A sensitised mutagenesis screen in the mouse to explore the bovine genome: study of muscle characteristics.

Meat yield and quality are closely related to muscle development. The muscle characteristics mainly take place during embryonic and postnatal phases. Thus, genetic control of muscle development in early stages represents a significant stake to improve product quality and production efficiency. In bovine, several programmes have been developed to detect quantitative trait loci (QTL) affecting growth, carcass composition or meat quality traits. Such strategy is incontestably very powerful yet extremely cumbersome and costly when dealing with large animals such as ruminants. Furthermore, the fine mapping of the QTL remains a real challenge. Here, we proposed an alternative approach based on chemical mutagenesis in the mouse combined with comparative genomics to identify regions or genes controlling muscle development in cattle. At present, we isolated seven independent mouse lines of high interest. Two lines exhibit a hypermuscular phenotype, and the other five show various skeletomuscular phenotypes. Detailed characterisation of these mouse mutants will give crucial input for the identification and the mapping of genes that control muscular development. Our strategy will provide the opportunity to understand the function and control of genes involved in improvement of animal physiology.

Two lineage boundaries coordinate vertebrate apical ectodermal ridge formation.

Proximal-distal outgrowth of the vertebrate limb bud is regulated by the apical ectodermal ridge (AER), which forms at an invariant position along the dorsal-ventral (D/V) axis of the embryo. We have studied the genetic and cellular events that regulate AER formation in the mouse. In contrast to implications from previous studies in chick, we identified two distinct lineage boundaries in mouse ectoderm prior to limb bud outgrowth using a Cre/loxP-based fate-mapping approach and a novel retroviral cell-labeling technique. One border is transient and at the limit of expression of the ventral gene En1, which corresponds to the D/V midline of the AER, and the second border corresponds to the dorsal AER margin. Labeling of AER precursors using an inducible Cre showed that not all cells that initially express AER genes form the AER, indicating that signaling is required to maintain an AER phenotype. Misexpression of En1 at moderate levels specifically in the dorsal AER of transgenic mice was found to produce dorsally shifted AER fragments, whereas high levels of En1 abolished AER formation. In both cases, the dorsal gene Wnt7a was repressed in cells adjacent to the En1-expressing cells, demonstrating that signaling regulated by EN1 occurs across the D/V border. Finally, fate mapping of AER domains in these mutants showed that En1 plays a part in positioning and maintaining the two lineage borders.

Impaired stress response and reduced anxiety in mice lacking a functional corticotropin-releasing hormone receptor 1.

Corticotropin-releasing hormone (CRH) is a potent mediator of endocrine, autonomic, behavioural and immune responses to stress, and has been implicated in the stress-like and other aversive consequences of drug abuse, such as withdrawal from alcohol. Two CRH receptors, Crhr1 and Crhr2, have been identified in the mouse. Crhr1 is highly expressed in the anterior pituitary, neocortex, hippocampus, amygdala and cerebellum, and activation of this receptor stimulates adenylate cyclase. Here we show that in mice lacking Crhr1, the medulla of the adrenal gland is atrophied and stress-induced release of adrenocorticotropic hormone (ACTH) and corticosterone is reduced. The homozygous mutants exhibit increased exploratory activity and reduced anxiety-related behaviour under both basal conditions and following alcohol withdrawal. Our results demonstrate a key role of the Crhr1 receptor in mediating the stress response and anxiety-related behaviour.

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.

The mouse Pax2(1Neu) mutation is identical to a human PAX2 mutation in a family with renal-coloboma syndrome and results in developmental defects of the brain, ear, eye, and kidney.

We describe a new mouse frameshift mutation (Pax2(1Neu)) with a 1-bp insertion in the Pax2 gene. This mutation is identical to a previously described mutation in a human family with renal-coloboma syndrome [Sanyanusin, P., McNoe, L. A., Sullivan, M. J., Weaver, R. G. & Eccles, M. R. (1995) Hum. Mol. Genet. 4, 2183-2184]. Heterozygous mutant mice exhibit defects in the kidney, the optic nerve, and retinal layer of the eye, and in homozygous mutant embryos, development of the optic nerve, metanephric kidney, and ventral regions of the inner ear is severely affected. In addition, we observe a deletion of the cerebellum and the posterior mesencephalon in homozygous mutant embryos demonstrating that, in contrast to mutations in Pax5, which is also expressed early in the mid-hindbrain region, loss of Pax2 gene function alone results in the early loss of the mid-hindbrain region. The mid-hindbrain phenotype is similar to Wnt1 and En1 mutant phenotypes, suggesting the conservation of gene regulatory networks between vertebrates and Drosophila.

N-MYC amplification, loss of heterozygosity on the short arm of chromosome 1 and DNA ploidy in retinoblastoma.

