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Deafness, X-Linked 2 (DFNX2) via the POU3F4 Gene

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

Sequencing

Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
1553 POU3F4$540.00 81479 Add to Order
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 18 days.

Clinical Sensitivity

A small cohort study involving 32 familial nonsyndromic hearing loss cases showed that 15.6% of the causative variants were in the POU3F4 gene (Choi et al.2013). Another study involving 17 patients showing clinical features of classical DFN2 showed that 13 patients harbored variants in the POU3F4 gene (de Kok et al. 1996). The analytical sensitivity of this test is expected to be high because most POU3F4 mutations reported to date are expected to be detected by direct sequencing of genomic DNA.

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Deletion/Duplication Testing via aCGH

Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
600 POU3F4$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

X-linked deafness-2 (DFNX2), also referred to as conductive deafness with stapes fixation (DFN3), pertains to a progressive, mixed (includes conductive and sensorineural), prelingual, nonsyndromic hearing loss involving all sound frequencies (Merry et al. 1989; Waryah et al. 2011). This hearing disorder involves abnormalities involving the temporal bone of the inner ear, which is easily detected through computed tomography (CT) examination (Vore et al. 2005; Altay et al. 2008; Santos et al. 2014). The observed anomalies involving the bony labyrinth of the inner ear are usually described as pseudo-Mondini stage II dysplasia (Gharib et al. 2012; Parzefall et al. 2013). Other features of DFNX2 include partial hypoplasia of the cochlea, as well as dilation of the internal auditory canal. DFNX2 also presents with poor transmission of sound between the lateral aspect and the cochlear basal turn (Kumar et al. 2003; Chee et al. 2006). The perilymphatic fluid of the inner ear also creates an outward pressure on the oval window, which inhibits the transmission of vibrations across the stapes bone (Merry et al. 1989; Naranjo et al. 2010). The footplate of the stapes bone and the oval window also show abnormalities that compromise the movement of the ossicle chains of the inner ear (Piussan et al. 1995; Coate et al. 2012). All these structural abnormalities involving inner ear development result in progressive hearing loss.

Genetics

DFNX2 hearing loss disorder follows an X-linked pattern of inheritance and is caused by variants in the POU class 3 homeobox 4 (POU3F4) gene, which has been localized to chromosomal band Xq21.1 (Piussan et al. 1995). Hearing loss and deafness resulting from mutations in the POU3F4 gene affect males, who generally show severe progressive mixed hearing loss and lack or strong reduction of vertibular responses, whereas female carriers show milder audiologic abnormalities and no vestibular dysfunction (Vore et al. 2005).The POU3F4 gene encodes a 361-amino transcription factor that is generally described in terms of its two major components. One component harbors a 75-amino acid DNA-binding domain that is unique to the POU gene family; this domain enhances the binding capacity of DNA. The other component includes a 63-amino acid homeobox domain that commonly occurs among various transcription factors; this region efficiently recognizes specific motifs on the DNA strand for potential binding (Lee et al. 2009). The two DNA-binding domains are linked together by a 17-amino acid polypeptide chain. Most of missense mutations occurring in the POU3F4 gene involve one of these two DNA-binding domains (Vore et al. 2005; Waryah et al. 2011; Parzefall et al. 2013). On the other hand, deletions often involve a regulatory element situated approximately 400 kilobases upstream of the POU3F4 gene (de Kok et al. 1996; Robert-Moreno et al. 2010). Mutations occurring in the POU3F4 gene account for approximately 40% of X-linked deafness cases (Vore et al. 2005).

About 60 causative variants have been reported in the POU3F4 gene, which include around 30 missense mutations, 20 small and gross deletions, and a few insertions and other complex rearrangements (Bach et al. 1992; de Kok et al. 1996; Marlin et al. 2009; Waryah et al. 2011; Choi et al. 2013; Parzefall et al. 2013).

Testing Strategy

Full Sanger gene sequencing of the single coding exon of the POU3F4 gene is performed. The full coding region of the exon plus ~20 bp of flanking non-coding DNA on either side is sequenced. We will also sequence any single portion (Test #100) or portions of this exon (Test #200) in family members of patients with a known mutation or to confirm research results.

Indications for Test

Any individual who presents with bilateral, progressive, mixed, X-linked hearing loss can be offered the POU3F4 gene test. The individual should have completed otologic and audiologic tests, as well as ancillary testing such as CT imaging of the inner ear to determine the characteristic abnormality involving the temporal bone (Kumar et al. 2003; Altay et al. 2008). Audioprofiling may also assist in determining the rate of progressive hearing loss each year. Cascade testing or successive testing of family members to trace the inheritance pattern of the identified mutation may be offered.

