Molecular characterization of a null allele at locus DXS8378

Typing of X-chromosomal short tandem repeat (STR) loci in a deficiency paternity case revealed a single Mendelian incompatibility between a female child and her putative grandmother, consisting of an opposite homozygosity at locus DXS8378. The presence of a null allele due to a primer binding site mutation on the child's paternally inherited X chromosome was confirmed by amplification with newly designed DXS8378 external primers. Sequencing analysis showed a point mutation (C > T transition at position 168, according to GenBank accession G08098) in the binding site of the original DXS8378 reverse primer.     

Powerplex® Y 23 System: Molecular characterization of a null allele at locus DYS549

We observed a null allele pattern at locus DYS549 in a male subject from North-East Italy typed with the PowerPlex^® Y 23 System (Promega). To investigate whether this pattern was due to the presence of a microdeletion/mutation in primer binding sites or in the locus target region, the sample was amplified with our designed DYS549 primers obtained from GenBank sequence (GDB: 515022). After amplification, a normal hemizygous genotype at this locus was generated, thus indicating the presence of a point mutation in the binding site of the original primer set of PowerPlex^® Y 23 System (Promega). This was further confirmed by sequence analysis, carried out with the Big Dye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems), according to the manufacturer's instructions. Sequences were run on the ABI Prism 3130 Genetic Analyzer (Applied Biosystems) and analyzed using the Sequencing Analysis v.5.3.1 and the SeqScape v2.6 softwares (Applied Biosystems). Ascertainment of the frequency of null alleles generated from variations at primer binding sites of short tandem repeats loci is of great importance in forensic genetics.     

Uses of the NIST 26plex STR assay for human identity testing

Ongoing work at the U.S. National Institute of Standards and Technology has focused on the characterization of 26 autosomal STR loci for human identity testing. These 26 loci are in addition to the existing 13 U.S. core loci and those found in PowerPlex16 and Identifiler commercial STR typing kits. The amplification of the 26 loci has been optimized for degraded extracts in unique miniplex panels and also for reference samples as a single reaction 26plex assay. A study has been performed comparing genotypes obtained with the 26plex primers to those with miniplex panels for allele drop out and concordance. The forensic utility of the 26plex assay was evaluated for situations where additional loci are beneficial. The utility of this large multiplex was also tested in a case involving DNA extracted from degraded bone samples. The 26plex can serve as a low-cost assay (compared to commercially available kits) useful for both sorting comingled remains and providing additional markers for increased statistical support for samples that require “non-trio” family references for human identification.     

A DYS438 null allele observed in two generations of a large family

Typing of Y-chromosomal STR loci using the AmpFISTR^® Yfiler kit showed a DNA profile lacking the DYS438 allele in an Austrian Caucasoid brother pair.Buccal swabs were collected from additional males of two generations and different branches of the family. All family members investigated did not show a DYS438 allele in their Yfiler profile. Using the Powerplex^® Y system an allele with dramatically reduced peak height was amplified. Subsequent sequencing of the DYS438 locus exhibited a transition upstream of the repetitive region.     

Two examples of null alleles at the D19S433 locus due to the same 4 bp deletion in the presumptive primer binding site of the AmpFlSTR Identifiler kit

Two cases of allelic loss at the D19S433 locus after multiplex PCR with the AmpFlSTR Identifiler kit (Applied Biosystems) are described. In both cases the failure of PCR resulted in genetic inconsistencies due to opposite homozygosity. After singleplex PCR with published primers additional alleles were observed and Mendelian inheritance was restored. These PCR products were sequenced and in both cases the same 4 bp deletion near the 3′ end of the repeat region was detected in two alleles of different length. The frequency of these null alleles (two events in 1026 allelic transfers) amounts to 0.0019 (95% confidence limits: 0.0002-0.0070).     

