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

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Research Article, J Genet Disor Genet Rep Vol: 6 Issue: 1

Association Study of IVS8TGmTn Polymorphism and Cystic Fibrosis Disease in a Tunisian Population

Sahli Chaima1*, Hadj Fredj Sondess1, Dabboubi Rym1, Boussetta Khedija2, Maherzi Ahmed3 and Messaoud Taieb1
1Biochemistry Laboratory, Children’s Hospital Bechir Hamza, Research laboratory LR00SP03, Tunis, Tunisia
2Department of Pediatrics, Children’s Hospital Bechir Hamza, Tunis, Tunisia
3Department of Pediatrics, Hospital Mongi Slim, Tunis, Tunisia
Corresponding author : Chaima Sahli
Biochemistry Laboratory, Children’s Hospital Bechir Hamza, Research laboratory LR00SP03Bab Saadoun Square, Tunis, Tunisia
+ 216 53916943
Fax: + 216 71151626
[email protected]
Received: September 19, 2016 Accepted: December 31, 2017 Published: January 03, 2017
Citation: Chaima S, Sondess HF, Rym D, Khedija B, Ahmed M, et al. (2017) Association Study of IVS8TGmTn Polymorphism and Cystic Fibrosis Disease in a Tunisian Population. J Genet Disor Genet Rep 6:1. doi: 10.4172/2327-5790.1000147


Background: Cystic fibrosis (CF) is the most common autosomal recessive disease in Caucasians, caused by mutation in cystic fibrosis transmembrane conductance regulator (CFTR). The analysis of some extra and intragenic markers within or closely linked to CFTR gene  is  useful  as  a  molecular  method  in  clinical  linkage  analysis.  Indeed, knowing that the molecular basis of CF is highly heterogeneous in our population is explained in the present study. The goal of the study was to examine the IVS8 TGmTn genotypes in a Tunisian sample of patients with CF disease and normal controls, and to compare the results with the findings from the literature.

Methods: We conducted a case–control study in a sample composed of 80 CF patients and 90 control subjects to investigate the possible effect of the polymorphism. The study of the IVS8 TGmTn polymorphism was performed using sequencing analysis.

Results: Our data revealed an association between the IVS8 TGmTn polymorphism and CF risk. There was a significant difference in the poly-T; TG repeats allele or genotype frequencies between cases and controls. Also, statistical analysis shows no association between F508del mutation and T5 variant. Furthermore, new TG9 repeat was reported in CF patients.

Conclusion: This study provides additional evidence that TGmTn poly tract has important role in the development of CF. Further investigation, which genetic and functional studies of polymorphisms in genetic diseases will become of major interest in the future.



Keywords: Cystic fibrosis; IVS8 TGmTn polymorphism; CFTR gene; F508del mutation; Tunisian population


Cystic fibrosis; IVS8 TGmTn polymorphism; CFTR gene; F508del mutation; Tunisian population


Cystic fibrosis (CF; OMIM number: 219700) is the most common severe autosomal recessive disorder caused by alterations in the CF transmembrane conductance regulator (CFTR; OMIM number: 602421). The CFTR gene composed of 27 exons and 26 introns encodes a protein with 1480 amino acids that constitutes a cAMP (Cyclic adenosine monophosphate) regulated chloride channel of the apical membrane of epithelial cells [1,2]. To date, more than 2000 different mutations have been reported to the Cystic Fibrosis genetic analysis consortium [3]. The type of mutations and their distribution varies widely between different countries and/or ethnic groups [4,5]. The most common disease-causing mutation, F508del (c.1521_1523delCTT), is found in 70% of Caucasian CF patients whereas the frequency of other common mutations does not exceed 3% [6].
The length of the polythymidine tract in intron 8 (C.1210-12T (5- 9) of CFTR gene affects the splicing efficiency of intron 8. Three alleles have been characterized in IVS8: 9T, 7T and 5T. The shortest (5T) is associated with the highest rate of incomplete transcripts. mRNA without exon 9 results in CFTR proteins that will not mature and will therefore not function as chloride channels in the apical membrane of epithelial cells. IVS8-5T is considered equivalent to a “mild”CFTR mutation with incomplete penetrance and is associated with the highest level of non-functional CFTR protein. The polymorphic sequence of intron 8 also contains the TG repeat (C.1242-35-1242- 12GT(8-10)) adjacent to the variant poly T involved in exon 9 splicing, it has been found to condition the Tn-dependent splicing [7].
The IVS8TGmTn polymorphism was the subject of several studies in CF, CF related diseases and healthy cohorts revealing its role as a predisposing genetic factor. In Tunisia, it has been only studied in Congenital Bilateral Absence of the Vas Deferens (CBAVD) patients [8].
To further investigate the contribution of polymorphisms in the CFTR gene in Tunisian population, the present work aims at performing a comparative analysis of the genotype and allele frequency distributions of the poly-T, TG-repeats within the CFTR gene on mutant and wild type chromosomes.

