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Expansions of short tandem repeats are genetic variants that have been implicated in several neuropsychiatric and other disorders, but their assessment remains challenging with current polymerase-based methods 1-4 . Here we introduce a CRISPR-Cas-based enrichment strategy for nanopore sequencing combined with an algorithm for raw signal analysis. Our method, termed STRique for short tandem repeat identification, quantification and evaluation, integrates conventional sequence mapping of nanopore reads with raw signal alignment for the localization of repeat boundaries and a hidden Markov model-based repeat counting mechanism. We demonstrate the precise quantification of repeat numbers in conjunction with the determination of CpG methylation states in the repeat expansion and in adjacent regions at the single-molecule level without amplification. Our method enables the study of previously inaccessible genomic regions and their epigenetic marks.

In some insect species, females may base their choice for a suitable mate on male odor. In the red mason bee, Osmia bicornis, female choice is based on a male's odor bouquet as well as its thorax vibrations, and its relatedness to the female, a putative form of optimal outbreeding. Interestingly, O. bicornis can be found as two distinct color morphs in Europe, which are thought to represent subspecies and between which we hypothesize that female discrimination may be particularly marked. Here we investigated (i) if these two colors morphs do indeed represent distinct, reproductively differentiated populations, (ii) how odor bouquets of male O. bicornis vary within and between populations, and (iii) whether variation in male odor correlates with genetic distance, which might represent a cue by which females could optimally outbreed. Using GC and GC-MS analysis of male odors and microsatellite analysis of males and females from 9 populations, we show that, in Denmark, an area of subspecies sympatry, the two color morphs at any one site do not differ, either in odor bouquet or in population genetic differentiation. Yet populations across Europe are distinct in their odor profile as well as being genetically differentiated. Odor differences do not, however, mirror genetic differentiation between populations. We hypothesize that populations from Germany, England and Denmark may be under sexual selection through female choice for local odor profiles, which are not related to color morph though which could ultimately lead to population divergence and speciation.

A multifactorial aetiology with genetic and environmental factors is assumed for orofacial clefts. Submucous cleft palate (SMCP), a subgroup of cleft palates with insufficient median fusion of the muscles of the soft palate hidden under the mucosa, has a prevalence of 1:1,250-1:5,000. We described the prevalence of risk factors among 103 German patients with the subtype SMCP and genotyped 24 single nucleotide polymorphisms (SNPs) from 12 candidate genes for orofacial clefts. Analysis of risk factors yielded a positive history for maternal cigarette smoking during pregnancy in 25.2% of the patients, and this was significantly more frequent than in the normal population. The group of patients differed in allele frequencies at SNP rs3917192 of the gene TGFB3 (nominal P = 0.053) and at SNP rs5752638 of the gene MN1 (nominal P = 0.075) compared with 279 control individuals. Our results indicate a potential role of maternal smoking during pregnancy for the formation of SMCP. The analysis of genetic variants hints at the contribution of TGFB3 and MN1 in the aetiology of SMCPs., (© 2012 Eur J Oral Sci.)

Background: Association between genetic variants located on human chromosome 8q24.21 with an increased risk for prostatic carcinoma has been well established. POU5F1P1, a processed pseudogene homologous to the pluripotency factor OCT4, is the only sequence with coding capacity in this region. The objective of this study was to investigate the POU5F1P1 expression in prostatic carcinoma and carcinoma surrounding prostatic tissue., Methods: RT-PCR and real-time PCR was used to measure the expression of POU5F1P1 relative to the expression of HPRT1 in cell lines, prostatic carcinoma and carcinoma surrounding prostatic tissue. The structure of the POU5F1P1 mRNA and the promoter sequence were elucidated by 5'-RACE experiments. The POU5F1P1 protein was shown with immunohistochemistry on prostate tissue., Results: POU5F1P1 was found to be the only member of the POU5F1 family to be expressed in prostate with over-expression in prostatic carcinoma compared to surrounding prostatic tissue probably because of an increased density of expressing cells. The POU5F1P1 expression is driven by a variety of promoter structures scattered over a genomic region of 860 kB., Conclusions: The over-expression of POU5F1P1 in prostatic carcinoma in addition to its genomic location and the putative function of its gene product render POU5F1P1 a good candidate to harbour functional genetic variants which modulate prostatic cancer susceptibility.

The chromosomal region 7q was repeatedly found to be rearranged in prostate carcinoma. It harbors several well described candidate tumor suppressor and oncogenes. We addressed two genes with opposite roles in cancer; CAV1, a putative tumor suppressor gene at 7q31, and EZH2 at 7q36, which is believed to promote tumor progression. Our primary aim was to assess their expression changes in primary tumors, and then to elucidate the underlying mechanism, assuming that genomic alterations of either locus could affect the other gene as well. In 35 prostate tumor samples, compared with adjacent tissues, CAV1 was overall downregulated (P < 10(-06)), whereas EZH2 was significantly overexpressed (P < 10(-06)). The observed dysregulations were coincident in nearly 70% of the cases. Copy number changes occurred in few tumors. Loss of CAV1 DNA was only marginally associated with reduced expression (P = 0.07), however, and genomic amplification of EZH2 could not explain its upregulation. Through bisulfite sequencing of four tumor samples, CpG-hypermethylation was verified as an alternative mechanism for CAV1 silencing, as reported previously. Moreover, it could also be involved in the reactivation of EZH2.

