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Novel mutations in the SLC26A4 gene
International Journal of Pediatric Otorhinolaryngology 76 (2012) 1249–1254
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Novel mutations in the SLC26A4 gene Micol Busi a,*, Alessandro Castiglione a, Marina Taddei Masieri b, Anna Ravani b, Valeria Guaran c, Laura Astolfi c, Patrizia Trevisi a, Alessandra Ferlini b, Alessandro Martini d a
Audiology Department – C.so Giovecca 203, 44121 - University of Ferrara, Italy Department of Experimental and Diagnostic Medicine, Section of Medical Genetics, C.so Giovecca 203, 44121 – University of Ferrara, Italy c Biomedical Campus – Pietro d’Abano, via Orus 2b, 35128 – Padua University, Italy d ENT – Otosurgery Department – Via, Giustiniani 2, 35128 – Padua University, Italy b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 23 February 2012 Received in revised form 16 May 2012 Accepted 17 May 2012 Available online 18 June 2012
Objectives: Mutations in the SLC26A4 gene (7q22.3–7q31.1) are considered one of the most common causes of genetic hearing loss. There are two clinical forms related to these mutations: syndromic and non-syndromic deafness. The first one is named Pendred Syndrome (PS) when deafness is associated with thyroid goiter; the second is called DFNB4, when no other symptoms are present. Both are transmitted as an autosomal recessive trait, but simple heterozygotes can develop both forms of deafness. Actually it is thought that Pendred Syndrome occurs when both alleles of SLC26A4 gene are mutated; DFNB4 seems due to monoallelic mutations. PS and DFNB4 can be associated with inner ear malformations. In most of the cases (around 80%), these consist in Enlarged Vestibular Aqueduct (EVA). EVA can also be present without SLC26A4 mutations. Understanding the role of new SLC26A4 variants should facilitate clinical assessment, as well as diagnostic and therapeutic approaches. This investigation aims to detect and report genetic causes of two unrelated Italian boys with hearing loss. Methods: Patients and family members underwent clinical, audiological and genetic evaluations. To identify genetic mutations, DNA sequencing of SLC26A4 gene (including all 21 exons, exon-intron boundaries and promoter region) was carried out. Results: Both probands were affected by congenital, progressive and fluctuating mixed hearing loss. Temporal bone imaging revealed a bilateral EVA with no other abnormalities in both cases. Probands were heterozygotes for previously undescribed mutations in the SLC26A4 gene: R409H/IVS2+1delG (proband 1) and L236P/K590X (proband 2). No other mutations were detected in GJB2, GJB6 genes or mitochondrial DNA (mit-DNA). Conclusions: The IVS2+1delG and K590X mutations have not yet been described in literature but there is some evidence to suggest that they have a pathological role. The results underlined the importance of considering the complete DNA sequencing of the SLC26A4 gene for differential molecular diagnosis of deafness, especially in those patients affected by congenital, progressive and fluctuating mixed hearing loss with bilateral EVA. ß 2012 Elsevier Ireland Ltd. All rights reserved.
Keywords: SLC26A4 Pendred syndrome DFNB4 Enlarged vestibular aqueduct
1. Introduction Mutations in the PDS/SLC26A4 (solute carrier family 26, member 4) gene, that encodes pendrin and has been mapped to chromosome 7 with regional assignment to 7q22.3–7q31.1 [1,2],
* Corresponding author at: Audiology Department, University Hospital of Ferrara, C.so Giovecca 203, 44121 Ferrara, Italy. Tel.: +39 0532 237036; fax: +39 0532 237447. E-mail addresses: [email protected] (M. Busi), [email protected] (A. Castiglione), [email protected] (M. Taddei Masieri), [email protected] (A. Ravani), [email protected] (V. Guaran), laura.astolfi@unipd.it (L. Astolfi), [email protected] (P. Trevisi), fl[email protected] (A. Ferlini), [email protected] (A. Martini). 0165-5876/$ – see front matter ß 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ijporl.2012.05.014
are considered one of the most common causes of congenital hearing loss [3] and enlarged vestibular aqueduct (EVA) [4,5]. They are involved in about 10% of all hereditary hearing loss [6–8]: SLC26A4 mutations are the second genetic cause of hearing loss after GJB2 mutations [9]. Two clinical pictures arise from mutations in the SLC26A4 gene: (1) the syndromic form, called Pendred Syndrome (PS), characterized by hearing loss, goiter and eventually hypothyroidism, with/without EVA or other inner ear malformations; (2) the non syndromic form, called DFNB4 or non syndromic EVA (when EVA is present), characterized by hearing loss with/without EVA or other inner ear malformations [10–15]. Both are transmitted as an autosomal recessive trait, but simple heterozygotes can develop both forms of deafness, which is likely due to mutations in uninvestigated genes [16,17]. For
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example, the FOXI1 (5q34) gene encodes for a transcriptional activator that is required to develop normal hearing, sense of balance and kidney function. This activator allows the transcription of the SLC26A4 gene [16]. Furthermore, mutations in the inwardly rectifying K(+) channel gene KCNJ10 (1q23.2) are also associated with non syndromic hearing loss in carriers of SLC26A4 mutations with an EVA/PS phenotype [17]. Some patients with hearing loss and EVA do not have mutations of the SLC26A4 gene. This condition is known as ‘‘hearing loss associated with EVA’’ or ‘‘EVA Syndrome’’ [13–15]. EVA can also be associated with other syndromes, as BOR syndrome or Waardenburg syndrome [18]; but in these syndromes other genes are supposed to be involved [19–21]. PS seems to be linked to biallelic mutations of the SLC26A4 gene. DFNB4 seems to be linked to monoallelic SLC26A4 mutations [22,23]. The SLC26A4 gene, also referred to as the PDS gene, encodes a transmembrane protein (named pendrin) expressed in thyroid gland, inner ear and kidney, where it is involved in the transport of anions such as iodide, chloride and bicarbonate [7,22]. The gene is 57,175 bp long and consists of 21 exons [23]. Pendrin weighs 73 kDa and is comprised of 780 amino acids [24,25] distributed in 12 putative transmembrane domains with both the amino and carboxyl termini located inside the cytosol [7,25,26]. To date, more than 170 variations in SLC26A4 have been reported (http://www.healthcare.uiowa.edu/labs/pendredandbor); most of the mutations related to hearing issues are present in the exons 2, 10 and 19 but novel pathological variants are continuously reported. Different populations are affected by different mutations and founder mutations (a recurrent mutation that occurs on a single haplotype in a population) have been reported in a few cases [22]. The most common mutations in Caucasian population are L236P, T416P and IVS8+1G>A [9,13,27]. T410M, T721M, L445W and R409H seem to be more common in the Mediterranean area [28–30]. EVA is diagnosed by neuroimaging: high-resolution CT scanning of the ear is useful to detect bony abnormalities. Axial scans should be evaluated using the horizontal (or lateral) semicircular canal as a landmark. At this point, it is possible to highlight a defect in the vestibular aqueduct that appears like a triangular opening through the posterior temporal bone. To confirm the presence of an enlarged endolymphatic sac and duct the neuroradiological study should be completed by a highresolution MRI with T2-weighted images [10,11,26,31]. The vestibular aqueduct is defined as enlarged if its diameter is greater than 1.5 mm at the midpoint [10,32]. About 20–30% of all congenital hearing impaired patients show abnormalities of the inner ear [33]. EVA is the most common radiologically detectable inner ear malformation associated with sensorineural hearing loss [34]. According to literature, there are about 7% of patients with EVA and hearing loss (ranging from 0.64% to 13%). A female preponderance has been reported, with a male to female ratio of 2:3. Nevertheless, the prevalence of progressive hearing loss and EVA ranges from 18% to 65%. EVA is caused by the premature arrest in the development of one or more components of the inner ear, around the seventh week of gestation [34]. In some cases, EVA is accompanied by Mondini labyrinthine malformations [15,20,21,34]. There are different types of Mondini malformations. Usually, the Mondini malformation is a cochlear anomaly characterized by a fusion of the apical and middle turn (only one and a half turns of the normal two and one half turns). Less frequently, there is also a posterior labyrinth dysplasia (semicircular canals and/or vestibules can be involved). It should be noted that malformations are different from anatomical variations that do not cause functional disorders [26,31,32]. The aim of this paper is to detect genetic causes and illustrate clinical and radiographic findings in two unrelated Italian boys affected by hearing loss.
