Pain perception has evolved as a warning mechanism to alert organisms to tissue damage and dangerous environments1,2. In humans, however, undesirable, excessive or chronic pain is a common and major societal burden for which available medical treatments are currently suboptimal3,4. New therapeutic options have recently been derived from studies of individuals with congenital insensitivity to pain (CIP)5,6. Here we identified 10 different homozygous mutations in PRDM12 (encoding PRDI-BF1 and RIZ homology domain-containing protein 12) in subjects with CIP from 11 families. Prdm proteins are a family of epigenetic regulators that control neural specification and neurogenesis7,8. We determined that Prdm12 is expressed in nociceptors and their progenitors and participates in the development of sensory neurons in Xenopus embryos. Moreover, CIP-associated mutants abrogate the histone-modifying potential associated with wild-type Prdm12. Prdm12 emerges as a key factor in the orchestration of sensory neurogenesis and may hold promise as a target for new pain therapeutics9,10.
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The authors are grateful for the participation of the patients and their families in this study. The help of all contributing medical, technical and administrative staff is greatly appreciated. We thank S. Malik for her invaluable work with family A, J.R.P. Madrid and F. Axelrod for advice and discussion and M.F. Passarge for helpful suggestions on the text. D.L.H.B. is a senior Wellcome Clinical Scientist (ref. no. 095698z/11/z). This work was supported by Cambridge NIHR Biomedical Research Centre (Y.-C.C., F.S. and C.G.W.), Austrian Science Fond (P23223-B19 to M.A.-G.), the UK Medical Research Council (M.S.N. and S.S.S.), Association Belge contre les Maladies Neuromusculaires and EU FP7/2007-2013 (grant 2012-305121 (NEUROMICS) to J.B. and P.D.J.), Deutsche Forschungsgemeinschaft (CRC/SFB 1140 to C.B. and KU1587/4-1 to I. Kurth), Gebert-Rüf Stiftung (GRS-046/09 to R.C. and J.S.), and Friedrich-Baur Stiftung (J.S.).
The authors declare no competing financial interests.
Integrated supplementary information
Mutations segregate with the disease consistent with autosomal recessive inheritance. All affected probands are homozygous for the respective mutations. m: mutation; wt: wild type.
Multiple protein sequence alignment generated with the program ClustalW. Sequence comparison showed that the PRDM12 missense mutations (red arrows) observed in patients with CIP affect amino acids that are identical in PRDM12 and its orthologs. Orange bar: PR/SET domain; blue bars: ZF motifs; green bar: poly-alanine tract.
Supplementary Figure 3 Distribution of PRDM12 poly-alanine tract lengths within different ethnic groups.
The length distribution of the PRDM12 poly-alanine tract showed a similar spectrum in different populations. The number of chromosomes analysed is given below the diagrams.
Skin biopsy from the lower lip, patient P7. (a) Dermal nerve fibres do not cross the dermal-epidermal border (dotted line). Staining with pan-neuronal marker PGP9.5, scale bar: 50 μm. (b) Dermal nerve containing myelinated fibres. Co-staining with PGP9.5 and myelin marker protein MBP; scale bar: 25 μm. (c) Innervation of sweat glands. Co-staining with PGP9.5 and VIP, a marker for a subpopulation of autonomic neurons; scale bar: 25 μm.
(a) Prdm12 mRNA is contained in mouse dorsal root ganglia (DRG) but not in sympathetic ganglia (SG) at E13.5. Gapdh was used as loading control. A no reverse transcriptase control (No RT) was used to exclude amplification from genomic DNA. (b) Purified DRG neurons (obtained by treating DRG explant cultures with 5-Fluorouracil) but not cells from the mouse Schwann cell line (MSC80) express Prdm12. The purity of the cell preparation was proven by RT-PCR for the S100 gene (Schwann cell marker) and the identity of neurons was confirmed by RT-PCR for Tuj-1 (neuron-specific class III β-tubulin). E13.5 DRG tissue and liver were used as positive and negative controls, respectively.
Supplementary Figure 6 Electrophysiological properties of iPSC-derived sensory neurons and gene expression of hESC-derived sensory neurons.
