Postovulatory aging affects dynamics of mRNA, expression and localization of maternal effect proteins, spindle integrity and pericentromeric proteins in mouse oocytes.

STUDY QUESTION: Is the postovulatory aging-dependent differential decrease of mRNAs and polyadenylation of mRNAs coded by maternal effect genes associated with altered abundance and distribution of maternal effect and RNA-binding proteins (MSY2)? SUMMARY ANSWER: Postovulatory aging results in differential reduction in abundance of maternal effect proteins, loss of RNA-binding proteins from specific cytoplasmic domains and critical alterations of pericentromeric proteins without globally affecting protein abundance. WHAT IS KNOWN ALREADY: Oocyte postovulatory aging is associated with differential alteration in polyadenylation and reduction in abundance of mRNAs coded by selected maternal effect genes. RNA-binding and -processing proteins are involved in storage, polyadenylation and degradation of mRNAs thus regulating stage-specific recruitment of maternal mRNAs, while chromosomal proteins that are stage-specifically expressed at pericentromeres, contribute to control of chromosome segregation and regulation of gene expression in the zygote. STUDY DESIGN, SIZE, DURATION: Germinal vesicle (GV) and metaphase II (MII) oocytes from sexually mature C57B1/6J female mice were investigated. Denuded in vivo or in vitro matured MII oocytes were postovulatory aged and analyzed by semiquantitative confocal microscopy for abundance and localization of polyadenylated RNAs, proteins of maternal effect genes (transcription activator BRG1 also known as ATP-dependent helicase SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 4 (SMARCA4) and NOD-like receptor family pyrin domain containing 5 (NLRP5) also known as MATER), RNA-binding proteins (MSY2 also known as germ cell-specific Y-box-binding protein, YBX2), and post-transcriptionally modified histones (trimethylated histone H3K9 and acetylated histone H4K12), as well as pericentromeric ATRX (alpha thalassemia/mental retardation syndrome X-linked, also termed ATP-dependent helicase ATRX or X-linked nuclear protein (XNP)). For proteome analysis five replicates of 30 mouse oocytes were analyzed by selected reaction monitoring (SRM). MATERIAL AND METHODS: GV and MII oocytes were obtained from large antral follicles or ampullae of sexually mature mice, respectively. Denuded MII oocytes were aged for 24 h post ovulation. For analysis of distribution and abundance of polyadenylated RNAs fixed oocytes were in situ hybridized to Cy5 labeled oligo(dT)20 nucleotides. Absolute quantification of protein concentration per oocyte of selected proteins was done by SRM proteome analysis. Relative abundance of ATRX was assessed by confocal laser scanning microscopy (CLSM) of whole mount formaldehyde fixed oocytes or after removal of zona and spreading. MSY2 protein distribution and abundance was studied in MII oocytes prior to, during and after exposure to nocodazole, or after aging for 2 h in presence of H2O2 or for 24 h in presence of a glutathione donor, glutathione ethylester (GEE). MAIN RESULTS AND ROLE OF CHANCE: The significant reduction in abundance of proteins (P < 0.001) translated from maternal mRNAs was independent of polyadenylation status, while their protein localization was not significantly changed by aging. Most of other proteins quantified by SRM analysis did not significantly change in abundance upon aging except MSY2 and GTSF1. MSY2 was enriched in the subcortical RNP domain (SCRD) and in the spindle chromosome complex (SCC) in a distinct pattern, right and left to the chromosomes. There was a significant loss of MSY2 from the SCRD (P < 0.001) and the spindle after postovulatory aging. Microtubule de- and repolymerization caused reversible loss of MSY2 spindle-association whereas H2O2 stress did not significantly decrease MSY2 abundance. Aging in presence of GEE decreased significantly (P < 0.05) the aging-related overall and cytoplasmic loss of MSY2. Postovulatory aging increased significantly spindle abnormalities, unaligned chromosomes, and abundance of acetylated histone H4K12, and decreased pericentromeric trimethylated histone H3K9 (all P < 0.001). Spreading revealed a highly significant increase in pericentromeric ATRX (P < 0.001) upon ageing. Thus, the significantly reduced abundance of MSY2 protein, especially at the SCRD and the spindle may disturb the spatial control and timely recruitment, deadenylation and degradation of developmentally important RNAs. An autonomous program of degradation appears to exist which transiently and specifically induces the loss and displacement of transcripts and specific maternal proteins independent of fertilization in aging oocytes and thereby can critically affect chromosome segregation and gene expression in the embryo after fertilization. LIMITATION, REASONS FOR CAUTION: We used the mouse oocyte to study processes associated with postovulatory aging, which may not entirely reflect processes in aging human oocytes. However, increases in spindle abnormalities, unaligned chromosomes and H4K12 acetylated histones, as well as in mRNA abundance and polyadenylation have been observed also in aged human oocytes suggesting conserved processes in aging. WIDER IMPLICATIONS OF THE FINDINGS: Postovulatory aging precociously induces alterations in expression and epigenetic modifications of chromatin by ATRX and in histone pattern in MII oocytes that normally occur after fertilization, possibly contributing to disturbances in the oocyte-to-embryo transition (OET) and the zygotic gene activation (ZGA). These observations in mouse oocytes are also relevant to explain disturbances and reduced developmental potential of aged human oocytes and caution to prevent oocyte aging in vivo and in vitro. STUDY FUNDING/COMPETING INTERESTS: The study has been supported by the German Research Foundation (DFG) (EI 199/7-1 | GR 1138/12-1 | HO 949/21-1 and FOR 1041). There is no competing interest.

