The size of small polydisperse circular DNA (spcDNA) in angiofibroma-derived cell cultures from patients with tuberous sclerosis (TSC) differs from that in fibroblasts

Summary Cell cultures were derived from angiofibromas of three patients with tuberous sclerosis (TSC), from the unaffected skin of these patients, and from the skin of five healthy donors. The length distributions of the small polydisperse circular DNA (spcDNA) fraction of these cell cultures were then analyzed. Nearly half the spcDNA molecules from the angiofibroma cultures were longer than 0.4 m, whereas only about 7% exceeded this threshold in the spcDNA preparations from the skin fibroblast cultures. The percentage of the larger size class of spcDNA showed an increase at higher numbers of in vitro passages in all three types of cultures, but this effect was much more conspicuous in the angiofibroma-derived cultures than in those from the skin fibroblasts. An age-dependent increase in the overall amount of spcDNA was only seen in the angiofibroma-derived cultures. Our earlier finding of elevated amounts of spcDNA in angiofibroma cultures was confirmed in cultures from an additional TSC patient.     

A small deletion and an adjacent base exchange in a potential stem-loop region of the neurofibromatosis 1 gene

Summary A single-strand conformational polymorphism found in the DNA of a patient with neurofibromatosis 1 (NF1) was shown to be caused by a deletion of a CCACC or CACCT sequence and an adjacent transversion, located about 500 base pairs downstream from the region that codes for a functional domain of the NF1 gene product. This mutation could also be detected in the patient and in his affected daughter by heteroduplex analysis. The deletion removes the proximal half of a small potential stem-loop and interrupts the reading frame in exon 1. A severely truncated protein with a grossly altered carboxy terminus lacking one third of its sequence is expected to be formed from the mutant allele.     

Detection of a restriction site polymorphism within the human α-globin gene complex

Summary A mutant RsaI restriction endonuclease site of high frequency has been identified in individuals of German, Greek, Italian, and Turkish origin. The mutation was found within the -globin gene complex and is located 0.7 kb 5 to the -globin gene. In individuals of central European origin 34 out of 58 chromosomes exhibited the -gene linkage to the presence of the polymorphic site, and thus a preliminary estimate of the gene frequency for this allele would be 0.59. DNA analysis data of individuals derived from Mediterranean populations indicate a distribution of this polymorphic marker in similar frequencies.     

An RsaI polymorphism in the transcribed region of the neurofibromatosis (NF1)-gene

We describe an RsaI polymorphism in the transcribed region (exon 5) of the neurofibromatosis type 1 (NF1)-gene     

Analysis of human extrachromosomal DNA elements originating from different β-satellite subfamilies

By screening total human DNA with probes derived from the small polydisperse circular (spc) DNA fraction of cultured human cells, we identified three clones that carry long stretches of -satellite DNA. Further experiments have shown that the three sequences belong to at least two different -satellite subfamilies, which are characterized by different higher order subunits. Members of one of these subfamilies are located in the cytological satellites of all acrocentric chromosomes, whereas members of another are located on the short arms of the acrocentrics on both sides of the stalk regions and also in the centromeric regions of chromosomes 1 and 9. This is the first time that -satellite sequences obtained from the spcDNA of human cells have been assigned to -satellite subfamilies that are organized as long arrays of tandemly arranged higher order monomers. This indicates that -satellite sequences can be excised from their chromosomal loci via intrastrand-recombination processes.This paper is dedicated to Prof. Dr. Ulrich Wolf on his 60th birthday, January 1993     

Toward a survey of somatic mutation of the NF1 gene in benign neurofibromas of patients with neurofibromatosis type 1

Neurofibromatosis type 1 (NF1), a common autosomal dominant disorder caused by mutations of the NF1 gene, is characterized by multiple neurofibromas, pigmentation anomalies, and a variety of other possible complications, including an increased risk of malignant neoplasias. Tumorigenesis in NF1 is believed to follow the two-hit hypothesis postulated for tumor-suppressor genes. Loss of heterozygosity (LOH) has been shown to occur in NF1-associated malignancies and in benign neurofibromas, but only few of the latter yielded a positive result. Here we describe a systematic approach of searching for somatic inactivation of the NF1 gene in neurofibromas. In the course of these studies, two new intragenic polymorphisms of the NF1 gene, a tetranucleotide repeat and a 21-bp duplication, could be identified. Three tumor-specific point mutations and two LOH events were detected among seven neurofibromas from four different NF1 patients. Our results suggest that small subtle mutations occur with similar frequency to that of LOH in benign neurofibromas and that somatic inactivation of the NF1 gene is a general event in these tumors. The spectrum of somatic mutations occurring in various tumors from individual NF1 patients may contribute to the understanding of variable expressivity of the NF1 phenotype.     

