Imprinted genes are expressed either from the paternal or the maternal allele, because the other allele has been silenced in the mother's or father's germline. Imprints are characterized by DNA methylation at cytosine phosphate guanine sites. Recently, abnormal sperm parameters and male infertility have been linked to aberrant methylation patterns of imprinted genes in sperm DNA. However, these studies did not account for possible epigenetic heterogeneity in sperm. We have investigated whether spermatozoa are a homogeneous cell population regarding DNA methylation of imprinted genes. Swim-up sperm was obtained from 45 men with normal (n = 19) and abnormal (n = 26) sperm parameters. DNA methylation of the imprinted gene KCNQ1OT1 was measured in multiple pools of 10 spermatozoa by a highly sensitive pyrosequencing-based oligo-sperm methylation assay (OSMA). DNA methylation of four imprinted genes (KCNQ1OT1, MEST, H19 and MEG3) was further analysed by deep bisulfite sequencing, which allows analysis at the single-cell level. Using OSMA, we found a significantly increased variation in the DNA methylation values of the maternally methylated gene KCNQ1OT1 in samples with abnormal sperm parameters. DBS showed that normozoospermic samples had a homogenous pattern of DNA methylation, whereas oligoasthenozoospermic samples contained discrete populations of spermatozoa with either normal or abnormal methylation patterns. Aberrant methylation of H19 appears to occur preferentially on the maternally inherited allele. Our results demonstrate the presence of epigenetic mosaicism in the semen of oligoasthenozoospermic men, which probably results from errors in imprint erasure.
Imprinting of the human RB1 gene is due to the presence of a differentially methylated CpG island (CGI) in intron 2, which is part of a retrocopy derived from the PPP1R26 gene on chromosome 9. The murine Rb1 gene does not have this retrocopy and is not imprinted. We have investigated whether the RB1/Rb1 locus is unique with respect to these differences. For this, we have compared the CGIs from human and mouse by in silico analyses. We have found that the human genome does not only contain more CGIs than the mouse, but the proportion of intronic CGIs is also higher (7.7% vs. 3.5%). At least 2,033 human intronic CGIs are not present in the mouse. Among these CGIs, 104 show sequence similarities elsewhere in the human genome, which suggests that they arose from retrotransposition. We could narrow down the time points when most of these CGIs appeared during evolution. Their methylation status was analyzed in two monocyte methylome data sets from whole-genome bisulfite sequencing and in 18 published methylomes. Four CGIs, which are located in the RB1, ASRGL1, PARP11, and PDXDC1 genes, occur as methylated and unmethylated copies. In contrast to imprinted methylation at the RB1 locus, differential methylation of the ASRGL1 and PDXDC1 CGIs appears to be sequence dependent. Our study supports the notion that the epigenetic fate of the retrotransposed DNA depends on its sequence and selective forces at the integration site.
We have recently shown that the human Nuclear pore-associated protein (NPAP1)/C15orf2 gene encodes a nuclear pore-associated protein. This gene is one of several paternally expressed imprinted genes in the genomic region 15q11q13. Because the Prader-Willi syndrome is known to be caused by the loss of function of paternally expressed genes in 15q11q13, a phenotypic contribution of NPAP1 cannot be excluded. NPAP1 appears to be under strong positive Darwinian selection in primates, suggesting an important function in primate biology. Interestingly, however, in contrast to all other protein-coding genes in 15q11q13, NPAP1 has no ortholog in the mouse. Our investigation of the evolutionary origin of NPAP1 showed that the gene is specific to primate species and absent from the 15q11q13-orthologous regions in all nonprimate mammals. However, we identified a group of paralogous genes, which we call NPAP1L, in all placental mammals except rodents. Phylogenetic analysis revealed that NPAP1, NPAP1L, and another group of genes (UPF0607), which is also restricted to primates, are closely related to the vertebrate transmembrane nucleoporin gene POM121, although they lack the transmembrane domain. These three newly identified groups of genes all lack conserved introns, and hence, are likely retrogenes. We hypothesize that, in the common ancestor of placentals, the POM121 gene retrotransposed and gave rise to an NPAP1-ancestral retrogene NPAP1L/NPAP1/UPF0607. Our results suggest that the nuclear pore-associated gene NPAP1 originates from the vertebrate nucleoporin gene POM121 and--after several steps of retrotransposition and duplication-has been subjected to genomic imprinting and positive selection after integration into the imprinted SNRPN-UBE3A chromosomal domain.
Genomic imprinting is an epigenetic process by which the male and the female germline confer different DNA methylation marks and histone modifications onto specific gene regions, so that one allele of an imprinted gene is active and the other one is silent. Since the dosage of imprinted genes is important for normal development, growth and behaviour, the loss or duplication of the active allele can cause disease.
