Distributions of Distributions of Mutations Associated Mutations Associated with Sensorineural with Sensorineural Hearing Loss Hearing Loss 2006 National EHDI Conference 2006 National EHDI Conference Alan Shanske, M.D., FAAP, FACMG Alan Shanske, M.D., FAAP, FACMG Center for Craniofacial Disorders Center for Craniofacial Disorders Children’s Hospital at Montefiore Children’s Hospital at Montefiore Bronx, New York Bronx, New York February 2, 2006 February 2, 2006
Transcript
Distributions of Distributions of Mutations Associated Mutations Associated
with Sensorineural with Sensorineural Hearing LossHearing Loss
2006 National EHDI Conference2006 National EHDI ConferenceAlan Shanske, M.D., FAAP, FACMGAlan Shanske, M.D., FAAP, FACMGCenter for Craniofacial DisordersCenter for Craniofacial DisordersChildren’s Hospital at MontefioreChildren’s Hospital at Montefiore
Bronx, New YorkBronx, New YorkFebruary 2, 2006February 2, 2006
• In the past 12 months, I have not had In the past 12 months, I have not had a significant financial interest or a significant financial interest or other relationship with the other relationship with the manufacturer(s) of the product(s) or manufacturer(s) of the product(s) or provider(s) of the service(s) that will provider(s) of the service(s) that will be discussed in my presentation.be discussed in my presentation.
• This presentation will not include This presentation will not include discussion of pharmaceuticals or discussion of pharmaceuticals or devices that have not been approved devices that have not been approved by the FDA or of “off-label” uses of by the FDA or of “off-label” uses of pharmaceuticals or devices.pharmaceuticals or devices.
ChildhoodChildhood Ear infections, antibiotics, medical problemsEar infections, antibiotics, medical problems
Family historyFamily history
Clinical evaluation of Clinical evaluation of hearing losshearing loss
Physical examPhysical exam Dysmorphic featuresDysmorphic features Ear malformations or effusionsEar malformations or effusions Skin (NF2)Skin (NF2) Hair and eyes (Waardenburg)Hair and eyes (Waardenburg)
TestingTesting EKG (Jervell and Lange-Nielsen syndrome)EKG (Jervell and Lange-Nielsen syndrome) +/- urinalysis +/- urinalysis CT scan of temporal bonesCT scan of temporal bones Genetic testingGenetic testing
GJB2GJB2
Encodes connexin 26 (Cx26)Encodes connexin 26 (Cx26) Gap junction protein in the cochleaGap junction protein in the cochlea Maps to 13q12Maps to 13q12 2263 nucleotides, 680 amino acids2263 nucleotides, 680 amino acids Two exons; one coding exonTwo exons; one coding exon CpG island near Exon 1CpG island near Exon 1
GJB2GJB2
AR mutations account for 15 – AR mutations account for 15 – 40% of inherited SNHL in North 40% of inherited SNHL in North AmericaAmerica Carrier rate of 1:33 in EuropeansCarrier rate of 1:33 in Europeans Most common mutation in Most common mutation in
Caucasians: 35delGCaucasians: 35delG Mutation spectrum is known to Mutation spectrum is known to
differ by ethnic groupdiffer by ethnic group
Gap Junction ChannelsGap Junction Channels
