Chromosomal Copy Number Variations Research Articles

Non-invasive prenatal test findings in 41,819 pregnant women: results from a clinical laboratory in southern China.

This paper evaluated the clinical utility of massively parallel sequencing-based non-invasive prenatal testing (NIPT) for detecting trisomy 21 (T21), T18, T13, sex chromosome aneuploidies (SCA), and rare chromosome aneuploidies (RCA) among the data collected by a clinical laboratory in southern China. In a 3-year period between January 2017 and December 2019, over 40,000 pregnant women underwent NIPT clinical screening test for fetal T21, T18, T13, SCA, and RCA in our laboratory. NIPT samples were processed using the NextSeq CN500 platform. The positive results were confirmed by karyotyping, and chromosomal microarray analysis (CMA) or copy number variants (CNV) sequencing. Details of the pregnancy outcomes were collected via telephone interview. NIPT results were available for 41,819 cases; 691 positive cases were reported. The overall sensitivity for detection of T21, T18, T13, SCA, and RCA was 99.21, 100.00, 100.00, 98.55, and 100.00%, and the specificity was 99.95, 99.94, 99.98, 99.69, and 99.92%, respectively. The positive predictive values (PPVs) for detection of T21, T18, T13, SCA, and RCA were 85.62, 45.24, 40.00, 34.17, and 13.51%, respectively, and those for detection of 45,X, 47,XXY, 47,XXX, 47,XYY, and 46,XY(delX) 20.00, 59.18, 28.95, 61.54, and 25.00%, respectively. Regarding pregnancy outcomes, 92.38% of the pregnancies with confirmed aneuploidies were terminated, and 91.20% of those identified as having a false-positive result were carried to term. Among 252 unconfirmed cases, 24.60% of the pregnancies were terminated and 38.10% carried to term, while 37.30% declined interview. NIPT is widely used to screen fetal aneuploidies based on its high sensitivity and specificity. However, in this study, the PPVs of NIPT in terms of detecting T18, T13, XO, XXX and RCA were < 50%. In addition, more than one-third of NIPT-positive women did not accept invasive prenatal diagnosis. Confirmatory diagnosis is strongly recommended for women with positive NIPT outcomes before any further decision is made.

Whole-genome characterization of large-cell lung carcinoma: A comparative analysis based on the histological classification.

Background: According to the 2015 World Health Organization classification, large cell neuroendocrine carcinoma (LCNEC) was isolated from Large-cell lung cancer (LCLC) tumors, which constitutes 2%-3% of non-small cell lung cancer (NSCLC). However, LCLC tumors are still fairly vaguely defined at the molecular level compared to other subgroups. Materials and Methods: In this study, whole-genome sequencing (WGS) was performed on 23 LCLC and 15 LCNEC tumor specimens. Meanwhile, data from the TCGA (586 LUADs and 511 LUSCs) and U Cologne (120 SCLCs) were analyzed and compared. Results: The most common driver mutations were found in TP53 (13/23, 57%), FAM135B (8/23, 35%) and FAT3 (7/23, 30%) in LCLC, while their counterparts in LCNEC were TP53 (13/15, 87%), LRP1B (6/15, 40%) and FAT1 (6/15, 40%). Notably, FAM135B mutations only occurred in LCLC (P = 0.013). Cosmic signature analysis revealed widespread defective DNA mismatch repair and tobacco-induced mutations in both LCLC and LCNEC. Additionally, LCNEC had a higher incidence of chromosomal copy number variations (CNVs) and structural variations (SVs) compared with LCLC, although the differences were not statistically significant. Particularly, chromothripsis SVs was significantly associated with CNVs. Furthermore, mutational landscape of different subtypes indicated differences between subtypes, and there seems to be more commonalty between our cohort and SCLC than with other subtypes. SMARCA4 mutations may be specific driver gene alteration in our cohort. Conclusion: Our results support that LCLC and LCNEC tumors follow distinct tumorigenic pathways. To our knowledge, this is the first genome-wide profiling comparison of LCLC and LCNEC.

Establishment of linkage phase, using Oxford Nanopore Technologies, for preimplantation genetic testing of Coffin-Lowry syndrome with a de novo RPS6KA3 mutation.

