11.Hypoxic ventilatory depressionと思われる一症例について(第551回千葉医学会例会・第9回麻酔科例会・第18回千葉麻酔懇話会)

FISH analysis on metaphase nuclei (top panel) of cultured cells derived from peripheral blood leukocytes of the proband of family 2 by using BAC probes for 11p15.5-15.4 (RP11-11A9, 3,236,552-3,356,012, green) and 11q22.3 (RP11-179B7, 104,298,339-104,459,797, red). The green signal on both homologues is visible only at chr11p, demonstrating the presence of an in cis duplication and excluding an unbalanced translocation. FISH analysis on interphase nuclei (bottom panel) using the BACs RP11-699D10 (2.9–3.0 Mb, red) and RP11-11A9 (green), hybridizing within the duplication. Note that single and duplicated signals can be seen on the two homologues, respectively. The red-green-green-red order of the duplicated signals indicates that the duplication is inverted. (PDF 52 kb

Additional file 1: of Is ZFP57 binding to H19/IGF2:IG-DMR affected in Silver-Russell syndrome?

Table S1. List of CTCF, OCT4/SOX2 and ZFP57 binding regions and potential target sites in the H19/IGF2:IG-DMR. Genomic positions of the binding regions (if demonstrated) and potential target sites and motifs of all these factors have been listed. (PDF 86 kb

The elevated <i>Igf2</i> expression in adult brain regions, compared to peripheral tissues, is mainly due to a dramatic increase in <i>Igf2</i> expression from the maternal allele.

<p>(A) The top panels demonstrate the specificity and amplification efficiency of BN and F specific primer sets used in the analysis. The bottom panels show the data from quantitative real-time PCR analysis of <i>Igf2</i> mRNA expressed from paternal (Pat.) and maternal (Mat.) alleles in various peripheral tissues and brain regions obtained from adult male F1 rats (n = 4–7; F_{(19, 112)} = 25.66, <i>P</i> < 0.0001). Data analyses combined F1 offspring from both the F (dam) x BN (sire) cross and the reciprocal BN (dam) x F (sire) cross. <i>Igf2</i> mRNA level in each tissue was normalized to the 18S RNA level of that tissue. Data are represented as mean ± SEM of the ratio of <i>Igf2</i> mRNA expression relative to the paternal <i>Igf2</i> level from liver. **<i>P</i><0.01, ***<i>P</i><0.001 (B) Schematic representations of the % expression of <i>Igf2</i> mRNA from the paternal or maternal allele in each tissue analyzed from adult male F1 rats. (C) Quantitative real-time PCR analysis of the relative <i>Igf2</i> mRNA levels in liver, dHC, ACC, PrL/IL and BLA obtained from adult female F1 rats (n = 6–7; F_{(4, 28)} = 53.31, <i>P</i> < 0.0001). In each sample, <i>Igf2</i> mRNA level was normalized to the 18S RNA level. Data are expressed as mean ± SEM of the ratio of <i>Igf2</i> mRNA in the liver. **<i>P</i><0.01. (D) Quantitative real-time PCR analysis of <i>Igf2</i> mRNA expressed from paternal (Pat.) and maternal (Mat.) alleles in different brain regions of adult female F1 offspring from F (dam) x BN (sire) cross and the reciprocal BN (dam) x F (sire) cross, compared to the <i>Igf2</i> level expressed in the liver (n = 6–7; F_{(8, 46)} = 121.8, <i>P</i> < 0.0001). <i>Igf2</i> mRNA level in each region was normalized to the 18S RNA level in the same region. Data are represented as mean ± SEM of the ratio of <i>Igf2</i> mRNA expression relative to the paternal <i>Igf2</i> level from liver. **P<0.01. (E) Schematic representations of the % expression of <i>Igf2</i> mRNA from the paternal or maternal allele in each adult female tissue analyzed. (F) Quantitative real-time PCR analysis of <i>Igf2</i> mRNA expressed from different promoters (P1, P2 and P3) in liver, heart, dHC, ACC, PrL/IL and BLA of adult F1 offspring from F (dam) x BN (sire) cross and the reciprocal BN (dam) x F (sire) cross. Data are presented as mean ± SEM of <i>Igf2</i> mRNA expressed from each promoter as percentage of the total <i>Igf2</i> mRNA expressed in the region analyzed. (n = 5–7, P2: F_{(6, 37)} = 43.66, <i>P</i> < 0.0001; P3: F_(6,37) = 50.91, <i>P</i> < 0.0001). ***<i>P</i><0.0001. (G) Schematic representations of the % expression of P2-derived <i>Igf2</i> mRNA from the paternal or maternal alleles, analyzed by pyrosequencing (n = 5–7).</p

The expression of <i>Igf2</i> is significantly and dramatically higher in adult rat brain regions compared to peripheral tissues, and is mainly expressed from the maternal allele in the brain and from the paternal allele in peripheral tissues.

