Perinatal follow-up of children born after preimplantation genetic diagnosis between 1995 and 2014.

PURPOSE: We aim to evaluate the safety of PGD. We focus on the congenital malformation rate and additionally report on adverse perinatal outcome. METHODS: We collated data from a large group of singletons and multiples born after PGD between 1995 and 2014. Data on congenital malformation rates in live born children and terminated pregnancies, misdiagnosis rate, birth parameters, perinatal mortality, and hospital admissions were prospectively collected by questionnaires. RESULTS: Four hundred thirty-nine pregnancies in 381 women resulted in 364 live born children. Nine children (2.5%) had major malformations. This percentage is consistent with other PGD cohorts and comparable to the prevalence reported by the European Surveillance of Congenital Anomalies (EUROCAT). We reported one misdiagnosis resulting in a spontaneous abortion of a fetus with an unbalanced chromosome pattern. 20% of the children were born premature (< 37 weeks) and less than 15% had a low birth weight. The incidence of hospital admissions is in line with prematurity and low birth weight rate. One child from a twin, one child from a triplet, and one singleton died at 23, 32, and 37 weeks of gestation respectively. CONCLUSIONS: We found no evidence that PGD treatment increases the risk on congenital malformations or adverse perinatal outcome. TRIAL REGISTRATION NUMBER: NCT 2 149485.

In vitro efficacy of forodesine and nelarabine (ara-G) in pediatric leukemia.

Forodesine and nelarabine (the pro-drug of ara-G) are 2 nucleoside analogues with promising anti-leukemic activity. To better understand which pediatric patients might benefit from forodesine or nelarabine (ara-G) therapy, we investigated the in vitro sensitivity to these drugs in 96 diagnostic pediatric leukemia patient samples and the mRNA expression levels of different enzymes involved in nucleoside metabolism. Forodesine and ara-G cytotoxicities were higher in T-cell acute lymphoblastic leukemia (T-ALL) samples than in B-cell precursor (BCP)-ALL and acute myeloid leukemia (AML) samples. Resistance to forodesine did not preclude ara-G sensitivity and vice versa, indicating that both drugs rely on different resistance mechanisms. Differences in sensitivity could be partly explained by significantly higher accumulation of intracellular dGTP in forodesine-sensitive samples compared with resistant samples, and higher mRNA levels of dGK but not dCK. The mRNA levels of the transporters ENT1 and ENT2 were higher in ara-G-sensitive than -resistant samples. We conclude that especially T-ALL, but also BCP-ALL, pediatric patients may benefit from forodesine or nelarabine (ara-G) treatment.

Breakpoint sites disclose the role of the V(D)J recombination machinery in the formation of T-cell receptor (TCR) and non-TCR associated aberrations in T-cell acute lymphoblastic leukemia.

Aberrant recombination between T-cell receptor genes and oncogenes gives rise to chromosomal translocations that are genetic hallmarks in several subsets of human T-cell acute lymphoblastic leukemias. The V(D)J recombination machinery has been shown to play a role in the formation of these T-cell receptor translocations. Other, non-T-cell receptor chromosomal aberrations, such as SIL-TAL1 deletions, have likewise been recognized as V(D)J recombination associated aberrations. Despite the postulated role of V(D)J recombination, the extent of the V(D)J recombination machinery involvement in the formation of T-cell receptor and non-T-cell receptor aberrations in T-cell acute lymphoblastic leukemia is still poorly understood. We performed a comprehensive in silico and ex vivo evaluation of 117 breakpoint sites from 22 different T-cell receptor translocation partners as well as 118 breakpoint sites from non-T-cell receptor chromosomal aberrations. Based on this extensive set of breakpoint data, we provide a comprehensive overview of T-cell receptor and oncogene involvement in T-ALL. Moreover, we assessed the role of the V(D)J recombination machinery in the formation of chromosomal aberrations, and propose an up-dated mechanistic classification on how the V(D)J recombination machinery contributes to the formation of T-cell receptor and non-T-cell receptor aberrations in human T-cell acute lymphoblastic leukemia.

Preimplantation genetic testing for Neurofibromatosis type 1: more than 20 years of clinical experience.

Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder that affects the skin and the nervous system. The condition is completely penetrant with extreme clinical variability, resulting in unpredictable manifestations in affected offspring, complicating reproductive decision-making. One of the reproductive options to prevent the birth of affected offspring is preimplantation genetic testing (PGT). We performed a retrospective review of the medical files of all couples (n = 140) referred to the Dutch PGT expert center with the indication NF1 between January 1997 and January 2020. Of the couples considering PGT, 43 opted out and 15 were not eligible because of failure to identify the underlying genetic defect or unmet criteria for in vitro fertilization (IVF) treatment. The remaining 82 couples proceeded with PGT. Fertility assessment prior to IVF treatment showed a higher percentage of male infertility in males affected with NF1 compared to the partners of affected females. Cardiac evaluations in women with NF1 showed no contraindications for IVF treatment or pregnancy. For 67 couples, 143 PGT cycles were performed. Complications of IVF treatment were not more prevalent in affected females compared to partners of affected males. The transfer of 174 (out of 295) unaffected embryos led to 42 ongoing pregnancies with a pregnancy rate of 24.1% per embryo transfer. There are no documented cases of misdiagnosis following PGT in this cohort. With these results, we aim to provide an overview of PGT for NF1 with regard to success rate and safety, to optimize reproductive counseling and PGT treatment for NF1 patients.

High-resolution identification of balanced and complex chromosomal rearrangements by 4C technology.

Balanced chromosomal rearrangements can cause disease, but techniques for their rapid and accurate identification are missing. Here we demonstrate that chromatin conformation capture on chip (4C) technology can be used to screen large genomic regions for balanced and complex inversions and translocations at high resolution. The 4C technique can be used to detect breakpoints also in repetitive DNA sequences as it uniquely relies on capturing genomic fragments across the breakpoint. Using 4C, we uncovered LMO3 as a potentially leukemogenic translocation partner of TRB@. We developed multiplex 4C to simultaneously screen for translocation partners of multiple selected loci. We identified unsuspected translocations and complex rearrangements. Furthermore, using 4C we detected translocations even in small subpopulations of cells. This strategy opens avenues for the rapid fine-mapping of cytogenetically identified translocations and inversions, and the efficient screening for balanced rearrangements near candidate loci, even when rearrangements exist only in subpopulations of cells.

Characterization of a pediatric T-cell acute lymphoblastic leukemia patient with simultaneous LYL1 and LMO2 rearrangements.

Translocation of the LYL1 oncogene are rare in T-cell acute lymphoblastic leukemia, whereas the homologous TAL1 gene is rearranged in approximately 20% of patients. Previous gene-expression studies have identified an immature T-cell acute lymphoblastic leukemia subgroup with high LYL1 expression in the absence of chromosomal aberrations. Molecular characterization of a t(7;19)(q34;p13) in a pediatric T-cell acute lymphoblastic leukemia patient led to the identification of a translocation between the TRB@ and LYL1 loci. Similar to incidental T-cell acute lymphoblastic leukemia cases with synergistic, double translocations affecting TAL1/2 and LMO1/2 oncogenes, this LYL1-translocated patient also had an LMO2 rearrangement pointing to oncogenic cooperation between LYL1 and LMO2. In hierarchical cluster analyses based on gene-expression data, this sample consistently clustered along with cases having TAL1 or LMO2 rearrangements. Therefore, LYL1-rearranged cases are not necessarily associated with immature T-cell development, despite high LYL1 levels, but elicit a TALLMO expression signature.

Anti-oxidative function of follicular fluid HDL and outcomes of modified natural cycle-IVF.

High density lipoproteins (HDL) are the main cholesterol carriers in follicular fluid (FF), the natural environment of oocyte development. Additionally, HDL have critical biological functions such as anti-oxidative capacity, which have not been studied in reproduction. Therefore, this study aimed to investigate whether the anti-oxidative function of FF-HDL is associated with fertility outcomes. From 253 women undergoing modified natural cycle (MNC)- IVF at a single academic centre FF and plasma were collected (n = 375 cycles). Anti-oxidative function of FF was mainly attributable to HDL (n = 8; 83%). FF-HDL had a higher anti-oxidative function than plasma HDL (n = 19, P < 0.001) coinciding with increased vitamin E and sphingosine 1 phosphate content (P = 0.028 each). Proteomic analysis indicated no significant differences in major anti-oxidative proteins such as paraoxonase 1, apolipoprotein (apo) A-I or apoA-IV between FF-HDL and matched plasma-HDL (n = 5), while apoC-III, apoE and apoC-II were relatively lower in FF-HDL. Finally, FF-HDL anti-oxidative function was related to a decrease in the odds of the oocyte undergoing normal fertilization, an association that persisted after adjustment for confounders (odds ratio 0.97 (0.93-1), P = 0.041). In conclusion, FF-HDL has considerable anti-oxidative properties that might be relevant for embryo quality.

