Short Tandem Repeat Analysis Research Articles

Optimization and Validation of Candida auris Short Tandem Repeat Analysis.

ABSTRACTCandida auris is an easily transmissible yeast with resistance to different antifungal compounds. Outbreaks of C. auris are mostly observed in intensive care units. To take adequate measures during an outbreak, it is essential to understand the transmission route, which requires isolate genotyping. In 2019, a short tandem repeat (STR) genotyping analysis was developed for C. auris. To determine the discriminatory power of this method, we performed STR analysis of 171 isolates with known whole-genome sequencing (WGS) data using Illumina reads, and we compared their resolutions. We found that STR analysis separated the 171 isolates into four clades (clades I to IV), as was also seen with WGS analysis. Then, to improve the separation of isolates in clade IV, the STR assay was optimized by the addition of 2 STR markers. With this improved STR assay, a total of 32 different genotypes were identified, while all isolates with differences of >50 single-nucleotide polymorphisms (SNPs) were separated by at least 1 STR marker. Altogether, we optimized and validated the C. auris STR panel for clades I to IV and established its discriminatory power, compared to WGS SNP analysis using Illumina reads.IMPORTANCE The emerging fungal pathogen Candida auris poses a threat to public health, mainly causing outbreaks in intensive care units. Genotyping is essential for investigating potential outbreaks and preventing further spread. Previously, we developed a STR genotyping scheme for rapid and high-resolution genotyping, and WGS SNP outcomes for some isolates were compared to STR data. Here, we compared WGS SNP and STR outcomes for a larger sample cohort. Also, we optimized the resolution of this typing scheme with the addition of 2 STR markers. Altogether, we validated and optimized this rapid, reliable, and high-resolution typing scheme for C. auris.

ABCL-371 STR Loci Allele Imbalance at 9p24.1 in Primary Mediastinal B-Cell Lymphoma

Molecular factors that determine the prognosis of immunotherapy response in primary mediastinal B-cell lymphoma (PMBCL) are not yet described. PD-L1/L2 expression levels in tumor samples have demonstrated clinical utility for many solid tumors. PD-L1/L2 expression levels could be affected by chromosomal lesions on 9p24.1 (losses or gains). Short tandem repeat (STR) analysis of appropriate markers is the simplest way to detect these lesions. To analyze possible associations between STR loci allele imbalance (AI) on 9p24.1 and PD-L1 expression levels in tumor cells from PMBCL patients. Two STR Loci (gt) n and (ttat)m on 9p24.1 in tumor cells were analyzed for a cohort of 36 PMBCL patients admitted to the National Medical Research Center for Hematology (Moscow, Russia) from 2013 to 2022. For six patients, STR data were compared with PD-L1 expression assessed by immunohistochemistry. DNA was isolated from tumor biopsy samples taken at diagnosis. Control DNA samples were taken from the blood of patients in complete remission or from the buccal epithelium. STR analysis for AI was performed by PCR with original primers. The fragment analysis was performed on an ABI3130 Genetic Analyzer. Inclusion criteria: de novo diagnosed PMBCL patients. pretreatment. The heterozygosity level for the (gt)n locus was 31 of 36 patients (86%), and AI was found in 16 of 31 (51.6%). The heterozygosity level for the (ttat)m locus was 20 of 36 patients (56%), and AI was found in 9 of 20 (45%). One patient was homozygous for both loci. No patients were found with AI at one locus and a normal heterozygous variant at the second locus. PD-L1 expression in tumor cells was evaluated for six patients. In two patients with aberrant STR loci, PD-L1 expression was found in 75%-100% of tumor cells. None of the four patients with normal 9p24.1 STR markers had PD-L1-positive cells. The high incidence of AI in the 9p24.1 locus indicates its possible involvement in the pathogenesis of PMBCL. The association between AI and PD-L1 expression in tumor cells requires confirmation in an extended cohort of patients. This study was supported by Rakfond grant 2/2020.

Calculation and implementation of sample-wide stochastic thresholds for forensic genetic analysis of STRs and SNPs for massively parallel sequencing platforms

Capillary electrophoresis (CE) analysis of short tandem repeats (STRs) and single nucleotide polymorphisms (SNPs) use a stochastic threshold to consider the possibility of missing alleles (dropouts) or detecting additional alleles (drop-ins). In CE, this threshold may be approximately 200 RFU, and peak heights are assessed relative to this threshold. In next generation sequencing (NGS), also known as massively parallel sequencing (MPS), STRs are identified by their sequence, and specific alleles are identified by their repeat number and intra-allelic variation. Abundance is approximated by the number of sequence reads for each allele. The total number of reads generated for each marker in a sample depends on factors such as the numbers of samples pooled for sequencing, the number of markers in the assay, the integrity and quantity of the input DNA sample, and the inter-locus balance of the assay. For multiplexes that contain both autosomal and sex-linked markers, the biological sex of the sample also influences total reads per locus. To normalize these variables and better establish a robust stochastic threshold, a sample-wide metric is proposed for estimating the possibility of dropouts or drop-ins based on the variance of the inter-locus balance of the markers across a sample. The intuition is that samples with variable allele balance globally are more likely to have noisier data and therefore require more stringent read count thresholds. This method is robust to sequencing multiplexity, biological sex and manufacturing lot variation.

