Gérard Roizès

MethDB—a public database for DNA methylation data

Methylation of cytosine in the 5 position of the pyrimidine ring is a major modification of the DNA in most organisms. In eukaryotes, the distribution and number of 5-methylcytosines (5mC) along the DNA is heritable but can also change with the developmental state of the cell and as a response to modifications of the environment. While DNA methylation probably has a number of functions, scientific interest has recently focused on the gene silencing effect methylation can have in eukaryotic cells. In particular, the discovery of changes in the methylation level during cancer development has increased the interest in this field. In the past, a vast amount of data has been generated with different levels of resolution ranging from 5mC content of total DNA to the methylation status of single nucleotides. We present here a database for DNA methylation data that attempts to unify these results in a common resource. The database is accessible via WWW (http://www.methdb.de). It stores information about the origin of the investigated sample and the experimental procedure, and contains the DNA methylation data. Query masks allow for searching for 5mC content, species, tissue, gene, sex, phenotype, sequence ID and DNA type. The output lists all available information including the relative gene expression level. DNA methylation patterns and methylation profiles are shown both as a graphical representation and as G/A/T/C/5mC-sequences or tables with sequence positions and methylation levels, respectively.

Lack of association between polymorphisms of eight candidate genes and idiopathic dilated cardiomyopathy: The CARDIGENE study

OBJECTIVES The study investigated the potential role of eight candidate genes in the susceptibility to idiopathic dilated cardiomyopathy (IDC). BACKGROUND Idiopathic dilated cardiomyopathy has a familial origin in 20% to 25% of cases, and several genetic loci have been identified in rare monogenic forms of the disease. These findings led to the hypothesis that genetic factors might also be involved in sporadic forms of the disease. In complex diseases that do not exhibit a clear pattern of familial aggregation, the candidate gene approach is a strategy widely used to identify susceptibility genes. All genes coding for proteins involved in biochemical or physiological abnormalities of cardiac function are potential candidates for IDC. METHODS We studied 433 patients with IDC and 401 gender- and age-matched controls. Polymorphisms investigated were the I/D polymorphism of the angiotensin I-converting enzyme (ACE) gene, the T174M and M235T polymorphisms of the angiotensinogen (AGT) gene, the A-153G and A+39C polymorphisms of the angiotensin-II type 1 receptor (AGTR1) gene, the T-344C polymorphism of the aldosterone synthase (CYP11B2) gene, the G-308A polymorphism of the tumor necrosis factor-alpha (TNF) gene, the R25P polymorphism of the transforming growth factor beta1 (TGFB1) gene, the G+11/in23T polymorphism of the endothelial nitric oxide synthase (NOS3) gene and the C-1563T polymorphism of the brain natriuretic peptide (BNP) gene. RESULTS None of the polymorphisms were significantly associated with the risk or the severity of the disease. CONCLUSIONS We did not find evidence for an involvement of any of the 10 investigated polymorphisms in the susceptibility to IDC.

New BAGE (B melanoma antigen) genes mapping to the juxtacentromeric regions of human chromosomes 13 and 21 have a cancer/testis expression profile.

A first BAGE (B melanoma antigen) gene, BAGE1, was identified because it encodes a human tumour antigen recognised by a cytolytic T lymphocyte. Here, we characterised five new BAGE genes mapping to the juxtacentromeric regions of human chromosomes 13 and 21 and nine BAGE gene fragments mapping to the juxtacentromeric regions of chromosomes 9, 13, 18, and 21. Genes and gene fragments share extensive regions of 90–99% nucleotide identity. We analysed the expression of BAGE genes on 215 tumours of various histological types and on nine normal tissues. Similar to BAGE1, the new BAGE genes are expressed in melanomas, bladder and lung carcinomas and in a few tumours of other histological types. All the normal tissues were negative, with the exception of testis. Our results show that human juxtacentromeric regions harbour genes, which are transcribed and translated, in addition to gene fragments that are generally not expressed. We suggest that the pattern of expression restricted to cancer/testis is a feature of the few genes mapping to juxtacentromeric regions.