Materials and Methods

This study included eighty Tunisian CF patients aged between three days and 17 years with an average age of 5 months. Our demographic data indicates that 58% of patients were born to consanguineous couples. The selection of our patients was based on typical clinical manifestations of lung or/and gastrointestinal disease with high levels of sweat chloride concentration (higher than 60 mmol/L). The sweat test was conducted by pilocarpine iontophoresis (Exsudose method). A control group of ninety healthy subjects aged between one month and 15 years was also studied. All parents of our patients signed informed consents. This work was carried out in accordance with the Ethical Guidelines of the World Medical Association Declaration of Helsinki.
Genomic DNA was extracted from peripheral blood leukocytes by the salting-out salting-out method [9].
Determination of mutations in the CFTR gene: CFTR mutations were previously identified using denaturing high performance liquid chromatography (DHPLC), denaturing gradient gel electrophoresis (DGGE) and direct sequencing [10,11,12]. Different CFTR genotypes were determined: 65 patients (81.25%) with homozygous mutations (60% F508del/F508del, 18.75% E1104X/E1104X and 2.5%711+1G→T/711+1G→T) and 6 patients (7.5%) with double heterozygous mutations (5% F508del/G542X and 2.5% F508del/ N1303K). In 9 cases (11.25%) with one identified mutation (8.75% F508del/ undetermined and 2.5% E1104X/undetermined).
Polymorphism poly-T, TG-repeats analysis: Polymorphism poly-T, TG-repeats were studied by direct sequencing on ABI prism 310 (Applied biosystemes, California, USA) and was used to analyze the junction intron 8/exon 9. Genomic DNA was amplified by primers spanning the splicing acceptor site of exon 9 [13,14]. PCR products were purified with the kit Wizard SV Gel and PCR clean up system (Promega, Madison, WI) and were sequenced using the Big Dye Terminator Cycle Sequencing Reaction Kit (PE Applied BioSystem, Foster City, California, USA). Each single stranded product was concentrated using a centri-sep column (Applied Bio systems, Foster City, California, USA).
Statistical analysis: Analysis was performed using the Statistical Package for Social Sciences (SPSS 20.0 version). Differences between the means of the 2 continuous variables were evaluated by studenttest. Odds ratio (OR) and 95 % confidence intervals (95 % CI) were calculated as strength of association between alleles or genotypes and CF patients. A p value of 0.05 was considered statistically significant.

