Short Tandem Repeat Genotyping Research Articles

Partial hydatidiform moles with unclear histopathological profile - importance of genetic diagnostics.

The aim of the study was the genetic characterization of a set of cases with an unclear morphological profile of the placental tissue suspected of a partial hydatidiform mole. This work presents the results of a genetic analysis of a group of 10 patients with various clinical manifestations of reproductive loss, where a partial hydatidiform mole was suspected on the basis of a histopathological examination. The composition of the genome of the products of conception was determined by short tandem repeats (STR) genotyping using a commercial kit;Devyser Compact v3 (Devyser). Out of 10 analyzed cases, five had diandric monogynic triploid genome, characteristic for a partial mole. Aneuploidies of chromosomes 13, 18, 21, X and Y were excluded in four cases and Pataus syndrome was dia-gnosed in one case. In the case of an unclear histopathological profile, consultative DNA analysis (ideally STR genotyping) can significantly help the pathologist in the differential dia-gnosis of a partial mole. The histopathological profile of a partial hydatidiform mole may be in some cases incomplete and unclear, especially in the early weeks of gestation, which can lead to false negativity of the examination. On the other hand, other pathologies, for example aneuploides or digynic triploidy, may produce a histopathological profile similar to a partial mole, which leads to false positivity. Accurate dia-gnosis of a partial hydatidiform mole using molecular genetic methods contributes to the determination of adequate dispensary care for patients.

Nanopore sequencing of a forensic combined STR and SNP multiplex

Nanopore sequencing for forensic purposes has gained attention, as it yields added discriminatory power compared to capillary electrophoresis (CE), without the need for a high up-front capital investment. Besides enabling the detection of iso-alleles, Massively Parallel Sequencing (MPS) facilitates the analysis of Short Tandem Repeats (STRs) and Single Nucleotide Polymorphisms (SNPs) in parallel. In this research, six single-contributor samples were amplified by such a combined multiplex of 58 STR and 94 SNP loci, followed by nanopore sequencing using an R10.3 flowcell. Basecalling was performed using two state-of-the-art basecallers, Guppy and Bonito. An advanced alignment-based analysis method was developed, which lowered the noise after alignment of the STR reads to a reference library. Although STR genotyping by nanopore sequencing is more challenging, correct genotyping was obtained for all autosomal and all but two non-autosomal STR loci. Moreover, genotyping of iso-alleles proved to be very accurate. SNP genotyping yielded an accuracy of 99% for both basecallers. The use of novel basecallers, in combination with the newly developed alignment-based analysis method, yields results with a pronouncedly higher STR genotyping accuracy compared to previous studies

Application of next-generation sequencing to preimplantation genetic testing for recurrent hydatidiform mole patients

To study the application of next-generation sequencing on preimplantation genetic testing for recurrent hydatidiform mole patients. A total of ten recurrent hydatidiform mole patients aged 27-34years with a history of at least twice hydatidiform moles and no normal pregnancy were collected from 2019 to 2020. The diagnosis of hydatidiform mole type was clarified using short tandem repeat genotyping on products of conception, and whole-exome sequencing was applied for all patients and their partners. Seven recurrent hydatidiform mole patients with complete hydatidiform mole/partial hydatidiform mole type among previous hydatidiform mole tissues and no Pathogenetic/Likely pathogenetic/Uncertain significance variants in NLRP7/KHDC3L/MEI1/C11orf80 underwent a procedure of preimplantation genetic testing. Next-generation sequencing for analyzing the copy number variants and the numbers of heterozygous single nucleotide polymorphism was adopted to clarify the ploidy and parental origin of the embryo chromosomes in vitro. Embryos with biparental diploidy were selected for transfer. Seven patients have undergone the procedure of preimplantation genetic testing, and twenty-three embryos were obtained, among which 82.6% (n = 19) were identified transferrable and 17.4% (n = 4) were identified aneuploid. Two patients have delivered healthy babies and another is currently in the second trimester after transfer. Analyzing the copy number variants and the numbers of heterozygous single nucleotide polymorphism on the basis of next-generation sequencing can be utilized in the procedure of preimplantation genetic testing among part of recurrent hydatidiform mole patients. The current study is effective to reduce the occurrence of hydatidiform mole with improved clinical strategy, the advanced testing technology and analysis methods, as three of seven patients have conceived or delivered successfully.

