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Aberrant transcript produced by a splice donor site deletion in the TECTA gene is associated with autosomal dominant deafness in a Brazilian family
Gene 511 (2012) 280–284
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Gene journal homepage: www.elsevier.com/locate/gene
Methods Paper
Aberrant transcript produced by a splice donor site deletion in the TECTA gene is associated with autosomal dominant deafness in a Brazilian family Karina Lezirovitz a, b,⁎, Ana C. Batissoco a, Fernanda T. Lima c, Maria T.B.M. Auricchio a, Renata W. Nonose a, Simone R. dos Santos d, e, Luiza Guilherme d, e, Jeanne Oiticica b, Regina C. Mingroni-Netto a a
Human Genome Research Center, Department of Genetics and Evolutionary Biology, Biosciences Institute, University of São Paulo, São Paulo (SP), Brazil Otolaryngology Lab/LIM32, School of Medicine Clinics Hospital, University of São Paulo, São Paulo (SP), Brazil FUNCRAF, Foundation for Study and Treatment of Craniofacial Deformities, São Bernardo do Campo, SP, Brazil d Heart Institute (InCor), School of Medicine, University of São Paulo, São Paulo (SP), Brazil e Institute for Immunology Investigation, National Institute for Science and Technology, University of São Paulo, São Paulo (SP), Brazil b c
a r t i c l e
i n f o
Article history: Accepted 6 September 2012 Available online 17 September 2012 Keywords: Hearing loss, sensorineural TECTA protein, human RNA splicing Lymphoblastoid cell line
a b s t r a c t We ascertained a Brazilian family with nine individuals affected by autosomal dominant nonsyndromic sensorineural hearing loss. The bilateral hearing loss affected mainly mid-high frequencies, was apparently stable with an early onset. Microsatellites close to the DFNA8/DFNA12 locus, which harbors the TECTA gene, showed significant multipoint lod scores (3.2) close to marker D11S4107. Sequencing of the exons and exon–intron boundaries of the TECTA gene in one affected subject revealed the deletion c.5383+5delGTGA in the 5′ end of intron 16, that includes the last two bases of the donor splice site consensus sequence. This mutation segregates with deafness within the family. To date, 33 different TECTA mutations associated with autossomal dominant hearing loss have been described. Among them is the mutation reported herein, first described by Hildebrand et al. (2011) in a UK family. The audioprofiles from the UK and Brazilian families were similar. In order to investigate the transcripts produced by the mutated allele, we performed cDNA analysis of a lymphoblastoid cell line from an affected heterozygote with the c.5383+5delGTGA and a noncarrier from the same family. The analysis allowed us to identify an aberrant transcript with skipping of exon 16, without affecting the reading frame. One of the dominant TECTA mutations already described, a synonymous substitution in exon 16 (c.5331 Gb A), was also shown to affect splicing, resulting in an aberrant transcript lacking exon 16. Despite the difference in the DNA level, both the synonymous substitution in exon 16 (c.5331 Gb A) and the mutation described herein affect splicing of exon 16, leading to its skipping. At the protein level they would have the same effect, an in-frame deletion of 37 amino-acids (p.S1758Y/G1759_N1795del) probably leading to an impaired function of the ZP domain. Thus, like the TECTA missense mutations associated with dominant hearing loss, the c.5383+5delGTGA mutation does not have an inactivating effect on the protein. © 2012 Elsevier B.V. All rights reserved.
Abbreviations: TECTA gene, Homo sapiens alpha-tectorin precursor gene; DFNA8/ DFNA12 locus, autosomal dominant deafness locus number eight/autosomal dominant deafness locus number twelve; DFNA13, autosomal dominant deafness locus number thirteen; DFNA21, autosomal dominant deafness locus number twenty one; DFNA24, autosomal dominant deafness locus number twenty four; DFNA31, autosomal dominant deafness locus number thirty one; DFNA44, autosomal dominant deafness locus number forty four; DFNA49, autosomal dominant deafness locus number forty nine; COL11A2, Homo sapiens collagen, type XI, alpha 2 gene; CCDC50, Homo sapiens coiled-coil domain containing 50 gene; ZP domain, zona pellucid domain; ZA domain, zonadhesin domain; OHCs, outer hair cells; vWFD domain, von Willebrand factor-like domain; FUNCRAF, Foundation for Study and Treatment of Craniofacial Deformities, São Bernardo do Campo, SP, Brazil; CONEP, the Brazilian National Committee on Ethics in Research; RT-PCR, reverse transcription polymerase chain reaction; qPCR, quantitative polymerase chain reaction; ACTB, Homo sapiens beta-actin gene; GJB2, Homo sapiens gap junction protein, beta 2, 26kDa, gene; GJB6, Homo sapiens gap junction protein, beta 6, 30kDa, gene; D11S4107, Homo sapiens chromosome 11, locus TECTA, polymorphic microsatellite marker; Cis, as a prefix of Latin origin, meaning “on the same side [as]” or “on this side [of]”; PGRN gene, progranulin gene; rs10502247, Homo sapiens single nucleotide variation. ⁎ Corresponding author at: Otolaryngology Lab/LIM32, School of Medicine Clinics Hospital, Universidade de São Paulo, Av. Dr. Arnaldo, 455 2° andar sala 2209, 01246‐ 903, São Paulo (SP), Brazil. Tel.: + 55 11 3061 7166; fax: + 55 11 3088 0299. E-mail address: [email protected] (K. Lezirovitz). 0378-1119/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gene.2012.09.023
1. Introduction Nonsyndromic hereditary deafness is a highly genetic heterogeneous condition that might exhibit all mendelian patterns of inheritance as well as mitochondrial inheritance. More than a hundred autosomal loci have been mapped through linkage analysis. To date, 49 autosomal dominant nonsyndromic deafness loci (DFNAs) have been mapped and 27 genes have been identified (Van Camp and Smith, 2011). Autosomal dominant inheritance is usually associated with postlingual high frequency progressive hearing loss. However, in some cases, the impairment may involve mainly mid-frequencies, the latter pattern being observed associated with DFNA8/12, DFNA13, DFNA21, DFNA24, DFNA31, DFNA44 and DFNA49 loci (Brown et al., 1997; Häfner et al., 2000; Hildebrand et al., 2011; Kunst et al., 2000; Modamio-Høybjør et al., 2003; Moreno-Pelayo et al., 2003; Snoeckx et al., 2004). The causative genes in only three of those loci have already been identified: the TECTA gene lies in the DFNA8/12 locus, the COL11A2 lies in the DFNA13 locus and the CCDC50 gene lies in the DFNA44 locus.
