Mutations in TBC1D24, a Gene Associated With Epilepsy, Also Cause Nonsyndromic Deafness DFNB86
2014; Elsevier BV; Volume: 94; Issue: 1 Linguagem: Inglês
10.1016/j.ajhg.2013.12.004
ISSN1537-6605
AutoresAtteeq U. Rehman, Regie Lyn P. Santos‐Cortez, Robert J. Morell, Meghan C. Drummond, Taku Ito, Kwanghyuk Lee, Asma A. Khan, Muhammad Asim Raza Basra, Naveed Wasif, Muhammad Ayub, Rana A. Ali, Syed Irfan Raza, Deborah A. Nickerson, Jay Shendure, Michael J. Bamshad, Saima Riazuddin, Neil Billington, Shaheen N. Khan, Penelope L. Friedman, Andrew J. Griffith, Wasim Ahmad, Sheikh Riazuddin, Suzanne M. Leal, Thomas B. Friedman,
Tópico(s)Cellular transport and secretion
ResumoInherited deafness is clinically and genetically heterogeneous. We recently mapped DFNB86, a locus associated with nonsyndromic deafness, to chromosome 16p. In this study, whole-exome sequencing was performed with genomic DNA from affected individuals from three large consanguineous families in which markers linked to DFNB86 segregate with profound deafness. Analyses of these data revealed homozygous mutation c.208G>T (p.Asp70Tyr) or c.878G>C (p.Arg293Pro) in TBC1D24 as the underlying cause of deafness in the three families. Sanger sequence analysis of TBC1D24 in an additional large family in which deafness segregates with DFNB86 identified the c.208G>T (p.Asp70Tyr) substitution. These mutations affect TBC1D24 amino acid residues that are conserved in orthologs ranging from fruit fly to human. Neither variant was observed in databases of single-nucleotide variants or in 634 chromosomes from ethnically matched control subjects. TBC1D24 in the mouse inner ear was immunolocalized predominantly to spiral ganglion neurons, indicating that DFNB86 deafness might be an auditory neuropathy spectrum disorder. Previously, six recessive mutations in TBC1D24 were reported to cause seizures (hearing loss was not reported) ranging in severity from epilepsy with otherwise normal development to epileptic encephalopathy resulting in childhood death. Two of our four families in which deafness segregates with mutant alleles of TBC1D24 were available for neurological examination. Cosegregation of epilepsy and deafness was not observed in these two families. Although the causal relationship between genotype and phenotype is not presently understood, our findings, combined with published data, indicate that recessive alleles of TBC1D24 can cause either epilepsy or nonsyndromic deafness. Inherited deafness is clinically and genetically heterogeneous. We recently mapped DFNB86, a locus associated with nonsyndromic deafness, to chromosome 16p. In this study, whole-exome sequencing was performed with genomic DNA from affected individuals from three large consanguineous families in which markers linked to DFNB86 segregate with profound deafness. Analyses of these data revealed homozygous mutation c.208G>T (p.Asp70Tyr) or c.878G>C (p.Arg293Pro) in TBC1D24 as the underlying cause of deafness in the three families. Sanger sequence analysis of TBC1D24 in an additional large family in which deafness segregates with DFNB86 identified the c.208G>T (p.Asp70Tyr) substitution. These mutations affect TBC1D24 amino acid residues that are conserved in orthologs ranging from fruit fly to human. Neither variant was observed in databases of single-nucleotide variants or in 634 chromosomes from ethnically matched control subjects. TBC1D24 in the mouse inner ear was immunolocalized predominantly to spiral ganglion neurons, indicating that DFNB86 deafness might be an auditory neuropathy spectrum disorder. Previously, six recessive mutations in TBC1D24 were reported to cause seizures (hearing loss was not reported) ranging in severity from epilepsy with otherwise normal development to epileptic encephalopathy resulting in childhood