Comparative Genomic Hybridization Analysis Research Articles

(058) Evaluation of Pathway Expression Following Down-Regulation of NELL1, a Potential Gene of Interest in Peyronie’s Disease

Abstract Introduction Peyronie’s Disease (PD) is a superficial fibrosing disorder characterized by inelastic fibrous plaques within the tunica albuginea. We previously performed high-resolution array comparative genomic hybridization analysis of genomic DNA from men with both PD and Dupuytren’s Disease (DD), yielding candidate genes, including NELL1, with potential involvement in these conditions. NELL1 is a secreted growth factor that enhances osteoblastic differentiation and chondrogenesis and may contribute to calcification and inflammation in fibrotic plaques via its interaction with bone morphogenetic protein (BMP) within the TGF-β signaling pathway. Here, we report on pathway analysis following up- and down-regulation of NELL1 using CRISPR modification in an in vitro setting. Objective This study aimed to investigate the influence of NELL1 modification on downstream gene regulation, focusing on pathways that were significantly regulated following NELL1 down-regulation. Methods For down-regulation of NELL1, gRNAs were delivered to HEK293 cells with a nuclease deficient (d) dCas9-KRAB system adapted from Streptococcus pyogenes. Gene regulation was assessed using qRT-PCR. Results For down-regulation of NELL1, PCR analysis confirmed a 95% reduction in expression. Non-target vectors did not change NELL1 expression levels. RNA sequencing results found over 5,000 differentially expressed genes between, with significant changes in multiple pathways, including the HIF-1α pathway, which promotes keloid development by activating the TGF-β/Smad pathways. Additionally, we identified pathways related to ECM-receptor interaction, inflammation, apoptosis, and cell cycle regulation that are mediated by TGF-β/Smad signaling, which plays a crucial role in fibrosis. Our analysis also demonstrated upregulation of the PI3K-Akt pathway, which is involved in regulating type I collagen expression in response to TGF-β1. These results highlight the significance of NELL1 in fibrotic regulation and suggest its potential involvement in the development of PD. Conclusions We have created guides for and successfully down-regulated the expression of NELL1, a gene of interest in Peyronie’s disease. We have also demonstrated, through RNA sequencing, that changes in NELL1 lead to large changes in gene and pathway expression. The identified modified pathways, including HIF-1α-mediated, TGF-β/Smad and PI3K-Akt, provide valuable insights into the potential molecular mechanisms underlying NELL1's role in regulation of fibrosis, and further investigation of these pathways may reveal potential therapeutic targets for fibrosis-related disorders. Disclosure Any of the authors act as a consultant, employee or shareholder of an industry for: Contraline, Techfields Inc, Alto Neurosciences, Curebase.

Comparative genomic hybridisation and transcriptome microarray analysis in triple negative breast cancer: An INDIAN study.

Triple-negative breast cancer (TNBC), which accounts for approximately 15–20% of all breast cancers, defined as lack of expression of estrogen receptor, progesterone receptor and Her-2 neu receptors. TNBC has two subtypes basal like and non-basal like, the former characterised by aggressive biology with limited therapeutic options. This study explored molecular markers involved in pathogenesis of TNBC and investigated novel potential diagnostic and therapeutic targets by CGH array and transcriptome array. aCGH analysis in TNBC demonstrated genes amplified were 3888, number of pathway hits was 1554 and major pathways amplified was found to be WNT signalling pathway and Cadherin signalling pathway. Among all metastatic sites and remission, activation of WNT signalling pathway is commonly observed. TNBC exhibited 1486 copy number variations (CNVR) which is approximately 250 times higher than controls. More than 20 CNVR was observed in all chromosomes and more than 80 CNVR was observed in Chr 1 to Chr 4, Chr 7, Chr 11 and Chr X. Common CNVR associated with amplified regions in Chr 22, Chr 14, Chr 8 and Chr 2 was observed in TNBC and CNVR associated with Chr 22q11.22–23, Chr 6p21.32–33, Chr11q12.2, Chr14q32.22–23, Chr 8p11.22–23, was observed in metastatic disease. In transcriptome array analysis a total of 11,359 differentially expressed genes with fold change 2.0 were in observed in TNBC comprise of 7639 upregulated genes and 3720 downregulated genes. Further, with fold change 10, 1526 upregulated genes and 839 down regulated genes were identified. Panther pathway analysis identified the main pathways of upregulated genes were Wnt signalling pathway, Integrin signalling pathway and Cadherin signalling pathway. The main pathways of down regulated genes were Inflammation mediated by chemokine and cytokine signalling pathways. PPI network shows that COL12A1, COL6A3, FN1, MMP3, WNT5A were key upregulated genes and ITGB7, PTPRC, ITGA4, LCK and CD247 were key down regulated genes. Cytoscape analysis followed by multiple list comparator tool identified top 5 significant hub genes were FN1, MMP3, COLL11A1, COL12A1 and COL3A1. The significant pathway genes obtained by CGH array and transcriptome array when compared, exhibited 5 common genes COL4A1, FN1, COL6A3, COL5A2 and PCDH7. These genes were not overexpressed in Controls and therefore involved in pathogenesis of TNBC. Expressions of these genes were validated by studying protein expression by immunohistochemistry. FN1 and COL6A3 protein over expression predicted worse DFS in TNBC and can be considered as therapeutic targets at diagnosis to reduce the disease metastases. These findings provide new insights into the pathogenesis of TNBC and guide for selection of targets related to diagnosis, prognosis and prediction of treatment in TNBC.

