hearing loss is regarded as a common neurosensory disorder , and affects approximately 12 in 1000 live births ( 1 ) . hearing loss is a multifactorial defect caused by genetic , environmental factors or a combination of both ( 2 ) . the role of genetic factors in the pathogenesis of hearing loss far outweighs the environmental parameters , and about 60% of the hearing loss is caused by genetic changes ( 3 ) . the inheritance patterns of genetic hearing loss are autosomal recessive ( 77% ) , autosomal dominant ( 22% ) , x - linked ( 1% ) , and mitochondrial ( < 1% ) ( 4 ) . based upon the presence or absence of distinctive clinical features , hearing loss can be classified into syndromic ( 2030% ) and non - syndromic ( 7080% ) forms ( 5 ) . autosomal recessive non - syndromic hearing loss ( arnshl ) is the most frequent hereditary form of hearing loss ( hl ) , and displays a very allelic and locus heterogeneity ( 6 ) . to date , more than seventy loci have been mapped for arnshl by linkage analysis , and they are designated as dfnb followed by an identification number ( 7 ) . totally 40 genes have been identified in these loci ( 8) . in attempt to find candidate locus for arnshl , dfnb1 ( mim 220290 ) was initially identified by guilford et al . which accounts for large percentage of arnshl ( 9 , 10 ) . in the dfnb1 locus , mutations in gjb2 gene , encoding the gap junction beta-2 protein connexin 26 are estimated to be responsible for 1050% of arnshl in many populations ( 11 ) . other common loci are associated with arnshl including dfnb3 ( myo15a gene , mim 602666 ) , dfnb9 ( otof gene , mim 603681 ) and dfnb21 ( tecta gene , mim 603629 ) ( 8 , 12 ) . the role of dfnb3 locus in arnshl pathogenesis was initially identified in a population of villagers of bengkala , bali ( 13 ) . the myo15a gene within the dfnb3 locus encodes myosin xva , an unconventional myosin , is a motor protein in hair cells of cochlea that deliver whirlin protein to the tips of stereocilia , and play fundamental role in formation of stereocilia structure ( 9 ) . another gene , otof ( dfnb9 ) , encodes otoferlin , a large trans membrane protein , play an important role in exocytosis of synaptic vesicles at the synapse between inner hair cell and auditory nerve fibers ( 14 ) . it has been previously reported that otof mutations lead to a unique form of arnshl called auditory neuropathy ( 15 ) . the tecta ( dfnb21 ) , a highly polymorphic gene , encodes alpha - tectorin , a glycoprotein that interacts with beta - tectorin to form the noncollagenous matrix of the tectorial membrane ( 16 ) . the tecta gene mutation may be disrupt the structure of this matrix that leads to insufficient transmission and amplification of sound in the inner ear ( 16 ) . due to large number of consanguineous marriage in middle east compared to european and american population , arnshl is 23 times more common in the middle east ( 12 ) . iran , with a heterogeneous population due to various ethnicities and with high consanguineous marriage rate ( 38.6% ) , represents a worthy opportunity for genetic linkage analysis of arnshl ( 17 ) . therefore , we aimed to address the question of  whether dfnb3 , dfnb9 and dfnb21 loci can be contributed to arnshl or not ,  by genetic linkage analysis these loci in gjb2 negative families . in this descriptive study , 32 families with at least two affected children were identified from hormozgan province of iran , during 20132014 . hl screening for members of the families was performed by pure tone audiometric test and clinical examination . hl informational questionnaires were obtained from all families and the pedigrees were drawn based on the filled - out questionnaires and interview with the members of the families by genetic counselors . families with the following criteria were excluded from the study : syndromic hearing loss , exposure to known environmental risk factors such as trauma ototoxic drugs , rubella during pregnancy and excessive noise . based upon pedigree of families , sixteen families with 24 affected children were finally screened as arnshl . this study was approved by the ethics and human rights committee of hormozgan university of medical sciences , bandar abbas , iran and the informed consents and demographic characteristics were filled out by all participants before enrolment . peripheral blood samples ( 5 ml from each ) were taken from all members of the families and genomic dna was extracted from the whole blood using the salting - out method as previously described ( 18 ) . all affected individuals were screened for mutations in exon 2 of the gjb2 gene using direct sequencing