Gene

Official Gene Symbol OMIM ID
POU3F4 300039
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT

Disease

Name Inheritance OMIM ID
Deafness, X-Linked 2 304400

CONTACTS

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Geneticist
Citations
  • Altay H, Savas R, Ogüt F, Kirazli T, Alper H. 2008. CT and MRI findings in X-linked progressive deafness. Diagnostic and Interventional Radiology 14: 117-119. PubMed ID: 18814129
  • Bach I, Brunner HG, Beighton P, Ruvalcaba RH, Reardon W, Pembrey ME, van der Velde-Visser SD, Bruns GA, Cremers CW, Cremers FP, Ropers HH. 1992. Microdeletions in patients with gusher-associated, X-linked mixed deafness (DFN3). American Journal of Human Genetics 51: 38-44. PubMed ID: 1609803
  • Chee NW, Suhailee S, Goh J.  Clinics in diagnostic imaging (111): X-linked congenital mixed deafness syndrome. 2006. Singapore Medical Journal 47: 822-814. PubMed ID: 16924369
  • Choi BY, Park G, Gim J, Kim AR, Kim BJ, Kim HS, Park JH, Park T, Oh SH, Han KH, Park WY. 2013. Diagnostic application of targeted resequencing for familial nonsyndromic hearing loss. PLoS One 8: e68692. PubMed ID: 23990876
  • Coate TM, Raft S, Zhao X, Ryan AK, Crenshaw EB 3rd, Kelley MW. 2012. Otic mesenchyme cells regulate spiral ganglion axon fasciculation through a Pou3f4/EphA4 signaling pathway. Neuron 73: 49-63. PubMed ID: 22243746
  • de Kok YJ, Vossenaar ER, Cremers CW, Dahl N, Laporte J, Hu LJ, Lacombe D, Fischel-Ghodsian N, Friedman RA, Parnes LS, Thorpe P, Bitner-Glindzicz M, Pander HJ, Heilbronner H, Graveline J, den Dunnen JT, Brunner HG, Ropers HH, Cremers FP. 1996. Identification of a hot spot for microdeletions in patients with X-linked deafness type 3 (DFN3) 900 kb proximal to the DFN3 gene POU3F4. Human Molecular Genetics 5: 1229-1235. PubMed ID: 8872461
  • Gharib B, Esmaeili S, Shariati G, Mazloomi Nobandegani N, Mehdizadeh M. 2012. Recurrent bacterial meningitis in a child with hearing impairment, mondini dysplasia: A case report. Acta Medica Iranica 50: 843-845. PubMed ID: 23456530
  • Kumar G, Castillo M, Buchman CA. 2003. X-linked stapes gusher: CT findings in one patient. AJNR American Journal of Neuroradiology 24: 1130-1132. PubMed ID: 12812938
  • Lee HK, Song MH, Kang M, Lee JT, Kong KA, Choi SJ, Lee KY, Venselaar H, Vriend G, Lee WS, Park HJ, Kwon TK, Bok J, Kim UK. 2009. Clinical and molecular characterizations of novel POU3F4 mutations reveal that DFN3 is due to null function of POU3F4 protein. Physiological Genomics 39: 195-201. PubMed ID: 19671658
  • Marlin S, Moizard MP, David A, Chaissang N, Raynaud M, Jonard L, Feldmann D, Loundon N, Denoyelle F, Toutain A. 2009. Phenotype and genotype in females with POU3F4 mutations. Clinical Genetics 76: 558-563. PubMed ID: 19930154
  • Merry DE, Lesko JG, Sosnoski DM, Lewis RA, Lubinsky M, Trask B, van den Engh G, Collins FS, Nussbaum RL. 1989. Choroideremia and deafness with stapes fixation: a contiguous gene deletion syndrome in Xq21. American Journal of Human Genetics 45: 530-540. PubMed ID: 2491012
  • Naranjo S1, Voesenek K, de la Calle-Mustienes E, Robert-Moreno A, Kokotas H, Grigoriadou M, Economides J, Van Camp G, Hilgert N, Moreno F, Alsina B, Petersen MB, Kremer H, Gómez-Skarmeta JL. 2010. Multiple enhancers located in a 1-Mb region upstream of POU3F4 promote expression during inner ear development and may be required for hearing. Human Genetics 128: 411-419. PubMed ID: 20668882
  • Parzefall T, Shivatzki S, Lenz DR, Rathkolb B, Ushakov K, Karfunkel D, Shapira Y, Wolf M, Mohr M, Wolf E, Sabrautzki S, de Angelis MH, Frydman M, Brownstein Z, Avraham KB. 2013. Cytoplasmic mislocalization of POU3F4 due to novel mutations leads to deafness in humans and mice. Human Mutation 34: 1102-1110. PubMed ID: 23606368
  • Piussan C, Hanauer A, Dahl N, Mathieu M, Kolski C, Biancalana V, Heyberger S, Strunski V. 1995. X-linked progressive mixed deafness: A new microdeletion that involves a more proximal region in Xq21. American Journal of Human Genetics 56: 224-230. PubMed ID: 7825582
  • Robert-Moreno À, Naranjo S, de la Calle-Mustienes E, Gómez-Skarmeta JL, Alsina B. 2010. Characterization of new otic enhancers of the pou3f4 gene reveal distinct signaling pathway regulation and spatio-temporal patterns. PLoS One 5: e15907. PubMed ID: 21209840
  • Santos S, Domínguez MJ, Cervera J, Suárez A, Bueno A, Bartolomé M, López R. 2014. Hearing loss and enlarged internal auditory canal in children. Acta Otorrinolaringologica Espanola 65: 93-101. PubMed ID: 24534420
  • Vore AP, Chang EH, Hoppe JE, Butler MG, Forrester S, Schneider MC, Smith LL, Burke DW, Campbell CA, Smith RJ. 2005. Deletion of and novel missense mutation in POU3F4 in 2 families segregating X-linked nonsyndromic deafness. Archives of Otolaryngology Head and Neck Surgery 131: 1057-1063. PubMed ID: 16365218
  • Waryah AM, Ahmed ZM, Bhinder MA, Choo DI, Sisk RA, Shahzad M, Khan SN, Friedman TB, Riazuddin S, Riazuddin S. 2011. Molecular and clinical studies of X-linked deafness among Pakistani families. Journal of Human Genetics 56: 534-540. PubMed ID: 21633365
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TEST METHODS

