Performance of the SNPforID 52 SNP-plex assay in paternity testing

The performance of a multiplex assay with 52 autosomal single nucleotide polymorphisms (SNPs) developed for human identification was tested on 124 mother–child–father trios. The typical paternity indices (PIs) were 10⁵–10⁶ for the trios and 10³–10⁴ for the child–father duos. Using the SNP profiles from the randomly selected trios and 700 previously typed individuals, a total of 83,096 comparisons between mother, child and an unrelated man were performed. On average, 9–10 mismatches per comparison were detected. Four mismatches were genetic inconsistencies and 5–6 mismatches were opposite homozygosities. In only two of the 83,096 comparisons did an unrelated man match perfectly to a mother–child duo, and in both cases the PI of the true father was much higher than the PI of the unrelated man. The trios were also typed for 15 short tandem repeats (STRs) and seven variable number of tandem repeats (VNTRs). The typical PIs based on 15 STRs or seven VNTRs were 5–50 times higher than the typical PIs based on 52 SNPs. Six mutations in tandem repeats were detected among the randomly selected trios. In contrast, there was not found any mutations in the SNP loci. The results showed that the 52 SNP-plex assay is a very useful alternative to currently used methods in relationship testing. The usefulness of SNP markers with low mutation rates in paternity and immigration casework is discussed.     

Gebrauch X-chromosomaler Marker in der forensischen Genetik

Apart from some exceptions typing of autosomal short tandem repeats is normally sufficient for kinship testing in the standard trio situation consisting of the mother, child and putative father. Nevertheless, there are some constellations, such as deficiency and incest cases, which require X-chromosomal typing. However, application is only possible in X-chromosomal inheritance lineages. X-chromosomal haplotyping is suitable for proving distant kinship, such as aunt/niece or first cousins. As male individuals only have one X chromosome the haplotype can be directly recognized by typing the markers and both haplotypes of female individuals often can be deduced from pedigree analysis. X-chromosomal testing can be effective in incest cases or to solve cases in which two alleged fathers are related. In forensic stain analysis X-chromosomal analyses should be utilized when a female component has to be identified in a mixture contaminated with male components. A series of ethical aspects have to be considered when X-chromosomal markers are investigated.     

X-chromosome data for 12 STRs: Towards an Argentinian database of forensic haplotype frequencies

The analysis of X-chromosome STRs is useful in certain kinship cases for which autosomal markers provide insufficient statistical power. Particularly, powerful results are achieved in paternity cases with a daughter, when the alleged father is not accessible for analysis, contrarily to his unquestioned mother or daughter. However, representative haplotype frequencies for this type of markers are not available for some populations, as is the case of Argentina, which prevents the quantification of the proof in routine forensic analyses. In this work we present haplotype frequencies for the 12 X-chromosome STRs included in the Investigator Argus X-12 kit, as well as segregation data, obtained from the analysis of the genetic profiles of 457 father-daughter duos, which gave us information on 914 (unrelated) haplotypes from residents of all Argentinian provinces.     

Comparative evaluation of alternative batteries of genetic markers to complement autosomal STRs in kinship investigations: autosomal indels vs. X-chromosome STRs

Kinship investigations such as paternity are currently solved using sets of (commercially available) highly polymorphic autosomal short tandem repeats (STRs), which lead to powerful likelihood ratios (LR). Still, some difficult cases arise whenever the kinship is much more remote or if the alternative hypotheses are not correctly formulated due to the lack of information (for e.g. there is an unknown relationship between the alleged and the true fathers). In these situations, beyond the routinely used marker set, laboratories usually enlarge the number and/or the type of markers analysed. Among these, autosomal indels and X-chromosome STRs have gained popularity. The aim of this study was to compare the results obtained after complementing an initial set of autosomal STRs with indels or with X-chromosome-specific STRs in simulated paternity cases where the alleged father is a close relative of the real one. Results show that in paternity cases where a low number of incompatibilities are observed, the best strategy is to increase the number of autosomal STRs under analysis. Nevertheless, if these are not available, our study globally shows that in father–daughter duos, a set of 12 X-STRs is more advantageous than 38 highly diverse autosomal biallelic markers. Additionally, the usefulness of X-STRs was also evaluated in cases where only a close relative of the alleged parent (father or mother) is available for testing. For those situations where these markers have the power to exclude, strong LR values are obtained. In the remaining cases, LRs are usually weak and sometimes the results are more likely under the wrong kinship hypothesis.     

