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Previous Volume 352:1557-1564 April 14, 2005 Number 15 Next

Summary PDF PDA Full Text PowerPoint Slide Set Supplementary Material Editorial by Nadeau, J. H. Add to Personal Archive Add to Citation Manager Notify a Friend E-mail When Cited E-mail When Letters Appear Related Article PubMed Citation

SUMMARY

Five adult siblings presented with autosomal recessive sensorineural hearing loss: two had high-frequency loss, whereas the other three had severe-to-profound loss affecting all frequencies. Genetic evaluation revealed that a homozygous mutation in CDH23 (which encodes cadherin 23) caused the hearing loss in all five siblings and that a heterozygous, hypofunctional variant (V586M) in plasma-membrane calcium pump PMCA2, which is encoded by ATP2B2, was associated with increased loss in the three severely affected siblings. V586M was detected in two unrelated persons with increased sensorineural hearing loss, in the other caused by a mutation in MYO6 (which encodes myosin VI) in one and by noise exposure, suggesting that this variant may modify the severity of sensorineural hearing loss caused by a variety of factors.

We evaluated a family in which five siblings were affected by autosomal recessive sensorineural hearing loss. Despite the presumably shared cause of the disorder, there were clinically significant differences among the siblings in the severity of their hearing loss. We undertook this study to identify the cause of their sensorineural hearing loss and a potential genetic modifier of its severity. We then sought to determine whether this same modifier might account for variations in the severity of sensorineural hearing loss caused by other factors in unrelated persons.

Methods

Subjects

This study was approved by the institutional review board of the National Institute of Neurological Disorders and Stroke and the National Institute on Deafness and Other Communication Disorders, National Institutes of Health. All the participants gave written informed consent before participation. The participants were members of the LMG132 family, which is descended from European ancestors. Medical-history interviews, physical examinations, video nystagmography with caloric testing, and pure-tone and speech audiometry were performed. The Usher syndrome was ruled out by funduscopy and electroretinography.

Genetic Analysis

Genomic DNA was extracted from venous-blood samples (Puregene, Gentra Systems). DNA samples were genotyped for short tandem-repeat markers flanking known nonsyndromic recessive deafness loci, and all exons of CDH23 were sequenced essentially as described.³ Primer sequences and polymerase-chain-reaction amplification and sequencing conditions for ATP2B2, which encodes the plasma-membrane calcium pump PMCA2, are provided in Table 1 of the Supplementary Appendix (available with the full text of this article at www.nejm.org). Control DNA samples were obtained from Coriell Cell Repositories and consisted of Human Variation Panels HD01 through HD09, HD027, and HD100CAU (described by the repository as a panel of samples from "self-declared Caucasians"). Our laboratory collected additional normal control DNA samples from 14 unrelated persons with a variety of self-reported European ancestries.

PMCA2 Functional Assay

Expression vectors were constructed for the PMCA2a splice isoform of PMCA2, since the former is the predominant PMCA isoform expressed in hair bundles in bullfrogs and inner-ear neurosensory cells in rats.¹¹ Full-length human complementary DNA fragments encoding wild-type PMCA2a or PMCA2a with the V586M variant (in which methionine replaces valine at amino acid position 586) were sequenced in their entirety and subcloned into baculovirus expression vector pVL1392 (Invitrogen). Details of the cloning procedures are provided in the Methods section of the Supplementary Appendix. Preparation and amplification of recombinant baculovirus, expression of PMCA2a in Sf 9 cells, preparation of microsomes, and measurement of ATPase activity were performed as described elsewhere.¹² Equivalent amounts of the expressed wild-type and mutant proteins were used in their respective reactions. The free calcium concentration was calculated with the Maxchelator program (www.stanford.edu/~cpatton/maxc.html).