Neuroblastoma Patients' Outcome and Chromosomal Instability.

Chromosomal instability (CIN) induces a high rate of losses or gains of whole chromosomes or parts of chromosomes. It is a hallmark of most human cancers and one of the causes of aneuploidy and intra-tumor heterogeneity. The present study aimed to evaluate the potential prognostic role of CIN in NB patients at diagnosis. We performed array comparative genomic hybridization analyses on 451 primary NB patients at the onset of the disease. To assess global chromosomal instability with high precision, we focused on the total number of DNA breakpoints of gains or losses of chromosome arms. For each tumor, an array-CGH-based breakpoint instability index (BPI) was assigned which defined the total number of chromosomal breakpoints per genome. This approach allowed us to quantify CIN related to whole genome disruption in all NB cases analyzed. We found differences in chromosomal breakages among the NB clinical risk groups. High BPI values are negatively associated with survival of NB patients. This association remains significant when correcting for stage, age, and MYCN status in the Cox model. Stratified analysis confirms the prognostic effect of BPI index in low-risk NB patients with non-amplified MYCN and with segmental chromosome aberrations.

The KAT6B::KANSL1 Fusion Defines a New Uterine Sarcoma With Hybrid Endometrial Stromal Tumor and Smooth Muscle Tumor Features

Neoplasms harboring a KAT6B/A::KANSL1 fusion were initially reported as benign (leiomyomas) and malignant (leiomyosarcomas, low-grade endometrial stromal sarcomas [LG-ESSs]) uterine neoplasms. However, they may represent an emerging entity characterized by clinical aggressiveness contrasting with a rather reassuring microscopic appearance. Here, we aimed to confirm that this neoplasm is a distinct clinicopathologic and molecular sarcoma and identify criteria that should alert pathologists and lead to KAT6B/A::KANSL1 fusion testing in routine practice. Therefore, we conducted a comprehensive clinical, histopathologic, immunohistochemical, and molecular study, including array comparative genomic hybridization, whole RNA-sequencing, unsupervised clustering, and cDNA mutational profile analyses of 16 tumors with KAT6B::KANSL1 fusion from 12 patients. At presentation, patients were peri-menopausal (median, 47.5 years), and the primary tumors were located in the uterine corpus (12/12, 100%), with an additional prevesical location in 1 (8.3%) of 12 cases. The relapse rate was 33.3% (3/9). All tumors (16/16, 100%) showed morphologic and immunohistochemical features overlapping between leiomyoma and endometrial stromal tumors. A whirling recurrent architecture (resembling fibromyxoid-ESS/fibrosarcoma) was found in 13 (81.3%) of 16 tumors. All tumors (16/16, 100%) exhibited numerous arterioliform vessels, and 13 (81.3%) of 18 had large hyalinized central vessels and collagen deposits. Estrogen and progesterone receptors were expressed in 16 (100%) of 16 and 14 (87.5%) of 16 tumors, respectively. Array comparative genomic hybridization performed on 10 tumors classified these neoplasms as simple genomic sarcomas. Whole RNA-sequencing on 16 samples and clustering analysis on primary tumors found that the KAT6B::KANSL1 fusion always occurred between exons 3 of KAT6B and 11 of KANSL1; no pathogenic variant was identified on cDNA, all neoplasms clustered together, close to LG-ESS, and pathway enrichment analysis showed cell proliferation and immune infiltrate recruitment pathway involvement. These results confirm that the sarcomas harboring a KAT6B/A::KANSL1 fusion represent a distinct clinicopathologic entity, close to LG-ESS but different, with clinical aggressiveness despite a reassuring morphology, for which the KAT6B/A::KANSL1 fusion is the molecular driver alteration.

Terminal microdeletion of chromosome 18 in a Malaysian boy characterized with few features of typical 18q- deletion syndrome: a case report

