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. This variant was absent in a population of 194 unrelated cats spanning 44 different breeds. RT-PCR was performed on patient PRP samples as well as a clinically heathy sex- and age-matched control cat to verify ITGA2B variant consequence. Complete loss of ITGA2B transcript and protein expression was verified on RT-PCR and flow cytometry, explaining the underlying etiology of Glanzmann’s thrombasthenia, and likely macrothrombocytopenia, in this 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 template (at 20ng/µL), and 5.25-6.25µL of laboratory grade sterilized distilled water. PCR reactions were carried-out by a Mastercycler Nexus (Eppendorf, Hamburg, Germany) under the following conditions: 94°C for 1min, 30 cycles of 94°C denaturation for 30 seconds (s), 56°C annealing for 30s, 72°C extension for 2mins, and a final extension at 72°C for 5mins. A PCR clean-up step was performed for all samples with the ExoSAP-IT Express Kit (Thermofisher, Waltham, GA, USA) according to manufacturer’s protocol. Treated PCR products and their corresponding primer set were later diluted according to facility specifications and submitted for Sanger sequencing (UC Davis, DNA Sequencing Facility, Davis, CA, USA). Chromatograms of the resulting .ab1 files from the affected patient (Affected_ITGA2B_F/R files) and 20 geriatric healthy controls (Unaffected_#_ITGA2B_F files) were manually inspected and analyzed with the use of the Sequencher (Gene Codes, Ann Arbor, MI, USA) and SnapGene Viewer sequence analysis software (https://www.snapgene.com/) and are provided here.

File naming: PatientThrombopathyStatus_ParticipantNumber_GeneName_Forward/ReverseReaction_SequenceID.ab1

All provided .ab1 files with an "Unaffected" label are representative of sequences from a population of cardiovascularly healthy geriatric control patients; please refer to manuscript for further information.

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

Data files

Abstract

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