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

Succinyl-CoA:3-ketoacid coenzyme A transferase (SCOT): development of an antibody to human SCOT and diagnostic use in hereditary SCOT deficiency

AbstractSuccinyl-CoA:3-ketoacid CoA transferase (SCOT) is a key enzyme for ketone body utilization. Hereditary SCOT deficiency in humans (McKusick catalogue number 245050) is characterized by intermittent ketoacidotic attacks and permanent hyperketonemia. Since previously-available antibody to rat SCOT did not crossreact with human SCOT, we developed an antibody against recombinant human SCOT expressed in a bacterial system. The recombinant SCOT was insoluble except under denaturing conditions. Antibody raised to this polypeptide recognized denatured SCOT and proved useful for immunoblot analysis. On immunoblots, SCOT was easily detectable in control fibroblasts and lymphocytes but was detected neither in fibroblast extracts from four SCOT-deficient patients, nor in lymphocytes from two SCOT-deficient patients. These data indicate that immunoblot analysis is useful for diagnosis of SCOT deficiency in combination with enzyme assay

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

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