Cystic Fibrosis Gene Mutation Frequency Among a Group of Suspected Children in King Hussein Medical Center.

INTRODUCTION: Cystic fibrosis (CF) is a genetic multisystem disorder that affects mostly the lungs, but other organs such as liver, pancreas and intestine also affected. CF is inherited in an autosomal recessive manner and occurs in males and females equally. Cystic fibrosis Transmembrane Conductance Regulator (CFTR) mutations are classified into five classes. Class 1 (non-functional protein), class 2 (near-absence of mature CFTR protein at the apical cell membrane), class 3 (full-length CFTR protein incorporated into the cell membrane), class 4 (reduced conductance CFTR mutation), and class 5 (reduced amount of CFTR protein with normal function). Globally F508 mutation is the most common. AIM: The aim of this study was to determine the frequency of CFTR gene mutation in Jordanian populations attending a major hospital (KHMC).
MATERIAL AND METHODS: This is a retrospective study was conducted on 777 sera samples for patients clinically suspected to have cystic fibrosis over a six year period 1/1/2013-1/10/2018. The patient's age range between 1year and 33 years, of which 59.2% (460) were male and 40.8% (317) female. Blood samples were analyzed at Princess Iman Centre for Research and Laboratory Sciences at King Hussein Medical Centre. The samples were tested for 34 mutations of CFTR gene using CF Strip Assay VIENNA LAB Diagnostics GmbH, Austria by polymerase chain reaction (PCR).
RESULTS: A total of 777 patients samples were analyzed for cystic gene mutations. Twelve (12) mutations were identified. In 49 patients (6.3%) were heterozygous genotype mutant and 28 (3.6%) were homozygous. The most frequent mutation F508del was found in 32/77 (41.5%). 20 (25.9%) of them were heterozygous genotype mutant and 12 (15.6%) were homozygous genotype mutant. The second frequent mutation was N1303K with frequency rate 15.6% (12/77), 9 (11.7%) of them were heterozygous and 3 (3.9%) were homozygous. Regarding frequency of cystic fibrosis gene mutation depending on sex, 55.8% (43/77) of mutations were found in male, whereas 44.2% (34/77) in female.
CONCLUSION: Our findings suggest that cystic fibrosis in Jordan is not a rare disease, and found that the most frequent CFTR gene mutation was F508del, which is in keeping with results from other Mediterranean countries.

INTRODUCTION

Cystic fibrosis (CF) is a genetic multisystem disorder that affects mostly the lungs, but other organs such as liver, pancreas and intestine also affected. CF is inherited in an autosomal recessive manner and occurs in males and females equally (1). The disease is caused by a mutation in cystic fibrosis transmembrane conductance regulator (CFTR) gene that encode CFTR protein, which play important role for transporting chloride ions through membranes and lead to increased viscosity of the mucous in the lung, liver, pancreas and intestines (2). As a result the symptoms of cystic fibrosis includes chronic obstructive pulmonary disease (infections with Pseudomonas aeruginosa and Staphylococcus aureus), liver and pancreatic fibrosis (poor nutrient uptake and pancreatic damage that can lead to diabetes mellitus) (3). Cystic fibrosis distributed worldwide and most common (1 in 2500 white individuals) in North Europe and 1 in 2270 Ashkenazi Jews (4). The cystic fibrosis gene, CFTR was identified in 1989 by Riordan et al and Rommens et al (5), and it is located on the seventh human chromosome at the position 7q13 (6). Regarding CFTR gene mutation there are 1900 mutations were identified, of which 1500 are potentially cause of cystic fibrosis in patients suspected to have the disease. CFTR mutations are classified into five classes. Class 1 (non-functional protein), class 2 (near-absence of mature CFTR protein at the apical cell membrane), class 3 (full-length CFTR protein incorporated into the cell membrane), class 4 (reduced conductance CFTR mutation), and class 5 (reduced amount of CFTR protein with normal function) (7).Globally F508 mutation is the most common (6, 8, 9). Diagnosis of cystic fibrosis performed using CFTR molecular testing that is based on direct gene analysis procedures. There are two groups of methods for this purpose: first one is method for analysis of samples DNA for presence or absence of specific mutations, and the second is screening samples for any deviation from the standard sequence (10).

AIM

The aim of this study was to determine the frequency of CFTR gene mutation in Jordanian populations attending a major hospital (KHMC).