From Rabionet From Rabionet et al et al in TRENDS in Molecular Medicine Vol.8 No.5 May 2002in TRENDS in Molecular Medicine Vol.8 No.5 May 2002
Expression of Cx26, Cx30 Expression of Cx26, Cx30 and Cx31 in the Cochleaand Cx31 in the Cochlea
From Rabionet From Rabionet et al et al in TRENDS in Molecular Medicine Vol.8 No.5 May 2002in TRENDS in Molecular Medicine Vol.8 No.5 May 2002
Preliminary StudyPreliminary Study
Chart review of 107 patientsChart review of 107 patients Referred to CHAM for genetic Referred to CHAM for genetic
evaluation of SNHLevaluation of SNHL Data collected:Data collected:
EthnicityEthnicity Cx26 mutation statusCx26 mutation status mtDNA DNA analysis (nt 1555, 7445, mtDNA DNA analysis (nt 1555, 7445,
3243, sequencing of 12s rRNA)3243, sequencing of 12s rRNA) CT scan of temporal bonesCT scan of temporal bones
Available SamplesAvailable Samples
107 Samples obtained from IRB 107 Samples obtained from IRB approved research project looking approved research project looking for mtDNA point mutations in SNHLfor mtDNA point mutations in SNHL
192 Controls provided by Dr. Robert 192 Controls provided by Dr. Robert Burk from HPV studyBurk from HPV study
mtDNA and CT ResultsmtDNA and CT Results
one Puerto Rican patient: one Puerto Rican patient: A503G variant + mtDNA mutation at nt A503G variant + mtDNA mutation at nt
14651465 no patient had A1555G or T7445C no patient had A1555G or T7445C
associated with SNHLassociated with SNHL 31 patients had CT scan results:31 patients had CT scan results:
2 had EVA, one of which carries G79A2 had EVA, one of which carries G79A 1 had ? Mondini’s, 1 had prominence of 1 had ? Mondini’s, 1 had prominence of
cochlear aqueducts, 1 had diffuse cochlear aqueducts, 1 had diffuse atrophyatrophy
Project designProject design
1.1. Designing primers for PCRDesigning primers for PCR1.1. Overcoming the GC contentOvercoming the GC content
2.2. Primers for Exons 1&2, and CpG islandPrimers for Exons 1&2, and CpG island
3.3. Identifying sequence variants with Identifying sequence variants with SequencherSequencher
4.4. Examine for known SNPsExamine for known SNPs
5.5. Screening controls with Screening controls with PyrosequencingPyrosequencing
CpG Island PrimersCpG Island Primers CGCCAGGTTCCTGGCCGGGCAGTCCGGGGCCGGCGGGCTCACCTGCGTCGGGAGGAAGCGCGGCGGGGCCGGGGCGGGGGTCTCGGCGTTGGGGTCTCTGCGCTGGGGCTCCTGCGCTCCTAGGCGGGTCCTGGGCCGGGCGCCGCCGAGGGGCTCCGAGTCGGGGAGAGGAGCGCGCGGGCGCTGCGGGGCCGCAACACCTGTCTCCCGCCGTGGCGCCTTTTAACCGCACCCCACACCCCGCCTCTTCCCTCGGAGACTGGGAAAGTTACGGAGGGGGCGGCGCCGCGGGCGGAGCGCGCCCGGCCTCTGGGTCCTCAGAGCTTCCCGGGTCCGCGAACCCCCGACCGCCCCCGAAAGCCCCGAACCCCCCAAGTCCCCTTCGAGGTCCCGATCATCTCCTAGTTCCTTTGAGCCTCCTAGTTCCTTTGAGCC
Patient with a C-34T variant and a G79A polymorphism. Is there significance to these changes when they co-occur?
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 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 leads to a frameshift mutation, as seen on this chromatogram.
Start Codon
G139T homozygous
35delG
35delG common mutation in Caucasian population, found in two Puerto Rican patients and one of mixed Italian, Irish and Jewish decent.
