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Absence of Maternal Methylation in Biparental Hydatidiform Moles from Women with NLRP7 Maternal-Effect Mutations Reveals Widespread Placenta-Specific Imprinting.

Sanchez-Delgado M, Martin-Trujillo A, Tayama C, Vidal E, Esteller M, Iglesias-Platas I, Deo N, Barney O, Maclean K, Hata K, Nakabayashi K, Fisher R, Monk D - PLoS Genet. (2015)

Bottom Line: These epigenotypes are consistent with NLRP7 being a maternal-effect gene and involved in imprint acquisition in the oocyte.In addition, bioinformatic screening of the resulting methylation datasets identified over sixty loci with methylation profiles consistent with imprinting in the placenta, of which we confirm 22 as novel maternally methylated loci.These observations strongly suggest that the molar phenotypes are due to defective placenta-specific imprinting and over-expression of paternally expressed transcripts, highlighting that maternal-effect mutations of NLRP7 are associated with the most severe form of multi-locus imprinting defects in humans.

View Article: PubMed Central - PubMed

Affiliation: Imprinting and Cancer Group, Cancer Epigenetic and Biology Program, Institut d'Investigació Biomedica de Bellvitge, Hospital Duran i Reynals, Barcelona, Spain.

ABSTRACT
Familial recurrent hydatidiform mole (RHM) is a maternal-effect autosomal recessive disorder usually associated with mutations of the NLRP7 gene. It is characterized by HM with excessive trophoblastic proliferation, which mimics the appearance of androgenetic molar conceptuses despite their diploid biparental constitution. It has been proposed that the phenotypes of both types of mole are associated with aberrant genomic imprinting. However no systematic analyses for imprinting defects have been reported. Here, we present the genome-wide methylation profiles of both spontaneous androgenetic and biparental NLRP7 defective molar tissues. We observe total paternalization of all ubiquitous and placenta-specific differentially methylated regions (DMRs) in four androgenetic moles; namely gain of methylation at paternally methylated loci and absence of methylation at maternally methylated regions. The methylation defects observed in five RHM biopsies from NLRP7 defective patients are restricted to lack-of-methylation at maternal DMRs. Surprisingly RHMs from two sisters with the same missense mutations, as well as consecutive RHMs from one affected female show subtle allelic methylation differences, suggesting inter-RHM variation. These epigenotypes are consistent with NLRP7 being a maternal-effect gene and involved in imprint acquisition in the oocyte. In addition, bioinformatic screening of the resulting methylation datasets identified over sixty loci with methylation profiles consistent with imprinting in the placenta, of which we confirm 22 as novel maternally methylated loci. These observations strongly suggest that the molar phenotypes are due to defective placenta-specific imprinting and over-expression of paternally expressed transcripts, highlighting that maternal-effect mutations of NLRP7 are associated with the most severe form of multi-locus imprinting defects in humans.

No MeSH data available.


Related in: MedlinePlus

Description of NLRP7 mutations with methylation and expression profiling of imprinted loci.(A) Confirmation of recessive NLRP7 mutations in female patients and heterozygous status in the RHM samples. The asterisk (*) on the electropherogram highlights the position of the mutation. For patient 3 the position of the deletion is shown. (B) Circular heat map of the 616 Infinium array probes mapping to 36 ubiquitously imprinted DMRs. The inner circle represents the methylation values of androgenetic HMs, the middle circles normal placental biopsies and the outer circle the RHMs associated with maternal-effect NLRP7 mutations. (C) Confirmation of the methylation profile of the NLRP7 mutated RHMs at the NAP1L5, PEG10, RB1, L3MBTL1 and H19 DMRs by bisulphite PCR and subcloning. Each circle represents a single CpG dinucleotide on a DNA strand, a methylated cytosine (●) or an unmethylated cytosine (○). For clarity, only the first 10 CpG dinucleotides from each amplicon are shown with the letters in the parentheses indicating SNP genotype. (D) Allelic expression analysis of imprinted genes NAP1L5, HYMAI, PEG10 and PEG3 in control placenta samples (PL) and NLRP7-mutated moles (RHM).
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pgen.1005644.g001: Description of NLRP7 mutations with methylation and expression profiling of imprinted loci.(A) Confirmation of recessive NLRP7 mutations in female patients and heterozygous status in the RHM samples. The asterisk (*) on the electropherogram highlights the position of the mutation. For patient 3 the position of the deletion is shown. (B) Circular heat map of the 616 Infinium array probes mapping to 36 ubiquitously imprinted DMRs. The inner circle represents the methylation values of androgenetic HMs, the middle circles normal placental biopsies and the outer circle the RHMs associated with maternal-effect NLRP7 mutations. (C) Confirmation of the methylation profile of the NLRP7 mutated RHMs at the NAP1L5, PEG10, RB1, L3MBTL1 and H19 DMRs by bisulphite PCR and subcloning. Each circle represents a single CpG dinucleotide on a DNA strand, a methylated cytosine (●) or an unmethylated cytosine (○). For clarity, only the first 10 CpG dinucleotides from each amplicon are shown with the letters in the parentheses indicating SNP genotype. (D) Allelic expression analysis of imprinted genes NAP1L5, HYMAI, PEG10 and PEG3 in control placenta samples (PL) and NLRP7-mutated moles (RHM).

