Mutations in the PCNA-binding domain of CDKN1C cause IMAGe syndrome

Abstract

IMAGe syndrome (intrauterine growth restriction, metaphyseal dysplasia, adrenal hypoplasia congenita and genital anomalies) is an undergrowth developmental disorder with life-threatening consequences. An identity-by-descent analysis in a family with IMAGe syndrome identified a 17.2-Mb locus on chromosome 11p15 that segregated in the affected family members. Targeted exon array capture of the disease locus, followed by high-throughput genomic sequencing and validation by dideoxy sequencing, identified missense mutations in the imprinted gene CDKN1C (also known as P57KIP2) in two familial and four unrelated patients. A familial analysis showed an imprinted mode of inheritance in which only maternal transmission of the mutation resulted in IMAGe syndrome. CDKN1C inhibits cell-cycle progression, and we found that targeted expression of IMAGe-associated CDKN1C mutations in Drosophila caused severe eye growth defects compared to wild-type CDKN1C, suggesting a gain-of-function mechanism. All IMAGe-associated mutations clustered in the PCNA-binding domain of CDKN1C and resulted in loss of PCNA binding, distinguishing them from the mutations of CDKN1C that cause Beckwith-Wiedemann syndrome, an overgrowth syndrome.

Figure 1. Identity-by-descent analysis in a family with IMAGE syndrome

(a) In this large family (Family A) with IMAGE syndrome, 24 individuals were tested for genetic mutations in CDKN1C. All affected individuals we tested carried the c.825T>G mutation on the maternally inherited allele resulting in a F276V amino acid change. Unaffected carriers all inherited the mutations on the paternal alleles. (b) IBD analysis identified a region on chromosome 11 spanning from base pairs 2,685,916 to 19,809,755 (according to hg19) that was shared by affected family members (IV-10, V-1, V-2, V-5, V-6, V-7, V-12) and different from an unaffected sibling (V-13).

Figure 2. Localization of IMAGE syndrome mutations in CDKN1C

(a) All IMAGE syndrome mutations are localized to the region of the gene encoding the PCNA-binding domain. (b) Highly conserved amino acid residues (indicated by *) in the PCNA-binding domain of CDKN1C are conserved down to D. rerio. All IMAGE mutations affect highly conserved residues. A colon (:), indicates conservation between groups of strongly similar properties; a period indicates conservation between groups of weakly similar properties.

Figure 3. Phenotypic validation of IMAGE syndrome-associated mutations in Drosophila melanogaster

(a) Light microscope IMAGE of an adult wild type eye (ey>+). Scale bar, 25μm. (b) Eye-specific expression of human CDKN1C (ey>CDKN1C ^WT) did not affect final size. (c-g) Overexpression of IMAGE syndrome-associated human CDKN1C mutations F276V (c-d, ey>CDKN1C^F276V) or K278E (e-f, ey>CDKN1C^K278E) in the developing eye led to a gain-of-function phenotype of moderate (c, e) to severely (d, f) restricted eye growth. (g-h) Overexpression of human CDKN1C carrying Beckwith-Wiedemann syndrome-associated mutations, c.826delT (g, ey>CDKN1C^826delT) or L42P (h, ey>CDKN1C^L42P), did not affect eye growth.

Figure 4. CDKN1C is expressed in the developing human adrenal gland and IMAGE mutants lose PCNA-binding, altering ubiquitination of CDKN1C

(a) Taqman RT-PCR showed higher expression of CDKN1C in the adrenal gland at 7–8 weeks post conception compared to control samples (muscle, brain). Error bars represent 1 s.d. (b) Immunohistochemistry showed CDKN1C protein expression in the developing adrenal gland at 8 weeks post conception. Nuclear expression of CDKN1C was observed in a subset of cells in the subcapsular region (arrowhead) and developing definitive cortex (arrow). Expression of the steroidogenic enzyme CYP11A1 is shown, predominantly in the fetal zone of the developing gland. Nuclear counterstaining was performed with DAPI. Scale bar, 200 μm. (c) HEK293T cells transfected with flag-tagged wild type or IMAGE mutant (F276V or K278E) CDKN1C were immunoprecipitated with Flag antibody. Left panel: whole lysates for wild type and non-transfected cells. Right panel: Flag immunoprecipitated fraction binds with endogenous PCNA in wild type but not in IMAGE mutants F276V and K278E. Flag-BAP: Bacterial Alkaline Phosphatase. (d) Immunoprecipitation of HEK293T cells co-transfected with Flag-CDKN1C (wild type or F276V) and HA-Ubiquitin constructs. Top panel: Immunoblot with HA antibody identifies a band at ~63kDa (indicated by *) in the CDKN1C wild type that is not present in the F276V IMAGE mutant. Bottom panel: Immunoblot of Flag protein.

Figure 5. Missense mutations in CDK-binding domain and truncating mutations in CDKN1C cause Beckwith-Wiedemann Syndrome while missense mutations localized to the PCNA-binding domain result in IMAGE syndrome

Comparison between CDKN1C mutations resulting in IMAGE Syndrome (black arrowheads located above gene) or in Beckwith-Wiedemann Syndrome (below; red arrowheads = missense mutations; yellow arrowheads = truncating mutations). BWS mutations are either missense mutations primarily located in the cyclin-binding domain or truncating mutations, while IMAGE syndrome mutations are all missense mutations localized to a highly conserved region of the PCNA-binding domain.