Establishment and characterization of a new Chinese hepatocellular carcinoma cell line, Hep-X1.

Hepatocellular carcinoma (HCC) is a highly aggressive and heterogeneous disease. Cell lines are commonly employed as in vitro models for cell type studies. However, the success rate of HCC primary culture establishment is low. In this study, we successfully established a liver cancer cell line, Hep-X1. Primary culture and passage of surgically removed tissues were used to establish hepatoma cell lines. Morphological examination, short tandem repeat (STR) analysis, immunohistochemical staining, doubling time, karyotype analysis, plate tumor formation experiments, organoid culture, and in vivo tumor formation investigations in animals were used to identify the cell lines. A novel liver cancer cell line, Hep-X1, was established based on morphology, immunophenotype, cytogenetics, and STR analysis. The novel cell line can be a valuable model for studying primary liver cancer.

Development of a multiplex assay for detection of autosomal and Y-chromosomal STRs, assessment of the degradation state of mitochondrial DNA and presence of mitochondrial length heteroplasmies

The current focus in most routine forensic casework is detection of autosomal or gonosomal Short Tandem Repeats (STRs). With increasing degradation, STR analysis tends to be less successful up to complete failure. For challenging samples such as telogen hair roots and shafts, touch DNA samples or skeletal remains, mitochondrial DNA (mtDNA) analysis provides a powerful tool. Determination of DNA quantity is an important part in the casework workflow. Several ready-to-use kits are commercially available for nuclear DNA targets. However, quantification of mtDNA targets requires the establishment of an in-house method. Some assays even contain assessment of degradation, which alleviates the choice of target enrichment for sequencing through medium or small amplicons. As Sanger-type Sequencing (STS) still remains the golden standard in many laboratories, identification of heteroplasmies in C-tract regions prior to the sequencing reaction is advantageous. Firstly, primer selection can be expanded with primers binding near the C-tract and secondly, determination of the dominant variant is straightforward. All those quantity (nuclear and mtDNA) and quality (degradation and length heteroplasmies) evaluations usually require at least two separate reactions. Therefore, the aim of this project was the combination of all these targets in one multiplex assay using capillary electrophoresis to spare valuable sample extract. Amplification of representative autosomal and Y-chromosomal STRs allows estimate of success of (Y-)STR analysis. Simultaneously, five length heteroplasmies in the mitochondrial control region are targeted as well as three conservative regions of differing fragment lengths for assessment of the mitochondrial degradation state. Based on the outcome of this assay, forensic examiners can decide if STR analysis may be suitable. In case of absent STR peaks, appropriate proceeding of mtDNA sequencing can be determined.

Establishment of a co-analysis system for personal identification and body fluid identification: a preliminary report.

Analysis of genetic markers can provide clues for case investigation. Short tandem repeat (STR) detection and analysis are widely used for both personal identification and parentage testing. However, DNA analysis currently cannot provide sufficient information for body fluid identification. Tissue or cell sources of samples can be identified by detecting body fluid-specific mRNA markers, which have been studied thoroughly. Integrating STR profiling and mRNA expression patterns can provide more information than conventional methods for investigations and the reconstruction of crime scenes; this can be achieved by DNA/RNA co-extraction technology, which is economical, efficient, and suitable for low-template samples. Here, we propose a co-analysis system based on the PowerPlex 16 kit. This system can simultaneously amplify 25 markers, including 15 STRs, one non-STR amelogenin, and nine mRNA markers (three blood-specific, two saliva-specific, two semen-specific, and two housekeeping gene markers). The specificity and sensitivity of the co-analysis system were determined and aged and degraded samples were used to validate the stability of the co-analysis system. Finally, different DNA/RNA ratios and various carriers were evaluated. The results showed that the DNA/RNA co-analysis system correctly identified different types of body fluid stains. The STR profiles obtained using the co-analysis system were identical to those obtained using the PP16 kit, which demonstrates that the mRNA primers used did not affect STR profiling. Complete STR and mRNA profiles could be obtained from 1/8 portions of buccal swabs, 1/16 portions of swabs of blood and semen samples, 0.1 cm2 of blood samples, 0.25 cm2 of semen samples, and 1.0 cm2 saliva samples. Additionally, our findings indicate that complete STR and mRNA profiles can be obtained with this system from blood and semen samples when the DNA/RNA ratio is 1:1/32. This study suggests that the co-analysis system could be used for simultaneous personal identification and body fluid identification.

