Reduced PRC2 function alters male germline epigenetic programming and paternal inheritance.

BACKGROUND: Defining the mechanisms that establish and regulate the transmission of epigenetic information from parent to offspring is critical for understanding disease heredity. Currently, the molecular pathways that regulate epigenetic information in the germline and its transmission to offspring are poorly understood. RESULTS: Here we provide evidence that Polycomb Repressive Complex 2 (PRC2) regulates paternal inheritance. Reduced PRC2 function in mice resulted in male sub-fertility and altered epigenetic and transcriptional control of retrotransposed elements in foetal male germ cells. Males with reduced PRC2 function produced offspring that over-expressed retrotransposed pseudogenes and had altered preimplantation embryo cleavage rates and cell cycle control. CONCLUSION: This study reveals a novel role for the histone-modifying complex, PRC2, in paternal intergenerational transmission of epigenetic effects on offspring, with important implications for understanding disease inheritance.

BTB-ZF transcriptional regulator PLZF modifies chromatin to restrain inflammatory signaling programs.

Inflammation is critical for host defense, but without appropriate control, it can cause chronic disease or even provoke fatal responses. Here we identify a mechanism that limits the inflammatory response. Probing the responses of macrophages to the key sensory Toll-like receptors, we identify that the Broad-complex, Tramtrack and Bric-a-brac/poxvirus and zinc finger (BTB/POZ), transcriptional regulator promyelocytic leukemia zinc finger (PLZF) limits the expression of inflammatory gene products. In accord with this finding, PLZF-deficient animals express higher levels of potent inflammatory cytokines and mount exaggerated inflammatory responses to infectious stimuli. Temporal quantitation of inflammatory gene transcripts shows increased gene induction in the absence of PLZF. Genome-wide analysis of histone modifications distinguish that PLZF establishes basal activity states of early response genes to maintain immune homeostasis and limit damaging inflammation. We show that PLZF stabilizes a corepressor complex that encompasses histone deacetylase activity to control chromatin. Together with our previous demonstration that PLZF promotes the antiviral response, these results suggest a strategy that could realize one of the major goals of immune therapy to retain immune resistance to pathogens while curbing damaging inflammation.

Flexible and Scalable Full-Length CYP2D6 Long Amplicon PacBio Sequencing.

Cytochrome P450 2D6 (CYP2D6) is among the most important genes involved in drug metabolism. Specific variants are associated with changes in the enzyme's amount and activity. Multiple technologies exist to determine these variants, like the AmpliChip CYP450 test, Taqman qPCR, or Second-Generation Sequencing, however, sequence homology between cytochrome P450 genes and pseudogene CYP2D7 impairs reliable CYP2D6 genotyping, and variant phasing cannot accurately be determined using these assays. To circumvent this, we sequenced CYP2D6 using the Pacific Biosciences RSII and obtained high-quality, full-length, phased CYP2D6 sequences, enabling accurate variant calling and haplotyping of the entire gene-locus including exonic, intronic, and upstream and downstream regions. Unphased diplotypes (Roche AmpliChip CYP450 test) were confirmed for 24 of the 25 samples, including gene duplications. Cases with gene deletions required additional specific assays to resolve. In total, 61 unique variants were detected, including variants that had not previously been associated with specific haplotypes. To further aid genomic analysis using standard reference sequences, we have established an LOVD-powered CYP2D6 gene-variant database, and added all reference haplotypes and data reported here. We conclude that our CYP2D6 genotyping approach produces reliable CYP2D6 diplotypes and reveals information about additional variants, including phasing and copy-number variation.

Critical points for an accurate human genome analysis.

Next-generation sequencing is radically changing how DNA diagnostic laboratories operate. What started as a single-gene profession is now developing into gene panel sequencing and whole-exome and whole-genome sequencing (WES/WGS) analyses. With further advances in sequencing technology and concomitant price reductions, WGS will soon become the standard and be routinely offered. Here, we focus on the critical steps involved in performing WGS, with a particular emphasis on points where WGS differs from WES, the important variables that should be taken into account, and the quality control measures that can be taken to monitor the process. The points discussed here, combined with recent publications on guidelines for reporting variants, will facilitate the routine implementation of WGS into a diagnostic setting.

