Chromosomal Copy Number Variations Research Articles

Prenatal diagnosis and pregnancy outcomes in fetuses with ventriculomegaly

ObjectiveGenetic etiology plays a critical role in fetal ventriculomegaly (VM). However, the studies on chromosomal copy number variants (CNVs) in fetal VM are limited. This study aimed to investigate the chromosomal CNVs in fetuses with mild to moderate VM, and explore its genotype-phenotype correlation.MethodsA total of 242 fetuses with mild to moderate VM detected by prenatal ultrasound were enrolled in our study from October 2018 to October 2022. All cases underwent chromosomal microarray analysis (CMA) and G-banding simultaneously. All VM cases were classified different subgroups according to the maternal age, severity, VM distribution and presence/absence of other ultrasound abnormalities. The pregnancy outcomes and health conditions after birth were followed up. We also performed a pooled analysis regarding likely pathogenic and pathogenic CNVs (LP/P CNVs) for VM.ResultsThe detection rate of chromosomal abnormalities by karyotyping was 9.1% (22/242), whereas it was 16.5% (40/242) when CMA was conducted (P < 0.05). The total detection rate of chromosomal abnormalities by karyotyping and CMA was 21.1% (51/242). A 12.0% incremental yield of CMA over karyotyping was observed. The detection rate of total genetic variants in fetuses with bilateral VM was significantly higher than in fetuses with unilateral VM (30.0% vs. 16.7%, P = 0.017). No significant differences were discovered between isolated VM and non-isolated VM, or between mild and moderate VM, or between advanced maternal age (AMA) and non-AMA (all P > 0.05). 28 fetuses with VM were terminated and 214 fetuses were delivered: one presented developmental delay and one presented congenital heart disease. The VM cases with both positive CMA and karyotypic results had a higher rate of termination of pregnancy than those with either a positive CMA or karyotypic result, or both negative testing results (P < 0.001).ConclusionThe combination of CMA and karyotyping should be adopted to improve the positive detection rate of chromosomal abnormalities for VM. The total genetic abnormalities detected using both techniques would affect the final pregnancy outcomes. LP/P CNVs at 16p11.2, 17p13, and 22q11.21 were identified as the top three chromosomal hotspots associated with VM, which would enable genetic counselors to provide more precise genetic counseling for VM pregnancies.

Chromosome Transplantation: Opportunities and Limitations.

There are thousands of rare genetic diseases that could be treated with classical gene therapy strategies such as the addition of the defective gene via viral or non-viral delivery or by direct gene editing. However, several genetic defects are too complex for these approaches. These "genomic mutations" include aneuploidies, intra and inter chromosomal rearrangements, large deletions, or inversion and copy number variations. Chromosome transplantation (CT) refers to the precise substitution of an endogenous chromosome with an exogenous one. By the addition of an exogenous chromosome and the concomitant elimination of the endogenous one, every genetic defect, irrespective of its nature, could be resolved. In the current review, we analyze the state of the art of this technique and discuss its possible application to human pathology. CT might not be limited to the treatment of human diseases. By working on sex chromosomes, we showed that female cells can be obtained from male cells, since chromosome-transplanted cells can lose either sex chromosome, giving rise to 46,XY or 46,XX diploid cells, a modification that could be exploited to obtain female gametes from male cells. Moreover, CT could be used in veterinary biology, since entire chromosomes containing an advantageous locus could be transferred to animals of zootechnical interest without altering their specific genetic background and the need for long and complex interbreeding. CT could also be useful to rescue extinct species if only male cells were available. Finally, the generation of "synthetic" cells could be achieved by repeated CT into a recipient cell. CT is an additional tool for genetic modification of mammalian cells.

[Prenatal diagnosis of fetal microdeletion and microduplication syndromes among pregnant women with advanced maternal ages].