(a) IPSC-derived sensory neurons (from a parallel culture of those used for RT-qPCR analysis in Fig. 3c) expressed tetrodotoxin (TTX)-resistant nociceptor ion channels (Nav1.8) after 52 days in culture demonstrating the cells utility as a model of human DRG neurons. Voltage-gated sodium currents were elicited in whole-cell voltage-clamp by voltage steps from -120 mV to 0 mV in the absence (control, black), presence (drug, red), and after washout (recovery, blue) of 500 nM TTX (scale bar, 10 nA and 25 ms). (b) The electrophysiological recording confirmed the membrane expression of nociceptor ion channels as a TTX-resistant sodium current (Nav1.8) was observed following TTX application (control=557.1±78.9 pA/pF, TTX (500 nM)=113.9±19.4 pA/pF, wash=486.4±62.2 pA/pF; n=5). (c) HESC-derived sensory neurons expressed PRDM12 during differentiation. PRDM12 expression was quantified by RT-qPCR and was found to peak at the neural crest specification stage of development (Day 9). The differentiated cells were shown to also express canonical markers of sensory neurons, NGF receptor NTRK1 and ion channel SCN9A, indicating cell specification into sensory neuron phenotype.
No PRDM12 expression was observed by RT-PCR using cDNA from various tissues and leukocytes (Clontech Human MTC Panel I and II, cat. no.: 636742 and 636743). Amplification of GAPDH was used to ensure integrity of the cDNA. Functionality of PRDM12 primers was tested with cDNA from human DRG.
(a) Prdm12 expression overlapped with the sensory placode marker Islet1 (yellow and green arrowheads) but was absent from other cranial placodes such as lens (Six3). Whole-mount in situ hybridisation, lateral views of late tailbud-stage embryos (stage 26). Gene expression domains of cranial placodes (colored outlines) in Xenopus laevis are shown in the schematic drawing (lateral view, late tailbud stage (modified from25)). An asterisk marks Prdm12 expression in diencephalon. PN: expression in pronephros. (b) Inhibition of Myc-Prdm12 translation by specific morpholino. Embryos were co-injected with Myc-Prdm12 mRNA and Control MO (5, 10, 20 ng/embryo) or Prdm12 MO (5, 10, 20 ng/embryo) at 2-cell stage and protein extracts were obtained from 26-stage embryos. Western blotting analysis was performed with anti-Myc and anti-α-tubulin antibodies. (Note that in situ hybridisation is not suitable to measure Prdm12 knockdown as the morpholino affects protein translation but does not predictably change the rate of mRNA degradation.) (c) Knockdown of Prdm12 by Prdm12 MO only marginally affected lens placode markers Six3 and Pax6 and otic placode marker Pax8 in Xenopus embryos. Embryos injected with Control MO (20 ng/embryo) or Prdm12 MO (20 ng/embryo) were analyzed at late tailbud stage (stage 28) by whole-mount in situ hybridisation. For normal gene expression domains of cranial placodes see (a), PN: pronephros. The results were categorized and quantified (n≱40 alive embryos per condition). Differences between Control MO- and Prdm12 MO-treated embryos were assessed statistically: ns, not significant (two-sided Mann-Whitney U-test).
(a) Effects of PRDM12 mutations on protein expression. Transfected missense mutant and wild-type mouse Myc-Prdm12 showed similar levels in COS-7 cells. Prdm12 signals were normalized to α-tubulin and GFP (transfection control). The graphs represent mean values of n independent experiments (biological replicates) and error bars represent SD. Differences between control (wild type) and Prdm12 mutants were assessed statistically: ns, not significant (Welch’s t-test). (b) Localisation in HEK-293T cells. Wild-type and missense mutant human HA-PRDM12 protein labelled nuclei in a diffuse pattern. (c) Co-immunoprecipitation of Prdm12 with G9a. Myc-Prdm12 and FLAG-G9a were expressed in COS-7 cells and immunoprecipitated using anti-Myc antibody. For quantification, bound G9a was normalized to Prdm12 protein amount in the IP fraction and the G9a protein amount in the cell lysate. The graphs represent mean values of n independent experiments (biological replicates), and error bars represent SD. Differences between control (wild type) and Prdm12 mutants were assessed statistically: ns, not significant (Welch’s t-test). (d) Chromatin fractionation of COS-7 cells transfected with human HA-PRDM12. Wild type and mutant PRDM12 appeared in both fractions. Alpha-tubulin (soluble fraction) and acetylated histone 3 (chromatin fraction) were used as controls to determine fraction purity.
Animal cap cells from Xenopus embryos were co-microinjected with Myc-Prdm12 of Xenopus, mouse or human origin, Wnt8 and Chrd mRNA and cultured until mid-neurula stage (stage 15). A dose-dependent increase of H3K9me2 (compared to negative control) was observed between 0.5, 1 and 2 ng/embryo doses of Myc-Prdm12, irrespective of whether the frog protein or its mammalian orthologs were overexpressed.
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Chen, YC., Auer-Grumbach, M., Matsukawa, S. et al. Transcriptional regulator PRDM12 is essential for human pain perception. Nat Genet 47, 803–808 (2015). https://doi.org/10.1038/ng.3308