Genetic analysis of X-chromosomal short tandem repeat (X-STR) frequencies in Arab Iraqi male population

BackgroundThe X-chromosome short tandem repeat (STR) polymorphisms are a particular tool in the fields of human population genetics and personal identification. It was necessary in investigating complex kinship or deficiency cases in conditions where information on mitochondrial DNA (mtDNA) or Y chromosome polymorphisms have been used to explore their direct paternal line. This study aimed to investigate the allele frequency of (12X-STR) of 200 unrelated males from different region of Baghdad City to serve as a reference data base for individual identification in Iraqi population. ResultsTwelve X-STR loci (DXS7424, HPRTB, DXS8377, GATA31E08, DXS7423, DXS8378, DXS9895, DXS10074, DXS6809, DXS7133, DXS101, DXS6807) were successfully amplified by multiplex PCR and divided into four groups. According to measures of allele frequency, the higher alleles frequency were 16, 11, 46, 11, 14, 10, 15, 15.2, 35, 11, 25, and 11 while the lowest alleles frequency were 11, 9, 52,53, 7, 17, 14, 13, 12.2,17, 36, 15, 16, 22, 29, and 17 that observed at the 12 loci respectively. Forensic efficiency parameter for DXS8377 locus in the first group showed highest polymorphic allele in the Iraqi Arab population with the frequencies ranging from 0.005 to 0.16%. The power of discrimination (PD) value ranged from 0.663 for DXS7423 locus and 0.9066 for DXS8377 locus. In addition, the polymorphism information content (PIC) value ranged from 0.602974 for DXS7423 locus to 0.899206 for DXS8377 locus. ConclusionsOverall the X-STR markers become used as an important source of information beside the autosomal and Y-STR markers, especially for kinship testing and haplotype analysis.

Investigation of Linear Amplification Using Abasic Site-Containing Primers Coupled to Routine STR Typing for LT-DNA Analysis.

Obtaining a full short tandem repeat (STR) profile from a low template DNA (LT-DNA) still presents a challenge for conventional methods due to significant stochastic effects and polymerase slippage. A novel amplification method with a lower cost and higher accuracy is required to improve the DNA amount. Previous studies suggested that DNA polymerases without bypass activity could not perform processive DNA synthesis beyond abasic sites in vitro and our results showed a lack of bypass activity for Phusion, Pfu and KAPA DNA polymerases in this study. Based on this feature, we developed a novel linear amplification method, termed Linear Aamplification for double-stranded DNA using primers with abasic sites near 3′ end (abLAFD), to limit the replication error. The amplification efficiency was evaluated by qPCR analysis with a result of approximately a 130-fold increase in target DNA. In a LT-DNA analysis, the abLAFD method can be employed as a pre-PCR. Similar to nested PCRs, primer sets used for the abLAFD method were designed as external primers suitable for commercial multiplex STR amplification assays. The practical performance of the abLAFD method was evaluated by coupling it to a routine PP21 STR analysis using 50 pg and 25 pg DNA. Compared to reference profiles, all abLAFD profiles showed significantly recovered alleles, increased average peak height and heterozygote balance with a comparable stutter ratio. Altogether, our results support the theory that the abLAFD method is a promising strategy coupled to STR typing for forensic LT-DNA analysis.

Y-STR Kits and Y-STR Diversity in the South African Population

The South African population consists of four ethnic groups, i.e., Blacks, Coloreds, Indians, and Whites, and is considered the most diverse conglomeration of humans. In addition to autosomal short tandem repeat (STR) variation, an important tool to study population diversity is Y-chromosome (Y)-STR analysis. Y-STRs aid in forensic investigations and provide essential data about paternal lineage origins. Y-STR kits consisting of an array of stable and rapidly mutating markers offer crucial information on a given population's genetic and haplotype diversity. This review discusses the development of Y-STR kits over the years and highlights some prominent Y-STR studies conducted on the South African population. The earliest Y-STR kit developed was the Y-PLEX™6, with the most recent being the UniQTyper™ Y-10 Multiplex. The South African population studies show varying data, with the “minimal haplotype” having low discrimination capacity among the ethnic groups and the UniQTyper™ Y-10 showing high genetic diversity among the ethnic groups of the country. There is a dearth of Y-STR studies on the South African population. With the advent of new Y-STR kits with increased discriminatory markers, additional studies are required to represent the South African population in the Y-STR databases. Considering the diversity of the South African population, establishment of a local/regional population database would be beneficial. In addition, data on the origins and prevalence of mutations and silent alleles should be obtained from STR datasets generated during kinship investigations (specifically, parentage tests) so that detailed information about the frequencies of mutations, silent alleles, and uniparental disomy in the South African population at Y STR loci can be estimated.

