Hereditary α-tryptasemia (HαT) is an autosomal dominant genetic trait discovered in 2016 that affects 4-6% of the Western population, making it the most common cause of elevated basal serum tryptase (BST) levels. This is due to extra copies of the TPSAB1 gene encoding the α-tryptase isoform, and is associated with cutaneous flushing, pruritus, dysautonomia, functional gastrointestinal symptoms, and connective tissue anomalies (Lyons J. et al., 2016). The multigene tryptase locus on chromosome 16 includes TPSB2, encoding only β-tryptase, and TPSAB1, encoding either α- or β-tryptase, which are inherited as a haplotype and are primarily expressed by mast cells. Detection of germline HαT in patients aids in interpretation, prognosis, and clinical management of patients with elevated BST. In systemic mastocytosis (SM), the WHO classification recommends adjustment of the BST in the presence of HαT, where BST>20 ng/mL is a minor criterion for SM.

Here, we report the genotypes of patients undergoing droplet digital PCR (ddPCR)-based TPSAB1 copy number analysis from our reference laboratory since launch of the test in May 2024, and explored associated BST levels and KIT D816V mutation, if present.

Study approval was obtained under IRB_00077285. ddPCR was performed on DNA extracted from whole blood or bone marrow specimens. Targeted regions of TPSAB1, TPSB2, and a control gene, AP3B1, were amplified in oil-immersed droplets and detected based on three fluorescent allele-specific probes in a single well. Copy number calculations were based on the allelic ratio of TPSAB1 to AP3B1, and TPSB2 to AP3B1. Patient test results for total serum tryptase, quantitative KIT D816V, and TPSAB1 copy number analysis were collected internally from May 2024–July 2025. R statistical software and the tidyverse package (v2.0.0; Wickham H. et al., 2019) were used for data analysis and visualization.

We analyzed TPSAB1 copy number records from a total of 1,137 patients with the following demographics: 68% female (767/1137) ages 0-92 (median age 44); 32% male (364/1137) ages 0-85 (median age 46); 0.5% unspecified (6/1137) ages 27 and 55 or unknown; and an overall HαT positivity rate of 44% (n=500). No difference in HαT positivity was observed between males and females (46% in females versus 40% in males, p-value > 0.05, Yates' chi-squared test), however HαT+ patients were present at a significantly younger age in females (42.1 years versus 45.9 years, p < 0.01, ANOVA) compared to males (41.8 years versus 42.5 years, p > 0.5, ANOVA). We observed a total of 22 unique HαT genotypes, with 13 genotypes previously found during initial validation and 9 new ones, including not increased TPSAB1 (HαT-; 0α-6β, 1α-4β) and increased TPSAB1 (HαT+; 2α-4β, 3α-1β, 4α-3β, 4α-4β, 5α-1β, 5α-2β, and 7α-2β). The three most common HαT- genotypes were 1α-3β (26%), 0α-4β (15%), and 2α-2β (14%), while the three most common HαT+ genotypes were 2α-3β (19%), 3α-2β (19%), and 4α-2β (3%) which together made up more than 95% of all observed genotypes. Least common HαT genotypes include 2α-4β, 3α-1β, 4α-3β, 4α-4β, and 7α-2β which were each only observed once with a frequency of 0.08% (1/1137). 254 patients (22%) had KIT and BST results together. While average BST levels were not significantly different in the subgroup of HαT+ (n=123) versus HαT- (n=131) patients (21.6 µg/L versus 18.6 µg/L; p=0.657, Yates' chi-squared test), we observed a trend for increased tryptase levels in HαT+ patients. We found that 98% (120/122) of HαT+ patients lacking KIT D816V were within the predicted intervals for BST after adjusting for TPSAB1 copy numbers (Chovanec J., et al. 2023). Concurrent KIT D816V testing was present in 359 patients. KIT D816V was detected in 7% (26/359) of all HαT-tested samples with a median value of 0.77% VAF (observed range 0.03-40.25%). We observed fewer KIT D816V-positive patients that were also positive for HαT+(2/169, 1%) compared to those negative for HaT (24/190, 13%).

The data demonstrate the diversity within the tryptase locus with 22 total genotypes including 9 previously unobserved during validation. In our dataset, only 254 (22%) patients had concurrent KIT and BST results. In this cohort, we observed that additional copy numbers of α-tryptase positively correlated with BST level. In addition, the majority of HαT+ patients were predominantly negative for KIT D816V mutation and tended to have increased BST levels compared to HαT- patients.

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2025
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