8]. All subjects were analyzed with an amplification refractory mutation system assay (PCR/ARMS), in previous study as described by Sediki et al [6].
For the patients in whom a mutation could still not be identified on one or both of CFTR genes, complete sequencing of the CFTR coding region and its exon/intron junctions was aimed. The Amplification of all 27 CFTR exons, including flanking intronic regions, was performed using CFTR gene specific primers. Direct nucleotide sequencing was carried out using Big Dye terminator cycle sequencing ready reaction kit (Applied Biosystem, New Jersey, USA). Sequence analysis was performed on an ABI Prism 310 DNA sequencer (Applied Biosystem, New Jersey, USA).
Mutation (s) that seem(s) to be specific to the Algerian CF patients and present in our cohort will be explored by in silico study.
Prediction methods
Different bioinformatics tools exploring impact on protein were used to evaluate the possible functional alteration.
SIFT (Sorting Intolerant from Tolerant) is a sequence homology-based tool that sorts intolerant from tolerant amino acid substitutions and predicts whether an amino acid substitution in a protein will affect the protein function (http://sift.jcvi.org). SIFT scores are classified as intolerant (0.00-0.05), potentially intolerant (0.051-0.10), borderline (0.101-0.20), or tolerant (0.201-1.00) [9]. The higher a tolerance index, the less functional impact a particular amino acid substitution is likely to have.
PolyPhen-2 (Polymorphism Phenotyping v2) is a tool for prediction of possible impact of an amino acid substitution on the structure and function of a human protein (http://genetics.bwh. harvard.edu/pph2/). This prediction is based on straightforward empirical rules which are applied to the sequence, phylogenetic and structural information characterizing the substitution.
Align GVGD
Align-GVGD is a program that combines the biophysical characteristics of amino acids and protein multiple sequence alignments to predict where missense substitutions in genes of interest fall in a spectrum from enriched deleterious to enriched neutral (http://agvgd.iarc.fr/agvgd_input.php).
Align-GVGD is an extension of the original Grantham differences to multiple sequence alignments and true simultaneous multiple comparisons.
Mutation taster
MutationTaster (http://www.mutationtaster.org/) is a fast web-based application to evaluate DNA sequence variants using information from various sources combined and evaluated in a naive Bayes classifier.
I-Mutant 2.0
I-Mutant2.0 (http://folding.biofold.org/i-mutant/i-mutant2.0.html) is a support vector machine (SVM) based tool for automatic prediction of protein stability changes upon single point mutations.
Swiss PDB viewer software
The mutations were performed by Swiss PDB Viewer Software (http://www.expasy.org/spdb), Swiss Institute of Bioinformatics, Basel, Switzerland).

Results

Genotyping results
A total of 21 patients were screened, 14 of them were consanguineous (66.66%) and in one patient we have no information.
14 mutations were identified; a description of those identified mutations is reported in Tables 1 and 2. Reported by Cabet F the c.4340del (4472delT), is a novel mutation not previously described in any databases or in published data. Two patients had no mutations identified.
Table 1: Distribution of genotypes in 21 Algerian CF patients.
Table 2: Frequencies of CFTR mutations detected in 21 Algerian CF patients (N=42 CF chromosomes).
The most prevalent mutation were c.1521_1523delCTT (F508del), it accounted for 21.42% (9/42). The c.579+1G>T (711+1G>T) mutation was found as the second most common 14.25 % (6/42), Two mutations reached a frequency of 9.52% (4/42), the c.680T>G (L227R) and thec.1624G>T (G542X).
The c.3909C>G (N1303K), c.595C>T (H199Y),c.2988+1G>A (3120+1G>A), c.1673T>C(L558S), andc.2051_2052delAAinsG(2183 AA>G)mutations were found with a frequency of 4.76% (2/42).While, the c.4340del (4772delT), the c.868C>T (Q290X), c.1766+3A>C (1898+3A>C),c.1652G>A (G551D) and 1647T>G (S549R)mutations account for 2.38% (1/42).
The c.680T>G(L227R) in silico analysis results
This mutation is a changed of a Leucine (L) into an Arginine (R) at position 227 of the CFTR protein. The in silico analysis of the effect of this mutation shows that, the Leucine is conserved among eleven spices, PolyPhen-2 predicts the variation as probably damaging with a score of 1.00.
Furthermore, this mutation was categorized as ‘Disease causing (p-value: 1)’and ‘Deleterious (score:0)’ using MutationTaster and SIFT softwares. Analysis with GVGD Align shows that the c.680T>G, p.Leu227Arg (L227R) was considered as ‘most likely interfere with function.’ I-Mutant2.0 shows that the mutation affects the protein stability.