BackgroundThe 18q- deletion syndrome is a rare congenital chromosomal disorder caused by a partial deletion of the long arm of chromosome 18. The diagnosis of a patient with this syndrome relies on the family medical history, physical examination, developmental assessment, and cytogenetic findings. However, the phenotype of patients with 18q- deletion syndrome can be highly variable, ranging from almost normal to severe malformations and intellectual disability, and normal cytogenetic findings are common, thus complicating the diagnosis. Interestingly, only few characteristic features of typical 18q- deletion syndrome were found in the patient, despite sharing the same critical region. To our knowledge, this is the first report of a Malaysian individual with 18q- terminal microdeletion diagnosed with microarray-based technology.Case presentationHere we report a 16-year-old Malaysian Chinese boy, a product of a non-consanguineous marriage, who presented with intellectual disability, facial dysmorphism, high arched palate, congenital talipes equinovarus (clubfoot), congenital scoliosis, congenital heart defect, and behavioral problems. A routine chromosome analysis on 20 metaphase cells showed a normal 46, XY G-banded karyotype. Array-based comparative genomic hybridization was performed using a commercially available 244 K 60-mer oligonucleotide microarray slide according to the manufacturer’s protocol. This platform allows genome-wide survey and molecular profiling of genomic aberrations with an average resolution of about 10 kB. In addition, multiplex ligation-dependent probe amplification analysis was carried out using SALSA MLPA kit P320 Telomere-13 to confirm the array-based comparative genomic hybridization finding. Array-based comparative genomic hybridization analysis revealed a 7.3 MB terminal deletion involving chromosome band 18q22.3-qter. This finding was confirmed by multiplex ligation-dependent probe amplification, where a deletion of ten probes mapping to the 18q22.3-q23 region was detected, and further multiplex ligation-dependent probe amplification analysis on his parents showed the deletion to be de novo.ConclusionThe findings from this study expand the phenotypic spectrum of the 18q- deletion syndrome by presenting a variation of typical 18q- deletion syndrome features to the literature. In addition, this case report demonstrated the ability of the molecular karyotyping method, such as array-based comparative genomic hybridization, to assist in the diagnosis of cases with a highly variable phenotype and variable aberrations, such as 18q- deletion syndrome.

(153) Investigating the Role of NELL1 as a Gene of Interest in Peyronie’s Disease

Abstract Introduction Peyronie’s Disease (PD) is a superficial fibrosing disorder characterized by inelastic fibrous plaques within the tunica albuginea. We previously performed high-resolution array comparative genomic hybridization analysis of genomic DNA from men with both PD and Dupuytren’s Disease (DD), yielding candidate genes, including NELL1, with potential involvement in these conditions. NELL1 is a secreted growth factor that enhances osteoblastic differentiation and chondrogenesis and may contribute to calcification and inflammation in fibrotic plaques via its interaction with bone morphogenetic protein (BMP) within the TGF-β signaling pathway. Here, we describe the creation of lentiviral gRNA vectors for the up- and down-regulation of NELL1 in an in vitro setting, as well as PCR results for NELL1 regulation and preliminary results from RNA sequencing following CRISPR modification. Objective To determine the influence of NELL1 on extracellular matrix (ECM) deposition and downstream gene regulation. Methods For down-regulation of NELL1, gRNAs were delivered to HEK293 cells with a nuclease deficient (d) dCas9-KRAB system adapted from Streptococcus pyogenes. For upregulation, gRNAs were delivered to HEK293 cells expressing the dCas9-VPR system adapted from Streptococcus pyogenes. Gene regulation via these systems was assessed using qRT-PCR. ECM deposition was evaluated using primary antibodies again Collagen I, Collagen III, and Elastin, and the appropriate secondary antibodies. RNA sequencing and analysis was performed by the High-Throughput Genomics and Bioinformatic Analysis core at the University of Utah. Results For up-regulation, four vectors were tested, with the most effective vector demonstrating a 40-fold increase in NELL1 expression via PCR testing. For down-regulation, three vectors were tested, with the most effective demonstrating a 95% reduction in expression via PCR testing (Figure 1). Non-target vectors did not change NELL1 expression levels. RNA sequencing results found over 10,000 differentially expressed genes between up-regulated and non-target cell lines, with significant changes in multiple pathways, including TNF-α signaling. Similarly, over 5,000 differentially expressed genes were found between down-regulated and non-target cell lines, with significant changes in inflammatory and G2-M checkpoint pathways, among others. Ongoing experiments are also testing the amount of extra-cellular matrix (ECM) deposited by up- and down-regulated cells and their non-target controls, in order to determine if changes in NELL1 expression influence ECM deposition. Conclusions We have demonstrated the creation of effective guides for the up- and down-regulation of NELL1, a gene of interest in Peyronie’s disease. We have also demonstrated, through RNA sequencing, that changes in NELL1 lead to large changes in gene and pathway expression. Ongoing experiments will determine if changes in NELL1 expression also affect ECM deposition. Disclosure Any of the authors act as a consultant, employee or shareholder of an industry for: Vault Health.

Giant Sex Chromosomes in Omophoita Species (Coleoptera, Chrysomelidae): Structural and Evolutionary Relationships Revealed by Zoo-FISH and Comparative Genomic Hybridization (CGH)

The beetles of the subtribe Oedionychina (Chrysomelidae, Alticinae) are the only ones that have the atypical giant and achiasmatic sex chromosomes, which are substantially larger than the autosomes. Previous cytogenetic analyses suggest a large accumulation of repetitive DNA in the sex chromosomes. In this study, we examined the similarity of X and Y chromosomes in four Omophoita species and compared genomic differentiation to better understand the evolutionary process and the giant sex chromosomes origin. Intraspecific genomic comparation using male and female genomes of O. octoguttata and interespecific analyses using genomic DNA of O. octoguttata, O. sexnotata, O. magniguttis, and O. personata were performed. In addition, whole chromosome painting (WCP) experiments were performed with X and Y chromosome probes of O. octogutatta. CGH analysis revealed great genomic similarity between the sexes and a sex-specific region on the Y chromosome, and interspecific analysis revealed a genomic divergence between species. In contrast, WCP results revealed that the sex chromosomes of O. octoguttata have high intra- and interspecific similarity with the studied species. Our data support a common origin under the canonical evolution of the sex chromosomes in this group, as they have high genomic similarity between them.

