Human endometrial cell-type-specific RNA sequencing provides new insights into the embryo-endometrium interplay.

STUDY QUESTION: Which genes regulate receptivity in the epithelial and stromal cellular compartments of the human endometrium, and which molecules are interacting in the implantation process between the blastocyst and the endometrial cells? SUMMARY ANSWER: A set of receptivity-specific genes in the endometrial epithelial and stromal cells was identified, and the role of galectins (LGALS1 and LGALS3), integrin ß1 (ITGB1), basigin (BSG) and osteopontin (SPP1) in embryo-endometrium dialogue among many other protein-protein interactions were highlighted. WHAT IS KNOWN ALREADY: The molecular dialogue taking place between the human embryo and the endometrium is poorly understood due to ethical and technical reasons, leaving human embryo implantation mostly uncharted. STUDY DESIGN SIZE DURATION: Paired pre-receptive and receptive phase endometrial tissue samples from 16 healthy women were used for RNA sequencing. Trophectoderm RNA sequences were from blastocysts. PARTICIPANTS/MATERIALS SETTING METHODS: Cell-type-specific RNA-seq analysis of freshly isolated endometrial epithelial and stromal cells using fluorescence-activated cell sorting (FACS) from 16 paired pre-receptive and receptive tissue samples was performed. Endometrial transcriptome data were further combined in silico with trophectodermal gene expression data from 466 single cells originating from 17 blastocysts to characterize the first steps of embryo implantation. We constructed a protein-protein interaction network between endometrial epithelial and embryonal trophectodermal cells, and between endometrial stromal and trophectodermal cells, thereby focusing on the very first phases of embryo implantation, and highlighting the molecules likely to be involved in the embryo apposition, attachment and invasion. MAIN RESULTS AND THE ROLE OF CHANCE: In total, 499 epithelial and 581 stromal genes were up-regulated in the receptive phase endometria when compared to pre-receptive samples. The constructed protein-protein interactions identified a complex network of 558 prioritized protein-protein interactions between trophectodermal, epithelial and stromal cells, which were grouped into clusters based on the function of the involved molecules. The role of galectins (LGALS1 and LGALS3), integrin ß1 (ITGB1), basigin (BSG) and osteopontin (SPP1) in the embryo implantation process were highlighted. LARGE SCALE DATA: RNA-seq data are available at www.ncbi.nlm.nih.gov/geo under accession number GSE97929. LIMITATIONS REASONS FOR CAUTION: Providing a static snap-shot of a dynamic process and the nature of prediction analysis is limited to the known interactions available in databases. Furthermore, the cell sorting technique used separated enriched epithelial cells and stromal cells but did not separate luminal from glandular epithelium. Also, the use of biopsies taken from non-pregnant women and using spare IVF embryos (due to ethical considerations) might miss some of the critical interactions characteristic of natural conception only. WIDER IMPLICATIONS OF THE FINDINGS: The findings of our study provide new insights into the molecular embryo-endometrium interplay in the first steps of implantation process in humans. Knowledge about the endometrial cell-type-specific molecules that coordinate successful implantation is vital for understanding human reproduction and the underlying causes of implantation failure and infertility. Our study results provide a useful resource for future reproductive research, allowing the exploration of unknown mechanisms of implantation. We envision that those studies will help to improve the understanding of the complex embryo implantation process, and hopefully generate new prognostic and diagnostic biomarkers and therapeutic approaches to target both infertility and fertility, in the form of new contraceptives. STUDY FUNDING/COMPETING INTERESTS: This research was funded by the Estonian Research Council (grant PRG1076); Horizon 2020 innovation grant (ERIN, grant no. EU952516); Enterprise Estonia (grant EU48695); the EU-FP7 Marie Curie Industry-Academia Partnerships and Pathways (IAPP, grant SARM, EU324509); Spanish Ministry of Economy, Industry and Competitiveness (MINECO) and European Regional Development Fund (FEDER) (grants RYC-2016-21199, ENDORE SAF2017-87526-R, and Endo-Map PID2021-127280OB-100); Programa Operativo FEDER Andalucía (B-CTS-500-UGR18; A-CTS-614-UGR20), Junta de Andalucía (PAIDI P20_00158); Margarita Salas program for the Requalification of the Spanish University system (UJAR01MS); the Knut and Alice Wallenberg Foundation (KAW 2015.0096); Swedish Research Council (2012-2844); and Sigrid Jusélius Foundation; Academy of Finland. A.S.-L. is funded by the Spanish Ministry of Science, Innovation and Universities (PRE2018-085440). K.G.-D. has received consulting fees and/or honoraria from RemovAid AS, Norway Bayer, MSD, Gedeon Richter, Mithra, Exeltis, MedinCell, Natural cycles, Exelgyn, Vifor, Organon, Campus Pharma and HRA-Pharma and NIH support to the institution; D.B. is an employee of IGENOMIX. The rest of the authors declare no conflict of interest.

