Complex Kinship Cases Research Articles

X-STRs: Relevance in complex kinship cases

At present, interest in X chromosome has increased considerably in forensic casework investigations due to its singular transmission features. The aim of this work is to show the applicability of X-STRs in cases unable to be resolved by conventional typing approaches. Ten X-STRs were analyzed in two complex cases. In both situations X-STRs data allowed us to obtain conclusive results.

Analysis strategies to establish vWF intron 40 haplotypes

Sequencing of a 0.65 kb region in the intron 40 of the vWF gene demonstrated a complex variability. Five STRs named Pol K, F, P1, P2-a and P2-b and an indel polymorphisms (I) are present. We established a routine analysing method to puzzle out the Pol K/F/I/P haplotypes which does not require a sequencing procedure. To recognise the combined polymorphisms as haplotypes, we performed short and middle range PCRs in combination with Nde I and BsmA I restriction tests. Comparison of the amplicon and restriction fragment length reveals the most likely haplotypes of each person involved a kinship test. Furthermore, a SNP allele specific PCR was employed. Additional information can be achieved by typing Pol P2-a and P2-b. Establishing of intron 40 vWF haplotypes using the methods described here can greatly support the resolution of complex kinship cases. This statement is illustrated by demonstration of a family study.

Kinship determination using DNA markers

Autosomal and Y chromosome short tandem repeats (STRs) and mitochondrial DNA polymorphisms are the most commonly used molecular tools for determination of kinship. To report a revision of 1,120 kinship cases (paternity and others) analyzed in our laboratory. Revision of all kinship cases analyzed between years 2001-2006. Autosomal and Y chromosome STRs and mitochondrial DNA polymorphisms were analyzed in DNA extracted from blood samples. Paternity was excluded in 27.2% of cases. This figure did not change significantly along years. Most paternity exclusions were confirmed by the discordance in 5 genetic markers (30.5%), followed by exclusion of 4 and 6 genetic markers (20.3 and 20% respectively). Two studied cases were paternal and maternal exclusions, corresponding to a change of children between two families. In one case, the paternal line was assessed through Y chromosome markers, studying 16 STRs of this chromosome, positively confirming this paternal relationship. Another case was analyzed for maternal line using mitochondrial DNA analysis. In six cases, a genetic marker with a paternal-sibling mutation, was observed. The criteria for the determination of mutation was the finding of only one discordant marker between at least thirteen markers analyzed in each case. Also, an increase or decrease in one unit repeated in tandem (tetranucleotide) between the alleged father and the son was also required. One subject had a double mutation. In this case, paternity was confirmed analyzing thirteen autosomic STRs and five Y-STRs. The authors have acquired great expertise in kinship analysis and had established criteria to solve complex kinship cases.

Estudios de parentesco mediante marcadores del ADN: Experiencia en resolución de casos en los últimos seis años

Autosomal and Y chromosome short tandem repeats (STRs) and mitochondrial DNA polymorphisms are the most commonly used molecular tools for determination of kinship.To report a revision of 1,120 kinship cases (paternity and others) analyzed in our laboratory.Revision of all kinship cases analyzed between years 2001-2006. Autosomal and Y chromosome STRs and mitochondrial DNA polymorphisms were analyzed in DNA extracted from blood samples.Paternity was excluded in 27.2% of cases. This figure did not change significantly along years. Most paternity exclusions were confirmed by the discordance in 5 genetic markers (30.5%), followed by exclusion of 4 and 6 genetic markers (20.3 and 20% respectively). Two studied cases were paternal and maternal exclusions, corresponding to a change of children between two families. In one case, the paternal line was assessed through Y chromosome markers, studying 16 STRs of this chromosome, positively confirming this paternal relationship. Another case was analyzed for maternal line using mitochondrial DNA analysis. In six cases, a genetic marker with a paternal-sibling mutation, was observed. The criteria for the determination of mutation was the finding of only one discordant marker between at least thirteen markers analyzed in each case. Also, an increase or decrease in one unit repeated in tandem (tetranucleotide) between the alleged father and the son was also required. One subject had a double mutation. In this case, paternity was confirmed analyzing thirteen autosomic STRs and five Y-STRs.The authors have acquired great expertise in kinship analysis and had established criteria to solve complex kinship cases.

A multiplex PCR for 11 X chromosome STR markers and population data from a Brazilian Amazon Region

The analysis of X-STR polymorphisms has received the attention of several researchers, mainly due to its applicability to the investigation of complex kinship cases. Although many X-STRs have been validated for forensic use, little is known about the variations of these polymorphisms in different populations of the world. The present work describes a new multiplex system that allows the simultaneous analysis of 11 X-STR markers, for use both in paternity determination and more complex forensic cases. The loci investigated include DXS9895, DXS7132, DXS6800, DXS9898, DXS6789, DXS7133, DXS7130, HPRTB, GATA31E08, DXS7423, and DXS10011, which together afford a power of discrimination in the order of 0.999999. In addition, this work presents the genotyping results obtained for a sample of 324 individuals (182 males and 142 females) from the admixed population of Belém, Pará, located in the Brazilian Amazon Region.

DXS10079, DXS10074 and DXS10075 are STRs located within a 280-kb region of Xq12 and provide stable haplotypes useful for complex kinship cases

The evaluation of the short tandem repeat (STR) markers DXS10079, DXS10074 and DXS10075 was amended to establish a STR cluster spanning a genetic distance<1 cM. These three STRs are located within a 280-kb region at Xq12 and provide stable haplotypes useful for solving complex kinship cases. Theoretically, this cluster could give rise to 2,548 different haplotypes in the German population and the genotyping of 781 men revealed the presence of 172 haplotypes. Since the three STRs were shown to be in strong linkage disequilibrium (LD), haplotype frequencies cannot be computed on the basis of a single locus allele frequency alone but have to be estimated directly. Here, we present data on linkage, haplotype frequencies and LD in a German population. Further clusters from other regions of the X chromosome will be published in the future to cover the chromosome with a well-structured network of highly informative sites.

The development of two new STR multiplex systems

Here we show two new amplification systems, which were realised for the amplification of 80 highly polymorphic loci, in combination with an Infrared Automated DNA Sequencer LI-COR 4200 (LI-COR, Nebraska, USA). The two amplification systems were indicated as MU5 and MU6. The amplification system MU5 contained D12S391, F13A01, SE33 and Penta D loci, the amplification system MU6 the loci D2S1338, D19S433, F13B and Penta E. Both systems were performed using forward IRDye™800 labelled and reverse unlabelled primers, in combination with the QIAGEN Multiplex PCR kit (Qiagen, Germany). The extension of the set of STRs in our laboratory is useful to further improve our ability to resolve complex kinship cases (e.g. deficiency cases, incest, paternity tests with mutations). Moreover, these new multiplex systems can also be used for manual typing, because the STRs do not overlap in size.