Analysis of aborted fetal material using autosomal STR markers in forensic cases of sexual assault

Sexual violence represents a widespread social problem associated with serious lifelong consequences. In many cases, an outcome of sexual violence is the victim's unwanted pregnancy, usually ended in an abortion. The objective of this paper is to report five rape cases, including rapes of a minor and young woman, two incest cases and a case of human trafficking for sexual exploitation, where every case resulted in the victim's pregnancy. In each case, pregnancy was terminated in the first trimester or at the beginning of the second trimester in the relevant medical center or clinic. Fresh fetal blood or aborted tissue samples were delivered to our laboratory in order to perform paternity testing for the purpose of proving the crime. DNA extraction using Qiagen Dneasy™ Tissue Kit was optimized according to the sample type. Amplification of autosomal STR (Short Tandem Repeat) markers was performed using the PowerPlex®16 System. In two cases, mixtures of maternal and fetal DNA in the aborted fetal material were found. Using the LRmix Studio v.2.1.5 Software for interpreting DNA mixtures based on a probabilistic model, the likelihoods of maternal contribution and presence of fetal allelic variants inherited from the alleged father/suspect were calculated. Based on these results, we confirmed the presence of assumed fetal fractions (determined before software analysis) in the mixtures. In all cases, positive paternity proved the crime (probabilities of paternity >99.9999%). This cases report once again pointed out the importance of DNA analysis in the process of clarifying and solving forensic cases and demonstrated that the LRmix Studio v.2.1.5 Software can deal with complex cases such as sexual assaults.     

Assessment of application value of 19 autosomal short tandem repeat loci of GoldenEyeTM 20A kit in forensic paternity testing

This study was carried out to assess the application value of 19 autosomal short tandem repeat (STR) loci of GoldenEye^TM 20A kit, in which 13 combined DNA index system core STR loci and PentaE, PentaD, D2S1338, D19S433, D12S391, and D6S1043 of six STR loci could be used in forensic paternity testing in Chinese population. We amplified the genomic DNA from blood samples on FTA paper of 289 paternity testing cases by using the GoldenEye^TM 20A kit. The amplified products were detected by capillary electrophoresis, and then the genotypes of 20 genetic markers including 19 STR loci as well as Amelogenin for sex determination were analyzed by GeneMapper v3.2 and GeneMarker HID Software. The results of genotypes were compared to the three commonly used commercial kits including AmpF?STR Identifiler^TM, PowerPlex^TM16, and AmpF?STR Sinofiler^TM kits. Compared to the three other common commercial kits, the GoldenEye^TM 20A kit had higher value of combined paternity index in certainty of paternity or non-exclusion paternity cases, and more numbers of STR loci were excluded in exclusionary paternity cases. Our data in this study showed that the GoldenEye^TM 20A kit has a higher application value in forensic paternity testing and will be of help for kinship analysis.     

Allele frequencies of nine STR loci of Jewish and Arab populations in Israel

DNA typing of nine short tandem repeat (STR) loci was carried out on unrelated Israeli Jewish and Arab individuals. All loci were highly polymorphic and the distribution of the obtained genotypes did not deviate from Hardy-Weinberg equilibrium. A comparison between Jewish and Arab population data revealed statistically significant differences in allele frequency distributions for some of the loci. The results presented in this study enable the use of these nine STR loci for forensic, identification and paternity cases in the Jewish and the Arab populations of Israel. Received: 9 April 2001 / Accepted: 2 July 2001     

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.     

Lewis genotyping by the PCR-RFLP method in a Japanese population and its evaluation in forensic analysis

Lewis phenotyping of red blood cells has many problems such as the influence of many biological conditions, the change during the period from newborn to early childhood and mistyping by non-specific anti-Lewis antibodies. Therefore, it would be useful to determine the Lewis genotype. Recently a method of Le-genotyping by PCR-RFLP was established. We determined the frequencies of Lewis genotypes in a Japanese population and discuss the applicability to paternity tests and other forensic applications. The gene frequencies of Le, le1 and le2 in the Japanese population studied were 0.7032, 0.2358 and 0.0610 respectively. Out of 12 paternity cases where paternity was excluded by other markers, 3 alleged fathers could also be excluded by Lewis genotyping. The genotype from organs of a fetus from a 3-month pregnancy was Le/Le. The determination of Lewis genotypes could play a useful role as a genetic marker in paternity tests and forensic analyses. Received: 9 October 1996 / Received in revised form: 1 April 1997     

