Forms

Deafness, Autosomal Recessive 9 (DFNB9) via the OTOF Gene

  • Summary and Pricing
  • Clinical Features and Genetics
  • Citations
  • Methods
  • Ordering/Specimens
Order Kits
TEST METHODS

NGS Sequencing

Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
4203 OTOF$690.00 81479 Add to Order
Pricing Comment

Our most cost-effective testing approach is NextGen sequencing with Sanger sequencing supplemented as needed to ensure sufficient coverage and to confirm NextGen calls that are pathogenic, likely pathogenic or of uncertain significance. If, however, full gene Sanger sequencing only is desired (for purposes of insurance billing or STAT turnaround time for example), please see link below for Test Code, pricing, and turnaround time information.

For Sanger Sequencing click here.
Targeted Testing

For ordering targeted known variants, please proceed to our Targeted Variants landing page.

Turnaround Time

The great majority of tests are completed within 28 days.

Clinical Sensitivity

The clinical sensitivity of this test ranges from 2.3% to 10%, depending on the ethnicity of the patient. For example, pathogenic sequence variants in the OTOF gene account for 10% (16/160) of Japanese patients with severe to profound recessive nonsyndromic hearing loss who did not harbor causative variants in the GJB2 and SLC26A4 genes (Iwasa et a. 2013). On the other hand, pathogenic sequence variants in the OTOF gene may be responsible for 7% (7/52) of Brazilian families with prelingual autosomal recessive hearing loss (Romanos et al. 2009). In China, 6.8% (5/73) of families with temperature-sensitive auditory neuropathy were determined to have pathogenic sequence variants in the OTOF gene (Wang et al. 2010). In Turkey, 6.1% (3/49) of families with prelingual deafness and no detected pathogenic variants in the GJB2, GJB6, and MTRNR1 genes harbored disease-causing variants in OTOF (Duman et al. 2011). In Algeria, 6% (2/33) of families with recessive nonsyndromic hearing loss and no detected pathogenic variants in the GJB2 and TMC1 genes showed causative variants in the OTOF gene (Ammar-Khodja et al. 2015). In Korea, 3.2% (1/32) of families with nonsyndromic hearing loss and did not carry pathogenic sequence variants in the GJB2, SLC26A4, and POU3F4 genes or mitochondrial DNA were determined to have causative sequence variants in the OTOF gene (Choi et al. 2013). In Pakistan, disease-causing OTOF sequence variants have been detected in 2.3% (13/557) of patients with recessive congenital/prelingual severe to profound deafness (Choi et al. 2009).

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Deletion/Duplication Testing via aCGH

Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
600 OTOF$690.00 81479 Add to Order
Pricing Comment

# of Genes Ordered

Total Price

1

$690

2

$730

3

$770

4-10

$840

11-30

$1,290

31-100

$1,670

Over 100

Call for quote

Turnaround Time

The great majority of tests are completed within 28 days.

Clinical Features

Autosomal recessive deafness 9 (DFNB9) is characterized by two major features: prelingual, bilateral nonsyndromic hearing loss and less frequently, tempeture-sensitive nonsyndromic auditory neuropathy (TS-NSAN) (Iwasa et al. 2013). DFNB9-related deafness generally appears during the first two years of life as an auditory neuropathy, which is detected by electrophysiologic testing, wherein auditory brainstem responses (ABRs) are absent and otoacoustic emssions (OAEs) are present (Madden et al. 2012). However, OAEs eventually disappear, resulting in electrophysiologic testing that indicates a cochlear defect. It is important to distinguish auditory neuropathy from a cochlear defect because cochlear implants may be of marginal value in individuals with auditory neuropathy. TS-NSAN pertains to normal-to-mild hearing loss in the absence of fever or severe to profound hearing loss in the presence of fever. Hearing often returns to normal as fever resolves (Shearer et al. 2008). The global prevalence rate of DFNB9 remains unknown.

