Distributions of Mutations Associated with Sensorineural Hearing Loss

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Distributions of Mutations Associated with
Sensorineural Hearing Loss

• 2006 National EHDI Conference
• Alan Shanske, M.D., FAAP, FACMG
• Center for Craniofacial Disorders
• Childrens Hospital at Montefiore
• Bronx, New York
• February 2, 2006

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Faculty Disclosure Information

• In the past 12 months, I have not had a
  significant financial interest or other
  relationship with the manufacturer(s) of the
  product(s) or provider(s) of the service(s) that
  will be discussed in my presentation.
• This presentation will not include discussion of
  pharmaceuticals or devices that have not been
  approved by the FDA or of off-label uses of
  pharmaceuticals or devices.

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Congenital Hearing Loss

• Epidemiology
• 1/1000 infants affected
• Etiology
• 50 genetic
• 70 non-syndromic sensorineural hearing loss
  (SNHL)
• 77 autosomal recessive
• 52 loci known 34 identified
• 22 autosomal dominant
• Remainder are mitochondrial or X-linked

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Clinical evaluation of hearing loss

• History
• Prenatal
• Infections, medication exposure
• Neonatal
• Prematurity, hyperbilirubinemia, infections,
  medications
• Childhood
• Ear infections, antibiotics, medical problems
• Family history

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Clinical evaluation of hearing loss

• Physical exam
• Dysmorphic features
• Ear malformations or effusions
• Skin (NF2)
• Hair and eyes (Waardenburg)
• Testing
• EKG (Jervell and Lange-Nielsen syndrome)
• /- urinalysis
• CT scan of temporal bones
• Genetic testing

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GJB2

• Encodes connexin 26 (Cx26)
• Gap junction protein in the cochlea
• Maps to 13q12
• 2263 nucleotides, 680 amino acids
• Two exons one coding exon
• CpG island near Exon 1

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GJB2

• AR mutations account for 15 40 of inherited
  SNHL in North America
• Carrier rate of 133 in Europeans
• Most common mutation in Caucasians 35delG
• Mutation spectrum is known to differ by ethnic
  group

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Gap Junction Channels

• From Rabionet et al in TRENDS in Molecular
  Medicine Vol.8 No.5 May 2002

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Expression of Cx26, Cx30 and Cx31 in the Cochlea

• From Rabionet et al in TRENDS in Molecular
  Medicine Vol.8 No.5 May 2002

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Preliminary Study

• Chart review of 107 patients
• Referred to CHAM for genetic evaluation of SNHL
• Data collected
• Ethnicity
• Cx26 mutation status
• mtDNA DNA analysis (nt 1555, 7445, 3243,
  sequencing of 12s rRNA)
• CT scan of temporal bones

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Available Samples

• 107 Samples obtained from IRB approved research
  project looking for mtDNA point mutations in SNHL
• 192 Controls provided by Dr. Robert Burk from HPV
  study

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mtDNA and CT Results

• one Puerto Rican patient
• A503G variant mtDNA mutation at nt 1465
• no patient had A1555G or T7445C associated with
  SNHL
• 31 patients had CT scan results
• 2 had EVA, one of which carries G79A
• 1 had ? Mondinis, 1 had prominence of cochlear
  aqueducts, 1 had diffuse atrophy

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Project design

1. Designing primers for PCR
2. Overcoming the GC content
3. Primers for Exons 12, and CpG island
4. Sequencing PCR products
5. Identifying sequence variants with Sequencher
6. Examine for known SNPs
7. Screening controls with Pyrosequencing

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CpG Island Primers

• CGCCAGGTTCCTGGCCGGGCAGTCCGGGGCCGGCGGGCTCACCTGCGTC
  GGGAGGAAGCGCGGCGGGGCCGGGGCGGGGGTCTCGGCGTTGGGGTCTCT
  GCGCTGGGGCTCCTGCGCTCCTAGGCGGGTCCTGGGCCGGGCGCCGCCGA
  GGGGCTCCGAGTCGGGGAGAGGAGCGCGCGGGCGCTGCGGGGCCGCAACA
  CCTGTCTCCCGCCGTGGCGCCTTTTAACCGCACCCCACACCCCGCCTCTT
  CCCTCGGAGACTGGGAAAGTTACGGAGGGGGCGGCGCCGCGGGCGGAGCG
  CGCCCGGCCTCTGGGTCCTCAGAGCTTCCCGGGTCCGCGAACCCCCGACC
  GCCCCCGAAAGCCCCGAACCCCCCAAGTCCCCTTCGAGGTCCCGATCTCC
  TAGTTCCTTTGAGCC