Array Comparative Genomic Hybridization Analysis of Products of Conception in Recurrent Pregnancy Loss for specific anomalies detected by USG.

To evaluate the proportion of chromosomal abnormalities in recurrent pregnancy loss (RPL) assisted by array comparative genomic hybridization (aCGH) bright out with higher detection rate, more accuracy, and less sample failure as compared with conventional cytogenetic analysis. In this study, product of conception samples with abnormal USG findings of the fetus and clinical history of RPL were first processed for karyotyping and Fluorescence In Situ Hybridization analysis. Normal results given by Karyotype and FISH samples with major anomalies detected by Ultrasound with RPL were divided into six groups and aCGH was performed to detect the gain or loss and copy number variations (CNVs) of a particular gene present in chromosomal segments. Among a total of 300 POC samples, 100 abnormal samples were identified either by karyotype (n=70) or by FISH (n=30). From the remaining 200 samples, 5 showed the presence of maternal cell contamination excluded. aCGH analysis revealed (n=195) that 74 (38%) samples with copy number variations (CNVs) and two samples with variants of unknown clinical significance (VOUS) were clinically associated with the clinical findings and 121(62%) samples showed no change in CNVs. The most frequent CNVs were loss of chromosome regions at 2q33.1, 7q11.21, 15q11.1, 16p11.2, Xp22.33, and Yp11.32. CNVs at arr[GRCh37]7p22.3,p21.2(830852-15124702)×1,7q34q36.3(141464180_158909738)×3, 14.2Mbp deletion of 7p22.3p21.2 (SUN1 gene) and 17.4Mbp duplication of 7q34q36.3 (KCNH2, CNTNAP2, and SHH genes) in one sample, CNVs at arr[GRCh37]8p22.2q22.3 (86326349_105509986)×1, 2.48Mbp deletion of 8p22.2q22.3 (GRHL1 gene) were found in another sample.

An Update on Clinicopathological, Imaging, and Genetic Features of Angioleiomyoma.

Angioleiomyoma is a benign, pericytic (perivascular) neoplasm that primarily occurs in the subcutis or dermis of the extremities. The lesion typically presents as a small, firm, slow-growing, painful nodule. Magnetic resonance imaging reveals the lesion to be a well-defined, round to oval mass with signal intensity similar to or slightly hyperintense to that of skeletal muscle on T1-weightwed sequences. A dark reticular sign on T2-weighted sequences appears to be a characteristic feature of angioleiomyoma. Prominent enhancement is usually seen after intravenous contrast. Histologically, the lesion consists of well-differentiated smooth muscle cells with many vascular channels. Based on vascular morphologies, angioleiomyoma is classified into three subtypes: solid, venous, and cavernous. By immunohistochemistry, angioleiomyoma is diffusely positive for smooth muscle actin and calponin and variably for h-caldesmon and desmin. Conventional cytogenetic studies have demonstrated relatively simple karyotypes characterized by one or few structural rearrangements or numerical aberrations. In addition, metaphase comparative genomic hybridization analyses have revealed recurrent loss of 22q and gain of Xq. Angioleiomyoma can be successfully treated with simple excision, with a very low recurrence rate. Knowledge of this peculiar neoplasm is important because it can mimic a variety of benign and malignant soft-tissue tumors. This review provides an updated overview of the clinical, radiological, histopathological, cytogenetic, and molecular genetic features of angioleiomyoma.

Mosaic trisomy 21 at amniocentesis associated with a favorable fetal outcome and perinatal progressive decrease of the trisomy 21 cell line