In the sexually deceptive orchid genus Ophrys, reproductive isolation is based on the specific attraction of males of a single pollinator species, mostly bees, by mimicking the female sex pheromone of this species. Changes in the floral odor can lead to hybridization, introgression, and possibly speciation. We investigated hybrid swarms of O. lupercalis and O. iricolor on Sardinia using behavioral, electrophysiological (GC-EAD), chemical, morphological, and genetic methods (AFLPs). In behavioral experiments, approximately 20% of the flowers from both species and hybrids were attractive to the "wrong" or both pollinator species. Analysis of the EAD-active hydrocarbons in the floral odor showed an overlap in the two species, whereby hybrid individuals could not be separated from O. iricolor. The genetic analysis confirmed the hybridization of the species. Plants of O. iricolor and hybrids are genetically indistinguishable and form an O. iricolor × lupercalis hybrid population. Remaining plants of O. lupercalis will possibly be displaced by the O. iricolor × lupercalis hybrid population in the future. Our study showed that in deceptive orchids, variation in the pollinator attracting cues, in this case, scent, can be the first step for speciation and at the same time cause the displacement of a species.

The human genome is composed of long stretches of DNA with distinct GC contents, called isochores or GC-content domains. A boundary between two GC-content domains in the human NF1 gene region is also a boundary between domains of early- and late-replicating sequences and of regions with high and low recombination frequencies. The perfect conservation of the GC-content distribution in this region between human and mouse demonstrates that GC-content stabilizing forces must act regionally on a fine scale at this locus. To further elucidate the nature of these forces, we report here on the spectrum of human SNPs and base pair substitutions between human and chimpanzee. The results show that the mutation rate changes exactly at the GC-content transition zone from low values in the GC-poor sequences to high values in GC-rich ones. The GC content of the GC-poor sequences can be explained by a bias in favor of GC > AT mutations, whereas the GC content of the GC-rich segment may result from a fixation bias in favor of AT > GC substitutions. This fixation bias may be explained by direct selection by the GC content or by biased gene conversion.

The MSR1 gene at 8p22 has been suggested as a candidate gene for hereditary prostate cancer because germline variants have been found to be associated with the disease. Aside from a single nonsense mutation (R293X) that was found repeatedly at low frequencies in several samples, little evidence has been gained by follow-up studies to confirm the gene's relevance for prostate cancer. Prompted by reasonable support for a linkage to 8p22, we sought to determine the mutation spectrum of MSR1 in our family sample. Screening of 139 probands (representing 139 prostate cancer families) revealed 15 novel and a total of 20 sequence variants within the 10 coding exons and their intronic proximities. Aside from the known mutation c.877C>T (R293X) present in two of our families, we identified a second nonsense allele (c.251C>G; S84X) and a splice-site mutation (c.818-1G>A) that results in mRNA instability (each in a single pedigree). The novel missense alleles were c.703C>T (H235Y), c.856C>T (P286S), c.905C>T (P302L), c.1193C>G (A398G), and c.1289A>G (K430R). Of the eight variants that affect the encoded protein (splice site, nonsense, and missense), only R293X as well as the polymorphism c.823C>G (P275A) were additionally present at remarkable frequencies in further samples of sporadic prostate cancer and controls. Of note, carriers of R293X were equally frequent in 367 sporadic prostate cancer cases (1.9%) and in 197 controls (2.0%). To our knowledge, our study is the first to demonstrate further loss of function variants of MSR1 apart from R293X. Nevertheless, the low frequencies of deleterious alleles, in addition to an apparently moderate penetrance, does not support MSR1 as a major susceptibility gene in this family sample., (2005 Wiley-Liss, Inc.)

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.

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.

Neurofibromatosis type 1 (NF1), an autosomal dominantly-inherited disorder, is mainly characterized by the occurrence of multiple dermal neurofibromas and is caused by mutations in the NF1 gene, a tumor suppressor gene. The variable expressivity of the disease and the lack of a genotype/phenotype correlation prevents any prediction of patient outcome and points to the action of genetic factors in addition to stochastic factors modifying the severity of the disease. The analysis of somatic NF1 gene mutations in neurofibromas from NF1 patients revealed that each neurofibroma results from an individual second hit mutation, indicating that factors that influence somatic mutation rates may be regarded as potential modifiers of NF1. A mutational screen of numerous neurofibromas from two NF1 patients presented here revealed a predominance of point mutations, small deletions, and insertions as second hit mutations in both patients. Seven novel mutations are reported. Together with the results of studies that showed LOH as the predominant second hit in neurofibromas of other patients, our results suggest that in different patients different factors may influence the somatic mutation rate and thereby the severity of the disease., (Copyright 2003 Wiley-Liss, Inc.)

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.

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.

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.

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.

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., (Copyright 2000 Academic Press.)

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.

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.

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.

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.

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.

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.