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Biomedical subjects

H H Ropers

Publications and source records attributed to H H Ropers.

At least 19 recordsLinked to original sources
PubMed2005

Screening of ARX in mental retardation families: Consequences for the strategy of molecular diagnosis.

Mutations in the human ARX gene have been shown to cause nonsyndromic X-linked mental retardation (MRX) as well as syndromic forms such as X-linked lissencephaly with abnormal genitalia (XLAG), Partington syndrome and X-linked infantile spasm. The most common causative mutation, a duplication of 24 bp, was found in families with a variety of phenotypes, but not in the more severe XLAG phenotypes. The aim of the study was to access the frequency of ARX mutations in families with established or putative X-linked mental retardation (XLMR) collected by the European XLMR Consortium. We screened the entire coding region of ARX for mutations in 197 novel XLMR families by denaturing high-performance liquid chromatography, and we identified eight mutations (six c.428_451dup24, one insertion and one novel missense mutation p.P38S). To better define the prevalence of ARX mutations, we included previously reported results of 157 XLMR families. Together, these data showed the relatively high rate (9.5%) of ARX mutations in X-linked MR families and an expectedly low rate in families with affected brother pairs (2.2%). This study confirms that the frequency of ARX mutations is high in XLMR, and the analysis of ARX in MRX should not be limited to duplication.

Amino Acid Sequence↗
PubMed2002

A cryptic deletion of 2q35 including part of the PAX3 gene detected by breakpoint mapping in a child with autism and a de novo 2;8 translocation.

We report a de novo, apparently balanced (2;8)(q35;q21.2) translocation in a boy with developmental delay and autism. Cross species (colour) paint (Rx) and SKY FISH, forward and reverse chromosome painting, and FISH with subtelomeric probes were used to examine the patient's karyotype, but further rearrangements were not detected. FISH with region specific clones mapping near 2q35 and 8q21.2 breakpoints and STS mapping performed on the isolated derivative chromosomes were used to refine the location of the breakpoints further. A cryptic deletion of between 4.23 and 4.41 Mb in extent and involving at least 13 complete genes or transcription units was found at the breakpoint on 2q35. The deletion includes the promoter and 5' untranslated region of the paired box 3 (PAX3) gene. The child has very mild dystopia canthorum which may be associated with the PAX3 haploinsufficiency. The 8q21.2 breakpoint is within MMP16 which encodes matrix metalloproteinase 16. We postulate that the cryptic deletion and rearrangement are responsible for the patient's phenotype and that a gene (or genes) responsible for autism lies at 2q35 or 8q21.2. The results present a step towards identifying genes predisposing to autism.

Autistic Disorder↗
PubMed2001

UniGene cDNA array-based monitoring of transcriptome changes during mouse placental development.

The placenta is a highly specialized organ essential for embryonic growth and development. Here, we have applied cDNA subtraction between extraembryonic tissues of early- (day 7.5 of gestation) and late-stage embryos (day 17.5) to generate stage-specific cDNA pools that were used for screening of high-density mouse UniGene cDNA arrays containing 25,000 clones. A total of 638 clones were identified, 488 with the e7.5-specific probe and 150 with the e17.5-specific probe. Importantly, 363/638 (56.9%) of the hybridizing clones were not known to be expressed during placental development before. Differential regulation was confirmed by Northern blot and in situ hybridization for a total of 44/44 of positive clones. Thus, this combination of cDNA subtraction and array hybridization was highly successful for identification of genes expressed and regulated during placental development. These included growth factors and receptors, components of the transcriptional and translational machinery, cell cycle regulators, molecular chaperones, and cytoskeletal elements. The extensive in situ hybridization analysis revealed extraembryonic structures with a high density of differentially expressed genes, most strikingly the ectoplacental cone and the spongiotrophoblast. This large-scale identification of genes regulated during placentogenesis is extremely useful to further elucidate the molecular basis of extraembryonic development.

Animals↗
PubMed2001

MECP2 is highly mutated in X-linked mental retardation.

Following the recent discovery that the methyl-CpG binding protein 2 (MECP2) gene located on Xq28 is involved in Rett syndrome (RTT), a wild spectrum of phenotypes, including mental handicap, has been shown to be associated with mutations in MECP2. These findings, with the compelling genetic evidence suggesting the presence in Xq28 of additional genes besides RabGDI1 and FMR2 involved in non-specific X-linked mental retardation (MRX), prompted us to investigate MECP2 in MRX families. Two novel mutations, not found in RTT, were identified. The first mutation, an E137G, was identified in the MRX16 family, and the second, R167W, was identified in a new mental retardation (MR) family shown to be linked to Xq28. In view of these data, we screened MECP2 in a cohort of 185 patients found negative for the expansions across the FRAXA CGG repeat and reported the identification of mutations in four sporadic cases of MR. One of the mutations, A140V, which we found in two patients, has been described previously, whereas the two others, P399L and R453Q, are novel mutations. In addition to the results demonstrating the involvement of MECP2 in MRX, this study shows that the frequency of mutations in MECP2 in the mentally retarded population screened for the fragile X syndrome is comparable to the frequency of the CGG expansions in FMR1. Therefore, implementation of systematic screening of MECP2 in MR patients should result in significant progress in the field of molecular diagnosis and genetic counseling of mental handicap.