To assess the value of combined chromosomal karyotyping and chromosomal microarray analysis (CMA) and/or copy number variation sequencing (CNV-seq) for the prenatal diagnosis for women with advanced maternal ages, and to explore the challenges of prenatal genetic counseling brought by the types of fetal CNVs and uncertainty of related phenotypes. A retrospective analysis was carried out on 1 841 women with advanced maternal age who underwent interventional prenatal diagnosis at the Prenatal Diagnosis Center of Xiamen University Affiliated Women and Children's Hospital from January 2017 to December 2020. Routine chromosomal karyotyping analysis and CMA/CNV-seq detection were carried out. CMA/CNV-seq had detected pathogenic variants in 2 cases which had failed karyotyping analysis. Two hundred and twenty one fetal chromosomal abnormalities were detected by karyotyping analysis, among which 187 were detected by CMA/CNV-seq. CMA/CNV-seq analysis of 23 cases with balanced chromosome structural aberrations and 10 cases with low proportion mosaicisms (including a marker chromosome) had yielded a negative result. In addition, 26 cases (26/1 841, 1.4%) with pathogenic CNVs were discovered among those with a normal karyotype, of which 13 (50.0%) were recurrent CNVs associated with neurocognitive impairment, with 22q11.21 microdeletions and microduplications being the most common types (26.92%). The combination of karyotyping analysis and CMA/CNV-seq not only increased the rate of prenatal diagnosis, but also complemented with each other, which has facilitated genetic counseling and formulation of prenatal diagnosis strategy for the affected families.

Detection of chromosomal instability using ultrasensitive chromosomal aneuploidy detection in the diagnosis of precancerous lesions of gastric cancer.

The diagnosis of Precancerous Lesions of Gastric Cancer (PLGC) is challenging in clinical practice. We conducted a clinical study by analyzing the information of relevant chromosome copy number variations (CNV) in the TCGA database followed by the UCAD technique to evaluate the value of Chromosomal Instability (CIN) assay in the diagnosis of PLGC. Based on the screening of gastric cancer related data in TCGA database, CNV analysis was performed to explore the information of chromosome CNV related to gastric cancer. Based on the gastroscopic pathology results, 12 specimens of patients with severe atrophy were screened to analyze the paraffin specimens of gastric mucosa by UCAD technology, and to explore the influence of related factors on them. The results of CNV in TCGA database suggested that chromosome 7, 8, and 17 amplification was obvious in patients with gastric cancer. UCAD results confirmed that in 12 patients with pathologic diagnosis of severe atrophy, five of them had positive results of CIN, with a positive detection rate of 41.7%, which was mainly manifested in chromosome seven and chromosome eight segments amplification. We also found that intestinalization and HP infection were less associated with CIN. And the sensitivity of CIN measurement results was significantly better than that of tumor indicators. The findings suggest that the diagnosis of PLGC can be aided by UCAD detection of CIN, of which Chr7 and 8 may be closely related to PLGC.

Associations between genomic aberrations, increased nuchal translucency, and pregnancy outcomes: a comprehensive analysis of 2,272 singleton pregnancies in women under 35.

Regarding increased nuchal translucency (NT), the cutoff values used are heterogeneous in clinical practice, this study aims to assess the efficacy of prenatal detection for chromosomal abnormalities and pregnancy outcomes in fetuses with varying NT thicknesses, in order to provide data that supports informed prenatal diagnosis and genetic counseling for such cases. We included 2,272 pregnant women under 35 with singleton pregnancies who underwent invasive prenatal diagnosis between 2014 and 2022. The cohort comprised 2,010 fetuses with increased NT (≥2.5 mm) and 262 fetuses with normal NT but exhibiting a single soft marker. Prenatal diagnoses were supported by chromosomal microarray (CMA) and copy number variation sequencing (CNV-seq) analyses. The detection rates of numerical chromosomal abnormalities were 15.4% (309/2,010) and 17.3% (297/1,717) in the NT ≥2.5 and ≥ 3.0 groups, respectively. Pathogenic/likely pathogenic CNV incidence increased with NT thickness (χ2 = 8.60, p < 0.05), peaking at 8.7% (22/254) in the NT 4.5-5.4 mm group. Structural defects were found in 18.4% of fetuses with NT values between 2.5 mm and 2.9 mm. Chromosomal abnormality rates in the isolated increased NT groups of 2.5-2.9 mm and 3.0-3.4 mm were 6.7% (16/239) and 10.0% (47/470), respectively, with no statistical significance (χ2 = 2.14, p > 0.05). Fetuses with NT thickness between 2.5 and 2.9 mm combined with the presence of soft markers or non-lethal structural abnormalities exhibited a significantly higher chromosomal abnormality risk (19.0%) compared to fetuses with isolated increased NT ranging from 3.5 to 4.4 mm (13.0%). Pregnancy termination rates increased with NT thickness (χ2 = 435.18, p < 0.0001), ranging from 12.0% (30/249) in the NT 2.5-2.9 mm group to 87.0% (141/162) in the NT ≥ 6.5 mm group. CMA or CNV-seq exhibited good performance in identifying genomic aberrations in pregnancies with increased NT thickness. NT ranging from 2.5 mm to 2.9 mm elevated the risk of fetal chromosomal abnormalities, particularly when combined with other soft markers.

