Ludwine Messiaen

Preimplantation genetic diagnosis of Marfan syndrome with the use of fluorescent polymerase chain reaction and the Automated Laser Fluorescence DNA Sequencer

OBJECTIVEnTo develop and apply clinical preimplantation genetic diagnosis (PGD) for Marfan syndrome.nnnDESIGNnCase report.nnnSETTINGnCenters for medical genetics and reproductive medicine in university hospitals.nnnPATIENT(S)nOne couple in which the husband was affected with Marfan syndrome.nnnINTERVENTION(S)nThe couple underwent three intracytoplasmic sperm injection cycles.nnnMAIN OUTCOME MEASURE(S)nThe correct diagnosis was obtained for embryos in three PGD cycles.nnnRESULT(S)nAlthough all the PGD cycles were followed by ET, no pregnancy ensued.nnnCONCLUSION(S)nThis assay can provide a reliable and accurate preimplantation diagnosis of Marfan syndrome.

Molecular analysis of 1p36 breakpoints in two Merkel cell carcinomas

Merkel cell carcinoma (MCC) is a rare aggressive neuroendocrine tumor of the skin. Only little information is available on the genetic alterations occurring in this tumor. Cytogenetic studies thus far have not shown recurrent chromosomal changes, although various structural chromosome 1 rearrangements, including deletions, often leading to loss of distal 1p material appear to be frequent. We report on fluorescence in situ hybridization and loss of heterozygosity analyses of an MCC tumor and MCC cell line UISO. The present study has shown that two distinct regions in the most distal band 1p36 on the short arm of chromosome 1 can be implicated in MCC. One region at 1p36.3 was delineated by a distal deletion in the MCC tumor as a result of an unbalanced translocation, resulting in loss of all markers distal to ENO1. This region was previously shown to be deleted in different tumor types including neuroblastoma. In cell line UISO an insertion in 1p36.2 was identified. The insertion breakpoint indicates a second, more proximal, region on 1p involved in MCC. The insertion breakpoint was mapped within a cluster of repetitive tRNA and snRNA genes and thus could coincide with the constitutional 1p36 breakpoint previously reported in a patient with neuroblastoma. Genes Chromosomes Cancer 23:67–71, 1998.

Characterisation of two different nonsense mutations, C6792A and C6792G, causing skipping of exon 37 in the NF1 gene

Abstract Neurofibromatosis type 1 (NF1), characterised by peripheral neurofibromas, café-au-lait spots and iris Lisch nodules, is one of the most common inherited disorders. We have analysed exons 35 to 49 in 21 unrelated NF1 patients using reverse transcription-polymerase chain reaction and protein truncation analysis. In two unrelated patients we found skipping of exon 37 at the cDNA level. Sequence analysis of genomic DNA revealed the presence of a C6792A transversion in one patient and a C6792G transversion in a second patient; both transversions change the codon for tyrosine to a nonsense codon. Sequencing of the exonic sequences as well as the branch sites, and the 3′ and 5′ splice sites of exons 36, 37 and 38 did not reveal any additional sequence abnormality. This is the first report showing that nonsense mutations in the NF1 gene can induce the skipping of a constitutive exon.

Exon 10b of the NF1 gene represents a mutational hotspot and harbors a recurrent missense mutation Y489C associated with aberrant splicing.

Purpose: To analyze the spectrum and frequency of NF1 mutations in exon 10bMethods: Mutation and sequence analysis was performed at the DNA and cDNA level.Results: We identified nine exon 10b mitations in 232 unrelated patients. Some mutations were recurrent (Y488C and L508P), other were unique (1465–1466insC and ivs10b + 2deITAAG). Surprisingly, at the RNA level, Y489c cause skipping of the last 62 nucleotides of exon 10b. another recurrent mutation, L508P, is undetectable by the Protein Truncation Test.Conclusion: As exon 10b show the highest mutation rate yet found in any of the 60 NF1 exon, it should be implemented with priorityin mutation analysis.