Background: This study aimed to perform preimplantation genetic testing (PGT) for a female Coffin-Lowry Syndrome (CLS) patient with a de novo mutation (DNM) in RPS6KA3. It was challenging to establish the haplotype in this family because of the lack of information from affected family members. Hence, we explored a new and reliable strategy for the detection of the DNM in PGT, using Oxford Nanopore Technologies (ONT) and the MARSALA platform. Methods: We performed whole-exome sequencing (WES) on the proband and confirmed the pathogenic mutation by Sanger sequencing. The proband then underwent PGT to prevent the transmission of the pathogenic mutation to her offspring. We diverged from the conventional methods and used long-read sequencing (LRS) on the ONT platform to directly detect the mutation and nearby SNPs, for construction of the haplotype in the preclinical phase of PGT. In the clinical phase of embryo diagnosis, the MARSALA method was used to detect both the SNP-based haplotype and chromosome copy number variations (CNVs), in each blastocyst. Finally, a normal embryo was selected by comparison to the haplotype of the proband and transferred into the uterus. Sanger sequencing and karyotyping were performed by amniocentesis, at 17weeks of gestation, to confirm the accuracy of PGT. Results: Using WES, we found the novel, heterozygous, pathogenic c.1496delG (p.Gly499Valfs*25) mutation of RPS6KA3 in the proband. The SNP-based haplotype that was linked to the pathogenic mutation site was successfully established in the proband, without the need for other family members to be tested with ONT. Eight blastocysts were biopsied to perform PGT and were assessed with a haplotype linkage analysis (30 SNP sites selected), to give results that were consistent with direct mutation detection using Sanger sequencing. The results of PGT showed that three of the eight blastocysts were normal, without the DNM. Moreover, the patient had a successful pregnancy, after transfer of a normal blastocyst into the uterus, and delivered a healthy baby. Conclusion: The ONT platform, combined with the MARSALA method, can be used to perform PGT for DNM patients without the need for other samples as a reference.

P499: RNA-sequencing positional gene enrichment is a useful tool in resolving cases of X chromosome copy number variation

P499: RNA-sequencing positional gene enrichment is a useful tool in resolving cases of X chromosome copy number variation

A multicenter-retrospective cohort study of chromosome instability in lung cancer: clinical characteristics and prognosis of patients harboring chromosomal instability detected by metagenomic next-generation sequencing.

The usefulness of metagenomic next-generation sequencing (mNGS) in identifying the prognosis of lung cancer with chromosomal instability (CIN) remains unclear. We aimed to analyze clinical characteristics and prognosis of patients in patients harboring CIN. This retrospective cohort study included 668 patients diagnosed with suspected pulmonary infection or lung cancer whose samples underwent mNGS detection from January 2021 to January 2022. Difference between clinical characteristics were calculated by the Student's t-test and the chi-square test. The subjects were followed-up from registered to September 2022. Survival curves were analyzed by the Kaplan-Meier method. Of 619 bronchoalveolar lavage fluid (BALF) samples collected by bronchoscopy, 30 CIN-positive samples were confirmed as malignant on histopathology, with a sensitivity of 61.22%, a specificity of 99.65%, and an 83.17% accuracy [cut-off values were established by the receiver operating characteristic (ROC) area under the curve (AUC) =0.804]. In 42 patients with lung cancer, mNGS detected 24 patients as CIN-positive and 18 as CIN-negative. There were no differences between two groups including ages, pathologic types, stage and metastases. In 25 cases, we detected 523 chromosomal copy number variants (CNVs) with forms including duplication (dup), deletion (del), mosaic (mos), and whole chromosome amplification or loss. A total of 243 duplication variants and 192 deletion variants occurred in all chromosomes. Duplications occurred in most chromosomes except for Chr9 and Chr13, in which CNV tended to delete. The median overall survival (OS) in patients with Chr5p15 duplication was 32.4 months [95% confidence interval (CI), 10.35-54.45 months]. The median OS differed significantly between the 5p15dup+ group and the combined group (32.4 vs. 8.63 months, P=0.049). In 29 patients with unresected lung cancer, the median OS of 18 cases in the CIN-positive group was 32.4 months (95% CI, 14.2-50.6 months) and the median OS of 11 cases in the CIN-negative group was 35.63 months (95% CI, 21.64-49.62 months; Wilcoxon, P=0.227). Various forms of CIN detected by mNGS may predict prognosis of patients with lung cancer differentially. CIN with duplication or deletion deserves further study to guide clinical treatment.

Variation of Female Pronucleus Reveals Oocyte or Embryo Chromosomal Copy Number Variations.