Inherited microdeletions in the Angelman and Prader-Willi syndromes define an imprinting centre on human chromosome 15.

A subset of patients with Angelman and Prader-Willi syndrome have apparently normal chromosomes of biparental origin, but abnormal DNA methylation at several loci within chromosome 15q11-13, and probably have a defect in imprinting. Using probes from a newly established 160-kb contig including D15S63 (PW71) and SNRPN, we have identified inherited microdeletions in two AS families and three PWS families. The deletions probably affect a single genetic element that we term the 15q11-13 imprinting centre (IC). In our model, the IC regulates the chromatin structure, DNA methylation and gene expression in cis throughout 15q11-13. Mutations of the imprinting centre can be transmitted silently through the germline of one sex, but appear to block the resetting of the imprint in the germline of the opposite sex.

Cloning of the putative tumour suppressor gene for hereditary multiple exostoses (EXT1).

Hereditary multiple exostoses is an autosomal dominant disorder that is characterized by short stature and multiple, benign bone tumours. In a majority of families, the genetic defect (EXT1) is linked to the Langer-Giedion syndrome chromosomal region in 8q24.1. From this region we have cloned and characterized a cDNA which spans chromosomal breakpoints previously identified in two multiple exostoses patients. Furthermore, the gene harbours frameshift mutations in affected members of two EXT1 families. The cDNA has a coding region of 2,238 bp with no apparent homology to other known gene sequences and thus its function remains elusive. However, recent studies in sporadic and exostosis-derived chondrosarcomas suggest that the 8q24.1-encoded EXT1 gene may have tumour suppressor function.

Imprint switching on human chromosome 15 may involve alternative transcripts of the SNRPN gene.

Imprinting on human chromosome 15 is regulated by an imprinting centre, which has been mapped to a 100-kb region including exon 1 of SNRPN. From this region we have identified novel transcripts, which represent alternative transcripts of the SNRPN gene. The novel exons lack protein coding potential and are expressed from the paternal chromosome only. We have also identified intragenic deletions and a point mutation in patients who have Angelman or Prader-Willi syndrome due to a parental imprint switch failure. This suggests that imprint switching on human chromosome 15 may involve alternative SNRPN transcripts.

De novo deletions of SNRPN exon 1 in early human and mouse embryos result in a paternal to maternal imprint switch.