Several observations have pointed to a major pathogenic role of somatostatin depletion with respect to amyloid accumulation, which is often thought to be the crucial event in a cascade leading to Alzheimer's disease (AD). As methylation of CpG islands plays an important role in gene silencing, we studied the methylation status of the CpG islands in the promoters of somatostatin (SST) and in that of its receptor subtype in the cerebral cortex, SSTR4, in tissue samples from the middle temporal (Brodmann area 22) and superior frontal gyrus (Brodmann area 9) of 5 severely affected AD patients aged 72-94 years (Braak stages V-C or VI-C) and 5 non-demented controls aged 50-92 years. Bisulfite sequencing of DNA from cortical gray and infracortical white matter showed that the DNA methylation status at the promoters of SST and SSTR4 did not significantly differ between AD and control samples in any of the regions analyzed. We confirmed these results using deep bisulfite sequencing of PCR products from the SST promoter amplified from DNA from the cortical gray of the superior frontal gyrus of all AD patients and non-demented controls. We observed a trend toward increased DNA methylation with increasing age. In conclusion, deregulated somatostatin signaling in the AD cortices studied cannot be explained by hypermethylation of the SST or SSTR4 promoter CpG islands.
Genome-wide association studies have revealed numerous risk loci associated with diverse diseases. However, identification of disease-causing variants within association loci remains a major challenge. Divergence in gene expression due to cis-regulatory variants in noncoding regions is central to disease susceptibility. We show that integrative computational analysis of phylogenetic conservation with a complexity assessment of co-occurring transcription factor binding sites (TFBS) can identify cis-regulatory variants and elucidate their mechanistic role in disease. Analysis of established type 2 diabetes risk loci revealed a striking clustering of distinct homeobox TFBS. We identified the PRRX1 homeobox factor as a repressor of PPARG2 expression in adipose cells and demonstrate its adverse effect on lipid metabolism and systemic insulin sensitivity, dependent on the rs4684847 risk allele that triggers PRRX1 binding. Thus, cross-species conservation analysis at the level of co-occurring TFBS provides a valuable contribution to the translation of genetic association signals to disease-related molecular mechanisms.
Data transformations prior to analysis may be beneficial in classification tasks. In this article we investigate a set of such transformations on 2D graph-data derived from facial images and their effect on classification accuracy in a high-dimensional setting. These transformations are low-variance in the sense that each involves only a fixed small number of input features. We show that classification accuracy can be improved when penalized regression techniques are employed, as compared to a principal component analysis (PCA) pre-processing step. In our data example classification accuracy improves from 47% to 62% when switching from PCA to penalized regression. A second goal is to visualize the resulting classifiers. We develop importance plots highlighting the influence of coordinates in the original 2D space. Features used for classification are mapped to coordinates in the original images and combined into an importance measure for each pixel. These plots assist in assessing plausibility of classifiers, interpretation of classifiers, and determination of the relative importance of different features.
Maternal effect genes code for oocyte proteins that are important for early embryogenesis. Transcription in oocytes does not take place from the onset of meiotic progression until zygotic genome activation. During this period, protein levels are regulated posttranscriptionally, for example by poly(A) tail length. Posttranscriptional regulation may be impaired in preovulatory and postovulatory aged oocytes, caused by delayed ovulation or delayed fertilization, respectively, and may lead to developmental defects. We investigated transcript levels and poly(A) tail length of ten maternal effect genes in in vivo- and in vitro- (follicle culture) grown oocytes after pre- and postovulatory aging. Quantitative RT-PCR was performed using random hexamer-primed cDNA to determine total transcript levels and oligo(dT)16-primed cDNA to analyze poly(A) tail length. Transcript levels of in vivo preovulatory-aged oocytes remained stable except for decreases in Brg1 and Tet3. Most genes investigated showed a tendency towards increased poly(A) content. Polyadenylation of in vitro preovulatory-aged oocytes was also increased, along with transcript level declines of Trim28, Nlrp2, Nlrp14 and Zar1. In contrast to preovulatory aging, postovulatory aging of in vivo- and in vitro-grown oocytes led to a shortening of poly(A) tails. Postovulatory aging of in vivo-grown oocytes resulted in deadenylation of Nlrp5 after 12 h, and deadenylation of 4 further genes (Tet3, Trim28, Dnmt1, Oct4) after 24 h. Similarly, transcripts of in vitro-grown oocytes were deadenylated after 12 h of postovulatory aging (Tet3, Trim28, Zfp57, Dnmt1, Nlrp5, Zar1). This impact of aging on poly(A) tail length may affect the timed translation of maternal effect gene transcripts and thereby contribute to developmental defects.
