SNPs in paternity investigation: The simple future

Based on the 52 SNP-plex developed by the SNPforID Consortium, we designed two 10-plex to study single nucleotide polymorphisms (SNPs) for human identification and to establish its usefulness in paternity casework. This 20 autosomal SNP set was studied in 56 paternity investigation cases from South Portuguese resident population, also analyzed with 17 Short Tandem Repeats (STRs). Results obtained with both methodologies were consistent with each other, except for one case where the alleged father could not be excluded by SNPs. No mutation was found in the SNP loci, whereas a mismatch in STRs was detected. The use of SNPs as a complement to the analysis of autosomal STRs in paternity casework can result in paternity index and paternity probability values equivalent or higher than those obtained with more STR loci, but with lower costs. This study shows that instead of using additional STR loci, the analysis of 20 autosomal SNPs, as a complement technique to standard methodologies, is an appealing alternative in paternity investigation cases.     

Allele frequencies of six miniSTR loci (D10S1248, D14S1434, D22S1045, D4S2364, D2S441 and D1S1677) in a Spanish population

Allele frequencies and forensic parameters for six miniSTR autosomal loci (D10S1248, D14S1434, D22S1045, D4S2364, D2S441 and D1S1677) were obtained from a sample of 264 unrelated individuals from Spain. No significant deviations from Hardy-Weinberg expectations were found. Due to the small PCR products (<125 bp), the use of these non-CODIS (NC) miniSTRs can increase the probability that a degraded sample can be typed. Additionally, these systems can be used in routine paternity analyses where more markers are needed to increase the power of exclusion or in complex paternity cases (e.g. involving closely related individuals).     

Population data for MiniNC01 in a population sample from North-eastern Italy and their use in neoplastic tissues fixed in formalin and embedded in paraffin

To establish a database for the three MiniNC01 loci D10S1248, D14S1434, D22S1045 in a population sample from North-eastern Italy, 102 unrelated individuals were typed. DNA was amplified in a multiplex reaction with subsequent automatic detection using capillary electrophoresis. The obtained data give a contribution to the definition of Italian population miniSTRs allele frequencies for the three analysed loci. These three MiniSTRs were tested on 21 neoplastic tissues and the obtained genotypes were compared to those obtained from normal tissue. Only 3 cases (14.28%) gave a different genotype suggesting a better performance of these markers than traditional STRs.     

Resolving relationship tests that show ambiguous STR results using autosomal SNPs as supplementary markers

When using a standard battery of STRs for relationship testing a small proportion of analyses can give ambiguous results – where the claimed relationship cannot be confirmed by a high enough paternity index or excluded with fully incompatible genotypes. The majority of such cases arise from unknowingly testing a brother of the true father and observing only a small number of exclusions that can each be interpreted as one- or two-step mutations. Although adding extra STRs might resolve a proportion of cases, there are few properly validated extra STRs available, while the commonly added hypervariable SE33 locus is four times more mutable than average, increasing the risk of ambiguous results. We have found SNPs in large multiplexes are much more informative for both low initial probabilities or ambiguous exclusions and at the same time provide a more reliable genotyping approach for the highly degraded DNA encountered in many identification cases. Eight relationship cases are outlined where the addition of SNP data resolved analyses that had remained ambiguous even with extended STR typing. In addition we have made simulations to ascertain the frequency of failing to obtain exclusions or conclusive probabilities of paternity with different marker sets when a brother of the true father is tested. Results indicate that SNPs are statistically more efficient than STRs in resolving cases that distinguish first-degree relatives in deficient pedigrees.     

Pros and cons in the use of SNPs in forensic kinship investigation: a comparative analysis with STRs

Recent advances in single nucleotide polymorphisms (SNPs) research have raised the possibility that these markers could replace the forensically established short tandem repeats (STRs). In this work, we compare STRs and SNPs applicability for kinship investigation in terms of expected informative content and probability of occurrence of "difficult cases" (when isolated Mendelian incompatibilities between alleged father and child are found). Since SNPs have a much lower mutation rate than STRs, these difficulties were expected to occur less frequently if SNPs were used instead of STRs. The purpose of this paper is to make some simulations allowing the estimation of how often such difficult cases are expected to occur using both types of markers and how serious can be their impact in routine work. Our results demonstrate that a battery based exclusively on SNPs matching the informative power of current STR kits would be prone, if applied to routine paternity investigation, to the occurrence of cases where the statistical evidence would be inconclusive. We infer that the introduction of a SNP based strategy, as a substitute to the now classical STR approach poses statistical problems that must be carefully evaluated.     

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.     

