Gene 534 (2014) 236–239

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Gene journal homepage: www.elsevier.com/locate/gene

A novel P2RX2 mutation in an Italian family affected by autosomal dominant nonsyndromic hearing loss Flavio Faletra a,⁎, Giorgia Girotto b, Adamo Pio D'Adamo b, Diego Vozzi a, Anna Morgan b, Paolo Gasparini a,b a b

Institute for Maternal and Child Health, IRCCS “Burlo Garofolo”, Via dell'Istria, 34137 Trieste, Italy University of Trieste, Trieste, Italy

a r t i c l e

i n f o

Article history: Accepted 24 October 2013 Available online 6 November 2013 Keywords: P2RX2 DFNA41 p.Gly353Arg Mutation ADNSHL

a b s t r a c t Hereditary hearing loss (HHL) is a common disorder accounting for at least 60% of prelingual deafness. It is characterized by a large genetic heterogeneity, and despite the presence of a major gene, still there is a need to search for new causative mutations/genes. Very recently, a mutation within ATP-gated P2X(2) receptor (ligand-gated ion channel, purinergic receptor 2) gene (P2RX2) at DNFA41 locus has been reported leading to a bilateral and symmetrical sensorineural non-syndromic autosomal dominant HHL in two Chinese families. We performed a linkage analysis in a large Italian family with a dominant pattern of inheritance showing a significant 3.31 LOD score in a 2 Mb region overlapping with the DNFA41 locus. Molecular analyses of P2RX2 identified a novel missense mutation (p.Gly353Arg) affecting a residue highly conserved across species. Visual inspection of the protein structure as obtained from comparative modeling suggests that substitution of the small glycine residue with a charged bulky residue such as an arginine that is close to the ‘neck’ of the region responsible for ion channel gating should have a high energetic cost and should lead to a severely destabilization of the fold. The identification of a second most likely causative mutation in P2RX2 gene further supports the possible role of this gene in causing autosomal dominant HHL. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Hearing loss (HL) is the most common sensory disorder in humans and affects about 300 million people worldwide (Shearer et al., 2011). Roughly, one to three children in a thousand are born with hearing impairment (Parving, 1999). To date, more than 60% of prelingual hearing impairment is caused by genetic factors. There are two main forms of hereditary hearing loss (HHL): syndromic (SHL) (about 25%), in which the deafness is accompanied by other specific features, and non-syndromic hearing loss (NSHL) (about 75%), in which there are no additional abnormalities (Kemperman et al., 2002). Approximately 75–80% of NSHL forms are recessive, 20–25% are dominant (ADNSHL) while only 1–1.5% is X-linked (Smith et al., 2013). According to the HHL homepage (http://hereditaryhearingloss.org/), more than 140 loci for NSHL have been mapped, 80 genes identified and approximately 1000 causing mutations described (http://deafnessvariationdatabase.org; Shearer et al., 2011; Van Camp and Smith, 2012).

Abbreviations: HHL, hereditary hearing loss; NSHL, non-syndromic hearing loss; ADNSHL, autosomal dominant non-syndromic hearing loss; HL, hearing loss. ⁎ Corresponding author at: Medical Genetics, Institute for Maternal and Child Health, IRCCS “Burlo Garofolo, Via dell'Istria 65/1, Trieste, Italy. Tel.: +39 040 378 5538; fax: +39 040 378 5540. E-mail addresses: fl[email protected] (F. Faletra), [email protected] (G. Girotto), [email protected] (A.P. D'Adamo), [email protected] (D. Vozzi), [email protected] (P. Gasparini). 0378-1119/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gene.2013.10.052

In 2002 a large Chinese ADNSHL family mapping at DNFA41 locus was described (Blanton et al., 2002). After a locus refinement in 2005 (Yan et al., 2005), the causative gene, named P2RX2, was recently discovered in two unrelated Chinese families (Yan et al., 2013). P2X receptors mediate a variety of cellular responses, including short-term effects as excitatory postsynaptic responses in sensory neurons (North, 2002) and long-term (trophic) effects comprising cell proliferation, differentiation and death, growth of axons during development and regeneration (Heine et al., 2006). In particular, P2X receptors in the inner ear act in the control of the outer hair cell electromotility (Yu and Zhao, 2008), auditory neurotransmission (Housley et al., 1999), gap junctions (Zhu and Zhao, 2012), and K+ recycling (Zhu and Zhao, 2012). The P2RX2 gene encodes a receptor protein expressed in cochlear epithelial cells, including the sensory hair cells and supporting cells of the organ of Corti and the afferent spiral ganglion neurons. It assembles as a trimer, forming a channel gated by extracellular ATP. Here, we report the identification of a second P2RX2 missense mutation in a large Italian family mapping by linkage in a 2 Mb region within the DNFA41 locus. 2. Subjects and methods 2.1. Family data and audiograms A large Italian family (22 individuals analyzed with 9 affected) spanning four generations with a bilateral, sensorineural and progressive