The polymorphism ApoB/4311 in patients with myocardial infarction and controls : the ECTIM study

SummaryThe polymorphism affecting codon 4311 of the apolipoprotein B gene (ApoB/4311) was investigated in a large case-control study in two French and one Northern Irish geographically defined populations. Cases were recruited 3 to 9 months after a myocardial infarction (MI) and controls were randomly selected from the population. The polymorphism was assessed using allele-specific oligonucleotides (ASO). The genotype frequencies of the ApoB/4311 polymorphism did not differ in Northern Ireland and France and were in Hardy-Weinberg equilibrium in all groups; strong associations with three other polymorphisms of the ApoB gene (XbaI, EcoRI, VNTR(34 repeats)) were observed and it was possible to identify highly sensitive and specific markers of the ApoB/4311 rare variant. Homozygotes for the ApoB 4311 rare variant were slightly less frequent in cases than in controls: 22 (4.4%) and 35 (6.7%) respectively (population adjusted χ2=3.3 P<0.07), especially in Belfast: 6 (3.1%) and 12 (7.6%), respectively (P<0.06). Several lipid and lipoprotein parameters were measured. Consistently among control groups, rare homozygotes had lower mean levels of ApoB (P<0.02), triglycerides (P<0.02), and lipoprotein particles containing ApoE and ApoB (LpE:B; P<0.001) and a higher mean level of lipoprotein particles containing ApoAI and not ApoAII (LpAI; P<0.02) than heterozygotes and frequent homozygotes combined. The strong association between the ApoB/4311 polymorphism and LpE:B was also observed in patients with MI. When present in the homozygous form, the ApoB/ 4311 Asn→Ser variant is associated with a lipoprotein profile that is apparently favourable.

Polymorphisms of genes of the cardiac calcineurin pathway and cardiac hypertrophy

The study investigated the role of genetic polymorphisms in four genes of the calcineurin pathway on cardiac hypertrophy and dilated cardiomyopathy. The cardiac calcineurin pathway has been suggested to play a role in the development of cardiac hypertrophy in response to a number of physiological and pathological stimuli. Calcineurin, a heterodimeric protein composed of a catalytic and a regulatory subunit, activates the nuclear factor NFATC4 which after translocation to the nucleus associates with the transcription factor GATA4 to activate several cardiac genes involved in hypertrophic response. We have screened the genes encoding the four major components of the heart calcineurin pathway in 95 individuals and identified 27 polymorphisms. These polymorphisms were investigated in 400 selected subjects obtained from a population-based study (LOVE) in relation to echocardiographic parameters. A Gly/Ala substitution at position 160 of the NFATC4 protein (G160A) was associated with left ventricular mass and wall thickness (P=0.02 and 0.006, respectively, GA+AA vs GG), the minor allele (Ala) being associated with lower mean values of these parameters. The other polymorphisms identified by the gene screen were not associated with cardiac phenotypes. For the G160A polymorphism in NFATC4, genotype frequencies were compared between patients with dilated cardiomyopathy and controls obtained from the CARDIGENE Study. Allele A carriers were less frequent in the patient than in the control group (P=0.04). Although the strength of the associations was rather weak, these observations raise the hypothesis that the G160A polymorphism of the NFATC4 gene plays a role in the development of human cardiac hypertrophy.

STRUCTURAL ORGANIZATION AND POLYMORPHISM OF THE ALPHA SATELLITE DNA SEQUENCES OF CHROMOSOMES 13 AND 21 AS REVEALED BY PULSE FIELD GEL ELECTROPHORESIS

SummaryMore than 30 unrelated individuals were analysed by pulse field gel electrophoresis for the alphoid centromeric sequences of chromosomes 13 and 21. These individuals had DNA patterns all different from each other and were most probably heterozygous at both loci. When several nuclear families were analysed in this manner, segregation was shown to be Mendelian, and no recombination event was detected over the 150 meioses scored in this study. Alphoid DNA sequences, therefore, constitute highly polymorphic centromeric markers, which can be used in linkage analysis for loci close to the centromeres of chromosomes 13 and 21.

