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Background: Identification of imprinted genes, demonstrating a consistent preference towards the paternal or maternal allelic expression, is important for the understanding of gene expression regulation during embryonic development and of the molecular basis of developmental disorders with a parent-of-origin effect. Combining allelic analysis of RNA-Seq data with phased genotypes in family trios provides a powerful method to detect parent-of-origin biases in gene expression. Results: We report findings in 296 family trios from two large studies: 165 lymphoblastoid cell lines from the 1000 Genomes Project and 131 blood samples from the Genome of the Netherlands (GoNL) participants. Based on parental haplotypes, we identified > 2.8 million transcribed heterozygous SNVs phased for parental origin and developed a robust statistical framework for measuring allelic expression. We identified a total of 45 imprinted genes and one imprinted unannotated transcript, including multiple imprinted transcripts showing incomplete parental expression bias that was located adjacent to strongly imprinted genes. For example, PXDC1, a gene which lies adjacent to the paternally expressed gene FAM50B, shows a 2:1 paternal expression bias. Other imprinted genes had promoter regions that coincide with sites of parentally biased DNA methylation identified in the blood from uniparental disomy (UPD) samples, thus providing independent validation of our results. Using the stranded nature of the RNA-Seq data in lymphoblastoid cell lines, we identified multiple loci with overlapping sense/antisense transcripts, of which one is expressed paternally and the other maternally. Using a sliding window approach, we searched for imprinted expression across the entire genome, identifying a novel imprinted putative lncRNA in 13q21.2. Overall, we identified 7 transcripts showing parental bias in gene expression which were not reported in 4 other recent RNA-Seq studies of imprinting. Conclusions: Our methods

Contains fulltext : 191908.pdf (publisher's version ) (Open Access)

Contains fulltext : 167696.pdf (Publisher’s version ) (Closed access), Genomic imprinting is a mechanism in which gene expression varies depending on parental origin. Imprinting occurs through differential epigenetic marks on the two parental alleles, with most imprinted loci marked by the presence of differentially methylated regions (DMRs). To identify sites of parental epigenetic bias, here we have profiled DNA methylation patterns in a cohort of 57 individuals with uniparental disomy (UPD) for 19 different chromosomes, defining imprinted DMRs as sites where the maternal and paternal methylation levels diverge significantly from the biparental mean. Using this approach we identified 77 DMRs, including nearly all those described in previous studies, in addition to 34 DMRs not previously reported. These include a DMR at TUBGCP5 within the recurrent 15q11.2 microdeletion region, suggesting potential parent-of-origin effects associated with this genomic disorder. We also observed a modest parental bias in DNA methylation levels at every CpG analyzed across approximately 1.9 Mb of the 15q11-q13 Prader-Willi/Angelman syndrome region, demonstrating that the influence of imprinting is not limited to individual regulatory elements such as CpG islands, but can extend across entire chromosomal domains. Using RNA-seq data, we detected signatures consistent with imprinted expression associated with nine novel DMRs. Finally, using a population sample of 4,004 blood methylomes, we define patterns of epigenetic variation at DMRs, identifying rare individuals with global gain or loss of methylation across multiple imprinted loci. Our data provide a detailed map of parental epigenetic bias in the human genome, providing insights into potential parent-of-origin effects.

Contains fulltext : 138097.pdf (publisher's version ) (Closed access), Parent-of-origin (PofO) effects, such as imprinting are a phenomenon where the effect of variants depends on parental origin. Conventional association studies assume that phenotypic effects are independent of parental origin, and are thus severely underpowered to detect such non-Mendelian effects. Risk of orofacial clefts is influenced by genetic and environmental effects, the latter including maternal-specific factors such as perinatal smoking and folate intake. To identify variants showing PofO effects in orofacial clefts we have used a modification of the family-based transmission disequilibrium test to screen for biased transmission from mothers and fathers to affected offspring, biased ratios of maternal versus paternal transmission, and biased frequencies of reciprocal classes of heterozygotes among offspring. We applied these methods to analyze published genome-wide single-nucleotide polymorphism (SNP) data from approximately 2500 trios mainly of European and Asian ethnicity with non-syndromic orofacial clefts, followed by analysis of 64 candidate SNPs in a replication cohort of approximately 1200 trios of European origin. In our combined analysis, we did not identify any SNPs achieving conventional genome-wide significance (P<5 x 10(-8)). However, we observed an overall excess of loci showing maternal versus paternal transmission bias (P=0.013), and identified two loci that showed nominally significant effects in the same direction in both the discovery and replication cohorts, raising the potential for PofO effects. These include a possible maternal-specific transmission bias associated with rs12543318 at 8q21.3, a locus identified in a recent meta-analysis of non-syndromic cleft (maternal-specific P=1.5 x 10(-7), paternal-specific P=0.17). Overall, we conclude from this analysis that there are subtle hints of PofO effects in orofacial clefting.

