Identification of GP1BA Gene Mutations in Iraqi Patients with Bernard- Soulier Syndrome
Lina Mussa Kadhim1*, Safa A Faraj2, Muthana Ibrahim Maleek3
1Wasit University, College of Science, Biology Department,Iraq
2Wasit University, College of Medicine, MD Pediatrics hema-oncologist,Iraq
3Wasit University, College of Science, Biology Department, Iraq
*Corresponding email: [email protected]
ABSTRACT
Background: Bernard-Soulier syndrome (BSS) is a hereditary bleeding disorder distinguished by macrothrombocytopenia and prolonged bleeding time resulting from quantitative or qualitative defects in the GPIb/IX/V complex. The aim of this project was to detect GP1BA mutations in BSS patients.
Methodology: Twenty BSS patients (11 males and 9 females) and twenty healthy individuals (12 males and 8 females) were included in this study. The selected regions in the GP1BA gene were amplified and directly sequenced.
Results: DNA sequence analysis revealed three mutations in the GP1BA gene, one of which was novel.
Conclusion: BSS, like other autosomal recessive disorders, is prevalent in communities with high incidence of consanguineous marriage, emphasizing the importance of genetic analysis
for confirming the diagnosis and identifying carriers were usually asymptomatic.
Keywords: Bernard–Soulier syndrome, GPIb-IX-V, GP1BA.
Introduction
Bernard-Soulier syndrome (BSS) is an autosomal recessive bleeding condition discovered by Bernard and Soulier in 1948, caused by a defect in the platelet complex GPIb/IX/V on a quantitative or qualitative level [1]. It is estimated to occur in 1 per million individuals.
However, due to under-recognition and misdiagnosis, the prevalence may be much higher than the estimated [2,3]. The most common symptoms of BSS are epistaxis, ecchymosis, hemorrhage after trauma and heavy menstrual bleeding, hematuria and gastrointestinal bleeding rarely occur.
Prolonged bleeding time, macthrombocytroopenia, and low/absence GP1b/V/IX expression approve the diagnosis of BSS [4]. The GPIb-IX-V receptor regulates platelet adhesion to proteins in the subendothelial matrix after an endothelial injury, specifically von Willebrand factor (vWF)[5]. GPIb-IX-V is comprised of four polypeptide chains: GPIbα, GPIbβ, GPIX and GPV.
Typically, the absence of one of the GPIb-IX-V subunits greatly reduces the surface expression of the entire receptor, resulting in platelet adhesion defects and increased bleeding susceptibility in BSS patients [6]. The GPIb-IX-V subunits are encoded separately by different genes located at chromosomes 17p12 (GPIBA), 22q11.2 (GPIBB), 3q21 (GP9) and 3q29(GP5) [7].Various missense, nonsense, and frame shift mutations were detected in the GP1BA, GP1BB, and GP9 genes. Most mutations block the matched association of the complex subunits, this results in very low GPIb/IX/V expression on the platelet surface. In other situations, the receptor is normal or slightly reduced but incapable of attach to vWF[5].
Methodology
Study groups This study included twenty patients with BSS (11 males and 9 females) aged between (6-42) year with a history of mild to severe bleeding and twenty healthy individuals (12 males and 8 females) aged between (9-54) year. BSS was diagnosed depending on the clinical presentation and hematological indices.
Blood sampling
Three ml of blood were collected by vein puncture in EDTA anticoagulant tubes from all patients and control group.
DNA isolation
Genomic DNA was extracted from whole frozen blood for molecular studies according to the
protocol ReliaPrep™ Blood gDNA Miniprep System.
Amplification of genomic DNA
Polymerase chain reaction (PCR) was used to amplify selected regions of the GP1BA gene using primer pairs previously described by Afrasiabi et al. [8]. PCR primers sequences, positions and the size of each PCR product and PCR components are shown in tables (1,2). Thermocycling conditions were as follows: an initial denaturation at 95 °C for 5 min, followed by 30 cycles of denaturation for 30 sec at 95 °C, annealing for 30 sec at 66 °C , and extension for 30 sec at 72 °C, and a final extension step at 72oC for 7 min. PCR products were then fractionated on 1%
agarose gel with a ladder (100 bp) and visualized.
