Comparative Hybridization Research Articles

Identification of copy number alternation profiles in metastatic oral squamous carcinoma patients by using microarray-based comparative genomic hybridization: A study on Turkish patients

ObjectiveOral squamous cell carcinoma (OSCC) is a severe form of cancer affecting different anatomic sites of the oral cavity. OSCC ranks as the sixth most common cancer type with an increasing prevalence globally. However, the mechanisms of OSCC process at later stages are not well understood. In this study, we aimed to determine genetic alternations in metastatic OSCC patients to identify genomic changes occurred at metastatic phase of the disease. Material and MethodsThe Illumina CytoSNP-12 Array was used to determine copy number variations in OSCC cancer genome. Hybridization procedures were performed according to the manufacturer procedures (Illumina). Arrays were scanned on iScan System (Illumina). Data were analyzed using Illumina Genotyping module of Genome Studio software (version 1.2, Illumina). Multiple CNV algorithms and copy number alternations were accessed by Genome Studio. CNVs in whole genome were investigated by using a chromosomal heat map. ResultsWe reported that gains in 8q21.11-ter, 9p21.3, 13q14.11-ter, 13q13.3-ter and losses in 5q14.3-ter, 5q35 and 17p13.3–12 were associated with the development of OSCC. In addition, we also detected that deletion in 2q33.2-ter and 2q35–37.3 regions were also associated with OSCC metastasis process. ConclusionsOur results were also showed that gains in 11q13.3-q13.4 and 2q13.2 chromosomal regions could promote the metastatic OSCC process. We believe that results of the study will help to find new biomarkers for diagnosis at later stage of OSCC.

Improved Sugarcane-Based Fermentation Processes by an Industrial Fuel-Ethanol Yeast Strain.

In Brazil, sucrose-rich broths (cane juice and/or molasses) are used to produce billions of liters of both fuel ethanol and cachaça per year using selected Saccharomyces cerevisiae industrial strains. Considering the important role of feedstock (sugar) prices in the overall process economics, to improve sucrose fermentation the genetic characteristics of a group of eight fuel-ethanol and five cachaça industrial yeasts that tend to dominate the fermentors during the production season were determined by array comparative genomic hybridization. The widespread presence of genes encoding invertase at multiple telomeres has been shown to be a common feature of both baker's and distillers' yeast strains, and is postulated to be an adaptation to sucrose-rich broths. Our results show that only two strains (one fuel-ethanol and one cachaça yeast) have amplification of genes encoding invertase, with high specific activity. The other industrial yeast strains had a single locus (SUC2) in their genome, with different patterns of invertase activity. These results indicate that invertase activity probably does not limit sucrose fermentation during fuel-ethanol and cachaça production by these industrial strains. Using this knowledge, we changed the mode of sucrose metabolism of an industrial strain by avoiding extracellular invertase activity, overexpressing the intracellular invertase, and increasing its transport through the AGT1 permease. This approach allowed the direct consumption of the disaccharide by the cells, without releasing glucose or fructose into the medium, and a 11% higher ethanol production from sucrose by the modified industrial yeast, when compared to its parental strain.

Cross-species chromosome painting and repetitive DNA mapping illuminate the karyotype evolution in true crocodiles (Crocodylidae).

Crocodilians have maintained very similar karyotype structures and diploid chromosome numbers for around 100 million years, with only minor variations in collinearity. Why this karyotype structure has largely stayed unaltered for so long is unclear. In this study, we analyzed the karyotypes of six species belonging to the genera Crocodylus and Osteolaemus (Crocodylidae, true crocodiles), among which the Congolian endemic O. osborni was included and investigated. We utilized various techniques (differential staining, fluorescence in situ hybridization with repetitive DNA and rDNA probes, whole chromosome painting, and comparative genomic hybridization) to better understand how crocodile chromosomes evolved. We studied representatives of three of the four main diploid chromosome numbers found in crocodiles (2n = 30/32/38). Our data provided new information about the species studied, including the identification of four major chromosomal rearrangements that occurred during the karyotype diversification process in crocodiles. These changes led to the current diploid chromosome numbers of 2n = 30 (fusion) and 2n = 38 (fissions), derived from the ancestral state of 2n = 32. The conserved cytogenetic tendency in crocodilians, where extant species keep near-ancestral state, contrasts with the more dynamic karyotype evolution seen in other major reptile groups.

