Communicated by Jürgen Horst
A Novel Deletion in SMPX Causes a Rare form of X-Linked Progressive Hearing Loss in Two Families Due to a Founder Effect
Article first published online: 11 OCT 2012
© 2012 Wiley Periodicals, Inc.
Volume 34, Issue 1, pages 66–69, January 2013
How to Cite
Abdelfatah, N., Merner, N., Houston, J., Benteau, T., Griffin, A., Doucette, L., Stockley, T., Lauzon, J. L. and Young, T.-L. (2013), A Novel Deletion in SMPX Causes a Rare form of X-Linked Progressive Hearing Loss in Two Families Due to a Founder Effect. Hum. Mutat., 34: 66–69. doi: 10.1002/humu.22205
Contract grant sponsors: Canadian Foundation for Innovation (New Investigator Award no. 9384; Leaders Opportunity Fund no.13120); Canadian Institute of Health Research (CIHR 207714); Research and Development Corporation (RDC 207711); Genome Canada (Atlantic Medical Genetics and Genomics Initiative to T-L.Y.); CIHR-Regional Partnership Program (RPP) Fellowship (207716/207696 to N.A.).
- Issue published online: 20 DEC 2012
- Article first published online: 11 OCT 2012
- Accepted manuscript online: 21 AUG 2012 09:35AM EST
- Manuscript Accepted: 31 JUL 2012
- Manuscript Received: 27 APR 2012
- Canadian Foundation for Innovation. Grant Number: 9384
- Leaders Opportunity Fund. Grant Number: 13120
- Canadian Institute of Health Research. Grant Number: CIHR 207714
- Research and Development Corporation. Grant Number: RDC 207711
- CIHR-Regional Partnership Program. Grant Number: 207716/207696
- X-linked hearing loss;
- founder effect;
X-linked hearing loss is the rarest form of genetic hearing loss contributing to <1% of cases. We identified a multiplex family from Newfoundland (Family 2024) segregating X-linked hearing loss. Haplotyping of the X chromosome and sequencing of positional candidate genes revealed a novel point deletion (c.99delC) in SMPX which encodes a small muscle protein responsible for reducing mechanical stress during muscle contraction. This novel deletion causes a frameshift and a premature stop codon (p.Arg34GlufsX47). We successfully sequenced both SMPX wild-type and mutant alleles from cDNA of a lymphoblastoid cell line, suggesting that the mutant allele may not be degraded via nonsense-mediated mRNA decay. To investigate the role of SMPX in other subpopulations, we fully sequenced SMPX in 229 Canadian probands with hearing loss and identified a second Newfoundland Family (2196) with the same mutation, and a shared haplotype on the X chromosome, suggesting a common ancestor.
X-linked nonsyndromic deafness is relatively rare and is estimated to contribute to <1% of all cases of nonsyndromic deafness worldwide [Petersen et al., 2008] and accounts for approximately 5% of prelingual male deafness [Fraser, 1965; Petersen et al., 2008; Reardon, 1990]. Males often show earlier onset and more severe presentation than females due to X-inactivation [Dobyns, 2006]. Currently, there are five known loci for X-linked deafness (DFNX1, MIM# 304500; DFNX2, MIM# 304400; DFNX3, MIM# 300030; DFNX4, MIM# 300066; DFNX5, MIM# 300614). We identified a multiplex, extended pedigree (Family 2024) segregating nonsyndromic sensorineural hearing loss from the founder population of Newfoundland, Canada (Fig. 1A). Although rare, support for X-linked inheritance in Family 2024 was determined by the absence of male-to-male transmission, early-onset, and more severe phenotype in males than in females. Audiological data showed that males had a prelingual and severe, bilateral symmetrical sensorineural hearing loss that affected all frequencies; in contrast, females had a variable age of onset and severity.
Genomic DNA was isolated from peripheral leukocytes of 10 affected (III-3, III-7, IV-2, IV-27, IV-38, IV-41, V-4, V-9, VI-1, and VI-2) and 2 unaffected (V-5 and IV-43) family members from Family 2024 as previously described [Miller et al., 1988] and ethics approval obtained (Human Investigation Committee #01.186). A genotype scan of the X chromosome was performed on four affected family members (III-7; V-4; VI-1; VI-2) and one unaffected spouse (V-5) spanning three generations. Sixty-seven microsatellite markers were amplified and run on an ABI 3130xl or 3730 DNA Analyzer and analyzed using GeneMapper v4.0 (Applied Biosystems, Carlsbad, CA). Haplotype analysis of four affected individuals (III-7; V-4; VI-1; VI-2) revealed two shared regions on the X chromosome: (1) a 27.6 Mb region on Xp11.2–13.1 and (2) a 13.9 Mb on Xq22.1–23. Haplotyping extended family members (V-9; IV-41; IV-27) excluded the smaller region on Xq22.1–23 and two recombinations (V-9 and IV-41) reduced the 27.6 Mb region to 13.3 Mb, which spanned DFNX3–4 including 48 positional candidate genes (Supp. Table S1). Nucleotide numbering reflects cDNA numbering with +1 corresponding to the A of the ATG translation initiation codon in the reference sequence, according to journal guidelines (www.hgvs.org/mutnomen). The initiation codon is codon 1.
