Application of SNPs with low minor allele frequencies in missing person identification (MPI) through kinship analysis of DNA mixtures.

The need to identify a missing person (MP) through kinship analysis of DNA samples found at a crime scene has become increasingly prevalent. DNA samples from MPs can be severely degraded, contain little DNA and mixed with other contributors, which often makes it difficult to apply conventional methods in practice. This study developed a massively parallel sequencing-based panel that contains 1661 single-nucleotide polymorphisms (SNPs) with low minor allele frequencies (MAFs) (averaged at 0.0613) in the Chinese Han population, and the strategy for relationship inference from DNA mixtures comprising different numbers of contributors (NOCs) and of varying allele dropout probabilities. Based on the simulated dataset and genotyping results of 42 artificial DNA mixtures (NOC = 2-4), it was observed that the present SNP panel was sufficient for balanced mixtures when referenced to the closest relatives (parents/offspring and full siblings). When the mixture profiles suffered from dropout, incorrect assignments were markedly associated with relatedness, NOC and the dropout level. We, therefore, indicate that SNPs with low MAFs could be reliably interpreted for MP identification through the kinship analysis of complex DNA mixtures. Further studies should be extended to more possible scenarios to test the feasibility of this present approach.

A microhap panel for kinship analysis through massively parallel sequencing technology.

It is widely recognized that microhaps are powerful markers for different forensic purposes, mainly due to their advantages of both short tandem repeats and single nucleotide polymorphisms, including multiple alleles, low mutation rate, and absence of stutter peaks. In the present study, a panel of 60 microhap loci was developed and utilized in forensic kinship analysis as a preliminary study. Genotyping of microhap was performed by massively parallel sequencing and haplotypes were directly achieved from sequence reads of 73 samples from Chinese Han population. We observed that 49 out of 60 loci have effective number of alleles greater than 3.0 and 10 out of 60 have values above 4.0, with an average value of 3.5598. The heterozygosity values were in a range from 0.5840 to 0.8546 with an average of 0.7268 and the cumulative power of exclusion value of the 60 loci is equal to 1-4.78 × 10-18 . Moreover, we demonstrated the applicability of this method by different relationship inference problems, including identification of single parent-offspring, full-sibling, and second-degree relative. The results indicated that the assembled microhap panel provided more power for relationship inference, than commonly used short tandem repeats or single nucleotide polymorphism system.

Noninvasive prenatal paternity testing by maternal plasma DNA sequencing in twin pregnancies.

SNPs, combined with massively parallel sequencing technology, have proven applicability in noninvasive prenatal paternity testing (NIPPT) for singleton pregnancies in our previous research, using circulating cell-free DNA in maternal plasma. However, the feasibility of NIPPT in twin pregnancies has remained uncertain. As a pilot study, we developed a practical method to noninvasively determine the paternity of twin pregnancies by maternal plasma DNA sequencing based on a massively parallel sequencing platform. Blood samples were collected from 15 pregnant women (twin pregnancies at 9-18 weeks of gestation). Parental DNA and maternal plasma cell-free DNA were analyzed with custom-designed probes covering 5226 polymorphic SNP loci. A mathematical model for data interpretation was established, including the zygosity determination and paternity index calculations. Each plasma sample was independently tested against the alleged father and 90 unrelated males. As a result, the zygosity in each twin case was correctly determined, prior to paternity analysis. Further, the correct biological father was successfully identified, and the paternity of all 90 unrelated males was excluded in each case. Our study demonstrates that NIPPT can be performed for twin pregnancies. This finding may contribute to development in NIPPT and diagnosis of certain genetic diseases.

Noninvasive prenatal paternity testing using targeted massively parallel sequencing.

