Somatic NF1 mutational spectrum in benign neurofibromas: mRNA splice defects are common among point mutations.

Neurofibromas, benign tumors that originate from the peripheral nerve sheath, are a hallmark of neurofibromatosis type 1 (NF1). Although loss of heterozygosity (LOH) is a common phenomenon in this neoplasia, it only accounts for part of the somatic NF1 mutations found. Somatic point mutations or the presence of "two hits" in the NF1 gene have only been reported for a few neurofibromas. The large size of the NF1 gene together with the multicellular composition of these tumors has greatly hampered their molecular characterization. Here, we present the somatic NF1 mutational analysis of the whole set of neurofibromas studied by our group and consisting in 126 tumors derived from 32 NF1 patients. We report the identification of 45 independent somatic NF1 mutations, 20 of which are reported for the first time. Different types of point mutations together with LOH affecting the NF1 gene and its surrounding region or extending along the 17q arm have been found. Among point mutations, those affecting the correct splicing of the NF1 gene are common, coinciding with results reported on germline NF1 mutations. In most cases, we have been able to confirm that both copies of the NF1 gene are inactivated. We have also found that both somatic and germline mutations can be expressed at the RNA level in the neoplastic cells. Furthermore, we have observed that the study of more than one tumor derived from the same patient is useful for the identification of the germline mutation. Finally, we have noticed that the culture of neurofibromas and their fibroblast clearance facilitates LOH detection in cases in which it is difficult to determine.

Molecular characterization of the breakpoints of a 12-kb deletion in the NF1 gene in a family showing germ-line mosaicism.

Neurofibromatosis type 1 (NF1) is caused by deletions, insertions, translocations, and point mutations in the NF1 gene, which spans 350 kb on the long arm of human chromosome 17. Although several point mutations have been described, large molecular abnormalities have rarely been characterized in detail. We describe here the molecular breakpoints of a 12-kb deletion of the NF1 gene, which is responsible for the NF1 phenotype in a kindred with two children affected because of germline mosaicism in the unaffected father, who has the mutation in 10% of his spermatozoa. The mutation spans introns 31-39, removing 12,021 nt and inserting 30 bp, of which 19 bp are a direct repetition of a sequence located in intron 31, just 4 bp before the 5' breakpoint. The 5' and 3' breakpoints contain the sequence TATTTTA, which could be involved in the generation of the deletion. The most plausible explanation for the mechanism involved in the generation of this 12-kb deletion is homologous/nonhomologous recombination. Since sperm of the father does not contain the corresponding insertion of the 12-kb deleted sequence, this deletion could have occurred within the NF1 chromosome through loop formation. RNA from lymphocytes of one of the NF1 patients showed similar levels of the mutated and normal transcripts, suggesting that the NF1-mRNA from mutations causing frame shifts of the reading frame or stop codons in this gene is not degraded during its processing. The mutation was not detected in fresh lymphocytes from the unaffected father by PCR analysis, supporting the case for true germ-line mosaicism.

Prenatal diagnosis of neurofibromatosis type 1: from flanking RFLPs to intragenic microsatellite markers.

Even though the neurofibromatosis type 1 (NF1) gene was cloned more than 3 years ago, the process of identifying mutations has not been fruitful, and genetic counselling is mainly based on the use of linked markers. Since 1990, we have analysed 130 NF1 families and have performed six prenatal diagnoses. In each case, genetic counselling has relied on linked markers and informativity was achieved in all of them. The use of intragenic microsatellite polymorphisms (IVS27AAAT2.1, IVS27AC28.4, IVS27AC33.1, and IVS38GT53.0) has increased the informativeness in our series of NF1 families to an average of 90 per cent, providing accurate diagnosis and confirmation of the disease status.

Neurofibromatosis type 1 due to germ-line mosaicism in a clinically normal father.