2. Materials and methods 2.1. Clinical, audiological and imaging data A detailed family and clinical history was obtained from patients and their parents, in two unrelated Italian families (family 1 and family 2) carrying novel mutations in the SLC26A4 gene. Acquired and environmental factors, possibly related to hearing loss (HL), were investigated. The probands (proband 1 and proband 2) were two unrelated Italian boys affected by congenital mixed HL. Other clinical data were collected, including thyroid and renal function tests [35]. A complete audiological evaluation was carried out using microotoscopy, pure-tone (250–8000 Hz) audiometry for air and bone conduction, speech audiometry (or speech recognition threshold), impedenzometry (tympanometry and acoustic reflex threshold), behavioral audiometry and ABR. Hearing of probands was measured depending on the age of the patient. Speech recognition threshold (SRT) is the threshold of a person for speech at the lowest level at which the presence of speech signal can be heard or recognized or identified 50% of the time (patients have to comprehend the speech signal and then have to repeat the correct 50% speech stimulus). Severity of hearing impairment (HI) was classified as follows: <20 dBHL (decibel hearing level), normal hearing; 20–40 dBHL, mild HL; 41–70 dBHL, moderate HL; 71–95 dBHL, severe HL; and >95 dBHL, profound HL. The HL was bilateral in all affected individuals [36,37]. High resolution CT (computed tomography) and MRI (magnetic resonance imaging) were carried for all probands to obtain a radiological examination of temporal bone. Axial temporal bone CT and MRI findings were classified according to accepted criteria to define cochleovestibular malformations. EVA was defined if the diameter was greater than 1.5 mm at a midway point between the common crus and the external aperture. Mondini’s dysplasia or incomplete partition type II was defined as a cochlea consisting of one and a half turns, in which the middle and apical turns coalesce to form a cystic apex, accompanied by a dilated vestibule and EVA [11,36,37]. 2.2. Genetic and molecular analysis Informed consent was obtained from patients and parents according to current national rules and laws. Molecular genetic studies of the GJB2, GJB6, SLC26A4 genes and mitochondrial DNA (mit-DNA) were performed. Genomic DNA was extracted by standard protocols from peripheral blood leukocytes of all probands and other available members of the two families. PCR amplification of the coding 21 exons, their flanking and promoter regions of SLC26A4 gene was performed using specific primers. Amplification reactions were performed in a final volume of 25 ml containing 100 ng of genomic DNA, 200 mmol/l dNTPs, 10 mmol/l each primer, 1.5 mmol/l MgCl2, 1 U of Taq polymerase. After 5 min of denaturation at 94 8C, 35 PCR cycles were carried out, each cycle consisting of 45 s of denaturation at 94 8C, 45 s of annealing at 60 8C and 80 s of extension at 72 8C. Direct sequencing of the PCR products on both strands was performed on an ABI PRISM 3130xl sequencer, using ABI BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems by Life Technologies) [35]. 2.3. In silico analysis The evolutionary conservation of pendrin residues among eleven SLC26A4 orthologs was investigated at the ClustalW2 EMBLEBI web site (http://www.ebi.ac.uk/Tools/msa/clustalw2/). The pathogenicity of the intronic mutations was investigated using the NetGene2 Splice Site Prediction program (http://www.cbs.dtu.dk/
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services/NetGene2/). Other investigations were conducted using the Mobyle@Pasteur portal for bioinformatics analyses (http:// mobyle.pasteur.fr/cgi-bin/portal.py) [29]. 3. Results 3.1. Clinical, audiological and imaging data 3.1.1. Family 1 Case 1 reports a seven-year-old boy suffering from progressive bilateral hearing loss (proband 1). He is the first-born of an Italian family with no history of relevant diseases. Birth occurred at term by spontaneous vaginal delivery. No pre-, peri- or postnatal risk factors for hearing loss were identified. No other family member showed hearing impairment and thyroid diseases. Audiological tests revealed a bilateral and severe mixed hearing loss (with conductive and sensorineural components). The young patient, with otoscopically normal tympanic membranes and bilateral normal type A tympanograms, did not show a stapedial reflex at all frequencies in both ears. The degree of hearing loss was quite severe and the tonal audiogram depicted a downsloping, highfrequency bilateral hearing impairment (Fig. 1). On subsequent controls we found a progressive worsening in pure tone threshold. At the age of 4 years, we prescribed him behind-the-ear hearing digital aids. The patient’s aided threshold is about 40 dBHL at all frequencies in both ears. During speech tests, he correctly repeated 17/20 disyllabic words in open set. Phonological confusion occurs only for fricative and affricate consonants. The boy always uses an FM (frequency modulation) system during class. Furthermore, we performed genetic and neuroimaging investigations. The proband’s CT scans showed bilateral enlarged
Fig. 2. Proband 1 shows bilateral EVA (white arrows) on axial temporal bone CT scans (A, right ear = 2.3 mm; B, left ear 1.6 mm).
vestibular aqueduct, slightly more enlarged on the right side (Fig. 2). Thyroid function blood tests were normal (FT3: 4.37 pg/ml, FT4: 1.5 ng/dl, TSH: 0.573 mU/ml, TPO Ab: 3 IU/ml, anti-TSH receptor Ab: 0.3 IU/I). The survey was completed by ultrasound of the thyroid, kidney and urinary tract. Exams were all within normal ranges. 3.1.2. Family 2 Case 2 reports an 8-year-old hearing impaired boy (proband 2). He is the second-born of an Italian family with no history of relevant diseases. Birth occurred at 40 weeks of gestation and delivery was spontaneous. No pre-, peri- or postnatal risk factors for hearing loss were identified. The proband 2 presented a bilateral prelingual (less than 2 years of age) non syndromic hearing impairment, so he was bilaterally aided at the age of two years. A maternal aunt was diagnosed with severe hearing loss at the age of six years, probably due to an undocumented viral infection, as reported by family members. No others in the family 2 were affected by hearing loss and goiter.
