32
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

Newborn genetic screening for high risk deafness associated 2

Embed Size (px)

Citation preview

Page 1: Newborn genetic screening for high risk deafness associated 2

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

Page 2: Newborn genetic screening for high risk deafness associated 2

How ear work?

http://www.dailymail.co.uk/health/article-1278160/Deafness-cure-breakthrough-scientists-create-tiny-ear-hairs-stem-cells.html2

Page 3: Newborn genetic screening for high risk deafness associated 2

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

Page 4: Newborn genetic screening for high risk deafness associated 2

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

Page 5: Newborn genetic screening for high risk deafness associated 2

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

Page 6: Newborn genetic screening for high risk deafness associated 2

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

Page 7: Newborn genetic screening for high risk deafness associated 2

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

Page 8: Newborn genetic screening for high risk deafness associated 2

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

Page 9: Newborn genetic screening for high risk deafness associated 2

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

Page 10: Newborn genetic screening for high risk deafness associated 2

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

Page 11: Newborn genetic screening for high risk deafness associated 2

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

Page 12: Newborn genetic screening for high risk deafness associated 2

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

Page 13: Newborn genetic screening for high risk deafness associated 2

Targeted disorders

Hearing loss

Amino acid

disorders

Fatty acid oxidation disorders

Cystic fibrosis

Urea cycle disorders

Congenital heart

defects

Severe combined immunode

ficiency

13

Page 14: Newborn genetic screening for high risk deafness associated 2

14

Page 15: Newborn genetic screening for high risk deafness associated 2

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

Page 16: Newborn genetic screening for high risk deafness associated 2

16

Ethics approval

Collection of samples

Isolation of genomic DNA

Design of Tetra primer for ARMS-PCR

Validation by Sanger sequencing

Materials and methods

Page 17: Newborn genetic screening for high risk deafness associated 2

Ethics approval

17

Approval of ethics committee of theChinese PLA General Hospital was takenfor the participation of 1181 Chinesenewborns

Page 18: Newborn genetic screening for high risk deafness associated 2

18

Collection of samples

Collection of neonates blood samples were collected from umbilical cord with a specimen collection card

Page 19: Newborn genetic screening for high risk deafness associated 2

19

Page 20: Newborn genetic screening for high risk deafness associated 2

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

Page 21: Newborn genetic screening for high risk deafness associated 2

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

Page 22: Newborn genetic screening for high risk deafness associated 2

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

Page 23: Newborn genetic screening for high risk deafness associated 2

Validation of tetra primer ARMS PCR kitwith wild and mutant type DNA sampleswas done by Sanger sequencing

Validation of tetra primer ARMS-PCR

23

Page 24: Newborn genetic screening for high risk deafness associated 2

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

Page 25: Newborn genetic screening for high risk deafness associated 2

25

Genotyping of MTRNR1 mt.1555A>G point mutations

Page 26: Newborn genetic screening for high risk deafness associated 2

26

Genotyping of MTRNR1 mt.1494C>T point mutations

Page 27: Newborn genetic screening for high risk deafness associated 2

27

Genotyping of SLC26A4 c.919-2A>G point mutations

Page 28: Newborn genetic screening for high risk deafness associated 2

28

Genotyping for GJB2 c.235delC point mutations

Page 29: Newborn genetic screening for high risk deafness associated 2

Mutation frequency of the four point mutations in samples

29

Page 30: Newborn genetic screening for high risk deafness associated 2

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

Page 31: Newborn genetic screening for high risk deafness associated 2

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

Page 32: Newborn genetic screening for high risk deafness associated 2

32