Analysis of CDKN1C in fetal growth restriction and pregnancy loss

Introduction

Cyclin-dependent kinase inhibitor 1C (CDKN1C, also known as P57/kip2) (OMIM 600856) is a key negative regulator of cell proliferation that is encoded by a paternally imprinted (maternally expressed) gene on the short arm of chromosome 11 (11p15.4) in humans (Stampone et al., 2018).

Consistent with its role in growth and development, maternally-inherited loss-of-function variants in CDKN1C are found in approximately 5–10% of individuals with the “overgrowth” condition, Beckwith-Wiedemann Syndrome (BWS) (OMIM 130650) (Eggermann et al., 2014). Clinical features of BWS include macrosomia, hyperinsulinism and adrenal tumors.

In contrast, gain-of-function variants in CDKN1C have been shown to cause growth restriction as part of IMAGe syndrome (OMIM 614732) (Arboleda et al., 2012). IMAGe syndrome is characterised by fetal/Intrauterine growth restriction, Metaphyseal dysplasia, Adrenal hypoplasia and Genital anomalies (in males, usually relatively mild hypospadias and undescended testes) as well as additional features such as hearing loss and hypercalciuria (Bennett et al., 1993; Vilain et al., 1999).

To date, children with IMAGe syndrome have all been found to harbour pathogenic single nucleotide variants (SNVs) in a very specific region of the PCNA-binding domain of CDKN1C (Arboleda et al., 2012; Cabrera-Salcedo et al., 2017). These changes potentially lead to increased activity through increasing protein stability, thereby preventing cell cycle progression into S phase (Borges et al., 2015; Hamajima et al., 2013).

More recently, SNVs in the PCNA-binding domain of CDKN1C have been reported in families with maternally-inherited fetal growth restriction (FGR) without adrenal insufficiency and in familial Silver-Russell syndrome (SRS) (OMIM 180860) (Brioude et al., 2013; Kerns et al., 2014; Sabir et al., 2019). SRS is characterised by variable clinical features including fetal and post-natal growth restriction, relative macrocephaly, feeding difficulties and characteristic facies. SRS is also described as phenotypically and genotypically opposite to BWS and approximately half of the molecular anomalies are attributed to Chr11p15.5 imprinting clusters, including several individuals with maternal duplication of the locus containing CDKN1C (Bonaldi et al., 2011; Boonen et al., 2016; Schönherr et al., 2007). These findings suggest that the growth restriction phenotype associated with CDKN1C may be more variable and adrenal insufficiency is not always present.

The aim of this study was therefore to review published CDKN1C variants associated with FGR/IUGR phenotypes, to study CDKN1C expression in different tissues, and to analyse the critical region in CDKN1C in a range of growth restriction and adverse pregnancy phenotypes, with a hypothesis that severe restriction of feto-placental growth may, in some situations, result in pregnancy loss or recurrent miscarriage.

Methods

Results

Single nucleotide variants in CDKN1C

Review of available data revealed seven publications describing isolated individuals (7) or families (5) with IMAGe syndrome and adrenal insufficiency, who had pathogenic variants in a key region of the PCNA-binding domain of CDKN1C affecting codons 272, 274, 276, 278 and 279 (Figure 1, Table 2) (Arboleda et al., 2012; Bodian et al., 2014; Brioude et al., 2013; Hamajima et al., 2013; Kato et al., 2014; Kerns et al., 2014; Sabir et al., 2019). These codons are highly conserved amongst species (Figure 2). Multiple individuals from different ancestral backgrounds were found to have p.Asp274Asn, p.Lys278Glu or p.Arg279Leu changes.

Figure 1. Schematic diagram showing the structure of CDKN1C and the clustering of pathogenic variants associated with IMAGe syndrome and/or growth restriction.

Table 2. Reported variants in CDKN1C and associated phenotypes.