Hypothesis: For the Worst and for the Best, L1Hs Retrotransposons Actively Participate in the Evolution of the Human Centromeric Alphoid Sequences

A number of questions concerning the evolution and the function of the alpha satellite DNA sequences present at the centromere of all human chromosomes are still open. In this paper, we present data which could contribute to understanding these points.It is shown here that the alphoid sequences within which L1 elements are found are quite divergent from those of the homogeneous alphoid subsets present at each centromere where none has so far been detected. In addition, a number of L1s are detected close to the ends of the alpha satellite blocks. A fairly high proportion exhibit a polymorphism of presence/absence. Strikingly, several L1s localized at a distance from each other are always either present or absent simultaneously. This is interpreted as resulting from intrachromosomal recombination, through distant L1s, leading to deletion of several of them at once together with their surrounding alphoid sequences.The parameters determining which portion of the several megabases of alphoid sequences is actually involved in the centromeric function are not known. From the above data we suggest that the alpha satellite domain within which DNA sequences are recruited to form a centromere is both homogeneous in sequence and uninterrupted by L1s or any other retrotransposons. Conversely, non-centromere competent alphoid sequences would be both divergent and punctuated by scattered L1 elements, particularly at the borders of the alphoid blocks. On the grounds of these data and hypotheses, a model is presented in which it is postulated that accumulation of L1 insertions within a centromere competent alphoid domain is ruining this competence, the consequence being damage to or even loss of the centromere- forming capability of the chromosome. Restoration of fully centromere-forming competence is supposed to occur by two alternative means, either de-novo amplification of a homogeneous and uninterrupted alphoid domain or by unequal crossing over with a homologue harbouring a large competent one. If L1 retrotransposons are acting detrimentally to centromere integrity (for the worst), one must also consider them as having positive consequences on chromosomes by preventing their centromeres from swelling indefinitely by the addition of alphoid sequences (for the best). The data and ideas presented here fit well with those already put forward by Csink and Henikoff (1998) using the example of Drosophila.

SINE AND LINE WITHIN HUMAN CENTROMERES

A number of the Alu and Ll elements present within the centromeric regions of the human chromosomes have been analyzed by polymerase chain reaction amplification. The oligonucleotide primers were homologous to the 3′ end consensus sequences of either Alu or Ll in conjunction with an oligonucleotide primer homologous to alphoid sequences specific to different chromosomes. This allowed one to detect an unusual number of Alu and Ll polymorphisms at different loci. It is proposed that this results from molecular rearrangements which occur within the α-satellite DNA in which they are embedded (Marçais et al. J. Mol. Evol. 33:42–48, 1991) and not because the centromeric regions are targets for new insertions of such elements. The same analyses were made on cosmids and YACs originating from the centromeric region of chromosome 21 as well as on a collection of somatic hybrids containing chromosome 21 centromere as unique common human genetic material. The results were consistent with the above hypothesis.

Comparative study of the L1 family in the genus Mus: Possible role of retroposition and conversion events in its concerted evolution

The long interspersed repetitive family L1 was analysed in different species belonging to the genus Mus. It is shown to be highly conserved even in M.n. setulosus, which diverged from the other species around ten million years ago. The study of the linkage between diagnostic restriction sites in the various species and the sequence variations of different regions of the L1Md repeat shows that the L1 family undergoes concerted changes involving subsets of repeats. The rate at which this homogenization process occurs does not appear to be the same for all the subfamilies detected. The L1Md repeat in the twelfth intron of the serum albumin gene of Balb/c mice is shown to be a recent insertion. The role retroposon- and gene conversion-like events may play in the concerted evolution of the L1 family is discussed.

Analysis of mating systems in the schistosome-vector hermaphrodite snail Bulinus globosus by DNA fingerprinting

Bulinus globosus, one of the intermediate hosts for Schistosoma, is a hermaphrodite freshwater snail. Multilocus DNA fingerprinting was applied in order to investigate the mating system of this facultatively self-fertilizing species. By analysing DNA fingerprints of eight broods, we have shown that selfed and outcrossed individuals could be unambiguously distinguished by comparison with their parent fingerprints. First, selfed offspring patterns shared a higher number of bands with their mother than did outcrossed ones. Secondly, at least three paternal bands could be detected per out-crossed offspring pattern.