Initial clinical and molecular investigation of 20q13.33 microdeletion with 17q25.3/14q32.31q32.33 microduplication in Chinese pediatric patients.

Limited research has been conducted regarding the elucidation of genotype-phenotype correlations within the 20q13.33 region. The genotype-phenotype association of 20q13.33 microdeletion remains inadequately understood. In the present study, two novel cases of 20q13.33 microdeletion were introduced, with the objective of enhancing understanding of the genotype-phenotype relationship. Two unrelated patients with various abnormal clinical phenotypes from Fujian province Southeast China were enrolled in the present study. Karyotype analysis and chromosomal microarray analysis (CMA) were performed to investigate chromosomal abnormalities and copy number variants. The results of high-resolution G-banding karyotype analysis elicited a 46,XY,der(20)add(20)(q13.3) in Patient 1. This patient exhibited various clinical manifestations, such as global developmental delay, intellectual disability, seizures, and other congenital diseases. Subsequently, a 1.0-Mb deletion was identified in the 20q13.33 region alongside a 5.2-Mb duplication in the 14q32.31q32.33 region. In Patient 2, CMA results revealed a 1.8-Mb deletion in the 20q13.33 region with a 4.8-Mb duplication of 17q25.3. The patient exhibited additional abnormal clinical features, including micropenis, congenital heart disease, and a distinctive crying pattern characterized by a crooked mouth. In the present study, for the first time, an investigation was conducted into two novel cases of 20q13.33 microdeletion with microduplications in the 17q25.3 and 14q32.31q32.33 regions in the Chinese population. The presence of micropenis may be attributed to the 20q13.33 microdeletion, potentially expanding the phenotypic spectrum associated with this deletion.

Abstract 5049: Detecting chromosomal copy number variations and point mutations in Glioma using a single assay; sparing tissue while significantly reducing testing time and cost

Abstract We aimed to develop a single streamlined assay to detect all Glioma biomarkers. The current diagnosis of Gliomas incorporates molecular biomarkers that include whole chromosomal copy number variations (CNVs) and single nucleotide variations (SNVs). Testing CNVs and SNVs are traditionally done using different large platforms and are performed in large genomic facilities that have the suitable infrastructure. This creates delays in testing, where samples undergo lengthy referrals and the tissue is often split between institutions, which also adds substantially to the costs. We have previously used Nanopore sequencing to develop an assay to detect IDH SNVs from formalin-fixed paraffin-embedded (FFPE) tissue. Nanopore sequencing platform uses small inexpensive tools and can test for both CNVs and SNVs. However, combining both targets efficiently in FFPE tissue remains a challenge. While nanopore whole genome sequencing can detect CNVs, it does not allow for enough coverage to accurately detect small SNVs. Also, raw FFPE DNA is not compatible with the nanopore technology. To fix both challenges we used a PCR based approach that allows enrichment of the SNV targets, as well as creates new nanopore compatible DNA copies. To counteract the bias that PCR creates with uneven amplification, we used a SNP based method to call chromosomal gains and losses. Statistical analysis of probability showed that targeting 15 heterozygous SNPs per chr arm is sufficient for accurate CNV detection (error rate &amp;lt;0.1%). A total of 57 amplicons targeting CNVs on Chr 1,7, 10, 19, and EGFR gene; and IDH1 and IDH2 SNVs were pooled together in one assay and tested on 4 Glioma samples. Variant calling was performed with a custom Python script. All CNV targets were accurately detected on all samples. Losses in chr 1p, 19q and chr 10 were evident with a loss of heterozygosity (LOH) pattern on the SNP analysis. The gain in chromosome 7 was also observed as an allele gain 2:1 SNP pattern on the corresponding samples. The SNV statuses of these samples were also accurately detected when compared to reference results. Finally, the SNV and CNV calling analysis were streamlined using custom shell script, further reducing total turnaround time (including sequencing and data analysis) to less than a day for the entire batch of samples. Initial cost analysis shows &amp;lt;50% of the traditional testing costs for the assay. This work is the first to develop a streamlined single test to detect CNVs and SNVs in Gliomas using FFPE DNA on the nanopore sequencing platform. The shorter testing time, streamlined workflow, and low assay/capital cost could positively influence the care of brain cancer patients. Notably, nanopore-based sequencing tools can also be implemented in other types of tumor diagnosis, especially by smaller labs, helping to overcome many of the existing diagnostic challenges. Citation Format: Mashiat L. Mimosa, Jared T. Simpson, Karel Boissinot, Mora Tiab, Ramzi Fattouh, Rola M. Saleeb. Detecting chromosomal copy number variations and point mutations in Glioma using a single assay; sparing tissue while significantly reducing testing time and cost [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5049.