Validation of nine non-CODIS STR loci for forensic use in a population from Central Poland

The D7S1517, D3S1744, D12S391, D2S1360, D6S474, D8S1132, D5S2500, D10S2325 and D4S236613 are STR loci potentially useful for forensic purposes whose analysis has recently become facilitated by availability of a commercial kit. The purpose of the study was to evaluate the usefulness of these loci for forensic identification in a population of Central Poland. The distribution of alleles of the nine STRs was determined in sample of 353 unrelated individuals born in Central Poland and indices of forensic informativeness were calculated. The studied loci were highly informative and did not show departures from Hardy-Weinberg equilibrium. For the loci located on the same chromosomes (D2S1360, D3S1744 D4S2366, D5S2500, D7S1517, D8S1132, D12S391) as other loci commonly used for identification purposes (TPOX, D2S1338, D3S1358, FGA, D5S818, D7S820, D8S1179 and D12S391) appropriate pairwise analysis of linkage disequilibrium was performed. In all cases no statistically significant deviation from independence was found. We conclude that the studied STRs are informative and, when necessary, can be used to extend the results obtained with other STRs commonly analyzed for identification purposes, in particular the CODIS set.     

Distinguishing minisatellite mutation from non-paternity by MVR-PCR

Minisatellite variant repeat (MVR) mapping using the polymerase chain reaction (PCR) was devised to map the interspersion pattern of subtle variant repeats along minisatellite tandem arrays. MVR-PCR has revealed enormous diversity of allele structures at several loci, far more than can be resolved by allele length analysis. We have reported the application of MVR-PCR at minisatellite MS32 (D1S8) and MS31A (D7S21) in a paternity case lacking a mother and showed that it resulted in higher paternity probabilities than for a set of 12 other DNA markers including six STRs. Hypervariable minisatellites like MS32 and MS3lA can however, show significant germline mutation rates to new length alleles which can generate false exclusions in paternity cases although paternity cases showing mutant paternal alleles at more than one locus will be rare when several MVR loci are examined. Detailed knowledge of mutation processes coupled with MVR analysis of allele structure can help distinguish mutation from non-paternity. We now show how similar mutant alleles are to their progenitors using both real and simulated data, and demonstrate how MVR-PCR can be used to identify mutant paternal allele in paternity cases showing apparent exclusions.     

Supplementary markers for deficient immigration cases: Additional STRs or SNPs?

Analysis with commonly available STR kits can sometimes fail to produce sufficient information in immigration cases containing only one parent. In these cases, not only does paternity/maternity need to be assured, but also other possible relationships dismissed (e.g. avuncular relationships).We have taken more than 50 of these cases and investigated which type of additional marker produces the greatest benefits: 48 SNPs or 6 additional informative STRs (including 5 additional markers from the new European extended set). The results of this analysis show the SNPs to be of greater value.     

Allele frequencies data and statistic parameters for 13 STR loci in a population of the Brazilian Amazon Region

Allele frequencies for 13 short tandem repeat (D3S1358, vWA, D21S11, D18S51, D5S818, D13S317, D7S820, TH01, TPOX, D16S539, CSF1PO, D8S1179 and FGA) loci were determined in a sample of 325 unrelated individuals from the population of the Amazon of Belém, Brazil. These loci are the most commonly used in forensic and paternity testing. The forensic parameters investigated presented high values. The power of discrimination and the probability of exclusion for these 13 STRs are 99.999999999992% and 99.9998%, respectively. In conclusion, these 13 markers are suitable for forensic analysis and paternity tests of the Amazonian population.     

Population data of nine miniSTR loci in Koreans

For highly degraded DNA samples of forensic casework, new miniSTR systems have been developed to supplement the current STR systems. In the present study, nine miniSTR loci were analyzed in 300 unrelated Koreans using three multiplex PCR systems (multiplex I: D10S1248, D14S1434 and D22S1045; multiplex II: D1S1677, D2S441 and D4S2364; and multiplex III: D3S3053, D6S474 and D20S482), and allele frequencies and forensic parameters were calculated. These data demonstrated that D10S1248, D2S441, D22S1045, D14S1434, and D6S474 are as highly informative as the CODIS STRs suggesting that the miniSTRs could be useful for forensic analysis of degraded DNA.     

