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B Horsthemke

Publications and source records attributed to B Horsthemke.

At least 37 records · Page 2Linked to original sources
PubMed2000

De novo deletions of SNRPN exon 1 in early human and mouse embryos result in a paternal to maternal imprint switch.

Prader-Willi syndrome (PWS) is a neurogenetic disease characterized by infantile hypotonia, gonadal hypoplasia, obsessive behaviour and neonatal feeding difficulties followed by hyperphagia, leading to profound obesity. PWS is due to a lack of paternal genetic information at 15q11-q13 (ref. 2). Five imprinted, paternally expressed genes map to the PWS region, MKRN3 (ref. 3), NDN (ref. 4), NDNL1 (ref. 5), SNRPN (refs 6-8 ) and IPW (ref. 9), as well as two poorly characterized framents designated PAR-1 and PAR-5 (ref. 10). Imprinting of this region involves a bipartite 'imprinting centre' (IC), which overlaps SNRPN (refs 10,11). Deletion of the SNRPN promoter/exon 1 region (the PWS IC element) appears to impair the establishment of the paternal imprint in the male germ line and leads to PWS. Here we report a PWS family in which the father is mosaic for an IC deletion on his paternal chromosome. The deletion chromosome has acquired a maternal methylation imprint in his somatic cells. We have made identical findings in chimaeric mice generated from two independent embryonic stem (ES) cell lines harbouring a similar deletion. Our studies demonstrate that the PWS IC element is not only required for the establishment of the paternal imprint, but also for its postzygotic maintenance.

Adult↗
PubMed1999

Detection of homozygous deletions in tumors by hybridization of representational difference analysis (RDA) products to chromosome-specific YAC clone arrays.

Representational difference analysis (RDA), a subtractive hybridization method that enriches differences between complex genomes, can be used to isolate fragments deleted in tumor genomes. Usually, most of the clones obtained by this approach result from polymorphic fragments. Therefore, identification of homozygously deleted fragments, which can indicate the presence of tumor suppressor loci, is often tedious. To overcome this limitation, we devised a novel strategy in which labeled RDA products are hybridized in toto against membranes spotted with YAC clones covering a region of interest. In such a way, identified YAC clones provide positional information on homozygous deletions and loss of heterozygosity (LOH) regions. We have tested this approach with a tumor known to have a homozygous deletion within a region of LOH on chromosome 13. RDA was performed using representations generated with restriction enzymes Bgl II, Nco I and Xba I, and the difference products of each experiment were separately hybridized to chromosome 13 YAC filters. When collating the map positions of positive YACs from three different RDA experiments a cluster of hits clearly identified the region on chromosome 13 which comprised the homozygous deletion. This shows that our novel approach can be effective.

Bacterial Proteins↗
PubMed1999

Human TRH-degrading ectoenzyme cDNA cloning, functional expression, genomic structure and chromosomal assignment.

Thyrotropin-Releasing Hormone (TRH) is an important extracellular signal substance that acts as a stimulator of hormone secretion from adenohypophyseal target cells and fulfills many criteria for the function of a neuromodulator/neurotransmitter within the central and peripheral nervous systems. The inactivation of TRH-signals is catalysed by a highly specific ectoenzyme. Here, we characterize the human TRH-degrading ectoenzyme (TRH-DE) by primary sequence, functional expression, genomic structure and chromosomal assignment. By screening a cDNA-library constructed from human lung, 5.7 kb of cDNA were identified. The longest open reading frame predicts a type II integral membrane protein of 117 kDa. The extracellular domain contains the HEXXH + E motif that is characteristic of a certain family of Zn-dependent aminopeptidases. Within this family, the sequences of human and rat TRH-DE reveal an unusual high degree of conservation (96% identical residues). Specific enzymatic activity was observed after transfecting COS-7 cells with human TRH-DE cDNA yielding a Km for TRH hydrolysis of 29.7 microM. Northern blot analysis demonstrated a restricted tissue distribution with highest transcript levels in the brain. Using fluorescent in situ hybridization with the cDNA and a genomic lambda clone, respectively, we localized the TRH-DE gene to the long arm of human chromosome 12. Five independent P1 artificial chromosome clones were required to span the complete cDNA sequence and revealed that it is distributed on 19 exons. Interspecies Southern analysis suggests that the gene is present as a single copy in human, monkey, rat, mouse, dog, bovine, rabbit and chicken DNA. All of these data further the notion that the TRH-DE is not an ordinary enzyme but a specific neuropeptidase that has been highly conserved among species.

Amino Acid Sequence↗
PubMed1999

Molecular characterization of radiation- and chemically induced mutations associated with neuromuscular tremors, runting, juvenile lethality, and sperm defects in jdf2 mice.