Prader-Willi syndrome (PWS) is a neurogenetic disease characterized by infantile hypotonia, gonadal hypoplasia, obsessive behaviour and neonatal feeding difficulties followed by hyperphagia, leading to profound obesity. PWS is due to a lack of paternal genetic information at 15q11-q13 (ref. 2). Five imprinted, paternally expressed genes map to the PWS region, MKRN3 (ref. 3), NDN (ref. 4), NDNL1 (ref. 5), SNRPN (refs 6-8 ) and IPW (ref. 9), as well as two poorly characterized framents designated PAR-1 and PAR-5 (ref. 10). Imprinting of this region involves a bipartite 'imprinting centre' (IC), which overlaps SNRPN (refs 10,11). Deletion of the SNRPN promoter/exon 1 region (the PWS IC element) appears to impair the establishment of the paternal imprint in the male germ line and leads to PWS. Here we report a PWS family in which the father is mosaic for an IC deletion on his paternal chromosome. The deletion chromosome has acquired a maternal methylation imprint in his somatic cells. We have made identical findings in chimaeric mice generated from two independent embryonic stem (ES) cell lines harbouring a similar deletion. Our studies demonstrate that the PWS IC element is not only required for the establishment of the paternal imprint, but also for its postzygotic maintenance.

Mutations in a new gene, encoding a zinc-finger protein, cause tricho-rhino-phalangeal syndrome type I.

Tricho-rhino-phalangeal syndrome type I (TRPS I, MIM 190350) is a malformation syndrome characterized by craniofacial and skeletal abnormalities and is inherited in an autosomal dominant manner. TRPS I patients have sparse scalp hair, a bulbous tip of the nose, a long flat philtrum, a thin upper vermilion border and protruding ears. Skeletal abnormalities include cone-shaped epiphyses at the phalanges, hip malformations and short stature. We assigned TRPS1 to human chromosome 8q24. It maps proximal of EXT1, which is affected in a subgroup of patients with multiple cartilaginous exostoses and deleted in all patients with TRPS type II (TRPS II, or Langer-Giedion syndrome, MIM 150230; ref.2-5). We have positionally cloned a gene that spans the chromosomal breakpoint of two patients with TRPS I and is deleted in five patients with TRPS I and an interstitial deletion. Northern-blot analyses revealed transcripts of 7 and 10.5 kb. TRPS1has seven exons and an ORF of 3,843 bp. The predicted protein sequence has two potential nuclear localization signals and an unusual combination of different zinc-finger motifs, including IKAROS-like and GATA-binding sequences. We identified six different nonsense mutations in ten unrelated patients. Our findings suggest that haploinsufficiency for this putative transcription factor causes TRPS I.

Maternal methylation imprints on human chromosome 15 are established during or after fertilization.

Prader-Willi syndrome (PWS) is a neurogenetic disorder that results from the lack of transcripts expressed from the paternal copy of the imprinted chromosomal region 15q11-q13 (refs. 1,2). In some patients, this is associated with a deletion of the SNURF-SNRPN exon 1 region inherited from the paternal grandmother and the presence of a maternal imprint on the paternal chromosome. Assuming that imprints are reset in the germ line, we and others have suggested that this region constitutes part of the 15q imprinting center (IC) and is important for the maternal to paternal imprint switch in the male germ line. Here we report that sperm DNA from two males with an IC deletion had a normal paternal methylation pattern along 15q11-q13. Similar findings were made in a mouse model. Our results indicate that the incorrect maternal methylation imprint in IC deletion patients is established de novo after fertilization. Moreover, we found that CpG-rich regions in SNURF-SNRPN and NDN, which in somatic tissues are methylated on the maternal allele, are hypomethylated in unfertilized human oocytes. Our results indicate that the normal maternal methylation imprints in 15q11-q13 also are established during or after fertilization.

Association of a human G-protein beta3 subunit variant with hypertension.

Hypertension is a common disorder of multifactorial origin that constitutes a major risk factor for cardiovascular events such as stroke and myocardial infarction. Previous studies demonstrated an enhanced signal transduction via pertussis toxin-sensitive G proteins in lymphoblasts and fibroblasts from selected patients with essential hypertension. We have detected a novel polymorphism (C825T) in exon 10 of the gene encoding the beta3 subunit of heterotrimeric G proteins (GNB3). The T allele is associated with the occurrence of a splice variant, GNB3-s (encoding G beta3-s), in which the nucleotides 498-620 of exon 9 are deleted. This in-frame deletion causes the loss of 41 amino acids and one WD repeat domain of the G beta subunit. By western-blot analysis, G beta3-s appears to be predominantly expressed in cells from individuals carrying the T allele. Significant enhancement of stimulated GTPgammaS binding to Sf9 insect cells expressing G beta3-s together with G alpha(i)2 and G gamma5 indicates that this splice variant is biologically active. Genotype analysis of 427 normotensive and 426 hypertensive subjects suggests a significant association of the T allele with essential hypertension.