<p>(A-B) Quantitative real-time PCR analysis of the relative <i>Igf2</i> mRNA levels in liver, kidney, spleen, blood, heart, dorsal hippocampus (dHC), anterior cingulated cortex (ACC), prelimbic/infralimbic (PrL/IL), basolateral amygdala (BLA) and chorioid plexus (CP) using two independent pairs of primers (n = 3–9; for primer set I: F_{(9, 59)} = 39.08, <i>P</i> < 0.0001; for primer set II: F_{(9, 51)} = 103.4, <i>P</i> < 0.0001). In each sample, <i>Igf2</i> mRNA level was normalized to the 18S RNA level. Data are expressed as mean ± SEM of the ratio of <i>Igf2</i> mRNA in the liver. **<i>P</i><0.01, ***<i>P</i><0.001 (C) Western blot analysis of IGF-2 protein levels in the dHC, ACC, PrL/IL and BLA, compared to that in liver (top panel). A parallel, duplicated gel was used for coomassie staining to reveal protein loading in each sample (bottom panel). This experiment was repeated on 3 rats and showed similar data. (D) DNA sequence alignment of the (CA)_n repeat region of <i>Igf2</i> 3’UTR and of 5’UTR derived from P2 between Brown Norway (BN) and Fischer (F) rats. Genetic polymorphisms are highlighted. (E) cDNAs obtained from different tissues from adult F1 offspring of BN (dam) x F (sire) (BF) and F (dam) x BN (sire) (FB) rats as well as genomic DNA from BN, F, and their F1 rats were used as templates for PCR amplification of the (CA)_n repeat region. The PCR products were resolved by electrophoresis on 2% agarose gel. The 337 bp band represents the allele from the F rats, whereas the 310 bp band represents the allele from the BN rats. PCR products from reactions run in parallel in the absence of reverse transcriptase (RT-) were included to show that the observed bands of RT-PCR products were not due to gDNA contamination. Abbreviations of different tissues: dHC–dorsal hippocampus, BLA–basolateral amygdala, PrL/IL–prelimbic/infralimbic cortex, ACC–anterior cingulate cortex, CB–cerebellum, CP–choroid plexus, SC–spinal cord, LM–leptomeninges, Li–liver, Sp–spleen, He–heart, Ki-kidney, Bl-blood. These data were confirmed by 2 experimenters in a total of 5 independent experiments.</p

Inhibitory avoidance training results in an increase of <i>Igf2</i> mRNA levels in the dHC, ACC, and PrL/IL from both parental alleles at 20hr post-training.

<p>Adult male F1 rats were trained in IA, and 20hrs later, brains were collected from both trained (Tr) rats and control naïve (N) rats. Quantitative real-time PCR was used to analyze the total, paternal (Pat.) and maternal (Mat.) <i>Igf2</i> mRNA levels in the dHC (A, n = 6–7; for total <i>Igf2</i>: t₁₁ = 2.96, <i>P</i> = 0.013; for paternal <i>Igf2</i>: t₁₁ = 4.31, <i>P</i> = 0.0012; for maternal <i>Igf2</i>: t₁₁ = 4.12, <i>P</i> = 0.0017), ACC (B, n = 7–9; for total <i>Igf2</i>: t₁₄ = 2.54, <i>P</i> = 0.0235; for paternal <i>Igf2</i>: t₁₄ = 2.87, <i>P</i> = 0.0124; for maternal <i>Igf2</i>: t₁₄ = 4.44, <i>P</i> = 0.0006), PrL/IL (C, n = 7; for total <i>Igf2</i>: t₁₂ = 2.28, <i>P</i> = 0.04; for paternal <i>Igf2</i>: t₁₂ = 2.77, <i>P</i> = 0.017; for maternal <i>Igf2</i>: t₁₂ = 2.02, <i>P</i> = 0.066) and BLA (D, n = 6–7; for total <i>Igf2</i>: t₁₁ = 0.07, <i>P</i> = 0.94; for paternal <i>Igf2</i>: t₁₁ = 1.15, <i>P</i> = 0.27; for maternal <i>Igf2</i>: t₁₁ = 0.23, <i>P</i> = 0.82). <i>Igf2</i> mRNA level in each sample was normalized to that of the 18S RNA level. Data are presented as mean ± SEM of the ratio of <i>Igf2</i> levels in the brain regions taken from trained to naïve rats. Schematic representations of the % expression of <i>Igf2</i> mRNA from the paternal or maternal allele in each tissue analyzed. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001. (E) Analysis of the CpG methylation levels by methylation-sensitive pyrosequencing in <i>Igf2</i>/H-19 imprinting control region (ICR), differentially methylation region 2 (DMR2) and P2 promoter of <i>Igf2</i> gene in liver, CP, dHC and ACC, as well as in dHC and ACC from naïve (N) <i>versus</i> trained (Tr) rats. Data are presented as the mean ± SEM of the percentage of methylation across 15 CpG sites in the ICR region (F_{(5, 84)} = 1.18, <i>P</i> = 0.33), 19 CpG sites in the DMR2 region (F_{(5, 108)} = 6.28, <i>P</i> < 0.0001), and 11 CpG sites in the P2 region (F_{(5, 60)} = 8.71, <i>P</i> < 0.0001). ***<i>P</i><0.001.</p

