Transcriptional regulator PRDM12 is essential for human pain perception

Abstract

Pain perception has evolved as a warning mechanism to alert organisms to tissue damage and dangerous environments. In humans, however, undesirable, excessive or chronic pain is a common and major societal burden for which available medical treatments are currently suboptimal. New therapeutic options have recently been derived from studies of individuals with congenital insensitivity to pain (CIP). 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 neurogenesis. 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 therapeutics.

Figure 1. Identification of mutations in PRDM12.

(a) In Pakistani multiplex family A, SNP-based autozygosity mapping of four individuals (solid red circles) pinpointed a single candidate region on chromosome 9q33.2-34.13 (represented by reference SNP cluster (rs) numbers of flanking SNP markers and a red vertical bar next to the chromosome 9 ideogram). Although exome sequencing of the index patient from family A (open blue circle) yielded inconclusive results, one gene in the candidate region, PRDM12, harbored a candidate homozygous mutation in family B (solid blue circle). P1–P6, patients 1–6. (b) Exome sequencing of subjects from two families, family C and family D (solid blue circles). PRDM12 was the only gene containing potentially deleterious variants excluded from all databases (“Not in DB”) on both alleles (“AR model”) in both affected individuals (“Shared”). (c) Schematic representation of the PRDM12 protein and distribution of mutations. Amino acid numbering is shown along the bottom. PR/SET, PR domain; ZF, zinc finger motif; A, polyalanine tract. (d) Distribution of PRDM12 polyalanine tract lengths in the general population (176 individuals).

Figure 2. Phenotype of affected individuals with PRDM12 mutations.

(a) Mutilation of tongue and lips, corneal opacity, scarring and mutilation of distal phalanges. Patients P17 and P18 (family J) represented a milder phenotype with sequelae such as facial scratching and diabetes-like foot ulcers. Consent to publish images of the individuals was obtained. (b) Sural nerve biopsy specimens showing selective loss of small-caliber myelinated axons. The total numbers of myelinated fibers per square millimeter were 4,692 (P6), 4,438 (P10) and 9,609 (healthy control). Semithin sections were stained with toluidine blue; scale bars, 20 μm. (c) Skin biopsies labeled with PGP9.5 (pan-neuronal marker), calcitonin gene-related peptide (CGRP, labeling a subpopulation of nociceptive primary afferents) and vasoactive intestinal peptide (VIP, a marker for autonomic nerve fibers). Although ample intraepidermal nerve endings (red arrowheads) were observed in the biopsy from a healthy donor, nerve fibers did not cross the dermal-epidermal border (red dashed line) in the affected subject’s biopsy. In the biopsy from P11, dermal CGRP-immunoreactive nerve fibers were almost absent, and sweat glands were innervated by VIP-immunoreactive fibers, but at a reduced density. Scale bars, 50 μm (top two rows) or 20 μm (bottom row).

Figure 3. A role for Prdm12 in sensory neuron development.

(a) Whole-mount in situ hybridization of mouse embryos at E9.0 (left) identified expression of Prdm12 in neural folds (black arrowhead), which coincided with the earliest stage of neural crest cell delamination and migration (white arrowhead). In situ hybridization of whole embryos (middle) and transverse sections of cervical spinal cord (right) at E10.5 showed strong Prdm12 expression in DRG (black arrowheads). Scale bars: left, 250 μm; middle, 500 μm; right, 100 μm. (b) RT-PCR analysis confirmed Prdm12 expression throughout the whole period of DRG development and sensory neuron differentiation (E9.5–P14) and in mature DRG (P56). (c) Quantitative RT-PCR of human iPSC-derived sensory neurons showed that PRDM12 expression peaked during neural crest specification. Changes in the expression of pluripotency markers and canonical sensory neuron markers confirmed successful differentiation. The schematic drawing above the heat map illustrates the stages of development during the differentiation of sensory neurons. D, day of differentiation process. (d) Knockdown of Prdm12 by a specific morpholino (MO) in Xenopus embryos caused irregular staining for markers of cranial sensory placode development (Ath3, Ebf3 and Islet1). Embryos injected with control MO or Prdm12 MO were analyzed at the late tailbud stage (stage 28) by whole-mount in situ hybridization; yellow arrowheads, profundal placode; green arrowheads, trigeminal placode. Scale bars, 200 μm. Normal gene expression domains of cranial placodes in Xenopus laevis are shown in the schematic drawing at the top of the panel (lateral view, late tailbud stage; modified from ref. 25). The results were categorized and quantified (n ≥ 46 live embryos per condition). Statistical differences between expression in control MO–treated and Prdm12 MO–treated embryos are indicated. ***P < 0.001 (two-sided Mann-Whitney U-test).

Figure 4. Consequences of PRDM12 mutations.

(a) The hemagglutinin (HA)-tagged PRDM12 polyalanine expansion mutant showed lower expression levels in COS-7 cells than did wild-type PRDM12; expression was recovered by MG132. HA-PRDM12 signals were normalized to those of α-tubulin and GFP (transfection-efficiency control). The transfected mutant formed aggregates in the nucleus and cytoplasm in HEK-293T cells (bottom). Red, PRDM12 (fluorescent detection of the anti-HA tag); blue, nuclei (DAPI staining). (b) Wild-type Prdm12 induced robust dimethylation on H3K9 in Xenopus neurula, but CIP-associated missense mutants were functionless. Xenopus animal cap cells were microinjected with Myc-Prdm12 (wild type and mutant), Wnt8 and Chrd mRNA and cultured until mid-neurula stage (stage 15). H3K9me2 signals were normalized to Prdm12 (Myc) and total H3. (c) The p.His289Leu alteration impairs Prdm12-G9a interaction. Myc-Prdm12 and FLAG-G9a were expressed in COS-7 cells (Input) and immunoprecipitated using anti-Myc (IP: Myc). Bound G9a was normalized to amounts of Prdm12 in the immunoprecipitation (IP) fraction and G9a protein in the lysate. According to the structural model for the PRDM12 zinc finger domains, His289 (orange) is one of the residues (cyan) that coordinate the zinc ion. Prdm12ΔZF is an artificial mutant lacking zinc fingers. IgG, immunoglobulin G. (d) Mutation-altered residues Ile102 and Trp160 (orange) have hydrophobic interactions with other residues (cyan) in the core of the PRDM12-PR domain. Introduction of a polar side chain (p.Ile102Asn) or a disulfide-bond partner (p.Trp160Cys) into the hydrophobic core may affect the structure of the PR domain. The bar graphs in a–c represent mean values of n biological replicates, and error bars represent s.d. Statistical differences between control (wild type) and Prdm12 mutants are indicated. ns, not significant. *P < 0.05, **P < 0.01, ***P < 0.001 (Welch’s t-test).