NF1 mRNA biogenesis: effect of the genomic milieu in splicing regulation of the NF1 exon 37 region

We have studied the splicing regulation of NF1 exons 36 and 37. We show that they not only require an intact exonic Splicing Enhancer (ESE) within exon 37, but also need the genomic region stretching from exons 31 to 38. Any nucleotide change in two exon 37 third codon positions disrupts the ESE. The extent of exons 36 and 37 skipping due to a mutated ESE depends on the genomic context. This is a unique example of what may be a more general phenomena involved in the tuning of pre-mRNA processing and gene expression modulation in the chromosomal setting.     

Genetic and epigenetic alterations of the NF2 gene in sporadic vestibular schwannomas

Background

Mutations in the neurofibromatosis type 2 (NF2) tumor-suppressor gene have been identified in not only NF2-related tumors but also sporadic vestibular schwannomas (VS). This study investigated the genetic and epigenetic alterations in tumors and blood from 30 Korean patients with sporadic VS and correlated these alterations with tumor behavior.

Methodology/Principal Findings

NF2 gene mutations were detected using PCR and direct DNA sequencing and three highly polymorphic microsatellite DNA markers were used to assess the loss of heterozygosity (LOH) from chromosome 22. Aberrant hypermethylation of the CpG island of the NF2 gene was also analyzed. The tumor size, the clinical growth index, and the proliferative activity assessed using the Ki-67 labeling index were evaluated. We found 18 mutations in 16 cases of 30 schwannomas (53%). The mutations included eight frameshift mutations, seven nonsense mutations, one in-frame deletion, one splicing donor site, and one missense mutation. Nine patients (30%) showed allelic loss. No patient had aberrant hypermethylation of the NF2 gene and correlation between NF2 genetic alterations and tumor behavior was not observed in this study.

Conclusions/Significance

The molecular genetic changes in sporadic VS identified here included mutations and allelic loss, but no aberrant hypermethylation of the NF2 gene was detected. In addition, no clear genotype/phenotype correlation was identified. Therefore, it is likely that other factors contribute to tumor formation and growth.     

Combinatorial sequencing-by-hybridization: analysis of the NF1 gene

Neurofibromatosis type 1 (NF1), one of the most common autosomal dominant disorders, is caused by mutations in the NF1 gene. A variety of methods are currently used in clinical settings to define disease-causing mutations. We describe microarray-based combinatorial sequencing-by-hybridization (cSBH), which overcomes some disadvantages associated with other techniques. Sequence readout of 2 kb was achieved on a single slide, with detection of base substitutions, insertions and small deletions. In addition, cSBH analysis of the entire NF1 gene demonstrates reproducibility, efficiency and reduced time; therefore, representing an alternative to extensive DNA sequence characterization.     

Sex differences in mutational rate and mutational mechanism in the NF1 gene in neurofibromatosis type 1 patients

Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder with a prevalence of around 1 in 3500, affecting all ethnic groups. The clinical manifestations of the disease are variable, even among members of the same family, and affect a variety of tissues and cell types, including skin, iris, central and peripheral nervous systems and skeletal system. It has been reported that the majority of sporadic mutations in NF1 arise in paternally inherited alleles. We present here a collaborative study of the parental origin and type of mutation in individuals with de novo NF1, who account for up to a half of all cases of clinically diagnosed NF1. We have studied intragenic and extragenic markers in 470 NF1 families. In 32 of these families it was possible to assess the parental origin of a de novo NF1 mutation either by linkage analysis (in families with three generations) or by the detection of an intragenic deletion in a sporadic NF1 case. Eleven of these 32 families have three generations (the second and third generation being affected), with the mutation (not a large deletion) being of paternal origin in 82% of them (P < 0.05). In the other 21 families an intragenic deletion was detected, in 76% being in the maternal chromosome and in 24% in the paternal one (P < 0.05). Our results suggest that in NF1 the majority of deletions occur in oogenesis, while other types of mutations should account for the paternally derived NF1 mutations. Received: 26 June 1996 / Revised: 1 August 1996     

Interleukin 1B gene polymorphism is associated with baseline C-reactive protein levels in healthy individuals

C-reactive protein (CRP) is a sensitive marker of inflammation induced by both IL-6 and IL-1. Thus, genetic variation in these genes could be associated with the variety in C-reactive protein levels, and therefore with the severity of the entire inflammatory response. Even a subtle elevation in baseline CRP levels in healthy individuals has been found to significantly increase the risk for cardiovascular diseases. Therefore, to find out the possible role of pro-inflammatory cytokines in CRP baseline regulation we conducted a study of 338 healthy blood donors whose CRP levels were determined and whose single nucleotide polymorphisms of IL1A(C/T)-889, IL1B(C/T)-511, IL1B(C/T) + 3954, IL6(G/C)-174 and ILRN (a VNTR) both genotyped and haplotyped. The data revealed an association between CRP levels and the IL1B + 3954 genotype. Also, the bilocus haplotype IL1B-511*1/IL1B + 3954*2 was more frequent in subjects with below median CRP levels (< 0.72 mg/l), and composite genotype analysis of IL1B-511/IL1B + 3954 supported this finding. Our findings suggest that in healthy people, basal CRP levels are regulated by IL1B but not by IL6 genetics.     

