PMC

Clinical phenotype and pedigree of the patients. (A) SRS patient 1 at 5 months (top left) and 14 months (top right). BWS patient 1 at birth (bottom left) and BWS patient 2 (bottom right). (B) Three-generation pedigree of the BWS family. Carriers of the 160 kb microduplication are indicated. *Individuals unavailable to molecular analysis. **Paternal transmission of the microduplication in I-4 was inferred by allele-specific analysis of ICR2 methylation (see Results). Note that maternal transmission segregates with the BWS phenotype with complete penetrance.

Analysis of microsatellite 11p15.5 markers. (A) Electropherograms obtained for microsatellite loci D11S4046 and TH in the SRS family. The arrows indicate the alleles that are maternally transmitted to the propositus. F, father; M, mother; P, propositus. Note the presence of two maternal alleles and one paternal allele at D11S4046 and an allelic imbalance at TH in the propositus. (B) Electropherograms obtained by sequencing the rs11023840 SNP region in the SRS family. Note the increased dosage of the T allele in the propositus. (C) Co-segregation of the 11p15.5 microduplication with the disease phenotype in the BWS family. Electropherograms obtained for the microsatellite locus D11S4088. Note that I4, BWS patient 2 (II-4), BWS patient 3 (II-3) and BWS patient 1 (III-6) show increased dosage approximating a 2:1 ratio for the 213 bp allele (highlighted in dark gray).

Diagram representing the observed and predicted methylation and expression patterns at chromosome 11p15.5 in the subjects under study. The duplicated regions are depicted separately from the chromosomes and connected to the breakpoint by dotted lines. The inverted orientation of the microduplications is shown. P, paternally derived chromosome; M, maternally derived chromosome. Representative imprinted genes are shown (those paternally expressed are in blue and those maternally expressed are in red). Gene transcription is indicated by arrows. Light red color for CDKN1C represents decreased expression. No quantitative data are available for the expression of KCNQ1 in BWS patients 1–3 and KCNQ1OT1 in the individual I-4 of the BWS family (indicated with ?). Filled and open circles represent methylated and non-methylated ICRs, respectively.

Interaction of the KCNQ1OT1 RNA with chromatin. (A) qPCR analysis of ChRIP-purified RNA from four lymphoblast cell lines. The enrichment of the KCNQ1OT1 RNA is measured with primers located 500 bp downstream of the transcription start site. The histogram represents fold enrichment of the H3 Ab over IgG. CTRL, normal control individual; BWS P1, BWS patient 1; BWS P2, BWS patient 2; BWS P4, BWS patient with ICR2 hypomethylation and no microduplication. (B) Allele-specific PCR analysis of ChRIP RNA. rs463924 allele-specific qPCR analysis of the cell lines analyzed in (A). The histograms report as enrichment over IgG the results obtained with primers specific for the paternal allele and primers specific for the maternal allele. All the above data represent mean ± SD of at least two independent experiments. (C) Sequence analysis at SNP rs463924 (±3 bp) of the cDNA obtained from the ChRIP RNA. The DNA sequence of the normal control individual (CTRL DNA seq) is shown to demonstrate the heterozygosity. Differently from the control cell line used in Figure A, the G allele is paternal in the control sample used for the ChRIP analysis. Note that both parental alleles of the KCNQ1OT1 RNA interact with chromatin in the cells derived from the BWS patients carrying the 160 kb duplication.