Calculation of ATP2B2^V586M Frequencies

Some of the samples used to calculate ATP2B2^V586M frequencies were derived from members of the same families. To avoid duplicative counting of alleles that were identical by descent among members of the same family, we examined each pedigree to deduce the numbers of independent wild-type and ATP2B2^V586M alleles. If we could not determine whether two sampled alleles were identical or not identical by descent, we defined minimum and maximum possible values, respectively, which were used to calculate high and low composite estimates of the frequency of ATP2B2^V586M in the entire group of samples. Since the frequency of the ATP2B2^V586M allele was low and no homozygotes were detected, carrier frequency was approximated by doubling the allele frequency.

Case Report

Five affected offspring (42 to 55 years of age) of a consanguineous union in Family LMG132 had autosomal recessive, nonsyndromic sensorineural hearing loss, with normal vestibular and retinal function (Figure 1A). All five siblings had severe-to-profound high-frequency sensorineural hearing loss that had begun, according to their recollection, in the first decade of life, after the initial development of speech and language, and that had steadily progressed to current levels during the subsequent decade. However, there were two different phenotypes among the siblings: Subjects II-4 and II-6 had normal low-tone hearing, whereas Subjects II-1, II-9, and II-10 had severe-to-profound low-frequency loss that had begun in the first or second decade of life and that had been followed by steady progression to current levels during the subsequent few decades (Figure 1D). The latter group relied on sign language, lip-reading, hearing aids, or a cochlear implant for communication, in contrast to the two siblings whose intact low-frequency and midfrequency hearing permitted oral and auditory communication without hearing aids. There was no history of exposure to aminoglycoside antibiotics, ototoxic noise levels, head trauma, or systemic or otic infections that could account for the sensorineural hearing loss in the five affected siblings.

View larger version (31K): [in this window] [in a new window]   Figure 1. CDH23 Genotypes and Phenotypes of Members of Family LMG132. Panel A shows the pedigree of five affected siblings, the offspring of a consanguineous union (double line), who were homozygous for the F1888S mutation (S) of CDH23. The 25-year-old child of a first cousin of the siblings had nonsyndromic, congenital, severe-to-profound sensorineural hearing loss affecting all frequencies (not shown). She was a compound heterozygote for F1888S and a novel frame-shift mutation (8882_8883insT) in CDH23. Solid symbols indicate persons with nonsyndromic sensorineural hearing loss, and symbols with a slash indicate deceased family members. Panel B shows electropherograms of wild-type (Subject II-5), heterozygous (Subject II-2), and homozygous (Subject II-10) genomic nucleotide sequences with respect to the missense mutation F1888S in exon 42 (arrows). Panel C shows the alignment of cadherin 23 amino acid sequences including and flanking F1888 (arrowhead) from Homo sapiens (Hs), Mus musculus (Mm), Rattus norvegicus(Rn), Gallus gallus (Gg), Danio rerio (Dr), and Tetraodon nigroviridis (Tn) (GenBank accession numbers AAG27034 [GenBank] AAG52817 [GenBank] NP_446096 [GenBank] , XP_421595 [GenBank] , NP_999974 [GenBank] , and CAG04741 [GenBank] respectively). The alignment program ClustalW was used. Identical residues are indicated by dark shading and conservatively substituted residues by light shading. Amino acids are denoted by their single-letter codes. Panel D shows pure-tone air-conduction thresholds for the better-hearing ear of Subjects II-1 through II-11. Bone-conduction thresholds were consistent with the presence of sensorineural hearing loss (data not shown). Arrows indicate that there was no response to a stimulus at the highest tested level. Normative 90th percentile pure-tone thresholds are from International Organization for Standardization publication ISO 7029.13 dB HL denotes decibels hearing level. Audiometric profiles are grouped according to CDH23 and ATP2B2 genotypes, where S denotes the F1888S mutation and M the V586M variant.