Paternity evaluation in cases lacking a mother and nondetectable alleles

In parentage testing the formulae for computing paternity index and exclusion probability generally ignores the presence of nondetectable alleles at the loci tested. In contrast, it is now known that even when paternity testing is done with hypervariable DNA markers, nondetectable alleles should not be ignored. This work presents simple formulae needed with this consideration, to analyze paternity evaluation from DNA markers in cases where the mother of the disputed child is unavailable for testing. It is shown that even a modest frequency of nondetectable alleles (e.g., 2–5% per locus) may have a substantial impact on the paternity index when the child and/or the alleged father exhibits a single-banded DNA profile at a locus. Use of such formulae can generate a high probability of exclusion and a high paternity index when multiple independently segregating hypervariable DNA markers are used.     

Molecular analysis of the human orosomucoid gene ORM1*Q0köln responsible for incompatibility in a German paternity case

In a German paternity test, an alleged father was excluded only by reverse homozygosity of ORM1 phenotypes (mother ORM1 S, child ORM1 S and alleged father ORM1 F1) out of the 28 classical and DNA markers investigated. Without the ORM1 system the biostatistical probability of paternity was calculated to exceed 99.9999%. The intensity of the immunoprinted bands of the ORM1 protein for the child and alleged father after isoelectric focusing appeared to be reduced to about half. To identify a possible null allele, gene-specific amplification followed by single-strand conformation polymorphism and sequencing analyses were carried out. Deletion of one of the two copies of a 4 bp direct repeat sequence (GTCT) in exon 4 of the consensus sequence of ORM1*F1 was observed in the child and alleged father. Thus, the sharing of a rare mutant gene, ORM1*Q0_köln, increased the probability of paternity.     

Assessing paternities with inconclusive STR results: The suitability of bi-allelic markers

In paternity testing the genetic profiles of the individuals are used to compare the relative likelihoods of the alleged father and the child being related as father/offspring against, usually, being unrelated.In the great majority of the cases, analyses with the widely used sets of short tandem repeat markers (STRs) provide powerful statistical evidence favouring one of the alternative hypotheses. Nevertheless, there are situations where the final statistical result is ambiguous, mostly because the alleged father shows incompatible genotypes at a few loci along with a very high paternity index in the remaining systems. In these cases, the possibility that the alleged father is actually a close relative of the real one (son, father or brother) can reasonably be raised.In such cases, when the statistical evidence obtained is considered as insufficient, the common practice is to extend the set of analysed markers. In this context, many authors have suggested that bi-allelic markers, such as single nucleotide (SNP) or insertion/deletion (Indel) polymorphisms, are markers of choice, as they are incomparably less prone to mutation than STRs.In this work we address the soundness of this claim and the consequences of this strategy, analyzing the a priori odds both for (a) expected number of Mendelian incompatibilities, and (b) expected values for the final likelihood ratios. Moreover, one hundred real pairs of second degree relatives, typed for two sets of markers: 15 STRs plus 38 Indels, were used to simulate paternity testing. Our data show that, for the number of markers commonly considered, the results from an extended battery of SNPs or Indels should be interpreted with caution when relatives are possibly involved.     

Genetic profile characterization and segregation analysis of 10 X-STRs in a sample from Santander,Colombia

Ten X-chromosome short tandem repeats (X-STRs: DXS8378, DXS7132, DXS9898, DXS6809, DXS6789, DXS101, GATA172D05, HPRTB, DXS8377, and DXS7423) were analyzed in a sample of unrelated individuals (108 males and 110 females) from the Santander Department in Colombia. In this sample, gene diversities varied between 63.56%, for DXS8378, and 91.41%, for DXS8377. For this set of 10 X-STRs, a high discrimination power was obtained for both male (1 in 3 x 10(6)) and female (1 in 9 x 10(10)) samples and a high mean exclusion chance in father/daughter duos (99.993%) and in father/mother/daughter trios (99.9999%), demonstrating the usefulness of this set of markers in forensic and kinship analysis. Hardy-Weinberg equilibrium was tested in the female sample and no significant deviations were found. Pairwise analysis showed significant differences in the comparison with samples from Spain, Peru, and Argentina and with African American and Hispanic samples from New York. This same set of X-STRs was also typed in 51 mother/father/daughter trios, 43 mother/son duos, and in a single father/daughter pair. In total, four mutations were observed; one at DXS7132 and at DXS6809, and two at DXS8377. Two mutations were paternal and one maternal; and to a fourth mutation, it was not possible to define its origin.     

Mutations or exclusion: an unusual case in paternity testing

In an immigration case with the scope of family reunification, the DNA extracted from the saliva samples of the male child, the alleged mother and the putative father was typed with 22 autosomal short tandem repeat (STR) systems. In seven STR systems, the alleged mother could be excluded from maternity, and the case then had to be regarded as a deficiency case. Taking this fact into consideration, only two exclusions were found for the putative father, and the question arose whether there was an exclusion of the putative father or the existence of two mutations. Autosomal STR typing could not clarify the case, but the application of eight Y-chromosomal markers showed that the alleged father could be excluded from paternity.     

