WO2013090377A1 - Methods and compositions for tryptase genotyping - Google Patents

Abstract

Methods and compositions for determining the tryptase genotype of a subject. Such methods and compositions are useful, for example, for the diagnosis and/or stratification of subjects having, or at risk of developing, diseases associated with abnormal tryptase activity.

Description

METHODS AND COMPOSITIONS FOR TRYPTASE GENOTYPING

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No. 61/570,038, filed December 13, 2011 , U.S. Provisional Application No. 61/590,036, filed January 24, 2012, and French Patent Application Number 1261623 filed December 4, 2012. The contents of these applications are each hereby incorporated by reference in their entireties.

BACKGROUND OF THE INVENTION

Tryptases are a family of serine peptidases that are the major component of mast cell secretory granules. These enzymes are involved in both allergic inflammation and host defense. For example, tryptases have been shown to augment histamine- induced bronchoconstriction in isolated airways models, and tryptase inhibitors reduce late-phase antigen-induced bronchoconstriction in asthmatic subjects. Moreover, mice lacking tryptase genes are more likely to die from Klebsiella pneumoniae and have diminished eosinophil recruitment and helminth containment after Trichinella spiralis infection.

Human soluble tryptases are produced by the TPSD1, TPSAB1, and TPSB2 genes, however, only the TPSAB1 , and TPSB2 genes are capable of producing active tryptase isoforms. At the TPSAB1 locus the active βΐ and inactive a alleles compete, whereas at the TPSB2 locus the active βΙΙ or βΙΙΙ alleles and inactive plllfs alleles compete. Thus, a single individual can theoretically have 0, 1, 2, 3 or 4 alleles that encode an active tryptase enzyme (Trivedi et al., (2009) J. Allergy Clin Immunol, 12:5, 1099-1105).

Given the importance of tryptase activity in both allergic inflammatory disease and host defense, there is a need in the art for methods of determining the tryptase genotype of a subject and, hence, the number of active tryptase alleles present in that subject.

SUMMARY OF THE INVENTION

The invention provides methods and compositions for determining the tryptase genotype of a subject. The methods and compositions are particularly advantageous in that they allow for the rapid, reliable and reproducible determination of the number of active tryptase alleles present in that subject. Such methods and compositions are useful, for example, for the diagnosis and/or stratification of subjects having, or at risk of developing, diseases associated with abnormal tryptase activity.

Accordingly, in a first aspect, the invention provides a method of detecting an oc- tryptase allele in a nucleic acid sample. The method generally comprises: providing a nucleic acid sample comprising a portion of a human TPSAB1 genomic locus;

amplifying from the nucleic acid sample a first portion of the TPSAB1 genomic locus to produce a first amplicon, wherein the first amplicon comprises positions 831-841 or 823-834 of the TPSAB1 gene; amplifying from the first amplicon a second portion of the TPSAB1 genomic locus to produce a second amplicon, wherein the second amplicon comprises positions 831-841 or 823-834 of the TPSAB1 gene; and determining the presence of an intron 4 deletion in the second amplicon, wherein the presence of the intron 4 deletion is indicative of the presence of an oc-tryptase allele.

In certain embodiments, the nucleic acid sample is a genomic DNA sample.

In certain embodiments, the first and/or second amplicon is produced using oligonucleotide primers. In one preferred embodiment, the oligonucleotide primers used to produce the first amplicon do not overlap the oligonucleotide primers used to produce the second amplicon. In one preferred embodiment, the first amplicon is produced using oligonucleotide primers comprising the nucleic acid sequences set forth in SEQ ID No: 3 and 4. In one preferred embodiment, the second amplicon is produced using oligonucleotide primers comprising the nucleic acid sequences set forth in SEQ ID No: 5 and 6. The oligonucleotide primers can be linked to a label, (e.g., a fluorescent molecule).

In certain embodiments, the first and/or second amplicon is produced by polymerase chain reaction (PCR) amplification.

In certain embodiments, the determining step is performed using capillary electrophoresis.

In another aspect, the invention provides a method of detecting an inactive βΙΙΙ- tryptase allele in a nucleic acid sample. The method generally comprises: providing a nucleic acid sample comprising a portion of a human TPSB2 genomic locus; amplifying from the nucleic acid sample a first portion of the TPSB2 genomic locus to produce a first amplicon, wherein the first amplicon comprises nucleic acid positions 980 and 981 of the TPSB2 gene; amplifying from the first amplicon a second portion of the TPSB2 genomic locus to produce a second amplicon, wherein the second amplicon comprises nucleic acid positions 980 and 981 of the TPSB2 gene; and determining the presence of a BslI restriction site in the second amplicon, wherein the presence of the BslI restriction site is indicative of the presence of an inactive βΙΙΙ-tryptase allele.

In certain embodiments, the nucleic acid sample is a genomic DNA sample.

In certain embodiments, the first and/or second amplicon is produced using oligonucleotide primers. In one preferred embodiment, the oligonucleotide primers used to produce the first amplicon do not overlap the oligonucleotide primers used to produce the second amplicon. In one preferred embodiment, the first amplicon is produced using oligonucleotide primers comprising the nucleic acid sequences set forth in SEQ ID No: 7 and 3. In one preferred embodiment, the second amplicon is produced using oligonucleotide primers comprising the nucleic acid sequences set forth in SEQ ID No: 8 and 9. The oligonucleotide primers can be linked to a label, (e.g., a fluorescent molecule).

In certain embodiments, the first and/or second amplicon is produced by polymerase chain reaction (PCR) amplification.

In certain embodiments, the determining step is performed using capillary electrophoresis.