Adult↗
PubMed2001

Functional hemizygosity of PAFAH1B3 due to a PAFAH1B3-CLK2 fusion gene in a female with mental retardation, ataxia and atrophy of the brain.

We report on the molecular characterization of a translocation t(1;19)(q21.3;q13.2) in a female with mental retardation, ataxia and atrophy of the brain. Sequence analysis of the breakpoints revealed an ALU:-repeat-mediated mechanism of recombination that led to truncation of two genes: the kinase CLK2 and PAFAH1B3, the gene product of which interacts with LIS1 as part of a heterotrimeric G protein complex PAF-AH1B. In addition, two reciprocal fusion genes are present. One expressed fusion gene encodes the first 136 amino acids of PAFAH1B3 followed by the complete CLK2 protein. Truncated PAFAH1B3 protein lost its potential to interact with LIS1 whereas CLK2 activity was conserved within the fusion protein. These data emphasize the importance of PAF-AH1B in brain development and functioning and demonstrate the first fusion gene apparently not associated with cancer.

Abnormalities, Multiple↗
PubMed2001

Detection of mitochondrial single nucleotide polymorphisms using a primer elongation reaction on oligonucleotide microarrays.

We have developed a novel allele-specific primer elongation protocol using a DNA polymerase on oligonucleotide chips. Oligonucleotide primers carrying polymorphic sites at their free 3'end were covalently bound to glass slides. The generation of single-stranded targets of genomic DNA containing single nuclotide polymorphisms (SNPs) to be typed was achieved by an asymmetric PCR reaction or exonuclease treatment of phosphothioate (PTO)-modified PCR products. In the presence of DNA polymerase and all four dNTPs, with Cy3-dUTP replacing dTTP, allele-specific extension of the immobilized primers took place along a stretch of target DNA sequence. The yield of elongated products was increased by repeated reaction cycles. We performed multiplexed assays with many small DNA targets, or used single targets of up to 4.4 kb mitochondrial DNA (mtDNA) sequence to detect multiple SNPs in one reaction. The latter approach greatly simplifies preamplification of SNP-containing regions, thereby providing a framework for typing hundreds of mtDNA polymorphisms.

Alleles↗
PubMed2001

A translocation breakpoint cluster disrupts the newly defined 3' end of the SNURF-SNRPN transcription unit on chromosome 15.

Balanced translocations affecting the paternal copy of 15q11--q13 are a rare cause of Prader-Willi syndrome (PWS) or PWS-like features. Here we report on the cytogenetic and molecular characterization of a de novo balanced reciprocal translocation t(X;15)(q28;q12) in a female patient with atypical PWS. The translocation breakpoints in this patient and two previously reported patients map 70-80 kb distal to the SNURF-SNRPN gene and define a breakpoint cluster region. The breakpoints disrupt one of several hitherto unknown 3' exons of this gene. Using RT--PCR we demonstrate that sequences distal to the breakpoint, including the recently identified C/D box small nucleolar RNA (snoRNA) gene cluster HBII-85 as well as IPW and PAR1, are not expressed in the patient. Our data suggest that lack of expression of these sequences contributes to the PWS phenotype.

Adult↗
PubMed2001

X chromosome-specific cDNA arrays: identification of genes that escape from X-inactivation and other applications.

Mutant alleles are frequently characterized by low expression levels. Therefore, cDNA array-based gene expression profiling may be a promising strategy for identifying gene defects underlying monogenic disorders. To study the potential of this approach, we have generated an X chromosome-specific microarray carrying 2423 cloned cDNA fragments, which represent up to 1317 different X-chromosomal genes. As a prelude to testing cell lines from patients with X-linked disorders, this array was used as a hybridization probe to compare gene expression profiles in lymphoblastoid cell lines from normal males, females and individuals with supernumerary X chromosomes. Measurable hybridization signals were obtained for more than half of the genes represented on the chip. A total of 53 genes showed elevated expression levels in cells with multiple X chromosomes and many of these were found to escape X-inactivation. Moreover, the detection of a male-viable deletion encompassing three genes illustrates the utility of this array for the identification of small unbalanced chromosome rearrangements.

Alleles↗
PubMed2001

Genomic organization and expression of the doublesex-related gene cluster in vertebrates and detection of putative regulatory regions for DMRT1.