Thirteen candidate genes (Supp. Table S1) were sequenced based on mRNA expression within the cochlea (Morton cochlear cDNA library: http://www.brighamandwomens.org/bwh_hearing/human-cochlear-ests.aspx), protein function, previous association with hearing loss, and overlap with known DFNX loci [Robertson et al., 1994]. All sequencing variants were checked for cosegregation with the deafness phenotype and no pathogenic mutations were identified.
Two papers were recently published which identified SMPX as the causative gene at the DFNX4 locus in two Dutch families, [Schraders et al., 2011] one German family and one Spanish family [Huebner et al., 2011]. SMPX [MIM# 300226] was the third gene identified for X-linked hearing loss. DFNX1 was mapped in 1996 in a large British–American family with a congenital, profound, sensorineural, X-linked hearing loss [Reardon et al., 1991; Tyson et al., 1996] and PRPS1 [MIM# 311850] encoding phosphoribosyl pyrophosphate (PRPP) synthetase 1, identified as the responsible gene [Liu et al., 2010]. The transcription factor POU3F4 [MIM# 300039] was mapped as the DFNX2 gene (formerly DFN3) in a large Dutch kindred with progressive, mixed hearing loss with perilymphatic gusher during surgery of the stapes [Brunner et al., 1988; de Kok et al., 1995]. DFNX4 (DFN6) was mapped in a single Spanish family, with postlingual bilateral, sensorineural, and progressive hearing loss. The SMPX gene was originally cloned from skeletal muscle, encodes an 88-amino acid protein with two known isoforms (NM_014332.2 and NR_045617.1) and no known functional domains [Kemp et al., 2001; Patzak et al., 1999], and has an important role in protecting the sarcolemmar plasma membrane from being damaged by the mechanical stress that arises from continuous contraction of muscle cells [Ervasti, 2003]. The SMPX protein was suggested to have the same role protecting cells of the cochlea from the mechanical stress exerted during the process of hearing [Huebner et al., 2011; Schraders et al., 2011]. Absence or malfunction of the SMPX protein could result in cumulative damage of the inner hair cells, leading to a progressive hearing loss.
Although within the critical region determined in Family 2024, SMPX was low on our screening list as it is not expressed in the cochlea according the Morton cochlear cDNA library. Sequencing of the longest isoform of SMPX (NM_014332.2) revealed a novel base pair deletion in exon 3 (c.99delC:p.Arg34GlufsX47; Fig. 2A and B). This mutation segregated exclusively with the hearing loss phenotype of Family 2024 and was absent in dbSNP and in 117 ethnically matched population controls. This point deletion is predicted to cause a frameshift at amino acid residue R34 and creates a premature stop codon 47 amino acids downstream of the deletion. To test whether this premature stop codon would cause the mRNA to be degraded via nonsense mediated mRNA decay (NMD), total RNA was extracted from Epstein–Barr virus-transformed B lymphocytes from two affected males (III-7, V-9) and one affected female (IV-27) from Family 2024. RNA was isolated using Trizol reagent (Invitrogen by Life Technologies, Carlsbad, CA) and was followed by treatment with TURBO DNA-free DNase treatment (Ambion, Austin, TX). Complementary DNA (cDNA) synthesis was performed using superscript III Kit (Invitrogen by Life Technologies) and PCR was carried out using primers surrounding the c.99delC mutation in the SMPX gene. The SMPX protein is known to be highly expressed in skeletal muscle, heart, and inner ear tissue; but there is no previous evidence for the expression of SMPX in lymphoblastoid cells. We successfully amplified a 267 bp cDNA product from SMPX surrounding the c.99delC mutation (Fig. 2C) and determined that the mutant allele is expressed in lymphoblastoid cell cDNA (Fig. 2D), suggesting that the mRNA is not degraded and thus does not undergo nonsense mediated mRNA decay. The c.99delC mutation resides 33 bp upstream of the exon–exon junction of exon 3, the penultimate coding exon of SMPX. The mutant transcript is unlikely to undergo NMD as this deletion is less than 50–55 bp of the splicing-generated exon–exon boundary between exons 3 and 4 [Maquat, 2005]. To confirm that the mRNA does not undergo NMD, further quantitative experiments will be required. This mutation has been submitted to the Leiden Open Variation Database (LOVD, http://databases.lovd.nl/shared/individuals/00000133).