 
Results and Discussion

CDH23 Deafness in Family LMG132

All five affected siblings were homozygous for short tandem-repeat markers linked to CDH23 on chromosome 10q22.1 (data not shown). Genomic nucleotide-sequence analysis of CDH23 exons in the affected siblings revealed homozygosity for a point mutation (5663TC; GenBank accession number, AY010111 [GenBank] ) in exon 42, predicted to result in the substitution of serine for phenylalanine at amino acid position 1888 (F1888S; GenBank accession number, AAG27034 [GenBank] in the extracellular domain of cadherin 23 (Figure 1B). This phenylalanine residue is conserved in mouse, rat, and chicken cadherin 23 (Figure 1C) but is not located within the motifs involved in calcium-mediated intermolecular associations among cadherins.¹⁴ The CDH23^{F1888S/F1888S} genotype cosegregated with sensorineural hearing loss in Family LMG132, and the CDH23^F1888S mutation was not detected in 108 European ("Caucasian") control samples.

ATP2B2 as a Modifier of CDH23 Deafness in Family LMG132

A variety of recessive mutations of Cdh23 cause profound deafness and vestibular dysfunction in homozygous waltzer mice,¹⁵ whereas another allele of Cdh23, called ahl, underlies less severe, age-related hearing loss in many inbred mouse strains.¹⁶ The severity of this age-related hearing loss is significantly increased by heterozygosity for the dfw^2J deaf-waddler allele of Atp2b2,¹⁶ which encodes PMCA2, the predominant PMCA of hair bundles. This interaction has been attributed to a reduction in PMCA2 activity that results in a decrease in extracellular calcium concentrations around hair bundles, where calcium-dependent, cadherin-mediated adhesion is thought to occur.^17,18

We hypothesized that one or more alleles of ATP2B2 modify the severity of sensorineural hearing loss caused by CDH23^{F1888S/F1888S}. DNA samples from Subjects II-1, II-4, II-6, II-9, and II-10 were genotyped for short tandem-repeat markers linked to ATP2B2. The resulting haplotypes were consistent with a model in which a dominant allele of ATP2B2 (Figure 2A, black haplotype bar) exacerbates sensorineural hearing loss in a manner analogous to the interaction between the dfw^2J allele of Atp2b2 and the ahl allele of Cdh23 in mice. Nucleotide-sequence analysis of ATP2B2 exons in CDH23^F1888S homozygotes revealed that heterozygosity for a point mutation in exon 12 (2075GA; GenBank accession number, NM_001683 [GenBank] ) was linked to this haplotype. The 2075GA mutation is predicted to result in the substitution of methionine for valine at amino acid position 586 (V586M; GenBank accession number, NP_001674 [GenBank] ) in the T4–T5 intracellular catalytic loop of PMCA2 (Figure 2B). Molecular modeling of V586M based on the three-dimensional structure of the closely related sarcoplasmic reticulum calcium pump predicts that substitution with the sterically larger methionine side chain distorts packing underneath the ATP-binding interface or increases its projection from the external solvent-exposed surface of the nucleotide-binding domain (data not shown).