death. Two of our four families in which deafness segregates with mutant alleles of TBC1D24 were available for neurological examination. Cosegregation of epilepsy and deafness was not observed in these two families. Although the causal relationship between genotype and phenotype is not presently understood, our findings, combined with published data, indicate that recessive alleles of TBC1D24 can cause either epilepsy or nonsyndromic deafness. Hearing loss occurs in approximately 0.2% of newborns, and two-thirds of these cases appear to have a genetic cause.1Hilgert N. Smith R.J. Van Camp G. Function and expression pattern of nonsyndromic deafness genes.Curr. Mol. Med. 2009; 9: 546-564Crossref PubMed Scopus (125) Google Scholar The prevalence of hearing loss increases with age, and there are hundreds of syndromes that include deafness as one feature of a complex phenotype (OMIM, see Web Resources). Additionally, 114 loci have been genetically mapped for nonsyndromic deafness segregating as either a dominant (DFNA) or a recessive (DFNB) trait (Hereditary Hearing Loss Homepage, see Web Resources).1Hilgert N. Smith R.J. Van Camp G. Function and expression pattern of nonsyndromic deafness genes.Curr. Mol. Med. 2009; 9: 546-564Crossref PubMed Scopus (125) Google Scholar, 2Duman D. Tekin M. Autosomal recessive nonsyndromic deafness genes: a review.Front Biosci (Landmark Ed). 2012; 17: 2213-2236Crossref PubMed Scopus (103) Google Scholar For approximately half of these loci, the genes with mutations that result in nonsyndromic deafness have been identified. The wild-type alleles of these genes subserve a myriad of cellular functions that span the gamut from transcription factors to extracellular matrix proteins,2Duman D. Tekin M. Autosomal recessive nonsyndromic deafness genes: a review.Front Biosci (Landmark Ed). 2012; 17: 2213-2236Crossref PubMed Scopus (103) Google Scholar, 3Richardson G.P. de Monvel J.B. Petit C. How the genetics of deafness illuminates auditory physiology.Annu. Rev. Physiol. 2011; 73: 311-334Crossref PubMed Scopus (163) Google Scholar and one-third of them encode proteins that interact with actin and are crucial for mechanotransduction of sound in the inner ear.4Drummond M.C. Belyantseva I.A. Friderici K.H. Friedman T.B. Actin in hair cells and hearing loss.Hear. Res. 2012; 288: 89-99Crossref PubMed Scopus (41) Google Scholar, 5Kazmierczak P. Müller U. Sensing sound: molecules that orchestrate mechanotransduction by hair cells.Trends Neurosci. 2012; 35: 220-229Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar Despite unique anatomical structures and physiological functions described in the auditory system,6Schuknecht H.F. Pathology of the Ear, Second Edition. Lea & Febiger, Malvern1993Google Scholar, 7Vollrath M.A. Kwan K.Y. Corey D.P. The micromachinery of mechanotransduction in hair cells.Annu. Rev. Neurosci. 2007; 30: 339-365Crossref PubMed Scopus (168) Google Scholar there are only a few examples of genes that have a pattern of expression limited to the inner ear.8Verhoeven K. Van Laer L. Kirschhofer K. Legan P.K. Hughes D.C. Schatteman I. Verstreken M. Van Hauwe P. Coucke P. Chen A. et al.Mutations in the human alpha-tectorin gene cause autosomal dominant non-syndromic hearing impairment.Nat. Genet. 1998; 19: 60-62Crossref PubMed Scopus (294) Google Scholar, 9Verpy E. Masmoudi S. Zwaenepoel I. Leibovici M. Hutchin T.P. Del Castillo I. Nouaille S. Blanchard S. Lainé S. Popot J.L. et al.Mutations in a new gene encoding a protein of the hair bundle cause non-syndromic deafness at the DFNB16 locus.Nat. Genet. 