July 1, 2015;
Transcriptional regulator PRDM12 is essential for human pain perception.
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
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
emerges as a key factor in the orchestration of sensory neurogenesis and may hold promise as a target for new pain therapeutics.
Disease Ontology terms:
Xla Wt + prdm12 MO
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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). 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).
Figure 3 A role for Prdm12 in sensory neuron development. 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.
Figure 3 A role for Prdm12 in sensory neuron development. (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).
Supplementary Figure 8: Prdm12 expression and knockdown in Xenopus embryos. (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 method and server for predicting damaging missense mutations.