Molecular Diagnosis of Hearing Loss

Kerry K. Brown1, Heidi L. Rehm2

1 Department of Genetics, Harvard Medical School, Boston, Massachusetts, 2 Laboratory for Molecular Medicine, Partners Healthcare Center for Personalized Genetic Medicine, Cambridge, Massachusetts
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 9.16
DOI:  10.1002/0471142905.hg0916s72
Online Posting Date:  January, 2012
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This unit discusses an approach to identifying a genetic etiology in an individual with nonsyndromic hearing loss. The unit begins with a discussion of the decision‐making process that can be used to determine whether specific genes and/or a large gene panel should be used for molecular diagnosis of a patient presenting with nonsyndromic hearing loss. Next, two protocols are presented: (1) a full gene‐sequencing assay to identify mutations in the GJB2 gene (encoding connexin 26), the most common cause of congenital hearing loss, and (2) an assay to detect the presence of the GJB6‐D13S1830 deletion, a 342‐kb deletion that causes hearing loss in homozygosity or in combination with a single GJB2 mutation. Finally, the unit ends with a strategy for determining the clinical significance of the test results, which can be challenging given the extensive genetic heterogeneity associated with hearing loss. Curr. Protoc. Hum. Genet. 72:9.16.1‐9.16.16 © 2012 by John Wiley & Sons, Inc.

Keywords: hearing loss; deafness; connexin 26; GJB2; connexin 30; GJB6

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: GJB2 (Connexin 26) Gene Test
  • Basic Protocol 2: GJB6–D13S1830 (Connexin 30) Deletion Test
  • Guide to Data Interpretation
  • Time Considerations
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: GJB2 (Connexin 26) Gene Test

  • FailSafe PCR System with PreMix D (Epicentre Technologies, cat. no. FS99100)
  • 10 µM primers: Cx26‐1F, ‐1R, ‐2AF, ‐2BF, ‐2AR, and ‐2BR (Table 9.16.1)
  • 25 mM MgCl 2
  • 10 mM dNTP mix ( appendix 2D)
  • AmpliTaq Gold Taq DNA polymerase and 10× PCR buffer II (Applied Biosystems)
  • PCR‐grade H 2O
  • 50 ng/µl genomic DNA ( appendix 3B) to be tested
  • Thermal cycler and suitable reaction tubes
  • PCR cleanup kit (supplied by many companies, e.g., Qiagen or Agencourt Bioscience)
  • Software for sequence data analysis
  • Additional reagents and equipment for agarose gel electrophoresis (unit 2.7) and DNA cycle sequencing (unit 7.7)
    Table 9.6.1   MaterialsGJB2 Primers

    Name Forward primer sequence (5′→3′) Name Reverse primer sequence (5′→3′) Product length (bp)
    PCR primers
    Additional sequencing primers

Basic Protocol 2: GJB6–D13S1830 (Connexin 30) Deletion Test

  • FailSafe PCR System with PreMix D (Epicentre Technologies, cat. no. FS99100)
  • 25 µM primers: F, NLR, and BKR (Table 9.16.3)
  • PCR‐grade H 2O
  • 50 µl genomic DNA ( appendix 3B) to be tested
  • Thermal cycler and suitable reaction tubes
  • Additional reagents and equipment for agarose gel electrophoresis (unit 2.7)
    Table 9.6.3   MaterialsGJB6‐D13S1830 Primers

    Name Forward primer sequence (5′→3′) Name Reverse primer sequences (5′→3′) Product length

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Literature Cited

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