technique . to amplify the exon 2 of the gjb2 gene , the primers f ( 5-ctccctgttctgtcctagct-3 ) and r ( 5-ctcatccctctcatgctgtc-3 ) were used in the pcr procedure , which yield a 809 bp fragment . the pcr solutions contained 100 ng genomic dna , 10 pcr buffer , 10 pmol of each primers , 10 nmol of each deoxyribonucleotide triphosphates , 1.5 mmol mgcl2 and 1 u taq polymerase in a final volume of 25 l . initial denaturation at 95 c for 2 min was followed by 35 cycles of denaturation at 94 c for 30 sec and annealing at 59 c for 30 sec , an extension at 72 c for 30 sec and a final extension at 72 c for 5 min . the pcr products were separated on 1% agarose gel and visualized with ethidium bromide staining . the amplified products were analyzed using direct sequencing method ( macrogen , south korea ) . we used chromas software to analyze the chromatograms and then the sequencing results were compared with the human genome database and genbank . negative samples for gjb2 gene mutations were tested for linkage analysis of dfnb3 , dfnb9 and dfnb21 loci using str markers shown in table 1 . based upon strs physical distance in ncbi map viewer and ncbi unists a panel of 11 different str markers was genotyped for dfnb3 , dfnb9 and dfnb21 loci , according to the selection criteria including greater heterozygosity values , shorter amplicon and locating near the known locus . the pcr solutions contained 100 ng genomic dna , 10pcr buffer , 10 pmol of each primers , 10 nmol of each deoxyribonucleotide triphosphates , 1.5 mmolmgcl2 and 1u taq polymerase in a final volume of 25 l . a two - step touchdown thermal cycling was designed for each str markers based on the tm of both primers . pcr was started with an initial denaturation step 94 c for 5 min was followed by 7 cycles of denaturation ( 94 c , 15 sec ) , annealing ( 60 c , 40 sec ) and extension annealing ( 72 c , 40 sec ) followed by a final 25 cycles of denaturation ( 96 c , 40 sec ) , annealing ( 58 c , 40 sec ) , and extension ( 72 c , 40 sec ) and final extension at 72 c for 10 min . finally , the pcr products were separated by electrophoresis procedure on 10% polyacrylamide gels stained by silver nitrate . in this descriptive study , 32 families with at least two affected children were identified from hormozgan province of iran , during 20132014 . hl screening for members of the families was performed by pure tone audiometric test and clinical examination . hl informational questionnaires were obtained from all families and the pedigrees were drawn based on the filled - out questionnaires and interview with the members of the families by genetic counselors . families with the following criteria were excluded from the study : syndromic hearing loss , exposure to known environmental risk factors such as trauma ototoxic drugs , rubella during pregnancy and excessive noise . based upon pedigree of families , sixteen families with 24 affected children were finally screened as arnshl . this study was approved by the ethics and human rights committee of hormozgan university of medical sciences , bandar abbas , iran and the informed consents and demographic characteristics were filled out by all participants before enrolment . peripheral blood samples ( 5 ml from each ) were taken from all members of the families and genomic dna was extracted from the whole blood using the salting - out method as previously described ( 18 ) . all affected individuals were screened for mutations in exon 2 of the gjb2 gene using direct sequencing technique . to amplify the exon 2 of the gjb2 gene , the primers f ( 5-ctccctgttctgtcctagct-3 ) and r ( 5-ctcatccctctcatgctgtc-3 ) were used in the pcr procedure , which yield a 809 bp fragment . the pcr solutions contained 100 ng genomic dna , 10 pcr buffer , 10 pmol of each primers , 10 nmol of each deoxyribonucleotide triphosphates , 1.5 mmol mgcl2 and 1 u taq polymerase in a final volume of 25 l . initial denaturation at 95 c for 2 min was followed by 35 cycles of denaturation at 94 c for 30 sec and annealing at 59 c for 30 sec , an extension at 72 c for 30 sec and a final extension at 72 c for 5 min . the pcr products were separated on 1% agarose gel and visualized with ethidium bromide staining . the amplified products were analyzed using direct sequencing method ( macrogen , south korea ) . we used chromas software to analyze the chromatograms and then the sequencing results were compared with the human genome database and genbank . negative samples for gjb2 gene mutations were tested for linkage analysis of dfnb3 , dfnb9 and dfnb21 loci using str markers shown in table 1 . based upon strs physical distance in ncbi map viewer