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R Willemsen

Publications and source records attributed to R Willemsen.

At least 55 records · Page 3Linked to original sources
PubMed1997

Characterization of the full fragile X syndrome mutation in fetal gametes.

Fragile X syndrome results from the expansion of the CGG repeat in the FMR1 gene. Expansion has been suggested to be a postzygotic event with the germline protected. From an analysis of intact ovaries of full mutation fetuses, we now show that only full expansion alleles can be detected in oocytes (but in the unmethylated state). Similarly, the testes of a 13-week full mutation fetus show no evidence of premutations while a 17-week full mutation fetus exhibits some germ cells with attributes of premutations. These data discount the hypothesis that the germline is protected from full expansion and suggest full mutation contraction in the immature testis. Thus, full expansion may already exist in the maternal oocyte, or postzygotic expansion, if it occurs, arises quite early in development prior to germline segregation.

DNA Methylation↗
PubMed1997

Differential expression of FMR1, FXR1 and FXR2 proteins in human brain and testis.

Lack of expression of the fragile X mental retardation protein (FMRP) results in mental retardation and macroorchidism, seen as the major pathological symptoms in fragile X patients. FMRP is a cytoplasmic RNA-binding protein which cosediments with the 60S ribosomal subunit. Recently, two proteins homologous to FMRP were discovered: FXR1 and FXR2. These novel proteins interact with FMRP and with each other and they are also associated with the 60S ribosomal subunit. Here, we studied the expression pattern of the three proteins in brain and testis by immunohistochemistry. In adult brain, FMR1, FXR1 and FXR2 proteins are coexpressed in the cytoplasm of specific differentiated neurons only. However, we observed a different expression pattern in fetal brain as well as in adult and fetal testis, suggesting independent functions for the three proteins in those tissues during embryonic development and adult life.

Adult↗
PubMed1997

Prenatal diagnosis of the fragile X syndrome: loss of mutation owing to a double recombinant or gene conversion event at the FMR1 locus.

The fragile X syndrome, an X linked mental retardation syndrome, is caused by an expanded CGG repeat in the first exon of the FMR1 gene. In patients with an expanded repeat the FMR1 promoter is methylated and, consequently, the gene is silenced and no FMR1 protein (FMRP) is produced, thus leading to the clinical phenotype. Here we describe a prenatal diagnosis performed in a female from a fragile X family carrying a large premutation. In chorionic villus DNA of the male fetus the normal maternal CGG allele and a normal pattern on Southern blot analysis were found in combination with the FRAXAC2 and DXS297 allele of the maternal at risk haplotype. A second chorionic villus sampling was performed giving identical results on DNA analysis and, in addition, expression of FMRP was shown by immunohistochemistry. We concluded that the male fetus was not affected with the fragile X syndrome. Subsequent detailed haplotype analysis showed a complex recombination pattern resembling either gene conversion or a double crossover within a 20 kb genomic region.

Alleles↗
PubMed1997

Rapid antibody test for prenatal diagnosis of fragile X syndrome on amniotic fluid cells: a new appraisal.

Fragile X syndrome is caused by mutations in the FMR1 gene and is one of the most frequent forms of inherited mental retardation in males. Postnatal and prenatal diagnosis of fragile X syndrome is feasible by direct DNA analysis. A new approach to prenatal diagnosis of fragile X syndrome in amniotic fluid cells is described, using a rapid and simple antibody test on uncultured amniotic fluid cells. The test requires 1 ml of amniotic fluid and the results of this antibody test are available on the same day as the amniocentesis.

Amniocentesis↗
PubMed1996

The fragile X phenotype in a mosaic male with a deletion showing expression of the FMR1 protein in 28% of the cells.

The instability of the CGG repeat region of FMR1 is not restricted to the CGG repeat but expands to flanking sequences as well. A mosaic fragile X male is reported with a deletion of part of the CGG repeat and 30 bp immediately 3' of the repeat, thus confirming the presence of a hotspot for deletions in the CGG region of FMR1. The deletion, detected in 28% of his lymphocytes, did not impair the transcription and translation of FMR1, suggesting that regulatory elements are not present in the deleted region. The patient has the characteristic fragile X phenotype and assuming that the mosaic pattern detected in the lymphocytes reflects the mosaic pattern in brain, 28% expression of FMRP may not be sufficient for normal cognitive functioning.

Aged↗
PubMed1996

Association of FMRP with ribosomal precursor particles in the nucleolus.