In another aspect, the invention provides a method of determining the number of inactive tryptase alleles in a nucleic acid sample. The method generally comprises: detecting the presence or absence of an oc-tryptase allele in the sample using the methods of the invention; detecting the presence or absence of an inactive βΙΙΙ-tryptase allele in sample using the methods of the invention; and determining the sum of the number of oc- tryptase and inactive βΙΙΙ-tryptase alleles detected in the sample.

In another aspect, the invention provides a kit for genotyping a TPSAB1 genomic locus comprising four oligonucleotide primers, having the nucleic acid sequences set forth in set forth in SEQ ID Nos: 3, 4, 5 and 6.

In another aspect, the invention provides a kit for genotyping a TPSB2 genomic locus comprising four oligonucleotide primers, having the nucleic acid sequences set forth in set forth in SEQ ID Nos: 3, 7, 8 and 9.

In another aspect, the invention provides, an isolated oligonucleotide consisting of the nucleic acid sequence set forth in SEQ ID No. 3, 7, 8, or 9. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is schematic representation of allelic competition at the human TPSAB1 and TSPB2 genetic loci.

Figure 2 depicts the output from an exemplary TPSABl genotyping analysis using methods of the invention. A sample homozygous for alpha alleles exhibits a single peak of 124bp, a sample homozygous for beta alleles exhibits a single peak of 134bp, and a heterozygous sample exhibits peaks of 124bp and 134bp.

Figure 3 depicts the output from an exemplary TPSB2 genotyping analysis using methods of the invention. A sample homozygous for active beta alleles exhibits a single peak of 234bp, a sample homozygous for inactive plllfs alleles exhibits a single peak of 182bp, and a heterozygous sample exhibits peaks of 182bp and 234bp.

DETAILED DESCRIPTION

The invention provides methods and compositions for determining the tryptase genotype of a subject. The methods and compositions are particularly advantageous in that they allow for the rapid, reliable and reproducible determination of the number of active tryptase alleles present in that subject. Such methods and compositions are useful, for example, for the diagnosis and/or stratification of subjects having, or at risk of developing, diseases associated with abnormal tryptase activity.

I. Tryptase Alleles

Human soluble tryptases are produced by the TPSD1, TPSABl, and TPSB2 genes, however, only the TPSABl , and TPSB2 genes are capable of producing active tryptase isoforms. At the TPSABlloc s, the active βΐ and inactive a alleles compete, whereas at the TPSB2 locus, the active βΙΙ or βΙΙΙ alleles and inactive plllfs alleles compete. Thus in a single individual can have 0, 1, 2, 3 or 4 active alleles (see, for example, Trivedi et al., (2009) J. Allergy Clin Immunol, 12:5, 1099-1105, which is hereby incorporated by reference in its entirety).

The human TPSABl, and TPSB2 gene loci are found at chromosome 16pl3.3. The genomic DNA sequences and RNA transcripts of both loci are known. See for example, Unigene entries Hs.405479 for TPSABlaad Hs.592982 for TPSB2. Portions of exemplary TPSABl, and TPSB2 genes are set forth below in SEQ ID No:l and 2 respectively. At the TPSAB1 locus, βΐ and a alleles can be distinguished by the presence of a lObp or 1 lbp deletion in the sequence of intron 4 in a alleles relative to βΐ alleles. An exemplary intron 4 deletion (positions 831-841) is highlighted in bold in the TPSAB1 sequence set forth below in SEQ ID No:l . Another exemplary intron 4 deletion (positions 823-834) is indicated by square brackets in the TPSAB1 sequence set forth below in SEQ ID No :1.

At the TPSB2 locus, βΙΙ or βΙΙΙ alleles can be distinguished from the plllfs allele by the presence of a single base insertion (deoxycytidine) between positions 980 and 981 of the genomic sequence of the plllfs allele relative to the βΙΙ or βΙΙΙ alleles (see, for example, Trivedi et al., (2009) J. Allergy Clin Immunol, 12:5, 1099-1105, which is hereby incorporated by reference in its entirety). The deoxycytidine insertion in the plllfs allele creates a BslI restriction endonuclease site in plllfs allele that is not found in the βΙΙ or βΙΙΙ alleles. Hence, the plllfs allele can be further distinguished from the βΙΙ or pill alleles by Restriction Fragment Length Polymorpism (RFLP) using the BslI restriction enzyme (see, for example, Trivedi et al., (2009) J. Allergy Clin Immunol, 12:5, 1099-1105, and U.S. Patent No. 5,866,398, which are both hereby incorporated by reference in their entireties). An exemplary TPSB2 plllfs allele is set forth below (SEQ ID No:2) with the deoxycytidine underlined and the BslI restriction endonuclease site highlighted in bold.

(SEQ ID No:l)