Recurrent genetic alterations different from the alteration of the RB1 gene on chromosome 13q14 have been described in retinoblastoma, including structural alterations on the short arm of chromosome 1 and amplification of the N-MYC oncogene. These two genetic alterations are major prognostic factors in neuroblastoma, another embryonic neuro-ectodermal tumour. In order to assess the frequency of these alterations and their possible association with clinical parameters in retinoblastoma, we studied a series of 46 retinoblastoma tumour samples. Ploidy was assessed by flow cytometry, N-MYC copy number was evaluated by a spot-blot procedure using the pNb-1 probe and loss of heterozygosity was investigated by PCR analysis at mini- and microsatellites located on the short arm of chromosome 1. Most tumours were in the diploid or near diploid range; only one case exhibited tetraploidy. N-MYC amplification was observed in only one of the 45 tumours. Loss of heterozygosity on the short arm of chromosome 1 was observed in 9/43 tumours (21%); in particular, its incidence was higher in metastatic than in localised disease (P < 0.05). We suggest that alterations of one or several genes on chromosome 1p might play a role in the oncogenesis or progression of retinoblastoma. Analysis of the long term follow-up of these and additional patients should determine the prognostic value of this parameter.

Spectrum of germline mutations in the RB1 gene: a study of 232 patients with hereditary and non hereditary retinoblastoma.

Germline mutations in the RB1 gene confer hereditary predisposition to retinoblastoma. We have performed a mutation survey of the RB1 gene in 232 patients with hereditary or non hereditary retinoblastoma. We systematically explored all 27 exons and flanking sequences as well as the promotor. All types of point mutations are represented and are found unequally distributed along the RB1 gene sequence. In the population we studied, exons 3, 8, 18 and 19 are preferentially altered. The range of frequency of detection of germline mutations is about 20%, indicating that other mechanisms of inactivation of RB1 should be involved. The spectrum of mutations presented here should help to improve the clinical management of retinoblastoma and to understand the molecular mechanisms leading to tumorigenesis.

Abnormal methylation pattern in constitutive and facultative (X inactive chromosome) heterochromatin of ICF patients.

We have investigated the distribution of DNA methylation in chromosomes and nuclei of normal individuals and ICF (Immunodeficiency, Centromeric instability and Facial abnormalities) syndrome patients, using 5-methylcytosine monoclonal antibody. In this syndrome, DNA digestion with methyl-sensitive enzymes has previously shown a specific hypomethylation of classical satellites located in constitutive heterochromatin. The chromosome methylation pattern confirms this hypomethylation showing in addition a clear undermethylation of facultative heterochromatin (X inactive chromosome). Antibodies give, in normal and ICF chromosomes, a non-uniform labeling of euchromatin, generating a weak R-like banding pattern on chromosomes. This pattern reflects an unequal distribution of DNA methylation over the genome disclosing another aspect of chromosome organization. The breakpoints of chromosome rearrangements and the heterochromatin stretchings observed in ICF patients were analyzed by means of in situ hybridization. These chromosome modifications involve hypomethylated classical DNA satellite sequences. The underlying hypomethylation, associated with an abnormal chromatin organization, may predispose to chromosome instability.

Identification of germline mutations in the RB1 gene by denaturant gradient gel electrophoresis and polymerase chain reaction direct sequencing.

Germline mutations in the RB1 gene confer hereditary predisposition to retinoblastoma. The majority of these mutations occur de novo and differ from one patient to another. Cytogenetics and Southern blotting were shown to detect less than 15% of constitutional rearrangements. In this study we used the polymerase chain reaction (PCR) combined with denaturant gradient gel electrophoresis (DGGE) to detect point mutations or small deletions and insertions in a pool of 120 unrelated retinoblastoma patients. Partial DGGE analysis of the RB1 gene enabled us to identify sequence alterations generating stop codons, leading to amino acid substitution or affecting splice sites as well as several polymorphisms. Most of the nucleotide changes detected are flanked by direct repeats. The approach described here has proved to be a useful method for the detection of germline mutations in the RB1 gene.

Detection of sequence variations in the human insulin-receptor gene by parallel denaturing gradient gel electrophoresis.

We developed a parallel denaturing gradient gel electrophoresis (DGGE) protocol to maximize the detection of nucleotide variants in the DNA sequence coding for the mature insulin receptor and in splice site junctions. The melting behaviours of exons 2 to 22 and flanking intronic sequences were computer-simulated using two programs, MELT87 and SQHTX. The data obtained from computer analysis were used to select primers for amplification by polymerase chain reaction and optimal electrophoretic conditions. The ability of this protocol to detect nucleotide changes at the insulin-receptor locus was assessed by studying amplified DNA of a patient with leprechaunism whose insulin-receptor mutations were known and by screening the insulin-receptor gene for polymorphisms in a population of unrelated caucasian individuals. Our results demonstrate that this DGGE protocol is sensitive since it detected (1) sequence variants reported to be undetectable by means of parallel DGGE, (2) previously characterized insulin-receptor nucleotide variants, and (3) unreported polymorphisms at the insulin-receptor locus of caucasian individuals. It is also simple as perpendicular denaturing gradient gels are not required. Application of this protocol will facilitate the search for molecular defects underlying the pathogenesis of insulin resistance observed in genetic syndromes of severe insulin resistance as well as in other metabolic disorders. In addition, its ability to detect several regions of the insulin-receptor gene displaying a number of common polymorphic sites and being multiallelic will contribute to linkage studies in families with diabetic and/or insulin-resistant subjects.