Fetal sex determination in twin pregnancies using cell free fetal DNA analysis.

OBJECTIVE(S): We sought to develop an accurate sex classification method in twin pregnancies using data obtained from a standard commercial non-invasive prenatal test. STUDY DESIGN: A total of 706 twin pregnancies were included in this retrospective analytical data study. Normalized chromosome values for chromosomes X and Y were used and adapted into a sex-score to predict fetal sex in each fetus, and results were compared with the clinical outcome at birth. RESULTS: Outcome information at birth for sex chromosomes was available for 232 twin pregnancies. From these, a total of 173 twin pregnancies with a Y chromosome identified in non-invasive pregnancy testing were used for the development of a predictive model. Global accuracy for sex classification in the testing set with 51 samples was 0.98 (95% confidence interval [0.90,0.99]), with a specificity and sensitivity of 1 (95% confidence interval [0.82,1.00]) and 0.97 (95% confidence interval [0.84,0.99]), respectively. CONCLUSION: While non-invasive prenatal testing is a screening method and confirmatory results must be obtained by ultrasound or genetic diagnosis, the sex-score determination presented herein offers an accurate and useful approach to characterizing fetus sex in twin pregnancies in a non-invasive manner early on in pregnancy.

Cancer stem cells from human glioblastoma resemble but do not mimic original tumors after in vitro passaging in serum-free media.

Human gliomas harbour cancer stem cells (CSCs) that evolve along the course of the disease, forming highly heterogeneous subpopulations within the tumour mass. These cells possess self-renewal properties and appear to contribute to tumour initiation, metastasis and resistance to therapy. CSC cultures isolated from surgical samples are considered the best preclinical in vitro model for primary human gliomas. However, it is not yet well characterized to which extent their biological and functional properties change during in vitro passaging in the serum-free culture conditions. Here, we demonstrate that our CSC-enriched cultures harboured from one to several CSC clones from the human glioma sample. When xenotransplanted into mouse brain, these cells generated tumours that reproduced at least three different dissemination patterns found in original tumours. Along the passages in culture, CSCs displayed increased expression of stem cell markers, different ratios of chromosomal instability events, and a varied response to drug treatment. Our findings highlight the need for better characterization of CSC-enriched cultures in the context of their evolution in vitro, in order to uncover their full potential as preclinical models in the studies aimed at identifying molecular biomarkers and developing new therapeutic approaches of human gliomas.

Distribution patterns of segmental aneuploidies in human blastocysts identified by next-generation sequencing.

