Recent Advances in Imprinting Disorders.

Imprinting disorders (ImpDis) are a group of currently 12 congenital diseases with common underlying (epi)genetic etiologies and overlapping clinical features affecting growth, development and metabolism. In the last years it has emerged that ImpDis are characterized by the same types of mutations and epimutations, i.e. uniparental disomies, copy number variations, epimutations, and point mutations. Each ImpDis is associated with a specific imprinted locus, but the same imprinted region can be involved in different ImpDis. Additionally, even the same aberrant methylation patterns are observed in different phenotypes. As some ImpDis share clinical features, clinical diagnosis is difficult in some cases. The advances in molecular and clinical diagnosis of ImpDis help to circumvent these issues, and they are accompanied by an increasing understanding of the pathomechanism behind them. As these mechanisms have important roles for the etiology of other common conditions, the results in ImpDis research have a wider effect beyond the borders of ImpDis. For patients and their families, the growing knowledge contributes to a more directed genetic counseling of the families and personalized therapeutic approaches.

Genetic and epigenetic methylation defects and implication of the ERMN gene in autism spectrum disorders.

Autism spectrum disorders (ASD) are highly heritable and genetically complex conditions. Although highly penetrant mutations in multiple genes have been identified, they account for the etiology of <1/3 of cases. There is also strong evidence for environmental contribution to ASD, which can be mediated by still poorly explored epigenetic modifications. We searched for methylation changes on blood DNA of 53 male ASD patients and 757 healthy controls using a methylomic array (450K Illumina), correlated the variants with transcriptional alterations in blood RNAseq data, and performed a case-control association study of the relevant findings in a larger cohort (394 cases and 500 controls). We found 700 differentially methylated CpGs, most of them hypomethylated in the ASD group (83.9%), with cis-acting expression changes at 7.6% of locations. Relevant findings included: (1) hypomethylation caused by rare genetic variants (meSNVs) at six loci (ERMN, USP24, METTL21C, PDE10A, STX16 and DBT) significantly associated with ASD (q-value <0.05); and (2) clustered epimutations associated to transcriptional changes in single-ASD patients (n=4). All meSNVs and clustered epimutations were inherited from unaffected parents. Resequencing of the top candidate genes also revealed a significant load of deleterious mutations affecting ERMN in ASD compared with controls. Our data indicate that inherited methylation alterations detectable in blood DNA, due to either genetic or epigenetic defects, can affect gene expression and contribute to ASD susceptibility most likely in an additive manner, and implicate ERMN as a novel ASD gene.

IFPA meeting 2014 workshop report: Animal models to study pregnancy pathologies; new approaches to study human placental exposure to xenobiotics; biomarkers of pregnancy pathologies; placental genetics and epigenetics; the placenta and stillbirth and fetal growth restriction.

Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialized topics. At IFPA meeting 2014 there were six themed workshops, five of which are summarized in this report. These workshops related to various aspects of placental biology but collectively covered areas of animal models, xenobiotics, pathological biomarkers, genetics and epigenetics, and stillbirth and fetal growth restriction.

Distinct promoter methylation and isoform-specific expression of RASFF1A in placental biopsies from complicated pregnancies.

INTRODUCTION: Epigenetic changes in the placenta have been postulated to act as mediators between environmental influences and poor fetal growth. We assessed if genes with a plausible influence on growth could be aberrantly methylated in placental samples from pregnancies complicated by intrauterine growth restriction (IUGR). METHODS: A candidate gene approach was undertaken using a custom Illumina Goldengate® array on a collection of placental samples from growth restricted pregnancies and normally grown controls with confirmation using bisulphite pyrosequencing. RESULTS: The custom array analysis revealed that the promoter of RASSF1A was the only region with significant methylation differences between IUGR placentas and those from pregnancies with appropriate growth for gestational age (AGA). The RASSF1A promoter had increased levels of DNA methylation in IUGR samples compared to controls. Interestingly, the methylation difference was also observed in preeclamptic samples. Higher methylation was associated with a concomitant decrease in expression of the RASSF1 transcript A, but not other isoforms that originate from an alternative, nearby promoter interval. DISCUSSION: Our results do not support the hypothesis that altered DNA-methylation in the placenta is a mechanism generally involved in fetal growth restriction. A specific region corresponding to the promoter of RASSF1A does display methylation changes in placenta that could be used to identify at-risk pregnancies.