Gene expression profiles and chromosome 3 copy number divide uveal melanomas into two distinct classes correlating with prognosis. Using exome sequencing, we identified recurrent somatic mutations in EIF1AX and SF3B1, specifically occurring in uveal melanomas with disomy 3, which rarely metastasize. Targeted resequencing showed that 24 of 31 tumors with disomy 3 (77%) had mutations in either EIF1AX (15; 48%) or SF3B1 (9; 29%). Mutations were infrequent (2/35; 5.7%) in uveal melanomas with monosomy 3, which are associated with poor prognosis. Resequencing of 13 uveal melanomas with partial monosomy 3 identified 8 tumors with a mutation in either SF3B1 (7; 54%) or EIF1AX (1; 8%). All EIF1AX mutations caused in-frame changes affecting the N terminus of the protein, whereas 17 of 19 SF3B1 mutations encoded an alteration of Arg625. Resequencing of ten uveal melanomas with disomy 3 that developed metastases identified SF3B1 mutations in three tumors, none of which targeted Arg625.
The human RB1 gene is imprinted due to a differentially methylated CpG island in intron 2. This CpG island is part of PPP1R26P1, a truncated retrocopy of PPP1R26, and serves as a promoter for an alternative RB1 transcript. We show here by in silico analyses that the parental PPP1R26 gene is present in the analysed members of Haplorrhini, which comprise Catarrhini (Old World Monkeys, Small apes, Great Apes and Human), Platyrrhini (New World Monkeys) and tarsier, and Strepsirrhini (galago). Interestingly, we detected the retrocopy, PPP1R26P1, in all Anthropoidea (Catarrhini and Platyrrhini) that we studied but not in tarsier or galago. Additional retrocopies are present in human and chimpanzee on chromosome 22, but their distinct composition indicates that they are the result of independent retrotransposition events. Chimpanzee and marmoset have further retrocopies on chromosome 8 and chromosome 4, respectively. To examine the origin of the RB1 imprint, we compared the methylation patterns of the parental PPP1R26 gene and its retrocopies in different primates (human, chimpanzee, orangutan, rhesus macaque, marmoset and galago). Methylation analysis by deep bisulfite sequencing showed that PPP1R26 is methylated whereas the retrocopy in RB1 intron 2 is differentially methylated in all primates studied. All other retrocopies are fully methylated, except for the additional retrocopy on marmoset chromosome 4, which is also differentially methylated. Using an informative SNP for the methylation analysis in marmoset, we could show that the differential methylation pattern of the retrocopy on chromosome 4 is allele-specific. We conclude that the epigenetic fate of a PPP1R26 retrocopy after integration depends on the DNA sequence and selective forces at the integration site.
NLRP7 is a maternal effect gene as maternal mutations in this gene cause recurrent hydatidiform moles, spontaneous abortions and stillbirths, whereas live births are very rare. We have studied a patient with multiple anomalies born to a mother with a heterozygous NLRP7 mutation. By array-based CpG methylation analysis of blood DNA from the patient, his parents and 18 normal controls on Illumina Infinium HumanMethylation27 BeadChips we found that the patient had methylation changes (delta ß ? 0.3) at many imprinted loci as well as at 87 CpGs associated with 85 genes of unknown imprinting status. Using a pseudoproband (permutation) approach, we found methylation changes at only 7-24 CpGs (mean 15; standard deviation 4.84) in the controls. Thus, the number of abberantly methylated CpGs in the patient is more than 14 standard deviations higher. In order to identify novel imprinted genes among the 85 conspicuous genes in the patient, we selected 19 (mainly hypomethylated) genes for deep bisulfite amplicon sequencing on the ROCHE/454 Genome Sequencer in the patient and at least two additional controls. These controls had not been included in the array analysis and were heterozygous for a single nucleotide polymorphism at the test locus, so that allele-specific DNA methylation patterns could be determined. Apart from FAM50B, which we proved to be imprinted in blood, we did not observe allele-specific DNA methylation at the other 18 loci. We conclude that the patient does not only have methylation defects at imprinted loci but (at least in blood) also an excess of methylation changes at apparently non-imprinted loci.
The human retinoblastoma gene (RB1) is imprinted; the mouse Rb1 gene is not. Imprinted expression of RB1 is due to differential methylation of a CpG island (CpG85), which is located in the pseudogene PPP1R26P1 in intron 2 of RB1. CpG85 serves as promoter for an alternative RB1 transcript, which is expressed from the unmethylated paternal allele only and is thought to suppress expression of the full-length RB1 transcript in cis. PPP1R26P1 contains another CpG island (CpG42), which is biallelically methylated. To determine the influence of PPP1R26P1 on RB1 expression, we generated an in vitro murine embryonic stem cell model by introducing human PPP1R26P1 into mouse Rb1. Next generation bisulfite sequencing of CpG85 and CpG42 revealed differences in their susceptibility to DNA methylation, gaining methylation at a median level of 4% and 18%, respectively. We showed binding of RNA polymerase II at and transcription from the unmethylated CpG85 in PPP1R26P1 and observed reduced expression of full-length Rb1 from the targeted allele. Our results identify human PPP1R26P1 as a cis-repressive element and support a connection between retrotransposition of PPP1R26P1 into human RB1 and the reduced expression of RB1 on the paternal allele.
