Human imprinted chromosomal regions are historical hot-spots of recombination.

Human recombination rates vary along the chromosomes as well as between the two sexes. There is growing evidence that epigenetic factors may have an important influence on recombination rates, as well as on crossover position. Using both public database analysis and wet-bench approaches, we revisited the relationship between increased rates of meiotic recombination and genome imprinting. We constructed metric linkage disequilibrium (LD) maps for all human chromosomal regions known to contain one or more imprinted genes. We show that imprinted regions contain significantly more LD units (LDU) and have significantly more haplotype blocks of smaller sizes than flanking nonimprinted regions. There is also an excess of hot-spots of recombination at imprinted regions, and this is likely to do with the presence of imprinted genes, per se. These findings indicate that imprinted chromosomal regions are historical "hot-spots" of recombination. We also demonstrate, by direct segregation analysis at the 11p15.5 imprinted region, that there is remarkable agreement between sites of meiotic recombination and steps in LD maps. Although the increase in LDU/Megabase at imprinted regions is not associated with any significant enrichment for any particular sequence class, major sequence determinants of recombination rates seem to differ between imprinted and control regions. Interestingly, fine-mapping of recombination events within the most male meiosis-specific recombination hot-spot of Chromosome 11p15.5 indicates that many events may occur within or directly adjacent to regions that are differentially methylated in somatic cells. Taken together, these findings support the involvement of a combination of specific DNA sequences and epigenetic factors as major determinants of hot-spots of recombination at imprinted chromosomal regions

Excessive formation of hydroxyl radicals and aldehyde lipid peroxidation products in cultured skin fibroblasts from patients with complex I deficiency

Previous studies suggest oxygen free radicals ’ involvement in the etiology of cardiomyopathy with cataracts. To investigate the role of free radicals in the pathogenesis of the cardiomyopathy with cataracts and complex I deficiency, fibroblasts from patients were assessed for hydroxyl radical formation and aldehydic lipid peroxidation products with and without redox active agents that increase free radicals. The rate of hydroxyl radical formation in patient cells was increased over 2–10-fold under basal conditions, and up to 20-fold after menadione or doxorubicin treatment compared with normal cells. We also found an overproduction of aldehydes in patient cells both under basal conditions and after treatment. Both hydroxyl radicals and toxic aldehydes such as hexanal, 4-hydroxynon-2-enal, and malondialdehyde were elevated in cells from patients with three types of complex I deficiency. In contrast, acyloins, the less toxic conjugated products of pyruvate and saturated aldehydes, were lower in the patient cells. Our data provide direct evidence for the first time that complex I deficiency is associated with excessive production of hydroxyl radicals and lipid peroxidation. The resultant damage may contribute to the early onset of cardiomyopathy and cataracts and death in early infancy in affected patients with this disease. (J. Clin. Invest. 1997. 99:2877– 2882.) Key words: complex I deficiency • cardiomyopathy • cataracts • hydroxyl radicals • lipid peroxidatio

Interindividual variability and parent of origin DNA methylation differences at specific human Alu elements.

We investigated the CpG methylation of 19 specific members of Alu sub-families in human DNA isolated from whole blood, using an assay based on methylation-sensitive restriction endonuclease digestion of genomic DNA and \u27hot-stop\u27 polymerase chain reaction. We found significant interindividual variability in the level of methylation for specific Alu elements among the members of 48 three-generation families. Surprisingly, some of the elements also displayed quantitative parent of origin methylation differences; i.e. the mean level of methylation differed significantly when the insertions were transmitted through paternal versus maternal meiosis. Bisulfite sequence analysis of individual elements at such loci suggests, further, that maternal and paternal elements differ in the propensity of particular CpG sites to become unmethylated. Some individuals who exhibited high levels of methylation at specific Alu elements came from families in which more than one member also exhibited abnormal patterns of methylation at the differentially methylated regions of the IGF2/H19 or IGF2R loci, suggesting that there may be heritable differences between individuals in the fidelity with which allelic DNA methylation differences are established or maintained. Quantitative parental origin differences in methylation were identified only for Alu elements that lie in sub-telomeric or sub-centromeric bands of human chromosomes, whereas those assayed at intermediate positions did not exhibit any significant differences. The centromere/telomere restricted location of the methylation differences and the fact that none of these differences occur in regions of chromosomes known to contain transcriptionally imprinted genes suggest that maternal/paternal epigenetic modifications may play additional roles in processes other than transcriptional control

LD Analysis at Human 11p15.5 Imprinted Cluster

<div><p>(A) Population-specific metric LD map for about 1-Mb region containing imprinted genes at human 11p15.5 chromosome. Positions along the chromosome are shown in bp on the <i>x-</i>axis. Straight lines are representing the genome-wide slopes (LDU/Mb) corresponding to each population, as extrapolated from De La Vega et al. [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020101#pgen-0020101-b016" target="_blank">16</a>]. Note that LD extends less far in the region containing imprinted genes compared with a region of similar length from the rest of genome, in agreement with the interpretation of higher recombination in these areas (i.e., breakdown of LD has been converted to implied recombination rate and rendered graphically as red rectangular “hot spots”). Location of CpG islands in the region are depicted as shown in MapView; dark blue represents CpG islands larger than 500 bp, and light blue represents CpG islands over 200 bp. For both categories, G + C content is higher than 50% and the observed CpG/expected CpG content is higher than 0.6. The two black arrows correspond to the regions containing the primary germline imprints at <i>H19</i>/<i>IGF2</i> DMR (left arrow) and <i>KCNQ1OT1</i> DMR (right arrow), respectively. Both are located at regions exhibiting steps of LD and recombination hot-spots and are zoomed-in in (B) and (C). The red open arrows correspond to smaller steps, which are variable between populations and do not correspond with any hot-spot of recombination.</p><p>(B) The metric LD map for the region containing <i>H19</i>/<i>IGF2</i> DMR using data from the four populations (HapMap) and the set of CEPH individuals analyzed in this study. The three horizontal bars correspond to recombinants mapped at this region, one in maternal meiosis (red) and two in paternal meioses (blue). The blue oval shape corresponds to the <i>H19</i>/<i>IGF2</i> DMR.</p><p>(C) The metric LD map for the region containing <i>KCNQ1OT1</i> DMR using data from the four populations (HapMap). Two recombinants (horizontal blue bars) were mapped at this region in paternal meioses. The red oval shape corresponds to the <i>KCNQ1OT1</i> DMR.</p></div

