Transcriptional Regulator PRDM12 Is Essential for Human Pain
Total Page：16
File Type：pdf, Size：1020Kb
LETTERS Transcriptional regulator PRDM12 is essential for human pain perception Ya-Chun Chen1,2,52, Michaela Auer-Grumbach3,52, Shinya Matsukawa4, Manuela Zitzelsberger5, Andreas C Themistocleous6,7, Tim M Strom8,9, Chrysanthi Samara10, Adrian W Moore11, Lily Ting-Yin Cho12, Gareth T Young12, Caecilia Weiss5, Maria Schabhüttl3, Rolf Stucka5, Annina B Schmid6,13, Yesim Parman14, Luitgard Graul-Neumann15, Wolfram Heinritz16,17, Eberhard Passarge17,18, Rosemarie M Watson19, Jens Michael Hertz20, Ute Moog21, Manuela Baumgartner22, Enza Maria Valente23, Diego Pereira24, Carlos M Restrepo25, Istvan Katona26, Marina Dusl5, Claudia Stendel5,27, Thomas Wieland8, Fay Stafford1,2, Frank Reimann28, Katja von Au29, Christian Finke30, Patrick J Willems31, Michael S Nahorski1,2, Samiha S Shaikh1,2, Ofélia P Carvalho1,2, Adeline K Nicholas2, Gulshan Karbani32, Maeve A McAleer19, Maria Roberta Cilio33,34, John C McHugh35, Sinead M Murphy36,37, Alan D Irvine19,38, Uffe Birk Jensen39, Reinhard Windhager3, Joachim Weis26, Carsten Bergmann40–42, Bernd Rautenstrauss5,43, Jonathan Baets44–46, Peter De Jonghe44–46, Mary M Reilly47, Regina Kropatsch48, Ingo Kurth49, Roman Chrast9,50,51, Tatsuo Michiue4, David L H Bennett6, C Geoffrey Woods1,2 & Jan Senderek5 Pain perception has evolved as a warning mechanism to alert exome sequencing on the index patient of family A and the unre- organisms to tissue damage and dangerous environments1,2. lated single CIP patient from family B. Although exome sequenc- In humans, however, undesirable, excessive or chronic pain ing of the subject from family A yielded no obvious pathogenic is a common and major societal burden for which available variant in genes located in the autozygous region on chromosome 9, medical treatments are currently suboptimal3,4. New we observed a homozygous missense mutation in one of these posi- Nature America, Inc. All rights reserved. Inc. Nature America, therapeutic options have recently been derived from studies tional candidate genes, PRDM12 (NM_021619.2), in the patient from 5 of individuals with congenital insensitivity to pain (CIP)5,6. family B (Fig. 1a and Supplementary Fig. 1). We then performed Here we identified 10 different homozygous mutations in Sanger sequencing of PRDM12 for multiple individuals from family A © 201 PRDM12 (encoding PRDI-BF1 and RIZ homology domain- and identified a homozygous trinucleotide expansion of alanine containing protein 12) in subjects with CIP from 11 families. codons from 12 to 19 in the terminal exon in all affected subjects Prdm proteins are a family of epigenetic regulators that control (Fig. 1a and Supplementary Fig. 1). In an independent approach, we neural specification and neurogenesis7,8. We determined that carried out exome sequencing on two individuals from two unrelated Prdm12 is expressed in nociceptors and their progenitors and families with genetically unclassified CIP, family C and family D, and participates in the development of sensory neurons in Xenopus identified PRDM12 as the only gene that carried different biallelic embryos. Moreover, CIP-associated mutants abrogate the missense mutations in both subjects (Fig. 1b and Supplementary histone-modifying potential associated with wild-type Prdm12. Fig. 1). Subsequently, we screened PRDM12 in 158 individuals with Prdm12 emerges as a key factor in the orchestration of sensory autosomal recessive or isolated unexplained CIP or HSAN. We found neurogenesis and may hold promise as a target for new pain seven additional unrelated index patients with homozygous PRDM12 therapeutics9,10. mutations. The majority of the variants were missense mutations; however, affected individuals from one family (family J) had an We studied two families whose pedigrees were consistent with 18-alanine repeat mutation, one isolated subject (family E) carried autosomal recessive CIP. We excluded mutations in the known causa- a frame-shift mutation and another isolated individual (family K) tive genes for CIP11,12 and for clinically similar types of hereditary had an obligatory splice-site mutation (Fig. 1c and Supplementary sensory and autonomic neuropathy (HSAN; types IV and V)13–15. Fig. 1). This study was approved by the National Research Ethics SNP array–based autozygosity mapping in a consanguineous Service, NRES Committee East of England–Cambridge Central, family (family A) identified a single 11.5-Mb homozygous region the Munich University Medical Research Ethics Committee and the on chromosome 9q33.2-34.13 (Fig. 1a). As this large interval con- Medical University of Vienna Ethics Committee. Informed consent tained almost 150 genes (Supplementary Table 1), we performed was obtained from all study subjects or their legal representatives. A