GGGGCAGGCAGGGGGTCC TGGGGC TCAGAAGCGGAGGAGGTGGGCTGGAGGCCCCAGTGACCAACAGGCCC AGGGGAGTCGGGAGGTGGGGTGGACGGACCTGCAGGGACAGTGTCAGCGC TGAATGGGATGGAGAGCACAG GGAGCTGGGGCCGGGGATGAGACCATGGGGAGCTGGGGCTGGGGCTGGGACTAGTCCATGGGGAGCTGGGG CTGAAGTTGGGGGTGAGTCCATGGGGAGCTGGGCTGGGCCTCCTGGGGTTGCACCTGCACTCCTCTCTGC T CTTCCC TCTGCGTATGAAGCTCAGATCCCATGATAAGGAGGCACCTGCAGACCAGGGGACCTGCACGGACA GCCCCAGAGGTGGACATTGAGGAC TCGTAGGAGGACTTGGGTCTCATACGGCGGGTGGGGAGCAGGGCCCC TTCCTGGCTGAGGACACT TGGTGC TGTCCCCTCTCAAGGCTGTT TCCCCATC TGACAAAGGGGTCTCACGT GAGCCCCCAACCAAGTGAGTCGAGGAGGGCTGGCCCCACCCCCGTGGATTCGGAGTCCGTAGGAGGGGTGT CACCCGTCATGTCCCCACCCCGTGGGCACCTTCCCGTC TCTTGGAGCGTGGCCCATGGACATGAGTTCCTC ACCCGTGTCCCTCTTGGGGAAACAGGTTTCAGGAGCGACGGGTC TTGTAGCC TGGGGCAGCCAGGCCACC T GGGTGCAGCAATGCCTGAAGGCCTCCTGGCACCGAGACAGGGGCAGGAGCAGATCCCACCAGCGGGAAGGT GGTGGGTTCCAGTGCTGGGATCCACCAGCTGACAGGTGGAGCTGCCAGTC TCCAGTGC TCAGCCCTCAGCG GGGCCTGCCTGGCAGCCCCACACACAGAGGGCATCGGGGTGGCGGGGGCACGTGTTACACGGGGGCCCTGG GTCTGAGTCATCCACTTCCTCCGAGTCTGGATGGGAGGACCCAGCGCCCC TCCTCCGCCCCCTCCTGATC T GGAAGGATAAATGGGGAGGGGAGAGCCCACTGGGTAGAAGGAACAGGGAGCGGCCAGGGTAAGTCCCCAC T CTCAGAGACCCTGACATCAGCGTCACCTGGAGCAGAGTGGCCCAGCCTCAGACTCAGAGCACCAAGACCCA GGCCCGCAGGCC TGGACCCACCCCGGTCCCCCCGTCCCAGCTCCATTCTTCACCCCACAATCTGTAGCCCC CAGCCC TGCCCTGTGAGGCCCGGCCAGGCCCACGATGC TCCTCC TTGCTCCCCAGATGCTGAATCTGCTGC TGCTGGCGCTGCCCGTCC TGGCGAGCCGCGCC TACGCGGCCCCTGGTGAGTCCCAGCCGGGGTCCACCCTG CCCCTCACCACATTCCACAGGTCAGGGCCTGGGTGGGT TCTGGGGAGGTCGGGCTGGCCCCCACACAGGGA AGGGCTGGGCCCAGGCCTGGGGCTGCTTCCTGGTCCTGACCTGGCACCTGCCCCAGCCCCAGGCCAGGCCC TGCAGCGAGTGGGCATCGTCGGGGGTCAGGAGGCCCCCAGGAGCAAGTGGCCCTGGCAGGTGAGCCTGAGA GTCCACGGCCCATACTGGATGCAC TTCTGCGGGGGCTCCCTCATCCACCCCCAGTGGGTGCTGACCGCAGC GCACTGCGTGGGACCGTGAGTCTCCCGGGGCCTGGAGGGGTGGGGAAGGGCTGGATGTGAGCCCTGGCTCC CGGGTGCTCCTGGGGGCTGCCCAGGGCCCTGAGTGGGATCCTCCGCTGCCCAGGGACGTCAAGGATCTGGC CGCCCTCAGGGTGCAACTGCGGGAGCAGCACCTCTACTACCAGGACCAGCTGCTGCCGGTCAGCAGGATCA TCGTGCACCCACAGTTCTACACCGCCCAGATCGGAGCGGACATCGCCCTGCTGGAGCTGGAGGAGCCGGTG AACGTCTCCAGCCACGTCCACACGGTCACCCTGCCCCCTGCCTCAGAGACCTTCCCCCCGGGGATGCCGTG CTGGGTCACTGGCTGGGGCGATGTGGACAATGATGGTGGGTCTGGGGACAGTGGAGGTGGGGCCAGGGTCT TAGCCACAGCCCAGCCCCTGGGCTCC [CTCTGGGCTCC]AGGTGGGGGTTGCCCGGCCCCCTCCTCAGGCT GCACCCTCTTCCCCACCTGCAGAGCGCCTCCCACCGCCATTTCCTCTGAAGCAGGTGAAGGTCCCCATAAT GGAAAACCACATTTGTGACGCAAAATACCACCTTGGCGCCTACACGGGAGACGACGTCCGCATCGTCCGTG ACGACATGCTGTGTGCCGGGAACACCCGGAGGGACTCATGCCAGGTGGGCCCCGCCTGTCCCCCGCCCCCC GCCCCCCAACCCCCACTCCCAGGCCTGTTCGGCGAGCGCTGACCTCTGACCTTCCCAGGGCGACTCCGGAG GGCCCCTGGTGTGCAAGGTGAATGGCACCTGGCTGCAGGCGGGCGTGGTCAGCTGGGGCGAGGGCTGTGCC CAGCCCAACCGGCCTGGCATCTACACCCGTGTCACCTACTACTTGGACTGGATCCACCACTATGTCCCCAA AAAGCCGTGAGTCAGGCCTGGGTTGGCCACCTGGGTCACTGGAGGACCAACCCCTGCTGTCCAAAACACCA CTGCTTCCTACCCAGGTGGCGACTGCCCCCCACACCTT [ Dl ] CCCTGCCCCGTCCTGAGTGCCCCTTCCTG TCCTAAGCCCCCTGCTCTCTTCTGAGCCCCTTCCCCTGTCCTGAGGACCCTTCCCTATCCTGAGCCCCCTT CCCTGTCCTAAGCCTGACGCCTGCACCGGGCCCTCCAGCCCTCCCCTGCCCAGATAGCTGGTGGTGGGCGC TAATCCTCCTGAGTGCTGGACCTCATTAAAGTGCATGGAAATCACTGGTGTGCATCGCTGTGTTTCTGGTT GTGGATGTCACTGGGAGAGAAGGGGTCCAGGTGTGCTGAGGACACCTGCCACAGTGTGAGGTCCTAGCCCT CAAGGCACAGCCAGTCACCGTGGGACGGGGCCTCCTGGGCAGCCCTGGTCCCCGAGGCTGGCTTCTCCCCA CACGATGCATCCAGCATTCGGGTCACACAGAGCCACTCGGGCAACTCAGTTGATTATAAAGGACAGCCAGG TCCCTGCAACCGGGTCAAGACAGAGAATGGTCGCCGGGAGCCCCAGGGCTGCCCATCATGAGCCCCTACCC CACGCTTCCCACGAGCTCTTCTCCCGGCCCCTCTGTCCACTGCTTGTGCTTTGCCTAGTTGTTTGCTTTGA GACGGGATCTCGCTGTGTCATCCAGGCTGAAGTGCAGTGGTGTGATCAGGGCTCACTGCAGCCTTAACTCC TGGGCTCAAGCGATCCTCCCATCTTGGCCTCCCATATAGCTGGGCCACAGGCGTGAGCCACCACGCCCAGT TAATTTTTGTATTTTCAGTAGAGATGGGGTTTCGCCATGTTGGCCAGTCTGGTCTCGAACTCCTGACCTCA AGTGATCCGCCCGCCTCGGCCTCCCAAAGTGCTGGGATGACAGGCGTGAGCCACCGCACCCGGCCTGAGTT TGACATTTTCAAATTCATTTTGAGGTCTTTCTCTACATCAATAGGTGAGCCCTCTGCGTCTGGCGAGTGTT GCATTTTATCCCGGGCTCTTGTTTGCATTTTTTATTTGAACATGATTACACCCAGGAATGAAATGCGGGGC TGTTCTGGTTGAAAACAACTCTCTAAAGAAACATTCACTCTTTCCTTCCAACTGTCAGATGCAGAGATGTG CATTTAGTCTCTCCAATCTCTGCAAATGACCTCTGTCCTCACAAGGGGTGGACTCGACTCCCAGCGCCCTC TCCAGCCCCACGTGACCTCTGCCTCTGCAGCCCCTGAAGGCCCATCCCTC