Allelic alterations of STRs in archival paraffin embedded tissue as DNA source for paternity testing

Owing to a wrong name registered on ID card, the identity of a businessman who had been dead and cremated was suspected, which led his son failed to get legacy. In order to prove the parenthood, the son submitted the gastric cancer tissues surgically removed and embedded in a paraffin block as DNA source for paternity test. After extracting DNA with QIAamp DNA Blood Mini Kit, the 16 STR loci was amplified by two commercial kits of Sinofiler^® (ABI)and Powerplex 16 (Promega), respectively. Both of the STR profiles were similarly showing allelic imbalance pattern at some loci and an additional allele at locus D18S51. The cancerous tissues and adjacent normal tissues were then partitioned off from each other by microscopic analysis of H.E. stained sections and followed by DNA extracting and STR typing, respectively. The allelic alteration could not be found in normal tissues whereas it did in cancerous tissues whose STR profile showed complete loss of one allele (LOH) at loci D13S317 (allele 11 was lost), partial loss of one allele (pLOH) at loci D21S11, D7S820, D19S433, vWA, D12S391 and Amelogein and occurrence of an additional allele (allele 20 was added) at locus D18S51. The results demonstrated that the Paraffin Embedded cancer Tissue used as DNA source for forensic identification is possibly questionable because of their microsatellite instability (MSI) or loss of heterozygosity. It was suggested to partition the normal tissues from the cancer tissues by microscopic evaluation first and then analyzing DNA separately. Comparing the STRs profile of normal tissue with the son's blood sample, the final conclusion was acquired that the donor of the paraffin embedded tissues is the biological father of the son.     

New alleles and mutational events at 14 STR loci from different German populations

The molecular origin of DNA mutations and the mutation rates were analyzed at 14 short tandem repeat (STR) loci with samples from trio cases derived from 10 different German population samples. STR loci comprised of D2S1360, D3S1744, D4S2366, D5S2500, D6S474, D7S1517, D8S1132, D10S2325, D12S391, D18S51, D19S246, D20S480, D21S226, and D22S689. In a total of 488 meioses, 16 isolated genetic inconsistencies in 8 different STRs were observed, whereas no mutations were found at the other loci. The data of five mutations suggested the presence of silent or null alleles due to sequence variation in primer binding site. This could be confirmed for four suspected cases by the use of alternative primer sets and by DNA sequence analyses. Furthermore, this study revealed nine new allelic variants at five different loci.     

Kin terminology and the study of kinship: A case study on the Greek island of Syros (1750–1820)

This historical–anthropological study of kinship on the Greek island of Syros examines kin terminology as an indicator of relationships. These terms have been traced in a series of notarial acts dating from 1750 to 1820. Departing from the case study, certain elements of the system of kin terminology have been placed in a broader territorial and cultural context. The aim of this comparative analysis is to achieve a deeper understanding of kinship on the microlevel of the case study. From a methodological point of view, this article points out the need for interdisciplinary research on kinship. History, social anthropology, and ethnology could be possible partners in such an approach.     

Likelihood ratios for complex mixtures with relatives

Likelihood ratios used for the analysis of complex DNA mixtures depend on a number of modeling assumptions and parameter estimates. In particular, the LR does not give information about the relative weight of the separate contributors for hypotheses conditioned on several contributors. An alternative is to evaluate the observed LR with respect to likelihood ratios expected under the defense hypothesis. Further, a p-value corresponding to the LR can be calculated. The p-value is the probability of observing a LR equally large or larger than the one observed, if the defense hypothesis is true. In this paper we investigate the distribution of likelihood ratios for mixtures with drop-in and drop-out and related contributors. Disregarding a plausible close relative of the suspect as an alternative contributor may overestimate the LR against a suspect.     

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.     

DNA profile evidence in complex disputed paternity cases: The analysis of 300 real cases

In disputed paternity cases where the putative father is unavailable DNA from one or more of his relatives could be used. However, interpreting results is often difficult, because of the partial information regarding the parental genotype obtained from his relatives. We analyzed results obtained in 300 real paternity cases performed through close relatives of the real father (sib, half-sibs, one grandparent and/or uncle). DNA was typed with PowerPlex (Promega) and the LR estimated with the Software BDGen. As expected the higher LR values were achieved with sibs and half-sibs (in such cases where his/her mother was available for testing). The LR values were tight related to the number of uninformative loci, which varied between 0 and 13. In 10% of the reviewed cases, 10 or more non-informative loci were observed; all of them associated LR values below 0.01. Thus, providing evidence in favor of no relatedness.     

