The problem of single parent/child paternity analysis--practical results involving 336 children and 348 unrelated men

In a certain amount of paternity investigations, only DNA from child and alleged father is analyzed, thus increasing the possibility of false paternity inclusions. The aim of this study was to determine how many wrong paternity inclusions could be detected in a rather small geographical area comparing empirical results from 336 children and 348 men (13-15 STRs were investigated per person). This comparison between each child and all unrelated men (i.e. all putative fathers from the other cases) with an especially designed computer program resulted in 116,004 man/child pairs. Less than three excluding STRs were found in 1666 child/unrelated man pairs (1.44% of the comparisons). At least one unrelated man with only two or less STR mismatches could be determined for 322 children (95.8% of all investigated children). In 26 comparisons no STR mismatches between a child and an unrelated man were detected, thus at least one and up to three "second father(s)" under 350 men could be found for 23 children, if the mother is excluded. Paternity probabilities between 95.475% and 99.996% were calculated. Our results underline the difficulties in motherless paternity cases using only STR analysis and advise great precaution in assigning verbal predicates such as "paternity proven" in those investigations.     

Problems of assessing sibship probabilities by means of genetic analysis

Nowadays, kinship studies based only on STR analysis are a very common practice. Apart from regular paternity cases, there is a rising number of cases in which the type of sibship between two persons has to be determined. Very often the parents or further relatives are unavailable for testing, so that the probability e. g. for a half-sibship has to be calculated as a deficiency situation. 50 unrelated persons (Group 1), 50 full siblings (Group 2), and 50 half-siblings (Group 3) were genotyped using the 15 STRs comprised by the AmpFlSTR Identifiler multiplex PCR-kit to investigate the reliability of common statistics programs and the indicative value of the calculated probabilities. Using a commercially available statistics program, we calculated probabilities for the following hypotheses: 1. The persons investigated are full siblings; 2. the persons investigated are half-siblings; 3. the persons investigated are unrelated. After STR analysis, half-sibling probabilities between 0.03 %-97.56% for group 1, between 0.01%-78.46% for group 2, and between 24.28%-90.18% for group 3 were calculated. In addition, the authors present two complicated deficiency cases which could be resolved only by extensive DNA typing using X- and Y-chromosomal loci, respectively, as well as the investigation of further relatives. The results of this study suggest that typing of autosomal STRs alone is no sufficient tool for resolving cases of possible half-sibship, even if probabilities of more than 90.0% are obtained. Due to the results of our study we suggest the implementation of mandatory standards for the investigation and interpretation of complicated kinship cases.     

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.     

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.     

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.     

基于高通量测序进行无创产前亲子鉴定的可行性

目的初步探讨基于高通量测序进行STR分型的技术方法应用于无创产前亲子鉴定的可行性。方法选择13个STR基因座(6个常染色体STR基因座,6个Y染色体STR基因座,1个性别判定基因座),进行复合PCR扩增和高通量测序文库构建后,采用Ion PGM400高通量测序平台进行测序,并采用自主研发软件NGS-STR genotyper(perl脚本)进行STR分型,本文简称上述过程为NGS-STR分型。对13个母子配对混合样本(母亲:儿子=2%~50%)、1组家系样本进行了上述NGS-STR分型,旨在(1)了解其在混合样本中的灵敏度及分型情况;(2)了解其在无创产前亲子鉴定中的应用可能性。结果 (1)当混合样本中低组分(儿子)的比例超过8%,所有基因座均可检出低组分的STR信息;(2)对1例血浆样本进行NGS-STR分型,共计69.2%的基因座可检出胎儿的STR基因型信息,且所有检出基因座均符合孟德尔遗传规律。结论初步证明了NGS-STR分型技术具有进行无创产前亲子鉴定的可行性。     

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.     

