Hypervariable regions of DNA for parentage testing and individual identification

Summary Four kinds of DNA probes that recognize hypervariable regions (HVR) were studied for parentage testing and individual identification. Allele frequencies and their confidence intervals among unrelated Japanese individuals were obtained. Codominant segregation of the polymorphism was confirmed in family studies. Two a priori probabilities were calculated for each HVR locus: the exclusion probabilities for an alleged father/mother/ child trio and for an alleged parent/child duo, and the probabilities of matching of genotypes of two unrelated individuals or two siblings. The ease of availability of the probes and their highly discriminating polymorphic patterns mean they could be very useful for forensic purposes.     

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.     

Paternity testing with VNTR DNA systems

Summary Paternity testing using DNA polymorphism of variable numbers of tandem repeat (VNTR) regions with restriction fragment length polymorphism (RFLP) was implemented. HinfI-digested DNA was separated by electrophoresis in agarose gels and hybridized with radiolabelled probes detecting the VNTR-systems D2S44 (YNH24), D5S43 (MS8), D7S21 (MS31), D7S22 (g3), and D12S11 (MS43a). The intra gel variability of 970 duplicate investigations on the same gel of DNA from 122 individuals showed no differences exceeding 1.25 mm between the positions of the corresponding DNA fragments. The comparison of 1,624 DNA fragments from 342 mother/child pairs showed only one difference above 1.25 mm which was interpreted as a mutation. Based on these observations, we decided to consider an intra gel difference above 1.25 mm between the non-maternal DNA fragment of the child and the nearest DNA fragment of the putative father as an exclusion in paternity testing. This matching criterion was used for the comparisons of 1,197 DNA fragment differences in 247 pairs of children and putative fathers who had not been excluded by conventional marker systems. In all of these cases, the migration differences between the DNA fragments of non-excluded men and the DNA fragments of the children were less than 1.25 mm except in 6 cases (0.5%). The man/child differences in all of 227 false trios exceeded 1.25 mm in 2 or more of the 5 VNTR systems investigated. Matching criteria for inter gel comparisons in paternity testing were established. The frequency distribution of Hinfl digested DNA fragments of the 5 VNTR systems in 650 unrelated Danes is presented and the raw data is available.     

Haplotype analysis of a de novo allele at a vWF STR locus using flanking STR loci

We have detected an unusual allele at the vWF-Kimpton (vWF-K) loci in the DNA of a child (genotype: 1415) in a paternity trio case. One allele of the child's DNA was found to derive neither from the mother (1214) nor from the putative father (1314), whose paternity was established not only by conventional polymorphic markers (probability 0.99999) but also by the other 10 STRs and the D1S80 and HLA DQ alpha loci. Two STRs flanking vWF-K comprise vWF haplotypes, which allow the parental origin of the unusual allele to be determined. Sequencing of clones encompassing the three STRs showed that the unusual allele segregated with the paternal haplotype. The de novo allele of the child thus seemed to be generated from the longer allele (14) by gaining a single unit (TCTA) through slippage replication.     

DNA investigations on fetal material from paternity cases

Summary Three paternity cases have been investigated where DNA was extracted from fetuses (age: 8–10 weeks old) after interruption of pregnancy. In each case it was possible to clearly identify the putative father using 5 or 6 single locus probes (SLP's). Fetal bands (SLP) could be clearly identified from mixtures of placental and fetal DNA by comparison with the maternal and paternal bands. However, it was very difficult to resolve the fragment patterns of tissue mixtures with one multi locus probe (MLP), because of band overlap. Another advantage of using SLP's was that biostatistical calculations could be carried out and very informative Essen-Möller values for the probability of paternity were obtained.     

Paternity testing that involves a DNA mixture

Here we analyse a complex disputed paternity case, where the DNA of the putative father was extracted from his corpse that had been inhumed for over 20 years. This DNA was contaminated and appears to be a mixture of at least two individuals. Furthermore, the mother's DNA was not available. The DNA mixture was analysed so as to predict the most probable genotypes of each contributor. The major contributor's profile was then used to compute the likelihood ratio for paternity. We also show how to take into account a dropout allele and the possibility of mutation in paternity testing.     

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.     

Komplikationen in der Vaterschaftsbegutachtung durch reduzierten Untersuchungsauftrag

A paternity case is presented which led to difficult or possibly false interpretation depending on the genetic methods employed. Investigation of the son and putative father by STR analysis only led to an ambiguous result while RFLP typing definitely excluded the man from fatherhood. The additional investigation of the mother also led to the exclusion after STR typing only. This case clearly shows how the increasing tendency to lower the costs of an analysis by investigation of child and putative father only and/or use of only a minimum number of methods required can lead to difficult or possibly false conclusions.     

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.     

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.     

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.     

