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Differential expression of genes involved in Bengal macrothrombocytopenia (BMTCP)
Blood Cells, Molecules and Diseases 55 (2015) 410–414
Contents lists available at ScienceDirect
Blood Cells, Molecules and Diseases journal homepage: www.elsevier.com/locate/bcmd
Differential expression of genes involved in Bengal macrothrombocytopenia (BMTCP) Shahnaz Ali ⁎, Kanjaksha Ghosh, Shrimati Shetty National Institute of Immunohaematology (ICMR), 13th Floor, KEM Hospital, Parel, Mumbai 400 012, India
a r t i c l e
i n f o
Article history: Submitted 7 August 2015 Revised 18 September 2015 Accepted 18 September 2015 Available online 21 September 2015 Editor: Mohandas Narla Keywords: Bengal macrothrombocytopenia Suppressive subtractive hybridization Gene expression qRT-PCR GP1b/IX/V complex
a b s t r a c t Bengal macrothrombocytopenia (BMTCP) is a giant platelet disorder with mild to moderate thrombocytopenia, clinically characterized by mild bleeding symptoms to totally asymptomatic condition. The pathophysiological mechanism of this condition is not fully understood yet. In the present study, 5 subjects (P1–P5) with BMTCP whose platelet counts ranged between 36140X 109/l and mean platelet volume (MPV)13.5–16.1 fl were analyzed for differential gene expression of platelets by suppressive subtractive hybridization (SSH) technique. Four genes i.e. myotubularin related protein 9 (MTMR9), iron responsive element binding protein 2 (IREB2), alpha tubulin(TUBA) and tyrosine kinase ligand (TKL) were found to be differentially expressed in patient platelets as compared to that of normal healthy controls which was further confirmed by quantitative RT PCR analysis. The study highlights a multi-factorial etiology for BMTCP which is widely prevalent in the northeastern region of the Indian subcontinent. © 2015 Elsevier Inc. All rights reserved.
1. Introduction
2. Material and methods
Bleeding syndromes that arise through an inherited defect of platelet production and giant platelets constitute a heterogeneous group of rare platelet disorders [1,2]. These include BernardSoulier Syndrome (BSS) [3], an autosomal recessive bleeding disorder caused by a defect in the platelet glycoprotein GP1b/IX/V complex, MYH9 related disorders (MYH9-RD) [4,5] an autosomal dominant disorder caused by mutations of MYH9 gene and Mediterranean macrothrombocytopenia (MM) which are generally asymptomatic [6]. Sitosterolemia/phytosterolemia [7,8] caused by mutations either in ABCG5 and ABCG8 genes encoding the ATP-binding cassette protein called Sterolin [9,10] and a rare X linked GATA-1 associated MTCP which is associated with dyserythropoiesis [11] and finally asymptomatic MTCP reported in healthy donors from West Bengal [12,13]. Recent progress in the elucidation of the responsible genes for several inherited MTCPs led to advances in understanding the pathophysiology and pathogenesis of the disease, however, in approximately half of the cases with inherited MTCP, the molecular cause remains unknown, but it is known that multiple etiologies do exist for MTCP. In the present report, suppressive subtractive hybridization (SSH) [14] and quantitative RT-PCR techniques were applied to know the differential expression of genes in the patient's platelets as compared to normal healthy controls.
2.1. Patients
⁎ Corresponding author. E-mail address: [email protected] (S. Ali).
http://dx.doi.org/10.1016/j.bcmd.2015.09.005 1079-9796/© 2015 Elsevier Inc. All rights reserved.
We analyzed 10 patients (4 females and 6 males; age range 10–49 years), diagnosed with macrothrombocytopenia from unrelated families from India (Table 1). Only 5 patients were recruited for the SSH study (P1–P5) along with the controls. The study was approved by the Institute Ethical Committee. A written informed consent was obtained from all the patients and controls prior to the study. Diagnosis of BMTCP was confirmed by the following laboratory parameters — giant platelets without neutrophil inclusions with the count reduced on peripheral smear (Fig. 1), increased mean platelet volume (MPV) as seen in an automated counter (XT-2000i, Transasia Bio-Medical Ltd., India), normal prothrombin time (PT) and activated partial thromboplastin time (APTT), normal platelet aggregation with agonists ristocetin (normal dose: 1.5 mg/ml, low dose; 0.5 mg/ml), adenosine diphosphate (ADP) (5 μM), arachidonic acid (AA) (20 μM) and collagen (2 μg/mL), normal glycoprotein receptor study using fluorescein isothiocyanate (FITC) conjugated monoclonal antibody against GPIb (CD42b), GPIX (CD42b) and PE conjugated antibody against GPIIb/IIIa (CD41a) with appropriate IgG1 and IgG2a isotypic controls (BD Pharmingen TM, San Jose, CA, USA). Screening for anti platelet antibodies was negative in all the cases. The bleeding tendency was measured according to the World Health Organization (WHO) bleeding scale (grade 0, no bleeding; grade 1, petechiae; grade 2, mild blood loss; grade 3, gross blood loss and grade 4, debilitating blood loss.
S. Ali et al. / Blood Cells, Molecules and Diseases 55 (2015) 410–414
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Table 1 Clinical manifestations and platelet cell indices in patients from West Bengal, India. Sample no.
Patient ID
AOD/ sex
Platelet count (× 109/l) NR: 150–450
Mean platelet volume (fl) NR: 7–10
Peripheral blood smear
WHO Bleeding Scale
Platelet aggregometry (ADP, Risto, AA, Coll, APA)
Platelet receptor study (GPIb, GPIX GPIIb/IIIa)
1 2 3 4 5 6 7 8 9 10
BM34.01 (P1) BM35.01 (P2) BM36.01 (P3) BM47.01 (P4) BM48.01 (P5) BM101 (P6) BM102 (P7) BM103 (P8) BM104 (P9) BM105 (P10)
10/F 31/M 28/M 24/F 21/M 41/M 29/M 49/M 32/F 35/F
36 71 41 115 135 140 110 116 41 83
13.6 13.9 14.1 13.5 14.6 14.4 14.7 13.5 16 16.1
Giant platelets Giant platelets Giant platelets Giant platelets Giant platelets Giant platelets Giant platelets Giant platelets Giant platelets Giant Platelets
3 0 3 2 1 0 0 0 2 1
Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal
Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal
WHO Bleeding Scale: 0, no bleeding; 1, petechiae; 2, mild blood loss; 3, gross blood loss, ADP: adenosine diphosphate, Risto: ristocetin, AA: arachidonic acid, Coll: collagen, APA: anti-platelet antibody, AOD: age of diagnosis; NR: normal range.
