A novel ITGA2B double-cytosine frameshift variant (c.1986_1987insCC) leads to Glanzmann’s thrombasthenia in a cat

Glanzmann’s thrombasthenia is the most recognized congenital platelet disorder in small animals. The disorder affects ~1:1,000,000 people globally and is characterized by impaired platelet aggregation and clot retraction. Autosomal recessive, loss-of-function, variants in either ITGA2B or ITGB3 of the αIIbβ3 receptor explains the disease in humans. Physical exam and clinical pathology assessments were performed on a feline patient with idiopathic thrombocytopenia and thrombopathia. Patient blood samples were obtained for flow cytometry and platelet aggregometry analyses and patient phenotyping. Patient and validation cohort (n=20) DNA samples were extracted for Sanger sequencing of a previously identified ITGA2B (c.1986delC) variant. A novel c.1986_1987insCC autosomal recessive variant in ITGA2B was identified. This variant leads to a frameshift near the 3’ end of the gene, consequentially leading to complete truncation of the encoded protein’s calf-1, calf-2, and transmembrane domains...., , , # A novel ITGA2B double-cytosine frameshift variant (c.1986\_1987insCC) leads to Glanzmann’s thrombasthenia in a cat

Given thrombocytopathia of presumed genetic etiology, the patient (Affected_ITGA2B_F/R files) was genotyped for a previously identified ITGA2B (c.1986delC) variant in another feline patient with Glanzmann's thrombasthenia. A new primer set, flanking the entirety of ENSFCAG00000003056.6 exon 18, was designed using the Primer3Plus software and specificity confirmed using UCSC’s In-Silico PCR tool on the latest cat genome build (felCat9). Specific-confirmed DNA oligonucleotides (F 5’-GCGGGTGCTACTGCTGAAT-3’, R 5’-TGAAAAGGAGTTTGGAGCTGA-3’) were synthesized by IDT (CoralVille, IA, USA). Amplification of PCR targets was conducted via end-point PCR using the TaKaRa LA Taq with GC Buffer PCR Kit. A single 25µL PCR reaction was comprised of 0.25µL of TaKaRa LA Taq, 12.5µL of 2X GC Buffer II, 4µL of dNTP (2.5mM), 0.5µL of both forward and reverse primers (20mM), 1-2µL of DNA templa...