Multiallelic copy number variation in the complement component 4A (C4A) gene is associated with late-stage age-related macular degeneration (AMD).

BACKGROUND: Age-related macular degeneration (AMD) is the leading cause of vision loss in Western societies with a strong genetic component. Candidate gene studies as well as genome-wide association studies strongly implicated genetic variations in complement genes to be involved in disease risk. So far, no association of AMD with complement component 4 (C4) was reported probably due to the complex nature of the C4 locus on chromosome 6. METHODS: We used multiplex ligation-dependent probe amplification (MLPA) to determine the copy number of the C4 gene as well as of both relevant isoforms, C4A and C4B, and assessed their association with AMD using logistic regression models. RESULTS: Here, we report on the analysis of 2645 individuals (1536 probands and 1109 unaffected controls), across three different centers, for multiallelic copy number variation (CNV) at the C4 locus. We find strong statistical significance for association of increased copy number of C4A (OR 0.81 (0.73; 0.89);P = 4.4 × 10(-5)), with the effect most pronounced in individuals over 78 years (OR 0.67 (0.55; 0.81)) and females (OR 0.77 (0.68; 0.87)). Furthermore, this association is independent of known AMD-associated risk variants in the nearby CFB/C2 locus, particularly in females and in individuals over 78 years. CONCLUSIONS: Our data strengthen the notion that complement dysregulation plays a crucial role in AMD etiology, an important finding for early intervention strategies and future therapeutics. In addition, for the first time, we provide evidence that multiallelic CNVs are associated with AMD pathology.

Glucocorticoid-induced leucine zipper (GILZ) inhibits B cell activation in systemic lupus erythematosus.

OBJECTIVES: Systemic lupus erythematosus (SLE) is a serious multisystem autoimmune disease, mediated by disrupted B cell quiescence and typically treated with glucocorticoids. We studied whether B cells in SLE are regulated by the glucocorticoid-induced leucine zipper (GILZ) protein, an endogenous mediator of anti-inflammatory effects of glucocorticoids. METHODS: We conducted a study of GILZ expression in blood mononuclear cells of patients with SLE, performed in vitro analyses of GILZ function in mouse and human B cells, assessed the contributions of GILZ to autoimmunity in mice, and used the nitrophenol coupled to keyhole limpet haemocyanin model of immunisation in mice. RESULTS: Reduced B cell GILZ was observed in patients with SLE and lupus-prone mice, and impaired induction of GILZ in patients with SLE receiving glucocorticoids was associated with increased disease activity. GILZ was downregulated in naïve B cells upon stimulation in vitro and in germinal centre B cells, which contained less enrichment of H3K4me3 at the GILZ promoter compared with naïve and memory B cells. Mice lacking GILZ spontaneously developed lupus-like autoimmunity, and GILZ deficiency resulted in excessive B cell responses to T-dependent stimulation. Accordingly, loss of GILZ in naïve B cells allowed upregulation of multiple genes that promote the germinal centre B cell phenotype, including lupus susceptibility genes and genes involved in cell survival and proliferation. Finally, treatment of human B cells with a cell-permeable GILZ fusion protein potently suppressed their responsiveness to T-dependent stimuli. CONCLUSIONS: Our findings demonstrated that GILZ is a non-redundant regulator of B cell activity, with important potential clinical implications in SLE.

Hormonal evaluation in relation to phenotype and genotype in 286 patients with a disorder of sex development from Indonesia.