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The TECTA gene encodes the α-tectorin protein. The α-tectorin protein together with two other noncollagenous glycoproteins (β-tectorin and otogelin) and three different types of collagen (collagen II, collagen IX and collagen XI) compose the tectorial membrane of the organ of Corti, an acellular sheet of extracellular gel-like matrix with unique structure (Legan et al., 2005). The tectorial membrane plays two important roles in the cochlea: 1) it is in contact with mechanosensory bundles of outer hair cells (OHCs), enabling these bundles to deflect when sound vibrations promote the motion of the basilar membrane, allowing OHCs to increase the sensitivity and frequency selectivity of cochlear motions (Russell et al., 2007); and 2) transmits these motions to the bundles of inner hair cells that send auditory information to the brain (Legan et al., 2005). Alpha-tectorin is the major noncollagenous component of the tectorial membrane and is expressed at high levels only in the inner ear (Legan et al., 1997, 2005). It has several functional domains: the entactin (ENT)-like domain, four von Willebrand factor-like type D (vWFD) domains in the zonadhesin (ZA) domain, and the zona pellucida (ZP) domain (Balciuniene et al., 1999; Legan et al., 1997, 2000). Both autosomal dominant (DFNA8/12) and autosomal recessive (DFNB21) hearing loss have been associated with mutations in the TECTA gene (Mustapha et al., 1999). Inactivating mutations (usually truncating) are responsible for the recessive forms whereas non-truncating mutations cause hearing loss with autosomal dominant inheritance. To date, 33 different TECTA mutations associated with autossomal dominant hearing loss have been described, 20 of them were recently reported by Hildebrand et al. (2011). Here, we describe the results of linkage analysis and identification of the mutation c.5383 + 5delGTGA in the TECTA gene in a Brazilian pedigree, in which autosomal dominant nonsyndromic hearing loss is segregating. Besides, the characterization of the aberrant transcripts that resulted from the detected mutation is presented, investigated in a lymphoblastoid cell line obtained from a mutation carrier. 2. Patients and methods 2.1. Patients A large Brazilian family with nine cases of autosomal dominant nonsyndromic sensorineural hearing loss was ascertained in FUNCRAF (Foundation for Study and Treatment of Craniofacial Deformities) and referred to our Genetic Counseling Unit. This study was approved by CONEP, the Brazilian National Committee on Ethics in Research. After appropriate written informed consents were obtained, blood samples were obtained from 15 members of this family (Fig. 1a). In addition, 69 deaf probands from families with hearing loss and possible autosomal dominant inheritance, also referred to our Genetic Counseling Unit, were screened for the mutation found in the TECTA gene. DNA was extracted by standard techniques using phenol/chloroform, kits or using an equipment Autopure LS (Gentra Systems, Minneapolis, MN, USA). 2.1.1. Audiological evaluation After performing otoscopy, rinoscopy and oroscopy exams, pure tone audiometry was carried out to test for air conduction (250–8000 Hz) and bone conduction (250–4000 Hz). Audiograms were available for seven of the nine affected members (IV:7, V:2, V:4, V:5, VI:2, VI:4 and VI:5 — Fig. 1c) of the pedigree and unaffected individual V:1 (Fig. 1d). Syndromic features were ruled out after physical examination and complete anamneses. No symptoms of vestibular dysfunction were reported. Hearing loss was bilateral with early onset, apparently stable, with mainly mid-high frequencies affected (Fig. 1c). In one affected member (IV:7) a flat audiometric configuration was documented. It probably had prelingual onset in at least two cases, since delayed speech was observed (VI:2 and VI:4). Patients V:4, V:5, V:6, VI:5 and VI:8
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also referred hearing impairment beginning in their early childhood, but they were not clinically evaluated until adolescence or adulthood. Individuals (IV:7 and VI:3) presented hearing loss near the age of 10 to 12 years. Patient VI:3 manifested hearing impairment by the age of 18 years. Patient V:1 exhibited unilateral late onset hearing loss (around 27 years of age), which was clinically different from the phenotype observed in the other members of the pedigree. Thus, she was considered phenotypically as unknown for all the analyses. 3. Molecular analysis 3.1. Linkage analysis Fluorescent microsatellite markers close to known hearing loss loci were analyzed in the MegaBACE 1000 DNA Analysis System with the software Genetic Profiler version 2.2 (Amersham Biosciences, Buckingham-shire, UK). The majority of primers used to amplify these markers were kindly provided by Dr Edward R. Wilcox from the National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Rockville, USA. 3.2. Lod scores Multipoint lod scores were calculated with Merlin software (Abecasis et al., 2002) and two-point lod scores with MLINK from FASTLINK version 4.1 (Cottingham et al., 1993); disease allele frequency was set at 0.0001 and the allele frequency of each marker were estimated based on genotypes from married-in and unrelated individuals. The penetrance was estimated to be 89% based on data from the extended pedigree. 3.3. Sequencing of TECTA coding region The primers sequences used in mutation screening of the TECTA gene are described in Meyer et al. (2007), except for exons 5 and 17, of which the primers were designed using “Primer3” software. PCR fragments were purified and directly sequenced in both strands using the ABI BigDye Terminator v3.1 Cycle Sequencing Kit and the ABI 3730 DNA Analyzer (Applied Biosystems, Carlsbad, CA, USA). 3.4. Bioinformatic analyses To address the predicted effect of the splice donor site deletion detected in this study on splice efficiency, two softwares were used: NNSPLICE 0.9 version (http://www.fruitfly.org/seq_tools/splice.html) and NetGene 2 Server (http://www.cbs.dtu.dk/services/NetGene2). 3.5. Analysis of TECTA mRNA levels and c.5383 + 5delGTGA mutation splicing effects EBV-transformed lymphoblasts' were generated from one affected individual and one normal hearing individual from the family in order to study the TECTA gene transcripts. Total RNA was isolated from lymphoblasts using the RNeasy Protect Cell Mini Kit (Qiagen, Hilden, Germany). The RNA samples were treated with DNase I (Invitrogen, Paisley, UK) and submitted to cDNA synthesis with the Superscript First-strand Synthesis System for RT-PCR according to manufactures' protocol (Invitrogen, Paisley, UK). To verify if the mutant allele was expressed in comparison to the normal allele in a heterozygous patient carrying the c.5383 + 5delGTGA mutation, sequence analysis was done in cDNA, in the region of a synonymous polymorphism (rs10502247) located in exon 8 in cDNA. Sequencing was also performed in genomic samples. Quantitative PCR (qPCR) using SYBR green PCR Mastermix (Applied Biosystems, Foster City, CA) was performed to assess mRNA levels of TECTA, using a forward primer located in exon 10
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Fig. 1. (a) Pedigree showing the DFNA8/12 haplotypes linked to hearing loss within this family and the individuals' genotypes regarding the c.5383 + 5delGTGA mutation as well as the rs10502247 polymorphism. By segregation analysis, we conclude that the c.5383 + 5delGTGA mutation is in cis with the T allele from rs10502247. The individual represented in gray (V:1) showed unilateral hearing loss at the age of 27 years. Her clinical and haplotypic data support the hypothesis that she represents a phenocopy. (b) Multipoint lod scores obtained with Merlin software showing significant values (3.20) in TECTA gene map position only. (c) Audiograms of seven affected patients in different ages, showing no apparent correlation between age and severity of the impairment, characterizing nonprogressive hearing loss. (d) Audiograms from patient V:1 showing unilateral profound hearing loss.