Idiopathic male infertility is strongly associated with aberrant methylation of MEST and IGF2/H19 ICR1.

Aberrant imprinting in spermatozoa in a subset of infertile men has been postulated to be a risk factor for congenital diseases in children conceived via assisted reproduction techniques (ART). Studies in clinically well characterized large cohorts, however, have been missing. Using bisulfite sequencing, we determined the degree of methylation of the IGF2/H19 imprinting control region 1 (ICR1) and MEST differentially methylated regions in swim-up purified spermatozoa from 148 idiopathic infertile men and 33 normozoospermic controls. All control individuals had a high degree of IGF2/H19 ICR1 and a low degree of MEST methylation. Low sperm counts were clearly associated with IGF2/H19 ICR1 hypomethylation and, even stronger, with MEST hypermethylation. MEST hypermethylation, but not IGF2/H19 ICR1 hypomethylation was found in idiopathic infertile men with progressive sperm motility below 40% and bad sperm morphology below 5% normal spermatozoa. Ageing could be ruled out as a cause for the observed methylation defects. Sequence analysis of the CTCFL gene in peripheral blood DNA from 20 men with severe methylation defects revealed several polymorphisms, but no bona fide mutation. We conclude that idiopathic male infertility is strongly associated with imprinting defects at IGF2/H19 ICR1 and MEST, with aberrant MEST methylation being a strong indicator for sperm quality. The male germ cell thus represents a potential source for aberrant epigenetic features in children conceived via ART.

Biallelic loss of function of the promyelocytic leukaemia zinc finger (PLZF) gene causes severe skeletal defects and genital hypoplasia.

BACKGROUND: Deletions of 11q23 are associated with mental retardation, craniofacial dysmorphism, microcephaly and short stature. We present a patient with similar clinical findings, in addition to absence of the thumbs, hypoplasia of the radii and ulnae, additional vertebrae and ribs, retarded bone age and genital hypoplasia. METHODS: Genomic DNA from the patient was screened for chromosomal imbalances by array-based comparative genomic hybridisation. DNA sequence analyses and reporter gene assays were performed in order to identify candidate gene mutations. RESULTS: The patient has an approximately 8 Mbp de novo deletion on the paternal chromosome 11, which includes the promyelocytic leukaemia zinc-finger gene (PLZF, ZBTB16; OMIM 176797). The maternal PLZF allele harbours a recessive missense mutation (c.1849A-->G), which leads to the substitution of a highly conserved methionine by valine (p.Met617Val) within a zinc-finger motif. Taking into account specific alpha-helical propensities of Val and Met, this mutation is likely to destabilise the alpha helix of the zinc finger that forms the contact with the DNA duplex, thus affecting the biological function as shown by reporter-gene assays. DISCUSSION: The PLZF gene is one of five partners fused to the retinoic acid receptor alpha in acute promyelocytic leukaemia. We describe the first patient, to our knowledge, with a germline mutation of PLZF. Our findings as well as observations in Plzf-deficient mice indicate that PLZF is a key regulator of skeletal and male germline development. Furthermore, this case highlights the importance of searching for a recessive mutation on the non-deleted chromosome in patients with a microdeletion and atypical clinical findings.

The European Prader-Willi Syndrome Clinical Research Database: an aid in the investigation of a rare genetically determined neurodevelopmental disorder.