(SEQ ID No:2)

AGTCCATGGGGAGCTGGGCTGGGGCTGGGGGTGAGTCCATGGGGAGCTGGGCTGGGGCTGGGGGTGAGTCC ATGGGGAGCTGGGGCTGGGGCTGGGACTAGTCCATGGGCAGCTGGGCTGGGGCTGGGGGTGAGTCCATGGG GAGCTGGGCTGGGGCTGGGGGTGAGTCCATGGGGAGCTGGGCTGGGGTTGGGGGTGAGTCCATGGGGAGCT GGGCTGGGTCTCCTGGGGTTGCACCTGCACTCCTGTCTGCCCTTCCCTCTGCCGATGAAGCTCAGATCCCA TGATAAGGAGGCACCTGCAGACCAGGGGTCCTGCACGGACAGCCCCAGAGGTGGACATTGAGGACTCGTAG GAGGACTTGGGTCTCATACGGCGGGTGGGGAGCAGGGCCCCTTCCTGGCTGAGGACACTTGGTGCTGTCCC CTCTCAAGGCTGTTTCCCCATCTGACAAAGGGGTCTCATGTGAGCCTCCCACCAAGTGAGTCGAGGAGGGC TGGCGCCACCCCCGTGGATTCGGAGTCCGTAAGAGGGGTGTCACCCGTCATGTCCCCACCCCGTGGGCACC TTCCCGTCTCTTGGAGGGTGGCCCATGGACATGAGTTCCTCACCCCGTGTCCCTCTTGGGGAAACAGGTTT CAGGAGCGATGGGTCTTGTAGCCTGGGACAGCCAGGCCACCTGGGTGCAGCAATGCCTGAAGGCCTCCTGG CACCGAGACAGGGGCAGGAGCAGATCCCACCAGCGGGAAGGTGGTGGGTTCTAGTGCTGGGATCCACCAGC TGACAGGTGGAGCTGCCAGTCTCCAGTGCTCAGCCCTCAGCGGGGCCTGCCTGGCAGCCCCACACACAGAG GGCATCGGGGTGGCGGGGGCACGTGTTACACGGGGGCCCTGGGTCTGAGTCATCCACTTCCTCCGAGTCTG GATGGGAGGACCCAGCGCCCCTCCTCCGCCCCCTCCTGATCTGGAAGCATAAATGGGGAGGGGAGAGCCCA CTGGGTAGAAGGAACAGGGAGCGGCCAGGGTAAGTCCCCACTCTCAGAGACCCTGACATCAGCGTCACCTG GAGCAGAGTGGCCCAGCCTCAGACTCAGAGCACCAAGACCCAGGCCTGCAGGCCTGGACCCACCCCGGTCC CCCCGTCCCAGCTCCATTCTTCACCCCACAATCTGTAGCCCCCAGCCCTGCCCTGTGAGGCCCGGCCAGGC CCACGATGCTCCTCCTTGCTCCCCAGATGCTGAATCTGCTGCTGCTGGCGCTGCCCGTCCTGGCGAGCCGC GCCTACGCGGCCCCTGGTGAGTCCCAGCCGGGGTCCACCCTGCCCCTCACCACATTCCACAGGTCAGGGCC TGGGTGGGTTCTGGGGAGGTCGGGCTGGCCCCCACACAGGGAAGGGCTGGGCCCAGGCCTGGGGCTGCTTC CTGGTCCTGACCTGGCACCTGCCCCAGCCCCAGGCCAGGCCCTGCAGCGAGTGGGCATCGTTGGGGGTCAG GAGGCCCCCAGGAGCAAGTGGCCCTGGCAGGTGAGCCTGAGAGTCCGCGACCGATACTGGATGCACTTCTG CGGGGGCTCCCTCATCCACCCCCAGTGGGTGCTGACCGCAGCGCACTGCGTGGGACCGTGAGTCTCCCGGG GCCTGGAAGGGTGGGGAAGGGCTGGATGTGAGCCCTGGCTCCCGGGTGCTCCTGGGGGCTGCCCAGGGCCC TGAGTGGGATCCTCCGCTGCCCAGGGACGTCAAGGATCTGGCCGCCCTCAGGGTGCAACTGCGGGAGCAGC ACCTCTACTACCAGGACCAGCTGCTGCCGGTCAGCAGGATCATCGTGCACCCACAGTTCTACACCGCCCAG created by the insertion of a deoxycytidine between positions 980 and 981 of the genomic sequence of the plllfs allele.