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Exon 1 Primers

• CCCAAGGACGTGTGTTGGTCCAGCCCCCCGGTTCCCCGAGACCCACGCG
  GCCGGGCAACCGCTCTGGGTCTCGCGGTCCCTCCCCGCGCCAGGTTCCTG
  GCCGGGCAGTCCGGGGCCGGCGGGCTCACCTGCGTCGGGAGGAAGCGCGG
  CGGGGCCGGGGCGGGGGTCTCGGCGTTGGGGTCTCTGCGCTGGGGCTCCT
  GCGCTCCTAGGCGGGTCCTGGGCCGGGCGCCGCCGAGGGGCTCCGAGTCG
  GGGAGAGGAGCGCGCGGGCGCTGCGGGGCCGCAACACCTGTCTCCCGCCG
  TGGCGCCTTTTAACCGCACCCCACACCCCGCCTCTTCCCTCGGAGACTGG
  GAAAGTTACGGA

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Exon 2 Coding Region Primers

• TTATTATAGAGATTATATTTTAATGTTTTAAATGTATTTGATACATTACA
  AAATTATTTTAGTTACA
• AGCATATCATTAAAGCTATTCTTTATTATTACAAAATGCTTTTACAATGC
  TATTCTTGACAACAGG
• AAAATACTTACCCTCACTGAAATATGTGGAGTACCATTTTTTGGAAACCA
  TGTCAAGCATAATGGC
• AATATTCAGGTTCAATCTTCCTATAGATCTGCTCAATATTTATCTAAACC
  TTAGCTTCTATTCTTTT
• CACATGTTATTAGCTATATTTTCACTTAAAAAATTGGAGGCTGAAGGGGT
  AAGCAAACAAACTTT
• TGAAGTAGACAAAGCTCATCTTTAATCAACAGACTTTAGAGTCCAGTCTT
  TCCAAATCTGTTTTTA
• ACGACAGAAACTTCTCCCTCCCCTGCCCCATTTTGTCCTCCCCATTAAAT
  GGTACTGTGTCAATAAA
• ATTCCCAAGCGACCTCTTTAAATCAGCGTTCTTTCCGATGCTGGCTACCA
  CAGTCATGGAAAAGG
• AGATGTGTTGGACAGGCCTGTCATTACAGGTAGTAGTTGGTGGTACATCC
  AGTCTGTATTTCTTA
• CACAAAATTACATCTAAATATTTGACATGAGGCCATTTGCTATCATAAGC
  CATCACTAGGAACTTC
• TAGTCTGTCTCACTCGATTGAGGCTACAATGTTGTTAGGTGCTATGACCA
  CAATGAATACAACAG
• ACAGCCTCTCAGCTGTGCTGCAAAGTATTCATAACCAAAAGACCATATTT
  CAAATTAAATCATAGT
• AGCGAATGACATACCATTTACATATTACAATCTGAGCCTCTGAAACAGGG
  GGAACATATAATGGT
• ATCCAGAACATCTTTACATCAAAATAACCTATCATACTACAAAGTTTTCA
  CTTCCAAAAAGTGTAAC
• AGAGTTTAAGGCACTGGTAACTTTGTCCACTGTTAGAGATTAAAACTTCC
  AAAGCAAATGAAAGA
• ACCAATGTTCACCTTTAACGTGGGGAAAGTTGGCAAAAAGAACCCCAGGA
  GGACACCCAAACCTT
• CTCTGTGTCCTCTGTGGAACCTGGCTTTTTTCTCTTGTCCTCAGAGAAAG
  AAACAAATGCCGATAT
• CCTCTGTTTAAAATATGAAAGTACCTTACACCAATAACCCCTAACAGCCT
  GGGGTCTCAGTGGAAC
• TAACTTAAGTGAAAGAAAATTAAGACAGGCATAGAATTAGGCCTTTGTTT
  TGAGGCTTTAGGGG