and ncbi unists , we selected the strs and their primers . a panel of 11 different str markers was genotyped for dfnb3 , dfnb9 and dfnb21 loci , according to the selection criteria including greater heterozygosity values , shorter amplicon and locating near the known locus . the pcr solutions contained 100 ng genomic dna , 10pcr buffer , 10 pmol of each primers , 10 nmol of each deoxyribonucleotide triphosphates , 1.5 mmolmgcl2 and 1u taq polymerase in a final volume of 25 l . a two - step touchdown thermal cycling was designed for each str markers based on the tm of both primers . pcr was started with an initial denaturation step 94 c for 5 min was followed by 7 cycles of denaturation ( 94 c , 15 sec ) , annealing ( 60 c , 40 sec ) and extension annealing ( 72 c , 40 sec ) followed by a final 25 cycles of denaturation ( 96 c , 40 sec ) , annealing ( 58 c , 40 sec ) , and extension ( 72 c , 40 sec ) and final extension at 72 c for 10 min . finally , the pcr products were separated by electrophoresis procedure on 10% polyacrylamide gels stained by silver nitrate . from sixteen families ( 85 people ) who enrolled in this study , 41 individuals had arnshl ( 48.23% ) . the subjects divided into moderate ( 4160 db ) , severe ( 6180 db ) and profound ( 80 db ) hearing impairment according to world health organization ( who ) classification of hearing loss . the frequencies of the moderate , severe and profound hearing loss among affected subjects were 9.75% ( n=4 ) , 63.25% ( n=26 ) , and 26.8% ( n=11 ) respectively . we found two homozygous pathogenic variants in exon 2 of the gjb2 gene including 283 g > a and 29delt mutations in two studied families ( 12.50% ) . results of gjb2 gene sequencing a ) chromatogram of a wild type of gjb2 gene , b ) chromatogram of 283g > a mutation of gjb2 gene we failed to find linkage of the dfnb3 , dfnb9 and dfnb21 loci among fourteen gjb2 negative families . if at least three str markers ( fully informative ) of a dfnb locus did not show homozygosity among the affected individuals of a family , then the locus would be determined unlinked . all members of gjb2 negative families , including parents , deaf and healthy siblings were heterozygous for all studied str markers . as a sample , fig . 2 shows the pcr products for d2s2223 marker of dfnb9 locus on 10% polyacrylamide gel . ra families pcr products for d2s2223 marker of dfnb9 locus on 10% polyacrylamide gel , dna ladder 50bp were used . left to right : 1 father , 2 mother , 3 negative control , 4 and 5 deaf children and 6 a healthy child ; the result showed that all bands are heterozygous . pedigree of ra family and genotyping of str markers ( locus dfnb9 ) ( ldb map ) from sixteen families ( 85 people ) who enrolled in this study , 41 individuals had arnshl ( 48.23% ) . the subjects divided into moderate ( 4160 db ) , severe ( 6180 db ) and profound ( 80 db ) hearing impairment according to world health organization ( who ) classification of hearing loss . the frequencies of the moderate , severe and profound hearing loss among affected subjects were 9.75% ( n=4 ) , 63.25% ( n=26 ) , and 26.8% ( n=11 ) respectively . we found two homozygous pathogenic variants in exon 2 of the gjb2 gene including 283 g > a and 29delt mutations in two studied families ( 12.50% ) . results of gjb2 gene sequencing a ) chromatogram of a wild type of gjb2 gene , b ) chromatogram of 283g > a mutation of gjb2 gene we failed to find linkage of the dfnb3 , dfnb9 and dfnb21 loci among fourteen gjb2 negative families . if at least three str markers ( fully informative ) of a dfnb locus did not show homozygosity among the affected individuals of a family , then the locus would be determined unlinked . all members of gjb2 negative families , including parents , deaf and healthy siblings were heterozygous for all studied str markers . as a sample , fig . 2 shows the pcr products for d2s2223 marker of dfnb9 locus on 10% polyacrylamide gel . the pedigree of one of the unlinked family is shown in fig . ra families pcr products for d2s2223 marker of dfnb9 locus on 10% polyacrylamide gel , dna ladder 50bp were used . left to right : 1 father , 2 mother , 3 negative control , 4 and 5 deaf children and 6 a healthy child ; the result showed that all bands are heterozygous . pedigree of ra family and genotyping of str markers ( locus dfnb9 ) ( ldb map ) our results showed that gjb2 mutations ( 283g > a and 29delt ) were causes of hearing loss in 12.5% of families with arnshl and no evidence of linkage were found for any of dfnb3 , dfnb9 and dfnb21 loci . due to the large number of dnfb loci , direct identification of arnshl genetic background