Distribution of CTG repeats at the DMPK gene in myotonic distrophy patients and healthy individuals from the Mexican population

Myotonic dystrophy type 1 (DM1), the most common form of adult muscular dystrophy, is caused by anormal expansion of CTG trinucleotide repeats located in the 3′-untranslated region of the DMPK gene. The clinical features of DM1 are multisystemic and highly variable, and the unstable nature of CTG expansion causes wide genotypic and phenotypic presentations. In this study, we described to our knowledge for the first time the molecular diagnosis of myotonic dystrophy type 1 patients in the Mexican population, applying a fluorescent PCR method in combination with capillary electrophoresis analysis of the amplified products. We identified expanded alleles in 45 out of 50 patients (90%) with clinical features of myotonic disease. Furthermore, genotyping of 400 healthy subjects revealed the presence of 25 different alleles, ranging in size from 5 to 34 repeats. The most frequent allele was 13 CTG repeats (38.87%) and the frequency for alleles over 18 CTG repeats was 6.7%. Molecular test is essential for DM1 diagnosis and distribution of the CTG repeat alleles present in the Mexican population are significantly different from those of other populations.     

Progress towards identifying the neurofibromatosis (NF1) gene

Von Recklinghausen neurofibromatosis (NF1) is a common autosomal dominant disorder of humans. Linkage analysis has recently mapped the NF1 gene to the proximal long arm of chromosome 17. The identification of two NF1 patients with balanced translocations has now allowed the location of the gene to be narrowed to a few hundred kilobases of chromosome band 17q11.2, using a combination of somatic cell hybrid technology, linking clones and pulsed field gel electrophoresis.     

Mutation spectrum and splicing variants in the OPA1 gene

Optic atrophy type 1 (OPA1, MIM 165500) is a dominantly inherited optic neuropathy that features low visual acuity leading in many cases to legal blindness. We have recently shown, with others, that mutations in the OPA1 gene encoding a dynamin-related mitochondrial protein, underlie the dominant form of optic atrophy. Here we report that OPA1 has eight mRNA isoforms as a result of the alternative splicing of exon 4 and two novel exons named 4b and 5b. In addition, we screened a cohort of 19 unrelated patients with dominant optic atrophy by direct sequencing of the 30 OPA1 exons (including exons 4b and 5b) and found mutations in 17 (89%) of them of which 8 were novel. A majority of these mutations were truncative (65%) and located in exons 8 to 28, but a number of them were amino acid changes predominantly found in the GTPase domain (exons 8 to 15). We hypothesize that at least two modifications of OPA1 may lead to dominant optic atrophy, that is alteration in GTPase activity and loss of the last seven C-terminal amino acids that putatively interact with other proteins.     

Recurrence of a nonsense mutation in the NF1 gene causing classical neurofibromatosis type 1

Summary The gene responsible for von Recklinghausen neurofibromatosis (NF1) has recently been identified, and several point mutations and deletions have been described. The availability of intron-exon boundaries of several exons of the NF1 gene facilitates the search for mutations in affected patients. We have analysed 38 patients for mutations in exon 4 of the NF1 gene, and found one patient with a CT transition at base position 1087 of the cDNA, changing an arginine codon to a stop codon, at amino acid position 365. Sequencing of other members of the family, including both parents, did not show the mutation, confirming that this mutation is responsible for this sporadic NF1 case. As the mutation described here was previously identified in an independent case by others, this case represents a recurrence of this mutation and suggests that codon 365 might be a hot spot for mutations in the NF1 gene. Thus, a specific search for this mutation should be performed when studying NF1 sporadic or familiar cases for genetic analysis.     

A DNA enzyme that mimics the first step of RNA splicing

We have discovered an artificial DNA enzyme that mimics the first step of RNA splicing. In vitro selection was used to identify DNA enzymes that ligate RNA. One of the new DNA enzymes carries out splicing-related catalysis by specifically recognizing an unpaired internal adenosine and facilitating attack of its 2'-hydroxyl onto a 5'-triphosphate. This reaction forms 2',5'-branched RNA and is analogous to the first step of in vivo RNA splicing, in which a ribozyme cleaves itself with formation of a branched intermediate. Unlike a natural ribozyme, the new DNA enzyme has no 2'-hydroxyl groups to aid in the catalytic mechanism. Our finding has two important implications. First, branch-site adenosine reactivity seems to be mechanistically favored by nucleic acid enzymes. Second, hydroxyl groups are not obligatory components of nucleic acid enzymes that carry out biologically related catalysis.     

Elevated risk for MPNST in NF1 microdeletion patients

An NF1 microdeletion is the single most commonly reported mutation in individuals with neurofibromatosis type 1 (NF1). Individuals with an NF1 microdeletion have, as a group, more neurofibromas at a younger age than the group of all individuals with NF1. We report that NF1 microdeletion individuals additionally have a substantially higher lifetime risk for the development of malignant peripheral nerve sheath tumors than individuals with NF1 who do not have an NF1 microdeletion. This should be taken into account in the medical follow-up of individuals with an NF1 microdeletion.