Methods for detecting TCR-gamma gene rearrangement
View Patent ↗The invention provides methods for detection of TCR-γ nucleic acid in acellular body fluid. The methods can be used to detect the TCR-γ gene rearrangement in acellular body fluid. The detection of TCR-γ gene rearrangement is useful in determination of clonality of T-cell population. The invention is useful in the diagnosis of lymphoproliferative disorder.
1. A method for diagnosing an individual as having a lymphoproliferative disorder, said method comprising:
a) providing an acellular bodily fluid sample containing less than 1% cells from said individual, wherein said sample comprises TCR-γ nucleic acid;
b) determining the relative abundance of a plurality of V-γ/J-γgene rearrangements in said TCR-γ nucleic acid by HPLC or capillary electrophoresis to determine the presence or absence of at least one major arrangement; and
c) identifying said individual as having a lymphoproliferative disorder when said TCR-γ nucleic acid comprises at least one major arrangement.
2. The method of claim 1 , wherein major arrangements comprise at least 10% of said TCR-γ nucleic acid present in said acellular body fluid sample.
3. The method of claim 1 , wherein major arrangements comprise at least 25% of said TCR-γ nucleic acid present in said acellular body fluid sample.
4. The method of claim 1 , wherein major arrangements comprise at least 50% of said TCR-γ nucleic acid present in said acellular body fluid sample.
5. The method of claim 1 , wherein major arrangements comprise at least 80% of said TCR-γ nucleic acid present in said acellular body fluid sample.
6. The method of claim 1 , further comprises comparing the relative abundance of a plurality of V-γ/J-γ gene rearrangements in said TCR-γ nucleic acid in said sample with the relative abundance of a plurality of V-γ/J-γ gene rearrangements of reference TCR-γ nucleic acid.
7. The method of claim 6 , wherein said reference TCR-γ nucleic acid is derived from polyclonal T-cells.
8. The method of claim 6 , wherein said reference TCR-γ nucleic acid is derived from monoclonal T-cells.
9. The method of claim 1 , wherein said lymphoproliferative disorder is selected from the group consisting of: T-cell acute lymphoblastic leukemia (ALL), large granular lymphocytic leukemia, peripheral T-cell lymphoma, T-lymphoblastic lymphoma, T-cell prolymphocytic leukemia, γδ-hepatosplenic lymphoma, Sezary syndrome, and mycosis fungoides.
10. The method of claim 9 , wherein said lymphoproliferative disorder is T-cell acute lymphoblastic leukemia.
11. The method of claim 1 , wherein said acellular body fluid is plasma or serum.
12. The method of claim 1 , wherein said TCR-γ nucleic acid is genomic DNA or mRNA.
13. The method of claim 1 , wherein said method comprises amplifying said TCR-γ nucleic acid using at least one primer comprising the sequence of SEQ ID NO: 2, 3, or complements thereof.
14. The method of claim 1 , further comprising determining the size of nucleic acid comprising V-γ/J-γ gene rearrangements.
15. The method of claim 14 , wherein said determining the size comprises HPLC.
16. The method of claim 14 , wherein said determining the size comprises capillary electrophoresis.
17. The method of claim 1 , further comprising identifying said at least one major arrangement and comparing the identity of said major arrangement to known V-γ/J-γgene rearrangements.
18. The method of claim 17 , wherein the identity of said at least one major arrangement is determined using a nucleic acid probe specific for the junction of a portion of V-γ gene segments and a portion of J-γ gene segment.
19. A method of determining the clonality of a T-cell population of an individual comprising:
a) providing an acellular bodily fluid sample containing less than 1% cells from said individual, wherein said sample comprises TCR-y nucleic acid;
b) evaluating the nucleic acid from the acellular bodily fluid sample of said individual by HPLC or capillary electrophoresis to determine the relative abundance of a plurality of V-γ/J-γ gene rearrangements in said TCR-γ nucleic acid, and
c) identifying the individual as having a monoclonal T-cell population when at least one major arrangement is identified.
20. The method of claim 19 , wherein major arrangements comprise at least 10% of said TCR-γ nucleic acid in said acellular body fluid sample.
21. The method of claim 19 , wherein major arrangements comprise at least 25% of said TCR-γ nucleic acid in said acellular body fluid sample.
22. The method of claim 19 , wherein major arrangements comprises at least 50% of said TCR-γ nucleic acid in said acellular body fluid sample.
23. The method of claim 19 , wherein said major arrangement comprises at least 80% of said TCR-γ nucleic acid in said acellular body fluid sample.
24. The method of claim 19 , wherein said acellular body fluid is plasma or serum.
25. The method of claim 19 , wherein said TCR-γ nucleic acid is genomic DNA or mRNA.
26. The method of claim 19 , further comprising identifying at least one major arrangement and comparing the identity of at least one major arrangement to known V-γ/J-γ gene rearrangements.