Application of an established canine genotyping method to a sequence-based approach

The advancement and accessibility of high throughput sequencing platforms have transformed forensic biology, with sequence-based genotyping of various genetic markers being widely validated for use. A key requirement for the successful implementation of such an approach for genotyping short tandem repeat (STR) loci is backward compatibility with existing databases derived from conventional capillary electrophoresis (CE). Other advantages of a sequence-based approach include novel allele discovery and increased allelic diversity. While sequence-based STR genotyping of human material is now relatively common in forensic biology, this approach for genotyping other animal species for forensic applications has been slower to emerge.Here we present proof-of-concept and concordance data for the sequencing of a canine STR panel for forensic applications. Sequence-based genotyping using the Mini-DogFiler panel is presented herein, including a bioinformatics pipeline for genotype calling. Allele discovery is presented as well as backward compatibility of sequence-based data with CE-derived genotypes of the same samples using the DogFiler and/or the Mini-DogFiler panel(s). The recorded CE genotypes, which included domestic dog and wolf samples, were compared to those generated using the sequence-based approach. General database statistics were evaluated across samples and included a comparison of the number of unique genotypes and alleles observed when using CE genotyping as compared to sequencing. The results demonstrate this method as being concordant and backward compatible to traditionally generated CE data. With additional developmental validation work, this approach offers a unique and timely addition to the suite of tools available for use to support animal and wildlife forensic science.

Abstract 2986: Conditional reprogramming of primary head and neck tumor cells to establish consistent and diverse cell line models

Abstract Introduction Head and neck squamous cell carcinoma (HNSCC) most commonly arise in the oral cavity, pharynx, and larynx and exhibit diverse molecular characteristics and clinical behavior across sites. Current models of HNSCC largely rely on immortalized cell lines, which suffer from selection bias and clonality. Primary cell cultures, on the other hand, are difficult to maintain and are limited by their finite ability to proliferate. Here, we describe the establishment of a robust HNSCC cell line bank using a “conditional reprogramming” (CR) method which relies on treatment with a rho kinase inhibitor (Y-27632) and co-culture with irradiated fibroblast (J2 strain) feeder cells to support indefinite tumor cell survival. Methods HNSCC tumors were acquired on an IRB-approved prospective tissue banking protocol. Tumor tissue and blood were collected from each patient, and tumor cells were digested and cultured using previously described CR methods. DNA was collected from tumor tissue and CR cultures and short tandem repeat (STR) profiling was used to validate culture against patient tumor tissue. To verify that the cultures were of squamous origin, western blot analysis was used to detect cytokeratin 5, cytokeratin 6 and p63. Tumor cell phenotype and growth characteristics were examined with light microscopy. HPV testing was carried out on DNA from CR cultures and matched tumor samples using PCR and specific genotyping by oligonucleotide hybridization. HPV testing was correlated with clinical p16 testing results. Whole exome sequencing was carried out on a subset of blood/tumor/CR culture samples. Results Eighteen CR lines were successfully cultured and validated with STR genotyping from 25 sequentially procured tumors. 10/18 were derived from oral cavity squamous cell cancers (SCCs), 5/18 were derived from oropharyngeal SCCs, and 3/18 were derived from laryngeal SCCs. 18/18 lines were found to express p63 and either cytokeratin 5 or 6, verifying these cultures contained tumor cells of squamous origin. 4/5 tumors from oropharyngeal SCCs were p16-positive on clinical testing and considered HPV-mediated. These 4 tumors all tested positive for HPV DNA in both tumor samples and CR cultures. Preliminary comparison of exome sequencing results between CR cultures and primary tumors suggests that overall mutational profiles are preserved through the tumor “conditional programming” process. Tumor heterogeneity between original tumor and CR culture is being actively compared in ongoing analyses. Conclusion We have consistently generated primary tumor CR cultures from patients with HNSCC arising in three major anatomical subsites of HNSCC disease, including HPV-mediated tumors. CR methods can be readily applied to all HNSCC tumors regardless of disease site and molecular background, providing a translational research model that can capture the molecular and phenotypic breadth of HNSCC disease. Citation Format: Daniel Li, Carlos Thomas, Nitisha Shrivastava, Nicholas Gadsden, Nicole Kawachi, Bradley A. Schiff, Richard V. Smith, Nicolas F. Schlecht, Michael B. Prystowsky, Gregory Rosenblatt, Stelby Augustine, Chandan Guha, Evripidis Gavathiotis, Robert D. Burk, Vikas Mehta, Thomas J. Ow. Conditional reprogramming of primary head and neck tumor cells to establish consistent and diverse cell line models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2986.