2.2. Suppressive subtractive hybridization (SSH) 2.2.1. cDNA synthesis primer Prl16, 5′-TTTTGTACAAGCTT30-3′ was used for first strand CDNA synthesis. Adapter 1 (5′-GTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAGGT-3′) Adapter 2 (5′-TGTAGCGTGAAGACGACAGAAAGGGCGTGGTGCGGAGGGCGGT-3′) PCR primers: P1: 5′-GTAATACGACTCACTATAGGGC-3′. P2: 5′-TGTAGCGTGAAGACGACAGAA-3′. PN1: 5′-TCGAGCGGCCGCCCGGGCAGGT-3′. PN2: 5′-AGCGTGGTCGCGGCCGAGGT-3′. The primers and adaptors were obtained from Clonetech Laboratories Inc., USA. Total platelet mRNA was isolated from the patients and the control blood sample respectively using TRIzol [15] using platelet concentrate. SSH was performed by using PCR-Select TM cDNA subtraction
kit (Clonetech Laboratories Inc., USA) according to the manufacturer's protocol, the cDNA pool prepared from the affected individuals was used as the tester and the driver was the cDNA pool prepared from the normal individuals in the forward hybridization and vice versa in the reverse hybridization. 5 μg (10 μl) of total platelet RNA template and 0.5 μg (1 μl) of Prl16 primer were mixed and incubated at 65 °C for 15 min and quick chilled on ice for 5 min, the final reaction volume was made up to 50 μl by adding 5 μl of DTT (0.1 M), 2.5 μl of dNTP mix (2.5 mM each), 5 μl 10× RT buffer and 0.25 μl RNasin (40 U/Ul) and 22.25 μl of sterile water. Reverse transcription (RT) was done by adding 2 μl of AMV reverse transcriptase (40 U/Ul), the reaction was incubated at 42 °C for 1 h followed by heat inactivation of the RT reaction at 94 °C for 5 min and snap chilled on ice to synthesize the first strand cDNA. The second strand cDNA synthesis was done immediately by adding 20 μl of first strand cDNA, 10 μl of second strand buffer, 4 μl of dNTP mix (2.5 mM each) and 2 μl(5 U/μl) of second strand enzyme cocktail to final volume of 100 μl with sterile water, the reaction mix was incubated at 37 °C for
Fig. 1. Leishman's stained peripheral blood smears of BMTCP cases: (A) BM34.01, (B) BM35.01, (C) BM36.01, (D) BM47.01, (E) BM48.01, and (F) BM101.01.
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2 h followed by heat inactivation at 70 °C for 10 min and immediately placed on ice, 10 U of T4 DNA polymerase was added and the reaction mix was incubated at room temperature for 5 min and was stopped by heating at 70 °C for 10 min, the final product was purified using PCR clean up kit (Chromous Biotech, Bangalore, India). The ds cDNA from the tester and driver was digested with Rsa1 at 37 °C for 2 h and the digested cDNA products were ligated to adapter 1 and adapter 2 in separate ligation reactions with 250 ng of cDNA, 4 μl of 5× ligase assay buffer, 2 μl of each adapter (10 μM), 1 μl of T4 DNA ligase buffer and sterile water to a final volume of 20 μl, the reaction mix was incubated at 16 °C for overnight followed by heating at 70 °C for 5 min to inactivate the ligase, the adapter ligated cDNA was purified with PCR clean up kit. 600 ng of digested driver ds cDNA was added to each tube containing 20 ng of tester ds cDNA ligated with adapter 1 and adapter 2. The samples were mixed with 1× hybridization buffer, was denatured at 98 °C for 2 min and allowed to anneal for 10 h at 68 °C (1st hybridization), the samples from the first hybridization were then mixed and combined with heat denatured 150 ng of driver ds cDNA and allowed to hybridize for 10 h at 68 °C. The hybridization mix was diluted 1:10 times for PCR amplification. The PCR amplification was performed with the following parameters: at 94 °C for 2 min, (94 °C for 30 s, 55 °C for 30 s, 72 °C for 1 min) × 35 cycles, 72 °C for 1 min. The products were examined by electrophoresis on a 1.5% ethidium bromide gel. The PCR products were purified and their concentration was measured by a spectrophotometer. The T/A cloning(pUC cloning vector) was done by ligating the PCR products to HindIII adapters (2 μl) with 1 μl of T/A cloning vector (50 ng/μl), 10 μl of 2× ligase assay buffer, 1ul of quick ligase buffer and water to final volume of 20 μl. The mixture was incubated at 4 °C overnight. The recombinant plasmids were transformed into highly efficient DH5alpha competent cells. A 100 μl of transformants was grown into 8 cm × 8 cm agar plates containing 100 μg/ml ampicilin, 100 μmol/l LIPTG and 100 μg/ml X-gal at 37 °C for 20 h when the blue/ white staining could be clearly distinguished. White clones were counted, inoculated into 3 ml of LB liquid medium containing ampicilin and shaken overnight at 37 °C, the cDNA plates were screened to obtain 500 positive clones from the library and used directly as PCR templates to amplify the inserts in 50 μl reaction volume containing 5 μl of 10 × buffer, 3 μl of 25 mM Mgcl2, 1 μl of primers PN1 and PN2, 1 μl of 25 mM dNTPs mix, 0.5 μl of taq enzyme (5 U/μl) and 0.5 μl of bacterial culture. PCR consisted of an initial denaturation at 94°C for 5 min, followed by 25 cycles at 94 °C for 30 s, at 68 °C for 30 s and at 72 °C for 2 min. The PCR products were analyzed in 2% ethidium bromide agarose gel. The cDNA sequences were edited by the help of Seq Scape_v5.2 software to remove the vector and adaptor sequences, cDNA sequences were prepared in FASTA format in text file and batch subjected to the web application Megablast (http://blast.ncbi.nlm.nh.gov/Blast.cgi) available online for analysis.