Mentions: In this study, we determine the genome-wide methylation profiles of RHMs from four females with NLRP7 mutations using the high-density Illumina Infinium HumanMethylation450 (HM450k) BeadChip arrays, which simultaneously quantify methylation at ~2% of all CpG dinucleotides in the human genome. The RHM samples were from women with a variety of genetic lesions including siblings carrying the same homozygous non-synonymous missense mutation (c. 2078G>C; p.R693P), an individual homozygous for a deletion that removes exons 2–5 (c.-39-1769_2129+228del) and a female with compound heterozygous mutations (c.2018C>G, c.2161C>T; p.S673X, p.R721W) (Fig 1A). Our initial analysis focused on comparing the methylation profiles of four androgenetic moles and seven normal placental samples (three first trimester and four third trimester) with those obtained for the NLRP7-mutated samples (Fig 1B and S1A Fig). A total of 616 probes mapping to 36 known ubiquitous imprinted DMRs were assessed, with observations confirmed by both pyrosequencing and standard allele-specific bisulphite PCR and sub-cloning (Fig 1C, S1C and S2 Figs). These comprehensive analyses revealed that, while normal placental biopsies had partial methylation consistent with allelic methylation (with the exception of the fully methylated NNAT and GNAS-AS1 promoters) [18], the majority of maternally methylated DMRs presented with lack-of-methylation (LOM) in both androgenetic and NLRP7-associated HMs. The only exceptions were for the IGF1R and RB1 DMRs that maintain allelic methylation in both types of mole, whereas the SNURF DMR was maintained in some of the NLRP7-mutated samples. In addition, we observe some inter-individual differences. The FAM50B DMR maintained a partially methylated state in two androgenetic CHMs and in a RHM from one of the sisters with the NLRP7 p.R693P mutation. Surprisingly, this same RHM sample also showed imprinted methylation at the PLAGL1 and PEG10 DMRs (Fig 1C and S2 Fig). Furthermore a comparison of two different RHMs from patient 4 revealed a similar methylation profile with the exception that the PEG10 and SNURF DMRs presented allelic methylation in one of the moles (S1 Fig). The PEG10 DMR was previously reported to be largely unaffected in three familial RHM samples [14]. The only paternal DMR with probes present on this array that acquires methylation in the male germline and is partially methylated in placenta is the H19 DMR (also known as ICR1). Consistent with the two copies of the sperm genome, the androgenetic CHMs are fully methylated at this locus, whereas the RHM are partially methylated. In 3 cases allele-specific bisulphite PCR revealed that the methylation was on the paternal allele (Fig 1C). Quantitative pyrosequencing of bisulphite PCRs targeting the IG-DMR on chromosome 14, which also acquires methylation from sperm but does not have probes on the HM450k array platform, revealed a partially methylated profile in both control placenta and RHMs (S2 Fig). Similarly the MEG3 DMR, which is regulated in-cis by the IG-DMR, shows a similar partially methylated profile consistent with allelic methylation (Fig 1B). The ZBDF2 and ZNF597/NAA60 promoters were fully methylated in both androgenetic CHM and NLRP7 mutated RHMs. This is consistent with the presumption that these regions acquire methylation on the paternal allele during early development under the hierarchical influence of the maternally methylated GPR1-AS and ZNF597 DMRs, respectively [18].