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

BackgroundDiscordance 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 presentationTwo 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.ConclusionsThis 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.

Evaluation and SNP typing of DNA from ultraviolet-irradiated human bloodstains using TaqMan assay

When detecting DNA profiles from forensic materials, it is pivotal to know the extent of degradation and which DNA marker can be genotyped. Ultraviolet (UV) is one of the common external factors that causes DNA damage, through which, an attempt to reveal cardinal genetic information can be made. In this study, after irradiation with three different UV wavelengths, UV-damaged DNA in the bloodstains was analyzed with long and short TaqMan assays using real-time PCR. In addition, both short tandem repeat (STR) profiles and single nucleotide polymorphisms (SNPs) from the damaged DNA at different stages of UV exposure were also assessed. With increasing in UV irradiation cycles, there was a delay of the amplification curves accompanied with a decrease in the DNA amounts collected. Despite the amplification of STR genotype was not altered after 75 cycles of UVC irradiation, all 12 SNP loci could still be detected. Furthermore, a short-assay line was detected in the absence of an amplification of the evaluation curve. The results indicate that, although the DNA template might not be useful and suitable for analysis of STR profile, this approach is of some values in detecting SNPs.

Establishment and characterization of 38 novel patient-derived primary cancer cell lines using multi-region sampling revealing intra-tumor heterogeneity of gallbladder carcinoma

Gallbladder carcinoma (GBC) is a lethal biliary tract malignant neoplasm. Patient-derived primary cancer cell lines (PDPCs) are appropriate models to explore biological characteristics and potential therapeutics; however, there is a lack of PDPCs in GBC. In this study, we aimed to establish and characterize the GBC PDPCs, and further investigated the intra-tumor heterogeneity (ITH). Multi-region sampling (3–9 regions) of the operable tumor tissue samples was used to establish PDPCs. Short tandem repeat genotyping for cell authentication and karyotyping was performed, followed by whole-exome sequencing and RNA sequencing to assess the ITH at the genetic and transcriptional levels, respectively. Thirty-eight PDPCs were successfully established from seven GBC patients and characterized. ITH was observed with a median of 38.3% mutations being heterogeneous (range, 26.6–59.4%) across all patients. Similar with other tumor types, TP53 mutations were always truncal. In addition, there were three genes, KMT2C, CDKN2A, and ARID1A, with truncal mutations in at least two patients. A median of 370 differentially expressed genes (DEGs) was identified per patient. Distinct expression patterns were observed between major histocompatibility complex (MHC) class I and II genes. We found the expression of MHC class II genes in the PDPC samples was closely regulated by CIITA, while that of MHC class I genes were not correlated with CIITA expression. The PDPCs established from GBC patients can serve as novel in vitro models to identify the ITH, which may pave a crucial molecular foundation for enhanced understanding of tumorigenesis and progression.