Variants in FREM1 and trisomy 18 identified in a neonatal progeria patient.

Neonatal progeroid disorders are rare disorders with clinical features including low body mass index, proptosis, aged and dysmorphic facial features at the time of birth, prominent veins, sparse scalp hairs, and severe growth retardation. Very few cases have been identified with an unknown genetic cause. Here, we report clinical and genetic findings of a proband with hallmark features of neonatal progeria. Microarray comparative genomic hybridization, whole exome sequencing (WES) and Sanger sequencing were performed using standard methods. Array combined genome hybridization data revealed trisomy 18 in the proband (II-1), and WES data identified novel compound heterozygous variants (c.247C > T; p.H83Y and c.14769868InsA) in the FREM1 gene. We report a novel complex case of neonatal progeria with atrial septal defects, trisomy 18 without typical features of Edward syndrome. The phenotype of the patient was more consistent with neonatal progeria, thus we speculate it to be caused by the FREM1 variants.

Mycoplasma DnaK increases DNA copy number variants in vivo

The human microbiota affects critical cellular functions, although the responsible mechanism(s) is still poorly understood. In this regard, we previously showed that Mycoplasma fermentans DnaK, an HSP70 chaperone protein, hampers the activity of important cellular proteins responsible for DNA integrity. Here, we describe a novel DnaK knock-in mouse model generated in our laboratory to study the effect of M. fermentans DnaK expression in vivo. By using an array-based comparative genomic hybridization assay, we demonstrate that exposure to DnaK was associated with a higher number of DNA copy number variants (CNVs) indicative of unbalanced chromosomal alterations, together with reduced fertility and a high rate of fetal abnormalities. Consistent with their implication in genetic disorders, one of these CNVs caused a homozygous Grid2 deletion, resulting in an aberrant ataxic phenotype that recapitulates the extensive biallelic deletion in the Grid2 gene classified in humans as autosomal recessive spinocerebellar ataxia 18. Our data highlight a connection between components of the human urogenital tract microbiota, namely Mycoplasmas, and genetic abnormalities in the form of DNA CNVs, with obvious relevant medical, diagnostic, and therapeutic implications.

Giant uterine fibroids with chromosome 22 monosomy. A clinical case

We present a clinical case of giant uterine fibroids in this research, with the peculiarity of surgical treatment and the course of the postoperative period described. A series of genetic studies specific to uterine fibroids was performed, namely, mutations in the MED12 gene, overexpression of the HMGA2 gene, and chromosomal imbalance. We did not detect mutations of exon 2 of the MED12 gene and an increase in the expression of the HMGA2 gene in the patients myomatous node sample. The molecular karyotype arr(22)1 (chromosome 22 monosomy) was established by comparative genomic hybridization in the tissue of giant uterine fibroids.

Simultaneous Nbs1 and p53 inactivation in neural progenitors triggers high-grade gliomas.

Nijmegen breakage syndrome (NBS) is a rare autosomal recessive disorder caused by hypomorphic mutations of NBS1. NBS1 is a member of the MRE11-RAD50-NBS1 (MRN) complex that binds to DNA double-strand breaks and activates the DNA damage response (DDR). Nbs1 inactivation in neural progenitor cells leads to microcephaly and premature death. Interestingly, p53 homozygous deletion rescues the NBS1-deficient phenotype allowing long-term survival. The objective of this work was to determine whether simultaneous inactivation of Nbs1 and p53 in neural progenitors triggered brain tumorigenesis and if so in which category this tumour could be classified. We generated a mouse model with simultaneous genetic inactivation of Nbs1 and p53 in embryonic neural stem cells and analysed the arising tumours with in-depth molecular analyses including immunohistochemistry, array comparative genomic hybridisation (aCGH), whole exome-sequencing and RNA-sequencing. NBS1/P53-deficient mice develop high-grade gliomas (HGG) arising in the olfactory bulbs and in the cortex along the rostral migratory stream. In-depth molecular analyses using immunohistochemistry, aCGH, whole exome-sequencing and RNA-sequencing revealed striking similarities to paediatric human HGG with shared features with radiation-induced gliomas (RIGs). Our findings show that concomitant inactivation of Nbs1 and p53 in mice promotes HGG with RIG features. This model could be useful for preclinical studies to improve the prognosis of these deadly tumours, but it also highlights the singularity of NBS1 among the other DNA damage response proteins in the aetiology of brain tumours.