2001; 29: 345-349Crossref PubMed Scopus (133) Google Scholar In fact, many genes with mutations associated with nonsyndromic deafness do not encode inner-ear-cell-specific molecules but rather are expressed in a variety of organ systems.10Scott H.S. Kudoh J. Wattenhofer M. Shibuya K. Berry A. Chrast R. Guipponi M. Wang J. Kawasaki K. Asakawa S. et al.Insertion of beta-satellite repeats identifies a transmembrane protease causing both congenital and childhood onset autosomal recessive deafness.Nat. Genet. 2001; 27: 59-63Crossref PubMed Scopus (185) Google Scholar, 11Ahmed Z.M. Masmoudi S. Kalay E. Belyantseva I.A. Mosrati M.A. Collin R.W. Riazuddin S. Hmani-Aifa M. Venselaar H. Kawar M.N. et al.Mutations of LRTOMT, a fusion gene with alternative reading frames, cause nonsyndromic deafness in humans.Nat. Genet. 2008; 40: 1335-1340Crossref PubMed Scopus (50) Google Scholar, 12Rehman A.U. Morell R.J. Belyantseva I.A. Khan S.Y. Boger E.T. Shahzad M. Ahmed Z.M. Riazuddin S. Khan S.N. Riazuddin S. Friedman T.B. Targeted capture and next-generation sequencing identifies C9orf75, encoding taperin, as the mutated gene in nonsyndromic deafness DFNB79.Am. J. Hum. Genet. 2010; 86: 378-388Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar For example, ACTG1 (MIM 102560), encoding cytoplasmic γ-actin,13Zhu M. Yang T. Wei S. DeWan A.T. Morell R.J. Elfenbein J.L. Fisher R.A. Leal S.M. Smith R.J. Friderici K.H. Mutations in the gamma-actin gene (ACTG1) are associated with dominant progressive deafness (DFNA20/26).Am. J. Hum. Genet. 2003; 73: 1082-1091Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar and HGF (MIM 142409), encoding hepatocyte growth factor (HGF), are associated with nonsyndromic deafness DFNA20 (MIM 604717) and DFNB39 (MIM 608265), respectively, and show widespread expression.14Schultz J.M. Khan S.N. Ahmed Z.M. Riazuddin S. Waryah A.M. Chhatre D. Starost M.F. Ploplis B. Buckley S. Velásquez D. et al.Noncoding mutations of HGF are associated with nonsyndromic hearing loss, DFNB39.Am. J. Hum. Genet. 2009; 85: 25-39Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar An inference from these observations is that a mutation in any gene, no matter how widely expressed in the body, could be a cause of a phenotypically restricted human disorder.15Bolze A. Mahlaoui N. Byun M. Turner B. Trede N. Ellis S.R. Abhyankar A. Itan Y. Patin E. Brebner S. et al.Ribosomal protein SA haploinsufficiency in humans with isolated congenital asplenia.Science. 2013; 340: 976-978Crossref PubMed Scopus (138) Google Scholar, 16McCann K.L. Baserga S.J. Genetics. Mysterious ribosomopathies.Science. 2013; 341: 849-850Crossref PubMed Scopus (85) Google Scholar Genome-wide homozygosity mapping of DFNB in a large consanguineous Pakistani family (PKDF799) identified the locus DFNB86 on chromosome 16p with a maximum LOD score of 8.5.17Ali R.A. Rehman A.U. Khan S.N. Husnain T. Riazuddin S. Friedman T.B. Ahmed Z.M. Riazuddin S. DFNB86, a novel autosomal recessive non-syndromic deafness locus on chromosome 16p13.3.Clin. Genet. 2012; 81: 498-500Crossref PubMed Scopus (10) Google Scholar In this study, three additional consanguineous pedigrees (DEM4221, DEM4587, and DEM4476) from Pakistan were ascertained (inbreeding coefficients, Table S1, available online), and their recessive deafness was linked to DFNB86 with maximum LOD scores of 6.90, 3.26, and 5.97, respectively. The overlapping region of homozygosity of these four pedigrees spans 2.05 Mb and contains 121 annotated genes (Figure 1). Here, we report that recessive mutations in TBC1D24 (TBC1 domain family, member 24 [MIM 613577]) are the cause of the nonsyndromic deafness (DFNB86) segregating in these four families. Approval for this study was obtained from the institutional review boards of the Baylor College of Medicine and Affiliated Hospitals (Houston), the National Centre of Excellence in Molecular Biology (University of the Punjab, Lahore), and Quaid-I-Azam University (Islamabad) and from the Combined Neuroscience Institutional