Abstammungsbegutachtung mit der RFLP- und STR-Analyse

The combination of restriction fragment length polymorphism (RFLP) and short tandem repeat (STR) analyses for paternity analysis is presented. The two methods were compared by investigating 113 paternity cases. RFLP analysis was done using the single locus probes YNH24, MS31 and MS43A and for STR investigations the Identifiler Plus kit was employed. The lowest paternity probability obtained via RFLP analysis was 98.936% compared to 99.99844% when using STR analysis and the highest values were 99.9996 (RFLP) and >99.999999% (STR). Using 3 single locus DNA probes the paternity probability was <99.9% in 45.5% of the cases, while STR analysis always led to at least 99.9%. In 36 cases the father was excluded. Using STR analysis between 4 and 12 exclusions out of 15 investigated loci per case were observed. In 14 cases (39%) RFLP analysis alone did not yield the 3 exclusions necessary for exclusion of paternity. In summary it could be shown that in all cases both STR analysis alone and the combination of STR and RFLP investigations led to results which conformed to the requirements of the German guidelines.     

Further evidence of a silent plasminogen (PLG) allele in two paternity cases

Summary Routine paternity testing has yielded two different cases of an apparent inverse homozygosity in the plasminogen (PLG) system. In one case, the child presented the phenotype PLG A and his putative father the type PLG B. The alleged father could not be excluded from the paternity in 25 additional blood group marker systems (biostatistical probability of paternity W>99.75%). In the other case an incompatibility was found in a mother-child pair. Analysis of PLG was carried out by isoelectric focusing on neuraminidase-treated sera. In both cases the immunologic and functional detection showed weaker banding pattern of the affected PLG types. The assumption of a silent allele in the PLG system was confirmed by quantitative investigations. The allele frequency of PLG*Q0 in the South German population was estimated to be 0.0013. In the same sample the variant PLG A3 has been shown to be polymorphic.This paper is dedicated to Prof. Dr. Hartwig Cleve on the occasion of his 60th birthday     

The examination of the ITI system in disputed paternities

Summary 106 paternity cases with a total of 114 putative fathers were examined in the inter--trypsin inhibitor (ITI) system. Analysis was performed by isoelectric focusing (IEF) of untreated sera on polyacrylamide gels. From 39 paternity exlusions, determined in other genetic systems, 7 were confirmed in the ITI system. In 75 expertises the alleged man was not excluded from fatherhood; in 68 cases the probability of paternity was W > 99.73%. The practical exclusion rate in the ITI system was therefore calculated to be 10.45%. The theoretical exclusion rate was determined to be 19.3%. In one paternity case the alleged father and the child showed inverse homozygosity in the ITI system, while the man was not excluded from fatherhood in 28 additional marker systems. The calculated probability of his paternity was 99.99%. The assumption of an incomplete expression of the ITI phenotypes in infants is supported by a significant deviation between the observed and expected ITI distributions at population equilibrium.     

General formulas of the estimated likelihood ratio Y/X in the diagnosis of paternity of a deceased putative father

Summary When the putative father is dead his probable genotype, essential for estimate of likelihood ratio in the diagnosis of paternity, should be deduced from his relatives. In the present paper are described the general method for such deduction of probable genotype and the derivation of the formula of likelihood ratio. 10⁴ examples of the diagnosis of paternity of deceased father are examined using a Monte Carlo method and the distributions of relative frequencies of log(Y/X) are calculated for the true father and non-father. These results indicate that the present method of estimation of Y/X from relatives is quite useful for the diagnosis of paternity of a deceased putative father.     

Sibling incest and formulation of paternity probability: case report

Paternity determination of a fetus whose mother was admitted to an institution for the welfare and health of handicapped persons was requested of us by a doctor and lawyer of the institution. The fetus was recovered by a legal artificial abortion based on the Act on Maternity Health and Welfare (Japan) with the permission of the custodian. Commercially available MCT118, HLADQA, PM, and 9 STRs were tested for DNA samples from the fetus, the mother, her younger brother, her father, her grandfather, and 4 staff members of the institution. Only the brother was not excluded and the paternity probability was estimated at 99.857% on the basis of newly formulated expressions for multiallelic loci on the assumption of sibling incest. We concluded then that the fetus was fathered by the brother. DNA fingerprinting with multilocus and single locus minisatellite probes which were performed to confirm the paternity also support the conclusion. Bandsharing frequencies between the family members, however, did not necessarily reflect their actual kinship, which findings suggest that multilocus DNA fingerprinting requires further accumulation of data for consanguineous cases such as incest. Universal formulation for calculating paternity probability for a sibling incest case on the basis of multiallelic monolocus polymorphisms is also presented.     

Pränatale Paternitätsbestimmung mittels PCR-VNTR-Polymorphismen

A case of prenatal paternity diagnosis where the woman had been raped is presented. PCR-VNTR polymorphisms were investigated on material which was obtained by biopsy during the 10th week of pregnancy. After DNA extraction nine PCR-VNTR polymorphisms were examined and the husband of the pregnant woman could not be excluded as the biological father of the child with a high degree of certainty. The ethical aspects associated with this problem will be discussed.     

Prenatal paternity testing with DNA analyses

Summary Two PCR amplified loci and 3 single locus DNA probes were applied in a paternity case in which a married woman became pregnant after being raped. DNA analysis were performed using samples from the woman, her husband and amniotic fluid cells taken during the 16th week of pregnancy. The combined probability of paternity for her husband was calculated as 0.999997107. The application of PCR analyses and single locus DNA probes were considered to be extremely informative in prenatal paternity testing.     

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.     

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.