and a reverse primer located in exon 11. qPCR samples were analyzed in triplicates and normalized against beta-actin (ACTB) mRNA levels. Relative quantification was calculated according to the formula RQ = (Etarget) ΔCPtarget(control-sample) / (Eref) ΔCPref(control-sample), as described by Pfaffl (2001) . The experiment was performed three times in order to obtain mean and standard deviation values. In order to characterize the different aberrant transcripts yielded by the c.5383 + 5delGTGA allele by sequencing, the cDNA of TECTA gene was amplified using the following primer pairs: 15F 5′-ATGG CATGCAGAAGAGACCT-3′ (located in exon 15) 17R 5′-TCAAAACCGA GCTGGAAGAG-3′ (located in exon 17) or 14F 5′‐TCTCTGTGGCAAC TTCAACG-3′ (located in exon 14) and 18R 5′‐GATGTTGCCAGTGTT GTTGG-3′ (located in exon 18), 15F 5′-ATGGCATGCAGAAGAGACCT-3′ (located in exon 15) with intron16R 5′-GATGCATCAAGGTCGTTCC-3′ (located in intron 16). Following, PCR products were separated using agarose gel electrophoresis, excised from gel, purified and sequenced as described above. 4. Results and discussion A Brazilian family with nine cases of autosomal dominant nonsyndromic hearing loss was molecularly investigated. After excluding frequent genetic causes of hearing loss (mutations in GJB2 gene,
deletions of GJB6 gene and A1555G mitochondrial mutation), linkage analysis with markers from 40 out of the 47 known DFNA loci did not show significant results. However, microsatellites linked to the DFNA8/DFNA12 locus, which harbors the TECTA gene, showed significant multipoint lod scores (3.2) close to marker D11S4107 (Fig. 1b). This marker resides in intron 19 of the TECTA gene. Sequencing of the exons and exon–intron boundaries of the TECTA gene in one affected subject revealed a deletion on position +5 of intron 16, that leads to change of the last two bases of the donor splice site consensus sequence (c.5383 + 5delGTGA or IVS16 + 5_8delGTGA, shown in Figs. 2a and b). We found the c.5383 + 5delGTGA mutation in the heterozygous state segregating with hearing loss in the pedigree. Two bioinformatics' tools predicted that the c.5383+ 5delGTGA mutation causes the loss of the donor splice site of intron 16 (NNSPLICE 0.9 version and NetGene 2 Server). We also screened the c.5383 + 5delGTGA in a cohort of 69 probands from pedigrees with possible autosomal dominant inherited hearing loss. None of them presented the mutation. The phenotype associated with dominant TECTA mutations usually depends on the α-tectorin domain that is affected. Mutations that affect the vWFD2-D3 interrepeat connector or the vWFD4 repeat from the zonadhesin (ZA) domain are associated with high frequency hearing loss (Hildebrand et al., 2011). On the other hand, mutations laying in the other regions from the zonadhesin domain (ZA), entactin
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Fig. 2. (a) Sequence analysis of the exon–intron 16 of the TECTA gene showing normal sequence; (b) sequence analysis of the exon–intron 16 of the TECTA gene showing the c.5383 + 5delGTGA in heterozygosis; (c) sequence analysis of the exon 8 of TECTA gene in the genomic DNA of a c.5383 + 5delGTGA carrier showing the polymorphism rs10502247 in heterozygosis; (d) sequence analysis of the exon 8 of TECTA gene in the cDNA derived from lymphoblasts of a c.5383 + 5delGTGA carrier showing the polymorphism rs10502247, with similar intensity in its both alleles, suggesting no difference of expression between them. (e) Agarose gel electrophoresis showing in: lane 2 — PCR products of cDNA from a patient without the mutation, lane 3 — PCR products of cDNA from a carrier of the mutation showing an aberrant transcript of 350 bp. (f) Schematic representation of the different splice forms produced as a result of the normal allele and the c.5383 + 5delGTGA mutation in a heterozygote. Sequence chromatograms show the normal transcript (upper panel) and the aberrant transcript with skipping of exon 16 (lower panel) in the c.5383 + 5delGTGA patient. (g) Quantitative real time PCR analysis shows an 11% reduction of TECTA mRNA levels in lymphoblasts from the c.5383 + 5delGTGA patient compared to a non-carrier. Data are shown as the average and standard deviation of three experiments, each performed with all samples in triplicate.
domain or in zona pellucida (ZP) domain, with few exceptions, are associated with mid-frequency hearing loss (Hildebrand et al., 2011; Plantinga et al., 2006). In the majority of cases, when cysteine residues are affected, the loss is progressive and if other residues are affected, the loss is stable, but Hildebrand et al.(2011) described exceptional cases. The c.5383 + 5delGTGA mutation lies in the ZA-ZP interdomain and its carriers exhibit mainly mid-high frequency hearing loss (Fig. 1c). The c.5383 + 5delGTGA mutation was also one of the 20 novel mutations recently described (Hildebrand et al., 2011). Hildebrand et al. (2011) detected this mutation in cis with the p.Asn886Ser mutation in a family with dominant hearing loss from the United Kingdom, but the authors did not clarify whether p.Asn886Ser is pathogenic. No other mutation in the TECTA gene was found segregating with hearing loss in the Brazilian family. The audioprofile exhibited by the UK family is similar to the audioprofile in the Brazilian pedigree (Fig. 1c), but in the UK family, hearing loss was prelingual and progressive, while in the Brazilian family, age of onset varied between prelingual to late adolescence (18 years of age) and progression was not observed. Other mutations that affect the ZA-ZP interdomain have been described: Collin et al. (2008) found the synonymous change Leu1777Leu affecting splicing of exon 16 in a Dutch family segregating prelingual and stable hearing loss, though first symptoms of hearing impairment were reported in the age ranges from b1 to 30 years; Hildebrand et al. (2011) found other two mutations, p.Pro1791Arg and c.5383 + 2 T > G (the latter probably affects splicing of exon 16), both associated with prelingual onset, and a stable profile was reported for the last one. Thus, clinical presentation in the Brazilian family differs from the UK family, but resembles the clinical presentation of the Dutch family (Collin et al., 2008). This indicates that the hearing loss phenotype cannot be only attributed to the main mutation found, but also to genetic background. Other variants in the TECTA gene (i.e. p.Asn886Ser mutation
in cis with c.5383 + 5delGTGA in the UK family) or other genes could have had its contribution (Hildebrand et al., 2011). In order to characterize the aberrant transcripts produced by the mutated allele, lymphoblasts were obtained from two individuals, one affected heterozygote and one hearing control without the mutation. Total RNA was extracted from the lymphoblasts and cDNA was synthesized. The abrogation of the acceptor splice site of intron 16 caused by the c.5383 + 5delGTGA could hypothetically lead to two different aberrant transcripts, one skipping exon 16 and the other retaining intron 16. Indeed, a similar mutation was described affecting splicing (IVS6 + 5_8delGTGA) in the PGRN gene responsible for frontotemporal lobar degeneration in two unrelated patients (Skoglund et al., 2011). By investigating the frontal lobe brain tissue mRNAs of PGRN gene, the authors showed that this mutation caused an altered splicing pattern generating two aberrant transcripts, one skipping exon 6 and the other retaining intron 6. According to the hypothesis of the c.5383 + 5delGTGA mutation leading to a transcript skipping of exon 16, we amplified the cDNA samples using primers located in adjacent exons: forward primer in exon 15 with a reverse primer in exon 17. After agarose gel electrophoresis, the control sample showed a PCR product of 461 bp (Fig. 2e), as anticipated based on the wild-type sequence. However, the c.5383 + 5delGTGA sample exhibited an additional smaller PCR product of 350 bp (Fig. 2e). Sequencing of the smaller PCR product revealed that it lacks exon 16, as predicted (Fig. 2f). In order to test the second hypothesis, of the existence of an aberrant transcript with retention of intron 16, after amplifying exons 14 to 18 in the cDNA samples, we performed a nested PCR with a forward primer located in exon 15 and a reverse primer located in intron 16 of the TECTA gene. No transcript retaining intron 16 was found, which could be attributed to the degradation of the mRNA retaining intron 16 through nonsense mediated decay mechanism. Interestingly, sequencing of the cDNA with primers 14F and
