Biomedical subjects

Margaret R Wallace

Publications and source records attributed to Margaret R Wallace.

10 recordsLinked to original sources

PubMed2005

Tumorigenic properties of neurofibromin-deficient Schwann cells in culture and as syngrafts in Nf1 knockout mice.

Neurofibromatosis type 1 (NF1) is one of the most common dominantly inherited genetic diseases associated with the nervous system. Functional loss of the NF1 tumor suppressor is frequently associated with the generation of benign neurofibromas that can progress to malignancy. Recent evidence in genetic mouse models indicates that the development of neurofibromas requires a loss of Nf1 in the cells destined to become neoplastic as well as heterozygosity in nonneoplastic cells. We tested this hypothesis in a newly developed syngraft mouse model in which Nf1-/- Schwann cells isolated from knockout embryos were grafted into the sciatic nerves of Nf1+/- mice, corresponding to the genetic background of NF1 patients. Furthermore, we also characterized in vitro growth of these cells. We found that embryonic mouse Nf1-/- Schwann cells exhibit increased proliferation and less growth factor-dependence in vitro compared with heterozygous and wild-type counterparts. Moreover, Nf1-/- Schwann cells showed tumorigenic growth when implanted into nerve of adult Nf1 heterozygous mice. These findings support the conclusion that loss of Nf1 in embryonic mouse Schwann cells is sufficient for tumor development in the heterozygous environment of adult mouse nerve. In addition, this syngraft model provides a practical means for the controlled induction of neurofibromas, greatly facilitating localized application of therapeutic agents and gene delivery.

Animals↗

PubMed2005

Analysis of somatic NF1 promoter methylation in plexiform neurofibromas and Schwann cells.

Neurofibromatosis 1 (NF1) is an autosomal dominant disorder with the characteristic feature being the neurofibroma. It is believed that both NF1 alleles must be inactivated as the first step in tumorigenesis. However, often the somatic mutations are not identified, suggesting that epigenetic changes such as methylation could account for the "second hit" in some tumors. The literature reports that the region of the NF1 promoter surrounding the transcription start site is completely unmethylated in several normal tissues and some NF1-related dermal and plexiform neurofibromas. We analyzed the methylation state of the NF1 promoter in normal Schwann cells (the cell type clonally expanded in neurofibromas) and in NF1-related plexiform tumor samples with unidentified somatic mutations. In a region of 451 bp surrounding the transcription start site, a low level of methylation was found at several specific cytosines in 12 of 18 tumor samples. Overall, epigenetic silencing through methylation does not appear to be a major mechanism for the second hit. However, this study, which analyzed the largest number of NF1-related plexiform tumors and is the first to include Schwann cell-enriched tumor cultures, detected greater methylation than in any previous reports. This suggests that methylation, especially at potential transcription factor binding sites, is moderately perturbed in some plexiform neurofibromas and should be investigated further.

Cells, Cultured↗

PubMed2005

The A118G single nucleotide polymorphism of the mu-opioid receptor gene (OPRM1) is associated with pressure pain sensitivity in humans.

Responses to painful stimuli are characterized by tremendous interindividual variability, and genetic factors likely account for some proportion of this variability. However, few studies have identified genetic contributions to experimental pain perception in humans. This experiment investigated whether the A118G single nucleotide polymorphism of the mu-opioid receptor gene ( OPRM1 ) was associated with responses to three different experimental pain modalities in a sample of 167 healthy volunteers (96 female, 71 male). Responses to thermal, mechanical, and ischemic pain were assessed in all subjects, and genotyping of OPRM1 was performed, which revealed that the rare A118G allele occurred in 24 females (25%) and 12 males (17%). Statistical analyses indicated that subjects with a rare allele had significantly higher pressure pain thresholds than those homozygous for the common allele. Also, a sex by genotype interaction emerged for heat pain ratings at 49 degrees C, such that the rare allele was associated with lower pain ratings among men but higher pain ratings among women. These data indicate an association of a common single nucleotide polymorphism of OPRM1 with mechanical pain responses and that this genotype may be associated with heat pain perception in a sex-dependent manner. This study examines the association of the A118G SNP of OPRM1 to experimental pain sensitivity. The results indicate that the rare allele is associated with higher pressure pain thresholds. These results support previous contentions that OPRM1 may be a pain-relevant gene; however, replication of these findings is needed.

Adult↗

PubMed2005

Fine mapping of chromosome 17 translocation breakpoints > or = 900 Kb upstream of SOX9 in acampomelic campomelic dysplasia and a mild, familial skeletal dysplasia.

