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.     

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.     

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.     

Paternity exclusion power: Comparative behaviour of autosomal and X-chromosomal markers in standard and deficient cases with inbreeding

In paternity testing the informativeness of genetic markers is traditionally measured through the probability of finding, in randomly chosen individuals, inconsistencies with parent to child Mendelian rules of transmission. This statistic, called power of exclusion (PE), paternal exclusion chance or probability, can be defined for duos (mother not typed) or trios (random false fathers are matched against mother/child pairs) and performed both for autosomal and X-chromosomal markers (restricted to paternity testing involving daughters). PE is an a priori statistic, in the sense of not depending on the individual's genetic data of a case, being dependent however on the estimates of genetic markers allele (or haplotype) frequencies.We have studied the behaviour of this statistic in situations where the randomness assumption is not met, because either (a) the alleged – and false – father is related to the true one, or (b) there is a non-negligible level of background relatedness in the population.For the first case, we derived general (autosomal and X-chromosomal) PE formulas for duos and trios for any genealogy linking alleged father and child, highlighting that the PE of each marker only depends on a single kinship parameter associated with their pedigree. In this case we also estimate a lower bound for the number of extra markers needed to be analysed to achieve the same global power as for unrelated individuals. In the second situation, we demonstrate that for realistic values of the coancestry coefficient the decrease in PE due to population inbreeding is very moderate even when duos are analysed.In this work, beyond the aforementioned issues, we also discuss the suitability of assuming the pedigree father–daughter for calculating the X-PE, since X-markers are not the tool of choice in laboratorial routine when the alleged father is available for testing. Indeed, X-markers are particularly useful in situations where the alleged father is not available for testing but experts are able to type the mother or a daughter of his. Such increase of power is due to the paternal genealogies: half- and full-sisters, and grandmother–granddaughter, having a non-null X-PE even when only duos are analysed in contrast to what happens for autosomes. Algebraic expressions for these cases are also presented.     

Application of the new insertion–deletion polymorphism kit for forensic identification and parentage testing on the Czech population

Insertion-deletion polymorphisms (INDELs) are diallelic markers derived from a single mutation event. Their low mutation frequency makes them suitable for forensic and parentage testing. The examination of INDELs thus combines advantages of both short tandem repeats (STR) and single nucleotide polymorphisms (SNP). This type of polymorphisms may be examined using as small amplicon size as SNP (about 100 bp) but could be analyzed by techniques used for routine STR analysis. For our population study, we genotyped 55 unrelated Czech individuals. We also genotyped 11 trios to analyze DIPplex Kit (QIAGEN, Germany) suitability for parentage testing. DIPplex Kit contains 30 diallelic autosomal markers. INDELs in DIPplex Kit were tested with linkage disequilibrium test, which showed that they could be treated as independent markers. All 30 loci fulfill Hardy–Weinberg equilibrium. There were several significant differences between Czech and African populations, but no significant ones within European population. Probability of a match in the Czech population was 1 in 6.8?×?10¹²; combined power of discrimination was 99.9999999999%. Average paternity index was 1.13–1.77 for each locus; combined paternity index reached about 27,000 for a set of 30 loci. We can conclude that DIPplex kit is useful as an additional panel of markers in paternity cases when mutations in STR polymorphisms are present. For application on degraded or inhibited samples, further optimization of buffer and primer concentrations is needed.     

Evaluation of the Genplex SNP typing system and a 49plex forensic marker panel

Using a 52 SNP marker set previously developed for forensic analysis, a novel 49plex assay has been developed based on the Genplex typing system, a modification of SNPlex™ chemistry (both Applied Biosystems) using oligo-ligation of pre-amplified DNA and dye-labeled, mobility modified detection probes. This gives highly predictable electrophoretic mobility of the allelic products generated from the assay to allow detection with standard capillary electrophoresis analyzers. The loci chosen comprise the 48 most informative autosomal SNPs from the SNPforID core discrimination set supplemented with the amelogenin gender marker. These SNPs are evenly distributed across all 22 autosomes, exhibit balanced polymorphisms in three major population groups and have been previously shown to be effective markers for forensic analysis. We tested the accuracy and reproducibility of the Genplex system in three SNPforID laboratories, each using a different Applied Biosystems Genetic Analyzer. Genotyping concordance was measured using replicates of 44 standardized DNA controls and by comparing genotypes for the same samples generated by the TaqMan^®, SNaPshot^® and Sequenom iPLEX^® SNP typing systems. The degree of informativeness of the 48 SNPs for forensic analysis was measured using previously estimated allele frequencies to derive the cumulative match probability and in paternity analysis using 24 trios previously typed with 18 STRs together with three CEPH families with extensive sibships typed with the 15 STRs in the Identifiler^® kit.     

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.     

