Familial Mediterranean fever (FMF) is an autosomal recessive disorder characterized by attacks of fever and serositis. In this paper, we define a minimal co-segregating region of 60 kb containing the FMF gene (MEFV) and identify four different transcript units within this region. One of these transcripts encodes a new protein (marenostrin) related to the ret-finger protein and to butyrophilin. Four conservative missense variations co-segregating with FMF have been found within the MEFV candidate gene in 85% of the carrier chromosomes. These variations, which cluster at the carboxy terminal domain of the protein, were not present in 308 control chromosomes, including 162 validated non-carriers. We therefore propose that the sequence alterations in the marenostrin protein are responsible for the FMF disease.

A candidate gene for familial Mediterranean fever

Neurofibromatosis type 1 (NF1) is one of the most common autosomal dominant disorders and is caused by mutations in the NF1 gene. Mutation detection is complex due to the large size of the NF1 gene, the presence of pseudogenes and the great variety of possible lesions. Although there is no evidence for locus heterogeneity in NF1, mutation detection rates rarely exceed 50%. We studied 67 unrelated NF1 patients fulfilling the NIH diagnostic criteria, 29 familial and 38 sporadic cases, using a cascade of complementary techniques. We performed a protein truncation test starting from puromycin-treated EBV cell lines and, if no mutation was found, continued with heteroduplex, FISH, Southern blot and cytogenetic analysis. We identified the germline mutation in 64 of 67 patients and 32 of the mutations are novel. This is the highest mutation detection rate reported in a study of typical NF1 patients. All mutations were studied at the genomic and RNA level. The mutational spectrum consisted of 25 nonsense, 12 frameshift, 19 splice mutations, six missense and/or small in-frame deletions, one deletion of the entire NF1 gene, and a translocation t(14;17)(q32;q11.2). Our data suggest that exons 10a-10c and 37 are mutation-rich regions and that together with some recurrent mutations they may account for almost 30% of the mutations in classical NF1 patients. We found a high frequency of unusual splice mutations outside of the AG/GT 5 cent and 3 cent splice sites. As some of these mutations form stable transcripts, it remains possible that a truncated neurofibromin is formed.

Exhaustive mutation analysis of the NF1 gene allows identification of 95% of mutations and reveals a high frequency of unusual splicing defects.

Neurofibromatosis type 1 (NF1) is one of the mostcommon inherited disorders in humans and is causedby mutations in the NF1gene. To date, the majority ofthe reported NF1mutations are predicted to result inprotein truncation, but veryfew studieshavecorrelatedthe causative NF1mutation with its effect at the mRNAlevel. We have applied a whole NF1cDNA screeningmethodology to the study of 80 unrelated NF1 patientsand have identified 44 different mutations, 32 beingnovel, in 52 of these patients. Mutations were detectedin 87% of the familial cases, but in 51% of the sporadicones. At least 15 of the 80 NF1 patients (19%) hadrecurrent mutations. The study shows that in 50% of thepatients in whom the mutations were identified, theseresulted in splicing alterations. Most of the splicingmutations did not involve the conserved AG/GTdinucleotides of the splice sites. One frameshift, twononsense and two missense mutations were alsoresponsible for alterations in mRNA splicing. Thelocation and type of mutation within the NF1gene, andits putative effect at the protein level, do not indicateany relationship to any specific clinical feature of NF1.The high proportion of aberrant spliced transcriptsdetected in NF1 patients stresses the importance ofstudying mutations at both the genomic and RNA level.It is possible that part of the clinical variability in NF1could be due to mutations affecting mRNA splicing,which is the most common molecular defect in NF1.INTRODUCTIONNeurofibromatosis type 1 (NF1) (MIM 162200) is one of themost common inherited disorders in humans with a prevalenceof 1 in 3000 individuals. NF1 is an autosomal dominantdisorder fully penetrant at the age of 5 years, but with a vari-able clinical expression, even among members of the samefamily. The main clinical features of NF1 are cafe au lait spots(CLSs), cutaneous neurofibromas and Lisch nodules (1,2). TheNF1 gene was mapped to chromosome 17q11.2 and was posi-tionally cloned in 1990 (3–5). It spans 350 kb of genomicDNA, contains 60 exons (6,7) and is transcribed ubiquitouslyto an mRNA of 11–13 kb that encodes for a protein, neuro-fibromin, of 2818 amino acids (8). The only region of neuro-fibromin with a well-defined function and structure is a centraldomain with a high similarity to ras-specific GTPase-activating proteins (GAPs), called the GAP-related domain(GRD), which downregulates ras activity (9–11).The NF1 gene has one of the highest mutation ratesdescribed for any human disorder ( 1 10

