Published online September 1, 2006
PEDIATRICS Vol. 118 No. 3 September 2006, pp. 985-994 (doi:10.1542/peds.2005-2519)
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ARTICLE

Simultaneous Multigene Mutation Detection in Patients With Sensorineural Hearing Loss Through a Novel Diagnostic Microarray: A New Approach for Newborn Screening Follow-up

Phyllis Gardner, MDa, Eneli Oitmaab, Anna Messner, MDc, Lies Hoefsloot, PhDd, Andres Metspalu, MD, PhDe,f and Iris Schrijver, MDg

a Departments of Medicine
c Otolaryngology/Head and Neck Surgery and Pediatrics
g Pathology, Stanford University School of Medicine, Stanford, California
b Asper Biotech, Ltd, Tartu, Estonia
d Department of Human Genetics, Radboud University Medical Center Nijmegen, Nijmegen, the Netherlands
e Institute of Molecular and Cell Biology, University of Tartu/Estonian Biocentre, Tartu, Estonia
f MDC, UL, Tartu University Hospital, Tartu, Estonia


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
OBJECTIVE. The advent of universal newborn hearing screening in the United States and other countries, together with the identification of genes involved in the process of hearing, have led to an increase in both the need and opportunity for accurate molecular diagnosis of patients with hearing loss. Deafness and hearing impairment have a genetic cause in at least half the cases. The molecular genetic basis for the majority of these patients remains obscure, however, because of the absence of associated clinical features in ~70% (ie, nonsyndromic hearing loss) of patients, genetic heterogeneity, and the lack of molecular genetic tests that can evaluate a large number of mutations across multiple genes.

DESIGN. We report on the development of a diagnostic panel with 198 mutations underlying sensorineural (mostly nonsyndromic) hearing loss. This panel, developed on a microarray, is capable of simultaneous evaluation of multiple mutations in 8 genes (GJB2, GJB6, GJB3, GJA1, SLC26A4, SLC26A5 and the mitochondrial genes encoding 12S rRNA and tRNA-Ser[UCN]).

RESULTS. The arrayed primer extension array for sensorineural hearing loss is based on a versatile platform technology and is a robust, cost-effective, and easily modifiable assay. Because hearing loss is a major public health concern and common at all ages, this test is suitable for follow-up after newborn hearing screening and for the detection of a genetic etiology in older children and adults.

CONCLUSIONS. Comprehensive and relatively inexpensive genetic testing for sensorineural hearing loss will improve medical management for affected individuals and genetic counseling for their families.

Key Words: APEX • sensorineural hearing loss • newborn hearing screening • connexin • microarray • molecular diagnostic

Abbreviations: SNHL—sensorineural hearing loss • OMIM— Online Mendelian Inheritance in Man • APEX—arrayed primer extension • gDNA—genomic deoxyribonucleic acid • PCR—polymerase chain reaction

Hearing loss is a very common birth defect.1 In industrialized nations, ~1:1000 children are born deaf, and 1:300 children are born with a milder degree of hearing loss. Before adulthood, an additional 1:1000 become profoundly hearing disabled.2,3 Overall, hearing loss affects 6% to 8% of the population.1 Hearing loss can be because of environmental factors, genetic etiologies, or both. At least 50% of prelingual hearing loss is estimated to be because of genetic changes.4 The genetic basis of hearing loss, however, is exceptionally intricate.5 Genetic hearing loss can follow a pattern of autosomal dominant, autosomal or X-linked recessive, or mitochondrial inheritance. Different mutations in the same gene may cause dominant and recessive hearing loss, and in some cases deafness may be caused by mutations in 2 different genes from the same functional group. Additional clinical manifestations may enable the diagnosis of a syndrome. However, the molecular basis of hearing loss remains obscure in as many as 85% of cases where the hearing loss is because of a genetic cause. This constitutes a major unmet medical need. We have developed a microarray assay, which, by means of hybridization followed by single nucleotide extension, can determine the molecular genetic basis of nonsyndromic and/or syndromic sensorineural hearing loss (SNHL) because of mutations in several connexin genes, mutations in 2 genes of the SLC26 family, and mutations in 2 mitochondrial genes that contribute to hearing loss.

