The Latest Research Progress on Cell-Free DNA and Prospects of Its Forensic Application.

In recent years, with the continuous progress of DNA extraction and detection technology, cell-free DNA(cfDNA)has been widely used in the life science field, and its potential application value in forensic identification is becoming more and more obvious. This paper reviews the concept, formation mechanism, and classification of cfDNA, etc., and describes the latest research progress of cfDNA in personal identification of crime scene touch DNA samples and non-invasive prenatal paternity testing (NIPPT). Meanwhile, this paper summarizes the potential application of cfDNA in injury inference, and discusses the advantages and disadvantages of common cfDNA analysis methods and techniques, and its application prospects, to provide a new idea for the wide application of cfDNA in the field of forensic science.

A novel 193-plex MPS panel integrating STRs and SNPs highlights the application value of forensic genetics in individual identification and paternity testing.

Massively parallel sequencing (MPS) has emerged as a promising technology for targeting multiple genetic loci simultaneously in forensic genetics. Here, a novel 193-plex panel was designed to target 28 A-STRs, 41 Y-STRs, 21 X-STRs, 3 sex-identified loci, and 100 A-SNPs by employing a single-end 400 bp sequencing strategy on the MGISEQ-2000™ platform. In the present study, a series of validations and sequencing of 1642 population samples were performed to evaluate the overall performance of the MPS-based panel and its practicality in forensic application according to the SWGDAM guidelines. In general, the 193-plex markers in our panel showed good performance in terms of species specificity, stability, and repeatability. Compared to commercial kits, this panel achieved 100% concordance for standard gDNA and 99.87% concordance for 14,560 population genotypes. Moreover, this panel detected 100% of the loci from 0.5 ng of DNA template and all unique alleles at a 1:4 DNA mixture ratio (0.2 ng minor contributor), and the applicability of the proposed approach for tracing and degrading DNA was further supported by case samples. In addition, several forensic parameters of STRs and SNPs were calculated in a population study. High CPE and CPD values greater than 0.9999999 were clearly demonstrated and these results could be useful references for the application of this panel in individual identification and paternity testing. Overall, this 193-plex MPS panel has been shown to be a reliable, repeatable, robust, inexpensive, and powerful tool sufficient for forensic practice.

Utilizing Massively Parallel Sequencing (MPS) of Human Leukocyte Antigen (HLA) Gene Polymorphism to Assess Relatedness in Deficiency Parentage Testing.

In the realm of DNA testing with legal implications, the reliability and precision of genetic markers play a pivotal role in confirming or negating paternity claims. This study aimed to assess the potential utility of human leukocyte antigen (HLA) gene polymorphism through massively parallel sequencing (MPS) technology as robust forensic markers for parentage testing involving genetic deficiencies. It sought to redefine the significance of HLA genes in this context. Data on autosomal short tandem repeat (aSTR) mutational events across 18 paternity cases involving 16 commonly employed microsatellite loci were presented. In instances where traditional aSTR analysis failed to establish statistical certainty, kinship determination was pursued via HLA genotyping, encompassing the amplification of 17 linked HLA loci. Within the framework of this investigation, phase-resolved genotypes for HLA genes were meticulously generated, resulting in the definition of 34 inherited HLA haplotypes. An impressive total of 274 unique HLA alleles, which were classified at either the field 3 or 4 level, were identified, including the discovery of four novel HLA alleles. Likelihood ratio (LR) values, which indicated the likelihood of the observed data under a true biological relationship versus no relationship, were subsequently calculated. The analysis of the LR values demonstrated that the HLA genes significantly enhanced kinship determination compared with the aSTR analysis. Combining LR values from aSTR markers and HLA loci yielded conclusive outcomes in duo paternity cases, showcasing the potential of HLA genes and MPS technology for deeper insights and diversity in genetic testing. Comprehensive reference databases and high-resolution HLA typing across diverse populations are essential. Reintegrating HLA alleles into forensic identification complements existing markers, creating a potent method for future forensic analysis.

Analysis of data and common mutations encountered during routine parentage testing in Zimbabwe.

