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Despite recent progress in molecular karyotyping and clinical sequencing the cause of intellectual disability in a considerable subset of individuals affected by this phenotype remains elusive. As intellectual disability is also a feature of various imprinting disorders and some monogenic forms of intellectual disability are caused by epigenetic modifiers we hypothesized that changes in DNA methylation might be associated with or even causative in some cases of intellectual disability. Therefore, we performed a DNA methylation analysis of peripheral blood samples from 82 patients with intellectual disability and additional features using the HumanMethylation450 BeadChip. The findings were compared to that of 19 normal controls. Differentially methylated loci were validated by bisulfite pyrosequencing. On a global level, we failed to detect a robust DNA methylation signature segregating individuals with intellectual disability from controls. Using an individual approach, we identified 157 regions showing individual DNA methylation changes in at least one patient. These correlated to 107 genes including genes linked to conditions associated with intellectual disability, namely COLEC11, SHANK2, GLI2 and KCNQ2, as well as imprinted genes like FAM50B and MEG3. The latter was suggestive of an undiagnosed Temple syndrome which could be confirmed by diagnostic tests. Subsequent in-depth analysis of imprinted loci revealed DNA methylation changes at additional imprinted loci, i.e. PPIEL, IGF2R, MEG8 and MCTS2/HM13, in up to five patients. Our findings indicate that imprinting disorders are rare but probably under-diagnosed in patients with intellectual disability and moreover point to DNA methylation changes as potential alternative means to identify deregulated genes involved in the pathogenesis of intellectual disability., (Copyright © 2015 Elsevier Masson SAS. All rights reserved.)

Objective: To identify CpG sites differentially methylated in peripheral blood of men with idiopathic infertility due to impaired spermatogenesis as compared with fertile controls., Design: DNA methylation profiling on peripheral blood samples using the HumanMethylation450 BeadChip (Illumina) in patients and controls, single-nucleotide polymorphism (SNP) typing by Sanger sequencing., Setting: University institute in cooperation with genetic and infertility clinics., Patient(s): 30 infertile men with normal CFTR and AZF tests and karyotype, and 10 fertile male controls., Intervention(s): None., Main Outcome Measure(s): DNA methylation levels at CpG sites., Result(s): We identified 471 CpGs (287 genes) as differentially methylated between patients and controls. These were significantly enriched for the gene ontology functions MHC class II receptor activity and piwi-interacting (piRNA) binding. The latter was associated with two methylation-sensitive SNPs in the genes PIWIL1 and PIWIL2, respectively, which showed significant allele distribution skewing in the infertile cohort. We found that 445 (94.5%) of 471 differentially methylated CpGs were associated with SNPs, but 26 (15 genes) were not genomically templated, including the ENO1, MTA2, BRSK2, and LBX2 genes previously associated with fertility and spermatogenesis., Conclusion(s): Our study identifies surrogate DNA methylation markers for idiopathic infertility in peripheral blood and suggests that allele-specific DNA methylation differences at regulatory sites of genes involved in piRNA regulation are associated with disturbed spermatogenesis., (Copyright © 2014 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Objectives: To test the hypothesis that mutations of SYCP3 encoding synaptonemal complex protein 3, result in increased frequency of aneuploidies in humans., Methods: Mutation analysis of the PCR-amplified 8 coding exons and exon-intron boundaries of the SYCP3 gene was done by direct sequencing of DNA isolated from 35 aneuploid fetuses of women having a potentially increased likelihood for an underlying genetic predisposition for chromosomal non-disjunction., Results: Based on the results of conventional karyotyping, the 35 aneuploid fetuses of 33 women were divided into separate groups: 9 aneuploid conceptuses of couples with recurrent aneuploid conceptions (4 of the women 35 years or younger), 12 conceptuses with double/multiple aneuploidies (5 of the women 35 years or younger), and 14 conceptuses with single aneuploidies of women younger than 35 years (8 trisomies and 6 monosomies). No pathogenic mutations in the SYCP3 coding exons and the immediately flanking intronic sequences were found., Conclusions: Under the assumption that genetic predisposition for chromosomal non-disjunction leading to aneuploidy is most likely polygenic in nature, our data suggest that SYCP3 mutations are not one of the common causes in humans.

