The structure and paramutagenicity of the R-marbled haplotype of Zea mays.

Paramutation is the meiotically heritable silencing of a gene that can occur in particular heterozygous combinations. The R-marbled (R-mb) haplotype is paramutagenic: it causes paramutable r1 haplotypes like R-r to become heritably silenced. R-mb was found to comprise three distinct r1 genes arranged as direct repeats. The most distal gene of R-mb, Scm, contains a novel transposable element, Shooter (Sho). Excision of the Sho element early in aleurone development results in the characteristic "marbled" aleurone pigmentation pattern conferred by R-mb. The effect of gene copy number on the paramutagenic strength of R-mb was tested. Paramutagenic strength of R-mb is directly correlated with r1 gene copy number. Paramutagenic strength of R-mb is directly correlated with r1 gene copy number. Paramutagenic strength of R-mb was not affected by removal, through crossing over, of the Sho transposon. Finally, R-mb does not appear to contain the transposable element, Doppia, which is associated with paramutability of R-r, and has been suggested to play a role in paramutagenicity of another paramutagenic haplotype, R-stippled.     

Molecular Organization and Germinal Instability of R-Stippled Maize

The spotted seed allele R-stippled (R-st) is comprised of the following genetic components: strong seed color (Sc), inhibitor-of-R (I-R) and near-colorless seed (Nc). I-R is a mobile element that represses (Sc) expression irregularly. Germinal I-R losses produce progeny with fully colored seed. Southern blot analysis revealed four r-hybridizing segments in R-st and three, two or one in two sets of unequal crossover deletion products. By comparison to published reports of r gene structure, we maintain that each segment contains at least one r gene. The proximal r gene, Sc, confers strong seed color; the three distal r genes together produce near-colorless seed. R-st's seed spotting phenotype is correlated with the presence of a 3.3-kb insert in Sc identified as I-R. The level of the near-colorless phenotype is inversely correlated with the number of r genes present, suggesting involvement of a multiple copy silencing mechanism in their regulation. Phenotypic changes in R-st occurred primarily by unequal exchange between r genes. The locations of exchange positions showed a strong polarity, nearly all occurring in the 3' portions of the identified r genes.     

Structural features and methylation patterns associated with paramutation at the r1 locus of Zea mays.

In paramutation, two alleles of a gene interact and, during the interaction, one of them becomes epigenetically silenced. The various paramutation systems that have been studied to date exhibit intriguing differences in the physical complexity of the loci involved. B and Pl alleles that participate in paramutation are simple, single genes, while the R haplotypes that participate in paramutation contain multiple gene copies and often include rearrangements. The number and arrangement of the sequences in particular complex R haplotypes have been correlated with paramutation behavior. Here, the physical structures of 28 additional haplotypes of R were examined. A specific set of physical features is associated with paramutability (the ability to be silenced). However, no physical features were strongly correlated with paramutagenicity (the ability to cause silencing) or neutrality (the inability to participate in paramutation). Instead, paramutagenic haplotypes were distinguished by high levels of cytosine methylation over certain regions of the genes while neutral haplotypes were distinguished by lack of C-methylation over these regions. These findings suggest that paramutability of r1 is determined by the genetic structure of particular haplotypes, while paramutagenicity is determined by the epigenetic state.     

Characterization of copy number mutants of plasmid pSC101

We have characterized three copy number mutants of the plasmid pSC101. These mutations caused single amino acid substitutions at the 46th, 83rd and 115th codons in the rep gene and an increase in the copy number by 4- to 8-fold. Although the in vivo and in vitro repressor activities of these mutated Rep proteins were quite different from each other, the intracellular concentrations of the proteins were maintained at higher levels than the wild-type protein. It has been reported that excess amounts of Rep inhibit pSC101 replication (Ingmer and Cohen, 1993). This inhibitory activity of Rep was markedly decreased in all three mutants. When both the wild-type and one of the mutated rep genes were retained in the same plasmids, the copy number of these plasmids was decreased compared with plasmids retaining a single mutated rep gene. These results support the theory that the inhibitory activity of Rep for its own replication plays an important role in copy number regulation.     