We analyzed parentage data collected over a ten-year period in a Zimbabwean DNA testing laboratory. Parentage case types, prevalence, exclusion data, mutations rates and observed genotyping irregularities were analyzed. We report analysis results from 1303 cases. DNA extraction and STR typing was conducted using standard commercial kits. Paternity was the most requested test (87.37%) followed by the indirect biological kinship tests (7.01%). Duo paternity (motherless) was the most common paternity test for both regular and court cases. We observed 367 paternity exclusions from 1135 cases, giving an overall paternity exclusion rate of 32.33%. Maternity had the lowest exclusion rate (8.33%), with criminal cases having the highest paternity (61.11%) and maternity (33.33%) exclusion rates. The number of mismatched STR loci ranged from 2-12 for duo cases and 4-18 for the trio cases. FGA, D2S1338, D18S51 and D2S441 were the most informative markers for exclusion. We detected 30 mutations out of 837 cases with an estimated paternal and maternal mutation rate of 0.0021 and 0.0011 respectively. Triallelic patterns were only observed at the TPOX locus with allele 10 and 11 being the extra alleles transmitted. Our report provides forensic parameters which can improve parentage and forensic analysis in Zimbabwe.

A possible case of offspring sex manipulation as result of a biased adult sex ratio.

Although random meiosis should prevent the facultative adjustment of offspring sex ratio, theory predicts that females should produce more of the sex with the higher reproductive value. We reported a case of offspring sex ratio manipulation in grass wrens Cistothorus platensis. Males in better body condition would have higher reproductive value than females due to the potential for social polygyny and extra-pair fertilizations. On the other hand, local demography influences reproductive strategies in grass wrens as male abundance affects both social polygyny and extra-pair paternity frequencies. We evaluated whether females bias their brood sex ratio in response to adult sex ratio and nestling body condition (a proxy for female's prospects of producing high-quality males). Females raised more male offspring when males were less abundant in the population (female-biased adult sex ratio). However, we found no relationship between nestling body condition and brood sex ratio, suggesting that females did not bias the brood sex ratio towards males when able to raise nestlings in better body condition. Taken together, our results provide the first suggestive evidence that female birds can manipulate their offspring sex ratio in response to the adult sex ratio.

For the good of the children-Life and ethical values when undergoing paternal alienation and involuntary loss of paternity.

BACKGROUND AND AIM: Today there is an aspiration and desire for fathers to be caring masculinities that build long-term father-child relationships and emotional presence with their children. Previous research shows that life changes where fathers are deprived of the opportunity for equal parenting and close contact with their children affect the fathers' lives and mental health. The aim of this caring science study is thereby to gain a deeper understanding of life and ethical values when undergoing paternal alienation and experiencing involuntary loss of paternity. DESIGN, RESEARCH METHODS, AND PARTICIPANTS: The study has a qualitative design. The data collection was carried out in 2021 through individual in-depth interviews according to Kvale and Brinkmann. The five fathers who participated in the interviews had experiences of undergoing paternal alienation and involuntary loss of paternity. The interviews were analysed with a reflexive thematic analysis according to Braun and Clarke. RESULTS: Three main themes emerged. Putting yourself aside includes forgetting one's own needs and prioritising the children's and being the best version of oneself for them. In playing with the cards you have been dealt lies an acceptance of life as it has become and also a responsibility not to let the grief take over, by creating new patterns for everyday life and holding up hope. Keeping your dignity as a human being includes being heard, affirmed and consoled, and a form of re-awakening one's dignity as a human being. CONCLUSION: It is fundamental to understand the grief, longing and sacrifice that paternal alienation and involuntary loss of paternity cause human life and how every day can be a struggle to hold on to hope, find comfort and reconcile with the situation. The fundamental foundation that makes life worth living is love and responsibility for the good of the children.

Podcast RP Convida: Pai parceiro na amamentação

Com o tema "Pai parceiro na amamentação“, o podcast RP Convida da revista Residência Pediátrica (RP) apresenta o episódio especial em alusão ao Agosto Dourado. Nesta edição, a convidada é a dra. Maria Beatriz Reinert do Nascimento, da Sociedade Brasileira de Pediatria (SBP).

Podcast RP Convida: Pai empoderado compartilha amamentação

Tema do podcast da revista Residência Pediátrica (RP) “Pai empoderado compartilha amamentação”. Com a convidada, a dra. Rossiclei Pinheiro, do Departamento Científico de Aleitamento Materno da Sociedade Brasileira de Pediatria (SBP), explica como o pediatra pode atuar para fortalecer o vínculo e apoio paterno na prática da amamentação.

Podcast RP Convida: Amamentação: como o pai pode participar

Podcast da revista Residência Pediátrica (RP) aborda o tema “Amamentação: como o pai pode participar”, com apresentação da dra. Graciete Vieira, do Departamento Científico de Aleitamento Materno da Sociedade Brasileira de Pediatria (SBP). Segundo a especialista, a amamentação é um dos melhores investimentos em saúde, com múltiplos benefícios para as crianças. Nesse contexto, a participação paterna é um fator decisivo para o início e manutenção da prática.

