Analysis Of Single Nucleotide Polymorphism Inγ-glutamyl Hydrolase Gene In Children With Acute Leukemia

Objective:y-Glutamyl hydrolase (GGH) plays an important role on the intracellular polyglutamation, and is a lysosomal peptidase that catalyses0. the removal of y-linked polyglutamates, converting long-chain methotrexate polyglutamate (MTXPG) into short-chain MTXPG and ultimately to methotrexate (MTX), allowing MTX to be exported from the cells. High levels of GGH have been associated with cellular resistance to MTX. This study is to investigate the allelic frequencies and genotype distribution characters of GGH coding region single nucleotide polymorphisms (cSNPs) in Chinese Han children with acute leukemia (AL) and normal control children, and expecting to discover novel polymorphisms. The distributive difference of each genotype between AL children and control children was evaluated.Methods:Bone marrow samples from92children with AL and peripheral blood samples from124healthy children were obtained to prepare complementary DNAs (cDNAs). The cDNAs were analyzed for the polymorphisms in GGH gene by reverse transcriptase-polymerase chain reaction-denaturing gradient gel electrophoresis (RT-PCR-DGGE) and direct sequencing.Results:The polymorphisms of T16C (Cys6Arg), G91A (Ala31Thr), A268G (He90Val) and C452T (Thrl51Ile) in the coding region of GGH were firstly identified in Chinese Han population. The allele frequencies and genotype distributions of T16C, G91A, A268G and C452T in GGH were identified in the children with acute leukemia (AL) and healthy children. The allele frequencies of C452T in AL children and control children were8.7%versus10.1%; those of T16C were20%versus17.1%; those of G91A were20%versus16.7%and A268G were1.1%versus0.8%. There were no significant differences neither genotypes nor allele frequencies of the four cSNPs in GGH between AL patients and control children (P>0.05), suggested that these GGH gene polymorphisms were not associated with the susceptibility to AL in Chinese Han children.However the C452T allele frequency in Chinese Han children was found to be significantly different from those reported in Japanese population. Mexican population, and African-American population, respectively. Two novel missense mutations were identified in GGH. one of which was a T→G transition in the second nucleotide of codon64in exon2of GGH gene, resulted in substitution of leucine with tryptophan (Leu64Trp); another was a A→G transition in the second nucleotide of codon779in exon8of GGH gene, resulted in substitution of histidine with arginine (His260Arg).Conclusions:To our knowledge, this is the first report about the examination of GGH cSNPs by RT-PCR-DGGE and direct sequencing. Four known cSNPs are firstly identified in Chinese Han population, including T16C (Cys6Arg), G91A (Ala31Thr). A268G (Ile90Val) and C452T (Thrl51He), the allele frequencies and genotype distributions of these polymorphisms are identified. The difference of GGH C452T allele frequency between Chinese Han children and other races is firsty confirmed by using statistical method, which suggests that ethnic difference may exist in MTX efficacy. Two novel missense mutations are identified for the first time in GGH, including T191G (Leu64Trp) and A779G (His60Arg).