2.2.2. Quantitative real time PCR (qRT-PCR) analysis qRT-PCR was done to confirm the differential expression of selected clones identified by SSH. From the total population of clones we selected 4 genes depending on the occurrence of gene fragments in the SSH library for analysis: iron responsive element binding protein 2 (IREB2), Myotubularin related protein 9 (MTMR9) gene fragments were abundant in the SSH library, alpha tubulin (TUBA) and tyrosine kinase ligand (TKL) gene was selected for its involvement in the biology of platelet pathophysiology. Total RNA from each sample was used to create first strand cDNA synthesis by using OligodT primers, PCR was performed using TaqMan gene expression assay(Life Technologies, USA) for the respective gene primers and house-keeping human beta-actin gene primers, the reaction was run on ABI StepOne Real time PCR. The qRTPCR experiment was done in all the patients (P1–P10) and 5 controls from the same ethnic region of India i.e. West Bengal.
3. Results There was no history of consanguinity among the parents; one of the parents carried the same phenotype suggesting dominant mode of inheritance. SSH was used to isolate differentially expressed genes from BMTCP cases as compared to the normal controls. SSH library was represented by differentially expressed genes in BMTCP cases which are listed in Table 2. Fig. 2 represents the frequency of the gene fragments as obtained from the SSH library. Randomly chosen subsets of the up- and downregulated clones were sequenced; good quality sequences were aligned with MEGABLAST or BLAT. The sequences were deposited with the dbEST database (NCBI). Of the identified gene fragments, the sequences of certain gene fragments were repeated more than others, these genes (Table 2 and Fig. 2) are probably the most abundant genes in the obtained SSH cDNA libraries. It was previously shown that the frequency of a gene in the SSH library was correlated with the degree of the differential expression of the gene [16]. To verify the results of the SSH 4 genes were selected based on the occurrence of gene fragments/clones (abundant for MTMR9, IREB2) in the SSH Library (Table 2), TUBA and TKL for its role in platelet cytoskeleton structuring, verified their expression in all the samples. The quantitative RT-PCR was used to compare expression levels of selected genes in patients with low platelet count and giant platelets with normal controls with normal platelet count and normal platelet size. The expression levels of the selected genes were consistent with the expression profiles obtained SSH in all patients. alpha tubulin (TUBA), IREB2, MTMR9 and tyrosine kinase ligand (TKL) genes were expressed more in patients than in controls (Fig. 3). 4. Discussion Macrothrombocytopenia is a heterogeneous group of disorders and was considered to be rare until the introduction of automated cell counters. The widespread use of electronic counters in the 1990s made platelet counting a routine analysis, and several new forms of mild thrombocytopenia with trivial or no bleeding diathesis has since been discovered. The molecular basis of MTCP has not been fully elucidated yet. Although the defective genes, coding for membrane glycoproteins, cytoskeleton components and intracellular signaling pathways, as well as transcription factors have been identified in most cases, the pathophysiology of these disorders is often unknown. One of the most important ways to understand the pathology of MTCP is to clone and identify the differentially expressed genes. In recent years several cloning techniques for the differentially expressed genes have been established, including differential display PCR (DDPCR), representational difference analysis (RDA), SSH and cDNA microarray. Until now, these techniques have been widely applied in the study of pathology [17], immune response [18], embryo development [19] as well as the specific expression of tissue proteins [20]. SSH was first put forward by Diatchenko et al. in 1996 [21]. The basic principle is that common cDNAs in the paired materials are subtracted by subtractive hybridization and then suppressive PCR is carried out to amplify cDNA fragments specially expressed in the tester. The advantage of SSH was the design of two different adaptors and the introduction of suppression PCR with the result that the differentially expressed cDNA fragments were amplified. It allows for two subtractive hybridizations in the forward or reverse direction. Some mRNA expressed with low abundance could be detected which provided clues for further gene sequencing and identification. With the development and improvement of SSH technique, it has become one of the most effective techniques for cloning differentially expressed genes. Until now, no SSH studies have been done on MTCP patients. It is the most popular and most cost effective method to study gene expression in specific tissues or cell types over standard microarray, which is cost prohibitive for some investigators and requires large sample sizes for generating good quality data.
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Table 2 SSH library data from the patients from West Bengal, India. Description
No. of clones/gene fragments
Gene function
Accession no.
Homo sapiens proteasome Homo sapiens iron-responsive element binding protein 2 Homo sapiens tyrosine 3-monooxygenase/tryptophan 5-momooxygenase activation protein 18S ribosomal RNA gene Homo sapiens mRNA Homo sapiens mRNA Homo sapiens Kelch-like 5 protein (KLHL5) mRNA Phytophthora cinnamomi tub1 gene for alpha-tubulin Gamma glutamyltransferase 1 (Homo sapiens) Tyrosine kinase ligand (TKL) Transmembrane channel like-2 Vertebrate kinesin family Vertebrate myotubularin related protein 9 (MTMR9) Carcharhinus plumbeus T cell receptor gamma (TCRG) gene Human s-adenosylmethionine decarboxylase 1 Homo sapiens cDNA Dimethyl sulfoxide reductase Potassium channel modulatory factor 1 RNA binding motif protein 39b Human OGN gene
10 23 2 1 42 26 1 6 1 1 1 1 205 1 1 29 1 7 1 1
Proteasome Iron binding Tyrosine activation ribosomal Human cDNA-2 Human cDNA-3 Kelch like protein Alpha tubulin GGT Tyrosine kinase Transmembrane protien Kinesin family Myotubularin protein T-cell receptor SAM Human cDNA-4 Reductase Potassium channel protein RNA binding protein OGN gene
BC056249.1 DQ496102.1 BC001197.1 GU968788.1 CR933672.1 CR749596.1 AF272976 AM412177.1 AL832738.1 Y09669.1 BX571945.15 BX649292.16 BX510941.5 FJ854492.1 AK31043.1 AP012030.1 BX294395.15 CR550308.6 AL137848.5
Bold data indicates significant at Genes selected for qRT-PCR.