Triamcinolone acetonide and anecortave acetate do not stimulate uveal melanoma cell growth.

PURPOSE: Radiotherapy-induced radiation retinopathy can develop in over 40% of eyes treated for uveal melanoma. Triamcinolone acetonide (TA) and anecortave acetate (AA) can be used to treat radiation retinopathy. It is not known whether TA or AA has any effect on potentially still viable uveal melanoma cells in the choroid after radiotherapy. We therefore studied the effect of these drugs on the proliferation of uveal melanoma cell lines in vitro. Furthermore, as these drugs are supposed to counteract vascular leakage, we determined their effect on the expression and production of the proangiogenic vascular endothelial growth factor-A (VEGF-A), the antiangiogenic pigment epithelium-derived factor (PEDF), and thrombospondin-1 (TSP-1) in uveal melanoma cells. METHODS: Three uveal melanoma cell lines were treated in vitro with TA or AA. Cell proliferation was measured by counting cells and using the Water-Soluble Tetrazolium Salt-1 (WST-1) assay. VEGF-A and PEDF production was measured by ELISA, and intracellular expression of angiogenic-associated genes including VEGF-A, PEDF, and TSP-1 was determined by real-time quantitative RT-PCR. RESULTS: We found no effect of TA or AA on tumor cell growth or production of VEGF-A and PEDF in any of the three uveal melanoma cell lines tested. Regarding expression as measured by RT-PCR, TA had an inhibiting effect on TSP-1 in only one cell line, and no effect on VEGF-A or PEDF. AA showed a similar lack of effect. CONCLUSIONS: Since TA and AA do not stimulate uveal melanoma cell growth, it seems to be safe to use these drugs to treat radiation retinopathy after irradiation for uveal melanoma. Additional experiments using more cell lines or primary tumor cell cultures are needed to validate this conclusion. Furthermore, the results of our study suggest that TA does not exert its antileakage effect through downregulation of VEGF-A or upregulation of TSP-1 or PEDF in uveal melanoma cell lines. It is possible that TA and AA influence these pro- and antiangiogenic factors only under hypoxic circumstances. Further investigation is needed.

Integrated transcript and genome analyses reveal NKX2-1 and MEF2C as potential oncogenes in T cell acute lymphoblastic leukemia.

To identify oncogenic pathways in T cell acute lymphoblastic leukemia (T-ALL), we combined expression profiling of 117 pediatric patient samples and detailed molecular-cytogenetic analyses including the Chromosome Conformation Capture on Chip (4C) method. Two T-ALL subtypes were identified that lacked rearrangements of known oncogenes. One subtype associated with cortical arrest, expression of cell cycle genes, and ectopic NKX2-1 or NKX2-2 expression for which rearrangements were identified. The second subtype associated with immature T cell development and high expression of the MEF2C transcription factor as consequence of rearrangements of MEF2C, transcription factors that target MEF2C, or MEF2C-associated cofactors. We propose NKX2-1, NKX2-2, and MEF2C as T-ALL oncogenes that are activated by various rearrangements.

Gamma-secretase inhibitors reverse glucocorticoid resistance in T cell acute lymphoblastic leukemia.

Gamma-secretase inhibitors (GSIs) block the activation of the oncogenic protein Notch homolog-1 (NOTCH1) in T cell acute lymphoblastic leukemia (T-ALL). However, limited antileukemic cytotoxicity and severe gastrointestinal toxicity have restricted the clinical application of these targeted drugs. Here we show that combination therapy with GSIs plus glucocorticoids can improve the antileukemic effects of GSIs and reduce their gut toxicity in vivo. Inhibition of NOTCH1 signaling in glucocorticoid-resistant T-ALL restored glucocorticoid receptor autoupregulation and induced apoptotic cell death through induction of the gene encoding BCL-2-like apoptosis initiator-11 (BCL2L11). GSI treatment resulted in cell cycle arrest and accumulation of goblet cells in the gut mediated by upregulation of the gene encoding the transcription factor Krüppel-like factor-4 (Klf4), a negative regulator of the cell cycle required for goblet cell differentiation. In contrast, glucocorticoid treatment induced transcriptional upregulation of cyclin D2 (Ccnd2) and protected mice from developing the intestinal goblet cell metaplasia typically induced by inhibition of NOTCH signaling with GSIs. These results support a role for glucocorticoids plus GSIs in the treatment of glucocorticoid-resistant T-ALL.