亲权指数计算的新方法

目的介绍一种亲权指数（paternity index,PI）计算的新方法。方法假定亲代的等位基因都要经过一个转变的过程才发生分离并遗传给子代。每个亲代的等位基因与子代相同时,其转变概率为1;当不相同时,其转变概率为0。且每个亲代的等位基因都有1/2的机会遗传给子代。据此,可以计算出孩子从争议父或母亲获得等位基因的概率。而随机男子提供等位基因给孩子的概率为等位基因频率。相应地算出PI值公式中的分子（X）和分母（Y）值。结果推导得到了一个能够计算三联体、二联体和失踪孩子案PI值的通用计算公式。结论本PI计算公式在亲子鉴定PI值计算上具有实用价值。     

The potential contribution of MVR-PCR to paternity probabilities in a case lacking a mother.

Minisatellite variant repeat (MVR) mapping using the polymerase chain reaction (PCR) was applied to a paternity case lacking a mother to evaluate the paternity probability. After three flanking polymorphic sites at each of MS31A and MS32 loci were investigated from the child and alleged father, allele-specific MVR-PCR was performed using genomic DNA. It was confirmed that one allele in the child was identical to that in the alleged father at both loci. Mapped allele codes were compared with allele structures established from population surveys. No perfect matches were found although some motifs were shared with other Japanese alleles. The paternity index and probability of paternity exclusion at these two MVR loci were then estimated, establishing the power of MVR-PCR even in paternity cases lacking a mother.     

Mutation in the STR locus D21S1 1 of father causing allele mismatch in the child

We analyzed a case of paternity dispute with 15 autosomal STR loci and found a mismatch in one of the alleles of the locus D21S11 in the child. The composition of the alleles of this locus in the mother, suspicious father, and child were 29/32, 29/29, and 29/30, respectively. The combined paternity index (2.4 x 10(10)) and paternity probability (0.9999) suggest that the suspicious father is the biological father of the child. Further analysis of 6 Y chromosome STR loci revealed matching of all the Y chromosomal alleles of the child with that of the suspicious father. Since there was a perfect match of all the paternal alleles inherited (15 autosomal and 6 Y chromosomal) in the child with that of the suspicious father except the allele D21S11, it is suggested that this might be a case of mutation. Cloning and sequencing of all the alleles of the locus D21S11 of the suspicious father, mother, and the child helped in determining that the suspicious father contributed the mutated allele.     

Parentage of Hydatidiform Moles

We were presented with the STR (short tandem repeat) profiles from two separate paternity trios. Each trio consisted of a mother, an alleged father, and products of conception (POC) that contained a hydatidiform mole but no visible fetus. In both cases , antecedent pregnancies had followed alleged sexual assaults. Mole classification and pathogenesis are described in order to explain the analyses and statistical reasoning used in each case. One mole exhibited several loci with two different paternal alleles, indicating it was a dispermic (heterozygous) mole. Maternal decidua contaminated the POC, preventing the identification of paternal obligate alleles (POAs) at some loci. The other mole exhibited only one paternal allele/locus at all loci and no maternal alleles, indicating it was a diandric and diploid (homozygous) mole. In each case, traditional calculations were used to determine paternity indices (PIs) at loci that exhibited one paternal allele/locus. PIs at mole loci with two different paternal alleles/locus were calculated from formulas first used for child chimeras that are always dispermic. Combined paternity indices in both mole cases strongly supported the paternity of each suspect.     

Polymorphism of plasminogen in Tuscany (Italy)

The distribution of PLG phenotypes in the population of Tuscany (Central Italy) has been investigated by means of isoelectric focusing followed by immunofixation of desialyzed sera. In a random sample of 383 unrelated healthy blood donors registered at the Hospital of Pisa, three common phenotypes, PLG A, A-B, and B, and two rare variants were found. The allele frequencies calculated in our study were: PLG*A = 0.6749, PLG*B = 0.3225, and PLG*rare = 0.0026. The theoretical exclusion rate in cases of disputed paternity is 17.42%.     

