Title: Distributions of Mutations Associated with Sensorineural Hearing Loss
1Distributions 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
2Faculty 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.
3Congenital 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
4Clinical evaluation of hearing loss
- History
- Prenatal
- Infections, medication exposure
- Neonatal
- Prematurity, hyperbilirubinemia, infections,
medications - Childhood
- Ear infections, antibiotics, medical problems
- Family history
5Clinical 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
6GJB2
- 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
7GJB2
- 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
8Gap Junction Channels
- From Rabionet et al in TRENDS in Molecular
Medicine Vol.8 No.5 May 2002
9Expression of Cx26, Cx30 and Cx31 in the Cochlea
- From Rabionet et al in TRENDS in Molecular
Medicine Vol.8 No.5 May 2002
10Preliminary 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
11Available 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
12mtDNA 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
13Project design
- Designing primers for PCR
- Overcoming the GC content
- Primers for Exons 12, and CpG island
- Sequencing PCR products
- Identifying sequence variants with Sequencher
- Examine for known SNPs
- Screening controls with Pyrosequencing
14CpG Island Primers
- CGCCAGGTTCCTGGCCGGGCAGTCCGGGGCCGGCGGGCTCACCTGCGTC
GGGAGGAAGCGCGGCGGGGCCGGGGCGGGGGTCTCGGCGTTGGGGTCTCT
GCGCTGGGGCTCCTGCGCTCCTAGGCGGGTCCTGGGCCGGGCGCCGCCGA
GGGGCTCCGAGTCGGGGAGAGGAGCGCGCGGGCGCTGCGGGGCCGCAACA
CCTGTCTCCCGCCGTGGCGCCTTTTAACCGCACCCCACACCCCGCCTCTT
CCCTCGGAGACTGGGAAAGTTACGGAGGGGGCGGCGCCGCGGGCGGAGCG
CGCCCGGCCTCTGGGTCCTCAGAGCTTCCCGGGTCCGCGAACCCCCGACC
GCCCCCGAAAGCCCCGAACCCCCCAAGTCCCCTTCGAGGTCCCGATCTCC
TAGTTCCTTTGAGCC
15Exon 1 Primers
- CCCAAGGACGTGTGTTGGTCCAGCCCCCCGGTTCCCCGAGACCCACGCG
GCCGGGCAACCGCTCTGGGTCTCGCGGTCCCTCCCCGCGCCAGGTTCCTG
GCCGGGCAGTCCGGGGCCGGCGGGCTCACCTGCGTCGGGAGGAAGCGCGG
CGGGGCCGGGGCGGGGGTCTCGGCGTTGGGGTCTCTGCGCTGGGGCTCCT
GCGCTCCTAGGCGGGTCCTGGGCCGGGCGCCGCCGAGGGGCTCCGAGTCG
GGGAGAGGAGCGCGCGGGCGCTGCGGGGCCGCAACACCTGTCTCCCGCCG
TGGCGCCTTTTAACCGCACCCCACACCCCGCCTCTTCCCTCGGAGACTGG
GAAAGTTACGGA
16Exon 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
1735delG
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?
18Patient 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.
19(No Transcript)
20- 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
21HE Heterozygote
22HE Heterozygote HO Homozygote
23Results
- 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
24Conclusions
- 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
25Future studies
- Patient recruitment
- JMC NICU and nursery
- JMC audiology clinic
- CHAM Craniofacial Center
- Controls
- Hope for
- 50 cases/year 300 controls/year
26Future 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
27Future 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
28Reference 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/