Mol. Hum. Reprod. Advance Access originally published online on November 26, 2007
Molecular Human Reproduction 2008 14(1):33-40; doi:10.1093/molehr/gam079
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A recurrent intragenic genomic duplication, other novel mutations in NLRP7 and imprinting defects in recurrent biparental hydatidiform moles
1Department of Obstetrics and Gynecology, Baylor College of Medicine, One Baylor Plaza, Room 721E, Mailstop BCM225, Houston, TX 77030, USA 2Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030, USA 3Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA 4Department of Obstetrics and Gynecology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA 5Department of Obstetrics and Gynecology, Assiut University, Said, Assiut 71111, Egypt
6 Correspondence address. Tel: +1-713-798-4914; Fax: +1-713-798-5997; E-mail: iveyver{at}bcm.edu
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Abstract |
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A complete hydatidiform mole (CHM) is an abnormal pregnancy with hyperproliferative vesicular trophoblast and no fetal development. Most CHM are sporadic and androgenetic, but recurrent HM have biparental inheritance (BiHM) with disrupted DNA methylation at differentially methylated regions (DMRs) of imprinted loci. Some women with recurrent BiHM have mutations in the NLRP7 gene on chromosome 19q13.42. Using bisulfite genomic sequencing at eight imprinted DMRs on DNA from two BiHMs, we found a pattern of failure to acquire or maintain DNA methylation at DMRs (PEG3, SNRPN, KCNQ1OT1, GNAS exon 1A) that normally acquire CpG methylation during oogenesis, but not at H19, which acquires CpG methylation during spermatogenesis. Secondary imprints at the GNAS locus showed variable abnormal patterns with both gain and loss of CpG methylation. We found novel missense and splice-site mutations in NLRP7 in women with non-familial recurrent BiHM. We identified and characterized a homozygous intragenic tandem duplication including exons 2 through 5 of NLRP7 that results in a predicted truncated protein in affected women of three unrelated Egyptian kindreds, suggesting a founder effect. Our findings firmly establish that NLRP7 mutations are a major cause of BiHM and confirm presence of a complex pattern of imprinting abnormalities in BiHM tissues.
Key words: biparental/hydatidiform mole/imprinting/mutation/NLRP7
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary DataFundingAcknowledgementReferences |
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Hydatidiform moles are abnormally developing pregnancies with hyperproliferative vesicular trophoblast and defective fetal development. Sporadic HM are classified into complete hydatidiform mole (CHM) or partial hydatidiform mole (PHM). Sporadic CHM have a 46,XX or 46,XY karyotype but are androgenetic concepti in which all the genetic material is paternally derived (AnCHM), whereas PHM have a diandric triploid genome, with three copies of each chromosome, two of which are paternally inherited (Van den Veyver and Al-Hussaini, 2006). A rare class of highly recurrent hydatidiform moles is pathologically indistinguishable from AnCHM or PHM (Moglabey et al., 1999; Al-Hussaini et al., 2003; Panichkul et al., 2005). They are however not androgenetic or diandric but have normal diploid biparental inheritance (BiHM) (Helwani et al., 1999; Fisher et al., 2002; Judson et al., 2002; Hodges et al., 2003). DNA methylation analysis of differentially methylated regions (DMRs) at imprinted loci (Judson et al., 2002; El-Maarri et al., 2003) and gene expression studies (Fisher et al., 2002) showed generalized defective reprogramming of imprinting in BiHM tissues. Absent methylation of imprinted DMRs that normally acquire DNA methylation on the maternally inherited allele in BiHM tissues suggests a defect in establishment or maintenance, rather than in erasure of maternal imprints (Fisher et al., 2002; Judson et al., 2002; El-Maarri et al., 2003). One study concluded that the primary defect lies in failure to acquire or maintain DNA methylation marks in the maternal germline during oogenesis because DMRs that normally acquire a primary DNA methylation imprint on the paternally inherited allele show normal patterns (Judson et al., 2002). A second study also found gain of paternal-specific DNA methylation on the maternally inherited allele of loci, such as the H19 DMR, that normally remain unmethylated on this allele (El-Maarri et al., 2003). Hence, the nature of the imprinting defect in BiHM tissues appears complex and is still incompletely understood.