The characteristics of the human pronuclei (PNs), which exist 16-22 h after fertilization, appear to serve as good indicators to evaluate the quality of human oocyte and embryo, and may reflect the status of female and male chromosome composition. Here, a quantitative PN measurement method that is generated by applying expert experience combined with deep learning from large annotated datasets is reported. After mathematic reconstruction of PNs, significant differences are obtained in chromosome-normal rate and chromosomal small errors such as copy number variants by comparing the size of the reconstructive female PN. After integrating the whole procedure of PN dynamics and adjusting for errors that occur during PN identification, the results are robust. Notably, all positive prediction results are obtained from the female propositus population. Thus, the size of female PNs may mirror the internal quality of the chromosomal integrity of the oocyte. Embryos that develop from zygotes with larger female PNs may have a reduced risk of copy number variations.

59. Inferring copy number events in Glioblastoma using spatial gene expression data

<h3>Background</h3> Glioblastoma (GBM) is the most common malignant brain tumour in adults, with inevitable treatment resistance and poor outcomes. Genomic instability of GBM gives rise to large-scale chromosomal copy number variants (CNVs) that drive resistance. Genetically distinct clones in GBM are conventionally investigated using whole-genome sequencing (WGS), but recent bioinformatic approaches can infer CNVs using transcriptomics data only. <h3>Purpose</h3> To investigate whether patient-derived xenograft (PDX) mouse models of GBM exhibit clonal diversity as a function of space (across tumour regions) and time (early to late time points). <h3>Methods</h3> PDX data from 3 cell lines representing the tumour core, vascular regions, and infiltrating edge of one GBM patient's tumour were profiled at early and late time points in disease progression, using the 10X Genomics Visium platform for spatial transcriptomics. The computational tool inferCNV was used to identify CNVs. <h3>Results</h3> Successfully identified large-scale CNVs were concordant with events detected in matched WGS data. Additionally, we identified a minor subclone not detectable by WGS that expands in multiple PDX mice indicating it is pre-existing in the patient-derived cell lines. Further analysis revealed localization of this subclone to the tumour core region suggesting a microenvironment-specific adaptation. <h3>Conclusions</h3> We show that spatial gene expression data can be leveraged to infer CNVs in PDX models of GBM that correspond to known events from bulk WGS data. Subclones present below the limit of WGS detection can also be inferred. Charting the spatially resolved position of these clones in vivo supports the use of PDX models in GBM.

Combining bulk RNA-sequencing and single-cell RNA-sequencing data to reveal the immune microenvironment and metabolic pattern of osteosarcoma

Background: Osteosarcoma (OS) is a kind of solid tumor with high heterogeneity at tumor microenvironment (TME), genome and transcriptome level. In view of the regulatory effect of metabolism on TME, this study was based on four metabolic models to explore the intertumoral heterogeneity of OS at the RNA sequencing (RNA-seq) level and the intratumoral heterogeneity of OS at the bulk RNA-seq and single cell RNA-seq (scRNA-seq) level. Methods: The GSVA package was used for single-sample gene set enrichment analysis (ssGSEA) analysis to obtain a glycolysis, pentose phosphate pathway (PPP), fatty acid oxidation (FAO) and glutaminolysis gene sets score. ConsensusClusterPlus was employed to cluster OS samples downloaded from the Target database. The scRNA-seq and bulk RNA-seq data of immune cells from GSE162454 dataset were analyzed to identify the subsets and types of immune cells in OS. Malignant cells and non-malignant cells were distinguished by large-scale chromosomal copy number variation. The correlations of metabolic molecular subtypes and immune cell types with four metabolic patterns, hypoxia and angiogenesis were determined by Pearson correlation analysis. Results: Two metabolism-related molecular subtypes of OS, cluster 1 and cluster 2, were identified. Cluster 2 was associated with poor prognosis of OS, active glycolysis, FAO, glutaminolysis, and bad TME. The identified 28608 immune cells were divided into 15 separate clusters covering 6 types of immune cells. The enrichment scores of 5 kinds of immune cells in cluster-1 and cluster-2 were significantly different. And five kinds of immune cells were significantly correlated with four metabolic modes, hypoxia and angiogenesis. Of the 28,608 immune cells, 7617 were malignant cells. The four metabolic patterns of malignant cells were significantly positively correlated with hypoxia and negatively correlated with angiogenesis. Conclusion: We used RNA-seq to reveal two molecular subtypes of OS with prognosis, metabolic pattern and TME, and determined the composition and metabolic heterogeneity of immune cells in OS tumor by bulk RNA-seq and single-cell RNA-seq.

