The locations of the TP73 SNPs analyzed in this study are diagrammed in Figure . We first genotyped all AD cases and controls for the known BanI polymorphism in exon 5 of TP73. We observed ethnic differences in allele frequencies between Caucasians, Caribbean Hispanics and African-Americans in our population sample, with the highest frequency of the BanI+ allele in African-Americans, but using stringent diagnostic criteria we did not find a consistent association with AD (Table ). After segregating the subjects by APOE genotypes, we observed a weak but significant association of the BanI +/- genotype with AD (reference genotype BanI -/-), but no allele dosage effect was observed, i.e. the BanI+/+ genotype was not positively correlated with AD (Table ).
Genotype frequencies of the TP73 exon 5 (BanI) polymorphism in AD cases and controls
Odds ratios for AD as a function of the TP73 exon 5 (BanI) polymorphism stratified by the presence or absence of APOE ε4 alleles
We therefore searched for additional sequence variants in TP73
. As a screening panel, we chose genomic DNA from 12 people (24 chromosomes), including several individuals from each of the three ethnic groups. This analysis would not be expected to detect rare variants, but would detect common alleles. Sequencing of the exons encoding the DNA binding domain of p73 did not reveal any coding variants, although the Ban
I SNP, which is coding-neutral, was observed with a high frequency and served as a positive control. However, we did find a common SNP in the P3 promoter of TP73
. This variant was a G→A transversion at position minus-386 relative to the transcriptional start site of the ΔN-p73 isoform. A transcription factor binding site database search [15
showed that this SNP converted a variant consensus progesterone receptor binding site overlapping the -386A allele to a non-consensus sequence in the -386G allele (Fig. ). This SNP also overlapped with a p53 consensus binding half-site, but no second nearby half-site was found, suggesting that p53 binding to the P3 promoter [16
] is not likely to be affected by the -386G/A SNP.
To ask whether the -386G/A SNP might be associated with AD risk, we genotyped the series of cases and controls. These individuals were also genotyped for coding variants at the APOE
locus, which are known modifiers of AD risk [17
]. The results are shown in Table . The -386G/A allelic system was in Hardy-Weinberg equilibrium in the controls, and the -386G allele was significantly associated with AD in the group as a whole, both when the data were analyzed as unadjusted odds ratios, and after multivariate logistic regression analysis correcting for age and education.
Genotype frequencies of the -386G/A P3 promoter polymorphism in AD cases and controls
After stratifying by APOE genotype, the AD association of the -386G TP73 allele was seen in the groups with one or zero APOE ε4 alleles, but was not seen in the group of APOE ε4/ε4 homozygotes (Table ). Since homozygosity for APOE ε4 in itself conveys a high risk of AD, this result is not surprising.
Odds ratios for AD as a function of the -386G/A P3 promoter polymorphism stratified by the presence or absence of APOE ε4 alleles
We next asked whether allele frequencies of the -386G/A SNP might differ by ethnicity. As shown in Table , there was indeed a difference among the three ethnic groups, with the highest frequency of the -386G allele in African-Americans and the lowest frequency in European Caucasians. Consistent with the known history of ethnic admixture in the Caribbean, Hispanics showed an intermediate allele frequency. When we reexamined the AD associations stratifying by ethnicity, we found a significant association of homozygosity for the -386G allele with AD in the Hispanics, and a similar direction of the effect, but failure to achieve significance, in the smaller number of African-Americans (Table ). There were too few Caucasians with the -386G allele to allow conclusions.
Genotype frequencies of the -386G/A SNP in three ethnic groups
Odds ratios for AD as a function of the -386G/A P3 promoter polymorphism stratified by ethnicity
Although multiple interpretations of these results are possible, one hypothesis is that the -386G/A SNP, or closely linked polymorphisms, might influence AD risk via a functional effect on ΔNp73 expression in the brain. Small variations in the net expression of the ΔNp73 mRNA isoform in human brains would be hard to quantify, but examining allele-specific expression, in which one allele serves as an internal control for the other, is expected to be more sensitive. We therefore screened a series of 34 brains for heterozygosity at the TP73 exon 5 BanI polymorphism, which would allow mRNA from the two alleles to be distinguished by RFLP and direct sequencing. Since the collection of autopsy brains available to us was primarily, though not exclusively, from Caucasians, the allele frequency at this marker was low. Nonetheless, we found 4 brains, 1 fetal mid-gestation and 3 adult, which were heterozygous. Two of the adult brains were affected by AD and one was normal.
Among these 4 brains, 3 manifested approximately equal biallelic mRNA expression of TP73 mRNA, as indicated by comparison with genomic PCR controls, while one adult brain (the normal brain) showed a strong allelic expression bias (Figure ). This bias was reproducible in multiple independent RT-PCR reactions (Fig. ), and was also reproduced starting from two separate RNA preparations from this same brain. We utilized RFLP analysis since this method gives a simple read-out of the two alleles. Heteroduplex formation can cause artifacts in this method when the cDNA is over-amplified, so we kept the PCR cycle number to a minimum (35 cycles). Genomic controls were run in parallel using the same PCR conditions, and these gave highly reproducible RFLP results, which were taken as the standard for biallelic representation. Lastly, the results of direct sequencing, which is not sensitive to heteroduplex formation, were consistent with the RFLP results (Fig. ). From this analysis, we conclude that some human brains manifest an allelic bias for TP73 mRNA expression. Lastly, we established the genotype at the -386G/A P3 SNP in the 4 brains (Table ). The adult brain with strongly biased expression was indeed heterozygous for this marker, but heterozygosity was also found in the fetal brain, which showed approximately equal biallelic mRNA expression.
Figure 2 Allele-specific TP73 mRNA expression in human brains. In the BanI RFLP autoradiograms the T-allele (uncut) is indicated by the thick marker line and the C-allele (cut) by the thin marker. Additional smaller fragments ran at a lower position and are not (more ...)
Genotypes of the four informative brains examined for TP73 allele-specific mRNA expression
Lastly, we tested for major effects of the -386G/A SNP on P3 promoter activity by transfecting promoter-luciferase reporter plasmids into human SK-NSH-N neuroblastoma cells. No significant difference in basal promoter activity was seen in this assay (Fig. ).
Figure 3 Activity of allelic variants of the TP73 P3 promoter-luciferase constructs in SK-NSH-N neuroblastoma cells. Values are mean (bars) and standard deviation (vertical lines) of triplicate determinations of firefly luciferase activity, normalized to Renilla (more ...)