BACKGROUND: Recent advances in massively parallel sequencing (MPS) technology have provided efficient methods for noninvasive prenatal paternity testing (NIPAT). However, a well-accepted protocol has not been established. The present study developed an MPS-based approach for NIPAT and compared the performance of two recently reported methods for MPS data interpretation. STUDY DESIGN AND METHODS: We selected 1795 unlinked polymorphic single-nucleotide polymorphisms (SNPs) and performed paternity analysis in 34 real parentage test cases with maternal plasma samples using the Illumina HiSeq platform. Sequencing data were interpreted by the straightforward counting method for the identification of paternal alleles and mathematical algorithms for paternity index (PI) calculation, respectively. RESULTS: Based on the sequencing data from each family case, both of the two statistical approaches produced a significant separation between the biological father and 90 unrelated males (p < 0.0001) when sufficient effective loci were attained. Nevertheless, up to 30.82% of real paternal alleles were filtered by a predefined cutoff and resulted in insufficient effective loci, especially in plasma samples with low fetal fraction (approx. 90.60% were filtered). In contrast, the PI calculation model utilized all maternal homozygous SNPs as effective loci (approx. 40% of total SNPs) and successfully identified the correct biological father, with the log-transformed combined PI (Lg(CPI)) value varying from 68.23 to 158.01 in each family case. CONCLUSION: Our study illustrates that the Bayesian approach represents the better choice in NIPAT data interpretation. Further, the adoption of more informative markers (e.g., tri-allelic SNPs, tetra-allelic SNPs, and micro-haplotypes) or deeper sequencing is recommended for the improvement of the testing efficiency.

An SNP panel for the analysis of paternally inherited alleles in maternal plasma using ion Torrent PGM.

Researchers have sought to develop an effective protocol for paternity analysis using cell-free DNA (cfDNA) in maternal plasma. The use of massively parallel sequencing (MPS) technology for SNP testing is attractive because of its high-throughput capacity and resolution to single-base precision. In this study, we designed a customized SNP panel for cfDNA sequencing that includes 720 short amplicons (< 140 bp) targeting SNPs on the autosome and Y chromosome. The systemic performance was evaluated using the Ion Torrent PGM, indicating balanced coverage among most of the included loci, except for 78 poorly performing SNPs that were observed to have an inconsistent allele balance, lower coverage reads or high background signals. Then, the custom panel was used to perform cfDNA genotyping in maternal plasma from 20 pregnancies in the first and second trimesters (9 to 21 weeks). By establishing an allele fraction cutoff of 2.0%, 53 to 128 autosomal SNP loci were considered informative for paternal origin. Validation results in foetal samples showed that 49.43% to 100% of the real paternal alleles were accurately identified, with incorrect alleles encountered in 3 cases. The concentration of foetal cfDNA ranged from 4.28% to 10.70%. Our results show that this amplicon-based sequencing strategy could be utilized in analysing paternally inherited alleles in maternal plasma. However, further studies and optimization are required for a more detailed and accurate interpretation of the cfDNA sequencing results based on MPS technology.

Noninvasive fetal genotyping of paternally inherited alleles using targeted massively parallel sequencing in parentage testing cases.

BACKGROUND: Researchers have sought to develop a noninvasive protocol for paternity analysis that uses fetal cell-free DNA (cfDNA) in maternal plasma. Massively parallel sequencing (MPS) is expected to overcome this challenge because it enables the analysis of millions of DNA molecules at a single-base resolution. STUDY DESIGN AND METHODS: Seven women were involved in prenatal paternity testing cases. Before conventional invasive procedures, cfDNA was isolated from maternal plasma. Fetal tissues were then collected, as were blood samples from the alleged fathers. A custom array was designed that targeted 1497 regions containing single-nucleotide polymorphisms. These regions were massively parallel sequenced. RESULTS: In these seven cases, the mean nonmaternal allele fractions in maternal plasma ranged from 3.22% to 6.17%. Setting the allele fraction cutoff of 2.5%, 300 to 491 loci were considered informative for paternal origin and no genetic incompatibilities with the alleged fathers were found. These results were concordant with those of conventional short tandem repeat genotyping. Validation results performed using fetal samples showed that sequencing noise was completely filtered out, and 78.35% to 99.19% of the paternal alleles were accurately genotyped. The fetal cfDNA concentrations ranged from 7.12% to 13.81%, and the overall sequencing error rates ranged from 0.40% to 0.93%. CONCLUSION: In our study, we evaluate a straightforward method that can be used to identify paternal alleles based on analyses of paternal alleles and sequencing errors in maternal plasma. Our results support the notion that an MPS-based method could be utilized in noninvasive fetal genotyping and prenatal paternity analyses.

Analysis of 24 Y-STR haplotype data in a Chinese Han population from Guangdong Province.