B. Amplification

The methods of the invention generally involve amplifying a portion of a TPSABl and/or TPSB2 genomic loci. In general, amplification is done in two steps. The first step involves amplification of a first portion of the TPSABl and/or TPSB2 genomic loci from a nucleic acid sample (e.g., human genomic DNA) to produce a first amplicon. The second step involves amplification of a second portion of the TPSABl or TPSB2 genomic loci from the first amplicon to produce a second amplicon.

Any method of amplifying portions of the TPSABl or TPSB2 genomic loci can be employed in the methods of the invention including without limitation, PCR, primer extension and RNA polymerase mediated amplification (see e.g., US Patent No 7,405,062 which is hereby incorporated by reference in its entirety). In a preferred embodiment, the first and/or second portion of the TPSABl or TPSB2 genomic locus are amplified by PCR using oligonucleotide primers. PCR amplification methods are well known in the art (see e.g., US Patent No 4,965,188 which is hereby incorporated by reference in its entirety).

In certain embodiments, oligonucleotide primers are employed to amplify the TPSABl and/or TPSB2 genomic loci. Oligonucleotide primers can comprise DNA and/or RNA or variants thereof. Oligonucleotide primers are generally between about 8 and about 50 bases in length. The skilled artisan will appreciate that the only limitation on the choice of oligonucleotide primers is that the oligonucleotide primers must flank the sequences of the TPSABl and/or TPSB2 genes necessary to distinguish between the inactive and active alleles and be capable of amplifying those regions by PCR. In the case of the TPSABl gene, the oligonucleotide primers must flank the region of intron 4 that varies between βΐ and a alleles, as disclosed herein. In the case of the TPSB2 gene, the oligonucleotide primers must flank the region comprising the BslI restriction endonuclease site created by the insertion of a deoxycytidine between positions 980 and 981 of the genomic sequence of the plllfs allele, as disclosed herein. In a preferred embodiment, the sequences of oligonucleotide primers used to amplify the first amplicon of the TPSABl and/or TPSB2 genes do not overlap the sequences of the primers used to amplify the second amplicon of the TPSABl and/or TPSB2 genes. ATCGGAGCGGACATCGCCCTGCTGGAGCTGGAGGAGCCGGTGAACGTCTCCAGCCACGTCCACACGGTCAC CCTGCCCCCTGCCTCAGAGACCTTCCCCCCCGGGGATGCCGTGC TGGGTCACTGGCTGGGGCGATGTGGAC AATGATGGTGGGTCTGGGGACAGTGGAGGTGGGGCCAGGGTCTTAGCCACAGCCCAGCCCCTGGGCTCCC T CTGGGC TCCAGGTGGGGGTTGCCCGGCCCCCTCCTGAGGCTGCACCCTCT TCCCCACC TGCAGAGCGCCTC CCACCGCCATTTCCTCTGAAGCAGGTGAAGGTCCCCATAATGGAAAACCACATTTGTGACGCAAAATACCA CCTTGGCGCCTACACGGGAGACGACGTCCGCATCGTCCGTGACGACATGC TGTGTGCCGGGAACACCCGGA GGGACTCATGCCAGGTGGGCCCCGCCTGTCCCCCGCCCCCCGCCCCCCAACCCCCACTCCCAGGCCTGTTC GGCGAGCGCTGACCTCTGACCTTCCCAGGGCGACTCCGGAGGGCCCCTGGTGTGCAAGGTGAATGGCACC T GGCTGCAGGCGGGCGTGGTCAGCTGGGGCGAGGGCTGTGCCCAGCCCAACCGGCCTGGCATCTACACCCGT GTCACC TACTAC TTGGAC TGGATCCACCAC TATGTCCCCAAAAAGCCGTGAGTCAGGCCTGGGGTGTCCAC CTGGGTCACTGGAGAGCCAGCCCC TCCTGTTCAAAACACCACTGCTTCCTACCCAGGTGGCGAC TGCCCCC CACACC TTCCCTGCCCCGTCCTGAGTGCCCCT TCCTGTCCTAAGCCCCCTGC TCTCTTCTGAGCCCCTTCC CCTGTCCTGAGGACCCTTCCCCATCCTGAGCCCCCTTCCCTGTCCTAAGCCTGACGCC TGCACCGGGCCC T CCGGCCCTCCCC TGCCCAGGCAGC TGGTGGTGGGCGCTAATCCTCCTGAGTGCTGGACCTCATTAAAGTGC ATGGAAATCACTGGTGTGCATCGC TGTGTTTC TGGTTGTGGATGTCACTGGGAGAGAAGGGGTCCAGGTGT GCTGAGGACACC TGCCACAGTGTGAGGTCCTAGCCCTCAAGGCACAGCCAGTCACCGTGGGACGGGGCCTC CTGGGCAGCCCTGGTCCCCGAGGC TGGCTTCTCCCCACACGATGCATCCAGCATTCGGGTCACACAGAGCC ACTCGGGCAACTCAGTTGATTATAAAGGACAGCCAGGTCCCTGCAACCGGGTCAAGACAGAGAATGGTCAC CGGGAGCCCCAGGGCTGCCCATCACGAGCCCC TACCCCACGCTTCCCACGAGCTCT TC TCCCGGCCCCTCC GTCCAC TGCTTGTGCTTTGCCTAGTTGTTTGC TTTGAGACGGGATCTCGC TGTGTCATCCAGGC TGAAGTG CAGTGGTGTGAT CAGGGC TCACTGCAGCCTTAACTCCTGGGCTCAAGCGATCCTCCCATCTTGGCCTCCCA TATAGC TGGGCCACAGGAGTGAGCCACCACGCCCAGTTAATTTT TGTATT TTCAGTAGAGATGGGGTTTCG CCATGT TGGCCAGTCTGGTCTCGAACTCCTGACCTCAAGTGATCCGCCCGCC TCGGCC TCCCAAAGTGCTG GGATGACAGGCGTGAGCCACCGCACCCGGCCTGAGTTTGACATT TTCAAATTCATTTTGAGGTC TTTCTC T ACATCAATAGGTGAGCCC TCTGCGTCTGGCGAGTGTTGCATTTTATCCCGGGCTCTTGTTTGCATTTTATA TTTGAACATGAT TACACTCAGGAATGAAATGCGGGGCTGTTCTGGTTGAAAACAACTC TCTAAAGAAACAT TCACTC TTTCCT TCCAAC TGTTAGATGCAGAGATGTGCATTTAGTCTCCCGAATCTCTGCAAATGACCTC T GTCCTCACAAGGGGTGGACTCAAC TCCCAGTGCCCTCTCCAGCCCCACGTGACCTCTGCCTCTGCAGCCCC TGAAGGCCCATCCCT