In the present study, SSH and bacterial culture PCR were performed to construct a subtracted cDNA library specific for the BMTCP patients from West Bengal, India. Both forward and reverse hybridizations was performed using the cDNA pool prepared from the affected individuals, which was labeled as the ‘Tester’ and cDNA pool prepared from the unaffected individuals as the ‘Driver’. About five hundred clones were obtained from this cDNA library and bacterial culture PCR was used to screen the positive recombinants. These were then subjected to DNA sequencing analysis and 20 genes were found to express differentially in affected platelets compared with the normal platelets (Table 2 & Fig. 2). The expression profiles of 4 of the 20 genes were further confirmed by quantitative reverse transcription polymerase reaction (Fig. 3). The construction of this library and further analysis by qRT-PCR is therefore of significance in screening and identifying new genes associated with MTCP which would be beneficial to the elucidation of the molecular mechanism/pathology of inherited MTCP.
The myotubularins are a family of inositol polyphosphate 3phosphatases consisting of 16 different catalytically active and inactive proteins. They form complexes among themselves which increases the catalytic activity of these enzymes and also changes specificity for their respective substrates [22]. Though it is an inactive protein, yet other active myotubularin related proteins form dimers with this inactive protein which increases their enzymatic activity. Besides their role in membrane trafficking, they are also known to have a role in cell differentiation and cytoskeletal dynamics. Both the membrane and cytoplasmic cytoskeletal components of the platelet are regulated by intracellular enzymes, one such enzyme being tyrosine kinase. The activation takes place when the platelet glycoprotein receptors bind fibrinogen and other ligands [23]. The discoid shape of the resting platelets is maintained by multiple coils of tubulin polymers and they disappear during platelet activation, which results in the platelet shape change. The regulation of this process is not
Fig. 2. Profiles of differentially expressed genes in Bengal macrothrombocytopenia patients.
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Fig. 3. mRNA expression plot for different genes by qRT-PCR. Results shows that expression of MTMR9, IREB2, TUBA and TKL gene among different affected individuals (P1–P10) has increased when compared to unaffected control samples (C1–C5).
clear; however, presence of lys at position 40 of alpha tubulin seems to have an important role in maintaining the stability of microtubules [24]. IREB2 being an iron-responsive element is regulated mainly by iron and oxygen. The association of platelet cytoskeletal abnormalities with IREB2 is however not clear. The up-regulation of these genes might be involved in shaping the platelets and production of low platelet counts in a particular ethnic region which needs further study. Thus in conclusion, the study provides a list of candidate genes which provide new clues in understanding the molecular mechanisms involved in the pathogenesis of this disorder. Further characterization of the identified genes will provide useful information in understanding the pathophysiology of the disorder. Conflict of interest statement None. Acknowledgments We thank Chromous Biotech Pvt. Ltd. Bangalore, India for their technical support. References [1] J.G. Drachman, Inherited thrombocytopenia: when a low platelet count does not mean ITP, Blood 103 (2004) 390–398. [2] A.E. Geddis, K. Kaushanky, Inherited thrombocytopenias: towards a molecular understanding of disorders of platelet production, Curr. Opin. Pediatr. 16 (2004) 15–22. [3] J. Bernard, J.P. Soulier, Sur une nouvelle variete de dystrophie thrombocytairehemorragipare congenitale, Sem. Hôpitaux Paris 24 (1948) 3217–3223. [4] B. Rocca, F.O. Ranelletti, N. Maggiano, et al., Inherited macrothrombocytopenia with distinctive platelet ultra-structure and functional features, Thromb. Haemost. 83 (2000) 35–41. [5] M. Seri, A. Pecci, F. Di, et al., MYH9 related diseases: May–Hegglin anomaly, Sebastian syndrome, Fechtner syndrome and Epstein syndrome are not distinct entities but represent a variable expression of a single illness, Medicine (Baltimore) 82 (2003) 203–215. [6] W.E. Behrens, Mediterranean macrothrombocytopenia, Blood 46 (1975) 199–208.