The juvenile development and fertility-2 (jdf2) locus, also called runty-jerky-sterile (rjs), was originally identified through complementation studies of radiation-induced p-locus mutations. Studies with a series of ethylnitrosourea (ENU)-induced jdf2 alleles later indicated that the pleiotropic effects of these mutations were probably caused by disruption of a single gene. Recent work has demonstrated that the jdf2 phenotype is associated with deletions and point mutations in Herc2, a gene encoding an exceptionally large guanine nucleotide exchange factor protein thought to play a role in vesicular trafficking. Here we describe the molecular characterization of a collection of radiation- and chemically induced jdf2/Herc2 alleles. Ten of the 13 radiation-induced jdf2 alleles we studied are deletions that remove specific portions of the Herc2 coding sequence; DNA rearrangements were also detected in two additional mutations. Our studies also revealed that Herc2 transcripts are rearranged, not expressed, or are present in significantly altered quantities in animals carrying most of the jdf2 mutations we analyzed, including six independent ENU-induced alleles. These data provide new molecular clues regarding the wide range of jdf2 and p phenotypes that are expressed by this collection of recently generated and classical p-region mutations.

Alleles↗
PubMed1999

The EIF3S3 gene encoding the p40 subunit of the translation initiation factor eIF3 has eight exons and maps to the Langer-Giedion syndrome chromosome region on 8q24, but is not the TRPS1 gene.

We have mapped the gene encoding the p40 subunit of the eukaryotic translation initiation factor eIF3 (EIF3S3) close to the distal border of the minimal critical region for tricho-rhino-phalangeal syndrome type I (TRPS I) on human chromosome 8q24. Because this location makes EIF3S3 a candidate for the TRPS1 gene, we have determined the genomic structure of the EIF3S3 gene and searched for gene deletions and mutations in patients with TRPS I. The gene has eight exons and is transcribed from telomere to centromere. No deletion could be detected in 32 unrelated patients with an apparently normal karyotype. Sequence analysis of all exons in 15 unrelated patients did not reveal any point mutation either. Our data exclude EIF3S3 as the TRPS1 gene.

Blotting, Northern↗
PubMed1999

Genes and chromosomal breakpoints in the Langer-Giedion syndrome region on human chromosome 8.

The tricho-rhino-phalangeal syndrome type II (TRPS II, or Langer-Giedion syndrome) is an example of contiguous gene syndromes, as it comprises the clinical features of two autosomal dominant diseases, TRPS I and a form of multiple cartilaginous exostoses caused by mutations in the EXT1 gene. We have constructed a contig of cosmid, lambda-phage, PAC, and YAC clones, which covers the entire TRPS I critical region. Using these clones we identified a novel submicroscopic deletion in a TRPS I patient and refined the proximal border of the minimal TRPS1 gene region by precisely mapping the inversion breakpoint of another patient. As a first step towards a complete inventory of genes in the Langer-Giedion syndrome chromosome region (LGCR) with the ultimate aim to identify the TRPS1 gene, we analyzed 23 human expressed sequence tags (ESTs) and four genes (EIF3S3, RAD21, OPG, CXIV) which had been assigned to human 8q24.1. Our analyses indicate that the LGCR is gene-poor, because none of the ESTs and genes map to the minimal TRPS1 gene region and only two of these genes, RAD21 and EIF3S3, are located within the shortest region of deletion overlap of TRPS II patients. Two genes, OPG and CXIV, which are deleted only in some patients with TRPS II may contribute to the clinical variability of this syndrome.

Child↗
PubMed1999

A 5-kb imprinting center deletion in a family with Angelman syndrome reduces the shortest region of deletion overlap to 880 bp.

Imprinting on human chromosome 15q11-q13 is controlled by a bipartite imprinting center (IC) that maps to the SNRPN locus. Deletions of the IC result in an imprinting defect and Prader-Willi syndrome or Angelman syndrome (AS). We have now identified a 5-kb IC deletion in an English AS patient (AS-LO); this represents the smallest microdeletion found in AS and narrows down the shortest region of deletion overlap to 880 bp.

Angelman Syndrome↗
PubMed1999

A previously unrecognised phenotype characterised by obesity, muscular hypotonia, and ability to speak in patients with Angelman syndrome caused by an imprinting defect.

The clinical features of Angelman syndrome (AS) comprise severe mental retardation, postnatal microcephaly, macrostomia and prognathia, absence of speech, ataxia, and a happy disposition. We report on seven patients who lack most of these features, but presented with obesity, muscular hypotonia and mild mental retardation. Based on the latter findings, the patients were initially suspected of having Prader-Willi syndrome. DNA methylation analysis of SNRPN and D15S63, however, revealed an AS pattern, ie the maternal band was faint or absent. Cytogenetic studies and microsatellite analysis demonstrated apparently normal chromosomes 15 of biparental inheritance. We conclude that these patients have an imprinting defect and a previously unrecognised form of AS. The mild phenotype may be explained by an incomplete imprinting defect or by cellular mosaicism.

Angelman Syndrome↗
PubMed1999

Molecular mechanism of angelman syndrome in two large families involves an imprinting mutation.