A four-step mutation at D22S1045 in one complex paternity case when the brother of the alleged father hypothesis is evaluated

International Journal of Legal Medicine - We report one complex paternity case presenting a presumable paternal four-step STR mutation between the alleged father (AF) and child; the complexity of...     

Development and forensic validation of a new multiplex PCR assay with 12 X-chromosomal short tandem repeats

One multiplex system for the co-amplification of 12 X-chromosomal short tandem repeats (STRs) DXS7132, DXS8378, DXS6809, DXS7133, DXS6789, DXS7424, GATA172D05, HPRTB, DXS7423, GATA31E08, DXS101, DXS6807 and amelogenin was analysed in a sample of 200 (100 males and 100 females) unrelated healthy individuals living in Northern Italy. The χ²-test for genotype distribution of the X-chromosomal STRs showed no significant deviation from the Hardy–Weinberg equilibrium (HWE). Allele frequencies between female and male samples were not significantly different in all examined markers. In the kinship cases involving 40 family trios with daughter and 10 father/daughter duos, no mutation was detected. The combined power of discrimination (PDc) of the 12 X-STRs for both females and males was PDc > 0.999999.     

Microdeletion at 8q24.13 rather than multistep microsatellite mutation resulting in the genetic inconsistency at the D8S1179 locus in a true trio

International Journal of Legal Medicine - When using microsatellite loci for DNA paternity testing, genetic inconsistencies sometimes occur in true trios and duos and may be erroneously attributed...     

The Karimojong from Uganda: Genetic characterization using an X-STR decaplex system

The Karimojong, an African group from the Karamoja region of Northeast Uganda, were genetically analysed using a decaplex system for X chromosome short tandem repeats (X-STRs). A total of 255 individuals (117 males and 138 females) were genotyped for the following loci: DXS8378, DXS9898, DXS7133, GATA31E08, GATA172D05, DXS7423, DXS6809, DXS7132, DXS9902 and DXS6789. Allele frequencies and parameters for forensic evaluation were calculated for each STR. No association was found between any pairs of loci studied. DXS6789 was the most polymorphic marker in this sample, followed by DXS6809, with gene diversities of 84.79% and 83.94%, respectively. The less discriminating locus observed was DXS7133, with a gene diversity of 39.79%. High overall values of power of discrimination were obtained for female (1 in 1.8 × 10¹⁰) and male samples (1 in 1.6 × 10⁶), as well as high power of exclusion in father/mother/daughter trios (99.9997%), in father daughter duos (99.9862%) and in half sisters with same father (99.0331%). These results confirm the potential of this 10-plex in parentage testing and in human identification.     

The new guidelines for paternity analysis in Germany—how many STR loci are necessary when investigating duo cases?

The requirements in the new German guidelines for paternity analysis have not only changed according to the so-called Gendiagnostikgesetz, the new German law regulating human genetic as well as paternity analyses, but also regarding the minimal number of short tandem repeats (STRs) which should be investigated (15 STRs) and the minimal required average exclusion chance (99.999 %). Even in paternity analyses involving only two people (e.g., father and child or mother and child), this exclusion chance is mandatory. A retrospective analysis of 330 father–child cases from our routine investigations showed in 142 cases (43 %) an individual exclusion chance below 99.999 % when using 15 STRs as required, in our routine work provided by the Powerplex® 16 kit which is reported to have an average exclusion chance of 99.988 %. Therefore, these same 330 father–child pairs were additionally analysed using the Powerplex® 21 kit and 120 of these duos were additionally analysed using the Powerplex® ESX17 kit enabling the analysis of 20 or 16 loci respectively. Now, an individual exclusion chance of more than 99.999 % could be achieved in 95.5 % (Powerplex® 21; calculation without the results of D6S1043), 98.8 % (Powerplex® 21; calculation with the results of D6S1043, using allele frequencies established in this study for a German and a West African population) and 98.3 % (Powerplex® ESX17). These data clearly demonstrate that in duo cases, more than the required 15 STR loci have to be investigated to obtain sufficient results.     