Abstract 6944: Transcriptional programs underpinning reoccurring cancer cell states, plasticity and tumour phylogenies in clear cell kidney cancer

Abstract Intratumor heterogeneity (ITH) is a substrate for tumor evolution and confers amenability to stressors and therapies. Sources of ITH include subclonal mutations, microenvironmental factors, and plasticity whereby a cell has the ability to adopt different cell states. Clear cell kidney cancer (ccRCC) has a well-characterized genetic initiating event - VHL inactivation - resulting in activation of HIF-1 and HIF-2 transcription factors. Although VHL inactivation is consistently clonal in nature, ccRCC nonetheless exhibits extensive genetic and morphological ITH. This makes ccRCC an excellent model in which to study ITH. We performed single cell RNA-seq on multiregional samples from kidney tumors and macroscopically normal kidney tissue. We obtained 55 samples from 16 tumors and used the 10x Genomics platform to obtain gene expression data from ~150,000 single cells, including ~80,000 cancer and normal epithelial cells. We identified three cancer cell states, co-existing in all clear cell kidney cancer tumors analyzed and marked by different gene expression programs: 1) differentiated-like cells marked by proximal tubule cell genes characteristic of the cancer cell of origin; 2) epithelial-mesenchymal transition (EMT)-like cells and 3) injured-like cells. Differentiated markers and EMT markers were associated with good and poor prognosis, respectively, indicating that these cell subpopulations are clinically important. Notably, these cell states are also present in the normal proximal tubule although in different proportions to the tumor samples, which contain more cells in the EMT-like and injured-like states. This suggests that the core features of ITH are inherent to the cell of origin of the cancer, but that the cell state trajectories are dysregulated in the tumor. Furthermore, in tumors these states exist as a continuum, whereby transitional cells in between states appear to exist, suggesting that cancer cells are continually and dynamically transitioning between states. Lastly, we used chromosomal copy number variation (CNV) patterns to study genetic ITH. We examined the effect of 14q loss, which is associated with ccRCC metastasis, on gene expression. We found that 14q loss is associated with downregulation of HIF-1 transcriptional activity together with a reciprocal increase in HIF-2 activity, MYC activity and oxidative phosphorylation, highlighting potential mechanisms by which 14q loss may driver ccRCC metastasis. Citation Format: Olivia Lombardi, Ran Li, Lisa Browning, Faiz Jabbar, Hannah Evans, Peter J. Ratcliffe, David R. Mole. Transcriptional programs underpinning reoccurring cancer cell states, plasticity and tumour phylogenies in clear cell kidney cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6944.

Abstract 417: Chromosomal instability leads to durable tumor suppression upon macrophage-checkpoint disruption, with induction of anti-tumor IgG

Abstract Solid tumors generally exhibit chromosome copy number variation caused by chromosomal instability (CIN) in mitosis, and the resulting aneuploidy associates with poor prognosis in various cancer types and poor Tcell checkpoint blockade response in melanoma. In such contexts, however, macrophages and the SIRPa-CD47 checkpoint are understudied. Here, CIN is induced pharmacologically in poorly immunogenic B16F10 mouse melanoma cells, generating micronuclei plus diverse aneuploidy and skewing macrophages towards an anti-cancer phenotype based on markers and short-term tumor studies. Mice bearing CIN-afflicted tumors with wild-type CD47 levels survive only slightly longer compared to chromosomally stable controls, but long-term survival can be maximized when anti-tumor IgG opsonization is combined with adoptive transfer of macrophages with SIRPa blockade or with CD47 knockout of the B16F10. Multi-epitope, de novo anti-cancer IgG in survivors promote phagocytosis of CD47 knockout B16F10 cells by macrophages and suppress tumoroids in vitro and growth of tumors in vivo. An unexpected benefit of pairing CIN with maximal macrophage anti-cancer activity is thus an anti-cancer vaccination-like response that can lead to durable cures and potentiate cell-mediated acquired immunity. Citation Format: Dennis E. Discher. Chromosomal instability leads to durable tumor suppression upon macrophage-checkpoint disruption, with induction of anti-tumor IgG [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 417.