Analytical validation of an RI sample cartridge with the RapidHIT® ID system.

Evaluating the short tandem repeat (STR) in the field is important for the timely identification of a suspect. Several lines showed that the RapidHIT® ID system is reliable for DNA genotyping with buccal swabs and naked DNA. However, the application of this approach with blood samples has been poorly investigated. Because blood samples are among the most common forensic samples in our laboratory, further studies should be conducted. Here, we assessed the analytical performance of 19 STR loci with a newly developed RapidINTEL (RI) Sample Cartridge Kit by using the blood samples with known genotypes. Several commonly used substrates were included in the sensitivity study, and FTA cards proved to be the most promising sample carrier for blood storage and later identification. There was superior sensitivity and specificity with a 100% concordance rate for 0.5μL of blood or 7ng of genomic DNA. The performance for blood samples was comparable with that for the standard protocol. High success rate (90.57%) and high-concordance (100%) genotyping were automatically achieved over a wide range of operating conditions except for TH01. No contamination was observed throughout the study. Hematin, indigo, and humic acid had limited influence on the instrument system, while urea and melanin dramatically affected the genotyping results. Generally, the newly developed RI sample cartridge provided an alternative method for the STR genotyping of single-source blood samples over a wide range of operating conditions.

Comparison of the Allelic Alterations between InDel and STR Markers in Tumoral Tissues Used for Forensic Purposes

Background and objectives: Over the last two decades, human DNA identification and kinship tests have been conducted mainly through the analysis of short tandem repeats (STRs). However, other types of markers, such as insertion/deletion polymorphisms (InDels), may be required when DNA is highly degraded. In forensic genetics, tumor samples may sometimes be used in some cases of human DNA identification and in paternity tests. Nevertheless, tumor genomic instability related to forensic DNA markers should be considered in forensic analyses since it can compromise genotype attribution. Therefore, it is useful to know what impact tumor transformation may have on the forensic interpretation of the results obtained from the analysis of these polymorphisms. Materials and Methods: The aim of this study was to investigate the genomic instability of InDels and STRs through the analysis of 55 markers in healthy tissue and tumor samples (hepatic, gastric, breast, and colorectal cancer) in 66 patients. The evaluation of genomic instability was performed comparing InDel and STR genotypes of tumor samples with those of their healthy counterparts. Results: With regard to STRs, colorectal cancer was found to be the tumor type affected by the highest number of mutations, whereas in the case of InDels the amount of genetic mutations turned out to be independent of the tumor type. However, the phenomena of genomic instability, such as loss of heterozygosity (LOH) and microsatellite instability (MSI), seem to affect InDels more than STRs hampering genotype attribution. Conclusion: We suggest that the use of STRs rather than InDels could be more suitable in forensic genotyping analyses given that InDels seem to be more affected than STRs by mutation events capable of compromising genotype attribution.

Concordance and characterization of massively parallel sequencing at 58 STRs in a Tibetan population.

BackgroundMassively parallel sequencing (MPS) is a promising supplementary method for forensic casework in short tandem repeats (STRs) genotyping, owing to several advantageous features in comparison to traditional capillary electrophoresis (CE). However, the application of MPS in casework requires accessible datasets from the worldwide population to enrich the allele frequencies of sequence‐based STR genotypes.MethodsIn this study, we report the characterization of sequence‐based allele frequencies of 58 STRs from a Tibetan population comprising 120 unrelated individuals using the ForenSeq™ DNA Signature Prep Kit. A concordance study evaluating MPS and CE allele data was performed to ensure that MPS is compatible with current CE‐based forensic databases. The diversity of observed alleles, allele frequencies, and forensic parameters per locus by length (LB), sequence without flanking region (RSB), and sequence with flanking region (FSB) were analyzed and compared.ResultsThe concordance study demonstrated a concordance rate exceeding 99%. The combined random match probability (RMP) for the 26 A‐STRs was 2.04 × 10–29, 1.93 × 10–31, and 9.56 × 10–33 for LB, RSB, and FSB, respectively. Similar trends were observed in other forensic parameters resulting from the increase in the number of unique alleles available. A total of 111 and 113 unique haplotypes in the Y‐STR loci were observed when using length‐based and sequence‐based alleles, respectively. In addition, we identified 35 novel alleles at 25 loci and 25 polymorphisms in the flanking regions at 17 STRs.ConclusionsOur data suggest that MPS‐ and CE‐derived alleles are compatible. MPS‐based analysis of the STR data substantially increased the allele diversity and improved the forensic parameters, which clearly demonstrated the advantages of MPS in comparison to CE. With more pooled data and larger‐scale validation, MPS could play a valuable role in forensic genetics and might be an additional tool for routine casework.