III. Tryptase Genotyping Methods

A. Nucleic Acid Samples

The invention provides methods for genotyping the tryptase alleles encoded by human TPSAB1 and/or TPSB2 gene sequences in a sample. Any nucleic acid sample comprising TPSAB1 and/or TPSB2 gene sequences is suitable for use in the methods of the invention. In certain embodiments, the nucleic acid sample can be isolated genomic DNA, or fragments thereof. In other embodiments, the nucleic acid sample comprises nucleic acids that have been derived from genomic DNA by, for example, DNA or RNA polymerase-mediated amplification (e.g., polymerase chain reaction (PCR), primer extension, and/or RNA transcription). The skilled artisan will appreciate that the only limitation on the choice of nucleic acid sample is that the sample must contain the sequences of the TPSAB1 and/or TPSB2 genes necessary to distinguish between the inactive and active alleles. In the case of the TPSAB1 gene, the nucleic acid sample must include, at a minimum, the region of intron 4 that varies between βΐ and a alleles, as disclosed herein. In the case of the TPSB2 gene, the nucleic acid sample must include, at a minimum, the region comprising the BslI restriction endonuclease site

7 Exemplary oligonucleotide primers for amplifying the TPSABl and/or TPSB2 genes are set forth in Table 1.

Table 1. Exemplary oligonucleotide primers for amplifying the TPSABl and/or TPSB2 genes

In certain embodiments, one or more of the oligonucleotide primers are labeled. Any label that facilitates optical or autoradiographic detection of the oligonucleotide primers, or nucleic acid into which the primers are incorporated, can be used in the methods of the invention Suitable labels include, without limitation, radionuclides and fluorescent dyes (e.g., WellRED™ D4 (Integrated DNA Technologies)). The label(s) can be linked (either covalently or non-covalently) to one or both oligonucleotides used to amplify an amplicon. In a preferred embodiment, at least one labeled primer is used to generate the second amplicon.

C. Detection of TPSABl and/or TPSB2 alleles

After amplification of portions of the TPSABl and/or TPSB2 genomic loci, the genotype of the final amplicon (e.g., the second amplicon) is then determined.

In the case of the TPSABl locus, the second amplicon is screened for the presence of a deletion in intron 4. As discussed above, the presence of an intron 4 deletion identifies an allele as an a allele. Whereas, in the case of the TPSB2 locus, the second amplicon is screened for the presence of a single base insertion (deoxycytidine) between positions 980 and 981 of the genomic sequence of the plllfs allele relative to the βΙΙ or βΙΙΙ alleles. In general, the presence of the deoxycytidine insertion is determined by BslI restriction enzyme digestion of the TPSB2 locus second amplicon. In general, the presence of an intron 4 deletion in the TPSAB1 locus and a BslI restriction site in the TPSB2 locus is determined by measuring the size (e.g., in base pairs) of the second amplicons (in the case of the TPSB2 locus second amplicon, this is after digestion by BslI). Any method of nucleic acid size determination can be used in the methods of the invention including, without limitation, gel electrophoresis (e.g., polyacrylamide and/or agarose) and mass spectrometry. In a preferred embodiment, the size of the second amplicon is measured using capillary gel electrophoresis (e.g., using Beckman Coulter CEQ8000 Genetic Analysis System).

Once the TPSAB1 and TPSB2 genotype of a nucleic acid sample has been determined, the number of active and inactive alleles of can then be calculated by simple addition. For example, if a sample is heterozygous for a alleles at TPSAB1 and heterozygous for the plllfs allele at TPSB2 then the sample contains 2 active and 2 inactive alleles.

IV. Kits

In another aspect, the invention provides kits for genotyping TPSAB 1 and/or TPSB2 genomic loci. In the general, the kits of the invention include at least 2 pairs of nested oligonucleotide primers that can be used to amplify first and second amplicons of the TPSAB 1 and/or TPSB2 genomic loci according to the methods disclosed herein.