Previously, our group reported a five-generation family in which a balanced t(13;17) translocation is associated with a spectrum of skeletal abnormalities, including Robin sequence, hypoplastic scapulae, and a missing pair of ribs. Using polymerase chain reaction (PCR) with chromosome-specific markers to analyze DBA from somatic cell hybrids containing the derivative translocation chromosomes, we narrowed the breakpoint on each chromosome. Subsequent sequencing of PCR products spanning the breakpoints identified the breaks precisely. The chromosome 17 breakpoint maps approximately 932 kb upstream of the sex-determining region Y (SRY)-related high-mobility group box gene (SOX) within a noncoding transcript represented by two IMAGE cDNA clones. A growing number of reports have implicated chromosome 17 breakpoints at a distance of up to 1 Mb from SOX9 in some cases of campomelic dysplasia (CD). Although this multigeneration family has a disorder that shares some features with CD, their phenotype is significantly milder than any reported cases of (nonmosaic) CD. Therefore, this case may represent an etiologically distinct skeletal dysplasia or may be an extremely mild familial example of CD, caused by the most proximal translocation breakpoint from SOX9 reported to date. In addition, we have refined the breakpoint in a acampomelic CD case described elsewhere and have found that it lies approximately 900 kb upstream of SOX9.

Base Sequence↗

PubMed2003

The melanocortin-1 receptor gene mediates female-specific mechanisms of analgesia in mice and humans.

Sex specificity of neural mechanisms modulating nociceptive information has been demonstrated in rodents, and these qualitative sex differences appear to be relevant to analgesia from kappa-opioid receptor agonists, a drug class reported to be clinically effective only in women. Via quantitative trait locus mapping followed by a candidate gene strategy using both mutant mice and pharmacological tools, we now demonstrate that the melanocortin-1 receptor (Mc1r) gene mediates kappa-opioid analgesia in female mice only. This finding suggested that individuals with variants of the human MC1R gene, associated in our species with red hair and fair skin, might also display altered kappa-opioid analgesia. We found that women with two variant MC1R alleles displayed significantly greater analgesia from the kappa-opioid, pentazocine, than all other groups. This study demonstrates an unexpected role for the MC1R gene, verifies that pain modulation in the two sexes involves neurochemically distinct substrates, and represents an example of a direct translation of a pharmacogenetic finding from mouse to human.

Adolescent↗

PubMed2003

Delayed rectifier K currents in NF1 Schwann cells. Pharmacological block inhibits proliferation.

K+(K) currents are related to the proliferation of many cell types and have a relationship to second messenger pathways implicated in regulation of the cell cycle in development and certain disease states. We examined the role of K currents in Schwann cells (SC) cultured from tumors that arise in the human disease neurofibromatosis type 1 (NF1). Comparisons were made between whole cell voltage clamp recordings from normal human SC cultures and from neurofibroma cultures and malignant peripheral nerve sheath tumor (MPNST) cell lines. The outward K currents of normal and tumor cells could be divided into three types based on pharmacology and macroscopic inactivation: (1) "A type" current blocked by 4-aminopyridine, (2) delayed rectifier (DR) current blocked by tetraethylammonium, and (3) biphasic current consisting of a combination of these two current types. The DR K current was present in MPNST- and neurofibroma-derived SC, but not in quiescent, nondividing, normal SC. DR currents were largest in MPNST-derived SC (50 pA/pF vs. 2.1-4.9 pA/pF in dividing and quiescent normal SC). Normal SC cultures had significantly more cells with A type current than cultures of MPNST and the plexiform neurofibroma. Conversely, MPNST and plexiform neurofibroma cultures had significantly more SC with DR current than did normal cultures, and these DR currents were significantly larger. In addition, the plexiform neurofibroma culture had significantly more cells with DR current than the dermal neurofibroma culture. K currents in SC from normal NF1 SC cultures had current abundances similar to GGF-exposed normal SC and the plexiform neurofibroma. We have established a link between DR K current blockade via TEA analogs and inhibition of proliferation of NF1 SC in vitro. In addition, a farnysyl transferase inhibitor (FTI), a blocker of Ras activation, blocked cell proliferation without blocking K currents in all cultures except a plexiform neurofibroma, suggesting that regulation of proliferation in neoplastic and normal SC in vitro is complex.

4-Aminopyridine↗

PubMed2003

Cataracts in the Bichon Frise.

PURPOSE: To determine the clinical characteristics of possible inherited cataract in the Bichon Frise breed. These characteristics include the relative frequency, gender effects, site of first cataract formation, age of onset, relationship of age to cataract maturity, and other concurrent pre- and postoperative ophthalmic diseases. METHODS: Four different populations of Bichon Frise were examined. They included: (1) referred patients of the University of Florida (VMTH; 1990-2000); (2) patients from other universities and large institutions (VMDB; 1970-2000); (3) patients from the Canine Eye Registry Foundation (CERF; 1970-2000); and (4) special patients recruited from eye clinics and ACVO specialty practices (1995-2001). Blood samples were obtained from many patients for future DNA analyzes. Statistical comparisons between groups were by general linear and anova analyzes, and P < 0.05 was considered significant. RESULTS: The four populations of cataractous and total Bichon Frise dogs included: (1) UF-VMTH: 57 cataractous dogs; (2) VMDB: 406 dogs (28%) with cataracts; total dogs--1407; (3) CERF: 505 cataractous dogs (6%); total dogs--8222; and (4) ACVO: 223 cataractous dogs (57%); total dogs--391. In each population group, gender did not affect cataract distribution. Dogs between 2 and 8 years of age were most frequently affected, and initial cataract involvement affected equally the anterior and posterior cortices. Immature cataracts occurred more often in younger dogs, and hypermature cataracts were more frequently diagnosed in older dogs. Dogs with early cataracts were encountered more frequently in the CERF population. Pre- and postoperative retinal detachments (RD) were not infrequently diagnosed in the UF-VMTH and VMDB groups. RD in the UF-VMTH and VMDB groups occurred in 33% and 13% of the patients, respectively. CONCLUSIONS: Cataracts were first encountered in CERF and VMDB data between 1975 and 1979 and have increased since this time. As expected, both similarities and differences between the four different populations of Bichon Frise were encountered. Both sexes of dogs are equally affected. The anterior and posterior cortical areas of the lens were first involved and dogs 2-8 years of age are most frequently affected. Cataracts affected younger dogs in the CERF group and older cataractous dogs in the other populations. Cataract formation appears to be inherited in the Bichon Frise dog. The frequency of pre- and postoperative retinal detachments present higher risks for cataract surgery in this breed.