P-802 Distinct genetic impact of CRISPR/Cas9 gene correction in human embryos compared to induced pluripotent stem cells

Abstract Study question Does CRISPR/Cas9 gene editing have a distinct genetic impact in human embryos compared to induced pluripotent stem cells (iPSCs)? Summary answer IPSCs display a difference in repair events, such as template usage, compared to the germline which indicates a distinct genetic impact by CRISPR/Cas9 gene editing. What is known already CRISPR/Cas9 is utilized to induce targeted DNA editing. To introduce specific changes, such as mutational correction, a DNA template is generally administered, stimulating homology-directed repair. Recent human germline editing studies aiming for mutational repair created a moderate amount of embryos which solely carried wild-type alleles. Remarkably, no template use was observed, but instead loss-of-heterozygosity (LOH) was demonstrated. This is the presence of only one (the wild-type) allele, either caused by gene conversion events using the wild type allele or chromosome loss. Up to date, no iPSCs studies have evaluated LOH events when attempting to correct a heterozygous mutation. Study design, size, duration A guide RNA targeting the mutant allele, and a repair template containing the wild-type allele and a synonymous variant to track template usage were designed. For iPSCs targeting, the components were nucleofected after which DNA was extracted from the whole well (n = 3) or from individual colonies (n = 33). For human embryo targeting, the components were injected simultaneously with sperm into donated spare oocytes (n = 32). DNA was extracted from embryos after 3-6 days of in vitro culture. Participants/materials, setting, methods Sperm and renal cells, from which the iPSCs were derived, were donated by a patient with a heterozygous base pair substitution in PLCZ1 (c.136-1G>C) causing fertilization failure. To overcome fertilization failure, assisted oocyte activation was employed during ICSI. For the embryos, next-generation sequencing (NGS), short tandem repeat (STR) analysis and a whole genome single nucleotide polymorphism (SNP) assay were performed. For the iPSCs, NGS and a targeted SNP assay were carried out. Main results and the role of chance The genetic events in embryos originating from mutant sperm (n = 32) displayed following distribution: 19% showed the untargeted mutant allele, 56% showed additional mutagenesis and 25% showed only the wild-type allele. In the latter group, the template was never utilized, pointing to LOH. STR analysis indeed revealed LOH events of different lengths in these embryos. SNP analysis of one embryo, originating from mutant sperm but displaying the wild-type allele, did not demonstrate LOH (with a detection limit of 500bp). These findings suggest the occurrence of gene conversion, which rejects the formerly stated hypothesis that the observed LOH in human embryos can mostly be attributed to chromosome loss. In the iPSCs, whole-well (n = 3) and single colony (n = 33) data demonstrated a similar trend in genetic event distribution compared to the embryos, with the main difference that in 30% of the corrected reads/colonies, template use was observed. LOH was further analysed in nineteen single colonies. In all the colonies (3/3) corrected with the template, no LOH was present. When the colonies displayed additional mutagenesis, 7% (1/13) contained LOH events. In 33% (1/3) of the colonies showing only the wild-type allele (without signs of template use), LOH was observed. Limitations, reasons for caution SNP assays have a higher resolution compared to STR analysis, and enable distinction of LOH events between ‘gene conversion’ and ‘chromosome loss’. Therefore, more embryos and iPSC colonies will be analysed with SNP assays. However, informative SNPs define the resolution of our assay (currently 500 bp from Cas9 cut site). Wider implications of the findings Our results show that template usage seems to differ between human embryos and iPSCs, but that iPSCs do not solely rely on template use as well. This demonstrates that embryo experiments to study the genetic impact of CRISPR/Cas9 cannot fully be replaced by iPSCs experiments. Trial registration number Not Applicable

Abstract 3112: Metabolic hallmarks of rare renal cell carcinoma patient-derived xenograft models