View larger version (33K): [in this window] [in a new window]   Figure 2. V586M Allele of ATP2B2. Panel A shows ATP2B2-linked short tandem-repeat marker (D3S3611, D3S3601, and D3S3589) haplotypes and ATP2B2 genotypes of each member of generation II of Family LMG132. Solid symbols indicate persons with severe-to-profound sensorineural hearing loss across all frequencies, and shaded symbols persons with normal low-tone hearing thresholds and severe-to-profound sensorineural hearing loss at high frequencies. Subjects II-1, II-9, and II-10, all of whom had severe-to-profound hearing loss affecting all frequencies, were heterozygous for the haplotype that cosegregates with V586M (M) (black bars). The green, blue, and open bars represent different haplotypes. Panel B shows electropherograms of wild-type (Subject II-4) and heterozygous (Subject II-10) genomic nucleotide sequences with respect to the missense substitution V586M in exon 12 of ATP2B2 (arrow). Panel C shows the alignment of amino acid sequences including and flanking V586 (arrowhead) of ATP2B2 orthologues and paralogues: Homo sapiens (Hs) PMCA2, Mus musculus (Mm) PMCA2, Rattus norvegicus (Rn) PMCA2, Oreochromis mossambicus (Om) PMCA2, H. sapiens PMCA1, R. norvegicus PMCA1, Oryctolagus cuniculus (Oc) PMCA1, Sus scrofa (Ss) PMCA1, Bos taurus (Bt) PMCA1, H. sapiens PMCA3, M. musculus PMCA3, R. norvegicus PMCA3, Procambarus clarkii (Pc) PMCA3, H. sapiens PMCA4, M. musculus PMCA4, and R. norvegicus PMCA4 (GenBank accession numbers NP_001674 [GenBank] , NP_033853 [GenBank] , P11506 [GenBank] , P58165 [GenBank] , P20020 [GenBank] , P11505 [GenBank] , Q00804 [GenBank] , NP_999517 [GenBank] , NP_777121 [GenBank] , Q16720 [GenBank] , NP_796210 [GenBank] , XP_343840 [GenBank] , AAR28532 [GenBank] NP_001001396, NP_998781 [GenBank] , and NP_001005871, respectively). The alignment program ClustalW was used. Identical residues are indicated by dark shading, and conservatively substituted residues by light shading. Amino acids are denoted by their single-letter codes. Panel D shows the calcium ATPase activity of wild-type PMCA2a and PMCA2aV586M in the absence or presence of 300 nM calmodulin. Three ATPase experiments were performed for each expressed protein from three microsomal preparations of two different expressed-protein preparations. Representative data are shown from one of the three experiments. The slope and standard error of the determination of activity were calculated by linear regression.

 
The valine residue at position 586 is completely conserved among mouse, rat, and fish PMCA2 orthologues, and either valine or a conservatively substituted residue (isoleucine) is present at this position in all known PMCA1 and PMCA3 amino acid sequences (Figure 2C). Mouse and rat PMCA4 has methionine at this residue, but PMCA2 has a faster calcium-activation time than PMCA4.¹⁹ Since up-regulation and relocation of PMCA1 and PMCA4 to stereocilia do not rescue auditory function in dfw^2J deaf-waddler mice, the faster calcium-activation time of PMCA2 may be required for normal hearing.¹⁷ When expressed as a recombinant baculovirus protein in Sf 9 cells, human PMCA2a^V586M has approximately 50 percent of the calcium ATPase activity of wild-type PMCA2a (Figure 2D).

Studies of heterozygous deaf-waddler mice demonstrate that partial loss of PMCA2 activity is expected for an allele that modifies, but is not itself sufficient to cause, hearing loss. The dfw allele product contains a pathogenic amino acid substitution in the T2–T3 cytoplasmic loop; this product retains approximately 30 percent of wild-type PMCA2 activity.²⁰ Atp2b2^dfw/+ mice have normal hearing thresholds, whereas mice that are heterozygous for loss-of-function alleles (dfw^2J and dfw^3J) of Atp2b2 have functionally significant sensorineural hearing loss on the same genetic background.¹⁸ Analogous to Atp2b2^dfw, ATP2B2^V586M is not itself a dominant deafness-causing allele, since two siblings with normal hearing in Family LMG132 (Subjects II-5 and II-11) were ATP2B2^V586M heterozygotes (Figure 1D and Figure 2A).