Genes related to the Drosophila melanogaster doublesex and Caenorhabditis elegans mab-3 genes are conserved in human. They are identified by a DNA-binding homology motif, the DM domain, and constitute a gene family (DMRTs). Unlike the invertebrate genes, whose role in the sex-determination process is essentially understood, the function of the different vertebrate DMRT genes is not as clear. Evidence has accumulated for the involvement of DMRT1 in male sex determination and differentiation. DMRT2 (known as terra in zebrafish) seems to be a critical factor for somitogenesis. To contribute to a better understanding of the function of this important gene family, we have analyzed DMRT1, DMRT2, and DMRT3 from the genome model organism Fugu rubripes and the medakafish, a complementary model organism for genetics and functional studies. We found conservation of synteny of human chromosome 9 in F. rubripes and an identical gene cluster organization of the DMRTs in both fish. Although expression analysis and gene linkage mapping in medaka exclude a function for any of the three genes in the primary step of male sex determination, comparison of F. rubripes and human sequences uncovered three putative regulatory regions that might have a role in more downstream events of sex determination and human XY sex reversal.

Amino Acid Sequence↗
PubMed2001

DNA sequence comparison of human and mouse retinitis pigmentosa GTPase regulator (RPGR) identifies tissue-specific exons and putative regulatory elements.

Retinitis pigmentosa 3 (RP3) is a progressive retinal degeneration due to mutations in the X-linked RPGR gene. Transcription studies in human and mouse tissues have revealed ubiquitously expressed transcripts and also an exceptional high number of tissue-specific alternative splice variants. However, regulation of tissue-specific expression and splicing is unclear, but this is of particular interest as mutations in this ubiquitously expressed gene lead to severe retinal degeneration, while other tissues are unaffected. To elucidate the conservation pattern of RPGR and to identify additional tissue-specific exons and putative regulatory elements we performed comparative genomic sequencing of the human and mouse RPGR gene. Each of the genes spans a region of nearly 59 kb, and all previously identified exons are conserved between the two species. DNA sequence comparison identified 28 conserved sequence elements (CSEs) in introns, upstream of exon 1, within the promotor region, and downstream of the most 3' exon. Some of the intronic CSEs flank tissue-specific exons and therefore may represent important regulatory elements for alternative splicing. Comparative northern blot hybridization of ubiquitous and tissue-specific RPGR probes identified high molecular weight transcripts with similar expression patterns in both human and mouse. These transcripts range from 6 to 15 kb in size and suggest the presence of additional transcribed sequences within RPGR. Our cross-species sequence comparison enables us to define candidate regions that may explain these large transcripts and will therefore contribute to the understanding of RPGR expression and splicing.

Alternative Splicing↗
PubMed2001

MID1, mutated in Opitz syndrome, encodes an ubiquitin ligase that targets phosphatase 2A for degradation.

The gene MID1, the mutation of which causes X-linked Opitz G/BBB syndrome (OS, MIM 300000), encodes a microtubule-associated protein (MAP). We show that mutation of MID1 leads to a marked accumulation of the catalytic subunit of protein phosphatase 2A (PP2Ac), a central cellular regulator. PP2Ac accumulation is caused by an impairment of a newly identified E3 ubiquitin ligase activity of the MID1 protein that normally targets PP2Ac for degradation through binding to its alpha4 regulatory subunit in an embryonic fibroblast line derived from a fetus with OS. Elevated PP2Ac causes hypophosphorylation of MAPs, a pathological mechanism that is consistent with the OS phenotype.

Animals↗
PubMed2000

The retinitis pigmentosa GTPase regulator (RPGR) interacts with novel transport-like proteins in the outer segments of rod photoreceptors.

Mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene cause X-linked retinitis pigmentosa type 3 (RP3), a severe, progressive and degenerative retinal dystrophy eventually leading to complete blindness. RPGR is ubiquitously expressed, yet mutations in the RPGR gene lead to a retina-restricted phenotype. To date, all RP3 associated missense mutations that have been identified are located in the RCC1-homologous domain (RHD) of RPGR. To investigate the molecular pathogenesis of RP3, we screened retinal yeast two-hybrid libraries with the RHD of RPGR. We identified several alternatively spliced gene products, some with retina-restricted expression, that interact specifically with RPGR in vivo and in vitro. Thus, these proteins were named RPGR-interacting protein 1 (RPGRIP1) isoforms. They contain a C-terminal RPGR-interacting domain and stretches of variable coiled-coil domains homologous to proteins involved in vesicular trafficking. The interaction between RPGR and RPGRIP1 isoforms was impaired in vivo by RP3-associated mutations in RPGR. Moreover, RPGR and RPGRIP1 co-localize in the outer segment of rod photoreceptors, which is in full agreement with the retinitis pigmentosa phenotype observed in RP3 patients. The localization of RPGRIP1 at 14q11 makes it a strong candidate gene for RP16. These results provide a clue for the retina-specific pathogenesis in RP3, and hint towards the involvement of RPGR and RPGRIP1 in mediating vesicular transport-associated processes.

3' Untranslated Regions↗