OBJECTIVE: To evaluate the ability of next-generation sequencing (NGS) to detect pure and mosaic segmental aneuploidies in trophectoderm biopsies and to identify distribution patterns in whole blastocysts. DESIGN: Validation study. SETTING: Reference laboratory. PATIENT(S): Seventy couples with known karyotypes who had undergone preimplantation genetic screening with diagnoses at the blastocyst stage using array comparative genomic hybridization (aCGH). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Concordance rates for segmental and whole-chromosome aneuploidies determined between aCGH and NGS, and estimates of mosaicism levels of segmental aneuploidies in fixed blastocysts. RESULT(S): We used NGS with amplified DNA from trophectoderm biopsies in which segmental aneuploidies had been previously detected by array comparative genomic hybridization (aCGH). Single-cell fluorescent in situ hybridization (FISH) was then used as an independent form of analysis. The concordance rate between NGS and aCGH was 124 (98.4%) of 126 for the detection of segmental aneuploidies, and 48 (96.0%) of 50 for whole-chromosome aneuploidies. The overall concordance rate was 99.8% (2,276 of 2,280 chromosomes assessed). After FISH analyses with 41.4 ± 24.3 cells per blastocyst, 26 (92.9%) of 28 segmentals detected by aCGH and NGS were confirmed. The FISH analysis did not detect the segmentals in two blastocysts, in which all cells analyzed were euploid. CONCLUSION(S): This is the first report analyzing distribution patterns of segmental aneuploidies in trophectoderm biopsy by NGS. We have demonstrated that NGS allows the detection of pure and mosaic segmental aneuploidies with the same efficiency as aCGH. The FISH analysis confirmed the existence of these events in the trophectoderm and the inner cell mass.

Is endometrial receptivity transcriptomics affected in women with endometriosis? A pilot study.

Endometrial receptivity is still questioned today in women with endometriosis. The aim of this study was to assess the endometrial receptivity gene signature in patients with different stages of endometriosis by investigating transcriptomic modifications of their endometrium using the endometrial receptivity array (ERA) test. A prospective, interventional multicentre pilot trial was designed and implemented in two university-affiliated infertility units from Belgium and Spain. Gene expression microarray was used to diagnose the receptivity status by quantifying the expression of 238 specific genes directly related to human endometrial receptivity. Unsupervised hierarchical clustering showed no clustering of samples based on endometriosis stages. Two subgroups of samples clustered together corresponding on the day of the cycle in which the biopsy was taken (day 18 versus days 19-20). None of the 238 genes present in the ERA array were significantly over- or under- expressed in any of different stages of the disease compared with controls. Minimal differences were found when looking at the functional profile, suggesting that the possible effect from a clinical point of view may be meaningless. Endometrial receptivity gene signature during the implantation window does not vary significantly among patients with endometriosis even considering different stages compared with healthy women.

Transcriptomics of the human endometrium.

During the mid-secretory phase, the endometrium acquires the receptive phenotype, which corresponds to the only period throughout the endometrial cycle in which embryo implantation is viable. Endometrial receptivity is a crucial process and even more important in Assisted Reproductive Technologies (ART) where embryo-endometrial synchronization is coordinated through embryo transfer timing. Over the last decade, transcriptomic analyses performed on the human endometrium have shown that specific genomic signatures can be used to successfully phenotype different phases of the menstrual cycle including the receptive stage, independently of the histological appereance of the endometrial tissue. In this paper, we review current evidence demonstrating that endometrial transcriptomics objectively identifies the implantation window in a personalized manner, opening the field for the diagnosis of the endometrial factor in ART and moving to stratified medicine at this level, using microarray technology and soon high-throughput next generation sequencing coupled with functional and systems genomics approach.

Clinical management of endometrial receptivity.

The endometrial window of implantation (WOI), the cycle days during which normal embryo implantation can occur, has generally been assumed to begin on cycle day 19 or 20 of an idealized 28 days cycle and last for 4 to 5 days. Noyes et al took the first steps in defining the WOI by establishing a set of morphological criteria to evaluate endometrial development and receptivity, but recent studies have invalidated their use in the routine evaluation of infertility. Based on greater than 10 years of extensive research, our group has developed a molecular diagnostic tool (the endometrial receptivity array [ERA] test) based on the specific transcriptomic signature that identifies the receptive endometrium in natural and artificial (hormonal replacement therapy) cycles. The ERA test has shown that some patients have a delayed WOI, others have an advanced WOI, and others can have unusually short windows of receptivity. This identification and characterization of the WOI allows the personalization of the embryo transfer. In this review, we describe the ERA and our experience with its use in assessment of the endometrial receptivity in patients undergoing assisted reproduction.

The impact of using the combined oral contraceptive pill for cycle scheduling on gene expression related to endometrial receptivity.