Novel Frameshift Mutation in a Heterozygous Woman with Fabry Disease and End-Stage Renal Failure

UNLABELLEDnIt is generally accepted that Fabry disease (angiokeratoma corporis diffusum) is an X-linked disorder resulting from the deficient activity of the lysosomal enzyme alpha-galactosidase. In males, the enzymatic defect leads to accumulation of glycosphingolipids, particularly in the kidney which causes end-stage renal disease. We report here a woman who presented in 1987 with focal and segmental glomerulosclerosis and required hemodialysis 4 years later when her son was evaluated for proteinuria. In these patients morphologic, biochemical, and genetic investigations were performed to explore the possibility of a hereditary renal disorder. Ultrastructural examination of the sons renal biopsy specimen revealed lamellated osmiophilic inclusions in the glomeruli, typical of Fabry disease. Four months after kidney transplantation in the mother, a graft biopsy specimen also revealed dense lamellated inclusions on electron microscopy. The leukocyte alpha-galactosidase activity was 0.008 mumol/min.10(9) cells in the son and 0.070 in the mother (range 0.100-0.500 mumol/min.10(9) cells). The diagnosis of Fabry disease was confirmed in both patients by the identification by DNA sequencing of a novel mutation in the alpha-galactosidase gene: one single base pair deletion in exon 3 (7317delA).nnnIN CONCLUSIONn(1) end-stage renal disease may occur in heterozygous women with Fabry disease; (2) morphologic lesions due to glycosphingolipid accumulation may be observed in the renal allograft after transplantation, and (3) DNA analysis confirmed the diagnosis by demonstrating a frameshift mutation, which has as yet not been reported.

Refined genetic and physical mapping of BPES type II.

BPES is a genetic disorder including blepharophimosis, ptosis of the eyelids, epicanthus inversus and telecanthus. Type I is associated with female infertility, whereas type II presents without other symptoms. Both types I and II occur sporadically or are inherited as an autosomal dominant trait. We present a molecular genetic and cytogenetic study in a large four-generation Belgian family with BPES type II. Karyotype analysis on high-resolution banded chromosomes yielded normal results. Fluorescence in situ hybridization (FISH) with cosmid probes spanning 3q22-q24 revealed normal hybridization patterns. Sixteen polymorphic CA repeats encompassing region 3q13-q25 were analysed. Linkage analysis in this large four-generation family provides conclusive evidence for the presence of a BPES gene in this region. Two-point lod scores greater than 3.0 between the disease and the following markers were seen: D3S1589 (4.67), D3S1292 (3.52), D3S1290 (3.59) and D3S1549 (3.65). By FISH, D3S1290, D3S1292 and D3S1549 were assigned to chromosome 3q23 using YACs positive for these markers.

Carrier screening for cystic fibrosis in a prenatal setting.

Maternal prenatal cystic fibrosis (CF) screening was offered from September, 1997, to April, 1999, at the Ghent University Hospital, to couples undergoing prenatal diagnosis (amniocentesis) for reasons not related to CF. Fifteen minutes were devoted to explaining CF, CF screening, and the study protocol. The purpose was to assess the short- and long-term knowledge of CF, the attitude towards carrier screening, and carriership. A total of 314 couples entered the pilot study; 13 female CF carriers were identified. None of their partners carried an identifiable mutation. Our survey results show that information about CF and CF screening can be given effectively as part of antenatal care because most couples recalled important medical and genetic issues, valued the genetic test for CF, and seemed to cope well with the results. Risk estimates and actual numbers were more difficult to process and recall. From the small number of couples in which the woman alone was found to be a carrier, there was little or no evidence of marked distress.

Assignment of the cellular retinol-binding protein 2 gene (RBP2) to human chromosome band 3q23 by in situ hybridization.