Recent genome-wide association studies have identified single nucleotide polymorphisms (SNPs) associated with non-syndromic cleft lip with or without cleft palate (NSCL/P), and other previous studies showed distinctly differing facial distance measurements when comparing unaffected relatives of NSCL/P patients with normal controls. Here, we test the hypothesis that genetic loci involved in NSCL/P also influence normal variation in facial morphology. We tested 11 SNPs from 10 genomic regions previously showing replicated evidence of association with NSCL/P for association with normal variation of nose width and bizygomatic distance in two cohorts from Germany (N=529) and the Netherlands (N=2497). The two most significant associations found were between nose width and SNP rs1258763 near the GREM1 gene in the German cohort (P=6 × 10(-4)), and between bizygomatic distance and SNP rs987525 at 8q24.21 near the CCDC26 gene (P=0.017) in the Dutch sample. A genetic prediction model explained 2% of phenotype variation in nose width in the German and 0.5% of bizygomatic distance variation in the Dutch cohort. Although preliminary, our data provide a first link between genetic loci involved in a pathological facial trait such as NSCL/P and variation of normal facial morphology. Moreover, we present a first approach for understanding the genetic basis of human facial appearance, a highly intriguing trait with implications on clinical practice, clinical genetics, forensic intelligence, social interactions and personal identity.
In mice, the transcription factor, PLZF, controls the development of effector functions in invariant NKT cells and a subset of NKT cell-like, ?? T cells. Here, we show that in human lymphocytes, in addition to invariant NKT cells, PLZF was also expressed in a large percentage of CD8+ and CD4+ T cells. Furthermore, PLZF was also found to be expressed in all ?? T cells and in all NK cells. Importantly, we show that in a donor lacking functional PLZF, all of these various lymphocyte populations were altered. Therefore, in contrast to mice, PLZF appears to control the development and/or function of a wide variety of human lymphocytes that represent more than 10% of the total PBMCs. Interestingly, the PLZF-expressing CD8+ T cell population was found to be expanded in the peripheral blood of patients with metastatic melanoma but was greatly diminished in patients with autoimmune disease.
Uveal melanoma (UM) is a rare eye tumor. There are two classes of UM, which can be discriminated by the chromosome 3 status or global mRNA expression profile. Metastatic progression is predominantly originated from class II tumors or from tumors showing loss of an entire chromosome 3 (monosomy 3). We performed detailed EFS (embryonal Fyn-associated substrate) methylation analyses in UM, cultured uveal melanocytes and normal tissues, to explore the role of the differentially methylated EFS promoter region CpG island in tumor classification and metastatic progression.
Ménières disease (MD) is a chronic illness characterized by sensorineural hearing loss, recurring vertigo attacks, and tinnitus. It is possibly of multifactorial origin, although several families with autosomal dominant inheritance and reduced penetrance have been described. To elucidate the genetic basis of MD, patients and their families were investigated, and linkage analysis was performed.
Computer systems play an important role in clinical genetics and are a routine part of finding clinical diagnoses but make it difficult to fully exploit information derived from facial appearance. So far, automated syndrome diagnosis based on digital, facial photographs has been demonstrated under study conditions but has not been applied in clinical practice. We have therefore investigated how well statistical classifiers trained on study data comprising 202 individuals affected by one of 14 syndromes could classify a set of 91 patients for whom pictures were taken under regular, less controlled conditions in clinical practice. We found a classification accuracy of 21% percent in the clinical sample representing a ratio of 3.0 over a random choice. This contrasts with a 60% accuracy or 8.5 ratio in the training data. Producing average images in both groups from sets of pictures for each syndrome demonstrates that the groups exhibit large phenotypic differences explaining discrepancies in accuracy. A broadening of the data set is suggested in order to improve accuracy in clinical practice. In order to further this goal, a software package is made available that allows application of the procedures and contributions toward an improved data set.
To analyze the effects of delayed ovulation on embryonic development in mice, because intrafollicular oocyte development may be delayed during assisted reproductive technology (ART) treatment in humans.
Genomic imprinting is an epigenetic process by which the male and the female germ line confer specific marks (imprints) onto certain gene regions, so that one allele of an imprinted gene is active and the other allele is silent. Genomic imprints are erased in primordial germ cells, newly established during later stages of germ cell development, and stably inherited through somatic cell divisions during postzygotic development. Defects in imprint erasure, establishment, or maintenance result in a paternal chromosome carrying a maternal imprint or in a maternal chromosome carrying a paternal imprint. A wrong imprint leads to activation of an allele that should be silent or silencing of an allele that should be active. Since the dosage of imprinted genes is very important for development and growth, imprinting defects lead to specific diseases. Imprinting defects can occur spontaneously without any DNA sequence change (primary imprinting defect) or as the result of a mutation in a cis-regulatory element or a trans-acting factor (secondary imprinting defect). The distinction between primary and secondary imprinting defects is important for assessing the recurrence risk in affected families.
To examine the association between genomewide association study-based diabetes mellitus-related single-nucleotide polymorphisms (SNPs) and coronary artery calcification (CAC), a valid risk factor for coronary heart disease, in a large, unselected, population-based cohort.