Repeats expansions in ATXN2, NOP56, NIPA1 and ATXN1 are not associated with ALS in Africans.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized primarily by progressive loss of motor neurons. Although ALS occurs worldwide and the frequency and spectrum of identifiable genetic causes of disease varies across populations, very few studies have included African subjects. In addition to a hexanucleotide repeat expansion (RE) in C9orf72, the most common genetic cause of ALS in Europeans, REs in ATXN2, NIPA1 and ATXN1 have shown variable associations with ALS in Europeans. Intermediate range expansions in some of these genes (e.g. ATXN2) have been reported as potential risk factors, or phenotypic modifiers, of ALS. Pathogenic expansions in NOP56 cause spinocerebellar ataxia-36, which can present with prominent motor neuron degeneration. Here we compare REs in these genes in a cohort of Africans with ALS and population controls using whole genome sequencing data. Targeting genotyping of short tandem repeats at known loci within ATXN2, NIPA1, ATXN1 and NOP56 was performed using ExpansionHunter software in 105 Southern African (SA) patients with ALS. African population controls were from an in-house SA population control database (n = 25), the SA Human Genome Program (n = 24), the Simons Genome Diversity Project (n = 39) and the Illumina Polaris Diversity Cohort (IPDC) dataset (n = 50). We found intermediate RE alleles in ATXN2 (27–33 repeats) and ATXN1 (33–35 repeats), and NIPA1 long alleles (≥8 repeats) were rare in Africans, and not associated with ALS (p > 0.17). NOP56 showed no expanded alleles in either ALS or controls. We also compared the differences in allele distributions between the African and n = 50 European controls (from the IPDC). There was a statistical significant difference in the distribution of the REs in the ATXN1 between African and European controls (Chi-test p < 0.001), and NIPA1 showed proportionately more longer alleles (RE > 8) in Europeans vs. Africans (Fisher’s p = 0.016). The distribution of RE alleles in ATXN2 and NOP56 were similar amongst African and European controls. In conclusion, repeat expansions in ATXN2, NIPA1 and ATXN1, which showed associations with ALS in Europeans, were not replicated in Southern Africans with ALS.

Forensic features and genetic structure of 20 autosomal STR loci in the Han population of Ningde City, Southeastern China

Background Short tandem repeat (STR) loci are widely used in forensic medicine and population genetics. Aim To profile 20 autosomal STR loci using the SureID® 21 G Human STR Identification Kit. Subjects and methods Samples were obtained from 1412 unrelated Chinese Han individuals from Ningde City, Southeastern China and 20 autosomal STR loci were profiled using the SureID® 21 G Human STR Identification Kit. Results A total of 261 alleles were observed among 1412 unrelated individuals and the corresponding allelic frequencies ranged from 0.5464 to 0.0004. The combined power of discrimination and exclusion of the 20 autosomal STR loci were 0.99999999999999999922 and 0.999999340285752, respectively. There was no significant deviation from Hardy–Weinberg equilibrium (HWE) and minimal departure from linkage equilibrium (LE) for two pairwise combinations of loci after sequential Bonferroni correction. In the population comparison, phylogenetic analysis was performed between the Han population and other relevant populations on the basis of the shared autosomal STR genotyping. Moreover, the neighbor-joining tree and principal component analysis were analysed based on the Nei’s standard genetic distance. Conclusion The population comparison revealed that the structure of the Ningde Han population was similar to the structure of southern Han populations in China and was significantly different to the other Chinese ethnic groups, such as Kyrgyz, Uzbek, Kazakh, Uyghur, Manchu from Xinjiang and Mongols.