ATP2B2^V586M as a Modifier of Other Forms of Hearing Loss

To explore the phenotypic consequences of the ATP2B2^V586M allele further, we screened 57 affected members of unrelated families with various progressive hearing-loss phenotypes and identified ATP2B2^V586M in 1 subject. The affected ATP2B2^V586M heterozygote (Subject IV-6 described by Mohiddin et al.²¹) had age-related hearing loss associated with a dominant missense substitution of MYO6. She had low-frequency (0.25-, 0.5-, and 1-kHz) hearing loss that was more severe than would be predicted by linear regression estimates of sensorineural hearing loss as a function of age in her affected relatives (Figure 3A). We also identified three ATP2B2^V586M heterozygotes, all of European ancestry, among 128 (119 self-reported European) unaffected members of families with a variety of other phenotypes. Two of these ATP2B2^V586M carriers had normal hearing (Figure 3B and Figure 3C), but the third had a history of occupational and recreational noise exposure and high-frequency sensorineural hearing loss that was highly characteristic of noise-induced ototoxicity (Figure 3D). Four of the 125 subjects who did not carry the ATP2B2^V586M allele also had audiometric phenotypes consistent with noise-induced sensorineural hearing loss (data not shown). All four ATP2B2^V586M carriers with normal hearing (Subjects II-5 and II-11 of Family LMG132 [Figure 1D] and the two carriers described above [Figure 3B and Figure 3C]) reported that they had no history of noise exposure. Although we cannot conclusively correlate the sensorineural hearing loss in the carrier with noise exposure (Figure 3D) with the ATP2B2^V586M/+ genotype, it has been reported that heterozygosity for a null allele of Atp2b2 predisposes mice to noise-induced sensorineural hearing loss.⁸

View larger version (16K): [in this window] [in a new window]   Figure 3. Phenotypes of ATP2B2V586M Heterozygotes. Panel A shows the pure-tone air-conduction threshold responses as a function of age and grouped according to stimulus frequency for affected persons with a mutation of MYO6 that results in the substitution of arginine for histidine at amino acid position 246 (H246R). Open circles represent the hearing thresholds of a five-year-old affected person (Subject VI-6 described by Mohiddin et al.21), who also carried ATP2B2V586M, and closed circles the hearing thresholds of her affected relatives who were also carriers of H246R but who had wild-type ATP2B2. Cross-sectional age-related progression of sensorineural hearing loss among the H246R carriers who had wild-type ATP2B2 was approximated by linear regression analysis (dashed lines) of the thresholds.22 The arrow indicates an 8-kHz response threshold that is a 90-dB HL hearing level. dB HL denotes decibels hearing level. Panels B and C show pure-tone air-conduction thresholds for a 9-year-old girl and a 41-year-old woman, respectively, who were ATP2B2V586M/+; both had normal hearing and no significant history of noise exposure. Open circles indicate the right-side air-conduction threshold, and crosses indicate the left-side air-conduction threshold. Dotted lines indicate sex- and age-matched 90th-percentile air-conduction thresholds from International Organization for Standardization publication ISO 702913; normative threshold data are not available for children. Panel D shows pure-tone air-conduction thresholds for a 37-year-old man who was ATP2B2V586M/+ and who had mild and moderate-to-severe sensorineural hearing loss in his left and right ears, respectively. The notched configuration characteristic of noise ototoxicity is evident. Bone-conduction thresholds were consistent with sensorineural hearing loss (data not shown).

 
The allele frequency of ATP2B2^V586M was cumulatively estimated in the European members of Family LMG132 and these cohorts. The lowest and highest estimates of allele frequency for the entire group were 4 of 258 (1.6 percent; 95 percent confidence interval, 0.6 to 3.9 percent) and 5 of 218 (2.3 percent; 95 percent confidence interval, 1.0 to 5.2 percent), respectively. The differing estimates arose from ambiguities in the segregation, and thus the independence, of alleles within some pedigrees. The corresponding low and high heterozygous carrier frequencies were deduced to be 4 of 129 (3.1 percent; 95 percent confidence interval, 1.3 to 7.7 percent) and 5 of 109 (4.6 percent; 95 percent confidence interval, 2.0 to 10.3 percent), respectively. In agreement with these findings, we also detected ATP2B2^V586M in 4 of 87 normal "Caucasian" control samples from an independent source (Coriell Cell Repositories) (4.6 percent; 95 percent confidence interval, 1.9 to 11.2 percent). We did not detect ATP2B2^V586M in 87 normal Pakistani control samples, but we did detect ATP2B2^V586M in 3 of 84 DNA samples from a Human Diversity Panel (Coriell Cell Repositories) representing 10 ethnic backgrounds. All three ATP2B2^V586M/+ samples were from a subgroup of five Pima Indian samples in this panel, although we could find no literature on hearing loss in Pima Indians. These carrier frequencies are consistent with a potential role for ATP2B2^V586M or other alleles of ATP2B2 in the etiology of presbycusis.