[7] A.K. Bhattacharya, W.E. Connor, Beta-sitosteroleamia and xanthomatosis, A newly described lipid storage disease in two sisters, J. Clin. Investig. 53 (1974) 1033–1043. [8] D.C. Rees, M. Iolascon, S. Carella, et al., Stomatocytic haemolysis and macrothrombocytopenia Mediterranean stomatocytosis/macrothrombocytopenia) is the haematological presentation of phytosterolemia, Br. J. Haematol. 130 (2005) 297–309. [9] S.B. Patel, G. Salen, H. Hidaka, et al., The sitosterolemia locus is found at chromosome 2p21, J. Clin. Investig. 102 (1998) 1041–1044. [10] K.E. Berge, H. Tian, G.A. Graf, et al., Accumulation of dietary cholesterol in sitosterolemia caused by mutations in ABC transporters, Science 290 (2000) 1771–1775. [11] K.E. Nichols, J.D. Crispino, M. Poncz, et al., Familial dyserythropoietic anaemia and thrombo-cytopenia due to an inherited mutation in GATA-1, Nat. Genet. 24 (2000) 266–270. [12] H.V. Naina, S.C. Nair, D. Danial, et al., Asymptomatic constitutional macrothrombocytopenia among West Bengal donors, Am. J. Med. 112 (2002) 742–743. [13] H.V. Naina, S.C. Nair, S. Harris, et al., Harris syndrome — a geographic perspective, J. Thromb. Haemost. 3 (2005) 2581–2582. [14] L. Diatchenko, S. Lukyanov, Y.F. Lau, et al., Suppression subtractive hybridization: a versatile method for identifying differential expressed genes, Methods Enzymol. 303 (1999) 349–380. [15] P. Chomczynsky, N. Sacchi, Single-step method of RNA isolation by acid guanidinium thiocyanate-phenolchloroform extraction, Anal. Biochem. 162 (1987) 156–159. [16] L. Diatchenko, S. Romanov, I. Malinina, et al., Identification of novel mediators of NFkappaB through genome-wide survey of monocyte adherence-induced genes, J. Leukoc. Biol. 78 (2005) 1366–1377. [17] R. Sharma, S. Samantaray, N.K. Shukla, et al., Transcriptional gene expression profile of human esophageal squamous cell carcinoma, Genomics 81 (2003) 481–488. [18] J.L. Dunphy, A. Balic, G.J. Barcham, et al., Isolation and characterization of a novel inducible mammalin galectin, J. Biophys. Chem. 275 (2000) 32106–32113. [19] P. Li, J.L. Li, R. Hou, et al., Changes in gene expression profile of the left heart ventricle during growth in the rat, Sheng Li Xue Bao 55 (2003) 191–196. [20] H. Ito, H. Akiyama, H. Iguchi, et al., Molecular cloning and biological activity of a novel lysyl oxidase-related gene expressed in cartilage, J. Biol. Chem. 276 (2001) 24023–24029. [21] L. Diatchenko, L.Y.F. Lau, A.P. Campbell, et al., Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries, Proc. Natl. Acad. Sci. 93 (12) (1996) 6025–6030. [22] J. Zou, C. Zhang, J. Marjanovic, et al., Myotubularin-related protein (MTMR) 9 determines the enzymatic activity, substrate specificity, and role in autophagy of MTMR8, Proc. Natl. Acad. Sci. 109 (24) (2012) 9539–9544. [23] L.L. Stice, C. Vaziri, D.V. Faller, Regulation of platelet-derived growth factor signaling by activated p21Ras, Front. Biosci. 4 (1999) D72–D86. [24] C. Janke, J.C. Bulinski, Post-translational regulation of the microtubule cytoskeleton: mechanisms and functions, Nat. Rev. Mol. Cell Biol. 12 (2011) 773–786.
Contents lists available at ScienceDirect
Blood Cells, Molecules and Diseases journal homepage: www.elsevier.com/locate/bcmd
Differential expression of genes involved in Bengal macrothrombocytopenia (BMTCP) Shahnaz Ali ⁎, Kanjaksha Ghosh, Shrimati Shetty National Institute of Immunohaematology (ICMR), 13th Floor, KEM Hospital, Parel, Mumbai 400 012, India
a r t i c l e
i n f o
Article history: Submitted 7 August 2015 Revised 18 September 2015 Accepted 18 September 2015 Available online 21 September 2015 Editor: Mohandas Narla Keywords: Bengal macrothrombocytopenia Suppressive subtractive hybridization Gene expression qRT-PCR GP1b/IX/V complex
a b s t r a c t Bengal macrothrombocytopenia (BMTCP) is a giant platelet disorder with mild to moderate thrombocytopenia, clinically characterized by mild bleeding symptoms to totally asymptomatic condition. The pathophysiological mechanism of this condition is not fully understood yet. In the present study, 5 subjects (P1–P5) with BMTCP whose platelet counts ranged between 36140X 109/l and mean platelet volume (MPV)13.5–16.1 fl were analyzed for differential gene expression of platelets by suppressive subtractive hybridization (SSH) technique. Four genes i.e. myotubularin related protein 9 (MTMR9), iron responsive element binding protein 2 (IREB2), alpha tubulin(TUBA) and tyrosine kinase ligand (TKL) were found to be differentially expressed in patient platelets as compared to that of normal healthy controls which was further confirmed by quantitative RT PCR analysis. The study highlights a multi-factorial etiology for BMTCP which is widely prevalent in the northeastern region of the Indian subcontinent. © 2015 Elsevier Inc. All rights reserved.
1. Introduction
2. Material and methods
Bleeding syndromes that arise through an inherited defect of platelet production and giant platelets constitute a heterogeneous group of rare platelet disorders [1,2]. These include BernardSoulier Syndrome (BSS) [3], an autosomal recessive bleeding disorder caused by a defect in the platelet glycoprotein GP1b/IX/V complex, MYH9 related disorders (MYH9-RD) [4,5] an autosomal dominant disorder caused by mutations of MYH9 gene and Mediterranean macrothrombocytopenia (MM) which are generally asymptomatic [6]. Sitosterolemia/phytosterolemia [7,8] caused by mutations either in ABCG5 and ABCG8 genes encoding the ATP-binding cassette protein called Sterolin [9,10] and a rare X linked GATA-1 associated MTCP which is associated with dyserythropoiesis [11] and finally asymptomatic MTCP reported in healthy donors from West Bengal [12,13]. Recent progress in the elucidation of the responsible genes for several inherited MTCPs led to advances in understanding the pathophysiology and pathogenesis of the disease, however, in approximately half of the cases with inherited MTCP, the molecular cause remains unknown, but it is known that multiple etiologies do exist for MTCP. In the present report, suppressive subtractive hybridization (SSH) [14] and quantitative RT-PCR techniques were applied to know the differential expression of genes in the patient's platelets as compared to normal healthy controls.
2.1. Patients
⁎ Corresponding author. E-mail address: [email protected] (S. Ali).
http://dx.doi.org/10.1016/j.bcmd.2015.09.005 1079-9796/© 2015 Elsevier Inc. All rights reserved.