PowerPlex~(TM) 16体系在亲子鉴定中的应用评估

目的 评估PowerPlexTM16体系在亲子鉴定中的检验能力。方法 以633例亲子鉴定案例为基础,调查PowerPlexTM16体系15个STR基因座的群体遗传学数据资料,并对该体系在亲子鉴定中的排除能力及遗传稳定性进行评估。结果 879名无关个体共检出197个等位基因,739种基因型,累计个体识别力为1×10-30,累计非父排除率为0.999999999999987。633例亲子鉴定案件中有95例确定为排除亲权,平均排除指标为6个。18例表现出1个STR基因座突变的现象,1例表现出2个STR基因座突变的现象。结论 PowerPlexTM16体系应用于亲子鉴定是高效、可靠的。     

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。     

Two loci ‘exclusion’ of true paternity is due to genetic disorder in a child

We describe a paternity case with three genetic incompatibilities between a three-year-old boy and his putative father.STR analysis of 2 out of 25 markers revealed the absence of paternal alleles and presence of two maternal alleles at D2S441 and D2S1338 loci in the child. The rest 23 STR markers served to confirm paternity. In addition, we analyzed Y-STRs and determined the same haplotype in the child and his putative father.With massive parallel sequencing on HID Ion GeneStudio S5 System using Precision ID GlobalFiler NGS STR Panel v2 (Applied Biosystems) we confirmed the presence of two alleles of maternal origin at D2S441, D2S1338 loci and identified two maternal alleles at additional locus D2S1776 located on chromosome 2 in the child.Finally, we confirm paternity. Three loci ‘exclusion’ was due to maternal uniparental disomy of chromosome 2 in the child.     

A cautionary note on the evaluation of genetic evidence from uniparentally transmitted markers

The combination of the information obtained from lineage genetic markers, such as mitochondrial DNA (mtDNA) and the non-homologous region of Y-chromosome, with data resulting from meiotically recombining loci (diploid/autosomal or haplodiploid/X chromosome) into a single likelihood ratio has been recently proposed. In this work we challenge this proposal and demonstrate that while the genetic evidence obtained from loci which reshuffle at meiosis is appropriate for individual probability calculations, mtDNA and Y-chromosome data are not and, consequently, that joining the evidential value of the two types of markers is generally inconsistent and should be avoided. The assumption of non-involvement of relatives must be clearly and explicitly stated and its acceptance must be left to the court decision.     

Use of sibling pairs to determine the familial searching efficiency of forensic databases

Pairs of individuals tested at the 13 CODIS core STR loci to determine sibship were used as a source of familial data that was seeded into a larger data set of 12,000 plus DNA profiles simulating a CODIS-like offender database. To determine whether known sibs could be found in the larger database two methods were used: degree of allele sharing and a kinship matching approach. The allele sharing method detected 62 of 109 of the known sib pairs (57%) while kinship matching detected 90 of the sib pairs (83%). Although kinship matching was the more efficient method of the two, the number of false positives generated prior to finding a true match was inversely related to the likelihood of sibship suggesting that many true siblings would not be easily found in a large forensic database via familial searching techniques.     

Analysis strategies to establish vWF intron 40 haplotypes

Sequencing of a 0.65 kb region in the intron 40 of the vWF gene demonstrated a complex variability. Five STRs named Pol K, F, P1, P2-a and P2-b and an indel polymorphisms (I) are present. We established a routine analysing method to puzzle out the Pol K/F/I/P haplotypes which does not require a sequencing procedure. To recognise the combined polymorphisms as haplotypes, we performed short and middle range PCRs in combination with Nde I and BsmA I restriction tests. Comparison of the amplicon and restriction fragment length reveals the most likely haplotypes of each person involved a kinship test. Furthermore, a SNP allele specific PCR was employed. Additional information can be achieved by typing Pol P2-a and P2-b. Establishing of intron 40 vWF haplotypes using the methods described here can greatly support the resolution of complex kinship cases. This statement is illustrated by demonstration of a family study.     

短串联重复基因座突变率的分析研究

分析亲子鉴定案例进行中的STR基因座的突变率。采用chelex 100快速抽提DNA,DNA扩增,聚丙烯酰胺凝胶电泳、银染色法,参照标准品进行DNA分型。在300例已确定亲生关系的案例中,有11例出现STR基因座变异,其中D11S554基因座6例,D19S253基因座2例,SE33、D12S391、D13S631基因座各1例。结果提示:用STR基因座进行亲子鉴定,必须考虑STR基因座突变因素。