Optimization and validation studies of the Mentype Argus X-8 kit for paternity cases

The use of ChrX-STRs is enormous in forensic case as these have proven to be powerful tools, mainly in deficiency paternity cases when the disputed child is female, and also some special cases involving blood relatives, incest cases, fetal typing in abortion material. The Mentype^® Argus X-8 kit is a commercial multiplex system which contains Amelogenin for gender determination as well as gonosomal STR markers (DXS8378, HPRTB, DXS7423, DXS7132, DXS10134, DXS10074, DXS10101 and DXS10135). Validation studies were being performed on blood obtained from the volunteers in Turkish population. In this study, some parameters were taken under consideration for validation like DNA extraction using different protocols, quantitated by using commercially available Invitrogen Qubit Fluorometer, reaction volume validation of Master Mix and the analysis of female/male, female/female and male/male mixtures were performed. The conditions were optimized and validated using GenAmp 9700 and reducing reaction volume from 25 μl to 12.5 μl and 6.5 μl. After reducing the total volume of the reaction, the results were same and there was no effect on peak height and quality when analyzed on ABI 310 genetic analyzer. 2 paternity cases were also performed which gave the same power of discrimination as has been mentioned in Mentype^® Argus X-8 kit.     

Sequence variation at three X chromosomal short tandem repeats in Caucasian and African populations

Sequence variation for the X chromosome short tandem repeats (X-STRs) DXS9898, DXS6789 and GATA172D05 was studied in two major population groups, namely Caucasians and Africans. DXS6789 revealed two different subtype sequence polymorphisms: for shorter alleles, with less than 17 repeats, results showed a simple composition with the following structure: (TATG)_m-(TATC)_n. For longer alleles, a constant TATC insertion was observed at the beginning of the variable repeat unit. Additionally, alleles identical in size revealed structural variations regarding the TATG/TATC proportion. Africans showed a higher intra-allelic variation at this locus than the Caucasian population group. For all three loci, DXS9898, DXS6789 and GATA172D05, no unique structure was found among Africans and Caucasians.     

How often have X- and autosomal-STRs mutations equivocal parental origin been assigned?

Short tandem repeat markers (STRs) are widely applied in population, evolutionary, and forensic genetics, due to extensive polymorphism in the number of repetitive motifs. The primary mutational mechanism leading to changes in the length of STRs is thought to be polymerase template slippage. Mutation rates in STRs and corresponding parental assignment are usually assessed through the number of Mendelian incompatibilities observed in one-generational, parent(s)-child, pedigrees, and paternal mutations have been assumed to be preponderant over maternal ones. Notwithstanding, diploid and haplodiploid modes of genetic transmission may not allow for the unequivocal assigning of the mutation to the correct parental origin (either paternal or maternal), especially when genotyping methodologies of fragment length determination are employed. In this work, the frequency under which a mutation might be assigned to the wrong parental origin or be interpreted as having an ambiguous origin is analyzed for both diploid and haplodiploid modes of genetic transmission. Genotypic configurations were generated with Python™ programming language, considering parents-child trios for autosomal transmission, and parents-daughter trios for the X chromosomal one. One single-, one two- or one three-step mutation was simulated in each familial constellation, and the resulting genotypic configuration was analyzed regarding the parental assignment of the mutation. When considering autosomal transmission, the meiosis suffering mutation was randomly selected. Contrarily, differential analyses were performed for paternal and maternal mutations for X-chromosomal transmission. In this work, we show that the biases in the rates between paternal and maternal mutations differ for autosomal and X-chromosomal modes of transmission. In the differential analysis performed for the X-chromosomal STRs, it is possible to ascertain that the maternal and paternal meioses are subject to different biases, the latter being better estimated than the first. This work shows that simulated data, along with reliable and properly communicated real one, may be crucial for the correct modeling of biological processes, such as the mutation in STRs.     

How frequently are Autosomal and X-STRs multistep mutations perceived as single-step?

Short tandem repeats (STRs) incur in length mutations that involve the loss or gain of repeats. STR mutation rates are usually estimated considering the rates of observed Mendelian incompatibilities in one generation familial configurations. When considering multistep mutations, for the autosomal and X-chromosomal modes of genetic transmission, underestimations are inevitable when using this approach (MIA), due to the occurrence of mutational events deceptively perceived as involving fewer steps. The rate of this occurrence depends on the mode of genetic transmission considered, the parental origin of the mutation, the type of familial configuration considered, and the genotypic background of the progenitor(s). MIA biases were weighted and compared for the diploid and haplodiploid modes of transmission, using familial genotypic configurations (parent(s)-child duos and trios) generated resorting to Python™ and real population databases from Norway, Somalia, and Spain for 10 Aut-STRs and Argentina, Eastern Asia, and Northern Europe for 12 X-STRs. One two- or one three-step mutation was simulated in each of the 1,000,000 familial configurations. The frequency with which mutations could be interpreted as involving fewer steps, when the most parsimonious reasoning is employed, was computed. Results showed that the magnitude and type of biases depend on the type of familial data and the genetic mode of transmission considered, being higher in duos than in trios, both in autosomes and the X chromosome. Indeed, whether X- or Aut-STRs are analyzed, trios generally provide better estimates and should be favored over duos. The pooling of the two types of data is not advised. The greater the number of steps involved in the mutation, the worst the estimates obtained. In X-chromosomal analyzes, trios with a paternal mutation presented the best estimates and mother-daughter duos the worst; mother-son duos showed similar estimates to trios when a maternal mutation was considered.