In this study, we investigated the genetic polymorphisms of 24 Y-chromosomal short tandem repeat (Y-STR) loci in 885 unrelated Chinese Han male individuals from Guangdong Province, using a domestic AGCU Y24 STR kit. A total of 878 different haplotypes were observed at the 24 Y-STR loci; among them, 871 haplotypes were unique and 7 haplotypes occurred twice. The overall haplotype diversity was 0.99998 and the discrimination capacity was 99.2%. The gene diversity values ranged from 0.4354 at DYS438 to 0.9606 at DYS385a/b. Population relationships between the Guangdong Han population and seven other published Chinese populations were evaluated by Rst values and visualized in a two multi-dimensional scaling plot. The results showed the 24 Y-STR loci are highly polymorphic in Guangdong Han population and of great value in forensic application.

Analysis of 17 Y-STR loci haplotype and Y-chromosome haplogroup distribution in five Chinese ethnic groups.

To investigate genetic diversity in Chinese populations, 706 unrelated male individuals from five ethnic groups (Han, Korean, Hui, Mongolian, and Tibetan, respectively) were analyzed with 17 Y-chromosomal STRs. The haplotype diversity was 0.99985 in the combined data. A total of 675 distinct haplotypes were observed, of which 649 were unique. Y-chromosome haplogroups in the five groups were also predicted with Y-STR haplotypes. Genetic distance between the five studied ethnic groups and other published groups was analyzed by analysis of molecular variance and visualized in a multidimensional scaling plot. In conclusion, the 17 Y-STR loci are highly polymorphic markers in the five groups and hence are very useful in forensic application, population genetics, and human evolution studies.

[Mutations in a Large Pedigree with Y-STR Genetic Markers].

OBJECTIVE: To explore the mutation of Y-STR loci in meiotic allelic transmission in a large pedigree. METHODS: The oral swabs of 163 male individuals were collected from a Lin pedigree. Twenty-two Y-STR genetic markers were typed with AGCU Y24 fluorescent detection kit (AGCU Y24 system), which also contained 16 Y-STR markers included in Yfiler multiple amplification kit (Yfiler system). The genotyping results of Y-STR loci were compared between each two males in the pedigree. RESULTS: There were 20 and 30 kinds of haplotypes obtained with Yfiler and AGCU Y24 systems in 163 male individuals from the Lin pedigree, respectively. The rates referred to haplotype differences (RRHD) of these two typing systems between male pairs were 0.910 5 and 0.922 7, respectively. The average number of marker differences were 6.582 1 and 9.824 8, respectively. The RRHD increased along with the incidents of meiosis. CONCLUSION: Y-STR mutation leads to different Y-STR haplotypes among the male members in a paternal pedigree and the rate of difference increases along with the incidents of meiosis.

Epigenome-wide DNA methylation assay reveals placental epigenetic markers for noninvasive fetal single-nucleotide polymorphism genotyping in maternal plasma.

BACKGROUND: The use of DNA methylation difference between maternal blood cell and fetal (placental) DNA is one of the main areas of interest for the development of fetal epigenetics markers in maternal plasma. STUDY DESIGN AND METHODS: We employed a methylation array (HumanMethylation450 array, Illumina, Inc.) to identify novel biomarkers that are specially hypermethylated in placental DNA versus maternal blood cells in a genome-wide basis. Validation by bisulfite genomic sequencing was performed and the priority was given to potential targets that harbor differential methylated CpG sites overlapped with at least two methylation-sensitive restriction enzyme (MSRE) recognizing sites, as well as one polymorphic single-nucleotide polymorphism (SNP), within a short DNA stretch. Three candidate regions of PSMB8, SKI, and CHST11 gene were selected for developing a preliminary polymerase chain reaction assay with MSRE digestion of maternal plasma DNA. SNP genotypes were confirmed by direct sequencing. RESULTS: We identified 2944 and 5218 fetal-specific hypermethylated CpG sites in the first- and third-trimester placenta, respectively, of which 2613 were overlapped, suggesting a consistency of differential methylation during the whole pregnancy. The array results were confirmed by bisulfite genomic sequencing. The preliminary tests in maternal plasma showed that postdigestion hypermathylated versions of these candidate molecules were detectable only in pregnant women. We further revealed that methylated targets in maternal plasma possessed the fetal SNP genotypes. CONCLUSION: The present studies systematically identified hypermethylated sites in fetal tissues and preliminarily demonstrated that some of the fetal epigenetic markers that contain informative SNPs have great potential for noninvasive fetal genetic diagnosis.