Genetics

DFNB9 is an autosomal recessive hearing disorder that is caused by pathogenic sequence variants in the otoferlin (OTOF) gene, which is mainly expressed in the inner hair cells of the cochlea and the brain, and plays a major role in synaptic exocytosis at the auditory ribbon synapse (Roux et al. 2006). The OTOF gene is 21 kb in size, located in chromosome 2p23.1, and consists of 48 coding exons that encode two isoforms. The long isoform contains six C2 domains and a C-terminal domain, whereas the short isoform comprises only the last three C2 domains (Chaib et al. 1993; Yasunaga et al. 1999, 2000). To date, a total of about 110 pathogenic OTOF sequence variants have been reported, which include missense/nonsense, splicing, small deletions, small insertions, small indels, and gross deletions (Human Gene Mutation Database).

Testing Strategy

For this NextGen test, the full coding regions plus ~20 bp of non-coding DNA flanking each exon are sequenced for the gene listed below. Sequencing is accomplished by capturing specific regions with an optimized solution-based hybridization kit, followed by massively parallel sequencing of the captured DNA fragments. Additional Sanger sequencing is performed for any regions not captured or with insufficient number of sequence reads. All pathogenic, likely pathogenic, or variants of uncertain significance are confirmed by Sanger sequencing.

Indications for Test

The ideal OTOF test candidates are individuals who present with congenital, bilateral, severe-to-profound, autosomal recessive nonsyndromic hearing loss.

Gene

Official Gene Symbol OMIM ID
OTOF 603681
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT

Disease

Name Inheritance OMIM ID
Deafness, Autosomal Recessive 9 601071

Related Test

Name
Nonsyndromic Hearing Loss and Deafness Sequencing Panel

CONTACTS

Genetic Counselors
Geneticist
Citations
  • Ammar-Khodja F. et al. 2015. Molecular Genetics and Genomic Medicine. 3: 189-96. PubMed ID: 26029705
  • Chaib H. et al. 1996. Human Molecular Genetics. 5: 155-8. PubMed ID: 8789454
  • Choi B.Y. et al. 2009. Clinical Genetics. 75(3): 237-43. PubMed ID: 19250381
  • Choi B.Y. et al. 2013. PLoS ONE. 8: e68692. PubMed ID: 23990876
  • Duman D. et al. 2011. Genetic Testing and Molecular Biomarkers. 15:29-33. PubMed ID: 21117948
  • Human Gene Mutation Database (Bio-base).
  • Iwasa Y. et al. 2013. BMC Medical Genetics. 14: 95. PubMed ID: 24053799
  • Madden C. et al. 2002. Archives of Otolaryngology, Head and Neck Surgery. 128:1026-30. PubMed ID: 12220206
  • Romanos J. et al. 2009. Journal of Human Genetics. 54: 382–385. PubMed ID: 19461658
  • Roux I. et al. 2006. Cell. 127: 277-89. PubMed ID: 17055430
  • Shearer A.E. et al. 2008. OTOF-Related Deafness. In: Pagon RA, Adam MP, Bird TD, Dolan CR, Fong C-T, Smith RJ, and Stephens K, editors. GeneReviews™, Seattle (WA): University of Washington, Seattle. PubMed ID: 20301429
  • Wang D.Y. et al. 2010. BMC Medical Genetics. 11: 79. PubMed ID: 20504331
  • Yasunaga S. et al. 1999. Nature Genetics. 21: 363-9. PubMed ID: 10192385
  • Yasunaga S. et al. 2000. American Journal of Human Genetics. 67: 591-600. PubMed ID: 10903124
Order Kits
TEST METHODS

NextGen Sequencing using PG-Select Capture Probes

Test Procedure

We use a combination of Next Generation Sequencing (NGS) and Sanger sequencing technologies to cover the full coding regions of the listed genes plus ~20 bases of non-coding DNA flanking each exon.  As required, genomic DNA is extracted from the patient specimen.  For NGS, patient DNA corresponding to these regions is captured using an optimized set of DNA hybridization probes.  Captured DNA is sequenced using Illumina’s Reversible Dye Terminator (RDT) platform (Illumina, San Diego, CA, USA).  Regions with insufficient coverage by NGS are covered by Sanger sequencing.  All pathogenic, likely pathogenic, or variants of uncertain significance are confirmed by Sanger sequencing.