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18R revealed that both normal and affected samples showed an exon 15 without the first 15 nucleotides when compared to the databases Sequences (ENST00000392793, NM_005422.2). Indeed Legan et al. (1997) and Verhoeven et al. (1998) found both isoforms (with and without the first 15 nucleotides) while amplifying the cDNA in mouse. After analyses of the human genomic sequence, the authors concluded that both isoforms (with and without a RPLAP peptide) derived from the use of two different splice sites in exon 15 were synthesized in mouse and man. However, they did not sequence the human cDNA in order to identify these isoforms. To verify if the c.5383 + 5delGTGA mutation affects the splicing efficiency of the neighboring exons 15 and 17, RT-PCR analysis was performed with primers located in exon 14 (fw) and exon 18 (rev). No other aberrant transcript than the one with skipping of exon 16 was observed. Thus, these results support the hypothesis that the transcript which lacks exon 16 being the one responsible for the hearing impairment. Exon 16 has 111 nucleotides and encodes 37 aminoacids (1759–1795). Skipping of exon 16 leads to a frame-preserved transcript and the absence of the corresponding 37 aminoacids is predicted after translation. This corroborates the general trend that non-inactivating mutations in the TECTA gene usually lead to autosomal dominant hearing loss. Collin et al. (2008) studying aberrant transcripts due to a synonymous change in exon 16 of the TECTA gene in lymphoblasts of the affected individuals, also detected only the transcript lacking exon 16. The first aminoacid to be affected by the skipping of exon 16 and joining of exon 15 to exon 17 is the serine in position 1758, that is replaced by a tyrosine. The serine in position 1796 is preserved and the frame is also preserved after this residue. Thus, at the protein level, the mutation found by us would probably have a similar effect to that one described by Collin et al. (2008), p.S1758Y/TG1759_N1795del. According to Legan et al. (1997), the tectorins are probably synthesized as glycosylphosphatidylinositol-linked, membrane bound precursors, targeted to the apical surface of the inner ear epithelia by the lipid and then released into the extracellular compartment by the action of an endoproteinase. The 37 aminoacids predicted to be lacking are located N-terminally from the Zona Pellucida domain and include two consensus N-glycosylation sites, that may be important for the alpha-tectorin folding and for cell-extracellular matrix attachment. However, the impact of the ZA-ZP interdomain on the structure and function of the alpha-tectorin protein remains to be elucidated. To assess if there was a reduction in the expression of the TECTA gene in the c.5383 + 5delGTGA carriers, two experiments were performed: 1) sequencing after amplification with primers specific to cDNA to analyze the intensity of the alleles of the SNP rs10502247, located in exon 8 of TECTA gene and comparison to the sequence of the genomic DNA; 2) Quantitative real time RT-PCR in one carrier of the c.5383 + 5delGTGA and a healthy control. The sequence analysis of the polymorphism did not indicate difference in the amount of each transcript (Figs. 2c and d). Through segregation analysis, we were able to establish the phase between the SNP and the c.5383 + 5delGTGA mutation, the c.5383 + 5delGTGA mutation is in cis with the T allele (derived) from rs10502247. However, an 11% reduction in the overall expression of the TECTA transcripts in the mutation carrier, compared to the noncarrier, was observed after quantitative real time RT-PCR as shown in Fig. 2g. In conclusion, through lymphoblast cDNA analysis we described the aberrant transcript produced by a splice donor site deletion in the TECTA gene. The consequence of genomic alterations affecting splice sites might be, in theory, predictable; however performing in vivo splicing assays to detect aberrant transcripts is valuable to understand the functional consequence of this type of mutation. This splice mutation leads to skipping of exon 16 and is predicted to generate a protein with an in frame deletion of 37 aminoacids in the ZP domain of the protein. Although it is a splice site deletion, it
does not behave as an inactivating mutation, since the reading frame is preserved. This is in agreement with previously established genotype-phenotype correlations, according to which truncating/ inactivating mutations are usually responsible for autosomal recessive deafness and non-inactivating mutations are associated with autosomal dominant deafness. Acknowledgments The authors thank all family members for participation in this hearing loss study and laboratory fellows for technical and scientific support, including Dayane Cruz, Lilian Kimura, Ronaldo Serafim Abreu-Silva, Vitor Goés de Lima Dantas, Camila Juncansen, Katia Maria da Rocha, Martha Lima-Cozzo, Camila Camanzano and Meire Aguena. We also thank Dr Edward Wilcox for sending us the primers to amplify markers close to DFNA loci. This work was financially supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). References Abecasis, G.R., Cherny, S.S., Cookson, W.O., Cardon, L.R., 2002. Merlin—rapid analysis of dense genetic maps using sparse gene flow trees. Nat. Genet. 30 (1), 97–101 (Jan). Balciuniene, J., et al., 1999. Alpha-tectorin involvement in hearing disabilities: one gene–two phenotypes. Hum. Genet. 105 (3), 211–216 (Sep). Brown, M.R., et al., 1997. A novel locus for autosomal dominant nonsyndromic hearing loss, DFNA13, maps to chromosome 6p. Am. J. Hum. Genet. 61 (4), 924–927 (Oct). Collin, R.W., et al., 2008. Mid-frequency DFNA8/12 hearing loss caused by a synonymous TECTA mutation that affects an exonic splice enhancer. Eur. J. Hum. Genet. 16 (12), 1430–1436 (Dec, Epub 2008 Jun 25). Cottingham Jr., R.W., Idury, R.M., Schäffer, A.A., 1993. Faster sequential genetic linkage computations. Am. J. Hum. Genet. 53 (1), 252–263 (Jul). Häfner, F.M., et al., 2000. A novel locus (DFNA24) for prelingual nonprogressive autosomal dominant nonsyndromic hearing loss maps to 4q35-qter in a large Swiss German kindred. Am. J. Hum. Genet. 66 (4), 1437–1442 (Apr, Epub 2000 Mar 17). Hildebrand, M.S., et al., 2011. DFNA8/12 caused by TECTA mutations is the most identified subtype of nonsyndromic autosomal dominant hearing loss. Hum. Mutat. 32 (7), 825–834 (Jul). Kunst, H., et al., 2000. Non-syndromic autosomal dominant progressive non-specific midfrequency sensorineural hearing impairment with childhood to late adolescence onset (DFNA21). Clin. Otolaryngol. Allied Sci. 25 (1), 45–54 (Feb). Legan, P.K., Rau, A., Keen, J.N., Richardson, G.P., 1997. The mouse tectorins. Modular matrix proteins of the inner ear homologous to components of the sperm-egg adhesion system. J. Biol. 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DFNA49, a novel locus for autosomal dominant nonsyndromic hearing loss, maps proximal to DFNA7/DFNM1 region on chromosome 1q21-q23. J. Med. Genet. 40 (11), 832–836 (Nov). Mustapha, M., et al., 1999. An alpha-tectorin gene defect causes a newly identified autosomal recessive form of sensorineural pre-lingual non-syndromic deafness, DFNB21. Hum. Mol. Genet. 8, 409–412. Pfaffl, M.W., 2001. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 29 (9), e45 (May 1). Plantinga, R.F., de Brouwer, A.P., Huygen, P.L., Kunst, H.P., Kremer, H., Cremers, C.W., 2006. A novel TECTA mutation in a Dutch DFNA8/12 family confirms genotype-phenotype correlation. J. Assoc. Res. Otolaryngol. 7 (2), 173–181 (Jun, Epub 2006 Apr 25). Russell, I.J., et al., 2007. Sharpened cochlear tuning in a mouse with a genetically modified tectorial membrane. Nat. Neurosci. 10, 215–223. Skoglund, L., et al., 2011. Novel progranulin mutation detected in 2 patients with FTLD. Alzheimer Dis. Assoc. Disord. 25 (2), 173–178 (Apr–Jun). Snoeckx, R.L., et al., 2004. A novel locus for autosomal dominant non-syndromic hearing loss, DFNA31, maps to chromosome 6p21.3. J. Med. Genet. 41 (1), 11–13 (Jan). Van Camp, G., Smith, R., 2011. Hereditary hearing loss homepage. http:// hereditaryhearingloss.org (12/). Verhoeven, K., et al., 1998. Mutations in the human alpha-tectorin gene cause autosomal dominant non-syndromic hearing impairment. Nat. Genet. 19 (1), 60–62 (May).
