Balkan Journal of Medical Genetics

DETECTION OF ALLELIC VARIANTS OF THE POLE AND POLD1 GENES IN COLORECTAL CANCER PATIENTS
Pätzold LA, Bērziņa D, Daneberga Z, Gardovskis J, Miklaševičs E*
*Corresponding Author: Professor Dr. Edvīns Miklaševičs, Institute of Oncology, Riga Stradiņš University, Dzirciema iela 16, Riga LV1007, Latvia. Tel: +371-6770-4028. Fax: +371-6706-9545. E-mail: edvins. miklasevics@rsu.lv
page: 83

DISCUSSION

Pol ε and Pol δ are two enzymes with a significant protective role in DNA replication with their ability of proofreading. Allelic variants of the POLD1 and POLE genes could alter the function of these enzymes leading to more frequent faulty DNA replication. This can potentially lead to more cases of hereditary colorectal cancer. Our patients did not show the conversion of S478N in POLD1 or L424V in POLE. This could be explained by the difference in patient groups taken for analysis. The Latvian and British populations both have diverse gene pools, which are mostly not overlapping. As our sample size was quite large and the patients come from every region in Latvia, it represented a large portion of the Latvian gene pool. The genetic variability in Latvia is also large due to its history. We especially screened for these two genetic variants of the POLE and POLD1 genes in greater expectation of positive results, which would correspond to the previous findings in similar studies. There is great theoretical potential to detect genetic variants in other exons of Pol δ and Pol ε, but this patient group was not screened for genetic variants in other gene regions. In the future we should take a look at those other regions of these two genes, as genetic variation could have great influence on the function of these two enzymes. To diagnose HNPCC or other hereditary cancers, patients need to fulfill criteria that often include detailed information about their patients’ family and the history of diseases running in those families. According to Vanags et al. [8], in breast cancer patients, families corresponding to hereditary cancer criteria are larger than families of patients with BRCA1 genetic variants, which do not fit to hereditary cancer criteria due to lack of knowledge about their family history. The same is seen in the case of colorectal cancer patients. Patients diagnosed according to the Amsterdam I criteria are found less often than patients who are diagnosed according to Amsterdam II and Bethesda criteria and proven to carry pathogenic variants in MMR genes [9]. We expected to find, at least in the hereditary colorectal cancer group, pathogenic variants of POLE or POLD1. This would correspond to the findings of Palles et al. [2], and Jansen et al. [3], as this subgroup consists in part of HNPCC patients. On the contrary, we found and sequenced one allelic variant with an unknown significance (c.1384-5dupCCTA) on the POLD1 gene in a male patient, who belongs to the subgroup with an unknown genetic background. This genetic variant has not been described before. A duplication of four nucleotides was found at the 3’ end of intron 11, which covers the splicing site of intron 11 and exon 12 of this gene. The intron belongs to the U2-type introns, as do 95.0% of all introns, and it is spliced by major spliceosomes [10]. As this genetic variant is not listed on the National Center for Biotechnology Information (NCBI) single nucleotide polymorphism (SNP) database and has not been published elsewhere, we can only predict the potential effect of this allelic variant on the splicing mechanism. An intron needs to fulfill certain requirements to ensure accurate splicing. Important features are conserved length between the branch point sequence (BPS) and the 3’ end and also conserved sequences at the 5’ and 3’ ends of the intron [11,12]. This is important for the binding of the spliceosome, lariat formation and cleavage at the 3’ end. Reed [12] showed that long pyrimidine stretches between the BPS and the 3’ end enhance the first step of the splicing. However, he also showed that if the distance between the BPS and the 3’ end increases or if the sequence is altered, the second step of splicing is significantly slowed down and the efficiency decreases. This could lead to non splicing of the intron and exon. Therefore, the first step of the splicing reaction could be enhanced as the genetic variant c.1384-5dupCCTA has three more pyrimidine bases (CCT) between the BPS and the 3’ end. Moreover, the efficiency of the second step of the reaction might decrease, as the length of the sequence between the BPS and the 3’ end is increased and also the sequence changed [12]. Thus, we concluded that this genetic variant most probably is clinically significant, as there are possible modifications in the intron-exon splice site. Even though this speculation was supported by molecular biology, the late age of diagnosis of this patient is speaking against it. This is just an assumption because the enzyme generated by this particular DNA alteration needs to be assessed before we can clearly see the effects on splicing. As none of the other samples of our study group showed the same genetic variant in that particular region of the gene, it seems unnecessary to screen healthy control samples for this variation. As our sample size is quite large and the diversity of patients is high, no other result is expected from screening healthy controls. All in all, we could not detect or confirm the connection between the genetic variants in the POLD1 and POLE genes and colorectal cancer patients, but we detected a novel genetic variant with an unknown significance.

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