Renee Berry1, Leanne Payne1, William Dronfield4, Naser El-Lagta1,4, Lloyd D’orsogna1,3, Dianne De Santis1,4, Sam Salman1-3
1 Department of Immunology, PathWest, Murdoch, Western Australia
2 Clinical Pharmacology and Toxicology, PathWest, Crawley, Western Australia
3 Medical School, University of Western Australia, Crawley, Western Australia
4 Department of Biomedical Sciences, University of Western Australia, Crawley, Western Australia
Introduction:
There is increasing clinical awareness of Hereditary α-tryptasemia (HαT), a common autosomal dominant genetic trait caused by an increased copy number of the α-tryptase encoding sequence in the TPSAB1 gene. Normal individuals have 4 copies of tryptase genes; either 2 α/2 β, 1 α/3 β, or 0 α/4 β. Additional copies of the α-tryptase gene (usually either duplication or triplication) leads to a higher baseline mast cell tryptase and has become a key differential in patients under investigation for mastocytosis, a clonal proliferation of mast cells which can require a bone marrow aspirate and trephine to fully investigate. Therefore, genetic testing to identify the absolute number of gene copies of both α and β tryptase is required. The Department of Clinical Immunology at PathWest commenced investigating the use of a digital droplet PCR method developed by Lyons et al (Nat Genet. 2016) for the detection of α and β tryptase gene copy number.
Method:
Patients with previous HαT testing, which included α and β tryptase gene copy number results from ddPCR obtained from an external service provided were included in the validation. To quantify allelic α- and β -tryptase copy number, a ddPCR assay was evaluated using custom primers and probes for α- and β-tryptase based on the publication by Lyons et al. The assay was performed on native and restriction-endonuclease-treated genomic DNA using the PrimePCR ddPCR Copy Number reference AP3B1, according to the manufacturer’s specifications (Bio-Rad).
Results: There were 32 patients tested using the ddPCR method. All results were concordant with the key clinical result, with 8 samples without HαT and 24 with HαT. Only one sample had discordant genotype, with no change in the interpretation (both genotypes were HαT negative). The total number of α and β tryptase gene copies reported ranged from 3 to 8, with a single duplication the most common HαT genotype (84%).
Conclusion: The ddPCR assay accurately determined the α/β tryptase genotype. In addition to current standard of care, the addition of TPSAB1 CNV testing for patients with elevated BST can help with diagnostic clarity and may avoid the need for invasive investigations for a monoclonal mast cell disorder where HαT is identified.