We analyzed 10 patients (4 females and 6 males; age range 10–49 years), diagnosed with macrothrombocytopenia from unrelated families from India (Table 1). Only 5 patients were recruited for the SSH study (P1–P5) along with the controls. The study was approved by the Institute Ethical Committee. A written informed consent was obtained from all the patients and controls prior to the study. Diagnosis of BMTCP was confirmed by the following laboratory parameters — giant platelets without neutrophil inclusions with the count reduced on peripheral smear (Fig. 1), increased mean platelet volume (MPV) as seen in an automated counter (XT-2000i, Transasia Bio-Medical Ltd., India), normal prothrombin time (PT) and activated partial thromboplastin time (APTT), normal platelet aggregation with agonists ristocetin (normal dose: 1.5 mg/ml, low dose; 0.5 mg/ml), adenosine diphosphate (ADP) (5 μM), arachidonic acid (AA) (20 μM) and collagen (2 μg/mL), normal glycoprotein receptor study using fluorescein isothiocyanate (FITC) conjugated monoclonal antibody against GPIb (CD42b), GPIX (CD42b) and PE conjugated antibody against GPIIb/IIIa (CD41a) with appropriate IgG1 and IgG2a isotypic controls (BD Pharmingen TM, San Jose, CA, USA). Screening for anti platelet antibodies was negative in all the cases. The bleeding tendency was measured according to the World Health Organization (WHO) bleeding scale (grade 0, no bleeding; grade 1, petechiae; grade 2, mild blood loss; grade 3, gross blood loss and grade 4, debilitating blood loss.
S. Ali et al. / Blood Cells, Molecules and Diseases 55 (2015) 410–414
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Table 1 Clinical manifestations and platelet cell indices in patients from West Bengal, India. Sample no.
Patient ID
AOD/ sex
Platelet count (× 109/l) NR: 150–450
Mean platelet volume (fl) NR: 7–10
Peripheral blood smear
WHO Bleeding Scale
Platelet aggregometry (ADP, Risto, AA, Coll, APA)
Platelet receptor study (GPIb, GPIX GPIIb/IIIa)
1 2 3 4 5 6 7 8 9 10
BM34.01 (P1) BM35.01 (P2) BM36.01 (P3) BM47.01 (P4) BM48.01 (P5) BM101 (P6) BM102 (P7) BM103 (P8) BM104 (P9) BM105 (P10)
10/F 31/M 28/M 24/F 21/M 41/M 29/M 49/M 32/F 35/F
36 71 41 115 135 140 110 116 41 83
13.6 13.9 14.1 13.5 14.6 14.4 14.7 13.5 16 16.1
Giant platelets Giant platelets Giant platelets Giant platelets Giant platelets Giant platelets Giant platelets Giant platelets Giant platelets Giant Platelets
3 0 3 2 1 0 0 0 2 1
Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal
Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal
WHO Bleeding Scale: 0, no bleeding; 1, petechiae; 2, mild blood loss; 3, gross blood loss, ADP: adenosine diphosphate, Risto: ristocetin, AA: arachidonic acid, Coll: collagen, APA: anti-platelet antibody, AOD: age of diagnosis; NR: normal range.
2.2. Suppressive subtractive hybridization (SSH) 2.2.1. cDNA synthesis primer Prl16, 5′-TTTTGTACAAGCTT30-3′ was used for first strand CDNA synthesis. Adapter 1 (5′-GTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAGGT-3′) Adapter 2 (5′-TGTAGCGTGAAGACGACAGAAAGGGCGTGGTGCGGAGGGCGGT-3′) PCR primers: P1: 5′-GTAATACGACTCACTATAGGGC-3′. P2: 5′-TGTAGCGTGAAGACGACAGAA-3′. PN1: 5′-TCGAGCGGCCGCCCGGGCAGGT-3′. PN2: 5′-AGCGTGGTCGCGGCCGAGGT-3′. The primers and adaptors were obtained from Clonetech Laboratories Inc., USA. Total platelet mRNA was isolated from the patients and the control blood sample respectively using TRIzol [15] using platelet concentrate. SSH was performed by using PCR-Select TM cDNA subtraction
kit (Clonetech Laboratories Inc., USA) according to the manufacturer's protocol, the cDNA pool prepared from the affected individuals was used as the tester and the driver was the cDNA pool prepared from the normal individuals in the forward hybridization and vice versa in the reverse hybridization. 5 μg (10 μl) of total platelet RNA template and 0.5 μg (1 μl) of Prl16 primer were mixed and incubated at 65 °C for 15 min and quick chilled on ice for 5 min, the final reaction volume was made up to 50 μl by adding 5 μl of DTT (0.1 M), 2.5 μl of dNTP mix (2.5 mM each), 5 μl 10× RT buffer and 0.25 μl RNasin (40 U/Ul) and 22.25 μl of sterile water. Reverse transcription (RT) was done by adding 2 μl of AMV reverse transcriptase (40 U/Ul), the reaction was incubated at 42 °C for 1 h followed by heat inactivation of the RT reaction at 94 °C for 5 min and snap chilled on ice to synthesize the first strand cDNA. The second strand cDNA synthesis was done immediately by adding 20 μl of first strand cDNA, 10 μl of second strand buffer, 4 μl of dNTP mix (2.5 mM each) and 2 μl(5 U/μl) of second strand enzyme cocktail to final volume of 100 μl with sterile water, the reaction mix was incubated at 37 °C for
Fig. 1. Leishman's stained peripheral blood smears of BMTCP cases: (A) BM34.01, (B) BM35.01, (C) BM36.01, (D) BM47.01, (E) BM48.01, and (F) BM101.01.