False paternity with one or two mismatches using commercial STR kits

This study presents a case of false paternity where one or two mismatches were found by using three commercial STR kits. The analysis with the Identifiler kit yielded two mismatches at the loci D2S1338 and vWA. These data did not, however, enable us to exclude the alleged father, as the total number of excluding loci was less than three. Further STR loci were therefore employed to resolve the case. The PowerPlex 16 system yielded only one mismatch at the vWA locus previously found with the Identifiler kit. GenePhile G-Plex, on the other hand yielded two inconsistencies at D3S1744 and D18S536 (out of 15 loci in total). Since the disputed child was a female and we were not able to exclude the possible involvement of a close male relative, we choose to use Genephile X-Plex kit to finally resolve the case. Out of 13 loci tested, we found a complete match of the child's profile with the mother and eight mismatches with the alleged father, clearly indicating that the alleged father is not the biological father. This case emphasizes the usefulness of either Y-chromosome or X-chromosome DNA data for interpreting borderline paternity cases.     

The probability distribution of the number of loci indicating exclusion in a core set of STR markers

The distribution of the number of loci out of the 13 in the CODIS STR set that would show an exclusion (i.e., a genotype set incompatible either with the prosecution hypothesis or with Mendelian transmission) was estimated in different scenarios. The knowledge of this distribution would provide a framework against which casework evidence can be compared. I used allele frequencies in Iberian and in Italian populations to generate individual genotypes at random and to test in 1 million simulation replicates, how many of the 13 loci would give an exclusion in an individual identification case, a paternity case, and a double parenthood case. All three scenarios were tested under an expected overall exclusion, both for unrelated individuals and for cases in which the suspect or the alleged father was the brother of the real culprit or real father. Paternity and double parenthood cases were also tested in the true scenario, with exclusionary loci due to mutation. In individual identification cases, the average number of exclusionary loci was 11.95 with a minimum of 7. This STR set also showed sufficient power to resolve identification cases in which the evidence sample came from a suspect’s sib. False paternity cases yielded an average of 7.65 exclusionary loci and exclusions with only one (0.0108%) or two (0.14%) exclusionary loci were obtained only rarely. The cases of exclusion with one locus could lead to likelihood ratios in favour of paternity, while both true and false paternity cases with two exclusionary loci would often lead to non-conclusive likelihood ratios. The average number of exclusionary loci in a paternity case where the alleged father was the real father’s brother was 3.82, with a significant number of cases where no exclusions were obtained. Received: 15 September 1999 / Accepted: 31 January 2000     

Simultaneous typing of the STR loci,vWF and HumTPO,using discontinuous polyacrylamide gel electrophoresis

Analyses of short tandem repeat (STR) loci are very useful for improving the accuracy of paternity determination. Combined use of several STRs makes this test even more accurate. We have devised a simple but effective method which consists of the co-amplification of two STR loci (vWF and HumTPO) in a single test tube, separation of the PCR products using discontinuous polyacrylamide gel electrophoresis, and detection using sensitive silver staining. An appropriate combination of PCR primer pairs gave a high resolution and good separation of the two STR bands in the same lane on the same gel. Many combinations of STR loci have potential usefulness in paternity and individualization testing or population genetic studies in forensic practice.     