Exploration the global single-cell ecological landscape of adenomyosis-related cell clusters by single-cell RNA sequencing

Background: Adenomyosis (AM) is a common benign uterine disease that threatens the normal life of patients. Cells associated with microenvironmental immune ecology are crucial in AM, although they are not as well understood at the cellular level. Methods: Single-cell sequencing (scRNA-seq) data were used to construct an AM global single-cell map, to further identify relevant cell clusters and infer chromosomal copy number variation (CNV) in AM samples. The biological functions of cell clusters were explored and cellular evolutionary processes were inferred by enrichment analysis and pseudotime analysis. In addition, a gene regulatory network (GRN) analysis was constructed to explore the regulatory role of transcription factors in AM progression. Results: We obtained the expression profiles of 42260 cells and identified 10 cell clusters. By comparing the differences in cell components between AM patients and controls, we found that significant abundance of endometrial cells (EC), epithelial cells (Ep), endothelial cells (En), and smooth muscle cells (SMC) in AM patients. Cell clusters with high CNV levels possessing tumour-like features existed in the ectopic endometrium samples. Moreover, the Ep clusters were significantly involved in leukocyte transendothelial cell migration and apoptosis, suggesting an association with cell apoptosis and migration. En clusters were mainly involved in pathways in cancer and apoptosis, indicating that En has certain malignant features. Conclusion: This study identified cell clusters with immune-related features, investigated the changes in the immune ecology of the microenvironment of these cells during AM, and provided a new strategy for the treatment of AM.

Current Perspectives of Prenatal Cell-free DNA Screening in Clinical Management of First-Trimester Septated Cystic Hygroma

First-trimester septated cystic hygroma occurs in approximately 1 in 268 pregnancies and has long been associated with a markedly increased risk of fetal aneuploidy and, among euploid fetuses, an increased risk of structural anomalies primarily affecting the cardiac and skeletal systems. Invasive prenatal diagnosis - chorionic villus sampling and/or amniocentesis - encompasses the time-honored clinical tools for the next step in management following prenatal sonographic diagnosis of first-trimester septated cystic hygroma. Currently, prenatal cell-free DNA (cfDNA) screening for fetal aneuploidy with select microdeletions is gradually replacing the considerably less sensitive, and labor-intensive combined first-trimester screening. These new technologies have opened potential new venues in the clinical management of this ominous late first-trimester sonographic diagnosis. Advances in cfDNA technologies are now permitting detection of chromosomal copy number variants (CNV) larger than 7Mb across genome and select serious single-gene disorders (mainly impacting skeletal and neurological development), affecting quality of life and may benefit from medical and/or surgical management. This commentary will address the available non-invasive prenatal screening technologies, which clearly enhance immediate genetic analysis modalities applicable in the presence of the complex sonographic finding of first-trimester septated cystic hygroma.

Copy number variations: A novel molecular marker for papillary thyroid cancer

BackgroundWe aimed to screen tumor-associated functional genes on a large scale through copy number variation (CNV) analysis of papillary thyroid cancer (PTC). MethodsWe analyzed 74 tissue samples from 41 patients with thyroid nodules. The samples were subjected to whole-genome resequencing and then analyzed by the ‘WISECONDOR’ method. Potential chromosome CNV regions were identified between the different sample groups. ResultsOf the 74 samples from 41 patients, 28 were PTC tissue samples, 29 were para-carcinoma tissue samples, 13 were benign tumor tissue samples and 4 were para-benign tumor tissue samples. According to our findings, PTC can be identified by CNVs at the corresponding positions on chromosomes 5, 7, 8, 10, and 17. For carcinoma tissue, the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), accuracy and area under the curve (AUC) of the test method were 100%, 66.7%, 87.5%, 100.0%, 90.0% and 0.83 (95% confidence interval [CI], 0.67–1.00) and for para-carcinoma tissue, these values were 96.6%, 75.0%, 96.6%, 75.0%, 93.9% and 0.86 (95% CI, 0.60–1.00). ConclusionCNV analysis assays involving high-volume sequencing analysis can increase the identification of PTC, potentially avoiding errors caused by position deflection in sampling.

