De Novo Truncating Mutations in the Last and Penultimate Exons of PPM1D Cause an Intellectual Disability Syndrome

Abstract

Intellectual disability (ID) is a highly heterogeneous disorder involving at least 600 genes, yet a genetic diagnosis remains elusive in ∼35%-40% of individuals with moderate to severe ID. Recent meta-analyses statistically analyzing de novo mutations in >7,000 individuals with neurodevelopmental disorders highlighted mutations in PPM1D as a possible cause of ID. PPM1D is a type 2C phosphatase that functions as a negative regulator of cellular stress-response pathways by mediating a feedback loop of p38-p53 signaling, thereby contributing to growth inhibition and suppression of stress-induced apoptosis. We identified 14 individuals with mild to severe ID and/or developmental delay and de novo truncating PPM1D mutations. Additionally, deep phenotyping revealed overlapping behavioral problems (ASD, ADHD, and anxiety disorders), hypotonia, broad-based gait, facial dysmorphisms, and periods of fever and vomiting. PPM1D is expressed during fetal brain development and in the adult brain. All mutations were located in the last or penultimate exon, suggesting escape from nonsense-mediated mRNA decay. Both PPM1D expression analysis and cDNA sequencing in EBV LCLs of individuals support the presence of a stable truncated transcript, consistent with this hypothesis. Exposure of cells derived from individuals with PPM1D truncating mutations to ionizing radiation resulted in normal p53 activation, suggesting that p53 signaling is unaffected. However, a cell-growth disadvantage was observed, suggesting a possible effect on the stress-response pathway. Thus, we show that de novo truncating PPM1D mutations in the last and penultimate exons cause syndromic ID, which provides additional insight into the role of cell-cycle checkpoint genes in neurodevelopmental disorders.

Keywords: PPM1D; cell-cycle checkpoint; intellectual disability; stress-response pathway; syndrome; truncating mutation.

Figure 1

Photographs of Nine Individuals with a Truncating Mutation in PPM1D, De Novo Mutations in PPM1D, and Predicted Consequences at the Protein Level (A) Shared facial features including a broad forehead, upturned nose, broad mouth with thin upper lip, and low-set posteriorly rotated ears. Extremities show small hands and feet with brachydactyly and hypoplastic toenails. Individual 13 also had a confirmed diagnosis of Potocki-Shaffer syndrome. Parents provided informed consent for the publication of these photographs. (B) Schematic representation of the coding sequence of PPM1D (GenBank: NM_003620.3), including zoomed-in exons 5 and 6. All de novo PPM1D mutations identified are depicted according to their location in the coding sequence. Protein domain structures encoded by exons 5 and 6 are highlighted in color: red for the PPM-type phosphatase domain (in exon 5) and green for the nuclear localization signal (in exon 6). (C) Predicted protein sequences in individuals 1–14. The last part of the translated sequence of exon 5 is in blue, and the amino acids encoded by the first part of exon 6 are in orange. For individuals 1–14, the predicted mutant amino acids are depicted in red. Abbreviations are as follows: WT, wild-type; and aa, amino acid.

Figure 2

Functional Effects of PPM1D Mutations at the RNA and Protein Levels and Downstream Effects on p53 Activation and Cell Growth (A) Semiquantitative PCR using primers PPM1D_3 (forward: 5′-AACCTGACTGACAGCCCTTC-3′; reverse: 5′-ACCAGGGCAGGTATATGGTC-3′) on tissue-specific cDNA libraries shows PPM1D expression in fetal and adult brain. EBV-LCL cDNA is the positive control (+), and ddH₂O is the negative control (−). (B) Expression levels of PPM1D were quantified by qPCR using cDNA obtained from EBV-LCLs derived from individuals with a mutation in PPM1D (individuals 2 and 3). Experiments were performed in triplicate with two sets of primer pairs: PPM1D_1 (forward: 5′-TGCTTGTGAATCGAGCATTG-3′; reverse: 5′-CCCTGATTGTCCACTTCTGG-3′) and PPM1D_2 (forward: 5′-AAGTCGAAGTAGTGGTGCTCAG-3′; reverse: 5′-TCTTCTGGCCCCTAAGTCTG-3′). Analysis was performed with SDS software according to standard procedures, and GUSB expression was used as a calibrator (forward: 5′-AGAGTGGTGCTGAGGATTGG-3′; reverse: 5′-CCCTCATGCTCTAGCGTGTC-3′). There was no significant change in PPM1D mRNA expression between affected individuals and control lines. Abbreviations are as follows: n.s., not significant; Ind, individual; Con, control individual; and Avg, average. Results are presented as the average ± SD. (C) Radiation-induced activation of p53 was investigated in fibroblasts derived from individual 1, EBV-LCLs derived from individuals 2 and 3, healthy control cells, and the cancer cell line with active PPM1D (MCF7 as the positive control). Cells were exposed to gamma irradiation (5 Gy) from an X-ray source. Whole-cell lysates were generated from cells 30–60 min and 4 hr after irradiation and subjected to protein electrophoresis. Immunoblotting of electrophoresed lysates was performed with antibodies specific to p53 (9282S), phospho-Histone γH2AX (ser139) (9718S), and actin (I-19), or β-tubulin (D-10). Anti-rabbit, anti-mouse, and anti-rabbit secondary antibodies were incubated with the blot (1:5,000) for 1 hr at room temperature, and then exposure using enhanced chemiluminescent detection followed. Western blot analysis showed no difference between case and control cell lines but did show increased p53 activation in the PPM1D mutant breast cancer cell line MCF7. (D) Growth behavior of EBV-LCLs derived from individuals with a mutation in PPM1D (individuals 2 and 3) was compared with that of age- and sex-matched control EBV-LCLs. Cells were irradiated (UV light: 60 J/m²) and cultured in a concentration of 3 × 10⁵ cells/mL. Cell numbers were counted in triplicate after 48 hr, and the experiment was repeated three times. 48 hr after irradiation, the number of EBV-LCLs derived from individuals with a mutation in PPM1D (n = 2) was significantly lower than that of control cells (n = 2), whereas the growth of untreated cells was unaffected. Abbreviations are as follows: ^∗, p < 0.05; n.s., no significance; Ind, individual; Con, control individual; and Avg, average. Results are presented as the average ± SD.