Abstammungsbegutachtung mit der RFLP- und STR-Analyse

The combination of restriction fragment length polymorphism (RFLP) and short tandem repeat (STR) analyses for paternity analysis is presented. The two methods were compared by investigating 113 paternity cases. RFLP analysis was done using the single locus probes YNH24, MS31 and MS43A and for STR investigations the Identifiler Plus kit was employed. The lowest paternity probability obtained via RFLP analysis was 98.936% compared to 99.99844% when using STR analysis and the highest values were 99.9996 (RFLP) and >99.999999% (STR). Using 3 single locus DNA probes the paternity probability was <99.9% in 45.5% of the cases, while STR analysis always led to at least 99.9%. In 36 cases the father was excluded. Using STR analysis between 4 and 12 exclusions out of 15 investigated loci per case were observed. In 14 cases (39%) RFLP analysis alone did not yield the 3 exclusions necessary for exclusion of paternity. In summary it could be shown that in all cases both STR analysis alone and the combination of STR and RFLP investigations led to results which conformed to the requirements of the German guidelines.     

Genetic analysis of 20 autosomal STR loci in the Miao ethnic group from Yunnan Province,Southwest China

The genetic polymorphisms of 20 autosomal short tandem repeat (STR) loci included in the PowerPlex^® 21 kit were evaluated from 748 unrelated healthy individuals of the Miao ethnic minority living in the Yunnan province in southwestern China. All of the loci reached Hardy–Weinberg equilibrium. These loci were examined to determine allele frequencies and forensic statistical parameters. The genetic relationship between the Miao population and other Chinese populations were also estimated. The combined discrimination power and probability of excluding paternity of the 20 STR loci were 0.999 999 999 999 999 999 999 991 26 and 0.999 999 975, respectively. The results suggested that the 20 STR loci were highly polymorphic, which makes them suitable for forensic personal identification and paternity testing.     

Paternity testing after pregnancy termination using laser microdissection of chorionic villi

We report an unusual case of paternity testing from residues of chorionic villi 5 weeks after pregnancy termination. The autopsy of a 32-year-old female homicide victim revealed the presence of intact chorionic villi at the former placenta implantation site. Fetal cells were selectively isolated by laser-induced microdissection of the remaining villi to avoid contamination with maternal DNA. Simultaneous amplification of 12 STR loci in 2 PCR reactions resulted in a combined probability of paternity of 99.94%. This case demonstrates that laser-assisted microdissection and multiplex STR typing provide tools for paternity testing performed on endometrial mucosa long after the product of conception was removed by therapeutic abortion. Received: 2 May 2000 / Accepted: 7 November 2000     

Population data on 6 short tandem repeat loci in a sample of Caucasian-Mestizos from Colombia

Blood samples from 409–452 unrelated Colombian Caucasian-Mestizo individuals were amplified and typed for six short tandem repeat (STR) markers (HUMF13A01, HUMFES/FPS, HUMVWA, HUMCSF1PO, HUMTPOX, HUMTH01). The allele frequencies, genotype frequencies, heterozygocity, mean paternity exclusion chance, polymorphism information content, discrimination power, assumption of independence within and between loci and Hardy Weinberg equilibrium were determined. The results demonstrate that all markers conform to Hardy-Weinberg equilibrium expectations. In addition, the results demonstrate the assumption of independence within and between the loci analysed. The mean exclusion chance (MEC) was 0.9851 for all six STR loci analysed and the discrimination power (DP) was 0.9999973. Therefore, this Colombian population database can be used in identity testing to estimate the frequency of a multiple PCR-based locus DNA profile in forensic cases as well as in paternity testing. Received: 24 September 1998 / Received in revised form: 22 December 1998 / Accepted: 11 January 1999     

Fourteen non-CODIS autosomal short tandem repeat loci multiplex data from Taiwanese

Interest in the development of polymorphic short tandem repeat (STR) markers unlinked to the CODIS loci is growing among forensic practitioners. We developed a multiplex system in which14 autosomal STR (D3S1744, D4S2366, D8S1110, D12S1090, D13S765, D14S608, Penta E, D17S1294, D18S536, D18S1270, D20S470, D21S1437, Penta D, and D22S683) could be amplified in one single polymerase chain reaction. DNA samples from 572 unrelated Taiwanese Han subjects were analyzed using this 14 STR multiplex system. Thirty parent–child pairs of parentage testing cases with a combined paternity index (CPI) below 1,000 and 32 parent–child pairs with single-step mutations found in AmpFℓSTR Identifiler loci were also recruited for validation of the newly developed system. DNA sequencing was performed for novel STRs and novel alleles found in these subjects. The distributions of allelic frequencies for these autosomal STRs and sequence data, allele nomenclature for the STRs, and forensic parameters are presented. The discrimination power in our multiplex loci ranged from 0.6858 (D18S536) to 0.9168 (Penta E), with a combined discrimination power of 0.999999999. It provides additional power to distinguish the possible single-step mutations in parent–child pairs and improves the ability to prove parentage by increasing the CPI. The combined power of exclusion of these 14 loci in Taiwanese Han in this study was 0.9999995913. In conclusion, this 14-autosomal STRs multiplex system provides highly informative STR data and appears useful in forensic casework and parentage testing.     

