Identification of Molecular Defects in ITGA2B and ITGB3 Genes and Phenotypic Correlation in Pakistani Patients with Glanzmann Thrombasthenia

Background:

Glanzmann thrombasthenia (GT) is most common inherited platelet functional defect. It is an autosomal recessive disorder, characterized by a bleeding diathesis. Incidence is increased in locations where consanguineous marriages are common. The defect is caused by mutations in the genes encoding ITGA2B or ITGB3. This results in qualitative or quantitative abnormalities of the platelet receptor, αIIb-β3 integrin.

Objectives:

The aim of this study was to identify and correlate the mutations in GT patients with phenotype of the patient.

Subjects and methods:

20 patients with GT were enrolled in the study to identify the molecular defects and to correlate their phenotype with their genotype. CBC with peripheral film, PT, APTT and Fibrinogen levels were done initially. Platelet aggregation studies, flow cytometry, and mutation analysis was done by Sanger sequencing. Genomic DNA was extracted from peripheral blood by QIAamp DNA Blood mini kit (Qiagen) and Exon specific PCR was done for GT gene and Direct gene sequenced on automated ABI-3130 Genetic Analyzer (Applied Biosystems). For any variation wild type was matched on HGMD (Human Gene Mutation Database http://www.hgmd.cf.ac.uk/ac/index.php) and wild type color fasta sequence (http://pga.gs.washington.edu/). Pathogenecity score was evaluated by using software tools including :

Polyphen-2(http://genetics.bwh.harvard.edu/pph2/) ,SNP&GO(http://snps.biofold.org/snps-and-go/index.html), MUpro (http://mupro.proteomics.ics.uci.edu/) ,SIFT (http://sift.jcvi.org), Provean (http://provean.jcvi.org/about.php) .

All samples were sequenced at the Gene Sequencing Lab of NIBD (National Institute of Blood diseases and Bone Marrow Transplantation) Karachi.

Results:

Mutations were identified in all patients. Missense mutations were seen in most of the GT patients. The remaining mutations were heterogeneous and were distributed throughout the length of the gene.

Conclusions:

The severe type I GT was the most common subtype found in this study.Carrier detection and genetic counseling in these families is a potentially effective alternative for decreasing the burden of severe type of GT.