The epigenetic imprinting defect of patients with Beckwith-Wiedemann syndrome born after assisted reproductive technology is not restricted to the 11p15 region.

BACKGROUND: Genomic imprinting refers to an epigenetic marking resulting in monoallelic gene expression and has a critical role in fetal development. Various imprinting diseases have recently been reported in humans and animals born after the use of assisted reproductive technology (ART). All the epimutations implicated involve a loss of methylation of the maternal allele (demethylation of KvDMR1/KCNQ1OT1 in Beckwith-Wiedemann syndrome (BWS), demethylation of SNRPN in Angelman syndrome and demethylation of DMR2/IGF2R in large offspring syndrome), suggesting that ART impairs the acquisition or maintenance of methylation marks on maternal imprinted genes. However, it is unknown whether this epigenetic imprinting error is random or restricted to a specific imprinted domain. AIM: To analyse the methylation status of various imprinted genes (IGF2R gene at 6q26, PEG1/MEST at 7q32, KCNQ1OT1 and H19 at 11p15.5, and SNRPN at 15q11-13) in 40 patients with BWS showing a loss of methylation at KCNQ1OT1 (11 patients with BWS born after the use of ART and 29 patients with BWS conceived naturally). RESULTS: 3 of the 11 (27%) patients conceived using ART and 7 of the 29 (24%) patients conceived normally displayed an abnormal methylation at a locus other than KCNQ1OT1. CONCLUSIONS: Some patients with BWS show abnormal methylation at loci other than the 11p15 region, and the involvement of other loci is not restricted to patients with BWS born after ART was used. Moreover, the mosaic distribution of epimutations suggests that imprinting is lost after fertilisation owing to a failure to maintain methylation marks during pre-implantation development.

DNMT3B mutations and DNA methylation defect define two types of ICF syndrome.

ICF syndrome is a rare autosomal recessive disease characterized by variable immunodeficiency, centromeric instability, and facial abnormalities. Mutations in the catalytic domain of DNMT3B, a gene encoding a de novo DNA methyltransferase, have been recognized in a subset of patients. ICF syndrome is a genetic disease directly related to a genomic methylation defect that mainly affects classical satellites 2 and 3, both components of constitutive heterochromatin. The variable incidence of DNMT3B mutations and the differential methylation defect of alpha satellites allow the identification of two types of patients, both showing an undermethylation of classical satellite DNA. This classification illustrates the specificity of the methylation process and raises questions about the genetic heterogeneity of the ICF syndrome.

Delayed and incomplete reprogramming of chromosome methylation patterns in bovine cloned embryos.

Full-term development has now been achieved in several mammalian species by transfer of somatic nuclei into enucleated oocytes [1, 2]. Although a high proportion of such reconstructed embryos can evolve until the blastocyst stage, only a few percent develop into live offspring, which often exhibit developmental abnormalities [3, 4]. Regulatory epigenetic markers such as DNA methylation are imposed on embryonic cells as normal development proceeds, creating differentiated cell states. Cloned embryos require the erasure of their somatic epigenetic markers so as to regain a totipotent state [5]. Here we report on differences in the dynamics of chromosome methylation between cloned and normal bovine embryos before implantation. We show that cloned embryos fail to reproduce distinguishable parental-chromosome methylation patterns after fusion and maintain their somatic pattern during subsequent stages, mainly by a highly reduced efficiency of the passive demethylation process. Surprisingly, chromosomes appear constantly undermethylated on euchromatin in morulae and blastocysts, while centromeric heterochromatin remains more methylated than that of normal embryos. We propose that the abnormal time-dependent methylation events spanning the preimplantation development of clones may significantly interfere with the epigenetic reprogramming, contributing to the high incidence of physiological anomalies occurring later during pregnancy or after clone birth.

Direct analysis of chromosome methylation.

DNA methylation is a possible candidate for a genomic imprinting marker in mammals. This epigenetic modification of DNA satisfies several essential criteria for the identification of the parental origin of individual alleles and larger portions of the genome: DNA methylation is stably propagated in somatic cells during cell division, it is reversible, it may inactivate the target sequence, and male and female gametes have different methylation patterns (reviewed in ref. 1).

Whole-genome methylation scan in ICF syndrome: hypomethylation of non-satellite DNA repeats D4Z4 and NBL2.

