Identification of α and β tryptase genes in human mast cell lines
LAD2 cells, which are monozygous for chromosome 16, possess one α and one βII gene.34
Compared to α tryptase cDNA sequence M30038.1, LAD2 α gene (accession #FJ931116) exons contain synonymous SNPs c.309A>G and c.333A>C and nonsynonymous SNPs c.44–46GCG>CGG, c.253A>G, and c.508C>T, which lead to Arg15
Ala, and Pro170
LAD-2 βII gene (accession #FJ9311117) exons are identical to M33492.32
HMC-1 cells lack both tryptases identified in LAD2 cells, instead having two βI and two βIII genes. One HMC-1 βI cDNA is identical to M33494, whereas the other (accession #FJ9311118) contains SNP c.382G>A, which results in an amino acid change (Glu121
Of two HMC-1 βIII cDNAs identified, one matches AF099143 except for a one-base insertion (c.980_981insC, accession# FJ9311119).22
The other (accession #FJ9311120) contains synonymous SNP c.420C>T and non-synonymous SNP c.421A>G (Thr141
Ala and c.719G>A (Gly240
Discovery and characterization of a frame-shifted βIII tryptase allele
The c.980_981insC mutation identified in a HMC-1 βIII cDNA results in a frame-shifted tryptase (βIIIFS) with truncation of 109 amino acids () and creates a BslI restriction fragment length polymorphism, which we confirmed using PCR of genomic DNA followed by BslI digestion (data not shown). Databank mining revealed a small intestine partial cDNA (DC387615) that is identical to βIIIFS in regions of overlap. To establish whether βIIIFS genes are common, we determined carrier frequency in several populations using PCR-based genotyping, which reveals that βIIIFS prevalence varies strikingly, being present in 23% and 19%, respectively, of individuals of European and African ancestry, but in 0% of surveyed Chinese and Japanese.
FIG 2 Alignment of frame-shifted and full-length βIII tryptases. The Panel A chromatogram identifies the cytosine insertion (arrow) in βIIIFS. Red, blue, black, and green signify thymine, cytosine, guanine, and adenine, respectively. Panel B (more ...)
Modeling of βIII and frame-shifted βIII tryptases
Homology models of full-length βIII and βIIIFS
() show that the 109-residue truncation deletes βIIIFS
residues crucial for substrate specificity and catalysis. Specifically, the deleted segment includes “specificity triad” residues Asp188
, and Gly225
, which shape the pocket accommodating the substrate P1 side chain at the site of hydrolysis.36
Truncation removes nearly half of the catalytic domain, including the catalytic Ser195
, which is universally present in active serine peptidases. Therefore, translated βIIIFS
likely lacks enzyme activity.
FIG 3 Models of full-length and frame-shifted βIII tryptases. Full-length (A and C) and frame-shifted (B and D) models are shown with “specificity triad” Asp188, Gly215, and Gly225 in green, catalytic Ser195 in red, and N-glycosylation (more ...)
Tracking additional β-tryptase polymorphisms
The HMC-1 Glu121Lys variant of βI-tryptase is detected solely in the Europeans, where it is present in 7% of genomes. Indeed, every individual with this βI SNP also possesses the βIIIFS mutation, comprising a haplotype perhaps limited to individuals of European ancestry. Only 22% of genomes with a βIIIFS mutation also possess the βI Glu121Lys mutation, suggesting that the βIIIFS mutation, which is also present in Africans, occurred earlier in tryptase evolution. Homology modeling of this βI variant does not predict a functional change (data not shown). By contrast, the c.421A>G (Thr141Ala) βIII SNP is present in all four populations, and, indeed, is the dominant form, representing 90%, 91% and 70% of African, Asian and European βIII genes, respectively. Modeling of Thr141Ala βIII tryptase does not predict altered protease function. Based on the likely functional significance of its truncation, we prioritized βIIIFS for genotyping in population screens.
α/β Tryptase genotyping assay
We developed an assay that simultaneously identifies α and β tryptase alleles, including α, βI, βII, βIII and βIIIFS
in individual samples. Since there are two relevant loci (TPSAB1
), each individual inherits four alleles. All known α-tryptases uniquely feature a SNP (c.733G>C), which results in a Gly215
Asp mutation that distorts the substrate binding site and limits peptidase activity. 25, 26
βII tryptases are defined by another SNP (c.396C>G), which results in an Asn102
Lys mutation that eliminates a highly conserved glycosylation site.37
βI and βIII genes are detected by allele-specific SNPs c.158C and c.158G, respectively. This assay accurately genotypes two BACs (324 and 48, of established tryptase content) and two cell lines (LAD2 and HMC-1), whose tryptase gene content is determined here by sequencing individual cDNAs and genes.23
Distribution of alleles in populations of African, Asian and European ancestry
Using this assay, we identified alleles in 270 individuals with a range of ancestries. Overall, as revealed in and Table E1 in the Online Repository, βII is the most common allele (frequency 0.33). The least abundant allele type is βIII (including βIIIFS), being half as common (frequency 0.17) as βII (P <0.0001). Nonfunctional α frequency (0.26) does not differ substantially from that of functional βI (0.23). Further analysis by subgroups reveals striking population-specific differences. For example, α tryptase is nearly twice as common in Asians than Africans (JPT/CHB = 0.35/0.34 vs. YRI = 0.18; P <0.0001) and is slightly more common in Asians than in Europeans (JPT/CHB = 0.35/0.34 vs. CEU = 0.26; P = 0.01). Similarly, βII is over twice as prevalent in Asians as in Africans or Europeans (JPT/CHB = 0.55/0.49, YRI = 0.18, CEU = 0.26; P <0.0001). Conversely, βI and βIII are strikingly enriched in Africans and Europeans (0.36 and 0.17 in YRI, 0.27 and 0.18 in CEU) compared to Asians (JPT/CHB = 0.06/0.09 and 0.04/0.07; all P <0.0001). Africans and Europeans differ in frequency of α (0.18 vs. 0.26, respectively; P = 0.01) and βI (0.36 and 0.27, respectively; P = 0.02). Japanese and Chinese do not differ significantly with respect to any allele. Individual populations are in Hardy-Weinberg equilibrium; however, pooled populations depart from equilibrium (P <0.001 and <0.04 for TPSAB1 and TPSB2, respectively), as expected of polymorphic genes in reproductively separated populations.