For Sanger sequencing, Polymerase Chain Reaction (PCR) is used to amplify targeted regions.  After purification of the PCR products, cycle sequencing is carried out using the ABI Big Dye Terminator v.3.0 kit.  PCR products are resolved by electrophoresis on an ABI 3730xl capillary sequencer.  In nearly all cases, cycle sequencing is performed separately in both the forward and reverse directions.

Patient DNA sequence is aligned to the genomic reference sequence for the indicated gene region(s). All differences from the reference sequences (sequence variants) are assigned to one of five interpretation categories, listed below, per ACMG Guidelines (Richards et al. 2015).

(1) Pathogenic Variants
(2) Likely Pathogenic Variants
(3) Variants of Uncertain Significance
(4) Likely Benign Variants
(5) Benign, Common Variants

Human Genome Variation Society (HGVS) recommendations are used to describe sequence variants (http://www.hgvs.org).  Rare variants and undocumented variants are nearly always classified as likely benign if there is no indication that they alter protein sequence or disrupt splicing.

Analytical Validity

As of March 2016, 6.36 Mb of sequence (83 genes, 1557 exons) generated in our lab was compared between Sanger and NextGen methodologies. We detected no differences between the two methods. The comparison involved 6400 total sequence variants (differences from the reference sequences). Of these, 6144 were nucleotide substitutions and 256 were insertions or deletions. About 65% of the variants were heterozygous and 35% homozygous. The insertions and deletions ranged in length from 1 to over 100 nucleotides.

In silico validation of insertions and deletions in 20 replicates of 5 genes was also performed. The validation included insertions and deletions of lengths between 1 and 100 nucleotides. Insertions tested in silico: 2200 between 1 and 5 nucleotides, 625 between 6 and 10 nucleotides, 29 between 11 and 20 nucleotides, 25 between 21 and 49 nucleotides, and 23 at or greater than 50 nucleotides, with the largest at 98 nucleotides. All insertions were detected. Deletions tested in silico: 1813 between 1 and 5 nucleotides, 97 between 6 and 10 nucleotides, 32 between 11 and 20 nucleotides, 20 between 21 and 49 nucleotides, and 39 at or greater than 50 nucleotides, with the largest at 96 nucleotides. All deletions less than 50 nucleotides in length were detected, 13 greater than 50 nucleotides in length were missed. Our standard NextGen sequence variant calling algorithms are generally not capable of detecting insertions (duplications) or heterozygous deletions greater than 100 nucleotides. Large homozygous deletions appear to be detectable.   

Analytical Limitations

Interpretation of the test results is limited by the information that is currently available.  Better interpretation should be possible in the future as more data and knowledge about human genetics and this specific disorder are accumulated.

When Sanger sequencing does not reveal any difference from the reference sequence, or when a sequence variant is homozygous, we cannot be certain that we were able to detect both patient alleles.  Occasionally, a patient may carry an allele which does not amplify, due to a large deletion or insertion.   In these cases, the report will contain no information about the second allele.  Our Sanger and NGS Sequencing tests are generally not capable of detecting Copy Number Variants (CNVs).

We sequence all coding exons for each given transcript, plus ~20 bp of flanking non-coding DNA for each exon.  Test reports contain no information about other portions of the gene, such as regulatory domains, deep intronic regions or any currently uncharacterized alternative exons.

In most cases, we are unable to determine the phase of sequence variants.  In particular, when we find two likely causative mutations for recessive disorders, we cannot be certain that the mutations are on different alleles.

Our ability to detect minor sequence variants due to somatic mosaicism is limited.  Sequence variants that are present in less than 50% of the patient’s nucleated cells may not be detected.

Runs of mononucleotide repeats (eg (A)n or (T)n) with n >8 in the reference sequence are generally not analyzed because of strand slippage during PCR.

Unless otherwise indicated, DNA sequence data is obtained from a specific cell-type (usually leukocytes from whole blood).   Test reports contain no information about the DNA sequence in other cell-types.

We cannot be certain that the reference sequences are correct.

Rare, low probability interpretations of sequencing results, such as for example the occurrence of de novo mutations in recessive disorders, are generally not included in the reports.

We have confidence in our ability to track a specimen once it has been received by PreventionGenetics.  However, we take no responsibility for any specimen labeling errors that occur before the sample arrives at PreventionGenetics.

