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Serum mast cell tryptase levels are used as a diagnostic criterion and surrogate marker of disease severity in mastocytosis. Approximately 29% of the healthy population lacks α tryptase genes; however, it is not known whether lack of α tryptase genes leads to variability in tryptase levels or impacts on disease severity in mastocytosis. We have thus analyzed tryptase haplotype in patients with mastocytosis, computing correlations between haplotype and plasma total and mature tryptase levels; and disease category. We found: (1) the distribution of tryptase haplotype in patients with mastocytosis appeared consistent with Harvey-Weinberg equilibrium and the distribution in the general population;(2) the disease severity and plasma tryptase levels were not affected by the number of α or β tryptase alleles in this study; and (3) information about the tryptase haplotype did not provide any prognostic value about the severity of disease. Total and mature tryptase levels positively correlated with disease severity, as well as prothrombin time and partial thromboplastin time, and negatively correlated with the hemoglobin concentration.
An elevated baseline total tryptase level is a minor diagnostic criterion for systemic mastocytosis according to World Health Organization criteria. Tryptases are serine proteases produced by mast cells [2; 3]. Among members of the tryptase family in humans, including α, β, δ , ε  and γ (transmembrane) [6; 7], the two that are most abundantly expressed in and secreted by mast cells are α and β tryptases. Tryptases ε and γ are ≤50% identical to α and β tryptases, while δ-tryptase, though more closely related to α/β-tryptases, is truncated 40 amino acids shy of the C-terminus of α/β tryptases and appears to be less abundantly expressed [8; 9]. Human mature β tryptase is stored in mast cell granules and released upon activation while α tryptase is apparently processed only to the proenzyme stage and is constitutively secreted from mast cells along with β protryptase . Therefore, serum or plasma levels of mature β tryptase are found transiently elevated after a mast cell degranulation event such as anaphylaxis, while the levels of the precursors of α/β tryptase are reported to reflect the total body mast cell burden; and to be elevated in patients with mastocytosis when compared to the general population [11; 12; 13].
The genes encoding α and β tryptases are located in 2 loci in close proximity to each other on chromosome 16p13.3 . Mapping data suggests that α alleles compete with β alleles at one locus, while an adjacent locus contains β alleles exclusively (αβ versus ββ haplotype) . Accordingly, it has been shown that 20-29% of the normal population lacks α tryptase and has a ββ:ββ genotype [10; 14; 15].
The αβ tryptase haploytpe and female gender are associated with a small but statistically significant elevation of circulating tryptase levels in healthy individuals . It is, however, not known whether patients with mastocytosis have the same allelic distribution of α and β tryptase genes as the general population and whether those lacking the α tryptase gene have a corresponding decrease in circulating tryptase protein levels. The World Health Organization’s (WHO) diagnostic criteria for systemic mastocytosis do not take the α/β tryptase haplotype into account. Because of the potential impact of this haplotype on these criteria, we have examined the α/β tryptase genes in a cohort of patients with mastocytosis and evaluated the correlations of the α/β tryptase genotype with category of disease, serum tryptase levels and hematologic laboratory values.
Thirty one patients (13 males) with mastocytosis were examined after signing informed consent. All patients were diagnosed and classified into a disease category according to the World Health Organization’s criteria. The numbers of patients in each disease category in the order of increasing disease severity were as follows: 4 patients with pediatric-onset cutaneous mastocytosis (CM), 24 with indolent systemic mastocytosis (ISM) (9 with the subvariant smouldering systemic mastocytosis [SSM]), 2 with systemic mastocytosis with associated clonal hematologic non-mast-cell lineage disease (SM-AHNMD) and 1 with aggressive systemic mastocytosis (ASM).
Plasma was collected in EDTA tubes and separated with centrifugation for 10 minutes at 750g. Tryptase levels were measured by immunoassay, using G5 and G4 monoclonal anti-tryptase antibodies to capture mature (mainly β) and total tryptase (combination of α, and β protryptases and mature [primarily β] tryptases) respectively, as described . Direct ELISA measurement of α protryptase is not currently possible as there is no antibody that distinguishes α from β pro or mature tryptases. Determination of α and β tryptase haplotypes in the genomic DNA from peripheral blood leukocytes was performed by PCR using primers across the site of the intron 4 deletion in the α tryptase gene as described .