17q21.31 Microduplication Syndrome in a Patient with Autism Spectrum Disorder, Macrocephaly, and Intellectual Disability

The chromosome 17q21.31 microduplication syndrome is a rare genetic syndrome presenting with craniofacial dysmorphisms, psychomotor delay, microcephaly, behavioral disorders, and poor social interaction. Only ten patients have been reported in the literature until today. All patients share some specific features, including psychomotor delay, behavioral disorders, and autism spectrum disorder (ASD). Here, a new case of this syndrome is reported in an 11-year-old Caucasian child who presented the classical clinical features of the 17q21.31 microduplication syndrome in association with new clinical characteristics previously unreported. The Array-Comparative Genomic Hybridization (aCGH) revealed a partial duplication of the long arm of chromosome 17. A literature review of previously studied patients with 17q21.31 microduplication syndrome is reported.

Molecular and Functional Characterisation of a Novel Intragenic 12q24.21 Deletion Resulting in MED13L Haploinsufficiency Syndrome.

Background and Objectives: Heterozygous pathogenic variants in the MED13L gene cause impaired intellectual development and distinctive facial features with or without cardiac defects (MIM #616789). This complex neurodevelopmental disorder is characterised by various phenotypic features, including plagiocephaly, strabismus, clubfoot, poor speech, and developmental delay. The aim of this study was to evaluate the clinical significance and consequences of a novel heterozygous intragenic MED13L deletion in a proband with clinical features of a MED13L-related disorder through extensive clinical, molecular, and functional characterisation. Materials and Methods: Combined comparative genomic hybridisation and single-nucleotide polymorphism array (SNP-CGH) was used to identify the changes in the proband's gDNA sequence (DECIPHER #430183). Intragenic MED13L deletion was specified via quantitative polymerase chain reaction (qPCR) and Sanger sequencing of the proband's cDNA sample. Western blot and bioinformatics analyses were used to investigate the consequences of this copy number variant (CNV) at the protein level. CRISPR-Cas9 technology was used for a MED13L-gene-silencing experiment in a culture of the control individual's skin fibroblasts. After the MED13L-gene-editing experiment, subsequent functional fibroblast culture analyses were performed. Results: The analysis of the proband's cDNA sample allowed for specifying the regions of the breakpoints and identifying the heterozygous deletion that spanned exons 3 to 10 of MED13L, which has not been reported previously. In silico, the deletion was predicted to result in a truncated protein NP_056150.1:p.(Val104Glyfs*5), partly altering the Med13_N domain and losing the MedPIWI and Med13_C domains. After MED13L gene editing was performed, reduced cell viability; an accelerated aging process; and inhibition of the RB1, E2F1, and CCNC gene expression were found to exist. Conclusions: Based on these findings, heterozygous intragenic 12q24.21 deletion in the affected individual resulted in MED13L haploinsufficiency due to the premature termination of protein translation, therefore leading to MED13L haploinsufficiency syndrome.

GRM1 Gene Fusions as an Alternative Molecular Driver in Blue Nevi and Related Melanomas