Linkage to chromosome 19q13.42 (Moglabey et al., 1999; Sensi et al., 2000; Hodges et al., 2003; Panichkul et al., 2005) and autosomal recessive mutations in the NALP-like receptor (NLR) family, pyrin domain containing 7 genes (NLRP7, formerly called NALP7) on chromosome 19q13.42 have been found in a small number (Murdoch et al., 2006; Qian et al., 2007), but not all (Judson et al., 2002; Slim et al., 2005; Zhao et al., 2006) affected women, supporting genetic heterogeneity. NLRP7 encodes a member of the NLRP-family of CATERPILLER proteins that has not been extensively studied yet, but similar to other NLRPs, may play a role in innate immunity (Kinoshita et al., 2005; Drenth and van der Meer, 2006; Ting et al., 2006). Whether it has a direct role in the establishment of imprinting marks during oogenesis or in their maintenance in the early embryo is currently unknown and it has been proposed that the imprinting abnormalities seen in BiHM are secondary to a more generalized immune-driven disruption in oogenesis or early embryo implantation (El-Maarri and Slim, 2006; Murdoch et al., 2006). Nevertheless, the methylation defects are very specific for imprinted loci, and DNA methylation at non-imprinted genes, as well as allelic methylation at genes subject to X-inactivation, is intact (Djuric et al., 2006). Affected women themselves also have normal DNA methylation patterns at imprinted loci (El-Maarri et al., 2005).
We report novel missense mutations, splice-site mutations and a genomic duplication in NLRP7 that results in a truncated protein in women with recurrent BiHM. We also find, in two BiHM tissues, deficient CpG methylation at imprinted DMRs that normally acquire a primary CpG methylation imprinting mark on the maternal allele during oogenesis, but normal haplomethylated patterns at the H19 DMR, which acquires its DNA-methylation imprinting mark on the paternally inherited allele during spermatogenesis. Our data at secondary DNA-methylation imprints at the complex imprinted GNAS locus show differences with two previous studies (Judson et al., 2002; El-Maarri et al., 2003). This supports that autosomal recessive NLRP7 mutations in women lead to complicated patterns of abnormal imprinting in trophoblast tissues of their BiHM pregnancies that likely originate both in oogenesis and post-zygotically.
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Materials and methods |
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Subjects and samples
Subjects were from two previously described Egyptian families (RHM 1 and 2) with recurrent BiHM (Al-Hussaini et al., 2003; Panichkul et al., 2005), but additional affected and unaffected individuals have been included since our prior reports (Al-Hussaini et al., 2003; Panichkul et al., 2005). HM42 and HM43 are sisters from a new Caucasian family RHM3. Updated pedigrees are in the online Supplementary Fig. S1. Six other single affected women, HM19 (Pakistani), HM33 (Egyptian), HM24, HM70 and HM73 (all Mexican Latino) were also included. Some of these have been previously reported (Panichkul et al., 2005). Written informed consent was obtained from all subjects with BiHM, participating family members and control subjects (with normal term pregnancies or sporadic AnCHM) prior to collection of clinical information, venous blood samples, molar pregnancy tissue and control term placenta (TP) samples according to a protocol approved by the Baylor College of Medicine Institutional Review Board for Human Subject Research. Epstein Barr-virus transformed lymphoblastoid cell lines (LCL) were established from a portion of the venous blood samples. DNA was extracted from peripheral blood leukocytes (PBL) and LCL using the PureGene purification kit (Gentra Systems, Inc., Minneapolis, MN, USA) according to the manufacturer's protocol.
Bisulfite treatment and DNA methylation analysis
Aliquots of 1 µg of genomic DNA from trophoblast tissue samples BiHM70 and BiHM73 (from subjects HM70 and HM73, respectively), AnCHM and TP (TP1, TP2 and TP3) were sheared by repeated up-and-down pipetting, denatured in 3 M NaOH for 10 min at 37°C and subsequently incubated with 30 µl 10 mM hydroquinone and 520 µl 3 M sodium bisulfite at 50°C for 16 h (Frommer et al., 1992). After purification using a Qiaquick DNA purification kit (Qiagen Inc., Valencia, CA, USA), another denaturization step using 5 µl of fresh 3 M NaOH for 5 min and repurification, the eluted samples were PCR amplified using primers specific for the DMRs of the studied imprinted genes, as previously described (Judson et al., 2002), but with minor modifications. Primer sequences are in the online Supplementary Table S1A). PCR products were cloned into the pCR2.1-TOPO vector (Invitrogen, Carlsbad, CA, USA), transformed into competent cells and between 8–20 individual clones from at least two independent reactions were picked for sequencing with M13 primers.