Absence of Maternal Methylation in Biparental Hydatidiform Moles from Women with NLRP7 Maternal-Effect Mutations Reveals Widespread Placenta-Specific Imprinting.

Sanchez-Delgado M, Martin-Trujillo A, Tayama C, Vidal E, Esteller M, Iglesias-Platas I, Deo N, Barney O, Maclean K, Hata K, Nakabayashi K, Fisher R, Monk D - PLoS Genet. (2015)

Description of NLRP7 mutations with methylation and expression profiling of imprinted loci.(A) Confirmation of recessive NLRP7 mutations in female patients and heterozygous status in the RHM samples. The asterisk (*) on the electropherogram highlights the position of the mutation. For patient 3 the position of the deletion is shown. (B) Circular heat map of the 616 Infinium array probes mapping to 36 ubiquitously imprinted DMRs. The inner circle represents the methylation values of androgenetic HMs, the middle circles normal placental biopsies and the outer circle the RHMs associated with maternal-effect NLRP7 mutations. (C) Confirmation of the methylation profile of the NLRP7 mutated RHMs at the NAP1L5, PEG10, RB1, L3MBTL1 and H19 DMRs by bisulphite PCR and subcloning. Each circle represents a single CpG dinucleotide on a DNA strand, a methylated cytosine (●) or an unmethylated cytosine (○). For clarity, only the first 10 CpG dinucleotides from each amplicon are shown with the letters in the parentheses indicating SNP genotype. (D) Allelic expression analysis of imprinted genes NAP1L5, HYMAI, PEG10 and PEG3 in control placenta samples (PL) and NLRP7-mutated moles (RHM).
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4636177&req=5

pgen.1005644.g001: Description of NLRP7 mutations with methylation and expression profiling of imprinted loci.(A) Confirmation of recessive NLRP7 mutations in female patients and heterozygous status in the RHM samples. The asterisk (*) on the electropherogram highlights the position of the mutation. For patient 3 the position of the deletion is shown. (B) Circular heat map of the 616 Infinium array probes mapping to 36 ubiquitously imprinted DMRs. The inner circle represents the methylation values of androgenetic HMs, the middle circles normal placental biopsies and the outer circle the RHMs associated with maternal-effect NLRP7 mutations. (C) Confirmation of the methylation profile of the NLRP7 mutated RHMs at the NAP1L5, PEG10, RB1, L3MBTL1 and H19 DMRs by bisulphite PCR and subcloning. Each circle represents a single CpG dinucleotide on a DNA strand, a methylated cytosine (●) or an unmethylated cytosine (○). For clarity, only the first 10 CpG dinucleotides from each amplicon are shown with the letters in the parentheses indicating SNP genotype. (D) Allelic expression analysis of imprinted genes NAP1L5, HYMAI, PEG10 and PEG3 in control placenta samples (PL) and NLRP7-mutated moles (RHM).
Mentions: In this study, we determine the genome-wide methylation profiles of RHMs from four females with NLRP7 mutations using the high-density Illumina Infinium HumanMethylation450 (HM450k) BeadChip arrays, which simultaneously quantify methylation at ~2% of all CpG dinucleotides in the human genome. The RHM samples were from women with a variety of genetic lesions including siblings carrying the same homozygous non-synonymous missense mutation (c. 2078G>C; p.R693P), an individual homozygous for a deletion that removes exons 2–5 (c.-39-1769_2129+228del) and a female with compound heterozygous mutations (c.2018C>G, c.2161C>T; p.S673X, p.R721W) (Fig 1A). Our initial analysis focused on comparing the methylation profiles of four androgenetic moles and seven normal placental samples (three first trimester and four third trimester) with those obtained for the NLRP7-mutated samples (Fig 1B and S1A Fig). A total of 616 probes mapping to 36 known ubiquitous imprinted DMRs were assessed, with observations confirmed by both pyrosequencing and standard allele-specific bisulphite PCR and sub-cloning (Fig 1C, S1C and S2 Figs). These comprehensive analyses revealed that, while normal placental biopsies had partial methylation consistent with allelic methylation (with the exception of the fully methylated NNAT and GNAS-AS1 promoters) [18], the majority of maternally methylated DMRs presented with lack-of-methylation (LOM) in both androgenetic and NLRP7-associated HMs. The only exceptions were for the IGF1R and RB1 DMRs that maintain allelic methylation in both types of mole, whereas the SNURF DMR was maintained in some of the NLRP7-mutated samples. In addition, we observe some inter-individual differences. The FAM50B DMR maintained a partially methylated state in two androgenetic CHMs and in a RHM from one of the sisters with the NLRP7 p.R693P mutation. Surprisingly, this same RHM sample also showed imprinted methylation at the PLAGL1 and PEG10 DMRs (Fig 1C and S2 Fig). Furthermore a comparison of two different RHMs from patient 4 revealed a similar methylation profile with the exception that the PEG10 and SNURF DMRs presented allelic methylation in one of the moles (S1 Fig). The PEG10 DMR was previously reported to be largely unaffected in three familial RHM samples [14]. The only paternal DMR with probes present on this array that acquires methylation in the male germline and is partially methylated in placenta is the H19 DMR (also known as ICR1). Consistent with the two copies of the sperm genome, the androgenetic CHMs are fully methylated at this locus, whereas the RHM are partially methylated. In 3 cases allele-specific bisulphite PCR revealed that the methylation was on the paternal allele (Fig 1C). Quantitative pyrosequencing of bisulphite PCRs targeting the IG-DMR on chromosome 14, which also acquires methylation from sperm but does not have probes on the HM450k array platform, revealed a partially methylated profile in both control placenta and RHMs (S2 Fig). Similarly the MEG3 DMR, which is regulated in-cis by the IG-DMR, shows a similar partially methylated profile consistent with allelic methylation (Fig 1B). The ZBDF2 and ZNF597/NAA60 promoters were fully methylated in both androgenetic CHM and NLRP7 mutated RHMs. This is consistent with the presumption that these regions acquire methylation on the paternal allele during early development under the hierarchical influence of the maternally methylated GPR1-AS and ZNF597 DMRs, respectively [18].