Activating mutations in GNAQ, GNA11, CYSLTR2, and PLCB4 genes are regarded as the main oncogenic drivers of blue nevi (BN) and blue malignant melanocytic tumors. Here we report 4 cases of blue melanocytic neoplasms devoid of these mutations but harboring GRM1 gene fusions. In this short series, there was no gender predominance (sex ratio, 1). The mean age at diagnosis was 40 years (range, 12-72). Tumors were located on the face (n = 2), forearm (n = 1), and dorsum of the foot (n = 1). Clinically, a plaque-like pre-existing BN was found in 2 cases, including a deep location; another case presented as an Ota nevus. Two cases were diagnosed as melanoma ex-BN, one as an atypical BN, and one as a plaque-like BN. Microscopic examination revealed a dermal proliferation of dendritic melanocytes in a sclerotic stroma. A dermal cellular nodule with atypia and mitotic activity was observed in 3 cases. Genetic investigation by whole exome RNA sequencing revealed MYO10::GRM1 (n = 2) and ZEB2::GRM1 (n = 1) fusions. A GRM1 rearrangement was identified by fluorescence in situ hybridization in the remaining case. SF3B1 comutations were present in the 2 melanomas, and both had a MYO10::GRM1 fusion. Array comparative genomic hybridization was feasible for 3 cases and displayed multiple copy number alterations in the 2 melanomas and limited copy number alterations in the atypical BN, all genomic profiles compatible with those of classical blue lesions. GRM1 was overexpressed in all cases compared with a control group of blue lesions with other typical mutations. Both melanomas rapidly developed visceral metastases following diagnosis, with a fatal outcome in one case and tumor progression under palliative care in the other. These data suggest that GRM1 gene fusions could represent an additional rare oncogenic driver in the setting of BN, mutually exclusive of classical canonical mutations, especially in plaque-type or Ota subtypes.

Comparison of clinical and preimplantation genetic testing for aneuploidy outcomes between in vitro fertilization and intracytoplasmic sperm injection in sibling mature oocytes from high-risk patients: A retrospective study.

To evaluate the influence of insemination methods on clinical outcomes by assessing preimplantation genetic testing for aneuploidy (PGT-A) outcomes in embryos obtained using in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) in sibling mature oocytes from high-risk patients. This retrospective study involved 108 couples with nonmale or mild male factor infertility who underwent split insemination cycles from January 2018 to December 2021. PGT-A was performed using trophectoderm biopsy, array comparative genome hybridization, or next-generation sequencing with 24-chromosome screening. Mature oocytes were divided into IVF (n = 660) and ICSI (n = 1028) groups. The normal fertilization incidence was similar between the groups (81.1% vs. 84.6%). The total number of blastocysts biopsied was significantly higher in the IVF group than in the ICSI group (59.3% vs. 52.6%; p = 0.018). However, euploidy (34.4% vs. 31.9%) and aneuploidy (63.4% vs. 66.2%) rates per biopsy and clinical pregnancy rates (60.0% vs. 58.8%) were similar between the groups. Implantation (45.6% vs. 50.8%) and live birth or ongoing pregnancy (52.0% vs 58.8%) rates were slightly higher in the ICSI group than in the IVF group and miscarriage rate per transfer was slightly higher in the IVF group than in the ICSI group (12.0% vs 5.9%); however no significant difference was observed. IVF and ICSI using sibling mature oocytes had similar clinical outcomes, and euploidy and aneuploidy rates in couples with nonmale and mild male factor infertility. These results suggest that IVF is a useful option, along with ICSI, as an insemination method in PGT-A cycles, especially in high-risk patients.

Clinical, pathological, and comprehensive molecular analysis of the uterine clear cell carcinoma: a retrospective national study from TMRG and GINECO network

BackgroundUterine clear cell carcinomas (CCC) represent less than 5% of uterine cancers. Their biological characteristics and clinical management remain uncertain. A multicenter study to explore both clinical and molecular features of these rare tumors was conducted.MethodsThis multicenter retrospective national study was performed within the French TMRG (Rare Gynecologic Malignant Tumors) network. Clinical data and, when available, FFPE blocks were collected. Clinical features, treatments, and outcome (progression-free survival (PFS) and overall survival (OS)) were analyzed and correlated to the protein (tissue micro-array), RNA (Nanostring nCounter® technology), and DNA (array-Comparative Genomic hybridization and target-next generation sequencing) levels using the tumor samples available.ResultsSixty-eight patients with uterine CCC were enrolled, 61 from endometrial localization and 5 with cervix localization. Median age at diagnosis was 68.9 years old (range 19–89.7). Most tumors were diagnosed at an early stage (78% FIGO stage I–II). Hysterectomy (performed in 90%) and lymph node dissection (80%) were the most frequent surgical treatment. More than 70% of patients received external beam radiotherapy and 57% received brachytherapy. Nearly half (46%) of the patients received chemotherapy. After a median follow-up of 24.7 months, median PFS was 64.8 months (95 CI [5.3–124.4]) and median OS was 79.7 (IC95 [31.0–128.4]). Low hormone receptor expression (13% estrogen-receptor positive), frequent PI3K pathway alterations (58% PTEN loss, 50% PIK3CA mutations), and P53 abnormalities (41%) were observed. Mismatch repair deficiency was identified in 20%. P16 expression was associated with shorter PFS (HR = 5.88, 95 CI [1.56–25], p = 0.009). Transcriptomic analyzes revealed a specific transcriptomic profile notably with a high expression of immune response-associated genes in uterine CCC displaying a very good overall prognosis.ConclusionsUterine CCC reported to be potentially MSI high, hormone receptors negative, and sometimes TP53 mutated. However, some patients with immune response-associated features and better prognosis may be candidate to treatment de-escalation and immunotherapy.