Sequence analysis of the imprinted UBE3A gene in a 3-year-old girl suspected of having Angelman syndrome had revealed a de novo 3bp in frame deletion predicted to encode a protein lacking the amino acid G538 (based on sequence NM_130838). In order to assess the clinical relevance of this unknown variant, we determined the parental origin and the functional consequences of the deletion. We separated the two chromosomes 15 by microdissection of metaphase spreads and used cytogenetic and molecular markers to demonstrate that the deletion is on the maternal chromosome. For determining the functional consequences of the deletion, we modelled the structure of the deletion mutant based on the wildtype X-ray structure and simulated the molecular dynamics of the wildtype and mutant protein in complex with UcbH7. Our simulations showed that deletion of G538 destroys a network of salt bridges between highly conserved residues in the catalytic cleft of UBE3A. In conclusion, our results strongly suggest that the 3bp deletion is a loss-of-function mutation of the maternal UBE3A allele that has caused Angelman syndrome in our patient. Our study may serve as a paradigm to determine the parental origin of a de novo mutation.
Recent data have revealed that the paradigmatic tumour suppressor gene RB1 on chromosome 13 is preferentially expressed from the maternal allele. Imprinted expression of RB1 is linked to a differentially methylated CpG island in intron 2 of this gene (CpG 85). On the paternal chromosome, CpG 85 is unmethylated and acts as a weak promoter of an alternative RB1 transcript. Paternal mRNA levels are probably reduced as the result of transcriptional interference of the regular promoter and the alternative promoter on this chromosome. CpG 85 is part of a truncated processed pseudogene (KIAA0649P) that integrated into the RB1 gene prior to the speciation of extant primates. It is plausible that differential penetrance and variation of age at diagnosis, which have been observed in patients with hereditary and non-hereditary retinoblastoma, respectively, are a consequence of imprinted expression of the RB1 gene. Interestingly, RB1 is imprinted in the same direction as CDKN1C, which operates upstream of RB1. The imprinting of two components of the same pathway indicates that there has been strong evolutionary selection for maternal inhibition of cell proliferation.
As first shown more than 100 years ago, fertilization of an aged (overripe) egg increases the rate of malformations and embryonic loss in several vertebrates, including possibly humans as well. Since the molecular events in aging eggs may be similar in these species, we established in the frog Xenopus tropicalis a defined protocol for delayed fertilization of eggs. A three-hour delayed fertilization led to a dramatic increase in malformation and mortality. Gene expression profiling revealed that 14% of the polyadenylated maternal transcripts were downregulated upon aging. These transcripts were not degraded, but rather deadenylated as shown for specific maternal mRNAs. The affected transcripts are characterized by a relatively short 3UTR and a paucity of cytoplasmic polyadenylation elements (CPE) and polyadenylation signals (PAS). Furthermore, maternal mRNAs known to be deadenylated during egg maturation as well as after fertilization were preferentially deadenylated in aged eggs. Taken together our analysis of aging eggs reveals that unfertilized eggs are in a dynamic state that was previously not realized. On the one hand deadenylation of transcripts that are typically deadenylated during egg maturation continues and this implies overripeness of the aged egg in the truest sense of the word. On the other hand transcripts that normally are deadenylated after fertilization loose their poly(A) in the aged egg and this implies that the egg awaiting fertilization starts processes that are normally only observed after fertilization. Based on our novel finding we postulate that the imbalance of the polyadenylated maternal transcripts upon egg aging contributes to the loss of developmental potential. Based on this hypothesis the developmental consequences of downregulation of specific transcripts can be analyzed in future.
As shown by genome-wide association studies single-nucleotide polymorphisms (SNPs) within intron 1 of the FTO gene are associated with the body mass index and type II diabetes, although the functional significance of these SNPs has remained unclear. Using primer extension assays, we have determined the ratio of allelic FTO transcript levels in unspliced heterogeneous nuclear RNA preparations from blood of individuals heterozygous for SNP rs9939609. Allelic expression ratios of the neighboring RPGRIP1L gene were investigated in individuals who were heterozygous for SNP rs4784319 and heterozygous or homozygous for rs9939609. In each of five individuals, the FTO transcripts containing the A (risk) allele of rs9939609 were more abundant than those with T allele (mean 1.38; 95% confidence interval 1.31-1.44). Similar results were obtained in a fibroblast sample. We also observed skewed allelic expression of the RPGRIP1L gene in blood, but skewing was independent of the FTO genotype. Our data suggest that increased expression of FTO is associated with increased body mass.
