ISFG: Recommendations on biostatistics in paternity testing

The Paternity Testing Commission (PTC) of the International Society for Forensic Genetics has taken up the task of establishing the biostatistical recommendations in accordance with the ISO 17025 standards and a previous set of ISFG recommendations specific to the genetic investigations in paternity cases. In the initial set, the PTC recommended that biostatistical evaluations of paternity are based on a likelihood ratio principle – yielding the paternity index, PI. Here, we have made five supplementary biostatistical recommendations. The first recommendation clarifies and defines basic concepts of genetic hypotheses and calculation concerns needed to produce valid PIs. The second and third recommendations address issues associated with population genetics (allele probabilities, Y-chromosome markers, mtDNA, and population substructuring) and special circumstances (deficiency/reconstruction and immigration cases), respectively. The fourth recommendation considers strategies regarding genetic evidence against paternity. The fifth recommendation covers necessary documentation, reporting details and assumptions underlying calculations. The PTC strongly suggests that these recommendations should be adopted by all laboratories involved in paternity testing as the basis for their biostatistical analysis.     

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

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 greater than 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.     

Scientific standards for studies in forensic genetics

Forensic molecular genetics has evolved from a rapidly developing field with changing technologies into a highly recognized and generally accepted forensic science, leading to the establishment of national DNA databases with DNA profiles from suspects and convicted offenders. DNA evidence has taken a central role by carrying a significant weight for convictions, as well as by excluding innocent suspects early on in a criminal investigation. Due to this impact on the criminal justice system, guidelines for research in forensic genetics have been introduced already since many years. The most important issues regarding the selection and definition of typing systems both for paternity testing and for forensic identification, the criteria for technical and biostatistical validation, as well as the use of mitochondrial DNA analysis are summarized and discussed.     

Empirical paternity exclusion rates

We have developed an empirical method of estimating paternity exclusion rates for any genetic system by counting exclusions among fictitious "nonfather" paternity cases generated from true paternity disputes. Especially for multiallele multiloci systems, this technique has advantages over traditional formulae methods in that it replaces tedious (and, in some cases, impossible) computations with simple data file manipulations, and it avoids introducing quantities such as gametic disequilibrium and recombination fraction that are difficult to measure. Exclusion rates for standard and one-parent paternity cases are given for three racial groups (Caucasians, blacks, and Hispanics) in four genetic systems (HLA, ABO, MNSs, and Rh). Beside the method, of interest are our findings of higher HLA exclusion rates (93.25% for Caucasians, 94.62% for blacks, and 95.82% for Hispanics) compared with rates previously reported and high combined exclusion rates (89.59% for Caucasians, 91.65% for blacks, and 92.54% for Hispanics) in one-parent paternity cases.     

Laboratory evidence of unsuspected parental consanguinity among cases of disputed paternity

A search was conducted to find evidence of possible incestuous unions between the biologic parents of children involved in 2500 paternity cases. Suspicion was raised when either (1) a mother and her child possessed identical HLA phenotypes, or (2) the child appeared to be possibly homozygous for one maternal haplotype (i.e., one of the child's HLA haplotypes was a blank). These mother-child HLA-haplotype dualisms (MHDs) occurred in 5% of all cases. Frequency of exclusion of the accused men in cases demonstrating MHD, was compared with the remaining paternity cases.No significant difference was found in overall exclusion rates between MHD cases and controls when exclusion produced by HLA and red cell antigen systems were observed. However, there was a greater rate of exclusion in MHD cases when comparing exclusions produced by red cell antigen systems regardless of whether HLA tests excluded paternity (p < 0.025). MHD cases involving teenaged mothers differed from control cases in frequency of exclusion of paternity only on the basis of red cell antigen phenotyping (p < 0.005).The HLA system's usefulness in paternity testing is diminished when there is MHD; multiple, independently-inherited systems are relatively more useful in these circumstances.The search method detects only half of potential incest cases; proof of incest requires more extensive testing for homozygosity among other polymorphisms. Since calculations of likelihood of paternity are inappropriate in cases involving close consanguinity, detection and follow up studies are important.Data suggest that one-fifth of MHD cases may involve first degree consanguinity and that the incest rate among paternity cases may be as high as 2%.     

