MFUM-BrTNBC-1, a Newly Established Patient-Derived Triple-Negative Breast Cancer Cell Line: Molecular Characterisation, Genetic Stability, and Comprehensive Comparison with Commercial Breast Cancer Cell Lines

Triple-negative breast cancer (TNBC) is a breast cancer (BC) subtype that accounts for approximately 15–20% of all BC cases. Cancer cell lines (CLs) provide an efficient way to model the disease. We have recently isolated a patient-derived triple-negative BC CL MFUM-BrTNBC-1 and performed a detailed morphological and molecular characterisation and a comprehensive comparison with three commercial BC CLs (MCF-7, MDA-MB-231, MDA-MB-453). Light and fluorescence microscopy were used for morphological studies; immunocytochemical staining for hormone receptor, p53 and Ki67 status; RNA sequencing, qRT-PCR and STR analysis for molecular characterisation; and biomedical image analysis for comparative phenotypical analysis. The patient tissue-derived MFUM-BrTNBC-1 maintained the primary triple-negative receptor status. STR analysis showed a stable and unique STR profile up to the 6th passage. MFUM-BrTNBC-1 expressed EMT transition markers and displayed changes in several cancer-related pathways (MAPK, Wnt and PI3K signalling; nucleotide excision repair; and SWI/SNF chromatin remodelling). Morphologically, MFUM-BrTNBC-1 differed from the commercial TNBC CL MDA-MB-231. The advantages of MFUM-BrTNBC-1 are its isolation from a primary tumour, rather than a metastatic site; good growth characteristics; phenotype identical to primary tissue; complete records of origin; a unique identifier; complete, unique STR profile; quantifiable morphological properties; and genetic stability up to (at least) the 6th passage.

Individual Identification with Short Tandem Repeat Analysis and Collection of Secondary Information Using Microbiome Analysis

Forensic investigation is important to analyze evidence and facilitate the search for key individuals, such as suspects and victims in a criminal case. The forensic use of genomic DNA has increased with the development of DNA sequencing technology, thereby enabling additional analysis during criminal investigations when additional legal evidence is required. In this study, we used next-generation sequencing to facilitate the generation of complementary data in order to analyze human evidence obtained through short tandem repeat (STR) analysis. We examined the applicability and potential of analyzing microbial genome communities. Microbiological supplementation information was confirmed for two of four failed STR samples. Additionally, the accuracy of the gargle sample was confirmed to be as high as 100% and was highly likely to be classified as a body fluid sample. Our experimental method confirmed that anthropological and microbiological evidence can be obtained by performing two experiments with one extraction. We discuss the advantages and disadvantages of using these techniques, explore prospects in the forensic field, and highlight suggestions for future research.

Sequence variations, flanking region mutations, and allele frequency at 31 autosomal STRs in the central Indian population by next generation sequencing (NGS)

Capillary electrophoresis-based analysis does not reflect the exact allele number variation at the STR loci due to the non-availability of the data on sequence variation in the repeat region and the SNPs in flanking regions. Herein, this study reports the length-based and sequence-based allelic data of 138 central Indian individuals at 31 autosomal STR loci by NGS. The sequence data at each allele was compared to the reference hg19 sequence. The length-based allelic results were found in concordance with the CE-based results. 20 out of 31 autosomal STR loci showed an increase in the number of alleles by the presence of sequence variation and/or SNPs in the flanking regions. The highest gain in the heterozygosity and allele numbers was observed in D5S2800, D1S1656, D16S539, D5S818, and vWA. rs25768 (A/G) at D5S818 was found to be the most frequent SNP in the studied population. Allele no. 15 of D3S1358, allele no. 19 of D2S1338, and allele no. 22 of D12S391 showed 5 isoalleles each with the same size and with different intervening sequences. Length-based determination of the alleles showed Penta E to be the most useful marker in the central Indian population among 31 STRs studied; however, sequence-based analysis advocated D2S1338 to be the most useful marker in terms of various forensic parameters. Population genetics analysis showed a shared genetic ancestry of the studied population with other Indian populations. This first-ever study to the best of our knowledge on sequence-based STR analysis in the central Indian population is expected to prove the use of NGS in forensic case-work and in forensic DNA laboratories.

Investigation of Mosaicism Detected in STR Typing

Short tandem repeats (STRs) are the most popular markers for human identification in forensics. These markers can be easily analyzed through a multiplex polymerase chain reaction and electrophoresis and provide high discrimination power. However, in STR analysis, several atypical phenomena can be observed such as allelic dropouts, drop-ins, or imbalance, which may be due to DNA polymerase slippage or DNA degradation effects. The observed atypical STR profiles can also provide information for mixed DNA samples or chromosomal abnormalities. In this study, we report a case of mosaicism detected in routine casework of paternity testing. Hair samples from a phenotypically normal male were tested, and the result presented a typical STR profile of a female for the amelogenin gene (XX). Through chromosome analysis using peripheral blood, it was found that 45,X/46,XY mosaicism resulted in the discrepancy between the genotype and the phenotype. In addition, the amount of Y chromosome detected was particularly low in hair compared to that in blood. This study shows that mosaicism can make interpretation difficult during STR analysis and suggests that sample types and repeated analysis should be considered even for routine STR testing.

