Introduction

Genomic imprinting is an epigenetic modification that causes genes to be expressed according to their parent of origin. Although less than 100 imprinted genes have been identified in human and mice, many imprinted genes appear to have a critical role in prenatal growth and development [1]. Molecular genetic analysis of rare human imprinting disorders has played a critical role in elucidating the mechanisms of genomic imprinting. In particular, studies of the imprinting disorder Beckwith-Wiedemann syndrome (BWS MIM 130650) have provided important insights into the structure and function of imprinting centres [2]. BWS is a congenital overgrowth syndrome, characterised by prenatal and postnatal overgrowth, macroglossia and anterior abdominal wall defects. Additionally, variable features include organomegaly, neonatal hypoglycaemia, hemihypertrophy, urogenital abnormalities and in about 5% of children, embryonal tumours (most frequently Wilms' tumour). The genetics of BWS are complex, but involve mutation or altered expression of several closely linked genes associated with cell cycle and growth control in the imprinted 11p15.5 chromosomal region. Imprinted genes most frequently implicated in the aetiology of BWS include the paternally expressed IGF2, KCNQ1OT1 (LIT1) genes and the maternally expressed H19 and CDKN1C (P57KIP2) genes. KCNQ1OT1 and H19 transcripts are not translated but the IGF2 gene product is an important prenatal growth factor and the CDKN1C protein is a candidate tumour suppressor that negatively regulates the cell cycle [3]. The majority of BWS cases are sporadic and result from epimutations of the distal (IC1) or proximal (IC2) 11p15.5 imprinting centres (see [4] and references within). IC1 is a differentially methylated region (DMR) about 5kb upstream of H19 that has an “insulator function” regulated by the zinc finger transcription factor, CTCF. The insulator is methylation sensitive, such that when CTCF binds to the unmethylated maternal allele, the IGF2 promoters do not have access to (are insulated from) enhancers downstream of H19. Methylation on the paternal allele prevents CTCF from binding, thus permitting interaction between the IGF2 promoters and the enhancers [5]. About 5–10% of sporadic BWS cases have hypermethylation of the H19 DMR and in these cases IGF2 shows loss of imprinting (LOI) and biallelic expression [6]. The second imprinting centre, IC2 is a DMR located in intron 10 of the KCNQ1 gene and is known as KvDMR1. The unmethylated paternal allele permits transcription of the antisense transcript KCNQ1OT1 (also known as LIT1) and silencing of genes including KCNQ1 and CDKN1C. Maternal methylation at the KvDMR1 is thought to prevent transcription of the KCNQ1OT1 gene and enable expression of CDKN1C. Loss of methylation (LOM) at the KvDMR1 is seen in up to 50% of sporadic BWS and is associated with biallelic expression (loss of imprinting) of KCNQ1OT1 and silencing of maternal CDKN1C expression [7]–[9]. Apparent hypomethylation of IC2 may, in rare cases, result from a germline IC2 deletion [10]. However, most BWS patients with IC2 methylation defects appear to have an epimutation of unknown cause (although there is an increased risk of BWS with IC2 epimutation in children conceived by assisted reproductive technologies) [11]–[13]. In order to gain insights into the factors responsible for IC2 imprinting defects, we studied a family with BWS that displayed evidence of IC2 epimutations through a trans mechanism.

Materials and Methods

Results

Case Report

A family with complex consanguinity (Figure 1) was ascertained after the diagnosis of two children with BWS. Both pregnancies were complicated by polyhydramnios and raised hHCG levels. Both affected children were born by Caesarean section. At birth Child 1 (V-1 in Figure 1) was macrosomic (4.55 kg at 38 weeks gestation) and was noted to have macroglossia, an omphalocele, ear creases, right inguinal hernia, undescended testis and neonatal hypoglycaemia in the first two days after birth. Similarly Child 2 (V-2) was macrosomic (3.633 kg at 35 weeks gestation) with macroglossia, ear lobe creases and neonatal hypoglycaemia that was difficult to control. Subsequently a third child was born without features of BWS (between the second and third children a probable hydatidiform mole was diagnosed). Molecular studies demonstrated loss of maternal allele KvDMR1 (IC2) methylation in both affected children, but the unaffected sibling had normal methylation. H19 methylation status was normal in both affected children and MLPA analysis demonstrated no evidence of an IC1 or IC2 deletion. Genotyping revealed no evidence of paternal uniparental disomy and linkage analysis with microsatellite markers flanking IC2 (TH and D11S4088) demonstrated that the two children had inherited opposite maternal and paternal 11p15.5 alleles. These findings were consistent with IC2 epimutation resulting from a trans imprinting defect.