Paternity probability when a relative of the father is an alleged father

When scientists use DNA evidence in court, coancestry effects such as population structure and relatedness are usually ignored. In paternity cases, only if a particular man has the child's paternal allele at a certain locus, can he not be excluded in the paternity dispute. However, it is certainly true that close relatives will be far more likely to have the child's paternal allele than will random members of the reference population. In particular, the probability that the true father's brother has the paternal allele is very much greater than that for any other relationship. In this paper, the authors describe a method for inference in a case where the true father may be a relative of the alleged father. This paper also reports that most current methods overstate the probability that the alleged father is the father.     

Paternity testing: blood group systems and DNA analysis by variable number of tandem repeat markers

Two recent paternity cases are reported. In the first case of paternity exclusion, deoxyribonucleic acid (DNA) restriction fragment length polymorphisms (RFLPs) on variable number of tandem repeat (VNTR) loci with multiple alleles were informative, as well as established systems of red blood antigens, red cell enzymes, serum proteins, and human leukocyte antigens. In the second case, in which both the alleged father and the first wife were deceased, the paternal genotype was determined by using genetic markers from the second wife and four children, which then were compared with the paternal alleles of the child in question, the plaintiff in this case. The high probability of paternity (0.999,998,7) made us conclude that the man probably was the actual father. The DNA analysis by VNTR probes appears to be quite valuable in the study of paternity cases.     

Plasminogen (PLG): a useful genetic marker for paternity examinations

The genetically determined polymorphism of plasminogen (PLG) was analyzed by isoelectric focusing on polyacrylamide gels. For analysis neuraminidase-pretreated sera were used. PLG was developed functionally by activation with urokinase and subsequent lysis of casein in an agar overlay. In a random sample of 957 unrelated healthy individuals from Southern Germany, three common phenotypes, PLG1, 2-1, and 2, and five rare variants were found. The allele frequencies were: PLG*1 = 0.7174, PLG*2 = 0.2780, and PLG*Var = 0.0046. The theoretical exclusion rate in cases of disputed paternity is 16.5%.     

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.     

Molecular analysis of the human esterase D gene ESD(*)Q0(yonago) responsible for incompatibility in a Japanese paternity case

In a Japanese paternity test, an alleged father was excluded only by reverse homozygosity of esterase D (ESD) phenotypes (mother, ESD 1; child, ESD 1; alleged father, ESD 2) out of 43 classical and DNA markers investigated. To solve the aberrant inheritance of the ESD phenotypes observed between them, fragments for all eight coding exons amplified by polymerase chain reaction (PCR) were subjected to DNA analysis. The child and alleged father shared a null allele, originating from ESD(*)1. It was characterized by having TGA for the stop codon instead of TCA for serine at codon 63. Thus, the sharing of a rare null gene, ESD(*)Q0(yonago), increased the probability of paternity.     

Establishing paternity using minisatellite DNA probes when the putative father is unavailable for testing

A paternity case involving a putative father who had died a few years earlier in an automobile accident was referred to the laboratory for testing. The child and his mother, the deceased's parents, and nine of the deceased's siblings were available for analysis. As previously reported, paternity testing using red blood cell groups, human leukocyte antigens (HLA), red blood cell enzymes, serum proteins, and immunoglobulin allotypes gave a cumulative paternity index of 43,300 and a combined probability of paternity equal to 99.998%. RFLP analysis using Hinf I and Sau 3A single digests and the minisatellite deoxyribonucleic acid (DNA) probes 15.1.11.4 and 6.3 showed no exclusion of paternity and gave nearly conclusive evidence that the putative father was the biological father of the child.     

Population studies and validation of paternity determinations by six microsatellite loci

A single locus system of 6 microsatellite markers was evaluated for paternity testing. A nonradioactive method based on peroxidase labeling of a DNA probe was used to estimate the allele frequency of markers D1S216, D3S1217, D7S480, D9S157, D13S153, and D16S422 by genotyping 1134-1698 chromosomes. The number of detected alleles were 22, 15, 23, 10, 16, and 19, respectively, and the allele frequency varied from 0.001 to 0.317. The genotype of 87 families, consisting of mother, father, and child was determined. The probability that a random individual will give a positive paternity was evaluated. We conclude that the markers can be reliably typed and give sufficient and reliable information for paternity testing.     

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.     

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.     

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.     

