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Newborn genetic screening for high risk deafness associated 2



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  • 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
  • 2. How ear work? 2
  • 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. 3
  • 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 4
  • 5. Epidemiology 10 9 8 Percentage % 7 6 5 9.5 4 7.4 3 2 4.25 4.34 2001 2004 1 0 WHO Deafness Fact Sheet 2013 2008 2015 5
  • 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 6
  • 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 7
  • 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. 8
  • 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. 9
  • 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. 10
  • 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 11
  • 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. 12
  • 13. Severe combined immunode ficiency Congenital heart defects Hearing loss Amino acid disorders Targeted disorders Urea cycle disorders Fatty acid oxidation disorders Cystic fibrosis 13
  • 14. 14
  • 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. 15
  • 16. Materials and methods Ethics approval Collection of samples Isolation of genomic DNA Design of Tetra primer for ARMS-PCR Validation by Sanger sequencing 16
  • 17. Ethics approval Approval of ethics committee of the Chinese PLA General Hospital was taken for the participation of 1181 Chinese newborns 17
  • 18. Collection of samples Collection of neonates blood samples were collected from umbilical cord with a specimen collection card 18
  • 19. 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 20
  • 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 21
  • 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 23
  • 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 24
  • 25. Genotyping of MTRNR1 mt.1555A>G point mutations 25
  • 26. Genotyping of MTRNR1 mt.1494C>T point mutations 26
  • 27. Genotyping of SLC26A4 c.919-2A>G point mutations 27
  • 28. Genotyping for GJB2 c.235delC point mutations 28
  • 29. Mutation frequency of the four point mutations in samples 29
  • 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. 30
  • 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 31
  • 32. 32