FIG 4 Tryptase allele frequencies in HapMap project populations from Yoruba in Ibadan Nigeria (African), Centre d’Etude du Polymorphisme Humain collection from Utah (European), Beijing Han (Chinese), and Tokyo Japanese. Overall allele frequencies are (more ...)
Estimation of linkage disequilibrium
To explore linkage between specific alleles at TPSAB1 and TPSB2, we calculated two measures of linkage disequilibrium (r2 and D'), which is strongly positive when populations are pooled (r2 = 0.83, D’ = 0.85, P <0.0001) or considered individually (r2/D’ is 0.94/0.99, 0.63/0.96, 0.80/0.99, 0.90/0.91 in CEU, YRI, CHB, and JPT, respectively; all P <0.0001).
Tryptase haplotypes and their corresponding frequencies in each population are shown in and Table E2 in the Online Repository. Given five alleles and two loci, 15 haplotypes are possible but only seven are encountered. The two major haplotypes (), defined as frequency ≥15%, are α–βII and βI–βIII, which together represent 77% of all chromosomes. Indeed, α-βII and βI-βIII are the only haplotypes present in all populations. Consistent with observed allele frequencies, α-βII is much more common in Asians than in Africans and Europeans (JPT/CHB = 0.68/0.71, YRI = 0.36, CEU = 0.44; P <0.0001), whereas βI-βIII exhibits the opposite trend (JPT/CHB = 0.09/0.14, YRI = 0.34, CEU = 0.36; P <0.0001). Africans and Europeans do not differ in frequency of the two major haplotypes, as is also true of Japanese vs. Chinese populations.
Tryptase haplotype frequencies in four populations. Haplotypes with overall frequency ≥ 0.15 and < 0.15 are defined as major and minor, respectively.
In addition to the two major haplotypes, we found five minor haplotypes () with frequencies <15%, some of which are enriched in specific populations. For example, βII-βII is much more common in Asians than in others. Indeed, in the Japanese, βII–βII is more prevalent than βI–βIII (P = 0.04), which is a major haplotype in the overall survey but is present in just 9% of Japanese chromosomes. The results also suggest that minor haplotype α–βI is “European” whereas βI–βII and βI–βI are “African”. The βI-βIIIFS haplotype is found solely in the Africans and Europeans, and does not differ between them.
The haplotype data also indicate that α and βIII (including βIIIFS
) are restricted to TPSAB1
, respectively, which is consistent with maps of tryptase loci cloned into BACs.22, 23
Thus, no α–α or βIII-βIII chromosomes are detected. Furthermore, the α-tryptase gene is almost always co-inherited with βII at the neighboring locus (linkage frequency = 0.94; 95% CI 0.91–0.98). More strikingly, βIII and βIIIFS
are 100% linked to βI at the neighboring locus. On most chromosomes, βI and βII are restricted to TPSAB1
, respectively, but in 16% of chromosomes βII and βI apparently occupy the adjacent locus and both can form haplotypes with themselves (i.e., βII-βII and βI-βI).
Testing of haplotype predictions in family trios
Modes of inheritance of alleles based on the seven predicted haplotypes are fully Mendelian in all YRI and CEU trios, thereby validating the haplotype predictions made on the basis of statistical association of alleles (see Fig E1 in the Online Repository).
Population-selective differences in active tryptase gene dosage
Strikingly, not a single Asian (JPT and CHB) sample possesses the βIIIFS deficiency allele (). However, Asians are enriched for the other known deficiency allele, α. Consequently, overall frequency of dysfunctional alleles (α + βIIIFS) in Europeans, Chinese, and Japanese is not statistically different, and the Africans have the lowest frequency (; P = 0.007 YRI vs. CEU; P <0.0001 YRI vs. JPT or CHB). Analysis of individuals with respect to number of inherited active alleles reveals dramatic skewing between populations (). Specifically, 30% of Africans inherit four active alleles compared to just 2% of Japanese (P <0.0001). Four active alleles are found in 13% and 9%, respectively, of Europeans and Chinese (P = 0.002 for both YRI vs. CEU and YRI vs. CHB). At the other extreme, 21% of Africans inherit just two active tryptase alleles, compared to 41%, 47%, and 40% of Europeans, Chinese, and Japanese, respectively (P = 0.03 YRI vs. JPT; P = 0.006 YRI vs. CEU; P = 0.003 YRI vs. CHB). Europeans, Chinese, and Japanese do not differ significantly with respect to number of inherited active tryptases.
Active tryptase gene dosage in four populations. *, **, *** represent P values < 0.05, < 0.001, and < 0.0001, respectively.
Protection against total tryptase deficiency
The results suggest that dysfunctional tryptase alleles are always co-inherited with functional alleles. Despite overall high prevalence of dysfunctional alleles, and the existence of dysfunctional alleles at TPSAB1 and TPSB2, we find no individual chromosomes with two dysfunctional alleles (i.e., theoretical haplotypes α–α, α–βIIIFS, and βIIIFS-βIIIFS). Thus, individuals with fewer than two functional alleles are notably absent. The observed strong linkage of deficiency and functional alleles protects from inheritance of less than two functional alleles.