Bottom Line: These epigenotypes are consistent with NLRP7 being a maternal-effect gene and involved in imprint acquisition in the oocyte.In addition, bioinformatic screening of the resulting methylation datasets identified over sixty loci with methylation profiles consistent with imprinting in the placenta, of which we confirm 22 as novel maternally methylated loci.These observations strongly suggest that the molar phenotypes are due to defective placenta-specific imprinting and over-expression of paternally expressed transcripts, highlighting that maternal-effect mutations of NLRP7 are associated with the most severe form of multi-locus imprinting defects in humans.

View Article: PubMed Central - PubMed

Affiliation: Imprinting and Cancer Group, Cancer Epigenetic and Biology Program, Institut d'Investigació Biomedica de Bellvitge, Hospital Duran i Reynals, Barcelona, Spain.

ABSTRACT
Familial recurrent hydatidiform mole (RHM) is a maternal-effect autosomal recessive disorder usually associated with mutations of the NLRP7 gene. It is characterized by HM with excessive trophoblastic proliferation, which mimics the appearance of androgenetic molar conceptuses despite their diploid biparental constitution. It has been proposed that the phenotypes of both types of mole are associated with aberrant genomic imprinting. However no systematic analyses for imprinting defects have been reported. Here, we present the genome-wide methylation profiles of both spontaneous androgenetic and biparental NLRP7 defective molar tissues. We observe total paternalization of all ubiquitous and placenta-specific differentially methylated regions (DMRs) in four androgenetic moles; namely gain of methylation at paternally methylated loci and absence of methylation at maternally methylated regions. The methylation defects observed in five RHM biopsies from NLRP7 defective patients are restricted to lack-of-methylation at maternal DMRs. Surprisingly RHMs from two sisters with the same missense mutations, as well as consecutive RHMs from one affected female show subtle allelic methylation differences, suggesting inter-RHM variation. These epigenotypes are consistent with NLRP7 being a maternal-effect gene and involved in imprint acquisition in the oocyte. In addition, bioinformatic screening of the resulting methylation datasets identified over sixty loci with methylation profiles consistent with imprinting in the placenta, of which we confirm 22 as novel maternally methylated loci. These observations strongly suggest that the molar phenotypes are due to defective placenta-specific imprinting and over-expression of paternally expressed transcripts, highlighting that maternal-effect mutations of NLRP7 are associated with the most severe form of multi-locus imprinting defects in humans.

No MeSH data available.


Related in: MedlinePlus