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Case Reports
. 2015 Nov;64(11):3951-62.
doi: 10.2337/db15-0477. Epub 2015 Jul 9.

A Missense Mutation in PPP1R15B Causes a Syndrome Including Diabetes, Short Stature, and Microcephaly

Baroj Abdulkarim  1 , Marc Nicolino  2 , Mariana Igoillo-Esteve  1 , Mathilde Daures  3 , Sophie Romero  3 , Anne Philippi  3 , Valérie Senée  3 , Miguel Lopes  1 , Daniel A Cunha  1 , Heather P Harding  4 , Céline Derbois  5 , Nathalie Bendelac  6 , Andrew T Hattersley  7 , Décio L Eizirik  1 , David Ron  4 , Miriam Cnop  8 , Cécile Julier  9
Affiliations
Case Reports

A Missense Mutation in PPP1R15B Causes a Syndrome Including Diabetes, Short Stature, and Microcephaly

Baroj Abdulkarim et al. Diabetes. 2015 Nov.

Abstract

Dysregulated endoplasmic reticulum stress and phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) are associated with pancreatic β-cell failure and diabetes. Here, we report the first homozygous mutation in the PPP1R15B gene (also known as constitutive repressor of eIF2α phosphorylation [CReP]) encoding the regulatory subunit of an eIF2α-specific phosphatase in two siblings affected by a novel syndrome of diabetes of youth with short stature, intellectual disability, and microcephaly. The R658C mutation in PPP1R15B affects a conserved amino acid within the domain important for protein phosphatase 1 (PP1) binding. The R658C mutation decreases PP1 binding and eIF2α dephosphorylation and results in β-cell apoptosis. Our findings support the concept that dysregulated eIF2α phosphorylation, whether decreased by mutation of the kinase (EIF2AK3) in Wolcott-Rallison syndrome or increased by mutation of the phosphatase (PPP1R15B), is deleterious to β-cells and other secretory tissues, resulting in diabetes associated with multisystem abnormalities.

