Transcript Gap Junction Channels

Distributions of Mutations
Associated with Sensorineural
Hearing Loss
2006 National EHDI Conference
Alan Shanske, M.D., FAAP, FACMG
Center for Craniofacial Disorders
Children’s Hospital at Montefiore
Bronx, New York
February 2, 2006
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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
Clinical evaluation of hearing
loss

History

Prenatal


Neonatal


Prematurity, hyperbilirubinemia, infections, medications
Childhood


Infections, medication exposure
Ear infections, antibiotics, medical problems
Family history
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
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

GJB2

AR mutations account for 15 – 40% of
inherited SNHL in North America
Carrier rate of 1:33 in Europeans
 Most common mutation in Caucasians: 35delG
 Mutation spectrum is known to differ by ethnic
group

Gap Junction Channels
From Rabionet et al in TRENDS in Molecular Medicine Vol.8 No.5 May 2002
Expression of Cx26, Cx30 and Cx31
in the Cochlea
From Rabionet et al in TRENDS in Molecular Medicine Vol.8 No.5 May 2002
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

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
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 ? Mondini’s, 1 had prominence of
cochlear aqueducts, 1 had diffuse atrophy

Project design
1.
Designing primers for PCR
1.
2.
2.
3.
4.
5.
Overcoming the GC content
Primers for Exons 1&2, and CpG island
Sequencing PCR products
Identifying sequence variants with
Sequencher
Examine for known SNPs
Screening controls with Pyrosequencing
CpG Island Primers
CGCCAGGTTCCTGGCCGGGCAGTCCGGGGCCGGCGG
GCTCACCTGCGTCGGGAGGAAGCGCGGCGGGGCCGG
GGCGGGGGTCTCGGCGTTGGGGTCTCTGCGCTGGGG
CTCCTGCGCTCCTAGGCGGGTCCTGGGCCGGGCGCC
GCCGAGGGGCTCCGAGTCGGGGAGAGGAGCGCGCGG
GCGCTGCGGGGCCGCAACACCTGTCTCCCGCCGTGG
CGCCTTTTAACCGCACCCCACACCCCGCCTCTTCCC
TCGGAGACTGGGAAAGTTACGGAGGGGGCGGCGCCG
CGGGCGGAGCGCGCCCGGCCTCTGGGTCCTCAGAGC
TTCCCGGGTCCGCGAACCCCCGACCGCCCCCGAAAG
CCCCGAACCCCCCAAGTCCCCTTCGAGGTCCCGATC
TCCTAGTTCCTTTGAGCC
Exon 1 Primers
CCCAAGGACGTGTGTTGGTCCAGCCCCCCGGTTCCC
CGAGACCCACGCGGCCGGGCAACCGCTCTGGGTCTC
GCGGTCCCTCCCCGCGCCAGGTTCCTGGCCGGGCAG
TCCGGGGCCGGCGGGCTCACCTGCGTCGGGAGGAAG
CGCGGCGGGGCCGGGGCGGGGGTCTCGGCGTTGGGG
TCTCTGCGCTGGGGCTCCTGCGCTCCTAGGCGGGTC
CTGGGCCGGGCGCCGCCGAGGGGCTCCGAGTCGGGG
AGAGGAGCGCGCGGGCGCTGCGGGGCCGCAACACCT
GTCTCCCGCCGTGGCGCCTTTTAACCGCACCCCACA
CCCCGCCTCTTCCCTCGGAGACTGGGAAAGTTACGG
A
TTATTATAGAGATTATATTTTAATGTTTTAAATGTATTTGATACATTACAAAATTATTTTAGTTACA
AGCATATCATTAAAGCTATTCTTTATTATTACAAAATGCTTTTACAATGCTATTCTTGACAACAGG
AAAATACTTACCCTCACTGAAATATGTGGAGTACCATTTTTTGGAAACCATGTCAAGCATAATGGC
AATATTCAGGTTCAATCTTCCTATAGATCTGCTCAATATTTATCTAAACCTTAGCTTCTATTCTTTT
CACATGTTATTAGCTATATTTTCACTTAAAAAATTGGAGGCTGAAGGGGTAAGCAAACAAACTTT
TGAAGTAGACAAAGCTCATCTTTAATCAACAGACTTTAGAGTCCAGTCTTTCCAAATCTGTTTTTA
ACGACAGAAACTTCTCCCTCCCCTGCCCCATTTTGTCCTCCCCATTAAATGGTACTGTGTCAATAAA
ATTCCCAAGCGACCTCTTTAAATCAGCGTTCTTTCCGATGCTGGCTACCACAGTCATGGAAAAGG
AGATGTGTTGGACAGGCCTGTCATTACAGGTAGTAGTTGGTGGTACATCCAGTCTGTATTTCTTA
CACAAAATTACATCTAAATATTTGACATGAGGCCATTTGCTATCATAAGCCATCACTAGGAACTTC
TAGTCTGTCTCACTCGATTGAGGCTACAATGTTGTTAGGTGCTATGACCACAATGAATACAACAG
ACAGCCTCTCAGCTGTGCTGCAAAGTATTCATAACCAAAAGACCATATTTCAAATTAAATCATAGT
AGCGAATGACATACCATTTACATATTACAATCTGAGCCTCTGAAACAGGGGGAACATATAATGGT
ATCCAGAACATCTTTACATCAAAATAACCTATCATACTACAAAGTTTTCACTTCCAAAAAGTGTAAC
AGAGTTTAAGGCACTGGTAACTTTGTCCACTGTTAGAGATTAAAACTTCCAAAGCAAATGAAAGA
ACCAATGTTCACCTTTAACGTGGGGAAAGTTGGCAAAAAGAACCCCAGGAGGACACCCAAACCTT
CTCTGTGTCCTCTGTGGAACCTGGCTTTTTTCTCTTGTCCTCAGAGAAAGAAACAAATGCCGATAT