Abstract Renal medullary carcinoma (RMC) and fumarate hydratase (FH)-deficient renal cell carcinoma (RCC) are rare but highly aggressive malignancies that are often refractory to the therapies used for the more common RCCs. FH-deficient RCC most often occurs in individuals with hereditary leiomyomatosis and renal cell carcinoma syndrome (HLRCC) and is characterized by loss of FH activity, a key metabolic enzyme. Bevacizumab plus erlotinib (B+E) is a preferred first-line therapy for FH-deficient RCC. RMC is characterized by loss of SMARCB1, which is involved in ATP-dependent chromatin remodeling. Platinum-based chemotherapy consisting of either cisplatin or carboplatin plus paclitaxel is the preferred first-line therapy for RMC. We developed patient-derived xenograft (PDX) models of tumors exposed to the commonly used first-line therapies to allow functional characterization of the metabolic hallmarks of treatment-experienced RMC and FH-deficient RCC. Methods: Resected tumor tissue was immediately implanted into NSG male and female mice. After tumor engraftment, animals were euthanized, and the tumor tissue implanted into another set of NSG mice. PDX tissues were confirmed to be genetically identical to the original patient tissue by short tandem repeat analysis. Histology was confirmed to be identical between original patient tumor and PDX by a trained clinical pathologist. Four tumors from each PDX model and four normal human kidney tissue samples were analyzed using reverse phase protein array (RPPA). Expression of proteins shown to be highly expressed by RPPA were further validated by Western blots. Results: We successfully generated one PDX model for RMC and one for FH-deficient RCC, respectively. The RMC model was generated from a 28-year old male with sickle cell trait, who had previously received cisplatin plus paclitaxel followed carboplatin plus paclitaxel. His lesion was characterized as ypT3apN1pMx and histology was consistent with RMC. The FH-deficient RCC model was generated from a 24-year-old female with HLRCC, who had been previously treated with B+E. Her lesion was characterized as pT3apN1pM1 and histology consistent with FH-deficient RCC. Germline testing revealed a R233H pathogenic mutation in the FH gene. RPPA revealed higher expression of hexokinase II (10x HLRCC, 16x RMC), lactate dehydrogenase A (27x HLRCC, 17x RMC), and glutaminase (4x HLRCC) in PDX tissue compared to normal kidney tissue. Western blots confirmed higher expression of glutaminase 1 in both HLRCC and RMC compared to normal kidney. Furthermore, we observed substantially higher hypoxia inducible factor 2α (HIF2α) protein expression in PDX tissue from FH-deficient RCC compared to RMC PDX tumors and normal kidney. Conclusions: Our study revealed distinct metabolic features in PDX models of FH-deficient RCC following treatment with B+E, and of platinum-experienced RMC, these differences may confer targetable vulnerabilities. Citation Format: Manuel Ozambela, Alberto Pieretti, Graciela M. Nogueras Gonzalez, Tapati Maity, Lei Wang, Carolyn De La Cerda, Breanna Alonzo, Luis Segarra, Angelita Alaniz, Priya Rao, Nizar Tannir, Jose A. Karam, Christopher G. Wood, Pavlos Msaouel, Niki M. Zacharias. Metabolic hallmarks of rare renal cell carcinoma patient-derived xenograft models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3112.

Abstract 207: MET inhibition enhances the effect of radiation in MET mutated non-small cell lung cancer brain metastasis patient derived xenografts

Abstract Objective: The MET receptor is mutated in 3-4% and amplified in 1-6% of patients with non-small cell lung cancer. The most common MET mutation is in exon 14, which results in deletion of the intracellular juxtamembrane domain of the receptor, leading to enhanced signaling. Mutation at Asp-1000 in the juxtamembrane region has also been reported and can lead to inhibition of apoptosis and cell proliferation. MET is involved in multiple pathways associated with radiation response. Therefore, we investigated the effects of inhibiting MET in combination with radiation in two preclinical non-small cell lung cancer (NSCLC) brain metastasis patient derived xenograft (PDX) models. Methods: Surgically obtained tissue was implanted subcutaneously into immunodeficient mice. Histology and DNA loci were compared between original tumor and PDX. DNA sequencing was performed on tumors for mutation analysis. In vivo growth responses to the MET inhibitor capmatinib or savolitinib with and without radiation were assessed. Radiation was delivered in 10 daily fractions of 2 Gy. Drug was administered by oral gavage daily 1 hour prior to radiation at a dose of 2.5 mg/kg for savaolitnib or 20 mg/kg for capmatinib for 10 days. Immunohistochemistry (IHC) was performed to evaluate MET signaling and proliferation. Results: PDXs were successfully established from two patients with MET mutated NSCLC brain metastases: a lung adenocarcinoma with a MET exon 14 skipping mutation and a lung sarcomatoid carcinoma with adenocarcinoma component with an Asp-1000 frameshift mutation. Morphologically, strong retention of cytoarchitectural features was observed between original patient tumors and PDXs. Short tandem repeat analysis confirmed 100% matching of alleles between patient tumors and PDXs. DNA sequencing confirmed METex14 and MET Asp-1000 frameshift mutation. In the METex14 PDX, savolitinib alone significantly inhibited tumor growth with a growth inhibition value of 46% (p<0.01). Combination of savolitinib and radiation significantly delayed growth compared to vehicle control, savolitinib alone or radiation alone (p<0.001). The absolute growth delay was 42.4 days for savolitinib plus radiation treatment compared to 9.3 days for savolitinib alone and 28.2 days for irradiation. In the MET Asp-1000 PDX, capmatinib and radiation significantly delayed growth compared the other treatment arms (p<0.01). IHC demonstrated inhibition of phospho-MET and pS6 and a decrease in Ki67 with MET inhibition. Conclusion: Inhibition of MET enhanced the effect of radiation in our two preclinical in vivo MET mutated NSCLC brain metastasis models. Additional studies are currently underway evaluating the mechanisms of radiation sensitization and the efficacy of this combination in MET amplified PDX models. Citation Format: Kwangok P. Nickel, Shrey Ramesh, Saahil Javeri, Nitin Somasundaram, Nan Sethakorn, Randall J. Kimple, Andrew M. Baschnagel. MET inhibition enhances the effect of radiation in MET mutated non-small cell lung cancer brain metastasis patient derived xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 207.