Age Distribution↗

PubMed2002

RT-PCR splicing analysis of the NF1 open reading frame.

Neurofibromatosis 1 (NF1) is an autosomal dominant condition whose molecular diagnosis is challenging because of the large size of the gene and the vast number of unique NF1 gene mutations. Some splicing and nonsense mutations have been shown to cause exon skipping. Recently, temperature-induced abnormal splicing has been found in NF1 in ex-vivo tissues. This prompted us to investigate the entire NF1 transcript for such aberrant splicing. We found several novel exon skips that appeared de novo or were present initially and increased in aged/cooled blood: exon 20, exons 20 and 21 combined, exon 33, exon 34, exon 37, exon 40, exon 45, exons 43 and 45 combined, part of exon 43, and the first codon of exon 12b. Some aberrant splice forms were undetectable when blood was drawn into Qiagen PAXgene tubes, rather than EDTA vacutainers, and we demonstrate how these aberrant splicing events are a potential pitfall for RNA-based NF1 mutation characterization. The same reverse transcription/polymerase chain reaction strategy was used to screen for novel NF1 alternative splicing in Schwann cells and seven other tissues. Even though no Schwann-specific alternative exons were identified, we found minor novel splicing isoforms differentially expressed such as skips of exon 37 and exon 40. Skipping of exon 43, part of exon 43, and the first codon of exon 12b were found in all tissues analyzed. These forms suggest greater tissue-based variability in the NF1 message than was previously thought and may indicate minor amounts of heterogeneity at the protein level.

Alternative Splicing↗

PubMed2002

NF1 mutations and molecular testing.

Neurofibromatosis 1 is a progressive autosomal dominant condition caused by mutations in the NF1 gene on chromosome 17. The condition shows clinical variable expressivity, with varying features even between family members who share the same mutation. Furthermore, it is impossible to precisely predict the severity and course of the condition, a source of frustration for families and physicians. Neurofibromatosis 1 is also heterogeneous at the mutation level, with more than 300 independent mutations having been reported in this gene. The mutation data have accumulated slowly owing to the variability of the mutation types and the size and complexity of the gene. This is also reflected in the lack of a simple, inexpensive, highly accurate DNA-based test for neurofibromatosis 1 at present. This article reviews current NF1 mutation spectrum and testing, discussing and illustrating mutation mechanisms and pathogenetic effects, as well as factors affecting DNA testing and interpretation/diagnosis.

Child↗

PubMed2002

The Chediak-Higashi protein interacts with SNARE complex and signal transduction proteins.

BACKGROUND: Chediak-Higashi syndrome (CHS) is an inherited immunodeficiency disease characterized by giant lysosomes and impaired leukocyte degranulation. CHS results from mutations in the lysosomal trafficking regulator (LYST) gene, which encodes a 425-kD cytoplasmic protein of unknown function. The goal of this study was to identify proteins that interact with LYST as a first step in understanding how LYST modulates lysosomal exocytosis. MATERIALS AND METHODS: Fourteen cDNA fragments, covering the entire coding domain of LYST, were used as baits to screen five human cDNA libraries by a yeast two-hybrid method, modified to allow screening in the activation and the binding domain, three selectable markers, and more stringent confirmation procedures. Five of the interactions were confirmed by an in vitro binding assay. RESULTS: Twenty-one proteins that interact with LYST were identified in yeast two-hybrid screens. Four interactions, confirmed directly, were with proteins important in vesicular transport and signal transduction (the SNARE-complex protein HRS, 14-3-3, and casein kinase II). CONCLUSIONS: On the basis of protein interactions, LYST appears to function as an adapter protein that may juxtapose proteins that mediate intracellular membrane fusion reactions. The pathologic manifestations observed in CHS patients and in mice with the homologous mutation beige suggest that understanding the role of LYST may be relevant to the treatment of not only CHS but also of diseases such as asthma, urticaria, and lupus, as well as to the molecular dissection of the CHS-associated cancer predisposition.

14-3-3 Proteins↗