Analysis of chromosomal copy number variations among 163 fetuses with echogenic bowel by using CNV-seq technology

To assess the value of low-depth whole-genome copy number variation sequencing (CNV-seq) for the analysis of chromosomal copy number variations among fetuses with echogenic bowel (EB). A total of 163 fetuses were included in this study. Amniotic fluid (162 cases) or chorionic villi (1 case) were collected and subjected to CNV-seq for the analysis of CNVs. Thirteen (8.0%) pathogenic CNVs were detected, including 9 (5.5%) aneuploidies and 4 (2.4%) CNVs. The detection rate of the isolated EB group and combined EB group were 1.7% (1/58) and 11.4% (12/105), respectively. There was a significant difference between the two groups (P < 0.05). A Xp22.1 duplication was detected in both groups, and the fetuses were predicted as female DMD carriers and born healthy. Nine cases of aneuploidies and 2 (likely) pathogenic CNVs were identified in the combined EB group, all of them have warranted induced labor. The prevalence of chromosomal aneuploidies and pathogenic CNVs in fetuses with combined EB was much higher than isolated EB, and most of them may warrant termination of pregnancy. Compared with isolated EB, more attention should be paid to combined EB, for which prenatal diagnosis and genetic counseling should be carried out in time.

Genome-wide analysis reveals allelic variation and chromosome copy number variation in paromomycin-resistant Leishmania donovani.

In the absence of adequate diagnosis and treatment, leishmaniasis remains a major public health concern on a global scale. Drug resistance remains a key obstacle in controlling and eliminating visceral leishmaniasis. The therapeutic gap due to lack of target-specific medicine and vaccine can be minimized by obtaining parasite's genomic information. This study compared whole-genome sequence of paromomycin-resistant parasite (K133PMM) developed through in vitro adaptation and selection with sensitive Leishmania clinical isolate (K133WT). We found a large number of upstream and intergenic gene variations in K133PMM. There were 259 single nucleotide polymorphisms (SNPs), 187 insertion-deletion (InDels), and 546 copy number variations (CNVs) identified. Most of the genomic variations were found in the gene's upstream and non-coding regions. Ploidy estimation revealed chromosome 5 in tetrasomy and 6, 9, and 12 in trisomy, uniquely in K133PMM. These contain the genes for protein degradation, parasite motility, autophagy, cell cycle maintenance, and drug efflux membrane transporters. Furthermore, we also observed reduction in ploidy of chromosomes 15, 20, and 23, in the resistant parasite containing mostly the genes for hypothetical proteins and membrane transporters. We chronicled correlated genomic conversion and aneuploidy in parasites and hypothesize that this led to rapid evolutionary changes in response to drug induced pressure, which causes them to become resistant.

Identification of partial trisomy 13q in two unrelated patients using single-nucleotide polymorphism array and literature overview

BackgroundPartial trisomy 13q is a less common chromosomal abnormality with a great clinical variability, among them, isolated partial trisomy 13q is extremely rare. Here, we report two new unrelated cases of partial trisomy 13q in Chinese families aiming to emphasize the genotype–phenotype correlation in partial trisomy 13q.MethodsEnrolled in this study were two unrelated cases of partial 13q trisomy from two families in Quanzhou region South China. Karyotpe and single-nucleotide polymorphism (SNP) array analysis were employed to identify chromosome abnormalities and copy number variants in the families.ResultsA 72.9-Mb duplication in 13q14.11q34 region was identified using SNP array analysis in Patient 1 with an intellectual disability, developmental delay, seizures, gastric perforation, and other congenital malformations from a family with paternal inv(13)(p12q14.1). SNP array detection in Patient 2 revealed a 92.4-Mb duplication in 13q12.11q34 region combined with an 8.4-Mb deletion in Xq27.3q28 region with intellectual disability, developmental delay, cleft palate, and duplication of the cervix and the vagina. No chromosomal abnormality was elicited from the parents of Patient 2.ConclusionsIn this study, we presented two new unrelated cases of partial trisomy 13q with variable features in Chinese population, which may enrich the spectrum of the phenotypes partial trisomy 13q and further confirm the genotype–phenotype correlation.