Identification and Characterization of Nine Novel X-Chromosomal Short Tandem Repeats on Xp21.1, Xq21.31, and Xq23 Regions

The application of X-chromosomal short tandem repeats (X-STRs) has been recognized as a powerful tool in complex kinship testing. To support further development of X-STR analysis in forensic use, we identified nine novel X-STRs, which could be clustered into three linkage groups on Xp21.1, Xq21.31, and Xq23. A multiplex PCR system was built based on the electrophoresis. A total of 198 unrelated Shanghai Han samples along with 168 samples from 43 families was collected to investigate the genetic polymorphism and forensic parameters of the nine loci. Allele numbers ranged from 5 to 12, and amplicon sizes ranged from 146 to 477 bp. The multiplex showed high values for the combined power of discrimination (0.99997977 in males and 0.99999999 in females) and combined mean exclusion chances (0.99997918 and 0.99997821 in trios, 0.99984939 in duos, and 0.99984200 in deficiency cases). The linkage between all pairs of loci was estimated via Kosambi mapping function and linkage disequilibrium test, and further investigated through the family study. The data from 43 families strongly demonstrated an independent transmission between LGs and a tight linkage among loci within the same LG. All these results support that the newly described X-STRs and the multiplex system are highly promising for further forensic use.

Application of Restriction Site-Associated DNA Sequencing (RAD-Seq) for Copy Number Variation and Triploidy Detection in Human

At present, low-pass whole-genome sequencing (WGS) is frequently used in clinical research and in the screening of copy number variations (CNVs). However, there are still some challenges in the detection of triploids. Restriction site-associated DNA sequencing (RAD-Seq) technology is a reduced-representation genome sequencing technology developed based on next-generation sequencing. Here, we verified whether RAD-Seq could be employed to detect CNVs and triploids. In this study, genomic DNA of 11 samples was extracted employing a routine method and used to build libraries. Five cell lines of known karyotypes and 6 triploid abortion tissue samples were included for RAD-Seq testing. The triploid samples were confirmed by STR analysis and also tested by low-pass WGS. The accuracy and efficiency of detecting CNVs and triploids by RAD-Seq were then assessed, compared with low-pass WGS. In our results, RAD-Seq detected 11 out of 11 (100%) chromosomal abnormalities, including 4 deletions and 1 aneuploidy in the purchased cell lines and all triploid samples. By contrast, these triploids were missed by low-pass WGS. Furthermore, RAD-Seq showed a higher resolution and more accurate allele frequency in the detection of triploids than low-pass WGS. Our study shows that, compared with low-pass WGS, RAD-Seq has relatively higher accuracy in CNV detection at a similar cost and is capable of identifying triploids. Therefore, the application of this technique in medical genetics has a significant potential value.

Utilization of Short Tandem Repeat Analysis to Resolve Specimen Mislabeling in the Anatomic Pathology Laboratory

Introduction/Objective A mislabeled specimen is an example of preanalytical error that can have significant consequences on patient care. These errors can be difficult to detect and resolve. One method to confirm genetic identity is short tandem repeat (STR) analysis, which is utilized in forensic investigations, paternity studies, and post- hematopoietic stem cell transplantation monitoring. Herein we present application of STR analysis to resolve a suspected specimen mislabeling prior to receipt in our anatomic pathology laboratory. Methods/Case Report DNA was extracted from paraffin embedded tissues. Chimerism testing was performed by STR analysis using the Globalfiler (ThermoFisher Scientific) and analyzed by Chimermarker (Softgenetics) automated chimerism software. Results (if a Case Study enter NA) Colon biopsies were received for a single patient (#1) with two requisition forms. Each specimen (A-F) was labeled with the patient’s name, with specimens A-D noted on first page of requisition and specimens E-F on the second requisition page. After the case was signed out, the lab was contacted looking for biopsy results on another patient (#2) who was seen on the same day as patient #1. Review of all the patients seen in the endoscopy suite on the given date raised suspicion that specimens E-F from the second page of the requisition actually pertained to patient #2. STR analysis performed on specimens confirmed that specimens E-F were genetically distinct from those labelled A-D. Tissue from a subsequent biopsy on patient #2 was analyzed by STR testing, which was identical to STR results performed on specimens E-F. Conclusion Here we utilized STR testing to resolve a suspected mislabeled specimen, allowing the appropriate diagnosis to be attributed to the correct patient. This is a unique application of a common method, which could be implemented in anatomic pathology laboratories to resolve cases of specimen mix-ups.