Genetic Analysis of TPOX, CSF1PO, D3S1358, D8S1179, vWA, D5S818, and TH01 Short Tandem Repeats Loci in Nias Population, Indonesia

BACKGROUND: Nias is an island located off the western coast of Sumatra, Indonesia. Nias is situated above the Eurasian and Indo-Australian subduction zone plates. This makes it prone to earthquakes and tsunamis. Genetic analysis and genetic variation of short tandem repeats (STR) locus are not widely known. These data are valuable for individual identification and paternity testing. METHODS: Seven STR loci (TPOX, CSF1PO, D3S1358, D8S1179, vWA, D5S818, and TH01) were analyzed using 25 healthy and unrelated persons Nias population. Allele frequency, power of discrimination (PD), expected heterozygosity, and probability of exclusion (PE) were calculated. RESULTS: We found 40 alleles. The allele with highest frequency was alleles 9 at the TH01 loci. While the lowest frequency were allele 9 at the CSF1PO loci, allele 12 at the TPOX loci, alleles 17 and 18 at the D8S1179 loci, and alleles 16 and 20 at the vWA loci. The highest Expected Heterozygosity, PD, and PE at the D8S1179 loci. The highest number of alleles is also at D8S1179 loci. All loci followed the Hardy–Weinberg equilibrium (p > 0.05). The PD values for all tested loci ranged from 80.6 to 94.5%. CONCLUSION: We report the allele frequencies and forensic statistical parameters of seven STR loci (TPOX, CSF1PO, D3S1358, D8S1179, vWA, D5S818, and TH01) in the Nias population, which can be used as a forensic database reference for Nias populations.

Aberrant hypomethylation at imprinted differentially methylated regions is involved in biparental placental mesenchymal dysplasia

BackgroundPlacental mesenchymal dysplasia (PMD) is a morphological abnormality resembling partial hydatidiform moles. It is often associated with androgenetic/biparental mosaicism (ABM) and complicated by Beckwith–Wiedemann syndrome (BWS), an imprinting disorder. These phenomena suggest an association between PMD and aberrant genomic imprinting, particularly of CDKN1C and IGF2. The existence of another type of PMD containing the biparental genome has been reported. However, the frequency and etiology of biparental PMD are not yet fully understood.ResultsWe examined 44 placental specimens from 26 patients with PMD: 19 of these were macroscopically normal and 25 exhibited macroscopic PMD. Genotyping by DNA microarray or short tandem repeat analysis revealed that approximately 35% of the macroscopic PMD specimens could be classified as biparental, while the remainder were ABM. We performed a DNA methylation analysis using bisulfite pyrosequencing of 15 placenta-specific imprinted differentially methylated regions (DMRs) and 36 ubiquitous imprinted DMRs. As expected, most DMRs in the macroscopic PMD specimens with ABM exhibited the paternal epigenotype. Importantly, the biparental macroscopic PMD specimens exhibited frequent aberrant hypomethylation at seven of the placenta-specific DMRs. Allelic expression analysis using single-nucleotide polymorphisms revealed that five imprinted genes associated with these aberrantly hypomethylated DMRs were biallelically expressed. Frequent aberrant hypomethylation was observed at five ubiquitous DMRs, including GRB10 but not ICR2 or ICR1, which regulate the expression of CDKN1C and IGF2, respectively. Whole-exome sequencing performed on four biparental macroscopic PMD specimens did not reveal any pathological genetic abnormalities. Clinical and molecular analyses of babies born from pregnancies with PMD revealed four cases with BWS, each exhibiting different molecular characteristics, and those between BWS and PMD specimens were not always the same.ConclusionThese data clarify the prevalence of biparental PMD and ABM-PMD and strongly implicate hypomethylation of DMRs in the pathogenesis of biparental PMD, particularly placenta-specific DMRs and the ubiquitous GRB10, but not ICR2 or ICR1. Aberrant hypomethylation of DMRs was partial, indicating that it occurs after fertilization. PMD is an imprinting disorder, and it may be a missing link between imprinting disorders and placental disorders incompatible with life, such as complete hydatidiform moles and partial hydatidiform moles.