The ICF (immunodeficiency, centromeric instability and facial abnormalities) syndrome is a rare recessive disease characterized by immunodeficiency, extraordinary instability of certain heterochromatin regions and mutations in the gene encoding DNA methyltransferase 3B. In this syndrome, chromosomes 1 and 16 are demethylated in their centromere-adjacent (juxtacentromeric) heterochromatin, the same regions that are highly unstable in mitogen-treated ICF lymphocytes and B cell lines. We investigated the methylation abnormalities in CpG islands of B cell lines from four ICF patients and their unaffected parents. Genomic DNA digested with a CpG methylation-sensitive restriction enzyme was subjected to two-dimensional gel electrophoresis. Most of the restriction fragments were identical in the digests from the patients and controls, indicating that the methylation abnormality in ICF is restricted to a small portion of the genome. However, ICF DNA digests prominently displayed multicopy fragments absent in controls. We cloned and sequenced several of the affected DNA fragments and found that the non-satellite repeats D4Z4 and NBL2 were strongly hypomethylated in all four patients, as compared with their unaffected parents. The high degree of methylation of D4Z4 that we observed in normal cells may be related to the postulated role of this DNA repeat in position effect variegation in facio- scapulohumeral muscular dystrophy and might also pertain to abnormal gene expression in ICF. In addition, our finding of consistent hypomethylation and overexpression of NBL2 repeats in ICF samples suggests derangement of methylation-regulated expression of this sequence in the ICF syndrome.

Abnormal methylation does not prevent X inactivation in ICF patients.

DNA undermethylation is a characteristic feature of ICF syndrome and has been implicated in the formation of the juxtacentromeric chromosomal abnormalities of this rare syndrome. We have previously shown that in female ICF patients the inactive X chromosome (Xi) is also undermethylated. This result was unexpected since female ICF patients are not more severely affected than male patients. Here we show that CpG island methylation is abnormal in some ICF patients but in other ICF patients, the difference in methylation pattern between Xi and Xa (active X) is maintained. The consequences of Xi undermethylation on gene expression were investigated by enzyme assays. They showed that significant gene expression did not correlate with CpG island methylation status. The widespread Xi undermethylation does not affect overall Xi replication timing and does not prevent Barr body formation suggesting that a normal methylation pattern is not required for normal chromatin organization of Xi. Molecular investigation of some X-chromosome intron regions showed that the methylation changes in ICF female patients extend to non CpG islands sequences. Our results suggest that the genetic alteration of DNA methylation in ICF syndrome has little consequence on X chromosome gene expression and chromatin organization.

Chromosome instability and immunodeficiency syndrome caused by mutations in a DNA methyltransferase gene.

The recessive autosomal disorder known as ICF syndrome (for immunodeficiency, centromere instability and facial anomalies; Mendelian Inheritance in Man number 242860) is characterized by variable reductions in serum immunoglobulin levels which cause most ICF patients to succumb to infectious diseases before adulthood. Mild facial anomalies include hypertelorism, low-set ears, epicanthal folds and macroglossia. The cytogenetic abnormalities in lymphocytes are exuberant: juxtacentromeric heterochromatin is greatly elongated and thread-like in metaphase chromosomes, which is associated with the formation of complex multiradiate chromosomes. The same juxtacentromeric regions are subject to persistent interphase self-associations and are extruded into nuclear blebs or micronuclei. Abnormalities are largely confined to tracts of classical satellites 2 and 3 at juxtacentromeric regions of chromosomes 1, 9 and 16. Classical satellite DNA is normally heavily methylated at cytosine residues, but in ICF syndrome it is almost completely unmethylated in all tissues. ICF syndrome is the only genetic disorder known to involve constitutive abnormalities of genomic methylation patterns. Here we show that five unrelated ICF patients have mutations in both alleles of the gene that encodes DNA methyltransferase 3B (refs 5, 6). Cytosine methylation is essential for the organization and stabilization of a specific type of heterochromatin, and this methylation appears to be carried out by an enzyme specialized for the purpose.

Chromosome methylation patterns during mammalian preimplantation development.

DNA methylation patterns were evaluated during preimplantation mouse development by analyzing the binding of monoclonal antibody to 5-methylcytosine (5-MeC) on metaphase chromosomes. Specific chromosome patterns were observed in each cell stage. A banding pattern predominated in chromosomes at the one-cell stage. Banding was replaced at the two-cell stage by an asymmetrical labeling of the sister chromatids. Then, the proportion of asymmetrical chromosomes decreased by one-half at each cell division until the blastocyst stage, and chromosomes became progressively symmetrical and weakly labeled. Our results indicate that chromosome demethylation is associated with each DNA replication and suggest that a passive mechanism predominates during early development.

Localization of 5-methylcytosine in metaphase chromosomes of diploid and triploid pacu fish, Piaractus mesopotamicus (pisces, characiformes).

The distribution of 5-methylcytosine (5-MeC) was investigated in fish chromosomes by indirect immunofluorescence using a highly specific 5-MeC monoclonal antibody. Diploid and artificially produced triploid specimens of the pacu fish, Piaractus mesopotamicus, were analyzed. The strong immunofluorescent signals were coincident with the heterochromatic regions of both diploids and triploids in a pattern that matched the C-banding pattern. In the euchromatin, heterogeneous labeling was observed along the chromatids. The weakness of this labeling hindered comparison of the fluorescence labeling of homologous chromosomes from diploid and triploid individuals. However, no striking differences were observed. The possibility that the euchromatin labeling by the 5-MeC antibody is related to the occurrence of mildly repetitive sequences in the genome of Piaractus is discussed.

cDNA cloning, tissue distribution and chromosomal localization of the human ID4 gene.