The 1998-1999 collaborative exercises and proficiency testing program on DNA typing of the Spanish and Portuguese Working Group of the International Society for Forensic Genetics (GEP-ISFG)

A total of 28 laboratories (labs) submitted results for the 1998 collaborative exercise and the proficiency testing program of the Spanish and Portuguese Working Group of the International Society for Forensic Genetics (GEP-ISFG) group. This number increased to 46 labs in 1999. Six bloodstains were submitted, each one with 200 microl soaked in cotton except the sample no. 6 submitted for DNA quantification which had 2 microl. One of the samples was a mixed stain. A paternity testing case and a criminal case in the 1998 trial (GEP'98) and two paternity testing cases in 1999 (GEP'99) were included and the statistical evaluation of the evidence was requested in both cases. In the GEP'99 trial, a theoretical paternity testing case was included. A total of 52 DNA genetic markers were used by the participants in the GEP'98 trial, which increased to 101 in GEP'99. Despite this increasing number of participating labs, results remained quite satisfactory. All the labs used PCR-based DNA polymorphisms with an increasing number of markers, obtaining good results. SLPs were used by a decreasing number of labs but the results indicated a good level of expertise despite the different protocols used.Good results were also obtained for mtDNA despite the difficulties presented by the samples due to the presence of length heteroplasmy in some samples in both trials. The detection of heteroplasmy should, however, be improved.Similar conclusions were reached for both, the paternity and the criminal case by all the labs. Common methodologies for the statistical evaluation of the paternity case were used and the paternity index and the probability of paternity (with an a priori value of 0.5) reported by most of the labs. Also, a great uniformity was found in the evaluation of the criminal case despite the lack of a specific hypothesis in the design of the exercise. Some errors in statistical programs or in calculations were detected in a theoretical paternity case included in the GEP'99 trial for statistical analysis.     

Sequence analysis of a new short tandem repeat locus D17S2266E located in the human growth hormone gene cluster HumGH@

D17S2266E is a new, variable genetic marker exhibiting polymorphism of the number of repeats of four- and two-nucleotide motifs. This study, carried out on a group of 250 unrelated persons from various regions of Poland, revealed the presence of 24 different alleles ranging in size from 232 to 290 base pairs. Analysis of the sequenced fragments demonstrated that the alleles consisted of two flanking regions and two variable blocks that were separated by a consensus sequence. There were (AAAG)(5)(AG)(1)(AAAG)(3-4) repeats in the first block, and [(AAAG)(2)(AG)(1)](0-1)[(AAAG)(6)(AG)(1)](0-1)(AAAG)(n) repeats in the second block. On the basis of the allele frequencies in the population, we were able to do biostatistical calculations, which gave the following results: expected heterozygosity 0.8947 +/- 0.0137, power of discrimination 0.9793, polymorphism information content 0.8837, probability of exclusion (PE) 0.7859, PE for motherless cases 0.6473, and an average paternity index of 4.7470. These biostatistical parameters show that the marker D17S2266E can find a wide range of applications in forensic testing.     

Correlation of paternity index with probability of exclusion and efficiency criteria of genetic markers for parternity testing

The statistical correlation between the chance of paternity exclusion and paternity index is explored to derive a new criterion of judging the efficiency of an array of genetic determinations for parentage diagnosis. The theoretical basis is illustrated with allele frequency data on genetic markers used in a paternity testing protocol to examine the possible effects of changing the genetic systems on the prospect of identifying fathers by genetic markers.     