Fig. 1. Pure tone audiometry of proband 1 with bilateral EVA and two SLC26A4 mutations (R409H/IVS2+1delG).
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Fig. 3. Pure tone audiometry of proband 2 with bilateral EVA and two SLC26A4 mutations (L236P/K590X).
Physical examination, otoscopy and tympanometry of proband 2 were normal. Pure-tone audiometry revealed a bilateral profound mixed hearing loss with a mild conductive component at middle and low frequencies (Fig. 3). Acoustic reflex were absent at all frequencies in both ears. The patient’s aided threshold is about 50 dBHL at all frequencies in both ears. Evaluation of speech processing at threshold levels has resulted in obtaining a speech recognition threshold (SRT). In this case unaided SRT is about 100 dBSPL (decibel sound pressure level) and aided SRT is about 50 dBSPL. During the speech test, he correctly repeated 16/20 disyllabic words in open set. Speech development was normal for the chronological age, with phonetic inventory in spontaneous speech sample being complete, except for fricative consonants. Following a detailed history and examination, he underwent genetic testing, including searching for GJB2, GJB6 and mitochondrial-DNA mutations. Imaging evaluation of temporal bone was performed by means of high resolution computer tomography (CT) and magnetic resonance imaging (MRI). Axial temporal bone CT (Fig. 4) and MRI showed bilateral EVA. Genetic investigation has been completed by searching for SLC26A4 mutations. Thyroid hormones levels dosage (FT3: 4.87 pg/ml, FT4: 1.47 ng/ dl, TSH: 1.32 mU/ml) were in the norm. The survey was completed by ultrasonography of the thyroid, kidney and urinary tract. Exams were all within normal ranges.
Fig. 4. Proband 2: on axial temporal bone CT scans, the vestibular aqueducts are enlarged bilaterally (white arrows), measuring 3.8 mm at the transverse midpoint in the right ear (A) and 2.9 mm at the transverse midpoint in the left ear (B).
3.2. Genetic and molecular analysis 3.2.1. Family 1 The proband 1 and all family members underwent molecular genetic analysis of the GJB2, GJB6 and SLC26A4 genes. Molecular genetics characterization has been performed as already described. Genetic analysis of the patient (proband 1) has revealed a compound heterozygous genotype for the R409H (exon 10) and IVS2+1delG (intron 2) mutations in the SLC26A4 gene. The father is a simple heterozygote for the R409H mutation and the mother is a simple heterozygote for the IVS2+1 delG mutation. They show normal hearing. The proband sister, who underwent prenatal genetic diagnosis, has a normal genotype (wt/wt). At 4 months of age the ABR results showed a normal auditory response. The family tree is shown in Fig. 5. 3.2.2. Family 2 The proband 2 underwent genetic testing, including searching for GJB2, GJB6 and mitochondrial-DNA mutations. After axial temporal bone CT and MRI, genetic investigation has been completed by searching for SLC26A4 mutations. The young patient has no mutations in GJB2, GJB6 and mitochondrial-DNA, but molecular analyses identified him as a compound heterozygote for two mutations in the SLC26A4 gene:
Fig. 5. Family tree of the proband 1 (R409H/IVS2 + 1delG) is compatible with autosomal recessive inheritance.
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Fig. 6. Family tree of the proband 2 (L236P/K590X) is compatible with autosomal recessive inheritance. A maternal aunt was diagnosed with severe hearing loss at the age of six years, probably due to an undocumented viral infection, as reported by family members.
Fig. 8. This figure illustrates the twelve transmembrane domains and the intracellular N-terminus and C-terminus of human pendrin; the C-terminal domain includes STAS domain (proximal and distal portion) and PKA site (in the distal portion) [38]; the approximate location of the K590X mutation is indicated by the black arrows.
Fig. 7. Multiple sequence alignment of pendrin from human, dog, rat, mouse, pig, monkey, chimpanzee, frog, zebrafish, chicken and cow. Amino acid residue K590 is conserved among orthologs.
L236P/K590X (exon 6 and exon 16). The family tree is shown in Fig. 6. 3.3. In silico analysis The evolutionary conservation of pendrin residues among eleven SLC26A4 orthologs was investigated at the ClustalW2. The SLC26A4 gene is conserved in human, dog, rat, mouse, pig, monkey, chimpanzee, frog, zebrafish, chicken and cow (Fig. 7). The splice site prediction program used indicated that IVS2+1delG would significantly affect splicing decreasing the score of the splice donor site from 0.90 in the wild type to 0.0 in the mutated variant. 4. Discussion and conclusions All probands have bilateral EVA and SLC26A4 mutations in both alleles. We found no other mutations in GJB2, GJB6 and mit-DNA. The clinical histories do not support other causes of deafness (infections, drugs, prematurity). The genetic tests revealed different genotypes with a quite similar phenotype: two compound heterozygotes (R409H/IVS2+1delG, L236P/K590X) with congenital progressive mixed hearing loss. The two studied probands with bilateral EVA showed a conductive component of hearing loss at 0.25, 0.5 and 1 kHz and alterations in stapedial reflexes; the conductive component matched the sensorineural hearing loss at 4 kHz (Figs. 1 and 3). These findings should suggest
a physical explanation, such as the third window phenomenon, for example [34]. No thyroid abnormalities were found, although we are aware that hearing impairment and thyroid dysfunction can develop at different times. R409H and L236P are well-known pathogenic SLC26A4 mutations responsible for autosomal recessive hearing loss [9,13,27–30]. The IVS2+1 delG mutation has never previously been described, but clinical and genetic data suggest a significant pathological role. Firstly the deletion of the intron 2 alters a canonical splice donor site. It is expected to have a pathogenic effect, overall if compared to the audiological findings. The mutation involves a partial conserved nucleotide; mutations in similar positions can result in defective splicing leading to variation in levels of normal or aberrant transcribed in different tissues. The family 1 tree is compatible with autosomal recessive inheritance (Fig. 5). The K590X mutation has never been described and it is expected to have pathological significance due to the stop codon determining an amino acid sequence of 589 amino acids (instead of 780) and leading to a shorter protein. The lysine (K) at position 590 is well preserved in different species (Fig. 7); furthermore the truncating mutation K590X should lead to the loss of most of the Cterminal domain, including the distal part of the sulfate transporter and antisigma factor antagonist (STAS) domain and the putative protein kinase A (PKA) phosphorylation site (Fig. 8). Elimination of the distal part of the (STAS) domain with the putative protein kinase A (PKA) phosphorylation site can result in: (1) a cytoplasmatic localization with a complete loss of function or (2) in a residual membrane insertion with a partial loss of function of pendrin [38]. Even in this case, the family 2 tree shows a pattern compatible with autosomal recessive inheritance (Fig. 6). In conclusion, our data suggest a significant pathological role for the IVS2+1 delG and K590X mutations even if a residual protein function for both mutations is possible. This aspect could explain the entity of hearing loss and the absence of thyroid dysfunction, even if differences in symptom onset time are possible. Further studies are needed to assess the genotype/phenotype correlations. Consent Written informed consent was obtained from the families for publication of these cases and any accompanying images.