Mutation analysis by direct sequencing
Primers were designed for amplification of all coding exons and flanking intronic sequences of NLRP7 using the genomic sequence from GeneID 199713 (NT_011109
[GenBank]
). PCR was performed for 35 cycles on 50 ng of genomic DNA using standard conditions. Primer sequences and amplification conditions are in the online Supplementary Table S1B. Purified PCR products were directly sequenced in forward or reverse orientation, and all identified mutations were verified by sequencing in both orientations of a separate PCR product. Sequences were compared with the reference genomic sequences (NT_011109
[GenBank]
region: 60126690-60150679) for mutation detection. Identified sequence variants and mutations were named according to the Human Genome Variation Society recommendations (den Dunnen and Antonarakis, 2000). To describe mutations, we chose reference transcript variant 2 of NLRP7 (NM_206828
[GenBank]
) to be consistent with prior reports (Murdoch et al., 2006; Qian et al., 2007), but we note that the alternate reference transcript variant 1 (NM_139176
[GenBank]
) is longer by 87 nucleotides, contains the alternatively spliced exon 10 (which is not present in NM_206828
[GenBank]
), but lacks the first 84 nucleotides of exon 5 because of alternative splicing. For comparison, we provide the online Supplementary Table S2 which lists the identified mutations according to the two alternate reference transcripts.
To evaluate identified mutations in controls, restriction enzyme digestion of PCR products was performed with BccI or MnlI and fragments were separated by gel electrophoresis.
RNA isolation, reverse transcription and RT–PCR
Total RNA was isolated using RNABee (IsoTex Diagnostics, Inc., TX, USA) from LCL cultures. First-strand cDNAs were synthesized from 2.5 µg of total RNA using oligo-dT primers and Powerscript II (Invitrogen, Carlsbad, CA, USA) reverse transcriptase. PCR amplification of reverse-transcribed cDNA was performed with Taq polymerase in the presence of 1.5 mM MgCl2 and Q solution (Qiagen Inc.) (primer sequences are in the online Supplementary Table S1C). Selected RT–PCR products were subsequently cloned into pCR2.1-TOPO for sequencing.
Southern analysis
Aliquots of 5 µg of genomic DNA samples from affected women, heterozygous controls and affected controls were digested with each of the restriction enzymes EcoRI, HincII, HindIII, XhoI and XcmI, electrophoresed through a 0.8% (w/v) agarose gel and blotted onto a Magnacharge nylon membrane Hybond-N+ (Amersham Biotech Inc., UK), which was hybridized with 
32P random-labeled NLRP7 cDNA probe (GenBank ID: BC109125
[GenBank]
) and visualized by autoradiography after stringent washing.
Quantitative real-time PCR
Quantitative real-time PCR (qPCR) for duplication detection within the NLRP7 locus was performed using SYBR-green chemistry on 20 ng of genomic DNA as previously described (del Gaudio et al., 2006; Wang et al., 2007). Primer sequences were designed using Primer Express software (Applied Biosystems, Foster City, CA, USA). Each reaction was performed in triplicate using the ABI7450 real-time PCR system, absolute gene copy number was determined by the standard curve method (Applied Biosystems user bulletin #2), data were normalized against an endogenous reference gene (GAPDH), and a melting curve analysis was performed to verify PCR product specificity. Primer sequences are provided in online Supplementary Table S1D.
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary DataFundingAcknowledgementReferences |
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Loss of DNA methylation at maternally methylated DMRs in BiHM
We examined the DNA methylation at DMRs of imprinted loci on DNA extracted from freshly evacuated tissues from the fourth and fifth recurrent molar pregnancy, respectively, of HM70 and of HM73, two unrelated Latino women from Mexico with recurrent BiHM, who carry mutations in NLRP7 (see below). The chorionic villi were carefully cleaned from contaminating decidual tissue and maternal blood under a dissecting stereoscope prior to DNA extraction. Biparental inheritance of the tissues was then confirmed by genotyping with ten genome-wide polymorphic short tandem repeat (STR) markers as described (data not shown) (Panichkul et al., 2005).
