An extensive molecular analysis of the CF transmembrane regulator (
Cystic fibrosis (CF) is a severe life threatening genetic disease most common among Caucasians with an incidence ranging from 1 in 2500 to 1 in 3600 [
So far, more than 1900 different
Recently, CF has been diagnosed in the Middle East ranging from 1 in 2,500 to 1 in 16,000 with different mutation frequencies according to the ethnic origin of populations [
The aim of this study was to determine the CF mutation spectrum among the Palestinian patient population. Samples from 60 unrelated CF patients residing in the West Bank and Gaza were collected and their respective CF mutations were determined. Consequently, the mutation spectrum was compared with other ethnic groups residing in the Arabic population.
A total of 60 unrelated Palestinian CF patients, 19 of them residing in the West Bank and 41 residing in Gaza, participated in this study of which 34 are males and 26 are females. Most of the participants (97%) were children less than 18 years old. The criteria for inclusion in this study were based on the clinical diagnosis. Typical pulmonary and/or gastrointestinal tract manifestations and/or elevated sweat chloride values (>60 mEq/L, Table
Patients data and sweat chloride concentrations.
# | Age (years) | Sex | Region | Sweat chloride values (mEq/L) |
---|---|---|---|---|
P1 | 1 | M | Gaza | 113 |
P2 | 3 | M | Gaza | 85 |
P3 | 8 | M | Gaza | 111 |
P4 | 11 | M | Gaza | 70 |
P5 | 11 | F | Gaza | 60 |
P6 | 5 | F | Gaza | 100 |
P7 | 8 | F | Gaza | 66 |
P8 | 3 | M | Gaza | 103 |
P9 | <1 | M | Gaza | 81 |
P10 | 1 | M | Gaza | NR |
P11 | 7 | M | Gaza | QNS |
P12 | 10 | M | Gaza | 72 |
P13 | 8 | M | Gaza | 90 |
P14 | 4 | M | Gaza | 60 |
P15 | 6 | M | Gaza | NR |
P16 | 7.5 | M | Gaza | 90 |
P17 | 7 | M | Gaza | 96 |
P18 | 6 | F | Gaza | 76 |
P19 | 4 | F | Gaza | 114 |
P20 | 4 | F | Gaza | 118 |
P21 | 1 | M | Gaza | 77 |
P22 | 1 | F | Gaza | 114 |
P23 | 10 | M | Gaza | 100 |
P24 | 3 | M | Gaza | 126 |
P25 | 2 | M | Gaza | 129 |
P26 | 9 | F | Gaza | 105 |
P27 | 18 | M | Gaza | 42 |
P28 | 11 | F | Gaza | 128 |
P29 | 6 | M | Gaza | 135 |
P30 | 5 | F | Gaza | QNS |
P31 | 13 | M | Gaza | 106 |
P32 | 15 | M | Gaza | 60 |
P33 | 3 | M | Gaza | 107 |
P34 | 9 | M | Gaza | 46 |
P35 | 17 | F | Gaza | 64 |
P36 | 1.5 | F | Gaza | 129 |
P37 | 7 | M | Gaza | NR |
P38 | 2 | F | Gaza | QNS |
P39 | 13 | F | Gaza | 54 |
P40 | 10 | M | Gaza | 62 |
P41 | 4 | M | Gaza | 70 |
P42 | 7 | M | West Bank | 117 |
P43 | 18.5 | M | West Bank | 121 |
P44 | 5 | M | West Bank | 105 |
P45 | 13 | M | West Bank | 100 |
P46 | 12 | M | West Bank | NR |
P47 | 12 | M | West Bank | 110 |
P48 | 7 | M | West Bank | 108 |
P49 | 8 | F | West Bank | 105 |
P50 | 7 | F | West Bank | NR |
P51 | 15 | F | West Bank | NR |
P52 | 2 | F | West Bank | NR |
P53 | 10 | F | West Bank | 87 |
P54 | 12 | F | West Bank | 104 |
P55 | 2.5 | F | West Bank | 110 |
P56 | 12 | F | West Bank | 112 |
P57 | 31 | M | West Bank | 120 |
P58 | 3 | F | West Bank | 103 |
P59 | 15 | F | West Bank | 45 |
P60 | 30 | M | West Bank | NR |
NR: no result, used to indicate nonreported results.
QNS: quantity not sufficient, used to indicate insufficient sweat samples to perform the sweat test.
Genomic DNA was extracted and purified from whole EDTA-blood by the automated extraction apparatus Autopure LS (Qiagen) using the PureGene DNA Purification Kit (Qiagen).
Amplification of the coding region and flanking introns of the
For each patient, all PCR products were pooled equimolary before they entered the Nextera sample preparation protocol (Nextera XT DNA Sample Prep Kit (Illumina, Inc., San Diego, CA)), followed by 250 bp single-end sequencing on a MiSeq instrument (Illumina, Inc., San Diego, CA). All 60 samples were labeled using 60 different index tags (Nextera, Epicentre Biotechnologies).