Follicle-stimulating hormone (FSH) mediated by its receptor (FSHR) is pivotal for normal gametogenesis. Inactivating FSHR mutations are known to cause hypergonadotropic hypogonadism with disturbed follicular maturation in females. So far, only very few recessive point mutations have been described. We report on a 17-year-old female with primary amenorrhea, hypergonadotropic hypogonadism and disturbed folliculogenesis. Chromosome analysis detected a seemingly balanced translocation 46,XX,t(2;8)(p16.3or21;p23.1)mat. FSHR sequence analysis revealed a novel non-synonymous point mutation in exon 10 (c.1760C>A, p.Pro587His), but no wild-type allele. The mutation was also found in the father, but not in the mother. Furthermore, molecular-cytogenetic analyses of the breakpoint region on chromosome 2 showed the translocation to be unbalanced, containing a deletion with one breakpoint within the FSHR gene. The deletion size was narrowed down by array analysis to approximately 163 kb, involving exons 9 and 10 of the FSHR gene. Functional studies of the mutation revealed the complete lack of signal transduction presumably caused by a changed conformational structure of transmembrane helix 6. To our knowledge, this is the first description of a compound heterozygosity of an inactivating FSHR point mutation unmasked by a partial deletion. This coincidence of two rare changes caused clinical signs consistent with FSH resistance.
The GFI1 gene encodes a transcriptional repressor, which regulates myeloid differentiation. In the mouse, Gfi1 deficiency causes neutropenia and an accumulation of granulomonocytic precursor cells that is reminiscent of a myelodysplastic syndrome. We report here that a variant allele of GFI1 (GFI1(36N)) is associated with acute myeloid leukemia (AML) in white subjects with an odds ratio of 1.6 (P < 8 x 10(-5)). The GFI1(36N) variant occurred in 1806 AML patients with an allele frequency of 0.055 compared with 0.035 in 1691 healthy control patients in 2 independent cohorts. We observed that both GFI1 variants maintain the same activity as transcriptional repressors but differ in their regulation by the AML1/ETO (RUNX1/RUNX1T1) fusion protein produced in AML patients with a t(8;21) translocation. AML1/ETO interacts and colocalizes with the more common GFI1(36S) form in the nucleus and inhibits its repressor activity. However, the variant GFI1(36N) protein has a different subnuclear localization than GFI1(36S). As a consequence, AML1/ETO does not colocalize with GFI1(36N) and is unable to inhibit its repressor activity. We conclude that both variants of GFI1 differ in their ability to be regulated by interacting proteins and that the GFI1(36N) variant form exhibits distinct biochemical features that may confer a predisposition to AML.
C15orf2 (Chromosome 15 open reading frame 2) is an intronless gene, which is located in the Prader-Willi syndrome (PWS) chromosomal region on human chromosome 15. Mice do not have an orthologous gene. Here we show that expression of C15orf2 in the fetal human brain is imprinted. Using Western blot and immunohistological studies we have obtained evidence that C15orf2 protein is present in several regions of the brain. Previously published phylogenetic studies as well as population genetic studies based on complex haplotypes as described here suggest that C15orf2 is under positive Darwinian selection. These results indicate that C15orf2 might have an important role in human biology and that a deficiency of C15orf2 might contribute to PWS.
The Prader-Willi syndrome (PWS) is caused by a 5-6 Mbp de novo deletion on the paternal chromosome 15, maternal uniparental disomy 15 or an imprinting defect. All three lesions lead to the lack of expression of imprinted genes that are active on the paternal chromosome only: MKRN3, MAGEL2, NDN, C15orf2, SNURF-SNRPN and more than 70 C/D box snoRNA genes (SNORDs). The contribution to PWS of any of these genes is unknown, because no single gene mutation has been described so far. We report on two patients with PWS who have an atypical deletion on the paternal chromosome that does not include MKRN3, MAGEL2 and NDN. In one of these patients, NDN has a normal DNA methylation pattern and is expressed. In another patient, the paternal alleles of these genes are deleted as the result of an unbalanced translocation 45,X,der(X)t(X;15)(q28;q11.2). This patient is obese and mentally retarded, but does not have PWS. We conclude that a deficiency of MKRN3, MAGEL2 and NDN is not sufficient to cause PWS.
Genomic imprinting is an epigenetic process leading to parent-of-origin-specific DNA methylation and gene expression. To date, approximately 60 imprinted human genes are known. Based on genome-wide methylation analysis of a patient with multiple imprinting defects, we have identified a differentially methylated CpG island in intron 2 of the retinoblastoma (RB1) gene on chromosome 13. The CpG island is part of a 5-truncated, processed pseudogene derived from the KIAA0649 gene on chromosome 9 and corresponds to two small CpG islands in the open reading frame of the ancestral gene. It is methylated on the maternal chromosome 13 and acts as a weak promoter for an alternative RB1 transcript on the paternal chromosome 13. In four other KIAA0649 pseudogene copies, which are located on chromosome 22, the two CpG islands have deteriorated and the CpG dinucleotides are fully methylated. By analysing allelic RB1 transcript levels in blood cells, as well as in hypermethylated and 5-aza-2-deoxycytidine-treated lymphoblastoid cells, we have found that differential methylation of the CpG island skews RB1 gene expression in favor of the maternal allele. Thus, RB1 is imprinted in the same direction as CDKN1C, which operates upstream of RB1. The imprinting of two components of the same pathway indicates that there has been strong evolutionary selection for maternal inhibition of cell proliferation.