Aliquots of DNA from these tissues, a sporadic confirmed androgenetic CHM (AnCHM), two different TPs and one PBL sample were used for bisulfite genomic sequencing of eight imprinted DMRs. The DMRs of paternally expressed genes PEG3, SNRPN and KCNQ1OT1, which normally acquire CpG methylation on the maternally inherited silenced allele, were mostly unmethylated in the two BiHM samples (Fig. 1A). The SNRPN DMR showed a few methylated clones in AnCHM, which was unexpected but also present in a previous study (Judson et al., 2002). The DMR 5' to H19 which acquires DNA methylation on the paternally inherited alleles during spermatogenesis showed a normal haplomethylated DNA methylation pattern in BiHM, whereas it was completely methylated in AnCHM tissues. This is similar to reported findings in BiHM tissue from a patient with recurrent biparental HM not linked to 19q13.42 (Judson et al., 2002), but is different from the increased DNA methylation on the maternal allele seen in another study on BiHM tissues from women with mutations in NLRP7 (El-Maarri et al., 2003).
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We also analyzed four DMRs at the complex GNAS imprinted locus (Fig. 1B). The maternal methylation at the GNAS exon 1A (1A) DMR is thought to be a primary imprint established in oogenesis (Judson et al., 2002). The pattern observed at the 1A DMR was exactly as expected with absence of methylation in both BiHMs and in the AnCHM, but with haplomethylation in TP and PBL. Methylation on the paternal allele of the NESP55 DMR is a secondary imprint, i.e. dependent on the correct establishment of the primary maternal methylation imprint at 1A (Judson et al., 2002). As expected, the NESP55 DMR was completely methylated in both BiHM tissues and in the AnCHM, likely secondary to the absence of methylation at 1A. Interestingly, all 19 sequenced clones of one TP sample were methylated and all 9 clones of the PBL sample were unmethylated, suggesting that even in normal tissues, methylation patterns at this locus are not always as predicted. At the GNAS antisense (AS) DMR, the results were also complex. Bisulfite sequencing of both TPs showed only a few unmethylated clones. In contrast to a theoretically predicted loss of methylation in AnCHM and BiHM, our data on AnCHM and BiHM70 were more consistent with haplomethylation, although all 14 sequenced BiHM73 clones were completely methylated. Furthermore, CpG methylation at the DMR in the XL
S first exon (XLS), which normally shows methylation on the maternal allele, i.e. established later in post-zygotic development, also showed a mixed pattern. Whereas the pattern was consistent with predicted haplomethylation in PBL and TP, and absent methylation in AnCHM, haplomethylation was found in both BiHM tissues. The complexity of the bisulfite sequencing data may have resulted in part from the presence of chimerism for both parental alleles in some of the cloned bisulfite-PCR products. This may explain the unexpected results in the control PBL and TP samples for some of the analyzed loci at the analyzed GNAS DMRs. We were not able to analyze multiple single nucleotide polymorphisms (SNPs) to determine the presence of chimeric clones.
Mutation analysis of NLRP7 in women with BiHM
Homozygosity mapping with STR markers in 19q13.42 on additional subjects from the BiHM pedigrees, as well as detailed analysis with 6 SNPs at the revised centromeric boundary of the candidate region confirmed that NLRP7, which during the course of our studies was reported to be mutated in other women with BiHM (Murdoch et al., 2006), was included in the region of homozygosity in affected patients in this study (see online Supplementary Figs S2 and S3).
We then PCR-amplified and sequenced all coding exons and flanking introns of NLRP7 on LCL-derived genomic DNA of 11 affected individuals with recurrent BiHM and their unaffected family members from a total of eight families of different ethnic backgrounds.
We identified a homozygous missense mutation c.2248C>G (p.L750V) in subject HM73 (Fig. 2A) that changes a conserved leucine to valine in the second leucine-rich repeat (LRR) of NLRP7. Individual HM70 is a compound heterozygote for the c.2248C>G (p.L750V) mutation and for a c.2810+2T>G mutation, which changes the invariant T of the GT splice donor sequence of exon 9. We further found a homozygous splice donor mutation c.2471+1G>A in subject HM19, who is of Pakistani origin, i.e. identical to one identified in an unrelated Pakistani patient (Murdoch et al., 2006).
