Isochores and replication time zones: a perfect match.

The mammalian genome is not a random sequence but shows a specific, evolutionarily conserved structure that becomes manifest in its isochore pattern. Isochores, i.e. stretches of DNA with a distinct sequence composition and thus a specific GC content, cause the chromosomal banding pattern. This fundamental level of genome organization is related to several functional features like the replication timing of a DNA sequence. GC richness of genomic regions generally corresponds to an early replication time during S phase. Recently, we demonstrated this interdependency on a molecular level for an abrupt transition from a GC-poor isochore to a GC-rich one in the NF1 gene region; this isochore boundary also separates late from early replicating chromatin. Now, we analyzed another genomic region containing four isochores separated by three sharp isochore transitions. Again, the GC-rich isochores were found to be replicating early, the GC-poor isochores late in S phase; one of the replication time zones was discovered to consist of one single replicon. At the boundaries between isochores, that all show no special sequence elements, the replication machinery stopped for several hours. Thus, our results emphasize the importance of isochores as functional genomic units, and of isochore transitions as genomic landmarks with a key function for chromosome organization and basic biological properties.

A comparison of the variability spectra of two genomic loci in a European group of individuals reveals fundamental differences pointing to selection or a population bottleneck.

Knowledge about the variability spectra of neutrally evolving sequences in a population is a prerequisite for the identification of genes, which may have been under positive selection during recent human evolution. Here, we report the results of a re-sequencing project of a presumably neutrally evolving chromosome 22 locus with a severely reduced recombination frequency in a group of 24 individuals of German origin. The comparison of these data with the results of a similar analysis of a chromosome 17 locus revealed striking differences, although the same group of individuals was used. For the chromosome 17 locus two well-separated groups of sequences, a positive value of Tajima's D and a TMRCA of 700,000 years were observed. In contrast, the sequences from the chromosome 22 locus were found to be relatively homogeneous, with no deep splits between subgroups; the obtained value for Tajima's D was negative and the TMRCA was only 260,000 years. These discrepancies may be explained by selection or demographic processes. Regarding demography, the most plausible explanation is the assumption of a severe bottleneck in the history of the European population: in the case of the chromosome 17 locus two ancient lineages passed this bottleneck; for the chromosome 22 locus it was only one ancient lineage.

A candidate gene approach within the susceptibility region PCaP on 1q42.2-43 excludes deleterious mutations of the PCTA-1 gene to be responsible for hereditary prostate cancer.

OBJECTIVE: The Prostate Carcinoma Tumor Antigen-1 (PCTA-1) is located at the prostate cancer susceptibility locus on chromosome 1q42.2-43 (PCaP). In this candidate gene approach, we searched for deleterious mutations within the PCTA-1 gene and its promoter. MATERIALS AND METHODS: Seventy-seven familial prostate cancer cases from 36 German and French pedigrees were screened for germline mutations in the PCTA-1 gene using enzymatic mutation detection (EMD). Putative missense mutations were genotyped by RPLP and ddNTP primer extension assays in 88 controls to assess allele frequencies and haplotypes. RESULTS: Several sequence variants were found but none of the findings indicated a deleterious mutation. Three affected brothers showed an intronic variation, which may interfere with correct splicing. Four non-conservative SNPs were characterized, coding for the amino acid alterations Y19F, C36R, V56M and S184R. All exchanges were found in controls with common allelic frequencies of at least 28%. Haplotype definition including six SNPs within the PCTA-1 gene revealed a complete linkage disequilibrium. Low haplotype diversity leads to a predominance of only two peptide variants of the PCTA-1 protein, coded by 95% of all chromosomes. CONCLUSIONS: PCTA-1 is not a classical high risk gene with deleterious mutations predisposing to hereditary prostate cancer. Its contribution to prostate cancer susceptibility as a low risk factor in sporadic disease has to be assessed in larger samples by association studies.

Long-range sequence composition mirrors linkage disequilibrium pattern in a 1.13 Mb region of human chromosome 22.

Association studies, the most powerful tool for the identification of genes underlying complex traits, depend on the observation of linkage disequilibrium (LD) between marker alleles and the trait. The LD pattern of the human genome which determines the regional density of required markers is non-uniform, with regions of long-range LD over several hundred kilobases and regions where LD extends only over a few kilobases. Studying LD in the NF1 gene region we encountered a transition from long-range to short-range LD which coincides with a switch in the isochore pattern. This observation prompted us to investigate the regional variation in the extent of LD more systematically and we selected an isochore transition within the MN1/PITPNB gene region on chromosome 22q12.1. Long-range LD characterizes the GC-poor (40% GC) parts of the sequences. No LD can be observed between closely spaced markers throughout the whole range of the GC-rich (50% GC) parts. In both cases, the NF1 and the MN1/PITPNB gene region, a clear-cut transition of the long-range GC content precisely coincides with a change in the extent of observable LD. The results can be explained by a 72-fold lower recombination frequency in the GC-poor, compared to the GC-rich isochores. Although recombination is not the only factor governing LD, our findings can help to predict levels of LD and marker densities required for association studies on the basis of regional GC content.