The fragile X syndrome, one of the most common forms of inherited mental retardation, is caused by an expansion of a polymorphic CGG repeat upstream the coding region of the FMR1 gene. These expansions are associated with hypermethylation of the FMR1 gene, which results in the absence of the gene product, the FMR1 protein (FMRP). The physiological function of FMRP remains to be determined. We studied the ultrastructural localization of FMRP at the electron microscopical level using the immunogold technique. FMRP is associated with ribosomes attached to the endoplasmic reticulum and with ribosomes free in the cytoplasm. In addition, FMRP is found in the nucleus where the protein is associated with the granular component of the nucleolus. The cellular function of FMRP is hypothesized in relation to its subcellular distribution.

Animals↗
PubMed1996

Expression of the ubiquitin-conjugating DNA repair enzymes HHR6A and B suggests a role in spermatogenesis and chromatin modification.

RAD6, a member of the expanding family of ubiquitin-conjugating (E2) enzymes, functions in the so-called "N-rule" protein breakdown pathway of Saccharomyces cerevisiae. In vitro, the protein can attach one or multiple ubiquitin (Ub) moieties to histones H2A and B and trigger their E3-dependent degradation. Rad6 mutants display a remarkably pleiotropic phenotype, implicating the protein in DNA damage-induced mutagenesis, postreplication repair, repression of retrotransposition, and sporulation. RAD6 transcription is strongly induced upon UV exposure and in meiosis, suggesting that it is part of a damage-induced response pathway and that it is involved in meiotic recombination. It is postulated that the protein exerts its functions by modulating chromatin structure. Previously, we have cloned two human homologs of this gene (designated HHR6A and HHR6B) and demonstrated that they partially complement the yeast defect. Here we present a detailed characterisation of their expression at the transcript and protein levels. Both HHR6 proteins, resolved by 2-dimensional immunoblot analysis, are expressed in all mammalian tissues and cell types examined, indicating that both genes are functional and constitutively expressed. Although the proteins are highly conserved, the UV induction present in yeast is not preserved, pointing to important differences in damage response between yeast and mammals. Absence of alterations in HHR6 transcripts or protein upon heat shock and during the cell cycle suggests that the proteins are not involved in stress response or cell cycle regulation. Elevated levels of HHR6 transcripts and proteins were found in testis. Enhanced HHR6 expression did not coincide with meiotic recombination but with the replacement of histones by transition proteins. Immunohistochemistry demonstrated that the HHR6 proteins are located in the nucleus, consistent with a functional link with chromatin. Electron microscopy combined with immunogold labeling revealed a preferential localisation of HHR6 in euchromatin areas, suggesting that the protein is associated with transcriptionally active regions. Our findings support the idea that both HHR6 genes have overlapping, constitutive functions related to chromatin conformation and that they have a specific role in spermatogenesis, involving Ub-mediated histone degradation.

Animals↗
PubMed1996

Molecular and biochemical analysis of protective protein/cathepsin A mutations: correlation with clinical severity in galactosialidosis.

Mutations in the gene encoding lysosomal protective protein/cathepsin A (PPCA) are the cause of the lysosomal disorder galactosialidosis (GS). Depending on age of onset and severity of the symptoms, patients present with either an early infantile (EI), a late infantile (LI), or a juvenile/adult (J/A) form of the disease. To study genotype-phenotype correlation in this disorder, we have analyzed the mutations in the PPCA gene of eight clinically different patients. In two EI and one J/A patient, we have identified four novel point mutations (Val104Met, Leu208Pro, Gly411Ser and Ser23Tyr), that prevent phosphorylation and, hence, lysosomal localization and maturation of the mutant precursors. Two amino acid substitutions (Phe412Val and Tyr221Asn) are shared by five LI patients. These mutations appear to be pathognomonic for this phenotype, and determine the clinical outcome depending on whether they are present together or in combination with other mutations. The latter include a single base deletion and a novel amino acid change (Met378Thr), which generates an additional glycosylation site. Within the LI group, patients carrying the Phe412Val mutation are clinically more severe than those with the Tyr221Asn substitution. This is in agreement with the biochemical behavior of the Asn221-mutant protein, that is, like the Phe412Val protein, phosphorylated, routed to lysosomes and proteolytically processed, but its intralysosomal stability is intermediate between that of wild-type PPCA and Val412-PPCA. Overall, these results may explain the clinical heterogeneity observed in GS patients and may help to correlate mutant allelic combinations with specific clinical phenotypes.

Alleles↗
PubMed1996

Variable FMR1 gene methylation of large expansions leads to variable phenotype in three males from one fragile X family.