Evaluation of the paternity probability on an application of minisatellite variant repeat mapping using polymerase chain reaction (MVR-PCR) to paternity testing

Minisatellite variant repeat (MVR) mapping using polymerase chain reaction (PCR) was applied to a practical case of paternity testing to evaluate the paternity probability. In order to obtain single allele mapping by allele-specific MVR-PCR, three flanking polymorphic sites for each of the MS31A and MS32 loci were investigated and all three individuals were typed as heterozygous for at least one flanking polymorphic site at each locus. Allele-specific MVR-PCR was then performed using genomic DNA. It was confirmed that one allele in the child was identical to that from the mother and the other one in the child was identical to that from the alleged father. Mapped allele codes were also compared with those in the database by dot-matrix analysis, and no identical allele was found although some motifs were shared with Japanese alleles. The paternity index and the probability of paternity exclusion in the case at these two MVR loci were calculated using the presumed values of the allele frequencies. These studies seem to illustrate the practical value of MVR mapping of MS31A and MS32 loci in paternity testing.     

Population study and mutation analysis for 28 short tandem repeat loci in southwest Chinese Han population

Short tandem repeat (STR) system is the most widely used genetic markers in modem forensic practice. Because of the relatively unstable molecular structure, STRs show a high mutation rate. In the current study, we report 169 mutation events of 13 CODIS and 15 non-CODIS STR loci that were found in 5569 cases of trios and duos paternity test. Our result indicated that locus-specific mutation rate varied among different populations, geometric means of the longest run of perfect repeats (LRPR) and heterozygosity. Along with previous published data, a forensic dataset for allele frequencies and locus-specific mutation rates of 13 CODIS and 15 non-CODIS STR loci from southwest Chinese Han population has been established. The mutation rate data have important implications in interpreting forensic individual identification and paternity testing.     

Calculation of likelihood ratio Y/X in diagnosis of paternity using computer methods

Summary A new computational method using a Monte Carlo technique is described for the calculation of plausibility of paternity in blood group systems.In this study gene frequencies of a blood group system are simulated by the range of the seven digit random numbers. By using a Monte Carlo method, four random numbers are generated and converted into paternal and maternal genotypes. Then the genotype of the child is determined according to the law of inheritance, and finally genotypes of the father, mother and child are converted into phenotypes.Repeating this process more than one hundred thousand times, the phenotypic frequencies of child-mother-father combinations (trio) and the likelihood ratio of paternity in any blood group system are calculated for all phenotypic combinations of the trios.This method is much easier than methods reported previously, and is sufficiently accurate.     

Application of single-locus hypervariable region DNA probes to deficiency cases in paternity testing

Summary Seven DNA probes which recognize single-locus hypervariable region (HVR) were applied to a paternity test in which the putative father and his wife were deceased. Three legitimate children, an illegitimate child and her mother were available for analysis. The cumulative paternity index of the illegitimate child derived from 15 conventional blood group markers was 18.71 and from 7 DNA probes 92,572.08, that is, 4,948 times higher than the former. Thus the DNA analyses gave nearly conclusive evidence that the putative father was the biological father of the child. The application of highly discriminating polymorphisms of DNA which recognize single HVR loci is considered to be extremely informative in cases of disputed parentage.     

False paternity with one or two mismatches using commercial STR kits

This study presents a case of false paternity where one or two mismatches were found by using three commercial STR kits. The analysis with the Identifiler kit yielded two mismatches at the loci D2S1338 and vWA. These data did not, however, enable us to exclude the alleged father, as the total number of excluding loci was less than three. Further STR loci were therefore employed to resolve the case. The PowerPlex 16 system yielded only one mismatch at the vWA locus previously found with the Identifiler kit. GenePhile G-Plex, on the other hand yielded two inconsistencies at D3S1744 and D18S536 (out of 15 loci in total). Since the disputed child was a female and we were not able to exclude the possible involvement of a close male relative, we choose to use Genephile X-Plex kit to finally resolve the case. Out of 13 loci tested, we found a complete match of the child's profile with the mother and eight mismatches with the alleged father, clearly indicating that the alleged father is not the biological father. This case emphasizes the usefulness of either Y-chromosome or X-chromosome DNA data for interpreting borderline paternity cases.     

Estimating mutation rates from paternity casework

We present a statistical methodology for making inferences about mutation rates from paternity casework. This takes account of a number of sources of potential bias, including hidden mutation, incomplete family triplets, uncertain paternity status and differing maternal and paternal mutation rates, while allowing a wide variety of mutation models. An object-oriented Bayesian network is used to facilitate computation of the likelihood function for the mutation parameters. This can process either full or summary genotypic information, both from complete putative father–mother–child triplets and from defective cases where only the child and one of its parents are observed. We use a dataset from paternity casework to illustrate the effects on inferences about mutation parameters of various types of biases and the mutation model assumed. In particular, we show that there can be relevant information in cases of unconfirmed paternity, and that excluding these, as has generally been done, can lead to biased conclusions.