Expression analysis of the centromeric domain genes. (A) Allele-specific expression of KCNQ1OT1. DNAs and RNAs extracted from lymphoblast cells of BWS patient 1, BWS patient 2 and normal control individuals were analyzed by PCR and RT–PCR. The parental alleles of KCNQ1OT1 were discriminated by typing for the SNPs G/A rs462402 and G/A rs463924 that were present in the duplicated (5′) region of KCNQ1OT1 and T/C rs760419 and A/G rs4930005 that were present in the non-duplicated (3′) region of KCNQ1OT1. The electropherograms obtained from sequencing of the PCR products over the SNPs are shown. The red arrows indicate the presence of both parental alleles and the blue arrows the presence of a single allele in the RT–PCR product. Note that the 5′ part of KCNQ1OT1 is expressed from both parental alleles, whereas the 3′ part is expressed from only one allele in BWS patient 1 and BWS patient 2. (B) Analysis of CDKN1C expression. The level of CDKN1C RNA was assessed by quantitative real-time RT–PCR in the skin fibroblasts derived from eight age-matched normal control individuals (diamonds), BWS patient 2 (square) and two BWS patients with loss of ICR2 methylation (triangles). CDKN1C values were normalized to the expression of GAPD. Note that CDKN1C expression in BWS patient 2 is 20-fold lower than the average level found in normal control individuals and is comparable to that of BWS patients with ICR2 hypomethylation and no microduplication.

Structural characterization of the 11p15.5 microduplications. (A) Genomic profiles of SRS patient 1 and BWS patient 1 chromosome 11p15.5 as determined by SNP array-CGH. A screenshot of the UCSC Genome Browser showing that the relevant genomic region is present at the top and the normalized CN detected with the 11p15.5 probes is reported in the lower part of the figure. The analysis of BWS patient 2 produced results very similar to those of BWS patient 1 (data not shown). Note that the duplication of the SRS patient is 1.2 Mb long and includes the entire imprinted gene cluster, whereas the duplication of the BWS family is 160 kb long and includes the KCNQ1 exons 12–15, ICR2 (dark gray triangle pointing upward) and the most centromeric 20 kb of KCNQ1OT1. ICR1 is indicated by a light gray triangle pointing downward. (B) Characterization by FISH of the 11p15 region duplicated in the patients under study. SRS patient 1, left panel: metaphase FISH using the BAC probes RP11-937011 (chr11:2 522 537–2 709 016, red) and RP11-876C12 (11q22.3, green). SRS patient 1, middle panel: FISH analysis of interphase nuclei using the BAC probes RP11-937011 (red) and RP11-876C12 (green). The 11p15.5 probe detects a duplicated signal (arrowed), whereas the 11q22.3 probe shows normal signal. SRS patient 1, right panel: FISH analysis of interphase nuclei using the BAC probes RP11-937O11 (green) and RP11-81K4 (chr11:2 755 275–2 927 014, red). Single and duplicated signals are detected on the two homologs. The order of the duplicated signal (arrowed): red-green-green-red indicates that the microduplication is inverted with the RP11-937O11 probe in the middle. Patient BWS 1, left panel: metaphase FISH using the fosmid probe G248P81952E2 (chr11:2 660 932–2 698 197, red) and the BAC probe RP11-876C12 (11q22.3, green). Patient BWS 1, middle panel: FISH analysis of interphase nuclei using the fosmid probe G248P81952E2 (red) and the BAC probe RP11-876C12 (green). The 11p15.5 probe detects a duplicated signal (arrowed), whereas the 11q22.3 probe shows normal signal. Patient 2, right panel: FISH analysis of interphase nuclei using the fosmid probes G248P81952E2 (red) and G248P800755G6 (chr11:2 759 661–2 803 886, green). Single and duplicated signals are detected on the two homologs. The order of the duplicated signal (arrowed): green- red-red-green indicates that the microduplication is inverted with the G248P81952E2 probe in the middle.

