Research

Cochlea, Pune is primarily involved in deafness rehabilitation of 0-6 year age group children who are born deaf. Early detection, intervention, application of hearing aid or undergoing cochlear implant before age of six is very critical for a child to have a possibility of speech development. Our research is focused on following areas:
 
Epidemiology of hearing loss in children in Pune district of state of Maharashtra and State of Goa
Speech analysis of children pre or post cochlear implant
Molecular diagnostics of prelingual deafness
Pedigree analysis of the family of deaf children
Refining of hearing aids

Details of the collaborative project between Cochlea, Pune and Dept. of Biotechnology, MES Abasaheb Garware College

Collaborative project was for the period of 2014 – 2016. Consumables for the project worth Rs. 10000-00 was provided by Cochlea Pune and instrumentation set up of MES abasaheb Garware college was used to carry out the project.
 
Total 3 postgraduate and 5 undergraduate students of biotechnology had worked on the project under the guidance of Dr. Prashant S. Duraphe. Titles and the abstract of the project are enlisted below.
 

1) Epidemiology and rehabilitation of prelingual Deafness in children attending special school



Name of student on the project: Ms. Jyostna Galande
Duration of project: 01-01-2015 – 30-06-2015
Objectives: 
a) Speech analysis (correlation of age, start of therapy, hearing aid, cochlear implant, BERA)
Data collection of children attending Swaranaad school and Cochlea clinic
b) Pedigree analysis for deafness inheritance 
Deafness history in children’s family for analysis of inheritance pattern  
c) Genetic basis of deafness (PCR-RFLP based mutation analysis of connexin 26 gene)
Collection of sample for DNA isolation (Buccal swab), DNA Extraction, DNA analysis for quantity and quality, Designing of primers for mutations in Connexin 26 gene amplification by PCR RFLP, Genetic diagnosis of deafness by analysis of connexin 26 expression pattern.
 
Abstract:
Mutations in the Cx26 gene have been shown to cause autosomal recessive nonsyndromic hearing loss (ARNSHL) at the DFNB1 locus on chromosome 13q12. Using gene specific primer- PCR and RFLP, screening of the 35delG mutation in GJB2 gene of 16 deaf families was designed. To that end non invasive DNA isolation protocol from buccal cells was optimized. The isolated DNA was characterized by agarose gel electrophoresis and spectrophotometric analysis for assessing the integrity and purity of isolated genomic DNA. Genetic inheritance of deafness within families was studied by pedigree analysis of 23 families. Out of 23 families, there were 21 consanguineous cases. The expected phenotype was observed in first, second, third generation.  
 

2) A semi-nested PCR RFLP based method for identification of mutations in connexin 26 gene that cause autosomal recessive nonsyndromic hearing loss

Name of student on the project: Ms. Kajal Patel
Duration of project: 01-01-2016 – 15-04-2016
Objectives: 
a) Primer design for various mutations in connexin 26 gene (35delG, W24X, I33T and V37I)
b) Genetic basis of deafness (PCR-RFLP based mutation analysis of connexin 26 gene)
Collection of sample for DNA isolation (Buccal swab), DNA Extraction, DNA analysis for quantity and quality, Designing of primers for mutations in Connexin 26 gene amplification by PCR RFLP
 
Abstract 
 
Mutations in the Cx26 gene have been shown to cause autosomal recessive nonsyndromic hearing loss (ARNSHL) at the DFNB1 locus on chromosome 13q12. Using PrimerBLAST primers for four mutations (35delG, W24X, I33T and V37I) were designed. To that end noninvasive DNA isolation protocol from buccal cells was optimized. The isolated DNA was characterized by agarose gel electrophoresis and spectrophotometric analysis for assessing the integrity and purity of isolated genomic DNA. A semi-nested PCR method for 35delG mutation detection was partially optimized.  
 