Spinal neurofibromatosis without café-au-lait macules in two families with null mutations of the NF1 gene.

Spinal neurofibromatosis (SNF) is considered to be an alternative form of neurofibromatosis, showing multiple spinal tumors and café-au-lait macules. Involvement of the neurofibromatosis type 1 (NF1) locus has been demonstrated, by linkage analysis, for three families with SNF. In one of them, a cosegregating frameshift mutation in exon 46 of the NF1 gene was identified. In the present study, we report four individuals from two families who carry NF1 null mutations that would be expected to cause NF1. Three patients have multiple spinal tumors and no café-au-lait macules, and the fourth has no clinical signs of NF1. In the first family, a missense mutation (Leu2067Pro) in NF1 exon 33 was found, and, in the second, a splice-site mutation (IVS31-5A-->G) enlarging exon 32 by 4 bp at the 5' end was found. The latter mutation has also been observed in an unrelated patient with classical NF1. Both NF1 mutations cause a reduction in neurofibromin of approximately 50%, with no truncated protein present in the cells. This demonstrates that typical NF1 null mutations can result in a phenotype that is distinct from classical NF1, showing only a small spectrum of the NF1 symptoms, such as multiple spinal tumors, but not completely fitting the current clinical criteria for SNF. We speculate that this phenotype is caused by an unknown modifying gene that compensates for some, but not all, of the effects caused by neurofibromin deficiency.

An isochore transition in the NF1 gene region coincides with a switch in the extent of linkage disequilibrium.

Whole-genome association studies will be a powerful tool to identify genes responsible for common human diseases. A crucial task for association-mapping studies is the evaluation of the relationship between linkage disequilibrium (LD) and physical distance for the genomic region under study. Since it is known that the extent of LD is nonuniformly distributed throughout the human genome, the required marker density has to be determined specifically for the region under study. These regions may be related to isochores and chromosomal bands, as indicated by earlier cytogenetic findings concerning chiasma distribution in meiosis. Therefore we analyzed the neurofibromatosis type 1 (NF1) gene region on chromosome 17q11.2, which is characterized by a nonuniform LD pattern and an L1-to-H2 isochore transition. Long-range LD within the NF1 gene was found to extend over 200 kb (D' = 0.937) in the L1 isochore, whereas, in the neighboring H2 isochore, no LD is apparent between markers spaced by 26 kb (D' = 0.144). Recombination frequencies derived from the LD are at.00019 (high LD) and.01659 (low LD) per megabase, the latter identical to the average value from segregation analysis. The boundary between these regions coincides precisely with a transition in the GC content of the sequences, with low values (37.2%) in the region with long-range LD and high values (51%) in the other. Our results suggest a correlation between the LD pattern and the isochores, at least in the NF1 region. If this correlation can be generalized, the marker densities required for association studies have to be adjusted to the regional GC content and may be chosen according to the isochores.

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.

Tumor antigen HuR binds specifically to one of five protein-binding segments in the 3'-untranslated region of the neurofibromin messenger RNA.

3'-untranslated regions of various mRNAs have been shown to contain sequence motifs which control mRNA stability, translatability, and efficiency of translation as well as intracellular localization. We aimed to identify protein binding regions of the long and highly conserved 3'UTR of the mRNA coding for neurofibromin, a well-known tumor suppressor protein, whose genetic deficiency causes the autosomal dominant disease neurofibromatosis type 1 (NF1). We discovered five RNA fragments that were able to undergo specific binding to proteins from cell lysates (NF1-PBRs, NF1-protein-binding regions). Additionally we identified the Elav-like protein HuR binding to NF1-PBR1. HuR interacts with AU-rich elements in the 3'UTR of many protooncogenes, cytokines, and transcription factors, thereby regulating the expression of these mRNAs on the posttranscriptional level. Transfection assays with a CAT reporter construct revealed reduced expression of the reporter, suggesting that HuR may be involved in the fine-tuning of the expression of the NF1 gene.

Characterization of an alphoid subfamily located near p-arm sequences on human chromosome 22.

The centromeric heterochromatin of all human chromosomes is composed of tandemly repeated alpha satellite DNA. Here we describe another alphoid subfamily that maps to human chromosome 22 as determined by FISH. The alphoid sequences were isolated from three YAC-clones carrying DNA from the pericentromeric region of the short arm of human chromosome 22 and limited amounts of alphoid DNA. This property enabled us to map the members of the subfamily to the border of the centromeric region and the short arm of the chromosome. The new alphoid subfamily may contribute to the closure of the gap remaining between the centromeric and short-arm maps of human chromosome 22.