In certain embodiments, kits of the invention comprise four oligonucleotide primers, having the sequences set forth in SEQ ID Nos: 3, 4, 5 and 6. In other embodiments, kits of the invention comprise four oligonucleotide primers, having the sequences set forth in set forth in SEQ ID Nos: 3, 7, 8 and 9.

Kits of the invention can comprise any additional reagents needed for genotyping a TPSAB 1 and/or TPSB2 genomic locus. Suitable additional reagents include, without limitation, buffers, DNA or RNA polymerases (e.g., thermostable DNA polymerases), deoxynucleotides, control DNA samples, and instructions for use. V. Exemplification

The present invention is further illustrated by the following examples which should not be construed as further limiting. The contents of Sequence Listing, figures and all references, patents and published patent applications cited throughout this application are expressly incorporated herein by reference.

EXAMPLE 1: TPSAB1 Genotyping Assay

A. Preamplification of the TPSABlGene

A first amplicon comprising the TPSAB1 gene was amplified from human genomic DNA (50ng/ul) using a Qiagen Multiplex PCR kit containing the 2X

Mastermix (including Multiplex PCR Buffer and HotStar Taq DNA Polymerase), RNase free water and Q Solution according to the manufacturer's instructions. The primers used for amplification were as follows:

5'- AGGCCCCAGTGACCAACA - 3' (TPSAB1-FP) (SEQ ID No:3)

5' - AGGTCCAGCACTCAGGAGGA - 3' (TPSRP) (SEQ ID No:4)

Each PCR reaction was 26 uL and contained:

12.5 uL of 2X Mastermix

2.5 uL of the primer stock solution (2 uM of each primer)

2.5 uL Q Solution

6.5 uL RNase-free water

The PCR reaction was performed in a thermocycler according to the following protocol:

After amplification, the first amplicon was diluted in IX TE. Using a new 96 well plate and multi-channel pipette, 4 of each amplified sample was diluted with 295 μΕ IX TE buffer and pipette mixed thoroughly to create a preliminary dilution sample. Using a new 96 well plate and multi-channel pipettors, 10 μΕ of each preliminary dilution sample was diluted with 290 μΕ of IX TE buffer and pipette mixed thoroughly to create the allele amplification samples. These samples were used as templates for amplification of a second amplicon comprising TPSAB1 alleles (see below).

B. Amplification of TPSAB1 Alleles

A second amplicon comprising TPS AB 1 alleles was amplified from the first amplicon (see above) using Promega GoTaq® Hot Start DNA Polymerase containing the 5X Colorless Flexi Reaction Buffer, 25mM MgC12 and GoTaq® Hot Start DNA Polymerase according to the manufacturer's instructions. The primers used for amplification were as follows:

5'- GCGATGTGGACAATGATGGTGGGTCTGG - 3' (TPSAB1FP) (SEQ ID No:5)

5' - GAAGAGGGTGCAGCCTGAGGAGG - 3' (TPSAB1RP) (SEQ ID No: 6)

The forward primer was labeled at the 5' end with WellRED™ D4 (Integrated

DNA Technologies) to allow detection of amplified products

Each PCR reaction was 25 uL and contained:

14.6 uL of Nuclease-free water

5 uL of the 5X Colorless Hot Start Flexi Reaction Buffer

0.5 uL dNTPs

2.6 uL MgCL2

0.2 uL oligos

0.1 uL GoTaq Hot Start DNA polymerase

The PCR reaction was erformed in a thermoc cler accordin to the followin rotocol:

After amplification, 5 μΐ_^ of each amplified sample was diluted in 35ul of IX TE.

C. TPSAB1 Amplicon Analysis

lul of the second amplicon (see above) was analyzed using a Beckman Coulter CEQ8000 Genetic Analysis System according to manufacturer's instructions.

Specifically lul of sample was added to 39 uL fragment analysis stock solution comprising 38.5 uL Sample Loading Solution (SLS) and 0.5 uL GenomeLab DNA Size Standard 400. Samples were run under the following conditions:

Figure 2 depicts the output from a typical experiment. A sample homozygous for alpha alleles exhibits a single peak of 124bp, a sample homozygous for beta alleles exhibits a single peak of 134bp, and a heterozygous sample exhibits peaks of 124bp and 134bp.

EXAMPLE 2: TPSB2 Genotyping Assay

A. Preamplification of the TPSB2 Gene

A first amplicon comprising the TPSB2 Gene was amplified from human genomic DNA (50ng/ul) using a Qiagen Multiplex PCR kit containing the 2X

Mastermix (including Multiplex PCR Buffer and HotStar Taq DNA Polymerase), RNase free water and Q Solution according to the manufacturer's instructions. The primers used for amplification were as follows:

5'- TGGGACTAGTCCATGGGCA - 3' (TPSB2FP) (SEQ ID No:7)

5' - AGGTCCAGCACTCAGGAGGA - 3' (TPSRP) (SEQ ID No:3)

Each PCR reaction was 26 uL and contained:

12.5 uL of 2X Mastermix

2.5 uL of the primer stock solution (2 uM of each primer)

2.5 uL Q Solution

6.5 uL RNase-free water

The PCR reaction was performed in a thermocycler according to the following protocol:

Step 7 Hold 4°C

After amplification, the first amplicon was diluted in IX TE. Using a new 96 well plate and multi-channel pipette, 4 μΕ of each amplified sample was diluted with 295 μΕ IX TE buffer and pipette mixed thoroughly to create a preliminary dilution sample. Using a new 96 well plate and multi-channel pipettors, 10 μΕ of each preliminary dilution sample was diluted with 290 μΕ of IX TE buffer and pipette mixed thoroughly to create the allele amplification samples. These samples were used as templates for amplification of a second amplicon comprising TPSB2 alleles (See below).