15个STR在单亲亲子鉴定中的排除分析

目的评估15个短串联重复(DODIS)基因座和Penta D、Penta E)在单亲亲子鉴定中的亲权排除率。方法 以644个随机个体构成207046个假想的家系,然后统计不排除或只有一个基因座排除亲权关系的例数。结果有27个假想家系15个STR均不排除亲权关系,排除率为0.999869。有384个假想家系只有一个基因座排除,占0.185％。结论用15个STR认定单亲案的亲权关系时要谨慎。出现单个基因座排除时要检测更多的遗传标记,以区分是否系突变。     

Validation of the STR DXS7424 and the linkage situation on the X-chromosome

X-linked microsatellite markers have proven to be powerful tools for parentage testing, mainly in deficiency paternity cases when the disputed child is female. However, only a small number of X-linked short tandem repeats (STRs) have been comprehensively described for forensic applications to date.We present sequence and population genetic data of the DXS7424 STR (GDB-G00-577-633) which is a trinucleotide repeat polymorphism representing 12 alleles of 147-180 bp in length. DXS7424 is located at Xq22 and closely linked to DXS101, corresponding to a genetic localisation of 104.9-121 cM from Xp-tel.PCR fragment length measurements and sequencing were carried out using the automatic gene analyser ABI 310 (Applied Biosystems).The population of 764 unrelated Germans checked for this STR exhibited the following features: polymorphism information content (PIC) = 0.780; heterozygosity (Het) = 0.843; mean exclusion chance (MEC = 0.766. Kinship tests revealed a typical X-linked inheritance. In 300 meioses under investigation, mutations were not found. Significant deviations from the Hardy-Weinberg equilibrium (HWE) were not established.Linkage studies confirmed closely linkage to DXS101. Additional we found linkage disequilibrium between DXS7424 and DXS101. This requires to use the established haplotype frequencies in kinship testing.     

Autosomal and Y-STR mutations in Lithuanian population

The new approach is given in this work combines autosomal and Y-chromosomal short tandem repeats (STRs) data in paternity testing with father/son pairs taking into account mutation events. Our results show that Y-STRs can identify nonpaternity cases with high accuracy but need to be validated with autosomal STR to establish paternity. Autosomal and Y-STR mutations give disputing questions in paternity testing as well.     

联合应用常染色体STR和X-STR标记鉴定单亲疑难案例

目的通过对常染色体和X染色体遗传标记的检测,探讨单亲疑难案例的鉴定策略。方法提取3个单亲鉴定案例的6份血样,采用Goldeneye 20A试剂盒和AGCU21＋1试剂盒检测常染色体上39个STR基因座,采用自主研制的16重X-STR扩增系统检测X染色体上16个STR基因座。结果用Goldeneye 20A试剂盒检测后发现每个单亲案例均有一个基因座不符合遗传规律,当常染色体STR基因座增加到39个时,案例1累计出现3个矛盾基因座;案例2和案例3均没有出现新的矛盾基因座。X染色体STR分型结果显示案例1有8个矛盾基因座,案例2和案例3无矛盾基因座,与常染色体分型结论相符。结论对于出现单基因座不符合遗传规律的母女、母子、父女单亲案例鉴定,不仅可以增加新的常染色体STR检测,也可以增加X染色体STR的检测,这样在相互验证的同时也能获得更加可靠的鉴定意见。     

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.     

A report of the 1997, 1998 and 1999 Paternity Testing Workshops of the English Speaking Working Group of the International Society for Forensic Genetics