Nucleotide variant	Protein change	Isolated/Familial	Phenotype	Reference
c.815T>G	p.Ile272Ser	Familial (3)	IMAGe	Hamajima et al., 2013
c.820G>A	p.Asp274Asn	Isolated	IMAGe	Arboleda et al., 2012
c.820G>A	p.Asp274Asn	Isolated	IMAGe	Kato et al., 2014
c.820G>A	p.Asp274Asn	Isolated	IMAGe (Glucocorticoid)	Kato et al., 2014
c.826T>G	p.Phe276Val	Familial (7)	IMAGe	Arboleda et al., 2012
c.827T>C	p.Phe276Ser	Isolated	IMAGe	Arboleda et al., 2012
c.832A>G	p.Lys278Glu	Isolated	IMAGe	Arboleda et al., 2012
c.832A>G	p.Lys278Glu	Isolated	IMAGe (Mineralocorticoid)	Bodian et al., 2014
c.836G>C	p.Arg279Pro	Isolated	IMAGe	Arboleda et al., 2012
c.836G>T	p.Arg279Leu	Familial (9)	Silver-Russell	Brioude et al., 2013
c.836G>T	p.Arg279Leu	Familial (2)	Silver-Russell	Sabir et al., 2019
c.842G>T	p.Arg281Ile	Familial (15)	IUGR, short stature, IGT/DM	Kerns et al., 2014

Abbreviations: DM, diabetes mellitus; IGT, impaired glucose tolerance; IMAGe, intrauterine growth restriction, metaphyseal dysplasia, adrenal hypoplasia congenita, genital anomalies; IUGR, intrauterine growth restriction. Numbers in parentheses refer to the number of affected individuals in each kindred.

Figure 2. Amino-acid conservancy in the “hot-spot” region of CDKN1C.

Red arrowheads represent codons that are mutated in IMAGe syndrome, FGR/IUGR or Silver-Russell syndrome. Yellow asterisks represent complete conservation amongst the species shown.

Variants in CDKN1C associated with familial Silver-Russell syndrome or growth restriction but normal adrenal function were found towards the carboxyl-terminal region of this “hot-spot” domain (p.Arg279Leu, p.Arg281Ile) (Figure 1).

Analysis of population data from the gnomAD browser showed a complete absence of variants in the key codons listed. Very rare heterozygous SNVs were found that are predicted to cause p.Ala277Val (11:2905355G>A;1 in 10512 alleles) and p.Ala283Val (11:2905337G>A; rs776541692; 1 in 1158 alleles) changes. Of note, these codons are two of the lesser-conserved amino acids within this “hot-spot” region (Figure 2).

Expression of CDKN1C in human tissue

RNA-Seq analysis of CDKN1C in a panel of human tissues showed highest expression in the placenta (Figure 3; Human Protein Atlas data, https://www.proteinatlas.org/ENSG00000129757-CDKN1C/tissue), with strong expression also in adipose tissue, ovary, adrenal, endometrium and kidney. Immunohistochemistry in the Human Protein Atlas repository shows strong staining in the nuclei of both decidual and trophoblastic cells (https://www.proteinatlas.org/ENSG00000129757-CDKN1C/tissue/placenta).

Figure 3. RNA expression of CDKN1C in placenta and different adult human tissues.

Data reproduced and modified with permission from the Human Protein Atlas (www.proteinatlas.org) (Uhlen et al., 2015). TPM = Transcripts Per Million.

Discussion

CDKN1C is now well-established as a key regulator of cell cycle and growth through G1 phase cell cycle arrest. Although loss-of-function of CDKN1C is known to cause macrosomia as part of Beckwith-Wiedemann Syndrome, it is only in the past eight years that “gain-of-function” variants in CDKN1C have been shown to cause growth restriction and IMAGe syndrome. These findings demonstrate clearly how opposite effects in protein function can have opposite phenotypes (Arboleda et al., 2012; Eggermann et al., 2014). Sometimes these features affect not just growth but also endocrine systems (e.g. adrenal tumours (BWS)/adrenal hypoplasia (IMAGe); congenital hyperinsulinism (BWS)/diabetes mellitus (one family with growth restriction)).

Review of published literature confirms that CDKN1C SNVs associated with IMAGe syndrome or growth restriction are all located within a “hot-spot” of the PCNA-binding domain of the protein. The exact function of this region is not clear, and the crystal structure of this region of CDKN1C has not yet been solved. Studies to date suggest pathogenic variants may increase protein stability or reduce the rate of degradation, thereby enhancing the negative effects of CDKN1C on cell cycle progression (Borges et al., 2015; Hamajima et al., 2013). The very localised nature of these variants clearly demonstrates a key role for this region in CDKN1C function.