Qualitative and Quantitative Analysis of the Fate of DNA in the Digestive Tract of Mice

Objective: Qualitative and quantitative examination of DNA degradation during the digestion process in the mouse gut through PCR, qPCR and short tandem repeat (STR) analysis.Methods: Human blood leukocytes were gavage into the digestive tract of mice. GAPDH, TH01, TPOX and D7S820 genes in the contents of the stomach and small intestine were analyzed through PCR and qPCR at various time 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.Results: The PCR results showed DNA bands 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 significant decrease in DNA concentrations during 40-120 minutes in mouse stomach after gavage. At 120 min, 85.62±8.10% of the DNA was degraded while the half-life of exogenous DNA degradation in mouse stomach was 70.50±5.46 min. At various time of digestion , almost no target genes were detected in the mouse small intestinal chyme. STR analysis showed a decrease in allele numbers with the advancement of bowl in the small intestine of mice.Conclusions: The degradation of exogenous DNA was higher in mouse stomach during first two hours while almost complete degradation was noted in the small intestine of mice within 40 min.

Screening for Fetal Aneuploidy and Sex Chromosomal Anomalies in a Pregnant Woman With Mosaicism for Turner Syndrome—Applications and Advantages of Cell-Based NIPT

Background: Cell-free NIPT and cell-based NIPT are risk-free testing options using maternal blood samples to screen for fetal aneuploidies, but the methods differ. For cell-free NIPT, the fetal fraction of cell-free DNA in plasma is analyzed with a high background of maternal DNA. In contrast, for cell-based NIPT, a limited number of the rare, intact fetal cells are isolated for the genetic analysis. This case demonstrates the differences regarding testing for fetal sex-chromosomes anomalies (SCAs) between these two tests.Materials and Methods: A pregnant woman with mosaicism for Turner syndrome opted for NIPT in first trimester. For the cell-free NIPT analysis, DNA extraction, genome-wide massive parallel sequencing, and data analysis were carried out as described by the kit manufacturer (Illumina©, San Diego, CA, USA). For cell-based NIPT, the first sample gave no result, but the woman consented to repeat cell-based NIPT. After whole genome amplification and STR analysis, fetal DNA from three individual fetal cells was subjected to chromosomal microarray (aCGH, Agilent oligoarray, 180 kb).Results: Fetal fraction was 7%, and cell-free NIPT showed 2 copies of chromosomes 13, 18, and 21 and a decreased proportion of chromosome X, suggestive of fetal Turner syndrome. In contrast, the cell-based NIPT result showed no aneuploidy and two X-chromosomes in the fetus.Conclusion: cell-based NIPT may provide a non-invasive testing option to screen for SCAs in women with mosaicism for monosomy-X in blood, where cell-free NIPT cannot discriminate whether the X-loss is maternal or fetal.

Verification of a Cryptic T(Y;15) Translocation in a Male With an Apparent 45,X Karyotype

BackgroundA rare disease is that an individual with a non-chimeric karyotype of 45, X develops into a male. We explored the genetic aetiology of an infertility male with an apparent 45, X karyotype, which was subsequently verified as cryptic translocation between chromosomes Y and 15. MethodsPeripheral blood sample was collected from the patient and subjected to a range of genetic testing, including conventional chromosomal karyotyping, short tandem repeat (STR) analysis for azoospermia factor (AZF) region, fluorescence in situ hybridization (FISH) with specific probes for CSP X/CSP Y, CSP Y/D15Z1/PML and SRY/D15Z1/PML, and chromosomal microarray analysis (CMA) for genomic copy number variations (CNVs). ResultsThe patient was found to have an apparent 45,X karyotype. STR analysis showed that he possessed a short arm of the Y chromosome, including the SRY gene but the absence of a long arm of the Y chromosome, including AZFa+b+c and Yqter. A FISH assay using CSP X and CSP Y probes showed a green signal at the centromere of the X chromosome and a red signal for the Y centromeric sequence on a D-group-sized chromosome. By FISH assaying with D15Z1 and CSP Y probes, chromosomes 15 and Y centromeric signals appeared closely on a single chromosome, as ascertained by the PML control probe. A further FISH assay with D15Z1 and SRY probes revealed a signal of the SRY gene at the end of one arm of chromosome 15. The result of the CMA indicated a deletion with an approximate size of 45.31 Mb spanning from Yq11 to Yter. ConclusionAlthough the 45,X male patient did not harbour an intact Y chromosome, his genome contained the SRY gene derived from the translocation of the Yp, which probably triggered the male differentiation and development. Imbalanced translocations of Yp to other chromosomes can result in short stature and infertility among patients. Delineation of the genetic aetiology can guide early intervention and assisted reproduction in adulthood.