This study explored the association between 7 polymorphisms in the folate metabolic pathway, chromosome damage in peripheral blood lymphocytes (as BNMN frequency), and the condition of being mother of a DS child with complementary non-linear approaches: supervised neural networks (ANNs), and the semantic connectivity map.
Through TWIST system, we established a consistent possibility to predict the status of being a MDS on the basis of 6 selected variables (Table 3) with 90% specificity, sensitivity and global accuracy (Table 4), this meaning that the selected variables contained specific information on the occurrence of a DS pregnancy. In particular, the BNMN frequency, as well as RFC1 80AA, MTHFR 677TT, MTR 2756AA, TYMS 1494 6bp del +/+ and TYMS 28bp 3R/3R polymorphisms resulted the most important variables for discriminating between MDS and control mothers (Table 3). Most of these variables had been previously associated with DS risk by means of genetic association studies .
The present study represents the first attempt to use ANNs to understand the complex relationship between folate metabolism and maternal risk for having a DS child. Though we achieved good results using ANNs for a small dataset, results are not necessarily generalizable to a larger population but need to be validated independently in future studies. A look at Table 1 shows that some of the genotypes, such as for example the MTHFR 1298CC one, were not present in our cohort while others were present only in a few subjects, indicating the need of further analyses in a larger group. Within this context we made our database available online (see additional file 1) so that other research groups can use the data to increase their own datasets. Moreover, we welcome any further analysis of our database with other methods of forecasting, including logistic regression methods, neural networks, or support vector machines. In addition, present results have been obtained in an Italian case-control cohort and are not generalizable to other populations.
Through the connectivity map (Figure 4) we established the connections between the studied variables and the condition of being MDS or control mother.
Considering individual findings the connectivity map showed several connections already known in the literature, as well as novel ones. Particularly, a connection resulted between MTHFR 677C>T and 1298A>C polymorphisms, for example among the MTHFR 677TT and the MTHFR 1298AA genotypes. This connection can be explained on the basis of a strong linkage disequilibrium existing between the two polymorphisms. MTHFR works as a dimer protein and the combined presence of the T allele at position 677 and of the C allele at position 1298 impairs the stability of the dimer. As a consequence, the 677T allele is in strong linkage with the 1298A one, while the 677T-1298C haplotype is rare and selected negatively .
Similarly, the connectivity map (Figure 4) showed a connection between TYMS 28bp repeats and 1494 6bp deletion polymorphisms. For example, the TYMS 1494 6bp +/+ genotype resulted connected with the TYMS 28bp 2R/2R one, and the TYMS 1494 6bp -/- genotype with the TYMS 28bp 3R/3R one. Again, linkage disequilibrium is known between these two polymorphisms. A previous study by us, performed in a large cohort of white non-Hispanic Americans, revealed that haplotypes containg both the 6bp deleted (-) allele and the 2R allele are rare . A possible biological explanation could be that the 6bp deletion is likely to impair the stability of the TYMS mRNA, while the 2R allele is associated with reduced transcription of the TYMS gene. Therefore, their combined presence on the same haplotype could seriously impair TYMS production .
The connectivity map (Figure 4) revealed that three variables are closely connected with the condition of being MDS: the RFC1 80GG genotype, the BNMN frequency, and the TYMS 2R/3R genotype. On the contrary, the RFC1 80AA genotype, the TYMS 3R/3R genotype and the MTR 2756AA genotype, are closely connected to the condition of being a control mother. The association between the RFC1 80G allele and increased risk for having a DS child has been often observed in Italian populations. We first reported a possible role for the RFC1 80G>A polymorphism, in interaction with MTHFR 677C>T and 1298A>C variants, in affecting DS risk in Italy, suggesting a causative role for the RFC1 80G allele and a protective role for the RFC1 80A one . Subsequently, in a larger case-control study, others observed an independent association between the RFC1 80GG genotype and increased risk for having a DS child in Italy . They also confirmed the protective and interactive role for the RFC1 80A allele previously observed by us . Overall, based on these studies, we concluded that in the Italian population the RFC1 80G allele could increase the risk for having a DS child, while the RFC1 80A allele could be protective . Interestingly, the connectivity map confirmed this observation, showing a connection between the RFC1 80GG genotype and the condition of being MDS, as well as a connection between the RFC1 80AA genotype and the condition of being control mother.
The BNMN frequency is another variable connected with the condition of being MDS. Several previous studies by us [11, 13, 14] revealed a statistically significant increased BNMN frequency in MDS respect to control mothers, leading us to formulate the hypothesis that MDS could have an increased tendency to chromosome malsegregation events during somatic mitotic divisions. More recently others have suggested that women who have a DS child in young age could be characterized by an elevated frequency of mitotic malsegregation events during embryogenesis .
The MTR 2756AA genotype resulted to be connected to the condition of being a control mother as well as to the MTRR 66AA genotype (Figure 4). As shown in figure 1, MTR and MTRR physically interact during folate metabolism, being MTRR required for the maintenance of MTR in its active state. In 2003, Bosco and co-workers observed association between the MTR 2756G allele and increased DS risk in Italy, arguing for a protective role for the 2756A allele. They also reported an interaction between MTR 2756A>G and MTRR 66A>G polymorphisms in increasing DS risk . We recently observed interactions between MTR 2756AA and MTHFR 677TT genotypes in increasing DS risk in Italy . However, as shown by the connectivity map (Figure 4), these two variables are not directly connected, and many other factors might affect their interaction.
A very interesting finding of the present study is in the central role played by TYMS polymorphisms in the connectivity map. Indeed, the TYMS 6bp +/- genotype resulted to be the principal hub of the system, the TYMS 28bp 2R/3R genotype was connected to the condition of being MDS, and the TYMS 28bp 3R/3R genotype to that of being a control mother (Figure 4). TYMS shifts the folate metabolic pathway from DNA methylation toward DNA synthesis (Figure 1). Both TYMS and MTHFR compete for the same substrate: 5,10-methyleneTHF, the first for DNA synthesis, the latter for DNA methylation purposes. The connectivity map (Figure 4) shows several connections between TYMS and MTHFR polymorphisms that can be explained by the following observation: given that both enzymes utilize the same substrate, polymorphisms reducing MTHFR enzyme activity might shift pools of 5,10-methyleneTHF from DNA methylation toward DNA synthesis, whereas polymorphisms affecting TYMS activity might shift the pathway from DNA synthesis toward DNA methylation . We recently observed interaction between MTHFR and TYMS polymorphisms in increasing DS risk, suggesting that an impaired balance between DNA synthesis and methylation processes could favour chromosome malsegregation events . The results from the present study (Table 3 and Figure 4) seriously argue in favour of a pivotal role for TYMS polymorphisms in DS risk.