ObjectiveWe present mosaic trisomy 21 at amniocentesis associated with a favorable fetal outcome and perinatal progressive decrease of the trisomy 21 cell line. Case reportA 33-year-old woman underwent elective amniocentesis at 17 weeks of gestation because of anxiety, and the karyotype of cultured amniocytes was 47,XX,+21[4]/46,XX[13]. In 17 colonies of cultured amniocytes, four colonies had 47,XX,+21, while the other 13 colonies had 46,XX. Simultaneous array comparative genomic hybridization (aCGH) analysis on uncultured amniocytes revealed the result of arr (21) × 3 [0.32] consistent with 32% mosaicism for trisomy 21. Repeat amniocentesis performed at 25 weeks of gestation revealed 47,XX,+21[4]/46,XX[24] with four colonies of 47,XX,+21 and 24 colonies of 46, XX on cultured amniocytes, and arr 21q11.2q22.3 × 2.25 by aCGH, 19.2% mosaicism for trisomy 21 (20/104 cells) by interphase fluorescence in situ hybridization (FISH), and no uniparental disomy (UPD) 21 by quantitative fluorescence polymerase chain reaction (QF-PCR) on uncultured amniocytes. The parental karyotypes were normal, and prenatal ultrasound was unremarkable. A phenotypically normal 2815-g female baby was delivered at 38 weeks of gestation. Cytogenetic analysis on the cord blood, umbilical cord and placenta revealed the karyotype of 47,XX,+21[10]/46,XX[30]. 47,XX,+21[5]/46,XX[35] and 47,XX,+21[38]/46,XX[2], respectively. QF-PCR analysis on the DNA extracted from parental bloods, uncultured amniocytes, cord blood, umbilical cord and placenta confirmed a paternal origin of trisomy 21. When follow-up at age two months, the neonate was phenotypically normal, the peripheral blood had a karyotype of 47,XX,+21[6]/46,XX[34], and no trisomy 21 signals by interphase FISH was found on 100 buccal mucosal cells. When follow-up at age 13 months, the neonate was phenotypically normal, and the peripheral blood had a karyotype of 47,XX,+21[3]/46,XX[37]. ConclusionMosaic trisomy 21 at amniocentesis can be a transient and benign condition, and the abnormal trisomy 21 cell line may decrease and disappear after birth.

Mosaic 46,XY,der(15)t(6;15)(q25.1;p12)/46,XY at amniocentesis in a pregnancy associated with a favorable fetal outcome and postnatal decrease of the aneuploid cell line with the unbalanced translocation

ObjectiveWe present mosaic 46,XY,der(15)t(6;15)(q25.1;p12)/46,XY at amniocentesis in a pregnancy associated with a favorable fetal outcome and postnatal decrease of the aneuploid cell line with the unbalanced translocation. Case reportA 34-year-old primigravid woman underwent amniocentesis at 17 weeks of gestation because of advanced maternal age. Amniocentesis revealed a karyotype of 46,XY,add(15)(p12)[17]/46,XY[5]. A second amniocentesis at 19 weeks of gestation revealed a karyotype of 46,XY,der(15)t(6;15)(q25.1;p12)[12]/46,XY[8], and array comparative genomic hybridization (aCGH) analysis on the DNA extracted from uncultured amniocytes revealed arr arr 6q25.1q27×2-3 with 40% mosaic level. She was referred for genetic counseling. Prenatal ultrasound and the parental karyotypes were normal. A third amniocentesis at 24 weeks of gestation revealed a karyotype of 46,XY,der(15)t(6;15)(q25.1;p12)[23]/46,XY[1], and in uncultured amniocytes, aCGH analysis revealed arr 6q25.1q27×2.5, interphase fluorescence in situ hybridization (FISH) revealed 51% mosaicism (51/100 cells) for partial trisomy 6q and quantitative fluorescence polymerase chain reaction (QF-PCR) analysis determined maternal origin of the aberrant chromosome and excluded uniparental disomy (UPD) 15 and UPD 6. A fourth amniocentesis at 27 weeks of gestation revealed a karyotype of 46,XY,der(15)t(6;15)(q25.1;p12)[21]/46,XY[5], and in uncultured amniocytes, aCGH analysis revealed arr 6q25.1q27×2.46, and interphase FISH revealed 35% mosaicism (35/100 cells) for partial trisomy 6q. At 39 weeks of gestation, a healthy 3028-g male baby was delivered without any phenotypic abnormality. The karyotypes of cord blood, umbilical cord and placenta were 46,XY,der(15)t(6;15)(q25.1;p12)[2]/46,XY,der(15)t(6;15)(q25.1;p12)[29]/46,XY[11] and 46,XY, respectively. When follow-up at age one month, the neonate was phenotypically normal, the peripheral blood had a karyotype of 46,XY (40/40 cells), and FISH analysis on 105 buccal mucosal cells detected five cells with partial trisomy 6q compared with 2% mosaicism (2/100 cells) in the normal control. ConclusionMosaicism for an unbalanced translocation with a normal cell line without UPD at amniocentesis can be a transient and benign condition, and can be associated with a favorable fetal outcome and postnatal decrease of the aneuploid cell line.

Mosaic trisomy 21 at amniocentesis in a twin pregnancy associated with a favorable fetal outcome, maternal uniparental disomy 21 and postnatal decrease of the trisomy 21 cell line