Prenatal screening and diagnosis for a fetus with mosaic sex chromosome abnormality

To carry out prenatal screening and diagnosis for a woman with advanced maternal age. Non-invasive prenatal testing (NIPT) was carried out to determine the risk of fetal chromosome aneuploidy. Aminiocentesis was proceeded for fetal chromosomal karyotyping and copy number variation sequencing (CNV-seq). The fetus was subjected to systematic ultrasound screening in the second trimester. NIPT has indicated there was a loss of fetal sex chromosome. Karotyping of the amniocyte showed a mosaic sex chromosome abnormality 45,X[53]/46,X,+mar[7]. The result of fetal DNA CNV-seq was seq[GRCh37]del(Yq11.1q12) chrY: g.13 104 553-28 819 361del, seq[GRCh37]del(Yp11.32p11.2) chrY: g.10 001-9 873 915del (mosaic ratio: 30%). Ultrasonography discovered that the fetus had renal dysplasia and male external genitalia. The karyotypes of the couple were both normal. Multiple genetic tests should be carried out for fetus with a high risk for chromosome aneuploidies signaled by NIPT. It is difficult to predict the post-natal phenotype for fetuses with mosaic sex chromosomal aneuploidies. The couple should be carefully counseled upon genetic counseling.

Positive predictive value estimates for noninvasive prenatal testing from data of a prenatal diagnosis laboratory and literature review

ObjectiveSince 2011, noninvasive prenatal testing (NIPT) has undergone rapid expansion, with both utilization and coverage. However, conclusive data regarding the clinical validity and utility of this testing tool are lacking. Thus, there is a continued need to educate clinicians and patients about the current benefits and limitations in order to inform pre- and post-test counseling, pre/perinatal decision making, and medical risk assessment/management.MethodsThis retrospective study included women referred for invasive prenatal diagnosis to confirm positive NIPT results between January 2017 and December 2020. Prenatal diagnosis testing, including karyotyping, chromosomal microarray analysis (CMA) were performed. Positive predictive values (PPVs) were calculated.ResultsIn total, 468 women were recruited. The PPVs for trisomies 21, 18, and 13 were 86.1%, 57.8%, and 25.0%, respectively. The PPVs for rare chromosomal abnormalities (RCAs) and copy number variants (CNVs) were 17.0% and 40.4%, respectively. The detection of sex chromosomal aneuploidies (SCAs) had a PPV of 20% for monosomy X, 23.5% for 47,XXX, 68.8% for 47,XXY, and 62.5% for 47,XYY. The high-risk groups had a significant increase in the number of true positive cases compared to the low- and moderate-risk groups.ConclusionsT13, monosomy X, and RCA were associated with lower PPVs. The improvement of cell-free fetal DNA screening technology and continued monitoring of its performance are important.

P-113 Association of Novel Mutations of TSPY1 Gene With Spermatogenic Failure Among Men

Abstract Study question Are the novel mutations of TSPY1 gene associated with idiopathic male infertility in men? Summary answer Sporadic TSPY1 gene mutation is one of the major causes of idiopathic male infertility among men. What is known already As per the published research, there are several studies including AZF micro-deletion, sex chromosome copy number variation but surprisingly studies on Y chromosome genes are not that much available. Due to the genetic cross-talks of different regulatory mechanisms involved in the spermatogenesis process, male infertility still remains idiopathic. Contradictory outcome of the analyses intrigues the association of the variations of TSPY1 gene with male infertility. Previous analyses did not reveal implication of TSPY1 gene as etiologic factor of male infertility. Study design, size, duration This was a population-based case-control study that includes 184 cases of spermatogenic failure and 197 age-matched fertile men as controls and the study was conducted between March, 2018 and February, 2021 to infer whether the genetic mutations are significantly associated with spermatogenic failure. Participants/materials, setting, methods The samples were collected from the Institute of Reproductive Medicine (IRM) and Genome-the fertility centre. All the selected individuals agreed to participate in the study and provided blood samples. We isolated DNA from the blood samples, designed specific primers and went for PCR. We performed Sanger’s dideoxy sequencing of TSPY1 gene reading frame followed by in silico analyses of detected variations to interpret their intuitive effects on transcript and protein level of TSPY1 gene. Main results and the role of chance We found one novel deletion namely MN734578 (NC_000024.10:g.9468830del) and one novel insertion MN719944 (NC_000024.10:g.9468815_9468816insA). Both these mutations exhibited strong association with male infertility. MN734578 showed compelling confederacy with male infertility increasing the risk factor against the odd 19.023 (P value 0.0027). Here the mutation was found in 8 out of 184 case individuals. MN719944 was found to be extremely correlated with male infertility increasing the risk factor by 14.547 folds (P value 0.0117). This mutation is found in 6 out of 184 case individuals. Both the mutations were not not found in the control individuals. In silico analyses using different software SIFT, PROVEAN, REGULATIONSPOTTER, HSF and SpliceAid suggest prospective disruption in splice sites and alteration in different isoforms of TSPY1 transcripts and amino acid sequence in TSPY1 protein. We calculated Odds ratios with respective 95% confidence interval (CI) by performing Fisher’s exact test. After Bonferroni's correction, a two-tailed P-value of less than 0.02 was considered statistically significant. Limitations, reasons for caution The origin of the novel mutations is unknown and the outcome does not provide mechanistic details how do de novo mutations imperil the process of spermatogenesis. Wider implications of the findings The study provides evidence in favour of implication of TSPY1 gene in male fertility. The outcome sheds light to get insight into the issue of unexplained male infertility in Bengali will help to manage fertility problem in men. Trial registration number Not applicable

