Upload
satyender-kumar
View
314
Download
3
Tags:
Embed Size (px)
Citation preview
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
How ear work?
http://www.dailymail.co.uk/health/article-1278160/Deafness-cure-breakthrough-scientists-create-tiny-ear-hairs-stem-cells.html2
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.
Hearing loss
3
Hearing loss can be categorized by whichpart of the auditory system is damaged
Conductive hearing loss
Sensorineuralhearing loss
Mixed hearing loss
Types of hearing loss
4
4.25 4.34
7.4
9.5
0
1
2
3
4
5
6
7
8
9
10
2001 2004 2008 2015
Pe
rce
nta
ge %
Epidemiology
WHO Deafness Fact Sheet 2013 5
According to the estimates of WHO, 278 million people have disabling hearingimpairment.The prevalence of deafness in India, 63 million people (6.3%) suffer fromsignificant auditory loss.
Contd.
WHO Deafness Fact Sheet 20136
Causes of Deafness Malformation of outer ear, ear canal, or middle
ear structuresFluid in the middle ear from coldsEar infectionAllergiesPoor Eustachian tube functionPerforated eardrumBenign tumorsImpacted earwaxNoiseGenetic factors
7
Genetics of Deafness in India• In, India about 30% of hearing loss may be genetic or
hereditary.World
GJB2
SLC26A4
GJB6
DFNB1
DFNB30
MTRNR1
India
GJB2
DFNB2
SLC26A4
TMC1
Ghosh M, Vijaya R, Kabra M. Genetics of deafness in India. Indian Journal of Pediatricts 200;71: 531-533.8
Mutation
A mutation is a permanent change in the DNAsequence of a gene.
Mutations in a gene's DNA sequence can alter theamino acid sequence of the protein encoded by thegene.
9
Point mutation
A point mutation, or single base substitution, is a typeof mutation that causes the replacement of a singlebase nucleotide with another nucleotide of the geneticmaterial, DNA or RNA.
The term point mutation also includes insertions or deletionsof a single base pair.
10
PCR restriction fragment length polymorphism (RFLP) analysis
Direct sequencing
Real-time PCR analysis
Mass spectrometry
Amplification-Refractory Mutation System (ARMS-PCR ) technique
Methods for the individual detection of specific point mutations
11
Newborn genetic screening is ahealth program that identifiestreatable genetic disorders innewborn infants.
Early intervention to treat thesedisorders can eliminate or reducesymptoms that might otherwisecause a lifetime of disability.
Newborn genetic screening
12
Targeted disorders
Hearing loss
Amino acid
disorders
Fatty acid oxidation disorders
Cystic fibrosis
Urea cycle disorders
Congenital heart
defects
Severe combined immunode
ficiency
13
14
Deafness
GJB2c.235delC
SLC26A4 c.919-2A>G
MTRNR1
mt.1555A>G/mt.1494C>T
Objectives
Han B, Zong L, Li Q, Zhang Z, Wang D, Lan L, Zhang J, Zhao Y, Wang Q. Newborn genetic screening for high risk deafness-associated mutations with a new Tetra-primer ARMS PCR kit. International jounal of Pediatric Otorhinolaryngology 2013;77: 1440-1445.
A cost effective method for the screening deafness associated mutations at early age
15
16
Ethics approval
Collection of samples
Isolation of genomic DNA
Design of Tetra primer for ARMS-PCR
Validation by Sanger sequencing
Materials and methods
Ethics approval
17
Approval of ethics committee of theChinese PLA General Hospital was takenfor the participation of 1181 Chinesenewborns
18
Collection of samples
Collection of neonates blood samples were collected from umbilical cord with a specimen collection card
19
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
Two allele specific amplicons were separated by gel electrophoresis
Both the wild type and mutant type ampliconswere simultaneously amplified with PCR reaction
Two inner primers in opposite direction
2- different non-specific outer primers (F1, R1)
2- different specific inner primers (F2, R2)
21
Design of Tetra primer ARMS-PCR
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
Validation of tetra primer ARMS PCR kitwith wild and mutant type DNA sampleswas done by Sanger sequencing
Validation of tetra primer ARMS-PCR
23
All samples were distinguished on 2.5% gel electrophoresis as shown in figures 1-4
For MTRNR1 mt.1555A>G and mt.1494C>T two conditions of Mutation type and Wild type were identified
For the GJB2 c.235delC and SLC26A4 c,919-2A>G three situations of Wild type, Homozygous and Heterozygous
genotypes were identified
The genotypic data and procedures were validated by analysis of DNA samples of 1181 newborns
Results
24
25
Genotyping of MTRNR1 mt.1555A>G point mutations
26
Genotyping of MTRNR1 mt.1494C>T point mutations
27
Genotyping of SLC26A4 c.919-2A>G point mutations
28
Genotyping for GJB2 c.235delC point mutations
Mutation frequency of the four point mutations in samples
29
Conclusion
Collection of samples and isolation of genomic DNAwas done from 1181 newborn samples.
Screening and validation methods forGJB2, SLC26A4, mtDNA 12S rRNA mutations werecarried out.
No false positive results were found.
A rapid, reproducible and cost effective detection ofdeafness gene mutation without special equipmentwas developed.
Detection of the 4-high risk deafness associatedmutations with only 4 single tube PCR reaction.
30
For future aspects
Larger-scale epidemiological studies might bepossible about hereditary hearing loss to addmore screening targets and to improve moleculardiagnosis and genetic counseling
31
32