A novel method for simultaneous detection of hematological tumors and infectious pathogens by metagenomic next generation sequencing of plasma

BackgroundMetagenomic next-generation sequencing (mNGS) is valuable for pathogen identification; however, distinguishing between infectious diseases and conditions with potentially similar clinical manifestations, including malignant tumors, is challenging. Therefore, we developed a method for simultaneous detection of infectious pathogens and cancer in blood samples. MethodsPlasma samples (n = 244) were collected from 150 and 94 patients with infections and hematological malignancies, respectively, and analyzed by mNGS for pathogen detection, alongside human tumor chromosomal copy number variation (CNV) analysis (≥5Mbp or 10Mbp CNV region). Further, an evaluation set, comprising 87 plasma samples, was analyzed by mNGS and human CNV analysis, to validate the feasibility of the method. ResultsAmong 94 patients with hematological malignancy, sensitivity values of CNV detection for tumor diagnosis were 69.15 % and 32.98 % for CNV region 5Mbp and 10Mbp, respectively, with corresponding specificities of 92.62 % and 100 % in the infection group. Area under the ROC curve (AUC) values for 5Mbp and 10Mbp region were 0.825 and 0.665, respectively, which was a significant difference of 0.160 (95 % CI: 0.110–0.210; p < 0.001), highlighting the superiority of 5Mbp output region data. Six patients with high-risk CNV results were identified in the validation study: three with history of tumor treatment, two eventually newly-diagnosed with hematological malignancies, and one with indeterminate final diagnosis. ConclusionsConcurrent CNV analysis alongside mNGS for infection diagnosis is promising for detecting malignant tumors. We recommend adopting a CNV region of 10Mbp over 5Mbp for our model, because of the lower false-positive rate (FPR).

[Analysis of X chromosome inactivation and prenatal diagnosis for a Chinese pedigree with loss of heterozygosity at Xq22.1q22.3].

To explore the correlation between skewed X chromosome inactivation (XCI) and clinical phenotype of a Chinese pedigree with loss of heterozygosity at Xq22.1q22.3. A pedigree diagnosed at Taizhou Hospital on November 10, 2021 was selected as the study subject. G-banded chromosomal karyotyping and copy number variation sequencing (CNV-seq) were carried out to analyze the amniotic fluid and peripheral blood samples from the couple. XCI was detected by PCR amplification of CAG repeats in exon 1 of androgen receptor gene before and after the digestion with methylation-sensitive restriction enzyme Hpa II. Correlation between the genotype and clinical phenotype was analyzed. The karyotypes of the pregnant woman and the fetus were both determined as 46,X,del(X)(q22), and the result of CNV-seq was seq[hg19]del(X)(q22.1q22.3) chrX: g.10046000_105740000del, suggesting that both had harbored a 5.28 Mb deletion on the X chromosome. No obvious abnormality was found in the husband. XCI analysis showed that the activity ratio of the two X chromosomes of the pregnant woman and her fetus was 0 : 100. The X chromosome harboring the q22.1q22.3 deletion was completely inactivated, and the inactivated X chromosome of the fetus was derived from its mother. The fetus has harbored a maternally derived inactivated X chromosome del(X)(q22) , and its phenotype is closely associated with the activity of the abnormal X chromosome. Pedigree XCI analysis combined with the clinical phenotype has facilitated recognition of the maternal phenotype and prognosis of female fetus with loss of heterozygosity at Xq22.1q22.3.

[Genetic analysis and prenatal diagnosis for a Chinese pedigree affected with co-morbid Ornithine carbamoyl transferase deficiency and MECP2 duplication syndrome].

To explore the genetic basis for a Chinese pedigree affected with co-morbid Ornithine carbamoyl transferase deficiency (OTCD) and MECP2 duplication syndrome. A proband who was admitted to the Neonatal Intensive Care Unit of Gansu Provincial Maternal and Child Health Care Hospital on December 19, 2017 was selected as the study subject. High-throughput sequencing and multiplex ligation-dependent probe amplification (MLPA) were carried out for her pedigree, and short tandem repeat-based linkage analysis and chromosome copy number variation sequencing (CNV-seq) were used for the prenatal diagnosis. The proband, a 3-day-old female, was found to harbor heterozygous deletion of exons 7-9 of the OTC gene. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), the variant was classified as likely pathogenic (PVS1+PM2_Supporting+PP4). The proband was diagnosed with OTCD , which was in keeping with her acute encephalopathy and metabolic abnormalities (manifesting as hyperammonemia, decreased blood citrulline, and increased urine orotic acid). Prenatal diagnosis was carried out for the subsequent pregnancy. The fetus did not harbor the exons 7-9 deletion of the OTC gene, but was found to carry a duplication in Xq28 region (which encompassed the whole region of MECP2 duplication syndrome) and was positive for the SRY sequence. The same duplication was also found in the proband and her mother. Considering the possible existence of X-chromosome inactivation, the proband was diagnosed with two X-linked recessive disorders including OTCD and MECP2 duplication syndrome, and the fetus was determined as a male affected with the MECP2 duplication syndrome. Discoveries of the pathogenic variants underlying the OTCD and MECP2 duplication syndrome have enabled clinical intervention, treatment, genetic counseling and prenatal diagnosis for this pedigree.