Y-STR DNA analysis of 154 female child sexual assault cases in the Philippines

The laboratory evaluated 154 sexual assault cases from four Child Protection Units in the Philippines involving female child victims aged from 2?years to 18?years old. All child victims sought medical attention within 72?h after sexual contact. In 130 cases, the child victim knew the alleged offender and identified them during the interview with the social worker. Penile ejaculation was reported by 68 child victims with varying reports of washing after contact. Overall, 84 child victims admitted having wiped their genitalia prior to the collection of biological samples for DNA testing. Laboratory personnel examined vaginal smears in only 109 cases using a light microscope and reported 23 samples to be positive for sperm cells. Using the PowerPlex? short tandem repeat of the Y chromosome (Y-STR) DNA multiplex system, male DNA was detected in vaginal swab samples from 63 child victims. In 39 cases, positive amplification at 11 Y-STR DNA markers consistent with a single male DNA profile was observed. Twenty-eight of these full single Y-STR DNA profiles were found to be unique when searched in worldwide Y-STR DNA population databases (~40,000 haplotypes), eight haplotypes matching Filipinos and/or Asian haplotypes and one Y-STR DNA profile only matching European, Caucasian, and Latin American haplotypes. Y-STR DNA profiles generated will be compared with reference DNA profiles of alleged offenders once reference samples are submitted to the laboratory.     

The identification of war victims by reverse paternity is associated with significant risks of false inclusion

Since February 2001 the process of DNA identification of war victims in Croatia relies on the database of over 3,000 9-locus (D3S1358, vWA, FGA, TH01, TPOX, CSF1PO, D5S818, D13S317 and D7S820) STR genotypes of relatives of missing persons. Instead of a targeted approach to DNA typing, the genotype of each skeletal remains analysed is compared to all genotypes in the database to identify potential parents and children. Although this approach has significantly increased the pace of identification by DNA typing, non-targeted matching in a database containing several thousand genotypes considerably decreases the significance of inclusion, especially when identification is based on reverse paternity analysis. To support this statistical prediction we present 3 cases of 10 STR loci matches and 1 case of 11 STR loci matches between a child, child's mother and skeletal remains that did not originate from a father of that child.     

Genetic polymorphisms of 20 autosomal STR loci in the Vietnamese population from Yunnan Province,Southwest China

The genetic polymorphisms of 20 autosomal short tandem repeat (STR) loci included in the PowerPlex® 21 kit were evaluated in 522 healthy unrelated Vietnamese from Yunnan, China. All of the loci reached the Hardy–Weinberg equilibrium. These loci were examined to determine allele frequencies and forensic statistical parameters. The combined discrimination power and probability of excluding paternity of the 20 STR loci were 0.999999999999999999999991 26 and 0.999999975, respectively. Results suggested that the 20 STR loci are highly polymorphic, which is suitable for forensic personal identification and paternity testing.