Identification of the sequence variations of 15 autosomal STR loci in a Chinese population.

BACKGROUND: DNA sequence variation including base(s) changes and insertion or deletion in the primer binding region may cause a null allele and, if this changes the length of the amplified fragment out of the allelic ladder, off-ladder (OL) alleles may be detected. AIM: In order to provide accurate and reliable DNA evidence for forensic DNA analysis, it is essential to clarify sequence variations in prevalently used STR loci. SUBJECTS AND METHODS: Suspected null alleles and OL alleles of PlowerPlex16® System from 21,934 unrelated Chinese individuals were verified by alternative systems and sequenced. RESULTS: A total of 17 cases with null alleles were identified, including 12 kinds of point mutations in 16 cases and a 19-base deletion in one case. The total frequency of null alleles was 7.751 × 10(-4). Eight hundred and forty-four OL alleles classified as being of 97 different kinds were observed at 15 STR loci of the PowerPlex®16 system except vWA. All the frequencies of OL alleles were under 0.01. CONCLUSION: Null alleles should be confirmed by alternative primers and OL alleles should be named appropriately. Particular attention should be paid to sequence variation, since incorrect designation could lead to false conclusions.

Complete paternal uniparental isodisomy for Chromosome 2 revealed in a parentage testing case.

BACKGROUND: Uniparental disomy (UPD) is a rare cytogenetic event that has previously been reported mostly via genetic analysis of patients with phenotypes of recessive diseases. The incidence of UPD of any chromosome is estimated to be approximately1:3500 live births. CASE REPORT: In a case of disputed paternity involving a phenotypically normal male child, mother-child exclusions were observed at five short tandem repeat markers, which were all located on Chromosome 2. Ten additional dinucleotide repeat markers spanning both arms of Chromosome 2 were investigated. The results revealed that the child was homozygous for all markers tested with all alleles originating from a single paternal Chromosome 2, which was consistent with paternal UPD for Chromosome 2. CONCLUSION: This case and other previous reports demonstrate that UPD poses a high risk for false exclusion and incorrect expert opinion. Furthermore, this case highlights that a conclusion of exclusion of paternity or maternity should not be postulated if multiple genetic incompatibilities are located on the same chromosome because of the occurrence of UPD.

Polymorphism analysis and evaluation of 19 STR loci in the Han population of Southern China.

BACKGROUND: The knowledge of allele and genotype frequencies is an essential prerequisite to the use of any human polymorphism in forensic medicine. AIM: To study the genetic polymorphism and evaluate the 19 STR loci using forensic medicine. SUBJECTS AND METHODS: Nineteen STR loci, which include D19S433, D5S818, D21S11, D18S51, D6S1043, D3S1358, D13S317, D7S820, D16S539, CSF1PO, Penta D, vWA, D8S1179, TPOX, Penta E, TH01, D12S391, D2S1338 and FGA, were amplified simultaneously with Goldeneye(TM) DNA ID system 20A kit for 1161 unrelated Han individuals in Southern China. The PCR products were separated with the arrayed capillary electrophoresis. The allele frequency distribution and several parameters commonly used in forensic science were statistically analysed. RESULTS: The observed heterozygosity (Hobs) of these 19 STR loci ranged from 0.6072-0.9070; the expected heterozygosity (Hexp) ranged from 0.6052-0.9117; the power of discrimination (PD) ranged from 0.7836-0.9662; the power of exclusion (PE) ranged from 0.3479-0.8227 for trio paternity cases and 0.1959-0.6988 for duo paternity cases; the polymorphic information content (PIC) ranged from 0.5443-0.9050. CONCLUSION: The results indicate that 19 STR loci are polymorphic among the Han population in Southern China. This set of polymorphic STR loci is a useful tool in forensic paternity test and anthropological study.

[Age estimation using content of sjTREC in human peripheral blood].