The human cellular retinol-binding protein 2 (RBP2) is responsible for the uptake and/or intracellular transport of vitamin A in the small intestinal epithelium. Demmer et al. (1987) mapped the RBP2 gene to mouse chromosome 9 closely linked (within 3 cM) to the highly homologous cellular retinol-binding protein 1 (RBP1). They mapped both genes on human chromosome 3 by using mouse-human somatic cell hybrids. Here we report a refined mapping of RBP2 to human chromosome band 3q23. We mapped the RBP2 coding sequence on 2 partially overlapping PACs (300-F 16 and 204-O 7; Fig. 1C) which were recently found to contain respectively RBP1 and the coatomer beta) subunit gene (COPB2) (De Baere et al., 1998). This finding confirms the hypothesis of the close linkage of RBP1 and RBP2 in humans (Demmer et al., 1987) and moreover, provides evidence that these genes are tightly linked in an interval of less than 100 kb. Based upon its function and the mapping data, the RBP2 gene can be considered as a putative candidate gene for the blepharophimosis syndrome (BPES) which has been mapped to 3q23 by cytogenetic analyses (Fukushima et al., 1991), radiation hybrid mapping (Lawson et al., 1995), linkage analyses (Amati et al., 1996; Messiaen et al., 1996) and physical mapping (Piemontese et al., 1997).

Assignment of the cellular retinol-binding protein 1 gene (RBP1) and of the coatomer beta subunit gene (COPB2) to human chromosome band 3q23 by in situ hybridization

The human cellular retinol-binding protein 1 (RBP1) is responsible for the intracellular transport of vitamin A to the final site of action. Rocchi et al. (1989) mapped RBP1 to chromosome 3q21→q22 using radioactive in situ hybridization. Based upon its function and the mapping data, the RBP1 gene has been considered as a putative candidate gene for the blepharophimosis syndrome (BPES) (Messiaen et al., 1996) which has been mapped to 3q23 by cytogenetic analyses (Fukushima et al., 1991), radiation hybrid mapping (Lawson et al., 1995), linkage analyses (Messiaen et al., 1996) and physical mapping (Piemontese et al., 1997). Here we report a more precise assignment of RBP1 to 3q23 using FISH and Southern blot analysis. FISH experiments were performed on metaphase chromosomes of a BPES patient carrying a balanced t(3;4)(q23;p15.2) translocation (Fukushima et al., 1991) using PAC 300-F 16 containing the coding sequence of this gene. The results provided strong evidence for the exclusion of RBP1 as a candidate gene for BPES (unpublished data). The coatomer beta) subunit gene (COBP2) is a beta) subunit of the Golgi coatomer complex, that constitutes the coat of nonclathrin coated vesicles (Harrison-Lavoie et al., 1993). COBP2 has previously been assigned by radiation hybrid mapping to human chromosome 3, between D3S1615–D3S1316 by Généthon, between D3S3528–D3S1316 by SHGC and between D3S1576–D3S1309 by WICGR (Schuler et al., 1996). In this report we map COBP2 to 3q23. Based upon its chromosome position, it can be considered as a candidate gene for BPES.

Assignment of SHOX2 (alias OG12X and SHOT) to human chromosome bands 3q25 -> q26.1 by in situ hybridization.

Human SHOX2 (alias OG12X and SHOT) is a recently identified homeobox gene of which the mouse homologue is expressed during heart and craniofacial development (Semina et al., 1998; Blaschke et al., 1998). It was mapped to human chromosome 3q22→q26 by radiation hybrid mapping (Semina et al., 1998) and to 3q25→q26 by FISH (Blaschke et al., 1998). Based upon the mapping to 3q22→q26 and the expression pattern of its mouse cognate, Semina et al. (1998) considered it as a candidate gene for the blepharophimosis syndrome (BPES) and the Cornelia de Lange syndrome, both mapped to this region. Here we report the mapping of SHOX2 to human chromosome band 3q25→q26.1. This finding is consistent with the mapping data of Blaschke et al. (1998) and allows us to exclude SHOX2 as a candidate gene for BPES which has been mapped to 3q23 by cytogenetic analyses (Fukushima et al., 1991), radiation hybrid mapping (Lawson et al., 1995), linkage analyses (Messiaen et al., 1996) and physical mapping (Piemontese et al., 1997).