Triallelic Pattern at INRA23 and BM1824 Loci Observed in a Sicilian Cattle
In this work we report an unusual double three-allelic dinucleotide pattern occurred in a Sicilian cattle at INRA023 and BM1824 autosomal loci (respectively located into the chromosome 3 and 1) during an investigation of identity verification. Triallelic profiles can find genetic reasons in several events such as translocations, chromosome duplication, meiotic non-disjunction events like trisomy as well as mosaicism and chimerism. A detailed overview of the main genetic anomalies described in cattle are given in Table 1 . Some elucidations concerning the genetic mechanism which underlines infrequent triallelic pattern will be provided in our study. According to Clayton et al. , triallelic pattern can be classified in two different types based on the peak intensities. Type I pattern happens during development and it is the more frequent. It shows generally three alleles of uneven intensity as results of a somatic mutation at a heterozygous locus. Moreover the area of the highest peak should be the result of the sum of the peak areas of the two other minor alleles. On the other hand, type II triallelic pattern shows usually three peaks of similar height indicating chromosomal rearrangement at a heterozygous locus such as localized chromosomal duplication or non-disjunction events. In our study the uneven peak intensities seem to show a type I-like pattern even if the sum of the peak areas of the minor alleles are not comparable to the unmutated allele with a difference for both loci estimated in ~1000 (2895 vs 1880 and 5941 vs 5021, respectively for INRA023 and BM1824) (Table 2). Type II patterns in humans were extensively described in literature [5,17]. Among these, Lukka et al.  described a case of a large chromosomal duplication event in TPOX locus confirming genotyping, by the presence of two dinucleotide STRs just adjacent to the locus. In his study he displayed an electropherogram for the two microsatellites analogous to ours. In light of this, our results appear to be difficult to be inferred and included in type I/II group.
Fragments Size, Peak Height in RFU and Peaks Area are referred to each peak for the corresponding locus.
Current literatures concerning unusual STR typing are referred to human since they employ simple or complex tetra- repeat (CODIS system); no data are available for three-allelic pattern in dinucleotide microsatellite markers in cattle till now. It is plausible that proneness to produce high-frequency stutter bands in di-repeat rather than tri- tetra- or penta- can make hard the assignment to pattern type; allele-specific stutter could be also included within the true allele peak enhancing its height. Furthermore the high intensity of some peaks could be due to its preferential amplification, masking a type II pattern. These conditions could be taken into account particularly for INRA023 which shows a more homogenous peak heights (2:1:2 with Peak 2 / Peak 1 ratio ~55%) if compared to BM1824 (2:3:1 with Peak 3 / Peak 2 ratio ~43%). Defined assignment criteria related to the ratio of the peak areas and their intensity values could help in type I/II inclusion. As stated above, many genetic reasons are involved in unusual threeband profile. Chimerism is defined as the existence of more than one genetically-distinct population of cells originated from more than one zygote. Exchanging of blood between twin fetuses by placental anastomoses are not uncommon in cattle and are known as chimeras (e.g. freemartins cattle). In the same way, dispermic chimerism which is the consequence of a double fertilization of two separate ova by two sperm was also reported as source of three-allele patterns in forensic genotyping . However we were prone to rule them out because of the presence of only 2 triallelic loci over 11 standard peaks. Beside any evidence of mixture or contaminated samples in other markers, the more accredited genetic reasons of unusual STRs typing in this work are represented by i) autosomal or germinal mosaicism ii) meiotic non-disjunction alone (e.g. trisomy) or followed by previous aberration (e.g. Robertsonian centric fusion) and iii) chromosomal duplications. Mosaicism denotes the co-presence of two or more genetically-distinct populations of cells derived from a single zygote. Defective mitosis can create two daughter cells, one of which with a higher number of chromosomes resulting in two different cell lines.Moreover mechanisms including meiotic non-disjunction and anaphase lag are reported to cause mosaicism. Unfortunately we can’t confirm if the mutational events occurred during embryogenesis step without typing germline tissue even if, according to Brinkmann et al. , mutations in germline cells are inclined to change the number of repeats by one mainly.
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