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Paternal deletion of the 11p15.5 centromeric-imprinting control region is associated with alteration of imprinted gene expression and recurrent severe intrauterine growth restriction.

De Crescenzo A, Sparago A, Cerrato F, Palumbo O, Carella M, Miceli M, Bronshtein M, Riccio A, Yaron Y - J. Med. Genet. (2012)

Bottom Line: Heterogeneous molecular defects affecting the 11p15.5 imprinted gene cluster are associated with the opposite growth disorders Beckwith-Wiedemann Syndrome (BWS) and Silver Russell syndrome (SRS).Maternal deletions of the centromeric domain usually result in BWS, but paternal deletions have been so far associated with normal phenotype.DNA methylation and gene expression analyses showed that the deletion led to an imprinting alteration restricted to the centromeric domain and resulting in silencing of KCNQ1OT1 and activation of CDKN1C and PHLDA2.

View Article: PubMed Central - PubMed

Affiliation: Department of Environmental Science, Second University of Naples, Caserta, Italy.

ABSTRACT

Background: Heterogeneous molecular defects affecting the 11p15.5 imprinted gene cluster are associated with the opposite growth disorders Beckwith-Wiedemann Syndrome (BWS) and Silver Russell syndrome (SRS). Maternal deletions of the centromeric domain usually result in BWS, but paternal deletions have been so far associated with normal phenotype. Here we describe a case of recurrent severe Intra-Uterine Growth Restriction (IUGR) with paternal transmission of an 11p15.5 60 kb deletion.

Methods and results: Chromosome microarray (CMA), PCR and DNA sequencing analyses showed that two fetuses conceived by a normal couple inherited from their father a 60 kb deletion encompassing the Imprinting Control Region of the 11p15.5 centromeric domain. The two fetuses died in utero with severe growth restriction. PCR amplification of parental DNAs indicated that the father carried the mutation in the mosaic state. DNA methylation and gene expression analyses showed that the deletion led to an imprinting alteration restricted to the centromeric domain and resulting in silencing of KCNQ1OT1 and activation of CDKN1C and PHLDA2.

Conclusions: Our data demonstrate that the phenotype associated with 11p15.5 deletions is strongly influenced by the size of the region involved and indicate imprinting defects leading to CDKN1C and PHLDA2 activation as cause of severe IUGR.

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Related in: MedlinePlus

Structural characterisation of the 11p15.5 microdeletion. (A) Genomic profiles of fetus 2 and the parents at chromosome 11p15.5 as determined by SNP array. The extension of the deletion described in this study is indicated by a red line; those of previously described deletions are indicated by a grey line: the dotted ends represent the undefined borders of these deletions. A schematic diagram showing the relevant genes of the region is present at the bottom. The green rectangles represent the exons, the arrows indicate the transcription orientation, the orange box the IC2. (B) Pedigree of the family with relevant 11p15.5 genotypes. Fetus 1 and fetus 2 are indicated as II-2 and II-3, respectively. The informative SNPs present in the deleted region in the individuals investigated are shown. (C) Electropherogram showing the DNA sequence of the deletion breakpoint in fetus 1. The extreme nucleotides at the breakpoint are highlighted.
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JMEDGENET2012101352F1: Structural characterisation of the 11p15.5 microdeletion. (A) Genomic profiles of fetus 2 and the parents at chromosome 11p15.5 as determined by SNP array. The extension of the deletion described in this study is indicated by a red line; those of previously described deletions are indicated by a grey line: the dotted ends represent the undefined borders of these deletions. A schematic diagram showing the relevant genes of the region is present at the bottom. The green rectangles represent the exons, the arrows indicate the transcription orientation, the orange box the IC2. (B) Pedigree of the family with relevant 11p15.5 genotypes. Fetus 1 and fetus 2 are indicated as II-2 and II-3, respectively. The informative SNPs present in the deleted region in the individuals investigated are shown. (C) Electropherogram showing the DNA sequence of the deletion breakpoint in fetus 1. The extreme nucleotides at the breakpoint are highlighted.