Table 1. Primers sequences and positions
Primer Seq. Position Product (bp)
GpIb_A1-F 5`-GAGAGAAGGACGGAGTCGAG-3` 2761–2780 3218–3199
457 GpIb_A1-R 5`-GGTTGTGTCTTTCGGCAGGT-3`
GpIb_A2-F 5`-CTGTGAGGTCTCCAAAGTGG-3` 3125–3144 3585–3566
460 GpIb_A2-R 5`-TAGCCAGACTGAGCTTCTCC-3`
GpIb_A3-F 5`-AAGGCAATGAGCTGAAGACC-3` 3511–3530 4108–4087
597 GpIb_A3-R 5`-CTTGTGTTGGATGCAAGGAG-3`
GpIb_A4-F 5`-CACAAGCCTGATCACTCCAA-3` 4394–4413 4978–4959
584 GpIb_A4-R 5`-TTCTCTCAAGGTCCCCAAAC-3`
Table 2. PCR reaction components
Component Component
Master Mix (Ready-to-use):TaqDNA, polymerase ,dNTPs, MgCl2 12.5 μl
Forward primer 1 μM
Reverse primer 1 μM
DNA template 2 ng/μl
Nuclease free water 8.5
Final volume 25
Aliquot per single rxn 23 μl of master mix per tube and add 2 μl of template
PCR products sequencing
The PCR products were directly sequenced using an automated sequencer (ABI3730XL Genetic Analyzer; Applied Biosystems) by(Macrogen Corporation – Korea). Geneious software was used for analysis of data and determining sequence variation between samples.
Statistical analysis
The Hardy-Weinberg equilibrium (H-WE) of the detected genotypic distribution was demonstrated using chi-square statistic. WinPepi software was used to determine odds ratio (OR), etiological fraction (EF) and preventive fraction (PF), confidence interval (C・I) of odds ratio at 95%. The differences for all relations were calculated using a two-tailed P-value at a significant level less than 0.05.
Results
This study included 20 patients with BSS (11 males and 9 females) aged between (6-42) year.
Consanguineous marriages in parents were reported in 60% of patients. The main clinical manifestations were epistaxis, easy bruising, menorrhagia and ecchymosis. Initial laboratory tests revealed variable thrombocytopenia, prolonged bleeding time (BT), and large platelets on
peripheral blood smear.
Direct sequencing analysis of twenty BSS patients revealed three mutations in the GP1BA gene, two were previously reported and one novel alteration. The first c.482 C > T (rs6065) is a missense mutation lead to replacement of nucleotide C to T at position chr17:4933086 (GRCh38.p12) in exon 2 of GP1BA gene, leading to substitution of amino acid from threonine to methionine at position161, c.5263 A>T mutation (rs81663) is replacement of nucleotide A to T at position chr17:4932557 (GRCh38.p12) in intron 1 of GP1BA gene, and c.6040 T>G is a novel missense mutation, located at chr17: 4933334 (GRCh38.p12) in exon 2 of GP1BA gene lead to substitution of tyrosine to aspartic acid at position 244 of the GPIbα subunit (figure 1).
c.482C>T was presented with two alleles (C and T) and three genotypes (CC, CT and TT).
These genotypes showed deviation from HWE in BSS patients and control group, because there was significant difference between the expected and the observed frequencies of the genotype (p
≤ 0.05). Comparing BSS patients to control shown that genotype frequencies of CC (85 % vs.
90%) and CT (10% vs. 5%), while the third genotype (TT) was observed with a frequency 5% in BSS patients and control. The CC,CT and TT genotypes were non-significant difference (p higher than 0.05) in comparison between control and BSS patient in the Fisher's exact test. It was also noticed the frequency of mutant allele (T) show non-significant difference in BSS patients compared with control individuals (10 vs. 7.5%; OR = 1.37 ; PF = 0.1;EF= 0.02; 95% C.I. = 0.29 – 6.43), tables(3,4). c.5263 A>T was presented with two alleles (A and T) and three genotypes (AA, AT and TT). These genotypes were in agreement with HWE in BSS patients and controls as there was non- significant difference between the observed and expected frequencies of the genotype. Comparing BSS patients to control revealed that frequencies of AA genotype (90 % vs.
95%) and AT (10% vs. 5%). The frequencies of these genotypes were non-significant difference (p higher than 0.05) in comparison between control and BSS patient in the Fisher's exact test. The TT genotype was not observed in BSS and in control. The frequency of allele (A) in BSS patients compared with control individuals was (95 vs. 97.5%), while the frequency of mutant allele (T) was (5 vs. 2.5) in BSS patients and control respectively. The frequency of alleles were non- significant difference in comparison between BSS and control (p higher than 0.05),table (3).
c.6040 T>G was presented with two alleles (T and G) and three genotypes (TT, TG and GG).