Mutations in the GJB2 gene (Online Mendelian Inheritance in Man [OMIM] No. *121011; www.ncbi.nlm.nih.gov/Omim/), which encodes the gap junction protein connexin 26, represent the most common known cause of autosomal recessive nonsyndromic SNHL. These mutations are responsible for the hearing loss in up to half of these cases in the United States, Europe, Australia, and Israel and have been reported in other populations as well.6 A list of all published mutations, as well as common polymorphisms in GJB2, is available for clinical correlation and interpretation at the Connexin Deafness home page (www.davinci.es/deafness/). Although up to half of the individuals with autosomal recessive nonsyndromic SNHL have GJB2 mutations, between 10% and 50% carry only 1 mutation.6 GJB6 (OMIM No. *604418), the gene adjacent to GJB2 on chromosome 13 that encodes the connexin 30 protein, carries a common ~309 kilobase (kb) deletion, which causes nonsyndromic SNHL when homozygous or when present on the opposite allele of a GJB2 mutation.6,7 Other connexin genes with mutations that may contribute to SNHL include GJB3 (connexin 31) and GJA1 (connexin 43).

Mutations in 2 genes of the SLC26 anion transport gene family are also implicated as the etiology of hearing loss. The first are the SLC26A4 gene (OMIM No. *605646), mutations, which are associated both with autosomal recessive nonsyndromic SNHL and with Pendred syndrome.8–10 This is one of the most common forms of syndromic deafness, but likely underdiagnosed, because it is characterized by late-onset and reduced penetrance of some typical features, particularly the typically euthyroid goiter.9 Pendred syndrome is associated with Mondini dysplasia of the cochlea (the cochlea has an incomplete number of turns) with enlargement of the vestibular aqueduct. These clinical signs can be evaluated by computed tomography or MRI studies, but they are not always evident. The SLC26A5 gene (OMIM No. +604943), also in the SLC26 family, encodes the prestin protein. This gene has been reported to carry mutations associated with nonsyndromic SNHL.11

Mitochondrial DNA mutations are also included on the APEX array. Currently, it is estimated that these mutations are present in ~3% of patients with SNHL, but it is expected that this number will increase as genetic testing becomes more readily available and used.12,13 Mitochondrial DNA mutations can cause deafness by themselves but also predispose to irreversible hearing loss resulting from aminoglycoside ototoxicity.

We describe a molecular diagnostic assay, relying on a new use of the arrayed primer extension (APEX) technology,14 able to detect 198 different mutations associated with hereditary SNHL. First described by Shumaker et al15 in 1996 and subsequently converted to an array format,14 this type of assay is based on primer extension by a single nucleotide at the sites of interest. Because mutations in the connexin genes, the SLC26 anion transport genes, and the mitochondrial genes can be included and analyzed simultaneously, the SNHL microarray is expected to provide many more patients with a molecular diagnosis, which can facilitate hearing loss management and family counseling. This test constitutes a novel, much more comprehensive option in how we can diagnose children with hearing loss and is the first of its kind.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mutation Selection
The 198 mutations on the APEX microarray were selected and characterized from multiple sources including the following: (1) the Connexin Deafness home page (http://davinci.crg.es/deafness/), (2) mitochondrial mutation literature and the Mitomap database (www.mitomap.org/),12 (3) Hereditary Hearing Loss home page (http://dnalab-www.uia.ac.be/dnalab/hhh/), and (4) Human Gene Mutation Database (www.hgmd.cf.ac.uk/). The full set of mutations is listed in Table 1.


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  		TABLE 1 Complete List of gDNA Sequence Variants Detectable With the SNHL APEX Assay

 
Oligonucleotide Microchips
An overview of the development of the SNHL APEX array is presented in Fig 1. The appropriate wild-type gene sequences for both the sense and antisense directions (www.ncbi.nlm.nih.gov/Genbank/) were used as templates for oligonucleotide primer design. The 25-bp oligonucleotides have 6-carbon amino linkers at their 5' end and are obtained from MWG (Munich, Germany). Most of these oligonucleotides are designed to extend by 1 base pair (bp) in the wild-type and mutant sequence, except when deletions or insertions are present that have the same nucleotide in the 1 bp direction as the expected wild-type sequence. In such instances, oligonucleotides are designed to extend further into the deletion or insertion to enable accurate discrimination of the nucleotide change.


