Biomedical science overview and applications

Molecular Basis Glanzmann Thrombasthenia

An investigation of the molecular basis of Glanzmann Thrombasthenia using Polymerase Chain Reaction (PCR)

The objective of this project is to investigate the molecular basis of Glanzmann Thrombasthenia (GT) using polymerase chain reaction. There have been many mutations discovered in GT patients over the years in many studies. Thus using PCR to genotype patients is one of the most effective ways of discerning the genetic basis of the disease. The purpose of these sets of experiments is to determine if a mutation on the ITG?3 promoter, which occurs in a certain percentage of Glanzmann Thrombasthenia (GT) patients, can be reversed through site directed mutagenesis and if normal platelet functioning can resume. Normal platelet functioning will be assessed through detecting promoter region binding to the myc transcription factor through chromatin immunoprecipitation assays, also known as ChIP assays. We anticipate that the myc transcription factor will have enhanced binding upon site-directed mutagenesis, reversing the mutation occurring in Glanzmann Thrombasthenia. This we predict will lead to healthier platelet production and function. There is a basis for determining if the myc transcription factor binds to the ITG?3 gene. If the myc transcription factor does indeed bind to the gene's promoter region, we will have deciphered a molecular mechanism by which the disease Glanzmann Thrombasthenia occurs through mutations on the ITG?3 promoter region. If this set of experiments leads to healthier platelets, a bench-derived therapy for the treatment of Glanzmann Thrombasthenia may have been uncovered, which could translate into clinical treatments. Subsequent experiments may assist in coming up with these therapies.

Numerous studies have been conducted to determine mutations in certain populations of patients with a relatively high occurrence of GT through PCR, which are detailed here in terms of how and why the studies were conducted and the role of PCR. GT is an autosomal recessive bleeding syndrome affecting the megakaryocyte lineage and characterized by a lack of platelet aggregation. It is a moderate to severe hemorrhagic disorder with mainly mucocutaneous bleeding. The molecular basis is linked to quantitative and/or qualitative abnormalities of "IIb"3 integrin, the receptor that mediates the incorporation of platelets into an aggregate or thrombus at sites of vessel injury (Nurden, 2006).

Glanzmann first described this disease in 1918 as "hereditary hemorrhagic thrombasthenia." A prolonged bleeding time and an isolated, rather than clumped, appearance of platelets on a peripheral blood smear were early diagnostic criteria. In 1956, Braunsteiner and Pakesch reviewed disorders of platelet function and described thrombasthenia as an inherited disease characterized by platelets of normal size that failed to spread onto a surface and did not support clot retraction. The diagnostic features of GT including the absence of platelet aggregation as the primary feature were clearly established in 1964 by the classic report on 15 French patients. Those patients with absent platelet aggregation and absent clot retraction were subsequently termed as having type I disease; those with absent aggregation but residual clot retraction, type II disease; while variant disease was first established in 1987 (Nurden, 2006).

Genetic basis

A continually updated database is available on the Internet http://sinaicentral.mssm.edu/intranet/research/glanzmann: it currently contains a list of about 100 mutations giving rise to GT. The ?IIb and ?3 genes are both affected and while posttranslational defects predominate, mRNA stability can also be reduced. In brief, integrin synthesis occurs in the megakaryocytes with "IIb"3 complex formation in the endoplasmic reticulum (ER). Noncomplexed or incorrectly folded gene products fail to undergo processing in the Golgi apparatus and are rapidly degraded intracellularly. One exception is the ability of normally synthesized ?3 to complex with ?v and form "v"3 Deletions and insertions, nonsense and missense mutations are common causes of GT. Splice site defects and frameshifts are also widespread. Large deletions are rare. The ?IIb gene is composed of 30 exons. In an early and classic study, three Israeli-Arab kindreds were shown to possess a 13-bp deletion leading to a six-amino acid deletion in the ?IIb protein. The affected region, including Cys107, was postulated to be critical for posttranslational processing of ?IIb. Missense mutations in exons encoding the extracellular ? -- propeller region of ?IIb have shown how the extracellular calcium-binding domains of ?IIb are essential for "IIb"3 biogenesis. Site-directed mutagenesis involving various amino acid substitutions at position 324 of ?IIb, illustrated to what extent the GT phenotype depended on both the nature of the substituted amino acid and its replacement. Mutations affecting the membrane-proximal calf-2 domain showed that while this region was not essential for complex formation in the ER, it was necessary for transport into and/or through the Golgi apparatus. These are but a few selected examples of ?IIb defects.

The ?3 gene is composed of 15 exons and mutations are again widely distributed within the gene. An 11 bp deletion leading to protein termination shortly before the transmembrane domain of ?3 was first described in six Iraqi Jews with type I disease. This defect prevented normal membrane insertion of the integrin and also "v"3 expression, both in platelets and other cells. Although most ?3 mutations affect "IIb"3 and "v"3 expression, rare mutations allow "v"3 expression while preventing "IIb"3 processing.