OBJECTIVE: To determine and verify the correlation formula of age estimation using the content of signal joint T-cell receptor excision DNA circle (sjTREC) in human peripheral blood and to discuss its application value in forensic biological practice. METHODS: The samples of peripheral blood stains were collected from 30 healthy unrelated individuals whose ages were known. The DNAs were extracted from the samples stored at room temperature after 4 weeks. The content of sjTREC was measured by real-time fluorescent quantitative PCR technique, and the TATA box binding protein (TBP) was selected as reference genes. The age of each sample was predicted with the formula which was Age = -7.181 5 Y-42.458 +/- 9.42 (Y = dCtTBP-sjTREC), and the result was compared with the real age of each individual to determine the accuracy of the formula. RESULTS: sjTREC and TBP gene were detectable in all 30 samples of peripheral blood. The contents of sjTREC in human peripheral blood showed a decreasing tendency with aging. The accuracy rate for the age estimation by this method was 76.67%. CONCLUSION: The method for the age estimation with the content of sjTREC was simple, fast, sensitive, and good species specific with important potential application prospect.

Null alleles of the X and Y chromosomal amelogenin gene in a Chinese population.

The use of amelogenin locus typing as a gender marker incorporated in short tandem repeat (STR) multiplexes is a common practice in sex typing. Mutations in the X or Y homologue of the amelogenin gene can be misleading and result in serious mistakes in forensic applications and prenatal diagnosis. In these present studies, the amelogenin gene of 8,087 unrelated male individuals from Chinese Han population was genotyped with Powerplex(®)16 system. The samples that showed discordant results were taken for frequency calculation and further validated by re-amplification with different primer sets, Y-STR typing, and sequencing. Our results describe six amelogenin X-allele (AMELX) or amelogenin Y-allele (AMELY) null cases in these studied subjects with an overall prevalence of 0.074%. Further validation revealed point mutations in the amelogenin-priming sites associated with AMELX nulls (three cases, 0.037%) and deletions on the Y chromosome encompassing the AMELY and other Y-STR loci with three AMELY nulls (0.037%). These mutations and failure of the amplification of the AMELX and AMELY alleles have not been reported for the Chinese population. These and previous findings suggest that mutations in the amelogenin gene may result in amplification failure of the AMELX or AMELY allele, and an additional gender test for unambiguous sex determination may be needed.

Identification of missing persons through kinship analysis by microhaplotype sequencing of single-source DNA and two-person DNA mixtures.

In forensic applications, there is an increasing demand for the analysis of DNA profiles arising from missing person identification (MPI) cases. A specific DNA profile may originate from a single source or more than one contributor (i.e., a DNA mixture). When direct references are not available, indirect relative references can be used to identify missing persons by kinship analysis. As a novel kind of multiallelic marker, microhaplotypes have proven promising for relatedness determination and mixture deconvolution. Herein, we developed a large panel of 185 microhaplotype markers and demonstrated its application in different scenarios of relationship inference through a simulation study and real pedigree analysis, combined with probabilistic genotyping models for data interpretation. Based on single-source profiles, it was shown that the present microhaplotype panel was sufficient for pairwise close relative testing (parent/child, full-sibling and 2nd-degree relative). For more distant relatives (3rd-degree relatives), there was a clear improvement when data from one well-chosen extra relative were available. We further sought to evaluate the theoretical systematic effectiveness and actual performance of microhaplotype markers in identifying the contribution of a missing pedigree member to a two-person mixture (as a minor donor). It was observed that 100% correct assignments were made in the balanced mixtures (with no dropout) when referenced to close relatives. When the mixture profiles suffered from dropout, incorrect assignments of minor donors were markedly associated with relatedness and the dropout level. Meanwhile, the studied scenarios generally exhibited zero or very low false-positive rates, indicating a low probability of incorrectly assigning an unrelated contributor as a close relative of the reference. Our results indicate that microhaplotype data can be reliably interpreted for identifying missing persons through kinship analysis based on DNA profiles of single-source samples or two-person mixtures. Furthermore, this study could be extended to more complex scenarios, such as determining the relatedness of contributors in (or among) mixed DNA profiles, if combined with different statistical frameworks.

Detection and quantification of the age-related sjTREC decline in human peripheral blood.