The impact of chimerism on DNA-based human identification from skin surface cells of post-allogenic hematopoietic stem cell transplantation (HCST) patients

The use of biological traces recovered from touched or handled items increased with the advance of the forensic analysis system. Thus, DNA profiles obtained from touch DNA became a useful tool in forensic investigation. However, a chimeric person with more than one chromosomal population can be challenging for a forensic analyst. We investigated the genetic profile in blood, buccal swab, and skin swabs from twenty-four recipients aged 21–63 years who underwent a matched sibling allogeneic hematopoietic stem cell transplantation with no sign of skin graft versus host disease. Autosomal short tandem repeats genotyping was performed to evaluate chimerism status at 15 loci along with gender marker Amelogenin. According to our results, donor chimerism was detected in all recipient's blood samples, while in buccal swabs, five recipients showed no presence of donor-derived cells in their genotype. Epithelial cells swabbed from hand fingertips were not devoid of donor-derived cells since all recipients showed high chimerism (39.69%–96.66%) in their genotypes. A significant change in chimerism was seen among various types of biological samples (p<0.05). No correlations were observed between chimerism and recipient age, gender, or time after transplant (p> 0.05). The loci D21S11, D8S1179, and FGA were the most informative, whereas D13S317, Vwa, and TOPX were the least informative STR markers. We concluded that touch DNA from a person who has undergone a successful allogeneic HSCTs should not be considered as reliable evidence for human identifications. Therefore, necessary precautions must be taken to avoid false identification and miscarriage of justice.

Allelic diversity and forensic estimations of the Beijing Hans: Comparative data on sequence-based and length-based STRs

Short tandem repeat (STR) profiling is routinely used in forensic genetics. At present, STR analysis is mainly performed by capillary electrophoresis (CE). However, due to limitations associated with the CE method, STR genotyping has been limited to length polymorphisms only. Because next generation sequencing (NGS) is capable of providing full resolution STR data at the sequence variation level, the individual identification capability of forensic STR loci could be significantly improved. Here we present sequence-based STR data for the Beijing Han population in which 291 individuals were screened for 23 commonly used forensic STRs using the SeqTypeR24 CASE kit on an Ion PGM platform. In total, 234 length-based alleles and 356 sequence-based alleles, which included 22 novel core repeat sequences, were observed. The sequence-based matching probability and power of discrimination were superior to the length-based numbers for 16 loci bearing micro-variant alleles. Combined matching probability reached 8.2 × 10−29 for 23 STR loci at the sequence level. This was two orders of magnitude higher than the parameters at length level and provides a data base for sequence-based STR casework applications.

Lack of genetic homozygosity in prepubertal teratomas: divergent pathogenesis distinct from that of teratomas in adolescents