Review Board (protocol OH93-DC-0016) at the National Institutes of Health (Bethesda). Written informed consent was obtained from all family members participating in this study. Pure-tone audiometric evaluations of 4 of the 11 affected individuals from family PKDF799 revealed profound deafness (hearing threshold ≥ 90 dB) at all test frequencies, whereas obligate carriers had normal hearing thresholds.17Ali R.A. Rehman A.U. Khan S.N. Husnain T. Riazuddin S. Friedman T.B. Ahmed Z.M. Riazuddin S. DFNB86, a novel autosomal recessive non-syndromic deafness locus on chromosome 16p13.3.Clin. Genet. 2012; 81: 498-500Crossref PubMed Scopus (10) Google Scholar Genomic DNA samples from three affected individuals (IV-23 from family PKDF799, IV-11 from family DEM4221, and IV-6 from family DEM4476) were processed for whole-exome sequencing (WES; Figure 2). For family PKDF799, a TargetSeq Exome Enrichment Kit (Applied Biosystems) was used for capturing the whole exome (45.1 Mb), which was sequenced on an Applied Biosystems SOLiD5500 platform. For families DEM4221 and DEM4476, an EZ Exome v.3.0 kit (NimbleGen) was used for capturing ∼64 Mb of protein-coding plus untranslated expressed sequences, and massively parallel sequencing was performed on an Illumina HiSeq. Sequence reads generated from these libraries were filtered for quality and were mapped to the hg19 human reference genome (UCSC Genome Browser). For family PKDF799, mapping and variant calling were performed with LifeScope (Applied Biosystems). ANNOVAR was used for variant analysis.18Wang K. Li M. Hakonarson H. ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data.Nucleic Acids Res. 2010; 38: e164Crossref PubMed Scopus (7857) Google Scholar For families DEM4221 and DEM4476, sequence alignment and variant calling were performed with the Burrows-Wheeler Aligner and Genome Analysis Toolkit, respectively. Depth of coverage and the number of DNA variants in the three WES data sets are summarized in Table S2. Assuming locus homogeneity, and because there is significant evidence of linkage between markers for DFNB86 and the deafness segregating in these families, we focused our evaluation of the WES data sets only on DNA variants in the smallest DFNB86 linkage interval defined by meiotic recombinations (Figure 1). Additional criteria for filtering data for identifying the pathogenic variants were homozygosity for a variant and an allele frequency < 1% in the NHLBI Exome Sequencing Project (ESP) Exome Variant Server (EVS)19Tennessen J.A. Bigham A.W. O’Connor T.D. Fu W. Kenny E.E. Gravel S. McGee S. Do R. Liu X. Jun G. et al.Broad GOSeattle GONHLBI Exome Sequencing ProjectEvolution and functional impact of rare coding variation from deep sequencing of human exomes.Science. 2012; 337: 64-69Crossref PubMed Scopus (1213) Google Scholar and the 1000 Genomes Project. These variants were confirmed by Sanger sequence analysis of genomic DNA from family members, and allele frequencies were obtained with the use of ethnically matched control individuals (≥148 chromosomes, Table 1).Table 1Homozygous Variants Identified within the DFNB86 IntervalGeneVariantaCoordinates are based on the hg19 human reference sequence (UCSC Genome Browser).mRNA ChangebRefSeq accession numbers are shown in parentheses.Deduced Effect on ProteinbRefSeq accession numbers are shown in parentheses.FamilyMutation TypeAllele Frequency in EVScNHLBI Exome Sequencing Project (ESP) Exome Variant Server.Allele Frequency in Control ChromosomesCACNA1Hg.1257427G>Ac.3060G>A (NM_001005407.1)p.Ala1020Ala (NP_001005407.1)PKDF799synonymous3/12,2292/174IGFALSg.1841013C>Tc.1520G>A (NM_001146006.1)p.Arg507His (NP_001139478.1)DEM4476missense0/12,875-dArg507 is not conserved in mouse, rat, or dog. The substitution of His for Arg507 is predicted to be neutral