ObjectiveWe present mosaic trisomy 21 at amniocentesis in a twin pregnancy associated with a favorable fetal outcome, maternal uniparental disomy (UPD) 21 and postnatal decrease of the trisomy 21 cell line. Case reportA 36-year-old woman underwent elective amniocentesis at 16 weeks of gestation because of advanced maternal age, and an abnormal non-invasive prenatal testing (NIPT) result suggesting trisomy 21. Amniocentesis revealed the karyotype of 46, XX in co-twin A and the karyotype of 47,XY,+21[12]/46,XY[21] in co-twin B in the cultured amniocytes by in situ culture method. Simultaneous array comparative genomic hybridization (aCGH) analysis on uncultured amniocytes revealed the result of arr (21) × 3 [0.40] in co-twin B, consistent with 40% mosaicism for trisomy 21. Prenatal ultrasound was unremarkable, and the parental karyotypes were normal. Following genetic counseling, the parents decided to continue the pregnancy. At 36 weeks of gestation, a 2140-g female co-twin A and a 1800-g male co-twin B were delivered without any phenotypical abnormality. The karyotypes of the umbilical cord and placenta of co-twin B were 47,XY,+21[16]/46,XY[24] and 47,XY,+21 (40/40 cells), respectively. Quantitative fluorescence polymerase chain reaction (QF-PCR) analysis on the DNA extracted from parental bloods and umbilical cord, cord blood and placenta and peripheral blood at age five months of co-twin B confirmed a maternal origin of trisomy 21 and maternal uniparental isodisomy 21. aCGH analysis on the cord blood revealed the result of arr 21q11.2q22.3 × 2.25 consistent with 20%–25% (log2 ratio = 0.15–0.2) mosaicism for trisomy 21. When follow-up at age five months, the co-twin B was phenotypically normal. His peripheral blood had a karyotype of 47,XY,+21[3]/46,XY[37]. Interphase fluorescence in situ hybridization (FISH) on 100 buccal mucosal cells detected no trisomy 21 signals. The peripheral blood had uniparental isodisomy 21. ConclusionMosaic trisomy 21 at amniocentesis can be a transient and benign condition and should alert the possibility of UPD 21. The abnormal trisomy 21 cell line in mosaic trisomy 21 at amniocentesis may decrease and disappear after birth.

Molecular cytogenetic characterization of de novo concomitant distal 8p deletion of 8p23.3p23.1 and Xp and Xq deletion of Xp22.13q28 due to an unbalanced X;8 translocation detected by amniocentesis

ObjectiveWe present molecular cytogenetic characterization of de novo concomitant distal 8p deletion of 8p23.3p23.1 and Xp and Xq deletion of Xp22.13q28 due to an unbalanced X;8 translocation detected by amniocentesis. Case reportA 33-year-old primigravid woman underwent amniocentesis at 18 weeks of gestation because of a Down syndrome risk of 1/52 at the first-trimester maternal serum screening calculated from 0.29 multiples of the median (MoM) of pregnancy associated plasma protein-A (PAPP-A), 1.14 MoM of free β-hCG and 0.46 MoM of placental growth factor (PlGF). Amniocentesis revealed a karyotype of 45,X,add(8)(p23.1). The parental karyotypes were normal. Array comparative genomic hybridization (aCGH) analysis on the DNA extracted from cultured amniocytes revealed a 137-Mb deletion of Xp22.13q28 and a 10.53-Mb deletion of 8p23.3p23.1. The karyotype thus was 45,X,der(8)t(X;8)(p22.13;p23.1). Prenatal ultrasound revealed pericardial effusion and skin edema. The pregnancy was subsequently terminated, and a 568-g malformed fetus was delivered with hypertelorism and low-set ears. The cord blood had a karyotype of 45,X,der(8)t(X;8)(p22.13;p23.1). aCGH analysis of the cord blood revealed the result of arr [GRCH37 (hg19)] 8p23.3p23.1 (191,530–10,724,642) × 1.0, arr Xp22.13q28 (18,194,098–155,232,907) × 1.0. ConclusionaCGH analysis is useful elucidating the genetic nature of an aberrant chromosome with an additional maternal of unknown origin attached to a chromosome terminal region.

Ciliary phenotyping in renal epithelial cells in a cranioectodermal dysplasia patient with WDR35 variants.

Background: Cranioectodermal dysplasia (CED) is a skeletal autosomal recessive ciliopathy. The characteristic clinical features of CED are facial dysmorphisms, short limbs, narrow thorax, brachydactyly, ectodermal abnormalities, and renal insufficiency. Thus far, variants in six genes are known to be associated with this disorder: WDR35, IFT122, IFT140, IFT144, IFT52, and IFT43. Objective: The goal of this study was to perform cilium phenotyping in human urine-derived renal epithelial cells (hURECs) from a CED patient diagnosed with second-stage chronic kidney disease (CKD) and three unrelated and unaffected pediatric controls. Methods: Genetic analysis by WDR35 screening was performed in the affected individual. Cilium frequency and morphology, including cilium length, height, and width, were evaluated by immunofluorescence (IF) experiments in hURECs using two markers visualizing the ciliary axoneme (Acet-Tub and ARL13B) and the base of the cilium (PCNT). The IF results were analyzed using a confocal microscope and IMARIS software. Results: WDR35 analysis revealed the presence of a known nonsense p. (Leu641*) variant and a novel missense variant p. (Ala1027Thr). Moreover, comparative genomic hybridization analysis showed that the patient carries a microdeletion on chromosome 7q31.1. Ciliary phenotyping performed on hURECs showed morphological differences in the patient's cilia as compared to the three controls. The cilia of the CED patient were significantly wider and longer. Conclusion: The obtained results suggest that CED-related second-stage CKD might be associated with cilia abnormalities, as identified in renal epithelial cells from a CED patient harboring variants in WDR35. This study points out the added value of hURECs in functional testing for ciliopathies.