The decline of signal joint T-cell receptor rearrangement excision circles (sjTRECs) in human peripheral blood has been demonstrated to be age-related, which can be a potential marker for individual age determination. However, little is known about the quantitative relationship between the levels of sjTREC and age. The aim of the present study was to investigate the levels of sjTREC in peripheral blood leukocytes (PBLs) among different age groups in Chinese population, so as to clarify whether it could serve as a suitable marker for biological age estimation in forensic practice. sjTREC levels were measured by real-time quantitative PCR analysis in peripheral blood samples from individuals of known age (n = 248). The quantification results showed that sjTREC declined in human PBLs in an age-dependent manner (r = -0.8177, P < 0.01). The formula for age estimation based on peripheral sjTREC decline was Y = -24.921x - 39.932 ± 10.47 (Y age, year; X log sjTREC/TBP; 10.47: standard error). Furthermore, there was no difference between males and females with regard to sjTREC levels. These results suggest that assessment of sjTREC in PBLs might be a valuable additional tool in age determination, especially in cases where traditional morphologic information is absent or inefficient in forensic practice.

[Calculation of the avuncular index].

OBJECTIVE: To introduce the method of avuncular index (AI) calculation. METHODS: Identity by decent coefficient, coancestry coefficient and AI law were employed in identification of uncle-niece relationship, when autosomal STR loci were detected to determine controversial uncle-niece relationship. RESULTS: The results of AI calculation were coincidental using identity by descent coefficien, coancestry coefficient and AI law. CONCLUSION: The results are coincidental using three methods in the different situations. AI index is higher with participation of children's mother.

A case of heteropaternal superfecundation identified by microhap sequencing of maternal plasma cell-free DNA: A case of HS identified by microhap sequencing.

Heteropaternal superfecundation (HS) refers to the fertilization of two or more oocytes by spermatozoa from different male partners during the polyovulatory period. The present study reported a newly discovered case of HS in the 10th week of gestation, in a case of disputed paternity involving a pair of female twins and two alleged fathers (AF1 and AF2), based on a custom-designed microhap sequencing assay and R package relMix for data interpretation. The results suggested that the twins had different biological fathers, e.g., HS, and indicated the paternity of AF1 in relation to one of the twins while excluding AF2 with regard to both twins. Standard short tandem repeat (STR) analysis was employed to confirm the paternity of the heteropaternal twins. The reported case indicates that HS may occur in paternity cases with dizygotic twins, and microhap, as a novel type of highly polymorphic marker proved to be suitable for mixture deconvolution, should be able to resolve this question effectively and noninvasively at the early stage of pregnancy.

Mosaic duplication of 8q24.1q24.3 detected by chromosomal microarray but not karyotyping in two unrelated fetuses with cardiac defects.

BACKGROUND: Discordance between traditional cytogenetic and molecular cytogenetic tests is rare but not uncommon. The explanation of discordance between two genetic methods is difficult but especially important for genetic counseling, particularly for prenatal genetic diagnosis. CASE PRESENTATION: Two unrelated fetuses were diagnosed with cardiac defects by prenatal ultrasound examination, and invasive cordocentesis was performed to obtain cord blood samples for prenatal genetic diagnosis. For both fetuses, chromosomal microarray analysis (CMA) detected a novel approximately 27-Mb mosaic duplication with a high copy number of approximately six to seven copies on chromosome 8q24.1q24.3 that was not identified by karyotyping. To exclude artificial errors and validate laboratory detection results, multiple procedures including copy number variation sequencing, fluorescence in situ hybridization, and short tandem repeat and single-nucleotide polymorphism genotype comparison were performed, confirming the discordant results between CMA and karyotyping. The potential causes of discordance between CMA and karyotyping using fetal blood lymphocytes are discussed; we suggest that extrachromosomal DNA or cell-free DNA fragmentation originating from certain tumor tissues with 8q24.1q24.3 duplication might deserve further investigation. CONCLUSIONS: This study may be helpful for prenatal evaluation and genetic counseling for subsequent patients with similar mosaic 8q24.1q24.3 duplications. Additionally, more cases and further research are needed to understand whether mosaic 8q24.1q24.3 duplication is associated with certain genetic disorders and to investigate the causes of discordance between molecular and morphological methods.