Genetically defined individual reference ranges for tryptase limit unnecessary procedures and unmask myeloid neoplasms

Abstract

Serum tryptase is a biomarker used to aid in the identification of certain myeloid neoplasms, most notably systemic mastocytosis, where basal serum tryptase (BST) levels >20 ng/mL are a minor criterion for diagnosis. Although clonal myeloid neoplasms are rare, the common cause for elevated BST levels is the genetic trait hereditary α-tryptasemia (HαT) caused by increased germline TPSAB1 copy number. To date, the precise structural variation and mechanism(s) underlying elevated BST in HαT and the general clinical utility of tryptase genotyping, remain undefined. Through cloning, long-read sequencing, and assembling of the human tryptase locus from an individual with HαT, and validating our findings in vitro and in silico, we demonstrate that BST elevations arise from overexpression of replicated TPSAB1 loci encoding canonical α-tryptase protein owing to coinheritance of a linked overactive promoter element. Modeling BST levels based on TPSAB1 replication number, we generate new individualized clinical reference values for the upper limit of normal. Using this personalized laboratory medicine approach, we demonstrate the clinical utility of tryptase genotyping, finding that in the absence of HαT, BST levels >11.4 ng/mL frequently identify indolent clonal mast cell disease. Moreover, substantial BST elevations (eg, >100 ng/mL), which would ordinarily prompt bone marrow biopsy, can result from TPSAB1 replications alone and thus be within normal limits for certain individuals with HαT.

Graphical abstract

Figure 1.

TPSAB1 duplications occur at 16p13.3 and are linked to an expanded promoter associated with α^DUP-tryptase overexpression. (A) Location and orientation of the 15-kb tandem duplication of TPSAB1. (B) Alignment of α^DUP-, α^WT-, and β-tryptases with unique 5′ variants and size of promoter repeat regions indicated; (C) repeat motif within promoter regions. Relative total α- to β-tryptase gene expression (D) and α^DUP-tryptase relative to α^WT-tryptase gene expression (E) in ex vivo basophils and cultured primary mast cells. ∗P < .05; ∗∗P < .005.

Figure 2.

The expanded promoter at duplicated TPSAB1 is conserved and has increased basal activity. (A) Normalized fluorescent reporter activity in unstimulated MonoMac-6 cells transfected with promoters cloned from endogenous (α^WT) or replicated (α^DUP) TPSAB1 loci. (B) BST from individuals grouped by TPSAB1 replication number. (C-D) Relative allelic frequency (C) and ratios (D) of α^WT-tryptase (y-axis) and α^DUP-tryptase (x-axis) associated 5′ variants determined by ddPCR. Dashed lines indicate predicted α^WT:α^DUP ratios by genotype. ∗P < .05.

Figure 3.

Modeling serum tryptase levels based on genotype improves clinical utility. (A) Normalized (left) and total (right) read counts for reads aligning exactly to the 39-bp consensus sequences that identify β-, α^WT-, and α^DUP-tryptase. (B) BST levels among individuals with conserved 4n tryptase copy number (combined from TPSAB1 and TPSB2). (C) Regression analysis of relative expression levels of α^DUP-tryptase (y-axis) and α^WT-tryptase (x-axis) transcripts. (D) Prediction intervals for BST levels based on TPSAB1 replication number. (E) Prevalence of HαT, clonal MCD, and those without either among individuals referred with BST levels above the predicted upper limit of normal (>11.4 ng/mL). ∗P < .01; ∗∗P < .005; ∗∗∗P < .0001.

Figure 4.

Tryptase genotyping in the evaluation of patients with elevated BST. Stepwise approach to a patient workup based on BST level and tryptase genotype using the BST CALCULATER. Myeloid neoplasms often exist in the absence of elevated BST; this algorithm is intended only to aid in the correct interpretation of elevated BST when other indications for workup are absent/nonspecific. AD, autosomal dominant; NGS, next-generation sequencing; ULN, upper limit of normal. ∗At the time of this publication, this is the only Clinical Laboratory Improvement Amendments/College of American Pathologists–certified laboratory performing tryptase genotyping. ^†Only α-tryptase–encoding TPSAB1 replications are associated with elevated BST and require correction. ^‡BST elevation is not a requirement for any clonal neoplasm, and evaluation should be guided by clinical presentation and findings. ^§Allele-specific PCR or ddPCR should be used because of low allelic frequency.