Reads were aligned to the human genome (hg19/GRC37) using the CLCBio software package (CLC Genomics Workbench 6.0.2). All exons with a coverage lower than 20 were analysed by Sanger sequencing. We first filtered all variants classified as deleterious according to the
Mutations were confirmed with the INNO-LiPA
We applied an NGS screening strategy to efficiently identify causative mutations in the 60 unrelated Palestinian patients with a clinical diagnosis of CF. Approximately, 98% of all reads on the MiSeq were successfully mapped to the reference genome. The overall mean read depth in the target area was 344x. A read depth of 10x for 92% of the bases and 20x for 90% of the bases was obtained. Patient P9 had the best coverage with all amplicons covered with at least 20x whereas patient P7 had the lowest number of amplicons (13) with a coverage higher than 20x (data not shown).
We identified 17 different mutations in 40 patients which have previously been described as CF-causing mutations, including 3 splice sites, 5 missense mutations, 4 frame shift mutations, 3 stopcodons, and 2 exon spanning deletions (Table
c-notation | p-notation | Exon/intron | dbSNP # | # of patients Gaza | # of patients West Bank | Patient(s) |
---|---|---|---|---|---|---|
del exon2 | Exon 2 | 1 | 1 | 24 (het), 43 | ||
c.223C>T | p.(Arg75*) | Exon 3 | 121908749 | 1 | 13 | |
c.254G>A | p.(Gly85Glu) | Exon 3 | 75961395 | 4 | 44 (het), 56, 57 (het), 58 | |
c.1040G>C | p.(Arg347Pro) | Exon 8 | 77932196 | 1 | 33 | |
c.1209+1G>A | Intron 9 | 397508176 | 1 | 2 | ||
c.1393−1G>A | Intron 10 | 397508200 | 4 | 42, 47, 54, 55 (het) | ||
c.1521_1523delCTT | p.(Phe508del) | Exon 11 | 113993960 | 11 | 5 | 1, 3, 9, 11, 17, 19, 22 (het), 23, 24 (het), 28 (het), 29, 46, 48, 49 (het), 50, 52 |
c.1585−1G>A | Intron 11 | 76713772 | 2 | 26, 31 | ||
c.1624G>T | p.(Gly542*) | Exon 12 | 113993959 | 1 | 28 (het) | |
c.2051_2052delAAinsG | p.(Lys684Serfs*38) | Exon 14 | 121908799 | 2 | 8, 25 | |
c.2089_2090insA | p.(Arg697fs) | Exon 14 | 397508341 | 1 | 22 (het) | |
c.2988+1Kbdel8.6Kb | Exons 19, 20, 21 | 4 | 1 | 10, 20, 21, 36, 49 (het) | ||
c.3299A>C | p.(Gln1100Pro) | Exon 20 | 397508535 | 1 | 57 (het) | |
c.3793G>A1 | p.(Gly1265Arg) | Exon 23 | 1 | 16 | ||
c.3808G>A | p.(Asp1270Asn) | Exon 23 | 11971167 | 1 | 18 (het) | |
c.3846G>A | p.(Trp1282*) | Exon 23 | 77010898 | 2 | 55 (het), 60 | |
c.3909C>G | p.(Asn1303Lys) | Exon 24 | 80034486 | 2 | 44 (het), 51 | |
c.4251delA | p.(Glu1417fs) | Exon 27 | 397508706 | 1 | 6 |
1Previously undescribed mutation.
All mutations are homozygous except those indicated by (het).
In patients P4, P5, P7, P12, P14, P15, P27, P30, P32, P34, P35, P37, P38, P39, P40, P41, P45, P53, and P59 no mutation was detected.
In P18 only one mutation was detected.
This is the first study to investigate the
The differences in the rates of mutations identified in CF patients residing in the West Bank versus those in Gaza, are remarkable. For example, mutations c.1393-1G>A, p.(Gly85Glu), p.(Trp1282X), and p.(Asn1303Lys) were only present among Palestinians in the West Bank, while mutations p.(Lys684Serfs*38) and c.1585-1G>A were only present among Palestinians residing in Gaza (Figure
NGS technology combined with MLPA analysis proved to be a very efficient and cost-effective way to identify
The relatively low mutation detection rate of 67% of the CF patients tested in this study is most likely caused by clinical misdiagnosis depending on the sweat chloride test. Values of this test can be influenced by nutritional state, skin condition, age, and many other factors, resulting in false positive sweat chloride values as high as 15% [
In conclusion, we identified the most common CF mutations and their respective frequency in the Palestinian population. Not only is this knowledge important for the families themselves but also it is a prerequisite to set up a reliable and sensitive diagnostic test for CF in this population. Genetic testing in this area for recessive disorders is highly recommended because of the high rates of consanguineous marriages among the Palestinians (25%–65%) [
The authors declare that there is no conflict of interests regarding the publication of this paper.
The authors thank the company Fujirebio for providing them with INNO Lippa Kits. The authors also thank Dr. Abdalla Hasaballa for his help to recruit the patients.