Incorporating Tryptase Genotyping Into the Workup and Diagnosis of Mast Cell Diseases and Reactions

Abstract

The measurement of mast cell tryptase levels in serum has found utility in the diagnosis and management of both clonal mast cell disorders and severe mast cell-dependent systemic reactions in the form of anaphylaxis. A more recent discovery is that a majority of individuals with elevated basal serum tryptase levels have increased germline TPSAB1 gene copy number encoding α-tryptase. This genetic trait is referred to as hereditary α-tryptasemia (HαT) and affects nearly 6% of the general population. In clinical practice, the presence or absence of HαT should thus now be determined when defining what constitutes an abnormal serum tryptase level in the diagnosis of mastocytosis. Further, as rises in serum tryptase levels are used to support the diagnosis of systemic anaphylaxis, variability in baseline serum tryptase levels should be factored into how significant a rise in serum tryptase is required to confirm the diagnosis of a systemic allergic reaction. In practicality, this dictates that symptomatic individuals undergoing evaluation for a mast cell-associated disorder or reaction with a baseline serum tryptase level exceeding 6.5 ng/mL should be considered for tryptase genotyping in order to screen for HαT. This review provides detailed information on how to use the results of such testing in the diagnosis and management of both mastocytosis and anaphylaxis.

Keywords: Alpha-tryptase; Hereditary α-tryptasemia; HαT; Mastocytosis; Tryptase.

Figure 1.. Comparison of false-positive rates for serial BST measurements in patients with SM and HαT using the Threshold Ratio approach versus the 20% + 2 ng/mL algorithm.

Individual values for sequential BST measurements shown as a percentage of the respective algorithm. The 20% + 2 ng/mL over BST algorithm (20+2) and cut-off (black dotted line) are shown on the right, while threshold ratios are shown on then left for the same individuals. High Sensitivity (Sensi) threshold ratio indicated by bottom (blue dotted line), High Specificity (Speci) threshold ratio indicated by the top (green dotted line), and the threshold ratio optimized to maximize both sensitivity and specificity (Optimized) indicated by the middle (red dashed line). Adapted from O’Connell and Lyons, Curr Opin Allergy Clin Immunol. 2022 Apr 1;22(2):143–152.

Figure 2.. Prevalence of HαT among individuals with SM compared to controls from the U.S. and U.K. and E.U.

Prevalence data among healthy individuals or biorepositories from 4 independent studies (–25) and among systemic mastocytosis patients (SM) (–24) from 3 independent studies. Light grey bars indicate control population prevalence data, and black bars indicate prevalence data from SM cohorts. Adapted from O’Connell and Lyons, Curr Opin Allergy Clin Immunol. 2022 Apr 1;22(2):143–152.

Figure 3.. HαT is the common cause of elevated BST levels in the general population.

Estimated prevalence of elevated BST and associated causes in Western populations. Clonal disease includes mastocytosis and other myeloid neoplasms including acute and chronic myeloid leukemias, myelodysplastic syndromes, myeloproliferative variant hypereosinophilic syndrome and related disorders. Idiosyncratic cases include rare inherited causes such as families with pathogenic GATA2 or PLCG2 variants, as well as acquired causes including but not limited to certain helminth infections. BST – basal serum tryptase; HαT – hereditary-alpha tryptasemia; ACKD – advanced chronic kidney disease; ESRD – end-stage renal disease. Adapted from Lyons, Ann Allergy Asthma Immunol. 2021 Oct;127(4):420–426.

Figure 4.. Tryptase genotyping strategies.

(A) Two alleles in an individual with HαT – one allele with two β-tryptase sequences - one each at TPSAB1 and TPSB2 (top), and the second HαT-associated allele with 3 α-tryptase encoding TPSAB1 copies and a single β-tryptase encoding TPSB2 copy (bottom). Genomic DNA (gDNA) extracted from cells from this individual, containing these six α- or β-tryptase sequences is either amplified or restriction digested. The amplified gDNA can then either be (B) Sanger sequenced, or (C) treated with the restriction enzyme EcoRV and Southern blotted to perform relative quantitation of α- and β-tryptases. In both cases the ratio of α- to β-tryptases calculated would be 1:1. (D) Unamplified digested gDNA is assayed by droplet digital PCR (ddPCR), which allows for absolute copy number detection of α- and β-tryptase sequences, yielding the genotype determination 3α:3β. Adapted from Lyons, Immunol Allergy Clin North Am. 2018 Aug;38(3):483–495.

Figure 5.. Clinical work-up of patients with elevated BST incorporating tryptase genotyping.

*‘Red flags’ may include, but are not limited to: hepatosplenomegaly, lymphadenopathy, CBC abnormalities [e.g., thrombocytopenia, anemia, polycythemia, neutrophilia, eosinophilia (AEC > 1500 cells/mL)], severe and/or recurrent anaphylaxis (in particular idiopathic or Hymenoptera venom-induced), urticaria pigmentosa/Darier’s sign, eosinophilic tissue infiltration, premature osteopenia/osteoporosis or pathological fracture; †24-hour urinary N-methylhistamine and/or 2,3-dinor-11β PGF2α can also be sent where available; §HαT is diagnosed by the presence of increased α-tryptase encoding TPSAB1 copy number; ‡See Table 1 for predicted upper 99.5%CI of BST level based upon TPSAB1 replication. Adapted from Luskin et al., J Allergy Clin Immunol Pract. 2021 Jun;9(6):2235–2242.