Patients with mutations allowing "IIb"3 to be processed, but in whom integrin function is abolished, are of particular interest. In most of these patients, it is the ?3 gene that is affected. In brief, many of the variants have platelets with sufficient "IIb"3 to normally allow aggregation, but the activation-dependent expression of adhesive protein binding sites on the integrin does not occur. As well as providing information on the ligand-binding pocket on the extracellular domains, variant molecules have highlighted the role of the ?IIb and ?3 intracellular tails in integrin signaling and even for integrin trafficking. For some variants, clot retraction can occur even if aggregation is prevented. Finally, recent studies on two patients have revealed that disruption of disulfide bridges in the ?3 Epidermal Growth Factor (EGF) extracellular domains gives rise to a constitutively active integrin, able to spontaneously bind fibrinogen. Here, aggregation fails to occur because of the absence of free counter receptors allowing platelet to platelet bridging (Nurden, 2006).

The recent application of mutation screening on a national basis, first in Italy and then in India, has re-emphasized how a wide array of mutations can be found in GT patients within a single country. Interestingly, while 17 out of 21 candidate mutations were in the ?IIb gene of the Italian patients, ?3 mutations with emphasis on exon 4 appear to characterize the Indian patients (Nurden, 2006).

Glanzmann thrombasthenia is an autosomal-recessive bleeding disorder caused by absence or dysfunction of the platelet integrin "IIb"3 receptor. The genes coding ?IIb and ?3 are located on the long arm of chromosome 17 at a physical distance of about 3.2 Mbp. The first mutation causing GT was described in 1990 and since then more than 100 mutations have been reported (http://sinaicentral.mssm.edu/intranet/research / glanzmann/). Although most mutations are sporadic, founder mutations have been described in highly consanguineous populations, including Iraqi Jews and Arabs living in Israel and gypsies living in France. The identification of disease-causing mutations led to the development of assays for carrier testing and prenatal diagnosis, revealed different mechanisms of mutagenesis and abnormal gene expression, and provided important insights into the structure and function of "IIb"3. The recent elucidation of crystal structures of av?3 and "IIb"3 further elucidated the impact of natural mutations on the structure and function of "IIb"3. In this study molecular basis of GT in southern India in a cluster of 40 affected families was studied (Peretz et al., 2006).

Mutations were detected by the method of single strand conformation polymorphism (SSCP) analysis, followed by nucleotide sequencing and confirmation by restriction fragment length polymorphism (RFLP) assays. The sequences of the primers employed and the reaction conditions for PCR amplification of the exons, adjacent intronic regions, and 50 and 30 untranslated regions of the ?IIb and ?3 genes respectively. The primers used for amplification of exon 1 of the ?3 gene (forward: 5'- TCCCGCTGCG GGAAAAGCG; reverse: 5'-CTCCAAGTCCGCAACTTGAC) were designed on the basis of a reported sequence. For first strand cDNA amplification of a fragment containing part of exon 27 as well as exons 28 and 29, the following primers were used: forward 5'-CCCTGTACTG TGGTGCAGTG and reverse 5' -CTTCCACATGGCCAGGAC. For SSCP analysis, 0.1 ml 33P-dCTP (10 mCi/ml; Amersham, Buckinghamshire, UK) was added to the reaction mixture and 5 ml of amplification product were added to 10 ml of stop solution (95% formamide, 20 mM EDTA, and tracking dyes). The samples were denatured for 3 min at 901C and cooled on ice for 3 min. Each sample (5 ml) was loaded onto a mutation detection enhancement gel (MDE; FMC BioProducts, Rockland, ME) with or without 6% glycerol. The gel was run at 400 V for 5 -- 6 hr, vacuum dried for 60 min at 801C, and autoradiographed (Peretz et al., 2006).

For nucleotide sequencing, PCR products were purified using the High Pure PCR Product Purification Kit (Roche Diagnostics GmbH, Mannheim, Germany). Sequencing reactions were per- formed with the same primers used for the PCR reaction, using the ThermoSequenase radiolabeled terminator cycle sequencing kit (Amersham, Cleveland, OH). Sequencing reaction products were denatured for 5 min at 701C and separated on 6% polyacrylamide gels at 2500V for 2 -- 5 hr. The gels were dried and subjected to autoradiography. Alternatively, automated sequencing of the purified PCR products was performed on an Applied Biosystems Genetic Analyzer 3100 (www.PEBIO.com) (Peretz et al., 2006).