P-644 The mitochondrial DNA copy number in cumulus cells associate with age and AMH level but do not predict implantation potential of embryos

Abstract Study question is there correlation between the mtDNA copy number in cumulus cells and anti-Mullerian hormone level,female age, oocyte quality, embryo morphology, ploidy and blastocyst implantation rate? Summary answer A positive correlation of mtDNA quantity in CCs with the AMH,female age was revealed.There was no correlation between mtDNA quantity and embryo morphology,ploidy,implantation rate What is known already Recent studies have suggested that age-related decreased competence of oocytes may be due to low quantity of mtDNA copy number. Moreover, quantification of mtDNA in CCs may serve as an predictor of blastocysts viability. The purpose of this study is to investigate relative levels of mtDNA in the OCCCs in association with female age, ovarian reserve, embryo morphology, ploidy and blastocyst implantation rate. Study design, size, duration Prospective clinical study performed on 470 CCs retrieved from 72 advanced reproductive age patients undergoing ART treatment with intracytoplasmic sperm injection (ICSI) and preimplantation genetic testing for aneuploidy (PGT-A). Out of the 130 obtained blastocysts 56 embryos were diagnosed as aneuploid, and 74 as euploid. Presently, 51 frozen euploid embryos were transferred (FET). All transferred euploid blastocysts (n = 51) divided into 2 groups: 1 group (n = 21) - implanted embryos, 2 group (n = 30) - non-implanted. Participants/materials, setting, methods Inclusion criteria: age 35-45years;BMI: 18 – 24,9kg/m2; FSH ≤15IU/ml; normal female/male karyotype; non-smokers. Exclusion criteria: genital endometriosis III-IV;severe extragenital pathology; polycystic ovary syndrome; chronic endometritis; > 96% of sperm with abnormal morphology (WHO criteria). MtDNA was assessed by using a quantitative real-time polymerase chain reaction technique. DNA from the trophectoderm samples were amplified and subjected to aneuploidy analysis using array comparative genomic hybridization. In statistic analysis was used Pearson’s correlation coefficients and Fisher’s exact test; p < 0.05 was considered significant. Main results and the role of chance The median age was 37.8 years old (range, 35–45), the mean level of anti-Mullerian hormone (AMH) was 2.66±1.09, and the mean body mass index was 22.3±1.5. A positive correlation of the relative level of mtDNA in the CCs with the patients' age (p = 0.008) and AMH levels (p = 0.003) was revealed. There was no statistically significant correlation between mtDNA copy number and embryo morphology on day 5 (p = 0.7). There was a tendency to increase mtDNA copy number in group 1 vs. group 2, 390 and 299, respectively (p > 0.05). In this study we didn’t find relationship between median mtDNA content of CCs and embryos ploidy (356 vs. 325, in euploid (n = 74) and aneuploidy (n = 56) blastocyst, respectively, p > 0.05). Limitations, reasons for caution Our study was carried out in a relatively small subset of participants and obtained embryos. Correspondingly, the results obtained cannot be readily extrapolated on other groups of patients and need to be confirmed in larger trials. Wider implications of the findings This study showed that mtDNA quantification in CCs isn’t a useful biomarker for prediction embryos implantation potential or ploidy. MtDNA content in CCs correlated only with female age and AMH level. Trial registration number *