B. Amplification of TPSB2 p3fs Allele

A second amplicon comprising the TPSB2 P3fs allele was amplified from the first amplicon (see above) using Promega GoTaq® Hot Start DNA Polymerase containing the 5X Colorless Flexi Reaction Buffer, 25 mM MgC12 and GoTaq® Hot Start DNA Polymerase according to the manufacturer's instructions. The primers used for amplification were as follows:

5'- CGGGAGCAGCACCTCTACTA - 3' (TPSB3fsFP) (SEQ ID No:8)

5' - CCACCATCATTGTCCACATC - 3' (TPSB3fsRP) (SEQ ID No:9)

The forward primer was labeled at the 5' end with WellRED™ D4 (Integrated

DNA Technologies) to allow detection of amplified products

Each PCR reaction was 25 uL and contained:

14.6 uL of Nuclease-free water

5 uL of the 5X Colorless Hot Start Flexi Reaction Buffer

0.5 uL dNTPs

2.6 uL MgCL2

0.2 uL oligos

0.1 uL GoTaq Hot Start DNA polymerase

The PCR reaction was performed in a thermocycler according to the following protocol:

C. TPSB2 Amplicon Analysis

The second TPSB2 amplicon (see above) was digested with BslI at 55 °C for 3 hours. Subsequently, lul of the Bsll-digested second amplicon was analyzed using a Beckman Coulter CEQ8000 Genetic Analysis System according to manufacturer's instructions. Specifically lul of sample was added to 39 uL fragment analysis stock solution comprising 38.5 uL Sample Loading Solution (SLS) and 0.5 uL GenomeLab DNA Size Standard 400. Samples were run under the following conditions:

Figure 3 depicts the output from a typical experiment. A sample homozygous for active beta alleles exhibits a single peak of 234bp, a sample homozygous for inactive 3fs alleles exhibits a single peak of 182bp, and a heterozygous sample exhibits peaks of 182bp and 234bp.

Claims

We claim:

1) A method of detecting an oc-tryptase allele in a nucleic acid sample comprising:

(a) providing a nucleic acid sample comprising a portion of a human TPSAB1 genomic locus;

(b) amplifying from the nucleic acid sample a first portion of the TPSAB1 genomic locus to produce a first amplicon, wherein the first amplicon comprises positions 831-841 or 823-834 of the TPSAB1 gene;

(c) amplifying from the first amplicon a second portion of the TPSAB1 genomic locus to produce a second amplicon, wherein the second amplicon comprises positions 831-841 or 823-834 of the TPSAB1 gene; and

(d) determining the presence of an intron 4 deletion in the second amplicon, wherein the presence of the intron 4 deletion is indicative of the presence of an oc- tryptase allele.

2) A method of detecting an inactive βΙΙΙ-tryptase allele in a nucleic acid sample comprising:

(a) providing a nucleic acid sample comprising a portion of a human TPSB2 genomic locus;

(b) amplifying from the nucleic acid sample a first portion of the TPSB2 genomic locus to produce a first amplicon, wherein the first amplicon comprises nucleic acid positions 980 and 981 of the TPSB2 gene;

(c) amplifying from the first amplicon a second portion of the TPSB2 genomic locus to produce a second amplicon, wherein the second amplicon comprises nucleic acid positions 980 and 981 of the TPSB2 gene; and

(d) determining the presence of a BslI restriction site in the second amplicon, wherein the presence of the BslI restriction site is indicative of the presence of an inactive βΙΙΙ-tryptase allele.

3) The method of claim 1 or 2, wherein the nucleic acid sample is a genomic DNA sample.

4) The method of any one of the preceding claims, wherein the first and/or second amplicon is produced using oligonucleotide primers.

5) The method of claim 4, wherein the oligonucleotide primers used to produce the first amplicon do not overlap the oligonucleotide primers used to produce the second amplicon

6) The method of claim 1 , wherein the first amplicon is produced using oligonucleotide primers comprising the nucleic acid sequences set forth in SEQ ID No: 3 and 4.

7) The method of claim 1 , wherein the second amplicon is produced using oligonucleotide primers comprising the nucleic acid sequences set forth in SEQ ID No: 5 and 6.

8) The method of claim 2, wherein the first amplicon is produced using oligonucleotide primers comprising the nucleic acid sequences set forth in SEQ ID No: 7 and 3.

9) The method of claim 2, wherein the second amplicon is produced using oligonucleotide primers comprising the nucleic acid sequences set forth in SEQ ID No: 8 and 9.

10) The method of any one of claims 4-9, wherein one or more of the oligonucleotide primers are linked to a label.

11) The method of claim 10, wherein the label is a fluorescent molecule.

12) The method of any one of the preceding claims, wherein the first and/or second amplicon is produced by polymerase chain reaction (PCR) amplification.

13) The method of any one of the preceding claims, wherein the determining step is performed using capillary electrophoresis.

14) A method of determining the number of inactive tryptase alleles in a nucleic acid sample comprising:

(a) detecting the presence or absence of an oc-tryptase allele in the sample using the method of claim 1 ;

(b) detecting the presence or absence of an inactive βΙΙΙ-tryptase allele in sample using the method of claim 2; and

(c) determining the sum of the number of oc-tryptase and inactive βΙΙΙ-tryptase alleles detected in steps (a) and (b), respectively, thereby determining the number of inactive tryptase alleles in the nucleic acid sample.

15. A kit for genotyping a TPSAB1 genomic locus comprising four oligonucleotide primers, having the nucleic acid sequences set forth in set forth in SEQ ID Nos: 3, 4, 5 and 6.

16. A kit for genotyping a TPSB2 genomic locus comprising four oligonucleotide primers, having the nucleic acid sequences set forth in set forth in SEQ ID Nos: 3, 7, 8 and 9.

17. An isolated oligonucleotide consisting of the nucleic acid sequence set forth in SEQ ID No. 3, 7, 8, or 9.