Mentions: A microdeletion at chromosome 11p15.5 of 40–70 kb was first detected by chromosome microarray (CMA) (performed at Signature Genomics, Spokane, WA, using the Roche NimbleGen 135K oligonucleotide array as platform, and the SignatureChipOS Lot#: 528428A05 V.3.0 for the data analysis) in DNA extracted from amniocytes of fetus 2 (not shown). Fluorescence in situ hybridisation analysis of metaphase cells using the 11p15.5 BAC probe RP11-1061N1 (and the 11q12.1 probe RP11-872D17) showed one normal signal and one diminished signal in all cells examined (not shown), confirming the deletion. To assess the parental origin, and to determine more precisely the extent of the deleted region, the DNA of fetus 2 and the parents was further analysed by CMA using the Affymetrix Genome Wide Human Single Nucleotide Polymorphism (SNP) Array 6.0 and the Affymetrix Genotyping Console 3.0.2 software, as described.11 This analysis demonstrated the presence of a 52/58 kb deletion spanning from CN_582601 (Chr11: 2,684,979 on hg19) to CN_584828 (Chr11: 2,737,280) in the DNA of fetus 2 (figure 1A). Normal copy number was detected in parental DNAs with this technique. The genotypes derived from SNPs included in the deletion indicated that the mutation was on the paternally inherited chromosome (figure 1B). The amount of available DNA from fetus 1 was insufficient to perform a similar analysis. We, therefore, further refined the deletion boundaries on the DNA of fetus 2 by quantitative PCR, and used the obtained information to set up a PCR assay across the breakpoint. The primers were 5′-TGTTCAAGCTGTGGCCACTGG -3′ and 5′-GATGGAGTGTGGTGAGGCAC -3′ and the PCR conditions: 1.5 Mm MgCl2, 10% Dimethyl sulfoxide (DMSO), annealing temperature 60°C. By using this assay and DNA sequencing (from PRIMM, Italy), we demonstrated the presence of the microdeletion also in fetus 1 (figure 1C). The sequencing results demonstrated that the deletion spans from chr11:2679858 to chr11:2739436 (based on University of California Santa Cruz (UCSC) 2009 hg19 assembly), and includes the exon 11 of the KCNQ1 gene, the centromeric-imprinted control region (IC2) of the 11p15.5 imprinted gene cluster and the most 5′ 40 kb of the KCNQ1OT1 non-coding RNA gene (figure 1A). When parental DNAs were tested by PCR, a weak band corresponding to the region encompassing the deletion breakpoint was observed in the father, indicating somatic mosaicism (see online supplementary figure and supplementary data). All the cell and DNA samples investigated have been obtained after release of informed consent. Unfortunately, we could not analyse the DNA of the healthy sibling (II-1) because the parents did not consent for this test. However, we observe that the 11p15.5 60 kb deletion is not present in 1500 samples analysed with the same platform, as well as in the Database of Genomic Variants (http://projects.tcag.ca/variation/ and data not shown).


Paternal deletion of the 11p15.5 centromeric-imprinting control region is associated with alteration of imprinted gene expression and recurrent severe intrauterine growth restriction.

De Crescenzo A, Sparago A, Cerrato F, Palumbo O, Carella M, Miceli M, Bronshtein M, Riccio A, Yaron Y - J. Med. Genet. (2012)

Structural characterisation of the 11p15.5 microdeletion. (A) Genomic profiles of fetus 2 and the parents at chromosome 11p15.5 as determined by SNP array. The extension of the deletion described in this study is indicated by a red line; those of previously described deletions are indicated by a grey line: the dotted ends represent the undefined borders of these deletions. A schematic diagram showing the relevant genes of the region is present at the bottom. The green rectangles represent the exons, the arrows indicate the transcription orientation, the orange box the IC2. (B) Pedigree of the family with relevant 11p15.5 genotypes. Fetus 1 and fetus 2 are indicated as II-2 and II-3, respectively. The informative SNPs present in the deleted region in the individuals investigated are shown. (C) Electropherogram showing the DNA sequence of the deletion breakpoint in fetus 1. The extreme nucleotides at the breakpoint are highlighted.
© Copyright Policy - open-access
Related In: Results  -  Collection