Mapping of members of the low-copy-number repetitive DNA sequence family chAB4 within the p arms of human acrocentric chromosomes: characterization of Robertsonian translocations.

Members of the long-range, low-copy-number repetitive DNA sequence family chAB4 are located on nine different human chromosome pairs and the Y chromosome, i.e. on the short arms of all the acrocentrics. To localize the chAB4 sequences more precisely on the acrocentrics, chAB4-specific probes together with rDNA and a number of satellite sequences were hybridized to metaphase chromosomes of normal probands and of carriers of Robertsonian translocations of the frequent types rob(13q14q) and rob(14q21q). The results demonstrate that chAB4 is located on both sides of the rDNA on all the acrocentrics; the exact location, however, may be chromosome specific. Chromosome 22, most probably, is the only chromosome where chAB4 is found in the direct neighbourhood of the centromere. Fluorescence in situ hybridization analyses of metaphase chromosomes of carriers of rob(21q22q) revealed breakpoint diversity for this rare type of Robertsonian translocation chromosome. A direct involvement of chAB4 sequences in recombination processes leading to the Robertsonian translocations analysed in this study can be excluded.

Concerted evolution of members of the multisequence family chAB4 located on various nonhomologous chromosomes.

During the last years it became obvious that a lot of families of long-range repetitive DNA elements are located within the genomes of mammals. The principles underlying the evolution of such families, therefore, may have a greater impact than anticipated on the evolution of the mammalian genome as a whole. One of these families, called chAB4, is represented with about 50 copies within the human and the chimpanzee genomes and with only a few copies in the genomes of gorilla, orang-utan, and gibbon. Members of chAB4 are located on 10 different human chromosomes. FISH of chAB4-specific probes to chromosome preparations of the great apes showed that chAB4 is located, with only one exception, at orthologous places in the human and the chimpanzee genome. About half the copies in the human genome belong to two species-specific subfamilies that evolved after the divergence of the human and the chimpanzee lineages. The analysis of chAB4-specific PCR-products derived from DNA of rodent/human cell hybrids showed that members of the two human-specific subfamilies can be found on 9 of the 10 chAB4-carrying chromosomes. Taken together, these results demonstrate that the members of DNA sequence families can evolve as a unit despite their location at multiple sites on different chromosomes. The concerted evolution of the family members is a result of frequent exchanges of DNA sequences between copies located on different chromosomes. Interchromosomal exchanges apparently take place without greater alterations in chromosome structure.

A third neurofibromatosis type 1 (NF1) pseudogene at chromosome 15q11.2.

Sequences related to the neurofibromatosis type 1 (NF1) gene have been identified on several human chromosomes. In the centromeric region of chromosomes 14 and 15, two NF1 pseudogenes have been described. Sequence comparison between NF1-related exons amplified from two yeast artificial chromosome clones hybridizing to chromosomal region 15q11.2 and published NF1-related sequences localized at 15q11.2 suggested that a third NF1 pseudogene resides in this chromosomal region. The previous localization of an NF1-related locus to the telomeric part of chromosome 15 could not be confirmed by us. Our findings further support pericentromeric spreading of partial NF1 gene copies at chromosome 15q11.2 during evolution.

Evidence for the presence of the second allele of the neurofibromatosis type 1 gene in melanocytes derived from café au laitmacules of NF1 patients.

Neurofibromatosis type 1 (NF1) is an autosomal dominantly inherited disorder caused by mutations in the NF1 gene on 17q11.2. Melanocytes cultured from cafe au lait macules (CALM) of patients with NF1 were analysed for loss of heterozygosity (LOH) at the NF1 locus using a 3'-flanking and four intragenic markers. None of the informative samples showed LOH. In addition, the X-inactivation pattern of melanocytes from CALM (n = 4) and from the unaffected skin of the patients (n = 3) suggests a monoclonal origin of the cells isolated from skin biopsies up to 2 cm2 in size.

Mutational analysis and expression studies of the neurofibromatosis type 2 (NF2) gene in a patient with a ring chromosome 22 and NF2.

The case of a seriously disabled and retarded female patient with neurofibromatosis type 2 (NF2) is reported. She suffered from bilateral vestibular schwannomas, multiple intracranial meningiomas and neurinomas. The constitutional karyotype of the patient was 46, XX, r(22)/45,XX,-22. A constitutional G to A transition in the proximal 3' untranslated region of isoforms 1 and 2 was identified in the patient's NF2 gene and shown not to affect differential splicing or mRNA stability. The instability of the ring chromosome 22 with the associated loss of tumor suppressor genes on chromosome 22, in particular the loss of the NF2 gene, are assumed to have caused multiple tumorigenesis in this patient.