Results and Discussion

Genotypes and alleles frequencies of poly-T, TG repeats polymorphism in CF patients and controls were delineated in Table 1. A significant difference in genotype and allele frequencies between the two groups was noted (p<0.05) (Table 1).
Table 1: Frequencies of genotypes of TGmTn poly tract polymorphism.
The analysis of the variant Tn showed the presence of 6 different genotypes. Indeed, the homozygous genotype (T9/T9) was over represented in the CF group with 32.5% followed by heterozygous genotypes (T7/T9) and (T5/T7) with 26.3% and 18.8% respectively. However, the control group showed a high prevalence of genotype (T7/T7) with 47.8% followed by the heterozygous genotype (T7/T9) (24.4%). Concerning the distribution of TGm genotype, we noted 4 genotypes in CF group whose TG10/TG10 genotype (36.3%) was the most frequent. Therefore, the genotype TG10 / TG11 is more common in control group (46.7%) (Table 1).
The distribution of allelic poly T variant showed the presence of three repeat T5, T7 and T9. The last repeat is the most frequent in CF patient (48.12%). However, the T7 is higher in healthy subjects (67.77%) and the T5 allele was overrepresented.
The TGmTn haplotype frequency is showed in Figure 1; nine haplotype were identified with seven in CF patients and eight in control group. Indeed, TG10T9 in CF group was the most frequent (39.37%) followed by TG11T7 (26.87%). Nevertheless, TG11T7 and TG10T7 were the most common in the control group with 36.11% and 26.68% respectively.
Figure 1: Determination of TGmTn haplotype in CF and control groups.
The fact that the F508del mutation is the most frequent in our cohort, we have chosen to study its association with the analyzed polymorphism. We classified the CF population into two groups. The first group (n=45) composed of patients homozygous for the most frequent mutation F508del. The second group composed of 35 patients with compound heterozygous F508del/other mutation as well as homozygous or compound heterozygous for mutations other than F508del. Statistical analysis shows no association between F508del mutation and T5 variant.


This study is the first ever performed on the TGmTn poly tract polymorphism in a Tunisian population. Although our sample was small in size which somehow limited definite conclusions, it nevertheless provided sufficient base data to explore potentially interesting risk factor for CF in Tunisia in comparison with results found in other populations.
The distribution of allele and genotype frequencies of the TGmTn poly tract polymorphism showed a significant difference between patients and control (p<0.05). Our results are in agreement with recent reports for other populations indicating that T7 variant was the most prevalent in CF and control groups, such as Indian/ Iranian [2,15] Japanese [16] Chinese [17] and Brazilian populations [18]. Furthermore, we noted no association between T5 variant with the frequent mutation F508del in our population. These data are in accordance with similar studies in Iranian population where they also found no association [15].
In this study, we analyzed the distribution of TGm genotype between CF and control groups. Indeed, the TG10/TG10 genotype frequency was significantly higher in the CF patients group (36.3 %) than in the controls group (32.78 %) in which the TG10/TG11 genotype was the most common in control groups with 46.7%. In previous studies on Chinese and Caucasian control groups, it was revealed 57.2% and 67% frequency of TG11 repeat which is also the most higher in our control group with 76.7% [17,19,20]. While among our CF population the TG10 was the most frequent with 82.5%. These results are in agreement with a study later conducted in American population (78.3%) [21].
CFTR mutations can determine the CF severity. Patients with mutations of classes I, II and III have more severe clinical forms than those with mutations of IV, V and VI classes [22]. However, we can observe heterogeneity in severity of CF in patients with identical mutations in the CFTR gene [23]. Our study included only severe mutations from classes I and II, which may help to normalize for the influence of the disease-causing CFTR mutation on the course of the disease. In our study, the T5 variant was present in twenty-one patients with mutations of classes I and II. Indeed, this variant was found in association with TG10 and TG11. On the other hand, for the first time, we noticed that the T5 was associated with TG10 repeat. However, the T5TG10 genotype was identified in four chromosomes; is the first description of this pattern in CF population. In addition, this variant has been identified in two chromosomes associated with TG11. In fact, Huang et al. demonstrated that the T5 variant is associated only with TG12 and TG13 [17,23]. These data could be explained by the difference in ethnic origin, and the nature of the identified CF mutations.
In conclusion, this study of TGmTn poly tract polymorphism performed for the first time in a Tunisian CF and healthy population. We first showed that CF patients with T5 variant frequency was significantly higher in CF patients group and may possibly constitute a risk factor for CF. Second, we noticed that no significant difference was found between T5 variant and F508del mutation and further investigation, involving genetic and functional studies of polymorphisms in genetic diseases will become of major interest in the future.

Disclosure statement

The authors declare that they have no conflicts of interest concerning this article.


This work was funded by the Ministry of Higher Education and Scientific Research in Tunisia. The authors are grateful to Mr.Faouzi Rassass (Tunis, Tunisia), for her contribution in checking the manuscript language.



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