Clinical, cytogenomic, and molecular characterization of isodicentric Y-chromosome and prediction of testicular sperm retrieval outcomes in azoospermic and severe oligozoospermic infertile men.

Sex chromosome abnormalities are associated with male infertility. The aim of this study was to characterize the clinical, cytogenetic, and molecular findings of 12 infertile men with isodicentric Y-chromosome [idic(Y)] abnormalities diagnosed over a period of 13years. Chromosomal analyses of peripheral blood samples were done using standard procedures. Fluorescence in situ hybridization (FISH) analysis was performed on metaphase spreads of the patients. Multiplex polymerase chain reaction (PCR) using several sequence-tagged site (STS) primer sets within the long arm of Y-chromosome was used to detect AZF deletions.The breakpoints and copy number variations (CNV) were identified by array comparative genomic hybridization analysis (aCGH) analysis.The short-stature homeobox (SHOX) gene deletions were verified using multiplex ligation-dependent probe amplification (MLPA) analysis. Twelve infertile men were diagnosed cytogenetically with idic(Y). The karyotypes of two of the patients were non-mosaic, and the remaining karyotypes showed various degrees of mosaicism. SHOX gene deletion was found in two of the four patients with short stature, and the remaining two patients had shown a 45,X dominant cell line (33.3%). The most common breakpoints for idic(Yq) and idic(Yp) were found to be in Yq11.222 and Yp11.32, respectively. Semen analysis of ten patients (83.3%) demonstrated azoospermia, and the remaining two patients (16.7%) showed severe oligoasthenoteratozoospermia (OAT). In total, 33% (4/12) of idic(Y) patients with or without microsurgical testicular sperm extraction (microTESE) had sperm retrieval. Twelve patients with idic(Y) and different breakpoints of Y-chromosome were characterized using multiple detection strategies. Sperm retrieval outcomes of patients either with idic(Yp) or idic(Yq) showed the possibility to find sperm by microTESE.

Autologous Platelet Rich Plasma (PRGF) Preserves Genomic Stability of Gingival Fibroblasts and Alveolar Osteoblasts after Long-Term Cell Culture

Plasma rich in growth factors (PRGF) has several applications in dentistry that may require repeated applications of PRGF. Furthermore, it has been used for ex vivo expansion of human origin cells for their clinical application. One of the most relevant issues in these applications is to guarantee the genetic stability of cells. In this study, the chromosomal stability of gingival fibroblasts and alveolar osteoblasts after long-term culture was evaluated. Cells were expanded with PRGF or foetal bovine serum (FBS) as a culture medium supplement until passage 7 or 8 for gingival fibroblast or alveolar osteoblasts, respectively. A comparative genomic hybridization (CGH) array was used for the genetic stability study. This analysis was performed at passage 3 and after long-term culture with the corresponding culture medium supplements. The cell proliferative rate was superior after PRGF culture. Array CGH analysis of cells maintained with all the three supplements did not reveal the existence of alterations in copy number or genetic instability. The autologous PRGF technology preserves the genomic stability of cells and emerges as a safe substitute for FBS as a culture medium supplement for the clinical translation of cell therapy.

Comprehensive study of nine novel cases of TFEB-amplified renal cell carcinoma: an aggressive tumour with frequent PDL1 expression.

First described in 2014, renal cell carcinoma (RCC) with TFEB amplification (6p21) is a rare molecular subgroup whose diagnosis is challenging. The prognosis and therapeutic implications remain unclear. We report here the clinical, histological, immunohistochemical, and genetic features of nine novel cases. The pathological and immunohistochemical features were centrally reviewed by expert uropathologists. Fluorescence in situ hybridisation (FISH) confirmed the diagnosis and comparative genomic hybridisation (CGH) was performed to determine quantitative genomic alterations. We also performed an exhaustive review of the literature and compiled our data. TFEB-amplified RCC were locally advanced, with initial lymph node involvement in one case and liver metastasis in another case. They were high-grade eosinophilic tumours with papillary/pseudopapillary architecture, frequent positivity for melanocytic markers, and frequent PDL1 expression. FISH demonstrated high-level TFEB amplification in six cases. One case showed concomitant TFEB translocation. CGH analysis identified complex alterations with frequent losses of 1p, 2q, 3p, 6p, and frequent 6p and 8q gains. VEGFA coamplification was identified in all cases with a lower level than TFEB. The prognosis was poor, with five patients having lymph node or distant metastases. TFEB-amplified RCC is a rare molecular subgroup with variable morphology whose diagnosis is confirmed by FISH analysis. The complex alterations identified by CGH are consistent with an aggressive clinical behaviour. The coamplification of VEGFA and the expression of PDL1 could suggest a potential benefit from antiangiogenics and targeted immunotherapy in combination for these aggressive tumours.

Comparative Genomic Features of Streptococcus canis Based on Pan-Genome Orthologous Group Analysis According to Sequence Type.