The impact of intragenic recombination on phylogenetic reconstruction at the sectional level in Eucalyptus when using a single copy nuclear gene (cinnamoyl CoA reductase)

Low copy number nuclear genes have been found to be useful for phylogenetic reconstruction at different taxonomic levels. This study investigated the utility of a single copy gene, cinnamoyl CoA reductase (CCR), for resolving phylogenetic relationships at the sectional level within Eucalyptus. The monophyly of sections Exsertaria and Latoangulatae was explored, using section Maidenaria as an outgroup, and the impact of intragenic recombination on phylogenetic reconstruction examined. Phylogenetic analysis did not resolve monophyletic groups. Latoangulatae and Maidenaria were polyphyletic or paraphyletic. Exsertaria species formed a clade but included a single Latoangulatae species (E. major). Recombination analysis identified two intragenic recombination events that involved species from different sections, which have probably been facilitated by inter-sectional hybridisation. One of the events most likely occurred prior to speciation, with several Latoangulatae species having the recombinant allele. The other event may have occurred after speciation, since only one of two E. globulus samples possessed the recombinant allele. This is the first detailed report of intragenic recombination in both CCR and Eucalyptus, and between species of different sections of a plant genus. The occurrence of intragenic recombination may explain the anomalous positions of some species within the phylogenetic tree, and indicates that phylogenetic analysis of Eucalyptus using nuclear genes will be problematic unless recombination is taken into account.     

DNA copy number analysis of the DFNB1 hereditary hearing loss locus

Recessive mutations in the GJB2 gene and large deletions of the cis-regulatory element of this gene are the main causes of congenital nonsyndromic sensorineural hearing loss in many countries, including Russia. Large deletions represent 0.3–10% of all alleles in the DFNB1 locus in different populations and are usually observed in compound heterozygous state with intragenic mutations or are rarely observed in the homozygous or compound-heterozygous state with another large deletion. According to published studies, six large deletions exist, including three frequent deletions del(GJB6-D13S1830), del(GJB6-D13S1854), and del(GJB2-D13S175) and three rare deletions observed in single cases. The present study describes the results of the copy number analysis of the GJB2 regulatory region for the detection of unknown deletions in patients with a single heterozygous recessive intragenic mutation. Additionally, a quantitative analysis of GJB2 and GJB6 gene sequences in individuals bearing homozygous mutation in the GJB2 gene, which might also have mutation in the hemizygous state, is performed. The system for quantitative analysis of the region including the regulatory element of the GJB2 gene based on the MLPA® approach is developed. Moreover, a commercial kit of reagents is used for the detection of copy number of the GJB2 and GJB6 genes by the same method. As a result of the conducted analysis, no changes in copy number are detected in the explored regions. Obviously, if Russian patients have mutations in unidentified regulatory or other regions of the DFNB1 locus, frequency of such unidentified mutations is extremely rare.     

Control of replication of FII plasmids: comparison of the basic replicons and of the copB systems of plasmids R100 and R1

The copy numbers of the FII plasmids R1 and R100 were determined in four different ways and found to be identical. Deletion of one of the copy number control genes, copB, together with its promoter gives rise to plasmid copy mutants with an increased copy number. The increase was found to be 8- and 3.5-fold for plasmids R1 and R100, respectively. These deletion derivatives were found to be extremely sensitive to the presence of CopB activity from their own parent plasmid but not to that of the other plasmid. Hence, the CopB protein and its target are plasmid-specific and not FII-group-specific. These results are consistent with the high degree of nonhomology between plasmids R1 and R100 in a 250-bp region covering the distal part of the copB gene and the repA promoter region, which contains the target for the CopB protein.     

