Introduction Chapter on "Molecular Basis Glanzmann Thrombasthenia"

Introduction Chapter 8 pages (2810 words) Sources: 1+

[EXCERPT] . . . .

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).

Genotypic Analysis of Control and Patient Genomic DNA Samples. High molecular weight genomic DNA was extracted from whole blood by the method of Miller et al. For population screening and carrier detection, oligonucleotide primers were designed to flank the regions found to be defective in the Arab and Iraqi-Jewish thrombasthenic genes; these were then used for PCR amplification of genomic DNA from other thrombasthenic individuals within these two populations. Mutations were detected by dot-blot hybridization analysis of the PCR products with oligonucleotide probes (Newman, 1991).

Limitations for using PCR in molecular diagnosis in this study

Currently, five platelet alloantigen (alloAg) systems have been established (HPA-I, -2, -3, -4, -5). Three of these are expressed on the glycoprotein (GP) IIb-IIIa complex, HPA-1, HPA-3 and HPA-4, inherited in an autosomal codominant mode. Recent investigations of the molecular basis of these platelet alloantigen systems have shown that only one nucleic acid base substitution in the genes encoding for GP IIb and GP IIIa is responsible for the polymorphism. This substitution is reflected in a difference in restriction enzyme recognition allowing platelet alloantigen typing by restriction fragment length polymorphism (RFLP) analysis of DNA amplified by the polymerase chain reaction (PCR).To validate the PCR technology for platelet typing, we have compared PCR-RFLP with monoclonal-antibody-specific immobilization of platelet antigens (MAIPA). For this purpose, different Glanzmann thrombasthenic families and particularly heterozygous individuals were studied who are not lacking GP IIb-IIIa, as a model to detect the occurrence of discrepancies between these two technologies. In two families, differences were found between molecular biology and serological methods with the lack of expression of one antigen on the platelet membrane surface. In the first family, the abnormality is related to the HPA-1 alloantigen system with three informative members; in the second, the HPA-3 alloantigen system is concerned with two informative members. Considering these results, there may not always be a perfect correlation between molecular biology and serological methods, as an unknown molecular defect could interfere with the PCR results and lead to false platelet typing. In cases of suspected post-transfusion purpura (PTP) or fetuses at risk for neonatal alloimmune thrombocytopenia (NAITP), great care must be taken in the interpretation of the results if only PCR-RFLP techniques are used (Morel-Kopp, 1994).

Works Cited

D'Andrea, G. et al. (2002) Glanzmann's Thrombasthenia: Identification of 19 New Mutations in 30 Patients. Thromb Haemost 87: 1034 -- 42.

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