A GEP-ISFG collaborative study on the optimization of an X-STR decaplex: data on 15 Iberian and Latin American populations

In a collaborative work carried out by the Spanish and Portuguese ISFG Working Group (GEP-ISFG), a polymerase chain reaction multiplex was optimized in order to type ten X-chromosome short tandem repeats (STRs) in a single reaction, including: DXS8378, DXS9902, DXS7132, DXS9898, DXS6809, DXS6789, DXS7133, GATA172D05, GATA31E08, and DXS7423. Using this X-decaplex, each 17 of the participating laboratories typed a population sample of approximately 200 unrelated individuals (100 males and 100 females). In this work, we report the allele frequencies for the ten X-STRs in 15 samples from Argentina (Buenos Aires, Córdoba, Río Negro, Entre Ríos, and Misiones), Brazil (São Paulo, Rio de Janeiro, Paraná, and Mato Grosso do Sul), Colombia (Antioquia), Costa Rica, Portugal (Northern and Central regions), and Spain (Galicia and Cantabria). Gene diversities were calculated for the ten markers in each population and all values were above 56%. The average diversity per locus varied between 66%, for DXS7133, and 82%, for DXS6809. For this set of STRs, a high discrimination power was obtained in all populations, both in males (≥1 in 5?×?10⁵) and females (≥1 in 3?×?10⁹), as well as high mean exclusion chance in father/daughter duos (≥99.953%) and in father/mother/daughter trios (≥99.999%). Genetic distance analysis showed no significant differences between northern and central Portugal or between the two Spanish samples from Galicia and Cantabria. Inside Brazil, significant differences were found between Rio de Janeiro and the other three populations, as well as between São Paulo and Paraná. For the five Argentinean samples, significant distances were only observed when comparing Misiones with Entre Ríos and with Río Negro, the only two samples that do not differ significantly from Costa Rica. Antioquia differed from all other samples, except the one from Río Negro.     

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.     

Analysis of 49 autosomal SNPs in an Iraqi population

Forty-nine of the 52 autosomal single nucleotide polymorphisms (SNPs) in the SNPforID 52plex were typed in 101 unrelated Iraqis living in Denmark. No significant deviation from HWE was found in all but one of the 49 SNP systems and no significant pairwise linkage disequilibrium was observed for any SNP pair. When 18 worldwide populations were compared (including populations in Iraq, Turkey, Israel, Pakistan, India, China, Taiwan, Japan, Siberia, Algeria, Somalia, Uganda, Mozambique, Angola, Nigeria, Denmark, Portugal, Spain), a significant global F_ST value was obtained. All but six F_ST values were statistically significant when pairwise comparisons were performed between the 18 populations. The Iraqi population did not show significant difference from the population in Turkey and it grouped together with other Middle-Eastern populations when a multidimensional scaling plot was drawn based on the pairwise F_ST values. The combined mean match probability and the typical paternity index for trios were 8.3 × 10⁻²⁰ and 259,000, respectively, for the Iraqi population.     

Autosomal SNPs study of a population sample from North of Portugal and a sample of immigrants from the Eastern Europe living in Portugal

The use of autosomal single nucleotide polymorphisms (SNPs) for forensic research has been widely discussed in recent years, mainly because SNPs have important advantages compared to short tandem repeats (STRs).In this study a total of 131 non related individuals from the North of Portugal and 85 immigrant individuals from the Eastern Europe, mainly Ukrainians, equally non related and residing in Portugal, were typed for 52 loci included in the in the SNP for ID 52plex with the SNaPshot™ assay.     

Autosomal SNPs study of a population sample from Southern Portugal and from a sample of immigrants from Guinea-Bissau residing in Portugal

In recent years, autosomal single nucleotide polymorphisms (SNPs) have been comprehensively investigated in forensic research due to their usefulness in certain circumstances in complementing short tandem repeats (STRs) analysis, or even for use on their own when analysis of STRs fails. However, as with STRs, in order to properly use SNP markers in forensic casuistic we need to understand the population and forensic parameters in question. As a result of Portugal’s colonial history during the time of empire, and the subsequent process of decolonization, some African individuals migrated to Portugal, giving rise to large African and African-descendent communities. One of these groups is the community originating from Guinea-Bissau, that in 2014, was enumerated to consist of more than 17,700 individuals with official residency status, more than the third major city of Guinea-Bissau.In order to study the population and forensic parameters mentioned above for the two populations important to our casuistic, a total of 142 unrelated individuals from the South of Portugal and 90 immigrants from Guinea-Bissau (equally non related and all residing in Portugal) were typed with SNaPshot™ assay for all 52 loci included in the SNPforID 52plex.     

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.     

Mutation rate estimates for 13 STR loci in a large population from Rio Grande do Sul, Southern Brazil

Short tandem repeat (STR) polymorphisms have been extensively used in forensic genetics analysis. Knowledge about the locus-specific mutation rates of STRs improves forensic probability calculations and interpretations of diversity data. To incorporate single-locus diversity information into autosomal STR mutation rate estimations, 13 STR loci were studied during 2007–2009 in 10,959 paternity investigation cases from Rio Grande do Sul, the southernmost state of Brazil, covering an overall number of 284,934 allelic transfers. A total of 355 mutations were identified; 348 repeats were gains or losses of one step, three were gains or losses of two steps, and four were gains or losses of not stepwise mutation. The mutation rates ranged from 4.6?×?10^?5 to 2.3?×?10^?3, and the overall mutation rate estimate was 1.2?×?10^?3. The average of the paternal mutation rate (1.8?×?10^?3) was five times higher than the maternal rate (0.36?×?10^?3). The observed mutational features for STRs have important consequences for forensic applications, including the definition of criteria for exclusion in paternity testing and the interpretation of DNA profiles in identification analysis.     