Frequent alterations of visual pigment genes in adrenoleukodystrophy.

Both adrenoleukodystrophy (ALD) and red/green color blindness have been mapped to the distal long arm of the human X chromosome (Xq28). Color-vision defects are frequently associated with ALD, and study of the red and green visual pigment genes in eight ALD kindreds has shown frequent structural changes including deletions and possible intragenic recombinations. Such changes may reflect chromosomal events underlying both ALD and the associated visual defects and should help define both the structural gene responsible for ALD and physical genetic relationships in the Xq28 region.     

Multiplex real-time PCR for detection of deletions and duplications in dystrophin gene

Genetic testing of Duchenne and Becker muscular dystrophies (DMD/BMD) is a difficult task due to the occurrence of deletions or duplications within dystrophin (DMD) gene that requires dose sensitive tests. We developed three multiplex quantitative real-time PCR assays for dystrophin exon 5, 45, and 51 within two major hotspots of deletion/duplication. Each exon was co-amplified with a reference X-linked gene and the copy number of the target fragment was calculated by comparative threshold cycle method (delta deltaC(t)). We compared the performance of this method with previously described end-point PCR fluorescent analysis (EPFA) by studying 24 subjects carrying DMD deletions or duplications. We showed that Q-PCR is an accurate and sensitive technique for the identification of deletions and duplications in DMD/BMD. Q-PCR is a valuable tool for independent confirmation of EPFA screening, particularly when deletions/duplications of single exons occur or for rapid identification of known mutations in at risk carriers.     

Mouse Models of Genomic Syndromes as Tools for Understanding the Basis of Complex Traits: An Example with the Smith-Magenis and the Potocki-Lupski Syndromes

Each human''s genome is distinguished by extra and missing DNA that can be “benign” or powerfully impact everything from development to disease. In the case of genomic disorders DNA rearrangements, such as deletions or duplications, correlate with a clinical specific phenotype. The clinical presentations of genomic disorders were thought to result from altered gene copy number of physically linked dosage sensitive genes. Genomic disorders are frequent diseases (~1 per 1,000 births). Smith-Magenis syndrome (SMS) and Potocki-Lupski syndrome (PTLS) are genomic disorders, associated with a deletion and a duplication, of 3.7 Mb respectively, within chromosome 17 band p11.2. This region includes 23 genes. Both syndromes have complex and distinctive phenotypes including multiple congenital and neurobehavioral abnormalities. Human chromosome 17p11.2 is syntenic to the 32-34 cM region of murine chromosome 11. The number and order of the genes are highly conserved. In this review, we will exemplify how genomic disorders can be modeled in mice and the advantages that such models can give in the study of genomic disorders in particular and gene copy number variation (CNV) in general. The contributions of the SMS and PTLS animal models in several aspects ranging from more specific ones, as the definition of the clinical aspects of the human clinical spectrum, the identification of dosage sensitive genes related to the human syndromes, to the more general contributions as the definition of genetic locus impacting obesity and behavior and the elucidation of general mechanisms related to the pathogenesis of gene CNV are discussed.Key Words: Gene copy number variation, complex traits, phenotypic consequences, mouse models.     

Extrachromosomal Element Delta in DROSOPHILA MELANOGASTER. IX. Induction of Delta-Retaining Chromosome Lines by Mutation and Gene Mapping