OBJECTIVE: The objective of this study was to determine the aetiological spectrum of disorders of sex development (DSD) in a large cohort of underprivileged and undiagnosed patients from Indonesia. METHODS: A total of 286 patients with atypical external and/or internal genitalia were evaluated using clinical, hormonal, molecular genetic and histological parameters. RESULTS: The age (years) at presentation was 0-0·5 in 41 (14·3%), >0·5-12 in 181 (63·3%) and >12 in 64 cases (22·4%). 46,XY DSD was most common (68·2%, n = 195), 46,XX DSD was found in 23·4% (n = 67) and sex chromosomal DSD in 8·4% (n = 24). In 61·2% of 46,XX DSD patients, 17·9% of 46,XY DSD patients and all sex chromosome DSD patients (29·4% in total), a final diagnosis was reached based on genetic or histological gonadal tissue evaluation. 17-hydroxyprogesterone and androstenedione levels were the most distinctive parameters in 46,XX DSD patients. In 46,XY DSD, diagnostic groups were identified based on the external masculinization score: androgen action disorder (AAD), unknown male undermasculinization (UMU), and gonadal dysgenesis (GD). LH, FSH and testosterone levels were most informative especially in the older age group. HCG tests were of no additional value as no patients with androgen synthesis disorders were found. Hormonal profiles of patients with sex chromosome DSD and a Y-chromosome sequence containing karyotype showed high levels of LH and FSH, and low levels of AMH, inhibin B and testosterone compared with the normal male range. Gene mutations were found in all patients with CAH, but in only 24·5% and 1·8% of patients with AAD and UMU. In 32% of 46,XY GD patients, copy number variants of different genes were found. CONCLUSION: A stepwise diagnostic approach led to a molecularly or histologically proven final diagnosis in 29·4% of the patients. The most informative parameters were serum levels of 17-hydroxyprogesterone and androstenedione in 46,XX DSD patients, and serum LH, FSH and testosterone levels in 46,XY DSD patients.

GILZ overexpression inhibits endothelial cell adhesive function through regulation of NF-κB and MAPK activity.

Glucocorticoid-induced leucine zipper (GILZ) is an anti-inflammatory protein first identified in T lymphocytes. We recently observed that GILZ is highly expressed in synovial endothelial cells in rheumatoid arthritis. However, the function of GILZ in endothelial cells is unknown. To investigate the actions of GILZ in this cell type, we induced GILZ expression in HUVECs via transient transfection. GILZ overexpression significantly reduced the capacity of TNF-stimulated HUVECs to support leukocyte rolling, adhesion, and transmigration. These effects were associated with decreased expression of E-selectin, ICAM-1, CCL2, CXCL8, and IL-6. Experiments in a human microvascular endothelial cell line demonstrated that TNF-inducible NF-κB activity was significantly inhibited by overexpression of GILZ. Exogenous GILZ inhibited TNF-induced NF-κB p65 DNA binding, although this occurred in the absence of an effect on p65 nuclear translocation, indicating that the mechanism of action of exogenous GILZ in endothelial cells differs from that reported in other cell types. GILZ overexpression also inhibited TNF-induced activation of p38, ERK, and JNK MAPKs, as well as increased expression of the MAPK inhibitory phosphatase, MKP-1. In contrast, silencing endogenous GILZ in glucocorticoid-treated HUVECs did not alter their capacity to support leukocyte interactions. These data demonstrate that exogenous GILZ exerts inhibitory effects on endothelial cell adhesive function via a novel pathway involving modulation of NF-κB p65 DNA binding and MAPK activity. Induction of GILZ expression in endothelial cells may represent a novel therapeutic modality with the potential to inhibit inflammatory leukocyte recruitment.

Technical considerations for genotyping multi-allelic copy number variation (CNV), in regions of segmental duplication.