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2 h followed by heat inactivation at 70 °C for 10 min and immediately placed on ice, 10 U of T4 DNA polymerase was added and the reaction mix was incubated at room temperature for 5 min and was stopped by heating at 70 °C for 10 min, the final product was purified using PCR clean up kit (Chromous Biotech, Bangalore, India). The ds cDNA from the tester and driver was digested with Rsa1 at 37 °C for 2 h and the digested cDNA products were ligated to adapter 1 and adapter 2 in separate ligation reactions with 250 ng of cDNA, 4 μl of 5× ligase assay buffer, 2 μl of each adapter (10 μM), 1 μl of T4 DNA ligase buffer and sterile water to a final volume of 20 μl, the reaction mix was incubated at 16 °C for overnight followed by heating at 70 °C for 5 min to inactivate the ligase, the adapter ligated cDNA was purified with PCR clean up kit. 600 ng of digested driver ds cDNA was added to each tube containing 20 ng of tester ds cDNA ligated with adapter 1 and adapter 2. The samples were mixed with 1× hybridization buffer, was denatured at 98 °C for 2 min and allowed to anneal for 10 h at 68 °C (1st hybridization), the samples from the first hybridization were then mixed and combined with heat denatured 150 ng of driver ds cDNA and allowed to hybridize for 10 h at 68 °C. The hybridization mix was diluted 1:10 times for PCR amplification. The PCR amplification was performed with the following parameters: at 94 °C for 2 min, (94 °C for 30 s, 55 °C for 30 s, 72 °C for 1 min) × 35 cycles, 72 °C for 1 min. The products were examined by electrophoresis on a 1.5% ethidium bromide gel. The PCR products were purified and their concentration was measured by a spectrophotometer. The T/A cloning(pUC cloning vector) was done by ligating the PCR products to HindIII adapters (2 μl) with 1 μl of T/A cloning vector (50 ng/μl), 10 μl of 2× ligase assay buffer, 1ul of quick ligase buffer and water to final volume of 20 μl. The mixture was incubated at 4 °C overnight. The recombinant plasmids were transformed into highly efficient DH5alpha competent cells. A 100 μl of transformants was grown into 8 cm × 8 cm agar plates containing 100 μg/ml ampicilin, 100 μmol/l LIPTG and 100 μg/ml X-gal at 37 °C for 20 h when the blue/ white staining could be clearly distinguished. White clones were counted, inoculated into 3 ml of LB liquid medium containing ampicilin and shaken overnight at 37 °C, the cDNA plates were screened to obtain 500 positive clones from the library and used directly as PCR templates to amplify the inserts in 50 μl reaction volume containing 5 μl of 10 × buffer, 3 μl of 25 mM Mgcl2, 1 μl of primers PN1 and PN2, 1 μl of 25 mM dNTPs mix, 0.5 μl of taq enzyme (5 U/μl) and 0.5 μl of bacterial culture. PCR consisted of an initial denaturation at 94°C for 5 min, followed by 25 cycles at 94 °C for 30 s, at 68 °C for 30 s and at 72 °C for 2 min. The PCR products were analyzed in 2% ethidium bromide agarose gel. The cDNA sequences were edited by the help of Seq Scape_v5.2 software to remove the vector and adaptor sequences, cDNA sequences were prepared in FASTA format in text file and batch subjected to the web application Megablast (http://blast.ncbi.nlm.nh.gov/Blast.cgi) available online for analysis.
2.2.2. Quantitative real time PCR (qRT-PCR) analysis qRT-PCR was done to confirm the differential expression of selected clones identified by SSH. From the total population of clones we selected 4 genes depending on the occurrence of gene fragments in the SSH library for analysis: iron responsive element binding protein 2 (IREB2), Myotubularin related protein 9 (MTMR9) gene fragments were abundant in the SSH library, alpha tubulin (TUBA) and tyrosine kinase ligand (TKL) gene was selected for its involvement in the biology of platelet pathophysiology. Total RNA from each sample was used to create first strand cDNA synthesis by using OligodT primers, PCR was performed using TaqMan gene expression assay(Life Technologies, USA) for the respective gene primers and house-keeping human beta-actin gene primers, the reaction was run on ABI StepOne Real time PCR. The qRTPCR experiment was done in all the patients (P1–P10) and 5 controls from the same ethnic region of India i.e. West Bengal.
3. Results There was no history of consanguinity among the parents; one of the parents carried the same phenotype suggesting dominant mode of inheritance. SSH was used to isolate differentially expressed genes from BMTCP cases as compared to the normal controls. SSH library was represented by differentially expressed genes in BMTCP cases which are listed in Table 2. Fig. 2 represents the frequency of the gene fragments as obtained from the SSH library. Randomly chosen subsets of the up- and downregulated clones were sequenced; good quality sequences were aligned with MEGABLAST or BLAT. The sequences were deposited with the dbEST database (NCBI). Of the identified gene fragments, the sequences of certain gene fragments were repeated more than others, these genes (Table 2 and Fig. 2) are probably the most abundant genes in the obtained SSH cDNA libraries. It was previously shown that the frequency of a gene in the SSH library was correlated with the degree of the differential expression of the gene [16]. To verify the results of the SSH 4 genes were selected based on the occurrence of gene fragments/clones (abundant for MTMR9, IREB2) in the SSH Library (Table 2), TUBA and TKL for its role in platelet cytoskeleton structuring, verified their expression in all the samples. The quantitative RT-PCR was used to compare expression levels of selected genes in patients with low platelet count and giant platelets with normal controls with normal platelet count and normal platelet size. The expression levels of the selected genes were consistent with the expression profiles obtained SSH in all patients. alpha tubulin (TUBA), IREB2, MTMR9 and tyrosine kinase ligand (TKL) genes were expressed more in patients than in controls (Fig. 3). 4. Discussion Macrothrombocytopenia is a heterogeneous group of disorders and was considered to be rare until the introduction of automated cell counters. The widespread use of electronic counters in the 1990s made platelet counting a routine analysis, and several new forms of mild thrombocytopenia with trivial or no bleeding diathesis has since been discovered. The molecular basis of MTCP has not been fully elucidated yet. Although the defective genes, coding for membrane glycoproteins, cytoskeleton components and intracellular signaling pathways, as well as transcription factors have been identified in most cases, the pathophysiology of these disorders is often unknown. One of the most important ways to understand the pathology of MTCP is to clone and identify the differentially expressed genes. In recent years several cloning techniques for the differentially expressed genes have been established, including differential display PCR (DDPCR), representational difference analysis (RDA), SSH and cDNA microarray. Until now, these techniques have been widely applied in the study of pathology [17], immune response [18], embryo development [19] as well as the specific expression of tissue proteins [20]. SSH was first put forward by Diatchenko et al. in 1996 [21]. The basic principle is that common cDNAs in the paired materials are subtracted by subtractive hybridization and then suppressive PCR is carried out to amplify cDNA fragments specially expressed in the tester. The advantage of SSH was the design of two different adaptors and the introduction of suppression PCR with the result that the differentially expressed cDNA fragments were amplified. It allows for two subtractive hybridizations in the forward or reverse direction. Some mRNA expressed with low abundance could be detected which provided clues for further gene sequencing and identification. With the development and improvement of SSH technique, it has become one of the most effective techniques for cloning differentially expressed genes. Until now, no SSH studies have been done on MTCP patients. It is the most popular and most cost effective method to study gene expression in specific tissues or cell types over standard microarray, which is cost prohibitive for some investigators and requires large sample sizes for generating good quality data.