Construction and evaluation of a novel set of 90 microhaplotypes for forensic applications using NGS technology.

Microhaplotypes (MHs), small sets of linked single nucleotide polymorphisms (SNPs), are becoming a valuable tool for paternity testing, personal identification and other different forensic purposes due to their advantages of both short tandem repeats (STRs) and SNPs. However, only a small part of MHs with small segments have been developed and reported so far. And the current population genetic data of MHs are still insufficient. MHs with small segments possess unique advantages in mixture deconvolution, degradation material identification, noninvasive prenatal paternity testing and even medical tumor diagnostic applications. In the present study, a set of 90 autosomal MHs whose PCR amplicon lengths are from 90-150 bp, of which 58 MHs are less than or equal to 100 bp are selected, and assembled into an amplification multiplex system optimized for Ion S5™ System for forensic application. Genetic diversity study of 90 MHs in the populations from different intercontinental regions shows that the polymorphism information content (PIC) values of 83 MHs are greater than 0.4 in populations from East Asia (EAS), and the average PIC value of 90 MHs is greater than 0.5. A total of EAS populations shows the highest cumulative match probability (CMP) and cumulative probability of exclusion (CPE) values in five intercontinental populations. The CMP and CPE values of 90 MHs in EAS are 1.1688 × 10-54 and 0.999999999998954. The informativeness for assignment (In) values of the 90 MHs are calculated based on data from five intercontinental populations, and the In values of 20 MHs have greater than 0.1, indicating that the 20 MHs are high effectiveness in distinguishing different intercontinental populations, which can be used as candidate ancestry informative markers. Further, we have studied the polymorphisms of the 90 MHs based on 224 unrelated individuals of Henan Han population, China, and obtained the frequency data of the 90 MHs. In the Henan Han population, the effective number of alleles (Ae) of the 90 MHs ranges from 1.7649 (MH45) to 3.9792 (MH50), and the Ae values of 10 MHs reach to 3.0; the Ae values of 80 MHs are greater than 2, and the average Ae value for these MHs is 2.422. The average expected heterozygosity, observed heterozygosity, PIC, matching probability, discrimination power and probability of exclusion values of 90 MHs in the Henan Han population are 0.5788, 0.5851, 0.5039, 0.2608, 0.7392 and 0.2806, respectively. The CMP value of 90 MHs in the study population is less than 10-54, and their CPE value reaches 0.999999999999999923. Moreover, the results of the depth of coverage, allele coverage ratio and noise level indicate that the 90 MH amplification system has well sequencing performance, and the sequencing results are reliable. The results indicate the 90 MHs show higher polymorphisms in the study population. The present panel can be well used in paternity testing and individual identification in the study population and even the populations from EAS.

Maternal uniparental disomy of chromosome 21 as a cause of pseudo-exclusion from paternity.

Uniparental disomy (UPD) is a rare chromosomal condition, which apart from its importance in medical genetics can affect an outcome of parentage DNA testing, often causing pseudo exclusions. We describe a case of trio paternity test using 24 informative STR loci with potential exclusion at 2 systems located on chromosome 21. Consequent genotyping of an additional 25 autosomal and 27 Y-specific STRs revealed one other inconsistency, also located on this chromosome. All three inconsistent markers had the same heteroallelic state between the child and the biological mother providing evidence for maternal heterodisomy of chromosome 21. The case highlights the importance of considering UPD as a cause of genetic inconsistencies, especially when the inconsistent marker systems are located on the same chromosome.

Therapeutic exposures and pubertal testicular dysfunction are associated with adulthood milestones and paternity after childhood cancer.

