Third Generation Cytogenetic Analysis (TGCA): diagnostic application of long-read sequencing.

Unbalanced Structural Variants (uSVs) play important roles in the pathogenesis of several genetic syn- dromes. Traditional and molecular karyotyping are considered the first-tier diagnostic tests to detect macroscopic and cryptic deletions/duplications. However, their time-consuming and laborious experi- mental protocols protract diagnostic times from three to fifteen days. Long read sequencing approaches, such as Oxford Nanopore Technologies (ONT), have the ability to reduce time to results for the detection of uSVs with the same resolution of current state-of-the-art diagnostic tests. Here we compared ONT to molecular karyotyping for the detection of pathogenic uSVs of 7 patients with previously diagnosed causative CNVs of different sizes and allelic fractions. Larger chromosomal anomalies included trisomy 21 and mosaic tetrasomy 12p. Among smaller CNVs we tested two recip- rocal genomic imbalances in 7q11.23 (1.367 Mb), a 170 kb deletion encompassing NRXN1 and mosaic 6q27 (1.231 Mb) and 2q23.1 (408 kb) deletions. DNA libraries were prepared following ONT standard protocols and sequenced on the GridION device for 48 h. Data generated during runs were analysed in online mode, using NanoGLADIATOR. We were capable to identify all pathogenic CNVs with detection time inversely proportional to size and allelic fraction. Aneuploidies were called after only 30 minutes of sequencing, while 30 hours were needed to call CNVs < 500 kb also in mosaic state (44%). These results demonstrate the clinical utility of our approach that allows the molecular diagnosis of genomic disorders within a 30 minutes to 30 hours time-frame.

Cytogenomic characteristics of murine breast cancer cell line JC

Background Breast cancer (BC), one of the most frequent human tumors, is genetically and histologically heterogeneous. Treatment options can be adapted according to BC subtype. Still, research is necessary to characterize BC biology better and to study potential new treatment options. Murine BC-cell lines can be used as model systems in this respect. Results Here for the first time murine BC-cell line JC was cytogenomically characterized as being complex rearranged and near-tetraploid. Multicolor banding and array comparative genomic hybridization were applied and the result was in silico translated to the human genome. Conclusions Even though being commercially available, cell line JC was yet not much included in BC-research, most likely due to a lack of cytogenomic data. Thus, here comprehensive data is provided on chromosomal aberrations, genomic imbalances and involved breakpoints of JC cell line. Also JC could be characterized as a model for BC of luminal B type, basal-like tumor rather than for luminal A type.

Genomic imbalances in the placenta are associated with poor fetal growth

Background Fetal growth restriction (FGR) is associated with increased risks for complications before, during, and after birth, in addition to risk of disease through to adulthood. Although placental insufficiency, failure to supply the fetus with adequate nutrients, underlies most cases of FGR, its causes are diverse and not fully understood. One of the few diagnosable causes of placental insufficiency in ongoing pregnancies is the presence of large chromosomal imbalances such as trisomy confined to the placenta; however, the impact of smaller copy number variants (CNVs) has not yet been adequately addressed. In this study, we confirm the importance of placental aneuploidy, and assess the potential contribution of CNVs to fetal growth. Methods We used molecular-cytogenetic approaches to identify aneuploidy in placentas from 101 infants born small-for-gestational age (SGA), typically used as a surrogate for FGR, and from 173 non-SGA controls from uncomplicated pregnancies. We confirmed aneuploidies and assessed mosaicism by microsatellite genotyping. We then profiled CNVs using high-resolution microarrays in a subset of 53 SGA and 61 control euploid placentas, and compared the load, impact, gene enrichment and clinical relevance of CNVs between groups. Candidate CNVs were confirmed using quantitative PCR. Results Aneuploidy was over tenfold more frequent in SGA-associated placentas compared to controls (11.9% vs. 1.1%; p = 0.0002, OR = 11.4, 95% CI 2.5–107.4), was confined to the placenta, and typically involved autosomes, whereas only sex chromosome abnormalities were observed in controls. We found no significant difference in CNV load or number of placental-expressed or imprinted genes in CNVs between SGA and controls, however, a rare and likely clinically-relevant germline CNV was identified in 5.7% of SGA cases. These CNVs involved candidate genes INHBB, HSD11B2, CTCF, and CSMD3. Conclusions We conclude that placental genomic imbalances at the cytogenetic and submicroscopic level may underlie up to ~ 18% of SGA cases in our population. This work contributes to the understanding of the underlying causes of placental insufficiency and FGR, which is important for counselling and prediction of long term outcomes for affected cases.

Genomic imbalances in the placenta are associated with poor fetal growth

Background: Fetal growth restriction (FGR) is associated with increased risks for complications before, during, and after birth, in addition to risk of disease through to adulthood. Although placental insufficiency, failure to supply the fetus with adequate nutrients, underlies most cases of FGR, its causes are diverse and not fully understood. One of the few diagnosable causes of placental insufficiency in ongoing pregnancies is the presence of large chromosomal imbalances such as trisomy confined to the placenta; however, the impact of smaller copy number variants (CNVs) has not yet been adequately addressed. In this study, we confirm the importance of placental aneuploidy, and assess the potential contribution of CNVs to fetal growth.Methods: We used molecular-cytogenetic approaches to identify aneuploidy in placentas from N=101 infants born small-for-gestational age (SGA), typically used as a surrogate for FGR, and from N=173 non-SGA controls from uncomplicated pregnancies. We confirmed aneuploidies and assessed mosaicism by microsatellite genotyping. We then profiled CNVs using high-resolution microarrays in a subset of N=53 SGA and N=61 control euploid placentas, and compared the load, impact, gene enrichment and clinical relevance of CNVs between groups. Candidate CNVs were confirmed using quantitative PCR.Results: Aneuploidy was over 10-fold more frequent in SGA-associated placentas compared to controls (11.9% vs. 1.1%; p=0.0002, OR=11.4, 95% CI 2.5-107.4), was confined to the placenta, and typically involved autosomes, whereas only sex chromosome abnormalities were observed in controls. We found no significant difference in CNV load or number of placental-expressed or imprinted genes in CNVs between SGA and controls, however, a rare and likely clinically-relevant germline CNV was identified in 5.7% of SGA cases. These CNVs involved candidate genes INHBB, HSD11B2, CTCF, and CSMD3.Conclusions: We conclude that placental genomic imbalances at the cytogenetic and submicroscopic level may underlie up to ~18% of SGA cases in our population. This work contributes to the understanding of the underlying causes of placental insufficiency and FGR, which is important for counselling and prediction of long term outcomes for affected cases.