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.     

D20S161 data for three ethnic populations and forensic validation

In order to evaluate the forensic applicability of the STR locus D20S161 and construct a preliminary database, the genotype distributions and allele frequencies in five populations from three main ethnic groups were investigated, including Germans, Slovakians, African Americans, Japanese and Chinese. A total of 512 samples from unrelated individuals and 85 confirmed father/mother/¶child triplets were analyzed by PCR and allele determination was carried out by comparison with a sequenced human allelic ladder. The results showed that D20S161 typing was both precise and reliable. A total of 7 alleles was found in these populations and no evidence of deviation from Hardy-Weinberg equilibrium was observed. Pairwise comparisons between populations showed that there were significant differences in the distributions of the allele frequencies among the three main ethnic groups. No mutation events were observed from the confirmed father/mother/¶child triplets. With a maximum likelihood method, the mutation rate was indirectly estimated as 2.5 × 10^–5. These results suggest that D20S161 is a useful marker for forensic casework and paternity analysis.     

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.     

Complex kinship analysis with a combination of STRs,SNPs, and indels

Complex kinship analysis has been widely applied in disaster victim identification and criminal investigations. A larger number of genetic markers is required to improve the discrimination power of the system in complex kinship analysis compared to that in paternity testing, as distant relatives share fewer genetic segments. Genetic markers, including short tandem repeats (STRs), single-nucleotide polymorphisms (SNPs), and insertions-deletions (indels), play complementary roles in kinship analysis. Few studies have systematically analyzed the system discrimination power of a new combination of different types of genetic markers before using these markers in practice. Here, we tested the ability of a set of 56 STRs available in commercial panels on complex kinship analysis. We next introduced a combination marker set of STRs, indels, and SNPs and evaluated the system discrimination power of 72 indels + 52 SNPs to improve the weight of 56 STRs. Statistical analysis of complex kinship within third-degree kinship testing was performed to compare 56 STRs or 72 indels + 52 SNPs alone. True samples were assessed, including 99 full siblings, 112 uncle/aunt-nephew/niece, 43 grandfather/grandmother-grandson/granddaughter, 63 first cousins, and 5931 unrelated pairs. Simulation was also performed using 10,000 pairs of relatives and 10,000 unrelated individuals. The effectiveness of the three marker sets in kinship testing was ranked as follows: 56 STRs + 72 indels + 52 SNPs > 56 STRs > 72 indels + 52 SNPs. All three marker sets were powerful in first-degree kinship testing; 56 STRs and 56 STRs + 72 indels + 52 SNPs could distinguish most second-degree relatives from unrelated pairs. However, only a portion of third-degree relatives was correctly determined from unrelated individuals using 56 STRs + 72 indels + 52 SNPs. In relationship testing, 56 STRs and 56 STRs + 72 indels + 52 SNPs were powerful enough to distinguish first-degree relatives from second-degree or third-degree relatives. Our results provide a strategy and guidance applicable in forensic practice for complex kinship analysis by combining STRs, SNPs, and indels.     

Identification of the third/extra allele for forensic application in cases with TPOX tri-allelic pattern

Genotyping of polymorphic short tandem repeats (STRs) loci is widely used in forensic DNA analysis. STR loci eventually present tri-allelic pattern as a genotyping irregularity and, in that situation, the doubt about the tri-allele locus frequency calculation can reduce the analysis strength. In the TPOX human STR locus, tri-allelic genotypes have been reported with a widely varied frequency among human populations. We investigate whether there is a single extra allele (the third allele) in the TPOX tri-allelic pattern, what it is, and where it is, aiming to understand its genomic anatomy and to propose the knowledge of this TPOX extra allele from genetic profile, thus preserving the two standard TPOX alleles in forensic analyses. We looked for TPOX tri-allelic subjects in 75,113 Brazilian families. Considering only the parental generation (mother + father) we had 150,226 unrelated subjects evaluated. From this total, we found 88 unrelated subjects with tri-allelic pattern in the TPOX locus (0.06%; 88/150,226). Seventy three of these 88 subjects (73/88; 83%) had the Clayton's original Type 2 tri-allelic pattern (three peaks of even intensity). The remaining 17% (15/88) show a new Type 2 derived category with heterozygote peak imbalance (one double dose peak plus one regular sized peak). In this paper we present detailed data from 66 trios (mother + father + child) with true biological relationships. In 39 of these families (39/66; 59%) the extra TPOX allele was transmitted either from the mother or from the father to the child. Evidences indicated the allele 10 as the extra TPOX allele, and it is on the X chromosome. The present data, which support the previous Lane hypothesis, improve the knowledge about tri-allelic pattern of TPOX CODIS’ locus allowing the use of TPOX profile in forensic analyses even when with tri-allelic pattern. This evaluation is now available for different forensic applications.     

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.