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Competing interests The authors declare that they have no competing interests. Web resources The URLs for data presented herein are as follows: Online Mendelian Inheritance in Man (OMIM), http://www.ncbi.nlm. nih.gov/Omim/ and http://www.healthcare.uiowa.edu/labs/pendredandbor/. References [1] P. Coucke, G. Van Camp, O. Demirhan, Y. Kabakkaya, W. Balemans, P. Van Hauwe, et al., The gene for Pendred syndrome is located between D7S501 and D7S692 in a 1.7-cM region on chromosome 7q, Genomics 40 (1) (1997) 48–54. [2] M. Mustapha, S.T. Azar, Y.B. Moglabey, M. Saouda, G. Zeitoun, J. Loiselet, et al., Further refinement of Pendred syndrome locus by homozygosity analysis to a 0.8 cM interval flanked by D7S496 and D7S2425, J. Med. Genet. 35 (3) (1998) 202– 204. [3] N.E. Morton, Genetic epidemiology of hearing impairment, Ann. N.Y. Acad. Sci. 630 (1991) 16–31. [4] W. Reardon, C.F. OMahoney, R. Trembath, H. Jan, P.D. Phelps, Enlarged vestibular aqueduct: a radiological marker of pendred syndrome, and mutation of the PDS gene, Q. J. Med. 93 (2) (2000) 99–104. [5] S. Usami, S. Abe, M.D. Weston, H. Shinkawa, G. Van Camp, W.J. Kimberling, Nonsyndromic hearing loss associated with enlarged vestibular aqueduct is caused by PDS mutations, Hum. Genet. 104 (1999) 188–192. [6] G.R. Fraser, Association of congenital deafness with goitre (Pendred’s syndrome): a study of 207 families, Ann. Hum. Genet. 28 (1965) 411–422. [7] L.A. Everett, B. Glaser, J.C. Beck, J.R. Idol, A. Buchs, M. Heyman, et al., Pendred syndrome is caused by mutations in a putative sulphate transporter gene (PDS), Nat. Genet. 17 (4) (1997) 411–422. [8] B.Y. Choi, A.C. Madeo, K.A. King, C.K. Zalewski, S.P. Pryor, J.A. Muskett, et al., Segregation of enlarged vestibular aqueducts in families with non-diagnostic SLC26A4 genotypes, J. Med. Genet. 46 (12) (2009) 856–861. [9] S. Albert, H. Blons, L. Jonard, D. Feldmann, P. Chauvin, N. Loundon, et al., SLC26A4 gene is frequently involved in nonsyndromic hearing impairment with enlarged vestibular aqueduct in Caucasian populations, Eur. J. Hum. Genet. 14 (6) (2006) 773–779. [10] M.F. Mafee, D. Charletta, A. Kumar, H. Belmont, Large vestibular aqueduct and congenital sensorineural hearing loss, Am. J. Neuroradiol. 13 (2) (1992) 805–819. [11] A. Martini, F. Calzolari, A. Sensi, Genetic syndromes involving hearing, Int. J. Pediatr. Otorhinolaryngol. 73 (Suppl. 1) (2009) S2–S12. [12] X.C. Li, L.A. Everett, A.K. Lalwani, D. Desmukh, T.B. Friedman, E.D. Green, et al., A mutation in PDS causes non-syndromic recessive deafness, Nat. Genet. 18 (3) (1998) 215–217. [13] C. Campbell, R.A. Cucci, S. Prasad, G.E. Green, J.B. Edeal, C.E. Galer, et al., Pendred syndrome, DFNB4, and PDS/SLC26A4 identification of eight novel mutations and possible genotype–phenotype correlations, Hum. Mutat. 17 (5) (2001) 403–411. [14] S. Berrettini, F. Forli, F. Bogazzi, E. Neri, L. Salvatori, A.P. Casani, et al., Large vestibular aqueduct syndrome: audiological, radiological, clinical, and genetic features, Am. J. Otolaryngol. 26 (6) (2005) 363–371. [15] S. Fitoz, L. Sennarog˘lu, A. Incesulu, F.B. Cengiz, Y. Koc¸, M. Tekin, SLC26A4 mutations are associated with a specific inner ear malformation, Int. J. Pediatr. Otorhinolaryngol. 71 (3) (2007) 479–486. [16] T. Yang, H. Vidarsson, S. Rodrigo-Blomqvist, S.S. Rosengren, S. Enerback, R.J. Smith, Transcriptional control of SLC26A4 is involved in Pendred syndrome and nonsyndromic enlargement of vestibular aqueduct (DFNB4), Am. J. Hum. Genet. 80 (2007) 1055–1063. [17] T. Yang, J.G. Gurrola, H. Wu, S.M. Chiu, P. Wangemann, P.M. Snyder, et al., Mutations of KCNJ10 together with mutations of SLC26A4 cause digenic nonsyndromic hearing loss associated with enlarged vestibular aqueduct syndrome, Am. J. Hum. Genet. 84 (2009) 651–657.