     

Evaluation of forensic genetic efficiency parameters of 22 autosomal STR markers (PowerPlex® Fusion system) in a population sample of Arab descent from Jordan

Jordan is a country located in the Middle East, on the East Bank of the Jordan River. In this study, the PowerPlex® Fusion (PPF) system was used to determine the allele frequencies and forensic efficiency parameters of 22 autosomal STR loci. Autosomal STR information was collected from the blood samples of 500 individuals belonging to the Jordanian population of Arab descent. The PPF system (Promega Corporation) was used to amplify the 22 autosomal STRs and the amplified samples were analysed on the 3130xl Genetic Analyser using GeneMapper ID-X 1.2 software (Applied Biosystems). All the autosomal STR loci met the requirements of the Hardy-Weinberg equilibrium after Bonferroni correction. This study revealed that the most informative locus among the 22 STR loci (excluding Amelogenin and DYS39) was Penta E locus (power of discrimination (PD) = 0.99), while the least informative locus was TPOX locus (PD = 0.834). The combined matching probability (MP) of the 22 loci was 1.9 × 10^?28. These forensic genetic parameters indicated the practicality of analysing these 22 STRs in forensic DNA identification and paternity testing among individuals from the Jordanian Arab population.     

Applying massively parallel sequencing to paternity testing on the Ion Torrent Personal Genome Machine

Massively parallel sequencing (MPS) is a promising supplementary method for forensic genetics and has gradually been applied to forensic casework. In this study, we applied MPS to forensic casework on an Ion Torrent Personal Genome Machine to evaluate its performance in paternity testing with mismatched STR loci. A total of 15 samples from seven cases containing one mismatched locus by capillary electrophoresis typing were analyzed. Combined paternity index (CPI) and relative chance of paternity were calculated according to the International Society for Forensic Genetics guidelines and the Chinese national standards recommended for paternity testing. With simultaneous analysis of enough STR loci, the results support the certainty of paternity, and the mismatched alleles were considered to be mutations (CPI > 10,000). With the detection of allele sequence structures, the origins of the mutations were inferred in some cases. Meanwhile, nine STRs (CSF1PO, D1S1656, D2S441, D2S1338, D3S1358, D8S1179, D12S391, D21S11 and D4S2408) were found in an increased number of unique alleles and three new alleles in three STRs (D2S441, D21S11, and FGA) that have not been reported before were detected. Therefore, MPS can provide valuable information for forensic genetics research and play a promising role in paternity testing.     

Demonstration of false inclusion risks of duo parentage analyses in the Turkish population in light of parentage acceptance criteria

In routine parentage tests, trio analyses (father-mother-child) are preferred. Under certain circumstances, laboratories may have to perform duo analysis (without mother/father). However, duo analyses increase the risk of false inclusions. This paper aimed to evaluate the false inclusion risks of duo analyses in the Turkish population from the point of forensic applications and the Turkish judicial system. Children from 400 previously analysed cases were compared separately with fathers and mothers of other cases by using a computer programme. From the total 345,006 comparisons, in 16 comparisons, no Short Tandem Repeat (STR) mismatch was observed at 15 STR loci between the child and an unrelated parent. In other words, duo tests provided a coincidental second mother or father to 16 children. In almost all of these cases, the probabilities of paternity estimation values are greater than Turkish Judicial System’s parentage acceptance limit, which is 99.73%. According to results, we suggested that trio cases should be performed as much as possible and the parentage acceptance limit, which is 99.73%, should be re-evaluated by a law maker’s commission to prevent false inclusion parentage cases in Turkey.     

产前亲子鉴定方法研究(附23例报告)

目的对23个产前案例进行亲子鉴定。方法超声监视下行羊膜穿刺术,抽取羊水30~40ml。离心收集羊水沉渣后提取其基因组DNA,同时抽取其父母双方外周血基因组DNA。应用毛细管电泳技术和五色荧光复合扩增的方法,检测所有DNA样本的16个STR基因座基因型。结果所有羊水基因组DNA均来自独立个体,无母体DNA的污染。三联体分析显示23个案例中17例为肯定亲权关系,亲子关系概率均大于0.9999,6例确定为排除亲权关系,平均排除(位点数)指标为7.67个。二联体分析显示23个案例中17例肯定父权的平均亲子关系概率为0.9997以上,6例排除亲权关系的平均排除(位点数)指标为5个,但其中1例的排除位点只有1个。结论16个STR位点的多重荧光扩增方法在对羊水中母体DNA的污染程度进行评估的同时,可以准确、可靠的应用于产前亲子鉴定。在检测单亲鉴定案例时,若排除(位点数)指标小于2时必须补充母亲样本或增加检测的STR位点指标数,直至得出明确结论。