STUDY QUESTION: Does the combined oral contraceptive pill (COCP) change endometrial gene expression when used for cycle programming? SUMMARY ANSWER: COCP used for scheduling purposes does not have a significant impact on endometrial gene expression related to endometrial receptivity. WHAT IS KNOWN ALREADY: Controversy exists around COCP pretreatment for IVF cycle programming, as some authors claim that it might be detrimental to the live birth rate. Microarray technology applied to the study of tissue gene expression has previously revealed the behavior of genes related to endometrial receptivity under different conditions. STUDY DESIGN, SIZE, AND DURATION: Proof-of-concept study of 10 young healthy oocyte donors undergoing controlled ovarian stimulation (COS) recruited between June 2012 and February 2013. PARTICIPANTS/MATERIALS, SETTING, AND METHODS: Microarray data were obtained from endometrial biopsies from 10 young healthy oocyte donors undergoing COS with GnRH antagonists and recombinant FSH. In group A (n = 5), COCP pretreatment was used for 12-16 days, and stimulation began after a 5-day pill-free interval. Stimulation in group B (n = 5) was initiated on cycle day 3 after a spontaneous menses. Endometrial biopsies were collected 7 days after triggering with hCG. MAIN RESULTS AND THE ROLE OF CHANCE: No individual genes exhibited increased or decreased expression (fold change (FC) >2) in patients with prior COCP treatment (group A) compared with controls (group B). However, the results of the functional analysis showed a total of 11 biological processes that were significantly enriched in group A compared with group B (non-COCP). LIMITATIONS, REASONS FOR CAUTION: The Endometrial Receptivity Array (ERA) has only been validated on endometrial samples obtained in natural cycles and after hormonal replacement treatment (HRT). Therefore, it was not possible in this study to classify the endometrial samples as receptive or non-receptive. We used the ERA to focus on 238 genes that are intimately related to endometrial receptivity, thus simplifying the analysis and understanding of the data. WIDER IMPLICATIONS OF THE FINDINGS: Cycle scheduling is common in IVF units and is used to avoid weekend retrievals and/or to distribute evenly the workload for better efficiency. Our failure to detect any relevant changes in the genes related to the window of implantation when cycles were programmed with COCP pretreatment suggests that, despite controversial clinical results in previous studies, the use of COCPs in this way does not affect uterine receptivity adversely. STUDY FUNDING/COMPETING INTEREST(S): Funding for this study was provided by an unrestricted grant from Merck Sharp & Dohme. C.S. and A.P. are co-inventors (with Patricia Diaz-Gimeno) of the Endometrial Receptivity Array and hold the patent. The other authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: EudraCT registration number is 2011-003250-34.

Impact of final oocyte maturation using gonadotropin-releasing hormone agonist triggering and different luteal support protocols on endometrial gene expression.

OBJECTIVE: To use microarray technology to analyze endometrial gene expression after gonadotropin-releasing hormone agonist (GnRH-a) triggering with four different protocols of luteal support in comparison with results obtained after a human chorionic gonadotropin (hCG) trigger. DESIGN: Prospective, randomized, controlled trial. SETTING: University-affiliated private assisted-reproduction center. PATIENT(S): 25 healthy oocyte donors undergoing controlled ovarian stimulation. INTERVENTION(S): On day of final oocyte maturation, randomization to [1] GnRH-agonist triggering and luteal support with oral estradiol (2 mg/8 hours) and vaginal progesterone (200 mg/12 hours), [2] GnRH-a and a daily dose of 150 IU of recombinant LH from oocyte pickup, [3] GnRH-a and a single bolus of 60 µg of recombinant hCG on oocyte pickup, [4] GnRH-a and three doses of 20 µg of recombinant hCG separated by 48 hours, or [5] 250 µg of recombinant hCG for trigger and standard luteal support; with endometrial biopsy samples collected 7 days after triggering. MAIN OUTCOME MEASURE(S): Gene expression using the Endometrial Receptivity Array (ERA) and pathway and network analysis of study groups 1-4 compared with controls (group 5). RESULT(S): The 56 genes in group 1 (25 up-regulated and 31 down-regulated) exhibited altered expression compared with the 36 genes from group 2 (13 up-regulated and 23 down-regulated), 44 from group 3 (28 up-regulated and 16 down-regulated), and 30 (20 up-regulated and 10 down-regulated) from group 4. CONCLUSION(S): Differences were seen in endometrial gene expression related to the type of ovulation trigger and luteal support. However, gene expression after the GnRH-a trigger and modified luteal support adding LH/hCG activity more closely resembles the pattern seen in the hCG group. CLINICAL TRIAL REGISTRATION NUMBER: EudraCT 2011-003250-34.