Portuguese population and paternity investigation studies with a multiplex PCR--the AmpFlSTR Profiler Plus

A Portuguese Caucasian population of 146 unrelated individuals was studied. DNA samples were amplified by multiplex PCR for D3S1358, vWA, FGA, D8S1179, D21S11, D18S51, D5S818, D13S317 and D7S820 using the AmpFlSTR Profiler Plus PCR Amplification Kit (Perkin-Elmer). All loci met Hardy-Weinberg expectations. Forensic statistical parameters were according to those obtained by other authors. Statistical differences were observed concerning three loci when comparing the Portuguese Caucasian population and an Italian Caucasian population, although these differences mainly concern the less frequent alleles. Eighty-three paternity investigation cases were analysed. Exclusions in between three and nine loci were observed in all the 23 exclusion cases obtained. Most of the non-exclusion cases had probability of paternity > 99.9%. Two cases with an isolated genetic incompatibility between the alleged father and the child were detected, which may indicate probable mutation cases. These results demonstrate that the AmpFlSTR Profiler Plus is a suitable multiplex for paternity investigation in the Portuguese population.     

Analysis of global variability in 15 established and 5 new European Standard Set (ESS) STRs using the CEPH human genome diversity panel

The CEPH human genome diversity cell line panel (CEPH-HGDP) of 51 globally distributed populations was used to analyze patterns of variability in 20 core human identification STRs. The markers typed comprised the 15 STRs of Identifiler, one of the most widely used forensic STR multiplexes, plus five recently introduced European Standard Set (ESS) STRs: D1S1656, D2S441, D10S1248, D12S391 and D22S1045. From the genotypes obtained for the ESS STRs we identified rare, intermediate or off-ladder alleles that had not been previously reported for these loci. Examples of novel ESS STR alleles found were characterized by sequence analysis. This revealed extensive repeat structure variation in three ESS STRs, with D12S391 showing particularly high variability for tandem runs of AGAT and AGAC repeat units. The global geographic distribution of the CEPH panel samples gave an opportunity to study in detail the extent of substructure shown by the 20 STRs amongst populations and between their parent population groups. An assessment was made of the forensic informativeness of the new ESS STRs compared to the loci they will replace: CSF1PO, D5S818, D7S820, D13S317 and TPOX, with results showing a clear enhancement of discrimination power using multiplexes that genotype the new ESS loci. We also measured the ability of Identifiler and ESS STRs to infer the ancestry of the CEPH-HGDP samples and demonstrate that forensic STRs in large multiplexes have the potential to differentiate the major population groups but only with sufficient reliability when used with other ancestry-informative markers such as single nucleotide polymorphisms. Finally we checked for possible association by linkage between the two ESS multiplex STRs closely positioned on chromosome-12: vWA and D12S391 by examining paired genotypes from the complete CEPH data set.     

SNP genotyping by multiplex amplification and microarrays assay for forensic application

OBJECTIVE: Research on the application feasibility of SNP genotyping for forensic identification by microarrays. METHODS: Oligonucleotide microarrays which could detect 34 different SNPs were used. After hybridization and washing, the arrays were scanned and fluorescence intensities analyzed using Microarray software. Population studies on 34 SNP loci were carried out in a sample of 109 unrelated Chinese Han individuals using oligonucleotide microarrays for genotype detection. The method was also applied to cases. RESULTS: According to the results of population studies, no deviations from Hardy-Weinberg equilibrium could be found. Among the 34 loci, 3 SNPs were low informative, 4 were medium informative and 27 were high informative. The combination discrimination power (CDP) of the 31 optimal polymorphic SNPs was 0.9999999999979. The matching probability was 2.13 x 10(-12). The average exclusion probability in paternity testing for duos was 0.9609. The average exclusion probability in paternity testing for trios was 0.9970. CONCLUSION: The data and case application demonstrated that SNP typing by oligonucleotide probe microarrays was a useful technique for paternity testing and individual identification. Combined with the 28 SNPs loci distributed on HLA-DRB1 and ABO genes, the combination discrimination power (CDP) was 0.9999999999999910. The matching probability was 9.02 x 10(-15). The average exclusion probabilities in duos and in trios were 0.9894 and 0.9992, respectively. It may be concluded that the 59 SNPs loci yield the same power in forensic identification as CODIS STRs currently used.     

Results of the 2009 Paternity Testing Workshop of the English Speaking Working Group of the International Society for Forensic Genetics

Here we present the results of the 2009 Paternity Testing Workshop of the English Speaking Working Group of the International Society for Forensic Genetics. The exercise included paternity testing of blood samples from a mother, a child and two alleged fathers. The laboratories were encouraged to answer questions concerning their laboratory routines and a paper challenge was distributed in order to compare statistical calculations. A total of 62 laboratories participated. The laboratories used a total of 49 autosomal STRs and PCR-investigated VNTRs, 19 Y-chromosomal STRs, 8 X-chromosomal STRs, 7 VNTR systems investigated with RFLP, 49 autosomal SNPs and 11 mtDNA SNPs. The rate of typing and reporting errors was 0.1%.     