Discussion

In the current study the c.1521_1523delCTT (F508del) mutation is the most common mutation with a frequency of 21.42%. This frequency is comparable to the one reported by Cabet et al (20% in a sample of 27 CF patient) [5]. Loumi et al. reported a frequency of 16.7% [4] which is less than our finding but this can be explained by the limited size of our sample. The present results match with the literature which showed that a very low frequency occur in North African countries.
Besides, the c.1521_1523delCTT (F508del), the c.579+1G>T (711+1G>T) mutation was the second most frequent (14.28%). It was found in four patients, two patients were homozygous for this mutation (Table 1), while two were carried the c.579+1G>T (711+1G>T) mutation in compound heterozygosity with the c.152_1523delCTT (F508del). This mutation was described in CF families living in Quebec, but it was already found in previous Algerian’s studies [4,5,10].
Finding this splicing mutation in the current data provides additional evidence for its higher frequency in the Maghreb.
The fourth (and fifth) mutation were c.680T>G (L227R) (9.52%) and c.1624G>T (G542X) (9.52%). The c.680T>G (L227R) mutation was found in two CF patients, they were homozygous. Based on the literature, this mutation was first described in CF Algerian patient [11], it seems to be specific to the Algerian population. It is rare in other populations, there are 9/49.000 patients with this mutation in the CFTR2 international database [12].
The c.1624G>T (G542X) mutation was found on two CF patients; they were homozygous and from consanguine families. It is considered as the most common mutation, in the Mediterranean regions of Europe and Africa [13]. This mutation appeared in the ancient Phoenicia and it was probably introduced into the Mediterranean region by the migration of Phoenicians and spread along their maritime trades routes [13,14].
Slatkin’s and Rannala’s works showed that this mutation is 1.5-3 times older than c.1521_1523delCTT which appeared probably less than 10000 years ago [15].
The c.595C>T (H199Y) was found in only one patient who was homozygous for this mutation (Table 1). This mutation has been found for the first time in German CF patient. It is due to a nucleotide change C to A at the position 727 of CFTR protein that falls into the transmembrane MSD1. In our study, an in silico test showed that the previous change predicted as probably damaging.
Found in one CF patient, the c.3909C>G (N1303K) account for 4.76%. This mutation was described as the second most frequent in Loumi et al. work (8.3%) [4]. It is found in most of the Western and Mediterranean countries [16], but its frequency seems to be high in middle east specially in Lebanon and Palestine where it reached 20% [17,18].
The c.2988+1G>A (3120+1G>A) is a splicing mutation in intron 18, it was found in one homozygous CF patient. This mutation was particularly frequent in African CF chromosome [19,20], it also has been identified in four native African CF patients [21]. Furthermore, it has been established that c.2988+1G>A (3120+1G>A) is a predominant CF mutation in Saudi Arabia [22]. Three Greek CF families have been reported to harbor this mutation [22]. Finding this mutation in a CF Algerian patient is evidence that this mutation is present in diverse populations.
The missense mutation c.1673T>C (L558S) was found in one CF patient (4.76%). This mutation was reported for the first time in one of the CF gene of a Sicilian patient [11].
The c.2051_2052delAAinsG (2183AA>G) is a frameshift mutation in exon 14, found in homozygous patient. This mutation was first described in three Canadian patients [23] and later was shown to have a significant frequency in patients from Middle east and southern Europe.
The c.4340del (4472delT) was found in one CF patient who was compound heterozygous. It has been detected by Faiza Cabet. This novel mutation never reported or described in any databases. It is a deletion of a T nucleotide at the position 4472 of the CFTR gene that causes a stop codon 20 bases further.
The c.868C>T (Q290X) was detected in one CF patient. This mutation was first reported in a French CF patient [11].
The c.1766+3 A>C (1898+3A>C) was found in a compound heterozygous, this mutation is likely to result on RNA splicing defect. It was originally identified in a Moroccan CF chromosome [11]. This mutation seems to be rare in Caucasians and does not reach the 0.1% threshold of general population frequency to warrant screening in the US [24].