We present the results of the 1997, 1998 and 1999 Paternity Testing Workshops of the English Speaking Working Group of the International Society for Forensic Genetics. The numbers of participating laboratories were 24 (1997), 31 (1998) and 32 (1999). In 1997, all laboratories drew the correct conclusion that the alleged father was the biological father of the child. In 1998, the alleged father was the biological brother of the child and all laboratories excluded him. The scenario in 1999 was a deficiency case consisting of mother, child and the parents of the alleged father and all but one laboratory drew the correct conclusion.The percentage of laboratories routinely performing variable number of tandem repeat (VNTR) investigations using single locus probes (SLPs) and restriction fragment length polymorphism (RFLP) decreased from 83% in 1997 to 66% in 1999. In the three workshops, more than 90% of the laboratories used polymerase chain reaction (PCR) based systems. In 1999, 80% of the laboratories performing PCR, used commercially available short tandem repeat (STR) kits. Other commonly used systems were HLA and PolyMarker investigated with PCR. Conventional systems and RFLP investigations of VNTRs with multi loci probes (MLPs) were used routinely by approximately 20% of the participating laboratories.All laboratories submitting results in the three workshops used RFLP-based VNTRs or/and PCR based VNTRs/STRs. Inter-laboratory comparisons of the results showed a very high concordance. The overall coefficients of variation between the laboratories of the results of RFLP typing of the commonly used VNTRs D2S44, D7S21, D7S22 and D12S11 were 1.2-1.3%. Consistent results were obtained in the great majority of the systems investigated by PCR and typing errors counted for less than 0.3% of the PCR based results.     

二联体亲权鉴定风险分析

目的探讨二联体亲权鉴定时存在的风险。方法选取22组经Goldeneye~(TM) 20A试剂盒检测后只有一个或没有不符合基因座的无关个体对构建假想家系。对其增加检测STRtyper-10G和/或AGCU 21+1 STR系统直至所有组不符合基因座个数大于3个,累积父权指数(CPI)不大于0.000 1。以三种规则:(1)不符合基因座数大于3个;(2)CPI值小于0.000 1;(3)同时满足(1)和(2),作为排除依据,使用不同数量的基因座(19个、26个、39个和46个)进行检测,讨论无关个体对的排除情况是否存在差异。结果 22组无关个体对,使用19个基因座和39个基因座以上的检测系统达到排除结果的分别为0组和22组。结论二联体亲子鉴定,使用19个基因座进行检测仍存在结果错判,39个基因座以上的检测系统能更有效的避免二联体的鉴定风险。     

Paternity analysis in deficiency cases with related putative fathers: simulation of a deficiency analysis in 27 families

During the last few years, the number of privately ordered paternity investigations has increased considerably. Probably due to financial reasons in more and more cases only the putative father and the child are investigated. Additionally, very often only one method, such as STR analysis, is employed. This raises the question whether such a reduced analysis leads to reliable and clear results when investigating cases with related putative fathers. We investigated 165 individuals from 27 families using the AmpFlSTRIdentifiler multiplex PCR and calculated the paternity probabilities of the children to their biological fathers, uncles, grand fathers and other relatives. In more than 30% less than three exclusions between child and relative were detected. In five cases no exclusions were found between child and uncle, always leading to paternity probabilities >99.9%. These results show that the calculation of high probabilities (>99.9%) does not necessarily lead to the accurate conclusion of fatherhood. In many of our cases misleadingly the brother of the real father or another close relative would have been declared to be the biological father.     

Using STR,MiniSTR and SNP markers to solve complex cases of kinship analysis

In complex kinship investigation, miniSTRs and SNPs have been frequently used in order to increase the likelihood ratio (LR), when the results obtained for the most commonly used STR multiplexes were not informative enough. In this work, we describe the results obtained when using a battery of 23 STRs, 3 miniSTRs and 52 SNPs to investigate three complex paternity cases where the father was not available, and one paternity case with bone samples, from which no results could be obtained for STRs (including the 3 miniSTRs, D10S1248, D14S1434 and D22S1045). In all cases, the additional information provided by the SNPforID 52plex identification panel was enough to achieve conclusive results.     