BACKGROUND: Childhood cancer therapy may cause long-term effects. This cross-sectional study evaluated adulthood milestones in male childhood cancer survivors (CCS). METHODS: The study population comprised 252 male CCS with 6 to 42 years of survival diagnosed at the Children's Hospital in Helsinki (1964-2000) at the age of 0 to 17 years. Sex-, age-, and area of residence-matched population controls were randomly selected from the Finnish national registries. Data on moving away from the parental home, marital status, offspring, and adoption in CCS were compared with the population controls. We analyzed the influence of chemotherapy and radiation exposures and testicular dysfunction (ever nontestosterone-substituted serum follicle stimulating hormone >15 IU/L, luteinizing hormone >15 IU/L, testosterone <2 ng/mL (5 nmol/L), need of testosterone replacement therapy, or testicular volume <12 mL at the end of puberty) during pubertal maturation on long-term social outcomes. RESULTS: CCS moved away from their parental home as frequently as population controls (97.8% vs. 98.5%, p = .45). CCS were less likely to marry or live in a registered relationship (46.4% vs. 57.5%, p < .001), especially when diagnosed at a young age (<4 years). Among those married, the probability of divorce was similar between CCS and population controls (27.4% vs. 23.8%, p = .41). Survivors were less likely to sire a child (38.5% vs. 59.1%, p < .001) and more likely to adopt (2% vs. 0.4%, p = .015). Lower probability of paternity was associated with hematopoietic stem cell therapy, testicular radiation dose >6 Gy, pubertal signs of testicular dysfunction (nontestosterone-substituted serum follicle stimulating hormone >15 IU/L, luteinizing hormone  >15 IU/L, testosterone <2 ng/mL (5 nmol/L), or need of testosterone replacement therapy during puberty, or testicular volume <12 mL at the end of puberty) or azoospermia after puberty. CONCLUSIONS: This study emphasizes the value of pubertal monitoring of testicular function to estimate future probability of paternity. If no signs of dysfunction occurred during pubertal follow-up, paternity was comparable to population controls. Testicular radiation dose >6 Gy appeared to be the strongest risk factor for decreased paternity. PLAIN LANGUAGE SUMMARY: Treatment with intensive therapies, including hematopoietic stem cell therapy, testicular radiation dose >6 Gy, and signs of testicular dysfunction, during puberty are important risk factors for lower rates of fertility. Intensive therapies and testicular dysfunction itself do not similarly hamper psychosocial milestones in adulthood; cancer diagnosis at a very young age (<4 years) lower the probability of marriage. This study accentuates the importance of monitoring of pubertal development, emphasizing on testicular function, not only sperm analysis, to estimate future fertility among male childhood cancer survivors.

Paternity data reveal high MHC diversity among sires in a polygynandrous, egalitarian primate.

Evidence from human and nonhuman primates suggests that females avoid breeding with close kin and may choose mates based on MHC diversity, which can improve offspring survival. In despotic societies, female mate choice may be hindered by male sexual coercion, but in egalitarian societies, females may be less constrained. Among northern muriquis-an egalitarian, polygynandrous primate with male philopatry-analyses of new data on paternity and variation at microsatellite and MHC loci, combined with behavioural and life-history data, revealed that sires showed higher MHC diversity than expected by chance and were never close kin of dams, consistent with predictions of female mate choice and close inbreeding avoidance. However, females did not differentially reproduce with males who were more distantly related to them or more dissimilar at the MHC than expected by chance, nor with those who had more MHC alleles distinct from their own. The lack of male dominance may permit females to identify and reproduce preferentially with non-offspring males and with males who are more diverse at the MHC. Nonetheless, the absence of disassortative mating at the MHC and neutral loci suggests that female mate choice may be limited by other factors impacting male fertilization success.

Genetic inconsistency at the D6S1043 locus caused by microdeletion at 6q15.

In the practice of parentage testing, short tandem repeat (STR) genetic inconsistencies occasionally occur and are usually treated as genetic mutations. However, they arise for various reasons. To elucidate the reasons for their occurrence, this study investigates a typical trio. For the D6S1043 locus, the genotype of the biological mother comprised the heterozygous alleles "7,20"; that of the child, allele 20; and that of the alleged father, a heterozygous allele "11,13," revealing a 7-step mutation. Different kits were first used to verify the data. The locus map, primers, and core sequences were then analyzed. Ultimately, the STR and single nucleotide polymorphisms of 6q were tested to determine the microdeletion range. The results revealed that this was indeed a true trio, and the underlying cause of the genetic inconsistency at this locus was a microdeletion of approximately 0.74-1.78 Mb in 6q15. Overall, genetic inconsistencies detected during practical work, and particularly rare multi-step mutations, cannot be directly identified as STR mutations. Different tools should be used to examine the causes of genetic inconsistencies from various perspectives and improve the effectiveness of genetic evidence.

A posteriori parameters from paternity tests of a Mexican laboratory with the powerplex fusion system.