3) Study of mutations in connexin 26 gene that causes autosomal recessive nonsyndromic hearing loss in Indian population

Name of student on the project: Ms. Hriticka Choudhurry
Duration of project: 01-01-2016 – 15-04-2016
 
Objectives: 
a) Primer design for mutation in connexin 26 gene (IVS1+1G>A, W77R and W77X)
b) Genetic basis of deafness (PCR-RFLP based mutation analysis of connexin 26 gene)
Collection of sample for DNA isolation (Blood), DNA Extraction, DNA analysis for quantity and quality, Designing of primers for mutations in Connexin 26 gene amplification by PCR RFLP
 
Abstract: 
 
BACKGROUND: Hearing loss, most common sensorineural disorder that affects half the elderly population, can be categorised into non-syndromic hearing loss (NSHL) and syndromic hearing loss (SHL). Among the autosomal recessive genes that cause NSHL, Gap Junction protein beta 2 (GJB2) gene is the most important. The splice site mutation IVS1+1G>A, also called -3172G>A, in the splice donor site of intron 1 in GJB2 is considered as a disruptive mutation yielding no detectable mRNA. In the present study we carried out PCR and RFLP (restriction fragment length polymorphism) based analysis of 35delG mutation in GJB2 and its optimization. RFLP could not be carried out due to time constraints. Primer designing was carried out that can be used for PCR-RFLP based detection of IVS1+1G>A mutation.
 
RESULT: PCR optimization was not complete so as to obtain connexin 26 amplification corresponding to 35delG mutation. Connexin 26 homologs were found out in all possible organisms by database searches. Primers were designed such that they create a restriction enzyme site so as to detect the mutated gene in patients. It was found that CjeFIII enzyme can be used in case of PCR RFLP based detection of IVS1+1G>A mutation of GJB2 gene. 
 

4) A study of mutations in connexin 26 protein focusing on W77R and W77X mutations and primer designing for its detection using PCR

Name of student on the project: Mr. Aniruddha Deo
Duration of project: 01-01-2016 – 15-04-2016
 
Objectives: 
a) Primer design for various mutations in connexin 26 gene (W24X, W77R, W77X, I33T, V37I)
 
Abstract: 
 
Autosomal recessive non syndromic hearing loss (ARNSHL) is caused by mutations in the gap junction gene, GJB2, which codes for connexin26 human populations. The aim of this project was to study the mutations of connexin26 specifically W77R and W77X of GJB2 gene in humans and to design primers for the said mutations. Detailed literature survey was done of connexin 26 mutations and their detection methods. W24X, W77R and W77X are most frequent mutations in Indian subcontinent, among documented GJB2 mutations. Primers were designed for W77X, W77R, W24X, I33T and V37I. The primer design was done using primer3 and blast-primer. 
 

5) Isolation of DNA by salting out method for the study of prelingual deafness due to mutations in connexin 26

Name of student on the project: Ms. Akanksha Limaye
Duration of project: 01-01-2016 – 15-04-2016
 
Objectives: 
a) Genetic basis of deafness (PCR-RFLP based mutation analysis of connexin 26 gene)
Collection of sample for DNA isolation (Blood), DNA Extraction, DNA analysis for quantity and quality
 
Abstract 
 
The major cause for hearing impairment is identified to be hereditary. The mutation in gene 
gap junction beta 2 (GJB2) encoding Connexin 26 is the major cause of hereditary hearing loss. A laboratory experiment aimed to study the mutation of Connexin 26 was carried out. The method of Salting out for the extraction of DNA from blood of healthy individual was employed. A frame shift mutation, 35delG, was particularly reported to be responsible for more than 50 percent of all cases of childhood non-syndromic hearing loss in some populations. The integrity and purity of isolated DNA was checked using agarose gel electrophoresis and spectrophotometry respectively. A genomic DNA of sufficiently good quality may be used for downstream applications such as PCR/RFLP for connexin 26 mutation analysis in deafness diagnosis. 
The salting out method used for isolation was modified to increase the yield of the DNA. The literature survey was carried out for the modifications made. A comparative experimentation for detecting the appropriate methodology was done. The organic solvent method was used as the second method for the study and results were compared and checked.
 