Deletion/Duplication Testing Via Array Comparative Genomic Hybridization

Test Procedure

Equal amounts of genomic DNA from the patient and a gender matched reference sample are amplified and labeled with Cy3 and Cy5 dyes, respectively. To prevent any sample cross contamination, a unique sample tracking control is added into each patient sample. Each labeled patient product is then purified, quantified, and combined with the same amount of reference product. The combined sample is loaded onto the designed array and hybridized for at least 22-42 hours at 65°C. Arrays are then washed and scanned immediately with 2.5 µM resolution. Only data for the gene(s) of interest for each patient are extracted and analyzed.

Analytical Validity

PreventionGenetics' high density gene-centric custom designed aCGH enables the detection of relatively small deletions and duplications within a single exon of a given gene or deletions and duplications encompassing the entire gene. PreventionGenetics has established and verified this test's accuracy and precision.

Analytical Limitations

Our dense probe coverage may allow detection of deletions/duplications down to 100 bp; however due to limitations and probe spacing this cannot be guaranteed across all exons of all genes. Therefore, some copy number changes smaller than 100-300 bp within a targeted large exon may not be detected by our array.

This array may not detect deletions and duplications present at low levels of mosaicism or those present in genes that have pseudogene copies or repeats elsewhere in the genome.

aCGH will not detect balanced translocations, inversions, or point mutations that may be responsible for the clinical phenotype.

Breakpoints, if occurring outside the targeted gene, may be hard to define.

The sensitivity of this assay may be reduced when DNA is extracted by an outside laboratory.

Order Kits

Ordering Options


myPrevent - Online Ordering
  • The test can be added to your online orders in the Summary and Pricing section.
  • Once the test has been added log in to myPrevent to fill out an online requisition form.
REQUISITION FORM
  • A completed requisition form must accompany all specimens.
  • Billing information along with specimen and shipping instructions are within the requisition form.
  • All testing must be ordered by a qualified healthcare provider.

SPECIMEN TYPES
WHOLE BLOOD

(Delivery accepted Monday - Saturday)

  • Collect 3 ml -5 ml (5 ml preferred) of whole blood in EDTA (purple top tube) or ACD (yellow top tube). For Test #500-DNA Banking only, collect 10 ml -20 ml of whole blood.
  • For small babies, we require a minimum of 1 ml of blood.
  • Only one blood tube is required for multiple tests.
  • Ship blood tubes at room temperature in an insulated container. Do not freeze blood.
  • During hot weather, include a frozen ice pack in the shipping container. Place a paper towel or other thin material between the ice pack and the blood tube.
  • In cold weather, include an unfrozen ice pack in the shipping container as insulation.
  • At room temperature, blood specimen is stable for up to 48 hours.
  • If refrigerated, blood specimen is stable for up to one week.
  • Label the tube with the patient name, date of birth and/or ID number.

DNA

(Delivery accepted Monday - Saturday)

  • Send in screw cap tube at least 5 µg -10 µg of purified DNA at a concentration of at least 20 µg/ml for NGS and Sanger tests and at least 5 µg of purified DNA at a concentration of at least 100 µg/ml for gene-centric aCGH, MLPA, and CMA tests, minimum 2 µg for limited specimens.
  • For requests requiring more than one test, send an additional 5 µg DNA per test ordered when possible.
  • DNA may be shipped at room temperature.
  • Label the tube with the composition of the solute, DNA concentration as well as the patient’s name, date of birth, and/or ID number.
  • We only accept genomic DNA for testing. We do NOT accept products of whole genome amplification reactions or other amplification reactions.

CELL CULTURE

(Delivery preferred Monday - Thursday)

  • PreventionGenetics should be notified in advance of arrival of a cell culture.
  • Culture and send at least two T25 flasks of confluent cells.
  • Some panels may require additional flasks (dependent on size of genes, amount of Sanger sequencing required, etc.). Multiple test requests may also require additional flasks. Please contact us for details.
  • Send specimens in insulated, shatterproof container overnight.
  • Cell cultures may be shipped at room temperature or refrigerated.
  • Label the flasks with the patient name, date of birth, and/or ID number.
  • We strongly recommend maintaining a local back-up culture. We do not culture cells.
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