[18] S. Santos, L. Sgambatti, A. Bueno, G. Albi, A. Sua´rez, M.J. Domı´nguez, Enlarged vestibular aqueduct syndrome. A review of 55 paediatric patients, Acta Otorrinolaringol. Esp. 61 (5) (2010) 338–344. [19] Y. Noguchi, T. Ito, A. Nishio, K. Honda, K. Kitamura, Audiovestibular findings in a branchio-oto syndrome patient with a SIX1 mutation, Acta Otolaryngol. 131 (4) (2011) 413–418. [20] B.Y. Huang, C. Zdanski, M. Castillo, Pediatric sensorineural hearing loss. Part 2. Syndromic and acquired causes, Am. J. Neuroradiol. (2011) (Epub ahead of print). [21] G.A. Krombach, D. Honnef, M. Westhofen, E. Di Martino, R.W. Gu¨nther, Imaging of congenital anomalies and acquired lesions of the inner ear, Eur. Radiol. 18 (2) (2008) 319–330. [22] S.P. Pryor, A.C. Madeo, J.C. Reynolds, N.J. Sarlis, K.S. Arnos, W.E. Nance, et al., SLC26A4/PDS genotype–phenotype correlation in hearing loss with enlargement of the vestibular aqueduct (EVA): evidence that Pendred syndrome and nonsyndromic EVA are distinct clinical and genetic entities, J. Med. Genet. 42 (2) (2005) 159–165. [23] T. Ito, B.Y. Choi, K.A. King, C.K. Zalewski, J. Muskett, P. Chattaraj, et al., SLC26A4 genotypes and phenotypes associated with enlargement of the vestibular aqueduct, Cell. Physiol. Biochem. 28 (3) (2011) 545–552. [24] D.A. Scott, L.P. Karniski, Human pendrin expressed in Xenopus laevis oocytes mediates chloride/formate exchange, Am. J. Physiol. Cell Physiol. 278 (1) (2000) C207–C211. [25] I.E. Royaux, K. Suzuki, A. Mori, R. Katoh, L.A. Everett, L.D. Kohn, et al., Pendrin, the protein encoded by the Pendred syndrome gene (PDS), is an apical porter of iodide in the thyroid and is regulated by thyroglobulin in FRTL-5 cells, Endocrinology 141 (2) (2000) 839–845. [26] X. Ma, Y. Yang, M. Xia, D. Li, A. Xu, Computed tomography findings in large vestibular aqueduct syndrome, Acta Otolaryngol. 129 (7) (2009) 700–708. [27] K. Tsukamoto, H. Suzuki, D. Harada, A. Namba, S. Abe, S. Usami, Distribution and frequencies of PDS (SLC26A4) mutations in Pendred syndrome and nonsyndromic hearing loss associated with enlarged vestibular aqueduct: a unique spectrum of mutations in Japanese, Eur. J. Hum. Genet. 11 (12) (2003) 916–922. [28] N. Lo´pez-Bigas, S. Melchionda, R. de Cid, A. Grifa, L. Zelante, N. Govea, et al., Identification of five new mutations of PDS/SLC26A4 in Mediterranean families with hearing impairment, Hum. Mutat. 20 (1) (2002) 77–78. [29] A. Pera, M. Villamar, A. Vin˜uela, M. Gandı´a, C. Meda`, F. Moreno, et al., A mutational analysis of the SLC26A4 gene in Spanish hearing-impaired families provides new insights into the genetic causes of Pendred syndrome and DFNB4 hearing loss, Eur. J. Hum. Genet. 16 (8) (2008) 888–896. [30] I. Charfeddine, M. Mnejja, B. Hammami, A. Chakroun, S. Masmoudi, H. Ayadi, et al., Pendred syndrome in Tunisia, Eur. Ann. Otorhinolaryngol. Head Neck Dis. 127 (1) (2010) 7–10. [31] M. Boston, M. Halsted, J. Meinzen-Derr, J. Bean, S. Vijayasekaran, E. Arjmand, et al., The large vestibular aqueduct: a new definition based on audiologic and computed tomography correlation, Otolaryngol. Head Neck Surg. 136 (6) (2007) 972– 977. [32] S. Dipak, N. Prepageran, A.S. Sazila, O. Rahmat, R. Raman, Large vestibular aqueduct syndrome, Med. J. Malaysia 60 (4) (2005) 489–491. [33] G. Zalzal, T. Sharon, L. Vezina, P. Bjornsti, K. Grundfast, Enlarged vestibular aqueduct and sensorineural hearing loss in childhood, Arch. Otolaryngol. Head Neck Surg. 121 (1995) 23–28. [34] Q. Gopen, G. Zhou, K. Whittemore, M. Kenna, Enlarged vestibular aqueduct: review of controversial aspects, Laryngoscope 121 (2011) 1971–1978. [35] F. Gualandi, A. Ravani, A. Berto, A. Sensi, C. Trabanelli, F. Falciano, et al., Exploring the clinical and epidemiological complexity of GJB2-linked deafness, Am. J. Med. Genet. 112 (1) (2002) 38–45. [36] E. Cama, M.S. Alemanno, E. Bellacchio, R. Santarelli, M. Carella, L. Zelante, et al., Identification of a novel mutation in the SLC26A4 gene in an Italian with fluctuating sensorineural hearing loss, Int. J. Pediatr. Otorhinolaryngol. 73 (10) (2009) 1458–1463. [37] N. Yazdanpanahi, M.H. Chaleshtori, M.A. Tabatabaiefar, Z. Noormohammadi, E. Farrokhi, H. Najmabadi, et al., Two novel SLC26A4 mutations in Iranian families with autosomal recessive hearing loss, Int. J. Pediatr. Otorhinolaryngol. (2012) (Epub ahead of print). [38] A. Bizhanova, T.L. Chew, S. Khuon, P. Kopp, Analysis of cellular localization and function of carboxy-terminal mutants of pendrin, Cell. Physiol. Biochem. 28 (3) (2011) 423–434.