[Delta b], symbolized as [delta(b)], is retained by S(b) chromosome lines and transmitted through the females to their progeny. Transmission through the males is not directly demonstrable (Minamori 1969a). [delta r], symbolized as [delta(r)], is retained by S(r) chromosome lines and transmitted biparentally (Minamori 1971). The multiplication of delta is suppressed at low temperature. All descendant lines derived from S(b)-carrying or S(r)-carrying flies in which the presence of delta cannot be demonstrated gradually accumulate their specific delta factors over many generations (Minamori 1969b, 1972). The delta factors and the sensitive chromosomes are inseparably associated. This observation led to the assumption that delta may be a copy of a chromosomal gene or a certain agent integrated into the chromosome (Minamori 1972). This assumption was examined in the present study by experiments designed to induce delta-retaining sensitive chromosomes, and to map the gene(s) responsible for delta-retention and/or for sensitivity to the killing action of delta factor. One sensitive chromosome which retained [delta(b)] (S(b) chromosome) was obtained in the presence of [delta(b)] out of 2492 insensitive chromosomes which retained no delta; in addition one S(b) chromosome was obtained in the presence of [delta(r)] out of 2131 insensitives. The latter finding suggests that S(b) might be induced by a mutation caused by [delta(b)] or [delta(r)], but not by integration of either delta into the chromosome. Four S(b) chromosomes and one sensitive chromosome which retained [delta(r)] (S(r) chromosome) were obtained out of 1970 insensitives when males carrying the chromosome were fed an alkylating mutagen, ethyl methane sulfonate (EMS). The location of delta-retaining genes was examined by crossing-over experiments employing eight S(b) and five S(r) chromosomes. The genes on these chromosomes were found to be located in the same region or near one another. The gene for [delta(b)], symbolized as Da(b), and the gene for [delta(r)], symbolized as Da(r), are assumed to be multiple alleles of a locus at 2-24.9. The sensitivity of the chromosomes was modified appreciably by recombination; hence, the genes controlling this trait are assumed to be a polygenic system. The findings obtained in this study lead to the hypothesis that delta may be produced by a chromosomal gene (Da) and transmitted extrachromosomally.     

Copy number of DAZ genes in Slovenian and Bosnian general population

Deletions of two of four DAZ (Deleted in AZoospermia) gene copies located on the Y chromosome were associated with spermatogenic failure, but the information on DAZ copy number is still very scarce. The aim of this study was to determine the frequency of partial DAZ gene deletions and to analyze the existence of duplications in general Slovenian and Bosnian population. To answer these questions, we used real time PCR. We analyzed 100 male samples from Slovenian and Bosnian general population. The incidence of two DAZ gene copies was 6% (3/50) in Slovenian population. The incidence of more than four DAZ genes was 2% (1/50) in Slovenian population and 8% (4/50) in Bosnian population. Observed differences have not reached statistical significance. In conclusion we demonstrate that DAZ genes are not only prone to deletions but also to duplication events. Further studies are needed to estimate the prevalence of these mutations and its' relevance to male infertility.     

Genome-wide signatures of 'rearrangement hotspots' within segmental duplications in humans

The primary objective of this study was to create a genome-wide high resolution map (i.e., >100 bp) of 'rearrangement hotspots' which can facilitate the identification of regions capable of mediating de novo deletions or duplications in humans. A hierarchical method was employed to fragment segmental duplications (SDs) into multiple smaller SD units. Combining an end space free pairwise alignment algorithm with a 'seed and extend' approach, we have exhaustively searched 409 million alignments to detect complex structural rearrangements within the reference-guided assembly of the NA18507 human genome (18× coverage), including the previously identified novel 4.8 Mb sequence from de novo assembly within this genome. We have identified 1,963 rearrangement hotspots within SDs which encompass 166 genes and display an enrichment of duplicated gene nucleotide variants (DNVs). These regions are correlated with increased non-allelic homologous recombination (NAHR) event frequency which presumably represents the origin of copy number variations (CNVs) and pathogenic duplications/deletions. Analysis revealed that 20% of the detected hotspots are clustered within the proximal and distal SD breakpoints flanked by the pathogenic deletions/duplications that have been mapped for 24 NAHR-mediated genomic disorders. FISH Validation of selected complex regions revealed 94% concordance with in silico localization of the highly homologous derivatives. Other results from this study indicate that intra-chromosomal recombination is enhanced in genic compared with agenic duplicated regions, and that gene desert regions comprising SDs may represent reservoirs for creation of novel genes. The generation of genome-wide signatures of 'rearrangement hotspots', which likely serve as templates for NAHR, may provide a powerful approach towards understanding the underlying mutational mechanism(s) for development of constitutional and acquired diseases.     