by MutationTaster, PolyPhen-2, SIFT, LRT, and MutationAssessor.TBC1D24g.2546357G>Tc.208G>T (NM_001199107.1)p.Asp70Tyr (NP_001186036.1)PKDF799missense0/12,8750/682TBC1D24g.2546357G>Tc.208G>T (NM_001199107.1)p.Asp70Tyr (NP_001186036.1)DEM4221missense0/12,8750/682TBC1D24g.2547027G>Cc.878G>C (NM_001199107.1)p.Arg293Pro (NP_001186036.1)DEM4476missense0/12,8750/634PRSS27g.2762774C>Tc.720G>A (NM_031948.3)p.Ser240Ser (NP_114154.1)PKDF799synonymous61/12,8750/490PRSS27g.2762774C>Tc.720G>A (NM_031948.3)p.Ser240Ser (NP_114154.1)DEM4221synonymous61/12,8750/490SRRM2g.2819161_2819163delTCTc.7897_7899delTCT (NM_016333.3)p.Ser2633del (NP_057417.3)DEM4476in-frame deletioneThis deletion of three nucleotides is one of several similar indels previously reported in the EVS as common SNPs resulting in deletion or insertion of one or more serine residues in a stretch of 42 consecutive serines.49/12,3961/290THOC6g.3077605C>Tc.973C>T (NM_024339.3)p.Arg280TrpfDoes not cosegregate with deafness in family DEM4221. (NP_077315.2)DEM4221missense0/12,8750/148a Coordinates are based on the hg19 human reference sequence (UCSC Genome Browser).b RefSeq accession numbers are shown in parentheses.c NHLBI Exome Sequencing Project (ESP) Exome Variant Server.d Arg507 is not conserved in mouse, rat, or dog. The substitution of His for Arg507 is predicted to be neutral by MutationTaster, PolyPhen-2, SIFT, LRT, and MutationAssessor.e This deletion of three nucleotides is one of several similar indels previously reported in the EVS as common SNPs resulting in deletion or insertion of one or more serine residues in a stretch of 42 consecutive serines.f Does not cosegregate with deafness in family DEM4221. Open table in a new tab On the basis of the criteria listed above, analyses of the WES data sets for the three families revealed a total of seven homozygous DNA variants within the refined DFNB86 interval (Table 1). Five of the seven variants were not considered to be pathogenic. A synonymous variant, c.3060G>A (p.Ala1020Ala), in CACNA1H (MIM 607904) is a polymorphism in the Pakistani population. The c.1520G>A (p.Arg507His) variant in IGFALS (MIM 601489) was not studied further because the affected amino acid is not evolutionarily conserved and the substitution is predicted to not be detrimental by five in silico programs, including MutationTaster, PolyPhen-2, and SIFT. The synonymous variant c.720G>A (p.Ser240Ser) in PRSS27 (MIM 608018) was not detected in 490 ethnically matched control chromosomes but has an allele frequency of 0.47% in the EVS (Table 1). A transition mutation, c.973C>T (p.Arg280Trp) in THOC6 (MIM 615403), identified in family DEM4221 does not cosegregate with deafness, further refining the linkage interval of this family (Figure 1). TBC1D24 was the only gene in which homozygous pathogenic mutations were found in the exome data sets of all three families (Table 1). Deaf members of families PKDF799 and DEM4221 are homozygous for a transversion mutation, c.208G>T, predicted to cause a p.Asp70Tyr substitution. Deaf members of family DEM4476 are homozygous for c.878G>C (p.Arg293Pro) in TBC1D24. We also Sanger sequenced the coding exons of TBC1D24 in affected individuals from the fourth family, DEM4587, and found the c.208G>T (p.Asp70Tyr) mutation. The c.208G>T mutation occurs on a haplotype spanning 477 kb shared among three families (Table S3). The two missense alleles of TBC1D24 cosegregate with deafness in these families, are absent from the EVS and 1000 Genomes Project, and were not observed in at least 634 control chromosomes from ethnically matched individuals (Table 1). The substitutions p.Asp70Tyr and p.Arg293Pro affect residues conserved in orthologs of TBC1D24 from Drosophila melanogaster to Homo sapiens (Figure 3A). The p.Asp70Tyr substitution is predicted to