Mapping around the Xq13.1 breakpoints of two X/A translocations in hypohidrotic ectodermal dysplasia (EDA) female patients.

Cellular hybrids were obtained from a t(X;12) identified in a female patient with hypohidrotic ectodermal dysplasia (EDA). This rearrangement had the same Xq13.1 cytogenetic breakpoint as a t(X;9) found in a previously observed EDA patient. A comparative analysis of these two rearrangements with nine probes was performed at the molecular level. These probes could define three subregions: three are proximal, two are distal, and four are between the two breakpoints. These last probes should prove useful for cloning the gene.

Physical map around the retinoblastoma gene: possible genomic imprinting suggested by NruI digestion.

A long-range restriction map constructed around the retinoblastoma (RB) gene by means of PFGE analysis allowed further definition of chromosomal rearrangements with a breakpoint within the gene, as well as of submicroscopic deletions. A serendipitous observation was that the NruI restriction pattern differs according to the parental origin of the rearrangement.

Molecular detection of constitutional deletions in patients with retinoblastoma.

The recent cloning of the retinoblastoma (RB) gene as well as the identification of intragenic polymorphisms afford the necessary tools for the analysis of rearrangements using molecular hybridization. We searched for constitutional deletions by Southern blotting in 67 independent patients with normal karyotype comprising 15 familial and 52 sporadic cases. Among the latter, 33 were bilaterally and 19 unilaterally affected. We detected 6 deletions using cDNA probes covering almost all of the RB gene, as well as a genomic probe of the 5' part of the gene. With this approach, the incidence of detectable deletions was around 10%. No hot spots for deletion breakpoints were found. Asymptomatic carriers were detected in 2 families. The effectiveness of genetic counselling was largely improved by this approach.

[Genetics and genetic counseling: the retinoblastoma case]. / Génétique et conseil génétique: le cas du rétinoblastome.

Forty-percent of retinoblastomas are due to a mutation inherited as an autosomal dominant trait with a high penetrance. Cytogenetic forms of retinoblastoma have led to the location of the gene, to the identification of numerous chromosome 13 specific DNA polymorphisms, and to the cloning of the gene. Intragenic DNA polymorphisms are now known. Using Southern blot hybridization, study of the mutation is possible either by direct analysis or by an indirect approach using linkage with polymorphic genetic markers. The majority of cases cannot be examined by these techniques. Their value and limits are discussed.

Loss of heterozygosity suggests tumor suppressor gene responsible for primary hepatocellular carcinoma.

Primary hepatocellular carcinoma (PHC), epidemiologically associated with chronic hepatitis B virus (HBV) infection, has historically been felt to be caused by the activation or introduction of an oncogene. However, transforming sequences from human PHC have not been reproducibly isolated. In this paper, evidence is presented that suggests PHC may result instead from the loss of an anti-oncogene. Seven of 12 human primary liver tumors tested against a panel of restriction fragment length polymorphisms (RFLPs) demonstrated loss of constitutional heterozygosity for markers on chromosome 4. Tumor and nontumor liver tissue were typed for 11 chromosome 4 RFLPs. In addition, at least one RFLP on nine other chromosomes (1, 2, 6, 7, 9, 11, 13, 14, and 17) was tested for allelic loss. Seven of nine tumors constitutionally heterozygous for chromosome 4q markers showed allele loss in tumor tissue. Six of the seven samples were jointly informative for both 4p and 4q markers. Five of the six demonstrated loss for only 4q RFLPs. In one individual, in which two samples were taken from distant locations within the same tumor, both samples showed loss of the same alleles. Among the other chromosomes informative for allele loss, one tumor showed changes on 13q. No other changes were observed in RFLPs located on the eight other chromosomes tested. These results indicate that an anti-oncogene may be located on 4q and suggest a mechanism for PHC and other cancers seroepidemiologically related to virus infection. Liver cancer caused by chronic HBV infection or other environmental agents may be linked through genetic events responsible for the loss of a tumor suppressor locus (anti-oncogene) located on chromosome 4.

Regional mapping to 4q32.1 by in situ hybridization of a DNA domain rearranged in human liver cancer.

Recently, a unique cellular DNA segment, representing the normal allele counterpart of hepatitis B virus integration site, has been isolated. It has allowed the identification of a cellular domain in which rearrangements occur in approximately 10% of primary liver tumours. We here report on the assignment of this probe, D4S112, by in situ hybridization to band 4q32.1.