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Gene journal homepage: www.elsevier.com/locate/gene
Methods Paper
Aberrant transcript produced by a splice donor site deletion in the TECTA gene is associated with autosomal dominant deafness in a Brazilian family Karina Lezirovitz a, b,⁎, Ana C. Batissoco a, Fernanda T. Lima c, Maria T.B.M. Auricchio a, Renata W. Nonose a, Simone R. dos Santos d, e, Luiza Guilherme d, e, Jeanne Oiticica b, Regina C. Mingroni-Netto a a
Human Genome Research Center, Department of Genetics and Evolutionary Biology, Biosciences Institute, University of São Paulo, São Paulo (SP), Brazil Otolaryngology Lab/LIM32, School of Medicine Clinics Hospital, University of São Paulo, São Paulo (SP), Brazil FUNCRAF, Foundation for Study and Treatment of Craniofacial Deformities, São Bernardo do Campo, SP, Brazil d Heart Institute (InCor), School of Medicine, University of São Paulo, São Paulo (SP), Brazil e Institute for Immunology Investigation, National Institute for Science and Technology, University of São Paulo, São Paulo (SP), Brazil b c
a r t i c l e
i n f o
Article history: Accepted 6 September 2012 Available online 17 September 2012 Keywords: Hearing loss, sensorineural TECTA protein, human RNA splicing Lymphoblastoid cell line
a b s t r a c t We ascertained a Brazilian family with nine individuals affected by autosomal dominant nonsyndromic sensorineural hearing loss. The bilateral hearing loss affected mainly mid-high frequencies, was apparently stable with an early onset. Microsatellites close to the DFNA8/DFNA12 locus, which harbors the TECTA gene, showed significant multipoint lod scores (3.2) close to marker D11S4107. Sequencing of the exons and exon–intron boundaries of the TECTA gene in one affected subject revealed the deletion c.5383+5delGTGA in the 5′ end of intron 16, that includes the last two bases of the donor splice site consensus sequence. This mutation segregates with deafness within the family. To date, 33 different TECTA mutations associated with autossomal dominant hearing loss have been described. Among them is the mutation reported herein, first described by Hildebrand et al. (2011) in a UK family. The audioprofiles from the UK and Brazilian families were similar. In order to investigate the transcripts produced by the mutated allele, we performed cDNA analysis of a lymphoblastoid cell line from an affected heterozygote with the c.5383+5delGTGA and a noncarrier from the same family. The analysis allowed us to identify an aberrant transcript with skipping of exon 16, without affecting the reading frame. One of the dominant TECTA mutations already described, a synonymous substitution in exon 16 (c.5331 Gb A), was also shown to affect splicing, resulting in an aberrant transcript lacking exon 16. Despite the difference in the DNA level, both the synonymous substitution in exon 16 (c.5331 Gb A) and the mutation described herein affect splicing of exon 16, leading to its skipping. At the protein level they would have the same effect, an in-frame deletion of 37 amino-acids (p.S1758Y/G1759_N1795del) probably leading to an impaired function of the ZP domain. Thus, like the TECTA missense mutations associated with dominant hearing loss, the c.5383+5delGTGA mutation does not have an inactivating effect on the protein. © 2012 Elsevier B.V. All rights reserved.
Abbreviations: TECTA gene, Homo sapiens alpha-tectorin precursor gene; DFNA8/ DFNA12 locus, autosomal dominant deafness locus number eight/autosomal dominant deafness locus number twelve; DFNA13, autosomal dominant deafness locus number thirteen; DFNA21, autosomal dominant deafness locus number twenty one; DFNA24, autosomal dominant deafness locus number twenty four; DFNA31, autosomal dominant deafness locus number thirty one; DFNA44, autosomal dominant deafness locus number forty four; DFNA49, autosomal dominant deafness locus number forty nine; COL11A2, Homo sapiens collagen, type XI, alpha 2 gene; CCDC50, Homo sapiens coiled-coil domain containing 50 gene; ZP domain, zona pellucid domain; ZA domain, zonadhesin domain; OHCs, outer hair cells; vWFD domain, von Willebrand factor-like domain; FUNCRAF, Foundation for Study and Treatment of Craniofacial Deformities, São Bernardo do Campo, SP, Brazil; CONEP, the Brazilian National Committee on Ethics in Research; RT-PCR, reverse transcription polymerase chain reaction; qPCR, quantitative polymerase chain reaction; ACTB, Homo sapiens beta-actin gene; GJB2, Homo sapiens gap junction protein, beta 2, 26kDa, gene; GJB6, Homo sapiens gap junction protein, beta 6, 30kDa, gene; D11S4107, Homo sapiens chromosome 11, locus TECTA, polymorphic microsatellite marker; Cis, as a prefix of Latin origin, meaning “on the same side [as]” or “on this side [of]”; PGRN gene, progranulin gene; rs10502247, Homo sapiens single nucleotide variation. ⁎ Corresponding author at: Otolaryngology Lab/LIM32, School of Medicine Clinics Hospital, Universidade de São Paulo, Av. Dr. Arnaldo, 455 2° andar sala 2209, 01246‐ 903, São Paulo (SP), Brazil. Tel.: + 55 11 3061 7166; fax: + 55 11 3088 0299. E-mail address: [email protected] (K. Lezirovitz). 0378-1119/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gene.2012.09.023
1. Introduction Nonsyndromic hereditary deafness is a highly genetic heterogeneous condition that might exhibit all mendelian patterns of inheritance as well as mitochondrial inheritance. More than a hundred autosomal loci have been mapped through linkage analysis. To date, 49 autosomal dominant nonsyndromic deafness loci (DFNAs) have been mapped and 27 genes have been identified (Van Camp and Smith, 2011). Autosomal dominant inheritance is usually associated with postlingual high frequency progressive hearing loss. However, in some cases, the impairment may involve mainly mid-frequencies, the latter pattern being observed associated with DFNA8/12, DFNA13, DFNA21, DFNA24, DFNA31, DFNA44 and DFNA49 loci (Brown et al., 1997; Häfner et al., 2000; Hildebrand et al., 2011; Kunst et al., 2000; Modamio-Høybjør et al., 2003; Moreno-Pelayo et al., 2003; Snoeckx et al., 2004). The causative genes in only three of those loci have already been identified: the TECTA gene lies in the DFNA8/12 locus, the COL11A2 lies in the DFNA13 locus and the CCDC50 gene lies in the DFNA44 locus.