Bi-Directional Sanger Sequencing

Test Procedure

Nomenclature for sequence variants was from the Human Genome Variation Society (http://www.hgvs.org).  As required, DNA is extracted from the patient specimen.  PCR is used to amplify the indicated exons plus additional flanking non-coding sequence.  After cleaning of the PCR products, cycle sequencing is carried out using the ABI Big Dye Terminator v.3.0 kit.  Products are resolved by electrophoresis on an ABI 3730xl capillary sequencer.  In most cases, sequencing is performed in both forward and reverse directions; in some cases, sequencing is performed twice in either the forward or reverse directions.  In nearly all cases, the full coding region of each exon as well as 20 bases of non-coding DNA flanking the exon are sequenced.

Analytical Validity

As of March 2016, we compared 17.37 Mb of Sanger DNA sequence generated at PreventionGenetics to NextGen sequence generated in other labs. We detected only 4 errors in our Sanger sequences, and these were all due to allele dropout during PCR. For Proficiency Testing, both external and internal, in the 12 years of our lab operation we have Sanger sequenced roughly 8,800 PCR amplicons. Only one error has been identified, and this was due to sequence analysis error.

Our Sanger sequencing is capable of detecting virtually all nucleotide substitutions within the PCR amplicons. Similarly, we detect essentially all heterozygous or homozygous deletions within the amplicons. Homozygous deletions which overlap one or more PCR primer annealing sites are detectable as PCR failure. Heterozygous deletions which overlap one or more PCR primer annealing sites are usually not detected (see Analytical Limitations). All heterozygous insertions within the amplicons up to about 100 nucleotides in length appear to be detectable. Larger heterozygous insertions may not be detected. All homozygous insertions within the amplicons up to about 300 nucleotides in length appear to be detectable. Larger homozygous insertions may masquerade as homozygous deletions (PCR failure).

Analytical Limitations

In exons where our sequencing did not reveal any variation between the two alleles, we cannot be certain that we were able to PCR amplify both of the patient’s alleles. Occasionally, a patient may carry an allele which does not amplify, due for example to a deletion or a large insertion. In these cases, the report contains no information about the second allele.

Similarly, our sequencing tests have almost no power to detect duplications, triplications, etc. of the gene sequences.

In most cases, only the indicated exons and roughly 20 bp of flanking non-coding sequence on each side are analyzed. Test reports contain little or no information about other portions of the gene, including many regulatory regions.

In nearly all 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 for example 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 and cycle sequencing.

Unless otherwise indicated, the sequence data that we report are based on DNA isolated from a specific tissue (usually leukocytes). Test reports contain no information about gene sequences in other tissues.

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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