is laborious . however , previous literatures have suggested that dfnb1 ( gjb2 ) , dfnb3 ( myo15a ) , dfnb4 ( slc26a4 ) , dfnb7/11 ( tmc1 ) , dfnb9 ( otof ) and dfnb21 ( tecta ) loci are the most common cause of arnshl in various populations ( 8 , 12 ) . therefore , we investigated the mutations of gjb2 gene and linkage of dfnb3 , dfnb9 and dfnb21 loci in gjb2 negative families with arnshl in south of iran for the first time . we found that gjb2 gene mutations ( 283g > a and 29delt ) were associated with deafness in 2 out of 16 families ( 12.50% ) . our results are in accordance with the findings of previous studies published in this field ( 19 ) . the rates of gjb2 gene mutations in iranian patients with arnshl were 11%18.3% ( 19 , 20 ) . the gjb2gene mutations are ethnic - specific and the 35delg mutation prevalent in caucasians , 167delt in ashkenazi jews , and w24x in pakistan and indian population ( 10 , 21 , 22 ) . by contrast , the 35delg and r127h mutations in gjb2 gene are prevalent in iranian population ( 19 ) . though , the population they had studied was ethnically different from the south iranian population . we failed to find linkage of the dfnb3 , dfnb9 and dfnb21 loci among gjb2 negative families . similar to our findings , two studies reported that myo15a gene mutations are not associated with arnshl ( 17 , 23 ) . however , several reports revealed the association between myo15a gene mutations and hearing loss ( 2427 ) . in a study 8 out of 140 families ( 5.71% ) linked to the dfnb3 locus ( 9 ) . in a recent study on 40 iranian arnshl families from qom and markazi provinces , in addition , previous studies have reported that the myo15a gene mutations accounts for 5% and 5.5% of arnshl in pakistani and iranian population respectively ( 29 , 30 ) . dfnb9 ( otof ) is mapped to 2p23.3 and no family was linked to this locus in our study . similarly , in a study on 36 large arnshl families no linkage was found to dfnb9 ( 31 ) . conversely , the frequency of otof gene mutations have reported 2.3% ( 13/557 ) in pakistani families ( 32 ) , 5.0% in turkish ( 24 ) , 1.4% ( 1/73 ) in chinese ( 33 ) and 2.7% in iran ( 17 ) . in accordance to a study ( 28 ) we were not found evidence of linkage for dfnb21 ( tecta ) in our population . previous several studies on iranian families have reported different frequencies of dfnb21 mutations in various ethnics 2.76.6% ( 17 , 34 ) . gjb2 gene mutations affected 12.50% of families with arnshl in our population . in contrast to several previous studies , we failed to find linkage of the dfnb3 , dfnb9 and dfnb21 loci among gjb2 negative families . this may be a consequence of racial differences populations or using low number of subjects in the present study . in spite of the no linkage reported in this study , possible linkage of dfnb3 , dfnb9 and dfnb21 loci , and arnshl therefore , it seems that further studies on large - scale population will be needed to conclusively find linkage of dfnb3 , dfnb9 and dfnb21 loci , and arnshl . ethical issues ( including plagiarism , informed consent , misconduct , data fabrication and/or falsification , double publication and/or submission , redundancy , etc . ) have been completely observed by the authors .
background : autosomal recessive non - syndromic hearing loss ( arnshl ) is the most common hereditary form of deafness , and exhibits a great deal of genetic heterogeneity . so far , more than seventy various dfnb loci have been mapped for arnshl by linkage analysis . 
 the contribution of three common dfnb loci including dfnb3 , dfnb9 , dfnb21 and gap junction beta-2 ( gjb2 ) gene mutations in arnshl was investigated in south of iran for the first time.methods:in this descriptive study , we investigated sixteen large families with at least two affected individuals . after dna extraction , gjb2 gene mutations were analyzed using direct sequencing method . 
 negative samples for gjb2 gene mutations were analyzed for the linkage to dfnb3 , dfnb9 and dfnb21 loci by genotyping the corresponding short tandem repeat ( str ) markers using polymerase chain reaction ( pcr ) and polyacrylamide gel electrophoresis ( page ) methods.results:gjb2 mutations ( 283g > a and 29delt ) were causes of hearing loss in 12.5% of families with arnshl and no evidence of linkage were found for any of dfnb3 , dfnb9 and dfnb21 loci.conclusion:gjb2 mutations are associated with arnshl . we failed to find linkage of the dfnb3 , dfnb9 and dfnb21 loci among gjb2 negative families . 
 therefore , further studies on large - scale population and other loci will be needed to find conclusively linkage of dfnb loci and arnshl in the future .