Although the interaction of a heterozygous dfw allele with a hypomorphic Cdh23 allele in ahl strains of mice suggests that ATP2B2^V586M could act as a dominant modifier allele in humans, our results do not formally rule out a model in which it is a recessive modifier allele that, in combination with another haplotype in Family LMG132 (Figure 2A, green haplotype bars), exacerbates sensorineural hearing loss. It is possible that important sequence variants within noncoding regions of this allele were missed by our genomic sequencing protocol. Nonetheless, our study indicates that ATP2B2^V586M or other alleles of ATP2B2 may be general modifiers of a variety of human hearing-loss phenotypes that are due to genetic determinants, environmental factors, or combinations of these influences. Since CDH23 and MYO6 mutations and ototoxic noise directly affect sensory hair cells of the inner ear,^2,4,23 the effects of ATP2B2^V586M may be confined to sensorineural hearing loss characterized by pathologic processes affecting primarily the hair cell. Although audiometric differences in ATP2B2^V586M carriers are most obvious with respect to low-frequency hearing in Family LMG132 (Figure 1D) and in the family with a MYO6 mutation (Figure 3A), the lack of detectable high-frequency hearing in ATP2B2^V586M carriers in Family LMG132 (Subjects II-1, II-9, and II-10) and the sensorineural hearing loss in the ATP2B2^V586M carrier with noise exposure (Figure 3D) raise the possibility that ATP2B2^V586M can modify hearing loss at all frequencies. Additional studies are needed to address these questions and to provide accurate genetic, prognostic, lifestyle, and occupational (i.e., noise avoidance) counseling as well as communication-rehabilitation counseling based on ATP2B2 genotype results.

Supported by grants (GM28825 and DC04200, to Dr. Penniston) from the National Institutes of Health and by intramural research funds (1 Z01 DC000039-05, 1 Z01 DC000060-02, and 1 Z01 DC000064-02) from the National Institute on Deafness and Other Communication Disorders.

We are indebted to the families who participated in the study; to T. Friedman for support and critical review of the manuscript; to S. Ohliger for sequencing; to Z. Ahmed for genotyping; to M. Mastroianni and Y. Szymko-Bennett for audiologic evaluations; to R. Caruso, P. Sieving, and M. Kaiser for ophthalmologic evaluations; to M. Meltzer and D. Tripodi for the coordination of clinical evaluations; to E. Carafoli and E. Strehler for vectors; to B. Ploplis, P. Lopez, and R. Nashwinter for technical support; to E. Cohn, W. Kimberling, X.Z. Liu, and V. Street for materials and unpublished observations; and to D. Drayna for critical review of the manuscript.

Source Information

From the Section on Human Genetics (J.M.S., A.L., R.J.M.), the Section on Gene Structure and Function (Y.Y., A.J.G.), and the Hearing Section (A.C.M., A.J.G.), National Institute on Deafness and Other Communication Disorders, and the Cardiovascular Branch, National Heart, Lung, and Blood Institute (S.A.M., L.F.), National Institutes of Health, Rockville and Bethesda, Md.; the Department of Biochemistry and Molecular Biology (A.J.C., A.G.F.) and the Department of Anesthesiology (A.R.P.), Mayo Foundation, Rochester, Minn.; and the Neuroscience Center, Massachusetts General Hospital and Harvard Medical School, Boston (J.T.P.).

Address reprint requests to Dr. Griffith at the NIDCD, National Institutes of Health, 5 Research Ct., Rm. 2A-01, Rockville, MD 20850, or at griffita{at}nidcd.nih.gov.

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