Appropriate whole genome amplification and pathogenic loci detection can improve the accuracy of preimplantation genetic diagnosis for deletional α-thalassemia.

To improve the accuracy of preimplantation genetic testing (PGT) in deletional α-thalassemia patients. Article. fifty-two deletional α-thalassemia couples. Whole genome amplification (WGA), Next-generation sequencing (NGS) and PCR mutation loci detection. WGA, Single nucleotide polymorphism (SNP) and PCR mutation loci detection results; Analysis of embryo chromosome copy number variation (CNV). Multiple Displacement Amplification (MDA) and Multiple Annealing and Looping-Based Amplification Cycles (MALBAC) methods for PGT for deletional α-thalassemia. Blastocyst biopsy samples (n = 253) were obtained from 52 deletional α-thalassemia couples. The results of the comparison of experimental data between groups MALBAC and MDA are as follows: (i) The average allele drop-out (ADO) rate, MALBAC vs. MDA = 2.27% ± 3.57% vs. 0.97% ± 1.4%, P=0.451); (ii) WGA success rate, MALBAC vs. MDA = 98.61% vs. 98.89%, P=0.851; (iii) SNP haplotype success rate, MALBAC vs. MDA = 94.44% vs. 96.68%, P=0.409; (iv) The result of SNP haplotype analysis is consistent with that of Gap-PCR/Sanger sequencing results, MALBAC vs. MDA = 36(36/72, 50%) vs. 151(151/181, 83.43%), P=0; (v) Valid SNP loci, MALBAC vs. MDA = 30 ± 9 vs. 34 ± 10, P=0.02; (vi) The mean CV values, MALBAC vs. MDA = 0.12 ± 0.263 vs. 0.09 ± 0.40, P=0.916; (vii) The average number of raw reads, MALBAC vs. MDA =3244259 ± 999124 vs. 3713146 ± 1028721, P=0; (viii) The coverage of genome (%), MALBAC vs. MDA = 5.02 ± 1.09 vs. 5.55 ± 1.49, P=0.008. Our findings indicate that MDA is superior to MALBAC for PGT of deletional α-thalassemia. Furthermore, SNP haplotype analysis combined with PCR loci detection can improve the accuracy and detection rate of deletional α-thalassemia.

Comprehensive single-cell analysis reveals heterogeneity of fibroblast subpopulations in ovarian cancer tissue microenvironment

BackgroundOvarian cancer, as a highly malignant tumor, features the critical involvement of tumor-associated fibroblasts in the ovarian cancer tissue microenvironment. However, due to the apparent heterogeneity within fibroblast subpopulations, the specific functions of these subpopulations in the ovarian cancer tissue microenvironment remain insufficiently elucidated. MethodsIn this study, we integrated single-cell sequencing data from 32 ovarian cancer samples derived from four distinct cohorts and 3226 bulk RNA-seq data from GEO and TCGA-OV cohorts. Utilizing computational frameworks such as Seurat, Monocle 2, Cellchat, and others, we analyzed the characteristics of the ovarian cancer tissue microenvironment, focusing particularly on fibroblast subpopulations and their differentiation trajectories. Employing the CIBERSORTX computational framework, we assessed various cellular components within the ovarian cancer tissue microenvironment and evaluated their associations with ovarian cancer prognosis. Additionally, we conducted Mendelian randomization analysis based on cis-eQTL to investigate causal relationships between gene expression and ovarian cancer. ResultsThrough integrative analysis, we identified 13 major cell types present in ovarian cancer tissues, including CD8+ T cells, malignant cells, and fibroblasts. Analysis of the tumor microenvironment (TME) cell proportions revealed a significant increase in the proportion of CD8+ T cells and CD4+ T cells in tumor tissues compared to normal tissues, while fibroblasts predominated in normal tissues. Further subgroup analysis of fibroblasts identified seven subgroups, with the MMP11+Fib subgroup showing the highest activity in the TGFβ signaling pathway. Single-cell analysis suggested that oxidative phosphorylation could be a key pathway driving fibroblast differentiation, and the ATRNL1+KCN + Fib subgroup exhibited chromosomal copy number variations. Prognostic analysis using a large sample size indicated that high infiltration of MMP11+ fibroblasts was associated with poor prognosis in ovarian cancer. SMR analysis identified 132 fibroblast differentiation-related genes, which were linked to pathways such as platinum drug resistance. ConclusionsIn the context of ovarian cancer, fibroblasts expressing MMP11 emerge as the primary drivers of the TGF-beta signaling pathway. Their presence correlates with an increased risk of adverse ovarian prognoses. Additionally, the genetic regulation governing the differentiation of fibroblasts associated with ovarian cancer correlates with the emergence of drug resistance.