BACKGROUND: Intrachromosomal segmental duplications provide the substrate for non-allelic homologous recombination, facilitating extensive copy number variation in the human genome. Many multi-copy gene families are embedded within genomic regions with high levels of sequence identity (>95%) and therefore pose considerable analytical challenges. In some cases, the complexity involved in analyzing such regions is largely underestimated. Rapid, cost effective analysis of multi-copy gene regions have typically implemented quantitative approaches, however quantitative data are not an absolute means of certainty. Therefore any technique prone to degrees of measurement error can produce ambiguous results that may lead to spurious associations with complex disease. RESULTS: In this study we have focused on testing the accuracy and reproducibility of quantitative analysis techniques. With reference to the C-C Chemokine Ligand-3-like-1 (CCL3L1) gene, we performed analysis using real-time Quantitative PCR (QPCR), Multiplex Ligation-dependent Probe Amplification (MLPA) and Paralogue Ratio Test (PRT). After controlling for potential outside variables on assay performance, including DNA concentration, quality, preparation and storage conditions, we find that real-time QPCR produces data that does not cluster tightly around copy number integer values, with variation substantially greater than that of the MLPA or PRT systems. We find that the method of rounding real-time QPCR measurements can potentially lead to mis-scoring of copy number genotypes and suggest caution should be exercised in interpreting QPCR data. CONCLUSIONS: We conclude that real-time QPCR is inherently prone to measurement error, even under conditions that would seem favorable for association studies. Our results indicate that potential variability in the physicochemical properties of the DNA samples cannot solely explain the poor performance exhibited by the real-time QPCR systems. We recommend that more robust approaches such as PRT or MLPA should be used to genotype multi-allelic copy number variation in disease association studies and suggest several approaches which can be implemented to ensure the quality of the copy number typing using quantitative methods.

Molecular methods for genotyping complex copy number polymorphisms.

Genome structural variation shows remarkable complexity with respect to copy number, sequence content and distribution. While the discovery of copy number polymorphisms (CNP) has increased exponentially in recent years, the transition from discovery to genotyping has proved challenging, particularly for CNPs embedded in complex regions of the genome. CNPs that are collectively common in the population and possess a dynamic range of copy numbers have proved the most difficult to genotype in association studies. This is in some part due to technical limitations of genotyping assays and the sequence properties of the genomic region being analyzed. Here we describe in detail the basis of a number of molecular techniques used to genotype complex CNPs, compare and contrast these approaches for determination of multi-allelic copy number, and discuss the potential application of these techniques in genetic studies.

Correlating multiallelic copy number polymorphisms with disease susceptibility.

The human genome contains a significant amount of sequence variation, from single nucleotide polymorphisms to large stretches of DNA that may be present in a range of different copies between individuals. Several such regions are variable in >1% of the population (referred to as copy number polymorphisms or CNPs), and many studies have looked for associations between the copy number of genes within multiallelic CNPs and disease susceptibility. Associations have indeed been described for several genes, including the ß-defensins (DEFB4, DEFB103, DEFB104), chemokine ligand 3 like 1 (CCL3L1), Fc gamma receptor 3B (FCGR3B), and complement component C4 (C4). However, follow-up replication in independent cohorts has failed to reproduce a number of these associations. It is clear that replicated associations such as those between C4 and systemic lupus erythematosus, and ß-defensin and psoriasis, have used robust genotyping methodologies. Technical issues associated with genotyping sequences of high identity may therefore account for failure to replicate other associations. Here, we compare and contrast the most popular approaches that have been used to genotype CNPs, describe how they have been applied in different situations, and discuss potential reasons for the difficulty in reproducibly linking multiallelic CNPs to complex diseases.

Complex SNP-related sequence variation in segmental genome duplications.

There is uncertainty about the true nature of predicted single-nucleotide polymorphisms (SNPs) in segmental duplications (duplicons) and whether these markers genuinely exist at increased density as indicated in public databases. We explored these issues by genotyping 157 predicted SNPs in duplicons and control regions in normal diploid genomes and fully homozygous complete hydatidiform moles. Our data identified many true SNPs in duplicon regions and few paralogous sequence variants. Twenty-eight percent of the polymorphic duplicon sequences we tested involved multisite variation, a new type of polymorphism representing the sum of the signals from many individual duplicon copies that vary in sequence content due to duplication, deletion or gene conversion. Multisite variations can masquerade as normal SNPs when genotyped. Given that duplicons comprise at least 5% of the genome and many are yet to be annotated in the genome draft, effective strategies to identify multisite variation must be established and deployed.