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The TECTA gene encodes the α-tectorin protein. The α-tectorin protein together with two other noncollagenous glycoproteins (β-tectorin and otogelin) and three different types of collagen (collagen II, collagen IX and collagen XI) compose the tectorial membrane of the organ of Corti, an acellular sheet of extracellular gel-like matrix with unique structure (Legan et al., 2005). The tectorial membrane plays two important roles in the cochlea: 1) it is in contact with mechanosensory bundles of outer hair cells (OHCs), enabling these bundles to deflect when sound vibrations promote the motion of the basilar membrane, allowing OHCs to increase the sensitivity and frequency selectivity of cochlear motions (Russell et al., 2007); and 2) transmits these motions to the bundles of inner hair cells that send auditory information to the brain (Legan et al., 2005). Alpha-tectorin is the major noncollagenous component of the tectorial membrane and is expressed at high levels only in the inner ear (Legan et al., 1997, 2005). It has several functional domains: the entactin (ENT)-like domain, four von Willebrand factor-like type D (vWFD) domains in the zonadhesin (ZA) domain, and the zona pellucida (ZP) domain (Balciuniene et al., 1999; Legan et al., 1997, 2000). Both autosomal dominant (DFNA8/12) and autosomal recessive (DFNB21) hearing loss have been associated with mutations in the TECTA gene (Mustapha et al., 1999). Inactivating mutations (usually truncating) are responsible for the recessive forms whereas non-truncating mutations cause hearing loss with autosomal dominant inheritance. To date, 33 different TECTA mutations associated with autossomal dominant hearing loss have been described, 20 of them were recently reported by Hildebrand et al. (2011). Here, we describe the results of linkage analysis and identification of the mutation c.5383 + 5delGTGA in the TECTA gene in a Brazilian pedigree, in which autosomal dominant nonsyndromic hearing loss is segregating. Besides, the characterization of the aberrant transcripts that resulted from the detected mutation is presented, investigated in a lymphoblastoid cell line obtained from a mutation carrier. 2. Patients and methods 2.1. Patients A large Brazilian family with nine cases of autosomal dominant nonsyndromic sensorineural hearing loss was ascertained in FUNCRAF (Foundation for Study and Treatment of Craniofacial Deformities) and referred to our Genetic Counseling Unit. This study was approved by CONEP, the Brazilian National Committee on Ethics in Research. After appropriate written informed consents were obtained, blood samples were obtained from 15 members of this family (Fig. 1a). In addition, 69 deaf probands from families with hearing loss and possible autosomal dominant inheritance, also referred to our Genetic Counseling Unit, were screened for the mutation found in the TECTA gene. DNA was extracted by standard techniques using phenol/chloroform, kits or using an equipment Autopure LS (Gentra Systems, Minneapolis, MN, USA). 2.1.1. Audiological evaluation After performing otoscopy, rinoscopy and oroscopy exams, pure tone audiometry was carried out to test for air conduction (250–8000 Hz) and bone conduction (250–4000 Hz). Audiograms were available for seven of the nine affected members (IV:7, V:2, V:4, V:5, VI:2, VI:4 and VI:5 — Fig. 1c) of the pedigree and unaffected individual V:1 (Fig. 1d). Syndromic features were ruled out after physical examination and complete anamneses. No symptoms of vestibular dysfunction were reported. Hearing loss was bilateral with early onset, apparently stable, with mainly mid-high frequencies affected (Fig. 1c). In one affected member (IV:7) a flat audiometric configuration was documented. It probably had prelingual onset in at least two cases, since delayed speech was observed (VI:2 and VI:4). Patients V:4, V:5, V:6, VI:5 and VI:8
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also referred hearing impairment beginning in their early childhood, but they were not clinically evaluated until adolescence or adulthood. Individuals (IV:7 and VI:3) presented hearing loss near the age of 10 to 12 years. Patient VI:3 manifested hearing impairment by the age of 18 years. Patient V:1 exhibited unilateral late onset hearing loss (around 27 years of age), which was clinically different from the phenotype observed in the other members of the pedigree. Thus, she was considered phenotypically as unknown for all the analyses. 3. Molecular analysis 3.1. Linkage analysis Fluorescent microsatellite markers close to known hearing loss loci were analyzed in the MegaBACE 1000 DNA Analysis System with the software Genetic Profiler version 2.2 (Amersham Biosciences, Buckingham-shire, UK). The majority of primers used to amplify these markers were kindly provided by Dr Edward R. Wilcox from the National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Rockville, USA. 3.2. Lod scores Multipoint lod scores were calculated with Merlin software (Abecasis et al., 2002) and two-point lod scores with MLINK from FASTLINK version 4.1 (Cottingham et al., 1993); disease allele frequency was set at 0.0001 and the allele frequency of each marker were estimated based on genotypes from married-in and unrelated individuals. The penetrance was estimated to be 89% based on data from the extended pedigree. 3.3. Sequencing of TECTA coding region The primers sequences used in mutation screening of the TECTA gene are described in Meyer et al. (2007), except for exons 5 and 17, of which the primers were designed using “Primer3” software. PCR fragments were purified and directly sequenced in both strands using the ABI BigDye Terminator v3.1 Cycle Sequencing Kit and the ABI 3730 DNA Analyzer (Applied Biosystems, Carlsbad, CA, USA). 3.4. Bioinformatic analyses To address the predicted effect of the splice donor site deletion detected in this study on splice efficiency, two softwares were used: NNSPLICE 0.9 version (http://www.fruitfly.org/seq_tools/splice.html) and NetGene 2 Server (http://www.cbs.dtu.dk/services/NetGene2). 3.5. Analysis of TECTA mRNA levels and c.5383 + 5delGTGA mutation splicing effects EBV-transformed lymphoblasts' were generated from one affected individual and one normal hearing individual from the family in order to study the TECTA gene transcripts. Total RNA was isolated from lymphoblasts using the RNeasy Protect Cell Mini Kit (Qiagen, Hilden, Germany). The RNA samples were treated with DNase I (Invitrogen, Paisley, UK) and submitted to cDNA synthesis with the Superscript First-strand Synthesis System for RT-PCR according to manufactures' protocol (Invitrogen, Paisley, UK). To verify if the mutant allele was expressed in comparison to the normal allele in a heterozygous patient carrying the c.5383 + 5delGTGA mutation, sequence analysis was done in cDNA, in the region of a synonymous polymorphism (rs10502247) located in exon 8 in cDNA. Sequencing was also performed in genomic samples. Quantitative PCR (qPCR) using SYBR green PCR Mastermix (Applied Biosystems, Foster City, CA) was performed to assess mRNA levels of TECTA, using a forward primer located in exon 10
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Fig. 1. (a) Pedigree showing the DFNA8/12 haplotypes linked to hearing loss within this family and the individuals' genotypes regarding the c.5383 + 5delGTGA mutation as well as the rs10502247 polymorphism. By segregation analysis, we conclude that the c.5383 + 5delGTGA mutation is in cis with the T allele from rs10502247. The individual represented in gray (V:1) showed unilateral hearing loss at the age of 27 years. Her clinical and haplotypic data support the hypothesis that she represents a phenocopy. (b) Multipoint lod scores obtained with Merlin software showing significant values (3.20) in TECTA gene map position only. (c) Audiograms of seven affected patients in different ages, showing no apparent correlation between age and severity of the impairment, characterizing nonprogressive hearing loss. (d) Audiograms from patient V:1 showing unilateral profound hearing loss.