O-307 Aneuploidy in oocytes of women of advanced maternal age: impact on embryo development and evidence for a novel mechanism of meiotic error

Abstract Study question In oocytes of advanced maternal age (AMA) women, what is the association of aneuploidy with embryo development and what are the mechanisms leading to aneuploidy? Summary answer We confirmed precocious separation of sister chromatids as the main cause of aneuploidy. Known chromosome segregation errors explained 90.4% of the observed abnormal chromosome copy numbers in polar bodies. What is known already Meiotic chromosomal aneuploidies in oocytes correlate with AMA (>35 years), and can affect over half of oocytes in this age group. This underlies the rationale for polar body (PB) biopsy as a form of early PGT-A, as performed in the ‘ESHRE STudy into the Evaluation of oocyte Euploidy by Microarray analysis’ (ESTEEM) randomised controlled trial (RCT). So far, chromosome analysis of oocytes and PBs has shown that precocious separation of sister chromatids (PSSC), meiosis II (MII) nondisjunction (ND) and reverse segregation (RS) are the main mechanisms leading to aneuploidy in oocytes. Study design, size, duration Data was sourced from the ESTEEM study, a multicentre RCT from seven European centres to assess the clinical utility of PGT-A on PBs using array comparative genomic hybridisation (aCGH) in patients of AMA (36-40 years). This included data on the chromosome complement in PB pairs, and on embryo morphology in a subset of embryos, up to day 6 post insemination (dpi), from both the intervention (PB biopsy for PGT-A) and from control (no biopsy) arms. Participants/materials, setting, methods ESTEEM recruited 396 AMA patients: 205 in the intervention group and 191 in the control group. Complete genetic data from 693 PB pairs were analysed. Additionally, the morphology from 1034 embryos generated from fertilized oocytes (2 pronuclei) in the PB biopsy group and 1082 in the control group were compared using age-adjusted multilevel mixed-effect ordinal logistic regression for each day of embryo scoring and summarized with adjusted odds ratios (aOR) and 95% confidence intervals (CI). Main results and the role of chance Overall, 461/693 PB pairs showed abnormal segregation in 1162/10810 chromosomes. The main observed abnormal segregations were compatible with PSSC in meiosis I (MI) (n = 568/1162; 48,9%), ND of chromatids in MII or RS (n = 417/1162; 35.9%) and less frequently ND in MI (n = 65/1162; 5.6%). For 112 chromosomes (112/1162; 9,6%), we observed a chromosome copy number in PB1 and PB2 that is not explained by any of the known mechanisms causing aneuploidy in oocytes. We observed that embryos in the PGT-A arm of the RCT did not have a significantly different morphology between 2 and 6 dpi compared to the control group, indicating that polar body biopsy did not affect embryo quality. Following age-adjusted multilevel mixed-effect ordinal logistic regression models performed for each embryo evaluation day (using the before-mentioned embryo scoring subcategories), aneuploidy was associated with a decrease in embryo quality on day 3 (aOR 0.62, 95% CI 0.43-0.90), day 4 (aOR 0.15, 95% CI 0.06-0.39) and day 5 (aOR 0.28, 95% CI 0.14-0.58). Limitations, reason for caution RS cannot be distinguished from normal segregation or MII ND using aCGH. The observed segregations were based on the detected copy number of PB1 and PB2 only and were not confirmed by the analysis of embryos. The embryo morphology assessment was static and single observer. Wider implications of the findings Our finding of frequent unexplained chromosome copy numbers in polar bodies indicates that our knowledge of the mechanisms causing aneuploidy in oocytes is incomplete. It challenges the dogma that aneuploidy in oocytes is exclusively caused by missegregation of chromosomes during meiosis I and II. Study funding/competing interest(s) Data was mined from a study funded by ESHRE. Illumina provided microarrays and other consumables necessary for aCGH testing of polar bodies. None of the authors have competing interests. This work is dedicated to the memory of Professor Joep Geraedts. Trial registration number NCT01532284 Study funding Yes Funding source Funding by national/international organization(s)

P-709 Predictive heatmap in Preimplantation Genetic Testing for Monogenic disorders (PGT-M) and Structural Rearrangements (PGT-SR)