Figure 1
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  		FIGURE 1 Schematic depiction of the APEX development and process. The steps for development of the SNHL APEX array are demonstrated. For those steps in which gDNA was used, individual times are listed to reflect the time to assay completion in a research or clinical setting. Hands-on time of the assay is ~1 hour and 25 minutes.

 
The microarray slides are coated with 3-aminopropyl-trimethoxysilane plus 1,4-phenylenedi-isothiocyanate (Asper Biotech Ltd). The primers are diluted to 50 µM in 100 mM of carbonate buffer (pH 9.0) and spotted onto the activated surface with BioRad VersArray (BioRad Laboratories, Hercules, CA). The slides are subsequently blocked with 1% ammonia solution and stored at 4°C until needed. Before the APEX reactions, the slides are washed with 95°C distilled water and 100 mM of NaOH to reduce background fluorescence and to prevent rehybridization of unbound oligonucleotides to the APEX slide.

Genomic and Synthetic Template Samples
We designed 45-bp synthetic templates for each mutation site and collected 22 genomic DNA (gDNA) samples extracted from peripheral blood with a total of 39 sequence variants represented on the microarray from individuals known to carry such variants. Both genomic and synthetic templates that carry mutations present on the APEX array were used to validate each primer site. We continue to test gDNA on the array when samples with different mutations become available.

The synthetic templates were designed according to the mutant gene sequence in both the forward and reverse direction. The design was optimized for melting temperature (MWG). In our hands, synthetic templates have been demonstrated to be a valid alternative for genomic patient DNA in the APEX assay.16 For the gDNA samples, the SNHL genes of interest were amplified in predesigned amplicons. The 50-µL polymerase chain reaction (PCR) was optimized, purified, and fragmented as described previously.14 Before PCR amplification and subsequent microarray analysis, all of the gDNA samples were anonymized to ensure blind analysis. The results of our synthetic template validation were compared with the results obtained with the gDNA samples.

APEX Reactions
The APEX mix consists of 32 µL of fragmented PCR product, 5 U of Thermo Sequenase DNA polymerase (Amersham Pharmacia Biotech Inc, Milwaukee, WI), 4 µL of Thermo Sequenase reaction buffer (260 mM of Tris-HCl [pH 9.5] and 65 mM of MgCl2; Amersham Pharmacia Biotech Inc), and 1 µM of final concentration of each fluorescently labeled ddNTP: FL12-ddUTP, Cy3-ddCTP, Texas Red-ddATP, and Cy5-ddGTP (PerkinElmer Life Sciences, Wellesley, MA, and custom synthesis by Amersham Pharmacia Biotech Inc). The DNA is first denatured for 10 minutes at 95°C. The enzyme and the dyes are then added to the DNA, and the mixture is applied to prewarmed slides. The reaction is allowed to proceed for 10 minutes at 58°C, followed by 1 washing step with 0.3% Alconox (Alconox Inc, White Plains, NY) and 2 with distilled water for 90 seconds at 95°C. A small drop of antibleaching reagent (AntiFade SlowFade, Molecular Probes Europe BV, Leiden, Netherlands) is applied to the slides before imaging.

Analysis
For each mutation site, forward and reverse oligonucleotides are spotted onto the APEX array. To vastly reduce the possibility of interpreting any false signal as positive, the forward and reverse oligonucleotides are spotted in duplicate. Thus, every mutation site corresponds with 4 data points for final interpretation. This approach also enables distinction between homozygosity and heterozygosity. The array images are captured by a Genorama QuattroImager 003 detector (Asper Biotech Ltd) at a resolution of 20 µm. This imager combines a total internal reflection fluorescence-based excitation mechanism with a charge-coupled device camera.17 Sequence variants are explicitly identified using Genorama 3.0 genotyping software.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
We selected 198 sequence variants for a diagnostic mutation panel that is both comprehensively compatible with the currently characterized mutations involved in the etiology of SNHL and diagnostically practical in terms of the number of mutations included. The goal is to create an assay that provides a molecular diagnosis for patients affected with SNHL and allows carrier detection of recessive SNHL. Carriers have 1 expected heterozygous mutation in the entire array, whereas hearing impaired individuals are expected to carry 2 mutations when the SNHL is recessive and 1 mutation when inheritance is dominant. If a patient carries 2 mutations that are both present on the array, either compound heterozygosity at 2 mutation sites or homozygosity at 1 mutation site could be observed.