<i>Igf2</i> mRNA levels in the brain are down-regulated from both paternal and maternal alleles during postnatal development, with a relatively larger decrease of paternal <i>Igf2</i>.

<p>(A-B) Quantitative real-time PCR analysis of total <i>Igf2</i> level, <i>Igf2</i> expressed from paternal (Pat.) and maternal (Mat.) alleles, in the hippocampus (HPC; A) and anterior cingulate cortex (ACC; B), collected from postnatal day (PND) 1, 7, 17 and 24 of F1 rats, compared to <i>Igf2</i> levels of adult HPC (n = 5–7; for total <i>Igf2</i>: F_{(4, 25)} = 23.06, <i>P</i> < 0.0001; for paternal <i>Igf2</i>: F_{(4, 25)} = 24.42, <i>P</i> < 0.0001; for maternal <i>Igf2</i>: F_{(4, 25)} = 21.45, <i>P</i> < 0.0001) and ACC (n = 5–9; for total <i>Igf2</i>: F_{(4, 27)} = 22.52, <i>P</i> < 0.0001, <i>P</i> < 0.0001; for paternal <i>Igf2</i>: F_{(4, 27)} = 21.32, <i>P</i> < 0.0001; for maternal <i>Igf2</i>: F_{(4, 27)} = 14.75, <i>P</i> < 0.0001). Data analyses combined male and female F1 offspring from both the F (dam) x BN (sire) cross and the reciprocal BN (dam) x F (sire) cross. <i>Igf2</i> mRNA level in each region was normalized to the 18S RNA level of the same region. Data are represented as mean ± SEM of the ratio of <i>Igf2</i> mRNA levels in developmental HPC and ACC over adult HPC and ACC, respectively. ***<i>P</i><0.001 (C) Schematic representations of the % expression of <i>Igf2</i> mRNA from the paternal or maternal allele in each tissue analyzed.</p

Additional file 6: Figure S6. of Two maternal duplications involving the CDKN1C gene are associated with contrasting growth phenotypes

DNA methylation (top) and copy number (CN, bottom) analyses at 11p15 region in family 2, determined by MS-MLPA. The histograms represent the normalized DNA methylation of ICR1 and ICR2 and CN of the genomic region spanning from the NSD1 to KCNQ1 gene. The CN range that is considered normal is shadowed. Note that methylation of ICR2 is abnormally low while CN values of ICR2 and KCNQ1 exon 13-17 are abnormally high in the proband and his mother. (PDF 130 kb

Additional file 1: Figure S1. of Two maternal duplications involving the CDKN1C gene are associated with contrasting growth phenotypes

Comparative genomic hybridization analysis performed on DNA of the proband from family 1. The extension, the genomic localization (GRC h37/hg19) and the genes included in the duplication are shown. (PDF 54 kb

Additional file 7: Figure S7. of Two maternal duplications involving the CDKN1C gene are associated with contrasting growth phenotypes

Note that the haplotype associated with the duplication segregates from I-1 to II-2 and III-1 and that only the D11S4088 marker shows allelic imbalances in II-2 and III-1. (PDF 141 kb

Additional file 2: Figure S2. of Two maternal duplications involving the CDKN1C gene are associated with contrasting growth phenotypes

Single nucleotide polymorphism analysis of genomic DNA from relatives of family 1. I-1 = maternal grandfather, II-2 = mother, III-1 = first brother of the proband, and III-2 = second brother of the proband. Note that the duplication is present in II-2 and III-2 but not in I-1 and III1. (PDF 127 kb