BACKGROUND: Prader-Willi Syndrome (PWS) is a rare genetically determined neurodevelopmental disorder with a complex phenotype that changes with age. The rarity of the syndrome and the need to control for different variables such as genetic sub-type, age and gender limits clinical studies of sufficient size in any one country. A clinical research database has been established to structure data collection and to enable multinational investigations into the development of children and adults with PWS. METHODS: As part of a joint basic science and clinical study of PWS funded through Framework 6 of the European Union (EU), an expert multidisciplinary group was established that included clinicians involved in PWS research and clinical practice, expert database software developers, and representatives from two national PWS Associations. This group identified the key issues that required resolution and the data fields necessary for a comprehensive database to support PWS research. RESULTS: The database consists of six 'index' entry points and branching panels and sub-panels and over 1200 data 'fields'. It is Internet-based and designed to support multi-site clinical research in PWS. An algorithm ensures that participant data are anonymous. Access to data is controlled in a manner that is compatible with EU and national laws. The database determines the assessments to be used to collect data thereby enabling the combining of data from different groups under specifically agreed conditions. The data collected at any one time will be determined by individual research groups, who retain control of the data. Over time the database will accumulate data on participants with PWS that will support future research by avoiding the need for repeat data collection of fixed data and it will also enable longitudinal studies and treatment trials. CONCLUSION: The development of the database has proved to be complex with various administrative and ethical issues to be addressed. At an early stage, it was important to clarify the exact function of the database. It was agreed that it was primarily to support grant-funded research rather than clinical practice. The most complex issues that had to be addressed were concerned with data ownership and establishing the rules for data entry, retrieval and sharing that are compatible with data protection laws, and which are likely to be acceptable to participants and their families and to individual research groups.

Parental-origin-determination fluorescence in situ hybridization distinguishes homologous human chromosomes on a single-cell level.

The differentiation of homologous chromosomes as well as their parental origin can presently be conducted and determined exclusively by molecular genetic methods using microsatellite or SNP analysis. Only in exceptional cases is a distinction on a single-cell level possible, e.g. due to variations within the heterochromatic regions of chromosomes 1, 9, 16 and Y or the p-arms of the acrocentric chromosomes. In the absence of such polymorphisms, an individual distinction of the homologous chromosomes is not currently possible. Consequently, various questions of scientific and diagnostic relevance are unable to be answered. Based on the recently detected large-scale copy-number variations (LCV) or copy-number polymorphisms (CNP) spanning up to several megabase pairs of DNA, in this study, a molecular cytogenetic technique for the inter-individual differentiation of homologous chromosomes called parental-origin-determination fluorescence in situ hybridization (pod-FISH) is presented. All human chromosomes were covered with 225 LCV- and/or CNP-specific BAC probes, and one- to five-color chromosome-specific pod-FISH sets were created, evaluated and optimized. We demonstrated that pod-FISH is suitable for single-cell analysis of uniparental disomy (UDP) in clinical cases such as Prader-Willi syndrome caused by maternal UPD. A rare clinical case with a mosaic form of a genome-wide isodisomy was used to determine the detection limits of pod-FISH. Additionally we analyzed the informativeness of conventional microsatellite analysis for the first time and compared the results to pod-FISH. With this new possibility to study the parental origin of individual human chromosomes on a single-cell level, new doors for diagnostic and basic research are opened.

Imprinting in Prader-Willi and Angelman syndromes.

Imprinted genes are marked in the germline and retain molecular memory of their parental origin, resulting in allelic expression differences during development. Abnormalities in imprinted inheritance occur in several genetic diseases and cancer, and are exemplified by the diverse genetic defects involving chromosome 15q11-q13 in Prader-Willi (PWS) and Angelman (AS) syndromes. PWS involves loss of function of multiple paternally expressed genes, while mutations in a single gene, UBE3A, which is subject to spatially restricted imprinting, occur in some AS patients. Identification of mutations in the imprinting process in PWS and AS has led to a definition of an imprinting center (IC), involving the promoter (in PWS) or an alternative transcript of the SNRPN gene (in AS). The IC regulates initiation of imprint switching for all genes in a 2 Mb imprinted domain during gametogenesis. Imprinting mutations define a novel mechanism of genetic disease because they have no direct effect in the affected patient but, rather, it is the parental germline effect of an IC mutation that leads to disease in the offspring.

Epimutations in human disease.

Epigenetics is the study of genes during development. Gene expression states are set by transcriptional activators and repressors and locked in by cell-heritable chromatin states. Inappropriate expression or repression of genes can change developmental trajectories and result in disease. Aberrant chromatin states leading to aberrant gene expression patterns (epimutations) have been detected in several recognizable syndromes as well as in cancer. They can occur secondary to a DNA mutation in a cis- or trans-acting factor, or as a "true" or primary epimutation in the absence of any DNA sequence change. Primary epimutations often occur after fertilization and lead to somatic mosaicism. It has been estimated that the rate of primary epimutations is one or two orders of magnitude greater than somatic DNA mutation. Therefore, the contribution of epimutations to human disease is probably underestimated.