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International Journal of Pediatric Otorhinolaryngology journal homepage: www.elsevier.com/locate/ijporl
Novel mutations in the SLC26A4 gene Micol Busi a,*, Alessandro Castiglione a, Marina Taddei Masieri b, Anna Ravani b, Valeria Guaran c, Laura Astolfi c, Patrizia Trevisi a, Alessandra Ferlini b, Alessandro Martini d a
Audiology Department – C.so Giovecca 203, 44121 - University of Ferrara, Italy Department of Experimental and Diagnostic Medicine, Section of Medical Genetics, C.so Giovecca 203, 44121 – University of Ferrara, Italy c Biomedical Campus – Pietro d’Abano, via Orus 2b, 35128 – Padua University, Italy d ENT – Otosurgery Department – Via, Giustiniani 2, 35128 – Padua University, Italy b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 23 February 2012 Received in revised form 16 May 2012 Accepted 17 May 2012 Available online 18 June 2012
Objectives: Mutations in the SLC26A4 gene (7q22.3–7q31.1) are considered one of the most common causes of genetic hearing loss. There are two clinical forms related to these mutations: syndromic and non-syndromic deafness. The first one is named Pendred Syndrome (PS) when deafness is associated with thyroid goiter; the second is called DFNB4, when no other symptoms are present. Both are transmitted as an autosomal recessive trait, but simple heterozygotes can develop both forms of deafness. Actually it is thought that Pendred Syndrome occurs when both alleles of SLC26A4 gene are mutated; DFNB4 seems due to monoallelic mutations. PS and DFNB4 can be associated with inner ear malformations. In most of the cases (around 80%), these consist in Enlarged Vestibular Aqueduct (EVA). EVA can also be present without SLC26A4 mutations. Understanding the role of new SLC26A4 variants should facilitate clinical assessment, as well as diagnostic and therapeutic approaches. This investigation aims to detect and report genetic causes of two unrelated Italian boys with hearing loss. Methods: Patients and family members underwent clinical, audiological and genetic evaluations. To identify genetic mutations, DNA sequencing of SLC26A4 gene (including all 21 exons, exon-intron boundaries and promoter region) was carried out. Results: Both probands were affected by congenital, progressive and fluctuating mixed hearing loss. Temporal bone imaging revealed a bilateral EVA with no other abnormalities in both cases. Probands were heterozygotes for previously undescribed mutations in the SLC26A4 gene: R409H/IVS2+1delG (proband 1) and L236P/K590X (proband 2). No other mutations were detected in GJB2, GJB6 genes or mitochondrial DNA (mit-DNA). Conclusions: The IVS2+1delG and K590X mutations have not yet been described in literature but there is some evidence to suggest that they have a pathological role. The results underlined the importance of considering the complete DNA sequencing of the SLC26A4 gene for differential molecular diagnosis of deafness, especially in those patients affected by congenital, progressive and fluctuating mixed hearing loss with bilateral EVA. ß 2012 Elsevier Ireland Ltd. All rights reserved.
Keywords: SLC26A4 Pendred syndrome DFNB4 Enlarged vestibular aqueduct
1. Introduction Mutations in the PDS/SLC26A4 (solute carrier family 26, member 4) gene, that encodes pendrin and has been mapped to chromosome 7 with regional assignment to 7q22.3–7q31.1 [1,2],
* Corresponding author at: Audiology Department, University Hospital of Ferrara, C.so Giovecca 203, 44121 Ferrara, Italy. Tel.: +39 0532 237036; fax: +39 0532 237447. E-mail addresses: [email protected] (M. Busi), [email protected] (A. Castiglione), [email protected] (M. Taddei Masieri), [email protected] (A. Ravani), [email protected] (V. Guaran), laura.astolfi@unipd.it (L. Astolfi), [email protected] (P. Trevisi), fl[email protected] (A. Ferlini), [email protected] (A. Martini). 0165-5876/$ – see front matter ß 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ijporl.2012.05.014
are considered one of the most common causes of congenital hearing loss [3] and enlarged vestibular aqueduct (EVA) [4,5]. They are involved in about 10% of all hereditary hearing loss [6–8]: SLC26A4 mutations are the second genetic cause of hearing loss after GJB2 mutations [9]. Two clinical pictures arise from mutations in the SLC26A4 gene: (1) the syndromic form, called Pendred Syndrome (PS), characterized by hearing loss, goiter and eventually hypothyroidism, with/without EVA or other inner ear malformations; (2) the non syndromic form, called DFNB4 or non syndromic EVA (when EVA is present), characterized by hearing loss with/without EVA or other inner ear malformations [10–15]. Both are transmitted as an autosomal recessive trait, but simple heterozygotes can develop both forms of deafness, which is likely due to mutations in uninvestigated genes [16,17]. For
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example, the FOXI1 (5q34) gene encodes for a transcriptional activator that is required to develop normal hearing, sense of balance and kidney function. This activator allows the transcription of the SLC26A4 gene [16]. Furthermore, mutations in the inwardly rectifying K(+) channel gene KCNJ10 (1q23.2) are also associated with non syndromic hearing loss in carriers of SLC26A4 mutations with an EVA/PS phenotype [17]. Some patients with hearing loss and EVA do not have mutations of the SLC26A4 gene. This condition is known as ‘‘hearing loss associated with EVA’’ or ‘‘EVA Syndrome’’ [13–15]. EVA can also be associated with other syndromes, as BOR syndrome or Waardenburg syndrome [18]; but in these syndromes other genes are supposed to be involved [19–21]. PS seems to be linked to biallelic mutations of the SLC26A4 gene. DFNB4 seems to be linked to monoallelic SLC26A4 mutations [22,23]. The SLC26A4 gene, also referred to as the PDS gene, encodes a transmembrane protein (named pendrin) expressed in thyroid gland, inner ear and kidney, where it is involved in the transport of anions such as iodide, chloride and bicarbonate [7,22]. The gene is 57,175 bp long and consists of 21 exons [23]. Pendrin weighs 73 kDa and is comprised of 780 amino acids [24,25] distributed in 12 putative transmembrane domains with both the amino and carboxyl termini located inside the cytosol [7,25,26]. To date, more than 170 variations in SLC26A4 have been reported (http://www.healthcare.uiowa.edu/labs/pendredandbor); most of the mutations related to hearing issues are present in the exons 2, 10 and 19 but novel pathological variants are continuously reported. Different populations are affected by different mutations and founder mutations (a recurrent mutation that occurs on a single haplotype in a population) have been reported in a few cases [22]. The most common mutations in Caucasian population are L236P, T416P and IVS8+1G>A [9,13,27]. T410M, T721M, L445W and R409H seem to be more common in the Mediterranean area [28–30]. EVA is diagnosed by neuroimaging: high-resolution CT scanning of the ear is useful to detect bony abnormalities. Axial scans should be evaluated using the horizontal (or lateral) semicircular canal as a landmark. At this point, it is possible to highlight a defect in the vestibular aqueduct that appears like a triangular opening through the posterior temporal bone. To confirm the presence of an enlarged endolymphatic sac and duct the neuroradiological study should be completed by a highresolution MRI with T2-weighted images [10,11,26,31]. The vestibular aqueduct is defined as enlarged if its diameter is greater than 1.5 mm at the midpoint [10,32]. About 20–30% of all congenital hearing impaired patients show abnormalities of the inner ear [33]. EVA is the most common radiologically detectable inner ear malformation associated with sensorineural hearing loss [34]. According to literature, there are about 7% of patients with EVA and hearing loss (ranging from 0.64% to 13%). A female preponderance has been reported, with a male to female ratio of 2:3. Nevertheless, the prevalence of progressive hearing loss and EVA ranges from 18% to 65%. EVA is caused by the premature arrest in the development of one or more components of the inner ear, around the seventh week of gestation [34]. In some cases, EVA is accompanied by Mondini labyrinthine malformations [15,20,21,34]. There are different types of Mondini malformations. Usually, the Mondini malformation is a cochlear anomaly characterized by a fusion of the apical and middle turn (only one and a half turns of the normal two and one half turns). Less frequently, there is also a posterior labyrinth dysplasia (semicircular canals and/or vestibules can be involved). It should be noted that malformations are different from anatomical variations that do not cause functional disorders [26,31,32]. The aim of this paper is to detect genetic causes and illustrate clinical and radiographic findings in two unrelated Italian boys affected by hearing loss.