The imprinted domain in human 15q11-q13 is controlled by a bipartite imprinting centre (IC), which overlaps the 5 part of the paternally expressed SNURF-SNRPN gene. We have recently described two novel genes upstream of SNURF-SNRPN (PWRN1 and PWRN2), which are biallelically expressed in the testis. We have now found that PWRN1 represents an alternative 5 part of SNURF-SNRPN, and that its expression in the brain is imprinted. To determine when the locus is activated during spermatogenesis and which factors are involved in this process, we have mined gene-expression data of testicular biopsies from men with different types of spermatogenic failure. Whereas PWRN1-SNURF-SNRPN and PWRN2 are expressed in post-meiotic germ cells only, a hitherto undetected SNURF-SNRPN upstream transcript is expressed already at meiosis. Several epigenetic factors (eg, MBD1 and MBD2 isoforms, MBD3L1, SUVH39H2, BRDT, and EZH2) are upregulated at specific stages of spermatogenesis, suggesting that they play an important role in the epigenetic reprogramming during spermatogenesis.
Methylation of CpG islands (CGIs) plays an important role in gene silencing. For genome-wide methylation analysis of CGIs in female white blood cells and in sperm, we used four restriction enzymes and a size selection step to prepare DNA libraries enriched with CGIs. The DNA libraries were treated with sodium bisulfite and subjected to a modified 454/Roche Genome Sequencer protocol. We obtained 163 034 and 129 620 reads from blood and sperm, respectively, with an average read length of 133 bp. Bioinformatic analysis revealed that 12 358 (7.6%) blood library reads and 10 216 (7.9%) sperm library reads map to 6167 and 5796 different CGIs, respectively. In blood and sperm DNA, we identified 824 (13.7%) and 482 (8.5%) fully methylated autosomal CGIs, respectively. Differential methylation, which is characterized by the presence of methylated and unmethylated reads of the same CGI, was observed in 53 and 52 autosomal CGIs in blood and sperm DNA, respectively. Remarkably, methylation of X-chromosomal CGIs in female blood cells was most often incomplete (25-75%). Such incomplete methylation was mainly found on the X-chromosome, suggesting that it is linked to X-chromosome inactivation.
Although there is an increased frequency of low birth weight after assisted reproduction, the mechanisms underlying this association are unclear. We have proposed that some of the children conceived by intracytoplasmic sperm injection (ICSI) with low birth weight might have an epimutation (faulty methylation pattern) in one of the imprinted genes involved in fetal growth control, eg, KCNQ1OT1, PEG1, PEG3, GTL2, IGF2/H19 and PLAGL1. Using bisulfite DNA sequencing and sequence-based quantitative methylation analysis (SeQMA), we determined the methylation pattern of these genes in buccal smears from 19 ICSI children born small for gestational age (SGA, birth weight <3rd percentile) and from 29 term-born normal weight children after spontaneous conception. We detected clear hypermethylation of KCNQ1OT1 and borderline hypermethylation of PEG1 in one and the same ICSI child. The other children and the parents of the affected child have normal methylation patterns. Imprinting defects appear to be a rare finding in ICSI children born SGA. Methylation of the paternal KCNQ1OT1 and PEG1 alleles may be a previously unrecognized cause of SGA. The epimutations found in the SGA child, whose father had oligozoospermia, probably result from an imprint erasure defect in the paternal germ line and therefore appear to be linked to the fertility problem of the father and not to in vitro fertilization/ICSI.
We conducted a genome-wide association study involving 224 cases and 383 controls of Central European origin to identify susceptibility loci for nonsyndromic cleft lip with or without cleft palate (NSCL/P). A 640-kb region at chromosome 8q24.21 was found to contain multiple markers with highly significant evidence for association with the cleft phenotype, including three markers that reached genome-wide significance. The 640-kb cleft-associated region was saturated with 146 SNP markers and then analyzed in our entire NSCL/P sample of 462 unrelated cases and 954 controls. In the entire sample, the most significant SNP (rs987525) had a P value of 3.34 x 10(-24). The odds ratio was 2.57 (95% CI = 2.02-3.26) for the heterozygous genotype and 6.05 (95% CI = 3.88-9.43) for the homozygous genotype. The calculated population attributable risk for this marker is 0.41, suggesting that this study has identified a major susceptibility locus for NSCL/P.
Maternal diabetes and high-fat feeding during pregnancy have been linked to later life outcomes in offspring. To investigate the effects of both maternal and paternal hyperglycemia on offspring phenotypes, we utilized an autosomal dominant mouse model of diabetes (hypoinsulinemic hyperglycemia in Akita mice). We determined metabolic and skeletal phenotypes in wildtype offspring of Akita mothers and fathers.