Profiling the Genome-Wide Landscape of Short Tandem Repeats by Long-Read Sequencing.

Background: Short tandem repeats (STRs) are highly variable elements that play a pivotal role in multiple genetic diseases and the regulation of gene expression. Long-read sequencing (LRS) offers a potential solution to genome-wide STR analysis. However, characterizing STRs in human genomes using LRS on a large population scale has not been reported. Methods: We conducted the large LRS-based STR analysis in 193 unrelated samples of the Chinese population and performed genome-wide profiling of STR variation in the human genome. The repeat dynamic index (RDI) was introduced to evaluate the variability of STR. We sourced the expression data from the Genotype-Tissue Expression to explore the tissue specificity of highly variable STRs related genes across tissues. Enrichment analyses were also conducted to identify potential functional roles of the high variable STRs. Results: This study reports the large-scale analysis of human STR variation by LRS and offers a reference STR database based on the LRS dataset. We found that the disease-associated STRs (dSTRs) and STRs associated with the expression of nearby genes (eSTRs) were highly variable in the general population. Moreover, tissue-specific expression analysis showed that those highly variable STRs related genes presented the highest expression level in brain tissues, and enrichment pathways analysis found those STRs are involved in synaptic function-related pathways. Conclusion: Our study profiled the genome-wide landscape of STR using LRS and highlighted the highly variable STRs in the human genome, which provide a valuable resource for studying the role of STRs in human disease and complex traits.

New and effective method to develop primary hepatocytes from liver cancer patients

Liver cancer (LC) is one of the most common malignant tumors worldwide. Since the mechanism of LC pathogenesis and metastasis cannot be carried out directly on the human body, it is particularly important to establish human liver cancer cell lines for research in vitro. In this study, tissue block adherence method combined with cell clumps digestion method was used to establish primary human hepatocytes (PHHs) with a successful rate of 60% (45/75). Short tandem repeat (STR) analysis proved the cells were derived from its paired tissues. These cells from hepatocellular carcinoma (HCC) expressed NTCP and secreted ALB and AAT as detected by western blot, and expressed hepatocyte-specific membrane protein ASGR1 as detected by flow cytometry. Liver cancer biomarkers like CK7 in ICC (intrahepatic cholangiocarcinoma), AFP, and GPC3 in HCC expressed of different degree as detected by immunohistochemical analysis. These cells displayed typical liver cancer cell morphological characteristics and can passage stably. In conclusion, we developed an effective method to establish PHHs. Further studies are necessary to study if these cells maintaining other liver function and reproduce the physiology of the tumors and how these cells behavior in the drug development.

Measuring the process and rate of exogenous DNA degradation during digestion in mice

This study aimed to perform qualitative and quantitative examination of DNA degradation during the digestion process in the mouse gut through PCR, qPCR and short tandem repeat (STR) analysis. Human blood leukocytes were gavaged into the digestive tract in mice. GAPDH, TH01, TPOX and D7S820 genes in the contents of the stomach and small intestine were analyzed with PCR and qPCR at various times pre- and post-gavage. Through STR analysis, 21 human genomic DNA loci were analyzed. The half-life of DNA degradation, and the relationship between the average peak area and digestion time were determined. The PCR results showed bands of amplified genes at pre-gavage (0 min) and post-gavage (40, 80 and 120 min) from the mouse stomach contents, whereas no DNA bands from small intestinal chyme were observed after gavage. The qPCR results revealed a significant decrease in DNA concentrations during 40–120 min in the mouse stomach after gavage. At 120 min, 85.62 ± 8.10% of the DNA was degraded, and the half-life of exogenous DNA degradation in the mouse stomach was 70.50 ± 5.46 min. At various digestion times, almost no target genes were detected in the mouse small intestinal chyme. STR analysis showed a decrease in allele numbers with bowel advancement in the small intestine in mice. The degradation of exogenous DNA was higher in the mouse stomach during the first 2 h, and almost complete degradation was observed within 40 min after entering the small intestine in mice.