and a reverse primer located in exon 11. qPCR samples were analyzed in triplicates and normalized against beta-actin (ACTB) mRNA levels. Relative quantification was calculated according to the formula RQ = (Etarget) ΔCPtarget(control-sample) / (Eref) ΔCPref(control-sample), as described by Pfaffl (2001) . The experiment was performed three times in order to obtain mean and standard deviation values. In order to characterize the different aberrant transcripts yielded by the c.5383 + 5delGTGA allele by sequencing, the cDNA of TECTA gene was amplified using the following primer pairs: 15F 5′-ATGG CATGCAGAAGAGACCT-3′ (located in exon 15) 17R 5′-TCAAAACCGA GCTGGAAGAG-3′ (located in exon 17) or 14F 5′‐TCTCTGTGGCAAC TTCAACG-3′ (located in exon 14) and 18R 5′‐GATGTTGCCAGTGTT GTTGG-3′ (located in exon 18), 15F 5′-ATGGCATGCAGAAGAGACCT-3′ (located in exon 15) with intron16R 5′-GATGCATCAAGGTCGTTCC-3′ (located in intron 16). Following, PCR products were separated using agarose gel electrophoresis, excised from gel, purified and sequenced as described above. 4. Results and discussion A Brazilian family with nine cases of autosomal dominant nonsyndromic hearing loss was molecularly investigated. After excluding frequent genetic causes of hearing loss (mutations in GJB2 gene,
deletions of GJB6 gene and A1555G mitochondrial mutation), linkage analysis with markers from 40 out of the 47 known DFNA loci did not show significant results. However, microsatellites linked to the DFNA8/DFNA12 locus, which harbors the TECTA gene, showed significant multipoint lod scores (3.2) close to marker D11S4107 (Fig. 1b). This marker resides in intron 19 of the TECTA gene. Sequencing of the exons and exon–intron boundaries of the TECTA gene in one affected subject revealed a deletion on position +5 of intron 16, that leads to change of the last two bases of the donor splice site consensus sequence (c.5383 + 5delGTGA or IVS16 + 5_8delGTGA, shown in Figs. 2a and b). We found the c.5383 + 5delGTGA mutation in the heterozygous state segregating with hearing loss in the pedigree. Two bioinformatics' tools predicted that the c.5383+ 5delGTGA mutation causes the loss of the donor splice site of intron 16 (NNSPLICE 0.9 version and NetGene 2 Server). We also screened the c.5383 + 5delGTGA in a cohort of 69 probands from pedigrees with possible autosomal dominant inherited hearing loss. None of them presented the mutation. The phenotype associated with dominant TECTA mutations usually depends on the α-tectorin domain that is affected. Mutations that affect the vWFD2-D3 interrepeat connector or the vWFD4 repeat from the zonadhesin (ZA) domain are associated with high frequency hearing loss (Hildebrand et al., 2011). On the other hand, mutations laying in the other regions from the zonadhesin domain (ZA), entactin
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Fig. 2. (a) Sequence analysis of the exon–intron 16 of the TECTA gene showing normal sequence; (b) sequence analysis of the exon–intron 16 of the TECTA gene showing the c.5383 + 5delGTGA in heterozygosis; (c) sequence analysis of the exon 8 of TECTA gene in the genomic DNA of a c.5383 + 5delGTGA carrier showing the polymorphism rs10502247 in heterozygosis; (d) sequence analysis of the exon 8 of TECTA gene in the cDNA derived from lymphoblasts of a c.5383 + 5delGTGA carrier showing the polymorphism rs10502247, with similar intensity in its both alleles, suggesting no difference of expression between them. (e) Agarose gel electrophoresis showing in: lane 2 — PCR products of cDNA from a patient without the mutation, lane 3 — PCR products of cDNA from a carrier of the mutation showing an aberrant transcript of 350 bp. (f) Schematic representation of the different splice forms produced as a result of the normal allele and the c.5383 + 5delGTGA mutation in a heterozygote. Sequence chromatograms show the normal transcript (upper panel) and the aberrant transcript with skipping of exon 16 (lower panel) in the c.5383 + 5delGTGA patient. (g) Quantitative real time PCR analysis shows an 11% reduction of TECTA mRNA levels in lymphoblasts from the c.5383 + 5delGTGA patient compared to a non-carrier. Data are shown as the average and standard deviation of three experiments, each performed with all samples in triplicate.