Epigenetic mechanisms including DNA methylation are supposed to play a key role in fetal development. Here we have investigated fetal DNA-methylation levels of 27,578 CpG loci in 47 chorionic villi (CVS) and 16 amniotic cell (AC) samples. Methylation levels differed significantly between karyotypically normal AC and CVS for 2,014 genes. AC showed more extreme DNA-methylation levels of these genes than CVS and the differentially methylated genes are significantly enriched for processes characteristic for the different cell types sampled. Furthermore, we identified 404 genes differentially methylated in CVS with trisomy 21. These genes were significantly enriched for high CG dinucleotid (CpG) content and developmental processes associated with Down syndrome. Our study points to major tissue-specific differences of fetal DNA-methylation and gives rise to the hypothesis that part of the Down syndrome phenotype is epigenetically programmed in the first trimester of pregnancy.

For the rapid detection of common aneuploidies either PCR or Fluorescence in situ hybridisation (FISH) on uncultured amniotic fluid cells are widely used. There are different commercial suppliers providing FISH assays for the detection of trisomies affecting the Down's syndrome critical regions (DSCR) in 21q22. We present a case in which rapid FISH screening with different commercial probes for the DSCR yielded conflicting results. Chromosome analysis revealed a deletion of one chromosome 21 in q22 which explained the findings. Prenatally an additional small supernumerary marker chromosome (sSMC) was discovered as well, which could not be characterised. Postnatal chromosome analysis in lymphocytes of the infant revealed complex mosaicism with four cell lines. By arrayCGH the sSMC was provisionally described as derivative chromosome 21 which was confirmed by targeted FISH experiments.

Purpose: The study intended to define the underlying genetic defects for 21 index patients affected with different forms of anterior segment dysgenesis. Sequence analysis for the PAX6, PITX2, FOXC1, and CYP1B1 genes has been implemented for this purpose., Methods: Ten patients affected with Peters anomaly, 8 with Rieger anomaly, and 3 with aniridia were included in this study. All patients underwent a complete eye examination, including anterior segment evaluation, with slit-lamp microsocopy, fundoscopy, tonography, and gonioscopy. Twenty-one intronic primer pairs were used to amplify the coding exons of the FOXC1, CYP1B1, PITX2, and PAX6 genes for sequence analysis on an automated sequencer (ABI 3730)., Results: We were able to detect mutations in 5 of 21 patients with anterior segment malformations. We found mutations in individuals suffering from Rieger anomaly and aniridia, in CYP1B1 and PAX6, respectively. None of the 10 Peters anomaly patients had causative mutations in any of the 4 genes we screened., Conclusions: Our results suggest primary congenital glaucoma and the anterior segment dysgenesis disorders may share a common molecular pathophysiology in the CYP1B1 pathway.

Williams-Beuren syndrome (WBS) is a contiguous gene syndrome that occurs mainly sporadically, with an estimated frequency of 1:13,700 to 1:25,000 [Grimm and Wesselhoeft, 1980; Martin et al., 1984; Udwin, 1990]. The cases of monozygotic twins concordant for WBS and dizygotic twins discordant for the syndrome have been reported. In addition, a few familial cases have been described since 1993. The clinical diagnosis has been supported by molecular genetic findings in only two patients, however. We herein report on two families in which the WBS was inherited in girls from their mothers. All four patients showed the typical hemizygous deletion at 7q11.23 [46,XX, ish,del(7)(q11.23q11.23) (ELN/LIMK1/D7S-613x1, D7S486/D7S522x2)], but the clinical picture was strikingly variable within and between families., (Copyright 2001 Wiley-Liss, Inc.)

Microdissection in combination with reverse painting fluorescence in-situ hybridization (FISH) is a very effective method to identify breakpoints and rearrangements of derived chromosomes and reveal the chromosomal origin of marker chromosomes. We describe an innovation that allows a convenient, fast and safe isolation of microdissected fragments as currently available protocols. The microdissected chromosomes are harvested in a collection drop located in a movable micropipette adjusted to a second micromanipulator under microscopic observation. We used this technique to analyze several cytogenetic aberrations. In order to evaluate the efficiency of our microdissection procedure, we compared the results obtained with microdissection probes made from only one fragment with those obtained with more than six microdissected fragments. In all cases, the single-fragment microdissections were sufficient to provide probes.