Beyond traditional paternity and identification cases. Selecting the most probable pedigree

The paper extends on the traditional methodology used to quantify DNA evidence in paternity or identification cases. By extending we imply that there are more than two alternatives to choose between. In a standard paternity case the two competing explanations H(1): "John Doe is the father of the child and H(2): "A random man is the father of the child, are typically considered. A paternity index of 100000 implies that the data is 100000 more likely assuming hypothesis H(1) rather than H(2). If H(2) is replaced by "A brother of John Doe is the father", the LR may change dramatically. The main topic of this paper is to determine the most probable pedigree given a certain set of data including DNA profiles. In the previous example this corresponds to determining the most likely relation between John Doe and the child. Based on DNA obtained from victims of a fire, bodies found in an ancient grave or from individuals seeking to confirm their anticipated family relations, we would like to determine the most probable pedigree. The approach we present provides the possibility to combine non-DNA evidence, say age of individuals, and DNA profiles. The program familias, obtainable as shareware from http://www.nr.no/familias, delivers the probabilities for the various family constellations. More precisely, the information (if any) prior to DNA is combined with the DNA-profiles in a Bayesian manner to deliver the posterior probabilities. We exemplify using the well published Romanov data where the accepted solution emerges among 4536 possibilities considered. Various other applications based on forensic case work are discussed. In addition we have simulated data to resemble an incest case. Since the true family relation is known in this case, we may evaluate the method.     

On statistical analysis of forensic DNA: theory, methods and computer programs

Statistics plays an important role in evaluating the evidential weight of forensic DNA. In this paper, general statistical principles for forensic DNA analysis are presented. We introduce the theory and methods for the statistical assessment in kinship determination and DNA mixture evaluation. In particular, analytical formulas for testing for biological relationship among three individuals and for assessing the DNA mixture evidence in the case of multiple subdivided ethnic groups are developed. Two user-friendly computer programs are demonstrated to exhibit their wide applicability in tackling with complex kinship/paternity and mixture problems. The EasyDNA program can solve a complicated paternity case in 1 min.     

Apparently contradictory homozygosity of gc- and esd-markers in three generations (author's transl)]

In a paternity case we found contradictory blood group antigens in the serum- and enzyme-polymorphisms Gc and EsD of the child, her mother and grandparents. A possible accidental exchange of the child could be excluded by biostatistical calculations of the probabilities of motherhood, fatherhood and parenthood, and the descent from the parents was proven in both generations. The serological findings may be explained by the concurrent existence of silent genes or of genes that cannot be determined as yet. The genetic information Gc degrees descended from the grandfather and that for EsD degrees from the grandmother. Both were transmitted from the mother to the child. Thus, the case could be cleared by statistical considerations only.     

亲权指数和个体识别似然比计算软件的开发与应用

目的建立计算亲子鉴定亲权指数(PI)和个体识别似然比(LR)的计算软件。方法依据相关行业规范和文献中给出的计算方法,利用计算机语言Visual Basic 6.0编写程序。结果开发出适用于PI和LR的计算软件。结论该计算软件可以帮助工作人员提高计算效率,服务法医物证工作。     

Genotyping of five short tandem repeat loci via triplex and duplex PCR

We have developed a triplex PCR method for D3S1359, HumTH01 and HumTPO tetranucleotide loci and a duplex PCR method for HumFES/FPS and HumvWA31A tetranucleotide loci using high resolution polyacrylamide gel electrophoresis and silver staining. The methods were evaluated for paternity testing and individual identification and allele frequencies at these loci are reported for 189–3387 unrelated individuals in the Finnish population. The D3S1359 locus, especially, was found to be a highly informative locus. Seventeen alleles were found in the D3S1359 locus with a highest observed allele frequency of 0.199, a high exclusion power (PE) in paternity testing (0.78) and a high observed heterozygosity (0.89). The combined PE for these five loci was 0.99.     

Genetic variation of 13 STR loci in the four endogamous tribal populations of Eastern India

We have analysed 13 autosomal STR loci in four endogamous tribal populations from two eastern states (Orissa and Nagaland) of India. The Gadaba, Kuvi Khond and Lotha Naga populations have not been analysed for microsatellite genetic variation previously. The allele frequencies for all loci are within the range observed in the geographical region and racial background, though some alleles showed greater variation. Departures from the Hardy-Weinberg equilibrium were tested by three methods and two loci (THO1 and TPOX) showed significant departures for all measures in Gadaba and Lotha Naga populations. The exclusion probability and discrimination probability were high for all analysed loci in all populations. There is no evidence for association of alleles among the STR loci studied. This allele frequency information will be useful for forensic, paternity and population genetic studies.     