MATERIAL AND METHODS

Our publication was approved by the research ethics committee of the Royal Medical Services, Amman-Jordan. This is a retrospective study was conducted on 777 sera samples for patients clinically suspected to have cystic fibrosis over a six year period 1/1/2013-1/10/2018. The patient’s age range between 1year and 33 years, of which 59.2% (460) were male and 40.8% (317) female. Blood samples were analyzed at Princess Iman Centre for Research and Laboratory Sciences at King Hussein Medical Centre. The samples were tested for 34 mutations of CFTR gene using CF Strip Assay VIENNA LAB Diagnostics GmbH, Austria by polymerase chain reaction (PCR). DNA was isolated from blood, amplified using biotinylated primers, then hybridization of amplification products to strips containing allele-specific probes immobilized as array of parallel lines, and finally bound sequences were detected using streptavidin-alkaline- phosphatase and color substrate. For each sample 2 strips was used, strip A that cover 18 mutations, and strip B that cover 16 mutations. Each test strip contains a positive reaction of the uppermost control line that indicates the correct function of conjugate solution and color developer.

RESULTS

A total of 777 patients samples were analyzed for cystic gene mutations. Twelve (12) mutations were identified. These mutations were F508del, G85E. N1303K, A455E, W1282X, 2184delA, 2183AA>G, 3849, R117H, 2789+5G>A, R334W, and 3272- 26A. In 49 patients (6.3%) were heterozygous genotype mutant and 28 (3.6%) were homozygous. The most frequent mutation F508del was found in 32/77 (41.5%). 20 (25.9%) of them were heterozygous genotype mutant and 12 (15.6%) were homozygous genotype mutant. The second frequent mutation was N1303K with frequency rate 15.6% (12/77), 9 (11.7%) of them were heterozygous and 3 (3.9%) were homozygous Table 1. Regarding frequency of cystic fibrosis gene mutation depending on sex, 55.8% (43/77) of mutations were found in male, whereas 44.2% (34/77) in female. 3272-26A and R334W mutations were found only in female patients with 1.3% frequency rate for each. Male were more frequently had F508del mutation (21/43-48.8%) in comparison with female, whose more frequently had F508del mutation (11/34-32.4%) N1303K (7/34-20.5%) and G85E (5/34-14.7%) (Figure 1).

Table 1.

Frequency of cystic fibrosis gene mutations.

Mutation	Heterozygous genemutant		Homozygous genemutant		Total
	Number	Percentage	Number	Percentage
F508del	20	25.9%	12	15.6%	32(41.5%)
N1303K	9	11.7%	3	3.9%	12(15.6%)
G85E	5	6.5%	4	5.2%	9(11.7%)
W1282X	3	3.9%	3	3.9%	6(7.8%)
A455E	5	6.5%	0	0%	5(6.5%)
2183AA>G	2	2.6%	3	3.9%	5(6.5%)
2184delA	1	1.3%	0	0%	1(1.3%)
3849	2	2.6%	0	0%	2(2.6%)
2789+5G>A	0	0%	2	2.6%	2(2.6%)
R334W	0	0%	1	1.3%	1(1.3%)
3272-26A	1	1.3%	0	0%	1(1.3%)
R117H	1	1.3%	0	0%	1(1.3%)
Total	49	63.6%	28	36.4%	77(100%)

Figure 1.

Cystic fibrosis gene mutation in male and female.

DISCUSSION

Our study examined a 777 of Jordanian patients with clinical features suspected to have cystic fibrosis for 34 mutations of CTTR gene. In Jordan the first report about cystic fibrosis was in 1984 by Nazer HM (11). Our results shows that F508del mutation was the most frequent (41.5%), followed by N1303K (15.6%) and G85E (11.7%). Study conducted by F. Al Sheyab et al for 3 mutations (F508del, W1282X, N1303K) report that frequency of F508del mutation was 23.75%, W1282X mutation (15%) and N1303K not detected. Lowest frequency of F508del may be due to a small number of specimens used in their study (120) in comparison with our study (777) (12). The frequency of W1282X in our study was 7.8%. In the surrounding countries such as Palestine Laufer-Cahana A et al report that the mutations DF508, N1303K, W1282X were found in all Arab ethnic subgroups, which is in keeping with our results (13). In Lebanon study conducted by Farra C shows F508del mutation was the most common with 34% frequency rate (14). Comparing our results with those from western Arab countries such as in Algeria and Tunisia, most frequent mutation in Algeria reported by Fatima Zohra Sediki was F508del (18.75%) and this lower frequency in comparison with our results due to small number of sample size (24 families). In Tunisian population the frequency of F508del was 50.4% (15). In European population the most frequent mutation is F508del with frequency rate ranged between 43% and 87.2% (9). Studies from Latin America show that F508del was most common in Venezuela (27.7%) (16) and 66.7% in Brazil (17). As we mentioned earlier the variation in frequency rate of mutation may be due to a variation in sample size in each study, but our findings suggest that F508del was the most frequent.

CONCLUSION

Our findings suggest that cystic fibrosis in Jordan is not a rare disease, and found that the most frequent CFTR gene mutation was F508del, which is in keeping with results from other Mediterranean countries.