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Mutations affecting mRNA splicing are the most common molecular defects in patients with neurofibromatosis type 1

Neurofibromatosis type 1 (NF1), also called von Recklinghausen disease or peripheral neurofibromatosis, is a common autosomal dominant disorder characterised by multiple neurofibromas, cafe au lait spots, and Lisch nodules of the iris, with a variable clinical expression. The gene responsible for this condition, NF1, has been isolated by positional cloning. It spans over 350 kb of genomic DNA in chromosomal region 17q11.2 and encodes an mRNA of 11-13 kb containing at least 59 exons. NF1 is widely expressed in a variety of human and rat tissues. Four alternatively spliced NF1 transcripts have been identified. Three of these transcript isoforms (each with an extra exon: 9br, 23a, and 48a, respectively) show differential expression to some extent in various tissues, while the fourth isoform (2.9 kb in length) remains to be examined. The protein encoded by NF1, neurofibromin, has a domain homologous to the GTPase activating protein (GAP) family, and downregulates ras activity. The identification of somatic mutations in NF1 from tumour tissues strongly supports the speculation that NF1 is a member of the tumour suppressor gene family. Although the search for mutations in the gene has proved difficult, germline mutation analysis has shown that around 82% of all the fully characterised NF1 specific mutations so far predict severe truncation of neurofibromin. Further extensive studies are required to elucidate the gene function and the mutation spectrum. This should then facilitate the molecular diagnosis and the development of new therapy for the disease.

https://jmg.bmj.com/content/jmedgenet/33/1/2.full.pdf

Molecular genetics of neurofibromatosis type 1 (NF1).

Glioblastoma, the most aggressive primary brain tumor in humans, exhibits a large degree of molecular heterogeneity. Understanding the molecular pathology of a tumor and its linkage to behavior is an important foundation for developing and evaluating approaches to clinical management. Here we integrate array-comparative genomic hybridization and array-based gene expression profiles to identify relationships between DNA copy number aberrations, gene expression alterations, and survival in 34 patients with glioblastoma. Unsupervised clustering on either profile resulted in similar groups of patients, and groups defined by either method were associated with survival. The high concordance between these separate molecular classifications suggested a strong association between alterations on the DNA and RNA levels. We therefore investigated relationships between DNA copy number and gene expression changes. Loss of chromosome 10, a predominant genetic change, was associated not only with changes in the expression of genes located on chromosome 10 but also with genome-wide differences in gene expression. We found that CHI3L1/YKL-40 was significantly associated with both chromosome 10 copy number loss and poorer survival. Immortalized human astrocytes stably transfected with CHI3L1/YKL-40 exhibited changes in gene expression similar to patterns observed in human tumors and conferred radioresistance and increased invasion in vitro. Taken together, the results indicate that integrating DNA and mRNA-based tumor profiles offers the potential for a clinically relevant classification more robust than either method alone and provides a basis for identifying genes important in glioma pathogenesis.

https://cancerres.aacrjournals.org/content/canres/65/5/1678.full.pdf

Integrated array-comparative genomic hybridization and expression array profiles identify clinically relevant molecular subtypes of glioblastoma

The neurofibromatosis 1 gene seems to play essential roles at several different stages of life. During embryogenesis, it is involved in cardiac development while in the adult, neurofibromin (the corresponding protein) is mainly expressed in the nervous system, and therein, essentially in neurons, non-myelinating Schwann cells and oligodendrocytes. In addition, the NF1 gene is considered a tumor suppressor gene, since mutations have been associated with the occurrence of benign and malignant tumors in neuralcrest-derived tissues. Using reverse transcription-polymerase chain reaction (RT-PCR) analyses with primers located in exons 7 and 13, we have identified evidence of alternative splicing in this region of the NF1 gene. Cloning and sequencing of cDNA allowed the characterization of an isoform bearing an extra 30 bp sequence between exons 9 and 10a, leading to the insertion of 10 amino acids between residues 420 and 421 of neurofibromin. The insertion is conserved in the mouse. Examination of the pattern of expression of this isoform demonstrated a high level of expression in the central nervous system and an absence of expression in all the other normal tissues tested including peripheral nervous tissues derived from the neural crest. Analysis of brain tumors indicated a reduced expression of the alternative exon in medulloblastomas and oligodendrogliomas. The results presented here are consistent with tissue-specific expression of this alternative exon which we propose to call exon 9br.