The genetic aetiology of Silver-Russell syndrome.

Silver-Russell syndrome (SRS MIM180860) is a disorder characterised by intrauterine and/or postnatal growth restriction and typical facies. However, the clinical picture is extremely diverse due to numerous diagnostic features reflecting a heterogeneous genetic disorder. The mode of inheritance is variable with sporadic cases also being described. Maternal uniparental disomy (mUPD) of chromosome 7 accounts for 10% of SRS cases and many candidate imprinted genes on 7 have been investigated. Chromosome 11 has moved to the forefront as the key chromosome in the aetiology, with reports of methylation defects in the H19 imprinted domain associated with the phenotype in 35-65% of SRS patients. Methylation aberrations have been described in a number of other imprinted growth related disorders such as Beckwith-Wiedmann syndrome. This review discusses these recent developments as well as the previous work on chromosome 7. Other candidate genes/chromosomal regions previously investigated are tabled.

Physiological effects on demography: a long-term experimental study of testosterone's effects on fitness.

Understanding physiological and behavioral mechanisms underlying the diversity of observed life-history strategies is challenging because of difficulties in obtaining long-term measures of fitness and in relating fitness to these mechanisms. We evaluated effects of experimentally elevated testosterone on male fitness in a population of dark-eyed juncos studied over nine breeding seasons using a demographic modeling approach. Elevated levels of testosterone decreased survival rates but increased success of producing extra-pair offspring. Higher overall fitness for testosterone-treated males was unexpected and led us to consider indirect effects of testosterone on offspring and females. Nest success was similar for testosterone-treated and control males, but testosterone-treated males produced smaller offspring, and smaller offspring had lower postfledging survival. Older, more experienced females preferred to mate with older males and realized higher reproductive success when they did so. Treatment of young males increased their ability to attract older females yet resulted in poor reproductive performance. The higher fitness of testosterone-treated males in the absence of a comparable natural phenotype suggests that the natural phenotype may be constrained. If this phenotype were to arise, the negative social effects on offspring and mates suggest that these effects might prevent high-testosterone phenotypes from spreading in the population.

Imprinting control within the compact Gnas locus.

Mouse distal chromosome 2 was one of the earliest described imprinting regions. Maternal and paternal inheritance of the region is associated with opposite phenotypes affecting growth, development and behaviour. Mis-expression of proteins determined by the imprinted Gnas locus can account for the phenotypes. The imprinting domain in mouse distal chromosome 2 is small, comprising the Gnas locus. This locus is unusually complex, containing biallelic, maternally and paternally expressed transcripts that share exons. It also contains two germline differentially methylated regions that have the characteristics of imprinting control regions. One of these specifically controls the tissue-specific imprinting of the Gnas exon 1 transcript but does not affect the imprinting of other transcripts. Imprinting of other transcripts may be controlled by the other germline differentially methylated region by a mechanism involving antisense RNA.

Imprinted genes and their role in human fetal growth.

Growth is defined as the progressive increase in size and is listed as one of the eight main characteristics of life. In human gestation the most rapid growth phase is from 16 to 32 weeks when first there is both cell number and size increase and then from 32 weeks onwards there is continued size increase (Pollack and Divon, 1992). The mechanism of growth in utero is of fundamental interest to clinicians and scientists because of its implications for neonatal health. Growth is multifactorial in origin with both genetics and environment contributing equally large parts. Despite this complexity analysis of the candidate genes involved is possible using simple tissue biopsies at the relevant stages of development. Of particular interest in understanding fetal growth is the analysis of a group of genes that show a parent-of-origin effect known as genomic imprinting. Imprinted genes are not only found in eutherian (placental) and metatherian (marsupial) mammals but surprisingly also in plants. Nevertheless, their evolution in mammals appears to be linked primarily to placentation. It is thought to result from a potential conflict between the parents in terms of the drive to successfully propagate their own separate genes and the mother's added drive for her survival through the pregnancy to reproduce again. This means that the mother wants to restrict fetal growth and the father to enhance it.