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Table 2 SSH library data from the patients from West Bengal, India. Description
No. of clones/gene fragments
Gene function
Accession no.
Homo sapiens proteasome Homo sapiens iron-responsive element binding protein 2 Homo sapiens tyrosine 3-monooxygenase/tryptophan 5-momooxygenase activation protein 18S ribosomal RNA gene Homo sapiens mRNA Homo sapiens mRNA Homo sapiens Kelch-like 5 protein (KLHL5) mRNA Phytophthora cinnamomi tub1 gene for alpha-tubulin Gamma glutamyltransferase 1 (Homo sapiens) Tyrosine kinase ligand (TKL) Transmembrane channel like-2 Vertebrate kinesin family Vertebrate myotubularin related protein 9 (MTMR9) Carcharhinus plumbeus T cell receptor gamma (TCRG) gene Human s-adenosylmethionine decarboxylase 1 Homo sapiens cDNA Dimethyl sulfoxide reductase Potassium channel modulatory factor 1 RNA binding motif protein 39b Human OGN gene
10 23 2 1 42 26 1 6 1 1 1 1 205 1 1 29 1 7 1 1
Proteasome Iron binding Tyrosine activation ribosomal Human cDNA-2 Human cDNA-3 Kelch like protein Alpha tubulin GGT Tyrosine kinase Transmembrane protien Kinesin family Myotubularin protein T-cell receptor SAM Human cDNA-4 Reductase Potassium channel protein RNA binding protein OGN gene
BC056249.1 DQ496102.1 BC001197.1 GU968788.1 CR933672.1 CR749596.1 AF272976 AM412177.1 AL832738.1 Y09669.1 BX571945.15 BX649292.16 BX510941.5 FJ854492.1 AK31043.1 AP012030.1 BX294395.15 CR550308.6 AL137848.5
Bold data indicates significant at Genes selected for qRT-PCR.
In the present study, SSH and bacterial culture PCR were performed to construct a subtracted cDNA library specific for the BMTCP patients from West Bengal, India. Both forward and reverse hybridizations was performed using the cDNA pool prepared from the affected individuals, which was labeled as the ‘Tester’ and cDNA pool prepared from the unaffected individuals as the ‘Driver’. About five hundred clones were obtained from this cDNA library and bacterial culture PCR was used to screen the positive recombinants. These were then subjected to DNA sequencing analysis and 20 genes were found to express differentially in affected platelets compared with the normal platelets (Table 2 & Fig. 2). The expression profiles of 4 of the 20 genes were further confirmed by quantitative reverse transcription polymerase reaction (Fig. 3). The construction of this library and further analysis by qRT-PCR is therefore of significance in screening and identifying new genes associated with MTCP which would be beneficial to the elucidation of the molecular mechanism/pathology of inherited MTCP.
The myotubularins are a family of inositol polyphosphate 3phosphatases consisting of 16 different catalytically active and inactive proteins. They form complexes among themselves which increases the catalytic activity of these enzymes and also changes specificity for their respective substrates [22]. Though it is an inactive protein, yet other active myotubularin related proteins form dimers with this inactive protein which increases their enzymatic activity. Besides their role in membrane trafficking, they are also known to have a role in cell differentiation and cytoskeletal dynamics. Both the membrane and cytoplasmic cytoskeletal components of the platelet are regulated by intracellular enzymes, one such enzyme being tyrosine kinase. The activation takes place when the platelet glycoprotein receptors bind fibrinogen and other ligands [23]. The discoid shape of the resting platelets is maintained by multiple coils of tubulin polymers and they disappear during platelet activation, which results in the platelet shape change. The regulation of this process is not
Fig. 2. Profiles of differentially expressed genes in Bengal macrothrombocytopenia patients.
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Fig. 3. mRNA expression plot for different genes by qRT-PCR. Results shows that expression of MTMR9, IREB2, TUBA and TKL gene among different affected individuals (P1–P10) has increased when compared to unaffected control samples (C1–C5).
clear; however, presence of lys at position 40 of alpha tubulin seems to have an important role in maintaining the stability of microtubules [24]. IREB2 being an iron-responsive element is regulated mainly by iron and oxygen. The association of platelet cytoskeletal abnormalities with IREB2 is however not clear. The up-regulation of these genes might be involved in shaping the platelets and production of low platelet counts in a particular ethnic region which needs further study. Thus in conclusion, the study provides a list of candidate genes which provide new clues in understanding the molecular mechanisms involved in the pathogenesis of this disorder. Further characterization of the identified genes will provide useful information in understanding the pathophysiology of the disorder. Conflict of interest statement None. Acknowledgments We thank Chromous Biotech Pvt. Ltd. Bangalore, India for their technical support. References [1] J.G. Drachman, Inherited thrombocytopenia: when a low platelet count does not mean ITP, Blood 103 (2004) 390–398. [2] A.E. Geddis, K. Kaushanky, Inherited thrombocytopenias: towards a molecular understanding of disorders of platelet production, Curr. Opin. Pediatr. 16 (2004) 15–22. [3] J. Bernard, J.P. Soulier, Sur une nouvelle variete de dystrophie thrombocytairehemorragipare congenitale, Sem. Hôpitaux Paris 24 (1948) 3217–3223. [4] B. Rocca, F.O. Ranelletti, N. Maggiano, et al., Inherited macrothrombocytopenia with distinctive platelet ultra-structure and functional features, Thromb. Haemost. 83 (2000) 35–41. [5] M. Seri, A. Pecci, F. Di, et al., MYH9 related diseases: May–Hegglin anomaly, Sebastian syndrome, Fechtner syndrome and Epstein syndrome are not distinct entities but represent a variable expression of a single illness, Medicine (Baltimore) 82 (2003) 203–215. [6] W.E. Behrens, Mediterranean macrothrombocytopenia, Blood 46 (1975) 199–208.
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