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Newborn genetic screening for high risk deafness associated 2
1. Newborn genetic screening for high
risk deafness associated mutations
with a new Tetra-primer ARMS PCR
Presented by:
Satyender kumar
Department of Natural Products
1
3. Hearing loss
Degree of hearing loss
Hearing loss range (db HL)
Normal
-10 to 15
Slight
16-25
Mild
26-40
Moderate
41-55
Moderate severe
56-70
Severe
71-90
Profound
91+
Clark, J. G. (1981). Uses and abuses of hearing loss classification. Asha, 23, 493–500.
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4. Types of hearing loss
Hearing loss can be categorized by which
part of the auditory system is damaged
Conductive
hearing loss
Sensorineural
hearing loss
Mixed
hearing loss
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6. Contd.
According to the estimates of WHO, 278 million people have disabling hearing
impairment.
The prevalence of deafness in India, 63 million people (6.3%) suffer from
significant auditory loss.
WHO Deafness Fact Sheet 2013
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7. Causes of Deafness
Malformation of outer ear, ear canal, or middle
ear structures
Fluid in the middle ear from colds
Ear infection
Allergies
Poor Eustachian tube function
Perforated eardrum
Benign tumors
Impacted earwax
Noise
Genetic factors
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8. Genetics of Deafness in India
• In, India about 30% of hearing loss may be genetic or
hereditary.
World
India
GJB2
GJB2
SLC26A4
DFNB2
GJB6
SLC26A4
DFNB1
TMC1
DFNB30
MTRNR1
Ghosh M, Vijaya R, Kabra M. Genetics of deafness in India. Indian Journal of Pediatricts 200;71: 531-533.
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9. Mutation
A mutation is a permanent change in the DNA
sequence of a gene.
Mutations in a gene's DNA sequence can alter the
amino acid sequence of the protein encoded by the
gene.
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10. Point mutation
A point mutation, or single base substitution, is a type
of mutation that causes the replacement of a single
base nucleotide with another nucleotide of the genetic
material, DNA or RNA.
The term point mutation also includes insertions or deletions
of a single base pair.
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11. Methods for the individual detection
of specific point mutations
PCR restriction fragment length
polymorphism (RFLP) analysis
Direct sequencing
Real-time PCR analysis
Mass spectrometry
Amplification-Refractory Mutation System
(ARMS-PCR ) technique
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12. Newborn genetic screening
Newborn genetic screening is a
health program that identifies
treatable genetic disorders in
newborn infants.
Early intervention to treat these
disorders can eliminate or reduce
symptoms that might otherwise
cause a lifetime of disability.
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15. Objectives
A cost effective method for the screening deafness associated
mutations at early age
SLC26A4 c.9192A>G
MTRNR1
GJB2c.235delC
mt.1555A>G/mt
.1494C>T
Deafness
Han B, Zong L, Li Q, Zhang Z, Wang D, Lan L, Zhang J, Zhao Y, Wang Q. Newborn genetic screening for high risk deafnessassociated mutations with a new Tetra-primer ARMS PCR kit. International jounal of Pediatric Otorhinolaryngology 2013;
77: 1440-1445.
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16. Materials and methods
Ethics approval
Collection of samples
Isolation of genomic DNA
Design of Tetra primer for ARMS-PCR
Validation by Sanger sequencing
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17. Ethics approval
Approval of ethics committee of the
Chinese PLA General Hospital was taken
for the participation of 1181 Chinese
newborns
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18. Collection of samples
Collection of neonates blood samples
were collected from umbilical cord with a
specimen collection card
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20. Isolation of genomic DNA
DNA extraction
Genomic DNA was extracted according to
ARMS –PCR kit (BIOSINO, China)
The yield of every blood sample produced
>10 ng/μl genomic DNA
Testing of quality and quantity of
extracted DNA using UV at 260 nm and gel
electrophoresis, respectively
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21. Design of Tetra primer ARMS-PCR
2- different specific inner primers (F2, R2)
2- different non-specific outer primers (F1, R1)
Two inner primers in opposite direction
Both the wild type and mutant type amplicons
were simultaneously amplified with PCR reaction
Two allele specific amplicons were separated by
gel electrophoresis
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22. ARMS-PCR principle
You FM, Huo N, Gu YQ, Luo MC, Ma Y, Hane D, Lazo GR, Dvorak J, Anderson OD - BMC Bioinformatics (2008) 22
23. Validation of tetra primer ARMS-PCR
Validation of tetra primer ARMS PCR kit
with wild and mutant type DNA samples
was done by Sanger sequencing
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24. Results
The genotypic data and procedures were validated by analysis
of DNA samples of 1181 newborns
For the GJB2 c.235delC and SLC26A4 c,919-2A>G three
situations of Wild type, Homozygous and Heterozygous
genotypes were identified
For MTRNR1 mt.1555A>G and mt.1494C>T two conditions of
Mutation type and Wild type were identified
All samples were distinguished on 2.5% gel electrophoresis as
shown in figures 1-4
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30. Conclusion
Collection of samples and isolation of genomic DNA
was done from 1181 newborn samples.
Screening
and
validation
methods
for
GJB2, SLC26A4, mtDNA 12S rRNA mutations were
carried out.
No false positive results were found.
A rapid, reproducible and cost effective detection of
deafness gene mutation without special equipment
was developed.
Detection of the 4-high risk deafness associated
mutations with only 4 single tube PCR reaction.
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31. For future aspects
Larger-scale epidemiological studies might be
possible about hereditary hearing loss to add
more screening targets and to improve molecular
diagnosis and genetic counseling
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