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

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

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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

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4.25 4.34

7.4

9.5

0

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2

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4

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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

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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.

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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.

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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

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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

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Targeted disorders

Hearing loss

Amino acid

disorders

Fatty acid oxidation disorders

Cystic fibrosis

Urea cycle disorders

Congenital heart

defects

Severe combined immunode

ficiency

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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

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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

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

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Collection of samples

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

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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)

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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

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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

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Genotyping of MTRNR1 mt.1555A>G point mutations

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Genotyping of MTRNR1 mt.1494C>T point mutations

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

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Genotyping for GJB2 c.235delC point mutations

Mutation frequency of the four point mutations in samples

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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.

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For future aspects

Larger-scale epidemiological studies might bepossible about hereditary hearing loss to addmore screening targets and to improve moleculardiagnosis and genetic counseling

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