AbstractIn adults, both immature and mature ovarian teratomas show frequent genetic homozygosity consistent with tumorigenesis involving germ cells after meiosis I. Investigation into genetic zygosity of various teratomas in children has been limited. Thirteen sacrococcygeal, 12 ovarian, and 3 testicular teratomas in children 18 years or younger were retrieved from our departmental archives and histologically reviewed. Tumor and paired normal tissues were microdissected and subjected to short tandem repeat (STR) genotyping. DNA genotyping was informative in 12 sacrococcygeal teratomas, 8 ovarian teratomas, and 3 testicular teratomas. Sacrococcygeal teratomas included seven mature teratomas, four immature teratomas, and one mixed germ cell tumor with patient age ranging from 0 days to 3 years. All but two patients were female. Ovarian teratomas included five mature and three immature teratomas with patient age ranging from 2 to 18 years. Testicular teratomas included two mature teratomas and one immature teratoma with patient age ranging from 3 months to 3 years. All sacrococcygeal, testicular, and ovarian teratomas in patients younger than 4 years showed no evidence of genetic homozygosity by STR genotyping. In contrast, all four ovarian teratomas in patients older than 9 years showed either partial or complete homozygosity. In conclusion, unlike adolescent and adult ovarian teratomas, prepubertal sacrococcygeal and gonadal teratomas lack genetic homozygosity, supporting the hypothesis that teratomas before puberty develop at an early stage of germ cell development different from that of teratomas in adolescents and adults.To investigate genetic homozygosity in pediatric teratomas, this study analyses 13 sacrococcygeal, 12 ovarian, and 3 testicular teratomas in children by short tandem repeat (STR) genotyping. While genetic homozygosity is frequent in adult ovarian teratomas, no evidence of genetic homozygosity is seen in patients younger than 4 years, arguing pediatric teratomas arise at an earlier stage of germ cell development.

Genetic analysis of 12 X-short tandem repeats loci in a northern Thai population.

Short tandem repeats (STRs) are widely used as DNA markers in paternity testing and criminal investigations because of their high genetic polymorphism among individuals in population. However, many factors influence genetic variations of STRs. Therefore, understanding STR information within individual populations could provide database and scientifically reliable STR genotyping for forensic genetic purposes. We aimed to examine allele frequencies of X-STRs, including some forensic parameters, in a northern Thai population. A retrospective descriptive study was conducted by collecting X-STR data from unrelated individuals living in a northern region of Thailand. The allele frequency and forensic parameters - for example polymorphism information content (PIC), power of discrimination in females and males (PDf and PDm), mean exclusion chance (MEC) and haplotype frequency - were calculated. The Hardy-Weinberg equilibrium was analysed. A total of 132 alleles were observed, with corresponding allele frequency ranging from 0.0064 to 0.4904. The PIC of all loci was >0.6, representing high genetic polymorphism, except DXS8378 and DXS7423. Notably, DXS10135 was the most diverse loci with the highest PD and MEC, while DXS7423 was the least polymorphic marker with the lowest PD and MEC. The highest haplotype diversity in male data was on linkage group III (DXS10101-DXS10103-HPRTB) by 0.9895. The genetic distance analysis demonstrated that the northern Thai population had a close relationship with Taiwanese (DA = 0.023). There are no significant deviations among the Hardy-Weinberg equilibrium except DXS10148. This study has established a northern Thai X-STRs reference database to be used as a tool for forensic genetic purposes.

Evaluation of the InnoTyper21® system for the applications into trace and degraded DNA in the Korean population.

The InnoTyper 21® Human Identification kit consists of amelogenin and 20 bi-allelic Alus, retrotransposon markers existing abundantly in human genome. The InnoTyper 21® kit produces shorter amplicons (60-125 bp) than conventional short tandem repeat (STR) genotyping kit, then it is effective on the analysis of challengeable forensic samples including insufficient or highly degraded DNAs. Also, as the genotyping with InnoTyper21® kit is compatible with PCR and capillary electrophoresis, it is easy to incorporate into the workflow in forensic laboratories. In the internal validation of InnoTyper21® kit on sensitivity, degradation, and mixture studies for the evaluation in this study, we acquired full profiles on analyzing small concentration DNA (as low as 25 pg) and highly degraded DNA (up to 105 degradation index value). Through the Korean population study, forensic statistical parameters were investigated and a specific variant of T insertion in NBC51 was confirmed in six samples. Comparison of Korean population with five populations or 1000 Genomes Project data show Korean specific substructure. It is expected that the InnoTyper 21® kit will be used into the actual forensic cases, utilizing the population study investigated through this research.