Population studies regarding Human identification (HID) systems report a priori forensic parameters, but rarely they describe a posteriori parameters from concluded paternity tests. We analyzed data from the PowerPlex® Fusion System in 1503 paternity tests from a Mexican laboratory for five years (2016-2020). The motherless duo paternity tests (89.8%) were more frequent than the standard trio tests (10.2%). A notable increase in motherless tests was noted regarding our previous report (89.8% vs 77.3%), probably explained by the COVID-19 pandemic. The estimated exclusion frequency in Mexico ranged from 30.1 (trio) to 32.1% (duo). For paternity exclusions, we report the number of mismatches and the frequency at which each STR was involved. The PowerPlex® Fusion system showed more than five mismatches in 100% of the standard trio tests excluding paternity, and the majority of motherless-duo tests (98.1%). In positive paternity tests, PowerPlex® Fusion offered a higher combined paternity index (PI) (average 1.18 E + 10) regarding HID systems with 15 and 20 STRs, even without the inclusion of the Y-linked locus DYS391 to the kinship interpretation. Individual and global STR mutation rates were estimated from 17 paternal mutations (µ = 0.0017), the majority involving a single-step mutation (94.11%). Five independent null alleles were detected, most of them involving the Penta E locus (80%), which suggests caution to the users working with DNA databases or kinship analysis, to avoid false exclusions with Penta E. In brief, our results provide a better overview of a posteriori informativeness offered by the PowerPlex® Fusion system for paternity testing in Mexico.

Low-Pass Genome Sequencing-Based Detection of Paternity: Validation in Clinical Cytogenetics.

Submission of a non-biological parent together with a proband for genetic diagnosis would cause a misattributed parentage (MP), possibly leading to misinterpretation of the pathogenicity of genomic variants. Therefore, a rapid and cost-effective paternity/maternity test is warranted before genetic testing. Although low-pass genome sequencing (GS) has been widely used for the clinical diagnosis of germline structural variants, it is limited in paternity/maternity tests due to the inadequate read coverage for genotyping. Herein, we developed rapid paternity/maternity testing based on low-pass GS with trio-based and duo-based analytical modes provided. The optimal read-depth was determined as 1-fold per case regardless of sequencing read lengths, modes, and library construction methods by using 10 trios with confirmed genetic relationships. In addition, low-pass GS with different library construction methods and 1-fold read-depths were performed for 120 prenatal trios prospectively collected, and 1 trio was identified as non-maternity, providing a rate of MP of 0.83% (1/120). All results were further confirmed via quantitative florescent PCR. Overall, we developed a rapid, cost-effective, and sequencing platform-neutral paternity/maternity test based on low-pass GS and demonstrated the feasibility of its clinical use in confirming the parentage for genetic diagnosis.

Calculation of the Paternity Index for the Alleged Father Related to the Child's Mother.

OBJECTIVES: To derive the paternity index (PI) calculation formula of the alleged father (AF) when the AF is a relative (parent/child, siblings, grandparent/grandchild, uncle/nephew, first cousins) of the child's biological mother. METHODS: For the case when the AF is related to the child's biological mother, the existence of the relationship in the numerator and denominator hypothesis of PI was considered. The genotype frequency of the AF was calculated by using the frequency formula in which the mother's genotype was considered, while the random male in the denominator was substituted as another relative of the mother's same rank. The PI calculation formula was derived to eliminate the effect of the relationship between AF and the child's biological mother. RESULTS: When the AF and the biological mother have first, second and tertiary kinship, a more conservative PI was obtained from the PI calculation formula derived in this study compared with the PI calculation method which did not consider kinship. CONCLUSIONS: The calculation method provided in this study can eliminate the effect of the relation of the AF and mother on the PI in incest cases, to obtain more accurate and conservative identification conclusions.

General Formulas for Calculating Commonly Used Kinship Index.

OBJECTIVES: To derive general formulas for calculating commonly used kinship index (KI). METHODS: By introducing the Kronecker symbol, the formulas used to calculate the same KI under different genotype combinations were summarized into a unified expression. RESULTS: The general formulas were successfully derived for KI in various case situations, including the paternity index, full sibling index, half sibling index, avuncular index, grandpaternity index, first-cousin index, and second-cousin index between two individuals without or with the mother being involved; grandpaternity index between grandparents and a grandchild without or with the mother being involved; half sibling index between two children with two mothers being involved; full sibling index among three children; and half sibling index among three children with no, one, or two mothers being involved. CONCLUSIONS: The general formulas given in this study simplify the calculation of KIs and facilitate fast and accurate calculation through programming.