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Figure 1. Family pedigree (partial) to demonstrate complex consanguinity.

https://doi.org/10.1371/journal.pgen.1000423.g001

Molecular Genetic Analysis

In view of the history of consanguinity, an autosomal recessive disorder was suspected (either affecting both children or affecting the mother). Genetic linkage studies were undertaken by genotyping the two children and both parents on an Affymetrix 250k SNP array platform. Five regions of homozygosity (>2Mbases) were shared by the two children but these did not contain a gene known to be implicated in the establishment or maintenance of genomic imprinting. However inspection of the maternal genotypes revealed an ∼8 Mbase homozygous region containing NLRP2 and NLRP7 at 19q13.4. NLRP7 is a homologue of the mouse NLRP2 gene (NLRP7 is not present in the mouse) and the human NLRP2 gene. Sequencing of NLRP2 in the mother identified a homozygous frameshift mutation ((c.1479delAG, NM_017852; Figure 2) that was predicted (in the absence of nonsense-mediated RNA decay) to result in a truncated protein (p.Arg493SerfsX32) lacking 539 amino acids from the C-terminal that includes the LRR domain. The mutation was not detected in 542 ethnically matched control chromosomes but the father was heterozygous for the mutation, child 1 was homozygous for the mutation and the other two children were heterozygous. Mutation analysis of 11 additional families with BWS did not reveal any evidence of pathogenic NLRP2 mutations.

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Figure 2. Germline NLRP2 mutation.

The alignments of NLRP2 nucleotides c.1465–c.1492 are shown. (A) is wildtype sequence in a control, (B) is heterozygous NLRP2 mutation (c.1479delAG) in the father, and (C) is homozygous 2bp deletion (c.1479delAG) in the mother.

https://doi.org/10.1371/journal.pgen.1000423.g002

To determine if the trans imprinting defect extended beyond KvDMR1, we analysed methylation levels at the TND (6q24), SNRPN (15q13) and PEG1 (7q32) DMRs. Both affected siblings (and all controls) had normal methylation levels at the TND and SNRPN DMRs but Child 2 demonstrated partial loss of methylation at the PEG1 DMR (Figure 3).

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Figure 3. Loss of methylation at PEG1 DMR.

Electropherogram of amplification products of MS-PCR. U, unmethylated product; M, methylated product. The X-axis represents the calculated product size (in bp and also represented as the top number in the box). The Y axis represents the peak height (bottom number in the box). The methylated to unmethylated ratio was calculated as the area under the curve (middle number in the box) of methylated versus unmethylated amplified products. (A) Normal Control (ratio 0.81), (B) Child 2 with LOM at PEG1.

https://doi.org/10.1371/journal.pgen.1000423.g003

Discussion

We identified a homozygous frameshift mutation in the mother of two children with BWS caused by epimutations at IC2 (KvDMR1). Most cases of BWS due to loss of methylation of KvDMR1 are sporadic, but a handful of familial cases have been described with maternally inherited germline IC2 deletions [10]. However, in our family there was no evidence of a germline deletion by MLPA analysis [16] and the two affected children were shown to have inherited opposite maternal and paternal 11p15.5 alleles. This suggested that in this family, KvDMR1 LOM resulted from a trans and not a in cis effect. Germline NLRP2 mutations have not been reported previously, but mutations in ZFP57 and NLRP7 can cause imprinting disorders in which epimutations at imprinted loci result from a trans effect. Thus individuals homozygous for ZFP57 mutations presented with transient neonatal diabetes mellitus (TNDM). The major cause of TNDM is aberrant expression of imprinted genes at chromosome 6q24 and about 20% of cases have LOM at the TND differentially methylated region (DMR). Patients with homozygous ZFP57 mutations have LOM at the TND DMR, but also at other imprinted loci including KvDMR1 [15]. However, there was no evidence of germline ZFP57 mutations in our family. Germline NLRP7 mutations are associated with familial recurrent biparental complete hydatidiform mole (FHM) in which there is epigenetic abnormalities at DMRs in multiple imprinting regions [17]–[19]. Although FHM associated with NLRP7 mutations is inherited in an autosomal recessive manner, in contrast to ZFP57 mutation, homozygotes have normal genomic methylation but in female homozygotes there is a failure to establish methylation imprints in their germ cells leading to hydatidiform moles and reproductive wastage (male homozygotes do not have imprinting defects in their sperm). The methylation defects in FHM are specific for imprinted loci, and DNA methylation at non-imprinted genes and genes subject to X-inactivation is unaffected [18]. The human NLRP2 and NLRP7 genes are highly homologous and the two proteins consist of 1,062 and 1,009 amino acids respectively and have identical structure and about 64% amino acid identity. Only one of the two affected children with BWS was homozygous for a NLRP2 mutation and, by analogy with FHM caused by NLRP7 mutations, the BWS phenotype most likely results from homozygosity in the mother, such that familial BWS associated with NLRP2 mutations is inherited in a similar manner to NLRP7 associated FHM and not in a conventional autosomal recessive manner.