P-105 Genetic variants in the spermatogenic transcription regulators RFX2 and TAF7 enhance the incidence of azoospermia

Abstract Study question What are the genetic variants of meiotic regulator genes that are implicated in male infertility among men from West Bengal, India? Summary answer TAF7 C16T (MW827584 G &amp;gt; A) and RFX2 562delT (MZ560629delA) new variations may have a major impact on optimal fertility in men from West Bengal. What is known already According to published research, roughly 50% of reported infertility cases in India are due to male reproductive abnormalities. Because of the involvement of several gene regulatory networks in the spermatogenesis process, the genetic etiology of idiopathic male infertility remains a mystery. The AZF region of the Y chromosome microdeletion has been discovered as a prevalent cause of male infertility across all ethnicities. Infertility in men has also been linked to sex chromosomal copy number variation and autosomal gene polymorphisms. Study design, size, duration This is a case-control study to infer whether the genetic variants are significantly associated in favor of cases or in favor of controls. The study included 160 healthy male controls and 150 azoospermic cases submitted to the Institute of Reproductive Medicine in Kolkata (IRM). All of the subjects agreed to participate in the study and provided blood and sperm samples. The duration of the study is three years. Seminograms were made to filter azoospermic individuals. Participants/materials, setting, methods 2 ml of venous blood samples were taken from azoospermic men using the venipuncture procedure and stored in EDTA coated vacutainer tubes at -20°C for DNA isolation.QIAamp Blood Mini Kit was used to isolate genomic DNA. For PCR analysis, unique primers for TAF7 and RFX2 functional domains were created. Taq Dye Deoxy Terminator sequencing kit (Applied Biosystems, Foster City, USA) and ABI Prism 377 DNA sequencer were used to analyze PCR results. Main results and the role of chance We have found two significant genetic alterations in the coding region of TAF7 and RFX2 which are MW827584 G &amp;gt; A(C16T) and MZ560629delA(562delT) respectively. Both were found to have a strong association to the occurrence of azoospermia. For MW827584 G &amp;gt; A, minor allele ‘A’ has shown elevated risk of azoospermic against the odd 4.684 (95% C.I = 2.900-7.564, Relative risk =2.110, p value = &amp;lt;0.001). Fisher’s exact test was performed to calculate Odds ratios with respective 95% confidence interval (CI). After Bonferroni's correction, a two-tailed P-value of less than 0.02 was considered statistically significant throughout. Only the case sample cohort had the RFX2 deletion MZ560629 delA. Only 9 cases with this unique deletion in heterozygous form were found out of 150 azoospermic patients. This frameshift mutation results in the production of an early termination signal at exon 6, resulting in the creation of a truncated protein. The wild-type RFX2 protein has 729 amino acids but this frameshift mutation results in a 212-amino-acid RFX2 protein that lacks several secondary motifs and domains, rendering the wild-type function useless. In silico analysis of SIFT, PROVEAN, MUTATION TASTER and REGULATION SPOTTER predicted that these genetic variants are disease-causing. Limitations, reasons for caution There are some potential limitations to our research. A larger sample size investigation is needed to reconfirm the genetic association study. Moreover, confounding effects of allelic variation of those genetic modifiers may cause differences in the manifestation of fertility state along with the allelic variants of TAF7 and RFX2 genes. Wider implications of the findings This diagnostic technique will not only reduce the hazards connected with assisted reproductive technologies (ART), but it will also provide insight into previously unknown areas of infertility and enable clinicians in conducting a more thorough investigation of the patients. Trial registration number not applicable

Abstract 6171: A single-cell solution for solid tumors to detect mutations and quantify copy number variations