A cDNA e encoding the human Id4 protein has been isolated from an astrocytoma library. The predicted protein product shares 98% identity with the mouse Id4 protein and is markedly different from that already reported. By FISH analysis, the human ID4 gene was more precisely mapped to chromosome 6p22.3-p23. Northern blot analysis showed that ID4 is mainly expressed in thyroid, brain and fetal tissue and in some nervous system tumor cell lines.

alpha-satellite DNA methylation in normal individuals and in ICF patients: heterogeneous methylation of constitutive heterochromatin in adult and fetal tissues.

The methylation profile of ten alpha-satellites was investigated in normal individuals and in ICF (Immunodeficiency, Centromeric instability, Facial abnormalities) patients. Two out of three ICF patients showed modified methylation of these sequences, reproducing a placental profile. CENP-B boxes, the binding sites of centromeric protein B, were always skewed toward nonmethylation. Unexpected results were observed in normal individuals: in somatic adult tissues the methylation pattern of alpha-satellite DNA varied between chromosomes, and in fetal tissues these satellites were homogeneously undermethylated. Detailed methylation analysis of CENP-B boxes revealed that unmethylated alpha-satellite units coexist with thoroughly methylated regions. These observations showed that the two major components of constitutive heterochromatin are differently methylated in normal somatic and fetal tissues, since classical satellites are consistently methylated. The definite changes in the methylation profile of heterochromatin in somatic chromosomes and the asynchronous timing of methylation of classical and alpha-satellites during development may reflect specific roles of highly repeated sequences in genomic organization.

Undermethylation of Alu sequences in ICF syndrome: molecular and in situ analysis.

The methylation status of young Alu sequences was investigated in four ICF patients. In fibroblast and leukocyte DNAs, Alu repeats were either undermethylated (HhaI and HpaII digestion) or demethylated (BstUI digestion), in contrast with the methylated status of Alus in control subjects. The methylation profile exhibited in ICF patients reproduces the normal profile of placental or sperm DNA. High-sensitivity immunocytochemical detection of HhaI and HpaII restriction sites on metaphase chromosomes provided further evidence of this undermethylation. The DNA methylation defect in ICF patients, first detected in satellite DNAs (constitutive heterochromatin) and CpG islands of genes on the inactive X chromosome (facultative heterochromatin), thus includes Alu sequences that are widely distributed throughout the human genome.

The gene for the ligand binding chain of the human interferon gamma receptor.

In order to characterize the gene encoding the ligand binding (1(st); alpha) chain of the human IFN-gamma receptor, two overlapping cosmid clones were analyzed. The gene spans over 25 kilobases (kb) of the genomic DNA and has seven exons. The extracellular domain is encoded by exons 1 to 5 and by part of exon 6. The transmembrane region is also encoded by exon 6. Exon 7 encodes the intracellular domain and the 3' untranslated portion. The gene was located on chromosome 6q23.1, as determined by in situ hybridization. The 4 kb region upstream (5') of the gene was sequenced and analyzed for promoter activity. No consensus-matching TATA or CAAT boxes in the 5' region were found. Potential binding sites for Sp1, AP-1, AP-2, and CREB nuclear factors were identified. Compatible with the presence of the Sp1/AP-2 sites and the lack of TATA box, S1-nuclease mapping experiments showed multiple transcription initiation sites. Promoter activity of the 5' flanking region was analyzed with two different reporter genes: the Escherichia coli chloramphenicol acetyltransferase and human growth hormone. The smallest 5' region of the gene that still had full promoter activity was 692 base pairs in length. In addition, we found sequences belonging to the oldest family of Alu repeats, 2 - 3 kb upstream of the gene, which could be useful for genetic studies.

Chromosomal mapping and expression of the human cyr61 gene in tumour cells from the nervous system.