Limited evolutionary conservation of imprinting in the human placenta.

The epigenetic phenomenon of genomic imprinting provides an additional level of gene regulation that is confined to a limited number of genes, frequently, but not exclusively, important for embryonic development. The evolution and maintenance of imprinting has been linked to the balance between the allocation of maternal resources to the developing fetus and the mother's well being. Genes that are imprinted in both the embryo and extraembryonic tissues show extensive conservation between a mouse and a human. Here we examine the human orthologues of mouse genes imprinted only in the placenta, assaying allele-specific expression and epigenetic modifications. The genes from the KCNQ1 domain and the isolated human orthologues of the imprinted genes Gatm and Dcn all are expressed biallelically in the human, from first-trimester trophoblast through to term. This lack of imprinting is independent of promoter CpG methylation and correlates with the absence of the allelic histone modifications dimethylation of lysine-9 residue of H3 (H3K9me2) and trimethylation of lysine-27 residue of H3 (H3K27me3). These specific histone modifications are thought to contribute toward regulation of imprinting in the mouse. Genes from the IGF2R domain show polymorphic concordant expression in the placenta, with imprinting demonstrated in only a minority of samples. Together these findings have important implications for understanding the evolution of mammalian genomic imprinting. Because most human pregnancies are singletons, this absence of competition might explain the comparatively relaxed need in the human for placental-specific imprinting.

Imprinting of IGF2 P0 transcript and novel alternatively spliced INS-IGF2 isoforms show differences between mouse and human.

Genomic imprinting is limited to a subset of genes that play critical roles in fetal growth, development and behaviour. One of the most studied imprinted genes encodes insulin-like growth factor 2, and aberrant imprinting and DNA methylation of this gene is associated with the growth disorders Beckwith-Wiedemann and Silver-Russell syndromes and many human cancers. Specific isoforms of this gene have been shown to be essential for normal placental function, as mice carrying paternal null alleles for the Igf2-P0 transcript are growth restricted at birth. We report here the identification of three novel human transcripts from the IGF2 locus. One is equivalent to the mouse Igf2-P0 transcript, whereas the two others (INSIGF long and short) originate from the upstream INS gene that alternatively splices to downstream IGF2 exons. In order to elucidate the molecular mechanisms involved in the complex imprinting of these novel IGF2 transcripts, both the allele-specific expression and methylation for all the IGF2 promoters including P0 and the INSIGF transcripts were analysed in human tissues. Similar to the mouse, the human IGF2-P0 transcript is paternally expressed; however, its expression is not limited to placenta. This expression correlates with tissue-specific promoter methylation on the maternal allele. The two novel INSIGF transcripts reported here use the INS promoter and show highly restricted tissue expression profiles including the pancreas. As previously reported for INS in the yolk sac, we demonstrate complex, tissue-specific imprinting of these transcripts. The finding of additional transcripts within this locus will have important implications for IGF2 regulation in both cancer and metabolism.

Evolución a largo plazo de la esquizofrenia de inicio tardío: estudio de 5 años de seguimiento / Long-term outcome of late-onset schizophrenia: 5-year follow-up study

INTRODUCCIÓN: Hay controversia sobre si la esquizofrenia de inicio tardío es un precursor de deterioro cognitivo. OBJETIVOS: Examinar la evolución a largo plazo de un grupo de pacientes con esquizofrenia de inicio tardío. PACIENTES Y MÉTODO: Pacientes con inicio de esquizofrenia DSM-III-R a una edad de 50 años o más tarde, sin demencia, y un grupo control sano. Se llevó a cabo una evaluación basal (n = 27 y n = 34, respectivamente), tras 1 año y tras 5 años (n = 19 y n = 24, respectivamente) con medidas de psicopatología, cognición y funcionamiento general, y se compararon sus índices de deterioro y la incidencia de demencia. RESULTADOS: Nueve pacientes con esquizofrenia de inicio tardío y ninguno del grupo control presentaron demencia (5 tipo Alzheimer, 1 vascular y 3 demencia de tipo desconocido) a los 5 años de seguimiento. Parecía haber un subgrupo de pacientes con esquizofrenia de inicio tardío sin signos de demencia basal o al año de seguimiento que posteriormente se deterioraron. CONCLUSIONES: La esquizofrenia de inicio tardío puede ser un pródromo de demencia tipo Alzheimer. Se requieren más estudios longitudinales para determinar su estado nosológico (AU)

The imprinted region on human chromosome 7q32 extends to the carboxypeptidase A gene cluster: an imprinted candidate for Silver-Russell syndrome.