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The p.L750V missense mutation in exon 6 introduces a BccI restriction site and the c.2810+2T>G mutation introduces a MnlI site (Fig. 2A). PCR amplification followed by restriction enzyme digestion of control DNA samples failed to demonstrate these mutations in 260 control chromosomes from women of various ethnic groups, including 120 chromosomes from Latino individuals. We established that patient HM70 is a true compound heterozygote for the c.2248C>G and the c.2810+2T>G mutation by analyzing the DNA of the BiHM70 trophoblast tissue, which only contained the c.2810+2T>G mutation, confirming that these mutations were not on the same allele (data not shown).
We then performed RT–PCR on RNA extracted from LCL cultures of subject HM70 to determine whether the c.2810+2T>G mutation affects splicing of exon 9 to exon 10 (Fig. 2B). Products of the predicted size were generated with a forward primer in exon 7 (7F) and reverse primer in exon 8 (8R), and with forward primer 10F and reverse primers 11R from RNA of HM70 and unaffected controls. However, amplification with primers 9F and 10R resulted in two products when amplified from the reverse-transcribed cDNA of HM70: the expected 257 bp fragment and a fainter larger fragment of 419 bp. Direct sequencing of these fragments after gel-purification revealed the expected normally spliced exon 9 and exon 10 sequences for the 257 bp product, whereas the larger product included an additional 162 bp from intron 9. This abnormally spliced mRNA was not present in controls and is predicted to result in an in-frame insertion of 54 amino acids in the translated mutant protein. Interestingly, splice-site prediction software (NetGene2 at http://www.cbs.dtu.dk/services/NetGene2/) predicted that the next best ‘GT’ donor splice site was at position +183 in intron 9, whereas the sequence following nucleotide 162 in intron 9 is ‘GG’, which is not a canonical splice-donor site. We cannot rule out that splicing after nucleotide 182 in intron 9 occurs, but that the resulting mutant mRNA may be undetectable because it is predicted to contain a frameshift and premature stop codon upstream of the 3'-most exon of NLRP7 that may trigger nonsense-mediated mRNA decay.
Intragenic tandem duplication containing exons 2 through 5 in Egyptian women with recurrent BiHM
We were not able to find mutations in coding exons of NLRP7 in any of the affected women with BiHM from Egypt. To our surprise, we detected apparent heterozygous changes in exon 4 in DNA from these women which was inconsistent with our mapping data that indicated homozygosity by descent for the genomic region containing NLRP7. Suspecting a possible ancestral duplication of exon 4 with accumulated sequence alterations in the duplicated exon, we performed Southern analysis of genomic DNA from Egyptian women with BiHM and from selected family members, as well as unrelated BiHM patients and unaffected controls (Fig. 3A). Hybridization with a NLRP7 cDNA probe revealed novel fragments in genomic DNA of all Egyptian affected women and carriers, as well as missing fragments in DNA of all Egyptian affected women with all restriction enzymes tested. These changes tracked with the disease haplotypes and were not detected in DNA of non-Egyptian subjects with BiHM, unaffected non-carrier Egyptian family members or unrelated controls. The combined Southern data suggested the presence of an intragenic duplication that included at least exon 4 and possibly flanking exons 2, 3 and 5. Real-time qPCR on LCL-derived genomic DNA of subjects from Egyptian families and controls using primers that amplify exons 1 through 6 and selected introns of NLRP7 (Fig. 3B) showed the presence of two extra copies of exons 2 through 5 of NLRP7 in affected subjects HM34, 35, 28, 46 and 33 (subject/control ratio of 
2.00), whereas there was one extra copy of these exons in all carrier subjects of the RHM 1 and RHM2 kindreds (subject/control ratio of 1.5) (Fig. 3B). Exons 1 and 6 were not duplicated and PCR amplification of fragments in introns 1 and 5 established that the duplication involved a region of 4 kb from intron 1 to intron 5.
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PCR amplification with a forward primer in intron 4 and a reserve primer in intron 2 resulted in amplification of a 951 bp PCR product from genomic DNA of all homozygous affected women and heterozygous carriers, but not from unaffected family members (Fig. 3C) or 150 control DNA samples (300 chromosomes) of individuals from various ethnic groups, confirming that the duplication was intragenic and in tandem. Sequencing of this PCR product revealed that the recombination between intron 5 and intron 1 occurred in a region of identical sequence of 32 nucleotides (Fig. 3C).