The fragile X syndrome is caused by an expanded CGG repeat (> 200 units, full mutation) at the 5' end of the FMR1 gene, which is associated with methylation of a CpG island upstream of the FMR1 gene and down regulation of the transcription. We describe three related males with full mutations in the FMR1 gene, as defined by size, but with different percentages of unmethylated alleles (+/-90%, 35%, and 15%, respectively) as studied in leucocytes. Normal mental status was observed in the male who showed 90% lack of methylation, whereas his two cousins were retarded. The mentally normal male did show some minor facial features of the fragile X syndrome; the FMR protein was detectable in 75% of his leucocytes. In all three cases, the proportion of unmethylated FMR1 genes corresponded to the percentage of leucocytes showing FMR1 protein production. Our results indicated a direct relationship between methylation and the ability to produce FMR protein. These cases will be discussed in relation to the phenotypic effects of incompletely methylated full mutations in the FMR1 gene as observed by others.

Adult↗
PubMed1996

Human alpha-N-acetylgalactosaminidase (alpha-NAGA) deficiency: new mutations and the paradox between genotype and phenotype.

Up to now eight patients with alpha-NAGA deficiency have been described. This includes the newly identified patient reported here who died unexpectedly aged 1 1/2 years of hypoxia during convulsions; necropsy was not performed. Three patients have been genotyped previously and here we report the mutations in the other five patients, including two new mutations (S160C and E193X). The newly identified patient is consanguineous with the first patients reported with alpha-NAGA deficiency and neuroaxonal dystrophy and they all had the alpha-NAGA genotype E325K/E325K. Clinical heterogeneity among patients with alpha-NAGA deficiency is extreme. Two affected sibs, homozygotes for E325K, are severely affected and have the signs and symptoms of infantile neuroaxonal dystrophy, but prominent vacuolisation is lacking. The mildly affected patients (two families, three patients) at the opposite end of the clinical spectrum have clear vacuolisation and angiokeratoma but no overt neurological manifestations. Two of them are homozygous for the stop mutation E193X, leading to complete loss of alpha-NAGA protein. These observations are difficult to reconcile with a simple genotype-phenotype correlation and we suggest that factors or genes other than alpha-NAGA contribute to the clinical heterogeneity of the eight patients with alpha-NAGA deficiency. At the metabolic level, the patients with alpha-NAGA deficiency are similar. The major abnormal urinary oligosaccharides are sialylglycopeptides of the O linked type. Our enzymatic studies indicated that these compounds are not the primary lysosomal storage products.

Animals↗
PubMed1995

Lysosomal protective protein/cathepsin A. Role of the "linker" domain in catalytic activation.

Lysosomal protective protein/cathepsin A is a serine carboxypeptidase that forms a complex with beta-galactosidase and neuraminidase. The enzyme is synthesized as a 54-kDa precursor/zymogen and processed into a catalytically active 32- and 20-kDa two-chain form. We have expressed in baculovirus-infected insect cells the human one-chain precursor as well as the two separate subunits in order to establish the mode of catalytic activation of the zymogen and the assembly and activation of the two subunits. Infected insect cells synthesize large quantities of the exogenous proteins, which are glycosylated and secreted but not processed. Co-expression of the two subunits results in their assembly into a two-chain form of 34- and 20-kDa with negligible enzymatic activity. Limited proteolysis with trypsin of the 54-kDa precursor and the reconstituted 34- and 20-kDa form gives rise to a fully active 32- and 20-kDa product. These results enabled us to map the sites of proteolytic cleavage needed for full activation of the cathepsin A zymogen. They further indicate that the 34- and 20-kDa form is a transient processing intermediate that is converted into a mature and active enzyme by removal of a 2-kDa "linker" peptide from the COOH terminus of the 34-kDa subunit.

Amino Acid Sequence↗
PubMed1995

Rapid antibody test for fragile X syndrome.

Fragile X syndrome is the most common known cause of inherited mental retardation. Identification of patients and carriers of fragile X syndrome is usually done with a DNA test system but we have developed a rapid antibody to identify fragile X patients. This non-invasive test requires only 1 or 2 drops of blood and can be used for screening large groups of mentally retarded people and neonates for fragile X syndrome.

Antibodies, Monoclonal↗
PubMed1995

Normal phenotype in two brothers with a full FMR1 mutation.

The fragile X syndrome is associated with an expanding CGG repeat in the 5' untranslated region of the first exon of the FMR1 gene. Subsequent methylation of the promoter region inhibits expression of the FMR1 gene. In two clinically normal brothers large, expanded CGG repeats and cytogenetically visible fragile sites were found. The FMR1 promoter was unmethylated and both RNA and protein could be detected. This indicates that inactivation of the FMR1 gene and not repeat expansion itself results in the fragile X phenotype. We conclude that repeat expansion does not necessarily induce methylation and that methylation is no absolute requirement for the induction of fragile sites.

Adolescent↗
PubMed1995

Characterization of FMR1 proteins isolated from different tissues.