Imprinted gene(s) on human chromosome 7q32-qter have been postulated to be involved in intrauterine growth restriction associated with Silver-Russell syndrome (SRS) as 7-10% of patients have mUPD(7). Three imprinted genes, MEST, MESTIT1, and COPG2IT1 on chromosome 7q32, are unlikely to cause SRS since epigenetic and sequence mutation analyses have not shown any changes. One hundred kilobases proximal to MEST lies a group of four carboxypeptidase A (CPA) genes. Since most imprinted genes are found in clusters, this study focuses on analysing these CPAs for imprinting effects based on their proximity to an established imprinted domain. Firstly, a replication timing study across 7q32 showed that an extensive genomic region including the CPAs, MEST, MESTIT1, and COPG2IT1 replicates asynchronously. Subsequently, SNP analysis by sequencing RT-PCR products of CPA1, CPA2, CPA4, and CPA5 indicated preferential expression of CPA4. Pyrosequencing was used as a quantitative approach, which confirmed predominantly preferential expression of the maternal allele and biallelic expression in brain. CPA5 expression levels were too low to allow reliable evaluation of allelic expression, while CPA1 and CPA2 both showed biallelic expression. CPA4 was the only gene from this family in which an imprinting effect was shown despite the location of this family of genes next to an imprinted cluster. As CPA4 has a potential role in cell proliferation and differentiation, two preferentially expressed copies in mUPD patients with SRS syndrome would result in excess expression and could alter the growth profiles of these subjects and give rise to intrauterine growth restriction.

Maternal repression of the human GRB10 gene in the developing central nervous system; evaluation of the role for GRB10 in Silver-Russell syndrome.

The GRB10 gene encodes a growth suppressor and maps to human chromosome 7p11.2-p13. Maternal duplication (matdup) of this region has recently been associated with Silver-Russell syndrome (SRS), which is characterised by pre- and postnatal growth restriction, craniofacial dysmorphism and lateral asymmetry. Maternal uniparental disomy for chromosome 7 (mUPD7) occurs in approximately 7% of SRS patients. Exposure of a recessive allele due to isodisomy has been ruled out in five mUPD7 cases, suggesting genomic imprinting as the basis for disease. Assuming SRS patients with matdup of 7p11.2-p13 and mUPD7 share a common aetiology, this would implicate a maternally expressed gene from this interval, which is involved in growth inhibition. Murine Grb10 was identified as a maternally expressed gene by subtractive hybridisation using normal and androgenetic mouse embryos. Grb10 maps to the homologous region of proximal mouse chromosome 11, for which mUPD incurs reduced birthweight. A role for GRB10 in SRS was evaluated by determining its imprinting status in multiple human foetal tissues using expressed polymorphisms, and by screening the coding region for mutations in 18 classic non-mUPD7 SRS patients. Maternal repression of GRB10 was observed specifically in the developing central nervous system including brain and spinal cord, with biallelic expression in peripheral tissues. This is in contrast to mouse Grb10, and represents the first example of opposite imprinting in human and mouse homologues. While a role for GRB10 in mUPD7 SRS cases can not be ruled out on the basis of imprinting status, no mutations were identified in the patients screened.

Use of estrogens for the prevention and treatment of Alzheimer's disease.

This review examines the biological rationale for the use of estrogen replacement therapy (ERT) and the evidence for the efficacy of ERT in enhancing cognition, preventing Alzheimer's disease (AD) and treating AD in postmenopausal women. While the biological basis for ERT as a cognition enhancer is strong and multiply mediated, the clinical evidence for its use is not as compelling and must be weighed against possible side effects. Until the results of definitive large trials are available, the use of ERT alone or in combination with other treatments is worthy of consideration.