DOI: 10.25338/b8jw72
title: A novel ITGA2B double-cytosine frameshift variant (c.1986_1987insCC) leads to Glanzmann’s thrombasthenia in a cat

description: 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.
description: # 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 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 () 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.