These genotypes showed deviation from HWE in BSS patients. The TTgenotype showed a decreased frequency in BSS patients compared to controls (85 vs. 100%). The GG show non- significant difference between BSS and healthy subject in the two- tailed Fisher’s exact test (p >
0.05), the frequency of GG genotype increased in BSS (15% vs.0.0%), GG considered as a risk factor in BSS patients(OR=8.2). The heterozygote genotype (TG) was not observed in patients and healthy individuals. The mutant allele (G) show a significant difference between BSS patients and control (p ≤ 0.05) with frequency (15 vs. 0%) and consider risk factor associated with BSS patients (OR=15.2), tables(3,4).
Figure 1.Chromatograms of c.6040 T>G variant in GP1BA gene
Table 3. Genotype/allele frequencies for BSS patients and control Control (20) BSS (20)
HWE P value expected
N(%) observed
N(%) HWE
P value expected N(%)
observed N(%) GP1BA
gene SNP
NS 20
0 0 ND ND 20(100)
0 (0) 0 (0) 40(100)
0(0) 0.001
72.25))14.45 5.1(25.5) 0.45(2.25)
ND ND 17(85
0 (0) 3(15) 34(85)
6(15) ,c.6040
TAC>GAC TT TG GG T G
NS 19.01(95.05)
0,97(4.85) 0.01(0.05)
ND ND 19(95)
1(5) 0(0) 39(97.5)
1(2.5) NS
18.05(90.25) 1.9 (9.5) 0.05(0.25)
ND ND 18(90)
2(10) 0(0) 38(95)
2(5) ,c.5263
A>T, rs81663
AA AT TT A T
0.005 17.1(85.5)
2.77(13.85) 0.11(0.55)
ND ND 18(90)
1(5) 1(5) 37(92.5)
3(7.5) 0.05
16.2(81) 3.6(18)
0.2(1) ND ND 17(85)
2(10) 1(5) 36(90)
4(10) ,c.482 C >
T, rs6065 CC CT TT C T
HWE=Hardy-Weinberg equilibrium, ND=Not determined, N: Number, NS=Non- significant.
Table 4. Epidemiological parameters for BSS patients
95% CI p-
value PF
EF OR
GP1BA gene polymorphism
0.43-157 0.86 - 270 0.23
0.02 0.04
0.02 0.14
0.14 8.2
15 c.6040 T > G (GG)
(G)
0.19- 23.80 0.18 - 22.8 1
1 0.11
0.05 0.05
0.02 2.11
2.05 c.5263 A>T, rs81663 (AT)
(T)
0.19-23.8 0.06-15.9 0.29 - 6.43 1
1 1 0.11
0.13 0.1 0.05
0 0.02 2.11
1 1.37 c.482 C > T, rs6065 (CT)
(TT) (T)
OR: Odds ratio, EF: Etiological Fraction, PF: Preventive Fraction, Cl: Confidence interval.
Discussion
Direct sequence analysis revealed that there are three mutations in the GP1BA gene.The first c.482 C > T (rs6065) is a missense variant in exon 2 of GP1BAgene, leading to substitution of
amino acid from polar (threonine) to hydrophobic (methionine) at position161. Thr161Met dimorphism affects the conformation of the N-terminal flanking region and leucine-rich repeats (LRR) of GPIbα. The vWF binding region is found in the LRR (19-281 aa) of the extracellular domain of GPIbα and by this (Thr161Met) influences vWF binding [9]. c.5263 A>T(rs81663) is located in intron 1 of GP1BA gene. Intronic variant although not changing the predicted protein sequence, but have an important effect on gene expression by several ways, for example can impact alternative splicing by interfering with splice site recognition and can significantly affect gene expression[10]. Intron can increase transcript level by influencing the rate of transcription, transcripts stability and nuclear export. Furthermore, introns can improve the efficiency of mRNA translation [11].
c.6040 T>G is a novel missense variant, located in exon 2 of GP1BA gene, lead to substitution of neutral amino acid (tyrosine) to acidic amino acid (aspartic acid) at position 244 of the GPIbα subunit. A nucleotide variation that causes an amino acid replacement can alter the tertiary structure of the GPIb-IX-V complex and possibly change platelet adhesive functions[12]. c.6040 T>G substitution located in the leucine-rich repeat C-terminal region (221-281 aa) of GPIbα subunit[13]. The LRR sequences are necessary for stability of four subunits and suitable configuration of the GPIb-IX-V receptor and involved vWF binding site, therefore mutations of conserved leucine residues are especially deleterious. The variations within the LRR resulted in a more substantial alteration in the formation of GPIbα as well as a significant disturbance in the binding of ligands [14,15].
Conclusion
Genetic analysis revealed a various GP1BA gene alterations in BSS patients. Identification of mutations is critical for genetic diagnosis of the affected families in populations such as Iraq where consanguineous marriage is prevalent.