SRY mutation analysis by next generation (deep) sequencing in a cohort of chromosomal Disorders of Sex Development (DSD) patients with a mosaic karyotype.

BACKGROUND: The presence of the Y-chromosome or Y chromosome-derived material is seen in 4-60% of Turner syndrome patients (Chromosomal Disorders of Sex Development (DSD)). DSD patients with specific Y-chromosomal material in their karyotype, the GonadoBlastoma on the Y-chromosome (GBY) region, have an increased risk of developing type II germ cell tumors/cancer (GCC), most likely related to TSPY. The Sex determining Region on the Y gene (SRY) is located on the short arm of the Y-chromosome and is the crucial switch that initiates testis determination and subsequent male development. Mutations in this gene are responsible for sex reversal in approximately 10-15% of 46,XY pure gonadal dysgenesis (46,XY DSD) cases. The majority of the mutations described are located in the central HMG domain, which is involved in the binding and bending of the DNA and harbors two nuclear localization signals. SRY mutations have also been found in a small number of patients with a 45,X/46,XY karyotype and might play a role in the maldevelopment of the gonads. METHODS: To thoroughly investigate the presence of possible SRY gene mutations in mosaic DSD patients, we performed next generation (deep) sequencing on the genomic DNA of fourteen independent patients (twelve 45,X/46,XY, one 45,X/46,XX/46,XY, and one 46,XX/46,XY). RESULTS AND CONCLUSIONS: The results demonstrate that aberrations in SRY are rare in mosaic DSD patients and therefore do not play a significant role in the etiology of the disease.

Breakpoint characterization of a rare alpha0 -thalassemia deletion using targeted locus amplification on genomic DNA.

INTRODUCTION: The high-sequence homology of the α-globin-gene cluster is responsible for microhomology-mediated recombination events during meiosis, resulting in a high density of deletion breakpoints within a 10 kb region. Commonly used deletion detection methods, such as multiplex ligation-dependent probe amplification (MLPA) and Southern blot, cannot exactly define the breakpoints. This typically requires long-range PCR, which is not always successful. Targeted locus amplification (TLA) is a targeted enrichment method that can be used to sequence up to 70 kb of neighboring DNA sequences without prior knowledge about the target site. METHODS: Genomic DNA (gDNA) TLA is a technique that folds isolated DNA, ensuring that adjacent loci are in a close spatial proximity. Subsequent digestion and religation form DNA circles that are amplified using fragment-specific inverse primers, creating a library that is suitable for Illumina sequencing. RESULTS: Here, we describe the characterization of a rare 16 771 bp deletion, utilizing gDNA TLA with a single inverse PCR primer set on one end of the breakpoint. Primers for breakpoint PCR were designed to confirm the deletion breakpoints and were consequently used to characterize the same deletion in 10 additional carriers sharing comparable hematologic data and similar MLPA results. CONCLUSIONS: The gDNA TLA technology was successfully used to identify deletion breakpoints within the alpha-globin cluster. The deletion was described only once in an earlier study as the --gb , but as it was not registered correctly in the available databases, it was not initially recognized as such.

Rapid high-throughput analysis of DNaseI hypersensitive sites using a modified Multiplex Ligation-dependent Probe Amplification approach.

