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...

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…