GJB2 Mutations by Ethnicity for 107 Patients
0
5
10
15
20
25
30
Black Black/PR PR DR/PR DR Mexico Guyana India Pakistan Other
Ethnicity
Nu
mb
er o
f P
atie
nts
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
*
* Compound Heterozygote: 35delG + 167delTPR= Puerto RicoDR=Dominican Republic
Schematic of Connexin 26 domains with mutations and
polymorphisms included
Mutations, polymorphisms and variants exhibited in our study are circled
Mutations are shown in GreenGreen
Polymorphisms are shown in PurplePurple
Variants of unknown significance are shown in OrangeOrange
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
• one Dominican patient was one Dominican patient was homozygous for a mutation in GJB2 homozygous for a mutation in GJB2 (G139T)(G139T)
• GJB2 mutations occur in 1/33 GJB2 mutations occur in 1/33 European controls (35delG in 2-4%)European controls (35delG in 2-4%)
• only one Hispanic 35delG carrier in our only one Hispanic 35delG carrier in our controls; all other nucleotide changes controls; all other nucleotide changes were polymorphisms or novel variantswere polymorphisms or novel variants
ConclusionsConclusions
• GJB2 mutations occur less frequently in our GJB2 mutations occur less frequently in our minority populationminority population
• lower carrier frequencies may account for lower carrier frequencies may account for the lower rate of homozygous individuals in the lower rate of homozygous individuals in our populationour population
• possible synergistic interaction of possible synergistic interaction of heterozygous GJB2 mutations and a heterozygous GJB2 mutations and a mutation in another gene such as GJB6mutation in another gene such as GJB6
Future studiesFuture studies
Patient recruitmentPatient recruitment JMC NICU and nurseryJMC NICU and nursery JMC audiology clinicJMC audiology clinic CHAM Craniofacial CenterCHAM Craniofacial Center
Cx30Cx30 Adjacent to GJB2Adjacent to GJB2 Mutations are rareMutations are rare
May lead to AD late onset deafnessMay lead to AD late onset deafness Deletions Deletions
Homozygous → deafnessHomozygous → deafness Heterozygous in trans with GJB2 Heterozygous in trans with GJB2
mutation → deafnessmutation → deafness
Future DirectionsFuture Directions
SLC26A4SLC26A4 Encodes monovalent and divalent Encodes monovalent and divalent
anion transporter related proteins anion transporter related proteins (Pendrin)(Pendrin) Involved in fluid homeostasisInvolved in fluid homeostasis
Mutations cause Pendred syndrome Mutations cause Pendred syndrome (AR; defects of thyroid, kidney and (AR; defects of thyroid, kidney and inner ear)inner ear)
Often also see Enlarged Vestibular Often also see Enlarged Vestibular Aqueduct (EVA) or Mondini dysplasiaAqueduct (EVA) or Mondini dysplasia
Reference ListReference ListFor more information on this topic, see the following For more information on this topic, see the following
publications:publications:Marazita ML, et al., (1993) Genetic epidemiologic studies of early-onset 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-deafness in the U.S. school-age population. Am J Med Genet 46:486-491).491).
Kelsell DP, et al., (1997) Connexin 26 mutations in hereditary non-Kelsell DP, et al., (1997) Connexin 26 mutations in hereditary non-syndromal sensoineural deafness. Nature 387(6628):80-83.syndromal sensoineural deafness. Nature 387(6628):80-83.
Morton, C (2002) Genetics, genomics and gene discovery in the auditory Morton, C (2002) Genetics, genomics and gene discovery in the auditory system. Human Molecular Genetics 11(10):1229-1240.system. Human Molecular Genetics 11(10):1229-1240.
Rabionet R, et al., (2002) Connexin mutations in hearing loss, Rabionet R, et al., (2002) Connexin mutations in hearing loss, dermatological and neurological disorders. Trends in Mol Med dermatological and neurological disorders. Trends in Mol Med 8(5):205-212).8(5):205-212).
Pandya, A, et al., (2003) Frequency and distribution of GJB2 (connexin Pandya, A, et al., (2003) Frequency and distribution of GJB2 (connexin 26) and GJB6 (connexin 30) mutations in a large North American 26) and GJB6 (connexin 30) mutations in a large North American repository of deaf probands. Genet Med 5(4):295-303.repository of deaf probands. Genet Med 5(4):295-303.
Additional information may be found at:Additional information may be found at:http://davinci.crg.es/deafness/http://davinci.crg.es/deafness/