Genetic variability in a genomic region with long-range linkage disequilibrium reveals traces of a bottleneck in the history of the European population

The inference of the demographic history of populations from genetic variability data is not only of academic interest. It also provides background information for the identification of genes which may have played a role in human evolution or in the aetiology of human disease. To obtain a clear picture of this background, it is necessary to compare data obtained from a number of genomic loci. Due to its very low recombination rate, the NF1 gene region can be regarded as a further suitable locus. A combined resequencing and SNP typing project in a European population disclosed the presence of only two well separated subgroups of NF1 sequences. Statistical analysis revealed a bimodal distribution of the pairwise differences, a positive value of Tajima’s D and a TMRCA of 700,000 years for the whole sample, and pairwise differences indicative for a growing population and TMRCAs of 130,000 to 150,000 years for the subgroups. Together, the data lead to a model that the recent European population went through a bottleneck during the last 150,000 years of its history. Regarding the given timeframe, this bottleneck could either reflect a speciation event which led to the anatomically modern human (AMH), or a severe reduction of the population size during the emigration of AMHs out of Africa or the immigration into Europe.     

An isochore transition zone in the NF1 gene region is a conserved landmark of chromosome structure and function

The mammalian genome is organized as a mosaic of isochores, stretches of DNA with a distinct sequence composition. Isochores form the basis of the chromosomal banding pattern, which is tightly correlated with a number of structural and functional features. We have recently demonstrated that the transition from a GC-poor isochore to a GC-rich one in the NF1 gene region occurs within 5 kb and demarcates genomic regions with high and low recombination frequency. We now report that the same transition zone separates early replicating from late replicating chromatin on the molecular level. At the isochore transition the replication fork is stalled in mid-S phase and can be visualized by fiber-FISH techniques as a Y-shaped structure. The switch in GC content and in replication timing is conserved between human and mouse, emphasizing the importance of the transition zones as landmarks of chromosome organization and function.     

The second case of a t(17;22) in a family with neurofibromatosis type 1: sequence analysis of the breakpoint regions

A reciprocal t(17;22)(q11.2;q11.2) was found in a female patient with neurofibromatosis type 1 (NF1) and in her affected daughter. Sequence analysis of cloned junction fragments traversing the breakpoints allowed the identification of the structures involved in the rearrangement. Aberrant bands in Southern hybridizations of restriction enzyme-digested DNA of the patient pointed to the disruption of the NF1 gene in intron 31. Semispecific polymerase chain reaction analysis of the genomic DNA of the patient with the specific primer anchored at NF1 exon 31 was used to obtain the breakpoint-spanning fragment of the derivative chromosome 17. The intron 31 sequence turned out to be interrupted within a large irregular (AT) repeat. The chromosome 22-derived sequence of the der(17) junction fragment allowed us to identify cosmids of the corresponding region from a chromosome 22-specific cosmid library. With the support of the breakpoint-spanning cosmids, the chromosome 22 region upstream of the fragment carried by the der(17) was characterized. Primers deduced from the sequence of this upstream region were used in combination with a primer in NF1 intron 31 distal to the breakpoint on chromosome 17 to amplify the der(22) junction fragment. The structure of the junction sequences suggested that the translocation had arisen by unequal homologous recombination between (AT)-rich repeats on chromosome 22 and on chromosome 17 in intron 31 of the NF1 gene. However, our data support the assumption of additional rearrangements prior to, or in the course of, the recombination event, leading to a loss of the sequences between the involved (AT) repeats on chromosome 22. In the direct vicinity of these (AT) repeats, two members of a previously undescribed low-copy repetitive sequence have been found, copies of which are also present on human chromosome 13. Received: 27 August 1996 / Revised: 7 October 1996     

Single-cell PCR performed with neurofibroma Schwann cells reveals the presence of both alleles of the neurofibromatosis type 1 (NF1) gene

It is commonly held that Schwann cells (SC) are the progenitor cells of benign neurofibromas. To test for loss of heterozygosity (LOH) at the neurofibromatosis 1 (NF1) gene locus, three intragenic polymorphic markers were analyzed after polymerase chain reaction amplification, starting from 98 single SC isolated from primary cultures of neurofibromas of five informative NF1 patients. The patterns obtained did not provide evidence for LOH at the NF1 gene. LOH by nondisjunction, large deletions, or somatic recombination in SC seems not to be the mechanism of generation of neurofibromas.