Neurofibromatosis 1 (NF1) mRNAs expressed in the central nervous system are differentially spliced in the 5′ part of the gene

Various genomic alterations have been detected in glioblastoma Chromosome 7p, with the epidermal growth factor receptor locus, together with chromosome 10q, with the phosphatase and tensin homologue deleted in chromosome 10 and deleted in malignant brain tumors-1 loci, and chromosome 9p, with the cyclin-dependent kinase inhibitor 2A locus, are among the most frequently damaged chromosomal regions in glioblastoma In this study, we evaluated the genetic status of 32 glioblastomas by comparative genomic hybridization; the sensitivity of comparative genomic hybridization versus differential polymerase chain reaction to detect deletions at the phosphatase and tensin homologue deleted in chromosome 10, deleted in malignant brain tumors-1, and cyclin-dependent kinase inhibitor 2A loci and amplifications at the cyclin-dependent kinase 4 locus; the frequency of genetic lesions (gain or loss) at 16 different selected loci (including oncogenes, tumor-suppressor genes, and proliferation markers) mapping on 13 different chromosomes; and the possible existence of a statistical association between any pair of molecular markers studied, to subdivide the glioblastoma entity molecularly Comparative genomic hybridization showed that the most frequent region of gain was chromosome 7p, whereas the most frequent losses occurred on chromosomes 10q and 13q The only statistically significant association was found for 7p gain and 10q loss

Chromosomal abnormalities in human glioblastomas: gain in chromosome 7p correlating with loss in chromosome 10q

Aims : Medulloblastoma (MB) is the most common primitive neuroectodermal tumour (PNET) of the central nervous system. Although supratentorial PNET (sPNET) and MB are histologically similar, their clinical behaviour differs, sPNET being more aggressive than MB. The aim of this study was to determine whether sPNET and MB are genetically different entities.



Methods and results : We investigated 32 PNET primary tumour samples (23 MB and nine sPNET) and four PNET cell lines, for the presence of CDKN2A homozygous deletions at exon 1-α of p16/INK4 and exon 1-β of p14/ARF, and promoter hypermethylation of both genes. No homozygous deletion of either p16/INK4 or p14/ARF was demonstrated in any of the PNET primary tumour samples. Methylation of p16/INK4 was found in one of six sPNET and in one of 23 MB, while p14/ARF methylation was observed in three of six sPNET and in three of 21 MB. No methylation of p16/INK4 or p14/ARF was found in any of the PNET cell lines analysed. The three MB cell lines did not show p16/INK4 expression, and only the MB Daoy cell line (homozygously deleted at CDKN2A) presented loss of p14/ARF expression.



Conclusions : Our results in this limited series of central PNET show that p14/ARF is frequently involved in PNET carcinogenesis, with a higher frequency, but not statistically significant, for sPNET than for MB.

High promoter hypermethylation frequency of p14/ARF in supratentorial PNET but not in medulloblastoma.

La localisation chromosomique et l'analyse du chimerisme de 22 YACs ont ete faites par hybridation in situ fluorescente. Les sondes sont obtenues en amplifiant par PCR la partie humaine du YAC grâce a des amorces Alu. Le maximum d'amplification des differents elements inter-Alu est obtenu pour une temperature d'hybridation des amorces inferieure a la temperature optimale utilisee pour une specificite stricte. Dans ces conditions, l'ADN de levure contribue aussi a la formation de produits d'amplification et, puisqu'une forte competition est necessaire pour la suppression de toutes les sequences Alu, les produits de PCR obtenus a partir de la levure sans YAC se sont averes d'efficaces competiteurs.

G. Danglot

Papers

Neurofibromatosis 1 (NF1) mRNAs expressed in the central nervous system are differentially spliced in the 5′ part of the gene

Chromosomal abnormalities in human glioblastomas: gain in chromosome 7p correlating with loss in chromosome 10q

High promoter hypermethylation frequency of p14/ARF in supratentorial PNET but not in medulloblastoma.

Mapping of 22 YACs on human chromosomes by fish using yeast DNA Alu-PCR products for competition.

Duplication of the Ph-chromosome as a possible mechanism of resistance to imatinib mesylate in patients with chronic myelogenous leukemia.