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JMEDGENET2012101352F1: Structural characterisation of the 11p15.5 microdeletion. (A) Genomic profiles of fetus 2 and the parents at chromosome 11p15.5 as determined by SNP array. The extension of the deletion described in this study is indicated by a red line; those of previously described deletions are indicated by a grey line: the dotted ends represent the undefined borders of these deletions. A schematic diagram showing the relevant genes of the region is present at the bottom. The green rectangles represent the exons, the arrows indicate the transcription orientation, the orange box the IC2. (B) Pedigree of the family with relevant 11p15.5 genotypes. Fetus 1 and fetus 2 are indicated as II-2 and II-3, respectively. The informative SNPs present in the deleted region in the individuals investigated are shown. (C) Electropherogram showing the DNA sequence of the deletion breakpoint in fetus 1. The extreme nucleotides at the breakpoint are highlighted.
Mentions: A microdeletion at chromosome 11p15.5 of 40–70 kb was first detected by chromosome microarray (CMA) (performed at Signature Genomics, Spokane, WA, using the Roche NimbleGen 135K oligonucleotide array as platform, and the SignatureChipOS Lot#: 528428A05 V.3.0 for the data analysis) in DNA extracted from amniocytes of fetus 2 (not shown). Fluorescence in situ hybridisation analysis of metaphase cells using the 11p15.5 BAC probe RP11-1061N1 (and the 11q12.1 probe RP11-872D17) showed one normal signal and one diminished signal in all cells examined (not shown), confirming the deletion. To assess the parental origin, and to determine more precisely the extent of the deleted region, the DNA of fetus 2 and the parents was further analysed by CMA using the Affymetrix Genome Wide Human Single Nucleotide Polymorphism (SNP) Array 6.0 and the Affymetrix Genotyping Console 3.0.2 software, as described.11 This analysis demonstrated the presence of a 52/58 kb deletion spanning from CN_582601 (Chr11: 2,684,979 on hg19) to CN_584828 (Chr11: 2,737,280) in the DNA of fetus 2 (figure 1A). Normal copy number was detected in parental DNAs with this technique. The genotypes derived from SNPs included in the deletion indicated that the mutation was on the paternally inherited chromosome (figure 1B). The amount of available DNA from fetus 1 was insufficient to perform a similar analysis. We, therefore, further refined the deletion boundaries on the DNA of fetus 2 by quantitative PCR, and used the obtained information to set up a PCR assay across the breakpoint. The primers were 5′-TGTTCAAGCTGTGGCCACTGG -3′ and 5′-GATGGAGTGTGGTGAGGCAC -3′ and the PCR conditions: 1.5 Mm MgCl2, 10% Dimethyl sulfoxide (DMSO), annealing temperature 60°C. By using this assay and DNA sequencing (from PRIMM, Italy), we demonstrated the presence of the microdeletion also in fetus 1 (figure 1C). The sequencing results demonstrated that the deletion spans from chr11:2679858 to chr11:2739436 (based on University of California Santa Cruz (UCSC) 2009 hg19 assembly), and includes the exon 11 of the KCNQ1 gene, the centromeric-imprinted control region (IC2) of the 11p15.5 imprinted gene cluster and the most 5′ 40 kb of the KCNQ1OT1 non-coding RNA gene (figure 1A). When parental DNAs were tested by PCR, a weak band corresponding to the region encompassing the deletion breakpoint was observed in the father, indicating somatic mosaicism (see online supplementary figure and supplementary data). All the cell and DNA samples investigated have been obtained after release of informed consent. Unfortunately, we could not analyse the DNA of the healthy sibling (II-1) because the parents did not consent for this test. However, we observe that the 11p15.5 60 kb deletion is not present in 1500 samples analysed with the same platform, as well as in the Database of Genomic Variants (http://projects.tcag.ca/variation/ and data not shown).

Bottom Line: Heterogeneous molecular defects affecting the 11p15.5 imprinted gene cluster are associated with the opposite growth disorders Beckwith-Wiedemann Syndrome (BWS) and Silver Russell syndrome (SRS).Maternal deletions of the centromeric domain usually result in BWS, but paternal deletions have been so far associated with normal phenotype.DNA methylation and gene expression analyses showed that the deletion led to an imprinting alteration restricted to the centromeric domain and resulting in silencing of KCNQ1OT1 and activation of CDKN1C and PHLDA2.

View Article: PubMed Central - PubMed

Affiliation: Department of Environmental Science, Second University of Naples, Caserta, Italy.

ABSTRACT

Background: Heterogeneous molecular defects affecting the 11p15.5 imprinted gene cluster are associated with the opposite growth disorders Beckwith-Wiedemann Syndrome (BWS) and Silver Russell syndrome (SRS). Maternal deletions of the centromeric domain usually result in BWS, but paternal deletions have been so far associated with normal phenotype. Here we describe a case of recurrent severe Intra-Uterine Growth Restriction (IUGR) with paternal transmission of an 11p15.5 60 kb deletion.

Methods and results: Chromosome microarray (CMA), PCR and DNA sequencing analyses showed that two fetuses conceived by a normal couple inherited from their father a 60 kb deletion encompassing the Imprinting Control Region of the 11p15.5 centromeric domain. The two fetuses died in utero with severe growth restriction. PCR amplification of parental DNAs indicated that the father carried the mutation in the mosaic state. DNA methylation and gene expression analyses showed that the deletion led to an imprinting alteration restricted to the centromeric domain and resulting in silencing of KCNQ1OT1 and activation of CDKN1C and PHLDA2.

Conclusions: Our data demonstrate that the phenotype associated with 11p15.5 deletions is strongly influenced by the size of the region involved and indicate imprinting defects leading to CDKN1C and PHLDA2 activation as cause of severe IUGR.

Show MeSH
Related in: MedlinePlus