References
[1] Macêdo, M. B., Brito, Jde M., Macêdo, Pda S., & Brito, J. A. (2015). Primigravida with Bernard–Soulier syndrome: A case report. BMC Research Notes, 8(1), 178.
doi:10.1186/s13104-015-1145-5
[2] Mhawech, P., & Saleem, A. (2000). Inherited giant platelet disorders. Classification and literature review. American Journal of Clinical Pathology, 113(2), 176–190.
doi:10.1309/FC4H-LM5V-VCW8-DNJU
[3] Savoia, A., Balduini, C. L., Savino, M., Noris, P., Del Vecchio, M., Perrotta, S., . . . Iolascon, A. (2001). Autosomal dominant macrothrombocytopenia in Italy is most frequently a type of heterozygous Bernard–Soulier syndrome. Blood, The Journal of the American Society of Hematology. Blood, 97(5), 1330–1335. doi:10.1182/blood.v97.5.1330
[4] Hadjkacem, B., Gargouri, J., & Gargouri, A. (2011). Bernard Soulier syndrome: A genetic bleeding disorder. Advances in the study of genetic disorders, p. 393.
[5] Savoia, A., Pastore, A., De Rocco, D., Civaschi, E., Di Stazio, M., Bottega, R. (2011).
Clinical and genetic aspects of Bernard-Soulier syndrome: Searching for genotype/phenotype correlations. Haematologica, 96(3), 417–423. doi:10.3324/haematol.2010.032631
[6] Drouin, J., Carson, N. L., & Laneuville, O. (2005). Compound heterozygosity for a novel nine‐ nucleotide deletion and the Asn45Ser missense mutation in the glycoprotein IX gene in a patient with Bernard‐ Soulier syndrome. American Journal of Hematology, 78(1), 41–48.
doi:10.1002/ajh.20236
[7] Lanza, F. (2006). Bernard–Soulier syndrome (hemorrhagiparous thrombocytic dystrophy). Orphanet Journal of Rare Diseases, 1(1), 46. doi:10.1186/1750-1172-1-46
[8] Afrasiabi, A., Lecchi, A., Artoni, A., Karimi, M., Ashouri, E., Peyvandi, F., & Mannucci, P.
M. (2007). Genetic characterization of patients with Bernard–Soulier syndrome and their relatives from Southern Iran. Platelets, 18(6), 409–413. doi:10.1080/09537100701191323 [9] Ulrichts, H., Vanhoorelbeke, K., Cauwenberghs, S., Vauterin, S., Kroll, H., Santoso, S., &
Deckmyn, H. (2003). Von Willebrand factor but not α-thrombin binding to platelet glycoprotein Ibα is influenced by the HPA-2 polymorphism. Arteriosclerosis, Thrombosis, and Vascular Biology, 23(7), 1302–1307. doi:10.1161/01.ATV.0000079510.23517.43
[10] Rigau, M., Juan, D., Valencia, A., & Rico, D. (2019). Intronic CNVs and gene expression variation in human populations. PLOS Genetics, 15(1), e1007902.
doi:10.1371/journal.pgen.1007902
[11] Shaul, O. (2017). How introns enhance gene expression. International Journal of Biochemistry and Cell Biology, 91(B), 145–155. doi:10.1016/j.biocel.2017.06.016
[12] Reiner, A. P., Siscovick, D. S., & Rosendaal, F. R. (2001). Platelet glycoprotein gene polymorphisms and risk of thrombosis: Facts and fancies. Reviews in Clinical and Experimental Hematology, 5(3), 262–87; discussion 311. doi:10.1046/j.1468- 0734.2001.00045.x
[13] Chang, W. A., Sheu, C. C., Liu, K. T., Shen, J. H., Yen, M. C., & Kuo, P. L. (2018).
Identification of mutations in SLC4A1, GP1BA and HFE in a family with venous thrombosis of unknown cause by next-generation sequencing. Experimental and Therapeutic Medicine, 16(5), 4172–4180. doi:10.3892/etm.2018.6693
[14] Shen, Y., Romo, G. M., Dong, J. F., Schade, A., McIntire, L. V., Kenny, D. (2000).
Requirement of leucine-rich repeats of glycoprotein (GP) Ibα for shear-dependent and static binding of von Willebrand factor to the platelet membrane GP Ib–IX-V complex. Blood, The Journal of the American Society of Hematology. Blood, 95(3), 903–910. doi:10.1182/blood.V95.3.903.003k37_903_910
[15] Cauwenberghs, N., Vanhoorelbeke, K., Vauterin, S., & Deckmyn, H. (2000). Structural determinants within platelet glycoprotein Ibα involved in its binding to von Willebrand factor. Platelets, 11(7), 373–378. doi:10.1080/09537100020019157