Both FHM and ZFP57–TNDM are associated with imprinting aberrations at multiple loci, and we identified partial loss of methylation at the PEG1 DMR in one of the affected children. Nevertheless it seems that NLRP2 mutations have a less severe effect on imprinting than NLRP7 or ZFP57 inactivation. A subset of children with BWS and an IC2 epimutation display hypomethylation at multiple imprinting centres (DMRs) (in our series these children are more likely to have been conceived by assisted reproductive technologies) [14],[20]. To our knowledge, none of these cases have been familial and we did not identify NLRP2 mutation in the sporadic cases we studied. It appears that the establishment (or maintenance) of methylation at KvDMR1 is particularly sensitive to genetic and/or environmental insults. We note that one of the affected children demonstrated a partial loss of methylation at PEG1 DMR1 both by bisulphite sequencing and MS-PCR suggesting that NLRP2 mutations may be associated with an incomplete failure of imprinting establishment and/or a partial failure of maintenance methylation at this DMR. Interestingly, investigation of a mouse knockout of ZFP57 has suggested a role in both the establishment of germline methylation imprints and in the postfertilisation maintenance of methylation imprints [21].

NLRP2 and NLRP7 encode members of the NLRP (Nucleotide-binding oligomerization domain, Leucine rich Repeat and Pyrin domain) family of CATERPILLER proteins. NLRP family of cytoplasmic proteins comprises 14 members of similar structure that are principally encoded by two gene clusters on chromosome 11p15 (NLRP6, 10 and 14) and 19q13.4 (NLRP2, 4, 5, 7, 8, 9, 11, 12 and 13). Most of the family members are well conserved from C. elegans, D. melanogaster, rat, and mouse to human but there is no rodent homologue for NLRP7 and the gene is found in only a few genomes (human, primate and cow). Some NLRP proteins are components of the inflammasome that is implicated in the sensing of, and inflammatory reaction to, extracellular pathogens and intracellular noxious compounds [22]. Germline mutations in NLRP3 and NLRP12 are associated with familial cold autoinflammatory syndrome [23],[24]. NLRP2 was suggested to function as a modulator of macrophage NFKB activation and procaspase 1 [25], however we found that the two family member homozygous for a NLRP2 truncating mutation did not show any evidence of an immune or autoinflammatory disorder. Nevertheless most NLRP family proteins are widely expressed and not restricted to the immune system. In addition, many are expressed in human oocytes and embryos at an early stage of development. Thus Zhang et al. have reported that NLRP4, 5, 8, 9, 11, 12, 13, and 14 were highly expressed in oocytes and then gradually decreased in embryos with a very low level in day 5 embryos, whilst NLRP2 and NLRP7 progressively decreased from oocytes to day 3 embryos then showed a sharp increase on day 5 [26]. These observations are consistent with NLRP2 and NLRP7 having a similar role in early development/imprinting establishment. Although it has been suggested that FHM might result from an immune-related defect in oogenesis or early embryo development (with methylation changes being a secondary phenomenon) the specific association of the methylation defects with imprinted DMRs suggests a more direct role in the establishment or maintenance of imprinting marks. Such a view is supported by the identification of germline NLRP2 mutations in BWS and should prompt further investigation of the role of NLRP2 and NLRP7 in genomic imprinting. The apparent involvement of NLRP proteins in genome methylation and the sensing and inflammatory response to extracellular pathogens and intracellular noxious compounds is intriguing given the suggestion that cytosine methylation may have evolved as a host response to transposons [27]. It is interesting that the third child in the family we report was unaffected. Most mothers with NLRP7 mutations have recurrent molar pregnancies only, but at least three families have been reported in which affected women had liveborn offspring (see [28] and references within). In one family, three affected members had, in addition to the molar phenotype, several miscarriages and three term pregnancies [29]. In one of the term pregnancies the baby was born with severe intrauterine growth retardation, but grew into a healthy adult with normal methylation levels [30]. In another term pregnancy the baby was born with unilateral cleft lip and palate and later manifested idiopathic delayed mental and motor development [31]. Murdoch et al. detected a homozygous splice site mutation in NLRP7 in all three affected women of this family [32]. Thus homozygous NLRP7 mutations may be associated with clinical heterogeneity/incomplete penetrance, possibly resulting from genetic modifier or environmental effects. In the light of these observations and the apparently milder phenotypic effects of maternal NLRP2 inactivation than NLRP7 inactivation (Beckwith-Wiedemann syndrome and molar pregnancy respectively) it might be predicted that clinical heterogeneity/incomplete penetrance would be a feature of maternal NLRP2 inactivation. Although maternal NLRP2 mutations appear to be a rare cause of familial BWS, the identification of these cases is important, as the inheritance pattern differs from the autosomal dominant inheritance (with parent of origin effects) associated with other inherited forms of BWS. The inheritance of NLRP2-associated BWS has similarities to other autosomal recessive disorders in which homozygous mothers are well, but there is a high risk to their offspring (e.g. FHM and treated maternal phenylketonuria).

Acknowledgments

We thank the patients and their families for their help with this study.