domain or in zona pellucida (ZP) domain, with few exceptions, are associated with mid-frequency hearing loss (Hildebrand et al., 2011; Plantinga et al., 2006). In the majority of cases, when cysteine residues are affected, the loss is progressive and if other residues are affected, the loss is stable, but Hildebrand et al.(2011) described exceptional cases. The c.5383 + 5delGTGA mutation lies in the ZA-ZP interdomain and its carriers exhibit mainly mid-high frequency hearing loss (Fig. 1c). The c.5383 + 5delGTGA mutation was also one of the 20 novel mutations recently described (Hildebrand et al., 2011). Hildebrand et al. (2011) detected this mutation in cis with the p.Asn886Ser mutation in a family with dominant hearing loss from the United Kingdom, but the authors did not clarify whether p.Asn886Ser is pathogenic. No other mutation in the TECTA gene was found segregating with hearing loss in the Brazilian family. The audioprofile exhibited by the UK family is similar to the audioprofile in the Brazilian pedigree (Fig. 1c), but in the UK family, hearing loss was prelingual and progressive, while in the Brazilian family, age of onset varied between prelingual to late adolescence (18 years of age) and progression was not observed. Other mutations that affect the ZA-ZP interdomain have been described: Collin et al. (2008) found the synonymous change Leu1777Leu affecting splicing of exon 16 in a Dutch family segregating prelingual and stable hearing loss, though first symptoms of hearing impairment were reported in the age ranges from b1 to 30 years; Hildebrand et al. (2011) found other two mutations, p.Pro1791Arg and c.5383 + 2 T > G (the latter probably affects splicing of exon 16), both associated with prelingual onset, and a stable profile was reported for the last one. Thus, clinical presentation in the Brazilian family differs from the UK family, but resembles the clinical presentation of the Dutch family (Collin et al., 2008). This indicates that the hearing loss phenotype cannot be only attributed to the main mutation found, but also to genetic background. Other variants in the TECTA gene (i.e. p.Asn886Ser mutation
in cis with c.5383 + 5delGTGA in the UK family) or other genes could have had its contribution (Hildebrand et al., 2011). In order to characterize the aberrant transcripts produced by the mutated allele, lymphoblasts were obtained from two individuals, one affected heterozygote and one hearing control without the mutation. Total RNA was extracted from the lymphoblasts and cDNA was synthesized. The abrogation of the acceptor splice site of intron 16 caused by the c.5383 + 5delGTGA could hypothetically lead to two different aberrant transcripts, one skipping exon 16 and the other retaining intron 16. Indeed, a similar mutation was described affecting splicing (IVS6 + 5_8delGTGA) in the PGRN gene responsible for frontotemporal lobar degeneration in two unrelated patients (Skoglund et al., 2011). By investigating the frontal lobe brain tissue mRNAs of PGRN gene, the authors showed that this mutation caused an altered splicing pattern generating two aberrant transcripts, one skipping exon 6 and the other retaining intron 6. According to the hypothesis of the c.5383 + 5delGTGA mutation leading to a transcript skipping of exon 16, we amplified the cDNA samples using primers located in adjacent exons: forward primer in exon 15 with a reverse primer in exon 17. After agarose gel electrophoresis, the control sample showed a PCR product of 461 bp (Fig. 2e), as anticipated based on the wild-type sequence. However, the c.5383 + 5delGTGA sample exhibited an additional smaller PCR product of 350 bp (Fig. 2e). Sequencing of the smaller PCR product revealed that it lacks exon 16, as predicted (Fig. 2f). In order to test the second hypothesis, of the existence of an aberrant transcript with retention of intron 16, after amplifying exons 14 to 18 in the cDNA samples, we performed a nested PCR with a forward primer located in exon 15 and a reverse primer located in intron 16 of the TECTA gene. No transcript retaining intron 16 was found, which could be attributed to the degradation of the mRNA retaining intron 16 through nonsense mediated decay mechanism. Interestingly, sequencing of the cDNA with primers 14F and
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18R revealed that both normal and affected samples showed an exon 15 without the first 15 nucleotides when compared to the databases Sequences (ENST00000392793, NM_005422.2). Indeed Legan et al. (1997) and Verhoeven et al. (1998) found both isoforms (with and without the first 15 nucleotides) while amplifying the cDNA in mouse. After analyses of the human genomic sequence, the authors concluded that both isoforms (with and without a RPLAP peptide) derived from the use of two different splice sites in exon 15 were synthesized in mouse and man. However, they did not sequence the human cDNA in order to identify these isoforms. To verify if the c.5383 + 5delGTGA mutation affects the splicing efficiency of the neighboring exons 15 and 17, RT-PCR analysis was performed with primers located in exon 14 (fw) and exon 18 (rev). No other aberrant transcript than the one with skipping of exon 16 was observed. Thus, these results support the hypothesis that the transcript which lacks exon 16 being the one responsible for the hearing impairment. Exon 16 has 111 nucleotides and encodes 37 aminoacids (1759–1795). Skipping of exon 16 leads to a frame-preserved transcript and the absence of the corresponding 37 aminoacids is predicted after translation. This corroborates the general trend that non-inactivating mutations in the TECTA gene usually lead to autosomal dominant hearing loss. Collin et al. (2008) studying aberrant transcripts due to a synonymous change in exon 16 of the TECTA gene in lymphoblasts of the affected individuals, also detected only the transcript lacking exon 16. The first aminoacid to be affected by the skipping of exon 16 and joining of exon 15 to exon 17 is the serine in position 1758, that is replaced by a tyrosine. The serine in position 1796 is preserved and the frame is also preserved after this residue. Thus, at the protein level, the mutation found by us would probably have a similar effect to that one described by Collin et al. (2008), p.S1758Y/TG1759_N1795del. According to Legan et al. (1997), the tectorins are probably synthesized as glycosylphosphatidylinositol-linked, membrane bound precursors, targeted to the apical surface of the inner ear epithelia by the lipid and then released into the extracellular compartment by the action of an endoproteinase. The 37 aminoacids predicted to be lacking are located N-terminally from the Zona Pellucida domain and include two consensus N-glycosylation sites, that may be important for the alpha-tectorin folding and for cell-extracellular matrix attachment. However, the impact of the ZA-ZP interdomain on the structure and function of the alpha-tectorin protein remains to be elucidated. To assess if there was a reduction in the expression of the TECTA gene in the c.5383 + 5delGTGA carriers, two experiments were performed: 1) sequencing after amplification with primers specific to cDNA to analyze the intensity of the alleles of the SNP rs10502247, located in exon 8 of TECTA gene and comparison to the sequence of the genomic DNA; 2) Quantitative real time RT-PCR in one carrier of the c.5383 + 5delGTGA and a healthy control. The sequence analysis of the polymorphism did not indicate difference in the amount of each transcript (Figs. 2c and d). Through segregation analysis, we were able to establish the phase between the SNP and the c.5383 + 5delGTGA mutation, the c.5383 + 5delGTGA mutation is in cis with the T allele (derived) from rs10502247. However, an 11% reduction in the overall expression of the TECTA transcripts in the mutation carrier, compared to the noncarrier, was observed after quantitative real time RT-PCR as shown in Fig. 2g. In conclusion, through lymphoblast cDNA analysis we described the aberrant transcript produced by a splice donor site deletion in the TECTA gene. The consequence of genomic alterations affecting splice sites might be, in theory, predictable; however performing in vivo splicing assays to detect aberrant transcripts is valuable to understand the functional consequence of this type of mutation. This splice mutation leads to skipping of exon 16 and is predicted to generate a protein with an in frame deletion of 37 aminoacids in the ZP domain of the protein. Although it is a splice site deletion, it
does not behave as an inactivating mutation, since the reading frame is preserved. This is in agreement with previously established genotype-phenotype correlations, according to which truncating/ inactivating mutations are usually responsible for autosomal recessive deafness and non-inactivating mutations are associated with autosomal dominant deafness. Acknowledgments The authors thank all family members for participation in this hearing loss study and laboratory fellows for technical and scientific support, including Dayane Cruz, Lilian Kimura, Ronaldo Serafim Abreu-Silva, Vitor Goés de Lima Dantas, Camila Juncansen, Katia Maria da Rocha, Martha Lima-Cozzo, Camila Camanzano and Meire Aguena. We also thank Dr Edward Wilcox for sending us the primers to amplify markers close to DFNA loci. 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