be “damaging” to TBC1D24 function by MutationTaster, Mutation Assessor, LRT, SIFT, and PolyPhen-2. The p.Arg293Pro variant was assessed by SIFT as “tolerated” but was predicted to be “damaging” by the other four algorithms and is not reported in the EVS or 1000 Genomes Project. However, the EVS does contain two variants for the Arg293 codon: c.877C>T (p.Arg293Cys) and c.878G>A (p.Arg293His). In the ∼6,500 EVS exomes, a heterozygous p.Arg293Cys substitution was detected once among 12,838 chromosomes, whereas heterozygosity for p.Arg293His was detected seven times among 12,828 chromosomes. Both of these rare variants are predicted by in silico programs to be damaging to TBC1D24 function, suggesting a crucial role for Arg293 in the normal function of TBC1D24. Human TBC1D24 has eight exons, and the longest mRNA encodes a protein of 559 amino acids (Figure 3B). In mammals, TBC1D24 is one of 42 TBC (Tre-2, Bub2, Cdc16)-domain-containing family members.20Fukuda M. TBC proteins: GAPs for mammalian small GTPase Rab?.Biosci. Rep. 2011; 31: 159-168Crossref PubMed Scopus (143) Google Scholar TBC domains have approximately 200 residues that are conserved in amino acid sequence from yeast to human.21Gao X. Jin C. Xue Y. Yao X. Computational analyses of TBC protein family in eukaryotes.Protein Pept. Lett. 2008; 15: 505-509Crossref PubMed Scopus (9) Google Scholar The p.Asp70Tyr substitution is located within the TBC domain of TBC1D24 (Figure 3B). Although the crystal structure of TBC1D24 is not known, the sequence can be compared with that of other TBC domains that have X-ray crystal structures available. Comparison with the sequence of TBC1D4 suggests that the altered residue Asp70 is analogous to residue Asp950 of TBC1D4 (3QYB).22Park S.Y. Jin W. Woo J.R. Shoelson S.E. Crystal structures of human TBC1D1 and TBC1D4 (AS160) RabGTPase-activating protein (RabGAP) domains reveal critical elements for GLUT4 translocation.J. Biol. Chem. 2011; 286: 18130-18138Crossref PubMed Scopus (21) Google Scholar This residue is located in a loop between two helices, termed α3 and α4, in the TBC1D4 structure. The boundaries of this loop vary significantly between different TBC domains, and high B-factors indicate a relatively large degree of flexibility or disorder in this region. Although it is possible that the loop might be the site of interaction with a different region of TBC1D24 or a binding region for another protein, no specific roles for the loop have yet been identified. Some TBC-domain-containing proteins have been shown to function as GTPase-activating proteins (GAPs), which accelerate the intrinsic rate of GTP hydrolysis of specific Rab-GTPases.23Pan X. Eathiraj S. Munson M. Lambright D.G. TBC-domain GAPs for Rab GTPases accelerate GTP hydrolysis by a dual-finger mechanism.Nature. 2006; 442: 303-306Crossref PubMed Scopus (259) Google Scholar When the GTP of a Rab-GTPase is hydrolyzed to GDP by a GAP, the protein becomes inactive until the GDP is exchanged for GTP by another class of regulatory proteins termed guanine nucleotide exchange factors.20Fukuda M. TBC proteins: GAPs for mammalian small GTPase Rab?.Biosci. Rep. 2011; 31: 159-168Crossref PubMed Scopus (143) Google Scholar Thus, GAPs are involved in the regulation of numerous membrane-trafficking and sorting processes of vesicles by modulating the activity of Rab-GTPases.20Fukuda M. TBC proteins: GAPs for mammalian small GTPase Rab?.Biosci. Rep. 2011; 31: 159-168Crossref PubMed Scopus (143) Google Scholar A direct Rab-GTPase target of TBC1D24 has not been reported and might not exist. In yeast, the TBC-domain-containing protein Gyp1p (RefSeq accession number NP_014713.1) has been demonstrated to require an arginine at residue 343 for its Rab-GAP-stimulating activity.23Pan X. Eathiraj S. Munson M. Lambright D.G. TBC-domain GAPs for Rab GTPases accelerate GTP hydrolysis by a dual-finger mechanism.Nature. 