The endometrial receptivity array for diagnosis and personalized embryo transfer as a treatment for patients with repeated implantation failure.

OBJECTIVE: To demonstrate the clinical value of the endometrial receptivity array (ERA) in patients with repeated implantation failure (RIF), for guiding their personalized embryo transfer (pET) as a novel therapeutic strategy. DESIGN: Prospective interventional multicenter clinical trial. SETTING: University-affiliated infertility and private clinics. PATIENT(S): Eighty-five RIF patients and 25 comparison patients. INTERVENTION(S): Endometrial sampling and pET guided by ERA. MAIN OUTCOME MEASURE(S): A receptive (R) or nonreceptive (NR) endometrial status according to ERA. Pregnancy (PR) and implantation (IR) rates after pET. RESULT(S): The ERA test gave an R result of 74.1% in RIF patients versus 88% in control subjects. Clinical follow-up was possible in 29 RIF patients, in whom pET was performed, resulting in 51.7% PR and 33.9% IR. The IRs and PRs in the 6 months after the biopsy showed that pregnancy was not related to the local injury. Twenty-two RIF patients (25.9%) were NR, and in 15 of them a second ERA validated a displacement of the window of implantation (WOI). In eight of them, pET was performed on the day designated by the ERA, resulting in 50.0% PR and 38.5% IR. These results should be considered as preliminary. CONCLUSION(S): There is an increased percentage of WOI displacement in RIF patients compared with comparison group patients, leading to the concept of pET as a therapeutic strategy. Rescue of NR patients by pET in a displaced WOI results in similar PR and IR.

Profiling the gene signature of endometrial receptivity: clinical results.

This article highlights the need for methods to objectively diagnose endometrial receptivity as a factor contributing to infertility in female patients. The correct identification of the appropriate window of implantation in a given patient, by using endometrial receptivity biomarkers, can help to prevent reproductive failure resulting from misplaced timing of the endometrial window of implantation (WOI). Although to date no single, clinically relevant morphologic, molecular, or histologic marker capable of indicating endometrial receptivity status has been identified, global transcriptomic analysis of human endometria performed in the last decade has given us insights into a genomic signature that is capable of identifying endometrial receptivity. As a consequence, a genomic tool named the Endometrial Receptivity Array (ERA), based on a customized microarray, was developed, and along with it a specially trained bioinformatic prediction computer algorithm was created to identify WOI timing in the endometrium. This tool has proven more accurate and consistent than histologic (Noyes) dating at identifying the personalized WOI day, thus leading to the new clinical concept of personalized ET on the optimum day of endometrial receptivity, identified individually case by case.

The accuracy and reproducibility of the endometrial receptivity array is superior to histology as a diagnostic method for endometrial receptivity.

OBJECTIVE: To compare the accuracy and reproducibility of the endometrial receptivity array (ERA) versus standard histologic methods. DESIGN: A comparative prospective study (May 2008-May 2012). SETTING: University-affiliated infertility clinic. PATIENT(S): Eighty-six healthy oocyte donors, regularly cycling, aged 20-34 years with a body mass index (BMI) of 19-25 kg/m(2). INTERVENTION(S): Endometrial biopsies were collected throughout the menstrual cycle. For the accuracy study, 79 samples were grouped into two cohorts: the training set (n = 79) for ERA machine-learning training and dating, and a dating subset (n = 49) for comparison between histologic and ERA dating. For the reproducibility study, seven women underwent ERA testing and it was repeated in the same patients on the same day of their cycle 29-40 months later. MAIN OUTCOME MEASURE(S): Concordance of histologic and ERA dating related to LH as a reference, and interobserver variability between pathologists were statistically analyzed by the quadratic weighted Kappa index. The ERA reproducibility was tested and its gene expression visualized by principal component analysis. RESULT(S): For each pathologist, concordance against LH peak yielded values of 0.618 (0.446-0.791) and 0.685 (0.545-0.824). Interobserver variability between pathologists yielded a Kappa index of 0.622 (0.435-0.839). Concordance for ERA dating against LH peak showed a value of 0.922 (0.815-1.000). Reproducibility of the ERA test was 100% consistent. CONCLUSION(S): The ERA is more accurate than histologic dating and is a completely reproducible method for the diagnosis of endometrial dating and receptivity status.