_________________________________________________
___CTACAGGGGTTTCAAATGGTTGCATTTAAGGTCAGAATCTTTGTGTT
GGGAAATGCTAGCGACTGAGCCTTGACAGCTGAGCACGGGTTGCCTCATC
CCTCTCATGCTGTCTATTTCTTAATCTAACAACTGGGCAATGCGTTAAAC
TGGCTTTTTTGACTTCCCAGAACAATATCTAATTAGCAAATAACACAATT
CAGTGACATTCAGCAGGATGCAAATTCCAGACACTGCAATCATGAACACT
GTGAAGACAGTCTTCTCCGTGGGCCGGGACACAAAGCAGTCCACAGTGTT
GGGACAAGGCCAGGCGTTGCACTTCACCAGCCGCTGCATGGAGAAGCCGT
CGTACATGACATAGAAGACGTACATGAAGGCGGCTTCGAAGATGACCCGG
AAGAAGATGCTGCTTGTGTAGGTCCACCACAGGGAGCCTTCGATGCGGAC
CTTCTGGGTTTTGATCTCCTCGATGTCCTTAAATTCACTCTTTATCTCCC
CCTTGATGAACTTCCTCTTCTTCTCATGTCTCCGGTAGGCCACGTGCATG
GCCACTAGGAGCGCTGGCGTGGACACGAAGATCAGCTGCAGGGCCCATAG
CCGGATGTGGGAGATGGGGAAGTAGTGATCGTAGCACACGTTCTTGCAGC
CTGGCTGCAGGGTGTTGCAGACAAAGTCGGCCTGCTCATCTCCCCACACC
TCCTTTGCAGCCACAACGAGGATCATAATGCGAAAAATGAAGAGGACGGT
GAGCCAGATCTTTCCAATGCTGGTGGAGTGTTTGTTCACACCCCCCAGGA
TCGTCTGCAGCGTGCCCCAATCCATCTTCTACTCTGGGCGGTTTGCTCTG
GAAAAGACGAATGCACACAACACAGGAATCACTAGCTAGGACAGAACAGG
GAGACTTCTCTGAGTCTGGGTAAGC
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35delG
167delT
35delG and 167delT compound heterozygote of mixed
Jewish, Italian and Irish decent. Deletion alters
chromatogram alignment, which is corrected with
the deletion on the opposite chromosome. Both
35delG and 167delT lead to frameshift mutations.
C-34T variant
Patient with a C-34T variant and a G79A
polymorphism. Is there significance to these
changes when they co-occur?
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Patient from consanguineous Dominican family with
a G139T homozygous mutation, leading to
substitution of Valine for Glutamine at amino
acid 47, initiating a premature STOP.
Start Codon
G139T homozygous
35delG common mutation in Caucasian population,
found in two Puerto Rican patients and one of
mixed Italian, Irish and Jewish decent.
35delG leads to a frameshift mutation, as seen on
this chromatogram.
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(No Transcript)
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• Schematic of Connexin 26 domains with mutations
  and polymorphisms included
• Mutations, polymorphisms and variants exhibited
  in our study are circled
• Mutations are shown in Green
• Polymorphisms are shown in Purple
• Variants of unknown significance are shown in
  Orange
• Also seen in our study were 9 patients with
  C-34T, in the 5UTR, not previously described
• We noted sequence variations at nucleotide 765,
  with 65/35 C/T

http//ent.md.shinshu-u.ac.jp/deafgene25/nonsyndr
omic/ohtsuka.gif
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HE Heterozygote
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HE Heterozygote HO Homozygote
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Results

• one Dominican patient was homozygous for a
  mutation in GJB2 (G139T)
• GJB2 mutations occur in 1/33 European controls
  (35delG in 2-4)
• only one Hispanic 35delG carrier in our controls
  all other nucleotide changes were polymorphisms
  or novel variants

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Conclusions

• GJB2 mutations occur less frequently in our
  minority population
• lower carrier frequencies may account for the
  lower rate of homozygous individuals in our
  population
• possible synergistic interaction of heterozygous
  GJB2 mutations and a mutation in another gene
  such as GJB6

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Future studies

• Patient recruitment
• JMC NICU and nursery
• JMC audiology clinic
• CHAM Craniofacial Center
• Controls
• Hope for
• 50 cases/year 300 controls/year

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Future Directions

• Cx30
• Adjacent to GJB2
• Mutations are rare
• May lead to AD late onset deafness
• Deletions
• Homozygous ? deafness
• Heterozygous in trans with GJB2 mutation ?
  deafness

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Future Directions

• SLC26A4
• Encodes monovalent and divalent anion transporter
  related proteins (Pendrin)
• Involved in fluid homeostasis
• Mutations cause Pendred syndrome (AR defects of
  thyroid, kidney and inner ear)
• Often also see Enlarged Vestibular Aqueduct (EVA)
  or Mondini dysplasia

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Reference List

• For more information on this topic, see the
  following publications
• Marazita ML, et al., (1993) Genetic epidemiologic
  studies of early-onset deafness in the U.S.
  school-age population. Am J Med Genet
  46486-491).
• Kelsell DP, et al., (1997) Connexin 26 mutations
  in hereditary non-syndromal sensoineural
  deafness. Nature 387(6628)80-83.
• Morton, C (2002) Genetics, genomics and gene
  discovery in the auditory system. Human Molecular
  Genetics 11(10)1229-1240.
• Rabionet R, et al., (2002) Connexin mutations in
  hearing loss, dermatological and neurological
  disorders. Trends in Mol Med 8(5)205-212).
• Pandya, A, et al., (2003) Frequency and
  distribution of GJB2 (connexin 26) and GJB6
  (connexin 30) mutations in a large North American
  repository of deaf probands. Genet Med
  5(4)295-303.
• Additional information may be found at
• http//davinci.crg.es/deafness/