2006; 442: 303-306Crossref PubMed Scopus (259) Google Scholar Clustal Omega alignment of the TBC domains of yeast Gyp1p and human TBC1D24 shows that there is a glutamine rather than a catalytic arginine at residue 100 of TBC1D24, suggesting that TBC1D24 lacks Rab-GAP GTPase-stimulating activity.20Fukuda M. TBC proteins: GAPs for mammalian small GTPase Rab?.Biosci. Rep. 2011; 31: 159-168Crossref PubMed Scopus (143) Google Scholar However, ARF6 (ADP-ribosylation factor 6) was reported to be a partner of TBC1D24 and could provide GTPase activity.24Falace A. Filipello F. La Padula V. Vanni N. Madia F. De Pietri Tonelli D. de Falco F.A. Striano P. Dagna Bricarelli F. Minetti C. et al.TBC1D24, an ARF6-interacting protein, is mutated in familial infantile myoclonic epilepsy.Am. J. Hum. Genet. 2010; 87: 365-370Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar To begin to understand wild-type TBC1D24 function in normal hearing, we examined its pattern of expression, alternative transcript splicing, and immunolocalization of TBC1D24 in the mouse inner ear. Although not previously reported in the auditory system, human TBC1D24 is expressed in a variety of tissues, including the heart, liver, kidney, stomach, lungs, and brain.24Falace A. Filipello F. La Padula V. Vanni N. Madia F. De Pietri Tonelli D. de Falco F.A. Striano P. Dagna Bricarelli F. Minetti C. et al.TBC1D24, an ARF6-interacting protein, is mutated in familial infantile myoclonic epilepsy.Am. J. Hum. Genet. 2010; 87: 365-370Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar The NCBI Gene database lists eight splice variants composing two protein isoforms (a and b) of mouse Tbc1d24 (NCBI Gene ID 224617). Isoforms a and b are predicted to encode 561 amino acids (63.2 kDa) and 555 amino acids (62.6 kDa), respectively. We investigated mouse Tbc1d24 mRNA expression after preparing a cDNA library from P12 mouse inner-ear tissue. We used forward and reverse PCR primers complementary to the 5′ UTR and 3′ UTR of the full-length transcript to amplify and characterize splice isoforms of this gene (Figure S1). Agarose gel electrophoresis separated the PCR product into five bands of distinct sizes (data not shown) that were individually isolated, cloned, and Sanger sequenced. We detected nine alternatively spliced transcripts, including the previously reported isoforms a and b, of protein-coding exons of Tbc1d24 (Figure S1). Isoforms a–e include large exon 5, which encodes 57% of the full-length protein, including the translation initiation codon. However, exon 5 is not included in isoforms f–i. If translated, these shorter transcripts would utilize in-frame ATG codons that do not satisfy the consensus (−3A and +4G) for a Kozak start site,25Kozak M. The scanning model for translation: an update.J. Cell Biol. 1989; 108: 229-241Crossref PubMed Scopus (2809) Google Scholar which is not an absolute requirement for translation initiation. If these short isoforms are also expressed in humans, they would not be affected by the two TBC1D24 missense mutations associated with DFNB86 deafness. Immunoblot analysis of P12 mouse brain and cochlea lysates with the use of a commercially available TBC1D24 antibody (Abcam, ab101933) revealed a signal at 60 kDa, consistent with the expected size for the two previously reported TBC1D24 isoforms. There was also a signal at 20 kDa in untransfected COS-7 cells and in mouse brain and cochlear lysates. The 20 kDa signal might be the short isoform f, which includes the epitope recognized by TBC1D24 antibody ab101933 (Figure S1). To determine whether this antibody recognizes TBC1D24, we included a lysate from COS-7 cells transfected with an expression vector encoding pEGFP-TBC1D24 (full length) in the immunoblot analysis, and the expected product corresponding to EGFP-TBC1D24 (26.9 + 63.2 kDa) was detected at approximately 90 kDa (Figure S1). To investigate TBC1D24 localization in P30 mouse inner-ear, we immunolabeled26Choi B.Y. Kim H.M. Ito T. Lee K.Y. Li X. Monahan K. Wen Y. Wilson E. Kurima K. Saunders T.L. et al.Mouse model of enlarged vestibular aqueducts defines temporal requirement of Slc26a4 expression for hearing acquisition.J. Clin. Invest. 