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Fig. 1. Family pedigree: linkage analysis and segregation of the mutation has been performed in all family members.

form of ADNSHL (see Fig. 1) was enrolled in the study. Age of onset was in the second decade of life. After signing the informed consent, each participant completed a questionnaire about hearing and balance. All subjects were examined otoscopically, and oto-immittance measurements were obtained to assess middle ear status. Pure tone air and bone thresholds were determined at frequencies of 250, 500, 1000, 2000, 4000, and 8000 Hz, with intensities up to 120 dB (Fig. 2). The hearing loss could be classified as mild to profound in almost all affected family members, mainly affecting medium-high frequencies (1000 and 4000 Hz). The severity of the disease seems to be worse in older affected family members, suggesting a possible correlation with age. Despite their young age, the IV:1 and IV:2 individuals show the deepest HL among all family members (Fig. 2). TC and MRI of the temporal bones and the inner ear revealed no malformations.

2.2. Genotyping and linkage analysis Genotyping was performed using the Illumina HumanCytoSNP-12 BeadChip. Linkage calculation was performed using the Linkage Program Merlin (Abecasis et al., 2002). Mendelian errors and unlikely recombinants were computed by Merlin error check, Pedcheck and Pedstats softwares (Abecasis et al., 2002; O'Connell and Weeks, 1998; Wigginton and Abecasis, 2005). SNPs found by these “error-check”

procedures were set to missing for all family members. Parametric linkage analysis under dominant model (risk allele frequency 0.00001 and complete penetrance) was carried out using Merlin version 1.1.2.

2.3. Mutational analysis All 11 exons and flanking intronic regions of the P2RX2 gene were amplified by polymerase chain reaction (PCR). Primer sequences and PCR conditions are available on request. PCR products were purified and sequenced bidirectionally on ABI Prism 3130xl sequencer (Applied Biosystems) according to the manufacturer's instructions. PolyPhen2 (Adzhubei et al., 2010), Mutation Taster (Schwarz et al., 2010), PhyloP, GERP++, SIFT and LRT (Liu et al., 2013) were used to perform in silico prediction of the impact of nucleotide changes on the protein structure and function. The presence of the mutated allele was also checked in 100 DNA samples of ethnically matched healthy controls.

2.4. Protein modeling A three-dimensional model was built by comparative modeling using the Swiss PDB Viewer program (Johansson et al., 2012) and with

Fig. 2. Audiograms of all affected family members at various ages.

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the coordinates of the zebrafish P2X4 (PDB accession number 3H9V) as structural template. 3. Results Linkage analysis led to the identification of a region with a maximum LOD score of 3.31 spanning from rs11061441 (chr12:131808553) to the end of chromosome 12, encompassing 2 Mb and containing 42 annotated genes (Suppl. 1). This region overlaps with the previously described DFNA41 locus, containing the P2RX2 gene, recently demonstrated to be involved in causing ADNSHL in two Chinese families (Yan et al., 2013). The whole coding region of P2RX2 was analyzed by Sanger sequencing, identifying a nucleotide change G to C at position 1057 (NM_170682.2, chr12:133198121 c.G1057C, exon 10). This substitution leads to a missense mutation from a glycine to an arginine at position 353 of the protein (p.Gly353) (Fig. 3A). This mutated allele was not previously described nor is included in any mutation databases. It involves a residue highly conserved across species (Fig. 3B). PolyPhen2 (Adzhubei et al., 2010), Mutation Taster (Schwarz et al., 2010), PhyloP, GERP++, SIFT and LRT (Liu et al., 2013) in silico prediction analyses reported this change as damaging (0.99999; 1.000; 4.169; 3.54; 1 and 1 respectively). The p.Gly353Arg mutation was not found on 500 chromosomes from ethnically matched healthy controls and segregates with the disease in the family under study. To better understand the functional role of this missense mutation, a three dimensional model of the structure of P2X2 was built by comparative modeling using the coordinates of P2X4 from zebrafish which shares 45% of sequence identity with P2X2. The similarity increases further in the region around the locus identified (Fig. 4B). Visual inspection of the model itself (Fig. 4A) shows that Glycine 353 (corresponding to Glycine 350 in P2X4) is at the C-terminus of the TM2 helix. This residue should be directly embedded near or in the lipid channel bilayer. Thus, it might suggest that substitution of this small amino acid with a positively charged bulky residue such as an Arginine will result in a high energetic cost and lead to a severe destabilization of the fold causing structural distortions and/or anomalous interaction with the lipid bilayer. 4. Discussion Linkage analysis followed by molecular analysis of P2RX2 gene allowed us to demonstrate the involvement of this gene in a large Italian family affected by ADNSHL. This finding further supports the P2RX2 role and extends its relevance in causing hearing loss. The mutation (p.Val60Leu) recently described in the Chinese families leads to a progressive form of HHL progressing to all frequencies with an onset in the first or second decade of life (Yan et al., 2013). Moreover, Yan et al. demonstrated an exacerbation of the HL at high frequencies mediated by a noise exposure jointly with the p.V60L mutation. Likewise, almost all the patients of our family show audiometric profiles similar to those showed in the Chinese families with a more severe HL, mostly in the medium-high frequencies. In our family, the two younger individuals display an earlier onset and a more severe phenotype. This variability,