Abstract Study question What is the predicted cumulative live birth rate (CLBR) relative to AMH-level for women undergoing preimplantation genetic testing (PGT) for monogenic disorders and structural rearrangements? Summary answer Anti-müllerian hormone (AMH) and age allow to give an estimate of the CLBR in PGT and becomes poor when AMH is ≤ 2µg/L and age ≥42y. What is known already Ovarian response is an important contributory factor in PGT in order to obtain a sufficient number of embryos for genetic analysis. A proportion of the obtained embryos will be carrier of the disease tested, and another small proportion of the embryos will fail to have a genetic diagnosis hence will not be available for embryo transfer. The number of embryos not suitable for embryo transfer as a result of PGT varies between 25-81% according to the indication for PGT. The combination of female age and AMH are good predictors for CLBR in conventional IVF. Study design, size, duration This is a single-centre retrospective cohort study including 1,522 females undergoing 3,130 PGT cycles over a 6-year period (01/01/2015-31/12/2020) at a university-based referral centre for PGT. The principal outcome measure was CLBR per intention to treat (ITT), and the secondary outcome was live birth rate (LBR) per embryo transfer, in couples undergoing PGT-M and PGT-SR either by polymerase chain reaction (PCR), single nucleotide polymorphism (SNP) array, microarray-based comparative genomic hybridization (aCGH), or next-generation sequencing (NGS). Participants/materials, setting, methods Mean ±SD was applied for continuous outcomes, whereas (relative) frequency was applied for dichotomous outcomes. Multivariable logistic regression analysis was applied with dependent variable being the CLBR or LBR and the independent variables the AMH, female age, BMI, inheritance mode and PGT-technique. Best-fit (Akaike information criterion (AIC)) versus polynomials were analysed. Non-inferiority testing between different technologies was performed. A 3D-prediction mosaic (including AMH, age and CLBR) was created, which can be used for counseling purposes. Main results and the role of chance The mean female age is 32.46 years (SD 4.43), with a mean AMH level of 2.75 µg/L (SD 2.58) and a mean BMI of 24.24 (SD 4.43). Age and AMH significantly affect CLBR irrespective of the inheritance mode or PGT technique used. There is a gradual decline of CLBR from a female age of 25 years onwards reaching a critical threshold of less than 10% CLBR per ITT over the age of 42 with AMH levels ≤2 µg/L. Between cleavage stage biopsy techniques (PCR) and trophectoderm biopsy PGT techniques (SNP array, aCGH, NGS) used there was no significant difference in outcome per ITT, however per embryo transfer there is a significantly higher chance of live birth in SNP array, aCGH and NGS cycles compared to PCR cycles. Limitations, reasons for caution Despite the large sample size, the findings are confined by limited confounder adjustment. Wider implications of the findings In a PGT-program, couples need to be informed on the limited prognosis, if the combination of female age and AMH is unfavourable, in order to allow them to make other reproductive choices. The prediction mosaic produced in this study can be used as an insightful visual tool in counseling PGT-couples. Trial registration number Not applicable

Chromosomal alterations in sporadic medullary thyroid carcinoma and correlation with outcome.

Somatic copy number alterations (SCNA) involving either a whole chromosome or just one of the arms, or even smaller parts, have been described in about 88% of human tumors. This study investigated the SCNA profile in 40 well-characterized sporadic medullary thyroid carcinomas by comparative genomic hybridization array. We found that 26/40 (65%) cases had at least one SCNA. The prevalence of SCNA, and in particular of chromosome 3 and 10, was significantly higher in cases with a RET somatic mutation. Similarly, SCNA of chromosomes 3, 9, 10 and 16 were more frequent in cases with a worse outcome and an advanced disease. By the pathway enrichment analysis, we found a mutually exclusive distribution of biological pathways in metastatic, biochemically persistent and cured patients. In particular, we found gain of regions involved in the intracellular signaling and loss of regions involved in DNA repair and TP53 pathways in the group of metastatic patients. Gain of regions involved in the cell cycle and senescence were observed in patients with biochemical disease. Finally, gain of regions associated with the immune system and loss of regions involved in the apoptosis pathway were observed in cured patients suggesting a role of specific SCNA and corresponding altered pathways in the outcome of sporadic MTC.