At chromosome 11p15.5, the imprinting centre 1 (IC1) controls the parent of origin-specific expression of the IGF2 and H19 genes. The 5 kb IC1 region contains multiple target sites (CTS) for the zinc-finger protein CTCF, whose binding on the maternal chromosome prevents the activation of IGF2 and allows that of H19 by common enhancers. CTCF binding helps maintaining the maternal IC1 methylation-free, whereas on the paternal chromosome gamete-inherited DNA methylation inhibits CTCF interaction and enhancer-blocking activity resulting in IGF2 activation and H19 silencing. Maternally inherited 1.4-2.2 kb deletions are associated with methylation of the residual CTSs and Beckwith-Wiedemann syndrome, although with different penetrance and expressivity. We explored the relationship between IC1 microdeletions and phenotype by analysing a number of previously described and novel mutant alleles. We used a highly quantitative assay based on next generation sequencing to measure DNA methylation in affected families and analysed enhancer-blocking activity and CTCF binding in cultured cells. We demonstrate that the microdeletions mostly affect IC1 function and CTCF binding by changing CTS spacing. Thus, the extent of IC1 inactivation and the clinical phenotype are influenced by the arrangement of the residual CTSs. A CTS spacing similar to the wild-type allele results in moderate IC1 inactivation and is associated with stochastic DNA methylation of the maternal IC1 and incomplete penetrance. Microdeletions with different CTS spacing display severe IC1 inactivation and are associated with IC1 hypermethylation and complete penetrance. Careful characterization of the IC1 microdeletions is therefore needed to predict recurrence risks and phenotypical outcomes.
A block of single-nucleotide polymorphisms within intron 1 of the FTO (fat mass and obesity associated) gene is associated with variation in body weight. Previous works suggest that increased expression of FTO, which encodes a 2-oxoglutarate-dependent nucleic acid demethylase, leads to increased body weight, although the underlying mechanism has remained unclear. To elucidate the function of FTO, we examined the consequences of altered FTO levels in cultured cells and murine brain. Here we show that a knockdown of FTO in HEK293 cells affects the transcripts levels of genes involved in the response to starvation, whereas overexpression of FTO affects the transcript levels of genes related to RNA processing and metabolism. Subcellular localization of FTO further strengthens the latter notion. Using immunocytochemistry and confocal laser scanning microscopy, we detected FTO in nuclear speckles and--to a lesser and varying extent--in the nucleoplasm and nucleoli of HEK293, HeLa and MCF-7 cells. Moreover, RNA modification analyses revealed that loss of Fto affects the 3-methyluridine/uridine and pseudouridine/uridine ratios in total brain RNA. We conclude that altered levels of FTO have multiple and diverse consequences on RNA modifications and the transcriptome.
The Prader-Willi syndrome (PWS) region in 15q11q13 harbours a cluster of imprinted genes expressed from the paternal chromosome only. Whereas loss of function of the SNORD116 genes appears to be responsible for the major features of PWS, the role of the other genes is less clear. One of these genes is C15orf2, which has no orthologues in rodents, but appears to be under strong positive selection in primates. C15orf2 encodes a 1156 amino acid protein with six nuclear localisation sequences. By protein BLAST analysis and InterProScan signature recognition search, we found sequence similarity of C15orf2 to the nuclear pore complex (NPC) protein POM121. To determine whether C15orf2 is located at nuclear pores, we generated a stable cell line that inducibly expresses FLAG-tagged C15orf2 and performed immunocytochemical studies. We found that C15orf2 is present at the nuclear periphery, where it colocalizes with NPCs and nuclear lamins. At very high expression levels, we observed invaginations of the nuclear envelope. Extending these observations to three-dimensional structured illumination microscopy, which achieves an 8-fold improved volumetric resolution over conventional imaging, we saw that C15orf2 is located at the inner face of the nuclear envelope where it strongly associates with the NPC. In nuclear envelope isolation and fractionation experiments, we detected C15orf2 in the NPC and lamina fractions. These experiments for the first time demonstrate that C15orf2 is part of the NPC or its associated molecular networks. Based on our findings, we propose Nuclear pore associated protein 1 as the new name for C15orf2.
The individual risk for common diseases not only depends on genetic but also on epigenetic polymorphisms. To assess the role of epigenetic variations in the individual risk for obesity, we have determined the methylation status of two CpG islands at the POMC locus in obese and normal-weight children. We found a hypermethylation variant targeting individual CpGs at the intron 2-exon 3 boundary of the POMC gene by bisulphite sequencing that was significantly associated with obesity. POMC exon 3 hypermethylation interferes with binding of the transcription enhancer P300 and reduces expression of the POMC transcript. Since intron 2 contains Alu elements that are known to influence methylation in their genomic vicinity, the exon 3 methylation variant seems to result from an Alu element-triggered default state of methylation boundary definition. Exon 3 hypermethylation in the POMC locus represents the first identified DNA methylation variant that is associated with the individual risk for obesity.
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