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Figures

Figure 1
Figure 1
Imaging of brain and skeleton of patient 1. A: Growth chart, showing growth retardation. B: Coronal T2-weighted brain MRI at age 15 years, showing a moderate white matter rarefaction characterized by increased sulcal size and moderate enlargement of ventricular system. C and D: Skeletal radiographies at age 28 years, showing kyphoscoliosis with tall vertebral bodies and hyperlordosis.
Figure 2
Figure 2
Identification of a homozygous PPP1R15B-R658C mutation in two siblings with diabetes and consequences of the mutation on the protein. A: Sanger sequencing of a control subject and the two siblings with diabetes (filled symbols), presenting the homozygous mutation and its consequence on the cDNA and protein. B: PPP1R15B protein sequence, showing the alignment of a highly conserved 62 amino acid segment (hatched) located within the COOH-terminal functional core region (gray). Representative sequences aligned are PPP1R15B from human (PR15B_HUMAN, Q5SWA1) and mouse (PR15B_MOUSE, Q8BFW3); PPP1R15A from human (PR15A_HUMAN, O75807), mouse (PR15A_MOUSE, P17564), and drosophila (PR15A_DROME, Q9W1E4); and homologous proteins from a variety of viruses: African Swine fever virus (VF71_ASFB7, Q65212), Amsacta moorei entomopoxvirus L (NP_064975), Glossina pallidipes salivary gland hypertrophy virus (YP_001687092), Choristoneura occidentalis granulovirus (YP_654457), and Trichoplusia ni ascovirus 2c (YP_803309). The mutated arginine (R) at position 658 is part of the functional RVxF-ΦΦ-R motif (boxed and underlined on human PPP1R15B) that has been recognized in PP1-interacting proteins (33). Residues shown in red are fully conserved in selected species; residues that are the most critical for establishing contact with PP1 according to Chen et al. (32) are indicated by stars.
Figure 3
Figure 3
The R658C mutation destabilizes the PPP1R15B-PP1 complex and diminishes its phosphatase activity. HEK293T cells were transfected with an empty vector (GFP) or a GFP-tagged WT or R658C-mutated human PPP1R15B, alone or in combination with a mouse PP1A expression plasmid. PPP1R15B-PP1 complexes were immunoprecipitated from lysed cells with anti-GFP antibody. A: The recovery of PP1 in complex with PPP1R15B was examined by Western blotting using anti-PP1 antibody. Immunoprecipitated protein is shown on the left (IP) and the eluant is shown on the right (input). B: The holophosphatase activity was studied in an eIF2α dephosphorylation assay, incubating the indicated PPP1R15B-PP1 complexes purified from cells with in vitro phosphorylated eIF2α (eIF2αP) protein for 30 min and resolving the phosphorylated and nonphosphorylated eIF2α (eIF2α0) on Phos-tag gels. C: A time course of eIF2α dephosphorylation by WT and R658C-mutated PPP1R15B and PP1A complexes recovered by immunopurification from transfected HEK293T cells. Unphosphorylated eIF2α was loaded onto lane 1 as a reference. The blots are representative of three or more independent experiments with similar outcomes.
Figure 4
Figure 4
PPP1R15B is induced by ER stress in β-cells in a PERK-dependent manner, and PPP1R15B silencing induces eIF2α phosphorylation and ATF3 in β-cells. A: INS-1E cells were exposed to the chemical ER stressors CPA, tunicamycin (TU), or brefeldin A (BR) or to the FFAs oleate (OL) or palmitate (PAL) for 24 h (n = 5–6). B: INS-1E cells were exposed or not (CT) to CPA in the presence or absence of the PERK inhibitor GSK2606414 (PERKi). PPP1R15B mRNA expression was examined by real-time PCR and normalized to the reference gene GAPDH. C: INS-1E cells were transfected with control siRNA (siCT) or two different siRNAs targeting PPP1R15B (P1R15B1 and P1R15B2). After a 48-h transfection, the cells were treated for 16 h with CPA, OL, or PAL. PPP1R15B mRNA expression was examined by real-time PCR and normalized to the reference gene GAPDH (n = 4). eIF2α phosphorylation (P-eIF2α) (D and E) and ATF3 (F and G) expression were examined by Western blot. D and F are representative images of n = 4. E and G represent densitometric quantifications of D and F, respectively. P-eIF2α was corrected for total eIF2α. ATF3 expression was corrected for α-tubulin and expressed as fold of CT. Data are presented as means ± SE. *treated vs. control, §DMSO vs. PERKi, #siP1R15B vs. siCT by two-sided Student paired t test. *,§,#P < 0.05; **,##P < 0.01; ***,###P < 0.001.
Figure 5
Figure 5
Glucose-stimulated insulin secretion is blunted by PPP1R15B deficiency in β-cells. INS-1E cells were transfected with control siRNA (siCT) or two siRNAs targeting PPP1R15B (siP1R15B1 and siP1R15B2). After a 48-h transfection, insulin secretion was induced by 1.67 mmol/L or 16.7 mmol/L glucose or 16.7 mmol/L glucose + 10 μmol/L forskolin (16.7+FK). A: Cellular insulin content corrected for total protein. B: Insulin release as percent of insulin content (n = 5). **P < 0.01, ***P < 0.001 vs. 1.67 mmol/L glucose. #P < 0.05, ##P < 0.01, siP1R15B vs. siCT.
Figure 6
Figure 6
PPP1R15B deficiency sensitizes β-cells to FFA- and ER stress–induced apoptosis and activates the intrinsic pathway of apoptosis via DP5, PUMA, and Bim-S. INS-1E (A and B) or primary rat β-cells (C) were transfected with a control siRNA (siCT) or two different siRNAs targeting PPP1R15B (siP1R15B1 and siP1R15B2). After a 24-h transfection, the cells were exposed or not (CT) to CPA, oleate (OL), or palmitate (PAL) for 16 (A and B) or 24 h (C) (n = 4–5). D: Mitochondrial cytochrome c (Cyto C) release was detected by Western blot in the cytoplasmic fraction 48 h after PPP1R15B knockdown. The right lane shows a noncytoplasmic fraction that includes mitochondria (Mito). Cox IV was used as a mitochondrial control and β-actin as a cytoplasmic control. Activation of caspase-9 (Casp9) (E) and caspase-3 (Casp3) (F) was detected by Western blot 48 h after PPP1R15B knockdown. β-Actin and α-tubulin were used as loading controls. D, E, and F are representative blots of 4–5 experiments. The densitometry data were normalized to the highest value. DP5 (G) and PUMA (H) mRNA expression were measured by real-time PCR and corrected for the reference gene GAPDH (n = 4). Bim-S levels were measured by Western blot (Supplementary Fig. 2), corrected for α-tubulin, and expressed as fold of siCT (I) (n = 5). J: PPP1R15B was silenced alone or in combination with DP5, PUMA, or Bim, and apoptosis was examined by Hoechst 33342/propidium iodide staining (n = 3–4). *P < 0.05, **P < 0.01, ***P < 0.001, treated vs. control. #P < 0.05, ##P < 0.01, ###P < 0.001, siP1R15B vs. siCT. §P < 0.05, single vs. double knockdown.
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