Abstract Cancer is a highly heterogeneous disease characterized by the presence of chromosomal structural copy number variation (CNV) and driver gene mutation events, which appear somatically at the early stages of oncogenesis and can drive tumor progression. Previously, we have developed a high throughput single cell DNA analysis platform that leverages droplet microfluidics and a multiplex-PCR based targeted DNA sequencing approach. It demonstrates high sensitivity detection of SNVs and indels in the same cells and generation of high resolution maps of clonal architecture based on mutational profiling. Here we expand upon the same approach and create an algorithm to accurately quantify CNVs along with genetic mutations from the same single cells. To estimate the CNV the amplicon read counts were corrected for artefacts related to cell to cell variability due sequencing efficiencies along with amplicon to amplicon variability that occur due to amplification efficiency differences. We used a spiked in diploid cell line as a reference to normalize all the cells. The spiked cell line was identified using its mutational profile. We validated the system using cell lines with known CNVs and found that we could at least accurately estimate copy numbers ranging from 0 through 5. Some of the regions were also validated orthogonally using droplet digital PCR. Another set of validation was performed using well characterized cell lines from NIST. We enable multiple visualizations of the copy number estimates in karyotype plots and line plots projected on SNV clones. With improved biochemistry, panel design and novel data analysis algorithms we develop a complete solution to detect amplification or loss of function in oncogenes and tumor suppressors reliably. Integration of CNVs and SNVs facilitates more accurate reconstruction of tumor evolution to better understand cancer progression mechanisms as well for quality control of gene edited cells, to further advance cancer research and therapy. Citation Format: Saurabh Parikh, Khushali Patel, Alex Li, Anup Parikh. A single-cell solution for solid tumors to detect mutations and quantify copy number variations [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 6171.

Abstract 2127: Master regulator analysis of the tumor microenvironment and the distinctive tumor sub-populations in pancreatic ductal adenocarcinoma

Abstract By 2030, pancreatic ductal adenocarcinoma (PDAC) is projected to become the second leading cause of cancer-associated mortality in the United States. However, current strategies in targeting key driver mutations such as KRAS, CDKN2A, TP53, and SMAD4, have found limited translational success. As a result, the standard of care for PDAC patients continues to rely on cytotoxic chemotherapies. Master regulator (MR) proteins integrate upstream genomic alterations to generate a common downstream transcriptional signature characteristic of a tumor cell population. Importantly, aberrant MR protein activity is requisite to implementing and maintaining a pro-tumor state. Therefore, MR proteins serve as critical tumor dependencies and represent a promising class of therapeutic targets for cancer treatment. Here, we propose a novel, network-based approach to identify top MR proteins by utilizing a combination of ARACNe (Margolin et al, 2006) and VIPER (Alvarez et al, 2016) algorithms for the analysis of single-nuclei transcriptomic data. Specifically, we used VIPER to investigate the heterogeneity of the tumor microenvironment (TME) and to characterize the tumor sub-populations that co-exist in a patient PDAC specimen. Clustering analysis based on protein activity revealed six populations in the TME. The SingleR classifier subsequently annotated these clusters as myeloid and lymphoid immune, fibroblast, neuroendocrine, epithelial, and putative tumor cells. Next, we validated the putative tumor cells by performing InferCNV which detected widespread chromosomal copy number variations. We identified two distinct tumor subpopulations which were found to be enriched by the MR signatures of the Oncogenic Precursor (OP) and Lineage subtypes, previously demonstrated to represent more and less differentiated tumor cell populations, respectively (Laise et al, 2020). Interestingly, top MR proteins identified for the putative tumor cells using the single-nuclei transcriptomics closely corresponded with those identified by single-cell and previous bulk transcriptomics, providing orthogonal validation for the inferred protein activities. We characterized the diverse cell populations in the PDAC TME, delineated two distinct tumor cell subtypes, and demonstrated high concordance of inferred MR protein activity at the single-cell, single-nuclei, and bulk levels. These results demonstrate the feasibility of applying systems biology-based methods to identify MR proteins in an N = 1 context and represent a novel precision medicine approach in the treatment of PDAC patients. Future lines of inquiry center on identifying treatments against each tumor subpopulation via OncoTreat, an algorithm that leverages high-throughput drug perturbation data to predict effective therapies based on inferred ability to invert MR protein activity (Alvarez, 2018). Citation Format: Jinjie Ling, Lorenzo Tomassoni, Alvaro Curiel, Kenneth Olive, Andrea Califano. Master regulator analysis of the tumor microenvironment and the distinctive tumor sub-populations in pancreatic ductal adenocarcinoma [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 2127.