3q29 duplications: A cohort of 46 patients and a literature review.

Duplications of the 3q29 cytoband are rare chromosomal copy number variations (CNVs) (overlapping or recurrent ~1.6 Mb 3q29 duplications). They have been associated with highly variable neurodevelopmental disorders (NDDs) with various associated features or reported as a susceptibility factor to the development of learning disabilities and neuropsychiatric disorders. The smallest region of overlap and the phenotype of 3q29 duplications remain uncertain. We here report a French cohort of 31 families with a 3q29 duplication identified by chromosomal microarray analysis (CMA), including 14 recurrent 1.6 Mb duplications, eight overlapping duplications (>1 Mb), and nine small duplications (<1 Mb). Additional genetic findings that may be involved in the phenotype were identified in 11 patients. Focusing on apparently isolated 3q29 duplications, patients present mainly mild NDD as suggested by a high rate of learning disabilities in contrast to a low proportion of patients with intellectual disabilities. Although some are de novo, most of the 3q29 duplications are inherited from a parent with a similar mild phenotype. Besides, the study of small 3q29 duplications does not provide evidence for any critical region. Our data suggest that the overlapping and recurrent 3q29 duplications seem to lead to mild NDD and that a severe or syndromic clinical presentation should warrant further genetic analyses.

Multi-omics Analysis Reveals Immune Features Associated with Immunotherapy Benefit in Patients with Squamous Cell Lung Cancer from Phase III Lung-MAP S1400I Trial.

Identifying molecular and immune features to guide immune checkpoint inhibitor (ICI)-based regimens remains an unmet clinical need. Tissue and longitudinal blood specimens from phase III trial S1400I in patients with metastatic squamous non-small cell carcinoma (SqNSCLC) treated with nivolumab monotherapy (nivo) or nivolumab plus ipilimumab (nivo+ipi) were subjected to multi-omics analyses including multiplex immunofluorescence (mIF), nCounter PanCancer Immune Profiling Panel, whole-exome sequencing, and Olink. Higher immune scores from immune gene expression profiling or immune cell infiltration by mIF were associated with response to ICIs and improved survival, except regulatory T cells, which were associated with worse overall survival (OS) for patients receiving nivo+ipi. Immune cell density and closer proximity of CD8+GZB+ T cells to malignant cells were associated with superior progression-free survival and OS. The cold immune landscape of NSCLC was associated with a higher level of chromosomal copy-number variation (CNV) burden. Patients with LRP1B-mutant tumors had a shorter survival than patients with LRP1B-wild-type tumors. Olink assays revealed soluble proteins such as LAMP3 increased in responders while IL6 and CXCL13 increased in nonresponders. Upregulation of serum CXCL13, MMP12, CSF-1, and IL8 were associated with worse survival before radiologic progression. The frequency, distribution, and clustering of immune cells relative to malignant ones can impact ICI efficacy in patients with SqNSCLC. High CNV burden may contribute to the cold immune microenvironment. Soluble inflammation/immune-related proteins in the blood have the potential to monitor therapeutic benefit from ICI treatment in patients with SqNSCLC.

Selection of ethanol tolerant strains of Candida albicans by repeated ethanol exposure results in strains with reduced susceptibility to fluconazole.