BACKGROUND: Mapping DNaseI hypersensitive sites is commonly used to identify regulatory regions in the genome. However, currently available methods are either time consuming and laborious, expensive or require large numbers of cells. We aimed to develop a quick and straightforward method for the analysis of DNaseI hypersensitive sites that overcomes these problems. RESULTS: We have developed a modified Multiplex Ligation-dependent Probe Amplification (MLPA) approach for the identification and analysis of genomic regulatory regions. The utility of this approach was demonstrated by simultaneously analysing 20 loci from the ENCODE project for DNaseI hypersensitivity in a range of different cell lines. We were able to obtain reproducible results with as little as 5 x 10(4) cells per DNaseI treatment. Our results broadly matched those previously reported by the ENCODE project, and both technical and biological replicates showed high correlations, indicating the sensitivity and reproducibility of this method. CONCLUSION: This new method will considerably facilitate the identification and analysis of DNaseI hypersensitive sites. Due to the multiplexing potential of MLPA (up to 50 loci can be examined) it is possible to analyse dozens of DNaseI hypersensitive sites in a single reaction. Furthermore, the high sensitivity of MLPA means that fewer than 10(5) cells per DNaseI treatment can be used, allowing the discovery and analysis of tissue specific regulatory regions without the need for pooling. This method is quick and easy and results can be obtained within 48 hours after harvesting of cells or tissues. As no special equipment is required, this method can be applied by any laboratory interested in the analysis of DNaseI hypersensitive regions.

Rapid and reliable determination of transgene zygosity in mice by multiplex ligation-dependent probe amplification.

The ability to rapidly and unequivocally distinguish heterozygous from homozygous transgenic mice is an integral part of any breeding strategy. Here we describe a quick and simple protocol for determining the zygosity of transgenic mice at multiple loci in a single reaction. This involved the development of a multiplex ligation-dependent probe amplification (MLPA) probe mix to simultaneously measure common transgenic alleles such as Cre recombinase (Cre), neomycin (Neo), beta-galactosidase (LacZ) and enhanced green fluorescent protein (eGFP), as well as loci specific to the X and Y chromosome to allow sexing. Each reaction required as little as 100 ng of genomic DNA isolated from a tail biopsy using a simple procedure. Normalization against autosomal control loci resulted in 100% call accuracy, with no ambiguous results. This probe mix can be easily implemented in any laboratory with access to a PCR machine and a DNA sequencer, and can be rapidly adapted to genotype any additional loci of interest.

Rare variants in non-coding regulatory regions of the genome that affect gene expression in systemic lupus erythematosus.

Personalized medicine approaches are increasingly sought for diseases with a heritable component. Systemic lupus erythematosus (SLE) is the prototypic autoimmune disease resulting from loss of immunologic tolerance, but the genetic basis of SLE remains incompletely understood. Genome wide association studies (GWAS) identify regions associated with disease, based on common single nucleotide polymorphisms (SNPs) within them, but these SNPs may simply be markers in linkage disequilibrium with other, causative mutations. Here we use an hierarchical screening approach for prediction and testing of true functional variants within regions identified in GWAS; this involved bioinformatic identification of putative regulatory elements within close proximity to SLE SNPs, screening those regions for potentially causative mutations by high resolution melt analysis, and functional validation using reporter assays. Using this approach, we screened 15 SLE associated loci in 143 SLE patients, identifying 7 new variants including 5 SNPs and 2 insertions. Reporter assays revealed that the 5 SNPs were functional, altering enhancer activity. One novel variant was linked to the relatively well characterized rs9888739 SNP at the ITGAM locus, and may explain some of the SLE heritability at this site. Our study demonstrates that non-coding regulatory elements can contain private sequence variants affecting gene expression, which may explain part of the heritability of SLE.

Skewed X-inactivation is common in the general female population.

X-inactivation is a well-established dosage compensation mechanism ensuring that X-chromosomal genes are expressed at comparable levels in males and females. Skewed X-inactivation is often explained by negative selection of one of the alleles. We demonstrate that imbalanced expression of the paternal and maternal X-chromosomes is common in the general population and that the random nature of the X-inactivation mechanism can be sufficient to explain the imbalance. To this end, we analyzed blood-derived RNA and whole-genome sequencing data from 79 female children and their parents from the Genome of the Netherlands project. We calculated the median ratio of the paternal over total counts at all X-chromosomal heterozygous single-nucleotide variants with coverage ≥10. We identified two individuals where the same X-chromosome was inactivated in all cells. Imbalanced expression of the two X-chromosomes (ratios ≤0.35 or ≥0.65) was observed in nearly 50% of the population. The empirically observed skewing is explained by a theoretical model where X-inactivation takes place in an embryonic stage in which eight cells give rise to the hematopoietic compartment. Genes escaping X-inactivation are expressed from both alleles and therefore demonstrate less skewing than inactivated genes. Using this characteristic, we identified three novel escapee genes (SSR4, REPS2, and SEPT6), but did not find support for many previously reported escapee genes in blood. Our collective data suggest that skewed X-inactivation is common in the general population. This may contribute to manifestation of symptoms in carriers of recessive X-linked disorders. We recommend that X-inactivation results should not be used lightly in the interpretation of X-linked variants.