full list of author affiliations appears at the end of the paper. Received 29 September 2014; accepted 27 April 2015; published online 25 May 2015; doi:10.1038/ng.3308 NATURE GEnETICS ADVANCE ONLINE PUBLICATION 1 LETTERS Figure 1 Identification of mutations Family B a Italy b in . (a) In Pakistani multiplex Family A PRDM12 Pakistan Family C Family D family A, SNP-based autozygosity mapping Serbia Turkey of four individuals (solid red circles) Chr9 24 pinpointed a single candidate region on P6 p 23 21 chromosome 9q33.2-34.13 (represented 13 12 P7 P8 by reference SNP cluster (rs) numbers of 12 13 AR model flanking SNP markers and a red vertical 21 Not in DB Shared q (genes) bar next to the chromosome 9 ideogram). 22 31 P7 397 23 P3 P4 PRDM12 Although exome sequencing of the index rs942153 32 P8 452 37 33 PRDM12 patient from family A (open blue circle) rs578802 34 yielded inconclusive results, one gene in P1 P2 P5 14 Ala the candidate region, PRDM12, harbored a c d 1.4% 7 Ala candidate homozygous mutation in family B 4.0% (solid blue circle). P1–P6, patients 1–6. 13 Ala 8 Ala 41.5% 0.3% (b) Exome sequencing of subjects from 9 Ala two families, family C and family D (solid Asp31TyrSer58Lysfs*85IIe102Asn Trp160CysArg168CysGlu172Asp c.683-1G>A His289Leu Ala354_Ala359dupAla353_Ala359dup 3.4% 10 Ala blue circles). PRDM12 was the only 0.3% gene containing potentially deleterious PR/SET ZF ZF ZF A variants excluded from all databases 12 Ala 1 100 200 300 49.1% (“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). PRDM12 is a five-exon gene encoding a single protein isoform the point mutations was present in public SNP databases (1000 of 367 amino acids containing a PR domain (related to the SET Genomes, Exome Variant Server and dbSNP138) or in the institutional methyltransferase domain), three zinc fingers and a C-terminal exome data sets of Helmholtz Zentrum München and the Cambridge polyalanine tract (Fig. 1c). The observed point mutations were Biomedical Research Campus. These resources together allow for the distributed throughout the gene, altered strictly conserved protein interrogation of exome data from >20,000 individuals. We suspected residues and were predicted to interfere with normal protein function that the trinucleotide expansions to 18 and 19 alanine codons in (Supplementary Fig. 2, Supplementary Table 2 and Supplementary two of the families studied here would be deleterious, as other Note). In all 11 families studied, the PRDM12 mutations segregated as known recessive and X-linked polyalanine expansion diseases in expected for recessive disease alleles (Supplementary Fig. 1). None of humans manifest clinically when the number of repeats exceeds 15 (refs. 16,17). We found that the length a of PRDM12 polyalanine in the general P14 P7 P11 P6 P17 Nature America, Inc. All rights reserved. Inc. Nature America, 5 Figure 2 Phenotype of affected individuals with PRDM12 mutations. (a) Mutilation of tongue and lips, corneal opacity, scarring and © 201 P7 P10 P6 mutilation of distal phalanges. Patients P17 and P14 P18 (family J) represented a milder phenotype P18 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 Healthy control P11 b P6 P10 c 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 PGP9.5 subpopulation of nociceptive primary afferents) Healthy control and vasoactive intestinal peptide (VIP, a marker P6 for autonomic nerve fibers). Although ample 2,000 P10 ) intraepidermal nerve endings (red arrowheads) –2 Healthy control were observed in the biopsy from a healthy 1,500 donor, nerve fibers did not cross the dermal-epidermal border (red dashed line) in 1,000 CGRP/PGP9.5 the affected subject’s biopsy. In the biopsy from P11, dermal CGRP-immunoreactive nerve fibers 500 were almost absent, and sweat glands were Myelinated fibers (mm innervated by VIP-immunoreactive fibers, 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 but at a reduced density. Scale bars, 50 µm Diameter of myelinated fibers (µm) PGP9.5/VIP/DAPI (top two rows) or 20 µm (bottom row). aDVANCE ONLINE PUBLICATION NATURE GEnETICS LETTERS Trigeminal a b d Profundal (bp) Otic 300 Lens 100 Prdm12 Lateral line 300 Olfactory 100 Gapdh Epibranchial P0 P56 E11 P14 E9.0 E10.5 E10.5 E7.5 E8.5 E9.5 Control MO Prdm12 MO E10.5 E11.5 E11.5 E13.5 E14.5 E17.5 E10.5 E12.5 Embryo Neural DRG tube Figure 3 A role for Prdm12 in sensory c Neuroectoderm Neural crest cell Neuron Ath3 neuron development. (a) Whole-mount specification specification maturation in situ hybridization of mouse embryos at E9.0 (left) identified expression of D0 D2 D4 D6 D8 D10 D12 Prdm12 in neural folds (black arrowhead), Undifferentiated Neuroectodermal Neural Sensory which coincided with the earliest stage of stem cells cells crest cells neurons neural crest cell delamination and migration Ebf3 (white arrowhead).