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.

Investigating the "Fetal Side" in Recurrent Pregnancy Loss: Reliability of Cell-Free DNA Testing in Detecting Chromosomal Abnormalities of Miscarriage Tissue.

(1) Background: The aim of our study is to evaluate whether cell-free DNA testing can overlap the genetic testing of miscarriage tissue in women with early pregnancy loss (EPL) and length of recurrent pregnancy loss (RPL); (2) Methods: We conducted a prospective cohort study at the Pregnancy Loss Unit of the Fondazione Policlinico Universitario A. Gemelli (IRCCS), Rome, Italy between May 2021 and March 2022. We included women with EPL and length of RPL. Gestational age was >9 weeks + 2 days and <12 weeks + 0 days of gestation corresponding to a crown rump length measurement of >25 and <54 mm. Women underwent both dilation and curettage for the collection of miscarriage tissue and for blood sample collection. Chromosomal microarray analysis (CMA) on miscarriage tissues was performed by oligo-nucleotide- and single nucleotide polymorphisms (SNP)-based comparative genomic hybridization (CGH+SNP). Maternal blood samples were analyzed by Illumina VeriSeq non-invasive prenatal testing (NIPT) to evaluate the cell-free fetal DNA (cfDNA) and the corresponding fetal fraction and the presence of genetic abnormalities; (3) Results: CMA on miscarriage tissues revealed chromosome aneuploidies in 6/10 cases (60%), consisting of trisomy 21 (5 cases) and monosomy X (one case). cfDNA analysis was able to identify all cases of trisomy 21. It failed to detect monosomy X. A large 7p14.1p12.2 deletion concomitant to trisomy 21 was, in one case, detected by cfDNA analysis but it was not confirmed by CMA on miscarriage tissue. (4) Conclusions: cfDNA largely reproduces the chromosomal abnormalities underlying spontaneous miscarriages. However, diagnostic sensitivity of cfDNA analysis is lower with respect to the CMA of miscarriage tissues. In considering the limitations when obtaining biological samples from aborted fetuses suitable for CMA or standard chromosome analysis, cfDNA analysis is a useful, although not exhaustive, tool for the chromosome diagnosis of both early and recurrent pregnancy loss.

Intrachromosomal amplification 21: A driver of acute myeloid leukemia?

A 68-year-old woman was referred for anemia and circulating peripheral blood blasts. Bone marrow aspirate confirmed the diagnosis of acute myeloid leukemia (AML) with a background of variable dysplasia that was not otherwise sufficient to subclassify as AML with myelodysplasia-related changes (AML-MRC) (panel A; original magnification × 1000; Wright-Giemsa stain). By flow cytometry, the blasts were positive for dim/partial CD7, CD13, CD34, CD117, HLA-DR, partial MPO and TdT, and were negative for other B/T cell associated antigens. G-banding karyotype showed the presence of tandem duplication at the long arm of chromosome 21 (panel B; 46,XX,dup(21)(q21q22)). Single color fluorescence in situ hybridization probes targeting the 21q22.13-q22.2 region showed a homogeneously staining region on chromosome 21 containing >5 copies of RUNX1 (panels C and D; metaphase-interphase FISH), further confirmed by comparative genomic hybridization. Intrachromosomal amplification 21 (iamp21) is a well-recognized event in B lymphoblastic leukemia; however, it is rare in AML with <20 published cases, variably associated with morphologic dysplasia although it uniformly occurred in the setting of complex karyotypes. It was thus postulated to be a late event resulting from genome instability and chromothripsis. In our case, iAMP21 occurred as a sole abnormality, suggesting the possibility of a rather early role in leukemogenesis. N.A wrote the manuscript and compiled the genetics images. Z.C designed the cytology and flow cytometry images. Z.C revised the manuscript. The study received no funding from internal or external sources. The authors disclose no conflict of interests. The Institutional Review Board at the American University of Beirut exempted our single case report from review. Informed consent was explained to and signed by the patient. Data sharing is not applicable to this article as no new data were created or analyzed in this study.