2. Materials and methods 2.1. Clinical, audiological and imaging data A detailed family and clinical history was obtained from patients and their parents, in two unrelated Italian families (family 1 and family 2) carrying novel mutations in the SLC26A4 gene. Acquired and environmental factors, possibly related to hearing loss (HL), were investigated. The probands (proband 1 and proband 2) were two unrelated Italian boys affected by congenital mixed HL. Other clinical data were collected, including thyroid and renal function tests [35]. A complete audiological evaluation was carried out using microotoscopy, pure-tone (250–8000 Hz) audiometry for air and bone conduction, speech audiometry (or speech recognition threshold), impedenzometry (tympanometry and acoustic reflex threshold), behavioral audiometry and ABR. Hearing of probands was measured depending on the age of the patient. Speech recognition threshold (SRT) is the threshold of a person for speech at the lowest level at which the presence of speech signal can be heard or recognized or identified 50% of the time (patients have to comprehend the speech signal and then have to repeat the correct 50% speech stimulus). Severity of hearing impairment (HI) was classified as follows: <20 dBHL (decibel hearing level), normal hearing; 20–40 dBHL, mild HL; 41–70 dBHL, moderate HL; 71–95 dBHL, severe HL; and >95 dBHL, profound HL. The HL was bilateral in all affected individuals [36,37]. High resolution CT (computed tomography) and MRI (magnetic resonance imaging) were carried for all probands to obtain a radiological examination of temporal bone. Axial temporal bone CT and MRI findings were classified according to accepted criteria to define cochleovestibular malformations. EVA was defined if the diameter was greater than 1.5 mm at a midway point between the common crus and the external aperture. Mondini’s dysplasia or incomplete partition type II was defined as a cochlea consisting of one and a half turns, in which the middle and apical turns coalesce to form a cystic apex, accompanied by a dilated vestibule and EVA [11,36,37]. 2.2. Genetic and molecular analysis Informed consent was obtained from patients and parents according to current national rules and laws. Molecular genetic studies of the GJB2, GJB6, SLC26A4 genes and mitochondrial DNA (mit-DNA) were performed. Genomic DNA was extracted by standard protocols from peripheral blood leukocytes of all probands and other available members of the two families. PCR amplification of the coding 21 exons, their flanking and promoter regions of SLC26A4 gene was performed using specific primers. Amplification reactions were performed in a final volume of 25 ml containing 100 ng of genomic DNA, 200 mmol/l dNTPs, 10 mmol/l each primer, 1.5 mmol/l MgCl2, 1 U of Taq polymerase. After 5 min of denaturation at 94 8C, 35 PCR cycles were carried out, each cycle consisting of 45 s of denaturation at 94 8C, 45 s of annealing at 60 8C and 80 s of extension at 72 8C. Direct sequencing of the PCR products on both strands was performed on an ABI PRISM 3130xl sequencer, using ABI BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems by Life Technologies) [35]. 2.3. In silico analysis The evolutionary conservation of pendrin residues among eleven SLC26A4 orthologs was investigated at the ClustalW2 EMBLEBI web site (http://www.ebi.ac.uk/Tools/msa/clustalw2/). The pathogenicity of the intronic mutations was investigated using the NetGene2 Splice Site Prediction program (http://www.cbs.dtu.dk/
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services/NetGene2/). Other investigations were conducted using the Mobyle@Pasteur portal for bioinformatics analyses (http:// mobyle.pasteur.fr/cgi-bin/portal.py) [29]. 3. Results 3.1. Clinical, audiological and imaging data 3.1.1. Family 1 Case 1 reports a seven-year-old boy suffering from progressive bilateral hearing loss (proband 1). He is the first-born of an Italian family with no history of relevant diseases. Birth occurred at term by spontaneous vaginal delivery. No pre-, peri- or postnatal risk factors for hearing loss were identified. No other family member showed hearing impairment and thyroid diseases. Audiological tests revealed a bilateral and severe mixed hearing loss (with conductive and sensorineural components). The young patient, with otoscopically normal tympanic membranes and bilateral normal type A tympanograms, did not show a stapedial reflex at all frequencies in both ears. The degree of hearing loss was quite severe and the tonal audiogram depicted a downsloping, highfrequency bilateral hearing impairment (Fig. 1). On subsequent controls we found a progressive worsening in pure tone threshold. At the age of 4 years, we prescribed him behind-the-ear hearing digital aids. The patient’s aided threshold is about 40 dBHL at all frequencies in both ears. During speech tests, he correctly repeated 17/20 disyllabic words in open set. Phonological confusion occurs only for fricative and affricate consonants. The boy always uses an FM (frequency modulation) system during class. Furthermore, we performed genetic and neuroimaging investigations. The proband’s CT scans showed bilateral enlarged
Fig. 2. Proband 1 shows bilateral EVA (white arrows) on axial temporal bone CT scans (A, right ear = 2.3 mm; B, left ear 1.6 mm).
vestibular aqueduct, slightly more enlarged on the right side (Fig. 2). Thyroid function blood tests were normal (FT3: 4.37 pg/ml, FT4: 1.5 ng/dl, TSH: 0.573 mU/ml, TPO Ab: 3 IU/ml, anti-TSH receptor Ab: 0.3 IU/I). The survey was completed by ultrasound of the thyroid, kidney and urinary tract. Exams were all within normal ranges. 3.1.2. Family 2 Case 2 reports an 8-year-old hearing impaired boy (proband 2). He is the second-born of an Italian family with no history of relevant diseases. Birth occurred at 40 weeks of gestation and delivery was spontaneous. No pre-, peri- or postnatal risk factors for hearing loss were identified. The proband 2 presented a bilateral prelingual (less than 2 years of age) non syndromic hearing impairment, so he was bilaterally aided at the age of two years. A maternal aunt was diagnosed with severe hearing loss at the age of six years, probably due to an undocumented viral infection, as reported by family members. No others in the family 2 were affected by hearing loss and goiter.
Fig. 1. Pure tone audiometry of proband 1 with bilateral EVA and two SLC26A4 mutations (R409H/IVS2+1delG).
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Fig. 3. Pure tone audiometry of proband 2 with bilateral EVA and two SLC26A4 mutations (L236P/K590X).