Deduction of paternity index from DNA mixture

Determination of individual genotypes in DNA mixture remains a challenge in forensic science. Using an approach of mixture of distributions, this article provides formula for calculation of paternity index (PI) in cases where only tissue mixture of the mother and alleged father, the genotypes of the mother and child, but not that of the alleged father are available. The formula has been used to solve a real case using mother's vaginal tissue contaminated with semen from alleged father.     

HLA investigations in paternity cases showing incompatible homozygosity

Incompatible homozygosity is seen when either the putative father is homozygous for a gene which the child lacks or when the child is homozygous for a gene which is absent from the putative father.Sixty-nine cases of paternity with incompatible homozygosity between child and putative father are reported. These cases were investigated in the HLA system and the results obtained are presented.     

Study on the application of parent-of-origin specific DNA methylation markers to forensic genetics

In paternity test, especially in motherless cases, the allele inherited from father (obligatory gene, OG) often cannot be determined. The paternity exclusion probability (PE) of a genetic marker is reduced considerably. Therefore, it is necessary to develop a new technique, by which the parental origin of alleles can be determined without genealogical analysis. In this paper, we explored the possibility of using parent-of-origin specific DNA methylation markers to determine the parental origin of alleles, choosing the imprinted single nucleotide polymorphism (SNP) locus rs220028 (A/G) as a model system. We typed the SNP by mutagenically separated PCR (MS-PCR). The frequencies of alleles were A = 0.5085, G = 0.4915; the unbiased heterozygosity was 0.5020. In order to discriminate between the maternal allele and paternal allele, post-digestion MS-PCR, a novel PCR based methylation analysis and SNP typing technique was developed and performed on 18 heterozygous children, and the methylated maternal allele was detected specifically. As a pilot study on the use of epigenetic markers in forensic genetics, our results demonstrated the feasibility of using parent-of-origin specific DNA methylation markers to determine the parental origin of alleles.     

Intentional mixed buccal cell reference sample in a paternity case

We report a case where an alleged father (AF) attempted to substitute someone else's saliva sample for his reference sample in a paternity analysis. Buccal cells were collected from the AF and the child, and DNA analysis was performed using an autosomal STR loci (Identifiler). The profile from the AF showed extra peaks in some loci, as well as a much higher "X" allele peak relative to the "Y" allele peak at the amelogenin locus. After conducting reanalysis by another technician with another set of positive and negative controls, it was concluded that the only source of the mixed profile was by intentional introduction by the AF, at the time of sampling, of some foreign human biological material, most likely saliva from a woman. Owing to the inconclusive results, when the AF was called back to the lab and the peculiar results were explained to him, he admitted that he had introduced into his mouth saliva from another person in an attempt to be excluded as the father of the child. Although tampering with DNA reference samples is not common, some individuals may attempt to contaminate or otherwise adulterate specimens before DNA tests. Personnel responsible for sampling should be aware of this possibility and should try to establish procedures to avoid the problem.     

The application of minisatellite variant repeat mapping by PCR (MVR-PCR) in a paternity case showing false exclusion due to STR mutation

A boy and a girl with their mother brought a paternity suit against an alleged but deceased father. We tested six conventional genetic markers, the AmpliType PM+ DQA1 and twelve STR loci the children and mother together with the alleged paternal grandparents. We also DNA typed the bloodstain found later in the alleged father's medical record. Only the result at D3S1358 in a nineplex STR system excluded the alleged father from parentage of the boy, whereas all markers were inclusive for the girl. Accordingly, we performed sequence analysis at D3S1358 to confirm the presence of a paternal exclusion or mutation. The sequence analysis indicated that the boy's allele 17 could have originated from either of the alleged father's allele 16 or 18 by a single-step mutation associated with slippage mutation in STR loci. We carried out minisatellite variant repeat mapping by PCR (MVR-PCR) at loci D1S8 (MS32) and D7S21 (MS31A) and mapped allele haplotypes of all individuals except the deceased alleged father. The MVR-PCR analysis showed that the boy has no inconsistency with the relationship between the alleged grandparents, and was very effective at increasing the paternity index (PI) value. We conclude that there is biological relationship between not only the girl but also the boy and the alleged father.