Sequence variants were selected from a variety of databases including the Connexin Deafness home page, the Hereditary Hearing Loss home page, the Mitomap database, and the literature therein. The mutations represent the most common reported mutations across population groups and include mutations associated with nonsyndromic SNHL (the most frequently encountered form), as well as syndromic SNHL. Usher syndrome mutations were not included, because a separate APEX microarray is available for this group of disorders.18 The mutation list includes single nucleotide substitutions, which are, from a technical perspective, the most straight-forward to detect with the APEX reaction. It also includes insertions and deletions, including the ~309-kb deletion affecting GJB6 (Table 1).

A few controversial mutations of uncertain or evolving clinical significance are included in this prototype. Examples include V27I, M34T, V37I, and E114G in the GJB2 gene and IVS2-2A>G in the SLC26A5 gene.19 Sequence variants in the GJA1 gene, which encodes connexin 43, are typically conductive rather than sensorineural. The 2 mutations that have been associated with sensorineural deafness, L11F and V24A, may in fact be located in a closely related pseudogene.20 Nevertheless, as our knowledge of the implicated genes and mutations evolves, this array platform is highly advantageous because of its versatility. APEX arrays are very flexible, and mutations can easily be added or deleted in future versions. Representative results of the current SNHL array are presented in Fig 2. Figure 2A demonstrates results of several individuals at the array spots for the R143W mutation in the GJB2 gene, which is common in individuals of African descent.21 In Fig 2B, results from the grid position for the 35delG mutation are displayed. 35delG is the most commonly identified mutation in the GJB2 gene. This mutation causes a deletion of a single G in a string of 6 and is reliably identified by the SNHL array in both the forward and reverse direction.


Figure 2
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  		FIGURE 2 Examples of APEX mutation detection of a missense mutation (R143W) and a deletion (35delG) in the GJB2 gene. A, R143W. For a normal allele, a signal for the sense (S) oligonucleotide is expected in the C channel and for the antisense (AS) oligonucleotide in the G channel (C/G). For a mutant allele, a signal is expected for the sense oligonucleotide in the T channel and for the antisense oligo in the A channel (T/A). Row 1, Normal genotype for position R143. The normal C allele is present in S. In AS, the normal G allele is detected (C/G). Row 2, Heterozygous for R143W. The normal C and mutant T alleles are detected in S. In AS, the normal G allele and the mutant A allele are present (CT/GA). Row 3, Negative control (no DNA added to the APEX reaction). B, 35delG. For a normal allele, a signal for the sense oligonucleotide is expected in the G channel and for the antisense oligo in the C channel (G/C). For a mutant allele, a signal for the sense oligonucleotide is expected in the T channel and for the antisense oligo in the A channel (T/A). Row 1, Heterozygous for 35delG. In S, the normal G allele and the mutant T allele are detected. In AS, the normal C allele and the mutant A allele are identified (GT/CA). Row 2, Homozygous for 35delG. Row 3, Normal genotype for nucleotide position 35. Row 4, Negative control.

 
gDNA Controls for Validation
The SNHL APEX microarray was validated in a pilot study by means of 22 gDNA patient samples with a total of 39 sequence variants. Of these, 37 were unique. Two occurred twice. Of the 37 individual mutations, 18 were in the GJB2 gene, 1 in GJB6, and 18 in SLC26A4. The assay was shown to be highly specific, as no false-positive mutations were detected. Specificity, however, depends on the labels on the dNTPs, which are not interchangeable in this assay and should be evaluated and optimized for each APEX array. We found 1 unexpected homozygous result for mutation IVS1 + 1G>A in SNHL-9 (Table 2). The homozygous result was reproduced twice more with the same PCR product on APEX analysis. Unfortunately, the necessary inclusion of dUTP in the PCR amplification before APEX analysis precludes the same sample to be analyzed by direct DNA sequencing. On reamplifications of the original DNA for the purpose of another APEX analysis and a sequence analysis, both the DNA sequence and all of the performed APEX analyses demonstrated heterozygosity. By exclusion, we conclude that there was allele dropout in the original PCR amplification.22 This has not been observed during the development of any of the existing APEX arrays and only occurred this once in development of the SNHL array. Allele dropout is a very rare risk inherent to any method that uses PCR and unrelated to the validation of microarray positions or conditions of the APEX reaction itself. Nevertheless, taking into account this event, specificity (true-negatives/true-negatives + false-positives) was calculated as 97% (32/32 + 1). Because this is not a true false-positive, however, true calculated specificity should be considered to be 100%. In 2 instances, gDNA samples carried amino acid changes not present on the array (I203T in SNHL-1 and V653L in SNHL-22, as identified by sequencing). Sequence changes I203T in the GJB2 gene and V653L in the SLC26A4 gene were not included on the array, because V653L is a known polymorphism, and I203T is listed in the category of polymorphisms on the Connexin Deafness home page as "clinical significance not determined." In both cases, a sequence change of the same amino acids is, in fact, located on the microarray (I203K in GJB2 and V653A in SLC26A4, respectively). The 2 changes present in the gDNA samples did not result in false-positive signals on the APEX array, which further underscores specificity of the APEX assay. Sensitivity of the assay was 100%, and no false-negatives were observed.