Kinship analysis on single cells after whole genome amplification

Short Tandem Repeat (STR-) and Single Nucleotide Polymorphism (SNP-) genotyping have been extensively studied within forensic kinship analysis. Nevertheless, no results have been reported on kinship analysis after whole genome amplification (WGA) of single cells. This WGA step is a necessary procedure in several applications, such as cell-based non-invasive prenatal testing (cbNIPT) and pre-implantation genetic diagnosis (PGD). In cbNIPT, all putative fetal cells must be discriminated from maternal cells after enrichment from whole blood. This study investigates the efficacy and evidential value of STR- and SNP-genotyping methods for the discrimination of 24 single cells after WGA, within three families. Formaldehyde-fixed and unfixed cells are assessed in offspring-parent duos and offspring-mother-father trios. Results demonstrate that both genotyping methods can be used in all tested conditions and scenarios with 100% sensitivity and 100% specificity, with a similar evidential value for fixed and unfixed cells. Moreover, sequence-based SNP-genotyping results in a higher evidential value than length-based STR-genotyping after WGA, which is not observed using high-quality offspring bulk DNA samples. Finally, it is also demonstrated that the availability of the DNA genotypes of both parents strongly increases the evidential value of the results.

Analysis of factors influencing the false-negative diagnosis of cervical/vaginal liquid based cytology

Objective: To investigate the possible influencing factors of false-negative diagnosis of cervical/vaginal liquid based cytology, and further improve the sensitivity of cervical/vaginal cytology. Methods: The results of cervical/vaginal cytology of outpatients and inpatients in Department of Obstetrics and Gynecology, Peking University First Hospital from July 2015 to December 2018 were analyzed retrospectively. Cytological false-negative cases were defined as the patients whose cytological results showed no intraepithelial neoplasia and malignant (NILM), but whose biopsy was diagnosed as cervical intraepithelial neoplasia (CIN) 2 or vaginal intraepithelial neoplasia (VAIN) 2 or above within 6 months of the diagnosis. The review of false-negative cytology smear was completed by two senior cytologists. Two-class logistic regression was used to evaluate the influence of age, location or number of lesion, and degree of lesion on the false-negative diagnosis of cytology. The reasons for the inconsistency of false-negative diagnosis were analyzed with the review results. Results: Among 1 009 cases of CIN2+ and VAIN2+ lesions, 180 cases (17.8%) showed NILM. After reviewing the smear, 123 cases (68.3%) were identified as NILM and 57 cases(31.7%) as abnormal. The false-negative rate was the highest (20.8%) in the patients with age≤30 years, and the risk was 8.85 times higher than the patients aged 31 to 60 years (P<0.001), 9.26 times than the patients aged≥60 years (P<0.001). The highest cytological false-negative rate was 50.0% for cervical polyps or intraductal lesions. The false-negative rate of vaginal wall or vaginal pedicle rupture was 13.0%; that of single cervical lesion was 22.3%; that of high-grade squamous intraepithelial lesion(HSIL) and adenocarcinoma in situ of cervix(AIS) was 13.7% and that of malignant lesions was 3.9%. The most common cell types in the reviewed abnormal cases were squamous cells in the middle surface layer (38.6%) and squamous cells in the outer bottom layer (24.6%). The abnormal cells in all smears was the most common distribution (59.7%), the number of abnormal cells in the smear was less than 10 (31.6%), nuclear enlargement and light staining were common (42.2%), and inflammatory lesions or keratotic changes in the background were most common (59.7%). Conclusions: Age of the patient, location or number of lesion, and degree of lesion are associated with false-negative diagnosis of cytology. Summarizing sampling experience and improving sampling skills will help reduce the occurrence of false-negative cases. Cytopathologists should examine slightly abnormal changes more carefully and learn how to further reduce the false-negative rate procactively.