Fig. 4. A) Sequence alignment of the region around the locus identified of P2X4 and P2X2 proteins showing a high similarity of residues. The black arrows indicate the position of the amino acid change in the two structures (Legend: green — identical residues, orange — similar residues, red — sequence mismatch); B) three dimensional model of the structure of P2X2 built by comparative modeling using the coordinates of P2X4 from zebra fish. In yellow, the glycine 353 is reported (corresponding to glycine 350 in P2X4) at the Cterminus of the TM2 helix while in green, the A344 that has already been considered as the ‘center’ of the gate of the ion channel gating.

also reported for some members of the two Chinese families, is not due to noise exposure (i.e. excluded in our cases) but may be due to other environmental factors or modifier genes/variants. P2X receptors are members of the ligand-gated ion channel family that open in response to extracellular ATP. Each receptor is made up of a trimer of subunits (P2X1-7) all of which share the common

Fig. 3. A) Electropherogram from a family member with hearing loss. B) Evolutionary conservation of the region of the P2X2 receptor protein including the variant site.

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structure of two transmembrane domains, a large extracellular loop and intracellular C- and N-termini. Receptors can be formed from triplets of identical subunits (homomeric) and can also exist as heteromers. The extracellular purinergic signaling through ATP-gated ion channels (P2X receptors) and G protein-coupled P2Y receptors influences the cochlear homeostasis and synaptic branching (Mammano, 2013) and the P2X2 protein is up-regulated as a result of exposure to noise (Wang et al., 2003). The two families reported by Yan et al. carry a mutation (Val60Leu) causing a substitution between two hydrophobic amino acids and abolishing the P2X2 response to ATP (Yan et al., 2013). In our family, a glycine residue, located in the TM2 region of the protein, is replaced with a arginine one. As already described (Jindrichova et al., 2009), the TM2 region appears to play different roles in the receptor functions, including channel assembly, gating, ion selectivity, and permeability of divalent ions. Gly350 (Gly353 in P2X2) is located in the ‘neck’ of the region of the protein responsible for ion channel gating being only 6aa away from A344 that has already been considered as the ‘center’ of the gate (Kawate et al., 2009). The substitution of a hydrophobic glycine, with a charged residue such as arginine, could destabilize protein fold and interaction with the membrane. Moreover, the ion channel gate defines the closest association of the TM2 helices and is flanked by important hydrophobic residues such as Val in P2X2 and Leu in P2X4. These findings strongly suggest that the p.Gly353Arg mutation should have a relevant impact on both protein structure and function. In conclusion, we have reported the identification of a new missense mutation (p.Gly353Arg) in P2RX2 gene further extending its role in causing ADNSHL. The identification of an Italian family affected by hearing loss due to a mutation in P2RX2 gene after the recent identification of a different missense mutation in two ADNSHL Chinese families strongly suggests that P2RX2 gene plays a relevant role in different ethnic groups. Moreover, being this mutation located within a different protein region can help in explaining the pathogenetic mechanisms of this gene in causing ADNSHL. Additional functional studies are needed to further investigate the role of Gly353Arg mutation. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.gene.2013.10.052. Acknowledgments We thank Annalisa Pastore of the MRC National Institute for Medical research in London for her help in interpreting the structural role of the mutation. Project funded by the Italian Ministry of Health (RC2006 and RF2011 to PG) and Telethon GGP09037 (to PG).

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