Patients with Angelman syndrome (AS) and Prader-Willi syndrome with mutations in the imprinting process have biparental inheritance but uniparental DNA methylation and gene expression throughout band 15q11-q13. In several of these patients, microdeletions upstream of the SNRPN gene have been identified, defining an imprinting center (IC) that has been hypothesized to control the imprint switch process in the female and male germlines. We have now identified two large families (AS-O and AS-F) segregating an AS imprinting mutation, including one family originally described in the first genetic linkage of AS to 15q11-q13. This demonstrates that this original linkage is for the 15q11-q13 IC. Affected patients in the AS families have either a 5.5- or a 15-kb microdeletion, one of which narrowed the shortest region of deletion overlap to 1.15 kb in all eight cases. This small region defines a component of the IC involved in AS (ie., the paternal-to-maternal switch element). The presence of an inherited imprinting mutation in multiple unaffected members of these two families, who are at risk for transmitting the mutation to affected children or children of their daughters, raises important genetic counseling issues.

Adolescent↗
PubMed1999

Imprinting-mutation mechanisms in Prader-Willi syndrome.

Microdeletions of a region termed the "imprinting center" (IC) in chromosome 15q11-q13 have been identified in several families with Prader-Willi syndrome (PWS) or Angelman syndrome who show epigenetic inheritance for this region that is consistent with a mutation in the imprinting process. The IC controls resetting of parental imprints in 15q11-q13 during gametogenesis. We have identified a larger series of cases of familial PWS, including one case with a deletion of only 7.5 kb, that narrows the PWS critical region to <4. 3 kb spanning the SNRPN gene CpG island and exon 1. Identification of a strong DNase I hypersensitive site, specific for the paternal allele, and six evolutionarily conserved (human-mouse) sequences that are potential transcription-factor binding sites is consistent with this region defining the SNRPN gene promoter. These findings suggest that promoter elements at SNRPN play a key role in the initiation of imprint switching during spermatogenesis. We also identified three patients with sporadic PWS who have an imprinting mutation (IM) and no detectable mutation in the IC. An inherited 15q11-q13 mutation or a trans-factor gene mutation are unlikely; thus, the disease in these patients may arise from a developmental or stochastic failure to switch the maternal-to-paternal imprint during parental spermatogenesis. These studies allow a better understanding of a novel mechanism of human disease, since the epigenetic effect of an IM in the parental germ line determines the phenotypic effect in the patient.

Adult↗
PubMed1999

A 28-kb deletion spanning D15S63 (PW71) in five families: a rare neutral variant?

Methylation analysis with probe PW71 (D15S63) is an established procedure to test patients suspected of having Prader-Willi syndrome or Angelman syndrome. Using this test, we have identified a 28-kb deletion spanning D15S63 in five independent families. Sequence analysis revealed identical breakpoints in all the families. The haplotype data are compatible with a common ancestral origin of the deletion in at least two families. The deletion was not found in 1, 000 unrelated controls. Although the deletion maps within the imprinting-center region, neither maternal nor paternal inheritance of the deletion appears to affect imprinting in proximal 15q. We conclude that the deletion is a rare neutral variant that can lead to false-positive results in the PW71-methylation test.

Adolescent↗
PubMed1999

The chromosome 15 imprinting centre (IC) region has undergone multiple duplication events and contains an upstream exon of SNRPN that is deleted in all Angelman syndrome patients with an IC microdeletion.

Imprinting of the Prader-Willi/Angelman syndrome region on human chromosome 15 is regulated by an imprinting centre (IC), which spans 5' exons of the gene encoding the small nuclear ribonucleoprotein N ( SNRPN ). The IC/ SNRPN transcripts are initiated at two alternative start sites, which share a high degree of sequence similarity with each other and with two newly identified sites 63 and >700 kb further upstream. Three of these sites are hypermethylated on the maternal chromosome, whereas one displays an oppositemethylation pattern. We have also identified novel splice variants of the IC/ SNRPN transcripts and hitherto undetected exons. One of these exons, which we designate u5, is deleted in all Angelman syndromepatients with a microdeletion of the IC. We conclude that elements of the IC region have undergone multiple duplication events and that u5 or a sequence close by may play a role in maternal imprinting.

Angelman Syndrome↗
PubMed1999

The ancestral gene for transcribed, low-copy repeats in the Prader-Willi/Angelman region encodes a large protein implicated in protein trafficking, which is deficient in mice with neuromuscular and spermiogenic abnormalities.

Transcribed, low-copy repeat elements are associated with the breakpoint regions of common deletions in Prader-Willi and Angelman syndromes. We report here the identification of the ancestral gene ( HERC2 ) and a family of duplicated, truncated copies that comprise these low-copy repeats. This gene encodes a highly conserved giant protein, HERC2, that is distantly related to p532 (HERC1), a guanine nucleotide exchange factor (GEF) implicated in vesicular trafficking. The mouse genome contains a single Herc2 locus, located in the jdf2 (juvenile development and fertility-2) interval of chromosome 7C. We have identified single nucleotide splice junction mutations in Herc2 in three independent N-ethyl-N-nitrosourea-induced jdf2 mutant alleles, each leading to exon skipping with premature termination of translation and/or deletion of conserved amino acids. Therefore, mutations in Herc2 lead to the neuromuscular secretory vesicle and sperm acrosome defects, other developmental abnormalities and juvenile lethality of jdf2 mice. Combined, these findings suggest that HERC2 is an important gene encoding a GEF involved in protein trafficking and degradation pathways in the cell.

Amino Acid Sequence↗