Possible pitfalls in motherless paternity analysis with related putative fathers

Nowadays, more and more paternity cases are carried out investigating only child and putative father, mostly for economical or private reasons. Usually, reliable results can be obtained and the putative father can be included or ruled out with a high certainty. Considerable problems might arise when a relative of the biological father is investigated as being the putative father. In this study, we investigated 164 persons from 27 families creating artificial deficiency cases using the AmpFlSTRIdentifiler kit, which amplifies 15 STRs simultaneously. We analyzed 93 child/biological father pairs and the corresponding uncles, respectively the brothers of the biological fathers. The average paternity probability for the biological father was 99.9699% (paternity index (PI): 3321.26); only in three cases the results were under 99.9%. In five out of 125 child/uncle pairs no STR mismatches were found and paternity probabilities between 99.9726% (PI 3652) and 99.9970% (PI 33,545) were calculated. The average number of excluding loci was 3.4, but in 31.2% of the cases only zero, one or two mismatches were found. When both putative fathers were genetically typed, the biological father usually had a statistically higher paternity probability. Nevertheless, the differences between probabilities for father and uncle were only small. These results show that a reliable investigation of deficiency cases (i.e. child and putative father) seems to be more difficult than generally assumed. Especially in cases with an unknown familiar background and/or when investigating foreigners for immigration purposes, the laboratory expert should include the mother, increase the number of investigated loci or include a second method such as RFLP-analysis, some serological systems or typing of X-chromosome specific STRs to further ascertain the results.     

Y-SNP-genotyping - a new approach in forensic analysis

Y-chromosomal DNA polymorphisms, especially Y-STRs are well established in forensic routine case work. The STRs are used for identification in paternity deficiency cases and stain analysis with complicate mixtures of male and female DNA. In contrast, Y-chromosomal SNPs are a new tool in forensic investigations. At present, Y-SNPs are mainly used in molecular anthropology for evolutionary studies. Nevertheless, these markers could also provide very useful information for the analysis of forensic cases. The aim of the presented study was to test Y-SNP-typing for stain analyses using different methods-SNaPshot and MALDI-TOF MS. Both methods are based on the principle of minisequencing. The selected Y-SNP markers are suited to define the most important European haplogroups.     

对Cofiler体系在亲子鉴定中应用价值的研究

将商品化 Profiler Plus和 Cofiler扩增检测试剂盒用于亲子鉴定案例和无关个体血样的分析。 186例亲子鉴定案中,母－子－假设父三联体 149例,假设父－子二联体 37例。在 149例含双亲和孩子的鉴定中,有 127例认定存在亲生血缘关系,其 RCP值平均为 99.98％;其余 22例排除了被检父为孩子生父,排除机率为 14.77％,排除指标平均为 6.1个。在 37例含单亲及孩子的鉴定中,有 23例认定存在亲生血缘关系,其 RCP值平均为 99.92％;其余 14例排除被检父为孩子生父,排除机率为 37.84％,平均有 5个不吻合的 STR位点出现。若特意省去三人组合案例中的母亲,将其作为单亲案件进行统计,仅检测 Profiler Plus体系的 9个 STR位点时,则 RCP值平均为 99.1959％,案例中 21.51％将不能得出明确的结论。同时检测 Profiler Plus和 Cofiler体系共 13个 STR位点,则案例中 97.31％能排除或认定生父,案例中 2.69％通过补充母亲样本能排除或认定生父。 Cofiler体系中 6个 STR位点的累积排除率达 0.9825。上述数据表明, Cofiler试剂盒在单亲鉴定案件中有着重要的应用价值。     

常染色体STR基因座三带型的观察与分析

目的观察亲子鉴定常用STR基因座的三带型现象，分析探讨其特点。方法分析11985例亲子鉴定（含29111个个体）的STR分型数据，筛选三带型事件，统计三带型的频率并分析三带型的特点。结果三带型的检出总频率为0．0721％（21／29111），其中D21Sll检出率最高，为0．0172％（5／29111）；D18S51检出率为0．0137％（4／29111）；FGA检出率为0．0103％（3／29111）。Typel三带型携带者随机传递一个等位基因给子代，Type2携带者可遗传两个等位基因给子代。结论三带型结果较少见，判读须慎重。     

单亲鉴定案例STR选择探讨和PI值计算方法评价

作者对STR位点的个体识别能力(DP)、杂合度(H)、非父排除率(PE)、多态信息含量(PIC)等进行统计学分析,探寻了适合中国人群单亲子鉴定、且有利于国内外DNA实验室数据交流的STR位点,并对现行的PI值计算方法进行了评价。