从亲子鉴定的问题看标准化规范化管理的重要性

近年来,亲子鉴定技术在我国发展较快,越来越多的人应用这一鉴定手段维护了自己的合法权益.但我们发现,我国目前对鉴定部门的管理却相对滞后,主要表现为实验室缺乏规范化管理、鉴定机构良莠不齐、技术标准不够完备以及缺乏实验室的质量检测体系等.这些问题如不解决,将不适应司法体制改革的趋势,阻碍鉴定技术科学、公正地为司法实践服务.因此,我们呼吁有关部门对这一问题加以重视、加强协作,使科学鉴定早日走上标准化、规范化管理的道路.     

Effect of the genetic markers Kell(K1), P1, Km(1), Tf(C

E Osterhaus  P Birkner 《Zeitschrift für Rechtsmedizin》1984,91(3):231-234

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。     

Subject index : Volume 25 (1984)

Biostatistical Parameters such as the paternal markers in the child (PM), individual exclusion change (IEC) probability of incidental involvement (Z), probability of paternity (POP), plausibility of paternity according to Essen-Möller (PLEM) and paternity index (PI) are defined and discussed in respect to their application in cases of disputed paternity. In order to calculate these parameters for up to 24 marker systems, including HLA under strict observation of the linkage disequilibrium, a computer programme in BASIC has been developed.     

Isolation and Individualization of Conceptus and Maternal Tissues from Abortions and Placentas for Parentage Testing in Cases of Rape and Abandoned Newborns*

Abstract: Abortion specimens are often submitted to forensic laboratories as the only piece of physical evidence in rape and incest cases. The recovery of conceptus tissues from this evidence permits the use of paternity testing to evaluate suspects. In cases of abandoned newborns, the recovery of maternal tissue from the placenta allows for the direct comparison of genetic profiles between the suspected mother and the biological mother. We report on the identification and isolation of conceptus tissues from embryonic‐ and fetal‐period abortions, and maternal tissues from delivered placentas, by gross and low‐magnification examination with manual dissection. Hundreds of single‐source samples have been successfully recovered by this method and short tandem repeat typed using standard forensic procedures. We additionally describe extraembryonic tissues that can be recovered and typed in the absence of the embryo proper. We conclude that an expertise and protocols can be developed by forensic laboratories for the routine analysis of this evidence.     

Mutations at Y-STR loci: implications for paternity testing and forensic analysis

Knowledge about mutation rates and the mutational process of Y-chromosomal short-tandem-repeat (STR) or microsatellite loci used in paternity testing and forensic analysis is crucial for the correct interpretation of resulting genetic profiles. Therefore, we recently analysed a total of 4999 male germline transmissions from father/son pairs of confirmed paternity (probability > or = 99.9%) at 15 Y-STR loci which are commonly applied to forensics. We identified 14 mutations. Locus specific mutation rate estimates varied between 0 and 8.58 x 10(-3), and the overall average mutation rate estimate was 2.80 x 10(-3) (95% CIL 1.72 x 10(-3)-4.27 x 10(-3)). In two confirmed father/son pairs mutation at two Y-STRs were observed. The probability of two mutations occurring within the same single germline transmission was estimated to be statistically not unexpected. Additional alleles caused by insertion polymorphisms have been found at a number of Y-STRs and a frequency of 0.12% was estimated for DYS19. The observed mutational features for Y-STRs have important consequences for forensic applications such as the definition of criteria for exclusions in paternity testing and the interpretation of genetic profiles in stain analysis. In order to further enrich our knowledge of Y-STR mutations we suggest the establishment of a Y-STR mutation database and ask the forensic community for data contribution.