DNA methylation and CN analyses at chromosome 11p15.5. (A) DNA methylation at ICR1 and ICR2 of the SRS family as determined by Southern blotting. About 10 μg of blood leukocyte DNA was double-digested with Csp6I/HpaII and hybridized with an ICR1-specific probe (left panel) or with BamHI/NotI and hybridized with an ICR2-specific probe (right panel), as reported previously (). The ratios (unme/me) between fast-migrating bands corresponding to the unmethylated allele and slow-migrating bands corresponding to the methylated allele are indicated below each panel. The values that differed significantly from controls are shown in bold. F, father; M, mother; P, propositus. Note that the propositus displays hypomethylation at ICR1 and hypermethylation at ICR2. (B) DNA methylation at ICR1 and ICR2 of the SRS family as determined by COBRA. About 2 μg of blood leukocyte DNA was treated with sodium bisulfite, PCR-amplified and incubated with the restriction enzyme BstUI (). The ratios (unme/me) between slow-migrating bands corresponding to the unmethylated allele and fast-migrating bands corresponding to the methylated allele are indicated below each panel. The values that differed significantly from controls are shown in bold. (C) DNA methylation at ICR1 and ICR2 of the BWS family as determined by MS-MLPA. The assay (ME030-B2 BWS/SRS, MRC-Holland) includes 11 probes that are specific for the BWS/SRS 11p15 region, contain a HhaI recognition site and provide information about the methylation status of the target sequence. The methylation index calculated from the results obtained with the ICR2- and ICR1-specific probes is shown in the upper and lower panels, respectively. Details on the chromosomal location of the probes can be found at http://www.mlpa.com/WebForms/WebFormProductDetails.aspx?Tag=tz2fAPIAupKyMjaDF\E\t9bmuxqlhe/Lgqfk8Hkjuss|&ProductOID=V3GEVpJWEXY|. The results relative to the 08745-L08765 probe have been omitted because they were not informative on the DNA methylation status due to the presence of an A > G SNP in the HhaI site. Note the slight ICR2 hypomethylation in III-6, II-4, I-4 and II-3. (D) CN analysis at 11p15.5 loci in the BWS family as determined by MS-MLPA. The histograms represent the normalized CN detected with 26 MS-MLPA probes (only the 11p15.5-specific probes were included) arranged according to the chromosomal location. The range of normal DNA dosages detected in normal control samples is shadowed. Note that CN values higher than the normal range (>1.2) were detected at ICR2 and KCNQ1 exons 12–15 in III-6, II-4, I-4 and II-3.

DNA methylation analysis of the ICR2 region. (A) Allele-specific DNA methylation analysis by bisulfite sequencing in SRS patient 1. DNA samples were treated with sodium bisulfite, amplified by PCR over the ICR2 region, cloned and sequenced. Each line corresponds to a single template DNA molecule, and each circle represents a CpG dinucleotide. Filled circles designate methylated cytosine and open circles correspond to unmethylated cytosines. The alleles were discriminated by typing for the SNP G/A rs11023840. Note that ICR2 is hypermethylated in patient 1 when compared with a control, but this is consistent with the microduplication of the methylated maternal allele. (B) Allele-specific DNA methylation analysis by bisulfite sequencing in BWS patients 1 and 2. DNA samples were analyzed as in (A). The maternal and paternal alleles were discriminated by typing for an insertion/deletion polymorphism and an SNP. Missing circles correspond to polymorphic CpGs. Although ICR2 is hypomethylated in all tested individuals, one allele is totally unmethylated and the other totally methylated in I-4, whereas the maternal allele is only partially methylated in BWS patients 1 and 2, indicating that the 160 kb duplication results in the alteration of the ICR2 imprinted methylation if maternally transmitted but does not affect imprinting when paternally inherited as likely happened in I-4. (C) Analysis of ICR2 methylation in lymphoblast cell clones as determined by COBRA. The DNAs derived from PBLs and lymphoblast clones of BWS patient 1, a BWS case with partial loss of ICR2 methylation due to mosaic UPD11, and a normal control were treated with sodium bisulfite, PCR-amplified and digested with restriction enzymes containing a CpG dinucleotide in their target sequence. The ratio (unme/me) between unmethylated and methylated alleles is indicated below each panel. Only three representative clones for each line are shown. The clones derived from the UPD11 case displayed complete loss of ICR2 methylation, whereas the PBLs from which they derived showed partial loss of methylation. The clones derived from a normal control maintained 50% methylation similar to the PBLs derived from the same individual. The clones derived from BWS patient 1 displayed partial ICR2 hypomethylation similar to their parental PBLs. The absence of selection indicates lack of epigenetic mosaicism in this patient.