Intra-individual Genomic Variation Analysis in Tissues (Blood vs. Testis) Through SNP Microarray: A Case Report of Two Patients with Idiopathic Sertoli Cell Only Syndrome (SCOS)

Background: Sertoli cell only syndrome (SCOS) or germ cell aplasia is characterized by the existence of only sertoli cells in the seminiferous tubule without any germ cells. SCOS is a multifactorial disorder but genetic factors play a major role in pathogenesis of idiopathic SCOS. Case Presentation: Two cases of idiopathic SCOS had been reported with no non-genetic factor in their medical history that could play a role in aetiology of SCOS. Also, two normal fertile males were recruited as controls in this study. For evaluation of genomic imbalance, karyotyping (G-banding), FISH, STS-PCR and SNP microarray were carried out. SNP microarray was carried out in DNA of peripheral blood for cases as well as controls. However, for cases, SNP microarray was conducted in DNA of testicular Fine needle aspiration cytology (FNAC). Conclusion: No chromosome abnormality and Yq microdeletion was found in cases as well as in controls. Microarray detected many CNVs and LOH that cover genes with spermatogenesis related function and PAR CNVs in both cases. Differential genomic variations were found in blood and testis for cases. Therefore, the evaluation of pathogenesis of idiopathic SCOS might be dependent on both tissue samples. The evaluation of genomic imbalances at both tissue levels should be done for a large cohort of patients.

Characterization of genomic imbalance in patients with balanced chromosomal rearrangements and abnormal phenotypes

Идентификация причин формирования аномалий развития у пациентов со сбалансированными хромосомными перестройками на сегодняшний день является актуальным и не в полной мере изученным направлением в цитогенетической практике, требующим разработки уникального подхода с использованием современных молекулярно-генетических технологий. Целью данного исследования явилась этиологическая диагностика геномного дисбаланса у пациентов со сбалансированными хромосомными перестройками и аномалиями развития. 20 пациентов с аномальным фенотипом и сбалансированными хромосомными перестройками, выявленными при стандартном кариотипировании, были обследованы методами хромосомного микроматричного анализа (ХМА) и FISH. Геномный дисбаланс (CNV) обнаружен в 13 случаях из 20, что составило 65%. Использование ХМА позволило установить, что в 69,2% случаев носительства сбалансированных хромосомных перестроек причиной аномального фенотипа являлось наличие микроделеций/микродупликаций, ассоциированных с точками разрывов при перестройках, а в 20,8% случаев наблюдались микроделеции/микродупликации на хромосомах, не задействованных в аберрациях. Несмотря на то, что ХМА позволил выявить причины аномалий развития в большинстве случаев, 7 пациентам (35%) не удалось установить окончательный молекулярно-цитогенетический диагноз. Более того, у некоторых пациентов выявленные CNVs не полностью отражают генотип-фенотип корреляцию, что требует проведения дополнительных молекулярно-генетических исследований. При отсутствии геномного дисбаланса при ХМА, а также при неполной генотип-фенотип корреляции, диагностика этиологии аномалий развития и патологического фенотипа должна быть продолжена молекулярными методами более высокого разрешения. Identification of genomic imbalances in cases with balanced chromosomal rearrangements and abnormal phenotype is a current trends in cytogenetics practice, requiring the development of unique approach using modern molecular genetic technologies. The aim of this study is diagnostics etiologies of the abnormal phenotype in patients with balanced chromosomal rearrangements. We report the investigations results of 20 patients with abnormal phenotype and balanced chromosomal rearrangements by conventional cytogenetic analysis. Genomic imbalances by microarray studies detected in 13 of 20 cases (65%). Most of CNVs was microdeletion or microduplication at a rearrangement breakpoint (69.2%) and 20.8% microdeletion/microduplication in other chromosomes.

Cytogenomic characterization of three murine malignant mesothelioma tumor cell lines

Background Malignant mesothelioma (MM) is a rare aggressive cancer primary located in pleura and lung. MMs can be divided into biphasic, epithelioid and sarcomatoid subtypes. In majority of cases MMs are induced by asbestos fiber exposure. As latency period after asbestos exposure ranges between ~ 10 and 60 years MMs are mainly observed in elder people. Human MM, being a rare tumor type, lacks detailed cytogenetic data, while molecular genetic studies have been undertaken more frequently. However, murine MM cell lines are also regularly applied to get more insight into MM biology and to test new therapy strategies. Results Here the murine MM cell lines AB1, AB22 and AC29 were studied by molecular cytogenetics and molecular karyotyping. Interestingly, yet there were no genetic or genomic studies undertaken for these already in 1992 established cell lines. The obtained data on genomic imbalances in these murine cell lines was translated into the human genome as previously reported based on human and murine genomic browsers. Conclusions It turned out that all three cell lines showed high similarities in copy number variants as observed typically in human MM. Also, all three cell lines were most similar to human epithelioid MMs, and should be used as models therefore.