Found in one CF chromosome the c.1652G>A (G551D) seems to be rare in our cohort, it is known as Celtic mutation. It is more common in north-west, in central Europe and in particular in UK with a frequency of 3.1%, Ireland (5.7%) and the Czech Republic (3.8%) [25].
The c.1647T>G (S549R) mutation was also found in one CF chromosome. This missense mutation was first reported in a non- Ashkenazi Jewish patient from Morocco [26]. It had been described also in Saudi Arabia and in the United Arabs Emirates [22,27].
Finally, two patients had no mutation. The inability to detect mutations patients may be partially explained by the fact that the currently most advanced CFTR genetic tests study only the coding region and the adjacent exon/intron junctions. Mutations located in the promoter regions as well as distant regulatory sequences are not routinely screened for, and may therefore be missed. In the present study, all exonic and intronic regions have been screened; the promoter region has not been done.
However, there is emerging evidence that genes other than CFTR may cause a disease clinically indistinguishable from CF. Mekus et al., works showed that, in a German family, no mutation could be identified in both CFTR genes of a CF patient, and his sister, who had inherited the same CFTR genes from their parents, was not affected [28].
CF is characterized not only by defective chloride secretion, but also by increased sodium absorption in the airways. Sodium transport is mediated through the amiloride sensitive epithelial sodium channel, ENaC, which is made of the 3 subunits SCNN1A, SCNN1B and SCNN1G. There is evidence that mutations in SCNN1B may cause CF-like disease in a small fraction (about 10%) of CF patients in whom a CFTR mutation cannot be found on both CFTR genes [29].
In the case reported by Mekus et al., Azad et al. identified heterozygosity for a hyperactive mutation in the SCNN1A gene [30]. The authors suggested that there might be a polygenic mechanism of disease involving CFTR and SCNN1A in some patients.
The in silico study of the c.680T>G(L227R) mutation with SIFT, showed that the amino acid change is not tolered; each amino acid has its own specific size, charge, and hydrophobicity-value.
The residue is located in a region annotated in the Uniprot database as a transmembrane domain. The wild-type residue was neutral, while the mutant residue is positively charged (Figure 1). This can disturb the (ionic) interactions with the other transmembrane helices.
Figure 1: Schematic structures of the original (left) and the mutant (right) amino acid. The backbone, which is the same for each amino acid, is colored red. The side chain, unique for each amino acid, is colored black.
The mutant residue is bigger than the wild-type residue (Mass: 174 vs. 131 Dalton, Volume: 173.4 vs.166.7 ų, Surface: 225 vs. 170 Ų). It is clearly shown in Figure 2; this can disturb either the contacts with the other transmembrane domains or/and with the lipid-membrane. The wild-type residue is more hydrophobic than the mutant residue which can affect the hydrophobic interactions within the core of the protein or with the membrane lipids.
Figure 2: A. Location of the Leucine at the position 227 of human CFTR protein on 3D model. Ribbon diagram of human CFTR protein was produced using Swiss PDB Viewer 4.0.4 software.
B. Location of the c.680T>G, p.Leu227Arg (L227R) mutation on 3D model of human CFTR protein. Ribbon diagram of human CFTR protein was produced using Swiss PDB Viewer 4.0.4 software.

Conclusion

In conclusion, this is to our knowledge the first study that includes a genetic exploration and bioinformatics analysis regarding CF in Algeria. The molecular analysis shows 14 mutations including a novel mutation. This work is evidence of a large mutational spectrum of CFTR gene mutation in Algeria. The information provided by our study is of interest in designing an appropriate diagnostic strategy. In other hand, the in silico analysis of the c.680T>G(L227R) mutation that is reported as Algerian ones, showed that this mutation seems to affect the hydrophobic interactions within the core of the protein or with the membrane lipids.
Our study suggests that the application of computational tools can provide an interesting approach to understand the impact of the mutations on protein function level.

Acknowledgments

The authors wish to thank patients and their respective physician for their full collaboration in these investigations.

Conflicts of Interest

The Authors declare no conflict of interest.

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