CDKN1C is strongly expressed in fetal adrenal development as shown by qPCR and immunohistochemistry (Arboleda et al., 2012), by microarray (Del Valle et al., 2017), and RNA-Seq (unpublished). Furthermore, analysis of RNA-Seq data from the Human Protein Atlas shows marked expression in the placenta (Figure 3). Immunohistochemistry shows strong nuclear staining in both decidual and trophoblastic cells. Therefore, gain-of-function of CDKN1C in the developing placenta could have a significant contribution to the growth restriction phenotype.

Further evidence for a potential role for CDKN1C in fetal growth restriction phenotypes has emerged with reports of CDKN1C variants in familial growth restriction and familial SRS (Brioude et al., 2013; Kerns et al., 2014; Sabir et al., 2019). Several features of IMAGe syndrome and SRS overlap, such as bi-frontal bossing and relative micrognathia. To date these children have not shown evidence of adrenal insufficiency. Whilst a lack of adrenal features could be due to underlying mosaicism or a somatic “rescue” event, as recently reported for variants in SAMD9 in the related condition MIRAGE syndrome (Buonocore et al., 2017; Narumi et al., 2016), the fact that several individuals in a family with the same CDKN1C SNV were affected provides strong evidence that the primary genomic event is influencing the phenotype rather than a rescue mechanism. Of note, review of the two SNVs associated with FGR and normal adrenal function reveals that they affect amino acids at the C-terminal region of the hotspot. In two families a charged arginine at codon 279 is replaced by a non-polar leucine, whereas a proline at this position is found in classic IMAGe syndrome. In another family, the arginine at position 281 is replaced by a non-polar isoleucine. In some situations, variants in amino-acids flanking critical motifs can be associated with milder phenotypes (Kyriakakis et al., 2017).

Despite these findings, we did not identify any CDKN1C variants in children with isolated (non-familial) SRS or IUGR/FGR. These results are similar to data from Brioude et al. who only reported a CDKN1C variant in familial SRS and did not find variants in 68 children with an isolated condition (Brioude et al., 2013). Whilst this does not exclude CDKN1C as a potential cause of isolated or sporadic SRS/FGR, it does suggest that it is not a common cause.

Identification of such strong expression of CDKN1C in the placenta lead us to consider whether gain-of-function/increased stability of CDKN1C could be associated with loss of pregnancy or recurrent miscarriage. This hypothesis was supported further by recent studies linking placental genes to pregnancy loss (Perez-Garcia et al., 2018); a potential role for placental CDKN1C expression in fetal growth and regulation by oestrogen (Chen et al., 2018; Gou et al., 2017; López-Abad et al., 2016; Unek et al., 2014); and the fact that paternal imprinting of the gene means that a pool of deleterious variants could be present in the population and cause pregnancy loss (together with live births) in women who carry this variant and inherit it from their father.

Analysis was therefore undertaken in a cohort of mothers who had recurrent miscarriages (often together with live birth(s)) and also from products of conception. However, no variants were found in the PCNA binding domain of CDKN1C in these cohorts.

This work has several limitations. Although 203 total individuals were studied, each sub group is still relatively small and rare CDKN1C variants might be discovered if the sample size is increased. The causes of recurrent miscarriage and fetal growth restriction are clearly diverse and can be influenced by many factors, and regulatory regions or enhancers (Perez-Garcia et al., 2018; Rai & Regan, 2006; Stalman et al., 2018; Wakeling et al., 2017). Nevertheless, this study does highlight the role of the key region of CDKN1C in human fetal growth restriction phenotypes, and starts to address the potential role of single gene growth restriction phenotypes in more common obstetric and fetal conditions.

Data availability

Data has been uploaded to OSF: http://doi.org/10.17605/OSF.IO/Y7KZV (Achermann, 2018). Representative sequencing data for chromatograms is shown in Supplementary Data 1. Data of coverage for next generation sequencing is shown in Supplementary Data 2–8.

Data are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).

Access for samples from the Baby Bio Bank is available and can be requested from the steering committee by emailing the Baby Bio Bank Manager (nita.solanky@ucl.ac.uk). More information about data access can be found on the website under the ‘Protocol for the management of the Baby Bio Bank’, section 15: https://www.ucl.ac.uk/child-health/research/genetics-and-genomic-medicine-programme/baby-biobank. An application form for the use of Baby Bio Bank resources must be completed prior to application (see appendix 2 in the protocol).