Clonal origin of tumor cells in a plexiform neurofibroma with LOH in NF1 intron 38 and in dermal neurofibromas without LOH of the NF1 gene.

LOH at the NF1 locus was investigated in 38 neurofibromas of 26 NF1 patients. Only in one of these tumors LOH was observed. In this plexiform neurofibroma of a NF1 patient with a constitutional one base-pair insertion in NF1 exon 4b, a non-random X-inactivation pattern was found, strongly suggesting a clonal origin of the tumor cells. The analysis of X-inactivation patterns allowed the classification of some of the other neurofibromas with regard to the detectability of clonal LOH. In 3 of 6 neurofibromas without LOH amenable to this analysis, a comparable X-inactivation pattern was found in constitutional and neurofibroma derived DNA. A clonal LOH would not have been detected in these tumors. However, we observed a nonrandom pattern in 3 of the 6 neurofibromas, suggesting a clonal origin of the tumor cells. LOH was not detected in these tumors, but could, however, have occurred by mutational events below the level of large somatic deletions, loss of a whole chromosome 17 or somatic recombination.

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.

A palindromic structure in the pericentromeric region of various human chromosomes.

The primate-specific multisequence family chAB4 is represented with approximately 40 copies within the haploid human genome. Former analyis revealed that unusually long repetition units ( > 35 kb) are distributed to at least eight different chromosomal loci. Remarkably varying copy-numbers within the genomes of closely related primate species as well as the existence of human specific subfamilies, which most probably arose by frequent sequence exchanges, demonstrate that chAB4 is an unstable genomic element, at least in an evolutionary sense. To analyze the chAB4 basic unit in more detail we established a cosmid contig and found it to be organized as inverted duplications of approximately 90 kb flanking a noninverted core sequence of approximately 60 kb. FISH as well as the analysis of chromosome-specific hybrid cell lines revealed a chromosomal localization of chAB4 on chromosomes 1, 3, 4, 9, Y, and the pericentromeric region of all acrocentrics. Furthermore, we can detect chAB4 sequences together with alpha satellites, beta satellites, and satellite III sequences within a single chromosome 22-specific YAC clone, indicating that chAB4 is located in close proximity to the centromere, at least on the acrocentrics. Hence, chAB4 represents an unstable genomic structure that is located just in the chromosomal region that is very often involved in translocation processes.

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.

On unequal allelic expression of the neurofibromin gene in neurofibromatosis type 1.

The autosomal dominantly inherited disease neurofibromatosis type 1 (NF1) is caused by mutations of a large gene comprising 59 exons, which code for a protein with 2818 amino acids called neurofibromin. Employing an expressed polymorphic site in exon 5 of the neurofibromin gene, the expression of its alleles was analysed quantitatively by scanning radioactive RT-PCR fragments of this exon prepared from the RNA of fibroblast cell cultures from 15 NF1 patients and of white blood cells from one NF1 patient. Thirteen of the RNA preparations yielded unequal amounts of the allelic messages. The deviations of the expression ratios (A2:A1) from 1.0 ranged from -0.9 to +25.8. The allelic messages were equally represented in the RNA preparations from five informative healthy donors. Apart from fibroblasts this phenomenon could also be detected in keratinocytes, melanocytes from normally pigmented skin and melanocytes from a café-au-lait spot of one patient. Only one of three patients affected by stop mutations exhibited unequal allelic expression. When nuclear RNA from 10 of the 13 patients was examined, equal amounts of the primary transcripts were found (average ratio A2/A1: 1.08 +/- 0.07 S.E.M.), indicating that unequal expression on the level of mRNA was not caused by mutations affecting transcriptional regulation. The ratio of the amount of neurofibromin to that of p120 GAP did not seem to be correlated with the extent of unequal allelic expression.

Analysis of an alternatively spliced exon of the neurofibromatosis type 1 gene in cultured melanocytes from patients with neurofibromatosis 1.

Neurofibromatosis type 1 (NF1) is characterized by clinical features that primarily affect tissues derived from the neural crest (neurofibromas, café-aulait macules). Because aberrant regulation of alternative splicing in the NF1 gene transcript may be of functional significance, cultured melanocytes from café-aulait macules (CALM), as an example of benign NF1 lesions, were examined for the expression of the different alternative splice products of this gene. Both kinds of NF1 messengers (type 1 and 2) were found not only in CALM melanocytes but also in keratinocytes, fibroblasts and blood cells. Except in blood cells, there was a predominance of the type 2 transcript. Melanocytes from NF1 patients and healthy donors showed similar expression patterns under several culture conditions. Our results suggest that the development of CALM does not correlate with a switch in the ratio of type 1 to type 2 NF1 messenger RNA.