CCTCTGTTTAAAATATGAAAGTACCTTACACCAATAACCCCTAACAGCCTGGGGTCTCAGTGGAAC
TAACTTAAGTGAAAGAAAATTAAGACAGGCATAGAATTAGGCCTTTGTTTTGAGGCTTTAGGGG
AGCAGAGCTCCATTGTGGCATCTGGAGTTTCACCTGAGGC
____________________________________________________CTACAGGGGTTTCAAATGGTTGCA
TTTAAGGTCAGAATCTTTGTGTTGGGAAATGCTAGCGACTGAGCCTTGACAGCTGAGCACGGGTT
GCCTCATCCCTCTCATGCTGTCTATTTCTTAATCTAACAACTGGGCAATGCGTTAAACTGGCTTTT
Exon 2 Coding Region Primers
TTGACTTCCCAGAACAATATCTAATTAGCAAATAACACAATTCAGTGACATTCAGCAGGATGCAA
ATTCCAGACACTGCAATCATGAACACTGTGAAGACAGTCTTCTCCGTGGGCCGGGACACAAAGC
AGTCCACAGTGTTGGGACAAGGCCAGGCGTTGCACTTCACCAGCCGCTGCATGGAGAAGCCGTC
GTACATGACATAGAAGACGTACATGAAGGCGGCTTCGAAGATGACCCGGAAGAAGATGCTGCTT
GTGTAGGTCCACCACAGGGAGCCTTCGATGCGGACCTTCTGGGTTTTGATCTCCTCGATGTCCT
TAAATTCACTCTTTATCTCCCCCTTGATGAACTTCCTCTTCTTCTCATGTCTCCGGTAGGCCACG
TGCATGGCCACTAGGAGCGCTGGCGTGGACACGAAGATCAGCTGCAGGGCCCATAGCCGGATGT
GGGAGATGGGGAAGTAGTGATCGTAGCACACGTTCTTGCAGCCTGGCTGCAGGGTGTTGCAGA
CAAAGTCGGCCTGCTCATCTCCCCACACCTCCTTTGCAGCCACAACGAGGATCATAATGCGAAA
AATGAAGAGGACGGTGAGCCAGATCTTTCCAATGCTGGTGGAGTGTTTGTTCACACCCCCCAGG
ATCGTCTGCAGCGTGCCCCAATCCATCTTCTACTCTGGGCGGTTTGCTCTGGAAAAGACGAATGC
ACACAACACAGGAATCACTAGCTAGGACAGAACAGGGAGACTTCTCTGAGTCTGGGTAAGC
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
C-34T variant
G79A
polymorphism
Patient with a C-34T variant and a G79A polymorphism. Is there
significance to these changes when they co-occur?
Start Codon
G139T homozygous
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.
35delG common mutation in Caucasian population, found in two
Puerto Rican patients and one of mixed Italian, Irish and Jewish
decent.
35delG
35delG leads to a frameshift mutation, as seen on this
chromatogram.
GJB2 Mutations by Ethnicity for 107 Patients
30
25
Number of Patients
20
*
15
10
T101C (M34T)(AD vs. Polym)
35delG (AR)
167delT (AR)
G139T (E47V)(AR)
C-15T (Polym)
G79A (V27I)(Polym)
G380A (R127H)(Polym)
A670C (K224Q)(Indeter)
A503G (K168R)(novel)
C684A (novel)
Negative
5
0
Black Black/PR
PR= Puerto Rico
DR=Dominican Republic
PR
DR/PR
DR
Mexico
Ethnicity
Guyana
India
Pakistan
Other
* Compound Heterozygote: 35delG + 167delT
K168R
Extracellular
Domain
Schematic of Connexin
26 domains with
mutations and
polymorphisms included
Mutations, polymorphisms and
variants exhibited in our study are
Transmembrane circled
Domain
Mutations are shown in Green
V27I
Polymorphisms are shown in
Purple
R127H
Variants of unknown significance
are shown in Orange
K224Q
Intracellular
Domain
http://ent.md.shinshuu.ac.jp/deafgene%25/nonsyndromic/ohtsuka.gif
Also seen in our study were 9
patients with C-34T, in the 5’UTR,
not previously described
We noted sequence variations at
nucleotide 765, with 65/35 C/T
12
10
8
6
4
2
Nucleotide Change
HE= Heterozygote
35
de
lG
H
E
H
E
A5
0
3G
H
E
T4
25
2T
C
68
G
C
H
E
H
E
1A
51
9A
10
G
79
A
H
E
H
E
0
G
Number of Patients
Control Data for 93 Hispanic Patients
Number of Patients
Control Data for 94 Black Patients
2.5
2
1.5
1
0.5
0
G
A
79
HE
G
A
79
HO
G
A
1
34
HO
C
1
0
T1
HE
G
8A
7
4
Nucleotide Change
HE = Heterozygote
HO= Homozygote
HE
G
9A
9
4
HE
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
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
Future studies

Patient recruitment
JMC NICU and nursery
 JMC audiology clinic
 CHAM Craniofacial Center



Controls
Hope for:

50 cases/year + 300 controls/year
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

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

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 46:486-491).
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/