Copy number variations due to large genomic deletion in X-linked chronic granulomatous disease

Mutations in genes for any of the six subunits of NADPH oxidase cause chronic granulomatous disease (CGD), but almost 2/3 of CGD cases are caused by mutations in the X-linked CYBB gene, also known as NAD (P) H oxidase 2. Approximately 260 patients with CGD have been reported in Japan, of whom 92 were shown to have mutations of the CYBB gene and 16 to have chromosomal deletions. However, there has been very little detailed analysis of the range of the deletion or close understanding of the disease based on this. We therefore analyzed genomic rearrangements in X-linked CGD using array comparative genomic hybridization analysis, revealing the extent and the types of the deletion genes. The subjects were five Japanese X-linked CGD patients estimated to have large base deletions of 1 kb or more in the CYBB gene (four male patients, one female patient) and the mothers of four of those patients. The five Japanese patients were found to range from a patient exhibiting deletions only of the CYBB gene to a female patient exhibiting an extensive DNA deletion and the DMD and CGD phenotype manifested. Of the other three patients, two exhibited CYBB, XK, and DYNLT3 gene deletions. The remaining patient exhibited both a deletion encompassing DNA subsequent to the CYBB region following intron 2 and the DYNLT3 gene and a complex copy number variation involving the insertion of an inverted duplication of a region from the centromere side of DYNLT3 into the deleted region.     

A deletion hot spot in the Duchenne muscular dystrophy gene

We have made a detailed study of a deletion hot spot in the distal half of the Duchenne muscular dystrophy (DMD) gene, using intragenic probe P20 (DXS269), isolated by a hybrid cell-mediated cloning procedure. P20 detects 16% deletions in patients suffering from either DMD or Becker muscular dystrophy (BMD), in sharp contrast to the adjacent intragenic markers JBir (7%) and J66 (less than 1%), mapping respectively 200-320 kb proximal and 380-500 kb distal to P20. Of the P20 deletions, 30% start within a region of 25-40 kb, the majority extending distally. P20 was confirmed to map internal to a distal intron of the DMD gene. This region was recently shown by both cDNA analysis (M. Koenig et al., 1987; Cell 50: 509-517), and field inversion electrophoresis studies (J.T. Den Dunnen et al., 1987, Nature (London) 329: 640-642) to be specifically prone to deletions. In addition, P20 detects MspI and EcoRV RFLPs, informative in 48% of the carrier females. Together, these properties make P20 useful for carrier detection, prenatal diagnosis, and the study of deletion induction in both DMD and BMD.     

Topological Constraints on Transvection between White Genes within the Transposing Element Te35b in Drosophila Melanogaster

The transposable element TE35B carries two copies of the white (w) gene at 35B1.2 on the second chromosome. These w genes are suppressed in a zeste-1 (z(1)) mutant background in a synapsis-dependent manner. Single-copy derivatives of the original TE35B stock give red eyes when heterozygous, but zeste eyes when homozygous. TE35B derivatives carrying single, double or triple copies of w were crossed to generate flies carrying from two to five ectopic w genes. Within this range, z(1)-mediated suppression is insensitive to copynumber and does not distinguish between w genes that are in cis or in trans. Suppression does not require the juxtaposition of even numbers of w genes, but is extremely sensitive to chromosomal topology. When arranged in a tight cluster, in triple-copy TE derivatives, w genes are nonsuppressible. Breakpoints falling within TE35B and separating two functional w genes act as partial suppressors of z(1). Similarly, breakpoints immediately proximal or distal to both w genes give partial suppression. This transvection-dependent downregulation of w genes may result from mis-activation of the X-chromosome dosage compensation mechanism.