Parent-of-origin (PofO) effects, such as imprinting are a phenomenon where the effect of variants depends on parental origin. Conventional association studies assume that phenotypic effects are independent of parental origin, and are thus severely underpowered to detect such non-Mendelian effects. Risk of orofacial clefts is influenced by genetic and environmental effects, the latter including maternal-specific factors such as perinatal smoking and folate intake. To identify variants showing PofO effects in orofacial clefts we have used a modification of the family-based transmission disequilibrium test to screen for biased transmission from mothers and fathers to affected offspring, biased ratios of maternal versus paternal transmission, and biased frequencies of reciprocal classes of heterozygotes among offspring. We applied these methods to analyze published genomewide single-nucleotide polymorphism (SNP) data from B2500 trios mainly of European and Asian ethnicity with non-syndromic orofacial clefts, followed by analysis of 64 candidate SNPs in a replication cohort of B1200 trios of European origin. In our combined analysis, we did not identify any SNPs achieving conventional genome

Item does not contain fulltext, Recent studies have identified PRDM9, a zinc finger (ZF) protein, as a key regulator of meiotic recombination. As both recurrent genomic disorders and chromosomal non-disjunction are known to be associated with specific unusual patterns of recombination, we hypothesized a possible link between PRDM9 ZF variation and susceptibility to microdeletion syndromes and/or trisomy. We sequenced the PRDM9 ZF domain in 271 parents of patients with de novo microdeletions of known parental origin (velocardiofacial syndrome, the 17q21.31 microdeletion syndrome, Prader-Willi/Angelman syndrome and Williams-Beuren syndrome), and in 61 parents of individuals with a supernumerary X chromosome. We compared PRDM9 ZF genotype frequencies between parents in whose germ line the de novo rearrangement occurred and their spouses. We observed a significantly increased frequency (p = 0.006) of PRDM9 variants in parents who transmitted de novo 7q11.23 deletions to their offspring. These data suggest that certain PRDM9 alleles may be associated with an increased susceptibility to recurrent 7q11.23 microdeletions that cause Williams-Beuren syndrome. However, as the majority of parents who transmitted a de novo microdeletion/supernumerary X chromosome to their offspring have the common AA genotype, we conclude that none of the rearrangements we have studied are dependent on specific non-A PRDM9 alleles.

1 september 2010, Contains fulltext : 87767.pdf (publisher's version ) (Open Access), The maternal and paternal genomes possess distinct epigenetic marks that distinguish them at imprinted loci. In order to identify imprinted loci, we used a novel method, taking advantage of the fact that uniparental disomy (UPD) provides a system that allows the two parental chromosomes to be studied independently. We profiled the paternal and maternal methylation on chromosome 15 using immunoprecipitation of methylated DNA and hybridization to tiling oligonucleotide arrays. Comparison of six individuals with maternal versus paternal UPD15 revealed 12 differentially methylated regions (DMRs). Putative DMRs were validated by bisulfite sequencing, confirming the presence of parent-of-origin-specific methylation marks. We detected DMRs associated with known imprinted genes within the Prader-Willi/Angelman syndrome region, such as SNRPN and MAGEL2, validating this as a method of detecting imprinted loci. Of the 12 DMRs identified, eight were novel, some of which are associated with genes not previously thought to be imprinted. These include a site within intron 2 of IGF1R at 15q26.3, a gene that plays a fundamental role in growth, and an intergenic site upstream of GABRG3 that lies within a previously defined candidate region conferring an increased maternal risk of psychosis. These data provide a map of parent-of-origin-specific epigenetic modifications on chromosome 15, identifying DNA elements that may play a functional role in the imprinting process. Application of this methodology to other chromosomes for which UPD has been reported will allow the systematic identification of imprinted sites throughout the genome.