6) Isolation of DNA by organic solvent method to study prelingual deafness caused due to mutation in connexin 26 protein

 
Name of student on the project: Ms. Aknkita Galgale
Duration of project: 01-01-2016 – 15-04-2016
 
Objectives: 
a)  Genetic basis of deafness (PCR-RFLP based mutation analysis of connexin 26 gene)
Collection of sample for DNA isolation (Blood), DNA Extraction, DNA analysis for quantity and quality
 
Abstract: 
Mutations in the gene gap junction beta 2 (GJB2) encoding connexin 26 (cx26) have been linked to sensorineural hearing loss either alone or as part of a syndrome. For this study, genomic DNA was isolated from human blood. The DNA was isolated by two different methods; one by using organic solvents and other by using salting out method. Organic solvent method was used for further studies because the DNA concentration obtained by this method was more than the salting-out method. DNA was characterized for quality and integrity by spectrophotometer and agarose gel electrophoresis respectively. The obtained DNA was contaminated with proteins and RNA, hence slight modifications were made in the original procedure. We could not amplify given DNA for connexin 26 gene specific primers by PCR due to impurities present in the isolated DNA and hence could not proceed further for RFLP analysis.
 

7) Optimization of DNA isolation from buccal cells for PCR RFLP based diagnosis of Connexin 26 mutations in prelingual deafness

Name of student on the project: Ms. Minal Ayachit, Ms. Susmita Bhadre
Duration of project: 01-01-2016 – 15-04-2016
 
Objectives: 
a) Genetic basis of deafness (PCR-RFLP based mutation analysis of connexin 26 gene)
Collection of sample for DNA isolation (Buccal swab), DNA Extraction, DNA analysis for quantity and quality
 
Abstract:
Non syndromic autosomal recessive hearing loss accounts for about 70-80% of the hearing loss in the world. Various mutations within the coding and non coding regions of multiple genes cause genetic impairment in the function. A mutation 35delG in the Gap junction beta 2 gene affects the function of connexin 26 is a major cause of deafness. This mutation can be detected by using a mutant primer in PCR RFLP process. This project deals with the optimization of DNA isolation from buccal cell swab. Optimization in simple terms is changing a set of parameters with respect to one control and finding out the method that may give the best result or maximum yield. The DNA was characterized by spectrophotometric analysis and agarose gel electrophoresis for its purity and integrity.
 



Figure 1: Pedigree analysis of deaf children attending Swaranaad pre-school
 
a) There are 13 families (consanguineous marriages) which has deaf child in 2nd generation                                 b) There are 4 families (consanguineous marriages) which have deaf children in 3rd generation.
c) There is 1 family (consanguineous marriage) which shows deafness in 2nd and 3rd generation.
d) There are two families (consanguineous marriages) which show deafness in 4th generation.


Figure 2: Primer design for connexin 26 mutation analyses in genetic diagnosis of deafness
Four prevalent mutations in Indian population namely W24X, I33T, V37I, and 35delG can be analyzed by semi nested PCR followed by RFLP analysis of the DNA isolated from deaf children and family.



Figure 3: Agarose gel electrophoresis of DNA isolated from buccal cells (A) and from blood (B) of the deaf children of Swaranaad pre-school.
 

Success Stories

  • photo
    Irfaan Mulani

    Irfaan did his preschool very well from the age 3 to 6yrs at Swaranaad. He got promoted successfully every year and was integrated into a main stream school in 2016

  • photo
    Shraddha Shilimkar

    Shraddha progressed from a hearing impaired child who could not speak at the age of 6 to a young girl, who is a blackbelt holder in Karate, a bharat natyam dancer and has scored 65% in her SSC exams this year.

  • photo
    Siddhant Gaikwad

    Swaranaad is proud of him as he was admitted directly to second standard, After the principal took his test, she said, “he knows all and more than we teach in the first standard, so why waste his year. He was detected when he was two yrs old. After the speech therapy he joined Swaranaad.

  • photo
    Om Babar
    Om came to Cochlea at the age of four years. He became confident as he was getting good academic training in ‘Swaranaad’ in a group of 6 children and developed language skills in Manoram in a group of 60 children.

  • photo
    Asawari Kulkarni
    Asawari bagged the ‘Best Student’ award in June 2015.She has scored 89% marks in her SSC exams from a school for normal children. She has passes the Sanskrit “ Madhyama’ exam and is a good Bharat Natyam dancer.

  • photo
    Hrishkesh Patil
    Hrishikesh can speak Marathi, English and Hindi. He has done M. Com., has won prizes in photography and is a hardcore trekker.Photos- compering, trekking photography prize.

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