Physical examination, otoscopy and tympanometry of proband 2 were normal. Pure-tone audiometry revealed a bilateral profound mixed hearing loss with a mild conductive component at middle and low frequencies (Fig. 3). Acoustic reflex were absent at all frequencies in both ears. The patient’s aided threshold is about 50 dBHL at all frequencies in both ears. Evaluation of speech processing at threshold levels has resulted in obtaining a speech recognition threshold (SRT). In this case unaided SRT is about 100 dBSPL (decibel sound pressure level) and aided SRT is about 50 dBSPL. During the speech test, he correctly repeated 16/20 disyllabic words in open set. Speech development was normal for the chronological age, with phonetic inventory in spontaneous speech sample being complete, except for fricative consonants. Following a detailed history and examination, he underwent genetic testing, including searching for GJB2, GJB6 and mitochondrial-DNA mutations. Imaging evaluation of temporal bone was performed by means of high resolution computer tomography (CT) and magnetic resonance imaging (MRI). Axial temporal bone CT (Fig. 4) and MRI showed bilateral EVA. Genetic investigation has been completed by searching for SLC26A4 mutations. Thyroid hormones levels dosage (FT3: 4.87 pg/ml, FT4: 1.47 ng/ dl, TSH: 1.32 mU/ml) were in the norm. The survey was completed by ultrasonography of the thyroid, kidney and urinary tract. Exams were all within normal ranges.
Fig. 4. Proband 2: on axial temporal bone CT scans, the vestibular aqueducts are enlarged bilaterally (white arrows), measuring 3.8 mm at the transverse midpoint in the right ear (A) and 2.9 mm at the transverse midpoint in the left ear (B).
3.2. Genetic and molecular analysis 3.2.1. Family 1 The proband 1 and all family members underwent molecular genetic analysis of the GJB2, GJB6 and SLC26A4 genes. Molecular genetics characterization has been performed as already described. Genetic analysis of the patient (proband 1) has revealed a compound heterozygous genotype for the R409H (exon 10) and IVS2+1delG (intron 2) mutations in the SLC26A4 gene. The father is a simple heterozygote for the R409H mutation and the mother is a simple heterozygote for the IVS2+1 delG mutation. They show normal hearing. The proband sister, who underwent prenatal genetic diagnosis, has a normal genotype (wt/wt). At 4 months of age the ABR results showed a normal auditory response. The family tree is shown in Fig. 5. 3.2.2. Family 2 The proband 2 underwent genetic testing, including searching for GJB2, GJB6 and mitochondrial-DNA mutations. After axial temporal bone CT and MRI, genetic investigation has been completed by searching for SLC26A4 mutations. The young patient has no mutations in GJB2, GJB6 and mitochondrial-DNA, but molecular analyses identified him as a compound heterozygote for two mutations in the SLC26A4 gene:
Fig. 5. Family tree of the proband 1 (R409H/IVS2 + 1delG) is compatible with autosomal recessive inheritance.
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Fig. 6. Family tree of the proband 2 (L236P/K590X) is compatible with autosomal recessive inheritance. A maternal aunt was diagnosed with severe hearing loss at the age of six years, probably due to an undocumented viral infection, as reported by family members.
Fig. 8. This figure illustrates the twelve transmembrane domains and the intracellular N-terminus and C-terminus of human pendrin; the C-terminal domain includes STAS domain (proximal and distal portion) and PKA site (in the distal portion) [38]; the approximate location of the K590X mutation is indicated by the black arrows.
Fig. 7. Multiple sequence alignment of pendrin from human, dog, rat, mouse, pig, monkey, chimpanzee, frog, zebrafish, chicken and cow. Amino acid residue K590 is conserved among orthologs.
L236P/K590X (exon 6 and exon 16). The family tree is shown in Fig. 6. 3.3. In silico analysis The evolutionary conservation of pendrin residues among eleven SLC26A4 orthologs was investigated at the ClustalW2. The SLC26A4 gene is conserved in human, dog, rat, mouse, pig, monkey, chimpanzee, frog, zebrafish, chicken and cow (Fig. 7). The splice site prediction program used indicated that IVS2+1delG would significantly affect splicing decreasing the score of the splice donor site from 0.90 in the wild type to 0.0 in the mutated variant. 4. Discussion and conclusions All probands have bilateral EVA and SLC26A4 mutations in both alleles. We found no other mutations in GJB2, GJB6 and mit-DNA. The clinical histories do not support other causes of deafness (infections, drugs, prematurity). The genetic tests revealed different genotypes with a quite similar phenotype: two compound heterozygotes (R409H/IVS2+1delG, L236P/K590X) with congenital progressive mixed hearing loss. The two studied probands with bilateral EVA showed a conductive component of hearing loss at 0.25, 0.5 and 1 kHz and alterations in stapedial reflexes; the conductive component matched the sensorineural hearing loss at 4 kHz (Figs. 1 and 3). These findings should suggest
a physical explanation, such as the third window phenomenon, for example [34]. No thyroid abnormalities were found, although we are aware that hearing impairment and thyroid dysfunction can develop at different times. R409H and L236P are well-known pathogenic SLC26A4 mutations responsible for autosomal recessive hearing loss [9,13,27–30]. The IVS2+1 delG mutation has never previously been described, but clinical and genetic data suggest a significant pathological role. Firstly the deletion of the intron 2 alters a canonical splice donor site. It is expected to have a pathogenic effect, overall if compared to the audiological findings. The mutation involves a partial conserved nucleotide; mutations in similar positions can result in defective splicing leading to variation in levels of normal or aberrant transcribed in different tissues. The family 1 tree is compatible with autosomal recessive inheritance (Fig. 5). The K590X mutation has never been described and it is expected to have pathological significance due to the stop codon determining an amino acid sequence of 589 amino acids (instead of 780) and leading to a shorter protein. The lysine (K) at position 590 is well preserved in different species (Fig. 7); furthermore the truncating mutation K590X should lead to the loss of most of the Cterminal domain, including the distal part of the sulfate transporter and antisigma factor antagonist (STAS) domain and the putative protein kinase A (PKA) phosphorylation site (Fig. 8). Elimination of the distal part of the (STAS) domain with the putative protein kinase A (PKA) phosphorylation site can result in: (1) a cytoplasmatic localization with a complete loss of function or (2) in a residual membrane insertion with a partial loss of function of pendrin [38]. Even in this case, the family 2 tree shows a pattern compatible with autosomal recessive inheritance (Fig. 6). In conclusion, our data suggest a significant pathological role for the IVS2+1 delG and K590X mutations even if a residual protein function for both mutations is possible. This aspect could explain the entity of hearing loss and the absence of thyroid dysfunction, even if differences in symptom onset time are possible. Further studies are needed to assess the genotype/phenotype correlations. Consent Written informed consent was obtained from the families for publication of these cases and any accompanying images.
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