FMR1 protein expression was studied in different tissues. In human, monkey and murine tissues, high molecular mass FMR1 proteins (67-80 kDa) are found, as shown in lymphoblastoid cells lines. The identity of these proteins was confirmed by their absence in tissues from patients with the fragile X syndrome and a FMR1 knock-out mouse. An Ile367Asn substitution in the FMR1 protein did not alter the translation, processing and localization of FMR1 proteins in lymphoblastoid cells from a patient carrying this mutation. All the high molecular mass FMR1 proteins isolated from normal lymphoblastoid cells and cells from the patient with the Ile367Asn substitution were able to bind RNA. However, the FMR1 proteins of the patient had reduced affinity for RNA binding at high salt concentrations. In some human, monkey and murine tissues low molecular mass FMR1 proteins (39-41 kDa) were found, which had the same N terminus as the 67-90 kDa isoforms, but differ in their C terminus and are therefore most likely the result of carboxy-terminal proteolytic cleavage. These low molecular mass FMR1 proteins did not bind RNA, in contrast with the high molecular mass FMR1 proteins. The significance of these low molecular mass proteins remains to be studied.

Animals↗
PubMed1995

Instability of the CGG repeat and expression of the FMR1 protein in a male fragile X patient with a lung tumor.

The molecular mechanism of the fragile X syndrome is based on the expansion of an CGG repeat in the 5' UTR of the FMR1 gene in the majority of fragile X patients. This repeat displays instability both between individuals and within an individual. We studied the instability of the CGG repeat and the expression of the FMR1 protein (FMRP) in several different tissues derived from a male fragile X patient. Using Southern blot analysis, only a full mutation is detected in 9 of the 11 tissues tested. The lung tumor contains a methylated premutation of 160 repeats, whereas in the testis, besides the full mutation, a premutation of 60 CGG repeats is detected. Immunohistochemistry of the testis revealed expression of FMR1 in the spermatogonia only, confirming the previous finding that, in the sperm cells of fragile X patients with a full mutation in their blood cells, only a premutation is present. Immunohistochemistry of brain and lung tissue revealed that 1% of the cells are expressing the FMRP. PCR analysis demonstrated the presence of a premutation of 160 repeats in these FMR1-expressing cells. This indicates that the tumor was derived from a lung cell containing a premutation. Remarkably, despite the methylation of the EagI and BssHII sites, FMRP expression is detected in the tumor. Methylation of both restriction sites has thus far resulted in a 100% correlation with the lack of FMR1 expression, but the results found in the tumor suggest that the CpGs in these restriction sites are not essential for regulation of FMR1 expression. This indicates a need for a more accurate study of the exact promoter of FMR1.

Base Sequence↗
PubMed1995

A biochemical and immunocytochemical study on the targeting of alglucerase in murine liver.

A current hypothesis is that functional glucocerebrosidase needs to be delivered to the lysosomes of tissue macrophages to guarantee successful enzyme therapy for Gaucher's disease. In this study, biochemical and immunohistochemical techniques were applied to identify in mice the localization of intravenously administered alglucerase (human modified placental glucocerebrosidase). Only in liver and spleen was a significant increase of glucocerebrosidase activity observed, with a maximum level at 15 minutes after enzyme infusion. The uptake of enzyme by liver was sufficiently high to allow more detailed studies on the (sub)cellular distribution of human alglucerase. The enzyme in liver is localized both in the endosomal-lysosomal system of the Kupffer cells and the endothelial cells lining the lumen of the sinusoids. Uptake by both of these types of cell is prevented by mannan. The results suggest that the cellular mechanisms responsible for improvement of Gaucher patients receiving alglucerase treatment is probably more complicated than previously recognized.

Animals↗
PubMed1994

The ER-TR4 monoclonal antibody recognizes murine thymic epithelial cells (type 1) and inhibits their capacity to interact with immature thymocytes: immuno-electron microscopic and functional studies.

The thymic stroma is heterogeneous with regard to cellular morphology and cellular function. In this study, we employed the monoclonal antibody ER-TR4 to characterize stromal cells at the ultrastructural level. To identify the labelled cell type, we used two techniques: immunogold labelling on ultrathin frozen sections and immunoperoxidase staining on thick "vibratome" sections. ER-TR4 reacted with thymic Type 1 epithelial cells (according to our classification). A dense labelling appears in the cytoplasm of cortical cells using the two techniques. Immunogold labelling identified small cytoplasmic vesicles whereas the cytoplasm and the cell membrane seem to be labelled with the immunoperoxidase technique. ER-TR4 also identified isolated thymic nurse cells (TNC), and was observed in vitro to inhibit the capacity of some type 1 epithelial cells to establish interactions with immature thymocytes. This finding supports the hypothesis that the factor is involved in the formation of lymphoepithelial interactions within thymic nurse cells, and thus in the relations that immature thymocytes establish with the thymic microenvironment.

Animals↗