RT–PCR amplification with a forward primer in exon 5 (5F) and reverse primer in exon 2 (2F) (Fig. 4A) yielded a 267 bp fragment from RNA of patient HM35, but not from control RNA (Fig. 4B). The sequence and size of the 267 bp RT–PCR product confirmed that exon 5 splices to exon 2, using the canonical splice donor and acceptor sites of both exons. Amplification with primers combinations 4F/5R, 5F/6R and 5F/7R followed by direct sequencing of PCR-products indicated that the mutant mRNA was detectable at low levels. Translation of the mutant mRNA is predicted to result in a truncated protein containing a frameshift after Val709 of six amino acids followed by a stop codon. This deletes the entire LRR region of NLRP7, indicating that this mutation results in loss-of-function.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary DataFundingAcknowledgementReferences |
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Most of the mutations in NLRP7 that we identified in women with recurrent BiHM disrupt the LRR, an important functional domain of NLRP7 that may play a role in protein–protein interactions or pattern recognition. The p.L750V changes a leucine to valine in the second LRR. Alignment of human NLRP7 with other proteins of the NLRP family and with NLRP7 sequences of the chimpanzee and cow genome indicates that this leucine is evolutionary conserved. Although leucine-to-valine substitutions are relatively conservative changes, they can be pathogenic (Ishiko et al., 2001). Furthermore, leucine residues are essential components of LRR motifs. The c.2810+2T>G splice-site mutation is also predicted to affect the LRR, whereas the intragenic duplication is predicted to result in a frameshift and truncated protein that, if translated, lacks the entire LRR domain. To the best of our assessment, women with this and other mutations we identified had only molar pregnancies and no other reproductive outcome (Al-Hussaini et al., 2003; Panichkul et al., 2005). This contrasts with other reports of spontaneous abortions, and even normal pregnancies interspersed with the recurrent BiHMs (Qian et al., 2007; Slim and Mehio, 2007). Whether the mutations we identified cause a more severe loss of function of NLRP7 than those previously reported, or whether these differences in reproductive outcome are due to genetic modifier alleles in the population is currently unknown.
NLRP7 belongs to the NLRP-family of CATERPILLER proteins and contains a NACHT associated domain (NAD), a NACHT domain and a LRR region with 9 LRRs. CATERPILLER proteins, especially NLRPs, are intracellular sensors of microbial and viral pathogens with some functional similarity to membrane-bound Toll-like receptors (TLRs) which recognize extracellular pathogens (Ting et al., 2006). Other members of the NLRP family of proteins are major components of the inflammasome, a recently discovered intracellular protein complex of macrophages and neutrophils that senses extracellular pathogens and intracellular noxious compounds and generates a caspase-1 dependent activation of the pro-inflammatory cytokine interleukin-1β (IL1β) (Drenth and van der Meer, 2006; Ogura et al., 2006). Studies in cultured cells suggest that NLRP7 may have an anti-inflammatory role as a feedback regulator of lipopolysaccharide-stimulated caspase-1-dependent IL1β secretion in lymphoid cells (Kinoshita et al., 2005), but its role in vivo has not been characterized.