Successful long-term growth hormone therapy in a girl with haploinsufficiency of the insulin-like growth factor-I receptor due to a terminal 15q26.2->qter deletion detected by multiplex ligation probe amplification.

CONTEXT: Microscopically visible heterozygous terminal 15q deletions encompassing the IGF1R gene are rare and usually associated with intrauterine growth retardation and short stature. The incidence of submicroscopic deletions is unknown, as is the effect of GH therapy in this condition. OBJECTIVE: The objective of the study was to describe the use of a novel genetic technique [multiplex ligation probe amplification (MLPA)] to detect haploinsufficiency of the IGF1R gene in a patient suspected of an IGF1R gene defect and evaluate the effect of long-term GH therapy. PATIENT: A 15-yr-old adolescent, born small for gestational age, showed persistent postnatal growth retardation, microcephaly, and elevated IGF-I levels. She had been treated with GH since the age of 5 yr. METHODS: MLPA and array comparative genomic hybridization (aCGH) were performed to examine gene copy number changes. Dermal fibroblast cultures were used for functional analysis. RESULTS: With MLPA, a deletion of one copy of the IGF1R gene was detected, defined by aCGH as a loss of 15q26.2->qter. IGF1R mRNA expression was decreased in fibroblasts. IGF-I binding and type 1 IGF receptor protein expression as well as activation of type 1 IGF receptor autophosphorylation and protein kinase B/Akt by IGF-I tended to be lower, but this did not reach statistical significance. GH treatment resulted in a good growth response and a normal adult height. CONCLUSIONS: MLPA and aCGH are useful tools to detect submicroscopic deletions of the IGF1R gene in patients born small for gestational age with persistent growth failure. The phenotype resembles that of a heterozygous inactivating IGF1R mutation. Long-term GH therapy causes growth acceleration in childhood and a normal adult height.

Loss of maternal EED results in postnatal overgrowth.

BACKGROUND: Investigating how epigenetic information is transmitted through the mammalian germline is the key to understanding how this information impacts on health and disease susceptibility in offspring. EED is essential for regulating the repressive histone modification, histone 3 lysine 27 tri-methylation (H3K27me3) at many developmental genes. RESULTS: In this study, we used oocyte-specific Zp3-Cre recombinase (Zp3Cre) to delete Eed specifically in mouse growing oocytes, permitting the study of EED function in oocytes and the impact of depleting EED in oocytes on outcomes in offspring. As EED deletion occurred only in growing oocytes and females were mated to normal wild type males, this model allowed the study of oocyte programming without confounding factors such as altered in utero environment. Loss of EED from growing oocytes resulted in a significant overgrowth phenotype that persisted into adult life. Significantly, this involved increased adiposity (total fat) and bone mineral density in offspring. Similar overgrowth occurs in humans with Cohen-Gibson (OMIM 617561) and Weaver (OMIM 277590) syndromes, that result from de novo germline mutations in EED or its co-factor EZH2, respectively. Consistent with a role for EZH2 in human oocytes, we demonstrate that de novo germline mutations in EZH2 occurred in the maternal germline in some cases of Weaver syndrome. However, deletion of Ezh2 in mouse oocytes resulted in a distinct phenotype compared to that resulting from oocyte-specific deletion of Eed. CONCLUSIONS: This study provides novel evidence that altering EED-dependent oocyte programming leads to compromised offspring growth and development in the next generation.