Imprinting defects on human chromosome 15.

The Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are two distinct neurogenetic diseases that are caused by the loss of function of imprinted genes on the proximal long arm of human chromosome 15. In a few percent of patients with PWS and AS, the disease is due to aberrant imprinting and gene silencing. In patients with PWS and an imprinting defect, the paternal chromosome carries a maternal imprint. In patients with AS and an imprinting defect, the maternal chromosome carries a paternal imprint. Imprinting defects offer a unique opportunity to identify some of the factors and mechanisms involved in imprint erasure, resetting and maintenance. In approximately 10% of cases the imprinting defects are caused by a microdeletion affecting the 5' end of the SNURF-SNRPN locus. These deletions define the 15q imprinting center (IC), which regulates imprinting in the whole domain. These findings have been confirmed and extended in knock-out and transgenic mice. In the majority of patients with an imprinting defect, the incorrect imprint has arisen without a DNA sequence change, possibly as the result of stochastic errors of the imprinting process or the effect of exogenous factors.

Increased prevalence of imprinting defects in patients with Angelman syndrome born to subfertile couples.

Recent case reports have suggested that infertility treatment with intracytoplasmic sperm injection (ICSI) may increase the risk of imprinting defects leading to Angelman syndrome (AS). Although imprinting defects account for only 4% of patients with AS, we have found four cases among 16 AS patients born to subfertile couples, who conceived with or without infertility treatment (25%; relative risk (RR) 6.25; 95% confidence interval (CI) 1.68 to 16.00). The risk in untreated couples with time to pregnancy (TTP) exceeding 2 years was identical to that of those treated by ICSI or by hormonal stimulation alone (RR 6.25; 95% CI 0.70 to 22.57). It was twice as high in couples who had received treatment and also had TTP >2 years (RR 12.5; 95% CI 1.40 to 45.13). Our findings suggest that imprinting defects and subfertility may have a common cause, and that superovulation rather than ICSI may further increase the risk of conceiving a child with an imprinting defect.

Detection of homozygous deletions in tumors by hybridization of representational difference analysis (RDA) products to chromosome-specific YAC clone arrays.

Representational difference analysis (RDA), a subtractive hybridization method that enriches differences between complex genomes, can be used to isolate fragments deleted in tumor genomes. Usually, most of the clones obtained by this approach result from polymorphic fragments. Therefore, identification of homozygously deleted fragments, which can indicate the presence of tumor suppressor loci, is often tedious. To overcome this limitation, we devised a novel strategy in which labeled RDA products are hybridized in toto against membranes spotted with YAC clones covering a region of interest. In such a way, identified YAC clones provide positional information on homozygous deletions and loss of heterozygosity (LOH) regions. We have tested this approach with a tumor known to have a homozygous deletion within a region of LOH on chromosome 13. RDA was performed using representations generated with restriction enzymes Bgl II, Nco I and Xba I, and the difference products of each experiment were separately hybridized to chromosome 13 YAC filters. When collating the map positions of positive YACs from three different RDA experiments a cluster of hits clearly identified the region on chromosome 13 which comprised the homozygous deletion. This shows that our novel approach can be effective.

Chromosomal gains and losses in uveal melanomas detected by comparative genomic hybridization.

Eleven uveal melanomas were analyzed using comparative genomic hybridization (CGH). The most abundant genetic changes were loss of chromosome 3, overrepresentation of 6p, loss of 6q, and multiplication of 8q. The smallest overrepresented regions on 6p and 8q were 6pter-->p21 and 8q24-->qter, respectively. Several additional gains and losses of chromosome segments were repeatedly observed, the most frequent one being loss of 9p (three cases). Monosomy 3 appeared to be a marker for ciliary body involvement. CGH data were compared with the results of chromosome banding. Some alterations, e.g., gains of 6p and losses of 6q, were observed with higher frequencies after CGH, while others, e.g., 9p deletions, were detected only by CGH. The data suggest some similarities of cytogenetic alterations between cutaneous and uveal melanoma. In particular, the 9p deletions are of interest due to recent reports about the location of a putative tumor-suppressor gene for cutaneous malignant melanoma in this region.