Statistical analyses were carried out by using non-parametric Spearman’s correlation test. For the purpose of correlating disease severity with other parameters, each disease category described above was assigned a numerical severity value from 1-5 according to extent of disease and prognosis, with cutaneous mastocytosis as 1 and aggressive systemic mastocytosis as 5. Results with p-values less than 0.05 were considered statistically significant.
Seven of 31 (23%) patients with mastocytosis lacked the α tryptase gene while 8 (26%) patients had 2 α tryptase alleles. This distribution is similar to that of the general population and is consistent with a population in Hardy-Weinberg equilibrium. These results suggest that patients with mastocytosis do not have a skewed distribution of α tryptase alleles.
There was a strong correlation between the severity of mastocytosis category and plasma total, and mature tryptase levels (Spearman’s r 0.81, p<0.001; and 0.55, p<0.01 respectively). There was, however, no correlation between the category of mastocytosis and tryptase genotype (Table 1).
Total and mature plasma tryptase levels were not significantly associated with the number of β alleles in tryptase genotypes (Figure 1). Although there appeared to be a trend of increasing mature tryptase levels with the number of β alleles, this was not statistically significant (p= 0.218).
Analysis of tryptase haplotype with respect to routine hematologic parameters also failed to show any significant correlation with WBC or platelet counts. Total and mature tryptase levels inversely correlated with hemoglobin (r= -0.36 and -0.37 respectively, p<0.05), and positively correlated with the prothrombin time (PT) (r=0.65, p<0.001 and r=0.56, p<0.01, respectively) and the partial thromboplastin time (PTT) (r=0.46 and 0.37; p<0.05 for both). These parameters did not correlate with the number of β alleles and the correlations of both PT and PTT with these tryptase levels were essentially unchanged after adjustment was made for the α/β tryptase genotype.
Baseline total tryptase levels in mastocytosis are thought to reflect enzymatically inactive α and β protryptases that are constitutively secreted, even though a portion of β-protryptase undergoes intracellular processing to yield mature tryptase and is then stored in secretory granules . Circulating protryptase levels thus appear to reflect the spontaneous secretion of both α and β protryptases, and possibly other tryptase precursors. Our results support the conclusion that while some patients with mastocytosis lack the α tryptase gene (ββ:ββ tryptase genotype), the deficiency of α tryptase does not appear to impact disease severity or total tryptase levels.
Min et al. have examined the effect of tryptase haplotype and gender on total tryptase levels in 106 healthy subjects, and found that the presence of an αβ haplotype was associated with a slight but statistically significant increase (0.5 ng/ml) above the mean in the serum level of total tryptase . As mature tryptase levels are normally undetectable in healthy subjects at baseline, this study could not correlate the number of beta alleles with the serum level of mature tryptase.
Analysis of our data also revealed a previously unreported correlation between tryptase levels (both total and mature) and PT and PTT, although neither parameter correlated with tryptase genotype. The exact mechanism of this correlation is beyond the scope of this paper, however, one possible explanation may be the partial fibrinogenolytic activity of tryptase . While the vast majority of circulating tryptase is enzymatically inactive protryptase, small quantities of mature tryptase released over time may increase fibrinogen degradation.
In conclusion, our data highlight the contribution of non-α protryptases (such as β protryptase) to baseline tryptase levels in patients with mastocytosis, and suggest that routine genotyping of tryptase alleles does not have any place in routine diagnostic workup of the patients with mastocytosis. The recent discovery of α tryptase deficiency thus should not lead to reconsideration of the WHO diagnostic criteria for systemic mastocytosis.
This work is supported by NIH grants HL024136 (G.H.C. and D.S.) and AI20487 (LBS), and the Division of Intramural Research, NIAID.
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