Contains fulltext : 80657.pdf (publisher's version ) (Closed access), BACKGROUND: Recurrent 15q13.3 microdeletions were recently identified with identical proximal (BP4) and distal (BP5) breakpoints and associated with mild to moderate mental retardation and epilepsy. METHODS: To assess further the clinical implications of this novel 15q13.3 microdeletion syndrome, 18 new probands with a deletion were molecularly and clinically characterised. In addition, we evaluated the characteristics of a family with a more proximal deletion between BP3 and BP4. Finally, four patients with a duplication in the BP3-BP4-BP5 region were included in this study to ascertain the clinical significance of duplications in this region. RESULTS: The 15q13.3 microdeletion in our series was associated with a highly variable intra- and inter-familial phenotype. At least 11 of the 18 deletions identified were inherited. Moreover, 7 of 10 siblings from four different families also had this deletion: one had a mild developmental delay, four had only learning problems during childhood, but functioned well in daily life as adults, whereas the other two had no learning problems at all. In contrast to previous findings, seizures were not a common feature in our series (only 2 of 17 living probands). Three patients with deletions had cardiac defects and deletion of the KLF13 gene, located in the critical region, may contribute to these abnormalities. The limited data from the single family with the more proximal BP3-BP4 deletion suggest this deletion may have little clinical significance. Patients with duplications of the BP3-BP4-BP5 region did not share a recognisable phenotype, but psychiatric disease was noted in 2 of 4 patients. CONCLUSIONS: Overall, our findings broaden the phenotypic spectrum associated with 15q13.3 deletions and suggest that, in some individuals, deletion of 15q13.3 is not sufficient to cause disease. The existence of microdeletion syndromes, associated with an unpredictable and variable phenotypic outcome, will pose the clinician with diagnosti

Contains fulltext : 71235.pdf (publisher's version ) (Open Access), BACKGROUND: Duplications and deletions in the human genome can cause disease or predispose persons to disease. Advances in technologies to detect these changes allow for the routine identification of submicroscopic imbalances in large numbers of patients. METHODS: We tested for the presence of microdeletions and microduplications at a specific region of chromosome 1q21.1 in two groups of patients with unexplained mental retardation, autism, or congenital anomalies and in unaffected persons. RESULTS: We identified 25 persons with a recurrent 1.35-Mb deletion within 1q21.1 from screening 5218 patients. The microdeletions had arisen de novo in eight patients, were inherited from a mildly affected parent in three patients, were inherited from an apparently unaffected parent in six patients, and were of unknown inheritance in eight patients. The deletion was absent in a series of 4737 control persons (P=1.1x10(-7)). We found considerable variability in the level of phenotypic expression of the microdeletion; phenotypes included mild-to-moderate mental retardation, microcephaly, cardiac abnormalities, and cataracts. The reciprocal duplication was enriched in nine children with mental retardation or autism spectrum disorder and other variable features (P=0.02). We identified three deletions and three duplications of the 1q21.1 region in an independent sample of 788 patients with mental retardation and congenital anomalies. CONCLUSIONS: We have identified recurrent molecular lesions that elude syndromic classification and whose disease manifestations must be considered in a broader context of development as opposed to being assigned to a specific disease. Clinical diagnosis in patients with these lesions may be most readily achieved on the basis of genotype rather than phenotype.

Contains fulltext : 53175.pdf (publisher's version ) (Closed access), We describe multiple individuals with mental retardation and overlapping de novo submicroscopic deletions of 15q24 (1.7-3.9 Mb in size). High-resolution analysis showed that in three patients both proximal and distal breakpoints co-localized to highly identical segmental duplications (>51 kb in length, > 94% identity), suggesting non-allelic homologous recombination as the likely mechanism of origin. Sequencing studies in a fourth individual provided base pair resolution and showed that both breakpoints in this case were located in unique sequence. Despite the differences in the size and location of the deletions, all four individuals share several major features (growth retardation, microcephaly, digital abnormalities, hypospadias and loose connective tissue) and resemble one another facially (high anterior hair line, broad medial eyebrows, hypertelorism, downslanted palpebral fissures, broad nasal base, long smooth philtrum and full lower lip), indicating that this represents a novel syndrome caused by haploinsufficiency of one or more dosage-sensitive genes in the minimal deletion region. Our results define microdeletion of 15q24 as a novel recurrent genomic disorder.

Udgivelsesdato: 2007/11