Using bacterial pan-genomes obtained through whole genome sequencing (WGS), coding DNA sequences (CDSs) can be clustered into pan-genome orthologous groups (POGs). We aimed to investigate comparative genomic features of Streptococcus canis based on POG analysis and to determine CDSs specific to prevalent sequence type (ST) 9. Twenty WGS datasets from S. canis strains, including invasive and non-invasive specimens, were retrieved from the National Center for Biotechnology Information Assembly database. Based on the WGS data, we performed comparative genome hybridization (CGH), pan- and core-genome prediction, Venn diagram testing with five ST9 strains, and phylogenetic analysis with ST determination. We compared the CDSs of seven ST9 and 13 non-ST9 strains. We observed genomic diversity based on CGH and Venn diagram analyses. The predicted pan- and core-genomes contained 4,772 and 1,403 genes, respectively. We found five clades consisting of different STs (ST1, ST44/2, ST13/14, ST21/15/41, and ST9) based on the phylogenetic tree. There were differences in four pathways (DNA restriction-modification system, DNA-mediated transposition, extracellular region, and response to oxidative stress) regulated by CDSs specific to ST9. Our findings describe genomic diversity in CGH and Venn diagram testing, pan- and core-genomes, five clades of genomes consisting of different STs, and unique CDS features associated with ST9.

Genetic investigation of syndromic forms of obesity

Syndromic obesity (SO) refers to obesity with additional phenotypes, including intellectual disability (ID)/developmental delay (DD), dysmorphic features, or organ-specific abnormalities. SO is rare, has high phenotypic variability, and frequently follows a monogenic pattern of inheritance. However, the genetic etiology of most cases of SO has not been elucidated. In this study, we investigated 20 SO patients by whole-exome sequencing (WES) trios to identify causal genetic variants. 4/20 patients had negative results for array comparative genomic hybridization (aCGH) analyses. In the remaining 15 patients, in addition to SNVs and indels, CNVs were also evaluated. Pathogenic/likely pathogenic (P/LP) SNVs/indels were detected in 6/20 patients (involving MED13L, AHDC1, EHMT1, MYT1L, GRIA3, and SETD1A), while two patients carried an inherited VUS. In addition, P/LP CNVs were observed in 3/15 patients (involving SATG2, KIAA0442, and MEIS2). All nine detected P/LP variants involved genes already known to lead to syndromic ID/DD; however, for only two genes (EHMT1 and MYT1L) is the link with obesity well established. This is the first study applying a comprehensive genomic investigation of an SO cohort, showing a high diagnostic yield (~47%). Additionally, our findings suggested that several known ID/DD genes may also predispose individuals to SO.

Congenital Aneuploidy in Klinefelter Syndrome with B-Cell Acute Lymphoblastic Leukemia Might Be Associated with Chromosomal Instability and Reduced Telomere Length.

Simple SummaryKlinefelter syndrome (KS) is a rare congenital aneuploidy characterized by inherited gain of one X chromosome (XXY). KS is associated with higher susceptibility to the development of cancer. Somatic acquired chromosomal aberrations and chromosomal instability are hallmarks of cancer and leukemia but little is known about the cellular mechanisms involved. The conducted research aimed to identify genomic mechanisms involved in chromosomal evolution mechanisms important for leukemic development. In the leukemic blasts of a patient with KS and B-cell acute lymphoblastic leukemia (B-ALL), we identified additional acquired chromosomal aberration and a significant reduction in the length of the chromosomal ends, i.e., telomeres. A literature review of KS patients with B-ALL revealed that the majority of these patients had acquired two or more additional chromosomal aberrations at B-ALL diagnosis. These data indicate that enhanced reduction in telomere length might be associated with chromosomal instability and may serve as a future target for therapy or prevention.Rare congenital aneuploid conditions such as trisomy 13, trisomy 18, trisomy 21 and Klinefelter syndrome (KS, 47,XXY) are associated with higher susceptibility to developing cancer compared with euploid genomes. Aneuploidy frequently co-exists with chromosomal instability, which can be viewed as a “vicious cycle” where aneuploidy potentiates chromosomal instability, leading to further karyotype diversity, and in turn, paving the adaptive evolution of cancer. However, the relationship between congenital aneuploidy per se and tumor initiation and/or progression is not well understood. We used G-banding analysis, array comparative genomic hybridization analysis and quantitative fluorescence in situ hybridization for telomere length analysis to characterize the leukemic blasts of a three-year-old boy with KS and B-cell acute lymphoblastic leukemia (B-ALL), to gain insight into genomic evolution mechanisms in congenital aneuploidy and leukemic development. We found chromosomal instability and a significant reduction in telomere length in leukemic blasts when compared with the non-leukemic aneuploid cells. Reviewing published cases with KS and B-ALL revealed 20 additional cases with B-ALL diagnostic cytogenetics. Including our present case, 67.7% (14/21) had acquired two or more additional chromosomal aberrations at B-ALL diagnosis. The presented data indicate that congenital aneuploidy in B-ALL might be associated with chromosomal instability, which may be fueled by enhanced telomere attrition.