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  		TABLE 2 Genomic DNA Samples Used for Mutation Evaluation on the APEX Array

 
Synthetic Validation Controls
Because validity of the approach had been demonstrated, in principle, in the previously created cystic fibrosis APEX assay,16 and because patient samples could not be obtained for the majority of mutations present on the APEX array, the detection of all of the mutations was validated with 45-mer synthetic oligomers. Their sequence was based on the wild-type sequence, but with incorporation of the mutation to be identified. This approach allows validation of the APEX reaction and mutation detection with the Genorama QuattroImager but cannot assess optimization of the PCR steps, which are required for gDNA samples, because synthetic templates are not amplified.

Thus, with this limited pilot validation series, sensitivity (true-positives/true-positives + false-negatives) was 100%, and specificity was 100% (discounting 1 PCR failure described above). The APEX reactions are entirely reproducible under our optimized testing conditions. Repeat testing at each site on the array for a heterozygous 35delG sample produced identical results in all 8 of the repeats performed. The results described constitute the outcome of a pilot study of the effectiveness of the APEX approach in the detection of mutations that cause SNHL. A larger study of blinded patient samples will be conducted to further demonstrate sensitivity, specificity, and clinical utility.

Assay Workflow
Quality and reproducibility of results, assay workflow, hands-on labor, other costs, and turn-around time are among the most important parameters that determine the applicability of a diagnostic assay. The APEX SNHL assay can be completed in ~6 hours (Fig 1). Individual steps include PCR amplification (3 hours), PCR product purification (20 minutes), DNA fragmentation (1 hour), isothermic APEX reaction (15 minutes), visualization of results (6 minutes), and analysis and initial interpretation of results (1 hour). Hands-on time required for the assay is ~1 hour and 25 minutes. Sample preparation and analysis are expected to be automated in the future. It is important to note that all of the steps up to the results display on the QuattroImager reader can be performed in parallel. Thus, the number of imagers per test site determines the number of samples that can be run per day. Given a basic analysis time of 6 or 7 minutes for each microarray, the throughput in 8 hours would be ~60 to 70 patients per individual imager.


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The APEX microarray technology has been applied by us for conditions such as cystic fibrosis16 and by others for Usher syndrome,18 for the detection of mutations in the ß globin and G6PD genes (the Thalassochip for Mediterranean populations),23 and for the detection of >400 mutations, associated with 5 distinct retinal phenotypes, in the ABCA4 gene.24 The APEX technology is accurate, reproducible, and technically robust, and as increasing indications are developed, APEX microarrays will become a "platform" technology. APEX is ideal for the molecular diagnosis of SNHL, which can be because of multiple mutations in multiple genes.

Because of the lack of a multigene diagnostic for SNHL, our experience with the cystic fibrosis APEX microarray, and the technical advantages of APEX technology, we developed the APEX SNHL microarray with 198 mutations from 6 nuclear and 2 mitochondrial genes, as presented in Table 1. These mutations represent the current knowledge about the most frequently involved genes underlying genetic SNHL. At present, molecular genetic SNHL testing in research and diagnostic laboratories is conducted by a variety of technologies that we believe are not as cost-effective, efficient, robust, or comprehensive as the APEX SHNL array. Most diagnostic laboratories currently only test for SNHL mutations in the GJB2 gene. Among screening and detection methods, DNA sequencing is the most comprehensive and definitive method, because almost all types of mutations can be detected. However, sequencing is both expensive and labor intensive and not well suited for application to numerous genes simultaneously. As a consequence, only 1 gene or a few genes may be tested (sequentially). In addition, to the advantage of more comprehensive testing, the APEX platform is flexible, and the chip can be adjusted to evolving research and clinical needs. More or different oligomers can easily be added to future versions of the APEX array, which will allow detection of newly identified mutations in known genes or in newly identified genes that contribute to hearing loss when affected by mutations.