2011; 121: 4516-4525Crossref PubMed Scopus (77) Google Scholar cryosections with the TBC1D24 antibody and counterstained them with DAPI and rhodamine phalloidin. The strong signal for TBC1D24 was observed in spiral ganglion cells, a collection of neurons critical for hearing and balance (Figure 4). In addition to a TBC domain, the only other predicted domain in TBC1D24 is a C-terminal TLDc domain (TBC, LysM, domain catalytic). Neither of the two TBC1D24 missense mutations associated with deafness alters the TLDc domain. There are four other human TLDc-domain-containing proteins, including NCOA7 and OXR1, both of which have been demonstrated to defend cells against oxidative stress.27Oliver P.L. Finelli M.J. Edwards B. Bitoun E. Butts D.L. Becker E.B. Cheeseman M.T. Davies B. Davies K.E. Oxr1 is essential for protection against oxidative stress-induced neurodegeneration.PLoS Genet. 2011; 7: e1002338Crossref PubMed Scopus (91) Google Scholar, 28Durand M. Kolpak A. Farrell T. Elliott N.A. Shao W. Brown M. Volkert M.R. The OXR domain defines a conserved family of eukaryotic oxidation resistance proteins.BMC Cell Biol. 2007; 8: 13Crossref PubMed Scopus (56) Google Scholar Interestingly, the mouse OXR1 TLDc domain alone is sufficient to protect granule cells of the cerebellum against oxidative stress.27Oliver P.L. Finelli M.J. Edwards B. Bitoun E. Butts D.L. Becker E.B. Cheeseman M.T. Davies B. Davies K.E. Oxr1 is essential for protection against oxidative stress-induced neurodegeneration.PLoS Genet. 2011; 7: e1002338Crossref PubMed Scopus (91) Google Scholar Clustal Omega alignment of the 163 residues of the TLDc domain of mouse OXR1 shows 66% similarity to the TLDc domain of human TBC1D24. Given the reported role of TLDc domains, an additional wild-type function of TBC1D24 might be to help safeguard spiral ganglion neurons against oxidative stress. Assuming that p.Asp70Tyr and p.Arg293Pro cause deafness as a result of an altered function of TBC1D24 in the spiral ganglion neurons of the inner ear, DFNB86 deafness might be an auditory neuropathy spectrum disorder.29Moser T. Predoehl F. Starr A. Review of hair cell synapse defects in sensorineural hearing impairment.Otol. Neurotol. 2013; 34: 995-1004Crossref PubMed Scopus (79) Google Scholar, 30Schoen C.J. Emery S.B. Thorne M.C. Ammana H.R. Sliwerska E. Arnett J. Hortsch M. Hannan F. Burmeister M. Lesperance M.M. Increased activity of Diaphanous homolog 3 (DIAPH3)/diaphanous causes hearing defects in humans with auditory neuropathy and in Drosophila.Proc. Natl. Acad. Sci. USA. 2010; 107: 13396-13401Crossref PubMed Scopus (78) Google Scholar, 31Starr A. Picton T.W. Sininger Y. Hood L.J. Berlin C.I. Auditory neuropathy.Brain. 1996; 119: 741-753Crossref PubMed Scopus (925) Google Scholar These disorders are audiologically defined by abnormal auditory brainstem response (ABR) waveforms and interpeak intervals in combination with normal otoacoustic emissions (OAEs).32Madden C. Rutter M. Hilbert L. Greinwald Jr., J.H. Choo D.I. Clinical and audiological features in auditory neuropathy.Arch. Otolaryngol. Head Neck Surg. 2002; 128: 1026-1030Crossref PubMed Scopus (180) Google Scholar These findings reflect an underlying lesion in the afferent auditory pathway, which includes the auditory nerve, the afferent synapse with
Referência(s)