Figure 1.
Deletion polymorphisms of the GSTT1 and GSTT2B genes.
The GSTT1 and GSTT2 genes are part of the Theta-class GST gene cluster on chromosome 22q11.23. GSTT1 and GSTT2 are separated by approximately 50 kb and share 55% sequence homology. GSTT1 is flanked by two 18 kb regions, HA5 and HA3, which are more than 90% homologous. In their central portion, HA3 and HA5 share a 403-bp sequence with 100% identity. HA5 and HA3 are direct repeats. The GSTT1 null allele arises by homologous recombination of the left and right 403-bp repeats, which results in a 54-kb deletion containing the entire GSTT1 gene. GSTT2 is positioned adjacent to the HA5 repeat. The duplicate copy of GSTT2 is a pseudogene named GSTT2B, because of an abnormal exon 2/intron 2 splice site that causes a premature translation stop. GSTT2 and GSTT2B are inverted repeats. The deletion of GSTT2B has a strong influence on GSTT2 gene expression, as described in the text.
Figure 2.
PCR assay for GSTT1 and GSTT2B deletion genotyping.
A four primer set was used in a single reaction to determine the presence or absence of the GSTT1 allele. (A); Primers A-L and A-R amplify a 459 bp sequence present in the GSTT1 gene, while primers O-L and O-R amplify a joint sequence of 1460 bp resulting from the deletion of GSTT1. When GSTT1 is not deleted, the DNA fragment (GSTT1) between the forward (O-L) and reverse primers (O-R) is too long for amplification and only the 459 bp sequence is amplified. Conversely, when GSTT1 is deleted, the 459 bp sequence is not amplified because absent while the 1460 bp fragment is now amplified. As a result of the PCR reaction, the only presence of a 459 bp band (lines 1–3 in the gel picture) or of a 1460 bp band (lines 7, 8) defines the WT/WT and the del/del genotypes respectively; while the presence of both fragments defines the heterozygous genotype (lines 4–6). Lane 9 was used as negative control. Solid lines represent genomic sequences; white rectangle represents the deleted sequence; small gray triangles indicate the 408 bp repeats flanking the GSTT1 gene. Expected PCR products are drawn as small gray bars. (B); A three primer set is used to determine the deletion polymorphism of GSTT2B in a single reaction. Primers 2B and 6857 amplify a 847 bp fragment detecting the presence of the GSTT2B gene, while primers 6857 and 6858 amplify a 505 bp sequence resulting from the deletion of GSTT2B. The 6857 primer is used for amplification of both fragments. When GSTT2B is not deleted, the DNA fragment (GSTT2B) between the 6857 and 6858 primers is too long for amplification and only the 847 bp fragment is amplified. Conversely, when the gene is deleted, the 505 bp fragment is amplified and the 847 bp fragment is not amplified. As a result of the PCR reaction we can have three possible polymorphisms: one single 847 bp (lines 1–3 in the gel picture) or one single 505 bp band (lines 6–8) represent the WT/WT and the del/del polymorphisms rispectively; while the contemporary presence of both bands represents the WT/del polymorphism (lines 4, 5). Lane 9 was used as a negative control. Solid lines represent genomic sequences; black rectangle represents the deleted sequence. Expected PCR products are drawn as small gray bars.
Table 1.
Genotype and allele frequencies of GSTT1, GSTT2B and GSTP1 polymorphisms and association with OSCC in Black and Mixed Ancestry populations.
Table 2.
Association of GSTT1, GSTT2B and GSTP1 polymorphisms with OSCC by smoking and drinking status in Black and Mixed Ancestry cases.
Table 3.
Haplotype frequencies for the GSTT1 and GSTT2B deletion polymorphisms in Black and Mixed Ancestry populations.