Development, characterization and experimental validation of a cultivated sunflower (Helianthus annuus L.) gene expression oligonucleotide microarray.

Oligonucleotide-based microarrays with accurate gene coverage represent a key strategy for transcriptional studies in orphan species such as sunflower, H. annuus L., which lacks full genome sequences. The goal of this study was the development and functional annotation of a comprehensive sunflower unigene collection and the design and validation of a custom sunflower oligonucleotide-based microarray. A large scale EST (>130,000 ESTs) curation, assembly and sequence annotation was performed using Blast2GO (www.blast2go.de). The EST assembly comprises 41,013 putative transcripts (12,924 contigs and 28,089 singletons). The resulting Sunflower Unigen Resource (SUR version 1.0) was used to design an oligonucleotide-based Agilent microarray for cultivated sunflower. This microarray includes a total of 42,326 features: 1,417 Agilent controls, 74 control probes for sunflower replicated 10 times (740 controls) and 40,169 different non-control probes. Microarray performance was validated using a model experiment examining the induction of senescence by water deficit. Pre-processing and differential expression analysis of Agilent microarrays was performed using the Bioconductor limma package. The analyses based on p-values calculated by eBayes (p<0.01) allowed the detection of 558 differentially expressed genes between water stress and control conditions; from these, ten genes were further validated by qPCR. Over-represented ontologies were identified using FatiScan in the Babelomics suite. This work generated a curated and trustable sunflower unigene collection, and a custom, validated sunflower oligonucleotide-based microarray using Agilent technology. Both the curated unigene collection and the validated oligonucleotide microarray provide key resources for sunflower genome analysis, transcriptional studies, and molecular breeding for crop improvement.

The genomics of the human endometrium.

The endometrium is a complex tissue that lines the inside of the endometrial cavity. The gene expression of the different endometrial cell types is regulated by ovarian steroids and paracrine-secreted molecules from neighbouring cells. Due to this regulation, the endometrium goes through cyclic modifications which can be divided simply into the proliferative phase, the secretory phase and the menstrual phase. Successful embryo implantation depends on three factors: embryo quality, the endometrium's state of receptivity, and a synchronised dialogue between the maternal tissue and the blastocyst. There is a need to characterise the endometrium's state of receptivity in order to prevent reproductive failure. No single molecular or histological marker for this status has yet been found. Here, we review the global transcriptomic analyses performed in the last decade on a normal human endometrium. These studies provide us with a clue about what global gene expression can be expected for a non-pathological endometrium. These studies have shown endometrial phase-specific transcriptomic profiles and common temporal gene expression patterns. We summarise the biological processes and genes regulated in the different phases of natural cycles and present other works on different conditions as well as a receptivity diagnostic tool based on a specific gene set profile. This article is part of a Special Issue entitled: Molecular Genetics of Human Reproductive Failure.

Enrichment of ultraconserved elements among genomic imbalances causing mental delay and congenital anomalies.