To investigate the role of SMPX in other unsolved families with hearing loss, we subsequently sequenced all exons and intron–exon boundaries of SMPX in 229 Canadian probands (NL = 127; The Hospital for Sick Children = 102) with sensorineural hearing loss and identified the c.99delC mutation in a second Newfoundland family (Family 2196, Fig. 1B). Genotyping markers spanning DFNX4 in these unrelated families indicate a common ancestor (Fig. 1C). Identifying two multiplex families with multiple affected males and females provided the opportunity to carefully examine the phenotype. The hearing loss in males appears to start in the first decade of life and affects all frequencies (Supp. Table S1A and B). Subjects VI-1 and V-9 (Family 2024) show hearing loss in the first decade of life and flat moderate hearing loss by age 2. Subject VI-1 passed the newborn hearing screening test, although this finding is not a definitive exclusion that the hearing loss in this case is not congenital, as he could have had a nondetectable mild loss when screened. Furthermore, the mid to high frequencies continue to deteriorate in the later years of the first and second decade (Supp. Fig. S1B and C) and, by the third and fourth decades, the high frequencies show a profound loss (Supp. Fig. S1C). The phenotype of males in Family 2196 (II-6 and V-1) are consistent with that of affected males in Family 2024; subject V-1 shows first-decade onset (Supp. Fig. S1D), and subject II-6 shows a severe hearing loss in low and mid frequencies with no measurable high-frequency hearing at age 74. Subject II-6 also reports that he had great difficulty with hearing in school and left school at age 8, which is consistent with the first-decade onset that was seen in subject V-1. In contrast, the phenotype in females from Family 2024 was highly variable. Subject VI-2 shows early onset of hearing loss, as demonstrated by a U-configuration or “cookie bite” at four years of age (Supp. Fig. S1E); her maternal parent's hearing loss (subject V-4) is gently sloping at age 35; subject IV-27 shows a steeply sloping loss at age 62. Successive audiograms of subject V-7 show late onset of hearing loss; this patient's hearing was normal at age 25, whereas mild-to-moderate unilateral right hearing loss appeared by age 28, and marked deterioration in both ears resulted in significant asymmetrical hearing loss by age 32. As SMPX is a muscle related protein, the previous studies have examined the potential for muscle weakness in mutation carriers. These studies have found that SMPX knockout mice show no overt muscle phenotype, suggesting a functional redundancy in muscle tissue [Palmer et al., 2001]. A study in humans also described a lack of muscle involvement with SMPX mutations, [Schraders et al., 2011] and no muscle weakness was noted in the proband or the proband's mother of Family 2024.
The complication of delayed diagnosis and treatment in SMPX mutation carriers is illustrated in subject III-7 (Family 2024) and subject II-6 (Family 2196), who both report functional difficulty by the end of the first decade. Both individuals left school at age 8 and are functionally illiterate, with low socioeconomic standing. Early identification of hearing loss and provision of a rehabilitative support system (hearing aids and FM hearing system) for school children has been successful in providing a favorable outcome for the patient. Frequent assessment of the hearing of children with early and severe loss is an important step to ensure that the hearing aid is appropriately adjusted as hearing loss progresses and one subject of Family 2024 has reported an improvement in hearing after he received a cochlear implant. The wide variability in the onset and severity of female patients makes it impossible to determine an appropriate monitoring schedule to ensure timely identification and treatment and may require frequent amplification device changes to meet hearing needs. The phenotype of the SMPX c.99delC carriers is similar to the phenotype of SMPX mutations carriers previously reported. [Huebner et al., 2011; Schraders et al., 2011; Weegerink et al., 2011] For example, males in previous studies of SMPX presented early, around 2 years of age, with bilateral slowly progressive hearing loss, whereas females with the same mutations showed a wide range of variability in the onset and severity of the disease much like that of the subjects observed in Families 2024 and 2196.
This study has identified a novel founder mutation in exon 3 (c.99delC) of SMPX that cosegregates in two Newfoundland families, and supports the previous association of SMPX to the DFNX4 locus. We also show that the c.99delC: p.Arg34GlufsX47 mutation in SMPX may undergo NMD, although further studies are needed to confirm this. It is also interesting to note that all the mutations associated with SMPX related hearing loss are null mutations resulting in a premature stop codon. Finally, we illustrate that this mutation may not play a significant role in the Canadian hearing loss population. Given that the Newfoundland population is of predominantly Northern European ancestry and these families have deep genealogies, this novel SMPX mutation may have originated in Northern Europe.
The project was conceived and experiments planned by T-L.Y, N.M., and N.A. Review of phenotypes and sample collection were performed by A.G., J.L.L., and T.S. Experiments were performed by N.M., N.A., T.B., T.S., and J.H. Data analyses were performed by T-L.Y, L.D., T.S., T.B., J.H., N.M., and N.A. The manuscript was written by T-L.Y., N.A., A.G., and L.D. All aspects of the study were supervised by T-L.Y.
We thank Family members for participating and Carol Negrijn and Mary Anderson for their involvement in gathering family information and coordinating testing. We also would like to thank the reviewers for their helpful comments and feel that the constructive criticism given during this process have greatly contributed to the quality of this manuscript.
Disclosure Statement: The authors declare no conflict of interest.
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