The expression of NLRP7 is however not restricted to the immune system. The mRNA is detected in a variety of tissues (Kinoshita et al., 2005), including oocytes, early preimplantation embryo, endometrium and uterus (Murdoch et al., 2006), and it was found to be up-regulated in germ cell tumors (Okada et al., 2004), suggesting that it may have a specific role in these reproductive organs. There is no rodent homologue for NLRP7 and the gene is found in only a few genomes (human, primate and cow), supporting that it may have a specialized function in those mammals, perhaps related to imprinting. Parent-of-origin specific reprogramming of imprinting is a complex process initiated during gametogenesis. In female mice, parental DNA methylation marks are first completely erased in primordial germ cells. This is followed in the growing oocyte by remethylation of maternally methylated DMRs. Post-zygotically, these imprinted DNA methylation marks are largely mitotically maintained and protected from the global DNA demethylation and remethylation of the rest of the genome. DNA methyltransferases, other chromatin-associated factors, microRNAs as well as motifs in the DNA sequence near imprinting control regions (ICRs) have all been implicated in the establishment and maintenance of imprinting marks, but it is incompletely understood which factors direct the recognition of the parental identity of imprinted alleles. The DNA methylation patterns observed in this study indicate that BiHM are characterized by failure to acquire primary DNA methylation imprints in the maternal germline, or maintain these acquired maternal DNA methylation patterns post-zygotically. For the H19 DMR which normally acquires primary paternal DNA methylation imprints, we found no evidence for increased methylation on maternally inherited alleles, although we could not establish the parental origin of the methylated clones. The results at DMRs with secondary imprints are more complex. While at the XLS DMR, our findings in BiHM and AnCHM are similar to those predicted and previously reported (Judson et al., 2002), they are nearly the opposite to what is predicted at the AS DMR. Combined with data from previously published reports (Judson et al., 2002; El-Maarri et al., 2003), this indicates that in addition to a failure to acquire or maintain primary maternal DNA methylation imprints set during oogenesis, there is a failure to correctly establish and/or maintain secondary post-zygotic imprinting marks in an unpredictable pattern in BiHM tissues.
Considering the role of NLRP7 in inflammation, it has been proposed that the defects in DNA methylation observed in BiHM trophoblasts are a secondary phenomenon associated with a more generalized immune-response-driven dysfunction of the oocyte, early embryo or intrauterine milieu. This could potentially explain other adverse reproductive outcomes seen in some women with recurrent BiHM (Slim and Mehio, 2007; Qian et al., 2007). It is however striking that the DNA methylation defects in BiHM tissues are highly specific for imprinted genes (Djuric et al., 2006) and do not affect other methylated loci, whereas women with BiHM have normal DNA methylation patterns themselves (El-Maarri et al., 2005). It is possible that NLRP7, in addition to its role in the regulation of the cellular immune response, may have been recruited to carry out a specialized different function in oogenesis and early pregnancy that involves correct establishment or maintenance of parent-of-origin-specific DNA methylation on imprinted genes. Several indirect observations support the possibility of a direct interaction of NLRP7 with DNA. First, although NLRP7 does not have a clear nuclear localization signal, the reprogramming of imprinting marks during meiosis and their propagation to daughter cells during mitosis occurs at stages in the cell cycle when the nuclear envelope is not present. Second, NLRP3 (CIAS1) was reported to bind nucleic acid (RNA), likely via its LRR (Kanneganti et al., 2006). Recent analysis of Cias1–/– mice has shown that it is activated in response to the synthetic compounds R837 and R848 (which resemble nucleic acids), to viral and bacterial ssRNA, to dsRNA (but not mammalian RNA) and to purines (Kanneganti et al., 2006; Mariathasan et al., 2006; Martinon et al., 2006). Third, the LRR domains of TLR7 and TLR8 bind RNA (Koski et al., 2004; Kariko et al., 2005), whereas the LRR domain of TLR9 recognizes unmethylated bacterial CpG motifs (Hemmi et al., 2000; Bauer and Wagner, 2002).
Further experiments will be needed to study the normal function of NLRP7 and to address whether it is primarily the establishment or maintenance of parent-of-origin-specific DNA methylation at DMRs at imprinted genes that is disrupted in BiHMs of women with NLRP7 mutations, or whether there is a more general inflammatory disturbance that affects oogenesis or embryogenesis, and secondarily imprinting marks.
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Supplementary Data |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary DataFundingAcknowledgementReferences |
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Supplementary data are available at http://molehr.oxfordjournals.org/.
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Funding |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary DataFundingAcknowledgementReferences |
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Post-doctoral fellowship from the Department of Obstetrics and Gynecology, Lin-Kou Medical Center, Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Tao-Yuan, Taiwan (to HHP); National Institutes of Health (HD045970) to IBV; the Baylor College of Medicine Mental Retardation and Developmental Disabilities Research Center of the National Institutes of Health (HD024064) to IBV; USDA CRIS award 6250-51 000-049 to IBV.
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Acknowledgement |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary DataFundingAcknowledgementReferences |
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We thank Zhiyin Yu for technical assistance.
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Footnotes |
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These two authors contributed equally to this work. 
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References |
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Submitted on September 8, 2007; resubmitted on October 22, 2007; accepted on October 25, 2007.
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