AIMS: To characterise the human cyr61 gene (cyr61H) and determine its chromosomal locality. To compare expression of cyr61H in human tumour cell lines with that of two other structurally related genes, novH (nephroblastoma overexpressed gene) and CTGF (connective tissue growth factor), that are likely to play a role in the control of cell proliferation and differentiation. METHODS: To isolate the human cyr61 gene, placental genomic and HeLa cDNA libraries were screened with murine cyr61 cDNA. The nucleotide sequence of the complete cyr61H cDNA was established. Both Southern blotting of a panel of somatic cell hybrids and in situ hybridisation on chromosomes were performed to map the cyr61H gene. Expression of cyr61H, novH, CTGF, and novH was analysed by northern blotting in both human neuroblastomas and glioblastoma cell lines. RESULTS: Genomic and cDNA clones encompassing the cyr61H gene were isolated and characterised. Comparison of mouse and human cyr61 sequences indicated that their genomic organisation is highly conserved. Alignment of coding sequences highlighted the conservation of cyr61 regions that might be critical for its biological function. The data showed that the cyr61H gene is assigned to chromosome 1p22.3 and that different levels of cyr61H, CTGF, and novH mRNA have been detected in several human tumour cell lines derived from the nervous system. CONCLUSIONS: The human cyr61 gene belongs to an emerging family of genes including CTGF/fisp12 and nov. The murine cyr61 encodes an extracellular cysteine rich protein that exhibits chemotactic activity, promotes attachment and spreading of cells, and potentiates the mitogenic effect of growth factors. Assignment of the cyr61H gene to chromosome 1p22.3 will allow studies to determine whether human pathologies derived from the nervous system or from other tissues are associated with chromosomal abnormalities involving this region. Although the coding regions of cyr61H, CTGF, and novH are highly homologous, a growing body of evidence suggests that expression of these genes is regulated differentially, and that a balance between expression of these genes might represent a key element in determining the stage of differentiation and/or the malignant potential of tumour cells.

Characterization and chromosomal location of two repeated DNAs in three Gerbillus species.

Two tandemly repeated DNA sequences of Gerbillus nigeriae (Rodentia) (GN1 and GN2) were isolated and characterized. Both share a 36bp repeated unit, which includes a 20bp motif also found in primate alphoid and other repeated DNAs. The localization of GN1 and GN2 sequences on metaphase chromosomes of three Gerbillus species, G. nigeriae, G. aureus and G. nanus, was studied by fluorescence in situ hybridization (FISH). In the G. nigeriae and G. aureus karyotypes, which were shown to possess large amounts of heterochromatin and to have undergone multiple rearrangements during evolution, both GN1 and GN2 sequences were observed at various chromosomal sites: centromeric, telomeric and intercalary. In contrast, the karyotypically stable G. nanus, which does not possess large amounts of heterochromatin and seems to be a more ancestral species, possesses only GN1 sequences, localized in the juxtacentromeric regions.

Mutation of a nuclear succinate dehydrogenase gene results in mitochondrial respiratory chain deficiency.

We now report a mutation in the nuclear-encoded flavoprotein (Fp) subunit gene of the succinate dehydrogenase (SDH) in two siblings with complex II deficiency presenting as Leigh syndrome. Both patients were homozygous for an Arg554Trp substitution in the Fp subunit. Their parents (first cousins) were heterozygous for the mutation that occurred in a conserved domain of the protein and was absent from 120 controls. The deleterious effect of the Arg to Trp substitution on the catalytic activity of SDH was observed in a SDH- yeast strain transformed with mutant Fp cDNA. The Fp subunit gene is duplicated in the human genome (3q29; 5p15), with only the gene on chromosome 5 expressed in human-hamster somatic cell hybrids. This is the first report of a nuclear gene mutation causing a mitochondrial respiratory chain deficiency in humans.

cDNA, gene structure, and chromosomal localization of human GAR1 (CNCG3L), a homolog of the third subunit of bovine photoreceptor cGMP-gated channel.

A unique glutamic acid-rich protein was previously identified in bovine rod photoreceptors (Sugimoto et al., 1991, Proc. Natl. Acad. Sci. USA 88: 3116-3119) and later suggested to be a third subunit (gamma) of the rod cGMP-gated cation channel (Chen et al., 1994, Proc. Natl. Acad. Sci. USA 91: 11757-11761). Here, we report on the characterization of the GAR1 gene encoding a human homolog of bovine gamma. Sequence analysis of cDNA clones encoding human gamma revealed an open reading frame predicting a protein of 299 amino acids (approximately 32 kDa), half the size of the bovine gamma subunit. Comparison of the N-terminal half of bovine gamma with the predicted human gamma sequence revealed 90% identity within the first 31 amino acids, and only 60% homology was found throughout the remainder of the protein sequence. As in bovine gamma, the predicted isoelectric point of the human protein is very acidic despite the absence of the bovine C-terminal glutamic acid-rich domain. The integrity of the cDNA sequence was confirmed by analysis of several overlapping genomic clones that span the GAR1 gene. The protein coding region of the gene consists of 12 exons spanning approximately 11 kb with exon sequence identical to that of the cDNA clones. PCR of somatic cell hybrid DNA with primer pairs that amplify a portion of the GAR1 gene (locus designation CNCG3L) demonstrate localization to chromosome 16. The location of the gene was further delimited by fluorescence in situ hybridization placing the gene at 16q13.(ABSTRACT TRUNCATED AT 250 WORDS)