A Novel External Auditory Canal Squamous Cell Carcinoma Cell Line Sensitive to CDK4/6 Inhibition.

To characterize cell line CAE606 derived from a squamous cell carcinoma (SCC) of the external auditory canal (EAC) and to show its usefulness as a model for testing candidate therapeutic agents. Preclinical translational research. Biomedical research institute. The cell line was initiated from a moderately differentiated T2N0M0 EAC SCC. We studied its histologic and genetic features as well as growth and invasion parameters. Sensitivity to cell CDK4/6 cell cycle inhibitor palbociclib was analyzed. CAE606 cells expressed heavy molecular weight cytokeratin, p63, and vimentin. The population doubling time was 25.8 hours, and the cells showed fast collective cell migration in a wound-healing assay. Short tandem repeat analysis confirmed it to be derived from the primary tumor of the patient. Next-generation sequencing revealed alterations in cell cycle regulation genes, including inactivating mutations in CDKN2A and TP53 and high-level amplification of CCND1 and EGFR. CAE606 showed a strong decrease of phospo-Rb expression upon exposure to the CDK4/6 inhibitor palbociclib, causing significant growth inhibition with an IC50 of 0.46 µM. This is the first report of a stable EAC SCC cell line. Its genetic features make it a useful tool for preclinical testing of new therapeutic agents for EAC SCC, particularly those targeting cell cycle regulation in combination with radio- and chemotherapy or other specific signaling pathway inhibitors.

Validation and beyond: Next generation sequencing of forensic casework samples including challenging tissue samples from altered human corpses using the MiSeq FGx system.

The proceeding developments in next generation sequencing (NGS) technologies enable increasing discrimination power for short tandem repeat (STR) analyses and provide new possibilities for human identification. Therefore, the growing relevance and demand in forensic casework display the need for reliable validation studies and experiences with challenging DNA samples. The presented validation of the MiSeq FGx system and the ForenSeq™ DNA Signature Prep Kit (1) investigated sensitivity, repeatability, reproducibility, concordance, pooling variations, DNA extraction method variances, DNA mixtures, degraded, and casework samples and (2) optimized the sequencing workflow for challenging samples from human corpses by testing additional PCR purification, pooling adjustments, and adapter volume reductions. Overall results indicate the system's reliability in concordance to traditional capillary electrophoresis (CE)‐based genotyping and reproducibility of sequencing data. Genotyping success rates of 100% were obtained down to 62.5 pg DNA input concentrations. Autosomal STR (aSTR) profiles of artificially degraded samples revealed significantly lower numbers of locus and allelic dropouts than CE. However, it was observed that the system still exposed drawbacks when sequencing highly degraded and inhibited samples from human remains. Due to the lack of studies evaluating the sequencing success of samples from decomposed or skeletonised corpses, the presented optimisation studies provide valuable recommendations such as an additional PCR purification, an increase in library pooling volumes, and a reduction of adapter volumes for samples with concentrations ≥31.2 pg. Thus, this research highlights the importance of all‐encompassing validation studies for implementing novel technologies in forensic casework and presents recommendations for challenging samples.

Investigation of X-STR haplotype diversity in the Australian Aboriginal population

ABSTRACT X-chromosome short tandem repeat (X-STR) analysis complements autosomal and Y-chromosome STR typing for complex kinship analysis and screening of candidate lists in familial searches. To statistically evaluate X-STR profiling results, Australian forensic laboratories require X-STR haplotype frequency databases for relevant populations. Our study is the first to report haplotype data for the Australian Aboriginal population. X-STR haplotypes were generated for 298 self-declared male Aboriginals using the Investigator® Argus X-12 Kit. Haplotype frequencies and parameters of forensic interest are presented. The discrimination power of a full haplotype was 98.7% and the cumulative power of discrimination was 0.9999999988 for males and 0.999999999999999 for females. MDS plots of pairwise FST genetic distances and STRUCTURE histograms identified some segregation of X-STR haplotypes between individuals from the Desert and Eyre regions as compared to the Spencer, Riverine and urban Adelaide regions. Comparison of Australian Aboriginal X-STR haplotypes to those of other global populations showed the most similarity to Europeans and least similarity with Asian populations. Ancestral X-STR haplotypes appeared most conserved in the outback Desert and Eyre regions where assimilation with Europeans is lower. Overall genetic structure was minimal and a single X-STR haplotype database combining all regions is considered appropriate for likelihood ratio calculation.