RFLP assays were designed to confirm the identified sequence alterations. The experimental details of these RFLP assays can be obtained upon request. Numbering of exons in the ?IIb and ?3 gene was done according to Heidenreich et al. [1990] and Villa-Garcia et al. [1994], respectively. For nucleotide numbering, the A nucleotide of the ATG start codon was designated 11 (cDNA ITGA2B and ITG?3 GenBank accession numbers NM_000419.2 and NM_000212.2, respectively). For amino acid numbering in the text, the classic system was used (the first amino acid of the mature ?IIb and ?3 proteins was designated 11). Both systems (one including the signal peptides (initiating Met designated 11) and one excluding the signal peptides) were employed (Peretz et al., 2006).

In another study, the molecular basis of GT was elucidated by screening for mutations in 30 GT patients. On the whole, 21 different candidate causal mutations, 17 in the IIb and 4 in the ?3 gene have been found. Only two (IIb Pro145Ala and IVS3(-3)-418del) have been previously reported. Nine mutations (42.9%) were likely to produce truncated proteins, whereas the remaining 12 were missense mutations that affected highly conserved residues in IIb and ?3 genes. Six mutations were found in different patients suggesting a possible founder effect. The wide spectrum of expressivity, ranging from mild to severe also among patients carrying the same mutations, provided evidence for a role of different loci or circumstantial factors. In conclusion, we have identified a spectrum of unreported mutations that may be of value to unravel the role of specific regions of IIb and ?3 genes (D'Andrea et al., 2002).

Isolation of DNA and PCR analysis were carried out according to standard procedures. A 9 ml volume of blood was drawn into 1 ml of 3.8% sodium citrate. For DNA extraction, peripheral blood leukocytes were separated by sedimentation and incubated overnight at 37° C. In a digestion buffer (100 mM NaCl 10 mM Tris-HCl, 25 mM EDTA, 1% SDS) containing 0.1 mg/ml of proteinase K. The nucleic acid was isolated by phenol/chloroform extraction and ethanol precipitation. Amplifications of all coding regions of IIb and 3 genes and intron/exon boundaries were achieved using sense and antisense oligonucleotides. For analysis of the IIb gene, oligonucleotides were numbered according to Heidenreich et al. For analysis of the ?3 gene, oligonucleotides were numbered according. (exon 1) and Zimrin et al. (exons 2-15). PCR was carried out on 50ul volume samples, in a Perkin Elmer-Cetus termal cycler (Perkin-Elmer Cetus, Norwalk ) (D'Andrea et al., 2002).

Although thrombasthenia is a rare disorder, its occurrence is increased in some regions of the world where intracommunity marriage and consanguinity are commonplace, resulting in increased expression of autosomal recessive traits. Investigators have been studying two populations having an unusually high frequency of Glanzmann disease, Iraqi Jews and Arabs living in Israel, and were able to distinguish the populations on the basis of immunodetectable GPIIIa and platelet surface vitronectin receptor (alpha v beta 3) expression. They describe molecular genetic studies based on use of the PCR that have allowed them to characterize platelet mRNA sequences encoding GPIIb and GPIIIa from patients in these populations. In six of six Iraqi-Jewish families studied, cDNA sequence analysis identified an 11-base deletion within exon 12 of the GPIIIa gene. This mutation produces a frameshift leading to protein termination shortly before the transmembrane domain of GPIIIa. In contrast, a 13-base deletion encompassing the splice acceptor site of exon 4 of the GPIIb gene was found in three of five Arab kindreds studied. This deletion results in forced alternative splicing to a downstream AG acceptor, producing a 6-amino acid deletion in the GPIIb protein, including a single cysteine residue (Newman 1991).

These nucleotide sequence variations were exploited to design a rapid, PCR-based oligonucleotide dot-blot hybridization test for both pre- and postnatal diagnosis of Glanzmann disease. These studies demonstrate the heterogeneity of Glanzmann thrombasthenia in different populations, and its homogeneity within geographically restricted populations, and offer insight into the requirements for integrin surface expression.. Platelet RNA was prepared from patient and control blood by using a modified version of the technique of Chomczynski and Sacchi. First-strand cDNA was synthesized from specified regions of GPIIb and GPIIIa using GPIIb -orGPIIIa- specific antisense primers and Moloney murine leukemia virus reverse transcriptase. cDNA was then amplified by the PCR according to the method of Newman and colleagues. In several experiments, both cDNA synthesis and PCR were performed with two different primer pairs in the same tube (duplex PCR), with one prime pair corresponding to GPIIb and the other corresponding to GPIIIa. This allowed assessment of relative abundance of mRNAs encoding these two glycoproteins in control and patient samples. After electrophoretic separation and analysis of PCR products on agarose gels, the appropriate bands were excised and recovered with Geneclean (Bio 101, La Jolla, CA). DNA was subjected to direct sequence analysis with T7 DNA polymerase (United States Biochemical) and standard dideoxynucleotide double-stranded sequencing techniques according to the manufacturer's directions. Occasionally, sequence analysis was performed on gel-purified PCR products that had been subcloned into the plasmid vector pGEM 5Zf (Promega Biotec) (Newman, 1991).