Distribution of Recombination Events at Human 11p15.5 Imprinted Cluster

<div><p>Positions of markers used for mapping recombinants in this region are indicated in Mb from the telomeric end (Tel) of the short arm. Imprinted genes are shown on the left side of the figure. Arrows correspond to direction and parental-specific origin of transcription: blue are paternally transcribed genes, red are maternally expressed genes, and black are genes with biallelic expression or unknown imprinting status. The two known germline imprints at this locus are shown by colored oval shapes on the left side of the figure: the blue oval corresponds to the paternally methylated <i>IGF2</i>/<i>H19</i> DMR and the red oval corresponds to the maternally methylated <i>KCNQ1OT1</i> DMR. Each vertical bar on the right side of the figure corresponds to a meiotic recombination event, delimitated by the nearest informative markers: Labeled in blue are crossovers in paternal meiosis, and labeled in red are recombinations in maternal meiosis.</p><p>An asterisk (*) represents an unidentified polymorphism found at <i>MUC5B</i> locus, and double asterisks (**) indicate an unidentified TaqI polymorphism found at <i>TH</i> locus (genotypes available through CEPH database—see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020101#s4" target="_blank">Materials and Methods</a>).</p></div

Pairwise LD Analysis and Pairwise FGT at the <i>H19</i>/<i>IGF2</i> DMR

<div><p>(A) Pairwise LD test between ten SNPs covering a 31-kb region containing <i>H19</i>/<i>IGF2</i> DMR shows a major breakdown of LD which corresponds to the LDU step shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020101#pgen-0020101-g003" target="_blank">Figure 3</a>B. Intensity of LD is coded in colors as shown.</p><p>(B) Pairwise FGT between the same ten SNPs. A “1” indicates recombination between that pair of loci (all four gametes) and “0” indicates only three types of gametes (recombination between the two loci is uncertain). Considering that a historical recombination would break the haplotype inside of which it appeared, at least eight haplotype blocks could be identified. The ten markers used for both analyses in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020101#pgen-0020101-g004" target="_blank">Figure 4</a>A and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020101#pgen-0020101-g004" target="_blank">4</a>B are the same as depicted in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020101#pgen-0020101-g003" target="_blank">Figure 3</a>B (CEPH)</p></div

LD Analysis at Human Chromosomal Regions Containing Imprinted Genes

<div><p>(A) Comparison of LDU values at imprinted versus control regions. Each LDU/Mb value obtained for a given imprinted bin was plotted against the LDU/Mb value of the corresponding control region. Note that most of the imprinted regions reach higher LDU values compared with their corresponding control regions (dotted line with slope 1 corresponds to virtual positions in cases with equal LDU/Mb values at imprinted and control regions).</p><p>(B) The number of haplotype blocks/Mb is higher at imprinted genes compared with their corresponding control regions (dotted line indicates equal values).</p><p>(C) The mean sizes of haplotype blocks are significantly smaller at imprinted regions versus flanking control regions (dotted line indicates equal sizes).</p><p>(D) There is a significant excess in the number of hot-spots of recombination at imprinted versus control regions (see text), and the total length of the hot-spots appears greater in imprinted regions than in control regions. Each value corresponds to the total length of all hot-spots of recombination (in base pairs [bp]) for a given imprinted or control region.</p></div

Reduction of Clofazimine by Mycobacterial Type 2 NADH:Quinone Oxidoreductase: A PATHWAY FOR THE GENERATION OF BACTERICIDAL LEVELS OF REACTIVE OXYGEN SPECIES*

The mechanism of action of clofazimine (CFZ), an antimycobacterial drug with a long history, is not well understood. The present study describes a redox cycling pathway that involves the enzymatic reduction of CFZ by NDH-2, the primary respiratory chain NADH:quinone oxidoreductase of mycobacteria and nonenzymatic oxidation of reduced CFZ by O2 yielding CFZ and reactive oxygen species (ROS). This pathway was demonstrated using isolated membranes and purified recombinant NDH-2. The reduction and oxidation of CFZ was measured spectrally, and the production of ROS was measured using a coupled assay system with Amplex Red. Supporting the ROS-based killing mechanism, bacteria grown in the presence of antioxidants are more resistant to CFZ. CFZ-mediated increase in NADH oxidation and ROS production were not observed in membranes from three different Gram-negative bacteria but was observed in Staphylococcus aureus and Saccharomyces cerevisiae, which is consistent with the known antimicrobial specificity of CFZ. A more soluble analog of CFZ, KS6, was synthesized and was shown to have the same activities as CFZ. These studies describe a pathway for a continuous and high rate of reactive oxygen species production in Mycobacterium smegmatis treated with CFZ and a CFZ analog as well as evidence that cell death produced by these agents are related to the production of these radical species