There are many excellent reasons to consider molecular diagnostic genetic testing in both children and adults with hearing loss.25 These include the ability to determine, by minimally invasive means, the etiology of nonsyndromic forms of hearing loss, which cannot be identified by other means. Syndromes that lack clinical features (other than hearing loss) early in life can also be identified. Once the etiology of the hearing loss has been identified, patients can benefit from accurate genetic counseling, and in some cases the treatment may be affected, with a substantial possible benefit for development. Furthermore, molecular testing is now much more informed and relevant because of the recent advances in hearing loss research, molecular diagnostic technologies, newborn screening, and treatment. Through Universal Newborn Hearing Screening, endorsed by the Joint Committee on Infant Hearing in 2000,26 hearing loss is now frequently identified in the neonatal period, throughout the United States. Universal Newborn Hearing Screening, however, is only 1 step in the optimum treatment of congenital SNHL. There are currently no universal guidelines for the tracking of infants who fail newborn hearing screens and the evaluation and treatment of those identified with hearing loss, including molecular diagnostic testing. Once the hearing loss is identified, early intervention with hearing aids and/or cochlear implant can significantly improve the ability to communicate and contribute to the quality of life for many hearing-impaired individuals.27

Microarray-based genetic testing for hereditary hearing loss will become clinically available in the very near future and can benefit patients through the potential of early, efficient, and definitive diagnosis of the underlying origin of the hearing loss. This type of more comprehensive testing, when cost-effective, is likely to become the standard of care. Microarray-based testing is expected to be ordered by pediatricians, otolaryngologists, geneticists, and other clinicians and could be integrated into more standardized follow-up after newborn hearing screening, where clinically indicated. It is important to stress that the selection of mutations included in this pilot study and other microarrays alike will, at least initially, be somewhat arbitrary. It needs to be emphasized that although a large number of variants can be included in microarrays, a negative result in a given patient does not indicate that the hearing loss is not of genetic origin. To optimally address the power and limitations of genetic testing for the identification of underlying causes of hearing loss, the explanation of results best takes place in the setting of genetic counseling.

Several hundred genes are involved in the biology of hearing and may contribute to hearing loss when mutations are present. In addition, although this array is intended to be comprehensive, more studies across different populations need to be conducted to better characterize mutation frequencies and identify those sequence variants, not currently known, that are frequent in distinct populations. Relevant founder effects may exist in ethnic groups or genetically isolated populations.

Despite the current limitations of any microarray for sensorineural hearing loss, however, we believe that the APEX SNHL microarray can meet the medical need for establishment of molecular causation in a large number of cases and possibly double the current mutation detection rate. When more information about mutation frequencies and distribution becomes available through larger diagnostic platforms, such as this APEX array, genotype-phenotype correlations can be improved, and an assessment of the clinical significance of individual mutations or their combinations will become more accurate.


   ACKNOWLEDGMENTS
 
This work was supported in part by a Pediatric Health Research Fund grant to Dr Messner by the Department of Pediatrics of Stanford University School of Medicine. Dr Metspalu is supported by Estonian MRE 0182582s03.

We thank Dr Tiina Kahre at the Molecular Diagnostics Centre of United Laboratories, Tartu University Clinics, Estonia, for the contribution of gDNA samples for validation of the SNHL APEX array. We are grateful to Dr Hannie Kremer at the Department of Otorhinolaryngology, Radbout University Medical Center, Nijmegen, the Netherlands, for helpful discussion.


   FOOTNOTES
 
Accepted Apr 11, 2006.

Address correspondence to Iris Schrijver, MD, Department of Pathology, L235, Stanford University Medical Center, 300 Pasteur Dr, Stanford, CA 94305. E-mail: ischrijver{at}stanfordmed.org

The authors have indicated they have no financial relationships relevant to this article to disclose.


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 METHODS
 RESULTS
 DISCUSSION
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