常染色体STR遗传标记在同胞鉴定中的应用

目的 探讨常染色体STR遗传标记用于鉴定两个体同胞关系的可行性。方法 用Power Plex~(TM)16体系15个STR基因座检测150对同胞个体和150对无关个体,ITO法计算同胞关系指数(PI_(FS))与同胞关系概率(W_(FS)),并比较两组W_(FS)值及两个体间等位基因匹配情况的差异,对前者进行组间差异的x~2检验。结果 100对(66.67％)同胞个体的W_(FS)大于0.9995;无关个体W_(FS)均小于0.8,其中100对(66.67％)W_(FS)小于0.27。同胞个体两个体间等位基因全相同的基因座个数为1～10个不等,平均5.49个,无关个体0～5个不等,平均1.33个;等位基因全不同的基因座个数,同胞个体0～6个不等,平均1.66个,无关个体2～11个不等,平均6.57个;等位基因半相同的基因座个数,同胞个体3～13个不等,平均7.85个,而无关个体1～13个不等,平均7.11个。经x~2检验,同胞个体和无关个体间全相同和全不同的基因座数差异均有极显著意义(P<0.001),半相同的基因座数差异无显著意义(P>0.05)。结论 PowerPlex~(TM)16体系可用于鉴定同胞关系。当两个体全不同基因座个数大于或等于6个,或全相同基因座数为0时,提示为无关个体;当两个体全不同基因座个数小于或等于1个,或全相同基因座数大于或等于6个时,提示为同胞。     

Identifiler^TM系统在亲子鉴定中的突变观察和分析

目的观察和分析IdentifilerTM系统15个短串联重复序列(STR)位点在亲子鉴定中的突变现象。方法用IdentifilerTM试剂盒检测2712例亲子鉴定案例。结果在2362例认定亲子关系的案例中,观察到51例中有1个STR位点发生突变。突变的位点包括D8S1179、D21S11、D7S820、CSF1PO、D3S1358、D13S317、D16S539、D2S1338、D19S433、vWA、D18S51、D5S818和FGA。其中以D21S11位点突变率最高(0.369%);突变的等位基因来自父亲36次,来自母亲7次,无法确定9次。结论STR位点突变是较为常见的现象,采用IdentifilerTM系统进行亲子鉴定,遇到1～2个STR位点不符合遗传规律时,有必要增加突变率低、稳定性好的STR位点进行复核。     

Mutations of short tandem repeat loci in Identifiler system

OBJECTIVE: To explore and analyze the mutations of 15 Short Tandem Repeat (STR) loci using Identifiler system in paternity identification. METHODS: 2712 cases of paternity testing were carried out using Identifiler PCR Amplification Kit. RESULTS: Of the 2362 paternity testing cases, mutations of single locus were observed in 51 cases. The mutation loci included D8S1179, D21S11, D7S820, CSF1PO, D3S1358, D13S317, D16S539, D2S1338, D19S433, vWA, D18S51, D5S818 and FGA, with the D21S11 locus having a highest mutation rate (0.369%). Thirty-six of the STR mutations were from paternal source, 7 from maternal source, and the rest (9) were undeterminable. The mutation rates at D21S11 were highest (0.369%). CONCLUSION: Mutations of STR loci are relatively common in human genome. Therefore, retesting of additional relatively stable STR loci with lower mutation rates is necessary when one or two loci exclusions are encountered in paternity testing.     

亲子鉴定中STR位点数选择及其应用价值研究

目的对在亲子鉴定中STR位点数的选择及其鉴定应用价值进行研究。方法将CODIS13个STR位点分为四个观测组,观测对象包括排除亲权的母亲-孩子-假设父亲三人组合102例,以及肯定亲权的母亲-孩子-假设父亲三人组合100例,通过310遗传分析仪对荧光复合扩增产物进行分型检测。结果各STR观察组出现的最低排除指标数与各观察组累积非父排除概率(CPE)值成一定正相关性,CPE值超过99.99%的两个STR观察组其出现的最低排除指标数为三个,同时这两个STR观察组在肯定亲权的案例分析中,其亲子关系概率值(RCP)值都超过了99.99%。结论对于亲子鉴定中的STR位点检测系统,其非父排除指标应为三个以上,其累积非父排除概率(CPE)值达到99.99%时,就可以认为该STR位点检测系统具有了相关的鉴定应用价值。