Genotyping Assay to Predict Gamma Glutamyl Hydrolase (GGH) Activity

Abstract

Single nucleotide polymorphisms (SNPs) in the gene encoding gamma glutamyl hydrolase (GGH) associated with reduced GGH activity are disclosed. The primary SNP is a change from a cytosine to a thymine at a position corresponding to nucleotide 511 of Genbank sequence accession no. NM 003878. Methods and kits for detecting these SNPs are provided, along with primers useful in detecting these SNP and for amplifying portions of the GGH gene containing these SNPs.

1 - 5 . (canceled) 
     
     
         6 . A method for determining the gamma glutamyl hydrolase (GGH) genotype of an individual comprising:
 a. isolating nucleic acid from the individual;   b. amplifying a region of the GGH gene from said nucleic acid which includes the position corresponding to nucleotide 511 of Genbank sequence accession no. NM 003878 (position 511 of SEQ ID No. 1); and   c. determining the identity of the nucleotide at said position.   
     
     
         7 . An isolated oligonucleotide molecule comprising a mutant allele of gamma glutamyl hydrolase (GGH) or a fragment thereof, which is at least ten consecutive bases long and contains a point mutation at the position corresponding to nucleotide 511 of Genbank sequence accession no. NM 003878 (position 511 of SEQ ID No. 1). 
     
     
         8 . The isolated oligonucleotide molecule of  claim 7 , wherein said point mutation is a thymine substitution for cytosine. 
     
     
         9 . The isolated oligonucleotide molecule of  claim 8 , wherein said oligonucleotide molecule has a sequence selected from the group consisting of SEQ ID No. 7 and SEQ ID No.8. 
     
     
         10 . The isolated oligonucleotide molecule of  claim 7 , wherein said oligonucleotide molecule is at least 15 bases long. 
     
     
         11 . The isolated oligonucleotide molecule of  claim 7 , wherein said oligonucleotide molecule is at least 20 bases long. 
     
     
         12 . The isolated oligonucleotide molecule of  claim 7 , wherein said oligonucleotide molecule is at least 50 bases long. 
     
     
         13 . The isolated oligonucleotide molecule of  claim 7 , wherein said oligonucleotide molecule is at least 100 bases long. 
     
     
         14 . A kit for determining the gamma glutamyl hydrolase (GGH) genotype of a subject comprising a carrier means having in close confinement therein at least two container means, wherein a first container means contains a first oligonucleotide molecule comprising a mutant allele of GGH or a fragment thereof, which is at least ten consecutive bases long and contains a point mutation at the position corresponding to nucleotide 511 of Genbank sequence accession no. NM 003878 (position 511 of SEQ ID No. 1), or a oligonucleotide molecule complementary thereto and a second container means containing a second oligonucleotide molecule encoding a wild-type allele of GGH, a fragment thereof, or an oligonucleotide molecule complementary thereto which is at least ten consecutive bases long and includes the position corresponding to nucleotide 511 of Genbank sequence accession no. NM 003878 (position 511 of SEQ ID No. 1). 
     
     
         15 . The kit of  claim 14  wherein said point mutation is a thymine substitution for cytosine. 
     
     
         16 . The kit of  claim 15  wherein said first oligonucleotide molecule has a sequence selected from the group consisting of SEQ ID No. 7 and SEQ ID No.8. 
     
     
         17 . A kit for determining the gamma glutamyl hydrolase (GGH) genotype of a subject comprising a carrier means having in close confinement therein at least two container means, wherein a first container means contains a first oligonucleotide primer at least ten consecutive bases long suitable for use in a polymerase chain reaction (PCR) corresponding to a portion of the GGH gene 5′ and adjacent to the position corresponding to nucleotide 511 of Genbank sequence accession no. NM 003878 (position 511 of SEQ ID No. 1) and a second container means containing a second oligonucleotide primer at least ten consecutive bases long suitable for use in a PCR corresponding to a portion of the GGH gene 3′ and adjacent to the position corresponding to nucleotide 511 of Genbank sequence accession no. NM 003878 (position 511 of SEQ ID No. 1), wherein said first oligonucleotide primer and said second oligonucleotide primer can be used to amplify a portion of the GGH gene comprising the position corresponding to nucleotide 511 of Genbank sequence accession no. NM 003878 (position 511 of SEQ ID No. 1) in a PCR. 
     
     
         18 . The kit of  claim 16  wherein said first oligonucleotide primer has a sequence selected from the group consisting of SEQ ID No. 3 and SEQ ID No. 5 and said second oligonucleotide primer has a sequence selected from the group consisting of SEQ ID No. 4 and SEQ ID No. 6.