BACKGROUND: The ultraconserved elements (UCEs) are defined as stretches of at least 200 base pairs of human DNA that match identically with corresponding regions in the mouse and rat genomes, albeit their real significance remains an intriguing issue. These elements are most often located either overlapping exons in genes involved in RNA processing or in introns or nearby genes involved in the regulation of transcription and development. Interestingly, human UCEs have been reported to be strongly depleted among segmental duplications and benign copy number variants (CNVs). However no comprehensive survey of a putative enrichment of these elements among pathogenic dose variants has yet been reported. RESULTS: A survey for UCEs was performed among the 26 cryptic genomic rearrangements detected in our series of 200 patients with idiopathic neurodevelopmental disorders associated to congenital anomalies. A total of 29 elements, out of the 481 described UCEs, were contained in 13 of the 26 pathogenic gains or losses detected in our series, what represents a highly significant enrichment of ultraconserved elements. In addition, here we show that these elements are preferentially found in pathogenic deletions (enrichment ratio 3.6 vs. 0.5 in duplications), and that this association is not related with a higher content of genes. In contrast, pathogenic CNVs lacking UCEs showed almost a threefold higher content in genes. CONCLUSIONS: We propose that these elements may be interpreted as hallmarks for dose-sensitive genes, particularly for those genes whose gain or loss may be directly implied in neurodevelopmental disorders. Therefore, their presence in genomic imbalances of unknown effect might be suggestive of a clinically relevant condition.

Identification of large rearrangements of the PCDH15 gene by combined MLPA and a CGH: large duplications are responsible for Usher syndrome.

PURPOSE: PCDH15, encoding protocadherin 15, is mutated in Usher syndrome type 1F (USH1F) patients. Not only point mutations, but also large deletions have been detected within this gene. However, the detection and characterization of gross deletions in the USH1F locus have been difficult. The purpose of the present work was to identify large genomic rearrangements of PCDH15 in a cohort of patients and to accurately identify the location of the junction breakpoints of the detected rearrangements. METHODS: A PCDH15 MLPA (multiplex ligation-dependent probe amplification) commercial kit was used, combined with a customized oligonucleotide array-based CGH analysis (aCGH), containing almost 20,000 probes tiling the nonrepetitive sequence of the PCDH15 gene. RESULTS: Two large intragenic rearrangements were identified-one deletion of 55 kb and one direct duplication of 82 kb-in 3 (13%) families from a cohort of 23 USH cases. The patients had been screened for mutations in the five known USH1 genes and were found to carry one or none of the pathogenic mutations in PCDH15. The exact breakpoints of both rearrangements were identified. CONCLUSIONS: This is the first time that large duplications have been associated with Usher syndrome. USH patients have not been extensively tested for large genomic rearrangements such as duplications and deletions. This type of mutation easily escapes detection by traditional PCR-based METHODS: Thus, a combination of PCR-based mutation screening, together with deletion and duplication analysis, is mandatory for the accurate screening of the PCDH15 gene in Usher patients.

Hypoxia promotes efficient differentiation of human embryonic stem cells to functional endothelium.

Early development of mammalian embryos occurs in an environment of relative hypoxia. Nevertheless, human embryonic stem cells (hESC), which are derived from the inner cell mass of blastocyst, are routinely cultured under the same atmospheric conditions (21% O(2)) as somatic cells. We hypothesized that O(2) levels modulate gene expression and differentiation potential of hESC, and thus, we performed gene profiling of hESC maintained under normoxic or hypoxic (1% or 5% O(2)) conditions. Our analysis revealed that hypoxia downregulates expression of pluripotency markers in hESC but increases significantly the expression of genes associated with angio- and vasculogenesis including vascular endothelial growth factor and angiopoitein-like proteins. Consequently, we were able to efficiently differentiate hESC to functional endothelial cells (EC) by varying O(2) levels; after 24 hours at 5% O(2), more than 50% of cells were CD34+. Transplantation of resulting endothelial-like cells improved both systolic function and fractional shortening in a rodent model of myocardial infarction. Moreover, analysis of the infarcted zone revealed that transplanted EC reduced the area of fibrous scar tissue by 50%. Thus, use of hypoxic conditions to specify the endothelial lineage suggests a novel strategy for cellular therapies aimed at repair of damaged vasculature in pathologies such as cerebral ischemia and myocardial infarction.

Novel genomic alterations and mechanisms associated with tumor progression in oligodendroglioma and mixed oligoastrocytoma.