BACKGROUND: The mutation rate of the neurofibromatosis type 1 (NF1) gene is one of the highest in the human genome, with about 50 percent of cases being due to new mutations. We describe a family in which neurofibromatosis type 1 occurred in two siblings with clinically normal parents, and we demonstrate germ-line mosaicism in the father. METHODS: We studied lymphocyte DNA from each member of the family and the father's spermatozoa for several polymorphic intragenic markers of the NF1 gene. Southern blots of DNA digested with several enzymes were hybridized with complementary DNA and individual NF1 exon probes to search for alterations in the gene. RESULTS: The affected siblings, with a clinically severe form of neurofibromatosis type 1, showed no inheritance of paternal alleles for a marker in intron 38 of the NF1 gene, whereas they received alleles from both parents for other NF1 markers. Analysis with probes from this region of the NF1 gene showed a 12-kb deletion of the NF1 gene, involving exons 32 to 39, in the affected offspring. Ten percent of the father's spermatozoa carried the same NF1 deletion, but the abnormality was not detected in DNA from his lymphocytes. CONCLUSIONS: The presence of the NF1 mutation in 10 percent of the clinically normal father's spermatozoa supports the hypothesis that most germ-line mutations occur in precursors of gametes. In cases of spontaneous mutation, analyzing the specific NF1 mutation in the father's sperm might help in the detection of mosaicism and thus facilitate genetic counseling about further pregnancies.

Novel alleles, hemizygosity and deletions at an Alu-repeat within the neurofibromatosis type 1 (NF1) gene.

Neurofibromatosis type 1 (NF1) (von Recklinghausen) is a common autosomal dominant disorder, characterised by the presence of peripheral neurofibromas, café-au-lait spots and Lisch nodules of the iris. Due to the high mutation rate at the NF1 locus, most patients are expected to have different mutations, limiting molecular analysis and genetic counseling to the identification of the mutation in each patient or family, or to the use of DNA polymorphisms. We have analysed an Alu-repeat polymorphic sequence (AAAT), located in intron 27 of the NF1 gene, in 70 NF1 and 40 CEPH families and we have detected several genetic and molecular abnormalities. In two families the NF1 individuals were hemizygous at the AAAT-repeat and/or at the CA-repeat of intron 27 of NF1, due to interstitial deletions, which include intron 27 to exon 37 of the NF1 gene. A 71-bp deletion at the Alu sequence was detected in non-NF1 chromosomes of members of three NF1 families. New alleles at the AAAT-repeat were found in one NF1 family and in three CEPH families giving a mutation rate for this AAAT-repeat of 0.36% per allele, which is one of the highest detected for a microsatellite locus.

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

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 C----T 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.

[Multiple hamartoma syndrome]. / Síndrome de hamartomas múltiples.

We describe the clinicopathological features of multiple hamartoma and neoplasia syndrome (Cowden's syndrome) in a 58 year-old man and in a 56 year-old woman. The typical mucocutaneous lesions of this disorder were present in both cases. Visceral hamartomas involving thyroid and gastrointestinal system (polyposis) were present in both patients. Dermal or submucous fibromas are proposed as an additional diagnostic criterium of Cowden's syndrome. Associated abnormalities and visceral involvement in multiple hamartoma syndrome are reviewed.

Localized pili canaliculi and trianguli.

A 3-year-old boy presented two patches of light, bright, smooth, uncombable hair that were morphologically different from the rest of the hairs. Examination under scanning electron microscopy showed the morphologic characteristics of pili canaliculi and trianguli. Clinical and ultrastructural features seemed to correspond with a previously undescribed localized form of pili canaliculi et trianguli. Differential diagnosis is discussed.

[Ito's hypomelanosis]. / Hipomelanosis de Ito.

Three additional cases of hypomelanosis of Ito (HI) are reported. HI is a syndrome manifested by irregular macular hypopigmented lesions, resembling the negative image of incontinentia pigmenti. Their cutaneous manifestations, associated abnormalities and the peculiar characteristics of the reported cases are discussed.

Panniculitis in Pseudomonas aeruginosa septicemia.

A 71-year-old man developed multiple subcutaneous nodules during Pseudomonas aeruginosa septicemia. The acute and simultaneous flare of inflammatory nodules in a septic patient appears to be rather specific in Pseudomonas infections. Histological vascular lesions are prominent in the subcutaneous nodules.