OC 45.4 - Investigating a family with severe inherited thrombocytopenia reveals a novel structural variant involving ANKRD26

Monday, June 24, 2024

15:30 – 15:45 ICT

Room: 110 A-C

Presenting Author(s)

AA

Ahmad Alcheikh, MBBS FRACP FRCPA

PhD candidate
Kolling Institute, Royal North Shore Hospital, New South Wales, Australia

Co-Author(s)

MM

Matthew McKay

Senior Researcher
Bowel Cancer Research, Kolling Institute, The University of Sydney, Sydney, NSW, Australia
Sydney, New South Wales, Australia
NS

Nisha Singh

Senior Scientist
Cytogenetics Laboratory, NSW Health Pathology – Northern Sydney, Royal North Shore Hospital, Sydney, NSW, Australia
Sydney, New South Wales, Australia
MM

Mark Molloy

Senior Researcher
Bowel Cancer Research, Kolling Institute, The University of Sydney, Sydney, NSW, Australia
Sydney, New South Wales, Australia
RB

Robert Bird

Director of Hematology
Princess Alexandra Hospital
Woolloongabba, Queensland, Australia
KM

Kirk Morris

Consultant Haematologist
Department of Cancer Care Services, Royal Brisbane and Womens Hospital, Brisbane Qld Australia
Brisbane, Queensland, Australia
Nina Raju, MSc

Haematologist
Queensland Health
Brisbane, Queensland, Australia
LL

Linda Lee

Senior Scientist
Molecular Genetics Department, NSW Health Pathology – Northern Sydney, Royal North Shore Hospital, Sydney, NSW, Australia
Sydney, New South Wales, Australia
CW

Chris Ward

Consultant Haematologist
Northern Blood Research Centre, Kolling Institute, Royal North Shore Hospital, Sydney, NSW, Australia
Sydney, New South Wales, Australia
WS

William Stevenson

Head of Department, Haematologist
Haematology Department, Royal North Shore Hospital, Sydney, Australia
Sydney, New South Wales, Australia
Marie-Christine Morel-Kopp, PhD

Senior Scientist
Northern Blood Research Centre, Kolling Institute of Medical Research, The University of Sydney, Sydney, Australia
St Leonards, New South Wales, Australia

Background: Dominant 5’UTR single nucleotide variants are the commonest cause of ANKRD26-related thrombocytopenia with increased leukaemia risk (ANKRD26-RT). We identified a pedigree demonstrating autosomal dominant severe thrombocytopenia in four members across two generations (mean platelet count 28x109/L) and a family history of leukaemia.

Aims: To investigate the cause of inherited thrombocytopenia in this family.

Methods: Optical genomic mapping, reverse transcription polymerase chain reaction (RT-PCR) and amplicon-based RNA-Seq were used for genetic characterisation. Stable pcDNA3.1 cell transfection, western blotting (WB) and mass spectrometry were used to detect protein expression.

Results: A targeted NGS panel for known causes of inherited thrombocytopenia did not identify a pathological variant. Genomic mapping suggested duplication of a 251.4 kb region encompassing ANKRD26 exons 6-34 and ABI1 exon 1 (Figure 1). Fusion transcripts were identified by by RT-PCR, sequenced and cloned into pcDNA3. RNA-Seq using ANKRD26 and ABI1::ANKRD26 primers showed a marked increase in total ANKRD26 mRNA transcripts from granulocytes, PBMC and platelets of patients compared to two normal controls (Figure 2). The commonest transcripts (94-98%) combined exon 1 of ABI1 with exons 7 or 6 of ANKRD26, while rare transcripts included portions of intron 6. None of the identified transcripts were in frame with ABI1 ATG start for protein coding. Transformed E. coli colonies harbouring wild-type and fusion constructs expressed a smaller ANKRD26 protein by mass spectrometry, beginning at amino acid ~ 873 of the canonical sequence suggesting use of an alternate internal ATG start site.

Conclusion(s): We identified a novel duplication-fusion variant in a family with severe inherited thrombocytopenia. Like standard ANKRD26-RT, ANKRD26 mRNA levels were increased. The ABI1::ANKRD26 transcripts are all out-of-frame and produce a substantially trunctated protein in bacteria, possibly explaining the inability to detect intact protein in a similar report of WAC::ANKRD26 fusion causing thrombocytopenia. Standard NGS panel testing may fail to identify these structural abnormalities.