Candida albicans is a commensal yeast that has important impacts on host metabolism and immune function, and can establish life-threatening infections in immunocompromised individuals. Previously, C. albicans colonization has been shown to contribute to the progression and severity of alcoholic liver disease. However, relatively little is known about how C. albicans responds to changing environmental conditions in the GI tract of individuals with alcohol use disorder, namely repeated exposure to ethanol. In this study, we repeatedly exposed C. albicans to high concentrations (10% vol/vol) of ethanol-a concentration that can be observed in the upper GI tract of humans following consumption of alcohol. Following this repeated exposure protocol, ethanol small colony (Esc) variants of C. albicans isolated from these populations exhibited increased ethanol tolerance, altered transcriptional responses to ethanol, and cross-resistance/tolerance to the frontline antifungal fluconazole. These Esc strains exhibited chromosomal copy number variations and carried polymorphisms in genes previously associated with the acquisition of fluconazole resistance during human infection. This study identifies a selective pressure that can result in evolution of fluconazole tolerance and resistance without previous exposure to the drug.

Limited ability of increased sequencing depth in detecting cases missed by noninvasive prenatal testing: a comparative analysis of 3 clinical cases

Increased sequencing depth can improve the detection rate of noninvasive prenatal testing (NIPT) for chromosome aneuploidies and copy number variations (CNVs). However, due to the technical limitations of NIPT, false-positives and false-negatives are inevitable. False-positives for aneuploidy and CNVs have been widely reported, but few missed cases have been reported. In this study, we report 3 patients missed by NIPT, which were still missed after increasing the sequencing depth. To verify the detection efficiency of the platform, the results of NIPT in 32,796 patients treated in Yulin Women and Children Health Care Hospital from 2020 to 2022 were retrospectively analyzed. Data on false-negative cases found by postnatal follow-up or amniocentesis were collected, and the sequencing data, pregnancy examination data, and postnatal follow-up results of these missed patients were summarized. Five patients missed by NIPT were found, and they were missed again by retesting or increasing the sequencing depth. Except for hypospadias found in 1 patient, ultrasonography of the other 4 patients showed no obvious abnormalities during the whole pregnancy. Our results suggest that pregnant women should be fully informed of the benefits and limitations of NIPT before undergoing the examination to avoid unnecessary medical disputes.

Prenatal diagnosis of dent disease type I with a nonsense pathogenic variant in CLCN5: a case study

IntroductionDent disease type I is a rare X-linked recessive renal tubular disease resulting from pathogenic variants in the CLCN5 gene. Due to the rarity of Dent disease type I and the diversity of its phenotypes, its clinical diagnosis is complex and poses a challenge to clinicians.MethodsA foetus and a child from a 36-year-old pregnant woman with a birth history of abnormal children were enrolled in this study. Pregnant women undergo amniocentesis for prenatal diagnosis at the gestational age of 12+ 3 weeks. Chromosomal microarray (CMA) analysis and whole-exome sequencing (WES) were employed to investigate the chromosomal copy number and single gene variants. Literature retrieval and data analysis were performed for genotype and phenotype collection analysis.ResultsNo chromosomal abnormalities or CNVs were detected in the entire family through karyotype and familial CMA analyses. WES identified a nonsense pathogenic variant in CLCN5 of the X chromosome, c.1942 C > T (exon 11, NM_000084), which was inherited from his mother, who exhibited regular clinical features.ConclusionThis study suggests that children with low-molecular-weight proteinuria and hypercalciuria should undergo prompt genetic testing to exclude Dent disease.

Inherent genome instability underlies trisomy 21-associated myeloid malignancies.

Constitutional trisomy 21 (T21) is a state of aneuploidy associated with high incidence of childhood acute myeloid leukemia (AML). T21-associated AML is preceded by transient abnormal myelopoiesis (TAM), which is triggered by truncating mutations in GATA1 generating a short GATA1 isoform (GATA1s). T21-associated AML emerges due to secondary mutations in hematopoietic clones bearing GATA1s. Since aneuploidy generally impairs cellular fitness, the paradoxically elevated risk of myeloid malignancy in T21 is not fully understood. We hypothesized that individuals with T21 bear inherent genome instability in hematopoietic lineages that promotes leukemogenic mutations driving the genesis of TAM and AML. We found that individuals with T21 show increased chromosomal copy number variations (CNVs) compared to euploid individuals, suggesting that genome instability could be underlying predisposition to TAM and AML. Acquisition of GATA1s enforces myeloid skewing and maintenance of the hematopoietic progenitor state independently of T21; however, GATA1s in T21 hematopoietic progenitor cells (HPCs) further augments genome instability. Increased dosage of the chromosome 21 (chr21) gene DYRK1A impairs homology-directed DNA repair as a mechanism of elevated mutagenesis. These results posit a model wherein inherent genome instability in T21 drives myeloid malignancy in concert with GATA1s mutations.