Genetic marker test for lupus
View Patent ↗Disclosed is a genetic testing method for diagnosing systemic lupus erythematosus (SLE) in a human subject. The method is related to amplifying nucleic acids from human tissue samples and detecting the presence or absence of variant alleles of a gene encoding poly(ADP-ribosyl)transferase expression (PARP), which are diagnostic of SLE or indicate a genetic predisposition for developing SLE. Also disclosed are useful oligonucleotide primers, primer sets and genetic testing kits for detecting a genetic predisposition for developing SLE.
1. A genetic testing method for systemic lupus erythematosus (SLE) in a human subject, comprising:
a) collecting a tissue sample from a human subject;
b) amplifying nucleic acids from said tissue sample to obtain amplification products, said nucleic acids comprising a genomic sequence of human chromosome 1 between microsatellite markers D1S2860 and D1S213; and
c) detecting in the amplification products the presence or absence of a twelve-fold CA dinucleotide repeat sequence consisting of (SEQ. ID. NO.:6) located between said microsatellite markers D1S2860 and D1S213, wherein the presence of said twelve-fold CA dinucleotide repeat is diagnostic of SLE in a subject having SLE symptoms or indicates a genetic predisposition to develop SLE in a subject not presenting SLE symptoms.
2. The method of claim 1 , further comprising:
detecting in the amplification products the presence or absence of an eighteen-fold CA dinucleotide repeat sequence consisting of (SEQ. ID. NO.:7) located between said microsatellite markers D1S2860 and D1S213, wherein the absence said eighteen-fold CA dinucleotide repeat is diagnostic of SLE in a subject having SLE symptoms or indicates a genetic predisposition to develop SLE in a subject not presenting SLE symptoms.
3. The method of claim 1 , wherein the tissue sample is a blood sample.
4. The method of claim 1 , wherein an oligonucleotide primer is used in amplifying said nucleic acids.
5. The method of claim 4 , wherein said primer has a nucleotide sequence GAT TCC CCA TCT CTC TTT CTT T (SEQ. ID. NO.:1) or a fragment thereof at least 18 nucleotides long, or AAA TTG TGG TAA TGA CTG CA (SEQ. ID. NO.: 2) or a fragment thereof at least 18 nucleotides long.
6. The method of claim 4 , wherein an oligonucleotide primer comprising nucleotide sequence GAT TCC CCA TCT CTC TTT CTT T (SEQ. ID. NO.: 1) or a fragment thereof at least 18 nucleotides long, is used in amplifying said nucleic acids.
7. The method of claim 4 , wherein an oligonucleotide primer comprising nucleotide sequence AAA TTG TGG TAA TGA CTG CA (SEQ. ID. NO.: 2) or a fragment thereof at least 18 nucleotides long, is used in amplifying said nucleic acids.
8. The method of claim 4 , wherein said oligonucleotide primer is labeled with a fluorescent dye.
9. The method of claim 8 , wherein said dye is selected from the group consisting of the with Chemical Abstract Service Registry Number CAS 163795-75-3 (SYBR Green I), quinolinium ,4-[(3-methyl-2(3H)-benzoxazolylidene) methyl]-1-[3-(triemthylammonio) propyl]-, diiodide (YO-PRO-1), 1,1′-(4,4,8,8-tetramethyl-4,8-diazaundecamethylene)-bis [4-[3-methyl-2,3-dihydro(benzo-1,3-thiazole)-2-methylidene]]quinolinium tetraiodide (thiazole orange), 6-carboxy-2′,4′7′,4,7-hexachlorofluorescein (Hex), 6-carboxyfluorescein (FAM) and 4,7,2′,7′-tetrachloro-6-carboxyfluorescein (TET).
10. A genetic testing method for systemic lupus erythematosus (SLE) in a human subject, comprising:
a) collecting a tissue sample from a human subject;
b) amplifying nucleic acids from said tissue sample to obtain amplification products, said nucleic acids comprising a genomic sequence of human chromosome 1 between microsatellite markers D1S2860 and D1S213; and
c) detecting in the amplification products the presence or absence of an eighteen-fold CA dinucleotide repeat sequence consisting of (SEQ. ID. NO.:7) located between said microsatellite markers D1S2860 and D1S213, wherein the absence of said eighteen-fold CA dinucleotide repeat is diagnostic of SLE in a subject having SLE symptoms or indicates a genetic predisposition to develop SLE in a subject not presenting SLE symptoms.
11. The method of claim 10 , wherein the tissue sample is a blood sample.
12. The method of claim 10 , wherein an oligonucleotide primer is used in amplifying said nucleic acids.
13. The method of claim 12 , wherein said primer has a nucleotide sequence GAT TCC CCA TCT CTC TTT CTT T (SEQ. ID. NO.: 1) or a fragment thereof at least 18 nucleotides long, or AAA TTG TGG TAA TGA CTG CA (SEQ. ID. NO.:2) or a fragment thereof at least 18 nucleotides long.
14. The method of claim 12 , wherein an oligonucleotide primer comprising nucleotide sequence GAT TCC CCA TCT CTC TTT CTT T (SEQ. ID.:NO. 1) or a fragment thereof at least 18 nucleotides long, is used in amplifying said nucleic acids.
15. The method of claim 12 , wherein an oligonucleotide primer comprising nucleotide sequence AAA TTG TGG TAA TGA CTG CA (SEQ. ID. NO.:2) or a fragment thereof at least 18 nucleotides long, is used in amplifying said nucleic acids.
16. The method of claim 12 , wherein said oligonucleotide primer is labeled with a fluorescent dye.
17. The method of claim 16 , wherein said dye is selected from the group consisting of the dye with Chemical Abstract Service Registry Number CAS 163795-75-3 (SYBR Green I), quinolinium, 4-[(3-methyl-2(3H)-benzoxazolylidene) methyl]- 1-[3-(triemthylammonio) propyl]-, diiodide (YO-PRO- 1), 1,1′-(4,4,8,8-tetramethyl-4,8-diazaundecamethylene)-bis[4-[3-methyl-2,3-dihydro(benzo-1,3-thiazole)-2-methylidene]]quinolinium tetraiodide (thiazole orange), 6-carboxy-2′,4′7′, 4,7-hexachlorofluorescein (Hex), 6-carboxyfluorescein (FAM) and 4,7,2′,7′-tetrachloro-6-carboxyfluorescein (TET).