Combined 1p/19q deletions are very prevalent in oligodendrogliomas (OGs) and, to a lesser extent, in oligoastrocytomas (OAs). These losses are associated with responsiveness to therapy. Using array-based comparative genomic hybridization, we screened for recurrent genomic alterations in OG and oligoastrocytoma subtypes on chromosome 19. Concomitant 1p/19q loss was detected in most of the tumors with allelic loss, but array-based comparative genomic hybridization revealed some tumors to have unrelated 1p/19q arm losses, suggesting alternative mechanisms of loss to that related to the reported t(1;19) translocation. Analyses of 1p/19q loss by fluorescence in situ hybridization and loss of heterozygosity assays and correlations of genomic data with the Ki-67 proliferation marker were also performed. Four 1q (or 19p) and 2 1p (or 19q) fluorescence in situ hybridization probe signals together with homozygosity of the 1p/19q microsatellites suggested a hypothetical mechanism of genome duplication consecutive to the loss of the derivative chromosome der(1p;19q) from the t(1;19)(1q;19p) translocation. This genome duplication was frequent in high-grade OGs and was strongly correlated with Ki-67 expression; thus, it could be related to tumor progression. Finally, in addition to the frequent 1p/19q loss, we report a novel 17q amplified region in OGs with BIRC5 as one of the possible candidate target genes of the amplicon.

Estrogen receptor status could modulate the genomic pattern in familial and sporadic breast cancer.

PURPOSE: Familial breast cancer represents 5% to 10% of all breast tumors. Mutations in the two known major breast cancer susceptibility genes, BRCA1 and BRCA2, account for a minority of familial breast cancer, whereas families without mutations in these genes (BRCAX group) account for 70% of familial breast cancer cases. EXPERIMENTAL DESIGN: To better characterize and define the genomic differences between the three classes of familial tumors and sporadic malignancies, we have analyzed 19 BRCA1, 24 BRCA2, and 31 BRCAX samples from familial breast cancer patients and 19 sporadic breast tumors using a 1-Mb resolution bacterial artificial chromosome array-based comparative genomic hybridization. RESULTS: We found that BRCA1/2 tumors showed a higher genomic instability than BRCAX and sporadic cancers. There were common genomic alterations present in all breast cancer groups, such as gains of 1q and 16p or losses of 8ptel-p12 and 16q. We found that the presence/absence of the estrogen receptor (ER) may play a crucial role in driving tumor development through distinct genomic pathways independently of the tumor type (sporadic or familial) and mutation status (BRCA1 or BRCA2). ER(-) tumors presented higher genomic instability and different altered regions than ER+ ones. CONCLUSIONS: According to our results, the BRCA gene mutation status (mainly BRCA1) would contribute to the genomic profile of abnormalities by increasing or modulating the genome instability.

Multiple myeloma primary cells show a highly rearranged unbalanced genome with amplifications and homozygous deletions irrespective of the presence of immunoglobulin-related chromosome translocations.

BACKGROUND AND OBJECTIVES: Multiple myeloma (MM) is a malignant plasma cell neoplasia in which genetic studies have shown that genomic changes may affect almost all chromosomes, as shown by fluorescence in situ hybridization (FISH) and comparative genomic hybridization (CGH). Our objective was the genomic characterization of CD 138 positive primary MM samples by means of a high resolution array CGH platform. DESIGN AND METHODS: For the first time, a high resolution array CGH with more than 40,000 probes, has been used to analyze 26 primary MM samples after the enrichment of CD138-positive plasma cells. RESULTS: This approach identified copy number imbalances in all cases. Bioinformatics strategies were optimized to perform data analysis allowing the segregation of hyperdiploid and non-hyperdiploid cases by array CGH. Additional analysis showed that structural chromosome rearrangements were more frequently seen in hyperdiploid cases. We also identified the same Xq21 duplication in nearly 20% of the cases, which originated through unbalanced chromosome translocations. High level amplifications and homozygous deletions were recurrently observed in our series and involved genes with meaningful function in cancer biology. INTERPRETATION AND CONCLUSIONS: High resolution array CGH allowed us to identify copy number changes in 100% of the primary MM samples. We segregated different MM subgroups based on their genomic profiles which made it possible to identify homozygous deletions and amplifications of great genetic relevance in MM.